International Journal of Innovative Pharmaceutical ... · medicinal importance eg. curry leaf,...
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RESEARCH ARTICLE Nupur Sinha et.al / IJIPSR / 3 (3), 2015, 151-163
Department of Biotechnology ISSN (online) 2347-2154
Available online: www.ijipsr.com March Issue 151
ANTIOXIDANT ACTIVITY, FREE RADICAL SCAVANGING
CAPACITY AND PHYTOCHEMICAL SCREENING OF SOME
SELECTED INDIAN PLANTS OF MEDICINAL IMPORTANCE
1Nupur Sinha*,
2Deepti Dua,
3Diksha Wadhwa
Amity Institute of Biotechnology, Amity University, Greater Noida Expressway, Sector-125,
Noida-201303, INDIA
Corresponding Author
Nupur Sinha
Department of Biotechnology
Amity Institute of Biotechnology,
Amity University, Greater Noida Expressway,
Sector-125, Noida-201303, INDIA
Email: [email protected]
Phone: +91 93110 52874
International Journal of Innovative
Pharmaceutical Sciences and Research www.ijipsr.com
Abstract
The present study was carried out to determine the relative antioxidant activity of selected Indian plants of
medicinal importance eg. curry leaf, cannabis, neem, methi, beet root and spring onion. They were extracted
with methanol and antioxidant enzyme assays such as superoxide dismutase (SOD), catalase (Cat) and
glutathione peroxidase (Gpx) were assayed in the methanolic extracts of the plants. Non enzymatic
parameter glutathione (GSH) content was also determined in these plants in addition to DPPH and FRAP
assay which was done to check their free radical scavenging potential. Phytochemical screening of these
plants revealed the presence of flavonoids, saponins, alkaloids, tannins and terpenoids. Only flavanoid was
present in curry leaf extract whereas spring onion extract showed the presence of alkaloids only. DPPH free
radical scavenging effects of these extracts were determined spectrophotometricaly. The highest radical
scavenging effect was observed in curry leaf extract with IC50 = 0.015mg/ml. A rapid evaluation for total
antioxidant potential by free radical scavenging capacity was done by Scan Dot Blot assay. DPPH staining
method demonstrated the different plants exhibited different free radical scavenging capacity. Antioxidant
power of different plants was also assessed by FRAP assay which depends upon the reduction of ferric to
ferrous ions using TPTZ. Highest reducing potential was shown by curry leaf extract and lowest by spring
onion extract. The greater amount of alkaloids in the plant extracts lead to more potent free radical
scavenging effect as shown by curry leaf extract. The lowest values of antioxidant enzyme were observed in
spring onion which also exhibited the lowest radical scavenging capacity.
Keywords: Antioxidants, medicinal plants, radical scavenger, TPTZ, flavanoids, DPPH, FRAP.
RESEARCH ARTICLE Nupur Sinha et.al / IJIPSR / 3 (3), 2015, 151-163
Department of Biotechnology ISSN (online) 2347-2154
Available online: www.ijipsr.com March Issue 152
INTRODUCTION
The recent growth in the knowledge of free radicals and antioxidants in biology is producing
medical revolution that provides the new age of health and disease management [1]. Free radicals
are defined as the atoms, molecules or ions having unpaired electrons in their valence shell [2].
These are capable of existing independently. In human body many processes such as metabolic
processes, inflammatory processes occur, which leads to the formation of free radicals. Free
radicals are also formed by cigarette smoke, radiations, environmental, pollution, drugs,
overdoses of medicines and preservatives. These are capable of reacting with cellular components
and can cause damage to the proteins, lipids, carbohydrates and nucleotides in the tissue [3,4] thus
leading to cell damage and destruction [5]. Free radicals released during oxidative stress pose the
major endogenous damage in the biological system. This type of damage is often associated with
various degenerative diseases and disorders such as cancer, cardiovascular disease,
immunofunction decline and ageing. Several neurological disorders such as Parkinson disease,
Alzheimer disease, Depression etc. which causes dysfunction in brain [6,7]. Antioxidants are the
substances that protect the cells against the destructive effects of the free radicals. Thus
antioxidants are capable of scavenging free radicals. These are capable of donating electrons to
other molecules to stabilize themselves in order to protect against the destructive effects of free
radicals [8]. Substantial evidence has accumulated and indicated key roles for reactive oxygen
species (ROS) and other oxidants in causing numerous disorders and diseases. The evidence has
brought the attention of scientists to an appreciation of antioxidants for prevention and treatment
of diseases, and maintenance of human health [9]. Human body has an inherent anti-oxidative
mechanism and many of the biological functions such as the anti-mutagenic, anti-carcinogenic,
and anti-aging responses originate from this property [10,11]. Antioxidants stabilize or deactivate
free radicals, often before they attack targets in biological cells [12]. Recently interest in naturally
occurring antioxidants has considerably increased for use in food, cosmetic and pharmaceutical
products, because they possess multifacetedness in their multitude and magnitude of activity and
provide enormous scope in correcting imbalance [13,14]. Since very old times, herbal
medications have been used for relief of symptoms of disease [15]. Despite the great advances
observed in modern medicine in recent decades, plants still make an important contribution to
health care. Much interest, in medicinal plants however, emanates from their long use in folk
medicines as well as their prophylactic properties, especially in developing countries. Large
RESEARCH ARTICLE Nupur Sinha et.al / IJIPSR / 3 (3), 2015, 151-163
Department of Biotechnology ISSN (online) 2347-2154
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number of medicinal plants has been investigated for their antioxidant properties. Natural
antioxidants either in the form of raw extracts or their chemical constituents are very effective to
prevent the destructive processes caused by oxidative stress [16]. Although the toxicity profile of
most medicinal plants have not been thoroughly evaluated, it is generally accepted that medicines
derived from plant products are safer than their synthetic counterparts [17,18]. It has been
mentioned the antioxidant activity of plants might be due to their phenolic compounds [19].
Flavanoids are a group of polyphenolic compounds with knownproperties which include free
radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action
[20]. Some evidence suggests that the biological actions of these compounds are related to their
antioxidant activity [21]. An easy , rapid and sensitive method for the antioxidant screening of
plant extracts is free radical scavenging assay using 1.1-diphenyl -2-picryl hydrazyl (DPPH)
stable radical and ferric reducing ability (FRAP assay) of different medicinal plants
spectrophotometrically. In presence of an antioxidant, DPPH radical obtains one more electron
and the absorbance decreases and in FRAP assay, in presence of antioxidants, ferric ions are
reduced to ferrous ions [22,23]. In the present study antioxidant enzyme (SOD, Catalase, Gpx)
activities were checked in various plants of medicinal importance. Non- enzymatic parameter,
glutathione (GSH) content, DPPH free radical scavenging activity and ferric reducing ability of
different plant extracts were carried out to check antioxidant potential of these plants. In addition,
a comparative study of free radical scavenging potential of methanolic extracts of individual
plants and polyherbal formulation by mixing these plants extracts were done to check if the
mixture is beneficial as antioxidant. We have also found the relationship of total flavanoid
contents with antioxidant enzymes activity. In the longer term, plant species identified as having
high levels of antioxidant activity in vitro may be of value in the design of further studies to
unravel novel treatment strategies for disorders associated with free radicals induced tissue
damage.
MATERIALS AND METHODS
COLLECTION OF PLANT MATERIAL
The leaves of six medicinal plants namely; Curry leaves (Murraya koenigii), Cannabis (Cannabis
sativa), Neem (Azadirachta indica), Methi (Trigonella foenum), Beet Root (Beta vulgaris) and
Spring Onion (Allium cepa) were collected from IARI Pusa, New Delhi. Plant materials were
dried at room temperature and ground in a mortar. Fifty grams of each plant powder was extracted
RESEARCH ARTICLE Nupur Sinha et.al / IJIPSR / 3 (3), 2015, 151-163
Department of Biotechnology ISSN (online) 2347-2154
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in 500 ml of methanol by maceration below 50ºC. After preparation of individual plant extracts,
equal volumes of the three plant extracts, in which maximum enzyme activities and antioxidant
potential was observed, were mixed to prepare polyherbal formulation. To check their total
antioxidant potential and free radical scavenging activity.
BIOCHEMICAL ENZYME ASSAYS
Superoxide dismutase (SOD) activity was determined in the extracts using phenazine
methosulphate, nitroblue tetrazolium, NADH & pyrophosphate buffer (pH 8.3). The inhibition by
SOD of reduction of NBT to blue coloured chromogen in presence of PMS & NADH was
measured at 560nm. One unit of enzyme activity was defined as enzyme concentration required
for inhibition of the absorbance at 560nm of chromogen production by 50% in 1 min under assay
conditions and expressed as specific activity in unit of SOD per min per mg of protein [24].
Catalase (CAT) activity was measured in the extracts by using buffered substrate containing
H2O2. Total catalase was determined in the cells treated with 6-OHDA as well as cells treated
with 6-OHDA + lycopene extract by measuring the decrease in absorbance at 240nm and enzyme
activity is expressed as µmoles of H2O2 oxidised per min per mg protein [25]. . Glutathione
peroxidise (Gpx) was assayed using GSH, 0.32M Sod phosphate buffer and DTNB (dithiobis
nitro benzoic acid) and measured at 412nm [26]. Estimation of GSH content in the extract was
done in the supernatant using phosphate buffer pH7.4 and Dithiobis nitrobenzoic acid (DTNB)
[27].
FREE RADICAL SCAVANGING ASSAY
DPPH free radical scavenging activity and FRAP assay was done on cells treated with 6-OHDA
and cells treated with 6-OHDA as well as 6-OHDA + Lycopene to check total antioxidant
potential of lycopene.20, 15.
The percentage of antioxidant activity (AA%) of each substance was
assessed by DPPH free radical assay. The measurement of the DPPH radical scavenging activity
was performed according to methodology described by Brand-Williams et al. [28]. The samples
were reacted with the stable DPPH radical in an ethanol solution. The reaction mixture consisted
of g sample, absolute ethanol DPPH radical solution in ethanol. When DPPH reacts with an
antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from
deep violet to light yellow) were read [Absorbance (Abs)] at 517 nm .The mixture of ethanol and
sample served as blank. The control solution was prepared by mixing ethanol and DPPH radical
solution .The scavenging activity percentage (AA%) was determined according to Mensor et
al.[29].
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The DPPH free radical scavenging activity was calculated using the following formula: %
scavenging = [Absorbance of control - Absorbance of test sample/Absorbance of control] X 100
Free radical scavenging activity was also determined by FRAP (ferric reducing ability of plasma)
method, which depends upon the reduction of ferric tripyridyltriazine {Fe(III)-TPTZ} complex to
ferrous tripyridyltriazine {Fe(II)-TPTZ}.Ferrous has intensive blue colour which can be
monitored at 593nm. FRAP value are obtained by comparing the absorbance change at 593nm in
test reaction mixtures with those containing range with antioxidant mixtures. Frap Assay was
done using TPTZ by method of Benzie ae al.[30].
PROTEIN QUANTIFICATION
Quantification of protein in 100µl supernatant was done by Lowry’s Method by using Folin
Ciocalteau Reagent [31]. Data were analysed by ANOVA and means compared by the least
significant difference test at 0.05 level of confidence.
PHYTOCHEMICAL SCREENING
Phytochemical screening of TLM for the presence of alkaloids, anthraquinones, cardiac
glycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids was carried
out.
Test for alkaloids
Wagner reagent (1.27 g I2 + 2 g KI in 100 ml distilled water) is added to a fraction of plant
extract. Formation of reddish brown precipitate indicates the presence of alkaloids.
Test for saponins
The ability of saponins to produce emulsion with oil was used for the screening test [32]. 10 ml of
the plant extract was mixed with 5 ml of distilled water and shaken vigorously for froth
formation. 3 drops of olive oil were mixed with froth, shaken vigorously and observed for
emulsion development.
Test for flavonoids
5 ml of dilute ammonia solution was added to 10 ml of plant extract followed by few drops of
concentrated H2SO4. Presence of flavonoids was confirmed by yellow colouration [33].
Test for tannins
A few drops of 0.1% FeCl3 was added in the plant extract and observed for colour change;
brownish green or a blue-black colouration was taken as evidence for the presence of tannins [34].
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Department of Biotechnology ISSN (online) 2347-2154
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STATISTICAL EVALUATION
The data were subjected to student t test for comparison between groups. The values are
expressed as mean ± SEM. Significance level was set at P<0.05, P<0.01, P<0.001.
RESULTS AND DISCUSSION
The present study showed the comparative levels of antioxidant enzymes, SOD, Cat and Gpx in
methanolic extracts of leaves of curry leaf, cannabis, neem, methi, spring onion and beet root.
Superoxide radical is considered a major biological source of reactive oxygen species [34].
Although superoxide anion is a weak oxidant, it gives rise to generation of powerful and
dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative
stress [35]. The superoxide radical scavenging effect of methanolic extract of different plant
leaves were analysed in terms of inhibition of formazan and SOD activity was calculated in terms
of units/mg of protein. The SOD activities were in the order of Curry Leaf > Cannabis > Neem >
Methi > Beet Root > Spring Onion (Figure 1). The highest superoxide scavenging activity was
found in curry leaf extract and lowest in spring onion extract (84.1±0.3 and 24.2±2 units/mg
respectively). Thus our results demonstrate that methanolic extract of curry leaf behave as
powerful superoxide anion scavenger that may include therapeutic use against oxidative stress.
Catalase activity which was determined by the scavenging activity for hydrogen peroxide by
methanolic extracts of different plants exhibited the same pattern as shown by superoxide
scavenging assay. The Cat activities were in the order of Curry Leaf > Cannabis > Neem > Methi
> Beet Root > Spring Onion (Figure 2). The highest hydrogen peroxide scavenging activity was
found in curry leaf extract and lowest in spring onion extract (1.9±0.05 and 0.3±0.01
µmoles/min/mg protein respectively).
Fig.1: Superoxide dismutase activity (SOD activity (units/mg protein)
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Fig. 2: Catalase Activity (µmoles/min/mg Protein)
The glutathione peroxidase (Gpx) activities were in the order of Curry Leaf > Cannabis > Neem >
Methi > Beet Root > Spring Onion (Figure 2). The highest peroxidase activity was found in curry
leaf extract and lowest in spring onion extract (0.5±0.05 and 0.04±0.01 µmoles/min/mg protein
respectively).
Fig. 3: Glutathione-peroxidase Activity (µmoles/min/mg Protein)
Fig.4: Glutathione Content (µg/mg protein)
Glatathione content was also reported to be highest in methanolic extract of curry leaf (65.4±0.5
µg/mg protein) and it was reported to be lowest in methanolic extract of spring onion (10.6 ±0.2
µg/mg protein).
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The free radical scavenging activity, exhibited by percentage inhibition of DPPH radical, in the
methanolic extracts of different plants decreased in the following order: Curry Leaf > Cannabis >
Neem > Methi > Beet Root > Spring Onion. Highest scavenging activity was shown by
methanolic extract of curry leaf (95.1±0.7) and lowest scavenging activity was exhibited by
methanolic extract of spring onion (31±0.4). (Figure 5). This also showed that total antioxidant
potential was highest in curry leaf extract.
Fig.5: Free Radical Scavanging Assay by DPPH
Free radical scavenging activity was also determined by the FRAP (ferric reducing ability of
plasma) method, which depends upon the reduction of ferric tripyridyltriazine (Fe (III)-TPTZ)
complex to the ferrous tripyridyltriazine (Fe (II)-TPTZ) by a reductant at low pH. Fe (II)-TPTZ
has an intense blue color and can be monitored at 593 nm. FRAP values are obtained by
comparing the absorbance change at 593 nm in test reaction mixtures with those containing range
with antioxidant mixtures. The free radical scavenging activity, exhibited by percentage rediction
of Ferric to ferrous ion, in the methanolic extracts of different plants decreased in the following
order: Curry Leaf > Cannabis > Neem > Methi > Beet Root > Spring Onion. Highest scavenging
activity was shown by methanolic extract of curry leaf (91.1±0.5) and lowest scavenging activity
was exhibited by methanolic extract of spring onion (31±0.7) (Figure 6).
Fig.6: FRAP Assay (Ferric reducing ability in mM/dry extract)
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Comparative study of DPPH and FRAP assays of curry leaf, which showed, maximum
scavenging activity with the mixture of all the methanolic extracts (polyherbal formulation),
showed that the activity was lesser in polyherbal formulation as compared to curry leaf extract
(Figure 6). Results showed that some component in the formulation is present which is inhibiting
the DPPH and FRAP scavenging activity.
Fig. 7: Comparative study of DPPH and FRAP Assay of Curry Leaf Extract and Mixture of
all the Methanolic Extracts (Polyherbal Formulation)
Table 1: Flavanoids, Tanin, Saponin and Alkaloids content in the studied plant methanolic
extracts.
Polyphenols are the major plant compounds with antioxidant activity. This activity is believed to
be mainly due to their redox properties which play an important role in adsorbing and neutralizing
free radicals, quenching singlet and triplet oxygen or decomposing peroxides. Methanolic extract
of curry leaf showed the presence of flavanoids only and cannabis extract showed the presence of
flavanoids and tannin. Neem extract showed the presence of flavanoids, saponin and alkaloids
wheras methi extract showed the presence of flavanoids, tannin and saponin. Beet root extract
showed the presence of all the components tested by us whereas spring onion extract showed the
presence of only alkaloids. The result of the present study showed that the methanolic extract of
Plant Extracts Flavanoids Tannin Saponin Alkaloids
Curry Leaf + - - -
Cannabis + + - -
Neem + - + +
Methi + + + -
Beet Root + + + +
Spring Onion - - - +
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curry leaf which contain only flavanoids, exhibited the greatest antioxidant activity. The high
scavenging property of curry leaf extract may be due to hydroxyl groups existing in the phenolic
compounds’ chemical structure that can provide necessary component as a radical scavenger.
These results strongly suggest that phenolics are important components of the plants especially
flavanoids and some of its pharmacological effects could be attributed to the presence of its
valuable constituents. Free radicals are often generated as byproducts of biological reactions or
from exogenous factors. The involvements of free radicals in the pathogenesis of a large number
of diseases are well documented. A potent scavenger of free radicals may serve as a possible
preventative intervention for the diseases. All of the extracts in this research exhibited different
extent of antioxidant activity. Curry leaf extract showed highest scavenging of DPPH free radical.
This may be related to high levels of flavanoids in the methanolic extract of curry leaf.
ACKNOWLEDGEMENT
We are thankful to Dr. C. Tandon, Director, Amity Institute of Biotechnology, Amity University,
Noida for providing requisite facilities for the research work. We are highly grateful to Council of
Science & Technology (U.P.) for the financial support granted for completion of the research
work.
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