J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240 DOI 10.

1007/s13562-011-0051-8

ORIGINAL ARTICLE

Antioxidant activities of different parts of Gnetum gnemon L.

Dayana Wazir & Syahida Ahmad & Radzali Muse & Maziah Mahmood & M. Y. Shukor

Received: 25 June 2010 / Accepted: 3 March 2011 / Published online: 27 April 2011 # Society for Plant Biochemistry and Biotechnology 2011

Abstract Analyses on biological activities of Gnetum gnemon were done to determine the total phenolic and antioxidants of the plant. Four parts of G. gnemon were used in this study, which were leaf, bark, twig, and seeds of the plant. All parts were extracted in methanol, ethanol, hexane, chloroform and hot water using reflux. The total phenolic content of the plant extracts were determined by using Folin-Ciocalteu method. The results demonstrated that the bark from hot water extract showed the highest total phenolic at 10.710.01 mg GAE/ FDW, while the lowest was chloroform extract of seed at 2.150.01 mg GAE/ FDW. The antioxidant activity of the plant extracts were determined by using DPPH and FRAP assays, respectively. The DPPH results showed that all plant extracts demonstrated weak free radical scavenging activity tested at the final concentration of 300 g/ml. In contrast, the methanolic twig extract showed strong reducing power activity (FRAP) at 83.551.05%, while the hot water seed extract showed the least activity at 41.864.22% tested at the final concentration of 300 g/ml. However, there were no correlation between total phenolics and both antioxidant assays tested. Keywords Gnetum gnemon . Melinjo . Antioxidant . Different polarity
D. Wazir : R. Muse : M. Mahmood : M. Y. Shukor (*) Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia e-mail: yunus@biotech.upm.edu.my S. Ahmad Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

Abbreviations FRAP Ferric-Reducing Antioxidant Power DPPH 2,2-diphenyl-1-picrylhydrazyl TBARS thiobarbituric acid reactive substance TAE tannic acid equivalent

Introduction Phytochemicals are bioactive non-nutrient chemical compounds found in plant, fruit, vegetables and grains. It is known that plant produce phytochemicals to protect itself, but recently, natural phytochemicals have gained a lot of attention as these phytochemicals have shown tremendous advantages to human health (Mehta et al. 2010; Rai et al. 2010a; Rai et al. 2010b; Sharma et al. 2009). Some of the well-known phytochemicals are flavonoids, phenolic compounds, lycopenes and carotenoids. Phytochemicals may act as antioxidant, anti-microbial, enzyme stimulant, hormone analogs and also as novel source of drugs (Lampe and Chang 2007). One of the phytochemicals, stillbenoids and its derivatives are known to have antioxidant property. Stilbenes are produced mainly in grapes and wine. Resveratrol, one of the stilbene compounds, has been reported to have high antioxidant activity than other stilbenes. Studies have shown that resveratrol modulates lipid metabolism, protects low-density lipoproteins against oxidative and free radical damage (Brito et al. 2002; Fremont et al. 1999).However, piceatannol, another stilbene derivative, has been hypothesized to possess anti-oxidant properties because of its structural similarities to resveratrol. Many investigators have hypothesized that the additional hydroxyl group of piceatannol makes it more reactive and is therefore a more potent free radical scavenger compared to resveratrol

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240

235

(Wallace et al. 1978). Research has demonstrated that pterostilbene scavenges for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals with an EC50 value of approximately 30 M (~7.68 g/ml) ( Manickam et al. 1997). EC50 or known as effective concentration are the antioxidant dose needed to cause 50% inhibition in the assay. Further investigations have demonstrated that pterostilbene protects against lipid peroxidation by reducing thiobarbituric acid reactive substance (TBARS) production by 61% in normal human fibroblasts (Stivala et al. 2001). Other investigators have measured the rate constants of pterostilbene with peroxyl radicals and demonstrated that pterostilbene acted only as a mild antioxidant in a homogeneous solution (Amorati et al. 2004). Patricio et al. (2003) showed that stilbene from methanol extract of Yucca periculosa F. Baker demonstrated scavenging properties towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) in TLC and spectrophotometric assays. The occurrence of various stilbenoids has been reported in G. gnemon and other Gnetum species (Huang et al. 2001). Iliya et al. (2003a, b) stated several stilbenoids derived from the roots of G. gnemon show antioxidant activity towards lipid peroxide inhibition and super oxide scavenging activity. Four stilbene derivatives such as gnemonols K and L (resveratrol trimers), M (isorhapontigenin dimer) and gnemonoside K (glucoside of resveratrol trimes) were isolated from the roots of G. gnemon. The scavenging activity for superoxide and lipid peroxide inhibition activity of those stilbenoids showed good activity in the assay tested. Apart from that, three new stilbenes; gnemonols D, E and F, have been isolated from the roots of G. gnemon. The effects of these compounds on lipid peroxide inhibition and scavenging ability for superoxides in a xanthine-xanthine oxidase system have shown potent antioxidant activity (Iliya et al. 2003b). However, the antioxidant properties of G. gnemon extracted using different polar solvents have not been investigated so far. G. gnemon is a gymnosperm species which is categorized under the genus of Gnetum, order of Gnetales and family of Gnetaceae. There are other non-specific scientific names for this species such as G. gnemon var. sylvistris .L, G. acutatum Miq. , G. gnemon var. ovalifolium (Poir) Blume and G. vinosum Elmer. Local names for this plant vary at different places such as belinjo (East Java), voe, khalet (Cambodia), maninjau (Malaysia), melindjo (Singapore) and gnetum, joint fir, or Spanish joint fir (English). Seven species occur in northern South America, two in western tropical Africa and the remaining twenty-one in tropical Asia. There are several varieties of G. gnemon including the tree form (var. gnemon) and the shrub form (vars. brunonianum, griffithii and tenerum) (Harley and Craig 2008). The seeds when cooked or roasted can be processed commercially into a crispy but slighty bitter snack which is famous among Indonesians. The young leaves and young

shoots are commonly used as a vegetable (salad) or ulam in South East Asia. It can be cooked with dried fish or pork and coconut milk. Fiber from the inner bark of the tree can be extracted and made into ropes. Gnetaceae are known to contains stilbenoids and have been used as folk medicine as well as food (Burkhill 1994) and also as resources for the development of new drugs ( Iliya et al. 2003a). According to Pandhair and Sekhon (2006), plants exposed to biotic and abiotic stresses generate more reactive oxygen species (ROS) than their capacity to scavenge them. This is also true in humans and researchers have demonstrated that free radical such as ROS have been implicated in degenerative or pathological process such as aging (Ames et al. 1993; Harman 1995), cancer, heart diseases and Alzheimers disease (Ames 1983; Gey 1990). Antioxidants are inhibitor of lipid peroxidation. This is important in the defence of living cells against oxidative damage (Jovanic 1994). Enzymatic antioxidants including superoxide dismutase, peroxidase, catalase and glutathione reductase have been known to detoxify either by quenching toxic compounds or regenerating antioxidants involving reducing power (Ames et al. 1993). The interest on natural antioxidants from plants is dramatically increasing as they have been associated with the prevention of cancer and cardiovascular disease (Virgili et al. 2001; Thadani et al. 2007) and as a replacement for synthetic antioxidants; the latter are being restricted due to their carcinogenicity (Velioglu et al. 1998). A variety of solvents have been applied to extract bio-active ingredients with various polarities (Akaha et al. 2003). Different solvents such as water, alcohol, acetone and ether are used to extract bio-active compounds from natural products due to their broad solubility into these solvents. Water is generally applied to extract high polar ingredients such as carbohydrates, glycosides and amino acids while methanol is frequently used to extract specific bio-active ingredients from various natural resources. Some studies has reported that type of solvents with varying polarities and pH is one of the important factors to determine the yield of chemical extraction apart from extraction time, temperature, chemical compositions and physical characteristics of the samples (Xu and Chang 2007). Thus, information obtained on the efficiency of various solvents in extracting antioxidants would be valuable and highly relevant.

Materials and method Plant materials Samples of Gnetum gnemon were collected from Bukit Ekspo, Taman Pertanian Universiti, Universiti Putra Malaysia (UPM), Serdang, Malaysia. The collections were done from

236

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240

January 2007 until the 31st of December 2008. Samples were identified and validated by the Faculty of Forestry, UPM, with the voucher number DW01/09. The samples had an average length for leaves from 12.0 to 7.0 cm with widths ranging from 5.0 to 6.5 cm, sticks from 19.0 to 52.0 cm with widths ranging from 0.2 to 0.4 cm, seed from 2.0 to 3 cm long with widths ranging from 1.2 to 1.5 cm and barks from 0.2 to 0.3 cm in thickness. Samples were washed under running tap water and wiped dry. The samples were then freeze dried (50C), and stored in a refrigerator (20C) until use. Extraction of G. gnemon parts Solvents of different polarity (methanol, ethanol, hexane, chloroform and boiling water) were used to determine the yield of soluble constituents from samples of G. gnemon. The extraction technique is a modified form of the Crozier method (Crozier et al. 1997). Briefly, 0.5 g of freeze-dried powders of each part (leaf, seeds, bark and stick) of G. gnemon was weighed and placed into a 100 ml conical flask. Forty ml of different polarity of solvents (v/v) was mixed with 10 ml of 6 M HCl. The mixture was placed in a sample flask (250 ml) and refluxed for 2 h at 90C. Then the mixture was filtered using Whatman No.1 filter paper (Whatman, England), and taken to dryness by using a vacuumed rotary evaporator (Buchii, Switzerland) at 40C. The method of extraction using boiling water was modified from Halici et al. (2005). Two grams of freezedried samples were extracted with 40 ml of boiling water at 100C for 12 min while stirred. The extracts were then filtered through a layer of Whatman No.1 filter paper (Whatman England). The filtered samples were vacuumdried in a rotary evaporator at 40C in order to get the crude extract. The crude extracts were then redissolved in 5 ml of respective solvent to be used in the test analysis. Total phenolic assay Total soluble phenolics content of G. gnemon in different solvent polarity treatments were determined with the FolinCiocalteu reagent according to the method of Slinkard and Singleton (1977). Five hundred microliters of G. gnemon crude extracts was added to 2.5 ml of Folin-Ciocalteu reagent (diluted 1: 10 v/v) and 2.0 ml of sodium carbonate (7.5% g/v). The mixture was vortexed and then incubated at 30C for 90 min. All samples were prepared in triplicate and in the dark. The absorbance for each samples were measured at 765 nm. The amount of total phenolic compound was calculated as mg of gallic acid equivalents (GAE) from the gallic acid standard curve and expressed as mg gallic acid equivalent (GAE) /g freeze dried weight (FDW) of the plant material.

DPPH radical scavenging assay The free radical scavenging activity of G. gnemon samples were measured using the 1,1-diphenyl-1-picryl-hydrazyl (DPPH) methods (Burits and Bucar 2000; Cuendet et al. 1997). One milliliter of G. gnemon crude extracts at different concentrations (100, 150, 200, 250 and 300 g/ml) was mixed with 5 ml of 0.004% (v/v) DPPH that was dissolved in methanol. The absorbance was measured at 517 nm. The above procedures were repeated with BHT using ascorbic acid and -tocopherol as positive controls. Experiments were carried out in triplicate. The percent of DPPH discoloration of the samples was calculated according to the formula: Scavenging activity% A0 A1 100% A0

A0 A1

negative control (absorbance of solution with no samples) positive control (absorbance of solution with samples)

Ferric-reducing antioxidant power assay The ferric reducing property of the G. gnemon extracts was determined using the assay described by Yen and Chen (1995). One milliliter of sample extracts was mixed with 2.5 ml of potassium phosphate buffer (0.2 M, pH 6.6) and 1 g/100 ml potassium ferricyanide. The mixture was incubated at 50C for 20 min. Trichloroacetic acid (10%) was added to the mixture to stop the reaction. Equal volume of distilled water was added followed by 0.5 ml ferric chlorate (0.1 g/100 ml) (FeCl3). The procedure was carried out in triplicate and allowed to stand for 30 min before measuring the absorbance at 700 nm. The above procedures were repeated with BHT using ascorbic acid and tocopherol as positive controls. The percentage of antioxidant activity in FRAP assay of the samples was calculated according to the formula: Antioxidant Activity% A1 A0 100% A1

A0 A1

negative control (Absorbance of the control reaction) positive control (Absorbance in the sample)

Results and discussion The content of extractable phenolic compound in the extracts, expressed in gallic acid equivalents (GAE)/g

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240

237

freeze dried weight (FDW), varied between 2.15 0.006 mg/g FDW and 10.710.008 mg/g FDW (Fig. 1). The highest total phenolic content was obtained from the boiling water extract from bark and sticks while the lowest was from the chloroformic extract from seed. The total phenolic contents of leaf, bark, stick and seed extracted by different solvents ranged from 3.86 to 8.70 mg GAE/ FDW, 3.25 to 10.71 mg GAE/ FDW, 3.13 to 10.3 mg GAE/ FDW and 1.15 to 6.49 mg GAE/ FDW, respectively. Overall, the total phenolic content of seed of G. gnemon was less affected by the different solvents used as there was no significant difference in the yield obtained (p>0.05) while there was a significant difference (p<0.05) in terms of total phenolic contents in the leaf, bark and stick extracts. These results suggest that boiling water extraction gave the highest yield among other solvents for extracting total phenolic content from the bark and sticks of G. gnemon. On the other hand, ethanolic extraction gave the highest yield for extracting total phenolics from leaf and seed of G. gnemon. Others species of Gnetaceae such as Gnetum bucholzianum Welw. and Gnetum africanum Wlw. showed total phenolic activity of 546.973.01 and 543.76 0.74 TAE (mg 100 g1), respectively, using the tannic acid equivalent (TAE) method (Odukoya et al. 2007). Several publications often relate total phenolic activity to antioxidant activity (Santos-Buelga and Scalbert 2000; Rice-Evans et al. 1996; Siddhuraju 2007; Sharma et al. 2009). Odukoya et al. (2007) stated that the variation in antioxidant activity in plants may be due to the differences in the structure of phenolics compound related to their hydroxylation and methylation patterns and the increased lag time as a result of phenols interacting with lipoproteins ( Odukoya et al. 2007) According to Pinelo et al. (2005), total phenols that is capable of being extracted with polar solvents (methanol, ethanol and boiling water) can vary largely as function of the sourced material with values of 1.03103 g/100 g of solid for Gevuina avellana hulls to 3.9 g/100 g of solid found in buckwheat extract. High amount of total phenolic
Total Phenolic (mg GAE/g FDW) 12 10 8 6 4 2 0 Leaf Bark Stick Sample Extract Seed MeOH EtOH Hexane Chloroform Boiling Water

Fig. 1 Total phenolic content of G. gnemon leaf, bark, stick and seed extracts using methanol, ethanol, hexane, chloroform and boiling water

in boiling water extraction of bark and stick of G. gnemon were also reported by Lim and Murtijaya (2007) in a smilar studies with Phyllanthus amarus. The intense heat from boiling water was able to release cell wall phenolics or bound phenolics due to the breakdown of cellular constituents, thus causing more polyphenols to be extracted (Toor and Savage 2006). Moreover, the high temperature from boiling water also increases the solubility of phenols (Amin et al. 2006). Besides, they also stated that one of the factors of antioxidant depletion may due to operations such as peeling, cutting and slicing as it may induce rapid oxidation of natural antioxidants. Reduction of DPPH by antioxidants results in the loss of absorbance. Thus, the degree of discolouration of the solution indicates the scavenging efficiency of the added substances. Extract from G. gnemon gave moderate results for DPPH radical scavenging activity. Figure 2 shows free radical scavenging activity of G. gnemon extract (leaf, bark, stick and seed) using different polarity of solvents (methanol, ethanol, hexane, chloroform and boiling water) at concentrations of 300 g/ml of sample extract. The results showed that the stick extracted with methanol gave the highest scavenging activity at 38.6790.745% while the lowest was bark extracted using hexane at 4.7680.176. The DPPH radical scavenging activity of bark, seed, stick and leaf extracted by different solvent ranged from 4.760.13% to 31.670.28%, 9.382.35% to 37.730.53%, 19.180.40% to 38.680.75%, 20.680.24% to 26.380.13%, respectively. These results suggest that chloroformic, ethanolic and methanolic extraction gave the highest DPPH radical activity in leaf, bark, stick and seed of G. gnemon, respectively. Based on this result, methanolic extraction of G. gnemon is more efficient than boiling water extraction in the case of bark, stick and seeds. This result is in agreement with Oki et al. (2002) which stated that the value of DPPH scavenging activity of red-hulled rice were 3 times higher using methanol than water. Likewise, studies by Pinelo et al. (2005) showed the DPPH scavenging activity of grape pomace was higher in methanol and ethanol solvents compared to water extraction. Kato et al. (2009) reported on the isolation of the stilbenoids from the seed of G. gnemon and their DPPH activites were investigated. Gnetin L, gnetin C, gnemonoside A, gnemonoside C, gnemonoside D and resveratrol from the seed tested could maintain DPPH radical scavenging effects even after 5 h, and their ED50 values decreased with time. Kato et al. (2009) also reported that the delayed oxidative activity of compounds could be because of the donation of multiple electrons to the DPPH single radical. In this assay, the ability of the extracts to reduce iron (III) to iron (II) was determined and compared to that of ascorbic acid, butylated hydrotoluene (BHT) and alphatocopherol, which are known for their strong reducing properties. Figure 3 shows the reductive potential activity

238
100 90 80 70 60 50 40 30 20 10 0
BC BB W SM LC LB W BM BE BH SB W Se e Se dM ed Se FE ed Se H e Alp Se dC ha -to edB W co ph ero l As co BHT rbi cA cid LM LE LH SE SH SC

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240

12 Gallic acid equivalent (mg/g) 10 8 6 4 2 0 0 10 20 30 40 50 60 70 Reducing power activity (%) 80 90 100 R2 = 0.0328

Scavenging effects (%)

Sample Extracts

Fig. 4 Correlation of reducing power activity and total phenolic activity of G. gnemon extracts from different polarity solvent

Fig. 2 Free radical scavenging activity of methanol (M), ethanol (E), hexane (H), chloroform (C), boiling water (BW) extracts of G.gnemon leaf (L), bark (B), stick (S) and seed parts by 1,1-diphenyl-2picrylhydrazyl radicals (DPPH) at 300 ug/ml. Bars indicate standard deviation of triplicate determination (n=3)

of G. gnemon extract (leaf, bark, stick and seed) using different solvent polarity (methanol, ethanol, hexane, chloroform and boiling water) at 300 g/ml of sample extract. Extracts from G. gnemon gave higher results of reductive activity than radical scavenging activity. The highest reducing power activity was from stick extracted with methanol (83.551.05%) while the lowest were boiling water extract of seed (41.864.22%). The reductive activity content of leaf, bark, stick and seed extract in different polarity of solvents ranged from 77.707.54 to 62.031.04, 72.821.62 to 46.891.393, 83.551.05 to 62.130.52, 74.494.65 to 41.8604.219, respectively. These results suggest that boiling water, ethanolic, methanolic, chloroformic and hexanoic extraction from leaf, bark, stick and seed, respectively, gave the highest yields among all solvents in the ferric reducing activity in G. gnemon. This study showed that the antioxidant power in FRAP assay could be referred to as the reducing ability of G. gnemon extracts since this assay gave higher activity compared to the DPPH assay.
Antioxidant activity (%) 100 90 80 70 60 50 40 30 20 10 0
SE SH SC SB Se W ed Se M ed Se E ed Se H e Alp Se dC ha -to edBW co ph ero l As co rbi BHT cA cid BE BH BC BB W SM LE LH LC LB W LM BM

Different studies have indicated that the electron donation capacity (reflecting the reducing power) of bioactive compounds is associated with antioxidant activity Siddhuraju et al. (2002). The presence of reductants, such as antioxidant substances in the samples, causes the reduction of the ferricyanide complex (Fe3+) to the ferrous form (Fe2+). Ferrous ions formed can be monitored by measuring the formation of Perls Prussian blue at 700 nm (Chung et al. 2002) Figures 4 and 5 showed the correlation of total phenolic and antioxidant of G. gnemon extracts in different polarity of solvents. The extracts showed no correlation between antioxidant activity and total phenolics. Correlation of reducing power activity, free radical scavenging activity and total phenolic activity of G. gnemon extracts from different polarity solvent gave a correlation coefficient values (R2) of 0.0328 and 0.0043, respectively. This indicates that the total phenolic activity in G. gnemon extracts have no correlation with the antioxidant activity in the extracts. This is probably because the samples used are crude extract, and due to the impurities in the extract, this may affect the scavenging activity of G. gnemon. G. gnemon extracts using different polarity of solvents showed antioxidant activity in several antioxidant assays such as total phenolic, DPPH radical scavenging and FRAP assays. The results showed that samples extracts using polar
12 Gallic acid equivalent (mg/g) 10 8 6 4 2 0 0 20 40 60 80 100 Free radical scavenging activity (%) R2 = 0.0043

Sample Extracts

Fig. 3 Reductive potential of methanol (M), ethanol (E) hexane (H), chloroform (C), boiling water (BW) extracts of G. gnemon leaf (L), bark (B), stick (S) and seed parts at 300 g/ml. Bars indicate standard deviation of triplicate determination (n=3)

Fig. 5 Correlation of free radical scavenging activity and total phenolic activity of G. gnemon extracts from different polarity solvent

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240

239 Iliya I, Ali Z, Tanaka T, Iinuma M, Furusawa M, Nakaya K, Shirataki Y, Murata J, Darnaedi D, Matsuura N, Ubukata M (2003b) Three new trimeric stilbene from Gnetum gnemon. Chem Pharm Bull 51:8588 Jovanic SV (1994) Flavonoids as antioxidants. J Am Chem Soc 116:48464851 Kato E, Tokunaga Y, Sakan F (2009) Stilbenoids isolated from the seeds of melinjo (Gnetum gnemon L.) and their biological activity. J Agric Food Chem 57:25442549 Lampe JW, Chang JL (2007) Interindividual differences in phytochemical metabolism and disposition. Semin Cancer Biol 17:347353 Lim YY, Murtijaya J (2007) Antioxidant properties of Phyllanthus amarus extracts as affected by different drying methods. LWT 40:16641669 Manickam M, Ramanathan JMA, Farboodniay JPN, Chansouria RAB (1997) Anti-hyperglycemic activity of phenolics from Pterocarpus marsupium. J Nat Prod 60:609610 Mehta S, Rai PK, Rai DK, Rai NK, Rai AK, Bicanic D, Sharma B, Watal G (2010) LIBS-based detection of antioxidant elements in seeds of Emblica officinalis. Food Biophysics 5(3):186192 Odukoya OA, Inya-Agha SI, Segun FI, Sofidiya MO, Hori O (2007) Antioxidant activity of selected Nigerian green leafy vegetables. Am J Food Technol 2:169175 Oki T, Masuda M, Kobayashi M, Nishiva Y, Furuta S, Suda I, Sato T (2002) Polymeric procyanidins as radical scavenging components in red hulled rice. J Agric Food Chem 50:75247529 Pandhair V, Sekhon BS (2006) Reactive oxygen species and antioxidants in plants: an overview. J Plant Biochem Biotechnol 15:7178 Patricio TJ, Guillermo A, Alfonso RV, Ana MG, Juan CM, Eduardo A, Carlos LC (2003) Antioxidant and insect growth regulatory activities of stilbenes and extracts from Yucca periculosa. Phytochemistry 64:463473 Pinelo M, Rubilar M, Jerez M, Sineiro J, Nunez MS (2005) Effects of solvent, temperature and solvent-to-solid on the phenolic content and anti-radical activity of extracts from different components of grape pomace. J Agric Food Chem 53:2111 2117 Rai PK, Jaiswal D, Rai DK, Sharma B, Watal G (2010a) Effect of Curcuma longa freeze dried rhizome powder with milk in STZ induced diabetic rats. Indian J Clin Biochem 25(2):175181 Rai PK, Jaiswal D, Rai DK, Sharma B, Watal G (2010b) Antioxidant potential of oral feeding of Cynodon dactylon extract on diabetes induced oxidative stress. J Food Biochem 34(1):7892 Rice-Evans CA, Miller NM, Paganda G (1996) Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 20:93956 Santos-Buelga C, Scalbert A (2000) Proanthocyanidins and tannin like compounds nature, occurrence, dietary intake and effects on nutrition and health. J Sci Food Agric 80:10941117 Sharma RK, Chatterji S, Rai DK, Mehta S, Rai PK, Singh RK, Watal G, Sharma B (2009) Antioxidant activities and phenolic contents of the aqueous extracts of some Indian medicinal plants. J Med Plants Res 3(11):944948 Siddhuraju P (2007) Antioxidant activity of polyphenolic compounds extracted from defatted raw and dry heated Tamarindus indica seed coat. LWT 40:982990 Siddhuraju P, Mohan PS, Becker K (2002) Studies on the antioxidant activity of Indian Laburnum (Cassia stula L.): a preliminary assessment of crude extracts from stem bark, leaves, flowers and fruit pulp. Food Chem 79:6167 Slinkard K, Singleton VL (1977) Total phenol analysis: automation and comparison with manual methods. Am J Ecol Viticulture 28:4955 Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, Forti L, Pagnoni UM, Albini A, Prosperi E, Vannini V (2001)

solvents have antioxidant activities against various antioxidant systems in vitro. This indicates that this plant has a potential to be used as a source of natural antioxidant and also as a food supplement.
Acknowledgements The authors would like to thank Graduate School of Studies,UPM for GRF fellowship and The Department of Biochemistry, Faculty Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), Malaysia for the laboratory facilities.

References
Ames BN (1983) Dietary carcinogen and anticarcinogens: oxygen radicals and degenerative diseases. Science 221:12561264 Ames BN, Shigena MK, Heagen TM (1993) Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci 90:79157922 Akaha PA, Ezike AC, Nwafor SV, Okoli CO, Emwerem NM (2003) Evaluation of the anti-asthmatic property Asystasia gangetica leaf extracts. J Ethnopharmacol 89:2536 Amin I, Norazaidah Y, Hainida KIE (2006) Antioxidant activity and phenolic content of raw and blanched Amaranthus species. Food Chem 94:4752 Amorati R, Lucarini M, Mugnaini V, Pedulli GF (2004) Anti-oxidant activity of hydroxystilbene derivatives in homogenous solution. J Org Chem 69:71017107 Brito P, Almeida LM, Dinis TCP (2002) The interaction of resveratrol with ferrylmyoglobin and peroxynitrite; protection against LDL oxidation. Free Radic Biol Med 36:621631 Burkhill HM (1994) The useful plants of West Africa. Vol 2, Royal Botanic Gardens Kew, pp 168 Burits M, Bucar F (2000) Antioxidant activity of Nigella sativa essential oil. Phytotheraphy Res 14:323328 Chung YC, Chang WW, Chao WW (2002) Antioxidative activity and safety of the 50% ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1. J Agr Food Chem 50:24542458 Crozier A, Jensen E, Lean MJ, Morags M (1997) Quantitative analysis of flavonoids by reversed-phase high-performance liquid chromatography. J Chromatogr A 761:315321 Cuendet M, Hostettmann K, Potterat O (1997) Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helv Chim Acta 80:11441152 Gey KF (1990) The antioxidant hypothesis of cardiovascular disease: epidemiology and mechanisms. Biochem Soc Trans 18:10411045 Fremont L, Belguendouz L, Delpal S (1999) Antioxidant activity of resveratrol and alcohol-free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. Life Sci 64:25112521 Halici M, Odabasoglu F, Suleyman H, Cakir A, Aslan A, Bayir Y (2005) Effects of water extract of Usnea longissima on antioxidant enzyme activity and mucosal damage caused by indomethacin in rats. Phytomedicine 12:656662 Harley IM, Craig RE (2008) Species profiles for Pacific Island agroforestry, www.tradiotionaltree.org. Accessed on 2008 Harman D (1995) Role of antioxidant nutrient in aging: overview. Age 18:5162 Huang KS, Li RL, Wang YH, Lin M (2001) Three new stilbene trimers from the lianas of Gnetum hainanense. Planta Med 67:6164 Iliya I, Ali Z, Tanaka T, Iinuma M, Furusawa M, Nakaya K, Murata J, Darnaedi D, Matsuura N, Ubukata M (2003a) Stilbene derivaties from Gnetum gnemon Linn. Phytochemistry 62:601606

240 Specific structural determinants are responsible for the antioxidant activity and the cell effects of resveratrol. J Biol Chem 276:2258622594 Thadani MB, Patel VH, Subhash R (2007) In vitro antioxidant activities of Stevia rebaudiana leaves and callus. J Food Compos Anal 20:323329 Toor RK, Savage GP (2006) Effect of semi-drying on the antioxidant components of tomatoes. Food Chem 94:9097 Velioglu YS, Mazza G, Gao L, Oomah BD (1998) Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J Agric Food Chem 46:41134117

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240 Virgili F, Scaccini C, Parker L, Rimbach G (2001) Cardiovascular disease and nutritional phenolics. In: Pokorny J, Yanishlieva N, Gordon M (eds) Antioxidant in food. Woodhead Publishing Ltd, Cambridge, pp 8799 Wallace JW, Porter PL, Chopin J (1978) C-Glycosylflavones in Gnetum gnemon. Phytochemistry 17:1809181 Xu BJ, Chang SKCA (2007) Comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. J Food Sci 72:159166 Yen GC, Chen HY (1995) Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agric Food Chem 43:2732