Europen Journl of Food Siene nd Tehnology Pulished y Europen Centre for Reserh Trining nd Development UK (www.ejournls.org) EFFECTS OF EXTRACTION SOLVENTS ON PHYTOCHEMICALS AND ANTIOXIDANT ACTIVITIES OF WALNUT (JUGLANS REGIA L.) GREEN HUSK EXTRACTS Qing Zhng College of Life Sienes, Shnxi Norml University, Linfen 041004, Chin ABSTRACT: The effets of extrtion solvents (hexne, ethyl-ette, etone, ethnol, methnol, nd wter) on the ontent of phytohemils inluding totl polyphenols, flvonoids nd ondensed tnnins, s well s ntioxidnt tivities of wlnut green husk were investigted. The results showed tht extrtion solvents signifintly ffeted phytohemils ontent nd ntioxidnt tivities of wlnut green husk. The etone, ethnol, nd methnol extrts hd the higher ontent of phytohemils, nd they exhiited stronger ntioxidnt tivities, followed y ethyl-ette nd wter extrts, nd the lowest for hexne extrt. These results indited tht seletive extrtion from wlnut green husk, y n pproprite solvent, is importnt for otining frtions with high ntioxidnt tivity, whih will e useful for the developing nd pplition of wlnut green husk. KEYWORDS: Wlnut Green Husk, Extrtion Solvent, Phytohemils, Antioxidnt Ativity. INTRODUCTION The wlnut (Juglns regi L.) is tree trditionlly ultivted for its vlule wood nd fruits. Not only dry fruit re used ut lso green wlnuts, shells, kernels, rk, green wlnut husks nd leves hve een used in oth foods, osmeti nd phrmeutil industry (Stmpr et l., 2006). All those produts n e used lso s signifint soure of different ompounds expressing ntioxidnt nd ntimiroil potentil, s well ntiprolifertive, ntinoieptive, ntisthmti, heptoprotetive, ntidieti, ntifertility, nti-inflmmtory, lipolyti nd mny others properties positively ffeted humn helth (Almeid et l., 2008; Oliveir et l., 2008; Zhng et l., 2009; Crvlho et l., 2010; Tjmul et l., 2014). Wlnut s green husk is one of the mjor wste produts from the wlnut prodution tht nowdys hs sre use. Some studies hve demonstrted tht they re rih in nturl iotive ompounds (Cosmulesu et l., 2010), nd possess mny iotivities inluding ntioxidnt tivity nd ntimiroil pity ginst different pthogeni Grm positive teri (Olivier et l., 2008; Crvlho et l., 2010; Fernndez-Agullo et l., 2013). For this reson, extrting iotive ompounds in wlnut green husk is si for further nlysis nd its development. Extrt solvents is one of the most importnt ftors ffeting the hemil omposition nd iologil tivity of plnt extrts (Turkmen et l., 2006; Reey et l., 2012; Cheok et l., 2012). Then, the im of this study ws to nlyze the effet of the extrtion solvents of vrying polrity (hexne, ethyl ette, etone, ethnol, methnol, 15
Europen Journl of Food Siene nd Tehnology Pulished y Europen Centre for Reserh Trining nd Development UK (www.ejournls.org) wter) on phytohemil ontents, ntioxidnt tivity of wlnut green husk extrts to provide referene for the omprehensive development nd utiliztion of wlnut green husk. MATERIALS AND METHODS Regents nd Stndrds: Glli id nd 2, 4, 6-Tri (2-pyridyl)-s-trizine (TPTZ) is purhsed from Fluk (Switzerlnd). 2,2 -zino-is (3-ethylenothizoline-6-sulfoni id) dimmonium slts (ABTS), 2,2-Diphenyl-1-pirylhydrzyl (DPPH), tehin nd rutin were from Sigm (USA). All other hemils nd regent used in the experiments were of nlytil grde. Preprtion of Extrts: Fresh wlnut green husk were grounded efore the extrtion proess, nd then twenty-five grms of wlnut green husk were extrted with 250 ml solvents nd kept in shker t 25 C for 1 h. And then the mixture ws entrifuged t 5000 rpm/min t 4 C for 10 min. The preipittion extrted with 250 ml solvent one gin nd mixed superntnts. The extrts were vuum evported to dryness t 40 o C, nd reonstituted with methnol to finl volume of 25 ml. Extrtion solvent used in our experiment ontins hexne, ethyl ette, etone, ethnol, methnol, nd wter. Determintion of Totl Polyphenoli Content (TPC): Polyphenoli ontent of extrts ws determined y method (Reey et l., 2012). Briefly, n liquot (0.1 ml) extrt ws mixed with 2.8 ml of distilled wter nd 0.1 ml of Folin-Ciolteu regent (1.0 mol/l), nd then the solution ws mixed nd inuted t room temperture for eight minute. Following tht, 2 ml of 7.5% sodium ronte (N2CO3) solution ws dded nd shken thoroughly. The mixture ws inuted for 2 h in the drk t room temperture nd the sorne ws determined t 765 nm. Glli id ws used for lirtion of the stndrd urve nd totl phenoli ontent ws expressed s milligrm glli id equivlent per grm smple. Determintion of Totl Flvonoid Content (TFC): The level of totl flvonoid ws mesured s desried y Reey et l. (2012) with some modifitions. Briefly, n liquot (1 ml) extrt ws mixed with 0.3 ml of 3% NNO3 solution nd inuted for 6 min. After tht, 0.3 ml of 10% Al(NO3)3 ws dded nd the solution ws kept t room temperture for 6 min. Finlly, mix the solution with 4 ml of 4% NOH solution nd dd wter to 10 ml. After 20 minute of inution, the sorne of the mixture t 510 nm ws mesured. Rutin ws used for lirtion of the stndrd urve, nd flvonoid ontent ws expressed s milligrm rutin equivlents per grm smple. Determintion of Condensed Tnnin Content (CTC): The ontent of ondensed tnnin ws mesured using Vnillin-hydrohlori id method (Reey et l., 2012). Briefly, 1.0 ml of extrt ws mixed with 5.0 ml of vnillin-solution dissolving in methnol (0.5 g vnillin dissolves in methnol whih ontining 4% hydrohlori id). The mixture ws kept in the drk t room temperture for 20 min nd the sorne ws mesured t 500 nm. The ontent of ondensed tnnin ws expressed s tehin milligrm equivlents per grm smple. 16
Europen Journl of Food Siene nd Tehnology Pulished y Europen Centre for Reserh Trining nd Development UK (www.ejournls.org) DPPH Rdil Svenging Assy: DPPH rdil svenging ssy ws mesured following the method of Xu et l. (2012) with some modifitions. Extrt ws serilly diluted to different onentrtions nd 0.5 ml of diluted extrt mixed with 2.5 ml of 60 μmol/l DPPH solution dissolving in methnol. The mixture ws shken thoroughly nd inuted in the drk t room temperture for 30 min, nd the sorne ws mesured t 517 nm. The svenging rte of DPPH rdil ws lulted ording to the formul given elow: DPPH rdil svenging ility (%) = [A0-(A1-A2)]/A0 100, where A0 is the sorne of the ontrol in whih methnol sustitutes extrt nd A1 is the result of the mixture of the extrt nd DPPH rdils while A2 is the sorne of the mixture of the extrt nd 2.5 ml methnol to eliminte the olor effet of the extrt. The DPPH rdil svenging tivity of ws mesured y IC50 vlue whih represents the effetive onentrtion of the extrt t whih DPPH rdil svenging ility up to 50%. ABTS Rdil Svenging Assy: ABTS rdil svenging ssy ws determined ording to the method of Xu et l. (2012). Briefly, ertin qulity of ABTS nd potssium persulfte ws dissolved in wter to keep the finl onentrtions of the two sustnes to e 7 mmol/l nd 2.45mmol/L respetively. The mixture ws kept in the drk for 16~24 h to mke the ABTS rdil working solution nd its sorne t 734 nm ws djusted to 0.700±0.050. The ABTS rdil svenging ility ws mesured y dding 50 μl of diluted extrt to 1.9 ml of ABTS rdil working solution nd the sorne t 734 nm fter 6 min ws reorded. The svenging rte nd IC50 vlue were lulted using the eqution desried for DPPH ssy. Ferri Reduing Antioxidnt Power (FRAP) Assy: The reduing ility ws determined y using FRAP ssy desried y Xu et l. (2012) with slight modifitions. Briefly, the FRAP regent ws freshly prepred from 300 mm sodium ette uffer (ph 3.6), 10 mm TPTZ solution in 40 mm HCl nd 20 mm FeCl3 solution in proportions of 10:1:1 (v/v), respetively. The FRAP regent ws prepred fresh dily nd ws wrmed to 37 o C in wter th prior to use. Then 0.1 ml of smples ws mixed with 1.8 ml of FRAP regent nd 3.1 ml ultrpure wter. The sorption of the retion mixture ws mesured t 593 nm fter inution for 30 min t 37 o C. Inresed sorne of the retion mixture indited inresed reduing power. RESULTS AND DISCUSSION Effets of Solvents on Phytohemils Content: The ontents of phytohemils of different solvent extrts were showed in Figure 1. Depending on the solvent used, the totl polyphenol ontent rnged from 0.34 to 6.27 mg/g. Aetone extrt hd the highest polyphenol ontent, followed y ethnol, methnol, wter, ethyl ette nd n-hexne. Among them, n-hexne extrt ws the lowest one nd ounted for one-tenth of etone extrt. The extrtion of totl flvonoids ws lso influened signifintly y extrting solvent (p < 0.05), nd its ontents vried from 0.19 to 0.71 mg/g, respetively for hexne nd ethnol. With regrd to totl flvonoid ontent, solvents ould e sequened in the 17
Europen Journl of Food Siene nd Tehnology Pulished y Europen Centre for Reserh Trining nd Development UK (www.ejournls.org) following deresing order: ethnol>ethyl ette>methnol>etone>wter>hexne. As regrd to the ondensed tnnins, the ontent vried from 1.39 to 3.18 mg/g nd their order ws methnol>ethyl ette>ethnol>etone>wter>hexne. As previously reported y phytohemil ontent (mg/g) 8 6 4 2 0 TPC TFC CTC hexne ethyl ette etone ethnol methnol wter extrtion solvent Reey et l. (2012), these results indites tht the different solvents, s the differenes in polrity, dispersiility nd penetrility, ould seletively extrt different phytohemils. Besides, the levels of totl phenolis, flvonoids, nd ondensed tnnins lso ould possily e influened y dieletri onstnt, hemil struture of orgni solvents (Cheok et l., 2012) s well s hemil properties of plnt phytohemils (Jyprksh et l., 2003). Figure 1. The ontents of phytohemils of different extrts Effets of Solvents on DPPH nd ABTS Svenging Ailities: Due to the differenes in the ompositions nd ontents of the phytohemils, the extrts showed the different ntioxidnt ilities. DPPH nd ABTS rdils svenging ilities of the extrts diluted to series of onentrtions hs een tested in our study nd IC50 vlue of eh smple ould e lulted through regression eqution. The lower IC50 vlue represents higher svenging ilities. The Tle 1 showed tht there were signifint differenes of DPPH rdil svenging ilities of different solvent extrts. Ethnol extrt of whih IC50 vlue ws 54.9 μg/ml owned the highest DPPH rdil svenging ility, followed y methnol nd etone. Wter nd hexne extrts hd poor DPPH rdil svenging ility, of whih IC50 vlues were 103.0 nd 143.8 μg/ml, respetively. 18
Europen Journl of Food Siene nd Tehnology Pulished y Europen Centre for Reserh Trining nd Development UK (www.ejournls.org) Tle 1. The svenging tivity of different extrts on DPPH rdils DPPH Extrts Regression eqution Correltion oeffiient (R 2 ) IC50/µg ml -1 Methnol y = 39.371Ln(x) + 164.04 0.9959 55.2 Ethnol y = 42.769Ln(x) + 174.16 0.9927 54.9 Aetone y = 32.829Ln(x) + 141.3 0.9905 62.0 Ethyl y = 646.94x + 3.6 0.9992 71.7 ette Hexne y = 449.88x - 14.705 0.9893 143.8 Wter y = 23.263Ln(x) + 102.87 0.9933 103.0 All of these extrts possessed some ABTS rdil svenging ilities in tht their phytohemil omponents nd ontents might hve some equivlene regrding to ABTS rdil svenging ility. Different from the results of DPPH (Tle 2), IC50 vlues of etone extrt ws 324.8 μg/ml, exhiiting the highest ABTS rdil svenging ility, followed y methnol, ethnol nd ethyl ette extrts, nd the lowest for wter extrts. However, IC50 vlues of hexne extrt did not gin in it s the highest mount. Tle 2. The svenging tivity of different extrts on ABTS rdils ABTS Extrts Regression eqution Correltion oeffiient (R 2 ) IC50/µg ml -1 Methnol y = 126.72x + 2.1131 0.9890 377..9 Ethnol y = 124.64x - 0.2056 0.9869 402.8 Aetone y = 123.96x + 9.7354 0.9897 324.8 Ethyl y = 126.72x + 2.1131 0.9791 377.9 ette Hexne y = 29.739x + 0.0079 0.9963 - Wter y = 106.18x + 3.3769 0.9988 439.1 Ferri Reduing Antioxidnt Power (FRAP): Figure 2 showed tht different wlnut green husk extrts exhiited different ferri reduing power, ut there ws signifint differene (p < 0.05) in the reduing power mong different extrts. The reduing power of extrts rnged from 0.024 to 0.509. The reduing power of etone extrts ws the highest, followed y ethnol, methnol, ethyl ette, nd wter extrts, while the lowest for hexne extrts. The result suggested tht extrts of wlnut green husk hd remrkle poteny to donte eletron to retive free rdils, onverting them into more stle non-retive speies nd terminting the free rdil hin retion. 19
Europen Journl of Food Siene nd Tehnology Pulished y Europen Centre for Reserh Trining nd Development UK (www.ejournls.org) 0.6 0.5 FRAP vlue (As) 0.4 0.3 0.2 0.1 0 hexne ethyl ette etone ethnol methnol wter extrtion solvent Figure 2. The reduing power of different solvent extrts CONCLUSIONS The extrting solvents signifintly ffeted phytohemils ontent nd ntioxidnt tivities of wlnut green husk extrts. Our findings indited tht s ompred to other extrts, ethnol, methnol, etone extrts hd the higher totl polyphenol, flvonoid nd ondensed tnnins ontent nd no signifint differene ws found mong them. Consistent with results desried ove, ethnol, methnol, etone extrts showed the higher ntioxidnt ilities in DPPH, ABTS rdils svenging ssys nd FRAP ssy. In onlusion, these results indited tht seletive extrtion from nturl soures, y n pproprite solvent, is importnt for otining frtions with high ntioxidnt tivity nd the development nd utiliztion of wlnut green husk. REFERENCES Almeid, I.F., Fernndes, E., Lim, J.L.F.C., Cost, P.C. nd Bhi, M.F. (2008). Wlnut (Juglns regi) lef extrts re strong svenger of pro-oxidnt retive speies. Food Chemistry, 106: 1014 1020. Crvlho, M., Ferreir, P.J., Mendes, V.S., Silv, R., Pereir, J.A., Jerónimo, C. nd Silv, B.M. (2010). Humn ner ell ntiprolifertive nd ntioxidnt tivities of Juglns regi L. Food nd Chemil Toxiology, 48: 441 447. Cheok, C,Y., Chin, N.L., Yusof, Y.A. nd Lw, C.L. (2012). Extrtion of totl phenoli ontent from Grini mngostn Linn. hull. I. Effets of solvents nd UV Vis spetrophotometer sorne method. Food nd Bioproess Tehnology, 5: 2928 2933. Cosmulesu, S., Trndfir, I., Ahim, G., Botu, M., Biu, A. nd Grui, M. (2010). Phenolis of green husk in mture wlnut fruits. Notule Botnie Horti Agrootnii Cluj-Npo, 38(1): 53 56. 20
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