RAJIV GANDHI UNIVERSTITY OF HEALTH SCIENCE BANGALORE, KARNATAKA.

M PHARM SYNOPSISYEAR OF ADMISSION-JUNE 2008

TITLE OF THE SYNOPSIS

“ANTIBACTERIAL ACTIVITY OF FEW PHYTOCONSTITUENTS AGAINST RESISTANT STRAINS OF GRAM-POSITIVE AND GRAM-

NEGATIVE BACTERIA”.

BYSREEDHAR.S

M PHARM, PART-IDEPARTMENT OF PHARMACOLOGY

UNDER THE GUIDANCE OF

Mr.SYED MANSOOR AHAMED M. Pharm (Ph.D).Asst. Professor

DEPARTMENT OF PHARMACOLOGY

INSTITUTIONSREE SIDDAGANAGA COLLEGE OF PHARMACY

B H ROAD, TUMKUR-572102 KARNATAKA.

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

ANNEXURE - II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name and address of the Candidate

Sreedhar.Ss/o S.Mohan,Do.no: 14/811,Nakash street, Sangampet,Kadapa(Dt),A.P

2. Name of the Institution Sree Siddaganga College of Pharmacy B.H. Road, Tumkur-572102

3. Course of Study and Subject M Pharm – Pharmacology

4. Date of Admission June 2008

5. Title of the Topic:

“ANTIBACTERIAL ACTIVITY OF FEW PHYTOCONSTITUENTS AGAINST RESISTANT STRAINS OF GRAM-POSITIVE AND GRAM-

NEGATIVE BACTERIA”.

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6.0

BRIEF REVIEW OF THE INTENDED WORK :

6.1 GENERAL DISCUSSION:

Infectious diseases are the leading cause of death worldwide. Antibiotic resistance has

become a global concern. The clinical efficacy of many existing antibiotics is being

threatened by the emergence of multi drug resistance pathogens. Many infectious

diseases have been known to be treated with herbal remedies throughout the history of

mankind1.

Even though pharmacological industries have produced a number of new antibiotics in

the last three decades, resistance to these drugs by microorganisms has increased. In

general, bacteria have the genetic ability to transmit and acquire resistance to drugs,

which are used as therapeutic agents. Such a fact is cause for concern, because number of

patients in hospital who have suppressed immunity, and due to new bacterial strains,

which are multi resistant. Consequently, new infections can occur resulting in high

mortality.

The problem of microbial resistance is growing and the outlook for the use of

antimicrobial drug in the future is still uncertain. Therefore, actions must be taken to

reduce this problem, for example, to control the use of antibiotic, develop research to

better understand the genetic mechanism of resistance, and to continue studies to develop

new drugs, either synthetic or natural. The ultimate goal is to offer appropriate and

efficient antimicrobial drugs to the patient2.

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There is a continuous and urgent need to discover new antimicrobial compounds with

diverse chemical structures and novel mechanism of action for new and re-emerging

infectious diseases. Therefore, researchers are increasingly turning their attention to folk

medicine, looking for new leads to develop better drugs against microbial infections. The

increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic

microbial infectious agents has led to screening of several medicinal plants for their

potential antimicrobial activity1.

The purpose of this study is to evaluate some Indian medicinal plants phytoconstituents

for their antimicrobial activity against resistant gram-positive and gram-negative bacteria.

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6.2 NEED FOR THE STUDY :

According to World Health Organization, medicinal plants are the best source to obtain a

variety of newer herbal drugs. About 80% of individuals from developed countries use

traditional medicine, which has compounds derived from medicinal plants. Therefore,

such plants should be investigated to better understand their properties, safety and

efficacy.

The use of plant extracts and phytochemicals, both with known antimicrobial properties,

can be of great significance in therapeutic treatments. In last few years, a number of

studies have been conducted in different countries to prove such efficiency3. Despite the

existence of potent antibiotics and antifungal agents, resistant or multi-resistant strains

are continuously appearing, imposing the need for a permanent search and development

of new drugs. This revival of interest in plant-derived drugs is mainly due to the current

widespread belief that “green medicine” is safe and more dependable than the costly

synthetic drugs, many of which have adverse side effects4.

Swertiamarin, Andrographolide, Bixin, Piperin, Lupeol and Nicotine are known for their

antibacterial activity against several strains of gram positive and gram-negative bacteria5,

6. But there is no published data on its antibacterial activity against resistant strains. The

aim of this study is to evaluate the antibacterial activity of few phytoconstituents against

resistant strains of gram-positive and gram-negative bacteria.

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6.3. REVIEW OF LITREATURE:

6.31 Swertiamarin:

It is a main chemical constituent isolated from Enicostemma axillare raynal

belonging to the family Gentianaceae. Swertiamarin is a secoiridoid glycoside present in

most of the plant belonging to the family Gentianaceae. Swertiamarin is one of the

important constituents of many crude drugs, which are marketed in Japan and other

countries and these crude drugs are normally evaluated by there high Swertiamarin

content9. Swertiamarin has very low toxicity and is safe and effective antispastic agent. It

showed sedative effect in mice, pigeons and rabbits and induced sleep in mice8. Its

antibacterial, brine shrimp lethality and anticholinergic activites11 were also reported.

Swertiamarin showed potent antinocciceptive and anti-inflammatory properties in rats

when given orally at 100 and 200 mg/kg b.w7.

Reported biological activities:

The alcoholic extract of Enicostemma littorale exhibited potent anti-inflammatory in

carrageenan induced inflammation and cotton pellet granuloma methods in rats7.

The methanol extract of Enicostemma littorale exhibited antitumor activity against

Dalton’s ascetic lymphoma in albino mice10.

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6.32 Andrographolide:

It is a main chemical constituent isolated from bitter shrub Andrographis paniculata

belonging to the family Acanthaceae12.It is well known as kalmegh and forms the

principle ingredient of a household medicine called Alui extensively used in Bengal. It is

used to relieve griping, irregular stools, loss of appetite, dyspepsia.roots and leaves are

febrifuge, stomachic,tonic and anthelmintic. Green leaves used as a tonic, alternative in

syphilitic cachexia and foul syphilitic ulcers.

Reported biological activities:

Immunomodulatory activity of andrographolide14.

Antiinflammatory activity of andrographolide15

Hepatoprotective effect of andrographolide against hexachlorocyclohexane-induced

oxidative injury16.

6.33 Piperine:

It is major plant alkaloid present in shrub Piper nigrum and Piper longum belonging to

the family Piperaceae13. It is used as stomachic, laxative, anthelmintic, carminative,

diseases of spleen, tumors, ascites, aphrodisiac, leprosy and jaundice.

Root and fruits are used as liver tonic, abortifacient, inflammatory and diuretic.

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Reported biological activities:

Anti-inflammatory and anti-arthritic effects of piperine17.

Piperine inhibits eosinophil infiltration and airway hyper responsiveness by suppressing

T cell activity and Th2 cytokine production in the ovalbumin-induced asthma model 18 .

Piperine activates testicular apoptosis in adult rats19.

6.34 Lupeol:

It is a major triterpenoid obtained from crude ethanol extract from the leaves of

Dendropanax querceti belonging to the family Araliaceae. It exhibits cytotoxic effect.

Reported biological activities:

Lupeol, a dietary triterpene, inhibited growth, and induced apoptosis through down-

regulation of DR3 in SMMC7721 cells20.

Lupeol Inhibits Proliferation of Human Prostate Cancer Cells by Targeting {beta}-

catenin Signaling21.

The triterpenoid lupeol attenuates allergic airway inflammation in a murine model 22 .

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6.35 Bixin:

It is carotenoid pigment fouind in the seeds of tropical plant Bixa orellana belonging to

the family Bixaceae. It is also called as Annatto a colouring agent which is commercially

used for colouring cheese. It is used as hair dye which is having anti inflammatory and

provitamin A properties extracted from seeds13. Parts of the plants can be used to make

medicinal remedies for such conditions as sunstroke, tonsillitis, burns, leprosy, pleurisy,

apnoea, rectal discomfort, headaches, heart disease, dysentery and jaundice.

Reported biological activities:

Evaluation of the clastogenicity and anticlastogenicity of the carotenoid bixin in human

lymphocyte cultures23.

Antioxidant action of bixin against cisplatin-induced chromosome aberrations and lipid

peroxidation in rats24.

Protective effect of curcumin, ellagic acid and bixin on radiation induced genotoxicity25.

6.36 Nicotine:

It is a major alkaloid isolated from an erect glandular pubescent herb Nicotiana tobacum

belonging to the family Solanaceae.It is used as tonic emetic, carminative, anthelmintic,

bronchitis, asthma, caries of the teeth, skin diseases, Scorpion bites, inflammation,

Scabies, wounds and disinfectant. The leaves are narcotic, sedative, emetic, ulcers and

painful tumours12.

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Reported biological activities:

Effect of nicotine on lipid profile, peroxidation & antioxidant enzymes in female rats

with restricted dietary protein26.

Transdermal nicotine patch failed to improve postoperative pain management27.

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6.4 OBLECTIVE OF THE STUDY:

Authentication and collection of plant materials.

Isolation of phytoconstituents

Collection of test organism and developing resistance in them.

Antimicrobial activity of the phytoconstituents by cup plate method.

Determination of MIC of the extracts.

MATERIALS AND METHODS:

7.1 Plant material:

These phytoconstituents will be isolated from their respective plants, using standard

methods mentioned in the literature7, 28, 29.

7.2 Standard drugs:

Pure Ofloxacin, Pure Cefixime.

7.3 Bacterial strains:

The test organisms used in this study includes five gram-positive bacteria and five gram-

negative bacteria. The bacteria will be grown in nutrient broth at 370C and maintained at

40C. Bacteria will be made resistant to standard drugs and used.

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7.4 METHOD:

For the antimicrobial assay, the in vitro cup diffusion method is adopted. Nutrient agar

media will be used to cultivate the bacteria. Peptone water (1%) will be used for fresh

culture of all bacteria and maintained by periodic sub culturing of fresh nutrient agar

medium. phytoconstituents will be dissolved in dimethyl formamide to give 1 mg/ml

solution. 100 ml portions of molten agar (450) will be inoculated with the desired volume

of fresh culture of the test organism to give an inoculate of approximately 106 cells/ml.

The agar will be poured on to plates and after solidification, cups (8 mm dia) were made.

To each of these cups 0.1 ml of aliquots of the test solution will be placed per cup. For

assaying antibacterial activity, plates will be incubated at 37+_10 for 24 h. The diameter

of zone of inhibition will be measured as an average of maximum dimensions of zones

around the disc30and Minimum Inhibitory Concentration (MIC) is measured. All the

experiments will be repeated six times and average values will be recorded.

7.5. Dose the study require any investigation or investigation to be conducted on patient

or other humans or animals?

“Not applicable”

7.4. Has Ethical clearance been obtained from your institution in case of 7.5?

“Not applicable”

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8.0 EXPERMENTAL DESIGN:

Sl.

NoPhytoconstituents

Resistant Micro-

organisms used

Incubation

time

Observation

(mm)

1 SwertiamarinGram positive and

Gram negative

24 h Zone of inhibition

and MIC

2 Andrographolide Gram positive and

Gram negative

24 h Zone of inhibition

and MIC

3 Bixin Gram positive and

Gram negative

24 h Zone of inhibition

and MIC

4 PiperineGram positive and

Gram negative

24 h Zone of inhibition

and MIC

5 LupeolGram positive and

Gram negative

24 h Zone of inhibition

and MIC

6 NicotineGram positive and

Gram negative

24 h Zone of inhibition

and MIC

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Standard drug

(Oflaxacin 1-2

μg/ml)31

Gram positive and

Gram negative

24 h Zone of inhibition

and MIC

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Standard drug

Cefixime

(2μg/ml)32

Gram positive and

Gram negative

24 h Zone of inhibition

and MIC

MIC: Minimum Inhibitory Concentration.

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9.0 REFERENCES:

1. Parekh J, Chanda SV. Invitro antimicrobial activity and phytochemical analysis of

some Indian medicinal plants. Turkey J Biol 2007; 31: 53-8.

2. Nascimento G, Locatelli J, Paulo C, Silva GL. Antibacterial activity of plant extracts

and phytochemicals on antibiotic-resistant bacteria. Brazilian J Microbiol. 2000; 31.

3. Prusti A, Mishra SR, Sahoo S, Mishra SK. Antibacterial activity of some medicinal

plants. Ethnobotanical leaflets 2008; 12: 227-30.

4. Parekh J, Chanda SV. Screening of aqueous and alcoholic extracts of some

medicinal plants for antibacterial activity. Indian J Pharm Sci 2006; 68: 835-8.

5. Hiremath SP, Badami S, Swamy HKS, Biradar JS. Antimicrobial activity of various

extracts of Acalypha indica. Indian J Microbiol 1993; 33: 75-7.

6. Ashraful AM, Rowshanul HM, Farjana N, Matiar R, Rezaul KM. Antimicrobial

Activity of Akanda (Calotropis gigantea) on Some Pathogenic Bacteria. Bangladesh

J Sci. Ind. Res 2008; 43: 397-404.

7. Sadique J, Chandra T, Thenmozhi V, Elango V. The anti-inflammatory activity of

Enicostemma littorale and Mollugo Cerviane. Biochem. Med Metab Biol 1987; 37:

107-76.

8. Kavimani S, Manisenthikumar KT.Effect of methanolic extract of Enicostemma

littorale on daltons ascititc lymphoma. J Ethnopharmacol 2000; 71: 349-52.

9. Harumi T, Kimie N, Fumihika Y. Analysis of Swertiamarin in Swertia herb and

preparations containing this crude drug by capillary electrophoresis. Analytical

Sciences 2001; 17: 885-8.

10. Mandal S, Jain R, Mukhopadhyay S. Naturally occuring iridoids with

pharmacological activity. Indian J Pharm Sci 1998; 60: 123-7.

11. Yamahara J, Kobayashi M, Mastsuda H, Aoki S. anticholinergic action of Swertia

japonica and an active constituents. J Ethnopharmacol 1991; 33: 31-5.

12. Kirtikar KR, Basu BD. Indian Medicinal Plants, 2nd ed. Dehradun: International book

distributors, 1987. Vol 3. p. 1884-5, 2126-9, 1798-9.

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13. Handa SS, Kaul MK. Supplement to Cultivation and Utilization of Medicinal Plants,

Jammu-Tawi: Regional research laboratory, 1996. p. 3-6, 542-3, 308-9.

14. Naik SR, Hule A. Evaluation of Immunomodulatory Activity of an Extract of

Andrographolides from Andographis paniculataPlanta Med. 2009 Mar 4.

15. Bao Z, Guan S, Cheng C, Wu S, Wong SH, Kemeny DM, Leung BP, Wong WS. A

novel antiinflammatory role for andrographolide in asthma via inhibition of the

nuclear factor-kappaB pathway. Am J Respir Crit Care Med. 2009 ;179:657-65.

16. Trivedi NP, Rawal UM, Patel BPHepatoprotective effect of andrographolide against

hexachlorocyclohexane-induced oxidative injuryIntegr Cancer Ther. 2007 ;6 :271-

80.

17. Bang JS, Oh DH, Choi HM, Sur BJ, Lim SJ, Kim JY, Yang HI, Yoo MC, Hahm DH,

Kim KS. Anti-inflammatory and anti-arthritic effects of piperine in human

interleukin-1beta-stimulated fibroblast-like synoviocytes and in rat arthritis models.

Arthritis Res Ther. 2009 ;11 :R49.

18. Kim SH, Lee YC. Piperine inhibits eosinophil infiltration and airway

hyperresponsiveness by suppressing T cell activity and Th2 cytokine production in

the ovalbumin-induced asthma model. J Pharm Pharmacol. 2009 ;61:353-9.

19. D'Cruz SC, Vaithinathan S, Saradha B, Mathur PP. Piperine activates testicular

apoptosis in adult rats. J Biochem Mol Toxicol. 2008 ;22:382-8.

20. Zhang L, Zhang Y, Zhang L, Yang X, Lv Z. Lupeol, a dietary triterpene, inhibited

growth, and induced apoptosis through down-regulation of DR3 in SMMC7721

cells. Cancer Invest. 2009 ;27:163-70.

21. Saleem M, Murtaza I, Tarapore R, Suh Y, Adhami VM, Johnson JJ, Siddiqui IA,

Khan N, Asim M, Hafeez BB, Shekhani MT, Li B, Mukhtar H. Lupeol Inhibits

Proliferation of Human Prostate Cancer Cells by Targeting {beta}-catenin Signaling.

Carcinogenesis. 2009 Feb 20.

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22. Vasconcelos JF, Teixeira MM, Barbosa-Filho JM, Lúcio AS, Almeida JR, de

Queiroz LP, Ribeiro-Dos-Santos R, Soares MB. The triterpenoid lupeol attenuates

allergic airway inflammation in a murine modelInt Immunopharmacol. 2008;8:1216-

21.

23. Júnior AC, Asad LM, Oliveira EB, Kovary K, Asad NR, Felzenszwalb I

Antigenotoxic and antimutagenic potential of an annatto pigment (norbixin) against

oxidative stress. Genet Mol Res. 2005 ;4:94-9.

24. Silva CR, Greggi Antunes LM, Bianchi ML. Antioxidant action of bixin against

cisplatin-induced chromosome aberrations and lipid peroxidation in rats. Pharmacol

Res. 2001 ;43:561-6.

25. Thresiammaz KC, George J, Kuttan R. Protective effect of curcumin, ellagic acid

and bixin on radiation induced genotoxicityJ Exp Clin Cancer Res. 1998 ;17:431-4.

26. Chattopadhyay K , Chattopadhyay BD. Effect of nicotine on lipid profile,

peroxidation & antioxidant enzymes in female rats with restricted dietary protein.

Indian J Med Res. 2008 ;127:571-6

27. Turan A , White PF, Koyuncu O, Karamanliodlu B, Kaya G, Apfel CC. Transdermal

nicotine patch failed to improve postoperative pain management. Anesth Analg.

2008 ;107:1011-7.

28. Ram P. Rastogi, Mehrotra. B.N. Compendium of Indian medicinal plants,

Publications and Information directorate, New Delhi, 1970.Vol 2. p.310.

29. Pulok.Dr. Mukherjee. K. Quality control of Herbal drugs, 3rd ed. Business Horizons

Publications, New Delhi, 2008. p.224, 263, 677.

30. Hiremath SP, Badami S, Swamy HKS. Antibacterial and antifungal activities of

Striga densiflora and Striga Orobanchioides. Indian J Pharm Sci 1996; 58: 174-8.

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31. Fu K P, Lafredo S C, Foleno B, Isaacson D M, Barrett J F,Tobia A J et al. In vitro

and in vivo antibacterial activities of levofloxacin (l-ofloxacin), an optically active

ofloxacin. Antimicrob Agents Chemother. 1992 ; 36: 860-6

32. Cynthia CK, Juan SM, John AW. Antibacterial Activities of Cefpodoxime, Cefixime,

and Ceftriaxone. Antimicrob Agents Chemother. 1988; 32: 1896-8.

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10 Signature of the candidate:

11 Remarks of the Guide: Recommended

12 Name and Designation of:

12.1 Guide: Mr. SYED MANSOOR AHAMED. M. Pharm ,(Ph.D).Asst.Professor Department of Pharmacology

12.2 Signature:

12.3 Co-Guide:

12.4 Signature:

12.5 Head of the Department: K.P. Shivalinge Gowda. M. Pharm, (Ph.D).Asst Professor and HOD.Department of Pharmacology.

12.6 Signature

13 13.1 Remarks of the Chairman and Principal

Forwarded to the University for approval

13.2 Signature DR. S BADAMIPrincipal

Sree Siddaganga College of Pharmacy ,B H Road, Tumkur-572102

.

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