Interntionl Food Reserh Journl 20(6): 3117-3123 (2013) Journl homepge: http://www.ifrj.upm.edu.my Effets of extrtion solvent system, time nd temperture on totl phenoli ontent of henn (Lwsoni inermis) stems 1,2 Tn, M. C., 3 Tn, C. P. nd 4,* Ho, C. W. 1 Deprtment of Biotehnology, Fulty of Applied Sienes, UCSI University, No. 1, Jln Menr Gding, UCSI Heights, Chers 56000, Kul Lumpur, Mlysi 2 Deprtment of Nutrition nd Dietetis, Fulty of Mediine nd Helth Sienes, Universiti Putr Mlysi, 43400 UPM Serdng, Selngor Drul Ehsn, Mlysi 3 Deprtment of Food Tehnology, Fulty of Food Siene nd Tehnology, University Putr Mlysi, 43400 UPM Serdng, Selngor Drul Ehsn, Mlysi 4 Deprtment of Food Siene nd Nutrition, Fulty of Applied Sienes, UCSI University, No. 1, Jln Menr Gding, UCSI Heights, Chers 56000, Kul Lumpur, Mlysi Artile history Reeived: 25 Deemer 2012 Reeived in revised form: 17 Septemer 2013 Aepted: 18 Septemer 2013 Keywords Henn (Lwsoni inermis) stems Totl phenoli ontent (TPC) Solvent extrtion Folin-iolteu ssy Astrt Henn plnt (Lwsoni inermis) is n Indin mediinl plnt used in trditionl mediine for the tretment of vrious diseses, esides its populrity s nturl dye to olour hnd nd hir. Reserh in the reent pst hs umulted enormous evidene reveling henn plnt to e n exellent soure of ntioxidnts suh s totl phenolis. In this study, the extrtion of totl phenolis from henn stems ws evluted using the Folin-Ciolteu ssy. A set of single ftor experiments ws rried out for identifying the optimum ondition of eh independent vrile ffeting totl phenoli ontent (TPC) extrtion effiieny of henn stems, nmely the solvent type, solvent onentrtion (v/v, %), extrtion time (min) nd extrtion temperture ( o C). Generlly, high extrtion yield ws otined using queous etone (out 40%) s solvent nd the extrtion yield ould further e inresed using prolonged time of 270 min nd higher inution temperture of 55 C. Under these optimized onditions, the experimentl mximum yield of TPC of 5554.15 ± 73.04 mg GAE/100 g DW ws otined. All Rights Reserved Introdution Henn (Lwsoni inermis), n Indin mediinl plnt, is perennil shru ntive to Indi, North Afri, Asi nd Austrli (Wyk nd Wink, 2004). It hs een reorded tht different prts of henn plnt re rih soure of vrious iotive priniples nd hs een used in trditionl mediine (Dsgupt et l., 2003). Reent study on phytohemil ontent in henn hs shown tht it is rih in phenoli ntioxidnts suh s lwsone, flvonoids, tnnins nd oumrins (Khre, 2007). Phenoli ompounds, yli derivtives of enzene with one or more hydroxyl groups ssoited with the romti ring, ount for one of the lrgest nd most widely distriuted group of phytohemils (Andjelkovi et l., 2006). They vry onsiderly in struture with over 8000 nturlly-ourring ompounds hving een identified (Blsundrm et l., 2005). They my exhiit wide rnge of physiologil properties, suh s nti-llergeni, nti-rtherogeni, nti-inflmmtory, nti-miroil, nti-virl, rdioprotetive nd vsodiltory effets (Blsundrm et l., 2005; Trt et l., 2007). The enefiil effets derived from phenoli ompounds in humn life hve een ttriuted to their ntioxidnt tivity tht minly due to their redox properties, whih llow them to t s reduing gents, hydrogen dontors, singlet oxygen quenhers nd metl-heltors (Rie-Evns et l., 1996). Therefore, phenoli ompounds hve een used in mny ntioxidnt pity ssys efore testing the properties of iologil system. Extrtion is the first importnt step in the reovery nd purifition of tive ingredients from plnt mterils. The im of n extrtion proess should e to provide for the mximum yield of sustnes nd of the highest qulity whih onsist of high onentrtion of trget ompounds nd ntioxidnt power of the extrts (Spigno et l., 2007). Mny tehniques hve een developed to extrt phenolis, suh s onventionl solvent extrtion, mirowve-ssisted, ultrsound-ssisted nd superritil fluid extrtion, mong whih solvent extrtion (solid-liquid nd liquid-liquid extrtion tehniques) is the most ommonly used, nd hs *Corresponding uthor. Emil: who@usi.edu.my Tel: +603 9101 8880; Fx: +603 9102 3606
3118 Tn et l./ifrj 20(6): 3117-3123 proven to e relile nd effiient method (Chirinos et l., 2007; Bnik nd Pndey, 2008). The effiy of solvent extrtion is ffeted y mny ftors suh s the type of solvent, solvent onentrtion, time, temperture, ph, numer of steps, liquid-to-solid rtio nd prtile size of the plnt mteril (Ce nd Mzz, 2003). There re two most ommonly used optimiztion studies, the lssil single ftor experiments nd the response-surfe methodology (RSM). The former is one-ftor-t--time pproh, in whih only one ftor is vrying t time while ll others re kept onstnt. Herein we used the singleftor experiments, despite eing hving some drwks, suh s time-onsuming, expensive, possile intertion effets etween vriles nnot e evluted nd misleding onlusions my e drwn (Bs nd Boyi, 2007; Bezerr et l., 2008). However, single ftor experiments re le to provide fundmentl informtion on the rnges for signifint extrtion prmeters on the extrtion of phenoli ompounds from plnt mterils. Up to now, solvent extrtion of phenoli ntioxidnts from henn (Lwsoni inermis) stems using single ftor experiments hs not een reported. In onsidering the growing interest in ssessing ntioxidnt pity of herl mediine, this study ws therefore imed (1) to determine the est extrtion onditions (type of solvent, solvent onentrtion, extrtion time nd extrtion temperture) for henn stems, in order to mximize simultneously the yield of totl phenoli ontent (TPC) y single ftor experiments, nd (2) to quntify the extrted phenoli ontents in henn stems. This study provides n opportunity to evlute the potentil of henn stem s nturl soure of potent ntioxidnts with potentil mediinl vlue. In ddition, the optiml rnges (minimum nd mximum vlues) otined for ll extrtion prmeters in the present study n lso e served s key informtion for the sle-up extrtion of ntioxidnt ompounds from henn stems. Mterils nd methods Plnt mteril Henn (Lwsoni inermis), trditionl mediinl plnt speies, ws olleted from lowlnd eside the min ity mpus of UCSI University, Chers, Kul Lumpur. The speies hs een identified nd onfirmed y Forest Reserh Institute Mlysi (FRIM), Kul Lumpur, Mlysi. Chemils nd regents All hemils nd solvents used were of nlytil regent (AR) grde. Folin-Ciolteu s phenol regent, sodium ronte nhydrous (N 2 CO 3 ) ( 99.9% purity), solute etone (CH 3 COCH 3 ) ( 99.5% purity) nd methnol (CH 3 OH) ( 99.8% purity) were purhsed from Merk KGA, Drmstdt, Germny. Glli id (C 7 H 6 O 5 ) (98% purity) ws otined from Aros Orgnis, Belgium, USA. Asolute ethnol (C 2 H 5 OH) ( 95% purity) ws purhsed from R&M Chemils, Essex, UK. The distilled wter (reverse osmosis (RO) wter) used for the nlysis ws purified y Milli-Q Millipore wter purifition system (Millipore Corportion, Billeri, MA, USA). Smple preprtion The fresh henn plnts olleted were first thoroughly wshed upon rrivl t the lortory. The leves removed in order to otin the stems (pproximtely 400 g) whih were then ut into onstnt size of 0.5 2.0 m. The ut-stems were then evenly oven-dried for 24 hours t 40 C. After drying, dried stems were ground into fine powder (0.5 mm) y miller (Model MF 10 si, IKA Werke, Germny) t 4000 rpm. The dried ground smples were susequently vuum-pkged into nylonliner low-density polyethylene (LDPE) pouhes y vuum-pkging mhine (Model DZQ 400/500, Clrity, Chin) nd stored in seled ontiner (drk, dry nd room temperture environment) for extrtions. Solvent extrtion of phenoli ompounds from henn stems Solvent extrtion ws performed in temperture ontrolled wter th shker (Model WNB 7-45, Memmert, Germny) with useful volume of 14 L (internl dimensions: 365 315 150 mm) t onstnt shking speed of 130 rpm. Firstly, 2 g of dried-ground smples were weighed urtely nd pled into onil flsk mde up to 20 ml volume with extrting solvent (solvent-to-solid rtio of 10:1). The flsk ws then overed with prfilm (Pehiney plsti pkging, USA) nd wrpped with luminium foil (Dimond, USA) in order to provide drk environment, nd inuted for different lengths of times t the required temperture. After the extrtion, the flsk ws removed from the wter th shker nd ooled to room temperture y old running tp wter. The henn stem extrt ws filtered through snd ore glss funnel with Whtmn No. 1 filter pper (Whtmn Interntionl Ltd., Englnd, UK), nd the ler solution of rude extrt (filtrte) ws olleted in light-proteted mer ottle for the determintion of nlysis without further tretment. All the extrtions were rried out in replites.
Tn et l./ifrj 20(6): 3117-3123 3119 Experimentl design Extrtion onditions for TPC from henn stems, nmely solvent type, solvent onentrtion, extrtion time nd extrtion temperture, were determined y vrying one ftor t time while keeping the others onstnt for delimittion of the experimentl region. The fixed ftors were the prtile size of 0.50 mm nd the solvent-to-solid rtio of 10:1. Extrtion solvent type evlution Smples were extrted with 60% (v/v) methnol, 60% (v/v) ethnol, 60% (v/v) etone, distilled wter nd oiling distilled wter respetively, following the proedures s desried in Setion 2.4. The independent vriles were onstnt solvent omposition of 60% (v/v), extrtion time (180 min) nd extrtion temperture (25 C). The optiml extrtion solvent ws seleted upon the highest vlue of TPC (mg GAE/100 g DW). Extrtion solvent onentrtion evlution Aqueous etone seleted in Setion 2.5() ws used s the extrtion solvent for the following experiments. Phenolis were extrted from henn stem powder using different etone onentrtions, rnging from 20% (v/v) to 100% (v/v) y the sme proedure desried in Setion 2.4 while holding the other two independent vriles, whih were extrtion time (180 min) nd extrtion temperture (25 C) t onstnt level. The optiml solvent onentrtion ws seleted upon the highest vlue of TPC (mg GAE/100 g DW). Extrtion time evlution Bsed on the solvent type nd solvent onentrtion seletions in Setions 2.5() nd (), 40% (v/v) etone ws seleted s the optiml extrting solvent to extrt phenolis from henn stem powder. By repeting the sme proedure s desried in Setion 2.4, dried henn stem powder ws merted with 40% (v/v) etone nd inuted for different time periods rnging from 30 to 450 min t onstnt temperture of 25 C. The optiml extrtion time ws seleted upon the highest vlue of TPC (mg GAE/100 g DW). Extrtion temperture evlution Henn stem powder ws merted with the optiml solvent type nd solvent onentrtion seleted in Setions 2.5() nd (), whih ws 40% (v/v) etone. Smples were inuted t different tempertures rnging from 25 to 55 C for 270 min, whih ws the optiml time determined in Setion 2.5(). The extrtion proedure ws repeted s desried in Setion 2.4. The optiml extrtion temperture ws seleted upon the highest vlue of TPC (mg GAE/100 g DW). Determintion of totl phenoli ontent (TPC) Preprtion of solutions Folin-Ciolteu (F-C) regent (10-fold diluted) ws freshly prepred y topping up 10 ml of F-C regent to 100 ml with distilled wter in 100 ml volumetri flsk. Sodium ronte (N 2 SO 3 ) (7.5%, w/v) ws prepred y urtely weighing 7.5 g of nhydrous N 2 SO 3 with n nlytil lne (AB204-S, Mettler Toledo, Switzerlnd), dissolved with some distilled wter in 100 ml eker, nd topped up to totl volume of 100 ml with distilled wter in 100 ml volumetri flsk. Proedure The TPC of henn stem extrts ws determined spetrophotometrilly using Folin-Ciolteu s regent ording to the method desried y Lim et l. (2007) with slight modifitions. Briefly, the rude extrts otined from the extrt preprtion were first pproximtely diluted 40 times. Three hundred mirolitres (300 μl) of henn stem rude extrts were introdued into luminium foil-wrpped test tues followed y 1500 μl of 10-fold diluted Folin- Ciolteu s regent nd 1200 μl of 7.5% (w/v) sodium ronte solution. The ontents of the tues were mixed thoroughly y using vortex mixer (Model LMS, Jpn) for 10 s, overed with prfilm nd llowed to stnd in the drk for 30 min t room temperture. After 30 min, the sorne of the deep lue olourtion developed ws mesured ginst lnk t 765 nm using Uvi Light Spetrophotometer (Model XTD 5, Seomm, Frne). Blnk regent ws prepred y repling 300 μl of smple with equl mount of distilled wter. Mesurements were rried out in triplite nd lultions were sed on lirtion urve otined with glli id, whih ws y = 10.422x + 0.0042 (R 2 = 0.9977). The TPC were expressed s mg glli id equivlents (GAE) per 100 g of dry weight (DW). All nlyses were performed in triplite. Sttistil nlysis All experimentl results in this study were expressed s men vlues ± stndrd errors (SE) of six mesurements (n = 6). In these single ftor experiments, the signifint differenes (p < 0.05) mong tretment mens were determined y one-wy nlysis of vrine (ANOVA) with Tukey s test, using Minit sttistil softwre (Version 15.1.1.0, Minit In., PA, USA).
3120 Tn et l./ifrj 20(6): 3117-3123 Results nd Disussions Effets of vrious solvent types on extrtion of totl phenoli ompounds Solvent extrtion is the most ommon method used for isoltion of phenoli ntioxidnts, nd oth extrtion yield nd extrtion tivity re strongly dependent on the solvent. Antioxidtive potentil of phenoli ompounds is strongly ffeted y the polrity of solvent used in extrtion. Hene, the seletion of extrtion solvents is ritil for the omplex plnt smples. An extrtion solvent system is generlly seleted ording to the purpose of extrtion, polrity of the interested omponents, polrity of undesirle omponents, overll ost, sfety nd environmentl onern (Wng et l., 2008). Aqueous mixtures of etone, ethnol nd methnol hve een widely used for extrting phenoli omponents from otnil mterils, espeilly hers nd mediinl plnts (Trt et l., 2007; Wng et l., 2008). Figure 1 shows the TPC results of henn stem extrts from five types of solvents s desried in Setion 2.5 were used. Aqueous etone (60%, v/v) signifintly (p < 0.05) showed the highest extrtion pity for phenolis from henn stems in omprison to the other solvents in this order: etone (60%) > ethnol (60%) > methnol (60%) > distilled wter > oiling wter. Aetone, ethnol nd methnol seem to hve their distint speifiity in the extrtion of polyphenoli sustnes. Mny studies showed tht etone is the est solvent for pronthoynidins nd tnnins extrtion; ethnol effetively extrts flvonoids nd their glyosides, tehols nd tnnins; wheres etter yield for phenoli ids nd tehin is otined with methnol (Chirinos et l., 2007; Mne et l., 2007; Spigno et l., 2007; Trt et l., 2007). These fts re in ordne with polrity of the solvent used for the extrtion nd soluility of phenoli ompounds in them. It is interesting to note tht the polrity of etone, ethnol nd methnol is 0.355, 0.654 nd 0.762, respetively. Aqueous etone is good solvent for polr ntioxidnts nd more useful for extrting polyphenols from protein mtries, sine they pper to degrde the polyphenol protein omplexes. Menwhile, ethnol nd methnol re more effetive in extrting polyphenols linked to polr firous mtries (Chirinos et l., 2007; Al-Frsi nd Lee, 2008). In ft, the use of queous etone hs severl dvntges to the use of queous ethnol, queous methnol nd pure wter, for exmple higher extrtion effiieny, suggesting the use of queous etone s extrtion solvent for the following stges in this study. However, there is still need to hek if using different wter perentges in etone (%, 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 60% A e tone 60% Ethnol 60% Methnol dh₂o Boiling dh₂o Solvents Figure 1. Influene of solvent type on extrtion effiieny of totl phenoli ontent (TPC) from henn stems. Vlues re mens ± stndrd errors (SE) of six determintions (n = 6) from two extrt replite. Vlues mrked y different letters indite signifintly different (p < 0.05). 6000 5000 4000 3000 2000 1000 0 d v/v) is possile to inrese the extrtion effiieny of TPC from henn stems in the present experiment. Effets of solvent onentrtion on extrtion of totl phenoli ompounds The extrtion of phenoli ompounds from plnt mteril is diretly relted to the omptiility of the phenoli ompounds to the solvent nd thus, when the ompounds re well mthed in polrity with the solvent they will e esily extrted. The effets of etone onentrtion in the extrtion solvent on the ontent of phenolis in henn stem extrts re shown in Figure 2. Bsed on Figure 2, the etone onentrtions hd signifint effet (p < 0.05) on 20% 40% 60% 80% 100% Aetone Conentrtions (%, v/v) Figure 2. Influene of etone onentrtion on extrtion effiieny of totl phenoli ontent (TPC) from henn stems. Vlues re mens ± stndrd errors (SE) of six determintions (n = 6) from two extrt replite. Vlues mrked y different letters indite signifintly different (p < 0.05). 5000 4800 4600 4400 4200 4000 3800 3600 3400 3200 3000 30 90 180 270 360 450 Extrtion time (min) Figure 3. Influene of extrtion time on extrtion effiieny of totl phenoli ontent (TPC) from henn stems. Vlues re mens ± stndrd errors (SE) of six determintions (n = 6) from two extrt replite. Vlues mrked y different letters indite signifintly different (p < 0.05). d d e
Tn et l./ifrj 20(6): 3117-3123 3121 the extrtion effiieny of TPC from henn stems. As n e seen from Figure 2, the TPC s funtion of etone onentrtion follows prol shpe. TPC of henn stem extrts rehed mximum t 40% etone (v/v) (4796.78 ± 70.67 mg GAE/100 g DW) followed y signifint (p < 0.05) derese with further inrese in the onentrtion of the etone in the extrtion medium. Phenoli ompounds in henn stem extrts might present modertely polr profile. From Figure 2, it is evident tht ddition of ertin mount of wter in etone ontriutes to the retion of modertely polr medium tht ensures the extrtion of polyphenols nd thus improves the overll extrting effiieny. Aetone is low-polr solvent while wter is strong polr solvent, nd they n e lended with eh other in ny proportion. Hene, with the ddition of wter to etone, the polrity of omplex solvent will inrese ontinuously. So the quired rtio of more polr phenoli ompounds in henn stem extrts inreses with inresing wter ontent ording to like dissolves like priniple (Chirinos et l., 2007; Zhng et l., 2007). Another possile reson for the inresed effiieny with the presene of some wter might e due to the inrese in swelling of plnt mteril y wter, whih inreses the ontt surfe re etween the plnt mtrix nd the solvent (Hemwimon et l., 2007). A modertely polr solvent of 40% etone (v/v) ws hosen for the determintion of extrtion time nd extrtion temperture. Effets of extrtion time ourse on extrtion of totl phenoli ompounds Extrtion time ws nother importnt prmeter influening the extrtion of phenoli ompounds. Figure 3 showed tht TPC of henn stem extrts inresed grdully with inresing of the extrtion time from 30 min (4478.22 ± 17.08 mg GAE/100 g DW) up to 270 min (4739.71 ± 10.08 mg GAE/100 g DW) nd egn to deline shrply until rehing minimum of 4380.21 ± 78.51 mg GAE/100 g DW t 360 min. These phenomen ould e well explined y the Fik s seond lw of diffusion, prediting tht finl equilirium etween the solute onentrtions in the solid mtrix (plnt mtrix) nd in the ulk solution (solvent) might e rehed fter ertin time, leding to deelertion in the extrtion yield (Silv et l., 2007). Moreover, prolonged extrtion time inreses the hne of deomposition nd oxidtion of phenolis due to their long exposure to unfvourle environmentl ftors like temperture, light nd oxygen (Nzk nd Shhidi, 2004). On the other hnd, the inresed extrtion time is uneonomil nd time onsuming from 6105 5105 4105 3105 2105 1105 105 25 35 45 55 Extrtion temperture ( C) Figure 4. Influene of extrtion temperture on extrtion effiieny of totl phenoli ontent (TPC) from henn stems. Vlues re mens ± stndrd errors (SE) of six determintions (n = 6) from two extrt replite. Vlues mrked y different letters indite signifintly different (p < 0.05). the industrilistion point of view, lso potentilly inresing the loss of solvent y vporistion whih diretly ffets the loss of solvent-to-solid rtio of extrtion. Thus, extrtion time of 270 min ws seleted s the optimum point for the susequent step due to the prtil nd eonomil onsidertions, despite the higher yield of TPC. Effets of extrtion temperture on extrtion of totl phenoli ompounds The seletion of n pproprite extrtion temperture ws the finl step in series of single ftor experiments. The phenolis extrtion yield s funtion of the extrtion temperture is shown in Figure 4. Results indited tht signifint (p < 0.05) inrese in the extrtion of totl phenolis from 4894.65 ± 74.81 mg GAE/100 g DW to 5554.15 ± 73.04 mg GAE/100 g DW when inresing the temperture from 25 to 55 C. An exeption is inution t 35 C whih resulted in n insignifint (p > 0.05) derese in the totl phenolis extrtion, down to 4702.11 ± 43.75 mg GAE/100 g DW. This is due to the inresed soluility nd diffusion oeffiients of phenolis; deresed solvent visosity; s well s the enhned mss trnsfer nd penetrtion of solvent into the plnt mtrix (Al-Frsi nd Lee, 2008; Hemwimon et l., 2007; Silv et l., 2007; Wng et l., 2008), thus elerting the whole extrtion. On the other hnd, ording to Shi et l. (2003), heting might soften the plnt tissue nd weken the phenol-protein nd phenol polyshride intertions in the plnt mterils. Consequently, more phenolis would trnsfer to the solvent portion. Despite the positive effets of higher tempertures on the phenolis extrtion, this nnot e inresed indefinitely. Elevting the tempertures up to ertin level might e followed y their possile onurrent deomposition of ntioxidnts whih were lredy moilized t lower tempertures (Liyn-Pthirn nd Shhidi, 2005). Other thn tht, denturtion of
3122 Tn et l./ifrj 20(6): 3117-3123 memrnes nd possile degrdtion of polyphenoli ompounds used y hydrolysis, internl redox retions nd polymeriztions whih re detrimentl to the extrtion yield my hppen nd influene quntifition of iotive ompounds (Ad-Gri et l., 2007). Moreover, it ws lso reported y Ce nd Mzz (2003) tht ertin phenoli ompounds suh s flvonoid fmilies (minly nthoynin nd flvn-3-ol derivtives) re het sensile, hene n upper limit must e respeted to void degrdtion of the thermo-sensitive phenoli ompounds. Most importntly, sine 40% etone (v/v) is used for extrtion in this study, the temperture must not go very high s etone hs oiling point of 56.2 C. Therefore, very high extrtion temperture my evporte etone from the queous etone solution nd susequently hnges the etone-to-wter rtio. Mnipultion steps nd extrtion osts re expeted to inrese with inresing of the extrtion temperture. Considering the ove fts, moderte extrtion temperture t 55 C ws seleted s the optiml extrtion temperture for this optimiztion design, whih extrted the totl yield of 5554.15 ± 73.04 mg GAE/100 g DW of TPC. Reproduiility The reproduiility of the mesurements were ssessed for henn stem extrtions in six determintions (n = 6) from two extrt replite t four different prmeter omintions. It ws oserved tht the overll perentge reltive stndrd devition (% RSD) is very smll nd flls etween the nrrow rnges of 0.52 4.39%, onfirming the high reproduiility of this extrtion method. Conlusions In nutshell, the experimentl optimum onditions tht llow fst, quntittive nd mximum extrtions of TPC from henn stems were otined through the effetive lssil solvent extrtion method s well s single ftor experiments. Aetone ws found to e the est solvent for the extrtion. Susequently, the optimum onditions for mximum TPC ws found to e etone onentrtion 40% (v/v), extrtion time 270 min nd operting temperture 55 C. These optimum onditions n e useful for further optimiztion of the extrtion of phenoli ntioxidnts from henn (Lwsoni inermis) stems using response surfe methodology (RSM) in onjution with entrl omposite rottle design (CCRD). However, solvent extrtion give resonle reovery ut it poses some disdvntges like the solvent need to e evported, dding extr ost nd possile loss of qulity. Therefore, other methods should e onsidered to extrt phenoli ontents from plnt mterils. Referenes Ad-Gri, B., Berruet, L. A., Mrquez, D. M. L., Ferrer, I. C., Gllo, B. nd Viente, F. 2007. Optimiztion nd vlidtion of methodology sed on solvent extrtion nd liquid hromtogrphy for the simultneous determintion of severl polyphenoli fmilies in fruit juies. Journl of Chromtogrphy A 1154: 87-96. Al-Frsi, M. A. nd Lee, C. Y. 2008. Optimiztion of phenolis nd dietry fire extrtion from dte seeds. Food Chemistry 108: 977-985. Andjelkovi, M., Cmp, J. V., Meulener, B. D., Depemelere, G., Soiu, C., Verloo, M. nd Verhe, R. 2006. Iron-heltion properties of phenoli ids ering tehol nd glloyl groups. Food Chemistry 98: 23-31. Blsundrm, N., Ai, T. Y., Smnthmurthi, R., Sundrm, K. nd Smmn, S. 2005. Antioxidnt properties of plm fruit extrts. Asi Pifi Journl of Clinil Nutrition 4: 319-324. Bnik, R. M. nd Pndey, D. K. 2008. Optimizing onditions for olenoli id extrtion from Lntn mr roots using response surfe methodology. Industril Crops nd Produts 27(3): 241 248. Bs, D. nd Boyi, I. H. 2007. Modelling nd optimiztion I. Usility of response surfe methodology. Journl of Food Engineering 78: 836 845. Bezerr, M. A., Sntelli, R. E., Oliveir, E. P., Villr, L. S. nd Esleir, L. A. 2008. Response surfe methodology (RSM) s tool for optimiztion in nlytil hemistry. Tlnt 76 (5): 965-977. Ce, J. E. nd Mzz, G. 2003. Optimiztion of extrtion of nthoynins from lk urrnts with queous ethnol. Journl of Food Siene 68: 209-215. Chirinos, R., Rogez, H., Cmpos, D., Pedreshi, R. nd Lrondelle, Y. 2007. Optimiztion of extrtion onditions of ntioxidnt phenoli ompounds from mshu (Tropeolum tuerosum Ruíz & Pvón) tuers. Seprtion nd Purifition Tehnology 55(2): 217-225. Dsgupt, T., Ro, A. R. nd Ydv, P. K. 2003. Modultory effet of Henn Lef (Lwsoni inermis) on drug metolizing phse I nd phse II enzymes, ntioxidnt enzymes, lipid peroxidtion nd hemilly indued skin nd forestomh ppillomgenesis in mie. Moleulr nd Cellulr Biohemistry 245: 11-22. Hemwimon, S., Pvsnt, P. nd Shotipruk, A. 2007. Mirowve-ssisted extrtion of ntioxidtive nthrquinones from roots of Morind itrifoli. Seprtion nd Purifition Tehnology 54: 44-50. Khre, C. P. 2007. Indin mediinl plnts An illustrted ditionry. New York: Springer.
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