International Journal of Innovative Pharmaceutical ... · medicinal importance eg. curry leaf, cannabis, neem, methi, ... Phytochemical screening of TLM for the presence of alkaloids,

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.

mailto:[email protected]




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




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




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].




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].




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).





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)




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 - - - +




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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International Journal of Innovative Pharmaceutical ... · medicinal importance eg. curry leaf, cannabis, neem, methi, ... Phytochemical screening of TLM for the presence of alkaloids,