CHEMICAL AND BIOLOGICAL EVALUATION OF MEDICINAL …
Transcript of CHEMICAL AND BIOLOGICAL EVALUATION OF MEDICINAL …
CHEMICAL AND BIOLOGICAL EVALUATION OF
MEDICINAL PLANTS FOR THEIR ANTI
CARCINOGENIC ATTRIBUTES
By
AISHA ASHRAF
M.phil. (UAF)
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSPHY
IN
CHEMISTRY
DEPARTMENT OF CHEMISTRY
FACULTY OF SCIENCES
UNIVERSITY OF AGRICULTURE, FAISALABAD
PAKISTAN
2015
Declaration I hereby declare that contents of the thesis ―Chemical and biological evaluation of medicinal plants
for their anti carcinogenic attributes are product of my own research and no part has been copied from
any published source (except the references, standard mathematical or genetic modals/ equations/
formulae/ protocols etc). I further declare that this work has not been submitted for award of any
other diploma/ degree. The University may take action if the information provided is found inaccurate
at any stage (in case of any default, the scholar will be proceeded against as per HEC plagiarism
policy).
Aisha Ashraf
2004-ag-414
Dedicated to
My Loving Mother
My Affectionate Father
My Worthy Hazrat Abdul
Haq Haqqani (Damut Brkaat Hm)
iii
Acknowledgements I have no words to thank Almighty ALLAH, The beneficent, The merciful, The most
gracious, WHO is the entire and only source of knowledge and wisdom endowed to mankind
and who blessed me with the ability to do this work. It is the blessing of Almighty ALLAH
an His Prophet Hazrat Muhammad (Sallallaho Alaihe Wasallam) which enabled me to
achieve this goal. I would like to take this opportunity to convey my cordial gratitude and appreciation to my
supervisor Dr. Raja Adil Sarfraz, Assistant Professor, Department of Chemistry and
Biochemistry/ Incharge Central Hi-Tech Lab, University of Agriculture, Faisalabad. Without
whose keen interest, constant help and vigilant guidance, the completion of this thesis was
not possible. I am really indebted to him for his though provoking guidance, immense
intellectual input, patience and sympathetic behavior.
I would like to pay my deepest gratitude to members of my supervisory committee, Dr.
Muhammad Abid Rashid, Assistant Professor and Dr. Muhammad Shahid, Associate
Professor, Department of Chemistry and Biochemistry, University of Agriculture,
Faisalabad, for their valuable suggestions and kind help during accomplishment of my Ph.D.
I am also very much thankful to Higher Education Commission (HEC), Pakistan, for
awarding me the Indigenous PhD Fellowship and foreign scholarship under International
Research Support Initiative Program (IRSIP) to carry out part my research work in The
University of Texas, M.D. Anderson Cancer Center, Houston, Texas, United States of
America under kind supervision of Dr. Bharat B Aggarwal.
Words are too debilitate to express my appreciation and deep sense of gratitude to Fouzoa
Mohammad (Dental Hygenist) and Muhammad Abrar Sanaullah (Mechanical
Engineer), Houston, Texas, for their loving, caring and understanding behviour with me
during my IRSIP stay in USA. Their invigorating encouragement and affectionation can
never be repaid. Truly saying, I have no suitable words to express my gratitude them.
Special thanks to my fellows especially, Bazgah Ahmad, Qurat-ul-ain, Fiaza
Nazir, Maryum Aslam, Nadia Noor, Moin ud Din and Ayesha Mahmood for their
assistance, good company and friendly attitude.
I am deeply grateful to my worthy Hazrat Abdul Haq Haqqani (Damut Barkaat
Hm) for His prayers and strong moral support in my life matters and during the conduct of
my Ph.D. I might not have courage to complete my degree without His magnificent moral
support.
Words seem to be meaningless to express my obligation to my Affectionate Parents,
for their huge sacrifice, moral support, encouragement, patience, tolerance and prayers for
my health and success who enabled me to achieve this goal. I owe to thank my loving brother
Dr. Adnan Ashraf, for his moral support, encougement during conduct of my Ph.D and
especially the prayers during foriegn evaluation and publication of my thesis. I am thankful
to my younger borther Farhan Ashraf, who helped me a lot in my computer work, without
him it was not possible to complete my thesis. I am thankful to my dearest sister Rabia
Ashraf, who shared my responsibilities and did not burden me during my Ph D. My special
regards for my aunt Vajiha Irum and Uncle Dr. Muhammad Naeem Akhtar for their
encouragement during the studies.
Aisha Ashraf
Contents Chapters Titles Page
No.
Chapter 1 Introduction 1-6
1.1 History of medicinal plants 1
1.2 Traditional uses of medicinal plants 1
1.3 Therapeutic compounds from medicinal plants in modern era 2
1.4 Oxidation reactions and their side effects 2
1.5 Antioxidants and their functions 2
1.6 Need for natural antioxidants 3
1.7 Alarming death rate due to infectious diseases 3
1.8 Limitation of modern antibiotics 3
1.9 Need for natural antimicrobial agents 3
1.10 Cancer 4
1.11 Motives of cancer 4
1.12 Conventional cancer therapies and their side effects 4
1.13 Use of plants in anticancer activity 5
1.14 Medicinal flora of Pakistan 5
1.15 Need for project 5
1.16 Amis and objectives 6
Chapter 2 Review of literature 7-35
2.1 Euclayptus camaldulensis 7
2.1.1 Ethno botanical description 7
2.1.2 Distribution 7
2.1.3 Ethno medicinal application 7
2.1.4 Chemical evaluation 8
2.1.5 Biological evaluation 11
2.2 Viola betonicifolia 13
2.2.1 Ethno botanical description 13
2.2.2 Distribution 13
2.2.3 Ethno medicinal application 13
2.2.4 Chemical evaluation 13
2.2.5 Biological evaluation 15
2.3 Euphorbia royleana 17
2.3.1 Ethno botanical description 17
2.3.2 Distribution 17
2.3.3 Ethno medicinal application 18
2.3.4 Chemical evaluation 19
2.3.5 Biological evaluation 20
2.4 Psidium guajava 21
2.4.1 Ethno botanical description 22
2.4.2 Distribution 22
2.4.3 Ethno medicinal application 22
2.4.4 Chemical evaluation 23
2.4.5 Biological evaluation 26
2.5 Ziziphus mauritiana 29
2.5.1 Ethno botanical description 29
2.5.2 Distribution 30
2.5.3 Ethno medicinal application 30
2.5.4 Chemical evaluation 30
2.5.5 Biological evaluation 31
Chapter 3 Materials and methods 36-50
3.1 Materials 36
3.1.1 Chemicals and reagents 36
3.1.2 Instruments 37
3.1.3 Collection of plant materials 37
3.1.4 Plants used in current study 38
3.1.5 Human cancer cell lines employed to access the anticancer potential of
plant extracts
39
3.1.6 Strains of microorganisms used to esetimate the antimicrobial activity
of plant extracts
41
3.2 Extraction of dried plant material 41
3.3 Extraction of fresh plant material 42
3.4 Chemical evaluation 42
3.4.1 Total phenolic contents 42
3.4.2 Total flavonoid contents 43
3.4.3 High performance liquid chromatography analysis 43
3.4.4 Gas chromatography mass spectrometry 44
3.5 Biological evaluation 44
3.5.1 Free radical scavenging activity (DPPH assay) 44
3.5.2 Antitumour activity (Potato disc assay) 45
3.5.3 Antimicrobial activity (Disc diffusion assay) 46
3.6 Anticancer attributes of plant extracts 47
3.6.1 Anticancer activity (MTT assay) 49
3.6.2 Anti-inflammatory activity (Electrophoretic mobility shift assay) 49
3.7 Statistical analysis 50
Chapter 4 Results and discussions 51-111
4.1 Euclayptus camaldulensis 51
4.1.(a) Chemical evaluation 51
4.1.1 Total phenolic acnd total flavnoid contents of E. camaldulensis 51
4.1.2 High performance liquid chromatographic (HPLC) analysis of E.
camaldulensis
53
4.1.3 Gas chromatography mass spectrometric (GC-MS) study of E.
camaldulensis
54
4.1.(b) Biological evaluation 57
4.1.4 Free radical (DPPH) scavenging activity of E. camaldulensis 57
4.1.5 Antitumor activity of E. camaldulensis 58
4.1.6 Antimicrobial activity of E. camaldulensis 60
4.2 Viola betonicifolia 61
4.2.(a) Chemical evaluation 61
4.2.1 Total phenolic acnd total flavnoid contents of V. betonicifolia 61
4.2.2 High performance liquid chromatographic (HPLC) analysis of V.
betonicifolia
63
4.2.3 Gas chromatography mass spectrometric (GC-MS) study of V.
betonicifolia
64
4.2.(b) Biological evaluation 69
4.2.4 Free radical (DPPH) scavenging activity of V. betonicifolia 69
4.2.5 Antitumor activity of V. betonicifolia 71
4.2.6 Antimicrobial activity of V. betonicifolia 72
4.2.7 Anticancer activity of V. betonicifolia 73
4.2.8 Anti-inflammatory activity of V. betonicifolia 76
4.3 Euphorbia royleana 78
4.3. (a) Chemical evaluation 78
4.3.1 Total phenolic acnd total flavnoid contents of E. royleana 78
4.3.2 High performance liquid chromatographic (HPLC) analysis of E.
royleana
80
4.3. (b) Biological evaluation 81
4.3.3 Free radical (DPPH) scavenging activity of E. royleana 81
4.3.4 Antitumor activity of E. royleana 82
4.3.5 Antimicrobial activity of E. royleana 83
4.4. Psidium guajava 85
4.4.(a) Chemical evaluation 85
4.4.1 Total phenolic acnd total flavnoid contents of P. guajava 85
4.4.2 High performance liquid chromatographic (HPLC) analysis of P.
guajava
86
4.4.3 Gas chromatography mass spectrometric (GC-MS) study of P. guajava 87
4.4. (b) Biological evaluation 90
4.4.4 Free radical (DPPH) scavenging activity of P. guajava 90
4.4.5 Antitumor activity of P. guajava 91
4.4.6 Antimicrobial activity of P. guajava 92
4.4.7 Anticancer activity of P. guajava 94
4.4.8 Anti-inflammatory activity of P. guajava 97
4.5 Ziziphus mauritiana 99
4.5.(a) Chemical evaluation 99
4.5.1 Total phenolic acnd total flavnoid contents of Z. mauritiana 99
4.5.2 High performance liquid chromatographic (HPLC) analysis of Z.
mauritiana 101
4.5.3 Gas chromatography mass spectrometric (GC-MS) study of Z.
mauritiana
102
4.5.(b) Biological evaluation 104
4.5.4 Free radical (DPPH) scavenging activity of Z. mauritiana 104
4.5.5 Antitumor activity of Z. mauritiana 106
4.5.6 Antimicrobial activity of Z. mauritiana 107
4.5.6 Anticancer activity of Z. mauritiana 108
Chapter 5 Summary 112-115
Refrences 116-167
List of Tables
Serial
No.
Title Page
No.
2.1 Taxonomic classification of E. camaldulensis 9
2.2 Taxonomic classification of V. betonicifolia 14
2.3 Taxonomic classification of E. royleana 18
2.4 Taxonomic classification of P. guajava 23
2.5 Taxonomic classification of Z. mauritiana 32
3.1 Instruments used with their model and company 37
4.1.1 HPLC analysis of methanol, chloroform and hexane extracts of E.
camaldulensis for identification and quantification of phenolic compounds
54
4.1.2 Chemical components of methanol, chloroform and hexane extracts of E. camaldulensis analyzed by GC-MS
56
4.1.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of E. camaldulensis
58
4.1.4 Antimicrobial activity of methanol, chloroform and hexane extracts of E. camaldulensis
61
4.2.1 HPLC analysis of methanol, chloroform and hexane extracts of V.
betonicifolia for identification and quantification of phenolic compounds 64
4.2.2 Chemical components of methanol, chloroform and hexane extracts of V.
betonicifolia analyzed by GC-MS
65
4.2.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of V. betonicifolia
70
4.2.4 Antimicrobial activity of methanol, chloroform and hexane extracts of V. betonicifolia
72
4.3.1 HPLC analysis of methanol, hexane and aqueous extracts of E. royleana for identification and quantification off phenolic compounds
80
4.3.2 Scavenging (%) of stable free radicals (DPPH) by methanol, hexane and aqueous extracts of E. royleana
82
4.3.3 Antimicrobial activity of methanol, hexane and aqueous extracts of E. royleana
84
4.4.1 HPLC analysis of methanol, chloroform and hexane extracts of P. guajava for identification and quantification off phenolic compounds
87
4.4.2 Chemical components of methanol, chloroform and hexane extracts of P. guajava analyzed by GC-MS
88
4.4.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of P. guajava
91
4.4.4 Antimicrobial activity of methanol, chloroform and hexane extracts of P. guajava
93
4.5.1 HPLC analysis of methanol, hexane and chloroform extracts of Z. mauritiana for identification and quantification off phenolic compounds
101
4.5.2 Chemical components of methanol, chloroform and hexane extracts of Z. mauritiana analyzed by GC-MS
103
4.5.3 Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform and hexane extracts of Z. mauritiana
105
4.5.4 Antimicrobial activity of methanol, chloroform and hexane extracts of Z. mauritiana
107
List of Figures
Serial Title Page
No. No.
3.1 Photographs of indigenous medicinal plants used in current study 39
3.2 Human cancer cell lines used in current research work 40
3.3 A typical agar plate showing the antimicrobial activity of plant extracts 47
4.1.1 Total phenolic and total flavonoid contents of methanol, chloroform and 52
hexane extracts of E. camaldulensis
4.1.2 Antitumor activity of methanol, chloroform and hexane extracts of E.
59
camaldulensis
4.2.1 Total phenolic and total flavonoid contents of methanol, chloroform and 62
hexane extracts of V. betonicifolia
4.2.2 Antitumor activity of methanol, chloroform and hexane extracts of 73
V.betonicifolia
4.2.3 Anticancer activity Of (A) methanol, (B) chloroform and (C) hexane 76
extracts of V. betonicifolia leaves against KBM5, SCC4 and HCT116 cells
4.2.4 Anti-inflammatory activity of chloroform extract of V. betonicifolia 77
4.3.1
Total phenolic and total flavonoid contents of methanol and hexane extracts
of E. royleana 79
4.3.2 Antitumor activity of methanol and hexane extracts of E. royleana 83
4.4.1 Total phenolic and total flavonoid contents of methanol, chloroform and 86
hexane extracts of P. guajava
4.4.2 Antitumor activity of methanol, chloroform and hexane extracts of P. 92
guajava
4.4.3 Anticancer activity of (A) methanol, (B) chloroform and (C) hexane 96
extracts of P. guajava leaves against KBM5, U266 and HCT116 cells
4.4.4 Anti-inflammatory activity of hexane extract of P. guajava 98
4.5.1 Total phenolic and total flavonoid contents of methanol, chloroform and 100
hexane extracts of P. guajava
4.5.2 Antitumor activity of methanol, chloroform and hexane extracts of Z. 106
mauritiana
4.5.3 Anticancer activity of (A) methanol, (B) chloroform and (C) hexane 110
extracts of Z. mauritiana leaves against KBM5, U266 and SCC4 cells
Abstract
Cancer is terrible disease. It is second leading cause of mortality worldwide.
Conventional cancer therapies burdened the disease crippled patient with toxic side effects and
are also very expensive. Therefore, the demand to use alternative approaches in treatment of
cancer is increasing. Plant derived compounds due to their unique structure and sophisticated
mechanism of action play promising role in anticancer therapies. The main objective of current
study was to evaluate anticancer potential of medicinal flora of Pakistan.
The plants used in current study are frequently utilized in folk medicines for treatment of
many ailments in Pakistan. In present research work these traditional medicinal plants were
scientifically examined for their antioxidant, antitumor, antimicrobial, anticancer and anti-
inflammatory potential. Furthermore, chemical composition of the plant extracts was evaluated
by state of art chromatographic techniques such as UV/Vis spectrophotometer, HPLC and GC-
MS, revealed the presence of phenolics, flavonoids and broad range of other bioactive
components in them. Antioxidant activity was estimated by free radical scavenging activity
estimated by DPPH (1,1- diphenyl 2-picrylhydrazyl) assay. The outcome of antimicrobial
activity assay showed that E. coli was the resistant strain to most of tested plant extracts. Overall,
among the fungal strains, A. niger was the sensitive one. Furthermore, we examined antitumor
activity by potato disc assay. Chloroform extract of V. betnocifolia (IC50= 38.13 µg/mL)
exhibited maximum antitumor activity. We determined anticancer activity of different plant
extracts against human cancer cell lines (KBM, mylegeous leukemia; U266, multiple myeloma;
SCC4, tongue squamous carcinoma and HCT116, colon carcinoma cells) by MTT assay. The
extracts with maximum anticancer activity against human mylegeous leukemia (KBM5) cells
were examined for inhibition of inflammatory transcription factor, nuclear factor kappa B (NF-
kB). Surprisingly all the plant extracts inhibited TNF-α induced NF-kB activation.
1
CHAPTER 1 INTRODUCTION
1.1. History of medicinal plants
Plants are the earliest companion of mankind. They serve humanity chiefly by
granting food, shelter, oxygen and medicinal compounds (Mamedov, 2012). They have been
employed for medical issues since 60¸000 years ago. There are ample evidences from Indian,
Arabian, Chinese, Roman and other traditions about the use of medicinal plants to fulfill
health needs (Bensoussan et al., 1998; Saad et al., 2005; Mazid et al., 2012). This traditional
herbal knowledge had been conveyed to next generations, mainly by oral communications
(Shinwari, 2010). However, written documents, some original herbal medicines and
preserved monuments revealed the use of herbal remedies by ancient civilizations (Petrovska,
2012). In India the first oldest written herbal recipes were found about 5000 years ago,
engraved on Sumerian clay slab (Kelly, 2009).
250,000 species of medicinal plants persist on earth, out of which more than one
thousand plant species are assessed to have significant anticancer activity (Mukherjee et al.,
2001), but there is still huge amount of work to be done on these lines. Pivotal role of plants
in health scenario is attributed to assets of bioactive compounds in them (Jagadish et al.,
2009). Phytochemical components of plants include immense array of compounds which
might impediment or inhibit the inception of degenerative ailments (Robard et al., 1999;
Guilford and Puzetto, 2008; Mehta et al., 2010).
1.2. Traditional uses of medicinal plants
Traditionally plants had been used in natural remedies as teas, tinctures or powders
(Samuelsson, 2004). Traditional therapies are still on peak practice in many regions of world
(Abe and Ohtani, 2013). Most of the people in developing countries prefer herbal remedies,
due to their low cost, easy availability and safe mode of action. World Health Organization
(WHO) witnessed that 80% of population in Asian and African countries relies on herbal
formulations to fulfill their health needs (Kim et al., 2012).
2
1.3. Therapeutic compounds from plants in modern era
In modern era potential therapeutic compounds such as vincristine, camptothecin,
navellbine, etoposide (Pezzuto, 1997), taxol, vinblastine (Flescher, 2007), doxorubicin
(Norris et al., 2000), mechlorethamine (Vonderheid et al., 1989), teniposide (Postmus et al.,
2000), topotecan (Gordon et al., 2001), casticin (Shen et al., 2009), proanthocyanidin B2
(Avelar and Gouvea, 2012), cirsimaritin (Quan et al., 2010), protoapigenone (Chang et al.,
2008), apigenin (Yin et al., 2001), androsacin, curcumin, xanthohuskiside A, genistein,
resveratrol, gallocatechin gallate, lycopene, flavopiridol, anethole, anthroquinone, chrysin,
eugenol, epicatechin, cinnamic acid, ellagitannin, catechol and many others have been
isolated from medicinal plants and are used in targeted therapies against cancer,
inflammation and microbial infections (Norris et al., 2000; Gupta et al., 2011). In addition to
leading role in health scenario they are also used as potential agent in food preservation,
neutraceuticals, pharmaceuticals, fragrances, natural therapies, functional foods, cosmetics
(Dung et al., 2008).
1.4. Oxidation reactions and their side effects
Oxidation reactions in mitochondria bestow energy for biological processes that act
as soul of cell life, but on the other hand oxidant and oxidation reactions are major
contributors to induce many pathological conditions (Dalleau et al., 2013). When production
of reactive oxygen species is higher in body as compared to enzymetic and non-enzymetic
antioxidants then this imbalance leads to cell damage and escort a vast range of degenerative
diseases including cancer, atherosclerosis, hypertension, AIDS, parkinsonism, inflammation,
aging, cardiovascular diseases (Li et al., 2009; Makasci et al., 2010) and microbial infections
(Samec et al., 2010; Steer et al., 2002; Peuchant et al., 2004).
1.5. Antioxidants and their function
Antioxidants are ubiquitous components that can hinder the oxidation process by
adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or
decomposing peroxides (Khadri et al., 2010). In addition to leading role in health scenario
they are also used as potential agent in food preservation, neutraceuticals, pharmaceuticals,
fragrances, functional foods, and cosmetics (Dung et al., 2008).
3
1.6. Need for natural antioxidants
Synthetic antioxidants such as butylated hydroxyltoulene (BHT), butylated
hydroxylanisole (BHA), propyl gallate and citric acid are frequently used in processed foods
and medicinal materials (Ebrahimabadi et al., 2010). Nutritionists and toxicologists dampen
the use of synthetic antioxidants because of reported side effects such as liver enlargement,
increase microsomal enzyme activity and carcinogenesis (Gulcin et al., 2004; Dung et al.,
2008). Therefore, the quest for exploring antioxidants from natural sources have received
great attention all over the world (Oktay et al., 2003; Dalal et al., 2010). Thus in current
study effort was done to evaluate antioxidant potential of plants.
1.7. Alarming death rate due to infectious diseases
Infectious diseases are main cause of high mortality rate in developing countries.
About 50,000 people are dying worldwide every day because of infectious disease (Ahmad
and Beg, 2001).
1.8. Limitation of modern antibiotics
Although pharmaceutical industries have introduced wide range of novel antibiotics
in last few years, but resistance to these antibiotics has been boosted by microorganisms and
it has become alarming situation globally (Nascimento et al., 2000). The global emergence of
multidrug bacteria is restraining the efficacy of modern drugs and ultimately causes the
failure of treatment.
1.9. Need for natural antimicrobial agents
As the resistance to antibiotics had been increased by microorganisms, so there is an
intense need to explore alternative antimicrobial agents. Affirmative effects of plant food are
attributed chiefly to innovative profile of bioactive components in them (Cai et al., 2004).
Plant derived bioactive compounds are used not only for treatment of infections but also to
dampen microflora growth in food, and ultimately increase the expectation of life. Thus in
present study attempts were made to investigate antimicrobial efficacy of plants.
4
1.10. Cancer
Cancer is lethal disease. It is second leading cause of mortality worldwide
characterized by invasion, apoptotic death, angiogensis, overproliferation of cells,
dysregulayion of cell signaling pathways and metastasis (Aggarwal et al., 2006). Cancer cells
may assault tissues which are in close proximity and may broaden through blood flow and
lymphatic scheme to other parts of body (Nagarani et al., 2011).
It causes more than six million deaths each year in the world (Nisa et al., 2011).
Consistent with global cancer statistics issued by the American Cancer Society, daily 20,000
deaths were reported worldwide from cancer, in 2007. 27 million new cancer cases are
predicted in the world up to 2050 (American Cancer Society, 2007).
1.11. Motives of cancer
Chief motives of cancer are viruses, environmental exposure (e.g. Ionizing and UV
radiations), life style factors (e.g. high calorie diets, excessive use of tobacco), medication
(e.g. alkylating agents, immunosuppressants) and genetic factors, e.g. inherited mutations,
cancer causing genes (Dhanamani et al., 2011). There are ample evidences that inflammation
particularly the chronic inflammation plays critical role in cancer (Elinav et al., 2013). In
addition to inflammation, different NF-κB regulated genes are associated with cellular
transformation, proliferation, angiogensis, invasion, tumor cell survival and metastasis
(Aggarwal et al., 2004). Another study reported tumor promoting role of NF-kappaB in
inflammation based cancer (Pikarsky et al., 2004). Thus suppression of NF-κB pathway will
play distinctive role in cancer therapy (Gupta et al., 2011). The current research work was
conducted to examine influence of potent plant extracts on NF-κB signaling pathways.
1.12. Conventional cancer therapies and their side effects
Conventional modalities for treatment of cancer such as radiation therapy,
chemotherapy, immunodilution and surgery have limited success as evident by high
morbidity, toxicity, indiscriminate nature, permanent disfigurement (Nawab et al., 2011).
Therefore, the demand to use alternative approaches in treatment of cancer is increasing.
5
1.13. Use of plants in anticancer activity
Plant derived compounds due to their unique structure and sophisticated mechanism
of action play promising role in anticancer therapies.114,000 extracts from 35,000 plants had
been screened by National Cancer Institute (NCI) for anticancer drug discovery. Mounting
evidence has indicated use of plant extracts in clinical trials (Chrubasik et al., 2004). The
current study also deals with screening of plant extracts for their anticancer activity.
1.14. Medicinal flora of Pakistan
Pakistan is the sixth most populous country in the world. Flora of Pakistan is rich in
medicinal plants due to its diverse climatic and soil conditions and many ecological regions
(Ali et al., 2001). The country has about 6,000 species of flowering plants of which about
400 to 600 are medicinally imperative (Hamayun et al., 2005). A vast range of medicinal
plants is reported from different areas of Pakistan including Balochistan (Goodman and
Ghafoor, 1992), Swat and Chitral (Sher, 2001), Utror and Gabral Valleys (Hamayun et al.,
2005), Margalla Hills (Shinwari and Khan, 1999), Pirgarharh Hills, South Waziristan Agency
(Badshah et al., 1996), Cholistan desert (Ahmed et al., 2014), Kadhi areas of Khushab
(Qureshi et al., 2011), Nara Desert, Sindh (Qureshi and Bhatti, 2008) and many more areas
(Malik et al., 1990; Hamayun et al., 2003; Ahmad et al., 2009).
1.15. Need for project
Although a heap of work is done to explore chemical and biological potential of
medicinal plants, WHO is also spectator of 20,000 plant species studied for medicinal
purposes (Gulluce et al., 2006), but data is still insufficient and further information is needed
on multi functionalities of plants. Moreover, novel therapeutic applications of plant derived
products have impelled many researchers to explore pharmacological potential of plants to
increase life expectancy.
In continuation of our efforts (Ashraf et al., 2014; Ahmed et al, 2014) to explore
ethnomedicinal, phytochemical and bioactive potential of flora of Punjab, Pakistan, the
current study reports chemical and biological potential of plant materials indigenous to
Punjab, Pakistan. To the best of our knowledge, our study is first report on chemical
6
composition, antioxidant, antimicrobial, antitumor, anticancer and anti-inflammatory
activities of the investigated medicinal flora of Punjab, Pakistan.
1.16. Aims and objectives:
(i) Scientific assessment of traditional medicinal plants of Pakistan.
(ii) Investigation of plants with respect to variation in extracting solvents.
(iii) Chemical characterization of plant extracts using state of the art chromatographic
and spectrophotometric techniques.
(iv) Assessment of biological activities (antioxidant, antimicrobial and antitumor) of
plant extracts.
(v) Exploration of in vitro anticancer potential of selective plant extracts against
different human cancer cell lines.
(vi) Evaluation of potent extracts for inhibition of inflammatory nuclear factor kappa
B (NF-κB), which is chief mediator of carcinogenesis.
7
CHAPTER 2 REVIEW OF LITERATURE
2.1. Euclayptus camaldulensis:
2.1.1. Ethno botanical description:
Euclayptus camaldulensis is commonly known as ―Safeda‖ in Pakistan. It is
perennial, large, single-stemmed and evergreen tree. It is tall tree of 40 m height and 0.8 m
diameter. It has irregular branches which spread widely.
Leaves are present in alternate form on branches. Leaves are alternate, glabrous and
sickle like. They are 8-22 cm in length and 1-2 cm broad and are pale green in color. Roots
are very dispersal and deep.
Flowers are 5-10. They have white thread like stamens which are 5-6 mm in length.
Anthers are present in tiny encircling glands. Flowering intensity is changeable. Almost 45%
of flowers not succeed to reach maturity level (Dexter, 1978; Bren and Gibbs, 1986).
2.1.2. Distribution:
It is distributed in Pakistan (Shahwar et al., 2012), Kenya, Australia, Turkey (Basak
and Candan, 2010), North Africa (Ei-Ghorab et al., 2003), Senegal, Spain, Morocco,
Arizona, Sierra Leone, Egypt (El-Mageed et al., 2011), Iran (Gharekhani et al., 2012),
Argentina, California, Nigeria etc.
2.1.3. Ethno medicinal applications:
In Pakistan paste of E. camaldulensis stem and decoction of its leaves is traditionally
used for treatment of fever, sneezing, malarial shivering and cough (Qasim et al., 2014).
In addition to Pakistan, it is used all over the world in traditional therapies for
treatment of diarrhea, eczema, spasm, cold, hemorrhage, sore throat, stomach problems,
cough, vermifuge, boils, bronchitis, bladder infections, oral thrush, sores, athletes foot,
dysentery, skin infections, fever, asthma, laryngitis, trachagia, constipation and wound
infections (Bala, 2006; Lassack and Maccarthy, 2006; Musa et al., 2011; Ozturk et al., 2013).
8
2.1.4. Chemical Evaluation:
(i) Triterpenoids:
Siddiqui et al., 2000, reported one new triterpenoid from fresh leaves of E.
camaldulensis var. obtuse that was identified as amirinic acid. Maurya and Srivastava, 2012,
identified two triterpenoids (uroslic acid lactone and uroslic acid) in leaves of Euclayptus
tereticornis. Pentacyclic triterpenoids from other medicinal plants are also reported
(Alqahtani et al., 2013).
(ii) Polyphenols:
(a) Leaves:
Conde et al., 1997, investigated polyphenols in leaves of Euclayptus camaldulensis,
Euclayptus rudis and Euclayptus globulus and identified them as vanillin, ellagic acid, gallic
acid, protocatechuic acid, quercetin-3-arabinoside, rutin, quercetin-3,7-dirhamnoside,
kaempferol-3-arabinoside, and quercetin. Antibacterial activity of vanillin related hydrazone
derivatives was estimated by disc diffusion assay (Govindasami et al., 2011). Ellagic acid
exhibited photoprotective efficacy on collagen breakdown.Moreover, it prevents
inflammation in UV-B irradiated human skin cells (Bae et al., 2010). Gallic acid is known
for its antioxidant activities (Bhadonya et al., 2012).
Quercetin plays leading role against cancer, inflammation and allergy (Shaik et al.,
2006; Jagtap et al., 2009). Furthermore, the presence of flavones like apigenin and luteolin
was also examined. Apigenin plays promising role in cancer prevention (Shukla and Gupta,
2010).
(b) Bark:
Polyphenolic components from bark extracts of Euclayptus camaldulensis,
Euclayptus rudis and Euclayptus globules were reported (Conde et al., 1996). Gallic acid,
vanillic acid, protocatechuic acid, protocatechuic aldehyde and ellagic acid were found
common in three investigated species of Euclayptus.
9
Protocatechuic acid is narrated for its antioxidant efficacy (Li et al., 2011).
Protocatechuic aldehyde is documented for its anti-inflammatory, antioxidant and
neuroprotective efficacies (Chang et al., 2011; Zhao et al., 2013).
(c) Wood:
Conde et al., 1995, studied the polyphenolic components of wood extracts of
Euclayptus camaldulensis, Euclayptus rudis and Euclayptus globulus. For this purpose wood
of each of three Euclayptus species was extracted with methanol. Frequent use of methanol
for extraction is attributed to comparatively higher extraction of several bioactive
components in it (Akowuah et al., 2002; Bae et al., 2012; Caunii et al., 2012).
Table. 2.1. Taxonomic classification of E. camaldulensis
Taxonomic classification
Kingdom Plantae
Subkingdom Tracheobionta
Super division Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Myrtales
Family Myrtaceae
Genus Euclayptus
Species Euclayptus camaldulensis
10
Various compounds such as gallic acid, vanillin, vanillic acid, sinapaldehyde,
quercetin, syringaldehyde, naringenin and ellagic acid were detected. Syringaldehyde has
potent applications in food, cosmetic, pharmaceutical and textile industries (Ibrahim et al.,
2012).
(ii) Flavonoid glycosides:
Abd-Alla et al., 1986, identified flavonoid glycosides from leaves of E.
camaldulensis and named them as: kaempferol 3-glucoside, kaempfero 3-glucuronide,
apigenin 7-glucuronide, quercetin 3-glucoside, 3-glucuronide, 3-rutinoside, 3-rhamnoside
and quercetin 7-glucoside, luteolin 7-glucoside and luteolin 7-glucuronide. Flavonoid and
phenolic components from leaves of E. camaldulensis Dehn were extracted by using
microwave assisted extraction method (Gharekhani et al., 2012).
(iii) Volatile constituents:
Hydro distillation technique was used for isolation of volatile oil of fruit of E.
camaldulensis var. brevirostris (El-Ghorab et al., 2002). The volatile oil isolated from E.
camaldulensis were identified as aromadendrene, p-cymenene, cubenol,p-cymen-7-ol,
thymol, α-gurjunene and α-pinene.
Francisco et al., 2001, isolated oils from E. camaldulensis by two techniques:
supercritical CO2 extraction and hydro distillation. The oil attained by hydrodistillation
technique presented higher amounts of globulol , α-pinene, p-cymene, β-pinene, terpinen-4-
ol and 1,8-cineole. Previous study showed that p-cymene had efficacy against Escherichia
coli (Kisko et al., 2005). Biological potential of α and β-pinenes is well documented (Silva et
al., 2012).
(iv) Phytochemical components:
Adeniyi and Ayepola, 2008, screened Euclayptus torelliana and Euclayptus
camaldulensis leaf extracts for their phytochemical components. Presence of tannins, cardiac
glycosides and saponins was noticed in both the extracts.
11
Musa et al., 2011, reported the presence of tannin and saponin in water extract of E.
camaldulensis. Pyhtochemical screening of E. camaldulensis leaves showed the presence of
steroid, cardiac glycosides, volatile oils, saponin glycosides, tannins, phenols and saponins
(Babayi et al., 2004). Previous studies indicated the use of saponins in preparation of folk
medicines (Asl and Hosseinzadeh, 2008).
2.1.5. Biological evaluation:
(i) Antimicrobial activity:
Babayi et al., 2004, reported antimicrobial potential of E. camaldulensis leaf extract
against Bacillus subtilis, Staphylococcus aureus and Candida albicans. Essential oil of
Euclayptus citriodora was more effective against C. albicans than B. subtilis and S. aureus
(Luqman et al., 2008). Antimicrobial potential of E. camaldulensis may be attributed to
existence of bioactive components in it. Similar finding was reported by other scientists
(Puupponen-Pimia et al., 2000; Nohvnek et al., 2006).
Adeniyi and Auepola, 2008, extracted leaves of Euclayptus camaldulensis and
Euclayptus torelliana in methanol and examined antibacterial activity. Another research
group (Tambehar and Dahikar, 2011) reported the similar antibacterial potential of Indian
medicinal plants. Aljebourey, 2014, observed that Euclayptus microtheca extracts also have
potent activity against P. aeruginosa.
Antibacterial activity of E. camaldulensis water extract at various concentrations
12.5, 25 and 50 mg/mL was also explored against six bacterial strains. A direct link was
observed between concentration of plant extract and inhibition zone.
It is interesting to note that Akgul and Kaya, 2004, narrated the analogous behavior
for Turkish medicinal plants. Among the tested bacterial strains Salmonella typhi was the
most sensitive strain. These results were different from previous findings (Shuaib et al.,
2013).
(ii) Anticancer activity:
12
Hrubik et al., 2012, extracted E. camaldulensis leaves of ethyl acetate, methanol,
water and n-butanol. Maximum anticancer activity by MTT assay was observed against
MDA-MB-231 and MCF-7 with IC50 values of 26.7 and 34.4 µg/ml ethyl acetate extract.
Some previous studies also reported anticancer potential of natural products against MDA-
MB-231 and MCF-7 cell lines (Rahman et al., 2011; Lin et al., 2013).
Anticarcinogenic potential of leaves of E. camaldulensis extracted with aqueous
acetone was evaluated against MCF-7, HeLa, Hep-2, HCT-116, HepG-2 and Caco-2
carcinoma cells (Singab et al., 2011). The aqueous acetone extract decreased the growth of
carcinoma cells in a dose-dependent mode.
Another research group (Parsad et al., 2012) reported the similar relationship between
viability of cancer cells and dose of tested compound.
(iii) Antioxidant activity:
Singab et al., 2011, explored antioxidant activity of aqueous acetone extract of E.
camaldulensis leaves by DPPH, super oxide anion and hydroxyl radical scavenging tests.
High stable free radical (DPPH) scavenging activity was examined in methanol fraction.
These results were in accordance with previous findings (Sowndhararajan and Kang, 2013)
in which methanol extract of medicinal flora had exhibited stronger free radical scavenging
activities.
(iv) Spasmolytic activity:
Spasmolytic components of E. camaldulensis var obtuse leaves were reported by
Begum et al., 2000. The outcome of study revealed the isolation of one new and four already
known compounds. The new one was identified as triterpenoid camaldulin. The others were
known as ursolic acid lactone acetate, betulinic acid and ursolic acid lactone evaluated for
their spasmolytic potential.
Spasmolytic potential of other natural flora of Pakistan is also narrated in literature
(Gilani et al., 2008). Betulinic acid also plays potent role in programmed cell death (Tan et
al., 2003) human melanoma cells.
13
2.2. Viola betonicifolia:
2.2.1. Ethno botanical description:
Viola betonicifolia is known as ―Banafsha‖ in Pakistan. It is perennial herb which is 8
to 20 cm tall. It has no stem, leaves are basal, triangular, numerous with dark brownish
stipules. They have a smaller lamina than petiole.
Leaves are arrow shaped and deep green in color. Leaves may be up to 10 cm in
length. Flowers are light or deep purplish in color sometimes with lighter colored patches.
The flowers stand on a slender stalk. They have a long pedicel. Roots are slender and have no
branches.
2.2.2. Distribution:
It is distributed in Pakistan, Sri Lanka, India, Malaysia, Nepal, Australia and China
(Muhammad et al., 2012).
2.2.3. Ethno medicinal applications:
It is used in traditional therapies in different parts of world. In Pakistan it is
traditionally used as anticancer, astringent, febrifuge, diuretic, purgative, antipyretic and
diaphoretic. Furthermore, it is also used for treatment of bronchitis, boils, nervous disorders,
pneumonia, epilepsy, kidney diseases and lung troubles (Ilyas and Hamayun, 2005).
Traditional use of V. betonicifolia for treatment of kidney diseases, skin disorders,
pharyngitis, pneumonia, blood disorders and sinusitis is reported from different parts of
world (Bhatt and Negi, 2006; Tiwari et al., 2010).
2.2.4. Chemical evaluation:
Yang et al., 2011, identified fifteen chemical components of Viola tianshanica which
represented 89.67% of oil. The main components in essential oil of V. tianshanica were
hexadecanoate, dibutyl phthalate methyl, 2,3-pentanedione and palmitic acid. Muhammad et
al., 2013, for the first time reported a cinnamic acid derivative from V. betonicifolia in form
of off white needle. It was named as 2,4-dihydroxy, 5-methoxy-cinnamic acid.
14
Table. 2.2. Taxonomic classification of V. betonicifolia
Another research group (Akhbari et al., 2011) reported twenty five compounds in
essential oil of Viola odorat L. GC-MS study of Viola etrusca explored a wide range of
chemical components in which α-pinene, 2-pentyl furan, β-pinene, p-cymene, 1,8-cineole,
nonanal, camphor, borneol, methyl salicylate, farnesane and germacrene D were the potent
one (Flamini et al., 2003).
(i) Phytochemical components:
Phytochemical screening of V. betonicifolia revealed the presence of alkaloids,
flavonoids, saponins, tannins, phenolics and proteins in it (Muhammad et al., 2012). Total
phenolic and flavonoid contents were higher in ethyl acetate fraction of V. betonicifolia
followed by chloroform, butanol and aqueous fractions, respectively (Muhammad et al.,
2011).
Taxonomic classification
Kingdom Plantae
Subkingdom Tracheobionta
Super division Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Dilleniidae
Order Violales
Family Violaceae
Genus Viola
Species Viola betonicifolia
15
2.2.5. Biological evaluation:
(i) Antimicrobial activity:
Muhammad et al., 2013, described antimicrobial potential of crude methanol extract
and other fractions of V. betonicifolia. Antibacterial potential was estimated against seven
bacterial strains (Enterobacter aerogenes, Proteus mirabilis, Escherichia coli, Bacillus
cereus, Staphylococcus aureus, Salmonella typhi and Enterococcus fecalis).
However, in a recent scientific study, methanol extract of another specie of Violaceae
was found more potent against respiratory tract bacteria (Gautam et al., 2012). Viola tricolor
herb is also known for its antimicrobial efficacy (Witkowska-Banaszczak et al., 2005).
Aqueous fraction obtained from V. betonicifolia methanol extract was predominantly
active against C. albicans. Literature survey showed that some natural products have efficacy
to completely inhibit the growth of C. albicans (Manohar et al., 2001). C. albicans is very
common funal pathogen. In individuals which have impaired immune function, the infections
caused by C. albicans may be life threatening (Scherer and Magee, 1990).
(ii) Neuro pharmacological potential:
Muhammad et al., 2012, examined hexane extract of V. betonicifolia for its
neuropharmacological potential. The consequence of staircase test showed that animals fed
with hexane extract of V. betonicifolia showed significant anxiolytic activity. In addition to
it, muscle relaxing, antidepressant and sedative potential of V. betonicifolia hexane extract
was also demonstrated.
(iii) Analgesic and anti-inflammatory activity:
Muhammad et al., 2012, observed dose dependent activity of methanol extract of V.
betonicifolia in different pain models. But analgesic activity of this extract was totally
antagonized by antagonists injection.
Moreover, it was also observed that the mentioned extract considerably antagonized
the histamine and carrageenan induced inflammation. In addition to V. betonicifolia, wide
16
range of medicinal plants of Pakistan is reported for their anti-inflammatory activity (Zaidi et
al., 2012).
(iv) Larvicidal activity:
Larvicidal activity of V. betonicifolia extracts was assessed against Aedes aegypti
vector, which explored that ranking of extracts for larvicidal activity was as: chloroform >
ethyl acetate > methanol with LC50 values of 13.03, 16.00 and 61.30 µg/ml, respectively
(Muhammad et al., 2011). Dengue fever is serious health problem in different parts of world
and Aedes aegypti vector is liable for it propagation (Jawale et al., 2010).
(v) Antipyretic potential:
Pyrexia induced by yeast is termed as pathogenic fever and it may be due to synthesis
of prostaglandins (Moltz, 1993). Reduction in synthesis of prostaglandins may be a approach
for antipyretic activity. In addition to it different mediators of pyrexia and hindrance to these
mediators may also be a potential factor for antipyretic efficacy (Rawlins, 1973). But
Muhammad et al., 2012, administrated methanol extract of V. betonicifolia in yeast induced
hyperthermia to estimate its antipyretic potential and it showed a dose dependent effect
which was close to standard drug.
(vi) Antioxidant activity:
Antioxidant activity of V. betonicifolia was determined by DPPH assay in a dose
dependent mode (Muhammad et al., 2011). Maximum scavenging activity was examined in
chloroform fraction followed by ethyl acetate, butanol, water and hexane with IC50 values of
80, 82, 176, 496 and 500 ppm, respectively.
Ebrahimzadeh et al., 2010, reported IC50 value of 245.1 µg/mL for Viola odorata.
IC50 values of Viola tricolor evaluated by DPPH assay was in range of 13.46 to 284.7 µg/mL
(Goncalves et al., 2012). Nikolova et al., 2010, evaluated IC50 value of Turkish V. tricolor L.
above 200 µg/mL.
(vii) Cytotoxic activity:
17
Cytotoxic activity of crude methanol extract and different solvent fractions of V.
betonicifolia was estimated by brine shrimp lethality assay (Muhammad et al., 2012).
Surprisingly, methanol and ethyl acetate extracts exhibited 100% cytotoxicity.
Cytotoxic cyclotides of V. tricolor are also reported (Svangard et al., 2004). Deng et
al., 2013, investigated cytotoxic activities of another specie of Violaceae (Viola philippica)
by MTT assay. Lindholm et al., 2002, isolated cytotoxic cyclotides from Vioal odorata L.
and Viola arvensis Murr.
(viii) Antiglycation activity:
Cinnamic acid derivative (2,4-dihydroxy, 5-methoxy-cinnamic acid) of V.
betonicifolia exhibited antiglycation activity with IC50 value of 355 ± 7.56 µM. Ramkissoon
et al., 2013, examined a strong correlation between anti-glycation activities and bioactive
components of medicinal herbs.
2.3. Euphorbia royleana:
2.3.1. Ethno botanical description:
Euphorbia royleana is commonly known as ―Thor‖ or ―Danda thor‖ or ―Dozkhi
meva‖in different areas of Pakistan. It is well known as ―Cactus‖ in English. It is spiny,
succulent, glabrous, readily deciduous long shrub or small tree usually up to 5 m tall. It has
stout stalk with 5-7 cm thick fleshy branches with pairs of thorns on the margins.
Flowers are yellow green, 3-4 in clusters in leaf axils. E. royleana belongs to family
Euphorbeaceae, which is one of the largest family among flowering vegetation. This family
consists of more than 300 genera and almost 8000 species are reported from this family.
2.3.2. Distribution:
It is widely distributed in Pakistan (Sabeen and Ahmad, 2009), India (Bani et al.,
2000), China (Li et al., 2009), Nepal (Kunwar et al., 2010), Bhutan, Burma. Many members
of Euphorbiaceae are distributed in hot and dry climatic conditions, but some exist as herbs
and rainforest tress.
18
2.3.3. Ethno medicinal applications:
In Pakistan it is used in folk medicines for treatment of bladder stone, earache, loose
motions and paralysis. For this purpose little amount of plant extract mixed with salt, is drop
wise fed to babies for treatment of loose motions.
Moreover, a part of main stem body is cut and for few minutes, it is kept on the fire.
The extract from this process is used for treatment of earache. For removing urinary bladder
stone, people remove the bark of plant and eat jelly like matter in it. The above mentioned
recepies are used in folk medicines, indigenous to Abbotabad, Islamabad, Pakistan (Sabeen
and Ahmad, 2009).
Table. 2.3. Taxonomic classification of E. royleana
Taxonomic classification
Kingdom Plantae
Subkingdom Tracheobionta
Super division Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Euphorbiales
Family Euphorbeaceae
Genus Euphorbia
Species Euphorbia royleana
Latex of E. royleana is irritant. In traditional phytotherapies E. royleana latex is used
as purgative, however in large doses it may be acrid and emetic (Singh and Singh, 2012).
19
Different species of Euphorbiaceae are used in folk medicines for treatment of
inflammations, swollen belly, arthritis, insanity, infections, neuralgia, asthma, convulsions,
cough, rheumatism, bowel problems, pimples, tumors, wounds, gonorrhea, earache,
toothache, warts, diarrhea, constipation, ring worms, dysentery and jaundice (Kapoor, 1989;
Kirtikar and Basu, 1991; Cataluna, 1999; Damme, 2001; Elujoba, 2005; Ali and Qaiser,
2009; Hussain et al., 2010).
2.3.4. Chemical evaluation:
(i) Phytochemical components:
Sivaraj et al., 2012, screened the pytochemical components (terpenoids, flavonoids,
phenols, cardio glycosides, amino acids, carbohydrates, proteins, tannins, steriods, alkaloids,
sterols and saponins) of E. royleana and four other plant species.
Phytochemical profile of E. royleana and five more plants of Euphorbiaceae is also
documented (Tanvir et al., 1994). Glycosides and alkaloids were present in all the
investigated plants. Alkaloids are copious secondary metabolites in plants and stand for one
of the most widespread class of compounds endowed with multiple pharmacological
properties (Stevigny et al., 2005).
(ii) Bioactive components:
Husain et al., 1992, reviewed amyrin, succinic acid, ingenol, tetracosanol, taraxerol,
sitosterol, luepol, terpenes, phenolics, ellagic acid, stigmasterol, campesterol, diterpene,
octacosanol and cycloroylenol from E. royleana. Tiwari et al., 2008, isolated cycloart-24-en-
3β-ol from latex of E. royleana
Total phenolic contents determined (Shahwar et al., 2010) by Folin-Ciocaltu method
showed that chloroform extract of E. royleana had highest amount of phenolic contents.
Another study (Rastogi and Meharotra, 1993) narrated that poisonous and medicinal
properties of E. royleana latex are ascribed to presence of bioactive components like 7-
methoxy-3,4-benzocoumarin, epitaraxerol, euphol, taraxerol, m-hydroxy benzoic acid, 2,7-
dihydroxy3,4-benzocoumarin, ellagic acid, sitosterol, 7-hydroxy 3,4-benzocoumarin.
20
(ii) Diterpenes:
Li et al., 2009, reported twelve diterpenes from E. royleana aerial parts. Ten were
ingol lathyrane diterpenes and were reported for the first time, while two were known
ingenol derivatives. It was interesting to note that ingol type diterpenes were also examined
in Euphorbia antiquorum (Qi et al., 2014).
However, Yang et al., 2013, showed the existence of Jatropholane-type diterpenes in
Euphorbia sikkimensis. Euphorbeaceae is rich in diterpenes. Another specie of
Euphorbeaceae (Euphorbia paralias) is also known for diterpenes (Jakupovic et al., 1998).
Diterpenes from Euphorbia prolifera and Euphorbia microsciadia are also in literature
records (Ghanadian et al., 2012; Xu et al., 2013).
2.3.5. Biological evaluation:
(i) Anti-inflammatory activity:
Anti-inflammatory potential of latex of E. royleana was investigated in chronic and
acute test models in mice and rats (Bani et al., 2000). E. royleana latex extracted with ethyl
acetate represented the considerable reduction of oedema in mice and rats.
It is surprising to note that Euphorbia splendens also exhibited anti-inflammatory
activity by inhibition of oedema in mice and rats (Bani et al., 1996).
Furthermore, chemical components of Euphorbia kansui are known for their anti-
inflammatory activity (Yasukawa et al., 2000). Euphorbia nerifolia Linn. leaf extract was
described for their anti-inflammatory potential (Gaur et al., 2009).
Anti-inflammatory efficacy of Euphorbia hirta was also in literature records (Das et
al., 2010). Antiarithritic and immunosuppressive potential of E. royleana latex was also
reported (Bani et al., 2000; Bani et al., 2005).
(ii) Antiangiogenic activity:
21
Angiogensis play main role in development of cancer (Carmeliet and Jain, 2000).
Ingenol derivatives isolated from E. royleana aerial parts (Li et al., 2009) exhibited
antiangiogenic effects on zebra fish model.
Ingenol derivatives may inhibit angiogensis by stimulating different protein such as
angiostatin, platelet factor 4, prolactin 16 kd fragment, interferon, tissue inhibitor of
metalloproteinase-1, -2, -3 and endostatin (Nishida et al., 2006).
(iii) Antioxidant activity:
Shahwar et al., 2010, examined antioxidant potential of different solvent extracts of
E. royleana. Antioxidant activity determined by DPPH assay explored that ethyl acetate
extract (34.7± 0.8%) had maximum potential. Comparatively higher DPPH scavenging
activity (72.9.4± 0.78%) was observed in Euphorbia hirta leaf extract (Basma et al., 2011).
Other members of Euphorbiaceae (Euphorbia helioscopia, Euphorbia neriifolia,
Euphorbia resinifera, Euphorbia hetrophylla, Euphorbia tirucalli) also had potent
antioxidant potential (Jyothi et al., 2008; Sharma et al., 2011; Maoulainine et al., 2012;
Okeniyi et al., 2012; Nadia and Benmahdi, 2013).
(iv) Toxic effects:
Parsad et al., 2010, narrated toxic effect of latex of E. royleana on Heteropneustes
fossilis (cat fish), which is dwelling of fresh waters. Singh and Singh, 2012, evaluated
muricidal and piscicidal potential of Euphorbia royleana latex against Channa punctatus
(Snakehead fish) and Mus musculus (Swiss albino mice).
Molluscicidal efficacy of various fractions of Euphorbia royleana was estimated
against Lymnaea (Radix) acuminata Lamarck which is dwelling of fresh waters (Tiwari et
al., 2005).
Tiwari et al., 2008, isolated cycloart-24-en-3β-ol from latex of E. royleana and its
piscicidal potential was explored by using a fish (Channa punctatus), dwelling of fresh
water.
2.4. Psidium guajava:
22
2.4.1. Ethno botanical description:
Psidium guajava (family Myrtaceae) is common guava known as ―Amrood‖ in
Pakistan. It is a fruit tree generally 3-10m high and has many branches.
(a) Leaves:
The leaves of P. guajava are opposite with short petiole (3-10 mm) and have
prominent veins.
(b) Fruit:
The P. guajava fruit (3-6 cm long; 5cm diameter) is ovoid that resides a fleshy
pericarp, juicy pulp and a seed cavity with several number of white kidney shaped or
flattened hard seeds (Gutierrez et al., 2008; Okunrobo et al., 2010; Rai et al., 2010).
(c ) Root:
It has superficial root system with numerous deep roots, but lacking any discrete
taperoot. Root system is very wide and extends beyond the canopy (Shruthi et al., 2013).
(d) Flower:
P. guajava has white flowers which are fragrant. The flowers have incurved petals
with yellow anthers. P. guajava is a hard tree and can sustain in broad range of temperature,
however the average temperature of 23-28ºC is optimum for its yield. Rai et al., 2010,
reported that a tropical atmosphere and bursting sun shine is required for its healthy growth.
2.4.2. Distribution:
It is widely distributed in Pakistan, India, China, Mexico, Brazil, United States of
America, Bangladesh, Thiland, Malaysia, Africa, Peru, Europe and many other countries of
the world (Pathak and Ojha, 1993; Gutierrez et al., 2008; Elekwa et al., 2009; Joseph and
Priya, 2011).
2.4.3. Ethnomedicinal applications:
23
It has wide ethno medicinal applications, while anti-diarrheal is the major one
(Gutierrez et al., 2008). In Pakistan, it is traditionally used for treatment of cancer, pain,
microbial infection, inflammation, diabetes and coughs (Sabeen and Ahmad, 2009).
In district Chakwal of Pakistan, P. guajava leaves are boiled with water and this
decoction was used for treatment of high blood pressure (Sultana et al., 2006).
It is traditionally used in different forms for treatment of diarrhea, stomach ache,
gastroenteritis, insomnia, asthma, dysentery, skin sore, menstrual disorders, ulcers, vertigo,
edema, hysteria, swelling, nephritis, epilepsy, bowel disorders, cholera, convulsions, scabies,
hemorrhoids, dyspepsia, vomiting, wounds, piles and other ailments in all over the world
(Lutterodt, 1988; Nadkarni and Nadkarni, 1991).
Table. 2.4. Taxonomic classification of P. guajava
2.4.4. Chemical evaluation:
(i) Phenolic compounds:
Taxonomic classification
Kingdom Plantae
Subkingdom Tracheobionta
Super division Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Myrtales
Family Myrtaceae
Genus Psidium
Species Psidium guajava
24
P. guajava leaves have broad spectrum of phenolic components. Many phenolic
compounds such as gallic acid, protocatechuic acid (Okuda et al., 1984), caffeic acid (Liang
et al., 2005), ferulic acid (Zhu et al., 1997), chlorgonic acid (Qian and Nihorimbere, 2004;
Liang et al., 2005), ellagic acid (Misra and Seshadri, 1968) and guavin B (Okuda et al., 1984;
Zhu et al., 1997) are isolated from P. guajava leaves.
(b) Flavonoids:
Literature reports (Nakarni and Nadkarni, 1999; Arima and Dano, 2002; Michael et
al., 2002; Liang et al., 2005; Prabu et al., 2006) showed the isolation of many flavonoids
including quercetin, leucocyanidin, kaempferol, querecetin3-α-L-arabinofuranoside,
guaijaverin, mecocyanin from leaves and other parts of P. guajava plant.
Arima and Danno, 2002, reported four flavonoids from P. guajava leaves and
identified them as quercetin, morin-3-O-α-L-arabopyranoside, guaijavarin and morin-3-O-α-
L-lyxopyranoside.
Vargas et al., 2006, showed the significant quantities of quercetin, apigenin, luteolin,
myricetin and kaempferol in floral buds of P. guajava.
(ii) Carotenoids:
An other research group (Mercandante et al., 1999) isolated many carotenoids such as
β-carotene, lycopene, rubixanthin, lutein, neochrome, β-cryptoxanthin, phytofluene and
criptoflavin from P. guajava leaves and fruit. Reddy et al., 2012, explored β-carotene
contents (40,000µg) of P. guajava leaves.
(iii) Triterpenes:
P. guajava is marvelous source of triterpenes. Different triterpenes such as oleanolic
acid (Siddiqui et al., 2002), uroslic acid, β-sitosterol, 2α-hydroxyuroslic acid, guavacoumaric
acid, guavenoic acid, asiatic acid, 2-α-hydroxyuroslic acid, 2α-hydroxy-3β-p-E-
coumaroyloxyurs-12,18-dien-28-oic acid (Begum et al., 2002), arjunolic acid and jacoumaric
acid (Salib and Michael, 2004) are isolated from P. guajava leaves, fruit and other parts.
25
Sesqui-terpenes, triterpenoids, flavonoida, alcohols and minerals are reported in twigs (Okwu
and Ekeke, 2003).
(iv) Nutritive profile:
Conway, 2002, described the P. guajava contain 5 times higher vitamin C contents
than oranges. Nutriative evaluation of P. guajava fruit showed the presence of moisture,
carbohydrates, crude fiber , fats , protein , ash , calcium , iron, Vitamin A , thiamin , niacin
and riboflavin (Fujita et al., 1985; Conway, 2002; Kamath et al., 2008).
Nadkarni and Nadkarni, 1999, reported the presence of Mn in this plant combined
with malic, phosphoric and oxalic acids. Moreover, skin of P. guajava fruit is rich in ascorbic
acid (Charles et al., 2006). Aminu et al., 2012, reported the presence of vitamin C in stem
bark extract of P. guajava.
(v) Chemical composition essential oil:
Iwu, 1993, indicated the presence of β-sitosterol, nerolidiol, uroslic, guayavolic and
crategolic acids in essential oil of P. guajava leaves. An extensive array of bioactive
components including copaene, azulene, β-caryophyllene, limonene (Li et al., 1999),
euclayptol (Oliver-Bever, 1986), octanol, 3-phenylpropanol (Kenneth et al., 1970), α-
humulene, benzaldehyde, obtusinin (Jordan et al., 2003), ascorbic acid, amritoside (Fujita et
al., 1985), asorbigen (Radha and Chandrasekaran, 1997), meocyanin (Salib and Michael,
2004) were isolated from essential oil of P. guajava. Paninandy et al., 2000, described the
presence of 3-caryophyllene, caryophyllene oxide, 3-phenylpropyl acetate and nerolidol in
essential oil of P. guajava fruit.
(vi) Other bioactive components:
Reddy et al., 2012, reported alkaloids, tannins, phenolics, flavonoids, reducing
sugars, steroids, saponins, terpenoids in different extracts of P. guajava leaves. Shruthi et al.,
2013, described the existence of tannis in bark of P. guajava tree.
Michel et al., 2002, reported the presence of quercetin-3-O-β-D-(2"-
Ogalloyglucoside)-4'-O-vinylpropionate in guava seeds. Some other research groups
26
(Dweck, 2001; Hwang et al., 2002) narrated the existence of glykosen, protein and
saccharose in P. guajava fruits.
Nagar and Rao, 1981, reported cytokinins from P. guajava plant. Ascorbic acid (34
mg) and glutathione (50.4 mMoles) contents of P. guajava leaves are also in literature
records (Reddy et al., 2012).
Different active components such as phenol, 1,2-benzenedicarboxylic acid, diethyl
phthalate, phthalic acid, phytol, 2,5-bis(1,1-dimethylethyl), asarone, butyldodecyl ester and
mono(2-ethylhexyl) ester were isolated from ethyl acetate fraction of P. guajava root bark
(Velmurugan et al., 2012).
2.4.5. Biological evaluation:
(i) Antioxidant activity:
Stem bark extract of P. guajava exhibited free radical (DPPH) scavenging activity
(Aminu et al., 2012). Ogunlana and Ogunlana, 2008, evaluated antioxidant potential of P.
guajava by scavenging superoxide, hydrogen peroxide and stable free (DPPH) radicals.
Reddy et al., 2012, narrated maximum stable free (DPPH) radicals scavenging
activity in aqueous extract of P. guajava. However, another group of researcher (He and
Venanat, 2004), showed that inhibition of free radicals (DPPH) in ethanol extract of P.
guajava leaves were higher than aqueous extract.
Siow and Hui, 2013, demonstrated that antioxidant potential of fresh P. guajava is
higher than oven dried one. Zahidah et al., 2013, investigated that ferric reducing antioxidant
power (FRAP) and free radical (DPPH) scavenging ability of P. guajava leaves was greater
than P. guajava seeds.
Antioxidant efficacy of P. guajava may be attributed to bioactive components like
quercetin, gallic, ferulic, protocatechuic, ascorbic and caffeic acids in it (Thaipong et al.,
2005).
(ii) Antimicrobial activity:
27
Zahidah et al., 2013, evaluated antimicrobial activity of P. guajava leaves and seeds
against various microorganisms. In another study (Pandey and Shweta, 2011), antifungal
activity of different extracts of P. guajava fruits and leaves was assessed by agar well
diffusion assay.
Arima and Danno, 2002, isolated four compounds from P. guajava and evaluated
their antibacterial activity against Salmonella enteritidis and Bacillus cereus. S. enteritidis
was found more sensitive to isolated compounds. Velmurugan et al., 2012, studied antiviral
activity of ethyl acetate fraction of P. guajava root bark.
Sato et al., 2000, reported that methanolic extract of ripe P. guajava fruit had
fungicidal activity against Chaetomium funicola and Arthrinium sacchari. Ethanolic extract
of P. guajava ripe fruit showed significant activity against Escherichia coli and
Streptococcus mutans (Neira and Ramirez, 2005). Fungistatic activity of bark tincture of P.
guajava was observed against Candida albicans (Dutta and Das, 2000).
(iii) Anticancer activity:
(a) Prostate cancer:
Chen et al., 2007, studied inhibition in growth of prostate cancer (DU-145) cells by
aqueous extract of P. guajava leaves.
(b) Murine leukemia:
Manosroi et al., 2006, observed that essential oil of P. guajava leaves has potent
activity against murine leukemia (P388) cells with IC50 value of 0.037 mg/mL. The efficacy
of essential oil might be on the basis of monoterpenes in it (Cito et al., 2003).
(c) Colon cancer:
Acetone extract of P. guajava branches exhibited antiproliferative efficacy against
human colon carcinoma (HT-29) cells (Lee and Park, 2010). Ampasavate et al., 2010,
narrated that P. guajava leaves extracted with ethanol had no effect on promyeloid (HL60),
lymphoblastic (Molt4), erythroid (K562) and monocytic (U937) cells.
28
(d) Leukemia and ovarian cancer:
Levy and Carley, 2012, described the decrease in growth of Kasumi-1 leukemia and
OV2008 ovarian cancer cells by hexane extract of P. guajava leaves.
Another group of scientist (Fernandes et al., 1997) studied anticancer activity of
methanol extract of P. guajava against mice induced cancer that was inoculated B16
melanoma cells.
(e) Osteosarcoma, breast cancer and cervical cancer:
Sulain et al., 2012, extracted P. guajava leaves with different solvents and evaluated
their anticancer activity against breast cancer (MDA-MB-231), osteosarcoma (MG-63) and
cervical cancer (HeLa) cells. However, Kaileh et al., 2007, described that methanolic extract
of P. guajava is effective against human breast cancer (MCF-7) and murine ficrosarcoma
(L929sA).
A group of researchers (Sato et al., 2010) showed that anticancer activity of P.
guajava was due to potential bioactive components in its bark, fruit and leaves.
(iv) Antidiabetic activity:
Diabetes mellitus is becoming a stern risk to human health. Some researcher has
reported anti diabetic activity of fruit, leaf and bark extracts of P. guajava. Mukhtar et al.,
2004, screened the decoction P. guajava leaves for hypoglycemic potential on alloxan-
induced rats.
Moreover, significant reduction on LDL glycation by P. guajava aqueous leaf extract
was observed in a dose-dependant mode. Some research groups (Ojewole, 2005; Wang et al.,
2005) have narrated that tannins, pentacyclic triterpenoids, quercetin, flavonoids, guiajaverin
and other bioactive components present in P. guajava plant are responsible for hypotensive
and hypoglycemic effects.
(v) Antinociceptive activity:
29
Santos et al., 1998, examined that essential oil of P. guajava leaf and its component
α-pinene had considerable antinociceptive activity. Shaheen et al., 2000, extracted the P.
guajava leaves with different solvents (methanol, ethyl acetate and hexane) and evaluated
their antinociceptive potential on central nervous system of mice.
2.5. Ziziphus mauritiana:
2.5.1. Ethno botanical description:
Ziziphus mauritiana (Rhamnaceae) is commonly known as ―Ber‖ in Pakistan. It is
known as Indian jujube in English. It exists as small spreading tree (9-15 m long) or in form
of large shrub with densely, drooping branches (Marwat et al., 2009).
(i) Leaves:
Leaves of Z. mauritiana are ovate or eliptic oblong. Leaves (2.5 to 6 cm long; 2 to
4cm wide) grow on alternate sides of branches. The leaves have dark green glossy look on
the upper side with fine tooth on the margins (Marwat et al., 2009; Goyal et al., 2012).The
leaf has three conspicuous, depressed and longitudinal veins.
(ii) Fruit:
The fruit has variable size usually 1.25-2.5 cm long. Under favorable conditions fruit
may reach up to 6.25 cm in length and 4.5 cm in width. Fruit is ovate to oblong with fleshy,
juicy, crispy and delicious pulp.
The color of fruit is yellowish or light green. It is of red brown color in ripe (Mahajan
and Chopda, 2010). It has a single central seed which is very hard and oval in shape.
(iii) Seed:
The seed has two elptic kernals, each of them is 6 mm long (Anonymous, 1989).
(iv) Flower:
Z. mauritiana has 5 petteled greenish white small flowers with acrid smell. Flowers
are produced in axillary cyme or small clusters (7-20 flowers). They had lobes in calyx.
30
2.5.2. Distribution:
Z. mauritiana is extremely drought and hardy tree. It is restricted to drier tropics and
is natural vegetation of desert and rocky area in Pakistan and India (Goyal et al., 2012). It is
cultivated and self sown in different areas of word including China, Afghanistan, Malaysia,
Australia, Africa, Southern Florida, Colombia, Guatemala, Venezuelan, Belize, Ceylon.
2.5.3. Ethno medicinal applications:
Z. mauritiana is used in folk medicines all over the world. In Pakistan it is
traditionally used for treatment of dysentery, wounds, fever, abscesses and intestinal worms.
It is also used as blood purifier in form of herbal teas (Jabeen et al., 2009).
In Khushab, Punjab, Pakistan, women boil its leave with water and apply it on hairs
to get smooth, healthy, long and shining hairs.
Moreover, in the same area people use leaves of Z. mauritiana to remove spines from
any body part. For this purpose leaves are combined with wheat flour, oil and turmeric and
are somewhat warmed over fire to make poultice which is applied externally on the skin to
remove spines (Qureshi et al., 2011).
In North Western areas of Pakistan, people use leaves of Z. mauritiana for treatment
of abscesses. For this purpose fresh crushed leaves are mixed with little quantity of soap and
powdered loaf sugar and this paste is used for dressing of abscesses. The dressing is changed
after one day and this treatment is practiced for 3 to 4 days. As a consequence of this
treatment new abscesses will vanish and the older ones will burst (Marwat et al., 2009).
In addition to Pakistan, various segments of Z. mauritiana plant are used in folk
medicines in different areas of world for treatment of cough, headache, asthma, diarrhea,
biliousness, leucorrhoea, fever, smallpox, nausea, eye troubles, hoarseness of throat,
constipation, pulmonary ailments, urinary infections, dysentery, ulcers, abdominal pains in
pregnancy, vomiting and liver troubles (Nadkarni, 1986; Anonymous, 1989; Oudhia, 2003;
Mahajan and Chopda, 2009).
2.5.4. Chemical evaluation:
31
(i) Alkaloids:
Chebouat et al., 2013, analyzed the crude alkaloidal extract of Z. mauritiana and lead
to identification of ten alkaloids. Out of ten, three alkaloids (4-Methoxyquinoline, 2-Fluoro-
3-(1-hydroxy-2-(methylamino)ethyl) phenol, (2-(4-Amino-2-methylphenyl)-7-methyl-1H-
indol-3-yl)(4-chlorophenyl)methanone) were identified in flowers.
Schmeller and Wink, 1998, reviewed the use of alkaloids in modern medications.
Literature survey indicates potent role of vinca alkaloids in treatment of cancer (Noble, 1990;
Hill et al., 1993).
Panseeta et al., 2011, narrated that alkaloids isolated from Z. mauritiana root extracts
had antimycobacterial and antiplasmodial activities.
Srivastava and Srivastava, 1979, isolated zizogenin from stem of Z. mauritiana.
Jossang et al., 1996, studied mauritine J in Z. mauritiana root bark. Panseeta et al., 2011,
examined five alkaloids (mauritine L, nummularine B, hemsine A, nummularine H and
maurtine M.) from methanol extract of Z. mauritiana roots. Bhatt and Dhyani, 2013,
quantified alkaloids (2.2%) from Z. mauritiana.
(ii) Phenolic acids:
Memon et al., 2012, studied the phenolic acid profile of Z. mauritiana fruit extract.
They quantified ten phenolic acids including protocatechuic, vanillic, p-coumaric, chlorgonic
,ferulic and caffeic acids. In this study bound and free phenolic acids were examined, but in
previous literature surveys (San and Yildirim, 2010) only free phenolic acids were reported.
The variation in phenolic acid contents of different natural foods may be attributed to
difference in geographical factors (Alstyne et al., 1998; Kumar et al., 2013).
Samee et al., 2006, extracted sliced fruit of Z. mauritiana with different solvents and
demonstrated phenolics (148.0± 36.6 µg GAE/g) and ascorbic acid ( 488.3 ±7.3 µg/g)
contents. However, Choi et al., 2011, examined lower content of phenolics in fruit and seed
of sister Ziziphus species (Ziziphus jujuba).
32
(iii) Nutritive profile:
Nutritive evaluation (Morton, 1987; Pareek and Dhaka, 2008; Pareek et al., 2009) of
Z. mauritiana fruit indicated the presence of fat (0.07 g), carbohydrates (17g), ash (0.3-
0.59g), niacin 90.7-0.0873 mg), total sugars (1.4-6.2 g), Ca (25.6mg), flouride (0.1-0.2 ppm),
thiamine (0.02-0.024 mg), iron (0.76-1.8mg), protein (0.8 mg), phosphorus (26.8 mg), fiber
(0.60 g) and other components. Another research group (Gupta et al., 2012) examined total
ash, volatile matter, crude fiber and moisture contents in the Z. mauritiana leaves.
(iv) Nortriterpenes:
Ji et al., 2012, isolated six nortriterpenes from root extract of Z. mauritiana. Out of
six three were known (Ceanothic acid, betulinic acid and ceanothenic acid) and three were
novel (Zizimauritic acids A,B,C) nortriterpenes.
Table. 2.5. Taxonomic classification of Z. mauritiana
Taxonomic classification
Kingdom Plantae
Subkingdom Tracheobionta
Super division Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Rhamnales
Family Rhamnaceae
Genus Ziziphus
Species Ziziphus mauritiana
33
Previously, various cyclopeptide alkaloids, triterpenes, aliphatic components, steroids
and flavonoids are reported from this Ziziphus (Jossang et al., 1996; Singh et al., 2007;
Pandey et al., 2008).
(v) Other bioactive components:
Goyal et al., 2012, reviewed phytocomponents of Z. mauritiana including
jujubosides, betulonic acid, zizyberanalic acid, zizyberenalic acid, 2- hydroxybetulinic acid
and leanonic acid.
Gupta et al., 2012, reported the presence of alakloids, proteins, carbohydrates, amino
acids, phenolics, triterpenoids, saponins, glycosides and flavonoids in different extracts of
leaves of Z. mauritiana. Phytochemical analysis revealed the presence of glycosides, tannins,
phenol and saponins in Z. mauritiana leaves (Nijafi, 2013).
Bhatt and Dhyani, 2013, quantified saponins (92.65%), flavonoids (25.8%) and
tannins (5 µg/ml) and saponins (92.65%) from fruit of Z. mauritiana. These bioactive
components play leading role against cancer, AIDS, HIV infection, cardiovascular diseases
and wide range of other ailments (Mcmahon et al., 1995; Urquiaga and Leighton, 2000;
Mahajan and Chopda, 2009).
Flavonoids, phenolics and ascorbic acid contents of ripe and green Z. mauritiana
fruits were demonstrated (Das, 2012). Literature survey indicated antimicrobial efficacy of
flavonoiods, phenolics and ascorbic acid (Cushnie and Lamb, 2005; Tajkarimi and Ibrahim,
2011; Alves et al., 2013).
2.5.5. Biological evaluation:
(i) Antimicrobial activity:
Najafi, 2013, reported antimicrobial potential of Z. mauritiana leaves against two
bacterial strains in a dose dependant mode. Many recent studies on antimicrobial potential of
plant extracts are also narrated in dose dependent mode (Kuber et al., 2013; Ahmed et al.,
2014).
34
Antimicrobial potential of Z. mauritiana leaves and bark against five bacterial and
three fungal strains was evaluated (Mahesh and Singh, 2008). Antimicrobial activity of ripe
and green Z. mauritiana fruit against panel of eight microorganisms was described by Das,
2012.
Abalaka et al., 2010, extracted Z. mauritiana leaves with ethanol and evaluated their
antimicrobial activity. An Indian study demonstrated that E. coli is less susceptible to Z.
mauritiana leaves (Mahesh and Satish, 2008).
Ziziphus lotus exhibits considerable activity against E. coli (Naili et al., 2010).
Cyclopeptide alkaloids from the root bark extract of Z. mauritiana are documented for
antimycobacterial activites (Panseeta et al., 2011).
(ii) Antioxidant activity:
Antioxidant activity of two varieties (Narikeli kul and Local) of Z. mauritiana fruit
was assessed by DPPH assay (Bhuiyan et al., 2009).
The antioxidant potential may be attributed to broad spectrum of phenolic and
flavonoid contents in the Z. mauritiana fruit (Memon et al., 2012).
Lamien-Meda et al., 2008, determined antioxidant activity of wild Z. mauritiana fruit
along with other thirteen edible fruit species. Z. mauritiana fruit variety from Thiland is also
known for scavenging activity on DPPH radicals (Samee et al., 2006).
Antioxidant activity of five different solvent extracts of Z. mauritiana was evaluated
by reduction of NBT and inhibition of nitric oxide in a dose dependant mode (Gupta and
Singh, 2008). Previously some other research groups also used the dose dependant mode to
evaluate antioxidant efficacy of plant extracts (Sim et al., 2010). Olajuyigbe and Afolayan,
2011, reported potent antioxidant potential of Ziziphus mucronata bark extracts.
(iii) Anticancer activity:
Mishra et al., 2011, explored anticancer activity of seeds of Z. mauritiana against
three human carcinoma (cervical (HeLa), lymphoblastic leukemia (Molt-4), promyelocytic
leukemia (HL-60)) and one normal (human gingival fibroblast (HGF) cell line by MTT
35
assay. Furthermore, the result of flow cytometric analysis explored that Z. mauritiana extract
cause induction of apoptosis in HL-60 cells. Huang et al., 2007, examined that chloroform
fraction of Z. jujube not only induce apoptosis but also G2/M arrest in HepG2 cells.
Betulinic acid isolated from Z. mauritiana roots (Panseeta et al., 2011) is narrated for
its role against neck, head, lung, cervical and ovarian carcinoma (Pisha et al., 1995). Slight
modification of betulinic acid structure can generate valuable derivatives, which may play
significant role in development of antitumor drugs (Kim et al., 1998).
36
CHAPTER 3 MATERIAL AND METHODS
The research work demonstrated in this dissertation was carried out in the Central Hi
Tech Laboratory, Department of Chemistry and Biochemistry, University of Agriculture,
Faisalabad, Pakistan; Department of Experimental Therapeutics, Cytokine Research
Laboratory, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, United
States of America.
3.1. Materials:
3.1.1. Chemicals and reagents:
Aluminium chloride hexahydrate (Fluka chemicals, GmbH), potassium ferricyanide
(DAEJUNG Chemicals and metals, Korea), ferric chloride (BDH laboratory supplies, UK).
Trichloroacetic acid, 2,2′- diphenyl-1-picrylhydrazyl (DPPH), gallic acid (GA), quercitrin,
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and various reference
chemicals (gallic acid, vanillic acid, caffeic acid, syrengic acid, sinapic acid, ferulic acid, m-
coumaric acid, p-coumaric acid and chlorogenic acid etc.) and rifampicin were obtained from
Sigma-Aldrich (Saint Louis, MO, USA). Methanol, chloroform and hexane were purchased
from Merck KGaA (Darmstadt, Germany). Nutrient agar was obtained from Oxoid,
Hampshire, UK. Nutrient Broth was acquired from LAB M, Limited UK. Potato dextrose
agar and Sabarose Dextrose Broth were obtained from Applichem, Dermastadt, Germany.
0.45 µm filter paper was obtained from Biotech, Germany. Terbinafine was generously
provided by Saffron pharmaceuticals, Faisalabad, Pakistan. Folin-Ciocalteu’s phenol reagent,
sodium carbonate were acquired from Applichem, GmbH, Darmstadt, Germany. Sodium
dodecyl sulfate and N,N-Dimethylformamide were provided by Fisher Scientific, USA.
DPBS (Dulbeco’s phosphate buffered saline), Trypsin EDTA, 1X (0.25% trypsin, 2.2 Mm
EDTA), DMEM (Dulbeco’s modification of Eagle’s medium, with 4.5 g/L glucose L-
glutamine and sodium pyruvate), RPMI 1640with L-glutamine, Antibiotic-antimycotic
solution (10,000 I.U./mL Penicillin, 10,000 µg/mL Streptomycin, 25 µg/mL Amphotericin
B) were purchased from Mediatech Inc. Manassas, VA, USA. Ferric chloride was attained
from BDH laboratory supplies, England. Fetal bovine serum was obtained from Atlanta
Biologics. Human recombinant TNF-α purified from bacterial cells to homogeneity with a
37
specific activity of 5 × 107 units/mg was provided by Genentech (South San Francisco, CA).
Optical density of cells was measured by ELISA (Dynex Technologies, USA)
3.1.2. Instruments:
The instruments used for different analyses during the study along with their
company identification are listed in Table. 3.1.
Table. 3.1. Instruments with their model and company
Equipment Made
Electric balance Shimadzu, Japan
Blender Singer, FP-500
Rotary shaker Gallenkamp, England
Water bath Memmertt, Japan
Vacuum drying oven Memmertt, Germany
Stereomicroscope Olympus, Japan
Autoclave Omron, Japan
Ultra low freezer Sanyo, Germany
Laminar air flow Dalton, Japan
Hot air oven Memmertt, Germany
Spectrophotometer Lambda 25, Perkin Elmer, USA
Phosphor-Imager imaging device Molecular Dynamics, Sunnyvale, CA
ELISA Dynex Technologies, USA
GC-MS (Model 6890GC-597MSD) Phenomenex, Torrance
Simple microscope Nikon, Japan
Centrifuge (GR G/2) Jouan, USA
CO2 incubator Thermo scientific
HPLC LC-10A, SHIMADZU, JAPAN
3.1.3. Collection of plant materials:
38
Ethno botanical surveys were undertaken in various areas of Punjab, Pakistan, where
herbal therapies are in common practice.
The plants were selected and collected on the basis of frequent use in traditional
medicines for treatment of various ailments. Ethno medicinal information was basically
collected from herbal practitioners of different areas of Punjab, Pakistan, including
Lathianwala, Chuhdary wala and Faisalabad. In addition to it, dialogues, meetings,
discussions and interviews with rural knowledgeable people about traditional use of plants,
also provided substantial information. Ethno medicinal importance of selected plants was
further confirmed from literature surveys (Sabeen and Ahmad, 2009). The collected plant
materials were identified by by authentic herbarium techniques.
3.1.4. Plants used in current study:
(i) Euclayptus camaldulensis
(ii) Viola betonocifolia
(iii) Euphorbia royleana
(iv) Psidium guajava
(v) Ziziphus mauritiana
Euclayptus camaldulensis
39
Viola betonicifolia Euphorbia royleana
Psidium guajava Ziziphus mauritiana
Figure. 3.1. Photographs of selected Indigenous plants used in current study
3.1.5. Human cancer cell lines employed to access the anticancer potential of medicinal
plants:
40
KBM5 cells HCT116 cells
SCC4 cells U266 cells
Figure. 3.2. Human cancer cell lines (KBM5, myelogenous leukemia; HCT116, colon
carcinoma; SCC4, tongue squamous carcinoma and U266 multiple myeloma) used in
current study
KBM5 (Human myelogenous leukemia cells), U266 (Human multiple myeloma
cells), SCC4 (Human tongue squamous carcinoma cells) and HCT116 (Human colon
41
carcinoma cells) used in current study were obtained from American Type Culture Collection
(Manassas, VA) by The University of Texas, M.D. Anderson Cancer Center, Houston,
Texas, USA, where cell culturing experiments were carried out.
KBM5 and U266 cells were maintained in RPMI-1640. DMEM (Dulbeco’s
modification of Eagle’s medium) was used for sustaining SCC4 and HCT116. Both the
media were supplemented with 10% fetal bovine serum (Atlanta Biologicals), and antibiotic
(10,000 I.U/mL). Cultures were maintained in 75cm2 flasks in humidified (95% air)
incubator at 37ºC with 5% CO2.
3.1.6. Strains of microorganisms used to estimate antimicrobial activity of plant
extracts:
(i) Escherchia coli
(ii) Bacillus subtilis
(iii) Pasterula multocida
(iv) Aspergillus niger
(v) Fusarium solani
The pure cultures of bacterial and fungal strains were procured from Nuclear Institute for
Agriculture and Biology (NIAB), Faisalabad and characterized from Department of
Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan. These microbial
strains were utilized to demonstrate antimicrobial activity of selected plant extracts.
3.2. Extraction of dried plant materials:
Fresh leaves of Euclayptus camaldulensis, Viola betonicifolia, Psidium guajava and
Ziziphus mauritiana were rinsed with distilled water to remove dust and any particulate
matter. Leaves were spread separately on paper sheets in well ventilated room.
Dried leaves were ground with help of food processor (Singer, FP-500) into fine
powder. The powder was passed through seiver (0.25 mm). Sieved powdered material was
stored in tightly packed glass jars. Extraction was carried out according to reported method
(Sultana et al., 2009) with slight modification.
42
10 gm of leaf powder was extracted with 100 mL methanol on a rotary shaker at 350
rpm for 6 hour. Filtration was made with the help of Buchner funnel and Whatman No. 1
filter paper. Filtrate was evaporated in vacuum drying oven at 65°C to dryness. Dried extract
was scratched with the help of sterilized spatula. Dried extract was transferred to extract vials
and stored at -4°C for further use. Similar procedure was repeated with hexane and
chloroform. Temperature of vacuum drying oven was adjusted at 62°C and 70°C for
chloroform and hexane, respectively.
3.3. Extraction of fresh plant material:
Fresh plant material of Euphorbia royleana was washed with distilled water and cut
into cubes of 2X2X2 cm3 with stain less steel knife (GLOBAL, Japan). Cubes of fresh plant
material (stem) were immediately used for extraction according to modified method in
literature (Shofian et al., 2011).
20 gm of cubes of fresh plant material were placed in 500 mL flask, mixed with 200
mL of methanol, plugged with cotton swab and tightly wrapped with aluminum foil.
Extraction was carried out by using an orbital shaker at 350 rpm for 72 hours. After 72 hours
filtration was done with the help of Whatman No.1 filter paper. The filtrate was evaporated at
65°C using vacuum drying oven (Memmert GmbH, Germany) to get dry extract. Solvent free
extract was transferred to extract vials and stored at 4°C for further use. Similar practice for
extraction was done with hexane and water.
3.4. Chemical evaluation:
3.4.1. Total phenolic content:
Total phenolics were determined by Folin-Ciocalteu process (Slinkard and Singleton,
1997; Jagadish et al., 2009). Briefly, one mL of plant extract solution (800 µg/mL) was
mixed with 7.5 mL of double deionized water. Then 500 µL of Folin-ciocalteu reagent and
one mL of 5% (W/V) Na2CO3 solution was added and mixed thoroughly.
Mixture was incubated for 90 min at room temperature. Measurement of absorbance
was carried out at 760 nm by using UV-Vis spectrophotometer (Lambda EZ 201, Perkin
43
Elmer, USA). The same procedure was repeated for all the standard gallic acid solutions and
a standard curve was obtained with following equation:
Absorbance = 0.005 µg gallic acid + 0.074 (R2 = 0.99)
Total phenols of each extract, as gallic acid equivalents, were determined using its
absorbance measured at 760 nm as input to the obtained standard curve and its equation. All
tests were carried out in triplicate.
3.4.2. Total flavonoid contents:
Total flavonoids were determined by the AlCl3 method. Briefly, 2 mL of plant extract
solution (800 µg/mL) was mixed with 2 mL of aqueous AlCl3.6H2O (0.1 mol/L). Mixture
was incubated at room temperature for 10 min and absorbance was measured with UV-Vis
spectrophotometer at 417 nm.
The same procedure was repeated for all the standard quercetin solutions and a
standard curve was obtained with following equation:
Absorbance = 0.040 µg quercetin + 0.051 (R2 = 0.99)
Total flavonoids of each extract, as quercetin equivalents, were determined using its
absorbance measured at 417 nm as input to the obtained standard curve and its equation. All
tests were carried out in triplicate with modified method (Lamaison and Carnat, 1990;
Quettier-Deleu et al., 2000).
3.4.3. High Performance Liquid Chromatography (HPLC) analysis:
Gallic acid, caffeic acid, vanillic acid, chlorogenic acid, syringic acid, sinapic acid, m-
coumaric acid, p-coumaric acid, ferulic acid, p-Hydroxy benzoic acid, catechin and quercetin
were used as standard.
HPLC analysis was performed by hydrolysis of the test samples (50 mg) of each plant
extract dissolved in 24 mL methanol and was homogenized. 16 mL distilled water was added
followed by 10 mL of 6M HCl. The mixture was then thermostated for 2 hr at 95oC. The
44
final solution was filtered using 0.45 µm nylon membrane filter (Biotech, Germany) prior to
high performance liquid chromatography (HPLC) analysis (Pak-Dek et al., 2011).
The separation of plant samples on gradient was performed using shim-pack column.
The chromatographic separation was carried out using as mobile phase gradient : A (H2O:
Acetic acid- 94:6, pH = 2.27), B (acetonitrile 100%), 0-15 min = 15% B, 15-30 min = 45%
B, 30-45 min = 100% B with 1 mL/min flow rate using UV- visible detector at 280 nm
wavelength at room temperature at injection rate of 20 µL. The identification of each
compound was established by comparing the retention time and UV-Visible spectra of the
peaks with those previously obtained by injection of standards. The quantification was
performed by external calibration.
3.4.4. Gas Chromatography Mass Spectrometry (GC-MS) study:
The analysis of plant extracts (0.5 mg/mL) was performed using a GC-MS that had an
electron energy of 70Ev, ion source temperature of 230ºC and electron emission of 34.6 µA.
The temperature of analyzer was maintained at 150ºC. Helium was used as carrier gas at flow
rate of 1mL/min. The injector and interface temperature was set at 290ºC and 360ºC,
respectively. The oven temperature was programmed as 50ºC (1 min) to 310ºC (20 min), at
increasing rate of 6ºC/min. Compounds were identified on the basis of relative retention time
and comparison of their mass spectrum with the spectrum of the known components stored in
National Institute Standard and Technology (NIST) database of GC/MS system.
3.5. Biological Evaluation:
3.5.1. Free radical (DPPH) scavenging activity (DPPH assay):
Free radical scavenging capability of each extract solution on was determined on
DPPH (2, 2’- diphenyl 1-picrylhydrazyl) radicals. A stock solution of 1000 µg/mL was
prepared of each tested extract. Different dilutions (25, 50, 100, 200, 500 and 800 µg/mL) of
each extract were prepared from respective stock solution of that extract.
Briefly, 4 mL of of DPPH (0.1mM) was mixed with 1mL of each of plant extract
solution at different concentrations (25, 50, 100, 200, 500 and 800 µg/mL). Incubation of
reaction mixture was carried out in dark room for 30 minutes and the free radical scavenging
45
ability was estimated by measuring the absorbance at 515nm with UV-Vis
spectrophotometer.
The reaction was carried out in capped glass test tubes which were tightly wrapped
with aluminum foil from top to bottom. The DPPH radical stock solution was freshly
prepared every day for the reaction, and precautionary measures were taken to reduce the
loss of free radical activity during the experiment. All experiments were carried out in
triplicate with modified method (Ozturk et al., 2011). The inhibition percentage of DPPH
radicals were calculated as:
Inhibition (%) of DPPH radicals = Ac – As/ Ac X 100
Where Ac is absorbance of control reaction (reaction in which all reagents participate
except plant extract) and As is absorbance of sample (plant extract).
3.5.3. Antitumor activity (Potato disc assay):
Antitumor activities of each plant extract was evaluated by potato disc assay
(McLaughlin and Rogers, 1998; Mahmood et al., 2012). The growth medium was prepared
by adding 0.5 g sucrose, 0.8 g nutrient Nutrient Broth and 0.1 g yeast extract in 100 mL of
distilled water and autoclaved. Medium was allowed to cool and one loop of Agrobacterium
tumefaciens from storage culture was inoculated in growth medium using aseptic techniques.
The culture was vigorously shaken and then placed on orbital shaker for 48 hrs at 28°C.
Red skinned potatoes were surface sterilized in 10% sodium hypochlorite for 20
minutes. Ends of potatoes were removed and again immersed in sodium hypochlorite for 10
minutes. Cylinders of potatoes were made using sterile cork borer. These cylinders were
extensively washed with autoclaved distilled water and cut into discs with the help of
surgical blades sterilized by gamma irradiation (2.5 M Rads). Agar was prepared 1.5% by
dissolving 1.5g plane agar powder in 100 mL distilled water and autoclaved. Agar (25 mL)
was poured into sterilized petri plates and allowed to solidify in laminar air flow. 8 potato
discs were placed on each agar plate with the help of sterilized forceps (Rolzem international,
Pakistan). 50µL of inoculums was placed on surface of each disc and allowed to diffuse for
46
10 to 20 minutes. DMSO was used as negative control. Plates were wrapped with para film
and incubated at 27ºC for 21 days.
After 21 days staining of discs was made with Lugol solution (10 % KI+5 % I2) for
20 minutes and tumors were counted on each disc by using stereo microscope. The area
where no tumors were found became brown or blue because starch of potatos had absorbed
the dye, while the area of disc possessing tumor could not be stained and thus appeared
creamy white. Percent inhibition was calculated by the formula (Kanwal et al., 2010).
Percentage inhibition = 1- Number of tumors in the sample / Number of tumors in negative
control X 100
3.5.4. Antimicrobial activity:
3.5.4. (a). Microbial culture preparation:
The plant extracts were individually tested against panel of microorganisms. Bacterial
strains were cultured in Nutrient Broth and kept incubation at 37°C for 24 hours (Dhale and
Markandeya, 2011). Fungal strains were grown in Sabouraud Dextrose Broth at 28°C for 48
to 72 hours. The turbidity in broth medium showed the growth of fungus. The cultures were
stored in refrigerator at 2-8°C for further analysis.
3.5.4. (b): Disc diffusion assay:
The agar disc diffusion method (NCCLS, 1997) was used for determination of
diameters of inhibition zones made by each plant extract solution against tested bacterial and
fungal strains. The petri plates were washed and autoclaved. Briefly, 100 µL of suspension
containing 108 colony forming units (CFU/ mL) of bacteria cells and 10
4 spore/mL of fungi
were spread on petri plates containing Nutrient agar (NA) and Potato dextrose agar (PDA)
medium (50 mL media/ plate).
Sterile filter discs (4 mm) of wicks sheets were prepared and impregnated with 50 µL
of sample solution (20 mg/mL) of tested plant extract and were placed in inoculated petri
plates with the help of sterile forceps. Rifampicin (100 µg/mL) and Terbinafine (100
µg/mL) were used positive control in bacterial and fungal inoculated plates, respectively.
47
DMSO was used as negative control. The plates were incubated at 37°C for 24 hours and at
27°C for 48 hours for maximum bacterial and fungal growth, respectively. Antibacterial and
antifungal activities were evaluated by measuring diameter (Millimeter) of inhibition zones
with the help of digital zone reader.
Figure. 3.3. A typical agar plate showing antimicrobial activity in form inhibition zones
3.6. Anticancer attributes of plant extracts:
3.6. (a). Media preparation:
48
All the media components were heated at 37°C before media preparation. 5 mL of
Antibiotic-antimycotic solution (10,000 I.U./mL Penicillin, 10,000 µg/mL Streptomycin, 25
µg/mL Amphotericin B) and 50 mL of FBS (Fetal Bovine Serum) were added into 500 mL
of DMEM (Dulbeco’s modification of Eagle’s medium) and RPMI 1640, individually. The
media were stored at 2-8°C till further used.
3.6. (b): Defrosting of cells:
DMEM and RPMI-1640 were prewarmed up to 37°C at least one hour before use.
Cells (KBM5, U266, SCC4 and HCT116) were removed from liquid nitrogen and placed in
an incubator at 37°C. Contents were transferred from vial to sterile centrifuge tube. Volume
was made up to 10 mL by adding DMEM for SCC4 and HCT116 cells and RPMI-1640 for
KBM5 and U266 cells, respectively.
Cells were centrifuged for 5 min at 700 rpm. Supernatant was removed and pallet of
each cell type was suspended in 8 to 10 mL of respective media. Suspension of each cell type
was transferred into 25 mL of cell culture flasks, individually.
Flasks were labeled with cell type and date initials. Flasks were incubated (95%
humidified air 5% CO2 at 37ºC) for overnight.
3.6. (c). Trypsinisation of cells:
Cells (HCT116 and SCC4) were trypsinized when they were 80-100% confluent.
Media was removed and cells were washed twice using 5 mL of PBS (Phosphate Buffer
Saline) to partially remove dead cells as they are non-adherent to flask surface.
Pre-warmed trypsin-EDTA (1mL per 25 mL flask) was added and incubated (95%
humidified air 5% CO2 at 37ºC) for 1-2 min. Flask was tapped gentle to dislodge the cells
and then viewed under microscope. After the cells were completely disassociated from the
flask, 4 Ml of prepared media (DMEM) was added into flask. Media was pipetted up and
down several times to break up any cell lumps. Solution was divided into two fresh flasks.
DMEM media about 5 mL was added to each flask.
49
Flasks were labeled and kept in incubator (95% humidified air 5% CO2 at 37ºC).
After 24 hour media was removed from each flask to remove trypsin and fresh media was
added.
3.6. (d). Cell counting:
10 µL of cell suspension and 10 µL of trypan blue were mixed by pipetting up and
down few times. 10 µL of suspension was added to the prepared hameocytometer slide. Cells
were counted in counted in each of 5 squares and mean was calculated.
3.6.1. Anticancer activity (MTT assay):
Anticancer assay was performed as described previously (Prasad et al., 2010).
Briefly, 5,000 cells of HCT116, SCC4, KBM5 and U266 were seeded individually in Biolite
96-well (Thermo Scientific, Korea) plates and incubated (95% humidified air 5% CO2 at
37ºC) for 24 hr. Then cells were treated with different concentrations (10, 25, 50, 100 and
200 µg/mL) of plant extracts and incubated (95% humidified air 5% CO2 at 37ºC) for 72 hr.
Then 20 µL of 5 mg/mL MTT was added to each well.
After 2 hour incubation each well was supplemented with 100µL of lysis buffer. Cells
were further incubated for 6 hrs and optical density values were measured at 570 nm using an
MRX Revelation 96-well multiscanner (Dynex Technologies, USA). The IC50 values
(concentration at which 50% of cells were killed) were calculated from the graph plotted
concentration against percent cell viability.
3.6.2. Anti-inflammatory activity (Electrophoretic Mobility Shift Assay):
To assess the anti-inflammatory potential of potential extracts against KBM5 (Human
myelogenous leukemia) cells, we have determined the NF-κB activation in cancer cells
pretreated with respective plant extract. We isolated nuclei from treated-, untreated-, and
induced-cells and performed electrophoretic mobility shift assay (EMSA) as described
previously (Chainy et al., 2000).
In brief, nuclear extracts prepared from cancer cells were incubated with 32P end-
labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg of protein with 16 fmol of
50
DNA) from the HIV long terminal repeat (5′-TTGTTACAAGGGACTTTC CGCTG
GGGACTTTC CAGGGA GGCGT GG-3′, with NF-κB-binding sites) for 30 min at 37 °C.
The resulting protein-DNA complex was separated from free oligonucleotides on 6.6%
native polyacrylamide gels. The dried gels were visualized by Phosphor-Imager imaging
device (Molecular Dynamics, Sunnyvale, CA).
3.7. Statistical Analysis:
Three samples of each plant extract were assayed. Each sample was analyzed in
triplicate and data is reported as Mean ± S.D. Minitab software version 16 was applied to
perform analysis of variance (ANOVA) and to determine significant differences (P < 0.05).
51
CHAPTER 4 RESULTS AND DISCUSIONS
In this study five plants from four different families were collected from Punjab,
Pakistan. These plants were examined for their chemical components by using various
spectroscopic and chromatographic techniques such as, ultraviolet-visible (UV/Vis)
spectrometry, high performance liquid chromatography (HPLC) and gas chromatography
mass spectrometry (GC-MS). Biological activities (antioxidant, antitumor, antimicrobial,
anticancer and anti-inflammatory) of these plants were observed by different in vitro assays.
4.1. Euclayptus camaldulensis:
4.1. (a). Chemical Evaluation:
4.1.1. Total phenolic and flavonoid contents of E. camaldulensis :
Phenolic compounds are health benefactors. They as antioxidative agents (Ozturk et
al., 2010). In the current study Folin-Ciocalteu (FC) method was used to estimate total
phenolic contents of Euclayptus camaldulensis leaf extracts (methanol, chloroform and
hexane). FC method is inexpensive, reproducible and rapid method, that is extensively used
for measurement of total phenolic contents of plant extracts and is also narrated in various
pharmacopoeias (Cicco et al., 2009; Blainski et al., 2013). The results obtained are
represented in Figure. 4.1.1.
In FC method reaction of phosphomolybdate and phosphotungstate take place with
the phenolic compounds present in the investigated sample (Ramirez-Sanchez et al., 2010).
As a result of this reaction a blue pigment is generated that had extensive absorption of light
at 760 nm (Schofield et al., 2001).
Total phenolic components of E. camaldulensis leaf extracts were solvent dependent
and expressed as microgram gallic acid equivalents (µg GAE) per milligram of plant extract.
Figure. 4.1.1. shows that total phenolic contents in different extracts (methanol, chloroform
and hexane) varied widely ranging from 39.13 ± 0.30 to 148.68 ± 3.15 µg GAE/ mg of plant
extract. Methanol extract exhibited the highest total phenolic contents (148.68 ± 3.15 µg
52
GAE/ mg of plant extract), followed by chloroform (75.75 ± 2.87 µg GAE/ mg of
plant extract) and hexane (39.13 ± 0.30 µg GAE/ mg of plant extract) extracts. Our results
are in agreement with earlier study in which extracting solvents (methanol, chloroform and
hexane) exhibited the similar order (methanol > chloroform > hexane) for extraction of
phenolic compounds (Yeboah and Majinda, 2009). We observed higher extent of phenolics
in E. camaldulensis leaf extracts (methanol, chloroform and hexane), than previous reports
on E. camaldulensis wood and bark extracts for the same compounds (Conde et al., 1995;
Conde et al., 1996). It is interesting to note that Singab et al., 2011, demonstrated
comparatively higher level of phenolics in methanol fractions of E. camaldulensis leaves.
Figure. 4.1.1. Total phenolic and total flavonoid contets of E. camaldulensis extracts
(methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P <
0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per
milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are
expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of
plant extract)
0
20
40
60
80
100
120
140
160
Methanol Chloroform Hexane
E. camaldulensis extracts
Total phenolic contents (µg
GAE/mg of plant extract)
Total flavonoid contents (µg
QE/mg of plant extract)
53
Flavonoids (polyphenolic plant compounds) are known for pharmacological activities
(Robard et al., 1999). In current study flavonoid contents were determined using aluminum
chloride colorimetric assay. In this method reaction of aluminum chloride with adjacent keto
or hydroxyl groups of flavones or flavonols generates acid stable complexes (Chang et al.,
2002), which showed maximum absorption at 417 nm.
Flavonoid contents of different E. camaldulensis leaf extracts are mentioned in
Figure. 4.1.1. Significant difference (P < 0.05) was observed among flavonoid contents of
methanol, chloroform and hexane extracts. The content of flavonoids expressed as (µg QE/
mg of plant extract) varied from 6.09 ± 0.96 to 20.45 ± 1.74 µg QE/ mg of plant extract, as
shown in Figure. 4.1.1.
It is depicted from the results (Figure. 4.1.1) that methanol is the successful solvent
for extraction of flavonoid contents from E. camaldulensis leaves. This is similar to previous
findings in which methanol was used for extraction of flavonoids from medicinal plants
(Stankovic, 2011). Our results in current study are in strong agreement with another scientific
study (Hossain et al., 2011) who reported similar hierarchy (methanol > chloroform >
hexane) of solvents for extraction of flavonoids. As compared to our finding, Abu-Qatouseh
et al., 2013, explored relatively higher amount of flavoniods in another Euclayptus species.
4.1.2. High performance liquid chromatography (HPLC) analysis of E. camaldulensis:
The results of HPLC analysis of E. camaldulensis leaf extracts (methanol,
chloroform and hexane) are shown in Table. 4.1.1. Amount of phenolic compounds in three
extracts ranged from 0.12 ± 0.05 ppm to 5.86 ± 0.23 ppm. Syringic acid (0.63 ± 0.07 ppm)
was the major component of hexane extract.
Among the investigated phenolic compounds, gallic acid was the dominating
component of methanol and chloroform extracts. Maximum amount of gallic acid was
examined in methanol extract (5.86 ± 0.23 ppm).
Extent of gallic acid in chloroform extract (2.21 ± 0.36 ppm) was greater than hexane
extract (0.42 ± 0.15 ppm). Our results in current study are in accordance with previous
finding in which predominant amount of gallic acid was observed in ethanolic extract of
54
Egyptian E. camaldulensis (El-Ghorab et al., 2003). Furthermore, Sasikumar et al., 2001,
confirmed the presence of gallic acid in fresh leaves of E. camaldulensis.
Table. 4.1.1. High performance liquid chromatographic (HPLC) study of methanol,
chloroform and hexane extracts of E. camaldulensis for identification and
quantification of phenolic compounds (ppm)
Phenolic compounds Methanol extract Chloroform extract Hexane extract
Gallic acid 5.86 ± 0.23b 2.21 ± 0.36
d 0.42 ± 0.15
c
Syringic acid 1.75 ± 0.30d 1.97 ± 0.02
d 0.63 ± 0.07
c
p-Coumaric acid Nd 0.12 ± 0.05 Nd
Vanillic acid 4.53 ± 0.01ab
Nd Nd
Quercetin 0.69 ± 0.11e Nd Nd
p-Hydroxy benzoic acid 2.91 ± 0.01c 1.33 ± 0.04
a 1.01 ± 0.09
b
Catechin 0.44 ± 0.07e Nd Nd
Nd = Not detected. Values are Mean ± SD of triplicate determinations. (P < 0.05)
The least existing component was p-coumaric acid with chemical contribution of 0.12
± 0.05 ppm. Existence of p-coumaric acid is previously reported in specie of Euclayptus
(Rashwan, 2002). Some research groups demonstrated antioxidant and antibacterial
properties of p-coumaric acid (Lou et al., 2012; Kilic and Yesiloglu, 2013).
Katsuragi et al., 2010, reported biotransformation of p-coumaric acid by plant cell
cultures of Euclayptus perriniana. Quercetin, an importrant flavonoid, was identified in
methanol (0.69 ± 0.11 ppm) extract. Euclayptus globules is previously reported for existence
of quercetin (Proestos and Komaitis, 2013).
4.1.3. Gas chromatography mass spectrometry (GC-MS) study of E. camaldulensis:
The outcome of GC-MS study of E. camaldulensis leaf extracts (methanol,
chloroform and hexane) is presented in Table. 4.1.2. Chemical investigation showed
55
significant difference in composition of hexane extract from methanol and chloroform
extracts. Only palmitic acid was common in three extracts with maximum contribution
(31.06 %) in hexane extract. Considerable amount of palmitic acid was examined (Rencoret
et al., 2007) in other Euclayptus species (Euclayptus globulus, Euclayptus maidenii,
Euclayptus nitens, Euclayptus dunnii and Euclayptus grandis). Plamitic acid known as n-
hexadecanoic acid is also observed in other medicinal plants (Al-Shammari et al., 2012;
Rajeswari et al., 2012; Dubal et al., 2013). Aparna et al., 2012, evaluated anti-inflammatory
potential of palmitic aicd (n-hexadecanoic acid). An in vivo study reported that palmitic acid
regulates hypothalamic insulin resistance (Benoit et al., 2009). Al-Shammari et al., 2012,
showed that essential oil rich in hexadecanoic acid exhibits significant antimicrobial activity.
Furthermore, we examined that eucalyptol, 5-Hydroxymethylfurfural, spathulenol, β-
eudesmol, stearic acid and eicosane were common in methanol and chloroform extracts, as
shown in Table. 4.1.2. In comparison to previous study on essential oil of E. camaldulensis
fruit we obtained significantly higher amount of eucalyptol in both (methanol and
chloroform) the investigated extracts. Euclayptol is also an important component of other
aromatic plants (Vincenzi et al., 2002). Another study reported that eucalyptol plays
considerable role in reduction of contractile activity in cardiac muscles of experimental
organisms (Soares et al., 2005).
Chemical contribution of β-eudesmol in methanol and chloroform extract was 7.61 %
and 1.24 %, respectively. β-eudesmol is investigated for in vitro and in vivo antiangiogenic
efficacy (Tsuneki et al., 2005). Li et al., 2013, reported that β-eudesmol induces apoptosis in
HL60 cells.
Moreover, pyrogallol, o-cymene, heneicosane, L-lyxose, alloaromadendrene and
many other components were present in the investigated extracts (methanol, chloroform and
hexane) at varying degrees. o-cymene is also examined in essential oils (Romanenko and
Tkachev, 2006; Custodio et al., 2010). Pyrogallol is known for its antimicrobial potential
(Kocacaliskan et al., 2006).
Reports on chemical composition of E. camaldulensis leaves are scant in literature.
To the best of knowledge, our study is first report to explore chemical composition of
56
methanol, chloroform and hexane extracts of E. camaldulensis leaves. It is interesting to note
that each extract (methanol, chloroform and hexane) of E. camaldulensis leaves had good
profile of bioactive components with pharmacological potential.
Table. 4.1.2. Chemical constituents of methanol, chloroform and hexane extracts of E.
camaldulensis analyzed by Gas chromatography mass spectrometry (GC-MS)
Components Retention
time
Methanol
extract
Chloroform
extract
Hexane
extract
Composition (%)
Sec. isoamyl alcohol 5.28 ---- 6.61±0.12 ----
Eucalyptol 9.86 31.86±0.03 45.94±0.001 ----
o-cymene 9.69 ---- 0.63±0.04 ----
Catecholborane 13.33 ---- ---- 10.56±0.09
5-Hydroxymethylfurfural 13.52 2.05±0.10 4.30±0.07 ----
Pyranone 14.47 ---- ---- 1.17±0.04
o-methxy-p-vinylphenol 15.20 ---- ---- 15.27±0.002
Pyrogallol 16.12 11.91±0.01 ---- ----
Aromandendrene 17.53 7.69±0.20 ---- ----
Succinamic acid 17.70 ---- ---- 2.81±0.002
D-(+)-galactosamine 17.75 ---- ---- 1.26±0.15
Alloaromadendrene 17.89 2.49±0.03 ---- ----
Epiglobulol 19.44 1.43±0.14 ---- 1.92±0.03
Lactose 19.59 1.07±0.03 ---- ----
Spathulenol 19.70 3.58±0.02 1.37±0.01 ----
Ledol 19.82 11.88±0.02 ---- 0.73±0.23
L-Lyxose 19.83 ---- ---- 3.58±0.03
β -lactic acid 19.85 ---- ---- 2.75±0.07
Viridiflorol 19.96 3.07±0.03 ---- ----
β-eudesmol 20.40 7.61±0.01 1.24±0.11 ----
57
( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data
4.1.(b). Biological Evaluation:
4.1.4. Free radical (DPPH) scavenging activity of E. camaldulensis :
DPPH assay is robust method to assess antioxidant potential of plant extracts or pure
compounds (Gawron-Gzella et al., 2012). This assay is aimed at measuring the capacity of
plant extract to scavenge purple colored 2,2-diphenyl-1-picryl hydrazil and converting it to
yellow colored diphenylpicrylhydrazin (Saha et al., 2008).
Greater the extent of antioxidant compounds in plant extract, greater will be extent of
yellow colored diphenylpicrylhydrazin molecules in the test solution and higher will be the
antioxidant efficacy (Tepe et al., 2005).
In the current study we investigated antioxidant activity of E. camaldulensis leaf
extracts (methanol, chloroform and hexane) in concentration dependant mode (25 to 800
µg/ml). Increase in free radical scavenging activity (%) was observed with increase in
concentration of each extract (methanol, chloroform and hexane).
d-Gulopyranose 22.16 2.22±0.04 ---- ----
Hentriaconatane 22.34 ---- 1.28±0.26 ----
Carhydrine 22.56 4.42±0.13 ---- ----
3-Heptadecene 23.13 ---- 2.25±0.19 ----
D(+)-Raffinose pentahydrate 24.33 1.07±0.06 ---- ----
Palmitic acid 27.27 4.38±0.13 6.94±0.01 31.06±0.03
Stearic acid 31.15 1.34±0.05 1.32±0.03 ----
Docosene 31.73 ---- 0.58±0.22 ----
Palmityl chloride 32.60 ---- 1.93±0.01 ----
Eicosane 38.32 1.71±0.11 1.18±0.04 ----
Benzisothiazolinone 38.57 ---- ---- 2.22±0.13
Heneicosane 40.97 ---- 6.82±0.001 ----
Fatty alcohol 42.60 ---- 1.49±0.02 ----
Flunixin 43.38 ---- 1.16±0.17 ----
Stearyl iodide 43.49 ---- 1.21±0.09 ----
58
Table. 4.1.3. Scavenging (%) of free radicals (DPPH) by methanol, chloroform and
hexane extracts of E. camaldulensis
E. camald- ulensis
extracts
Scavenging (%) of free radicals at different concentrations (µg/mL) IC50
(mg/mL)
25 50 100 200 400 800
Methanol 23.64±1.92a
e
37.51±1.26c
a
55.66±1.51e
c
68.93±0.21f
d
79.22±0.95b
f
87.61±2.03a
b
0.08 Chloroform 21.74±1.06
b
a
30.28±2.49d
b
35.95±1.97d
f
57.11±5.13a
c
72.48±2.70d
e
74.93±3.24c
d
0.15 Hexane 13.12±2.32
d
c
20.28±1.83f
a
20.66±0.92b
a
28.51±1.48e
d
45.03±4.81c
b
63.29±3.62e
e
0.54 Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among concentrations tested.
Minimum percent inhibition (%) values for three extracts (methanol, chloroform and
hexane) were examined at 25 µg/mL, while maximum rate of inhibition (%) was noticed at
800 µg/mL. Gallic acid that was used as positive control in free radical (DPPH) scavenging
assay exhibited excellent free radical scavenging activity with IC50 value of 0.02 mg/mL. It is
depicted from the results (Table. 4.1.3) that among the three extracts, methanol extract was
examined as leading one with respect to inhibition (%) values.
Overall, among three extracts moderate free radical scavenging activity was observed
in chloroform extract. Hexane extract exhibited minimum scavenging of stable free radicals
at various doses (25 to 800 µg/mL). These results are in strong agreement with previous
finding (Yusri et al., 2012) in which hexane extract of a Malaysian medicinal plant was
reported as poor candidate for scavenging stable free radicals (DPPH) as compared to
chloroform and methanol extracts. Our results are in accordance with the fact that different
geographical conditions are innovative factor to effect phytochemical components and
related pharmacological properties of medicinal plants (Edoga et al., 2005).
4.1.5. Antitumor activity of E. camaldulensis (Potato Disc assay):
Agrobacterium tumefaciens induces neoplastic disease in plants (Galsky et al., 1981). The
consistency of this assay is based on the observation that some tumorigenic mechanisms are
same in animals and plants. Galsky et al., 1981, showed that inhibition of crown gall tumor
59
in potato tissues had good correlation with compounds and extracts active in 3PS leukemic
mouse assay.
The results of antitumor activity are shown in Figure. 4.1.2. Antitumor activity of
three extracts varied from 17.06 ± 1.20% to 90.09 ± 0.70%.
Figure. 4.1.2. Antitumor activity of methanol, chloroform and hexane extracts of E.
camaldulensis, estimated by potato disc assay. Data is represented as Mean ± SD for three
samples of each extract (methanol, chloroform and hexane) analyzed individually in
triplicates. (P < 0.05).
Maximum antitumor activity was observed in methanol extract (IC50 = 59.68 µg/mL)
followed by chloroform (IC50 = 112.5 µg/mL) and hexane (IC50 = 191.7 µg/mL) extracts.
The activity of three extracts (methanol, chloroform and hexane) was significantly (P < 0.05)
different from each other. This is in accordance with previous investigation in which
0
10
20
30
40
50
60
70
80
90
100
25 50 100 200 400 800
Inh
ibit
ion
of
tum
ors
(%)
Concentartion (µg/mL)
Methanol Chloroform Hexane
60
significant effect of extracting solvents was observed on A. tumefaciens induced tumors
(Mazid et al., 2011).
Mclaughlin and Rogers (1998) narrated that potato disc assay is inexpensive, animal-
sparing, rapid, statistically reliable and safe technique for preliminary screening of antitumor
agents. Many researchers used this technique for screening of antitumor potential of plants
(Islam et al., 2009; Bibi et al., 2011).
4.1.6. Antimicrobial activity of E. camaldulensis:
The methanol, chloroform and hexane extracts obtained from E. camaldulensis leaves
were tested against five microorganisms in order to assess their antimicrobial efficacies. The
results are shown in Table. 4.1.4. Among the bacterial strains, B. subtilis (21.78 ± 2.60 mm)
was more susceptible, while E. coli (10.06 ± 0.19 mm) was the resistant one. Hexane extract
showed no activity against E. coli and A. niger.
Tefsen et al., 2005, observed that outer membrane of gram negative bacteria like E.
coli is bestowed with lipopolysachrides which provide them protection by conferring a
negative charge on the surface. So the E. coli is less susceptible to examined extracts
(methanol, chloroform and hexane).
In case of fungi, F. solani (18.56 ± 0.05 mm) was found to be more sensitive to
methanol extract than A. niger (11.49 ± 0.86 mm). This finding is in line with previous study,
in which methanol extract of a plant was found to be more potent against F. solani than A.
niger (Alagesaboopathi, 2012). Significant antimicrobial activity of methanol extract might
be attributed to higher level of bioactive components in it, which have strong correlation with
antimicrobial activity (Si et al., 2006).
It is interesting to note that chloroform extract was more potent against A. niger
(16.16 ± 0.07 mm) as compared to F. solani (9.27 ± 1.46 mm). These results are different
61
Table. 4.1.4. Antimicrobial activity of methanol, chloroform and hexane extracts of E.
camaldulensis
Nt = not tested. Data is represented as Mean ± SD of triplicate determinations of each extracts (methanol, chloroform and hexane) against
each microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents
tested. *Standard antibiotic for bacteria; * *Standard antibiotic for fungi.
from previous finding (Shirurkar and Wahegaonkar, 2012) in which Euclayptus oil exhibited
equivalent activity against A. niger (19 mm) and F. solani (19 mm). Methanol and hexane
extracts were more effective against F. solani than A. niger. These results are in agreement
with previous investigation, in which a potent medicinal plant (Chromolaena odorata) was
found more active against F. solani than A. niger at the highest tested dose of extract (Llondu,
2014).
4.2. Viola betnocifolia
4.2. (a). Chemical Evaluation:
4.2.1. Total phenolic and flavonoid contents of V. betonicifolia extracts:
Total phenolic and total flavonoid contents in methanol, chloroform and hexane
extracts of Viola betonicifolia leaves were demonstrated by using the Folin-Ciocalteu and
E.
camaldulensis
leaf extracts
Diameter of inhibition zones (mm)
Bacterial Strains Fungal Strains
Escherichia
coli
Bacillus
subtilis
Pasteurella
multocida
Aspergillus
niger
Fusarium
solani
Methanol 10.06±0.19a 21.78±2.60
c 15.21±0.34
f 11.49±0.86
a 18.56±0.05
d
Chloroform 12.67±0.98b 18.43±1.54
a 0 ± 0 16.16±0.07
d 9.27±1.46
a
Hexane 0 ± 0 14.84±0.03f 17.99±0.76
e 0 ± 0 13.32±2.09
c
*Rifampicin 21.66±1.41c 24.66±0.47
b 23.33±1.69
c Nt Nt
**Terbinafine Nt Nt Nt 25.66±1.69c 24.00±0.82
b
62
aluminum chloride colorimetric methods, respectively. The results are presented in Figure.
4.2.1.
Total phenolic contents of three solvents ranged from 29.35 ± 1.66 (µg GAE /mg of
plant extract) to 155.78 ± 4.12 (µg GAE /mg of plant extract). Total flavonoid contents were
in range of 2.06 ± 0.70 (µg QE / mg of plant extract) to 16.30 ± 1.27 (µg QE /mg of plant
extract).
Figure. 4.2.1. Total phenolic and total flavonoid contents of V. betnocifolia extracts
(methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P <
0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per
milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are
expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of
plant extract).
0
30
60
90
120
150
180
Methanol Chloroform Hexane
V. betnocifolia extracts
Total phenolic contents (µg
GAE/mg of plant extract)
Total flavonoid contents (µg
QE/mg of plant extract)
63
It is evident from the results (Figure. 4.2.1) , that we observed higher extent of total
phenolic contents in each extract as compared to total flavonoid contents. This observation is
in line with scientific studies (Kaur and Mondal, 2014; Saeed et al., 2011) in which higher
extent of total phenolic contents was observed in medicinal plants as compared to flavonoid
contents.
It was observed that total phenolic and flavonoid contents vary significantly (P <
0.05) with variation in extracting solvent. These results are in strong agreement with previous
findings (Shabir et al., 2011) where significant difference (P < 0.05) was observed in total
phenolic and flavonoid contents for different extracts.
It was depicted from the results that three extracts shared the similar ranking
(methanol > chloroform > hexane) in case of Folin-Ciocalteu and aluminum chloride
colorimetric assays. These findings are in comparison with previous scientific studies in
which identical hierarchy of extracting solvents (methanol > chloroform > hexane) was
examined for measurement of total phenolic and total flavonoid contents (Yeboah and
Majinda, 2009). In contrast to previous literature (Muhammad and Saeed, 2011) on Viola
betonicifolia, we observed comparatively higher phenolic and lower flavonoid contents in
current study.
4.2.2. High performance liquid chromatography (HPLC) analysis of V. betonicifolia
extracts:
The extracts obtained from V. betonicifolia were investigated for the presence of
phenolic compounds by HPLC analysis, as presented in Table. 4.2.1. For sinapic acid,
methanol extract was the most rich (6.61 ± 0.40 ppm), followed by chloroform (3.20 ± 0.02
ppm) extract. Sinapic acid was absent in hexane extract. The extent of ferulic acid, gallic acid
and p-coumaric acid ranged from 0.37 ± 0.001 ppm to 4.52 ± 0.15 ppm for the three extracts.
Quercetin, was also detected in methanol extract (2.65 ± 0.003 ppm).
All the investigated compounds had distinct pharmacological properties. Zhao and
Hu, 2013, observed that gallic acid increases death rate of human cervical carcinoma cells.
Chemopreventive effects of gallic acid in mice model were also observed (Raina et al.,
64
2008). Antioxidant efficacy of p-coumaric acid is documented in literature records (Zang et
al., 2000).
Picone et al., 2013, reported that ferulic acid is potential candidate for inhibition of
oxidative stress. Some research group showed that ferulic acid and caffeic acid inhibit tumor
promotion in mouse skin (Huang et al., 1988).
Siger et al., 2013, narrated antioxidant potential of sinapic acid derivatives. Thus, the
biological activities (antioxidant, antimicrobial, anticancer, and anti-inflammatory) of V.
betonicifolia evaluated in current study may be correlated to potential phenolic compounds in
these extracts. To the best of our knowledge, phenolic compounds of V. betonicifolia are not
previously reported in literature.
Table. 4.2.1. High performance liquid chromatographic (HPLC) analysis of methanol,
chloroform and hexane extracts of V. betonicifolia for identification of phenolic
compounds (ppm)
Phenolic compounds Methanol extract Chloroform extract Hexane extract
Gallic acid 4.52 ± 0.15b Nd 0.29 ± 0.08
c
Sinapic acid 6.61 ± 0.40cd
3.20 ± 0.02be
Nd
p-coumaric acid 0.93 ± 0.03 Nd Nd
Ferulic acid 3.28 ± 0.21a 0.37 ± 0.001
b Nd
Quercetin 2.65 ± 0.003 Nd Nd
Nd = Not detected. Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among solvents (methanol, chloroform and hexane) used.
4.2.3. Gas chromatography mass spectrometry (GC-MS) study of V. betonicifolia
extracts:
The results of GC-MS analysis of methanol, chloroform and hexane extracts of V.
betonicifolia leaves were expressed in percentage (%) and are shown in Table. 4.2.2. It is
65
evident from the results that 24 compounds were identified in methanol extract. Total number
of compounds in chloroform and hexane extract were 37 and 31 respectively.
Table. 4.2.2. Chemical constituents of methanol, chloroform and hexane extracts of V.
betonicifolia analyzed by gas chromatography mass spectrometry (GC-MS)
aComponents bRT Methanol
extract
Chloroform
extract
Hexane
extract
Composition (%)
Butanone dimethyl acetal 4.34 0.80 1.9 0.31
1,3-Dihydroxyacetone dimer 6.79 1.27 ---- ----
Dicyclohexyl oxalate 8.41 ---- 0.49 ----
Glycerin 8.68 ---- 0.55 ----
Cyclopentanone,dimethylhydrazone 10.55 0.57 ---- ----
4,5-Diamino-6-hydroxypyrimidine 10.76 ---- 0.85 ----
2-Hydroxy-2,3-dimethylsuccinic acid 10.95 ---- 0.76 ----
Geraniol 11.13 ---- ---- 0.22
2,5-dihydropyrrrol 11.31 6.37 ---- ----
Pyranone 12.02 1.18 ---- ----
4H-Pyran-4-one, 2,3-dihydro-3,5-
dihydroxy-6-methyl
12.14 ---- 0.94 ----
Coumaran 13.32 0.52 ---- ----
5-Hydroxymethylfurfural 13.51 ---- 1.71 ----
1-Piperazineethanamine, 4-methyl- 13.56 0.72 ---- ----
2-Methoxy-4-vinylphenol 15.20 ---- 1.01 ----
Eugenol 15.93 ---- 0.12 ----
66
Piperazine 17.41 ---- 0.74 ----
Octadecane 18.08 ---- 0.11 ----
2,4-Di-t-butylphenol 18.35 ---- ---- 0.10
Nonadecyl pentafluoropropionate
18.53 ---- ---- 0.15
Tetracosane 18.77 ---- ---- 4.57
Lauric acid 18.96 ---- 0.53 ----
(E)-Stilbene 19.24 ---- 0.88 ----
Cetene 19.50 ---- ---- 0.08
1-Methyl-4-amino-4,5(1H)-dihydro-
1,2,4-triazole-5-one
19.82 ---- 1.15 ----
Butanoic acid, 2-hexylimino- 19.95 ---- 0.83 ----
Ar-tumerone 20.86 ---- ---- 0.72
Patchouli alcohol 21.22 ---- 0.33 ----
Curlone 21.56 ---- ---- 0.37
Octacosyl trifluoroacetate 22.08 ---- ---- 0.13
Mristic acid 22.31 1.02 2.07 0.69
Cis-pinane 24.33 0.55 ---- ----
3-Nonen-1-ol, (Z)- 24.34 ---- 2.25 ----
Phytol, acetate 25.51 ---- 0.68 0.26
Methyl palmitate 26.54 ---- 1.21 0.38
Palmitic acid 27.29 25.67 19.48 22.52
Heptadecyl heptafluorobutyrate 28.07 ---- ---- 0.41
Cyclohexanecarboxylic acid, cyclopentyl
ester
28.87 ---- ---- 0.18
67
L-(+)-Ascorbic acid 2,6-dihexadecanoate 29.43 0.16 ---- ----
Linoleic acid ethyl ester 30.10 ---- 0.64 ----
Methyl linolenate 30.22 ---- ---- 1.62
Phytol 30.42 1.07 2.43 0.18
Linoleic acid 30.74 16.99 0.63 19.61
α-linolenic acid 30.87 18.48 9.72 13.63
Stearic acid 31.16 6.65 5.48 ----
1-Heptacosanol 31.74 ---- ---- 1.536
p-Menth-8(10)-en-9-ol, cis- 32.80 ---- 0.82 ----
Eicosane 33.33 ---- ---- 0.15
Butyl 9,12,15-octadecatrienoate 34.28 ---- ----- 0.17
Funixin 35.47 ---- ---- 0.10
Pentacosane 35.98 ---- ---- 0.30
Eicosane 37.59 ---- ----- 0.13
1-chloroheptacosane 38.32 0.51 ----- ----
Octacosane 39.64 ---- ----- 0.35
Squalene 40.22 ---- ----- 0.10
Benzo quinoline, 2,4-dimethyl- 40.73 ---- 0.63 ----
2-Ethylacridine 41.00 ---- 22.15 ----
Silicic acid, diethyl bis(trimethylsilyl)
ester
42.22 ---- 3.65 ----
2,4 Dimethylbenzoquinoline 42.29 0.18 ----- ----
bis(trimethylsilyl) diethyl silicate 42.35 0.70 ---- ----
68
( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data
Various chemical components such as α-linolenic acid, lactose, stearic acid, geraniol,
eugenol, tetracosane, linoleic acid ethyl ester, cetene, patchouli alcohol, 2,6- dihexadeconate,
ascorbic acid, methyl linoleate, phytol, heptacosanol, funixin, α-amyrin, β-amyrin, β-
sitostenone, fucosterol, curlone, stigmasterol,α-tochopherol, β-tochopherol and many others
are observed at varying degrees in the investigated extratcts.
Extent of linoleic acid ethyl ester (0.64 %) in chloroform extract of V. betonicifolia
was lower than previously reported value (4.13 %) in an Indian medicinal plant
(Gopalakrishan et al., 2011). Linoleic acid ethyl ester is reviewed for its anti-inflammatory,
β-Tocopherol 43.01 ---- ----- 0.08
Thymol- TMS 43.38 1.24 ----- ----
α-Tocopherol 44.16 ---- ---- 1.53
Trimethyl[4-(1-methyl-1
methoxyethyl)phenoxy]silane
45.19 ---- 0.24 ----
5-Methyl-2-trimethylsilyloxy-
acetophenone
46.55 ---- 0.90 ----
4-Methyl-2-trimethylsilyloxy-
acetophenone
46.59 ---- 0.13 ----
Silicic acid, diethyl bis(trimethylsilyl)
ester
46.77 ---- 0.09 ----
2,4 DimethylBenzoquinoline 47.32 1.80 ----- ----
Stigmasterol 47.34 0.76 ----- ----
γ-Sitosterol 47.68 3.42 ---- ----
Fucosterol 47.97 0.19 1.92 ----
Arsenous acid, tris(trimethylsilyl) ester 48.43 ---- 0.38 ----
β-Amyrin 48.67 ---- ---- 5.58
α-Amyrin 49.66 ---- ---- 7.35
β-Sitostenone 50.62 5.45 ---- ----
Tris(tert-butyldimethylsilyloxy)arsane 52.09 ---- 1.59 ----
69
hepatoprotective, insectifuge, antieczemic, antiandrogenic, antiacne, antiarthritic, nematicide
and anticoronary activities.
α-amyrin and β-amyrin are potent triterpenes and are known for their antinociceptive
prospective (Otuki et al., 2005). Amount of β-amyrin (5.58 %) in hexane extract of V.
betonicifolia is different from previously reported value of β-amyrin (15.62 %) in petroleum
ether extract of an Egyptian medicinal plant (Ibrahim, 2012).
Singh et al., 2009, reported in vivo antihyperglycaemic potential of α-amyrin acetate.
Santos et al., 2012, examined in vivo hypolipidemic and antihyperglcemic efficacies of α-
amyrin and β-amyrin. We also examined 7.35 % of α-amyrin in hexane extract of V.
betonicifolia.
β-sitostenone (5.45 %) was potent component of methanol extract of V. betonicifolia.
Chen and Wang, 2010, isolated β-sitostenone from an important Chinese medicinal plant
along with other steroids.
Lee et al., 2003, isolated fucosterol from marine algae and evaluated it for
hepatoprotective and antioxidant activities. We also observed fucosterol in methanol and
chloroform extracts of V. betonicifolia as shown in Table. 4.2.2. Khanavi et al., 2012,
narrated fucosterol fraction of marine algae for cytotoxic activities against colon and breast
cancer cells.
Stilbenes are known for their antimicrobial attributes (Albert et al., 2011). Existance
of (E)- stilbene (0.88 %) in chloroform extract of V. betonicifolia was examined.
GC-MS analysis of chloroform extract revealed the presence of silicic acid, diethyl
bis(trimethylsilyl) ester. Extent of Silicic acid, diethyl bis(trimethylsilyl) ester is in
comparison with previously reported value for an Indian medicinal plant (Tyagi and Sharma,
2014).
4.2. (b). Biological Evaluation:
4.2.4. Free radical (DPPH) scavenging activity of V. betonicifolia extracts:
70
The free radical scavenging activity of methanol, chloroform and hexane extracts of
Viola betonicifolia was recorded in terms of percent inhibition (%) and results are shown in
Table. 4.2.3. It is evident from the results that this activity has linear correlation with
concentration (25 to 800 µg/mL) of each extract.
At higher concentration greater the extent of antioxidant components and higher will
the rate of percent inhibition (%) of free radicals (Saha et al., 2008). In current study the
three extracts (methanol, chloroform and hexane) effectively scavenged the DPPH radicals.
The chloroform extract exhibited the highest free radical scavenging activity, with percent
inhibition of (70.88 ± 0.45%) at the highest tested dose (800 µg/mL).
This is in accordance with previous study in which chloroform extract of Viola
betonicifolia was examined for maximum inhibition of stable free radicals (Muhammad and
Saeed, 2011). Lowest free radical scavenging activity was examined in hexane extract at
various tested doses (25 to 800 µg/mL). Similar observation had already been reported for
hexane extract of different medicinal plants of Pakistan (Bukhari et al., 2008; Khan et al.,
2012). Statistical analysis revealed significant difference (P < 0.05) in free radical scavenging
activities of different extracts at different concentrations.
Table. 4.2.2. Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform
and hexane extracts of V. betonicifolia
V. betonici-
folia
extracts
Scavenging (%) of free radicals at different concentrations (µg/mL) IC50
(mg/mL)
25 50 100 200 400 800 Methanol 12.35±0.56
b
a
20.70±0.82c
c
24.26±0.67f
d
25.70±1.93e
f
40.41±2.71d
e
42.02±0.22c
b
>1
Chloroform 19.44±0.08c
f
30.16±3.73d
e
36.75±2.21c
c
41.12±2.36f
b
58.46±1.51b
d
70.88±0.45e
a
0.23 Hexane 8.51 ±0.78
f
b
9.17 ±1.59a
d
12.22±0.90b
f
16.28±0.24b
a
30.68±0.37e
c
39.47±0.69d
e
>1 Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among concentrations tested.
71
4.2.5. Antitumor activity of V. betonicifolia extracts (Potato Disc Assay):
It is evident from the literature that some plant extracts exhibit a cell-type antitumor
efficacy (Rehman et al., 2009). In current study antitumor potential of methanol, chloroform
and hexane extracts of Viola betonicifolia was evaluated by potato disc assay, as shown in
Figure. 4.2.2. The three extracts were able to significantly (P < 0.05) inhibit the growth of A.
tumefaciens at varying extents.
Figure. 4.2.2. Antitumor activity of methanol, chloroform and hexane extracts of V.
betonicifolia. Data is represented as Mean ± SD for three samples of each extract (methanol,
chloroform and hexane) analyzed individually in triplicates. (P < 0.05).
We examined that inhibition of tumors (%) increase linearly with increase in dose of
extracts. This observation is strongly supported by previous study (Mahmood et al., 2012) in
which increase in crown gall tumor inhibition (%) was observed with increase in dose of
extracts. Maximum antitumor activity (IC50 = 38.13 µg/mL) was examined in chloroform
0
10
20
30
40
50
60
70
80
90
100
25 50 100 200 400 800
Inh
ibit
ion
of
tum
ors
(%)
Concentration (µg/mL)
Methanol Chloroform Hexane
72
extract. Antitumor activity of methanol extract (IC50 = 120.9 µg/mL) was greater than hexane
extract (IC50 = 196.4 µg/mL).
4.2.6. Antimicrobial activity of V. betonicifolia extracts:
The in vitro antimicrobial activity of methanol, chloroform and hexane extracts of V.
betonicifolia against employed microorganisms was assessed by disc diffusion assay. As can
be seen from Table. 4.2.4, the two extracts (methanol and hexane) illustrated considerable in
vitro antimicrobial potential against all the investigated microorganisms (Escherichia coli,
Bacillus subtilis, Pasteurella multocida, Aspergillus niger and Fusarium solani). However,
chloroform extract was inactive against E. coli, P. multocida and F. solani.
Table. 4.2.4. Antimicrobial activity of methanol, chloroform and hexane extracts of V.
betonicifolia
Nt = Not tested. Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, chloroform and hexane) against
each microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents
tested. *Standard antibiotic for bacteria; * *Standard antibiotic for fungi.
The diameter of inhibition zones ranged from 7.68 ± 0.09 mm to 20.10 ± 2.26 mm. It
is evident from the results that hexane extract showed good activity against most of tested
V.
betonicifolia
extracts
Diameter of inhibition zones (mm)
Bacterial Strains Fungal Strains
Escherichia
coli
Bacillus
subtilis
Pasteurella
multocida
Aspergillus
niger
Fusarium
solani
Methanol 9.97±0.57ab
13.23±1.52cd
11.05±0.83a 0 ± 0 10.46±1.24
e
Chloroform 0 ± 0 8.74 ±0.07ae
0 ± 0 12.74±0.92cd
0 ± 0
Hexane 16.65±1.30bd
20.10±2.26c 7.68±0.09
f 17.37±0.15
cd 0 ± 0
*Rifampicin 21.66±1.41e 24.66±0.47
b 23.33±1.69
cd Nt Nt
**Terbinafine Nt Nt Nt 25.66±1.69af
24.00±0.82ac
73
microbial strains. Potent antimicrobial activity of hexane extract might be attributed to
presence of α and β amyrin in it, as represented in GC-MS analysis (Table. 4.2.2).
Johann et al., 2007, explored that α and β amyrin and their derivatives have
distinctive antifungal activities. It was examined that α and β amyrin, α and β amyrin
forminate and α and β amyrin acetate exhibited potential antifungal activities against all the
investigated Candida species.
Surprisingly, α and β amyrin forminate was found as effective as fluconazole in
inhibiting the adhesion of C. albicans to buccal epithelial cells. A. niger was found more
sensitive to hexane extract than F. solani. These results are in line with previous study in
which A. niger was examined more susceptible to Carica papaya extracts than F. solani
(Llondu, 2011). However, methanol extract was effective against F. solani than A. niger.
This observation is in strong agreement with earlier scientific study in which aqueous exrtact
of an important plant of Pakistan was found more active against F. solani than A. niger
(Ahmed et al., 2014).
4.2.7. Anticancer activity of V. betonicifolia extracts:
The relationship between doses of V. betonicifolia leaf extracts (methanol, chloroform
and hexane) and their cell growth inhibiting effects on KBM5, myelogenous leukemia;
SCC4, tongue squamous carcinoma and HCT116, colon carcinoma cells was evaluated by
MTT assay.
The incubation of cell lines (KBM5, SCC4 and HCT116) with V. betonicifolia leaf
extracts decreased the viability of these cell lines. Similar observation was noticed in
previous study in which decrease in viability of lung (A549), colon (HCT116), breast
(MCF7) and liver (HepG2) cancer cells was noticed with increase in concentration of Suadi
plant extracts (Elsharkawy and Alshathly, 2013).
Based on the results of MTT assay the cell survival rate (%) was calculated for each
concentration of extracts (Figure. 4.2.3). Overall the three extracts of V. betonicifolia leaves
were active against the tested cell lines (KBM5, SCC4 and HCT116). The results in Figure.
4.2.3. (A) revealed that the methanol extract had poor effects on viability (%) of cancer cell
74
lines (KBM5, SCC4 and HCT116) as compared to chloroform and hexane extracts, whose
results are represented in Figure. 4.2.3. (B) and Figure. 4.2.3. (C), respectively.
The chloroform extract revealed remarkable decrease in viability of KBM5 cells with
IC50 value of 0.001 mg/mL followed by hexane (IC50 = 0.002 mg/mL) and methanol (IC50 =
0.067 mg/mL) extracts.
In case of SCC4 cells maximum anticancer activity was observed in hexane (IC50 =
0.096 mg/mL) extract followed by chloroform (IC50 = 0.103 mg/mL) and methanol (IC50 =
0.155 mg/mL) extracts. HCT116 cells were more susceptible to chloroform extract (IC50 =
0.063 mg/mL), while resistant to methanol extract (IC50 = 0.183 mg/mL). Moderate
anticancer activity was observed by hexane extract (IC50 = 0.094 mg/mL) against HCT116
cells.
75
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 SCC4 HCT116
A
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 SCC4 HCT116
B
76
Figure. 4.2.3. Anticancer activity of (A) methanol (B) chloroform and (C) hexane extracts
of V. betonicifolia against KBM5 (Human myelogenous leukemia), SCC4 (Human tongue
squamous carcinoma) and HCT116 (Human colon carcinoma) cell lines. Values are
represented as Mean ± SD of quadruplicate determinations. (P < 0.05).
4.2.8. Anti-inflammatory activity of V. betonicifolia chloroform extract:
The inflammatory transcription factor is an important target for prevention of cancer
(Gupta et al., 2011). Many investigations had demonstrated that plant extracts play promising
role for in vitro NF-κB inhibition. In current study we examined the NF-κB inhibitory
potential of chloroform extract of V. betonicifolia leaves.
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 SCC4 HCT116
C
77
Figure. 4.2.3. Anti-inflammatory activity of chloroform extract of V. betonicifolia. Lane 1
and lane 2 show unstimulated Myelogenous leukemia (KBM5) cells. In lane 3 cells were
treated with TNF-α alone. In other lanes (4-6) cells were pre-incubated with chloroform
extract of V. betonicifolia (10, 25 and 50 µg/mL) for 12 hours and then treated with 0.1 nM
TNF-α for 30 minutes. Nuclear extracts were prepared and assayed for NF-κB activation by
EMSA.
78
In this assay cells were treated with increasing doses of V. betonicifolia chloroform
extract, and subsequently stimulated with TNF-α. Then equal amounts of nuclear extracts
from cells (Human mylegenous leukemia) were investigated by EMSA for NF-κB activity.
As indicated in Figure. 4.2.4, activation of NF-κB almost completely inhibited by
pretreatment of mylogenous leukemia cells with 50 µg/mL. It was postulated that anti-
inflammatory potential of chloroform extract might be attributed to unique profile of
bioactive components in it, as shown in Table. 4.2.2. Various components (Table. 4.2.2) of
chloroform extract are known for their anti-inflammatory activity (Srinivasan et al., 2007;
Boots et al., 2008; Chao et al., 2009).
4.3. Euphorbia royleana:
4.3.(a). Chemical Composition:
4.3.1. Total phenolic and flavonoid contents of E. royleana:
Total phenolic contents (TPC) of methanol, aqueous and hexane extracts of fresh E.
royleana ranged from 30.65 ± 0.46 and 63.68 ± 0.43 µg GAEs/ mg of plant extract,
respectively. Surprisingly, highest extent of phenolic compounds (63.68 ± 0.43 µg GAEs/mg
of plant extract) was observed in hexane extract followed by methanol extract. Our results in
current study are in strong agreement with previous findings in which higher phenolic
contents were observed for hexane extract followed by methanol extract (Aris et al., 2009). A
recent scientific study (Rahman et al., 2013) narrated that phenolic contents of hexane extract
of an Indian medicinal plant was higher than that of methanolic extract.
It is interesting to note that FC reagent may also react with some non phenolic
substances in reaction mixture (Ikawa et al., 2003; Everette et al., 2010) and results in
unusuall higher extent of total phenolic contents. It is worthy to note that phenolics include
diverse range of compounds which are often polar in nature, however, due to non polar
linkages they are extracted with less polar and non polar solvents (Mokhtarpour et al., 2014).
Furthermore, steric hindrance in polar compounds, awards them non polar naturte. In contrast
to previous studies on phenolic contents of Euphorbiaceace, we observed comparitvely
higher extent of phenolic contents in hexane extract of fresh Euphorbia royleana (Shahwar et
79
al., 2010). To the best of our knowledge, there is no literature report about phenolic contents
of fresh Euphorbia royleana.
Methanol, hexane and aqueous extracts of fresh E. royleana exhibited significant (P <
0.05) amount of flavonoid contents as represented in Figure. 4.3.1. Maximum amount of
flavonoids (µg QE/ mg of plant extract) was examined in hexane extract (47.47 ± 0.71). This
is in line with previous investigation, in which maximum amount of flavonoids was observed
in hexane extract of an endemic Indian medicinal plant (Fidrianny et al., 2014). Flavonoid
contents of fresh Euphorbia royleana extracts are in comparison with other medicinal flora
of Pakistan (Naz and Bano, 2013).
Figure. 4.3.1. Total phenolic and total flavonoid contents of E. royleana extracts (methanol,
hexane and aqueous). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total
phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant
extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram
quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract).
0
10
20
30
40
50
60
70
80
90
Methanol Hexane
Total phenolic contents (µg
GAE/mg of plant extract)
Total flavonoid contents (µg
QE/mg of plant extract)
E. royleana extracts
80
4.3.2. High performance liquid chromatography (HPLC) analysis of E. royleana:
HPLC is frequently used technique for identification of phenolic compounds (Brum
et al., 2013). The identification of phenolic compounds in methanol, hexane and aqueous
extracts of E. royleana was done by comparing the HPLC chromatograms of these
compounds with HPLC chromatogram of standard compounds at the same conditions. The
data is shown in Table. 4.3.1.
Methanol extract was found to be the highest in ferulic acid (4.59 ± 0.23 ppm). Another
specie of Euphorbeaceae (Euphorbia Tirucalli) is also known for existence of ferulic acid
(Araujo et al., 2014).
Table. 4.3.1. High performance liquid chromatographic (HPLC) analysis of methanol,
and hexane extracts of E. royleana for identification and quantification phenolic
compounds (ppm)
Phenolic compounds Methanol extract Hexane extract Aqueous extract
Gallic acid 2.62 ± 0.18bd
1.01 ± 0.51a 2.13 ± 0.01
bd
Chlorogenic acid 1.08 ± 0.40ab
0.16 ± 0.001bc
1.49 ± 0.02ab
Ferulic acid 4.59 ± 0.23cf
0.51 ± 0.09ad
5.13 ± 0.05cf
Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate significant difference
(P < 0.05) among solvents (methanol and hexane) used.
In addition to ferulic acid, gallic acid and chlorogenic acid were also detected in two
extracts at varying degrees. Our results in current finding are in accordance with previous
scientific studies in which presence of phenolic acids was reported in different members of
Euphorbeaceae (Loh et al., 2009; Liu et al., 2011; Rakhimov et al., 2011; Jahan et al., 2013).
It is interesting to note that extent of phenolic compounds in current study is different
from those detected in other members of Euphorbeaceae. This might be attributed to the fact
that different plant species within the same family own a unique chemical profile (Cai et al.,
2004).
81
Phenolic compound profile of hexane extract of E. royleana is in agreement with
current scientific studies (Hussain et al., 2014; Sharma and Vig, 2014) which reported the
existence of phenolic acids in hexane extracts of medicinal flora. To the best of our
knowledge, our study is first report on phenolic profile of Euphorbia royleana.
4.3. (b). Biological Evaluation:
4.3.3. Free radical (DPPH) scavenging activity of E. royleana:
The stable free radical (DPPH) has been extensively used as tool to assess antioxidant
efficacies of plant extracts or other antioxidant substances (Szabo et al., 2007). The two
extracts (methanol and hexane) showed the significant (P < 0.05) free radical scavenging
activity through all concentrations, as shown in Table. 4.3.2.
Minimum scavenging activities by methanol (18.22 ± 1.11%) and hexane (16.09 ±
0.51%) extracts were examined at 25 µg/mL. However, when concentration increased, free
radical scavenging activities were also increased and maximum scavenging activities by
methanol (56.78 ± 1.37%) and hexane (60.52 ± 0.39%) extracts were exhibited at 800
µg/mL.
It is surprising to note that hexane extract exhibited higher percent inhibition (%) values than
methanol extract. These results were in strong agreement with previous finding in which
hexane extract of an important medicinal plant was examined for the higher inhibition (%) of
DPPH radicals as compared to methanol extract (Sim et al., 2010). Another study by Dhar et
al., 2013, demonstrated that hexane extract of phytococktail had high free radical (DPPH)
scavenging activity than methanol extract. Our observation was supported by some other
group of scientists who narrated high free radical (DPPH) scavenging activity of hexane
extract (Bae et al., 2012; Rahman et al., 2013).
The free radical scavenging activities for methanol and hexane extracts determined in
the current study were in comparison with that reported for chloroform, n-butanol and ethyl
acetate extracts of Euphorbia royleana (Shahwar et al., 2010). Some research groups
(Turkmen et al., 2006; Zhao et al., 2006) reported that nature of extracting solvent plays
critical role in demonstrating the antioxidant potential of plants.
82
Table. 4.3.2. Scavenging (%) of stable free radicals (DPPH) by methanol and hexane
extracts of E. royleana
E.royleana extracts
Scavenging (%) of free radicals at different concentrations (µg/mL) IC
50 (mg/mL)
25 50 100 200 400 800 Methanol 18.22±1.11
c
a
23.46±0.64d
b
28.22±1.46a
c
33.51±1.30f
d
46.71±1.25b
e
56.78±1.37e
f
0.58 Hexane 16.09±0.51
c
d
24.89±0.68d
f
31.19±0.38c
b
39.38±1.19a
e
50.96±0.44e
c
60.52±0.39f
a
0.38 Aqueous 11.18±0.77a
b 17.47±1.49
b
e
20.18±0.96d
a
25.95±1.51e
f
36.68±0.29f
c
51.46±0.46c
d
1.13 Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among concentrations tested. Mean with different subscript letters in the same column indicate significant
difference (P < 0.05) among solvents tested.
4.3.5. Antitumor activity of E. royleana:
Euphorbia royleana is traditionally used for the treatment of paralysis, bladder stone,
earache, inflammation and other diseases (Sabeen and Ahmad, 2013). However, antitumor
potential of this plant was not scientifically reported. So in current study, antitumor activity
of fresh Euphorbia royleana was examined by potato disc assay.
To achieve the goal, different doses (25 to 800 µg/mL) of methanol, hexane and
aqueous extracts of fresh Euphorbia royleana were evaluated against Agrobacterium
tumefaciens growth. The results (Figure. 4.3.2) of this assay showed that tumor inhibition
(%) linearly increased with increase in dose of each extract. This trend was in agreement with
previous finding (Kanwal et al., 2011).
We observed comparatively higher tumor inhibition in hexane extract at all the tested
doses (25 to 800 µg/mL). Maximum antitumor activity was observed in hexane extract (IC50
= 267.5 µg/mL). These results are in strong agreement with previous finding (Mazid et al.,
2011) in which hexane extract (IC50 = 200 µg/disc) of an important medicinal plant was
reported for higher inhibition of A. tumefaciens induced tumors as compared to methanol
extract (IC50 > 400 µg/disc). Effect of extracting solvents and doses on tumor inhibition was
statistically significant (P < 0.05).
83
Figure. 4.3.2. Antitumor activity of methanol, aqueous and hexane extracts of E. royleana.
Data is represented as Mean ± SD for three samples of each extract (methanol and hexane)
analyzed individually in triplicates. (P < 0.05).
4.3.6. Antimicrobial activity of E. royleana:
The results of antimicrobial potential of methanol and hexane extracts of fresh
Euphorbia royleana are presented in Table. 4.3.3.
The three extracts exhibited poor activities against Escherichia coli and Pasteurella
multocida. Our results in current study were found better than previous finding which
demonstrated that Euphorbia trigona extracts had no effect on growth of E. coli and B.
subtilis (Upadhyay et al., 2013).
0
10
20
30
40
50
60
70
80
90
100
25 50 100 200 400 800
Inh
ibit
ion
of
tum
ors
(%)
Concentartion (µg/mL)
Methanol Hexane
84
Minimum antibacterial was examined against E. coli (Gram negative) with inhibition
zone of 5.67 ± 0.57 mm. These results are in agreement with previous finding in which
similar inhibition zone (5 mm) was observed against gram negative bacteria by methanol
extract of vegetable (Ullah et al., 2013). Hexane extract strongly inhibited the growth of
Bacillus subtilis. The most susceptible fungal strain was Aspergillus niger (14.00 ± 1.00
mm). Despite of the antifungal activity of our extracts, chloroform extract from the aerial
parts of Euphorbia trigona also exhibited considerable antifungal activity (Upadhyay et al.,
2013).
Table. 4.3.2. Antimicrobial activity of methanol, aqueous and hexane extracts of E.
royleana
nt = Not tested. Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, hexane and aqueous) against each
microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents tested.
*Standard antibiotic for bacteria; * *Standard antibiotic for fungi.
Overall the order of antimicrobial activities of two extracts was as: hexane >
methanol. Hexane extract exhibited distinctive antimicrobial activity, as compared to
methanol extract. Higher antimicrobial activity of hexane extract was also in line with
literature records (Akhter, 2008; Alaribe et al., 2011; Zakaria et al., 2011).
E. royleana
extracts
Diameter of inhibition zones (mm)
Bacterial Strains Fingal Strains
Escherichia
coli
Pasteurella
multocida
Bacilus
subtilis
Aspergillus
niger
Fusarium
solani
Methanol 5.67±0.57b 8.33 ±0.57
c 10.67±0.57
b 11.66±0.57
c 10.00±1.00
a
Hexane 7.00±1.00b 10.33±0.57
c 12.00±0.57
b 14.00±1.00
b 10.33±0.57
a
Aqueous 6.00±1.00b 8.00±1.00
c 8.33±0.00
c 9.33±0.57
a 8.66±0.57
a
*Rifampicin 21.66±1.41a 23.33±1.69
b 24.66±0.47
a Nt Nt
**Terbinafine Nt Nt Nt 25.66±1.69d 24.00±0.82
b
85
In another study, Akinyele et al., 2011, screened crude n-hexane extract of a husk
against forty five strains of Vibrio pathogens and twenty five other bacterial isolates.
Surprisingly, hexane extract exhibited activity against 21 bacterial and 38 of test Vibrio
species. Extracts from other Euphorbia species (Euphorbia hirta, Euphorbia milii,
Euphorbia nerifolia, Euphorbia heterophylla) were also found active against some bacterial
and fungal strains (Bakkiyaraj and Pandiyaraj, 2011; Ekundayo and Ekekwe, 2013;
Venkatanagaraju and Goli, 2014).
4.4. Psidium guajava:
4.4. (a). Chemical Evaluation:
4.4.1. Total phenolic and flavonoid contents of P. guajava extracts:
Phenolic and flavonoid compounds act as antioxidants by scavenging free radicals,
chelating metal ions, donating hydrogen and quenching singlet oxygen (Aminu et al., 2012).
In addition to it they play major role against cancer, microbial infections and vast range of
degenerative ailments (Quettier-Deleu et al., 2000; Jagadish et al., 2009). So while
investigating pharmaceutical activities of P. guajava leaves, it was quite necessary to explore
the extent of these bioactive components.
Total phenolic and flavonoid contents (TPC and TFC) of three extracts (methanol,
chloroform and hexane) were represented as µg gallic acid equivalent/mg of plant extract and
µg qurecetin equivalents/mg of plant extract, respectively. The results are shown in Figure.
4.4.1.
The results showed that extraction of TPC with three solvents was as follows: methanol
(83.34 ± 0.49) > chloroform (71.49 ± 0.48) > hexane (53.24 ± 2.05), while the order of
solvents in case of TFC was methanol (53.39 ± 0.89) > chloroform (32.76 ± 1.15) > hexane
(21.26 ± 1.49). Difference in amount of phenolics and flavonoids in different solvents is
acknowledged to nature of solvent (Bae et al., 2012).
Total phenolic and flavonoid contents of methanol extract were higher than previous
study (Aminu et al., 2012). However, Qian and Nihorimbere, 2004, investigated significantly
higher phenolic contents in polar extract of P. guajava leaves.
86
Figure. 4.4.1. Total phenolic and total flavonoid contents of P. guajava extracts (methanol,
chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P < 0.05). Total
phenolic contents are expressed as microgram gallic acid equivalents per milligram of plant
extract (µg GAE/ mg of plant extract). Total flavonoid contents are expressed as microgram
quercetin equivalents per milligram of plant extract (µg QE/ mg of plant extract).
4.4.1. High performance liquid chromatography (HPLC) analysis of P. guajava
extracts:
Phenolic compounds detected in methanol, chloroform and hexane extracts of P. guajava
are represented in Table. 4.4.1. The common phenolic compound of three extracts (methanol,
chloroform and hexane) was quercetin. Caffeic acid, ferulic acid and vanillic acid were also
examined. Gallic acid was observed in methanol (5.62 ± 0.29 ppm) and chloroform (3.21 ±
0.57 ppm) extracts.
0
20
40
60
80
100
Methanol Chloroform Hexane
P. guajava extracts
Total phenolic contents (µg
GAE/mg of plant extract)
Total flavonoid contents (µg
QE/mg of plant extract)
87
Previously, Verza et al., 2008, quantified considerably higher extent of gallic acid in P.
guajava leaves. Wu et al., 2009, examined that gallic acid present in P. guajava leaves,
exhibit inhibitory effects on glycation process. Another study (Kato et al., 2001) showed that
gallic acid derivative of P. guajava stimulated production of cytokines by Th2 cells. Lin and
Yin, 2012, determined that P. guajava extract rich in quercetin, caffeic acid and ferulic acid
exhibited significant in vivo renal protective effects.
Rattanachaikunsopon and Phumkhachorn, 2010, studied that quercetin isolated from P.
guajava leaves exhibited significant antibacterial activity against E. coli, P. fluorescens, V.
cholerae, S. aureus, L. monocytogenes, B. hermosphacta, S. enterica and B.
stearothermophilus.
Table. 4.4.1. High performance liquid chromatographic (HPLC) study of methanol,
chloroform and hexane extracts of P. guajava for identification and quantification of
phenolic compounds (ppm)
Phenolic compounds Methanol extract Chloroform extract Hexane extract
Gallic acid 5.62 ± 0.29bc
3.21 ± 0.53d Nd
Caffeic acid 0.70 ± 0.14 Nd Nd
Chlorogenic acid 2.35 ± 1.03 Nd Nd
Ferulic acid 1.00 ± 0.19d 1.17 ± 0.26
e Nd
Vanillic acid 2.72 ± 0.11 Nd Nd
Quercetin 11.84 ± 0.002be
5.09 ± 0.08ac
3.61 ± 0.47d
Nd = Not detected. Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among solvents (methanol, chloroform and hexane) used.
4.4.3. Gas chromatography mass spectrometry (GC-MS) study of P. guajava extracts:
Chemical components of methanol, chloroform and hexane extracts are shown in
Table. 4.4.2. Pyrogallol (35.18%), vitamin E (30.70%) and palmitic acid (30.73%) were the
major chemical constituents of methanol, hexane and chloroform extracts, respectively.
88
Pyrogallol has wide range of pharmacological properties, including inhibition of
proinflammatory cytokines (Nicolis et al., 2008).
Table. 4.4.2. Chemical components of methanol, chloroform and hexane extracts of P.
guajava analyzed by gas chromatography mass spectrometry (GC-MS)
aComponents bRT Methanol
extract
Chloroform
extract
Hexane
extract
Hydroxydimethylacetic acid 5.19 ---- 3.018 ----
Tridecyl trifluoroacetate 13.88 ---- 2.543 ----
n-cetane 14.47 ---- 2.229 ----
Farnesan 15.59 ---- 3.396 ----
Pyrogallol 16.10 35.18 ---- ----
α-Copaene 16.42 11.92 ---- 2.18
Caryophyllene 17.21 6.36 ---- 8.43
Aromandendrene 17.35 3.48 ---- ----
Dodecyliodide 17.83 ---- 3.170 ----
Heneicosane 17.83 ---- ---- 6.28
Alloarmadendrene 17.89 2.52 2.182 ----
γ-Muurolene 18.04 2.30 ---- ----
Eicosane 18.08 ---- 3.637 ----
Tetracosane 18.09 ---- ---- 2.77
β-Bisabolene 18.41 2.23 ---- ----
β –chamigrene 18.58 ---- 6.492 ----
89
( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data
Vitamin E also plays significant role against cancer (Constantinou et al., 2008). Carr
et al., 2000, reported antiatherogenic potential of vitamin E. Singh et al., 2005, narrated that
vitamin E is potential candidate against inflammation. Effectiveness of vitamin E against
atherosclerosis is also evidenced by literature (Upston et al., 2003).
Extent of palmitic acid (n-hexadecanoic acid) in methanol and chloroform extracts of
P. guajava leaves is higher than previously reported value for P. guajava essential oil
(Khadhri et al., 2014).
α-Calacorene 19.11 ---- 3.260 ----
Cetene 19.50 ---- 2.238 ----
Caryophyllene oxide 19.83 19.71 ---- ----
α-Bulnesene 19.83 ---- ---- 11.37
Epiglobulol 20.13 2.64 ---- ----
Ledol 20.14 ---- ---- 2.38
cis-Thujopsene 20.40 3.56 ---- ----
Copaene 20.69 ---- ---- 3.27
Culmorin 21.15 1.43 ---- ----
E-15-Heptadecenal 23.13 ---- 17.78 ----
cis-Z-α-Bisabolene epoxide 23.48 1.86 ---- ----
Palmitic acid 27.27 5.05 30.73 ----
Stearic acid 31.15 1.76 8.86 ----
Hexachlorodisiloxane 32.55 ---- 2.95 ----
Squalene 40.22 ---- ---- 15.36
Vitamin E 44.12 ---- ---- 30.70
γ-Sitosterol 47.63 ---- ---- 17.26
90
Other main components were caryophyllene oxide, copaene, alloaromadendrene,
caryophyllene, palmitic acid, sitosterol, α-bulnesene, α-copaene and squalene. Caryophyllene
oxide is used as preservative in cosmetics, food stuff and drugs. Antifungal potential of
caryophyllene oxide is also documented (Yang et al., 1999).
Existence of caryophyllene oxide in P. guajava hexane extract is previously described
Presence of α-copaene in fruit extract of P. guajava is reported in a scientific study (Meckes
et al., 1998). β-caryophyllene oxide induce apoptosis in human prostate cancer cells (Ryu et
al., 2012). Li et al., (1999), reported comparatively higher level of copaene and
caryophyllene in essential oil from P. guajava leaves.
α-bulnesene (11.37%) an effective component of hexane extract of P. guajava as
indicated in Table. 4.4.2. Some research groups (Hsu et al., 2006; Tsai et al., 2007) examined
inhibitory prospective of α-bulnesene on platelet-activating factor.
Our results differ with regard to chemical constituents from previously reported
results for hexane extract of P. guajava from India (Nisha et al., 2011). Analysis of
methanol, chloroform and hexane extracts of P. guajava leaves by rapid and reliable
technique (GC-MS) led to identification of bioactive components with therapeutic
background.
4.3. (b). Biological evaluation:
4.4.2. Free radical (DPPH) scavenging activity of P. guajava extracts:
It is evident from our results (Table. 4.4.3) that inhibition of free radicals (%) is
elevated with rise in concentration of investigated extracts (methanol, chloroform and
hexane). This observation is supported by previous study in which a linear relationship was
observed between inhibition (%) of free radicals (DPPH.) and dose of plant extract (Sim et
al., 2012).
The free radical (DPPH.) scavenging activity of methanol extract was observed to be
higher than that of chloroform and hexane extracts, respectively. Maximum free radical
scavenging activity (93.19 ± 1.43%) seen in methanol extract of P. guajava was found to be
higher than alcoholic extract of P. guajava root bark (75.6%) (Kuber et al., 2013). This
91
difference might be attributed to different geographical conditions; the key factor to effect
phytochemical components and related pharmacological properties of medicinal plants
(Edoga et al., 2005). Scavenging activity of methanol extract (IC50 = 0.09 mg/mL) is in
comaprison with gallic acid (IC50 = 0.02 mg/mL) that was used as positive control.
Table. 4.4.3. Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform
and hexane extracts of P. guajava
P.guajajva
extracts Scavenging (%) of free radicals at different concentrations (µg/mL) IC
50
(mg/mL) 25 50 100 200 400 800
Methanol 25.35±0.88b
d
27.45±1.03d
a
52.37±3.02f
c
71.34±1.70c
a
92.67±0.99e
f
93.19±1.43a
f
0.09 Chloroform 20.34±2.67
d
a
31.03±4.42c
b
31.72±1.89d
b
50.97±3.23a
d
54.54±2.15b
e
75.31±1.14f
c
0.20 Hexane 16.23±0.63
a
f
18.10±0.66b
e
29.43±1.93c
b
38.05±1.51e
c
50.09±4.87d
a
59.90±3.31b
d
0.42 Values are Mean ± SD (Standard deviation) of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among concentrations tested. Means with different subscript letters in the same column indicate significant
difference (P < 0.05) among solvents tested.
Scavenging efficacy of hexane extract on DPPH stable free radicals was lower than
chloroform and methanol extracts at all the tested doses (25 to 800 µg/ml). This finding is in
agreement with previous study in which hexane extract of a Pakistani medicinal plant was
reported for poor free radical scavenging activity than methanol extract (Iqbal et al., 2013).
However, contrary to our observation, Fidrianny et al., 2102, reported comparatively
different values of DPPH inhibition in hexane extract of P. guajava leaves.
4.4.3. Antitumor activity of P. guajava extracts:
In this trial different concentrations (25 to 800 µg/mL) of methanol, chloroform and
hexane extracts of P. guajava were evaluated for their antitumor activity by bench top
potato disc assay (Figure. 4.4.2).
The results indicated that percent tumor inhibition was elevated with rise in
concentration of P. guajava extracts (methanol, chloroform and hexane). These results are in
accordance with previous scientific reports (Hussain et al., 2007; Rehman et al., 2009) on
antitumor activity of medicinal flora of Pakistan, which showed decline in A. tumefacien
92
induced tumors with increase in concentration of plant extracts. The order of antitumor
activity of three extracts was as: hexane (IC50 = 65.02 µg/mL) > chloroform (IC50 = 160.7
µg/mL) > methanol (IC50 = 337.4 µg/mL). This observation is in agreement with previous
finding in which hexane extract was reported for higher anticancer activity as compared to
polar extracts (Shylesh et al., 2005).
Figure. 4.4.2. Antitumor activity of methanol, chloroform and hexane extracts of P. guajava.
Data is represented as Mean ± SD for three samples of each extract (methanol, chloroform
and hexane) analyzed individually in triplicates. (P < 0.05).
4.4.5. Antimicrobial activity of P. guajava extracts:
The results of antimicrobial activity assay are given in Table. 4.4.4. The maximal
inhibition zones for microbial strains sensitive to P. guajava extracts (methanol, chloroform
and hexane) were in range of 10.42 ± 0.91 mm to 19.33 ± 0.18 mm. Overall, the methanol
0
10
20
30
40
50
60
70
80
90
100
25 50 100 200 400 800
Inh
ibit
ion
of
tum
ors
(%)
Concentartion(µg/mL)
Methanol Chloroform Hexane
93
extract of P. guajava exhibited stronger antimicrobial potential than chloroform and hexane
extracts, respectively. Hexane extract was inactive against the tested bacterial strains (E. coli,
B. subtilis and P. multocida).
Table. 4.4.4. Antimicrobial activity of methanol, chloroform and hexane extracts of P.
guajava
Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, chloroform and hexane) against each microbial
strain. Means with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents tested.
*Standard antibiotic for bacteria; * *Standard antibiotic for fungi.
We observed that B. subtilis was more susceptible to chloroform extract than E. coli.
These results are consistent with previous study (Kuber et al., 2013) on root bark extract of
P. guajava in which B. subtilis was reported as sensitive strain while E. coli was the resistant
one. Furthermore, according to another report (Zahidah et al., 2013) P. guajava leaves had
distinctive antimicrobial potential against B. subtilis as compared to E. coli. It is interesting
to note that methanol extract was more effective against E. coli (13.74 ± 1.11 mm) as
compared to B. subtilis (10.42 ± 0.91 mm) and P. multocida (12.66 ± 1.52 mm). This
observation in strong agreement with previous finding (Zubair et al., 2011) in which
P. guajava
extracts
Diameter of inhibition zones (mm)
Bacterial Strains Fungal Strains
Escherichia
coli
Bacillus
subtilis
Pasteurella
multocida
Aspergillus
niger
Fusarium
solani
Methanol 13.74±1.11b 10.42±0.91
bc 12.66±1.52
d 15.21±0.14
cf 0 ± 0
Chloroform 11.91±0.34d 13.00±1.25
c 18.19±0.07
b 19.33±0.18
d 0 ± 0
Hexane 0 ± 0 0 ± 0 0 ± 0 11.36 ±0.73e 12.02 ±0.06
c
*Rifampicin 21.66±1.41e 24.66±0.47
a 23.33±1.69
c Nt Nt
**Terbinafine Nt Nt Nt 25.66±1.69a 24.00±0.82
f
94
methanol extract of an important medicinal plant was reported as successful candidate
against E. coli than B. subtilis and P. multocida, respectively.
In case of fungi, methanol and chloroform extracts of P. guajava were more efficient
against A. niger than F. solani. These results are in accordance with previous study (Leeja
and Thoppil, 2007) in which methanol extract of an Indian medicinal plant exhibited greater
antifungal activity against A. niger as compared to F. solani.
Hexane extract was more efficient against F. solani (12.02 ± 0.06 mm) than A. niger
(11.36 ± 0.73 mm). This is in agreement with another scientific report on medicinal plant of
Pakistan, (Jabeen et al., 2008) citing F. solani as more sensitive strain than A. niger.
Antimicrobial activity of P. guajava leaves might be attributed to presence of potent
antimicrobial compounds (morin-3-O-α-L- arabopyranoside, quercetin, morin-3-O-α-L-
lyxopyranoside and guaijavarin) in them (Arima and Danno, 2002).
4.4.4. Anticancer activity of P. guajava extracts:
Antiproliferative activity of methanol, hexane and chloroform extracts of P. guajava
against four different cell lines (KBM5, U266 and HCT116) was evaluated by MTT assay
(Figure. 4.4.3) whereby mitochondrial dehydrogenase enzyme in metabolically active cells
converts yellow tetrazolium salt to dark blue formazan and the intensity of blue color predicts
cell viability (Mena-Rejon et al., 2009).
Hexane extract exhibited maximum decrease in cell viability (%) with IC50 values of
0.022, 0.020 and 0.005 mg/mL for KBM5, U266 and HCT116 cells, respectively. These
results are in line with previous finding (Bibi et al., 2011) in which hexane extract (IC50 =
1.10 mg/mL) of a potent medicinal plant of Pakistan was found more effective against human
malignant melanoma (HT144) cells as compared to methanol extract (IC50 = 10.69 mg/mL).
Maximum anticancer activity of hexane extract might be attributed to presence of
tetracosane, α-copaene, γ-sitosterol, vitamin E and squalene in it, as shown in Table. 4.4.2.
95
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 U266 HCT116
A
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 U266 HCT116
B
96
Figure. 4.4.2. Anticancer activity of (A) methanol (B) chloroform and (C) hexane extracts of
P. guajava against KBM5 (Human myelogenous leukemia), U266 (Human multiple
myeloma) and HCT116 (Human colon carcinoma) cell lines. Values are represented as Mean
±SD of quadreplicate determinations. (P < 0.05)
These vital bioactive components exhibit anticancer potential by different
mechanisms including suppression of signaling pathways, apoptosis induction and cell cycle
arrest (Ryu et al., 2012; Sundarraj et al., 2012).
Jurasz et al., 2004, showed that tumor cell induced platelets aggregation is necessary
for the survival of tumor cells and its successful metastasis. α-bulnesene present in hexane
extract inhibits platelet aggregation (Hsu et al., 2012). IC50 values lower than 0.03 mg/mL
enabled the hexane extract to fulfill criteria of American National Cancer Institute, to attain
attention for purification (Suffness and Pezzuto, 1990).
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 U266 HCT116
C
97
Chloroform extract exhibited maximum cytotoxic activity against HCT116 (IC50 =
0.006 mg/mL) followed by U266 (IC50 = 0.026 mg/mL) and KBM5 (IC50 = 0.041 mg/mL)
cells Anticancer activity of chloroform extract may be attributed to significant amount of
palmitic acid (30.98%) in it. Harada et al., 2002, reported apoptotic effect of palmitic acid in
human leukemic cells.
Palmitic acid is also known for DNA topoisomerase I inhibition in human lung
adenocarcinoma epithelial cells (Karna et al., 2012). The ranking of anticancer activity in
case of methanol extract was as: HCT116 (IC50 = 0.019 mg/mL) > KBM5 (IC50 = 0.051
mg/mL) > U266 (IC50 = 0.089 mg/mL). IC50 values of methanol extract were comparable
with previously reported values for methanol extract of P. guajava from Malaysia (Sulain et
al., 2012).
It is interesting to note that, we observed similar ranking of extracts (hexane >
chloroform > methanol) is same in case of anticancer (Section. 4.4.7) and antitumor (Section.
4.4.5) assays. This observation is supported by findings of some research groups (Galsky et
al., 1980; Galsky et al., 1981; Karpas, 1982) that strongly correlated the antitumor activity
assessed by potato disc to in vitro or in vivo anticancer activity.
4.4.8. Anti-inflammatory activity of P. guajava hexane extract:
In this section we investigated whether P. guajava hexane extract can exhibit anti-
inflammation and modulate the NF-κB activation. Our results indicated that TNF-α induced
the NF-κB activation, whereas P. guajava extract alone has no effect.
The pretreatment of KBM5 cells with this extract suppressed TNF-α-induced NF-κB
activation in a dose-dependent manner (Fig. 4.4.4). TNF-α-induced NF-κB activation was
completely inhibited at 25 μg/mL dose of P. guajava hexane extract. Treatment with hexane
extract under these conditions had little effect on cell viability.
To the best of our knowledge, this is the first report to suggest that P. guajava hexane
extract can down-modulate the NF-κB activation induced by proinflammatory cytokines in
cancer cells.
98
Figure. 4.4.4. Anti-inflammatory activity of hexane extract of P. guajava. Lane 1 and lane 2
showed unstimulated Myelogenous leukemia (KBM5) cells. In lane 3 cells were treated with
TNF-α alone. In other lanes (4-6) cells were pre-incubated with chloroform extract of P.
guajava (10, 25 and 50 µg/mL) for 12 hours and then treated with 0.1 nM TNF-α for 30 min.
Nuclear extracts were prepared and assayed for NF-κB activation by EMSA.
99
Chainy et al., 2000, reviewed that cancer patients have higher levels of
proinflammatory cytokines and activated NF-κB, which is a major mediator of inflammatory
pathways. Therefore, anti-inflammatory agents that can modulate the NF-κB activation and
inflammatory pathways can play a major role in cancer treatment.
There are more than 80 % drugs derived from natural products that are serving mankind
for thousands of years (Kim et al., 2012). Significant amount of Vitamin E (Table. 4.4.2) in
hexane extract may be responsible for inhibition of NF- κB activation (Calfee-Mason et al.,
2002). Similarly garcinol, anethole and many other plant derived components have been
shown to inhibit NF-κB activity and to modulate inflammatory pathways (Chainy et al.,
2000; Kim et al., 2012).
4.5. Ziziphus mauritiana:
4.5.(a). Chemical Evaluation:
4.5.1. Total phenolic and flavonoid contents of Z. mauritiana:
Phenolic and flavonoids are filed as potent antioxidants (Duthie and Morrice, 2012).
They possess wide spectrum of biological and pharmacological activities (Huang et al.,
2010). They are also documented as successful candidates against oxidative stress based
diseases, microbial infections and malignancy (Pandey and Rizvi, 2009).
Total phenolic and flavonoid contents (TPC and TFC) of methanol, chloroform and
hexane extracts of Z. mauritiana leaves were presented in Figure. 4.5.1. Significant
differences (P < 0.05) were observed among TPC and TFC of three extracts. Chloroform
extract presented the highest extent of total phenolics (84.69 ± 0.92 µg GAE/mg of plant
extract). Overall the total phenolic contents of three extracts were in following order:
chloroform (84.69 ± 0.92 µg GAE/mg of plant extract) > methanol (77.88 ± 1.10 µg
GAE/mg of plant extract) > hexane (56.22 ± 1.46 µg GAE/mg of plant extract). This finding
is in accordance with previous scientific study on a potent medicinal plant, in which
Shahriar et al., 2013, examined maximum extent of phenolics in chloroform extract
followed by methanol and hexane extracts, respectively. Yusri et al., 2012, also observed
100
the identical ranking of solvents (chloroform > methanol > hexane) for extraction of
phenolic compounds.
The phenolic contents for Z. mauritiana leaf extracts observed in our study
were higher than those of previously reported for Z. mauritiana fruit (Memon et al., 2012).
Another study reported lower extent of phenolics in seed extract of Z. mauritiana (San et al.,
2013). However, Dureja and Dhiman, 2012, reported higher level of total phenolic and
flavonoid contents in fruits of Z. mauritiana.
Figure. 4.5.1. Total phenolic and total flavonoid contents of Z. mauritiana extracts
(methanol, chloroform and hexane). Values are Mean ± SD of triplicate determinations. (P <
0.05). Total phenolic contents are expressed as microgram gallic acid equivalents per
milligram of plant extract (µg GAE/ mg of plant extract). Total flavonoid contents are
expressed as microgram quercetin equivalents per milligram of plant extract (µg QE/ mg of
plant extract).
0
20
40
60
80
100
Methanol Chloroform Hexane
Z. mauritiana extracts
Total phenolic contents (µg
GAE/mg of plant extract)
Total flavonoid contents (µg
QE/mg of plant extract)
101
In contrast to our study recently another research group (Lamien-Meda et al.,
2008) reported high phenolic and flavonoid contents in Z. mauritiana fruit extracts.
Variation in phenolic and flavonoid contents might be attributed to changes in variety and
climate.
4.5.2. High performance liquid chromatographic (HPLC) analysis of Z. mauritiana
extracts:
HPLC technique was used to identify and quantify the individual phenolic
compounds in three solvent extracts (methanol, chloroform and hexane) of Z. mauritiana
leaves. The results are shown in Table. 4.5.1.
Caffeic acid was the most abundant phenolic compound in the tested extracts
(methanol, chloroform and hexane). Moreover, chlorogenic acid, ferulic acid, p-coumaric
acid and sinapic acid were also detected at varying degrees in three extracts.
Table. 4.5.1. High performance liquid chromatographic (HPLC) study of methanol,
chloroform and hexane extracts of Z. mauritiana for identification and quantification of
phenolic compounds (ppm)
Phenolic
compounds
Methanol
extract
Chloroform
extract
Hexane
extract
Caffeic acid 4.41 ± 0.11ab
2.13 ± 0.00ef
1.92 ± 0.02ce
Chlorogenic acid 2.39 ± 1.03be
1.22 ± 0.16d 0.17 ± 0.10
a
Sinapic acid 0.78 ± 0.19 Nd Nd
p-coumaric acid 1.16 ± 0.07b 1.04 ± 0.13
b Nd
Ferulic acid 0.89 ± 0.12 Nd Nd
nd = Not detected. Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate
significant difference (P < 0.05) among solvents (methanol, chloroform and hexane) used.
Our results were in strong agreement with previous findings;
Muchuweti et al., 2005, detected the presence of p-coumaric acid, caffeic acid and ferulic
102
acid in Z. amuritiana fruit segment. Memon et al., 2012, reported the presence of p-coumaric
acid, o-coumaric acid and ferulic acid in fruit extract of Z. mauritiana, but they did not find
other phenolic compounds i.e. caffeic acid sinapic acid and chlorogenic acid detected in
current research work. All of the identified phenolic compounds have good pharmacological
properties (Zang et al., 2000; Raina et al., 2008; Zhao and Hu, 2013).
4.5.3. Gas chromatography mass spectrometry (GC-MS) study of Z. mauritiana
extracts:
The data for GC-MS analysis is shown in Table. 4.5.2. It is indicated from the results
that methyl sterate (15.59%) and α-Linolenic acid (26.45%) were the major components of
methanol and hexane extracts, respectively. Other, significant components in methanol
extract were palmitic acid (13.57%), squalene (12.09%), phytol (9.78%), methyl palmitate
(7.81%), linoleic acid methyl ester (5.98%) and vitamin E (2.35%). Considerable quantities
of palmitic acid (16.26%), squalene (12.83%), α-tochopherol (3.92%), γ-sitosterol (2.72%),
phytol (2.52%), trans-geranylgeraniol (2.34%), octacosane (2.04%), methyl palmitate
(1.01%) and myristic acid (0.73%) were observed in hexane extract. We also observed
considerable quantities of α-tochopherol (10.01%), stearic acid (5.82%), vitamin E (5.41%),
uneicosane (4.79%), α- nonadecylene (3.77%), bacchotricuneatin C (3.48%), myristic acid
(2.80%) and lauric acid (1.66%) in chloroform extract.
Reports on chemical composition of Z. mauirtiana leaf extracts are scarce in
literature. However, similar to our results, Memon et al., 2012, reported the presence of
palmitic acid and lauric acid in fruit extracts of Z. mauritiana. Furthermore, palmitic acid,
lauric acid, myristic acid and stearic acid were also detected in Z. mauritiana flowers
collected from Brazil (Alves et al., 2005). All the mentioned fatty acids were also reported in
current study.
On the other hand, Chebouat et al., 2013, presented absolutely different chemical
components in crude alkaloidal extract of Z. mauritiana plant. Squalene which was present in
considerable amount in methanol and hexane extracts was also reported from seed oil of Z.
mauritiana fruit (Memon et al., 2012). Squalene is known for its chemo preventive effects
against colon carcinoma cells (Rao et al., 1998).
103
Previous studies reported vitamin E from fruit of Z. mauritiana (Nyanga et al., 2013).
Presence of vitamin E in methanol and chloroform extracts of Z. mauritiana leaves is also
examined (Table. 4.5.2). Anticancer effect of vitamin E analogues has been demonstrated in
many in vivo and in vitro studies (Anderson et al., 2005). Saturated isomer of vitamin E
suppress proliferation in wide range of carcinoma cells including colon, breast, lung, liver,
stomach, prostate and pancreas (Sakai et al.,2006; Comitato et al., 2010; Yang et al., 2010).
Table. 4.5.2. Chemical components of methanol, chloroform and hexane extracts of Z.
mauritiana analyzed by Gas chromatography mass spectrometry (GC-MS)
aComponents
bRT Methanol
extract
Chloroform
extract
Hexane
extract
Composition (%)
Diglycerol 8.68 0.30±0.01 ---- ----
2,3 dihydro benzofuran 13.31 0.60±0.12 ---- ----
1,2 diacetate glycerol 13.81 1.44±0.09 ---- ----
Uneicosane 18.76 ---- 4.79±0.34 ----
Lauric acid 19.00 ---- 1.66±0.05 ----
Myristic acid 22.35 ---- 2.80±0.13 0.73±0.51
E-15-Heptadecenal 23.13 ---- 12.31±0.01 ----
Phytol acetate 23.34 ---- ---- 1.02±0.16
Methyl palmitate 26.55 7.81±0.11 2.83±0.25 1.01±0.43
Palmitic acid 27.28 13.57±0.02 38.55±0.08 16.26±0.01
Hentriaconate 27.57 ---- 3.25±0.14 ----
Linoleic acid, methyl ester 30.08 5.98±0.40 ---- 0.45±0.38
Phytol 30.14 9.78±0.19 ---- 2.52±0.15
Methyl state 30.19 15.59±0.01 2.31±0.07 0.53±0.06
Linoleic acid 30.73 4.75±0.08 ---- 1.37±0.04
α-Linolenic acid 30.87 14.21±0.32 ---- 26.45±0.01
Stearic acid 31.21 1.94±0.45 5.82±0.28 ----
α-Nonadecylene 31.73 ---- 3.77±0.04 ----
Archidic acid methyl ester 33.65 1.60±0.02 ---- ----
104
( ---- ) = not detected. Compounds are identified on the basis of comparison of retention time and mass spectra in NIST data
4.5.(b). Biological Evaluation:
4.5.4. Free radical (DPPH) scavenging activity of Z. mauritiana extracts:
Free radicals are major contributor of vast range of degenerative diseases (Pham-Huy
et al., 2008). Pharmacological evaluation of extracts is incomplete without assessment of
their free radical scavenging activity. Thus in the present study stable free DPPH radicals
were used to explore free radical scavenging activity of methanol, chloroform and hexane
extracts of Z. mauritiana leaves.
In our findings, trend for free radical scavenging activity of three extracts was as:
methanol > chloroform > hexane (Table. 4.5.3) which agreed with previous scientific report
anothter group of researcher (Yusri et al., 2012).
It is depicted from the results (Table. 4.5.3) that Z. mauritiana methanol extract had
free radical scavenging power with value of 91.04 ± 15.6% at 800 µg/mL. Moderately high
Carbromal 33.78 0.76±0.05 ---- ----
Bacchotricuneatin C 34.14 ---- 3.48±0.16 ----
3-methyl piperidine 34.21 0.48±0.07 ---- ----
o-methyl delta-tochopherol 36.71 ---- ---- 0.47±0.09
Octacosane 38.33 ---- ---- 2.04±0.08
Cyclobarbital 38.95 0.61±0.13 ---- ----
Squalene 40.23 12.09±0.35 ---- 12.83±0.26
Trans-Geranylgeraniol 41.74 ---- ---- 2.34±0.35
2,4- Dimethyl Benzoquinoline 42.47 ---- ---- 2.28±0.03
α-tochopherol 44.16 ---- 10.01±0.16 3.92±0.01
Vitamin E 44.17 2.35±0.09 5.41±0.22 ----
4-Chloro-2-
trifluoromethylbenzoquinoline
45.93 ---- ---- 1.74±0.01
Thymol TMS 47.51 1.26±0.03 ---- ----
γ-sitosterol 47.65 ---- ---- 2.72±0.48
17-Hydroprogesterone 54.12 ---- ---- 3.42±0.02
105
free radical scavenging activity (54.49 ± 1.11%) as assessed by DPPH assay was examined in
chloroform extract of Z. mauritiana leaves at highest investigated dose (800 µg/mL), while
lowest value of antioxidant activity was found in hexane extract at various tested doses (25 to
800 µg/mL). Overall the order of antioxidant activity of three extracts estimated by DPPH
assay was as: methanol > chloroform > hexane.
Our results differed from previously reported (Bhuiyan et al., 2009; Abalaka et al.,
2011) values of antioxidant powers for Z. mauritiana. This difference might be due to
genotypic differences in different verities grown under different geological and climatic
conditions (Capocasa et al., 2008).
Furthermore, we used dose dependent (25 to 800 μg/mL) manner to estimate DPPH
scavenging efficacy of three extracts.
Table. 4.5.3. Scavenging (%) of stable free radicals (DPPH) by methanol, chloroform
and hexane extracts of Z. mauritiana
Z. mauritia-
na extracts Scavenging (%) of free radicals at different concentrations (µg/mL) IC
50
(mg/mL) 25 50 100 200 400 800
Methanol 25.13±0.04f
c
29.10±0.74e
a
42.04±1.72a
e
59.79±0.25e
d
85.41±0.84b
b
91.04±1.56c
e
0.11 Chloroform 18.62±1.13
b
a
20.57±0.89d
d
26.72±2.68c
b
41.3±1.50e
f
43.17±1.05a
e
54.49±1.11f
b
0.63 Hexane 14.89±1.42
c
d
17.02±3.27f
b
18.53±0.95e
a
22.43±1.08a
d
35.65±1.91c
f
48.99±0.88e
c
> 1 Values are Mean ± SD of triplicate determinations. Mean with different superscript letters in the same row indicate significant
difference (P < 0.05) among concentrations tested. Mean with different subscript letters in the same column indicate significant
difference (P < 0.05) among solvents tested.
It was examined that antioxidant activity was directly associated with dose of Z.
mauritiana leaf extracts. This is in accordance with previous scientific studies in which
highest antioxidant activity was reported at the maximum concentration of plant extract
(Gawron-Gzella et al., 2012).
106
4.5.5. Antitumor activity of Z. mauritiana extracts:
Bench top potato disc assay was used to assess potential of methanol, chloroform and hexane
extracts of Z. mauritiana leaves against plant pathogen A. tumefaciens which is involved in
induction of neoplastic disease (Crown gall tumor) in dicotyledonous plants. It is depicted
from the results (Fig. 4.5.2) that three extracts significantly decreased the growth of A.
tumefaciens.
Tumor inhibition by three extracts was observed in dose dependent mode (25 to 800 µg/mL).
We observed minimum tumor inhibition in methanol, chloroform and hexane extracts at 25
µg/mL.
Figure. 4.5.1. Antitumor activity of methanol, chloroform and hexane extracts of Z.
mauritiana. Data is represented as Mean ± SD for three samples of each extract (methanol,
chloroform and hexane) analyzed individually in triplicates. (P < 0.05).
0
10
20
30
40
50
60
70
80
90
100
25 50 100 200 400 800
Inh
ibit
ion
of
tum
ors
(%)
Concentartion(µg/mL)
Methanol Chloroform Hexane
107
Overall the order of antitumor activity of three extracts was as: chloroform (IC50 =
70.74 µg/mL) > hexane (IC50 = 188.5 µg/mL) > methanol (IC50 = 596.4 µg/mL). Variation in
antitumor approach of different extracts may be due to difference in secondary metabolites in
each extract (Kuete et al., 2008).
4.5.6. Antimicrobial activity of Z. mauritiana :
Antimicrobial activity of Z. mauritiana leaf extracts was investigated by disc diffusion assay
and results are summarized in Table. 4.5.4. It is evident from the results that three extracts
exhibited significant (P < 0.05) antimicrobial potential against all the investigated strains.
Table. 4.5.4. Antimicrobial activity of methanol, chloroform and hexane extracts of Z.
mauritiana
Nt = Not tested. Data is represented as Mean ± SD of triplicate determination of each extracts (methanol, chloroform and hexane) against
each microbial strain. Mean with different superscript letter in the same column indicate significant difference ( P < 0.05) among solvents
tested. *Standard antibiotic for bacteria; * *Standard antibiotic for fungi.
Diameter of inhibition zones ranged from 9.00 ± 0.00 mm to 23.39 ± 1.25 mm.
Among all the bacterial strains largest inhibition zone (21.66 ± 0.33 mm) was observed
against B. subtilis. However, Nagumanthri et al., 2012, reported comparatively smaller
Z. mauritiana
leaf extracts
Diameter of inhibition zones (mm)
Bacterial Strains Fungal Strains
Escherichia
coli
Bacillus
subtilis
Pasteurella
multocida
Aspergillus
niger
Fusarium
solani
Methanol 12.3±0.1b 21.7±0.3
d 0 ± 0 23.4±1.3
e 16.7±0.1
b
Chloroform 0 ± 0 0 ± 0 10.8±0.2b 19.5±1.0
d 0 ± 0
Hexane 9.0±0.0c 13.3±1.6
e 0 ± 0 0 ± 0 0 ± 0
*Rifampicin 21.7±1.4e 24.7±0.5
f 23.3±1.7
d Nt Nt
**Terbinafine Nt Nt Nt 25.7±1.7a 24.0±0.8
c
108
inhibition zone against B. subtilis by methanol extract of Indian Z. mauritiana. In contrast to
our results, Abalaka et al., 2012, reported larger zone of inhibition against E. coli by Z.
mauritiana leaves. These differences might be attributed to the difference in extraction
techniques used to prepare the extracts and different geological conditions as well (Turkmen
et al., 2007; Edoga et al., 2005). Furthermore, among fungal strains we observed larger zone
of inhibition (23.39 ± 1.25 mm) by methanol extract against A. niger as compared to previous
study (Das, 2012).
4.5.7. Anticancer activity of Z. mauritiana:
The anticancer activity of Z. mauritiana leaf extracts (methanol, chloroform and
hexane) against cultured KBM-5 (myelogenous leukemia), U266 (multiple myeloma) and
SCC-4 (tongue squamous) cancer cells was estimated by using MTT assay. Cells were
exposed to increasing doses of Z. mauritiana leaf extracts (methanol, chloroform and hexane)
ranging from 10 to 200 µg/mL for 72 hour at 37ºC. Then viability of cancer cells (KBM5,
U266 and SCC4) was assessed and described in terms of relative absorbance of treated cells,
in contrast to control cells.
The results are expressed in Figure. 4.5.3. It is evident from the results that viability
of the three investigated cell lines (KBM5, U266 and SCC4) decreased with increase in dose
of Z. mauritiana leaf extracts (methanol, chloroform and hexane). It was examined that Z.
mauritiana leaf extracts (methanol, chloroform and hexane) were active against all these cell
lines.
Maximum death of myelogenous leukemia (KBM5), multiple myeloma (U266) and
tongue squamous carcinoma (SCC4) cells in each extract (methanol, chloroform and hexane)
was observed at 200 µg/mL.
The chloroform extract showed pronounced anticancer activity with IC50 values of
0.016, 0.022 and 0.043 mg/mL for myelogenous leukemia (KBM5), multiple myeloma
(U266) and tongue squamous carcinoma (SCC4) cells, respectively.
109
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 U266 SCC4
A
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 U266 SCC4
B
110
Figure. 4.5.3. Anticancer activity of (A) methanol (B) chloroform and (C) hexane extracts of
Z. mauritiana against KBM5 (Human myelogenous leukemia), U266 (Human multiple
myeloma cells) and SCC4 (Human tongue squamous carcinoma) cell lines. Values are
represented as Mean ±SD of quadreplicate determinations. (P < 0.05).
Potent anticancer activity of chloroform extract might be acknowledged to
remarkably high concentration of phenolics in it as shown in, Figure. 4.5.1. Literature survey
has indicated that these bioactive components exhibited anticancer activities by apoptosis
induction, anti-angiogenesis, topoisomerase inhibition, upregulation of p53, cell cycle arrest
and various other pathways (Luk et al., 2005; Sagar et al., 2006; Han et al., 2008).
Hexane extract exhibited moderate anticancer activity with IC50 values of 0.1, 0.069
and 0.094 mg/mL for KBM5 (myelogenous leukemia), U266 (multiple myeloma) and tongue
squamous carcinoma (SCC4) cells. Poor anticancer activity was investigated in methanol
extract with potent IC50 values for proliferating cells (KBM5, 0.148; U266, 0.046 and SCC4,
0
20
40
60
80
100
120
0 10 25 50 100 200
Cel
l via
bil
ity (
%)
Dose (µg/mL)
KBM5 U266 SCC4
C
111
0.163 mg/mL). Similar results by Z. mauritiana polar extract against HL-60 were studied
previously by Mishra et al., 2011.
112
CHAPTER 5 SUMMARY
The present research work was carried out in the Central Hi Tech Laboratory,
Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan.
A total of five species from four families were collected from different areas of Pakistan to
explore chemical composition (total phenolic, total flavonoid contents, phenolic compounds
and chemical profile) and biological activities (antioxidant, antimicrobial, antitumor,
anticancer and anti-inflammatory) of investigated medicinal plants. Efforts were made to
evaluate effect of extracting solvents on chemical composition and biological activities of
tested medicinal plants.
Among the investigated plant extracts, chloroform extract of V. betonicifolia
presented the maximum amount of total phenolic contents (155.78 ± 4.12 µg GAE/ mg of
plant extract). Aluminium complexation assay used for assessment of total flavonoid contents
revealed that methanol extract of P. guajava (53.39 ± 0.89 µg QE/ mg of plant extract) is
leading one, while hexane extract of E. camaldulensis (6.09 ± 0.96 µg QE/ mg of plant
extract) was at least position.
In HPLC analysis, a total of nine standard phenolic compounds (Gallic acid, caffeic
acid, vanillic acid, chlorogenic acid, syringic acid, sinapic acid, m-coumaric acid, p-coumaric
acid and ferulic acid) were run. However, all (nine) of the analyzed phenolic compounds
were not detected in the same sample. The result of HPLC analysis showed the existence of
different phenolic compounds at varying degrees in different plant extracts. In E.
camaldulensis gallic acid (5.86 ± 0.23 ppm) was examined at maximum extent, while p-
coumaric acid (0.12 ± 0.05 ppm) was the minor one. Sinapic acid (6.61 ± 0.40 ppm) was the
major phenolic compound of V. betonicifolia examined in methanol extract. Ferulic acid
(0.37 ± 0.001 ppm) noticed in chloroform extract of V. betonicifolia was the minimum
contributor of phenolic profile. Among all the investigated phenolic compounds, quercetin
was observed as major constituent of P. guajava phenolic profile. Extent of phenolic acids in
E. royleana ranged from 0.16 ± 0.001 ppm to 4.59 ± 0.23 ppm. HPLC analysis of Z.
mauritiana demonstrated the chlorogenic acid of hexane extract as the poor contributor of
phenolic acid profile. Overall the major extent of phenolic acids was observed in highly polar
solvent (methanol).
113
The out come of GC-MS study revealed the innovative chemical spectrum of each
extract. Chemical constituent of methanol, chloroform and hexane extracts of E.
camaldulensis ranged from 0.58% to 45.94%. Euclayptol was the major and common
constituent of chloroform and methanol extracts with chemical contribution of 45.94% and
31.86%, respectively. Ledol (0.73%) was the least existing component of hexane extract of
E. camaldulensis. Palmitic acid (25.67%) was the major component of V. betonicifolia, while
the β-tochopherol (0.08%) and cetene (0.08%) were the least existing components with equal
chemical contribution. Chemical constituents of P. guajava leaf extracts (methanol,
chloroform and hexane) varied from 1.43% to 35.18%. Pyrogallol (35.18%) of methanol
extract was the leading constituent of P. guajava. Minor components of methanol,
chloroform and hexane extracts of P. guajava were culmorin (1.43%), alloaromadendrene
(2.18%) and α-copaene (2.18%), respectively. GC-MS analysis of Z. mauritiana leaf extracts
indicated palmatic acid as major constituent and diglycerol (0.30%) as minor one.
The analyzed plant extracts showed good free radical scavenging activities.
Maximum free radical scavenging activity (91.04 ± 1.56 %) was observed in methanol
extract of Z. mauritiana. Hexane extract of V. betonicifolia exhibited poor free radical
scavenging activity (39.47 ± 0.69 %).
Antimicrobial potential of the tested medicinal plant extracts was evaluated by disc
diffusion assay. Among the bacterial strains maximum inhibition zone was observed by
methanol extract of E. camaldulensis against B. subtilis. Minimum antibacterial activity was
observed in methanol extract of E. royleana against E. coli (5.67 ± 0.57 mm). Maximum
antifungal efficacy was observed in methanol extract of Z. mauritiana against A. niger (23.39
± 1.25 mm). Overall, among the bacterial strains, maximum activities were observed by most
of plant extracts against B. subtillis (Gram positive) and minimum against E. coli (Gram
negative). In case of fungi, A. niger was the sensitive strain while F. solani was the resistant
one against most of plant extracts.
Antitumor activity estimated by potato disc assay showed that tested extracts had
potential to inhibit the growth of Agrobacterium tumefacien. Maximum antitumor activity
(IC50 = 38.13 µg/mL) was observed in chloroform extract of V. betnocifolia, while methanol
extract of E. royleana (IC50 = 485.1 µg/mL) was poor candidate. Among three extracts
114
(methanol, chloroform and hexane) of Z. mauritiana chloroform extract exhibited maximum
ability to inhibit the growth of tumors (IC50 = 70.74 µg/mL). In case of E. camaldulensis
methanol extract (IC50 = 59.68 µg/mL ) was the potential candidate. Antitumor activity of P.
guajava ranged from (IC50 = 65.02 µg/mL ) to (IC50 = 337.4 µg/mL ).
The out come of MTT assay showed that human myelogenous leukemia (KBM5)
cells were the most sensitive to V. betonicifolia chloroform extract (IC50 = 0.001 mg/mL).
Hexane extract (IC50 = 0.020 mg/mL) of P. guajava was successful candidate against human
multiple myeloma (U266) cells. Maximum death of human tongue squamous carcinoma cells
(IC50 = 0.043 mg/mL) was observed by chloroform extract of Z. mauritiana. Human colon
carcinoma cells (HCT116) exhibited maximum susceptibility to hexane extract of P. guajava
(IC50 = 0.005 mg/mL). Methanol extract of V. betonicifolia was least effective (IC50 = 0.183
mg/mL) against colon carcinoma (HCT116) cells. Methanol extract of Z. mauritiana (IC50 =
0.148 mg/mL) was poor candidate against human myelogenous leukemia (KBM5) cells.
Human multiple myeloma (U266) cells were most resistant (IC50 = 0.089 mg/mL) to
methanol extract of P. guajava. Overall chloroform extract of most of plants was the best
candidate against cancer. Methanol extract of most of investigated plants exhibited poor
anticancer activity.
Plant extracts with potent anticancer activity against human myelogenous leukemia
(KBM5) cells were tested for inhibition of inflammatory transcription factor, nuclear factor
kappa B (NF-κB). The results of electrophoretic mobility shift assay (EMSA) demonstrated
that the investigated extracts had excellent anti-inflammatory activity. Hexane extract of P.
guajava proved best anti-inflammatory agent. It exhibited complete inhibition of TNF-α
induced NF-κB activation at 25 µg/mL in human myelogenous leukemia (KBM5) cells.
Chloroform extracts of V. betonicifolia also showed complete inhibition of TNF-α induced
NF-κB activation at 50 µg/mL.
It is evident from our results, that current study provided valuable information about
chemical composition, antioxidant, antimicrobial, antitumor and anticancer activities of
traditional medicinal plants of Punjab, Pakistan. The knowledge of phenolic composition in
different solvent extracts will help to explore their potential as source of natural medicinal
agent particularly the antioxidants. Moreover, the comparison of phenolic composition and
115
other chemical compounds among solvents of variable polarity will help to optimize the
solvent for extraction of phenolic compounds. The considerable antioxidant, antimicrobial,
antitumor and anticancer activities of different extracts may assist in preparation of herbal
drugs for treatment of microbial infections, cancer and oxidative stress related disorders. The
future work will be focused on the isolation of bioactive compounds from the potential
extracts followed by exploration of their mechanism of action against carcinogenesis.
116
REFRENCES
Abalaka, M.E., S.Y. Daniyan and A. Mann. 2010. Evaluation of the antimicrobial activities
of two Ziziphus species (Ziziphus mauritiana L. and Ziziphus spina Christi L.)
on some microbial pathogens. African Journal of Pharmacy and
Pharmacology. 4:135-139.
Abd-Alla, M.F., S.I. El-Negoumy, M.H. El-Lakanya and N.A.M. Saleh. 1980. Flavonoid
glycosides and the chemosystematics of Euclayptus camaldulensisis.
Phytochemistry. 19 (12) : 2629-2632.
Abe, R. and K. Ohtani. 2013. An ethnobotanical study of medicinal plants and traditional
therapies on Batan Island, the Philippines. Journal of Ethnopharmacology. 145
(2): 554-565.
Abu-Qatouseh, L.F., H. Boutennoune, L. Boussouf, K. Madani, P. Shihab and K. Al-Qaoud.
2013. In vitro susceptibility of Helicobacter pylori to urease inhibitory effects
of polyphenolic extracts of local herbs from Algteria. The International Arabic
Journal of Antimicrobial Agents. 3(4): 1-9.
Adeniyi, B.A. and Ayepola, O.O. 2008. The phytochemical screening and antimicrobial
activity of leaf extracts of Eucalyptus camaldulensis and Eucalyptus
torelliana (Myrtaceae). Research Journal of Medicicnal Plant. 2 (1):34-38.
Adesida, A. and E.O. Farombi. 2012. Free radical scavenging activities of guava extract in
vitro. African Journal of Medicine and Medicinal Sciences. 41: 81-90.
Aggarwal, B.B., G. Sethi, K.S. Ahn, S.K.Sandur, M.K.Pandey, A.B.Kunnumakkara, B.Sung
and H. Ichikawa. 2006. Targeting signal-transducer-and-activator-of-
transcription-3 for prevention and therapy of cancer: modern target but ancient
solution. Annals of the New York Academy of Sciences. 1091: 151-169.
Agarwal, M.K, M.L. Agarwal, M. Athar and S. Gupta. 2004. Tocotrienol-rich fraction of
palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis
independent of cell cycle association. Cell Cycle. 3: 205-211.
Agarwal, S.K., S.S. Singh, S. Verma and S. Kumar. 2000. Two new aliphatic compounds
from the leaves of Ziziphus mauritiana. Indian Journal of Chemistry. 39: 872-
874.
117
Ahmed, D., H. Baig and S. Zara. 2012. Seasonal variation of phenolics, flavonoids,
antioxidant and lipid peroxidation inhibitory activity of methanolic extract of
Melilotus indicus and its sub-fractions in different solvents. International
Journal of Phytomedicine. 4 (3) : 326-332.
Ahmed, M., A. Khaleeq and S. Ahmad. 2014a. Antioxidant and antifungal activity of
aqueous and organic extracts of liquorice. World Applied Science Journal. 30
(11): 1664-1667.
Ahmed, N., A. Mahmood, S.S. Tahir, A. Bano, R.N. Malik, S. Hassan and Aisha Ashraf.
2014b. Ethnomedicinal knowledge and relative importance of indigenous
medicinal plants of Cholistan desert, Punjab Province, Pakistan. Journal of
Ethnopharmacology. http://dx.doi.org/10.1016/j.jep.2014.7.007.
Ahmad, M.R., M. Qureshi, M.A. Arshad and M. Zafar. 2009. Traditional herbal remedies
used for the treatment of diabetes from district Attock (Pakistan). Pakistan
Journal of Botany. 41: 2777-2782.
Ahmad, I. and A.Z. Beg. 2001. Antimicrobial and phytochemical studies on 45 Indian
medicinal plants against multi-drug resistant human pathogens. Journal of
Ethnopharmacology. 74: 113-123.
Ahmed, O., S. Libsu and D. Moges. 2013. A study of antioxidant activities of guava
(Psidium guajava) and mangi (Mangifera indica) fruits. International Journal
of Integrative Sciences, Innovation and Technology. 2(3): 1-5.
Ahmad, A., V.S. Vuuren and A. Viljoen. 2014a. Unravelling the complex antimicrobial
interactions of essential oils--the case of Thymus vulgaris (thyme). Molecules.
19(3): 2896-28910.
Akgul, C. and I. Kaya. 2004. Potent antibacterial activity of oligo-3-aminopyridine against
Staphylococcus aureus and Enterococcus faecalis. Indian Journal of
Biochemistry and Biophysics. 41: 120-122.
Akhbari, M., H. Batooli and F.J. Kashi. 2012. Composition of essential oil and biological
activity of extracts of Viola odorata L. from central Iran. Natural Product
Research. 26 (9): 802-809.
118
Akhter, A., M.S. Rahman and M. Ahsan. 2008. Preliminary antimicrobial and cytotoxic
activities of n-Hexane extract of Jatropha pandurifolia. Latin American
Journal of Pharmacy. 27 (6): 918-921.
Akinyele, T.A., O.O. Okoh, D.A. Akinpelu and A.I. Okoh. 2011. In vitro antibacterial
properties of crude aqueous and n-hexane extracts of the husk of Cocos
nucifera. Molecules. 16 (3) : 2135-2145.
Akpuaka, A., M.M. Ekwenchi, D.A. Dashak and A. Dildar. 2013. Biological activities of
characterized isolates of n-hexane extract of Azadirachta indica A. juss
(Neem) leaves. New York Science Journal. 6 (6) : 119-124.
Aktaer, A., M.S. Rahman and M. Ahsan. 2008. Preliminary antimicrobial and cytotoxic
activities of n-hexane extract of Jatropha pandurifolia. Latin American
Journal Pharmacy. 27 (6) : 918-921.
Akowuah, G.A., A. Sadikun and A. Mariam. 2002. Flavonoid identification and
hypoglycemic studies of butanol fraction from Gynura procumbens.
Pharmaceutical Biology. 40: 405-410.
Albert, S., R. Horbach, H.R. Deising, B. Siewert and R. Csuk. 2011. Synthesis and
antimicrobial activity of (E) stilbene derivatives.Bioorganic and Medicinal
Chemistry. 19(7): 5155-5166.
Alagesaboopathi, C. 2012. Antimicrobial activity and phytochemical analysis of
Andrographis alata Nees from Southern India. International Journal of Pharm
Tech Research. 3(3): 1322-1328.
Ali, M., M. Arfan, K. Zaman, H. Ahmad, N. Akbar, I. Anis and M.R. Shah. 2011.
Antiproliferative activity and chemical constituents of Hypericum
oblongifolium. Journal of Chemical Society of Pakistan. 33: 772-777.
Ali, H. and M. Qaiser. 2009. The ethnobotany of Chitral valley, Pakistan with particular
reference to medicinal plants. Pakistan Journal of Botany. 41 (4) : 2009-2041.
Alaribe, C.S., F. Shode, H.A. Coker, G. Ayoola, A. Sunday, N. Singh and S. Iwuanyanwu.
2011. Antimicrobial activities of hexane extract and decussatin from stem
bark extract of Ficus congensis. International Journal of Molecular Sciences.
12 (4) : 2750-2756.
119
Alqahtani, A., K. Hamid, A. Kam, K.H. Wong, Z. Abdelhak, V.R. Naumovski, K. Chan ,
K.M. Li, P.W. Groundwater and G.Q.Li. 2013. The pentacyclic triterpenoids
in herbal medicines and their pharmacological activities in diabetes and
diabetic complications. Current Medicinal Chemistry. 20 (7): 908-931.
Al-Shammari, L.A., W.H.B. Hassan and H.M.A. Youssef. 2012.Chemical composition and
antimicrobial activity of the essential oil and lipid content of Carduus
pycnocephalus L. growing in Saudi Arabia. Journal of Clinical and
Pharmaceutical Research. 4(2): 1281-1287.
Alstyne, K.L.V., J. J. McCarthy, C. L. Hustead and D.O.Duggins. 1998. Geographic
variation in polyphenolic levels of Northeastern PaciÆc kelps and rockweeds.
Marine Biology. 133: 371-379.
Alves, R.J.Y., A.C. Pinto, A.V.M.D. Costa and C.M. Rezende. 2005. Zizyphus mauritiana
Lam. (Rhamnaceae) and the chemical composition of its floral fecal odor.
Journal of Brazilian Chemical Society. 16(30): 654-656.
Alves, M.J., I.C. Ferreria, H.J. Froufe, R.M. Abreu, A. Martins and M. Pintado. 2013.
Antimicrobial activity of phenolic compounds identified in wild mushrooms,
SAR analysis and docking studies. Journal of Applied Microbiology. 115 (2):
346-357.
American Cancer Society. 2007. Cancer Prevention & Early Detection Facts & Figures.
Atlanta, GA: American Cancer Society.
Aminu, M., M.S. Bello, O. Abbas, M. Aliyu, B.S. Malam, G. Auwalu, Hafsat, A.
Muhammad, M. Shafiu, N. N. Hussaina, A. Hasiya and A. San. 2012.
Comparative in vitro antioxidant studies of ethanolic extracts of Psidium
guajava stem bark and Telfairia occidentalis leaf. International Journal of
Modern Biochemistry. 1(1): 18-26.
Ampasavate, C., S. Okonogi and S. Anuchapreeda. 2010. Cytotoxicity of extracts from fruit
plants against leukemic cell lines. African Journal of Pharmacy and
Pharmacology. 4 (1): 13-21.
Anderson, S.I. and B.Y. Rubin. 2005. Tocotrienols reverse IKAP and monoamine oxidase
deficiencies in familial dysautonomia. Biochemcial and Biophysical Research
Communications. 336: 150-156.
120
Anonymous, 1989. The Wealth of India (Raw material), Vol XI: X-Z, (Council of Industrial
and Scientific Research, New Delhi) :111-124.
Aparna, V., K.V. Dileep, P.K. Mandal, P. Karthe, C. Sadasivan and M. Haridas. 2012. Anti-
inflammatory property of n-hexadecanoic acid: structural evidence and kinetic
assessment. Chemical Biology and Drug Design. 80 (3): 434-439.
Araujo, K.M.D., A.D. Lima, J.D.N. Silva, L.L. Rodrigues, A.G.N. Amorim, P.V. Quelemes,
R.C.D. Santos, J.A. Rocha, É.O.D. Andrades , J.R.S.A. Leite, J.M. Filho and
R.A.D. Trindade. 2014. Identification of phenolic compounds and evaluation
of antioxidant and antimicrobial properties of Euphorbia Tirucalli L.
Antioxidants. 3 (1) : 159-175.
Archana, B., N. Dasgupta and B. De. 2005. In vitro study of antioxidant activity of Syzygium
cumini fruit. Food Chemistry. 90, 727-733.
Arima, H. and G. Danno. 2002. Isolation of antimicrobial compounds from guava (Psidium
guajava L.). Bioscience, Biotechnology and Biochemistry. 66, 727–1730.
Aris, S.R.S., S. Mustafa, N. Ahmat and F.M. Jaafar. 2009. Phenolic content and antioxidant
activity of fruits of Ficus deltoidea var. angustifolia sp. The Malaysian
Journal of Analytical Sciences.13 (2) :146-150.
Ashraf, A., R.A. Sarfraz, M.A. Rashid and M. Shahid. 2014. Antioxidant, antimicrobial,
antitumor and cytotoxic activities of an important medicinal plant
(Euphorbia royleana) from Pakistan. Journal of Food and Drug Analysis.
Asl, M.N. and H. Hosseinzadeh. 2008. Review of pharmacological effects of Glycyrrhiza sp.
and its bioactive compounds. Phytotherapy Research. 22 (6): 709-724.
Avelar, M.M. and C.M.C.P. Gouvea. 2012. Procyanidin B2 cytotoxicity to MCF-7 human
breast adenocarcinoma cells. Indian Journal of Pharmaceutical Research. 74
(4): 351-355.
Babayi, H., I. Kolo, J. I. Okogun and U. J. J. Ijah. 2004. The antimicrobial activities of
methanolic extracts of Eucalyptus camaldulensis and Terminalia catappa
against some pathogenic microorganisms. Biokemistri. 16(2): 106-111.
Bae, H., G.K. Jayaprakasha, J. Jifon and B.S. Patil. 2012. Variation of antioxidant activity
and the levels of bioactive compounds in lipophilic and hydrophilic extracts
121
from hot pepper (Capsicum spp.) cultivars. Food Chemistry. 134 (4) : 1912-
1918.
Bae, H., G.K. Jayaprakasha, K. Crosby, J.L. Jifon and B.S. Patil. 2012. Influence of
extraction solvents on antioxidant activity and the content of bioactive
compounds in non-pungent peppers. Plant Foods for Human Nutrition. 67,
120-128.
Bakkiyaraj, S. and S. Pandiyaraj. 2011. Evaluation of potential antimicrobial activity of some
medicinal plants against common food-borne pathogenic microorganism.
International Journal of Pharma and Bio Sciences. 2 (2) : B484-B491.
Bala, S.A. 2006. Euphorbia hirta linn.: In some ethnomedicinal plants of the of the savanna
regions of west Africa: Description and phytochemicals. Bala SA(ed.). 1st
edn. The Triump Publishing Company Ltd, Gidan Saadu Zungur, Kano,
Nigeria. pp. 19-25.
Bani, S., A. Kaul, B.S. Jaggi, K.A. Suri, O.P. Suri and O.P.Sharma. 2000. Anti-inflammatory
activity of the hydrosoluble fraction of Euphorbia royleana latex. Fitoterapia.
71: 655-662.
Bani, S., A. Kaul, B. Khan, S.F. Ahmad, K.A. Suri, N.K. Satti, M. Amina and G.N. Qazi,
2005. Immunosuppressive properties of an ethyl acetate fraction from
Euphorbia royleana. Journal of Ethnopharmacology. 99 (2): 185-192.
Bani, S., D. Chand, K.A. Suri, O.P. Suri and O.P. Sharma. 1996. Antiinflammatory effects
of an ethyl acetate extract of Euphorbia royleana. Phytotherapy Research. 10 :
285-291.
Basak, S.S. and F. Candan. 2010. Chemical composition and in vitro antioxidant and
antidiabetic activities of Eucalyptus Camaldulensis Dehnh. essential oil.
Journal of Iranian Chemical Society. 7(1): 216-226.
Basma, A.A., Z. Zakaria, L.Y. Latha and S. Sasidharan. 2011. Antioxidant activity and
phytochemical screening of the methanol extracts of Euphorbia hirta L. Asian
Pacific Journal of Tropical Medicine. 4 (5) : 386-390.
Batick, M.J. 1984. Ethnobotany of Palms in the Neotropics. In; Prance GT, Kallunki JA,
editors, Advances in Economic Botany: Ethnobotany in the Neotropics. New
York Botanical Garden, New York, USA, pp. 9-23.
122
Begum, S., I. Sultana, B.S. Siddiqui , F. Shaheen and A.H.Gilani. 2000. Spasmolytic
constituents from Eucalyptus camaldulensis var. obtusa leaves. Journal of
Natural Products. 63 (9) : 1265-1268.
Begum, S., S.I. Hassan, B.S. Siddiqui, F. Shaheen, M.N. Ghayur and A.H. Gilani. 2002.
Triterpenoids from the leaves of Psidium guajava. Phytochemistry. 61: 399–
403.
Begum, S., S.I. Hassan and B.S. Siddiqui. 2004. Chemical constituents from the leaves of
Psidium guajava. Natural Product Research. 18:135–140.
Bensoussan, A., N.J. Talley, M. Hing, R. Menzies, A. Guo and M. Ngu. 1998. Treatment of
irritable bowel syndrome with Chinese herbal medicine: a randomized
controlled trial. JAMA. 280 (18) :1585-1589.
Benzie, I.F. and J.J.Strain. 1996. The ferric reducing ability of plasma (FRAP) as a measure
of antioxidant power: The FRAP assay. Analytical Biochemistry. 239, 70-76.
Benize, I.F. and J.J. Strain. 1999. Ferric reducing/antioxidant power assay: direct measure of
total antioxidant activity of biological fluids and modified version for
simultaneous measurement of total antioxidant power and ascorbic acid
concentration. Methods in Enzymology. 299:15-27.
Benoit, S.C., C.J. Kemp, C.F. Elias, W. Abplanalp, J.P. Herman, S. Migrenne, A.L. Lefevre,
C.C. Guglielmacci, C. Magnan, F.Yu, K. Niswender, B.G. Irani, W.L.
Holland and D.J. Clegg.2009. Palmitic acid mediates hypothalamic insulin
resistance by altering PKC-θ subcellular localization in rodents. The Journal
of Clinical Investigation. 119(9): 2577-2589.
Bhat, B.A., C. Elanchezhiyan and S. Sethupathy. 2012. In vitro antioxidative role Helicteres
isora (L). International Journal of Bioassays. 1 (12) :177-183.
Bhatt, S. and S. Dhyani. 2013. Quantification of secondary metabolites from Ziziphus
mauritiana lam. Bark. International Journal of Bio-Technology and Research.
3(2): 1-6.
Bhatt, V.P. and G.C.S Negi. 2006. Ethnomedicinal plant resources of Jaunsari tribe of
Garhwal, Himalaya, Uttaranchal. Indian Journal of Traditional Knowledge.
5(3): 331-335.
123
Bhuiyan, M.A.R. and M.Z. Hoque. 2010. Free radical scavenging activties of Ziziphus
mauritiana. Electronic Journal of Environmental, Agriculture and Food
Chemistry. 9(1): 199-206.
Blainski, A., G.C. Lopes and J.C.P.D. Mello. 2013. Application and analysis of the Folin
Ciocalteu method for the determination of the total phenolic content from
Limonium Brasiliense L. Molecules. 18: 6852-6865.
Bibi, G., I.U. Haq, N. Ullah, A. Mannan and B. Mirza. 2011. Antitumor, cytotoxic and
antioxidant potential of Aster thomsonii extracts. African Journal of Pharmacy
and Pharmacology. 5(2): 252-258.
Bibi, Y., S. Nisa, M. Zia, A. Waheed, S. Ahmed and M.F. Chaudhary. 2012. In vitro
cytotoxic activity of Aesculus indica against breast adenocarcinoma cell line
(MCF-7) and phytochemical analysis. Pakistan Journal of Pharmaceutical
Sciences. 25 (1): 183-187.
Boots, A.W., L.C. Wilms, E.L. Swennen, J.C. Kleinjans, A. Bast and G.R. Haenen. 2008. In
vitro and ex vivo anti-inflammatory activity of quercetin in healthy
volunteers. Nutrition. 24(7-8): 703-710.
Bren, L.J. and Gibbs, N.L. (1986) Relationships between flood frequency, vegetation and
topography in a river red gum forest. Australian Forest Research 16, 357-370.
Brum, T.F.D., M. Zadra , M. Piana, A.A. Boligon, J.K. Frohlich, R.B.D. Freitas , S.T.
Stefanello, A.L.F. Froeder , B.V. Belke, L.T. Nunes , R.D.S. Jesus, M.M.
Machado, J.B.T.D. Rocha, F.A.A.Soares and M.L. Athayde. 2013. HPLC
analysis of phenolics compounds and antioxidant capacity of leaves of Vitex
megapotamica (Sprengel) Moldenke. Molecules. 18 (7) : 8342-8357.
Bukhari, S.B., M.I. Bhanger and S. Memon. 2008. Antioxidative activity of extracts from
Fenugreek seeds (Trigonella foenum-graecum). Pakistan Journal of Analytical
and Enviornmental Chemistry. 9(2): 78-83.
Cai, Y., Q. Luo, M. Sun and H. Croke. 2004. Antioxidant activity and phenolic compounds
of 112 traditional Chinese medicinal plants associated with anticancer. Life
sciences 74: 2157-2184.
124
Calfee-Mason, K.G., B.T. Spear and H.P. Glauert. 2002. Vitamin E inhibits hepatic NF-
kappaB activation in rats administered the hepatic tumor promoter,
phenobarbital. The Journal of Nutrition. 132 (10): 3178-3185.
Capocasa, F., J. Scalzo, B. Mezzetti and M. Battino. 2008. Combining quality and
antioxidant attributes in the strawberry: The role of genotype. Food
Chemistry. 111(4):872-878.
Carr, A.C., B.Z. Zhu and B. Frezi. 2008. Potential antiatherogenic mechanism of ascorbate
(Vitamin C) and α-tocopherol (Vitamin E). Circulation Research.87:349-354.
Caunii, A., G. Pribac, I. Grozea, D. Gaitin and I. Samfira. 2012. Design of optimal solvent
for extraction of bio–active ingredients from six varieties of Medicago sativa.
Chemistry Central Journal. 6: 1-8.
Carmeliet, P. and R.K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature. 407
(6801) : 249-257.
Cataluna, R.S.M.K. 1999. The traditional use of the latex from Euphorbia tirucalli Linnaeus
(Euphorbiaceae) in the treatment of cancer in South Brazil. Second World
Congress on Medicinal and Aromatic Plants for Human Welfare Wocmap, 2
(501): 289-295.
Ceylan, E and D.Y.C. Fung. 2004. Antimicrobial activity of spices. Journal of Rapid
Methods and Automation in Microbiology.12: 1-55.
Chan, E.W.C., Y.Y. Lim and M. Omar. 2007. Antioxidant and antibacterial activity of leaves
of Etlingera species (Zingerberaceae) in Peninsular Malaysia. Food
Chemistry. 104: 1586-1593.
Chainy, G.B., S.K. Manna, M.M. Chaturvedi and B.B. Aggarwal. 2000. Anethole blocks
both early and late cellular responses transduced by tumor necrosis factor:
effect on NF-kappaB, AP-1, JNK, MAPKK and apoptosis. Oncogenes. 19:
2943-2950.
Chang, C.C., M.H. Yang, H.M. Wen HM and J.C. Chern. 2002. Estimation of total flavonoid
contents in propils by two complementary colorimetric methods. Journal of
Food and Drug Analysis. 10 (3) : 178-182.
Chang, H.L., Y.C. Wu, J.H. Su, Y.T. Yeh and S.S. Yuan. 2008. Protoapigenone, a novel
flavonoid, induces apoptosis in human prostate cancer cells through activation
125
of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase
1/2.The Journal of Pharmacology and Experimental Therapeutics. 325 (3):
841-849.
Chang, Z.Q., E. Gebru, S.P. Lee, M.H. Rhee, J.C. Kim, H.Cheng and S.C. Park. 2011. In
vitro antioxidant and anti-inflammatory activities of protocatechualdehyde
isolated from Phellinus gilvus. Journal of Nutrition Science and Vitaminol. 57
(1) :118-122,
Chao, P.C., C.C. Hsu and M.C. Yin. 2009. Anti-inflammatory and anti-coagulatory activities
of caffeic acid and ellagic acid in cardiac tissue of diabetic mice. Nutrition and
Metabolism. 6: 33.
Charles,W.W., E.S. Philip and W.C. Carl. 2006. Determination of organic acids and sugars in
P. guajava L. cultivars by high-performance liquid chromatography. Journal
of the Food and Agriculture. 33: 777–780.
Chen, K.C., C.L. Hsieh, C.C. Peng, H.M.H. Li, H.S. Chiang, K.D. Huang and R.Y. Peng.
2007. Brain derived prostate cancer DU-145 cells are effectively inhibited in
vitro by guava leaf extracts. Nutrition Cancer. 58: 93-106.
Chen, C.Y. and Y.D. Wang. 2010. Steroids from the whole plants of Leucaena leucocephala.
American Journal of Analytical Chemistry. 1: 31-33.
Chen, Y., C. Zhou, Z. Ge, Y. Liu, Y. Liu, W. Feng, S. Li, G. Chen and E. Wei. 2013.
Composition and potential anticancer activities of essential oils obtained from
myrrh and frankincense. Oncology Letters. 6(4): 1140-1146.
Chebouat, E., B. Dadamoussa, A. Kabouche, M. Allaoui, M. Gouamid, A. Cheriti and N.
Gherraf. 2013. Gas chromatography-mass spectrometry (GC-MS) analysis of
the crude alkaloid extract of Ziziphus mauritiana Lam., grown in Algerian.
Journal of Medicinal Plants Research. 7(20):1511-1514.
Choi, C.W., S.C. Kim, S.S. Hwang, B.K. Choi, H.J. Ahn, S.H. Park, S.K. Kim.
2002.Antioxidant activity and free radical scavenging capacity between
Korean medicinal plants and flavonoids by assay-guided comparison. Plant
Science. 163 (6) : 1161-1168.
Chrubasik, S., C. Conradt and B.D. Roufogalis. 2004. Effectiveness of Harpagophytum
extracts and clinical efficacy. Phytotheraphy Research. 18 (2): 187-189.
126
Cicco, N., M.T. Lanorte, M. Paraggio, M. Viggiano and V. Lattanzio. 2009. A reproducible,
rapid and inexpensive Folin–Ciocalteu micro-method in determining
phenolics of plant methanol extracts. Microchemical Journal. 91(1): 107-110.
Cito, A.M.G.L., A.A. Souza, J.A.D. Lopes, M.H. Chaves, F.B. Costa, A.S.A. Souza and
Amaral, M.P.M., 2003. Protium heptaphyllum March (Burceraceae resin)
chemical composition of essential oil and cytotoxic evaluation with respect to
Artemia salina Leach. Anais da Academia Brasileira de Ciencias. 52: 74-76.
Constantinou, C., A. Papas and A.I. Constantinou. 2008. Vitamin E and cancer: An insight
into the anticancer activities of vitamin E isomers and analogs. International
Journal of Cancer.123: 739- 752.
Comitato, R., G. Leoni, R. Canali, R. Ambra, K. Nesaretnam and F. Virgili. 2010.
Tocotrienols activity in MCF-7 breast cancer cells: involvement of ER beta
signal transduction. Molecular Nutrition and Food Research. 64: 327-332.
Conde, E., E. Cadahia and M.C.G. Vallejo. 1997. Low molecular weight polyphenols in
leaves of Euclayptus camaldulensis, E. globulus and E. rudis. Phytochemical
Analysis. 8 (4): 186-193.
Conde, E., E. Cadahia, M.C.G. Vallejo and M.B.F. Simon. 1995. Polyphenolic composition
of wood extracts from Eucalyptus camaldulensis, E. globulus and E. rudis.
Holzforschung. 49 (5): 411-417.
Conde, E.,E. Cadahia, R.D. Barra and M.C.G. Vallejo. 1996. Polyphenolic composition of
bark extracts from Eucalyptus camaldulensis, E. globulus and E. rudis.
European Journal of Wood and Wood Products. 54 (3): 175-181.
Conway, P. 2002. Tree Medicine: A Comprehensive Guide to the Healing Power of Over
170 Trees. 2001. Judy Piatkus (Publishers) Ltd, pp. 2173-2177.
Council of Europe. Determination of tannins in herbal drugs. In European Pharmacopoeia.
2007. 6th ed.; European Directorate for the Quality of Medicines: Strasbourg,
France. p. A286.
Cushine, T.P. and A.J. Lamb. 2005. Antimicrobial activity of flavonoids. International
Journal of Antimicrobial Agents. 26(5): 484-488.
Custodio, D.L., R.P. Burgo, B. Moriel, A.D.M. Barbosa, M.I. Rezende, J.F.D.S. Daniel, J.P.
Pinto, E. Bianchini and T.D.J. Faria. 2010. Antimicrobial activity of essential
127
oils from Pimenta pseudocaryophyllus and Tynanthus micranthus. Brazilian
Journal of Biology and Biotechnology. 53 (6): 1363-1369.
Dalal, M., V. Chinnusamy and K.C. Bansal. 2010. Isolation and functional characterization
of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites.BMC
Plant Biology. 10: 61.
Dalleau, S., M. Baradat, F. Guéraud and L. Huc. 2013. Cell death and diseases related to
oxidative stress: 4-hydroxynonenal (HNE) in the balance. Cell Death and
Differentiation.20: 1615-1630.
Damme, V.P.L.J. 2001. Euphorbia tirucalli for high biomass production, in: A. Schlissel and
D. Pasternak (Eds.), Combating desertification with plants, Kluwer Academic
Pub. pp. 169-187.
Das, P., S. Mekap, S. Pani, R. Sethi and P. Nayak. 2010. Pharmacological evaluation of anti–
inflammatory activity of Euphorbia hirta against carrageenan induced paw
edema in rats. Der Pharmacia Lettre. 2 (2) : 151-154.
Das, S. 2012. Antimicrobial and antioxidant activities of green and ripe fruits of Averrhoa
carambola linn. and Zizyphus mauritiana lam. Asian Journal of
Pharmaceutical and Clinical Research. 5: 102-105.
Dhar, P., P.K. Bajpai, A.B. Tayade, O.P. Chaurasia, R.B. Srivastava and S.B. Singh. 2013.
Chemical composition and antioxidant capacities of phytococktail extrtacts
from trans-Himalayan cold desert. BMC Complementary and Alternative
Medicine. 13: 259.
Dhanamani, M., S.L. Devi and S. Kannan. 2011. Ethnomedicinal plants for cancer therapy.
Hygia-Journal of Drugs and Medicine. 3 (1): 1-10.
Deng, W., B. Hu, C.L. Dai, Y.J. Wang, H.F. Chen, S.W. Zito, L.W. Fu and Z.S. Chen. 2013.
Anticancer activity of Oldenlandia diffusa and Viola philippica car. Journal of
Cancer Research Updates. 2: 87-94.
Dexter, B.D. (1978) Silviculture of the River Red Gum forests of the central Murray
floodplain. Proceedings of the Royal Society of Victoria 90, 175-194.
Dweck, A.C. 2001. A review of Psidium guajava. Malayan Journal of Medical Science. 8:
27–30.
128
Dubal, K.N., P.N. Ghorpade and M.V. Kale. 2013. Studies on bioactive compounds of
Tectaria coadunate (Wall. Ex hook. And Grev.). Asian Jouranl of
Pharmaceutical and Clinical Research. 6 (2): 186-187.
Dung, N.T., J.M. Kim and S.C. Kang. 2008. Chemical composition, antimicrobial and
antioxidant activities of the essential oil and the ethanol extract of
Cleistocalyx operculatus (Roxb.) Merr and Perry buds. Food and Chemical
Toxicology. 46 : 3632–3639.
Dureja, A.G and K. Dhiman. 2012. Free radical scavenging potential and total phenolic and
flavonoid content of Ziziphus mauritiana and Ziziphus nummularia fruit
extracts.International Journal of Green Pharmacy. 6: 187-192.
Duthie, G and P. Morrice. 2012. Antioxidant capacity of flavonoids in hepatic microsomes is
not reflected by antioxidant effects in vivo. Oxidative Medicine and Cellular
Longevity. 2012: 1-6.
Dutta, B.K. and T.K. Das. 2000. In vitro study on antifungal property of common fruit plants.
Biomedicine. 20:187–189.
Ebrahimabadi, A.H., E.H. Ebrahimabadi, Z. Djafari-Bidgoli, F.J. Kashi, A. Mazoochi and H.
Batooli. 2010. Composition and antioxidant and antimicrobial activity of the
essential oil and extracts of Staachys infata Benth from Iran. Food Chemistry.
119: 452-458.
Ebrahimzade, M.A., S.M. Nabvi, S.F. Nabvi, F. Bahramian and A.B. Bekhradina. 2010.
Antioxidant and free radical scavenging activity of H. officinalis l. var.
angustifolius, V. odorata, B. hyrcana and C. speciosum. Pakistan Journal of
Pharmaceutical Sciences. 23(1): 29-34.
Edoga, H.O., D.E. Okwu and B.O. Mbaebie. 2005. Phytochemicals constituents of some
Nigerian medicinal plants. African Journal of Biotechnology. 4: 685-688.
El-Ghorab, A.H., H.M. Fadel and K.F. El-Massry. 2003. The Egyptian Eucalyptus
camaldulensis var. brevirostris: chemical compositions of the fruit volatile oil
and antioxidant activity. Flavour and Frgrance Journal. 17 (4): 306-312.
Elinav, E., R. Nowarski, C.A. Thaiss, B. Hu, C. Jin and R.A. Flavell. 2013. Inflammation-
induced cancer: crosstalk between tumours, immune cells and
microorganisms. Nature Review Cancer.13:759-771.
129
Elekwa, I., S.C. Okereke and B.O. Ekpo. 2009. Preliminary phytochemical and antimicrobial
investigations of the stem bark and leaves of Psidium guajava L. Journal of
Medicinal Plants Research. 3: 45-48.
El-Mageed, A.A.A.,A.K. Osman, A.Q. Tawik and H.A. Mohammed. 2011. Chemical
composition of the essential oils of four Eucalyptus species (Myrtaceae) from
Egypt. Research Journal of Phytochemistry. 5 (2): 115-122.
Elsharkawy, E. and M. Alshathly. 2013. Anticancer activity of Lactuca steriolla growing
under desert conditions Northern region in Saudi Arabia. 2013. Journal of
Natural Sciences Research. 3 (2): 5-12.
Elujoba, A.A., O.M. Odeleye and C.M. Ogunyemi. 2005. Review-Traditional medicine
Development for medical and dental primary health care delivery system in
Africa. African Journal of Traditional, Complementary and Alternative
Medicine. 2(1): 46-61.
Ekundayo, E.O. and J. N. Ekekwe. 2013. Antibacterial activity of leaves extracts of Jatropha
curcas and Euphorbia heterophylla. African Journal of Microbiology
Research. 7 (44): 5097-5100.
Everette, J.D.,. Q.M. Bryant, A.M. Green, Y.A. Abbey, G.W. Wangila and R.B. Walker.
2010. Thorough study of reactivity of various compound classes toward the
Foli-Ciocalteu reagent. Journal of Agriculture and Food Chemistry. 58 (14):
Fidrianny, I., R. Hartai and N. Raveendaran. 2012. Antioxidant activity of ethylacetate
extract of red Psidium guajava leaves grown in Manoko, Lembang, Indonesia.
Indonesian Journal of Pharmacy. 23 (1): 36.
Fidrianny, I., I. Rahmiyani and K.R. Wirasutisna. 2013. Antioxidant capacities from various
leaves extracts of four varities mangoes using DPPH, ABTS assays and
correlation with total phenolic, flavonoid, carotenoid. International Journal of
Pharmacy and Pharmaceutical Sciences. 5(4): 189-194.
Fidrianny, I., A. Darmawati and Sukrasno. 2014. Antioxidant capacities from different
polarities extracts of cucurbitaceae leaves using FRAP, DPPH assays and
correlation with phenolic, flavonoid, carotenoid content. International Journal
of Pharmacy and Pharmaceutical Sciences. 6 (2) : 858-862.
130
Fernandes, M.R.V., A.E.C.S. Azzolini, M.L.L. Martinez, C.R.F. Souza, Y.M.L. Valim and
W.P. Oliveira. 2012. Physicochemical and antioxidant properties of spray
dried preparations from Psidium guajava L. Planta Medica. 78: 1370.
Flamini, G., P.L. Cioni and I. Morelli. 2003. Varibility of essential oil of Viola etrusca.
Annals of Botany. 91: 493-497.
Flescher, E. 2007. Jasmonates in cancer therapy. Concern Letters. 245: 1-10.
Folmer, F., M. Jaspars, G. Solano, S. Cristofanon, E. Henry, J. Tabudravu, K. Black, D.H.
Green, F.C. Kupper, W. Aalbersberg, K. Feussner, M. Dicato and M.
Diederich. 2009. The inhibition of TNF-a-induced NF-kB activation by
marine natural products, Biochemical Pharmacology. 78(6): 592-606.
Francisco, J.D.C., E.P. Jarvenpaa, R. Huopalahti and B. Sivik . 2001. Comparison of
Eucalyptus camaldulensis Dehn. oils from Mozambique as obtained by
hydrodistillation and supercritical carbon dioxide extraction. Journal of
Agriculture and Food Chemistry. 49 (5): 2339-2340.
Fujita, T., K. Massaharu, K. Tamotsu, Y. Kenji, O. Kejichi and S. Kiyoshi. 1985. Nutrient
contents in fruit and leaves of guava and in leaves of Japanese persimmon.
Seikatsu Eisei. 29: 206–209.
Galsky, A.G., R. Kozimor, D. Piotrowski and R.G. Powell. 1981. The crown-gall potato disk
Bioassay as a primary screen for compounds with antitumor activity. Journal
of National Cancer Institute. 67: 687-692.
Gaur, K., A.C. Rana, R.K. Nema, M.L. Kori and C.S. Sharma. 2009. Anti-inflammatory and
analgesic activity of hydro alcoholic leaves extract of Euphorbbia neriifolia
Linn. Asian Journal of Pharmaceutical and Clinical Research. 2 (1): 26-29.
Gautam, S.S., Navneet and S. Kumar. 2012. The Antibacterial and phytochemical aspects of
Viola odorata Linn. Extracts Against Respiratory Tract Pathogens.
Proceedings of the National Academy of Sciences, India Section B: Biological
Sciences. 82(4): 567-572.
Gawron-Gzella, A., M.D. Makuch and I. Matlawska. 2012. DPPH radical scavenging activity
and phenolic compound content in different leaf extracts from selected
balckberry species. Acta Biologica Cracoviensia. 54(2): 32-38.
131
Ghanadian, S.M., A.M. Ayatollahi, S. Afsharypour, S. Hareem, O.M. Abdalla and J.K.
Bankeu. 2012. Flavonol glycosides from Euphorbia microsciadia Bioss. with
their immunomodulatory activities. Iranian Journal of Pharmaceutical
Research. 11 (3): 925-93.
Gharekhani, M., M. Ghorbani and N. Rasoulnejad. 2012. Microwave-assisted extraction of
phenolic and flavonoid compounds from Eucalyptus camaldulensis dehn
leaves as compared with ultrasound-assisted extraction. Latin American
Applied Research. 42: 305-310.
Gilani, A.H, I.A. Bukhari, R.A. Khan, A.J. Shah, I. Ahmad and A. Malik. 2009. Presence of
blood-pressure lowering and spasmolytic constituents in Buddleja crispa.
Phytotherapy Research. 23(4) : 492-497.
Goodman, S.M. and A. Ghafoor. 1992. The ethno botany of Southern Balochistan, Pakistan
with particular reference to medicinal plants. Fieldia. 31: 1-84.
Gollahon, LS., Y. Jeong, V. Finckbone, K. Lee and J.S. Park. 2011. The natural product NI-
07, is effective against breast cancer cells while showing no cytotoxicity to
normal cells. The Open Breast Cancer Journal. 3: 31-44.
Goncalves, A.F.K., R.B. Friedrich, A.A. Boligon, M. Piana, R.C.R. Beck and M.L. Athayde.
2012. Antioxidant capacity, total phenolic contents and HPLC determination
of rutin in Viola tricolor (L) flowers. Free Radicals and Antioxidants. 2(4):
32-37.
Gopalakrishnan, S., K. Saroja and J.D. Elizabeth. 2011. GC-MS analysis of the methanolic
extract of the leaves of Dipteracanthus patulus (Jacq.) Nees. Journal of
Chemical and Pharmaceutical Research. 3(3): 477-480.
Gopalakrishnan, K. and R. Udayakumar. 2014. Antimicrobial activity of Marsilea
quadrifolia (L.) against some selected pathogenic microorganisms. British
Microbiology Research Journal. 4(9): 1046-1056.
Gordon, A.N., J.T. Fleagle, D. Guthrie, D.E. Parkin, M.E. Gore and A.J. Lacave. 2001.
Recurrent epithelial ovarian carcinoma: A randomized phase III study of
pegylated liposomal Doxorubicin versus Topotecan. Journal of Clinical
Oncology. 19: 3312-3322.
132
Goyal, M., B.P. Nagori and D.Sasmal. 2012. Review on ethnomedicinal uses,
pharmacological activity and phytochemical constituentsof Ziziphus
mauritiana Z. jujuba Lam., non Mill. Spatula DD. 2 (2): 107-116.
Govindasami, T., A. Pandey, N. Palanivelu and A. Pandey. 2011.Synthesis, Characterization
and antibacterial activity of biologically important vanillin related hydrazone
derivatives. International Journal of Organic Chemistry. 1:71-77.
Greten, F.R., L. Eckmann, T.F. Greten, J.M. Park, Z.W. Li, L.J. Egan, M.F. Kagnoff and M.
Karin. 2004. IKKbeta links inflammation and tumorigenesis in a mouse model
of colitis-associated cancer. Cell. 118:285-296.
Guil-Guerrero, J.L., A.D. Delgado, M.C.M.Gonzalez and M.E.T. Isasa. 2004. Fatty acids and
carotenes in some ber (Ziziphus jujube Mill.) varieties. Plant Foods for Human
Nutrition. 59:23-27.
Guilford, J.M. and J.M. Pezzuto. 2008. Natural products as inhibitors of carcinogenesis.
Expert Opinion on Investigational Drugs. 17: 1341-1352.
Gulcin, I., O.I. Kufrevioglu, M. Oktay and M.E. Buyukokuroglu. 2004.Antioxidant,
antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.).
Journal of Ethnopharmacology. 90: 205-215.
Gulluce, M., A. Aslan, M. Sokmen, F. Sahin, A. Adiguzel, G. Agar and A. Sokmen. 2006.
Screening the antioxidant and antimicrobial properties of the lichens
:Parmelia saxatilis, Platismatia glauca, Ramalina pollinaria, Ramalina
polymorpha and Umbilicaria nylanderiana. Phytomedicine. 13: 515–521.
Gundampati, R.K. and M.V. Jagannadham. 2012. Molecular docking based inhibition of
trypanothione reductase activity by Taxifolin novel target for antileishmanial
activity. Journal of Applied Pharmaceutical Sciences. 2 (10): 133-136.
Gupta, M.K., A.K. Bhandari and R.K. Singh. 2012. Pharmacognostical evaluations of the
leaves of Ziziphus mauritiana. 3 (3): 818-821.
Gupta, S.C., J.H. Kim, R. Kannappan, S. Reuter, P.M. Dougherty and B.B. Aggarwal. 2011.
Role of nuclear factor-κB-mediated inflammatory pathways in cancer-related
symptoms and their regulation by nutritional agents. Experimental Biology
and Medicine. 236 (6): 658-671.
133
Gutierrez, R.M.P., S. Mitchell and R.V. Solis. 2008. Psidium guajava: A review of its
traditional uses, phytochemistry and pharmacology. Journal of
Ethnopharmacology. 117: 1-27.
Hajimahmood, M., M. Hanifeh, M.R. Oveisi, N. Sadeghi and B. Jannat. 2008. Determination
of total antioxidant capacity of green teas by the ferric reducing / antioxidant
power assay. Iranian Journal of Enviornemental Health Science and
Engineering. 5(3): 167-172.
Hamayun, M., S.A. Khan, I. Iqbal, G. Rehman, T. Hayat and M.A. Khan. 2005.
Ethnobotanical profile of Utror and Gabral Valleys, District Swat, Pakistan.
Ethnobotanical Leaflets.
Han, H.J., N.H. Tan, G.Z. Zeng, J.T. Fan, H.Q. Huang, C.J. Jia, Q.S. Zhao, Y.J. Zhang, X.J.
Hao and L.Q. Wang. 2008. Natural inhibitors of DNA topoisomerase I with
cytotoxicities. Chemistry and Biodiversity. 5 (7): 1364-1368.
Hasan, M.F., R. Das, A. Khan, M.S. Hossain and M. Rahman. 2009. The determination of
antibacterial and antifungal activities of Polygonum hydropiper (L.) root
extract. Advances in Biological Research. 3 (1-2): 53-56.
Hassine, D.B., M. Abderrabba, Y. Yvon, A. Lebrihi, F. Mathieu, F. Couderc and J. Bouajila
2012. Chemical composition and in vitro evaluation of the antioxidant and
antimicrobial activities of Eucalyptus gillii essential oil and extracts.
Molecules. 17, 9540-9558.
Harada, H., U. Yamashita, H. Kurihara, E. Fukushi, J. Kawabata and Y. Kamei. 2002.
Antitumor activity of palmitic acid found as a selective cytotoxic substance in
a marine red alga. Anticancer Research. 22(5):2587-2590.
Hayouni, E.A., M. Abedrabba, M. Bouix and M. Hamdi. 2007. The effects of solvents and
extraction method on the phenolic contents and biological activities in vitro
of Tunisian Quercus coccifera L. and Juniperus phoenicea L. fruit extracts.
Food Chemistry.105: 1126-1134.
Hemalatha, A., K. Girija, C. Parthiban, C. Saranya and P. Anantharaman. 2013. Antioxidant
properties and total phenolic content of a marine diatom, Navicula clavata and
green microalgae, Chlorella marina and Dunaliella salina. Advances in
Applied Science Research. 4 (5): 151-157.
134
Heo, B.G., Y.S. Park, S.U. Chon, S.Y. Lee, J.Y. Cho and S. Gorinstein. 2007. Antioxidant
activity and cytotoxicity of methanol extracts from aerial parts of Korean
salad plants. Biofact. 30(2):79-89.
He, Q. and N. Venant. 2004. Antioxidant power of phytochemicals from Psidium guajava
leaf. Journal of Zhejiang University Science. 5(6): 676-683.
Hill, S.A., S.J. Lonergan, J. Denekamp and D.J. Chaplin. 1993. Vinca alkaloids: Anti-
vascular effects in a murine tumour. European Journal of Cancer. 29(9): 1320-
1324.
Hossain, M.A., M.D. Shah, C. Gnanaraj and M. Iqbal. 2011. In vitro total phenolics,
flavonoids contents and antioxidant activity of essential oil, various organic
extracts from the leaves of tropical medicinal plant Tetrastigma from Sabah.
Asian Pacific Journal of Tropical Medicine. 4 (9): 717-721.
Hossain, M.A., K.A.S.A.Raqmi, Z.H.A.Mijizy, A.M. Weli and Q.A. Riyam. 2013. Study of
total phenol, flavonoids contents and phytochemical screening of various
leaves crude extracts of locally grown Thymus vulgaris. Asian Pacific Journal
of Tropical Biomedicine. 3 (9): 705-710.
Hrubik, J.D., S.N. Kaisarevic, B.D. Glisic, E.D. Jovin, N.M.M. Dukic and R.Z. Kovacevic.
2012. Myrtus communis and Euclayptus camaldulensis cytotoxicity on breast
cancer cells. Zbornik Matice Srpske za Prirodne Nauke. 123: 65-73.
Hsu, H.C., W.C. Yang, W.J. Tsai, C.C. Chen, H.Y. Huang and Y.C. Tsai. 2006. Alpha-
bulnesene, a novel PAF receptor antagonist isolated from Pogostemon cablin.
Biochemical and Biophysical Research Communications. 345, 1033-1038.
Hunag, M.T., R.C. Smart, C.Q. Wong and A.H. Conney. 1998. Inhibitory effect of curcumin,
chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse
skin by 12-O-tetradecanoylphorbol-13-acetate.Cancer Research. 48 (21):
5941-5946.
Huang, W.Y., Y.Z. Cai and Y. Zhang. 2010. Natural phenolic compounds from medicinal
herbs and dietary plants: potential use for cancer prevention. Nutrition Cancer.
62: 1-20.
135
Husain, A., O.P. Virmani, S.P. Popali, L.N. Mishra, M.M. Gupta, G.N. Srivastava, Z.
Abraham and A.K. Singh. 1992. Dictionary of indian medicinal plants, central
institute of medicinal and aromatic plants. Lucknow, india, 546 pp.
Hussain, A., M. Zia and B. Mirza. 2007. Cytotoxic and antitumor potential of Fagonia
cretica L. Turkish Journal of Biology. 31: 19-24.
Hussain, K., M.F. Nisa, A. Majeed, K. Nawaz and K.H. Bhatti. 2010. Ethnomedicinal survey
for important plants of JalalpurJattan district Gujrat, Punjab, Pakistan.
Ethnobotanical Leaflets.14: 807-825.
Hwang, J.S., Y.P. Yen, M.C. Chang and C.Y. Liu. 2002. Extraction and identification of
volatile components of guava fruits and their attraction to Oriental fruit fly,
Bactrocera dorsalis (Hendel). Plant Protection Bulletin. 44: 279–302.
Ibrahim, A.T. 2012. Chemical composition and biological activity of extracts from Salvia
bicolor Desf. growing in Egypt. Molecules. 17(10): 11315-11334.
Ibrahim, M.N.M., R.B. Sriprasanthi, S. Shamusdeen, F. Adam and S. Bhawani. 2012. A
concise review of the natural existance, synthesis, properties and applications
of syringaldehyde. Bioresoirces. 7 (3): 4377-4399.
Ikawa, M., T.D. Schaper, C.A. Doolard and J.J. Sansner. 2003. Utilization of Folin–Ciocalteu
phenol reagent for the detection of certain nitrogen compounds. Journal of
Agriculture and Food Chemistry. 51 (7): 1811-1815.
Ilyas, I. and M. Hamayun. Studies on the traditional uses of plants if Malam Jabba valley,
District Swat, Pakistan. Ethnobotanical Leaflets.
Iqbal, S., U. Younas, K.W. Chan, M.Z.U. Haq and M. Ismail. 2012. Chemical composition of
Artemisia annua L. leaves and antioxidant potential of extracts as a function
of extraction solvents. Molecules. 17: 6020-6032.
Iqbal, T., A.I. Hussain, S.A.S. Chatha, S.A.R. Naqvi, and T.H. Bokhari. 2013. Antioxidant
activity and volatile and phenolic profiles of essential oil and different extracts
of wild mint (Mentha longifolia) from the Pakistani flora. Hindawi Publishing
Corporation. 2013: 6.
Islam, M.D., M.M. Aktar, M.D.S. Parvez, M.D.J. Alam and M.F. Alam. 2013. Antitumor and
antibacterial activity of a crude methanol leaf extract of Vitex negundo.
Archives of Biological Science Belgrade. 65 (1): 229-238.
136
Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, pp. 786-789.
Jabeen, R., M. Shahid, A. Jamil and M. Ashraf. 2008. Microscopic evaluation of the
antimicrobial activity of seed extracts of Moringa oleifera. Pakistan Journal of
Botany. 40(4): 1349-1358.
Jabeen, A., M.A. Khan, M. Ahmad, M. Zafar and F. Ahmad. 2009. Indigenous uses of
economically important flora of Margallah Hills National Park, Islamabad,
Pakistan. African Journal of Biotechnology. 8(5): 763-784.
Jadhav, V., S. Deshmukh and S. Mahadkar. 2013. Evaluation of antioxidant potential of
Clitoria ternatea L. International Journal of Pharmacy and Pharmaceutical
Sciences. 5 (2): 595-599.
Jagadish, L.K., V.V. Krishnan, R. Shenbhagaraman and V. Kaviyarasan. 2009. Comparative
study on the antioxidant, anticancer and antimicrobial property of Agaricus
bisporus Imbach before and after boiling. African Journal of Biotechnology.
8 (4): 654-661.
Jagtap, S., K. Meganathan, V. Wagh, J. Winkler, J. Hescheler and A. Sachinidis. 2009.
Chemoprotective mechanism of the natural compounds, epigallocatechin-3-O-
gallate, quercetin and curcumin against cancer and cardiovascular
diseases.Current Medicicnal Chemistry. 16 (12): 1451-1462.
Jahan, N., K.U. Rahman, S. Ali and M.R. Asi. 2013. Phenolic acid and flavonol contents of
gemmo-modified and native extracts of some indigenous medicinal plants.
Pakistan Journal of Botany. 45 (5) : 151-1519.
Jakupovic, J., T. Morgenstern, J.A. Marco and W. Berendsohn. 1998. Diterpenes from
Euphorbia paralias. Phytochemistry. 47 (8) : 1611-1619.
Jawale, C., R. Kirdak and L. Dama. 2010. Larvicidal activity of Cestrum nocturnum on
Aedes aegypti. Bangladesh Journal of Pharmacology. 5(1): 39-40.
Ji, C.J., G.Z. Zeng, J. Han, W.J. He, Y.M. Zhang and N.H. Tan. 2012. Zizimauritic acids A–
C, three novel nortriterpenes from Ziziphus mauritiana. Bioorganic and
Medicinal Chemistry Letters 22 : 6377–6380.
Jimenez-Escrig, A., M. Rincon, R. Pulido and S.F. Calixto. 2001. Guava fruit (Psidium
guajava L.) as a new source of antioxidant dietary fiber. Journal of
Agriculture and Food Chemistry. 49(11): 5489-5493.
137
Jithesh, H. and Nirmala. 2013. Phytochemical analysis of leaef extracts of plant Acacia
nilotica by GCMS method. Advances in Biological Research. 7 (5): 141-144.
Johann, S., C. Soldi, J.P. Lyon, M.G. Pizzolatti and M.A. Resende. 2007. Antifungal activity
of the amyrin derivatives and in vitro inhibition of Candida albicans adhesion
to human epithelial cells. Letters in Applied Microbiology. 2207; 45: 148-153.
Jordan, M.J., C.A. Margaria, P.E. Shaw and K.L. Goodner. 2003. Volatile components and
aroma active compounds in aqueous essence and fresh pink guava fruit puree
(Psidium guajava L.) by GC–MS and multidimensional GC/GC-O. Journal of
Agriculture and Food Chemistry. 51: 1421–1426.
Joseph, B. and M.R. Priya. 2011. Review on nutritional, medicinal and pharmacological
prospects of guava (Psidium guajava Linn.). International Journal of Pharama
and Bio Sciences. 2 (10) : 53-69.
Jossang, A., A. Zahir and D. Diakite. 1996. Mauritine J, a cyclopeptide alkaloid from
Ziziphus mauritiana. Phytochemistry. 42: 565-567 (1996)
Jursaz, P., D. Alonso-Escolano and M.W. Radomski. 2004. Platelet–cancer interactions:
mechanisms and pharmacology of tumor cell-induced platelet aggregation.
British Journal of Pharmacology. 143, 819-826.
Jyothi, T.M., M.M. Shankariah, K. Prabhu, S. Lakshminarasu, G.M. Srinivasa and S.S.
Ramachandra. 2008. Hetroprotective and antioxidant activity of Euphorbia
tirucalli. Iranian Journal of Pharmacology and Therapeutics. 7 (1): 25-30.
Kamath, J.V., N. Rahul, C.K.A. Kumar and S.M. Lakshmi. 2008. Psidium guajava L: A
review. International Journal of Green Pharmacy. 2(1): 9-12.
Kang, H.G., S.H. Jeong and J.H. Cho. 2010. Antimutagenic and anticarcinogenic effect of
methanol extracts of sweet potato (Ipomea batata) leaves. Toxicological
Research. 26(1): 29-35.
Kanwal, S., N. Ullah, I.L. Haq, I. Afzal and B. Mirza. 2011. Antioxidant, antitumor activities
and phytochemical investigation of Hedera nepalensis K. Koch, an important
medicinal plant from Pakistan. Pakistan Journal of Botany. 43: 85-89.
Kapoor, L.D. 1989. Handbook of Ayurdev madical plants, In L. D. Kapoor, (ed.).Med.plants.
CRC Press.
138
Karna, S., W.B. Lim, J.S. Kim, S.W. Kim, H. Zheng, K.H. Bae, M.S. Cho, H.K. Oh, O.S.
Kim, H.R. Choi and O.J. Kim. 2012. C16 saturated fatty acid induced
autophagy in A549 cells through topoisomerase I inhibition. Food and
Nutrition Science. 3: 1220-1227.
Kato, K., S. Yamashita, S. Kitanaka and S. Toyoshima. 2001. Effect of gallic acid derivatives
on secretion of Th1 cytokines and Th2 cytokines from anti CD3-stimulated
spleen cells. Yakugaku Zasshi: Journal of the Pharmaceutical Society of
Japan. 121 (6): 451-457.
Katsuragi, H., K. Shimoda, N. Kubota, N. Nakajima, H. Hamada and H. Hamada. 2010.
Biotransformation of cinnamic acid, p-coumaric acid, caffeic acid and ferulic
acid by plant cell cultures of Eucalyptus perriniana. 74 (9): 1920-1924.
Kaur, S. and P. Mondal. 2014. Study of total phenolic and flavonoid contents, antioxidant
activity and antimicrobial properties of medicinal plants. Journal of
Microbiology and experimentation. 1 (1): 2-6.
Kelly, K. 2009. History of medicine. New York: Facts on file. pp. 29–50
Kenneth, S., L. Brekke, E. Johon and S. Donald. 1970. Volatile constituents in guava. Journal
of Agriculture and Food Chemistry. 18: 598-599.
Khadri, A., M. Neffati, S. Smiti, P. Fale, A.R.L. Lino, M.L.M. Serralherio and M.E.M.
Araujo. 2010. Antioxidant, antiacetylcholinesterase and antimicrobial
activities of Cymbopogon schoenanthus L. Spreng(lemon grass) from Tunisia.
LWT- Food Science and Technology. 43: 331-336.
Khadhri, A., R.E. Mokin, C. Almeida, J.M.F. Nogueira and M.E.M. Araujo. 2014.
Chemical composition of essential oil of Psidium guajava L. growing
inTunisia. Industrial Crops and Products. 52: 29-31.
Khan, B.A., N. Akhtar, A. Rasul, T. Mahmood, H.M.S. Khan, S.U. Zaman, M. Iqbal and G.
Murtaza. 2012. Investigation of the effects of extraction solvent/ technique on
the antioxidant activity of Cassia fistula L. Journal of Medicinal Plants
Research. 6(3):500-503.
Khanavi, M., B. Gheidarloo, N. Sadati, M.R.S. Ardekani, S.M.B. Nabavi, S. Tavajohi and
S.N. Ostad. 2012. Cytotoxicity of fucosterol containing fraction of marine
139
algae against breast and colon carcinoma cell line. Pharmacognosy Magzine.
8(29): 60-64.
Kiliç, I. and Yeşiloglu, Y. 2013. Spectroscopic studies on the antioxidant activity of p-
coumaric acid. Spectrochima Acta A Molecular and Biomolecular
Spectroscopy. 115:719-724.
Kim, D.S., J.M. Pezzuto and E. Pisha. 1998. Synthesis of betulinic acid derivatives with
activity against human melanoma. Bioorganic and Medicinal Chemistry
Letters. 8(13): 1707-1712.
Kim, J.H., S.C. Gupta, B. Park, V.R Yadav and B.B. Aggarwal. 2012. Turmeric (Curcuma
longa) inhibits inflammatory nuclear factor (NF)-κB and NF-κB-regulated
gene products and induces death receptors leading to suppressed proliferation,
induced chemo sensitization, and suppressed osteoclastogenesis. Molecular
Nutrition and Food Research. 56: 454-465.
Kim, Y.J. 2007. Antimelanogenic and antioxidant properties of gallic acid. Biological and
Pharmaceutical Bulletin. 30 (6): 1052-1055.
Kirtikar, K.R. and B.D. Basu. 1991. Indian Medicinal Plants, Periodical Experts Books
Agency, 2nd edition, Vol. 3, New Delhi.
Kisko, G. and S. Roller. 2005. Carvacrol and p-cymene inactivate Escherichia coli O157:H7
in apple juice. BMC Microbiolpgy. 5:36.
Kocacaliskan, I., I. Talan and I. Terzi. 2006. Antimicrobial activity of catechol and
pyrogallol as allelochemicals. Zeitchrift fur Naturforschung C Journal of
Biosciences. 61(9-10): 639-642.
Koksal, E., E. Bursal, E. Dikici, F. Tozoglu and I. Gulcin. 2010. Antioxidant activity of
Melissa officinalis leaves. Journal of Medicinal Plant Research. 5 (2) : 217-
222.
Koley, T.K., S. Walia, P. Nath, O.P. Awasthi and C. Kaur. 2011. Nutraceutical composition
of Zizyphus mauritiana Lamk (Indian ber): effect of enzyme-assisted
processing. Interntional Journal of Food Science and Nutrition. 62 (3): 276-
279.
140
Krishna, H. and A. Parashar. 2012. Phytochemical constituents and antioxidant activities of
some Indian Jujube (Ziziphus mauritiana Lamk.) cultivars. Journal of Food
Biochemistry. 37 (5): 571-577.
Kuber, B.R., M.R. Lakshmi, E. Deepika and P. Yamini. 2013. Phytochemical screening, in
vitro antibacterial and antioxidant activit of the Psidium guajava root bark.
International Journal of Current Microbiology and Applied Sciences. 2(10):
238-248.
Kuete, V., B. Ngameni, C.C.F. Simo, R.K. Tankeu, B.T. Ngadjui, J.J.M. Meyer, N. Lall and
J.R Kuiate. 2008. Antimicrobial activity of the crude extracts and compounds
from Ficus chlamydocarpa and Ficus cordata (Moraceae). Journal of
Ethnopharmacology. 120: 17-24.
Kumar, A., K.C. Singhal, R.A. Sharma, G.K. Vyas and V. Kumar. 2013. Total phenolic and
antioxidant activtiy of Catharanthus roseus in different geographical locations
of Rajasthan. Asian Journal of Experimental Biological Sciences. 4(1): 155-
158.
Kunwar, R.M., C. Burlakoti, C.L. Chowdhary and R.W. Bussmann. 2010. Medicinal plants
in farwest Nepal: Indigenous uses and pharmacological validity. Medicinal
and Aromatic Plant Science and Biotechnology. 1 (1) : 28-42.
Kwape, T.E. and P. Chaturvedi. 2012. Antioxidant activities of leaf extracts of Ziziphus
mucronata. International Journal of Food, Agriculture and Veterinary
Sciences. 2 (1): 62-669.
Lamaison, J.L. and A. Carnat. 1990. Teneurs en acide rosmarinique, en de rive s
hydroxycinnamiques totaux et activite´ s antioxydantes chez les Apiace´ es,
les Borraginace´ es et les Lamiace´ es me´ dicinales. Pharamceutica Acta
Helvetiae. 65: 315-320.
Lamien-Meda, A., C.E. Lamien, M.M.Compaore, R.N. Meda, M. Kiendrebeogo, B. Zeba,
J.F. Millogo and O.G. Nacoulma. 2008. Polyphenol content and antioxidant
activity of fourteen wild edible fruits from Burkina Faso. Molecules. 13(3):
581-594.
Leeja, L. and J.E. Thoppil. 2007. Antimicrobiall activity of Origanum majorana L. (Sweet
marjoram). Journal of Environmental Biology. 28(1): 145-146.
141
Lee, S., Y.S. Lee, S.H. Jung, S.S. Kang and K.H. Shin. 2003. Anti-oxidant activities of
fucosterol from the marine algae Pelvetia siliquosa. Archives of
Pharmaceutical Research. 26(9): 719-722.
Lee, S.B. and H.R. Park. 2010. Anticancer activity of guava (Psidium guajava L.) branch
extracts against HT-29 human colon cancer cells. Journal of Medicinal Plants
Research. 4(10): 891-896.
Leh, M., W.V. Berghe, E. Boone, T. Essawi and G. Haegeman. 2007. Screening of
indigenous Palestinian medicinal plants for potential anti-inflammatory and
cytotoxic activity. Journal of Ethnopharmacology. 113(3): 510-516.
Levy, A.S. and S.K. Carley. 2012. Cytotoxic activity of hexane extracts of Psidium guajava
L (Myrtaceae) and Cassia Alata L (Caesalpineaceae) in Kasumi-1 and
OV2008 cancer cell lines. Tropical Journal of Pharmaceutical Research. 11
(2): 201-207.
Liang, Q., H. Qian and W. Yao. 2005. Identification of flavonoids and their glycosides by
high-performance liquid chromatography with electrospray ionization mass
spectrometry and with diode array ultraviolet detection. Journal European of
Mass Spectrometry. 11: 93-101.
Li, J., F. Chen and J. Luo. 1999. GC–MS analysis of essential oil from the leaves of Psidium
guajava. Zhong Yao Cai. 22: 78-80.
Li, J., Y. Cheng, W. Qu, Y. Sun, Z. Wang ,H. Wang and B. Tian. 2011. Fisetin, a dietary
flavonoid, induces cell cycle arrest and apoptosis through activation of p53
and inhibition of NF-kappa B pathways in bladder cancer cells. Basic Clinical
Pharmacology and Toxicology. 108 92): 84-93.
Li, J.W., S.D.Ding and X.L. Ding. 2005. Comparison of antioxidant capacities of extracts
from five cultivars of Chinese jujube. Process Biochemistry. 40 :3607-3613.
Li, H., S.Park, B. Moon, Y.B. Yoo, Y.W. Lee and C. Lee. 2012. Targeted phenolic analysis
in Hericium erinaeum and its antioxidany activities. Food Science and
Biotechnology. 21 (3): 881-888.
Li, X.L., Y. Li, S.F. Wang, Y.L. Zhao, K.C. Liu, X.M. Wang and Y.P. Yang. 2009. Ingol and
ingenol diterpenes from the aerial parts of Euphorbia royleana and their
antiangiogenic activities. Journal of Natural Products. 72 (6): 1001-1005.
142
Li, X., X. Wang, D. Chen and S. Chen. 2011. Antioxidant activity and mechanism of
protocatechuic acid in vitro. Functional Foods in Health and Disease.7 : 232-
244.
Li, Y., T. Li, C. Miao, J. Li, W. Xiao and E. Ma. 2013. β-Eudesmol induces JNK-dependent
apoptosis through the mitochondrial pathway in HL60 cells. Phototherapy
Research. 27(3):338-343.
Lin, C.Y. and M.C. Yin. 2012. Renal protective effects of extracts from guava fruit (Psidium
guajava L.) in diabetic mice.Plant Foods for Human Nutrition. 67(3): 303-
308.
Lindholm, P., U. Goransson, S. Johansson, P. Claeson, J. Gullbo, R. Larsson, L. Bohlin and
A. Backlund. 2002. Cycylotides: a novel type of cytotoxic agents. Molecular
Cancer Therapeutics. 1(6): 365-369.
Liu, H., S. Xiao, Z. You, L. Zhong, Z. Lin and W. Zhe. 2011. Quantitative analysis of
quercetin in Euphorbia helioscopia L by RP-HPLC. Cell Biochemistry and
Biophysics. 61 (1): 59.
Llondu, E.M. 2011. Evaluation of some aqueous plant extracts used in the control of
paawpaw fruit (Carica papaya L.) rot fungi. Journal of Applied Biosciences.
37: 2419-2424.
Locatelli, C., F.B.F. Monteiro and T.B.C. Pasa. 2013. Alkyl esters of gallic acid as anticancer
agents: a review. European Journal of Medicinal Chemistry. 60: 233-239.
Loh, D.S., H.M. Er and Y.S. Chen. 2009. Mutagenic and antimutagenic activities of aqueous
and methanol extracts of Euphorbia hirta. Journal of Ethnopharmacology. 126
(3): 406-414.
Lou, Z., H. Wang, S. Rao, J. Sun, C. Ma and J.Li. 2012. p-Coumaric acid kills bacteria
through dual damage mechanisms. Food Control. 25(2): 550-554.
Lucky, O.O., K.E. Imafidon and A.A. Alabi. 2010. Phytochemical, Proximate and Metal
Content Analysis of the Leaves of Psidium guajava Linn (Myrtaceae),
International Journal of Health Research. 3(4), 217-221.
Luk, S.C.W., S.W.F. Siu, C.K. Lai, Y.J. Wu and S.F. Pang. 2005. Cell cycle arrest by a
natural product via G2/M checkpoint. International Journal of Medical
Sciences. 2(2): 64-69.
143
Luqman, S., G.R. Dwivedi, M.P. Darokar, A. Kalra and S.P.S. Khanuja. 2008. Antimicrobial
activityof Euclayptus citriodora essential oil. International Journal of
Essential Oil Therapeutics. 2 (2): 69-75.
Lutterodt, G.D. and A. Maleque. 1988. Effects on mice locomotor activity of a narcotic-like
principle from Psidium guajava leaves. Journal of Ethnopharmacology. 24(2-
3): 219-231.
Makasci, A.A., R. Mammadov, O.Dusen ans H.I. Isik. 2010. Antimicrobial and antioxidant
activities of medicinal plant species Ornithogalum alpigenum stapf. from
Turkey. Journal of Medicinal Plants Research Vol. 4 (16): 1637–1642.
Ma, E.L., Y.C. Li, H. Tsuneki, J.F. Xiao, M.Y. Xia, M.W. Wang and I. Kimura . 2008. Beta-
eudesmol suppresses tumour growth through inhibition of tumour
neovascularisation and tumour cell proliferation. Journal of Asian Natural
Product Research. 10 (1-2): 159-167.
Mahajan, R.T. and M.Z. Chopda. 2009. Phyto-Pharmacology of Ziziphus jujuba Mill- A
plant review. Pharmacognosy Review. 3(6): 320-329.
Mahesh, B. and S. Satish. 2008. Antimicrobial activity of some important medicinal plant
against plant and human pathogens. World Journal of Agriculture Sciences.
4(S): 839-843.
Mahmood, A., A. Mahmood and M. Mahmood. 2012. In vitro biological activities of most
common medicinal plants of family Solanaceae. World Applied Science
Journal. 17 (8): 1026-1032.
Mamedov, N. 2012. Medicinal plants studies: History, challenges and prospective. Medicinal
and Aromatic Plants. 1 (8): 1-2.
Mangal, M., P. Sagar, H. Singh, G.P.S. Raghava and S.M. Agarwal. 2013. NPACT:
Naturally occurring plant-based anti-cancer compound-activity-target
database. Nucleic Acid Research. 41: D1124-D1129.
Manohar, V., C. Ingram, J. Gray, N.A. Talpur, B.W. Echard, D. Bagchi and H.G. Preuss.
2001. Antifungal activities of origanum oil against Candida albicans.
Molecular and Cellular Biochemistry. 228 (1-2): 111-117.
144
Manosroi, J., P. Dhumtanom and A. Manosroi. 2006. Anti-proliferative activity of essential
oil extracted from Thai medicinal plants on KB and P388 cell lines. Cancer
Letter. 235: 114-120.
Maoulainine, B.M.L., A. Jelassi, I. Hassen and O.M.S.O.A. Boukhari. 2012. Antioxidant
properties of methanolic and ethanolic extras of Euphorbia helioscopia (L.)
aerial parts. Internationla Food Researh Journal. 19 (3) : 1125-1130.
Marwat, S.K., M.A. Khan, M.A. Khan, F.U. Rehman, M. Ahmad and M. Zafar. 2008.
Salvadora persica, Tamarix aphylla and Zizyphus mauritiana-three woody
plant species mentioned in Holy Quran and Ahadith and their ethnobotanical
uses in North Western part (D.I. Khan) of Pakistan. Ethnobotanical Leaflets.
12: 1013-1021.
Maurya, A. and S.K. Srivastav. 2012. Determination of ursolic acid and ursolic acid lactone
in the leaves of Eucalyptus tereticornis by HPLC. Journal of the Brazilian
Chemical Society. 23 (3):468-472.
Mazid, M.A., B.K. Datta, L. Nahar and S.D. Sarkar. 2011. Assessment of antitumour activity
of two Polygonum species using potato disc assay. Bangladesh
Pharmaceutical Journal. 14(1): 37-40.
McLaughlin, J.L and L.L Rogers. 1998. The use of biological assays to evaluate botanicals.
Drug Information Journal. 32: 513-524.
Mcmahon, J.B., M.J. Currens, R.J. Gulakowski, R.W.J. Buckheit,C. Lackman-Smith, Y.F.
Hallock and M.R. Boyd. 1995. Michellamine B., a novel plant alkaloid,
inhibits human immunodeficiency virus-induced all killing by at least two
distinct mechanisms. Antimicrobial Agents and Chemotherapy. 39 (2): 484-
488.
Meckes, M., F. Calzada, J. Tortoriello, J.L. Gonzalez and M. Martinez. 1998. Terpenoids
isolated from Psidium guajava hexane extract with depressant activity on
central nervous system. Phytotherapy Research. 10(7): 600-603.
Mehta, R.G., G. Murillo, R. Naithani and X. Peng. 2010. Cancer chemoprevention by natural
products: how far we have come. Pharmaceutical Research. 27: 950-961.
145
Memon, A.A., N. Memon, D.L. Luthria, A.A. Pitafi and M.I. Bhanger. 2012. Phenolic
compounds and seed oil composition of Ziziphus mauritiana L. fruit. Polish
Journal of Food and Nutrition Sciences. 62(1): 15-21.
Mena-Rejon, G., E. Caamal-Fuentesa, Z. Cantillo-Ciaub, R. Cedillo-Riveraa, J. Flores-
Guidoc and R. Moo-Puc. 2009. In vitro cytotoxicity of nine plants used in
Mayan. Journal of Ethnopharmacology. 121, 462-465.
Mercadante, Z., A. Steck and H. Pfander. 1999. Carotenoids from guava (Psidium guajava
Lin): Isolation and structure elucidation. Journal of Agriculture Food
Chemistry. 47(1): 145-151.
Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. Mclaughlin.
1982. Brine shrimp; a convenient general bioassay for active plant
constituents. Journal of Medicinal Plant Research. 45: 31-34.
Michael, H.N., J.Y. Salib and M.S. Ishak. 2002. Acylated flavonol glycoside from Psidium
guajava L. seeds, Pharmazie, 57(12):859-860.
Mishra, T., M. Khullar and A. Bhatia. 2011. Anticancer potential of aqueous ethanol seed
extract of Ziziphus mauritiana against cancer cell lines and ehrlich ascites
carcinoma. Evidence Based Complementary and Alternative Medicines. 2011;
2011: 1-11.
Misra, K. and T.R. Seshadri. 1968. Chemical components of the fruits of Psidium guajava.
Phytochemistry. 7: 641–645.
Mitsuhashi, S., A. Saito, N. Nakajima, H. Shima and M. Ubukata. 2008. Pyrogallol structure
in polyphenols is involved in apoptosis-induction on HEK293T and K562
cells. Molecules. 13(12): 2998-3006.
Miyazawa, M. and N.Tamura. 2007. Inhibitory compound of tyrosinase activity from the
sprout of Polygonum hydropiper L. (Benitade). Biological & Pharmaceutical
Bulletin. 30 (3): 595-597.
Mohdaly, A.A., M.A. Sarhan, I. Smetanska and A. Mahmoud. 2010. Antioxidant properties
of various solvent extracts of potato peel, sugar beet pulp and sesame cake.
Journal of Science Food and Agriculture. 90 (2): 218-226.
146
Mokhtarpour, A., A.A. Naserian, R. Valizadeh, M.D. Mesgaran and F. Pourmollae. 2014.
Extraction of phenolic compounds and tannins from Pistachio by products..
Annual Research and Review in Biology. 4 (8): 1330-1338.
Moltz, H. 1993. Fever: causes and consequences. Neuroscience and Biobehavioral Reviews.
17:237-269.
Morton, J. 1987. Breadfruit. In: Fruits of warm climates. Julia F. Morton, Miami, FL. P. 50-
58.
Muchuweti, M., G. Zenda , A.R. Ndhlala and A. Kasiyamhuru. 2005. Sugars, organic acid
and phenolic compounds of Ziziphus mauritiana fruit. European Food
Research and Technology.221: 570-574.
Mukherjee, A.K., S. Basu, N. Sarkar and A.C. Gosh. 2001. Advances in cancer therapy with
plant based natural products. Current Medicinal Chemistry. 8: 1467-1486.
Muhammad, N. and M. Saeed. 2011. Biological screening of Viola betonicifolia Smith whole
plant. African Journal of Pharmacy and Pharmacology. 5 (20): 2323-2329.
Muhammad, N. and M. Saeed and H. Khan. 2012. Antipyretic, analgesic and anti-
inflammatory activity of Viola betonicifolia whole plant. BMC
Complementary and Alternative Medicines. 2 (12): 29.
Muhammad, N., M. Saeed, M. Qayum and H. Khan. 2013. Antimicrobial screening of Viola
betonicifolia. Middle East Journal of Scientific Research. 15 (1): 55.
Mukhtar, H.M., S.H. Ansari, Z.A. Bhat, T. Naved and P. Singh. 2006. Antidiabetic activity
of an ethanol extract obtained from the stem bark of Psidium guajava
(Myrtaceae). Pharmazie. 61: 725–727.
Mu, Y.M., T. Yanase, Y. Nishi, A. Tanaka, M. Saito, C.H. Jin, C. Mukasa, T. Okabe, M.
Nomua, K. Goto and H. Nawata. 2001. Saturated FFAs, palmitic acid and
stearic acid, induce apoptosis in human granulosa cells. Endocrinology.
142(8): 3590-3597.
Nadia, B. and H. Benmahdi. 2013. Phytochemical study: antioxidant activity of Euphorbia
resinifera L. Advanced Techniques in Biology and Medicine. 1 (1) : 104.
Nadkarni, K.M.1986. Indian Materia Medica, (Popular Prakashan, Bombay) :1315-1319.
147
Nadkarni, K.M. and A.K. Nadkarni. 1991. Indian Materia Medica - with Ayurvedic, Unani-
Tibbi, Siddha, Allopathic, Homeopathic, Naturopathic and Home remedies.
Popular Prakashan Private Ltd., Bombay, India; P. 142-49.
Nadkarni, K.M. and A.K. Nadkarni. 1999. Indian Materia Medica with Ayurvedic, Unani-
Tibbi, Siddha, allophatic, homeopathic, naturopathic and home remedies.
Popular Prakashan Private Ltd., Bombay, India, pp. 142–149.
Nagarani, B., S. Debnath, S. Kumar C, C. Bhattacharjee and G.G. Kumar. 2011. Herbs used
as anticancer agents. International Research Journal of Pharmacy. 2 (1): 20-
24.
Naili, M.B., R.O. Alghazeer, N.A. Saleh and A.Y.A. Najjar. 2010. Evaluation of antibacterial
and antioxidant activities of Artemisia campestris (Astraceae) and Ziziphus
lotus (Rhamnacea). Arabian Journal of Chemistry. 3(2): 79-84.
Nagar, P.K. and T.R, Rao. 1981. Studies on endogenous cytokinins in guava (Psidium
guajava L.). Annals of Botany. 48, 845-852.
Nagumanthri, V., S. Rahiman, B.A. Tantry, P. Nissankararao and M.P. Kumar. 2012. In vitro
antimicrobial activity of Acacia nilotica, Ziziphus mauritiana, Bauhinia
variegate and Lantana camara against some clinical isolated strains. Iranian
Journal of Science and Technology. A2: 213-217.
Najafi, S. 2013. Phytochemical screening and antibacterial activity of leaf extract of Ziziphus
mauritiana Lam. International Research Journal of Applied and Basic
Sciences. 4(11):3274-3276.
Nambiar, V.S., H.M. Matela and A. Baptist. 2013. Total antioxidant capacity using ferric
reducing antioxidant power and 2, 2-diphenyl-1 picryl hydrazyl methods and
phenolic composition of fresh and dried drumstick (Moringa oleifera) leaves.
International Journal of Green Pharmacy. 7 (1): 66-72.
Nascimento, G.G.F., J. Locatelli, P.O. Freitas and G.L. Silva. 2000. Antibacterial activity of
plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazalian
Journal of Microbiology. 31 (4): 247-256.
Nawab, A., M. Yunus, A.A. Mehdi and S.Gupta. 2011. Evalution of anticancer properties of
medicinal plants from the Indian sub continent. Molecular and Cellular
Pharmacology. 3 (1): 21-29.
148
Naz, R. and A. Bano. 2013. Phytochemical screening, antioxidants and antimicrobial
potential of Lantana camara in different solvents. Asian Pacific Journal of
Tropical Diseases. 3 (6) : 480-486.
NCCLS (National Committee for Clinical Laboratory Standards). 1997. Performance
standards for antimicrobial disc susceptibility test (6th ed.) Approved
Standard.M2-A6, Wayne, PA.
Neira, G.A., G.M.B. Ramirez. 2005. Actividad antimicrobiana de extractos de dos especies
de guayaba contra Sterptococcus mutans by Escherichia coli. Actualidades
Biologicas. 27: 27–30.
Nicolis, E.,I. Lampronti,M.C. Dechecchi, M. Borgatti, A. Tamanini, N. Bianchi, V.
Bezzerri, I. Mancini, M.G. Giri, P. Rizzotti, R. Gambari and G. Cabrini.
2008. Role of nuclear factor-κB-mediated inflammatory pathways in cancer-
related symptoms and their regulation by nutritional agents. International
Immunopharmacology. 10: 1672-1680.
Nikolova, M., L. Evstatieva and T.D. Nguyen. 2011. Screening of plant extracts for
antioxidant properties. Botanica Serbica. 35(1): 43-48.
Nisa, S., Y. Bibi, A.Waheed, M. Zia, S. Sarwar, S. Ahmed and M.F. Chaudhary. 2011.
Evalution of anticancer activity of Debregeasia salicifolia extract against
estrogen receptor positive cell line. African Journal of Biotechnology. 10 (6):
990-995.
Nisa, S., Y. Bibi, M. Zia, A.Waheed and M.F. Chaudhary. 2013. Anticancer investigations
on Carissa opaca and Toona ciliata extracts against human breast carcinoma
cell line. Pakistan Journal of Pharmaceutical Sciences. 26 (5):1009-1012.
Nisha, K., M. Darshana, G. Madhu and M.K. Bhupendra. 2011. GC-MS Analysis and anti-
microbial activity of Psidium guajava (leaves) grown in Malva region of
India. International Journal of Drug Development and Research. 3 (4): 237-
245.
Nishida, N., H. Yano, T. Nishida, T. Kamura and M. Kojiro. 2006. Angiogenesis in cancer.
Journal of Vascular Health and Risk Management. 2 (3) : 213-219.
Noble, R.L. 1990. The discovery of the vinca alkaloids—chemotherapeutic agents against
cancer. Biochemistry and Cell Biology. 68(12) :1344-1351.
149
Norris, B., K.I. Pritchard, K. James, J. Myles, K. Bennett, S. Marlin, J. Skillings, B. Findlay,
T. Vandenberg, P. Goss, J. Latreille, L. Rudinskas, W. Lofters, M. Trudeau,
D. Osoba and A. Rodgers. 2000. Phase III Comparative Study of Vinorelbine
Combined With Doxorubicin Versus Doxorubicin Alone in Disseminated
Metastatic/Recurrent Breast Cancer: National Cancer Institute of Canada
Clinical Trials Group Study MA8. Journal of Clinical Oncology. 18 (12):
2385-2394.
Nyanga, L.K., M.J. Nout, E.J. Smid, T. Boekhout and M.H. Zwietering. 2013. Fermentation
characteristics of yeasts isolated from traditionally fermented masau (Ziziphus
mauritiana) fruits. International Journal of Food Microbiology. 166(3): 426-
433.
Ogunlana, O.E. and O.O. Ogunlana. 2001. In vitro assessment of the free radical scavenging
activity of Psidium guajava. Research Journal of Agriculture and Biological
Sciences. 4(6): 666-671.
Ojewole, J.A., 2006. Antiinflamatory and analgesic effects of Psidium guajava Linn
(Myrtaceae) leaf aqueous extract in rats and mice. Methods and Findings in
Experimental and Clinical Pharmacology. 28: 441-446.
Okeniyi, S.O., B.J. Adedoyin and S. Garba. 2012. Phytochemical screening, cytotxicity,
antioxidant and antimicrobial activties of stem and leave extracts of Euphorbia
heterophylla. Bulletion of Enviornment, Pharmacology and Life Sciences. 1
(8) : 87-91.
Oktay, M., I. Gulcin, O.I. Kufrevioglu. 2003. Determination of in vitro antioxidant activity of
fennel (Foeniculum vulgare) seed extracts. LWT- Food Science and
Technology. 36 (2) : 263-271.
Okuda, T., H. Tsutomu and Y. Kazufumi. 1984. Guavin B, an ellagitannin of novel type.
Chemical Pharmaceutical Bulletin. 32: 3787-3788.
Okwu, D.E. and O. Ekeke. 2003. Phytochemical screening and mineral composition of
chewing sticks in South Eastern Nigeria. Global Journal of Pure and Applied
Sciences. 9: 235-238.
150
Olajuyigbe, O.O. and A.J. Afolayan. 2011. Phenolic content and antioxidant property of the
bark extracts of Ziziphus mucronata Willd. subsp. mucronata Willd. BMC
Complementary and Alternative Medicine. 2011, 11:130.
Oliver, B.B. 1986. Medicinal Plants in tropical West Africa. Cambridge University Press,
Cambridge, pp. 457–461.
Olubunmi, A. and O.A.Gabriel. 2010. Epicuticular wax and volatiles of Kigelia pinnata leaf
extract. Ethnobotanical Leaflets.14: 797-806.
Osman, A.M.M., H.M. Bayoumi, S.E.A. Harthi, Z.A. Damanhouri and M.F.E. Shal. 2012.
Modulation of doxorubicin cytotoxicity by resveratrol in a human breast
cancer cell line. Cancer Cell International. 12 (47): 1-8.
Otuki, M.F., J. Ferreira, F.Y. Lima, S.C. Meyre, A. Malheiros, L.A. Muller, G.S. Cani, A.R.
Santos, R.A. Yunes and J.B. Calixto. 2005. Antinociceptive properties of
mixture of alpha-amyrin and beta-amyrin triterpenes: evidence for
participation of protein kinase C and protein kinase A pathways.The Journal
of Pharmacology and Experimental Therapeutics. 313(1): 310-318.
Oudhia, P. Research Note on Medicinal herb of Chhattirgarl, India having less known
traditional uses, IX (2003).
Ozkan, A. and A. Erdogan. 2013. Membrane and DNA damaging/protective effects of
eugenol, eucalyptol, terpinen-4-ol, and camphor at various concentrations on parental
and drug-resistant H1299 cells. Turkish Journal of Bioogyl. 37 (4): 405-413.
Ozturk, H., Kolak, U. and C. Meric. 2010. Antioxidant, anticholinesterase and antibacterial
activities of Jurinea consanguinea DC. Records of Natural Products. 5, 43-51.
Ozturk, M., I.Uysal, S.Gucel, E.Altundag, Y. Dogan and S. Baslar. 2013. Medicinal uses of
natural dye-yielding plants in Turkey. RJTA. 17: 69
Pareek, S. and R. S. Dhaka. 2008. Association analysis for quality attributes in ber. Indian
Journal of Arid Horticulture. 3:77-80.
Pareek, S., L. Kitinoja, R. A. Kaushik and R.Paliwal. 2009. Postharvest physiology and
storage of ber. Stew. Posthar. Rev. 5(5):1-10.
Pandey, K.B and S.I. Rizvi. 2009. Plant polyphenols as dietry antioxidants in human health
and diseases. Oxidative Medicine and Cellular Longevity. 2: 270-278.
151
Pandey, M.B., A.K. Singh, J.P. Singh, V.P. Singh and V.B. Pandey. 2008. Three new
cycylopeptide alkaloids from Ziziphus species. Journal of Asian Natural
Product Research. 10 (8): 709-713.
Paniandy, J.C., M.J. Chane and J.C. Pieribattesti. 2000. Chemical composition of the
essential oil and headspace solid-phase microextraction of the guava fruit
(Psidium guajava L.). Journal of Essential Oil Research. 12: 153–158.
Panseeta, P., K.. Lomchoey, S. Prabpai, P. Kongsaeree, A. Suksamrarn and S. Ruchirawat.
2011. Antiplasmodial and antimycobacterial cyclopeptide alkaloids from the
root of Ziziphus mauritiana. Phytochemistry. 72: 909–915.
Pathak, R.K. and C.M. Ojha. 1993. Genetic resources of guava, Vol. I, Fruit Crops, Part 1,
In; Advance in Horticulture [C]. Chadha KL, Pareek OP, editorss, Malhotra
Publishing House, New Delhi, pp. 143–147.
Petrovska, B.B. 2012. Historical review of medicinal plants’ usage Phramcognosy Reviews.
6 (11):1-5.
Pezzuto, J.M. 1997. Plant derived anticancer agents. Biochemical Pharmacology. 53: 121-
133.
Pham-Huy, L.A., H. He and C. Pham-Huy. 2008. Free radicals, antioxidants in disease and
health. International Journal of Biomedical Sceinces. 4: 89-96.
Picone, P., D. Nuzzo and C.M. Di. 2013. Ferulic acid: a natural antioxidant against oxidative
stress induced by oligomeric A-beta on sea urchin embryo. The Biological
Bulletin. 224(1): 18-28.
Pikarsky, E., R.M. Porat, I. Stein, R. Abramovitch, S. Amit, S. Kasem,P.E. Gutkovich, S.S.
Urielli, E. Galum and Y.N. Ben. 2004. NF-kappaB functions as a tumour
promoter in inflammation-associated cancer. Nature. 431: 461-466.
Pisha, E., H. Chai, I.S. Lee, T.E. Chaqwedera, N.R. Farnsworth, G.A. Cordell, C.W.
Beecher, H.H. Fong, A.D. Kinghorn and D.M. Brown. 1995. Discovery of
betulinic acid as a selective inhibitor of human melanoma that functions by
induction of apoptosis. Nature Medicine. 1(10): 1046-1051.
Peuchant, E., J. Brun, V. Rigalleau, L. Dubourg, M. Thomas and J. Danial. 2004. Oxidative
and antioxidative status in pregnant woman with either gestational or type 1
diabetes. Clinical Biochemistry. 37: 293-298.
152
Postmus, P.E., H.R. Haaxma, E.F. Smit, H.J. Groen, H. Karnicka, T. Lewinski, M.J. Van, M.
Clerico, A. Gregor, D. Curran, T. Sahmoud, A. Kirkpatrick and G. Giaccone.
2000. Treatment of brain metastases of small-cell lung cancer: comparing
teniposide and teniposide with whole-brain radiotherapy--a phase III study of
the European Organization for the research and treatment of cancer lung
cancer cooperative Group.Journal of Clinical Oncology. 18 (19): 3400-3408.
Prasad, S., J. Ravindran, B. Sung, M.K. Pandey and B.B. Aggarwal. 2010. Garcinol
potentiates TRAIL-induced apoptosis through modulation of death receptors
and antiapoptotic proteins. Molecular Cancer Therapeutics. 9, 856-868.
Prasad, M., A. Kumar, S.K. Srivastav and A..K. Srivastav. 2011. Euphorbia royleana, a
botanical affects of ultimobranchial gland of the catfish Heteropneustes
fossilis. Egyptian Journal of Biology. 13 : 14-20.
Proestos, C. and M. Komaitis. 2013. Analysis of naturally occurring phenolic compounds in
aromatic plants by RP-HPLC coupled to diode array detector (DAD) and GC-
MS after silylation. Foods. 2: 90-99.
Prabu, G.R., A. Gnanamani and S. Sadulla. 2006. Guaijaverin a plant flavonoid as potential
antiplaque agent against Streptococcus mutans. Journal of Applied
Microbiology. 101: 487–495.
Puupponen-Pimia, R., L. Nohynek, H.L. Alakomi and K.M.O. Caldentey. 2005. Bioactive
berry compounds-novel tools against human pathogens. Applied
Microbiology and Biotechnology. 67 (1): 8-18.
Qasim, M., Z. Abideen, M.Y. Adnan, R. Ansari, B. Gul and M.A. Khan. 2014. Traditional
ethno-botanical uses of medicinal plants from coastal areas of Pakistan.
Journal of Coastal Life Medicine. 2(1): 22-30.
Qian, H. and V. Nihorimbere. 2004. Antioxidant power of phytochemicals from Psidium
guajava leaf. Journal of Science. 5: 676-683.
Qi, W.Y., W.Y. Zhang, Y.Shen, Y.Leng, K.Gao and J.M.Yue. 2014. Ingol-type diterpenes
from Euphorbia antiquorum with mouse 11β-Hydroxysoteroid dehydrogenase
Type 1 Inhibition activity. Journal of Natural Product. 77 (6): 1452-1458.
Quan, Z., J. Gu, P. Dong, J. Lu, X. Wu, W. Wu, X. Fei, S. Li, Y. Wang, J. Wang and Y. Liu.
2010. Reactive oxygen species-mediated endoplasmic reticulum stress and
153
mitochondrial dysfunction contribute to cirsimaritin-induced apoptosis in
human gallbladder carcinoma GBC-SD cells.Cancer Letters. 295 (2): 252-
259.
Quettier-Deleu, C., B. Gressier, J. Vasseur, T. Dine, C. Brunet, M. Luyckx, M. Cazin, J.C.
Cazin, F. Bailleul and F. Trotin. 2000. Phenolic compounds and antioxidant
activities of buckwheat (Fagospyrum esculentum Moench) hulls and flour.
Journal of Ethnopharmacology. 72, 35-42.
Qureshi, R. and G.R. Bhatti. 2008. Ethnobotany of plants used by the Thari people of Nara
Desert, Pakistan. Fitoterapia. 79: 468-473.
Qureshi, R., M. Maqsood, M. Arshad and A.K. Chaudhry. 2011. Ethnomedicinal uses of
plants by the people of Kadhi areas of Khushab, Punjab, Pakistan. Pakistan
Journal of Botany. 43: 121-133.
Radha, A. and R. Chandrasekaran. 1997. X-ray and conformational analysis of arabinan.
Carbohydrate Research. 298: 105–115.
Rahman, H., K. Manjula, T. Anoosha, K. Nagaveni, M.C. Eswaraiah and D. Bardalai. 2013.
In vitro antioxidant activity of Citrullus lanatus seed extracts. Asian Journal
of Pharmaceutical and Clinical Research. 6 (3) : 152-157.
Rajeswari, G., M. Murugan and V.R. Mohan. 2013. GC-MS analysis of bioactive
components of Hugonia mystax L. (Linaceae). Research Journal of
Pharmaceutical, Biological and Chemical Sciences. 3(4): 301-308.
Rakhimov, R.N., N.G. Abdulladzhanova and F.G. Kamaev. 2011. Phenolic compounds from
Euphorbia canescens and E. franchetii. Chemistry of Natural Compounds. 47
(2): 286.
Raina, K., S. Rajamanickam, M. Singh, R. Agarwall and C. Agarwall. 2008.
Chemopreventive effects of oral gallic acid feeding on tumor growth and
progression in TRAMP mice. Molcular Cancer Thereapeutics. 7: 1258-1267.
Rai, P.K., S. Mata and G. Watal. 2010. Hypolipidaemic & hepatoprotective effects of
Psidium guajava raw fruit peel in experimental diabetes. The Indian Journal
of Medicinal Plant Research. 131: 820-824.
Rao, C.V., H.L. Newmark and B.S. Reddy. 1998. Chemopreventive effect of squalene on
colon cancer. Carcinogenesis. 19:287-290.
154
Ramirez-Sanchez, I., L. Maya, G. Ceballos and F. Villarreal. 2010. Flourescent detection of
(-)-epicatechin in microsamples from cacao seeds and cocoa products:
comparison with Folin-Ciocalteu method. Journal of Food Composition and
Analysis. 23 (8) : 790-793.
Ramkissoon, J.S., M.F. Mahomoodally, N. Ahmaed and A.H. Subratty. 2013. Antioxidant
and anti-glycation activities correlates with phenolic composition of tropical
medicinal herbs. Asian Pacific Journal of Tropical Medicine. 6(7): 561-569.
Rashwan, O.A. 2002. New phenylpropanoid glucosides from Eucalyptus maculate.
Molecules. 7:75-80.
Rastogi, R.P. and Mehrotra, B.N. 1993. Compendium of Indian medicinal plants,
Publications and Information Directorate, CSIR, New Delhi, vol. III, PP. 286.
Rattanachaikunsopon, P. and P. Phumkhachorn. 2010. Contents and antibacterial activity of
flavonoids extracted from leaves of Psidium guajava. Journal of Medicinal
Plants Research. 4(5): 393-396.
Rauha, J.P., S. Remes, M. Heinonen, A. Hopia, M. Kahkonen, T. Kujala, K. Pihlaja, H.
Vuorela and P. Vuorela. 2000. Antimicrobial effects of Finnish plant extracts
containing flavonoids and other phenolic compounds. International Journal of
Food Microbiology. 45 (1): 3-12.
Reddy, V.P., N. Shana and A. Urooj. 2012. Antioxidant activity of Aegle marmelos and
Psidium guajava leaves. International Journal of Medicinal and Aromatic
Plants. 2(1): 155-160.
Rehman, A.U., A. Mannan, S. Inayatullah, M.Z. Akhtar and M. Qayyum. 2009. Biological
evaluation of wild thyme (Thymus serpyllum). Pharmaceutical Biology. 47
(7): 628-633.
Rencoret, J., A. Gutierrez and J.C.D Rio. 2007. Lipid and lignin composition of woods from
different Eucalyptus species. Holzforschung. 61 (1):165-174.
Reyes-Gibby, C.C., X. Wu, M. Spitz, R. Kurzrock, M. Fisch, E. Bruera and S. Shete. 2008.
Molecular epidemiology, cancer-related symptoms, and cytokines pathway.
Lancet Oncology. 9:777-785.
Robard, K., D.P. Paul, T. Greg, P. Swatsitang and W. Glover. 1999. Phenolic compounds and
their role in oxidative processes in fruits. Food Chemistry. 66 (4) : 401-436.
155
Romanenko, E.P. and A.V. Tkachev. 2006. Identification by GC—MS of cymene isomers
and 3,7,7-trimethylcyclohepta-1,3,5-triene in essential oils. Chemistry of
Natural Compounds. 42(6): 699-701.
Ryu, N.H., K.R. Park and S.M. Kim. 2012. A hexane fraction of guava leaves (Psidium
guajava L.) induces anticancer activity by suppressing AKT/mammalian
target of rapamycin/ribosomal p70 S6 kinase in human prostate cancer cells.
Journal of Medicinal Food. 15 (3) : 231-241.
Saad, B., H. Azaizeh and O. Said. 2005. Tradition and perspectives of Arab herbal medicine:
A review. Evidence-based complementary and alternative medicine. 2(4) :
475-479.
Sabeen, M. and S.S. Ahmad. 2009. Exploring the folk medicinal flora of Abbotabad city,
Pakistan. Ethnobotanical leaflets.13: 810-833.
Saeed, N., M.R. Khan and M. Shabbir. 2011. Antioxidant activity, total phenolic and total
flavonoid contents of whole plant extracts Torilis leptophylla. BMC
Complementary and alternative medicine. 12: 221.
Sagar, S.M., D. Yance and R.K. Wang. 2006. Natural health products that inhibit
angiogenesis: a potential source for investigational new agents to treat cancer-
Part 1. Current Oncology. 13 (1): 14-26.
Saha, M.R., S.M.R. Hasan, R. Akter, M.M. Hossain, M.S. Alam and M.E.H. Mazumder.
2008. In vitro free radical scavenging activity of methanol extract of leaves of
Mimusops elengi Linn. Bangladesh Journal of Veterinary Medicine. 6 (2):
197-202.
Sakai, M., M. Okabe, H. Tachibana and K. Yamada. 2006. Apoptosis induction by gamma-
tocotrienol in human hepatoma Hep3B cells. The Journal of Nutritional
Biochemistry. 17: 672-676.
Samee, W., M. Engkalohakul, N. Nebbua, P. Direkrojanavuti, C. Sornchaithawatwong and
N. Kamkaen. 2006. Correlation analysis between total acid, total phenolic and
ascorbic acid contents in fruit extracts and their antioxidant activities.Thai
Pharm Health Science Journal. 1(3):196-203.
Samec, D., J. Gruz, M. Strnad, D. Kremer, I. Kosalec, R.J. Grubesic, K. Karlovic, A. Lucic
and J.P. Zegarac. 2010. Antioxidant and antimicrobial properties of Teucrium
156
arduini L. (Lamiaceae) flower and leaf infusions (Teucrium arduini L.
antioxidant capacity). Food and Chemical Toxicology. 48 (1): 113-119.
Samuelsson, G. 2004. Drugs of Natural Origin: a Textbook of Pharmacognosy, 5th Swedish
Pharmaceutical Press, Stockholm.
San, A.M.M., S. Thongpraditchote, P. Sithisarn and W. Gritsanapan. 2013. Total phenolics
and total flavonoids contents and hypnotic effect in mice of Ziziphus
mauritiana Lam. seed extract. Evidence-Based Complementary and
Alternative Medicine. 2013: 835-854.
San, B. and A. N. Yildirim. 2010. Phenolic, alpha-tocophérol, beta-carotène and fatty acid
composition for four promising jujube (Ziziphus jujuba miller) selections.
Journal of Food Composition and Analysis. 23:706-710.
Santos, F.A., J.T. Frota, B.R. Arruda, T.S.D. Melo, A.A.D.C.A.D. Silva, G.A.D.C. Brito,
M.H. Chaves and V.S. Rao. 2012. Antihyperglycemic and hypolipidemic
effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in
mice. Lipids in Health and Disease. 11:98.
Sasikumar, K., C. Vijayalakshmi and K.T. Parthiban. 2001. Allelopathic effects of four
Eucalyptus species on Redgram (Cajanus cajan L.). Journal of Tropical
Agriculture. 39:134-138.
Scherer, S. and P.T. Magee. 1990. Genetics of Candida albicans. Microbiological Reviews.
54(3): 226-241.
Schofield, P., D.M. Mbugua and A.N. Pell. 2001. Analysis of condensed tannins: a review.
Animal Feed Science and Technology. 91 (1-2) : 21-40.
Seruga, B., H. Zhang, L.J. Bernstein and I.F. Tannock. 2008. Cytokines and their relationship
to the symptoms and outcome of cancer. Nature Reviews Cancer. 8: 887-899.
Seyyednejad, S.M., H. Motamedi, F.D. Najvani and Z. Hassannejad. 2014. Antibacterial
effect of Euclayptus microtheca. International Journal of Enteric Pathogens.
2(2): 1-5.
Shen, J.K., H.P. Du, M. Yang, Y.G. Wang and J. Jin. 2009. Casticin induces leukemic cell
death through apoptosis and mitotic catastrophe. Annals of Hematology. 88
(8): 743-752.
157
Shabir, G., F. Anwar, B. Sultana, Z.M. Khalid, M. Afzal, Q.M. Khan and M. Ashrafuzzaman.
2011. Antioxidant and antimicrobial attributes and phenolics of different
solvent extracts from leaves, flowers and bark of Gold Mohar [Delonix regia
(Bojer ex Hook.) Raf. Molecules. 16: 7302-7319.
Sher, H. 2001. Medicinal and economic plants of alpine and sub alpine regions of district
Swat and Chitral, Pakistan, technical report submitted to IUCN-P. 23-56.
Shirurkar, D.D. and N.K. Wahegaonkar. 2012. Antifungal activity of selected plant derived
oils and some fungicides against seed borne fungi of maize. European
Journalof Experimental Biology. 2 (5): 1693-1696.
Shylesh, B.S., N.S. Ajikumaran and A. Subramoniam. 2005. Induction of cell-specific
apoptosis and protection from Dalton's lymphoma challenge in mice by an
active fraction from Emilia sonchifolia.Indian Journal of Pharmacology. 37
(4): 232-237.
Si, W., J. Gong, R. Tsao, M. Kalab, R. Yang and Y. Yin. 2006. Bioassay-guided purification
and identification of antimicrobial components in Chinese green tea extract.
Journal of Chromatogrgaphy A. 1125: 204-210.
Siddiqui, B., S. Imran, S. Hassan and S. Begum. 2002. Triterpenoids from Psidium guajava
leaves. Natural Product Letters. 16: 173–175.
Siddiqui, S.B., I. Sultana and S. Begum. 2000. Triterpenoidal constituents from Eucalyptus
camaldulensis var. obtusa leaves. Phytochemistry. 54 (8) : 861-866.
Siger, A., J. Czubinski, K. Dwiecki, P. Kachlicki and M.N. Kalucka. 2013. Identification and
antioxidant activity of sinapic acid derivatives in Brassica napus L. seed meal
extracts. European Journal of Lipid Science and Technology. 115(10): 1130-
1138.
Silva, A.C.R.D., P.M. Lopes, M.M.B.D. Azevedo, D.C.M. Costa, C.S. Alviano and D.S.
Aviano. 2012. Biological activities of α-pinene and β-pinene enantiomers.
Molecules. 17 : 6305-6316.
Sim, E.W., S.Y. Lai and P. Chang. 2012. Antioxidant capacity, nutritional and phytochemical
content of peanut (Arachis hypogaea L.) shells and roots. African Journal of
Biotechnoloy. 11(53): 11547-11551.
158
Sim, K.S., A.M.S. Nuresti and A.W. Norhanom. 2010. Phenolic content and antioxidant
activity of Pereskia grandifolia Haw. (Cactaceae) extracts. Pharmacognosy
Magzine. 6(23): 248-254.
Singab, A.N., N. Ayoub, E.A. Sayed, O. Martiskainen, J. Sinkkonen and K. Pihlaja. 2011.
Phenolic constituents of Euclayptus camaldulensis Dehnh, with potential
antioxidant and cytotoxic activities. Records of Natural Products. 5(4): 271-
280.
Singh, U., S. Devaraj and I. Jialal. 2005. Vitamin E, oxidative stress and inflammation.
Annual Review of Nutrition. 25: 151-174.
Singh, A.B., D.K. Yadav, R. Maurya and A.K. Srivastava. 2009. Antihyperglycaemic
activity of alpha-amyrin acetate in rats and db/db mice. Natural Product
Research. 23(9): 876-882.
Singh, A.K., M.B. Pandey, V.P. Singh and V.B. Pandey. 2007. Xylopyrine-A and
xylopyrine-B, two new peptide alkaloids from Zizyphus xylopyra. Natural
Product Research. 21(12): 1114-1120.
Singh, P. and A. Singh. 2012. Evaluation of latex extract of Euphorbia royleana for its
piscicidal and muricidal activties. World Journal of Agriculture Sciences. 8
(5) : 520-524.
Singh, U. and I. Jialal. 2004. Anti-inflammatory effects of alpha-tocopherol. Annals of New
York Academy of Sciences. 1031:195-203.
Siow, L.F. and Y.W. Hui. 2013. Comparison on the antioxidant properties of fresh and
convection oven-dried guava (Psidium guajava L.). International Food
Research Journal. 20(2): 639-644.
Sivaraj, R., A. Balakrishnan, M. Thenmozhi and R. Venckatesh. 2011. Preliminary
phhytochemical analysis of Aegle marmelos, Ruta graveolens, Opuntia
dellini, Euphorbia royleana and Euphorbia antiquorum. International Journal
of Pharmaceutical Sciences and Reseaerch. 2 (1) : 132-136.
Si, W., J. Gong, R. Tsao, M. Kalab, R. Yang and Y. Yin. 2006. Bioassay-guided purification
and identification of antimicrobial components in Chinese green tea extract.
Journal of Chromatography A. 1125: 204-210.
159
Shahriar, M., M.I. Hossain, F.A. Sharmin, S. Akhter, M.A. Haque and M.A. Bhuiyan. 2013.
In vitro antioxidant and free radical scavenging activity of Wathinia somnifera
root. Iosr Journal of Chemistry. 3(2): 38-47.
Shahwar, D., S.U. Rehman, N. Ahmad, S. Ullah and M.A. Raza. 2010. Antioxidant activties
of the selected plants from the family Euphorbiaceae, Lauraceae, Malvaceae
and Balsaminaceae. African Journal of Biotechnology. 9 (7) : 1086-1096.
Shahwar, D., M.A. Raza, S. Bukhari and G. Bukhari. 2012. Ferric reducing antioxidant
power of essential oils extracted from Eucalyptus and Curcuma species.
S1633-S1636.
Shaik, Y.B., M.L. Castellani, A. Perrella, F. Conti, V. Salini, S. Tete, B. Madhappan, J.
Vecchiet, M.A.D. Lutiis, A. Caraffa and G. Cerulli. 2006. Role of quercetin (a
natural herbal compound) in allergy and inflammation. Journal of Biological
Regulators and Homeostatic Agents. 20 (3-4): 47-52.
Sharma, P.V., R. Paliwal and S. Sharma. 2011. In vitro free radical scavenging and
antioxidant potential of ethanolic extract of Euphorbia neriifolia Linn.
International Journal of Pharmacy and Pharmaceutical Sciences. 3 (1) : 238-
242.
Sharma, S. and A.P. Vig. 2014. Preliminary phytochemical screening and in vitro antioxidant
activities of Parkinsonia aculeata Linn. BioMed Research International. 2014.
1-8.
Shinwari, Z.K. 2010. Medicinal plants research in Pakistan. Journal of Medicinal Plants
Research. 4(3): 161-176.
Shreedhara, C.S., R.H.N. Aswatha, S.B. Zanwar, F.P. Gajera and A.S. Zanwar. 2011. Free
radical scavenging activity and total phenolic contents of Ziziphus mauritiana
Lam. Pharmacologyonline.3: 868-879.
Shukla, S. and S. Gupta. 2010. Apigenin: A promising molecule for cancer prevention.
Pharmaceutical Research. 27 (6) : 962-978.
Shofian, N.M., A.A. Hamid, A. Osman, N. Saari, F. Anwar, M.S.P. Dek and M.R. Hairuddin.
2011.Effect of Freeze-Drying on the Antioxidant Compounds and Antioxidant
Activity of Selected Tropical Fruits. International Journal of Molecular
Sciences. 12: 4678-4692.
160
Slinkard, K. and V.L. Singleton. 1997. Total phenol analysis: automation and comparison
with manual methods. American Journal of Enology and Viticulture. 28 : 49-
55.
Soares, M.C, Damiani CE, Moreira CM, Stefanon I, Vassallo DV. 2005. Eucalyptol, an
essential oil, reduces contractile activity in rat cardiac muscle. Brazilian
Journal of Medical and Biological Research. 38(3): 453-461.
Sowndhararajan, K. and S.C. Kang. 2013. Free radical scavenging activity from different
extracts of leaves of Bauhinia vahlii Wight & Arn. Saudi Journal of Biological
Sciences. 20 (4): 319-325.
Srinivasan, M., A.R. Sudheer and V.P. Menon. 2007. Ferulicacid: Therapeutic potential
through its antioxidant property. Journal of Clinical Biochemistry and
Nutrition. 40(2): 92-100.
Srivastava, S.K. and S.D. Srivastava.1979. Structure of Zizogenin, a new sapogenin from
Ziziphus mauritiana. Phytochemistry. 18(10): 1758-1759.
Stankovic, M.S. 2011. Total phenolic content, flavonoid concentration and antioxidant
activity of Marrubium peregrinum L. extracts. Kragujevac Journal of Science.
33: 63-72.
Staszewski, M.V., A.M.R. Pilosof and R.J. Jagus. 2011. Antioxidant and antimicrobial
performance of different Argentinean green tea varieties as affected by whey
proteins. Food Chemistry. 125, 186-192.
Steer, P., J. Milligard, D.M. Sarabi, B. Wessby and T. Kahan. 2002. Cardiac and vascular
structure and function are related to lipid peroxidation and metabolism.
Lipids. 37: 231–236.
Stevigny, C., C. Bailly and J.Q. Leclercq. 2005. Cytotoxic and antitumor potentialities of
aporphinoid alkaloids. Current Medicinal Chemistry. 5: 173-182.
Sulain, M.D., K.E. Zazali and N. Ahmad. 2012. Screening on anti-proliferative activity of
Psidium guajava leaves extract towards selected cancer cell lines. Journal of
US-China Medical Science. 9(1): 30-37.
Sultana, B., F. Anwar and R. Przybylski. 2007. Antioxidant activity of phenolic components
present in barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica, and
Eugenia jambolana Lam. Trees. Food Chemistry. 14: 1106-1114.
161
Sultana, S., M.A. Khan, M. Ahmad and M. Zafar. 2006. Indigenous knowledge of folk herbal
medicines by the Women of District Chakwal, Pakistan. Ethnobotanical
Leaflets 10: 243-253.
Sundarasekar, J., G. Sahgal, S.A. Mubbarakh and S. Subramaniam. 2012. Potential
antioxidant activities of methanolic extracts of Spider lily (Hymenocallis
littoralis). Australian Journal of Crop Sciences. 7: 625-631.
Sundarraj, S., R. Thangam, V. Sreevani, K. Kaveri, P. Gunasekaran, S. Achiraman and S.
Kannan. 2012. γ-Sitosterol from Acacia nilotica L. induces G2/M cell cycle
arrest and apoptosis through c-Myc suppression in MCF-7 and A549 cells.
Journal of Ethnopharmacology. 14: 803-809.
Sun, J., B.R. Liu, W.J. Hu, L.X. Yu and X.P. Qian. 2007. In vitro anticancer activity of
aqueous extracts and ethanol extracts of fifteen traditional Chinese medicines
on human digestive tumor cell lines. Phytotherapy Research. 21 (11) 1102-
1114.
Sun, W., Q. Wang, B. Chen, J. Liu, H. Liu and W. Xu. 2008. Gamma-tocotrienol-induced
apoptosis in human gastric cancer SGC-7901 cells is associated with a
suppression in mitogen-activated protein kinase signalling. British Journal of
Nutrition. 99(6): 1247-1254.
Su, W., P. Li, L. Huo, C. Wu, N. Guo and L. Liu. 2011. Phenolic content and antioxidant
activity of Phymatopteris hastata. Journal of the Serbian Chemical Society.
76(11): 1485-1496.
Surveswaran, S., Y.Z. Cai, H. Corke and M. Sun. 2007. Systematic evaluation of
naturalphenolic antioxidants from 133 Indian medicinal plants. Food
Chemistry. 102: 938-953.
Svangard, E., U. Goransson, Z. Hcoaoglu, J. Gullbo, R. Larsson, P. Claesona nd L. Bohlin.
2004. Cytotoxic cyclotides from Viola tricolor. Journal of Natural Products.
67(2): 144-147.
Szabo, M.R., C. Iditoiu, D. Chambre and A.X. Lupea. 2007. Improved DPPH determination
for antioxidant activity spectrophotometric assay. Chemical Papers. 61(3):
214-216.
162
Tan, Y., R. Yu and J.M. Pezzuto. 2003.Betulinic acid-induced programmed cell death in
human melanoma cells involves mitogen-activated protein kinase activation.
Clinical Cancer Research. 9 (7): 2866-2875.
Tanvir, R., R. Nawaz, A.A. Zaidi and R. Shamshila. 1994. Phytochemical screening of
medicinal plants belonging to family Euphorbiaceae. Pakistan Veterinary
Journal.14 (3): 160-162.
Tambekar, D.H. and S.B. Dahikar. 2011. Antibacterial activity of some Indian ayurvedic
preprations against enteric bacterial pathogens. Journal of Advanced
Pharmaceutical Technology and Research. 2 (1): 24-29.
Tajkarimi, M. and S.A. Ibrahim. 2011. Antimicrobial activity of ascorbic acid alone or in
combination with lactic acid on Escherichia coli O157:H7 in laboratory
medium and carrot juice. Food Control. 22(6): 801-804.
Tefsen, B., J. Geursten, F. Beckers, J. Tommassen and H.D. Cock. 2013. Lipopolysaccharide
transport to the bacterial outer membrane in spheroplasts, Te Journal of
Biological Chemistry. 250 (6): 4504-4509.
Tepe B, Sokmen M, Akpulat HA and Sokmen A (2005). In vitro antioxidant activities of the
methanol extracts of four Helichrysum species from Turkey. Food Chemistry.
90: 685-689.
Thaipong, K., U. Boonprakob, L. Cisneros-Zevallos and D.H. Byrne. 2005. Hydrophilic and
lipophilic antioxidant activities of guava fruits. Southeast Asian Journal of
Tropical Medicine Public Health. 36: 254–257.
Tiwari, J.K., R. Ballabha and P. Tiwari. 2010. Ethnopaediatrics in Garhwal Himalaya,
Uttarakhand, India (Psychomedicine and medicine). New York Science
Journal. 3 (4): 123-126.
Tiwari, R. J. and R. N. S. Banafar. 1995. Studies on the nutritive constituents yield and yield
attributing characters in some ber (Zizyphus jujuba) genotypes. Indian Journal
of Plant Physiology. 38:88-89.
Tiwari, S., S.K. Singh and A. Singh. 2005. Toxicological effect and biochemical alterations
induced by different fractions of Euphorbia royleana latex in freshwater
harmful vector snail Lymnaea acuminata. Indian Journal of Experimental
Biology. 42 (12) : 1220-1225.
163
Tiwari, S., R.P. Pandey and A. Singh. 2008. Effect of cycloart-24-en-3β-ol from Euphorbia
royleana latex neuroenzyme AChE and oxidative metabolism of freshwater
fish, Channa Punctatus.African Journal of Traditional, Complementary and
Alternative Medicines. 5 (4) : 332-339.
Turkmen, N., F. Sari and Y.S. Velioglu. 2006. Effects of extraction solvents on concentration
and antioxidant activity of black and black mate tea polyphenols determined
by ferrous tartrate and Folin–Ciocalteu methods. Food Chemistry. 99 (4) :
835-841.
Turkmen, N., Y.S. Velioglu, F. Sari and G. Polat. 2007. Effect of extraction conditions on
measured total polyphenol contents and antioxidant and antibacterial activities
of black tea. Molecules. 12: 484-496.
Turkoglu, A., M.E. Duru, N. Mercan, I. Kivrak and K. Gezer. 2007. Antioxidant and
antimicrobial activities of Laetiporus sulphureus (Bull.), Murill. Food
Chemistry. 11: 267-273.
Tsai, Y.C., H.C. Hsu, W.C. Yang, W.J. Tsai, C.C. Chen and T. Watanabe. 2007. Alpha-
bulnesene, a PAF inhibitor isolated from the essential oil of Pogostemon
cablin. Fitoterapia. 78 (1): 7-11.
Tsiri, D., D. Aligiannis, K. Graikou, C. Spyropoulos and I. Chinou. 2008. Triterpenoids from
Eucalyptus camaldulensis Dehnh. tissue cultures. Helvetica Chemica Acta. 91
(11): 2110-2114.
Tsuneki, H., E.L. Ma, S. Kobayashi, N. Sekizaki, K. Maekawa, T. Sasaoka, M.W. Wang and
I. Kimura. 2005. Antiangiogenic activity of beta-eudesmol in vitro and in
vivo. European Journal of Pharmacology. 512 (2-3): 105-115.
Tyagi, R. and V. Sharma. 2014. A comparison of volatile compounds in different genotypes
of Sesamum indicum L. by GC-MS. International Journal of Pharmaceutical
Sciences and Research. 5(1): 249-258.
Ullah, M..O., M. Haque, K.F. Urmi, A.H.M. Zulfikar, E.S. Anita, M. Begum and K. Hamid.
2013. Anti-bacterial activity and brine shrimp lethality bioassay of methanolic
extracts of fourteen different edible vegetables from Bangladesh. Asian
Pacific Journal of Tropical Biomedicine. 3 (1): 1-7.
164
Upadhyay, M., N. Nashikkar, D. Begde, S. Bundale, M. Pise, J. Rudra and A. Upadhyay.
2013. Study of antimicrobial, antioxidant and antiquorum sensing properties
of Euphorbia trigona. Global Journal of Research on Medicinal Plants and
Indigenous Medicine. 2 (9) : 630-641.
Urquiaga, I. and F. Leighton, 2000. Plant polyphenol antioxidants and oxidative stress.
Biological Research. 33: 55-64.
Vargas, A.D., H.M. Soto, H.V.A. Gonzalez, E.M. Engleman and G.A. Martinez. 2006.
Kinetics of accumulation and distribution of flavonoids in guava (Psiduim
guajava). Agrociencia. 40: 109–115.
Velmurugan, S., M.M. Babu, S.M.J. Punitha, V.T. Viji and T.. Citarasu. 2012. Screening and
characterization of antiviral compounds from Psidium guajava Linn. Root
bark against white spot syndrome virus. Indian Journal of Natural Products
and Resources. 3: 208-214.
Venkatanagaraju, E. and D. Goli. 2014. Antimicrobial activity of Euphorbia milii leaves
extract. International Journal of Pharmacy Research and Science. 2(2): 135-
140.
Verma, S., A. Singh and A. Mishra. 2012. Taxifolin acts as type I inhibitor for VEGFR-2
kinase: stability evaluation by molecular dynamic simulation. Journal of
Applied Pharmaceutical Sciences. 2 (1) : 41-46.
Verza, S.G., C. Pavei and G.G. Ortega. 2008. Study of the specificity of cross-povidone
(PVPP) as binding agent in the quantification of polyphenolic compounds.
Journal of the Brazailian Chemical Society. 19(8): 1627-1633.
Vincenzi,D.M., M. Silano, D.A.Vincenzi, F. Maialetti and B.Scazzocchio. 2002.
Constituents of aromatic plants: eucalyptol. Fitoteratpia. 73(3): 269-275.
Vithlani, V.A. and H.V. Patel. 2010. Production of functional vinegar from Indian jujbe
(Ziziphus mauritiana) and its antioxidant properties. Journal of Food
Technology. 8(3): 143-149.
Vonderheid, E.C., E.T. Tan, A.F. Kantor, L. Sharger, B. Micaily and S.E.J. Van. 1989. Long-
term efficacy, curative potential, and carcinogenicity of topical
mechlorethamine chemotherapy in cutaneous T cell lymphoma.Journal of The
American Academy of Dermatology. 20 (3): 416-428.
165
Wang, B., Liu, H.C., Ju, C.Y., 2005. Study on the hypoglycemic activity of different extracts
of wild Psidium guajava leaves in Panzhihua area. SichuanDa Xue Xue Bao
Yi Xue Ban. 36: 858-861.
Witkowska-Banaszczak, E., W. Bylka, I. Matlawska, O. Goslinska and Z. Muszynski. 2005.
Antimicrobial activity of Viola tricolor herb. Fitoterapia. 76(5): 458-461.
Wood, L.J., L.M. Nail, A. Gilster, K.A. Winters and C.R. Elsea. 2006. Cancer chemotherapy-
related symptoms: evidence to suggest a role for proinflammatory cytokines.
Oncology Nursing Forum. 33:535-542.
Wu, J.W., C.L. Hsien, H.Y. Wang and H.Y. Chen. 2009. Inhibitory effects of guava (Psidium
guajava L.) leaf extracts and its active compounds on the glycation process of
protein. Food Chemistry. 113 (1): 78-84.
Xu, J., B. Yang, L. Fang, S. Wang, Y. Guo, T. Yamakuni and Y. Ohizumi. 2013. Four new
myrsinol diterpenes from Euphorbia prolifera. Journal of Natural Medicines.
67 (2): 333-338.
Yang, D., L. Michel, J.P. Chaumont and J.C. Millet. 1999. Use of caryophyllene oxide as an
antifungal agent in an in vitro experimental model of onychomycosis.
Mycopathologia. 148 (2): 79-82.
Yang, D.S., Y.L. Zhang, W.B. Peng, L.Y. Wang, Z.L. Li, X. Wang, K.C. Liu, Y.P. Yang,
H.L. Li and X.L. Li. 2013. Jatropholane-type diterpenes from Euphorbia
sikkimensis. Journal of Natural Product. 76 (2) : 265-269.
Yang, J., Z. Qu, Y.L. Xiao, G.F. Qiu, T. Zhang, Z.Y. Wu, H.R. He and X.M. Hu. 2011.
Chemical composition and antioxidant activity of the essential oil of endemic
Viola tianshanica. Natural Product Research. 25(17):1635-1640.
Yang, Z., H. Xiao, H. Jin, P.T. Koo, D.J. Tsang and C.S. Yang. 2010. Synergistic actions of
atorvastatin with gamma-tocpotrienol and celecoxib against human colon
cancer HT29 and HCT116 cells. International Journal of Cancer. 126: 852-
863.
Yasukawa, K., T. Akihisa, Z.Y. Yoshida and M. Takido. 2000. Inhibitory effect of euphol, a
triterpene alcohol from the roots of Euphorbia kansui, on tumour promotion
by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in
mouse skin. The Journal of Pharmacy and Pharmacology. 52 (1) : 119-124.
166
Yeboah, E.M. and R.R. Majinda. 2009. Radical scavenging activity and total phenolic
content of extracts of the root bark of Osyris lanceolata. Natural Product
Communication. 4 (1): 89-94.
Yin, F., A.E. Giuliano, R.E. Law and H.A.J. Van. 2001. Apigenin inhibits growth and
induces G2/M arrest by modulating cyclin-CDK regulators and ERK MAP
kinase activation in breast carcinoma cells. Anticancer Research. 21 (1A):
413-420.
Yumrutas, O. and S.D. Saygideger. 2012. Determination of antioxidant and antimutagenic
activities of Phlomis armeniaca and Mentha pulegium. Journal of Applied
Pharmaceutical Sciences. 2(1): 36-40.
Yun, K.T, D.J. Koh, S.H. Kim, S.J. Park, J.H. Ryu, D.G. Kim, J.Y. Lee and K.T. Lee. 2008.
Anti-inflammatory effects of sinapic acid through the suppression of inducible
nitric oxide synthase, cyclooxygase-2, and proinflammatory cytokines
expressions via nuclear factor-kappaB inactivation. Journal of Agriculture and
Food Chemistry. 56 (21): 10265-10272.
Yusri, N.M., K.W. Chan, S. Iqbal and M. Ismail. 2012. Phenolic content and antioxidant
activity of Hibiscus cannabinus L. seed extracts after sequential solvent
extraction. Molecules. 17: 12612-12621.
Yoo, Y.C., B.H. Shin, J.H. Hong, J. Lee, H.Y. Chee, K.S. Song and K.B. Lee. 2007. Isolation
of fatty acids with anticancer activity from Protaetia brevitarsis larva.
Archives of Pharmacal Research. 30(3):361-365.
Zahidah, W.Z.W.N., A. Noriham and M.N. Zainon. 2013. Antioxidant and antimicrobial
activities of pink guava leaves and seeds. Journal of Tropical Agriculture and
Food Science. 41(1): 53-62.
Zaidi, S.F., J.S. Muhammad, S. Shahyar, K. Usmanghani, A.H. Gilani, W. Jafri and T.
Sugiyama. 2012. Anti-inflammatory and cytoprotective effects of selected
Pakistani medicinal plants in Helicobacter pylori-infected gastric epithelial
cells. Journal of Ethnopharmacology. 141(1): 403-410.
Zakaria, N.A., D. Ibrahim, S.F. Shaida and N.A. Supardy. 2011. Phytochemical composition
and antibacterial potential of hexane extract from Malaysian red algae,
167
Acanthophora spicifera (Vahl) Borgesen.World Applied Sciences Journal. 15
(4) : 496-501.
Zang, L.Y., G. Cosma, H. Gardner, X. Shi, V. Castranova and V.Vallyathan. 2000. Effect of
antioxidant protection by p-coumaric acid on low-density lipoprotein
cholesterol oxidation. American Journal of Physiology, Cell Physiology.
279(4): 954-960.
Zhang, Z.R., A.M. Zaharna, M.M.K. Wong, S.K. Chiu and H.Y. Cheung. 2013. Taxifolin
enhances andrographolide-induced mitotic arrest and apoptosis in human
prostate cancer cells via spindle assembly checkpoint activation. PLoS ONE.
8 (1) : e54577, 1-16.
Zhao, B. and M. Hu. 2013. Gallic acid reduces cell viability, proliferation, invasion and
angiogenesis in human cervical cancer cells. Oncology Letters. 6(6): 1749-
1755.
Zhao, H., J. Dong , J. Lu, J. Chen, Y. Li, L. Shan, Y. Li, W. Fan and G. Gu. 2006. Effects of
extraction solvent mixtures on antioxidant activity evaluation and their
extraction capacity and selectivity for free phenolic compounds in Barley
(Hordeum vulgare L.). Journal of Agriculture and Food Chemistry. 54 (19) :
7277-7286.
Zhao, X., S. Zhai, M.S. An, Y.H. Wang, Y.F. Yang, H.Q. Ge, J.H. Liu and X.P. Pu. 2013.
Neuroprotective effects of protocatechuic aldehyde against neurotoxin-
induced cellular and animal models of Parkinson’s disease. PLoS ONE. 8 (10)
: e78220, 1-16.
Zhu, M., J.D. Phillipson, P.M. Greengrass, N.E. Bowery and Y. Cai. 1997. Plant
polyphenols: biologically active compounds or non-selective binders to
proteins. Phytochemistry. 44: 441–447.
Zubair, M., K. Rizwan, N. Rasool, N. Afshan, M. Shahid and V. Ahmed. 2011.
Antimicrobial potential of various extract and fractions of leaves of Solanum
nugrum. International Journal of Phytomedicine. 3: 63-67.
168