Separation and Purification Technology 48 (2006) 176–181

Extraction of polyphenols from grape seedsand concentration by ultrafiltration

Haseeb Nawaz a, John Shi b, Gauri S. Mittal a,∗, Yukio Kakuda c

a School of Engineering, University of Guelph, Guelph, Ont., Canadab Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ont., Canada

c Department of Food Science, University of Guelph, Guelph, Ont., Canada

Abstract

A solvent extraction method utilizing 50% ethanol and 50% water as solvent was used for the extraction of polyphenols from grape seeds.An additional ultrafiltlration step was also included to determine its beneficial effect. Various experimental conditions, such as solid to liquidratio (0.1–0.25 g/ml), number of extraction stages (single, double and triple) and membrane pore size (0.22 and 0.45 �m) were investigatedto optimize the extraction. When compared to a gallic acid standard, the extraction of grape seed polyphenols with a 0.2 g/ml solid to liquidratio, double stage extraction and 0.22 �m membrane pore size were the optimal conditions. Under these conditions, the maximum amounts ofpa©

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olyphenols (11.4% of the total seeds weight) were recovered from grape seeds. From the standpoint of solvent toxicity, extraction efficiencynd percentage of grape seed polyphenols recovered, this method proved to be adequate on all three counts.

2005 Elsevier B.V. All rights reserved.

eywords: Polyphenols; Ultrafiltration; Solvent extraction; Grape seed; Neutraceutical

. Introduction

The importance of polyphenols is due to their positiveontribution to cellular processes within the body. In terms ofharmacological activity, they protect against the oxidation ofigh-density lipids (HDLs) [1]. Hence, they help the body toetain important HDLs while helping to remove problematicow-density lipids (LDLs). Additionally, polyphenols alsoave anti-ulcer [2], anti-carcinogenic [3] and anti-mutagenicctivities [4]. The reason for these activities is the strongntioxidant nature of polyphenols, which is based on theirbility to absorb free radicals. Polyphenols are broadly dis-ributed in the plant kingdom and are the most abundantecondary metabolites found in plants [5]. These polypheno-ic substances or polyphenols include many classes of com-ounds ranging from phenolic acids, colored anthocyanins,imple flavonoids and complex flavonoids. Polyphenols rep-esent the third most abundant constituent in grapes andines after carbohydrates and fruit acids [6]. The most com-

∗ Corresponding author. Tel.: +1 519 824 4120x52431;

mon polyphenolic acids in grapes include cinnamic acids(coumaric, caffeic, ferulic, chlorogenic and neochlorogenicacids) and flavonoids include colorless flavan-3-ol (such ascatechin, epicatechin, their polymers and their ester formswith galactic acid or glucose) [5] (Fig. 1).

Oligomeric proanthocyanidins (OPCs) are a class ofpolyphenolic biflavanoids that are found in fruits and veg-etables. The highest concentration of these is in the seedsof grapes. OPCs are made up of two or more monomers,which are chemically bonded. For example, dimers are twomonomers, trimers are three monomers and tetramers arefour monomers bonded together. The dimeric procyanidinsare often referred to as the B-series and the trimeric pro-cyanidins as the C-series. Five different dimers (procyanidinB1, B2, B3, B4 and B5) and two trimers (C1 and C2) wereidentified from grape skin and seeds [7]. Catechin and epi-catechin are the two proanthocyanidin monomers. Dimers,trimers, tetramers, etc. are created when each of these twomonomers bind at the � or � position on their molecularstructures. Additionally, catechin and epicatechin can com-bine to create esters, such as catechin/epicatechin gallate.They can also bond with sugars and proteins to create glyco-

ax: +1 519 836 0227.

E-mail address: gmittal@uoguelph.ca (G.S. Mittal). sides and peptides. About 162 dimers, such as gallic acid

383-5866/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2005.07.006

H. Nawaz et al. / Separation and Purification Technology 48 (2006) 176–181 177

Fig. 1. Structures of major polyphenols identified in grape seed extract [5].

and glucose esters, can be created [8]. The polyphenoliccompounds are broadly distributed inside grapes. The com-position of polyphenols depends upon whether the extractionis performed on whole grape pulp, skin or seeds. The percent-age of the total extractable polyphenols in grape tissues are:10% or less in the pulp, 60–70% in the seeds and 28–35% inthe skin. The polyphenol content of seeds may range from 5to 8 wt% [9].

The extraction of polyphenols is dependant upon twoactions, the dissolution of each polyphenolic compound atthe cellular level in the plant material matrix, and their dif-fusion in the external solvent medium [10]. Previously, allextractions were performed with organic solvents. Theseextraction procedures were efficient, but the extracts were notsafe for human consumption due to potential toxic effectsfrom the residual solvent. At the present time, there is noreported grape seed extraction procedure utilizing ethanol asthe sole solvent. In the past, solvents, such as hexane andmethanol combinations [11], ethanol–benzene combinations[12], ethyl acetate [13] and sulfur dioxide have been used[14]. All these solvents are toxic to humans if consumed inlarge doses. These health concerns have sparked research intomethods that would reduce the use of organic solvents in theextraction procedure. Membrane processing is one method

Table 1Molecular weight of various polyphenols [15]

Polyphenol type Molecular weight (MW)

Catechin 290.3Epi-catechin 290.3Epi-catechin gallate 442.4Procyanidin dimer 578.5Procyanidin trimer 870Procyanidin tetramer 1160Kaempferol 286.2Gallic acid 170.1Quercetin 448.4Caffeic acid 180.2Coumaric acid 164.2Chlorogenic acid 354.3

that reduces the use of toxic organic solvents and concentratesthe final product. Solvents are still used with this method, butnot to the same extent as with organic solvent extraction.Membranes are used towards the end of the process, whenthe polyphenols are to be separated and concentrated fromthe solvent–solute complex.

Ultrafiltration (UF) is the most commonly used proce-dure to separate desirable components from a mixture. UF isdependent on the particle size, and typical rejected speciesinclude sugars, bio-molecules, polymers and colloidal parti-cles. The driving force for transport across the membrane isthe pressure differential. UF operate at 2–10 bar, although insome cases up to 25–30 bar have been used. UF performs feedclarification, concentration of rejected solutes and fractiona-tion of solutes. UF membranes can reject molecules withinthe molecular weight ranges of more than 1000 MW, a reasonwhy they are successful in separating polyphenols [15]. Themolecular weight of various polyphenols are in Table 1.

There are numerous advantages associated with mem-brane separation processes. Compared to mechanical separa-tions, membrane separation involves high purity, low energyrequirement, no additive, mild operating conditions, greaterseparation efficiency and easy scaling up. UF has been usedin the past for the removal of polyphenols from grape mustsor wines [16]. Therefore, the objective of this study was toextract polyphenols from grape seeds using an ethanol–watermt

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ixture, optimize the extraction procedure and then concen-rate the polyphenols by UF.

. Materials and methods

.1. Plant materials and chemicals

Whole grape seeds were collected from Joseph’s Estateines (Niagara, Ont., Canada) and milled to a fine pow-

er. Ethanol was of analytical reagent grade (Commerciallcohols Inc., Brampton, Canada). Folin–Ciocalteau reagent,

odium bicarbonate powder and gallic acid (Sigma Chemical,t. Louis, USA) were used for the analysis of total polyphe-ols.

178 H. Nawaz et al. / Separation and Purification Technology 48 (2006) 176–181

Fig. 2. Gallic acid standard curve based on the data collected (linear trendline in solid).

2.2. Gallic acid standard curve

The level of total polyphenols was determined usingFolin–Ciocalteau (F&C) colorimetric reaction method [17].A range of gallic acid concentrations from 0.0005 to 0.02 g/mlwas used to prepare the calibration curves. Half millilitre ofthe standard solution was added to 10 ml water and 0.5 mlF&C reagent. After 5 min, 8 ml of 7.5% sodium carbon-ate solution was added. The solution was allowed to sit for2 h, and then refrigerated over night for analysis the follow-ing day. Readings were taken at 765 nm using a ShimadruUV–vis recording spectrophotometer (model UV-260). Thegallic acid standard curve is shown in Fig. 2.

2.3. Organic solvent extraction and concentration

The optimal solid to liquid ratio should be the first opti-mization procedure undertaken. The extraction and concen-tration procedures are illustrated in Fig. 3. The solid to liquidratio (0.1–0.25 g/ml), number of extraction (single, doubleand triple) and ethanol as an organic solvent were used.The membrane pore sizes (0.22 and 0.45 �m) used, rejectedmolecules within the molecular weight value of 1000 MW[15]. The two selected membranes were Millipore type GS0.22 �m and Millipore Type HA 0.45 �m. The selection ofeebowt

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Fig. 3. Schematic representation of the extraction and concentration proce-dure.

for 5 min. After mixing, the solution was left in the dark toextract for 1 h. Once extracted, the analysis for polyphenolswas carried out as described for the gallic acid standard curve.The data are plotted in Fig. 4.

The following is the outline of the sample preparation:

1. About 40 g of milled grape seed was mixed with 200 ml50% ethanol solution. This was mixed for 5 min and thenallowed to extract in the dark for 1 h.

2. Top phase was filtered through Whatman filter paper #4(9.0 cm diameter).

3. Residue resuspended in 150 ml 95% ethanol solution.Mixed for 5 min and then allowed to extract in the darkfor 1 h.

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thanol as the organic solvent was based on the fact thatthanol when mixed with water improves the solubility of theioactive component when compared to pure water [18]. Theptimum solution consists of a mixture of 50% ethanol–50%ater. Additionally, ethanol is a safe biological solvent unlike

he aforementioned organic solvents.In determining the optimal solid to liquid ratio, the UF

tep was not included, since this step was important foretermining the extraction effectiveness in the later trials.dditionally, the comparison of the extraction efficiency with

nd without the UF step was used to determine its signifi-ance in the extraction procedure. The appropriate amount ofilled seeds were placed in 50% ethanol solution and mixed

ig. 4. Solid to liquid ratio curve based on the data collected (thick linehowing polynomial trend).

H. Nawaz et al. / Separation and Purification Technology 48 (2006) 176–181 179

4. Top phase filtered through Whatman filter paper #4(9.0 cm diameter) and added to filtered solution in step2.

5. Residue re-suspended in 150 ml 95% ethanol solution.Mixed for 5 min and then allowed to extract in the darkfor 1 h.

6. Top phase filtered through filter paper #4 and added tofiltered solution in step 4.

The single extraction was prepared using steps 1–2. Thedouble extraction was prepared using steps 1–4. The tripleextraction was prepared using steps 1–6. During the firstextraction the majority of the polyphenols were removed.Therefore, a stronger solvent was required for polyphenolextraction in the second and third extractions (95% ethanolsolution). A Millipore stirred ultrafiltration cell (model 8050lab-scale unit) was used for the UF process. The operatingpressure and temperature were set and maintained at 5 barand 23 ± 1 ◦C, respectively. The pressure produced the fluxthrough the membrane. The volume of the filtration cell was50 ml.

Since polyphenols are micromolecules, membranes withsmall enough pore sizes were selected to reduce the amountof unnecessary material in the final concentrate. Two selectedmembranes were Millipore type GS 0.22 �m and MilliporeType HA 0.45 �m. In all cases, the samples were ultra-fiwtF

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(solute) mass transfer during extraction are [19]:

−dm

dt= km(m − me) (1)

ds

dt= ks(s − se) (2)

where km is the moisture transfer coefficient (1 min), ks thesolute transfer coefficient (1 min), m the moisture content bydry basis, s the solute concentration (g/g), e for equilibriumand t is the time (min).

Integrating Eqs. (1) and (2) results in the following equa-tions:

ln

(m − me

m0 − me

)= ln Mr = −kmt (3)

ln

(s − se

s0 − se

)= lnSr = −kst (4)

Mr = m − me

m0 − me

(5)

Sr = s − se

m0 − me

(6)

where Mr is the moisture ratio, Sr the solute ratio and 0 is forinitial concentration.

The collected experimental data (Mr versus t) werei

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ltered using the filtration cell. The concentrate collectedas analyzed using the same procedure as the one utilized

o produce the gallic acid curve. The data are plotted inig. 5.

.4. Mass transfer during extraction

To determine mass transfer during extraction, periodiceasurements of residue weight and solution volume were

aken after every 15 min for each type of extraction (sin-le, double and triple). The equations for moisture and solid

ig. 5. Absorbance readings of the various experiment types based on theverage of the two trials.

nputted into Eq. (3) and km was calculated.

. Results and discussion

.1. Gallic acid standard curve

The following regression model expresses the absorbancef gallic acid standard as a function of concentration.

= 10.071 C + 0.314 (7)

here A is the absorbance at 765 nm and C is the gallic acidoncentration (g/ml).

.2. Effect of UF on yield of polyphenols

The UF of the ethanol extracts were performed to deter-ine whether it had a significant influence on the final

olyphenol concentration. The absorbance values were quiteigh in comparison to the absorbance values from otherxperiments (Fig. 4). This was due to the extra particles andnnecessary solutes that hinder the isolation of polyphenols.hus, the UF step definitely aids in the polyphenol concen-

ration.

.3. Effect of solid (solute) to liquid ratio on optimalxtraction

Fig. 4 indicates that the optimal solid to liquid ratio is.2 g/ml. A higher ratio results in excess solutes that cannot

180 H. Nawaz et al. / Separation and Purification Technology 48 (2006) 176–181

be efficiently penetrated by the ethanol solvent. The followingregression model expresses the absorbance at a specific solidto liquid ratio.

A = −2.70 C2 + 0.981 C + 0.795 (8)

Due to the water solubility of many polyphenols, hot watercould serve as an extraction solvent. This treatment will elimi-nate any safety concerns but it will complicate the extractionand concentration process. The presence of ethanol in the50/50 mixture will assist in penetrating the hydrophobic areasof the seed matrix and help to precipitate the soluble seed pro-teins that would interfere with the ultrafiltration procedure[20].

3.4. Effect of the number of extractions on the overallextraction of polyphenols

Fig. 5 illustrates the absorbances of extractions using twopore size membranes and three extraction stages. It appearsthat in all cases the triple extraction procedure results in alower absorbance due to the dilution of the polyphenols.In the first case (0.22 �m membrane), the double extrac-tion produced a higher concentration of polyphenols, whilein the second case (0.45 �m membrane) a double extractionytr

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3.6. Mass transfer during extraction

By analyzing Fig. 6a and b, km values were determined:

Single extraction : ln Mr = −0.0095t (10)

Double extraction : lnMr = −0.0007t (11)

Triple extraction : lnMr = −0.0002t (12)

The plots of the ln (moisture content) and ln (solid content)versus treatment time (Fig. 6a and b) show that the moistureand solute diffusion rates decreased with further extractions.There are essentially two mass transfers, the diffusion of sol-vent into the grape seed particles and diffusion of solute fromthe grape seed particles to the solvent. In the first mass trans-fer, the driving force is the pressure difference while in thesecond concerning the solute, the concentration difference isthe driving force. Therefore, it can be implied that a thirdextraction barely assists in the total process. It just furtherdilutes the polyphenols that only makes the separation pro-cess further cumbersome. Thus, the two-stage extraction isoptimal.

Fig. 6. (a) In variation of moisture and (b) solute content with time duringextraction at 23 ◦C.

ielded the same amount of polyphenols as the single extrac-ion. Therefore, a double extraction would be ideal, since itemoved most of polyphenols.

.5. Effect of membrane pore size on the extraction ofolyphenols

As noted earlier, the UF step played an important rolen the extraction process by concentrating the polyphenols.ccording to Fig. 5, the 0.22 �m membrane provided better

esults than the 0.45 �m membrane due to the fact that themaller pore size rejected greater amounts of unnecessaryarticles and solutes. The smaller the membrane utilized,he better the results would be, and the purer the polyphe-ol extract. The following regression model expresses thebsorbance of a double extraction at various membrane poreizes.

= −0.4663 Sm + 0.5526 (9)

here Sm is the membrane pore size (�m).Based on the gallic acid standard curve, a 0.01 g/ml con-

entration of polyphenols was achieved. Since a doublextraction was optimal, a solid to liquid ratio of 0.114 g/mlas achieved. As mentioned earlier, the polyphenol contentf seeds may range from 5 to 8 wt%. Based on the finalolyphenol concentration, the maximum amount of polyphe-ols was extracted. Future work could use membranes withmaller pore sizes to further concentrate polyphenols andurify polyphenol mixture.

H. Nawaz et al. / Separation and Purification Technology 48 (2006) 176–181 181

4. Conclusions

Ethanol as the extracting solvent was shown to be an effi-cient method of extracting polyphenols from grape seeds.With the concentration step of UF, the procedure providedhigh extraction rates, high extraction selectivity, short extrac-tion time and significant labor savings. Ideally, the solid toliquid ratio of the raw material should be 0.2 g/ml, accord-ing to mass transfer calculations and absorbance readings adouble extraction would be better, and a membrane with asmall pore size should be used (0.22 �m). With this combi-nation, it was possible to recover the maximum amount ofpolyphenols (11.4% of the total seeds weight) from the grapeseed. The temperature of extraction and the composition ofthe extracting phase even with the same solvent-system havea great effect on the extraction yield of polyphenols. Food andmedicinal industries that utilize polyphenols as antioxidantswould be benefited from this combination of extraction andconcentration.

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