RAAMAN NANJIAN* SRIRAM CHANDRASEKARAN ABSTRACTSRIRAM CHANDRASEKARAN *Professor, Natural Products and Tissue Culture Laboratory, Cent re for Advanced Studies in Botany, University

JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.625, ISSN: 2320-5083, Volume 3, Issue 6, July 2015

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IN VITRO ANTIOXIDANT AND ANTIPROLIFERATIVE ACTIVITIES OF WILD PLANT AND CALLUS EXTRACTS OF INDIGOFERA ASPALATHOIDES VAHL.EX.DC

RAAMAN NANJIAN*

SRIRAM CHANDRASEKARAN**

*Professor, Natural Products and Tissue Culture Laboratory, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India

**Research Scholar, Natural Products and Tissue Culture Laboratory, Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India

ABSTRACT

Indigofera aspalathoides Vahl. ex. DC, belongs to the family Fabaceae and it is

commonly called as wiry indigo. The present study was carried out to study the comparative

account on antioxidant potential and antiproliferative activity between the methanolic extracts

of wild plant and in vitro grown callus. Murashige and Skoog (MS) medium supplemented

with NAA (0.5 mg/L) showed maximum callus induction from root (92%) and nodal explants

(89%). The quantitative phytochemical analysis showed maximum total phenol (382 mg/g)

and total flavonoid content (324 mg/g) in methanolic extract of wild plant followed by callus

(370 mg/g and 318 mg/g). Antioxidant activities by different methods such as, 2,2’-azinobis

(3-ethylbenzo-thiazo-line-6- sulfonate) (ABTS•+) radical scavenging activity,

phosphomolybdenum activity, hydroxyl radical scavenging activity and iron chelating

activity showed maximum antioxidant potential in methanolic extract of wild plant followed

by methanolic extract of callus and standard. Antiproliferative activity of A375 cells showed

IC50 value of 86.29 µg/mL for methanolic extract of callus and 90.76 µg/mL for methanolic

extract of wild plant.

KEYWORDS: Indigofera Aspalathoides, Murashige and Skoog (MS) Medium, Callus

Induction, Antioxidant Activity, Antiproliferative Activity, A375 Cell Line.

INTRODUCTION

Medicinal plants play a key role in the human healthcare. About eighty percent of the

world populations rely on the traditional medicine, which is predominantly based on plant

materials. Large number of medicinal plants and their purified isolated metabolites have been

shown to have beneficial therapeutic potential (Agbar et al., 2008). The plant Indigofera

aspalathoides is commonly called as “Sivanar vembu” in tamil. In the traditional medicinal

system, leaves, flowers and tender shoots are said to posses cooling and demulcent effect.

They are used in the form of decoction for the treatment of leprosy and cancer (kirtikar and


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Basu, 1975). Rajkapoor et al. (2005) and Selvakumar et al. (2011) have reported that the

aqueous extract of I. aspalathoides contains mainly saponins, tannins, carbohydrates and

steroids that have the ability to counteract the adverse biological effect of antioxidant activity.

Natural antioxidants have been studied extensively for decades in order to find compounds

protecting against a number of diseases related to oxidative stress and free radicals. Phenolic

natural products such as flavonoids are of particular interest because of their antioxidant

activity through scavenging oxygen radicals. It has been reported that the ability of these

compounds for scavenging the free radicals, play an important role in many diseases such as

aging, cancer, cardiovascular diseases and inflammatory disorders (Anusuya and Manian,

2013). The plants contain chemical compounds that may be in one way or another

responsible for their healing properties and other functions. In developing countries, it is

estimated that about 80 % of the population rely on traditional medicine for their primary

healthcare (Gurav et al., 2007).

Antioxidants protect against reactive oxygen species (ROS) toxicity by prevention of

ROS formation, by scavenging the reactive metabolites, by the interruption of ROS attack

and converting them to less reactive molecules and by enhancing the resistance of sensitive

biological target to ROS attacks, by facilitating the repair caused by the ROS and by

providing co-factors for the effective functioning of other antioxidants (Sen, 1995).

Developments of life threatening diseases like cancer are linked to the availability of these

antioxidants (Bakasso et al., 2008). The present study was carried out to compare the total

phenol, flavonoid content and antioxidant potential between methanolic extracts of wild plant

and callus of I. aspalathoides.

MATERIALS AND METHODS

Collection of plant material

Fresh aerial plants were collected from Pachaimalai hills, Tambaram sanitorium,

Chennai, India and the plant was identified as Indigofera aspalathoides Vahl. ex. DC. by

Botanical Survey of India (BSI), Coimbatore.

Callus formation from I. aspalathoides

Healthy seeds of I. aspalathoides was surface sterilized with 1% sodium hypochlorite

for 15 min followed by 0.05% mercuric chloride for 3 min and finally rinsed with sterile

distilled water for 5 times and the contamination free seeds was inoculated on MS medium


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(Murashige and Skoog, 1962). The in vitro grown seedling explants such as node and root

was used for callus induction in MS medium amended with different growth hormones such

as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α- naphthalene acetic acid (NAA)

and 2,4-dichlorophenoxyacetic acid (2,4-D) with different concentrations was incubated at

25±2ºC. A 16/8 h (light/dark) photoperiod of cool white light (2000 lux) was provided.

Preparation of extract

Three hundred grams of shade dried whole plant and 10 g of in vitro grown callus

were powdered separately using a mechanical pulverizer. The powder was soaked with

methanol (1:5 w/v) for 72 h and the extract was filtered using Whatmann filter paper and

repeated for 3 times and the pooled extract was concentrated at 45ºC under reduced pressure

using rotary evaporator (IKA RV 10). The concentrated methanolic extracts of wild plant (1

2 g) and in vitro grown calli (3.5 g) was used for further analysis.

Determination of total phenolic content (Singleton and Rossi, 1965)

Two grams of the extracts was centrifuged at 10,000 rpm for 15 minutes at 4°C.

Twenty µL of the extract was prepared using the supernatant and made up with 3 mL of

distilled water. Folin- Ciocalteu phenol reagent (0.5 mL) was added to the tubes. The tubes

were placed in the incubator for 3 minutes at 45°C. After 3 minutes, 2 mL of 20 % Na2CO3

was added to the tubes and kept for incubation after which its absorbance was measured at

650 nm. The total phenol content of the extract was calculated using the formula,

C (GAE) = c x V/M

where, c = concentration of sample from the curve obtained (mg/mL), V = volume

used during the assay (mL), M = mass of the sample used during the assay (g).

Determination of total flavonoids

Flavonoid contents were determined by slightly modified spectrophotometry method

of Karadeniz et al. (2005). One g of dry powder was weighed and ground with 200 mL of 80

% aqueous methanol in a mortar and pestle. The ground sample was filtered and a clear

filtrate was obtained. About 0.5 mL aliquot of the extract was taken in a test tube, 3 mL of

distilled water and 0.3 mL of 5 % sodium nitrite were added. The solution was vortexed and

allowed to stand at room temperature for 5 minutes. To the above solution, 0.6 mL of 10 %

aluminium chloride was added. After 6 minutes, 2 mL of 1 M sodium hydroxide was added


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to the test tube. The solution was made up to 10 mL with distilled water. The absorbance was

read at 510 nm. The total flavonoid content was calculated as quercetin equivalent (mg QE/g)

using the formula,

X = (A.M0/A0.M)

where, A= absorption of sample, A0= absorption of standard (quercetin), M= weight

of sample (mg/mL), M0= weight of quercetin in solution (mg/mL)

Thin layer chromatography (TLC) (Deinstrop, 2000)

Chromatographic separations take advantage of the fact that different substances are

partitioned differently between two phases, a mobile phase and a stationary phase. It is used

to separate the compound present in the fractionized extract and also from callus extract. The

separation of the compound also depends on the usage of the solvent. The concentration (1

mg/mL) of the drug was spotted on the TLC plates and dried. It was then run with different

ratio of solvents. The spots were identified both in the UV light, far light and in the iodine

chamber. Then, Rf value was calculated as the distance travelled by the solute to the distance

travelled by the solvent. The compounds from the spots were scrubbed and used for further

screening

Rf = Distance travelled by the solute / Distance travelled by the solvent

ABTS•+ free radical scavenging activity (Re et al., 1999)

ABTS•+ was dissolved in deionised water to 7 mM concentration, and potassium

persulphate added to a concentration of 2.45 mM. The reaction mixture was left to stand at

room temperature overnight (2 h) in the dark before usage. The resultant intensely-coloured

ABTS•+ radical cation was diluted with 0.01 M PBS (phosphate buffered saline), pH 7.4, to

give an absorbance value of ~0.70 at 734 nm. The sample was diluted with methanol and

treated with ABTS•+ solution made up to a total volume of 1 mL. Absorbance was measured

spectrophotometrically at time intervals of 10 min after addition for a range of 10-100 µg/mL

concentrations for the extract. The assay was performed in triplicates. Fresh stocks of

ABTS•+solution were prepared every five days due to self-degradation of the radical.

Ascorbic acid was used as standard. The result of the assay was expressed relative to ascorbic

acid.

ABTS activity (%) = Abs (control) – Abs (test)/Abs (control) X 100


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where, Abs (control) = absorbance of control, Abs (test) = absorbance of extracts /

standard

Hydroxyl radical scavenging activity (Halliwell et al., 1987; Prieto et al., 1999)

The capacity to scavenge hydroxyl radicals was measured by modification of the

method. The hydroxyl radicals generates by iron- ascorbate-EDTA-H2O2, which then attack

deoxyribose to form thiobarbituric acid reactive substances (TBARS) which yield pink

chromogen at low pH while heating with trichloroacetic acid (TBA). The hydroxyl

scavengers compete with deoxyribose for hydroxyl radicals and decreases TBARS lead to

reduction in formation of pink chromogen. The reaction mixture contained 4 mM

deoxyribose, 0.3 mM ferric chloride, 0.2 mM EDTA, 0.2 mM ascorbic acid, 2 mM H2O2 and

various concentrations (10-100 µg/mL) of the samples were added. The tubes were

capped tightly and incubated for 30 min at 37°C. To the samples, reaction mixture was added

with 0.4 mL of 5% TBA and 0.4 mL of 1% TBA. The reaction mixture was kept in boiling

water bath for 20 min. The intensity of pink chromogen was measured

spectrophotometrically at 532 nm against blank sample. Quercetin was used as a positive

control. All tests were performed in triplicate. The hydroxyl radical scavenging activity of

sample reported as % inhibition of deoxyribose degradation and was calculated by following

equation:

% Inhibition = [(A0 − A1) / A0] × 100

where A0 was the absorbance of the control and A1 was the absorbance in the

presence of the sample or positive control.

Phosphomolybdenum assay (Prieto et al., 1999)

The total antioxidant capacities of the extracts in various concentrations (10-100

µg/mL) were evaluated by the Phosphomolybdenum assay based on the reduction of Mo

(VI) to Mo (V) by the extracts and subsequent formation of a green phosphate-Mo (V)

complex in acidic condition. An aliquot of 100 μL of sample solution was combined

with 1mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM am

monium molybdate) in a 4 mL vial. The vials were capped and incubated at 95ºC for 90 min i

n a water bath. After the samples had cooled to room temperature, the absorbance of the mixt

ure was measured at 695 nm against blank. The results reported (Ascorbic acid equivalent ant

ioxidant activity) are mean values expressed as g of ascorbic acid equivalents/100g extract.


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Ferrous iron chelating assay

The chelating activity of the ferrous ions (Fe2+) was measured according to the

method of Dinis et al. (1994). Briefly, 0.5 mL of different concentrations (10-100 µg/mL) of

the extracts was added to the solution of 2 mM FeCl2 (0.05 mL). The reaction was initiated

by the addition of 5 mM ferrozine (0.2 mL). The mixture was shaken vigorously and left at

room temperature for 10 min. Ferrozine reacted with the divalent iron to form stable magenta

complex which is soluble in water. Absorbance of the solution was then measured

spectrophotometrically at 562 nm. The percentage inhibition of ferrozine-Fe2+ complex

formation was calculated as:

Percentage of inhibition (%) = [(A0 − A1) / A0] × 100

where A0 was the absorbance of the control, A1 was the absorbance of EDTA

(positive control).

Cytotoxicity activity (Sahranavarid et al., 2012)

The MTT assay was used to measure the rate of cancer cell death. A quantity of 0.5-

1×104 cells/mL was seeded into a 96-well plate and after 24 or 48 h, the cells were washed

and maintained with different concentrations of samples (10-100 µg/mL) and incubated for

48 h at 37°C under 5% CO2 atmosphere. The concentration of samples prepared in DMSO,

which were serially diluted in complete culture medium, were added to the cells in triplicate.

After 48 h incubation, the medium in each well was replaced with MTT (3-[4,5-

dimethylthiazol-2-yl]-2,3- diphenyltetrazodium bromide). After 4 h, to the dye DMSO was

added to dissolve the formed violet formazan crystals. The formazan production is directly

proportional to the viable cell number and inversely proportional to the degree of

cytotoxicity. The plates were well shaked for 20 min and the optical density was measured at

570 nm and a reference at 630 nm with a microplate reader. Non-treated cells were used as

negative control and IC50 was calculated as the concentration of samples and 50% inhibition

of cell viability. The percentage of inhibition of the cells were measured using the formula,

Percentage of inhibition =

Mean OD of untreated cells (control) - Mean OD of treated cells

Mean OD of untreated cells (control) X 100


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DNA laddering assay (Solowey et al., 2014)

A quantity of 1 - 5 X 106 cells/mL in Dulbecco’s modified eagle medium (DMEM)

and Modified eagle medium (MEM) and F:12 (1:1) medium was seeded and incubated at

37°C under 5% CO2 atmosphere for 48 h. The IC50 concentration of the isolated compounds

was added and incubated (1.5 mg/mL, final concentration) for 48 h. The cells were

tripsinized, washed twice with PBS, resuspended in 4 mL of lysis buffer (15 mM Tris-HCl;

pH 7.4; 3 mM EDTA pH 8.0; 150 mM NaCl; 0.2% SDS; 10 �g/mL proteinase K and

50 �g/mL RNase) and incubated overnight at 37°C. DNA was extracted by the following

procedure: 4 mL of phenol and chloroform (1:1 ratio to lysis buffer volume) was added and

the solution was centrifuged at 3150 g for 5 min at room temperature. The supernatant was

collected and 1:1 volume of chloroform was added. The solution was centrifuged again at

3150 g and the supernatant was collected. NaCl concentration was adjusted to 0.5 M. Two

volumes of absolute cold (−20°C) ethanol was added and the solution was

incubated at −80°C for 1 h, for the DNA to precipitate. The precipitate was isolated by

centrifugation at 10,000 g for 30 min at 4°C. The pellet was washed twice with 70% cold

ethanol, air-dried and resuspended in 10 mM Tris, 1 mM EDTA, pH 8.0. 10 �g DNA of each

sample was loaded on a 1.5 % agarose gel.

RESULTS AND DISCUSSION

Callus induction from node and root explants

Healthy and surface sterilized seeds of I. aspalathoides, on MS basal medium without

any plant growth hormones, showed maximum of 95% growth of in vitro plantlets. The node

and root from the in vitro grown plantlets were used as explants for callus initiation and

production of secondary metabolites (Fig. 1). Vidoz et al. (2012) reported that cotyledons of

Lotononis bainesii showed 50% of growth on MS medium without the growth hormones.

According to Vipranarayana et al. (2012), seeds of Pterocarpus santalinus germinated on

half strength MS medium supplemented with GA3 showed maximum of 90% growth and in

the medium devoid of GA3, the germination was only 45%. MS basal medium

supplemented with NAA (0.5 mg/L) showed maximum (92%) of callus from root explants on

30 d and 89% of callus obtained from node explants on 45 d. Medium augmented with

different hormones and concentrations yielded 25% and 29% of callus from node and root

explants. NAA supplemented medium showed maximum callus formation when compared

with that of other hormones such as, 2, 4-D, IAA and IBA (Fig. 1). Cenkci et al. (2008)


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reported that medium supplemented with 0.5–20 µM NAA or 2, 4-D showed root explants

better responding and callusing percentages as compared to the cotyledons.

Total phenol and flavonoid estimation

The total phenolic content of methanolic extract of wild plant of I. aspalathoides was

382 mg/g followed by methanolic extract of callus was 370 mg/g equivalence to the gallic

acid used as standard (Fig. 2). The total flavonoid content of methanolic extract of wild plant

was 324 mg/g followed by methanolic extract of callus (318mg/g equivalence) and quercetin

was used as standard (Fig. 3). According to Philips et al. (2010), total phenolic contents of

ethanol and chloroform fractions of I. aspalathoides was 810 and 476 mg/g. Total flavonoids

of the two fractions (ethanol and chloroform) showed 55 and 15 mg/g.

Thin Layer Chromatography for the detection of phytochemicals present

TLC analysis for methanolic extracts of wild plant and callus was carried out with

solvent system toluene: ethyl acetate. Different spots were identified at UV light and with

Iodine vapor. No spot were detected in visible light (Fig. 4).

TLC profiles showed Rf values of 0.11, 0.15, 0.20, 0.33, 0.40, 0.44, 0.52, 0.64, 0.77

and 0.97 under 365 nm and 0.32, 0.39 under iodine vapor for methanolic extract of wild

plant. Methanolic extract of callus showed Rf values of 0.10, 0.14, 0.39, 0.43, 0.48, 0.79 and

0.89 under 365 nm and iodine vapor showed Rf values of 0.84 and 0.90.

According to Puratchikody and Swarnalatha (2011), TLC profile of methanolic

extract of stem of I. aspalathoides showed spots under 365 nm and 254 nm in the solvent

system methanol: chloroform: acetic acid: formic acid (90:5.0:2.5:2.5). Pavala rani

et al. (2013) have reported that ethanolic extract of leaf and aerial parts showed different

spots at different Rf values under 365 nm with the solvent system toluene: ethyl acetate:

acetic acid: methanol (2.5:7.0:0.25:0.25).

Antioxidant activity of I. aspalathoides

With regarding to ABTS•+ activity, methanolic extract of wild plant showed

maximum absorption of 98.84 µg/mL followed by methanolic extract of callus (96.23

µg/mL) which is found to be higher when compared with the standard ascorbic acid (79.72

µg/mL) at 100 µg/mL concentration. The IC50 was 19.16 µg/mL for methanolic extract of

wild plant and 20.78 µg/mL for methanolic extract of callus followed by 41.11 µg/mL for


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standard (Fig. 5). Hydroxyl radical scavenging activity showed maximum absorption of 95.71

µg/mL for methanolic extract of wild plant followed by methanolic extract of callus (92.85

µg/mL) and standard ascorbic acid (94.12 µg/mL) at 100 µg/mL concentration. The IC50

value for methanolic extract of wild plant was 38.89 µg/mL and 50 µg/mL for methanolic

extract of callus followed by standard (41.31 µg/mL) (Fig. 6). Philips et al. (2010) stated that

ethanol and chloroform fractions of I. aspalathoides showed IC50 value for ABTS•+ activity

as 12.1 µg/mL, 8.6 µg/mL and standard as 14.6 µg/mL and hydroxyl radical scavenging

activity of ethanol and chloroform fractions of I. aspalathoides showed IC50 value as 32.5

µg/mL, 21.85 µg/mL and standard as 67.8 µg/mL.

The reduction of Mo (VI) to Mo (V) showed maximum in methanolic extract of wild

plant (72%) followed by methanolic extract of callus (65.27%) which was less when

compared with that of standard (95.65%) at 100 µg/mL concentration (Fig. 7). Fe3+

reduction is often used as an indicator of electron donating activity, which is an important

mechanism of phenolic antioxidant action and can be strongly correlated with other

antioxidant properties. The reducing power of both methanolic extracts of wild plant and

callus was observed maximum (98.5 µg/mL and 92.53 µg/mL) followed by standard

Na2EDTA (91.04 µg/mL) at 100 µg/mL concentration. The IC50 value for methanolic extract

of wild plant was 21.61 µg/mL followed by methanolic extract of callus (31.40 µg/mL) and

standard (41.87 µg/mL) (Fig. 8). According to Thangavel et al. (2014), of the various types

of extracts of Indigofera cordifolia, ethanol extract had more reducing power than other

extracts used. Ebrahimzadeh et al. (2008) have reported that methanol extract of Melilotus

arvensis showed chelating activity of 80 µg/mL concentration.

Antiproliferative activity of A375 cells

Methanol extract of wild plant and callus of I. aspalathoides with the varying

concentrations from 10 μg/mL to 100 μg/mL showed anti-proliferative activity on A375 cell

lines. The normal structural morphology was altered on dose dependent when compared with

that of the normal cell line. IC50 value for the methanolic extract of callus was 86.29 μg/mL

followed by methanolic extract of wild plant (90.76 μg/mL) (Fig. 9, 10).

The DNA fragmentation assay was observed on the IC50 concentrations of the extracts

treated on A375 cells after 24 h incubation. The control cells in the lane 4 of A375 cells

showed intact DNA while the fragmentation was observed in other lanes treated with the

extracts. The DNA fragmentation pattern was observed because of the chromatin


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condensation and nuclear fragmentation which occurred during apoptotic cell death. The

fragmentation pattern showed maximum cell death with the methanolic extract of wild plant

followed by methanolic extract of callus treated on A375 cells (Fig. 11).

According to Ranjitha kumari et al. (2013), methanolic extract of root of

I. aspalathoides showed IC50 value of 50.62 μg/mL and 50.63 μg/mL against NCI h460 (lung

cancer) cells after 24 and 48 h incubation. IC50 value of the extract also showed induction of

DNA fragmentation of the cells treated for 24 and 48h and the maximum was observed after

48h incubation. Chanda and Nagani (2013) suggested that stem extract of

I. aspalathoides possess 95% anticancer activity against various skin disorders and cancer

including Ehrlich’s ascites carcinoma cancer.

CONCLUSION

In the present work, an effective study was carried out to evaluate the antioxidant

potential between methanolic extracts of wild plant and in vitro callus of

I. aspalathoides. The study revealed that the callus also showed maximum antioxidant

activity with that of wild plant due to the presence of secondary metabolites accumulation

present in them. Antiproliferative activity of the A375 cells showed that both methanolic

extracts of wild plant and callus balanced activity against the growth of the cells. Methanolic

extract of callus of I. aspalathoides showed potential inhibitory activity against oxidation due

to the presence of various secondary metabolites present in them.

ACKNOWLEDGEMENTS

One of the author (Sriram Chandrasekaran) thank to University Grants Commission,

New Delhi, for the award of UGC-BSR Herbal Science research fellowship.

REFERENCES

1. Agbar, Z.A.E., Shakya, A.K., Khalaf, N.A. and Haroon, M.A. 2008. Comparative antioxidant activity of some edible plants. Turk j Biol., 32: 193-196.

2. Anusuya, N. and Manian, S. 2013. Antioxidant and free radical scavenging potential of different solvent extracts of Indigofera tinctoria leaves. International Journal of Pharmacy and Pharmaceutical Sciences, 5: 142-147.

3. Bakasso, S., Lamien meda, A., Lamien, C.E., Kiendrebeogo, M., Millogo, J., Ouedraogo, A.G. and Nacoulma, O.G. 2008. Polyphenol contents and antioxidant activities of five Indigofera species (Fabaceae) from Burkina Faso. Pak. J. Biol. Sci., 11: 1429-1435.

4. Cenkci, S., Kargioglu, M., Dayan, S. and Konuk, M. 2008. In vitro propagation of an endangered plant species, Thermopsis turcica (Fabaceae). Biologia, 63: 652-657.

5. Chanda, S. and K. Nagani. 2013. In vitro and in vivo methods for anticancer activity evaluation and some Indian medicinal plants possessing anticancer properties: An Overview. Journal of Pharmacognosy and Phytochemistry, 2: 140-152.


11 www.jiarm.com

6. Deinstrop, E.H. 2000. “Applied Thin-Layer Chromatography”, Wiley-VCH, Weinheim. 320 p. 7. Dinis, T.C.P., Madeita, V.M.C. and Almeida, L.M. 1994. Action of phenolic derivatives

(acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys., 315: 161-169.

8. Ebrahimzadeh, M.A., Pourmorad, F. and Bekhradnia, A.R. 2008. Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. African Journal of Biotechnology, 7: 3188-3192.

9. Gurav, S., Vijay, G., Nandkishore, D. and Arun, P. 2007. Pharmacognosy, Phytochemistry, pharmacology and clinical applications of Gymnema sylverstre RcBr., 1: 414-433.

10. Halliwell, B., Gutteridge, J.M.C. and Aruoma, O.I. 1987. The deoxyribose method: A simple test tube assay for determination of rate constants for hydroxyl radicals. Anal. Biochem., 156: 215-219.

11. Karadeniz, F., Burdurulu, H.S., Koca, N. and Soyer, Y. 2005. Antioxidant activity of selected fruits and vegetables grown in Turkey. Journal of Agriculture and Food Chemistry, 29: 297-303.

12. Kirtikar, K.R. and Basu, B,D. (1975). Glossary of Indian Medical Plants. Volume I. M/S Periodical Experts, Second Edition. Jayed Press, New Delhi, 338 p.

13. Pavala rani, N., C. Moorthi, R. Senthamarai and K. Kathiresan. 2013. Identification and phytochemical evaluation of ethanolic extract of Indigofera aspalathoides (Shivanar vembu). International Journal of Pharmacy and Pharmaceutical Sciences, 5: 508-511.

14. Philips, A., Philip, S., Arul, V., Padmakeerthiga, B., Renju, V., Santha, S. and Sethupathy S. 2010. Free Radical Scavenging Activity of Leaf Extracts of Indigofera Aspalathoides – An in vitro Analysis. J. Pharm. Sci. and Res., 2: 322-328.

15. Prieto, P., Pineda, M. and Aguilar, M. 1999. Spectophotometric quantitative of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal. Biochem., 269: 337‐341.

16. Puratchikody, A. and S. Swarnalatha. 2011. Preliminary phytochemical investigation of methanolic stem extract of Indigofera aspalathoides Vahl. International Journal of Pharmaceutical Sciences Review and Research, 11: 116-118.

17. Rajkapoor, B., Murugesh, N., Chodon, D. and Sakthisekaran, D. 2005. Chemoprevention of N-nitrosodiethylamine induced phenobarbitol promoted liver tumors in rat by extract of Indigofera aspalathoides. Biol. Pharm. Bull., 28: 364-366.

18. Ranjitha kumari, B.D., Rajaperumal, S. and M. Nimmi. 2013. In vitro studies on antimicrobial and antioxidant effect of methanolic extract of Indigofera aspalathoides (Vahl ex DC) and its cytotoxic property against human lung cancer cell line NCI H460. European Journal of Experimental Biology, 3: 18-29.

19. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. 1999. Antioxidant activity applying an improved ABTS radical cation decolourisation assay. Free Radical Biology and Medicine, 26: 1231-1237.

20. Sahranavard, S., F. Naghibi and S. Ghaffari. 2012. Cytotoxic activity of extracts and pure compounds of Bryonia aspera. International Journal of Pharmacy and Pharmaceutical Sciences, 4: 541-543.

21. Selvakumar, S., Karrunkaran, C.M., Rao, M.R.K. and Balasubramanian, M.P. 2011. Inhibitory Effect of Indigofera aspalathoides on 20-methylcholanthrene-induced chemical carcinogenesis. Journal of carcinogenesis, 10: 1-7.

22. Sen, C.K. 1995. Oxygen toxicity and antioxidants: state of the art. Indian journal of physiol. Pharmacol., 39: 177-196.

23. Singleton, V.L. and Jr. Rossi, J.A. 1965. Colorunetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Amer. J. Enol. Viticult., 16: 144-158.

24. Solowey, E., M. Lichtenstein, S. Sallon, H. Paavilainen, E. Solowey and H. L. Galski. 2014. Evaluating medicinal plants for anticancer activity. The Scientific World Journal, 2014: 1-12.

25. Thangavel, D., Govindasamy, J. and Senthil Kumar, R. 2014. In vitro antioxidant and anticancer activities of various extracts of Indigofera cordifolia Roth. Journal of Pharmaceutical Biology, 4: 85-93.

26. Vidoz, M.L., Quesenberry, K.H., Real, D. and Gallo, M. 2012. Plant regeneration of Lotononis bainesii Baker (Fabaceae) through cotyledon and leaf culture. African Journal of Biotechnology, 11: 9724-9731.

27. Vipranarayana, S., Prasad, T.N.V.K.V. and Damodharam, T. 2012. In vitro seed germination and induction of enhanced shoot multiplication in Pterocarpus santalinus Linn.f: An endemic medicinal plant of Seshachalam hills, Tirumala. Int. J. Pure Appl. Sci. Technol., 9: 118-126.


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RAAMAN NANJIAN* SRIRAM CHANDRASEKARAN** ABSTRACTSRIRAM CHANDRASEKARAN** *Professor, Natural Products and Tissue Culture Laboratory, Cent re for Advanced Studies in Botany, University

RAAMAN NANJIAN* SRIRAM CHANDRASEKARAN ABSTRACTSRIRAM CHANDRASEKARAN *Professor, Natural Products and Tissue Culture Laboratory, Cent re for Advanced Studies in Botany, University