Biochemical Engineering Journal37(2007)271–278Design of optimal solvent for extraction of bio-activeingredients from mulberry leavesJong-Min Kim a,Sang-Mok Chang a,In-Ho Kim b,Young-Eun Kim c,Ji-Hwan Hwang c,Kyo-Seon Kim d,Woo-Sik Kim c,∗a Department of Chemical Engineering,Dong-A University,Saha-ku,Hadan2-dong840,Busan604-714,Republic of Koreab Department of Chemical Engineering,Choongnam University,Yusung-ku Kung-dong,Daejeon305-764,Republic of Koreac Department of Chemical Engineering,Kyunghee University,Yongin Kiheung Seochun1,Kyungki-Do449-701,Republic of Koread Department of Chemical Engineering,Kangwon National University,Chuncheon Hyoja2-Dong,Kangwon-Do200-701,Republic of KoreaReceived3October2006;received in revised form8April2007;accepted13May2007AbstractA method of designing solvents for the optimal extraction of bio-active ingredients from natural resources was developed using an alcohol–water binary solvent.The target bio-active ingredient of polyphenols,anti-oxidation and anti-tyrosinase ingredients exhibited different dependency of extraction efficiency on the alcohol species(methanol,ethanol,n-propanol and i-propanol)and composition of binary ing the solubility parameter,the extraction efficiency of the bio-active ingredients was correlated with the solvent polarity.As a result,the optimal solvent polarities for the extraction of polyphenols,anti-oxidation and anti-tyrosinase ingredients were predicted as38.5,37.33,and33.0[MPa1/2],respectively.These predictions also agreed well with the optimal solvent conditions of the water–alcohol mixtures depending on the alcohol species and composition. Plus,the correlation was confirmed with model solvents designed using other solvent species,including acetone and ethylene glycol.©2007Elsevier B.V.All rights reserved.Keywords:Extraction;Alcohol–water binary solvent;Bio-active ingredients;Solvent polarity1.IntroductionRecent studies on exploiting natural compounds for medicine and cosmetics have drawn much attention to the effective extrac-tion of the desired bio-active ingredients from natural products. Typically,various solvents of water,alcohols,acetone and ether, etc.are used to extract bio-active substances from natural prod-ucts due to their broad solubility propensity on solvents,where water is generally applied to extract high polar ingredients,such as carbohydrates,glycosides,and amino acids,while ether is used to extract low polar ingredients,such as aromatic com-pounds.Thereby,alcohol–water mixtures are used to extract out various ingredients having broad range of solubility propensity for the investigation of the specific functionality of the molecular compounds from extracted ingredients.For example,Doi et ed hexane and butanol to extract ingredients having low polarity from mulberry roots∗Corresponding author.Fax:+82312021946.E-mail address:wskim@khu.ac.kr(W.-S.Kim).[1],then investigated the specific functional compounds of prenyflavas,glycoside,iso-quercetine,and astragalin from extracted solution.Methanol is frequently used to extract spe-cific bio-active ingredients from various natural resources.As such,anti-inflammatory ingredients have been found in the methanol extraction from Culcasia scadens P.Beauv[2],anti-microbial compounds from Ceanothus americanus[3],and anti-histaminic compounds from Mentha spicata[4].Plus, ethylacetate and n-hexane have also been applied to extract bio-active ingredients.Thus,from the above previous studies, the solvent polarity would appear to be important for extract-ing specific functional ingredients from a natural resource. Thus,a variety of solvents,pure and mixtures,have been applied to extract bio-active ingredients with various polarities[5].A few studies have already examined the optimal extraction conditions for bio-active ingredients.For example,ethanol for the extraction of anti-oxidants from Spirulina platenis has been suggested as the best solvent among hexane,petroleum ether, and water,while the extraction temperature and time had a min-imal influence on the extraction of the anti-oxidants[6].Mean-1369-703X/$–see front matter©2007Elsevier B.V.All rights reserved. doi:10.1016/j.bej.2007.05.006272J.-M.Kim et al./Biochemical Engineering Journal37(2007)271–278while,Chandrika and Fereidoon attempted tofind the optimal solvent conditions for extracting polyphenolic compounds from wheat using mixture of methanol,ethanol and acetone with vary-ing the solvent composition,extraction temperature,and time [7].Although such previous studies have shown that the optimal solvent conditions for extraction can be found based on trials with various species of solvent and compositions,no system-atic method has yet been suggested for determining the optimal extraction solvent for natural resources.Accordingly,the present study is focused to develop a method for determining the optimal solvent conditions and designing a solvent for the optimal extraction of bio-active ingredients from mulberry leaf known to contain the active ingredients for anti-oxidation and anti-hyperpigmentation.Since the extrac-tion of specific ingredients from natural resources depends on the polarity of the solvent,as implied in previous studies,the extraction efficiency of the solvent for bio-active ingredients is investigated,along with the variation in the polarity of the solvent according to the species and composition of a binary alcohol–water solvent.Here,methanol,ethanol,n-propanol,and iso-propanol are used as the alcohol species for the binary mixture.Plus,ethylene glycol and acetone are used to design model solvents to confirm the relationship between the extrac-tion of bio-active ingredients and the solvent polarity.Based on the extraction of mulberry leaf,activities of ingredients spe-cific to anti-oxidation and anti-hyperpigmentation are used as references to evaluate the extraction efficiency of the solvent [8,9].2.Experimental2.1.ExtractionMulberry(Morus alba L.)leaf,purchased from a herbal market in Korea,was completely dried in a convection oven at60–80◦C for a couple of days,thenfinely pulverized using a milling machine.Next,the leaf powder was meshed with an aperture size of200m and kept in a desiccator. To extract the bio-active ingredients,2g of the leaf powder were added to10ml of a solvent made of a binary mix-ture of alcohol and water for1h in a hot bath at80◦C. The extracted solution was then separated from the solid leaf using a centrifuge(Hanil Science Industrial Co.Ltd.,HA-500, Korea)and the contents of the bio-active ingredients examined, including the polyphenolic compounds,anti-oxidants,and anti-tyrosinase.The solvent conditions for extracting the bio-active ingre-dients were varied by adjusting the alcohol composition and species in the alcohol–water binary mixture.Methanol,ethanol, n-propanol and iso-propanol were used for the binary mixture and their compositions were changed from0to100%.Plus,ethy-lene glycol and acetone were also applied to formulate a binary mixture solvent to evaluate the optimal extraction conditions for the solvent.For the present experiment,all chemicals were pur-chased from Sigma–Aldrich Chemical Co.(U.S.A.)and ACS grade.2.2.Assay of phenolic compoundsBased on the method of Goldstein and Swain[10],the total content of polyphenolic compounds in the extraction was eval-uated.First,the extraction was dilutedfifty times with distilled water(1/50,v/v),then100l of the diluted sample was com-pletely mixed with1250l of the Folin-Denis reagent(ACS grade,Fluka,Switzerland)that had been diluted ten times with distilled water(1/10,v/v).Thereafter,the mixture solution was incubated at25◦C for20min after adding250l of satu-rated sodium ing a UV spectrophotometer with a 760nm wavelength(JASCO,Model V-570,Japan),the content of polyphenolic compounds was estimated by comparing with a standard concentration curve for tannic acid that has an equiv-alent absorbance to the UV wavelength.The standard curve for tannic acid was prepared using the same procedure used to mea-sure the UV absorbance of the extracted sample.Thus,100l of a tannic acid solution was mixed with1250l of the same Folin-Denis reagent and250l of saturated sodium carbonate, then incubated at25◦C for20min.The UV absorbance of the tannic acid solution relative to the concentration resulted in the following standard curve:C P=A T−0.01980.00472(1)where C P is the tanic acid concentration[g ml−1]and A T is the UV absorbance.Eq.(1)was then used to estimate the polyphenol concentration equivalent to the tannic acid concentration.2.3.Anti-oxidation activityThe anti-oxidation activity of the extraction was evaluated based on the degree of scavenging1,1-diphenyl-2-picryhydrazyl (DPPH)free radicals[11].First,a free radical solution was pre-pared with0.15mM DPPH in2000l of ethanol and100l of a0.5%(v/v)Tween-20solution.The pH of the radical solu-tion was then adjusted to7.4using1800l of a0.1M Tris–HCl buffer.After adding10l of the extraction sample to the radical solution,the mixture was allowed to react for30min at room temperature,then the UV absorbance was measured at a wave-length of517nm.The blank solvent containing no extraction was used as a base reference for the anti-oxidation activity.The activity of the extraction(C AO)was expressed on a relative scale as:C AO(%)=A AO−A ROA RO×100(2)where A AO and A RO are the UV absorbances of the extraction sample and blank solvent,respectively.2.4.Anti-tyrosinase activityBased on the method suggested by Lee et al.[9],the mush-room tyrosinase inhibition of the ingredients was measured as indicative activity of anti-hyperpigmentation.The extraction sample was diluted50%(v/v)with methanol,then320l of the diluted sample was mixed with960l of0.83mM l-dopaJ.-M.Kim et al./Biochemical Engineering Journal37(2007)271–278273 and320l of a tyrosinase solution,containing125units ml−1and buffered with0.1M phosphate at pH6.8.The mixture wasquickly cooled to0◦C right after being incubated at37◦C for10min,then the UV absorbance of the solution at490nm wasmeasured.The blank mixture without tyrosinase was used as abase reference for the anti-tyrosinase activity.The activity of theextraction(C AT)was then expressed on a relative scale as:C AT(%)=A AT−A RTRT×100(3)where A AT and A RT are the UV absorbances of the extraction sample and blank mixture,respectively.3.Results and discussion3.1.Extraction of bio-active ingredientsIt is already known that most of the natural plant bio-active ingredients that are polyphenolic compounds,such as mulber-roside F,quercetine,catechin,and rutin,etc.in mulberry leaves, have broad solubility propensities due to their molecular polar-ities.Thus,solvents composed of a broad range of alcohol species and compositions were used for effective extraction of the bio-active ingredients from mulberry leaves.The extrac-tion efficiency of the solvents was evaluated based on three criteria:the total polyphenolic content,anti-oxidation activity (DPPH radical scavenging),and anti-tyrosinase activity(mush-room tyrosinase inhibition)of the extracted solution.As shown in Fig.1,the total content of polyphenols extracted from the mulberry leaves varied with the composition and species of alcohol in the binary mixture solvent.The extrac-tion efficiency of a propanol binary mixture(n-propanol–water and iso-propanol–water mixtures)was slightly maximized with an alcohol composition of about20%,then dramatically reduced when increasing the propanol composition.Meanwhile,with an ethanol–water mixture,the optimum condition for extraction appeared with an alcohol composition of about40%.In the case of a methanol–water mixture,the optimum condition for extrac-tion shifted further to an alcohol composition of60%although the extraction efficiency somewhatfluctuated with the alcohol composition,and the dependency of the extraction efficiency on the methanol composition was significantly diminished when compared to that with a propanol–watermixture.Fig.1.Extraction of polyphenol content by alcohol–water binary mixtures.The extraction of organic ingredients from plant leaves is directly related to the compatibility of the ingredients to the sol-vent;thus,when the ingredients are well matched in polarity with the solvent they will be easily extracted,otherwise,it will be hard to extract them.Therefore,based on the current experi-mental results,it was supposed that an optimal solvent condition of polarity could maximize the total content of polyphenol compounds extracted from mulberry leaves.Also,when using different species for a binary mixture,the solvent composition for optimal polarity was varied due to the distinct polarity of each solvent species.As a result,the optimal alcohol–water mixture appeared at a low composition with propanols and shifted to a high composition with methanol,as the methanol was more polar than the propanols,as displayed in Table1.Since the polar-ity range for the methanol–water mixture was smaller than that for the propanol–water mixture,the extraction efficiency of the methanol–water mixture was found to be much less sensitive to the composition than with any of the other alcohol–water mixtures.The anti-oxidation activity,equivalent capability of scav-enging DPPH free radicals,of the extracted solution was also evaluated under various solvent conditions,as shown in Fig.2. When methanol was used for the binary mixture,the maximum anti-oxidation activity of the extracted solution was obtained at about60%of methanol composition,yet this shifted to a lower alcohol fraction when using ethanol and propanols.It was inter-esting to note that the anti-oxidation activity of the extractedTable1Cohesive energies and solubility parameter of solventsSolvent Molecular weight(g mol−1)Molecular volume(cm3mol−1)δ(MPa1/2)δdδpδhδ(25◦C)δ(80◦C) Water18.0218.112.222.840.448.044.1 Methanol32.040.711.613.024.029.727.3 Ethanol46.158.712.611.220.026.124.0n-Propanol60.175.214.110.517.724.922.9iso-Propanol60.176.814.09.816.023.421.5 Ethylene glycol62.155.910.115.129.834.932.1 Acetone58.174.013.09.811.019.718.1274J.-M.Kim et al./Biochemical Engineering Journal 37(2007)271–278Fig.2.Extraction of bio-active ingredients for anti-oxidation by alcohol–waterbinary mixture.solution was more sensitive to the solvent conditions (species and composition of alcohol)than the polyphenol extraction effi-ciency.In particular,when using methanol for the binary water mixture,the anti-oxidation activity of the solution extracted with a high alcohol composition (above 80%)was dramati-cally reduced,while the extraction efficiency of polyphenolic compounds remained almost the same with only a slight drop from the maximum extraction efficiency.Consequently,it would appear that among the polyphenolic compounds in the extracted solution,the effective ingredients related to anti-oxidation activ-ity were quite polar and their solubility was very sensitive to the solvent polarity.However,the anti-tyrosinase activity of the extracted solution behaved quite differently when varying the alcohol species and composition of the solvent.As shown in Fig.3,when using methanol in the solvent,the activity of the solution continued to be enhanced when increasing the methanol fraction,up to an alcohol composition of around 80%,after which it slightly dropped,whereas in the cases of ethanol and propanols,theFig.3.Extraction of bio-active ingredients for anti-tyrosinase by alcohol–waterbinary mixture.optimal solvent condition was found at an alcohol composition of around 60%.This means that the bio-active ingredients related to anti-tyrosinase activity were more favorably extracted by a solvent with a lower polarity than the polyphenols and anti-oxidation ingredients.3.2.Design of solvents for extraction of bio-active ingredientsAs shown in the above experiment,the extraction efficiency of the bio-active ingredients was directly related to the polarity of the binary solvent,which was varied based on the alcohol fraction and species.Thus,an optimal solvent for the extraction of specific active ingredients could be designed if the relation-ship between the solvent polarity and the extraction efficiency were available.Hence,the solubility parameter [12]is intro-duced as a simple way of representing the polarity of a solvent and correlating the extraction efficiency of the polyphenol con-tent,anti-oxidation ingredients,and anti-tyrosinase ingredients with the polarity of the solvent.Actually,this parameter has already been frequently used to predict the miscibility and solu-bility of materials with a particular solvent [13,14].According to Hildebrand [12],the solubility parameter is directly dictated by the cohesive energy (E coh,i )composed of a linear combination of contributions from the dispersion interaction (E d,i ),polar inter-action (E p,i ),and hydrogen bonding interaction (E h ,i ),defined as:δi =E coh ,ii = E d,i +E p,i +E h,i i (4)Since the cohesion parameters are related to the correspondinginteraction energies as:E d,i +E p,i +E h,i =δ2d,i +δ2p,i +δ2h,i(5)the solubility parameter can be rearranged as:δi =δ2d,i +δ2p,i +δ2h,i(6)where δi is the solubility parameter [MPa 1/2]for species i andV i is the mole volume for species i .For pure solvents of current study,the values of the cohesion parameters,as summarized in Table 1,were then used to calculate the solubility parameters for the water and alcohol species,and the solubility parameters for the alcohol–water mixtures estimated based on a simple mixing rule as follows:δm = ix i δi (7)where δm is the solubility parameter for the alcohol–water mix-ture and x i is the volume fraction of species i in the mixture.In addition,the temperature adjustment for the solubility parameter is considered by Barton [12]: δ1δ2 2=T 2T 1(8)where T i indicates the extraction temperature [K].J.-M.Kim et al./Biochemical Engineering Journal37(2007)271–278275Fig.4.Correlations of extraction of bio-active ingredients form mulberry leaves with polarity of solvent:(a)content of polyphenols,(b)anti-oxidation ingredients and(c)anti-tyrosinase ingredients.As shown in Fig.4,the extraction efficiencies of the solvents for the bio-active ingredients in mulberry leaves,obtained using various alcohol species and compositions,correlated well with the single parameter of the solvent polarity.The optimal extrac-tion for polyphenolic compounds was achieved with a solvent polarity of38.5[MPa1/2](Fig.4(a)),corresponding to a36.9% methanol fraction,30.9%ethanol fraction,29.3%n-propanol fractions,and27.5%iso-propanol fraction in the binary mixture. Meanwhile,the optimal extraction for anti-oxidation ingredients was achieved with a solvent polarity of37.3[MPa1/2](Fig.4(b)), representing a slight increase in the alcohol fraction in the binary mixture(42%methanol,35.1%ethanol,33.3%n-propanol,and 31.2%iso-propanol fraction in the binary mixture).As such, these correlation results on the optimal solvent polarity for extraction would seem to explain why the optimal alcohol com-position varied with the alcohol species in Figs.1and2,and agreed well with the extraction efficiency profiles relative to the alcohol composition.However,the optimal solvent polarity for the anti-tyrosinase ingredients was about33.0[MPa1/2],which was lower than that for the polyphenols and anti-oxidation ingredients,as shown in Fig.4(c).From those experiment results,it would be inferred that the anti-tyrosinase ingredients were less polar and then optimally extracted out with the lower polarity of the solvent than anti-oxidation ingredients.As a result,this low polar condition for the optimum solvent meant a high alcohol fraction was required, such as a70.3%methanol,58.7%ethanol,55.7%n-propanol, and52.3%iso-propanol fraction in the binary mixture,to extract the anti-tyrosinase ingredients.The correlations of the extraction efficiency for bio-active ingredients with the solvent polarity were evaluated using model solvents with a broad polarity range.As summarized in Table2, ethylene glycol,acetone,methanol,and water were used to make the model solvents with polarities ranging from24.8to41.0 [MPa1/2],and applied to extract the bio-active ingredients,as shown in Fig.5.Despite the different species and composi-tions of the model solvents,their extraction efficiencies for the polyphenol content,anti-oxidation and anti-tyrosinase ingredi-ents were well matched with the correlations obtained using the alcohol–water binary mixtures.Furthermore,the polarities of quercetine and mulberroside F were estimated to confirm the above correlation using a func-276J.-M.Kim et al./Biochemical Engineering Journal 37(2007)271–278Table 2Solubility parameters of model solvents (at 80◦C)Solventδ(MPa 1/2)Symbols (for Fig.5)PolyphenolsAnti-oxidant Anti-tyrosinase25%ethylene glycol in water 41.10᭹42%ethylene glycol in water 39.0558%ethylene glycol in water 37.1247%acetone in water 31.8857%acetone in water 29.28♦27%acetone inmethanol24.81parison of extraction of bio-active ingredients by model solvents with correlation of extraction along with solvent polarity.tional group contribution theory [12,15],as these compounds,molecular structures shown in Fig.6,were the most active anti-oxidation and anti-tyrosinase compoundsfrom the mulberry leaves,respectively.Due to the unavailability of cohesive energyFig.6.Molecular structures of (a)quercetine and (b)mulberroside F that are most active ingredients for anti-oxidation and anti-tyrosinase,respectively,in mulberry leaves.data,the solubility parameter for quercetine and mulberroside F was predicted from the molar vaporization energy (g U i )and molar volume (g V i )of the functional group in each compound using the following equation [12]:δi = g U igV i1/2(8)As summarized in Table 3,when using the functional group data on the molar vaporization energy and molar volume with Eq.(8),the polarity of quercetine and mulberroside F was pre-dicted as 36.1and 34.5[MPa 1/2],respectively.Consequently,these polarity values for the compounds,which were consistent with the optimal solvent polarities for the extractions,provide a possible explanation why the optimal solvent conditions for the anti-tyrosinase extraction occurred at a lower solvent polarity than the anti-oxidation extraction in the correlations.The correlation of the extraction efficiency with the solvent polarity was also examined by comparing the contents of a target ingredient extracted under different solvent conditions.As shown in Fig.7,when extracted at three different solvent polarities (21.5,32.0,and 44.1[MPa 1/2]),the thin layer chro-matography spectra clearly revealed that the extraction with a solvent polarity of 32.0[MPa 1/2]contained a higher concen-tration of mulberroside F than any of the other extractions,as expected from the above correlation with the anti-tyrosinase ingredients.Table 3The molar vaporation energy and molar volume of functional groups for predic-tion of solubility parameters of quercetine and mulberroside F [9](at 25◦C)Groupg U(kJ mol −1)g V (cm 3mol −1)CH 3 4.7133.5CH 2 4.9416.1>CH 3.43−1.0CH 4.3113.5>C4.31−5.5Ring closure,five or more atoms1.0516Conjugation in ring,for each double bond1.67−2.2OH (disubstituted or on adjacent C atoms)21.913.0OH 29.810.0O 3.35 3.8CO17.410.8J.-M.Kim et al./Biochemical Engineering Journal37(2007)271–278277parison of concentration of mulberroside F extacted at three different solvent conditions.4.ConclusionsAn effective solvent for extracting bio-active ingredients, such as anti-oxidants and anti-tyrosinases,from mulberry leaves was identified by varying the solvent species and composi-tions.For the effective extraction of target ingredients with a specific activity from among many ingredients,the solvent con-dition was determined based on the polar propensity of the target ingredients.As such,the active anti-oxidation ingredi-ents and polyphenols with a high polar propensity required an alcohol–water binary solvent with a methanol fraction of about 60%for optimal extraction,whereas the anti-tyrosinase ingre-dients were most favorably drawn out by a binary solvent with an80%methanol fraction.In addition,due to the lower polar propensity of ethanol and propanols than methanol,their frac-tions of the binary mixture for the optimal extraction of the active ingredients were lower than the methanol fraction.This dependency of the optimal extraction on the solvent species and composition can be simply described by the sol-vent polarity represented by the solubility parameter.Based on correlating the extraction efficiency of the anti-oxidation and anti-tyrosinase ingredients with the solvent polarity,the opti-mal conditions for the solvent were predicted as a solubility parameter above38.0and33.0[MPa1/2],respectively.These predictions for the optimal solvent conditions for the extraction of the anti-oxidation and anti-tyrosinase ingredients were also consistent with model solvent conditions designed using acetone and ethylene glycol,and confirmed by the extracted contents of quercetine and mulberroside F,the most active anti-oxidation and anti-tyrosinase compounds in mulberry leaves,respectively.Accordingly,the correlations between the extracted contents of bio-active ingredients and the solvent polarity would appear to provide useful information on the optimal solvent conditions for the extraction of target ingredients with specific activity,which may prove to be helpful in the design of industrial processes and solvents.AcknowledgementThe authors are grateful for grants from the Korean Ministry of Health and 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