Optimization of extraction of phenolic antioxidants from peanut skins

Transcription

1 Journl of the Science of Food nd Agriculture J Sci Food Agric 85:33 38 (2005) DOI: 102/jsf.1933 Optimiztion of extrction of phenolic ntioxidnts from penut skins Vleri Nepote, 1 Nelson R Grosso 2 nd Crlos A Guzmán 1 1 Fcultd Ciencis Excts, Fisics y Nturles, Universidd Ncionl Cordob (IMBIV-CONICET), Av Velez Srsfield 1600, 5000 Cordob, Argentin 2 Quimic Biologic, Fcultd Ciencis Agropecuris, Universidd Ncionl Cordob (IMBIV-CONICET), CC 509, 5000 Cordob, Argentin Abstrct: This work ws conducted to optimize the extrction conditions for the best recovery of ntioxidnt compounds from penut skins. The extrcts from the penut skins were obtined by different extrction methods. The extrction conditions were: different ethnol proportions s the solvent (0, 30, 50, 70 nd 96% v/v in distilled wter), different penut skin prticle sizes (0 1, 1 2 nd 2 10 mm nd noncrushed skins), different proportions of solvent/skins (20, 30, 40, 50 nd 60 ml g 1 ), different extrction times (by mcertion nd shking) nd different numbers of extrctions. The different extrcts obtined unr different extrction conditions were compred with specil regrd to yield, totl phenolic compounds nd rdicl scvenging ctivity. The results showed tht the best livery of phenolic compounds ws reched using 70% ethnol, non-crushed penut skins, rtio of solvent/solid of 20 ml g 1, t 10 min shking nd three extrctions. The mximum yield of g g 1 ws record for phenolic compounds when extrcted t the optimum conditions Society of Chemicl Industry Keywords: Archis hypoge; ntioxidnt compounds; penut skins; phenolic compounds INTRODUCTION Argentin is mjor producer nd exporter of penuts, long with Chin, Indi nd USA, mong others. In 2001/2002 the Argentinen penut production ws bout tons. 1 Blnched penut is one of the most importnt products obtined. Blnched penuts re used to obtin products like slted fried penuts, penut butter, rosted penuts, etc. 2 Penut skins re by-product from the blnching process of penut kernels. In Argentin, penuts skins re used to feed cttle. However, their vlue could be incresed if other more vluble uses were found for tht wste. Recently, some works hve been unrtken to study ntioxidnt compounds obtined from Argentinen penut skins. 3 In those studies, the content of phenolic compounds ws found to be between nd g g 1. The ethnolic extrcts exhibited high rdicl-scvenging nd ntioxidnt ctivity, s monstrted in sunflower oil. Pronthocynidins hve been scribed s the most importnt phenolic nd ntioxidnt compounds in penut skins. 4 Recently, resvertrol ws found in penuts s well s in penut skins. 5 7 It hs been monstrted tht resvertrol possesses cncer chemopreventive ctivity in mice nd cts s n ntioxidnt nd ntimutgen. 8 It is lso ssocited with reduction in risk of crdiovsculr disese by inhibiting or ltering pltelet ggregtion nd cogultion, or modulting lipoprotein metbolism It hs been proved tht wine consumption in morte quntities is ssocited with significntly lower risk of crdiovsculr th, which could possibly be explined by higher consumption of wines contining resvertrol. 15 The purpose of this work ws to termine the best conditions of extrction for ntioxidnt compounds from penut skins t lbortory scle using ethnol wter mixtures s the extrction solvent. MATERIALS AND METHODS Mterils Penut skins from Argentinen penut (Archis hypoge cv Florunner, 2001 crop yer) were obtined by blnching nd were provid by the Compny Lorenzti, Ruestch y Ci, Ticino, Córdob, Argentin in July The penut skins were kept in seled plstic bg nd stored t 4 C until use. Corresponnce to: Nelson R Grosso, Quimic Biologic, Fcultd Ciencis Agropecuris, Universidd Ncionl Cordob (IMBIV- CONICET), CC 509, 5000 Cordob, Argentin E-mil: nrgrosso@gro.uncor.edu Contrct/grnt sponsor: CONICET Contrct/grnt sponsor: SECYT-UNC (Received 14 October 2003; revised version received 17 My 2004; ccepted 19 My 2004) Published online 9 September Society of Chemicl Industry. J Sci Food Agric /2004/$3 33

2 V Nepote, NR Grosso, CA Guzmán Optimiztion of ethnol wter mixtures s the extrction solvent Phenolic compounds were extrcted from penut skins nd ftted penut skins using different ethnolic solutions in distilled wter: 96, 70, 50, 30 nd 0% (v/v). To obtin the extrcts, the penut skins (2.5 g) were mcerted with the extrction solvent (50 ml) for 24 h t room temperture in drk room. The extrct ws filtered nd the filtrte ws evported to dryness unr vcuum in rotry evportor (Buchi R 110, Frwil, Switzerlnd) t 35 C. 3 The ftted penut skins were prepred using soxhlet pprtus for 16 h with n-hexne (Anedr, Sn Fernndo, Buenos Aires, Argentin). Yield of dry extrcted mtter It ws termined on dry penut skins bsis following the formul: g g 1 dry extrcted mtter = (g dry extrcted mtter)/(g dry penut skins). Totl phenolic compound The phenolic compound of the extrcts ws termined spectrophotometriclly using the Folin Cioclteu method ccording to Wtermn nd Mole. 16 Penut skin extrct (3 mg) ws dissolved in 10 ml of the extrcting solvent. This solution (0.1 ml) ws trnsferred into 10 ml volumetric flsk. Deionized wter (8.4 ml) nd 0.5 ml Folin Cioclteu regent (Anedr, Sn Fernndo, Buenos Aires, Argentin) were dd nd the contents of the flsk mixed thoroughly. After 1min,1mlN 2 CO 3 solution (20 g in 100 ml of wter) ws dd nd finlly m up to 10 ml with ionized wter. After 1 h, bsorbnce ws mesured with spectrophotometer (Spectronic 21, Busch nd Lomb, USA) t 760 nm. The concentrtion of totl phenolic compounds in extrcts of penut skins ws termined by comprison with the bsorbnce of phenol (Merck, Drmstdt, Germny) used t different concentrtion s stndrd. All tests were run in triplicte, nd nlyses of ll smples were run in duplicted nd verged. The yield of totl phenolic compound ws expressed in grms of totl phenolic extrcted per grm of dry penut skins. Rdicl-scvenging ctivity The rdicl-scvenging ctivity of the extrcts ws termined using diphenyl picryl hydrzyl rdicl (DPPH) (Aldrich, Milwkee, WI, USA) ccording to Schmed-Hirschmnn et l. 17 Ethnolic solution of penut skins extrct (5 µl; 300 µgml 1 ) ws dd to 1.5 ml ethnolic solution of DPPH rdicl (20 µgml 1 ) to give finl extrct concentrtion of 1 µgml 1. The mixture ws shken vigorously nd left for 5 min. The bsorbnce of the resulting solution ws mesured t 517 nm with spectrophotometer (Spectronic 21, Busch nd Lomb, USA). All tests were run in triplicte, nd nlyses of ll smples were run in duplicte nd verged. The rdicl-scvenging ctivity ws clculted using the formul: percentge inhibition = [1 (bsorbnce of DPPH nd smple bsorbnce smple)/(bsorbnce of DPPH) 100]. Optimiztion of penut skins prticle size Penut skins were crushed nd sifted in different prticle sizes: 0 1, 1 2 nd 2 10 mm. These penut skins (2.5 g) were extrcted with 50 ml of 70% (v/v) ethnol in wter using two extrction methods: by mcertion for 24 h nd by shking for 1 h, both t room temperture nd in drkness. The extrcts were filtered nd evported unr vcuum t 35 C. The yields of totl phenolic compounds extrcted from the penut skins were mesured ccording to the Folin Cioclteu method scribed bove. Optimiztion of the reltion solvent solid for the extrction Penut skins (2.5 g) were extrcted with 50, 75, 100, 125 nd 150 ml of 70% (v/v) ethnol in wter. The reltions were 20, 30, 40, 50 nd 60 ml extrction solvent g 1 penut skins, respectively. The penut skins were extrcted by mcertion for 24 h t room temperture nd in drkness. The yields of totl phenolic compounds extrcted were mesured ccording to the methodology scribed bove. Optimiztion of extrction contct time Penut skins (2.5 g) were extrcted with 50 ml of 70% (v/v) ethnol in wter t room temperture. Extrctions were veloped by mcertion nd shking (mgnetic shker, 2000 rpm) during different periods of time: 1, 10, 30, 60 nd 120 min. The yield of totl phenolic compounds extrcted were mesured ccording to the methodology scribed bove. Optimiztion of number of extrction stges Penut skins (2.5 g) were extrcted with 50 ml of 70% (v/v) ethnol in wter by shking (mgnetic shker, 2000 rpm) during 20 min t room temperture. According to the results obtined from the optimiztion of the extrction contct time, the extrction equilibrium stte ws reched fter 10 min. Owing to this, 20 min extrction ws chosen. The extrct ws filtered nd the residue ws extrcted gin unr the sme conditions in five stges. The yields of totl phenolic compounds extrcted from the skins were mesured ccording to the methodology scribed bove. Sttisticl nlysis Sttisticl nlyses were conducted with the InfoStt softwre (InfoStt softwre, version 1.1, 2002, Fcultd Ciencis Agropecuris, Universidd Ncionl Córdob, Córdob, Argentin) for replicted test dt. Anlyses of vrince were performed by one-wy nd two-wy ANOVA procedures. Significnt differences (α = 0.05) between of 34 J Sci Food Agric 85:33 38 (2005)

3 Extrction of phenolic ntioxidnts mens were termined by Duncn s test. Regression nlysis ws performed to termine the reltion between the rdicl-scvenging ctivity nd the content of totl phenolic compounds of the extrcts. Second orr polynomil regression eqution ws obtined for ech tretment: y = Ax 2 + Bx + C, where y = percentge inhibition (rdicl-scvenging ctivity) nd x = g totl phenolic compounds g 1 extrct. RESULTS AND DISCUSSION Optimiztion of ethnol wter mixtures s the extrction solvent The yields of dry extrcted mtter of penut skins nd ftted penut skins in reltion to the ethnol content in the extrction solvent re shown in Fig 1. In two-wy ANOVA (α = 0.05), the inpennt vribles were: A-ethnol content in the solvent, B-type of penut skins (penut skins nd ftted penut skins), nd the pennt vrible-yield of dry extrcted mtter. The two inpennt vribles nd the interction between both hd significnt effect on the pennt vrible. In other words, the effect on the yield of dry extrcted mtter of ethnol content in the solvent ws different in nonftted penut skins thn in ftted penut skins. When ethnol content incresed from 0 to 96% (v/v), the yield of dry extrcted mtter on penut skins incresed from ± 1 to ± 6 g g 1. However, the dry extrcted mtter from ftted penut skins hd mximum yield when 50 nd 70% (v/v) ethnol ws used (0.181 ± 6 nd ± 0 g g 1, respectively). This effect could be explined becuse lipids re prtilly soluble in ethnol. As consequence, when 96% ethnol ws used to extrct penut skins, it extrcted phenolic compounds nd some lipid components. Lipids were bsent in ftted penut skins, therefore 96% ethnol extrcted only phenolic compounds in less yield thn 50 or 70% ethnol in this mteril. In generl, no significnt differences were found in the yields of extrction between penut skins nd ftted penut skins using 50 nd 70% (v/v) ethnol in the solvent. Duh et l 18 compred the yield of extrction of penut hulls using methnol nd ethnol. Methnol extrcted ± 2.19 mg soluble constituents from 2.5 g dried penut hulls (0.042 g g 1 ) nd ethnol extrcted 49.7 ± 2.05 mg (0.020 g g 1 ). In our cse, the ethnol (96% v/v) extrcted ± 6 g g 1 soluble constituents from the penut skins nd ± g g 1 from the ftted penut skins. These results indicte tht penut skins contin bout nine times more ethnol soluble constituents thn penut hulls. The yields of totl phenolic compounds extrcted from penut skins nd ftted penut skins in reltion to the ethnol content in the solvent re presented in Fig 2. When the pennt vrible in the two-wy ANOVA ws the yield of totl phenolics compounds extrcted, the two inpennt vribles A nd B hd significnt effect on the pennt vrible but they did not present ny effect of interction. Thus, the effect on this pennt vrible of the ethnol content in the solvent showed no difference between non-ftted penut skins nd ftted penut skins. In generl, no significnt differences were found between penut skins nd ftted penut skins using the sme ethnol wter mixture in the extrction solvent (Duncn test). In both skins, the mximum vlues of extrction of g g 1 in penut skins nd g g 1 in ftted penuts skins were obtined with 30, 50 nd 70% (v/v) ethnol in the extrction solvent. Yen et l 19 found levels between 33.4 nd 71.3 mg totl phenolic compounds g 1 penut hulls ( g g 1 of totl phenolic compounds). The extrction ws veloped in two stges, with reltion of 50 ml methnol from 5 g of penut hulls in ech stge. The ethnol wter mixture used in this work extrcted between nd g g 1 totl phenolic compounds from the penut skins. Tht mens lmost twice the totl phenolic compounds obtined by Yen et l 19 using methnol with penut hulls. Rdicl-scvenging ctivity of the extrcts of penut skins nd ftted penut skins is shown in Fig 3. When the pennt vrible in the two-wy ANOVA g g -1 of dry extrcted mtter e d 0.15 d b c 0.05 Penut Skins b Deftted Penut Skins % ethnol in wter g g -1 of totl phenolic compound extrcted 0.14 c c 0.12 bc 0.08 b bc bc Penut Skins 0.02 Deftted Penut Skins % ethnol in wter Figure 1. Yield of dry extrcted mtter of penut skins nd ftted penut skins (g dry mtter extrcted g 1 dry penut skins) obtined by the ethnol wter mixture s extrction solvent. The sme letter indictes tht yields of dry extrcted mtter re not significntly different t α = Figure 2. Yield of totl phenolic compounds extrcted from penut skins nd ftted penut skins (g totl phenolic compounds extrcted g 1 dry penut skins) in reltion to the ethnol content in the extrction solvent. The sme letter indictes tht vlues of yield of totl phenolic compound re not significntly different t α = J Sci Food Agric 85:33 38 (2005) 35

4 V Nepote, NR Grosso, CA Guzmán % Inhibition bc c 20 bcd bc c 10 b Penut Skins Deftted Penut Skins % ethnol in wter Figure 3. Rdicl-scvenging ctivity of the extrcts of penut skins nd ftted penut skins (% inhibition) in reltion to the ethnol content in the extrction solvent. The sme letter indictes tht the vlues of percentge inhibition re not significntly different t α = e c 100 ws the percentge of inhibition, the inpennt vrible A (ethnol content in the solvent) hd significnt effect on the pennt vrible, but the inpennt vrible B (type of penut skins penut skins nd ftted penut skins) nd the interction between both did not hve significnt effect on the pennt vrible. In other words, the effect on this pennt vrible of the ethnol content in the solvent showed no difference between non-ftted penut skins nd ftted penut skins. This result ws similr to the one obtined for the yields of phenolic compounds (Fig 2) with mximum in the percentge of inhibition in both penut skins using 30, 50 nd 70% (v/v) ethnol in the solvent mixture. The mximum vlues were % for penut skins nd % for ftted penut skins. Yen et l 20 studied the scvenging effect of methnolic extrcts of penut hulls on DPPH. Their extrcts showed bout 90% inhibition when the concentrtion ws 1.5mgml 1. In our work, the concentrtion of the penut skins extrcts showed hlf the percentge of inhibition when the concentrtion of the extrcts ws 1 µgml 1, 1500 times less thn in penut hulls extrcts. As consequence, the penut skins extrcts re much more ctive thn penut hulls extrct. On the bsis of the results observed in Figs 1 3, the prtil conclusion is tht the best solvents to extrct high yield of totl phenolic nd ntioxidnt compounds were 50 nd 70% (v/v) ethnol in wter. However, when consiring the evportion time nd cost, the preferred solvent mixture ws 70% (v/v) ethnol in wter becuse the evportion time is lower. As consequence, the possibility of oxidtion of the extrcted phenolic compounds cresed. Becuse of this, the mixture ethnol wter (70% v/v) ws chosen for the optimiztion of prticle size, solvent volume, contct time nd extrction stges to extrct ntioxidnt components from penut skins. Correltion nd regression nlyses where y = percentge inhibition (rdicl-scvenging ctivity) nd x = g totl phenolic compounds g 1 extrct. The linel regression ws: y = 92.29x x (R 2 = 0.638). The Person coefficient = 0.78 These results showed good reltion between both vribles, nd they implied tht it is possible to predict the rdicl-scvenging ctivity in the extrct by knowing the content of totl phenolic compounds using the bove eqution. Optimiztion of penut skin prticle size The yield of totl phenolic compounds extrcted by mcertion nd shking from the different prticle sizes of penut skins is shown in Tble 1. From two-wy ANOVA (α = 0.05), inpennt vrible A-prticle size, inpennt vrible B-mcertion nd shking, nd the pennt vrible-yield of totl phenolics extrcted, it could be conclud tht the two inpennt vribles, nd the interction of both, hd significnt effect on the pennt vrible. The effect of the prticle size ws observed only in the extrction by mcertion; the penut skins between 0 nd 1 mm exhibited the lest vlues of totl phenolic compound extrcted (0.069 ± 1 g g 1 ). This vlue could be due to the compctness of the solid, resulting in smller contct surfce nd higher vlue of retined solvent (6.85 ml) in comprison to the lrgest prticle sizes (retined solvent between 5.00 nd 5.11 ml). Tht effect ws compensted when the extrction ws conducted by shking. In this cse, the prticle size did not produce n effect in the inpennt vrible. As consequence, the crushing of the penut skins did not produce n increse in the totl phenolic compound extrcted by single extrction stge using mcertion or shking. The mximum vlues obtined in yield of totl phenolic compounds extrcted were between (non-crushed penut skins, mcertion) nd g g 1 (2 10 mm prticle size, mcertion) with no significnt differences between these results (Duncn test). Optimiztion of reltion solvent solid for the extrction The yields of dry mtter nd totl phenolic compounds extrcted with different reltions solvent/penut skins Tble 1. Yields of totl phenolic compounds extrcted from different prticle sizes of penut skins by mcertion nd shking Yield of totl phenolic compounds extrcted Prticle size (mm) Mcertion Shking ± 1 b ± 4b b ± 9b ± 6b ± 0b ± 2b Non-crushed skins ± 2b ± 2b g totl phenolic compounds g 1 dry penut skins. b Mens ± stndrd error followed by the sme letter re not significntly different t α = J Sci Food Agric 85:33 38 (2005)

5 Extrction of phenolic ntioxidnts (v/w) re presented in Tble 2. The yields did not show significnt differences (one-wy ANOVA, Duncn test, α = 0.05). The increse of the solvent volume did not produce n increse in the totl phenolic compounds extrcted. The chosen volume solvent used to extrct non-crushed penut skins ws the minimum required to cover the skins, resulting in reltion 20 ml solvent g 1 penut skins, obtining ± 7 g of dry mtter extrcted nd ± 8 g totl phenolic compounds g 1 dry penut skins. Optimiztion of extrction contct time The yields of totl phenolic compounds extrcted from the penut skins t different contct times by shking nd mcertion re presented in Fig 4. From two-wyanova(α = 0.05, inpennt vrible A-mcertion or shking, inpennt vrible B- time, nd pennt vrible-yield of totl phenolics extrcted) it could be conclud tht the two inpennt vribles hd significnt effect on the pennt vrible, nd the interction between both hd no significnt effect on the pennt vrible. The mcertion process required 60 min for extrcting the sme quntity of totl phenolic compounds in comprison to the shking method, which required 10 min, six times less thn the mcertion method. The mximum yield of totl phenolic compounds extrcted with both methods ws between nd g g 1. Tble 2. Yields of dry mtter nd totl phenolic compounds extrcted from the penut skins with different rtios of solvent:penut skins (v/w) Rtio of solvent (ml): penut skins (g) Yield of dry mtter extrcted Yield of totl phenolic compounds extrcted b 20 c ± ± ± ± ± ± ± ± ± ± 0.31 g extrcted mtter g 1 dry penut skins. b g totl phenolic compounds g 1 dry penut skins. c Mens ± stndrd error followed by the sme letter within ech column re not significntly different t α = Optimiztion of number of extrction stges The yields of totl phenolic compounds extrcted with 70% (v/v) ethnol in wter from the penut skins in five stges re shown in Fig 5. The percentges of extrction of the totl phenolic compounds extrctble from the penut skins in ech stge were 64.6, 87.3, 94.8, nd 100% for single, two, three, four nd five stges, respectively. The totl phenolic compounds obtined with three stges ws ± 0 g g 1, nd with five stges ws ± 2 g g 1. As consequence, using more thn three stges is not recommend becuse the time nd solvent required to chieve 100% extrction would not be justified by such low difference in the yield of extrction g g -1 of totl phenolic compounds extrcted Mcertion Shking Extrction time (min) Figure 4. Yield of totl phenolic compounds extrcted from the penut skins (g totl phenolic compounds g 1 dry penut skins) t different contct time by shking nd mcertion. The sme letter indictes tht the vlues of percentge inhibition re not significntly different t α = g g -1 of totl phenolic compounds extrcted 0.12 phenolic compounds 0.08 extrcted per stge 0.06 ccumulted phenolic 0.04 compounds extrcted Stge number Figure 5. Yield of totl phenolic compounds extrcted from the penut skins (g totl phenolic compounds extrcted g 1 dry penut skins) by extrction stges. J Sci Food Agric 85:33 38 (2005) 37

6 V Nepote, NR Grosso, CA Guzmán In most of the works relted to the extrction of phenolic compounds from penut hulls, 19,21,22 the extrcts were obtined in two stges, by shking t room temperture overnight or fter 24 h contct. In our study, the mximum time required to obtin the ethnolic extrcts ws 10 min by shking, nd 1 h by mcertion t room temperture. CONCLUSION As conclusion from the results obtined in the extrction of the ntioxidnt compounds from penut skins with ethnolic solvent t lbortory level, the best method ws using 70% (v/v) ethnol in wter, non-crushed penut skins, by shking for 10 min nd in three stges. The phenolic compounds obtined for this procedure could be used s dditives in food products s nturl ntioxidnts to extend their shelf-life. Synthetic ntioxidnts, such s butylted hydroxynisole (BHA), butylted hydroxytoluene (BHT) nd propyl gllte (PG), re used in mny foods to prevent rncidity but there is growing concern for the potentil helth hzrds of synthetic ntioxidnts. One exmple is the work of Ito et l 23 tht reported BHA to be crcinogenic in niml experiments. The present reserch renews interest in the incresed use of nturlly occurring ntioxidnts. ACKNOWLEDGEMENTS We thnk the compny Lorenzti, Ruestch y Ci for the provision of penut skins. This work ws supported by CONICET nd SECYT-UNC. REFERENCES 1 CIARA, Anurio Estdístico Oleginoss. Producción y Exportciones Argentins Oleginoss y Derivdos. Cámr l Industri Aceiter l Repúblic Argentin, Buenos Aires (2002). 2 Woodroof JG, Penuts, Production, Processing, Products, 3th edn. Avi, Westport, CT, USA, pp (1983). 3 Nepote V, Grosso NR nd Guzmán CA, Extrction of ntioxidnt components from penut skins. Grss Aceites 53: (2002). 4 Msquelier J, Plnt extrct with pronthocynidins content s therpeutic gent hving rdicl scvenger effect nd use thereof. US Ptent 4,698,360 (1987). 5 Sobolev VS, Cole RJ, Dorner JW nd Ygen B, Isoltion, purifiction nd liquid chromtogrphy termintion of stilbene phytolexins in penuts. JAOAC Int 78: (1995). 6 Sobolev VS nd Cole RJ, Trns-resvertrol content in commercil penut nd penut products. J Agric Food Chem 47: (1999). 7 Snrs TH, McMichel RW Jr nd Hendrix KW, Occurrence of resvertrol in edible penuts. J Agric Food Chem 48: (2000). 8 Jng M, Ci L, Uni GO, Slowing KV, Thoms CF, Beecher CWW, Fong HHS, Frnsworth NR, Kinghorn AD, Meht RG, Moon RC nd Pezzuto JM, Cncer chemopreventive ctivity of resvertrol, nturl product rived from grpes. Science 275: (1997). 9 Arichi H, Kimur Y, Okud H, Bb K, Kozw M nd Arichi S, Effects of stilbene compounds of the roots of Polygonum cuspidtum Sieb. et Zucc. on lipid metbolism. Chem Phrm Bull 30: (1982). 10 Kimur Y, Okud H nd Arichi S, Effects of stilbenes on rchidonte metbolism in leukocytes. Biochim Biophys Act 834: (1985). 11 Kimur Y, Okud H nd Kubo M, Effects of stilbenes isoltes from medicinl plnts on rchidonte metbolism nd grnultion in humn polymorphonucler leukocytes. J Ethnophrmc 45: (1995). 12 Chung MI, Teng CM, Cheng KL, Ko FN nd Lin CN, An ntipltelet principle of Vertrum formosnum. Plnt Med 58: (1992). 13 Bertelli AAE, Giovnnini L, Ginnessi D, Migliori M, Bernini W, Fregoni M nd Bertelli A, Antiopltelet ctivity of synthetic nd nturl resvertrol in red wine. Int J Tiss Rect 17:1 3 (1995). 14 Pce-Ascik CR, Hhn S, Dimndis EP, Soles G nd Goldberg DM, The red wine phenolics trns-resvertrol nd quercetin block humn pltelet ggregtion nd eicosnoid synthesis: implictions for protection ginst coronry hert disese. Clin Chim Act 235: (1995). 15 Kltsky AL, Armstrong MA nd Friedmn GD, Alcohol nd mortlity. Ann Intern Med 117: (1992). 16 Wtermn PG nd Mole S, Anlysis of Phenolic Plnt Metbolites. Blckwell Scientific, Oxford, pp (1994). 17 Schmed-Hirschmnn G, Rzmilic I, Gutierrez MI nd Loyol JI, Proximte composition nd biologicl ctivity of food plnts gthered by Chilen Amerindins. Econ Bot 53: (1999). 18 Duh P, Yeh D nd Yen G, Extrction nd intifiction of n ntioxidnt component from penut hulls. JAOCS 69: (1992). 19 Yen G, Duh P nd Tsi C, Reltionship between ntioxidnt ctivity nd mturity of penut hulls. J Agric Food Chem 41:67 70 (1993). 20 Yen G nd Duh P, Scvenging effect of methnolic extrcts of penut hulls on free-rdicl nd ctive-oxygen species. JAgric Food Chem 42: (1994). 21 Yen G nd Duh P, Antioxidnt ctivity of methnolic extrcts of penut hulls from vrious cultivrs. JAOCS 72: (1995). 22 Duh P nd Yen G, Antioxidnt efficcy of methnolic extrcts of penut hulls in soyben nd penut oils. JAOCS 74: (1997). 23 Ito N, Hgiwr A, Shibt M, Ogiso T nd Fukushim S, Induction of squmous cell crcinom in the forestomch of F334 rts treted with butylted hydroxynisole. Gnn 73: (1982). 38 J Sci Food Agric 85:33 38 (2005)