Production of an Isoflavone Concentrate from Red Clover Flowers KUMAR

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1 JFS S: Sensory and Nutritive Qualities of Food Production of an Isoflavone Concentrate from Red Clover Flowers LEI EI XU,, ASHW SHWANI KUMAR UMAR,, KAREN LAMB AMB, AND LINDA LAYTON ABSTRACT CT: : Red clover flowers are a rich source of isoflavones ones in the forms of biochanin A, formononetin, genistein, and genistin. A simple process was developed eloped to reco ecover er isoflavones ones from red clover flowers as a nutraceutical prod- uct. Isoflav soflavones ones wer ere e 1st extracted in alkaline water and insoluble polyvinylpolypyrr yrrolidone (PVP) was then added to the extract for adsorbing isoflavones ones. The adsorbed isoflavones ones wer ere e subsequently eluted with ethanol and dried. The main steps of this process wer ere e optimized for the product yield and quality. The final product was an isoflavone one concentrate ate,, containing more than 20 wt% isoflavones ones,, which repr epresented esented a reco ecover ery of greater than 50% of the total isoflavone one amount in red clover flowers ers. Keywor eywords: red clover er,, isoflavones ones,, extraction, adsorption, elution Introduction Recent studies have shown that bioactive compounds such as isoflavones have many health benefits. As both antioxidants and phytoestrogens, isoflavones are able to prevent certain hormone-related cancers (Wei and others 1993; Adlercreutz 1995; Barnes 1997), reduce the risk of osteoporosis and cardiovascular diseases (Carrol 1991; Adlercreutz and Mazur 1997; Anderson and Carner 1997), and relieve menopausal symptoms (Kuzer 2000; Liu and others 2001). So far soybeans remain the main source of isoflavones, and soybased supplements are particularly rich in daidzein and genistein in the forms of aglycones, glucosides, and esters. Two types of isoflavone products are produced from soybeans: isoflavone concentrates and isoflavone-enriched soy protein isolates (Chang 2002). In the extraction and processing of these products, large quantities of organic solvents or expensive enzymes are used. To achieve high purity, chromatographic techniques are usually used following solvent extraction, which inevitably slows down the process. Membrane processing, on the other hand, is able to accelerate the concentration step of extracted isoflavones, but it alone cannot give a high purity product (Xu and others 2004). Tablets made of isoflavone concentrates may be taken directly as supplements. Hendrich and others (1994) have estimated that the anticarcinogenic dose of isoflavones for humans is 1.5 to 2.0 mg/kg body weight every day. In addition to all the health benefits, the soybased isoflavones also show stimulatory effects on breast tissue owing to their weak estrogenic activity (Atkinson and others 2004), which may affect their breast cancer fighting ability, thus limiting their potential medical applications. Lately, it has been found that red clover, a common plant that grows in pasturelands and hay fields, contains significant amounts of isoflavones in its flowers. Unlike soybeans, the predominant isoflavones in red clover are biochanin A and formononetin, which are methyl ether derivatives of genistein and daidzein, respectively. Although the compositional difference could affect the bioavailability of isoflavones, nutritional research shows that absorption in humans of isoflavones MS Submitted 5/18/05, Revised 6/28/05, Accepted 7/12/05. Authors are with Inst. for Chemical Process and Environmental Technology, Natl. Research Council, Montreal Rd. Campus, Ottawa, Ontario, Canada K1A 0R6. Direct inquiries to author Kumar ( ashwani.kumar@nrc-cnrc.gc.ca). S558 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 8, 2005 Published on Web 10/6/2005 from soy and red clover is similar (Tsunoda and others 2002). Further studies indicate that in contrast to conventional hormone replacement therapies using soy isoflavones, red clover derived isoflavones do not increase breast density, suggesting their potential use as an alternative to soy isoflavones in hormone replacement therapies (Atkinson and others 2004). Moreover, the idea of using isoflavones from red clover as an antioxidant ingredient in functional foods is supported by the relatively higher radical scavenging capacity of a red clover extract than that of a soy preparation (Kroyer 2004). Isoflavones from red clover can be extracted and isolated using the same techniques as in soy processing. A simple, economical, and efficient method is, however, still needed. The present study reports a new process to recover isoflavones from red clover flowers, including main steps of extraction, adsorption, and elution and drying. A powdery isoflavone concentrate was obtained as the final product with a high isoflavone recovery. Materials and Methods Extr xtraction of isoflavones ones from red clover er Approximately 9 kg dried red clover flowers were purchased from Frontier Natural Products (Norway, Iowa, U.S.A.). Flowers were ground in a 4-L Warring blender (Warring Commercial, Torrington, Conn., U.S.A.) for 2 min, and well mixed in a 20-L pail. Subsequent experiments for isoflavone extractions were performed with batches of 90 g ground material at different temperatures and ph conditions. For extraction at elevated temperatures, each batch of ground flowers was mixed with deionized water at a water-to-flowers ratio of 40 in a 4-L beaker, whereas water was preheated to the target temperature. The slurry was stirred on a Mirak digital stirrer SP72725 (Barnstead Intl., Dubuque, Iowa, U.S.A.) while the temperature during extraction was maintained. The beaker was covered with aluminum foil to minimize water loss by evaporation. Using a pipette and a coffee filter, duplicate 3 ml samples were taken from the extraction solution for isoflavone analyses at intervals of 15 min during first hour and 30 min in subsequent experiments. Extraction was also conducted at room temperature as a control. In all cases of alkaline extraction, the ph of each batch was adjusted and maintained by adding 4 M NaOH, and extractions were carried out at room temperature only Institute of Food Technologists Further reproduction without permission is prohibited

2 Adsorption of isoflavones ones by polyvinylpolypyrr yrrolidone (insoluble PVP) For this set of experiments, in each run red clover was 1st extracted at ph 10.0 for 2 h, and then the extract was strained through Tyler Nr 18 and Nr 400 sieves to separate the solid residue. Fresh water was poured evenly over the residue for washing. The amount of water was equivalent to that of the extract retained in the residue, and it was combined with the extract. A desired amount of insoluble PVP (Sigma Aldrich, St. Louis, Mo., U.S.A.) was added to the extract, and then the ph of the extract was decreased to 5.5 by adding 6 M HCl. The mixture was stirred for 15 min at room temperature. The PVP was recovered using an IEC CR-6000 swing basket centrifuge (Intl. Equipment Co., Needham, Mass., U.S.A.) at 2000 rpm for 10 min. To calculate the adsorbed amount of isoflavones, samples were analyzed before and after the adsorption. Adsorption experiments were conducted at different PVP-to red clover ratios ranging from 5% to 30% (w/w). Elution of isoflavones ones with ethanol In these experiments, the above procedure was followed for alkaline extraction at ph 10.0 and adsorption with 15% PVP. After centrifugation, PVP was collected and washed with amounts of 95% ethanol ranging from 5% to 20% of the volume of the original extract (~3.6 L) (Commercial Alcohols Inc., Brampton, Ontario, Canada). The PVP in ethanol was stirred for 15 min at room temperature. This ethanol solution was filtered through Whatman Nr 40 filter paper using a B chner funnel and its volume was measured in a graduated cylinder. Duplicate samples were then taken for isoflavone analyses. Reuse of PVP without regener egeneration The used PVP was eluted with 540 ml ethanol (equivalent to 15% of the volume of the extraction solution) and air-dried overnight in a fume hood. It was then used for adsorption of isoflavones in the next run. This cycle was repeated 4 times, and each time, duplicate samples were taken from the aqueous extract before and after the adsorption for isoflavone analyses to estimate the adsorbed amount. Production of isoflavone one concentrates Each production run was started with 90 g ground red clover flowers in 3.6 L water. The procedures of alkaline extraction, PVP adsorption, and ethanol elution were followed, where the ph for extraction was set at 10.0, the PVP level at 15%, and the volume of ethanol at 540 ml. Before ethanol elution, the PVP was washed with 360 to 720 ml water. The ethanol filtrate after the elution was evaporated under vacuum at 60 C in a Laborota 4000 evaporator (Heidolph Instruments, Cinnaminson, N.J., U.S.A.) to remove ethanol. The remaining aqueous portion was lyophilized for 48 h using a FreeZone 1 L freeze dryer (Labconco Corp., Kansas City, Mo., U.S.A.). The residue of red clover flowers after alkaline extraction was dried at 105 C overnight in an oven. Duplicate samples were taken from the dried residue, washing water, and spent extract after PVP adsorption, and triplicate samples from dried isoflavone concentrate for isoflavone analyses. Isoflav soflavone one analyses Each aqueous sample was prepared by mixing it with an equal amount of high-performance liquid chromatography (HPLC)-grade methanol containing 2% glacial acetic acid (v/v) in a 10-mL graduated cylinder with a stopper. All ethanol samples were analyzed as is. For the determination of isoflavone concentration, an HPLC-based method was used following the parameters set by Klump and others (2001). The key instrument was a Hewlett Packard 1100 HPLC system with a Zorbax SB-C18 reversed-phase column ( mm i.d.) and URLs and addresses are active links at a diode array UV detector (Agilent Technologies, Wilmington, Del., U.S.A.). The isoflavone standards were purchased from Indofine Chemical Co. (Somerville, N.J., U.S.A.). All analytical results of isoflavone determination were expressed as g/g for consistency, and t- test was used whenever statistical analyses were performed for sample comparison. Results and Discussion Isoflav soflavone one extraction from red clover Amounts of isoflavones in ground red clover flowers as determined by HPLC are listed in Table 1. Biochanin A and formononetin were present as 2 major isoflavones in our analysis of red clover, which was similar to reported data (Liu and others 2001; Tsunoda and others 2002). The total isoflavone content of 2590 g/g was higher than those reported for soybeans and soy products (Chang 2002). Although isoflavones are highly soluble in organic solvents such as alcohols, in an attempt to eliminate or reduce the use of these solvents, water was tested for isoflavone extraction in this study. Extraction was 1st done at elevated temperatures between 60 and 90 C, as most isoflavones had low water solubilities below 50 C. Due to the fast evaporation rate of water, no extraction experiments were done at higher than 90 C. The runs at room temperature were conducted only as a control. The extraction curves in Figure 1 show that in all runs at elevated temperatures, the isoflavone concentration rose steadily in the 1st 90 min, and then began to level off, indicating that equilibrium was reached. An ascending trend was observed in isoflavone extractability with the rising temperatures in different runs, as shown by the increasing isoflavone concentration in the extract at equilibrium. As expected, extraction at room temperature yielded only a small amount of isoflavones in the extract. The final isoflavone concentration in the extract at 90 C was 43 g/g, which is equivalent to an extractability of approximately 66% based on the total amount of isoflavones in ground flowers. The effects of ph on isoflavone extraction from red clover were also examined. It could be seen in Figure 2 that the isoflavone concentration in the extract continued to increase in the 1st 2 h and then leveled off, suggesting that alkaline extraction did not reach equilibrium faster than extraction at elevated temperatures. It is, however, obvious that the final isoflavone concentrations for high ph extracts were higher than those done at elevated temperatures. Although isoflavone concentration in the extract increased with ph, the effect Figure 1 Extraction of isoflavones from red clover at elevated temperatures. Error bars are based on duplicate runs. Vol. 70, Nr. 8, 2005 JOURNAL OF FOOD SCIENCE S559

3 Table 1 Isoflavone composition in red clover flowers Isoflavone type Content ( g/g) a Genistein 357A Genistin 320A Biochanin A 1252B Formononetin 661C Total 2590 a Means of 4 replicates. Values not sharing a common capital letter are significantly different from one another (P < 0.05). of ph was far greater on the extractability below ph 9.5. For example, the final isoflavone concentration in the extract increased from 51 to 63 g/g as extraction ph changed from 9.0 to 9.5, whereas it changed only slightly from ph 9.5 to The extraction curves at ph 10.0 and 10.5 almost overlapped between 120 and 180 min of extraction time (Figure 2). At ph 10.0, the final solution concentration of 66 g/g, indicated an almost complete extraction of all isoflavones in red clover, thus rendering any studies on higher ph unnecessary. Only 1 ph in the acidic range was investigated for isoflavone extraction, and in striking contrast to alkaline extraction, an even lower amount of isoflavones was extracted at ph 3.0 than the natural ph of aqueous extract of red clover (5.5). This shows that apparently isoflavone extraction in acidic medium is lower than that of basic medium. Based on the above results, ph 10.0 was chosen for isoflavone extraction in subsequent experiments, due to the high yield at this ph. Although a similar extractability was observed at ph 9.5, it was felt that in the vicinity of this value, even minor ph fluctuations might affect the yield significantly. Any ph value above 10.0 was not necessary for the extraction, as noted earlier. Adsorption of isoflavone one by y PVP Considering that red clover extract contained various components, an adsorbent could be used to separate isoflavones. We selected a synthetic polymer, cross-linked PVP, for isoflavone separation. This polymeric adsorbent, trade name Polyclar AT, has long been used in the beer industry to prevent hazing, because it is able to remove the polyphenols that bind and precipitate proteins. Xu and Diosady (2002) used insoluble PVP for phenolic removal in the process for making high quality canola protein isolates. Their tests were successful in the slightly acidic ph range. This was due to the ability of PVP to form hydrogen bonds with phenolic compounds. Therefore, in this study, we also tried this polymer to bind isoflavones because they are, by nature, phenolics as well. According to Xu and Diosady (2002), PVP did not work quite as well at high ph owing to the presence of excessive hydroxide ions, so after PVP addition, the ph of the extract was lowered to 5.5 in all subsequent adsorption experiments. As could be seen in Figure 3, PVP was effective in adsorbing extracted isoflavones. A 5% PVP (4.5 g) addition adsorbed 60% of the extracted amount. Increasing the PVP amounts by 3 times to 15% resulted in an appreciable gain of about 20 percentage points in adsorbed isoflavone to about 81%. However, even at 30% addition, there was still about 15% of the extracted amount that was not adsorbed. It indicated an equilibrium between the isoflavones adsorbed by PVP and those remaining in the extract, which can be characterized by a partition coefficient K =C PVP /C extract, where C PVP and C extract are concentrations of isoflavones adsorbed by PVP and remaining in the extract, respectively. Theoretically, K is a constant, but due to the errors in isoflavone analyses, particularly in samples with low isoflavone content, the K values determined at different PVP levels varied from 860 to 1240 (Table 2). It is apparent that these values were not significantly different, and K has a mean value of 1090 ± 140. The high K value indicated that the partition was greatly in favor of PVP; therefore this polymer would indeed make a good adsorbent for isoflavones. The estimated K value can be used in process scale-up to determine the amount of PVP needed to treat extracts of isoflavones. In the subsequent experiments, the PVP addition was fixed at 15% (13.5 g). Elution of isoflavone one and reuse of PVP Alcohols could be used to wash the adsorbed isoflavones off PVP as they have higher hydrogen-bonding propensities. Although methanol is used in isoflavone analyses, it is obviously not suitable for the production of isoflavones intended for health applications. Ethanol was therefore chosen for desorbing isoflavones from PVP. The objective was to maximize the isoflavone yield with a minimum amount of ethanol to reduce energy consumption in ethanol recovery. The results indicated that a small amount of ethanol (equivalent to 5% of the volume of the alkaline extract), eluted only about half of the adsorbed isoflavones after filtration (Figure 4). This low recovery was likely a result of PVP retaining some ethanol, which was approximately 5 times of dry PVP weight at different ethanol vol- Figure 2 Extraction of isoflavones from red clover at elevated phs. Error bars are based on duplicate runs. Figure 3 Adsorption of extracted isoflavones at different PVP levels. Error bars are based on duplicate runs. S560 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 8, 2005 URLs and addresses are active links at

4 Table 2 Estimated partition coefficient of isoflavones between water and PVP PVP level (%) K ( 10 3 ) a a Means of duplicate runs. umes. The overall loss of isoflavone as a result of retained volume could be significant if only a small volume of ethanol was used for elution. Therefore, use of higher ethanol volume for extraction would give higher isoflavone yields. An amount of ethanol equivalent to 15% of the volume of the alkaline extract recovered almost 90% of the adsorbed isoflavones. Further increase in ethanol use, however, did not increase the yield significantly, whereas energy consumption in recovering extra ethanol would be very significant. It was understood that cross-linked PVP was an expensive reagent; hence its reuse was sought in an attempt to make this process cost effective. It is desirable that the adsorbent be directly reusable after ethanol elution without further treatment, and the reusability of PVP was confirmed by the tests in this study. Figure 5 showed that while fresh PVP adsorbed about 80% of the extracted isoflavones, used PVP was able to adsorb more than 70%, indicating a retention of nearly 90% of its adsorption capacity. The direct reuse of PVP would save the cost of PVP regeneration, thus making the process more commercially attractive. Production of isoflavone one concentrates To improve the purity of the product, we explored washing PVP with water before elution of isoflavones with ethanol. As the alkaline extract was neutralized with hydrochloric acid, there could be significant amounts of salts and other compounds besides isoflavones in the retained liquid of wet PVP cake. These compounds would definitely contribute to the impurities of the product. It is shown in Table 3 that without washing PVP, a final product was obtained with a relatively low isoflavone content of less than g/g, or 5.0 wt%. Its purity was significantly improved to g/g (17.4 wt%) when the wet PVP cake was washed with an amount of water Table 3 Effect of washing of PVP on isoflavone content of the product Amount of Isoflavone content washing water (%) a ( 10 4 g/g) b A B C C 10 2 c 21.2C a As percentage of volume of the alkaline extract. b Means of triplicates. Values with different capital letters are significantly differently from one another. c Washed twice, each time with 10% amount. that was equivalent to 10% (360 ml) of the volume of the alkaline extract. Increasing the water amount to 15% raised the isoflavone content of the product to more than g/g, or 20 wt%. This seemed to be the limit for washing, as further increase in water amount did not make significant difference. Additional washing was not only unnecessary but also undesirable for both economics and adverse environmental impact. Table 4 lists the isoflavone distribution among main streams of the production process. Remarkably, about half of the total amount of isoflavones was recovered in a powdery form with a isoflavone content of g/g, or 20.8 wt%, qualifying it as an isoflavone concentrate. This isoflavone concentrate contained , , and g/g of biochanin A, formononetin, and genistein, respectively. Genistin was essentially undetectable, and its disappearance from the concentrate was probably due to the fact that it was an isoflavone glucoside, thus being relatively hydrophilic and not readily adsorbed by PVP. The isoflavone concentrate thus prepared could be used directly in many health applications or further processed to make purer products such as isolates. It was also noted from the data listed in Table 4 that about 15% of the total isoflavone amount remained in the dried residue after alkaline extraction. This finding seemed to contradict the earlier conclusion that nearly all isoflavones were extracted at ph This amount was likely a result of red clover residue retaining as much alkaline extract as 10 times its dry weight, and apparently the water washing step did not rid the wet residue cake of all the retained isoflavones. The spent aqueous extract after PVP adsorption account- Figure 4 Elution of isoflavones from PVP with different amounts of ethanol. Error bars are based on duplicate runs. URLs and addresses are active links at Figure 5 Reuse of PVP without regeneration. Error bars are based on duplicate runs. Vol. 70, Nr. 8, 2005 JOURNAL OF FOOD SCIENCE S561

5 Table 4 Isoflavone distribution of processing with red clover Mass Isoflavone Isoflavone Isoflavone Stream (g) content ( g/g) amount (mg) recovery (%) Red clover Dried residue Spent extract after PVP adsorption PVP washing water Isoflavone concentrate Unaccountable a n.a. b n.a. b a All product streams minus starting materials (red clover). b Not applicable. ed for approximately 20% of the total amount of isoflavones, which could be treated by reverse osmosis to further recover isoflavones and to recycle the wastewater. Washing PVP with water resulted in the loss of a very small amount of isoflavones (2.3%). The ethanol fraction retained in PVP after elution was largely responsible for an unaccountable amount of approximately 12% of the total. However, analytical uncertainty, particularly among samples low in isoflavones, would also contribute to the errors in material balance. In the overall processing, losses to various process steps involving transfer of extracts were negligible. Conclusions Red clover flowers contained high amounts of isoflavones, making it an ideal source for recovery in purer forms, such as isoflavone concentrates, for health applications. The 2 principal isoflavone components found in red clover were biochanin A and formononetin with lower amounts of genistein and genistin. Extractability of these isoflavones in water was generally very low at room temperature, however it improved considerably at elevated temperatures. These isoflavones were highly extractable at alkaline phs. The extracted isoflavones were readily adsorbed to cross-linked insoluble PVP at a slightly acidic ph (~5.5), which could be easily eluted with ethanol. A process based on the above treatments was able to deliver a concentrate with an isoflavone content of g/g, or 20 wt%, accounting for more than 50% of total amount of isoflavones in red clover. This concentrate could be used directly to make pills as isoflavone supplement or further purified to make isoflavone isolates. Acknowledgments Authors are thankful for the financial support of Natural Resources Canada through the Technology and Innovation (Biotechnology) program. NRCC nr References Adlercreutz H Phytoestrogens: Epidemiology and a possible role in cancer protection. Environ Health Perspect 103: Adlercreutz H, Mazur W Phytoestrogens and Western disease. Ann Med 29: Anderson JJB, Carner SC The effects of phytoestrogens on bones. Nutr Res 17: Atkinson C, Warren RML, Sala E, Dowsett M, Dunning A, Healey CS, Runswick S, Day NE, Bingham SA Red clover-derived isoflavones and mammographic breast density: a double-blind, randomized, placebo-controlled trial [IS- RCTN ]. Breast Cancer Res 6:R Barnes S The chemopreventive properties of soy isoflavonoids in animal models of breast cancer. Breast Cancer Res. Treatment 46: Carrol KK Review of clinical studies cholesterol-lowering response to soy protein. J Am Diet Assoc 91: Chang SKC Isoflavones from soybeans and soy foods. In: Shi J, Mazza G, LeMaguer M, editors. Functional foods: biochemical and processing aspects. Vol 2. Boca Raton, Fla.: CRC Press. p Hendrich S, Lee KW, Xu X, Wang HJ, Murphy PA Defining food components as new nutrients. J Nutr 124:1789S 92S. Klump SP, Allred MC, MacDonald JL, Ballam, JM Determination of isoflavones in soy and selected foods containing soy by extraction, saponification, and liquid chromatography: collaborative study. J Assoc Off Anal Chem Intern 84: Kroyer GT Red clover extract as antioxidant active and functional food ingredient. Innovative Food Sci Emerg Tech 5: Kuzer MS Hormonal effects of soy isoflavones: Studies in premenopausal and postmenopausal women. J Nutr 130:660S 1S. Liu J, Burdette JE, Xu H, Gu C, van Breemen RB, Bhat KPL, Booth N, Constantinou AI, Pezzuto JM, Fong HHS, Farnsworth NR, Bolton JL Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms. J Agric Food Chem 49: Tsunoda N, Pomeroy S, Nestel P Absorption in humans of isoflavones from and red clover is similar. J Nutr 132: Wei H, Wei LH, Frenkel K, Bowen R, Barnes S Inhibition of tumor promoter induced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr Cancer 20:1 12. Xu L, Diosady LL Removal of phenolic compounds in the production of highquality canola protein isolates. Food Res Intern 35: Xu L, Lamb K, Layton L, Kumar A A membrane-based process for recovering isoflavones from a waste stream of soy processing. Food Res Intern 37: S562 JOURNAL OF FOOD SCIENCE Vol. 70, Nr. 8, 2005 URLs and addresses are active links at