In Vitro Studies on Extracts of Pongamia pinnata (L)

1 downloads 0 Views 37KB Size Report
Department of Chemistry, Sri Sathya Sai Institute of Higher Learning, Prasanthi .... deepest gratitude to their Divine Chancellor, Bhagavan Sri Sathya. Sai Baba.
International Journal of Agriculture and Food Science Technology Volume 1, Number 1 (2010), pp. 7-11 © Research India Publications http://www.ripublication.com

In Vitro Studies on Extracts of Pongamia pinnata (L) Pierre Flowers as a Potent Antioxidant Dandamudi Rajesh Babu and G. Nageswara Rao* Department of Chemistry, Sri Sathya Sai Institute of Higher Learning, Prasanthi Nilayam Campus, Prasanthi Nilayam, Anantapur District, Andhra Pradesh-515134. India *Corresponding author E-mail address: [email protected]

Abstracts Ethanolic extract of the flowers of Pongamia pinnata and Et2O, EtOAc, nBuOH and Aq. soluble fractions obtained from the ethanolic extract were tested for their antioxidant activity. The extracts exhibited significant free radical scavenging, reducing power, ferric reducing antioxidant power, chelation with ferrous ions, hydrogen peroxide scavenging and superoxide scavenging activities. Differential pulse voltamograms of the extracts revealed the presence of many electroactive species. The total phenolic content of the extracts were determined as gallic acid equivalents and total flavonoids as quercetin equivalents. Pongamia pinnata flower extracts stand as potent antioxidants.

Keywords: Pongamia pinnata flowers, antioxidant activity, total phenolic content, chemical constituents, differential pulse voltammetry.

Introduction: Pongamia pinnata (L) Pierre (Leguminosae, Papilionaceae) is a deciduous tree widely distributed along South East Asia and Northern Australia. In India it is commonly known as ‘karanj’ or ‘karanja’1. Various parts of this plant have been used in traditional medicine for curing skin diseases, piles, tumors, ulcers etc2. The flowers of this plant have been found to possess antihyperglycemic and antilipidperoxidative properties3. Flavones, isoflavones, chalcones, furanoflavonoids and pyranoflavonoids were reported as main constituents from various parts of the investigated plant 4-9.

8

Dandamudi Rajesh Babu and G. Nageswara Rao

Materials and methods Pongamia pinnata (L) Pierre flowers were collected during full flowering season of April 2009 from the trees in the vicinity of the University Campus. Voucher specimen (No: DOC/09/PPF/01) was deposited in the herbarium of the Department of Biosciences, Prasanthinilayam campus, Sri Sathya Sai Institute of Higher Learning. Flowers were dried in shade and defatted by refluxing with petroleum ether (6080°C). Defatted material was extracted with distilled ethanol. The ethanolic extract was concentrated and fractionated with Et2O, EtOAc and n-BuOH. The remaining extract was labelled as Aq-Soluble fraction. Ethanolic extract and Et2O, EtOAc, nBuOH, Aq-Soluble fractions were used for the invitro analysis. Free radical scavenging capacity of the extracts was analysed by 1,1-diphenyl-2picrylhydrazyl (DPPH) assay as described by Aquino10. Ferric Reducing Antioxidant Power (FRAP) was estimated according to the method described by Iris Benzie and Strain11. Similarly, reducing power12, chelation with ferrous ions13, the ability to scavenge hydrogen peroxide14 and superoxide scavenging activity15 were also analysed. The total phenolic content was determined as gallic acid equivalents and total flavonoids as quercetin equivalents16. Differential pulse voltamograms were recorded using IVUM COMPACTSTAT potentiostat/galvanostat having a closed standard three electrode cell. A glassy carbon electrode was used as working electrode (BAS, Ø = 0.3 cm). Pt foil was used as counter electrode and Ag/AgCl 3M HCl was used as reference electrode. The working electrode was polished in an aqueous suspension of 0.3μm alumina and rinsed with deionised water. After every analysis the electrode was sonicated in 6 M HCl and then in methanol for 3 min17. The operating conditions were 60mV pulse amplitude and 0.030Vs-1 as scan rate. Current density of various fractions was plotted as a function of potential.

Results Table 1 summarises the total phenolic content, total flavonoid content and superoxide scavenging activity of the extracts. The diethyl ether and ethyl acetate fractions contained maximum phenolic and flavonoid content. The data of the various in vitro antioxidant studies are summarized in table 2. The diethyl ether and ethyl acetate fractions exhibited excellent free radical scavenging, reducing power, ferric-reducing antioxidant power, and hydrogen peroxide scavenging activity. The aqueous soluble fraction exhibited excellent chelation with ferrous ions. Differential pulse voltamograms in figure 1 reveal the presence of more than three species in diethyl ether and ethyl acetate fractions at a lower oxidative potential. These species may be responsible for the excellent DPPH and FRAP activities exhibited by these fractions.

In Vitro Studies on Extracts of Pongamia pinnata (L) Pierre Flowers

9

Table 1: Total phenolic content, total flavonoid content and superoxide scavenging activity of EtOH extract and fractions of Pongamia pinnata flowers. Sample

Total phenolic content (mg/g extract)a,b 0.214±0.01 0.472±0.03 0.478±0.02 0.213±0.02 0.140±0.01

Total flavonoid content (µg/g extract)a,c

Ethanol extract Et2O fraction EtOAc fraction n-BuOH fraction Aq.Soluble fraction BHT a Average of four determinations, mean±SE b Expressed as gallic acid equivalents. c Expressed as quercetin equivalents.

60.82±0.02 173.98±0.06 121.03±0.09 53.27±0.03 19.49±0.01

Superoxide Scavenging activity (%) at 1mg/ml concentration of extracta 46±5 29±8 60±8 80±7 46±3 73±7

Table 2: Antioxidant activities of the EtOH extract and fractions from P. pinnata flowers. Conc.(mg/ml)

EtOH Et2O EtOAc fraction extract fraction Free radical scavenging activity ( % Scavenged) BHT Standard(0.1mg/ml): 87.94±0.08 0.1 10.4±1.6 33.4±2.6 33.7±0.1 0.3 36.3±0.7 66.4±1.2 86.6±0.6 0.5 56.5±0.3 94.9±0.1 94.5±0.5 0.7 78.6±0.7 95.1±0.1 94.6±0.2 0.9 92.8±0.6 94.9±0.1 94.0±0.7

n-BuOH fraction

Aq. Soluble fraction

9.9±1.0 30.3±0.3 46.9±1.3 63.4±0.1 76.9±0.2

2.8±0.2 10.7±0.3 20.6±1.4 29.0±1.2 38.8±0.3

FRAP activity (absorbancy, 595nm) BHT Standard(1 mg/ml):1.416±0.018 1 0.262±0.008 0.746±0.015 2 0.540±0.081 1.328±0.016 3 0.789±0.055 1.404±0.002 4 1.027±0.009 1.421±0.003 5 1.169±0.022 1.418±0.003 6 1.269±0.006 1.421±0.001 7 1.327±0.001 1.409±0.001

0.917±0.008 1.325±0.028 1.401±0.008 1.408±0.004 1.405±0.001 1.406±0.001 1.407±0.001

0.197±0.011 0.452±0.018 0.661±0.009 0.827±0.010 0.993±0.009 1.117±0.025 1.225±0.014

0.287±0.005 0.378±0.012 0.457±0.015 0.551±0.015 0.639±0.013 0.741±0.011 0.816±0.007

Reducing power(absorbancy, 700nm) BHT Standard(0.2mg/ml):1.05±0.013 0.5 0.175±0.002 0.685±0.022 1.5 0.618±0.059 1.132±0.028 2.5 1.001±0.020 1.209±0.004 3.5 1.437±0.005 1.247±0.002 4.5 1.808±0.010 1.263±0.002 5.5 1.920±0.014 1.273±0.005 6.5 2.091±0.021 1.278±0.004

0.669±0.009 1.066±0.015 1.149±0.007 1.200±0.008 1.229±0.008 1.245±0.008 1.273±0.002

0.198±0.007 0.707±0.018 0.973±0.021 1.073±0.010 1.131±0.014 1.176±0.006 1.204±0.011

0.24±0.002 0.582±0.015 0.849±0.012 0.992±0.003 1.051±0.015 1.098±0.008 1.169±0.005

10

Dandamudi Rajesh Babu and G. Nageswara Rao

Conc.(mg/ml)

EtOH Et2O extract fraction Chelation with ferrous ions (% Chelation) Ascorbic acid Standard(0.3µg/ml): 65.42±0.38 0.5 1.0 1.5 2.0 2.5 3.0

9.4±0.9 15.0±3.6 36.0±3.1 52.8±0.3 66.5±0.9 70.1±1.3

EtOAc fraction

n-BuOH fraction

Aq. Soluble fraction

1.5±2.1 8.5±4.3 9.5±2.9 12.8±2.6 18.3±5.4 25.3±1.6

3.6±0.1 4.0±3.7 5.7±4.1 11.5±4.8 22.7±0.9 37.4±3.2

7.5±1.5 47.1±3.9 72.1±2.2 84.7±0.7 92.5±1.6 93.7±1.1

80.8±3.3 94.8±0.3 98.0±0.2 97.8±0.7 97.5±0.7 96.8±0.8

25.0±1.2 47.9±2.1 64.3±2.9 76.8±0.7 87.2±0.7 91.1±0.5

20.7±1.1 31.9±3.2 40.9±0.3 47.4±2.2 53.3±1.5 nd

1.8±1.7 2.5±2.3 5.4±1.7 5.2±4.1 7.2±1.0 11.8±5.3

H2O2 Scavenging activity (% Scavenged) TROLOX Standard (1.5mM): 95.±8.6) 0.5 38.5±3.1 84.3±0.9 1.0 63.0±1.1 90.2±0.1 1.5 81.5±0.5 92.2±0.4 2.0 92.2±1.0 97.0±0.4 2.5 96.5±1.5 99.4±0.5 3.0 98.7±1.0 100.0±0.2 nd- not determined Values are average of four determinations, mean ±SE

Our studies conclude that all the extracts and fractions of Pongamia pinnata flowers possess excellent antioxidant activities at various levels. Past work done on the toxicology of the ethanolic extract of the flowers showed that the extract produced no toxic effects upto 3gms/kg in rats even after 72 hours18. Pongamia pinnata is an important medicinal plant which grows widely throughout India. In the light of its excellent free radical scavenging and other antioxidant properties these flower extracts find excellent use as functional tonics. Differential pulse voltamograms of the extract and fractions showed good correlation between the number of electroactive species present and the antioxidant activities exhibited and hence can be used as a helpful tool in assessment of antioxidant activity.

Acknowledgements Authors express deepest gratitude to their Divine Chancellor, Bhagavan Sri Sathya Sai Baba. Financial assistance from UGC under Minor Research Project is thankfully acknowledged.

References [1] [2]

Sathyati, G.V., Gupta, A.K., Tandon, N., In. Medicinal Plants of India (vol 2), Indian Council of Medical Research, New Delhi, 1987, pp. 490-492. Parmar, B.S., Sahravat, K.L., Mukarjee, S.K., Pongamia glabra: constituents and uses. J. Sci. Ind. Res, 1976, 35, 608-611.

In Vitro Studies on Extracts of Pongamia pinnata (L) Pierre Flowers [3] [4] [5] [6] [7] [8]

[9] [10] [11] [12] [13]

[14] [15]

[16] [17] [18]

11

Punitha, R., and Manoharan, S., Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn.) Pierre flowers in alloxan induced diabetic rats, J. Ethnopharmacol.., 2006, 105, 39-46. Liya Li., Xiang Li., Chi Shi., Zhiwei Deng., Hongzheng Fu., Peter Proksh., and Wenhan Lin., Pongamones A–E, five flavonoids from the stems of a mangrove plant, Pongamia pinnata, Phytochemistry, 2006, 67, 1347-52. Tanaka, T., Linuma, M., Yuki, K., Fujy, Y., and Mizuno, M. Two New βHydroxychalcones from the root bark of Pongamia pinnata, Chem. Pharm. Bull., 1991, 39, 1473-75. Tanaka, T., Linuma, M., Yuki, K., Fujy, Y., and Mizuno, M., Flavonoids in root bark of Pongamia pinnata. Phytochemistry, 1992, 31, pp 993-998. Pavanaram, S.K., and Row, L.R. New flavones from Pongamia pinnata (L.) Merr.: identification of compound P, Nature, 1955, 176, 1177-1178. Kitagawa, I., Zang, R., Hori, K., Tsuchiya, K., and Shibuya, H., Indonesian Medicinal Plants. II. Chemical Structures of Pongapinones A and B, Two New Phenylpropanoids from the bark of Pongamia pinnata (Papilionaceae), Chem. Pharm. Bull., 1992, 40, 2041-2043. Yadav, P.P., Ahmed, G., and Mourya, R. Furanoflavonoids from Pongamia pinnata fruits. Phytochemistry, 2004, 65, pp 439-443. Aquino, R., Morelli, S., Lauro, M.R., Abdo, S., Saija, A., and Tomaino, A. Phenolic constituents and antioxidant activity of an extract of Anthurium versicolor leaves. Journal of Natural Products, 2001, 64, 1019. Benzie, I. F. F., and Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure of "Antioxidant Power". The FRAP assay. Anal. Biochem., 1996, 239, 70-76. Oyaizu, M. Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine, Jpn. J. Nutr., 1986, 44, 307–315. Guang-Rong Zhao., Heng-Ming Zhang., Ting-Xiang Ye., Zhi-Jun Xiang., Ying-Jin Yuan., Zhi-Xin Guo., and Li- Bin Zhao. Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B, Food Chem. Toxicol., 2008, 46, 73-81. Pazdzioch-Czochra, M.; Widenska, A. Spectrofluorimetric determination of hydrogen peroxide activity, Anal. Chim. Acta. 2002, 452, 177-184. · Martinez, A. C., Marcelo, E. L., Marco, A. O., and Moacyr., Differential responses of superoxide dismutase in freezing resistant Solanum curtibolum and freezing sensitive Solanum tuberosum subjected to oxidative and water stress. Plant Science, 2001, 160, 505–515. Mehmet Ozturk, Fatima Ayogmus-Ozturk, Mehmet Emin Duru, and Gulacti Topcu. Antioxidant activity of stem and root extracts of Rhubarb (Rheum ribes): An edible medicinal plant, Food Chem., 2007, 103, 623-630. Barros, L., Falcao, S., Baptista, P., Freire, C., Vilas-Boas, M., and Ferreira I.C.F.R., Antioxidant activity of Agaricus spp. mushrooms by chemical, biochemical and electrochemical assays. Food chemistry, 2008,111, 61-66. Annie Shirwaikar, Malini,S., Chandrika Kumari S., Protective effects of Pongamia pinnata flowers against cis-platin and gentamicin induced nephortoxicity in rats, Indian Journal of Experimental Biology, 2002, 40, 316320.

12

Dandamudi Rajesh Babu and G. Nageswara Rao