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 Gnetumgnemon were done to determine the total phenolic andantioxidants of the plant. Four parts of G. gnemon wereused in this study, which were leaf, bark, twig, and seeds ofthe plant. All parts were extracted in methanol, ethanol,hexane, chloroform and hot water using reflux. The totalphenolic content of the plant extracts were determined byusing Folin-Ciocalteu method. The results demonstratedthat the bark from hot water extract showed the highesttotal phenolic at 10.710.01 mg GAE/ FDW, while thelowest was chloroform extract of seed at 2.150.01 mgGAE/ FDW. The antioxidant activity of the plant extractswere determined by using DPPH and FRAP assays,respectively. The DPPH results showed that all plantextracts demonstrated weak free radical scavenging activitytested at the final concentration of 300 g/ml. In contrast,the methanolic twig extract showed strong reducing poweractivity (FRAP) at 83.551.05%, while the hot water seedextract showed the least activity at 41.864.22% tested atthe final concentration of 300 g/ml. However, there wereno correlation between total phenolics and both antioxidantassays tested.

Keywords Gnetum gnemon . Melinjo . Antioxidant .

Different polarity

AbbreviationsFRAP Ferric-Reducing Antioxidant PowerDPPH 2,2-diphenyl-1-picrylhydrazylTBARS thiobarbituric acid reactive substanceTAE tannic acid equivalent

Introduction

Phytochemicals are bioactive non-nutrient chemical compoundsfound in plant, fruit, vegetables and grains. It is known that plantproduce phytochemicals to protect itself, but recently, naturalphytochemicals have gained a lot of attention as thesephytochemicals have shown tremendous advantages to humanhealth (Mehta et al. 2010; Rai et al. 2010a; Rai et al. 2010b;Sharma et al. 2009). Some of the well-known phytochemicalsare flavonoids, phenolic compounds, lycopenes and carote-noids. Phytochemicals may act as antioxidant, anti-microbial,enzyme stimulant, hormone analogs and also as novel sourceof drugs (Lampe and Chang 2007).

One of the phytochemicals, stillbenoids and its deriva-tives are known to have antioxidant property. Stilbenes areproduced mainly in grapes and wine. Resveratrol, one ofthe stilbene compounds, has been reported to have highantioxidant activity than other stilbenes. Studies haveshown that resveratrol modulates lipid metabolism, protectslow-density lipoproteins against oxidative and free radicaldamage (Brito et al. 2002; Fremont et al. 1999).However,piceatannol, another stilbene derivative, has been hypoth-esized to possess anti-oxidant properties because of itsstructural similarities to resveratrol. Many investigatorshave hypothesized that the additional hydroxyl group ofpiceatannol makes it more reactive and is therefore a morepotent free radical scavenger compared to resveratrol

D. Wazir :R. Muse :M. Mahmood :M. Y. Shukor (*)Faculty of Biotechnology and Biomolecular Sciences,Universiti Putra Malaysia,43400 UPM Serdang,Selangor, Malaysiae-mail: yunus@biotech.upm.edu.my

S. AhmadInstitute of Bioscience, Universiti Putra Malaysia,43400 UPM Serdang,Selangor, Malaysia

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240DOI 10.1007/s13562-011-0051-8

(Wallace et al. 1978). Research has demonstrated thatpterostilbene 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 aseffective concentration are the antioxidant dose needed tocause 50% inhibition in the assay. Further investigationshave demonstrated that pterostilbene protects against lipidperoxidation by reducing thiobarbituric acid reactive substance(TBARS) production by 61% in normal human fibroblasts(Stivala et al. 2001). Other investigators have measured therate constants of pterostilbene with peroxyl radicals anddemonstrated that pterostilbene acted only as a mild anti-oxidant in a homogeneous solution (Amorati et al. 2004).Patricio et al. (2003) showed that stilbene from methanolextract of Yucca periculosa F. Baker demonstrated scavengingproperties towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) inTLC and spectrophotometric assays.

The occurrence of various stilbenoids has been reportedin G. gnemon and other Gnetum species (Huang et al.2001). Iliya et al. (2003a, b) stated several stilbenoidsderived from the roots of G. gnemon show antioxidantactivity towards lipid peroxide inhibition and super oxidescavenging activity. Four stilbene derivatives such asgnemonols K and L (resveratrol trimers), M (isorhapontigenindimer) and gnemonoside K (glucoside of resveratrol trimes)were isolated from the roots of G. gnemon. The scavengingactivity for superoxide and lipid peroxide inhibition activityof 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 effectsof these compounds on lipid peroxide inhibition andscavenging ability for superoxides in a xanthine-xanthineoxidase system have shown potent antioxidant activity (Iliyaet al. 2003b). However, the antioxidant properties of G.gnemon extracted using different polar solvents have notbeen investigated so far.

G. gnemon is a gymnosperm species which is categorizedunder the genus of Gnetum, order of Gnetales and family ofGnetaceae. There are other non-specific scientific names forthis species such as G. gnemon var. sylvistris .L, G. acutatumMiq. , G. gnemon var. ovalifolium (Poir) Blume and G.vinosum Elmer. Local names for this plant vary at differentplaces such as belinjo (East Java), voe, khalet (Cambodia),maninjau (Malaysia), melindjo (Singapore) and gnetum,joint fir, or Spanish joint fir (English). Seven species occurin northern South America, two in western tropical Africaand the remaining twenty-one in tropical Asia. There areseveral varieties of G. gnemon including the tree form (var.gnemon) and the shrub form (vars. brunonianum, griffithiiand tenerum) (Harley and Craig 2008).

The seeds when cooked or roasted can be processedcommercially into a crispy but slighty bitter snack which isfamous among Indonesians. The young leaves and young

shoots are commonly used as a vegetable (salad) or ulamin South East Asia. It can be cooked with dried fish or porkand coconut milk. Fiber from the inner bark of the tree canbe extracted and made into ropes. Gnetaceae are known tocontains stilbenoids and have been used as folk medicine aswell as food (Burkhill 1994) and also as resources for thedevelopment of new drugs ( Iliya et al. 2003a).

According to Pandhair and Sekhon (2006), plantsexposed to biotic and abiotic stresses generate morereactive oxygen species (ROS) than their capacity toscavenge them. This is also true in humans and researchershave demonstrated that free radical such as ROS have beenimplicated in degenerative or pathological process such asaging (Ames et al. 1993; Harman 1995), cancer, heartdiseases and Alzheimers disease (Ames 1983; Gey 1990).

Antioxidants are inhibitor of lipid peroxidation. This isimportant in the defence of living cells against oxidativedamage (Jovanic 1994). Enzymatic antioxidants includingsuperoxide dismutase, peroxidase, catalase and glutathionereductase have been known to detoxify either by quenchingtoxic compounds or regenerating antioxidants involvingreducing power (Ames et al. 1993).

The interest on natural antioxidants from plants isdramatically increasing as they have been associated withthe prevention of cancer and cardiovascular disease (Virgiliet al. 2001; Thadani et al. 2007) and as a replacement forsynthetic antioxidants; the latter are being restricted due totheir carcinogenicity (Velioglu et al. 1998).

Avariety of solvents have been applied to extract bio-activeingredients with various polarities (Akaha et al. 2003).Different solvents such as water, alcohol, acetone and etherare used to extract bio-active compounds from naturalproducts due to their broad solubility into these solvents.Water is generally applied to extract high polar ingredientssuch as carbohydrates, glycosides and amino acids whilemethanol is frequently used to extract specific bio-activeingredients from various natural resources. Some studies hasreported that type of solvents with varying polarities and pH isone of the important factors to determine the yield of chemicalextraction apart from extraction time, temperature, chemicalcompositions and physical characteristics of the samples(Xu and Chang 2007). Thus, information obtained on theefficiency of various solvents in extracting antioxidants wouldbe valuable and highly relevant.

Materials and method

Plant materials

Samples of Gnetum gnemon were collected from BukitEkspo, Taman Pertanian Universiti, Universiti Putra Malaysia(UPM), Serdang, Malaysia. The collections were done from

J. Plant Biochem. Biotechnol. (JulyDec 2011) 20(2):234240 235

January 2007 until the 31st of December 2008. Sampleswere identified and validated by the Faculty of Forestry,UPM, with the voucher number DW01/09. The sampleshad an average length for leaves from 12.0 to 7.0 cmwith widths ranging from 5.0 to 6.5 cm, sticks from 19.0to 52.0 cm with widths ranging from 0.2 to 0.4 cm, seedfrom 2.0 to 3 cm long with widths ranging from 1.2 to1.5 cm and barks from 0.2 to 0.3 cm in thickness.Samples were washed under running tap water and wipeddry. The samples were then freeze dried (50C), andstored 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 theyield of soluble constituents from samples of G. gnemon.The extraction technique is a modified form of the Croziermethod (Crozier et al. 1997). Briefly, 0.5 g of freeze-driedpowders of each part (leaf, seeds, bark and stick) of G.gnemon was weighed and placed into a 100 ml conicalflask. Forty ml of different polarity of solvents (v/v) wasmixed with 10 ml of 6 M HCl. The mixture was placed in asample flask (250 ml) and refluxed for 2 h at 90C. Thenthe mixture was filtered using Whatman No.1 filter paper(Whatman, England), and taken to dryness by using avacuumed rotary evaporator (Buchii, Switzerland) at 40C.

The method of extraction using boiling water wasmodified from Halici et al. (2005). Two grams of freeze-dried samples were extracted with 40 ml of boiling water at100C for 12 min while stirred. The extracts were thenfiltered through a layer of Whatman No.1 filter paper(Whatman England). The filtered samples were vacuum-dried in a rotary evaporator at 40C in order to get the crudeextract. The crude extracts were then redissolved in 5 ml ofrespective solvent to be used in the test analysis.

Total phenolic assay

Total soluble phenolics content of G. gnemon in differentsolvent polarity treatments were determined with the Folin-Ciocalteu reagent according to the method of Slinkard andSingleton (1977). Five hundred microliters of G. gnemoncrude extracts was added to 2.5 ml of Folin-Ciocalteureagent (diluted 1: 10 v/v) and 2.0 ml of sodium carbonate(7.5% g/v). The mixture was vortexed and then incubated at30C for 90 min. All samples were prepared in triplicateand in the dark. The absorbance for each samples weremeasured at 765 nm. The amount of total phenoliccompound was calculated as mg of gallic acid equivalents(GAE) from the gallic acid standard curve and expressed asmg 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 sampleswere 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 atdifferent concentrations (100, 150, 200, 250 and 300 g/ml)was mixed with 5 ml of 0.004% (v/v) DPPH that wasdissolved in methanol. The absorbance was measured at517 nm. The above procedures were repeated with BHT usingascorbic acid and -tocopherol as positive controls. Experi-ments were carried out in triplicate. The percent of DPPHdiscoloration of the samples was calculated according tothe formula:

Scavenging activity % A0 A1 A0

100%

A0 negative control (absorbance of solution with nosamples)

A1 positive control (absorbance of solution with samples)

Ferric-reducing antioxidant power assay

The ferric reducing property of the G. gnemon extracts wasdetermined using the assay described by Yen and Chen(1995). One milliliter of sample extracts was mixed with2.5 ml of potassium phosphate buffer (0.2 M, pH 6.6) and1 g/100 ml potassium ferricyanide. The mixture wasincubated at 50C for 20 min. Trichloroacetic acid (10%)was added to the mixture to stop the reaction. Equal volumeof distilled water was added followed by 0.5 ml ferricchlorate (0.1 g/100 ml) (FeCl3). The procedure was carriedout in triplicate and allowed to stand for 30 min beforemeasuring the absorbance at 700 nm. The above procedureswere repeated with BHT using ascorbic acid and -tocopherol as positive controls. The percentage of antiox-idant activity in FRAP assay of the samples was calculatedaccording to the formula:

Antioxidant Activity % A1 A0 A1

100%

A0 negative control (Absorbance of the control reaction)A1 positive control (Absorbance in the sample)

Results and discussion

The content of extractable phenolic compound in theextracts, expressed in gallic acid equivalents (GAE)/g

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

freeze dried weight (FDW), varied between 2.150.006 mg/g FDW and 10.710.008 mg/g FDW (Fig. 1).The highest total phenolic content was obtained from theboiling water extract from bark and sticks while the lowestwas from the chloroformic extract from seed. The totalphenolic contents of leaf, bark, stick and seed extracted bydifferent 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/ FDWand 1.15 to 6.49 mg GAE/ FDW, respectively.

Overall, the total phenolic content of seed of G. gnemonwas less affected by the different solvents used as there wasno significant difference in the yield obtained (p>0.05)while there was a significant difference (p /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 150 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles true /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PreserveEditing false /UntaggedCMYKHandling /UseDocumentProfile /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ]>> setdistillerparams> setpagedevice