International Journal of Biotechnology, Vol. 4 (6), 568-574, June, 2011Available online at http://ijbiotch.webs.com/ijb462011.htmISSN 2221-6766 © 2011 Academic Journals

Full Length Research Paper

In-vitro and In-vivo antioxidant activity and total phenolic content of Pongamia pinnata (L.) Pierre: An

important medicinal plant

Savita Sagwan, D. V. Rao and R. A. Sharma

Biotechnology lab, Lab no. 5, Department of Botany, University of Rajasthan, Jaipur. India

Accepted on 11 May, 2011

Pongamia pinnata (L.) Pierre (Family: Fabaceae), popularly known as “Karanj”, is a medium-sized glabrous tree which have immense medicinal value. In present study callus was raised from the internode. Maximum callus was obtained on MS medium supplemented with combination of NAA (6mg/L), BAP (1mg/L), TDZ (0.02mg/L) along with additives like citric acid (50mg/L) and ascorbic acid (100mg/L). The callus and different plant parts were used for total phenolic contents and antioxidant activity. The total phenol varied from 7.58 ± 0.15 to 12.2 ± 0.22 mg/gdw in the various extracts. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effect of the extracts was determined spectrophotometrically. The highest radical scavenging effect was observed in leaves of Pongamia pinnata with IC50 = 40µg/ml. The greater amount of phenolic compounds leads to more potent radical scavenging effect as shown by leaves extract of Pongamia pinnata.

Key words: Pongamia pinnata (L.), phenolic contents, antioxidant activity, DPPH.

INTRODUCTION

Plants are the basis of life on earth and are central to people’s live hoods. India is endowed with a rich wealth of medicinal plants. India recognizes more than 2500 plant species which have medicinal values (Kirtikar and Basu, 1995). A large number of medicinal plants are being exploited from the natural flora for the commercial production of drugs. In order to conserve the natural flora and meet increasing demand for plant based drugs, alternative methods such as tissue culture has become popular for the production of therapeutic compounds from uniformly vigorous plants (Heble and Roja, 1997; Hirai et al., 1997).

Our body is exposed to a large number of foreign chemicals everyday (Santhakumari et al, 2003). The most of which are man-made and our inability to properly metabolize them negatively affects our health by the generation of free radicals. Free radicals are also generated during normal metabolism of aerobic cells (Carmen and Florin, 2009; Ghaseme et al, 2009; Li et al, 2008; Hunag et al, 2005; Zaporozhets et al, 2004; Odukoya

*Correspondence author: savita.sagwan@gmail.com

et al, 2007). The oxygen consumption inherent in cells growth leads to the generation of series of oxygen free radicals. Highly active free radicals and their uncontrolled production are responsible for numerous pathological processes such as cell tumour (prostate and colon cancers) and coronary heart diseases (Karadenz et al, 2005; Barros et al, 2007; Chanwitheesuk et al, 2005; Marinova et al, 2005; Jagadish et al, 2009).

Various reactive species include- superoxide anions, hydrogen peroxide, and hydroxyl, nitric oxide and peroxynitrite radicals, which play an important role in oxidative stress related to the pathogenesis of various important diseases (Kratchanova et al., 2010, Halliwell and Gutteridge,1999, Finkel and Holbrook, 2000). These species cause the cellular damage by reacting with various biomolecules such as proteins, membrane lipids, enzymes and nucleic acid (Veerapur et al.,2010). This Damage is the major contributor of the production of free radicals in healthy individuals and is balanced by the antioxidative defense system.

The screening studies for antioxidant properties of medicinal and food plants have been performed increasingly for the last few decades in hope of finding an efficient remedy for several present-day diseases and means to delay aging symptoms (Halliwell, 2008). Antioxidants are important in the prevention of various

Sagwan et al., 2011 569

human diseases. Naturally occurring antioxidants in leafy vegetables and seeds, such as ascorbic acid, vitamin E, and phenolic compounds, possess the ability to reduce the oxidative damage associated with many diseases, including cancer, cardiovascular diseases, cataracts, atherosclerosis, diabetes, arthritis, immune deficiency diseases and ageing.(Pietta et al., 1998; Lee et al., 2000; Middleton et al., 2000).

Pongamia pinnata (L.) Pierre (Family:-Fabaceae), is a medicinal plant locally known as “ Karanj” in Hindi, and Indian beech in English, is found in the littoral regions of south-east Asia, Australia and Fiji (Chopra et al., 1998; Simin et al., 2002). Traditionally its bark is used in pile; leave are effective as medicated bath and rheumatic pains and the seeds are used in hypertension, bronchitis, whooping cough, skin diseases and rheumatic arthritis (Ballal, 2005; Tanaka et al., 1992; Carcache et al., 2003). Roots are used for cleaning gums, teeth, and ulcers also effective in gonorrhea (Rastogi and Malhotra , 2001; Chauhan and Chauhan ,2002). Flowers used for diabetes. In Ayurveda and Unani medicine, used as anti inflammatory, anti-plasmodial, anti-noneceptive, anti-hyperglycemic, anti-lipodoxidative, antidiarrheal, anti-ulcer, anti-hyper ammonic and antioxidant activity. In the present study, callus of Pongamia pinnata was raised and comparative study of phenolic content and antioxidant activity of root, stem, leaves, fruit and callus was carried out.

MATERIAL AND METHODS

Plant material and extract preparation

The root, stem, leaf, and seeds of Pongamia pinnata were collected from field-grown plants at nursery of University of Rajasthan and authenticated by Herbarium, Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India. Plant materials were washed with distilled water and dried at room temperature. The dried samples were manually ground to a fine powder. The plant extracts were prepared as described earlier (Aqil and Ahmad; 2007) with little modification. 50gm of dry plant powder were soaked in 500ml of methanol for 3-5 days with intermittent shaking. At the end of extraction, it was passed through Whatman filter paper No.1 (Whatman Ltd., England). This methanolic filtrate was concentrated under reduced pressure on rotary evaporator at 40°C for further use.

Chemicals

All the chemicals and growth regulators were used are analytical grade and purchased from Hi Media Pvt. Ltd. Mumbai, India.

Callus induction

Internodes were surface sterilized by 1 % Teepol for 15 min followed by 0.1 % mercuric chloride for 10 min, and then rinsed thoroughly with sterile distilled water. The internodes were inoculated in the MS medium (Murashige and Skoog, 1962) fortified with different concentrations of 2,

4-D and IAA. The pH of the media was adjusted to 5.8 before autoclaving. All media were autoclaved at 1.06 kg cm-2 and 121ºC for 15 min. The cultures were incubated in growth room at temperature of 25 ± 2 °C and 16-h photoperiod. 20 replicate cultures were established and each experiment was repeated twice and the cultures were observed at regular intervals.

Total phenols determination

Total phenolics content was determined according to the Folin-Ciocalteu method (Bray and Thorpe, 1954), using gallic acid as standards. Extract powders (1mg) were dissolved in 1ml of 50% methanol solution. Extract solution (0.5ml) was mixed with 0.5ml of 50% Folin-Ciocalteu reagent. After of 2-5 min, 1.0ml of 20% Na2Co3 was added to the mixture and incubated for 10 min at room temperature. The mixture was centrifuged at 150 g for 8 min and the absorbance of the supernatant was measured at 750nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram dry weight sample.

DPPH assay

The antioxidant activities were determined using 1,1-diphenyl-2-picryhydrazyl (DPPH) as a free radical. Experiments were initiated by preparing a 0.1 mM solution of DPPH and 1mg/ml solution of different plant parts extracts in methanol was prepared. Different concentrations (10, 50, 100,200,400,600,800,1000 µg/ml) of test plant samples were mixed with 2ml of DPPH. After 30 min of incubation at room temperature the reduction of the DPPH free radical was measured by reading the absorbance at 515nm. DPPH is a purple colored stable free radical; when reduced it becomes the yellow-colored diphenylpic-rylhydrazine. Initally, absorption of blank sample containing the same amount of methanol and DPPH solution was prepared and measured as control (Brand Williams, 1995). Ascorbic acid (Merck; M.W. 176.13) was used as standard. The experiment was carried out in triplicate. The inhibitory effect of DPPH was calculated according to the following formula:

Inhibition (%) = [(Absorbance of control – Absorbance of sample)/ Absorbance of control] × 100

The antioxidant activity of each sample was expressed in terms of IC50 (micromolar concentration required to inhibit DPPH radical formation by 50%, calculated from the inhibition curve (HSU, 2006; Fejes et al., 2000; Yokozawa et al., 1998).

RESULT AND DISCUSSION

Callus Induction

MS medium supplemented with combination of NAA, BAP and TDZ along with additives like citric acid and ascorbic acid for callus induction. Internodes showed maximum callus formation on MS medium with combination

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Figure 1:- Induction and proliferation of callus from internodes of pongamia pinnata (on MS medium supplemented with combination of NAA,BAP and TDZ along with additives like citric acid and ascorbic acid.)

A. Direct callus initiation after one week of cultureB. Callus after 4 weeks of cultureC. Callus after 6 weeks of cultureD. Callus after 8 weeks of culture

of auxin NAA (6mg/L) and cytokinin BAP (1mg/L), TDZ (0.02mg/L) along with additives like citric acid (50mg/L) and ascorbic acid (100mg/L). Similar result of using auxins, cytokinin and additives for callus induction was also observed by Keresa et al., 2009 in Iris adriatica Trinajstić ex Mitić. However in contrast to this Amiri et al., 2011, reported that only one auxin 2,4-D is responsible for maximum callus growth in Datura stramonium L.

The callus obtained from above observation wascompact and greenish yellow colored. Callus obtained after 8 weeks of culture as shown in figure-1 was further evaluated for phenolic content and antioxidant activity.

Total phenol contents of the extracts

Phenolic compounds are a class of antioxidant agents which act as free radical terminators (Shahidi and Wanasundara, 1992). Based on the absorbance values after reaction with Folin–Ciocalteu reagent, results of the colorimetric analysis are given in Table-1. The amount of total polyphenols was higher in leaf (12.2 ± 0.22 mg/gdw) and lower amount was recorded in callus (7.58 ± 0.15mg/gdw) as shown in figure-2.Similar result was also observed by Tanwer et al., 2010 in Spilanthes acemella Murr. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donars and singlet oxygen quenchers (Hasan et al., 2009).According to our study the high

contents of phenol in pongamia pinnata leaf can explain its high free radical scavenging activity.

Antioxidant activity

Free radicals are involved in many disorders like neurodegenerative diseases, cancer and AIDS. Antioxidants through their scavenging power are useful for the management of these diseases. DPPH stable free radical method and is relatively rapid and sensitive way which when encounters proton donors such as antioxidants, the radicals get quenched and absorbance gets reduced, and thus used to survey the antioxidant activity of a specific compound or plant extracts (Koleva et al., 2002, Qureshi et al., 2010).

From the Fig.5 and 6, it is found that IC50 of the leaf extract of Pongamia pinnata is 40µg/ml which indicates the remarkable antioxidant activity of the extract. The antioxidant activity may be due to the presence of phenolic hydroxyl or methoxyl groups, flavone hydroxyl, keto groups, free carboxylic groups and other structural features (Patt and Hudson., 1990, Demiray et al., 2009). From the Fig.3 and 4, it is found that IC50 of the stem of Pongamia pinnata250µg/ml which indicates that low level of antioxidant activity with compare to root, seed, callus and leaf ofPongamia pinnata. The free radical scavenging activity in the different plant part extracts decreased in the following order: Pongamia leaf > Pongamia root > Pongamia callus >

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Table: 1. Total phenolic content of different plant parts of Pongamia pinnata (L.).S.No Plant Parts Total Phenolic Content (mg/gdw)

1. Root 8.76 ± 0.092. Stem 10.24 ± 0.153. Leaf 12.2 ± 0.224. Seed 9.64 ± 0.115. Callus 7.58 ± 0.15

Each value in the table was obtained by calculating the average of three experiments ± standard deviation

0

2

4

6

8

10

12

14

Root Stem Leaf Seed Callus

Ph

eno

l (m

g/g

dw

)

Figure-2 . Phenolic contents of Pongamia pinnata (L.) in mg/gram dry weight.

Pongamia seed > Pongamia stem. Phenolic compounds are considered to be the most important antioxidative components of herbs and other plant materials, and a good correlation between the concentrations of plant phenolic and the total antioxidant capacities has been reported (Ozcan et al., 2009; Nahak and Sahu, 2010, Zaman et al., 2011).

CONCLUSION

Scavenging activity for free radicals of di-phenyl-2-picrylhydrazyl (DPPH) has been widely used to evaluate the antioxidant activity of natural products from plant and microbial sources. The result of the present study showed that the extract of Pongamia pinnata leaf, which contain highest amount of phenolic compounds, exhibited the greatest antioxidant activity. The high scavenging property of Pongamia pinnata leaf may be due to hydroxyl groups existing in the phenolic compounds chemical structure that can provide the necessary component as a radical scavenger. In present study a significant correlation was obtained between antioxidant activity and phenolic content indicating that phenolic compounds contribute significantly to antioxidant activity of the investigated different plant parts of pongamia pinnata.

Acknowledgement

Authors are thankful to Dr. Vinod kumar and Govind vyas , Institute of Applied Sciences and Biotechnology ( Chemind Biosolutions, Jaipur) for providing me necessary facilities, continuous encouragement and support.

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