ISOFLAVONE CHARACTERIZATION FROM LEGUMES OTHER THAN SOYBEANS. Koh L.W. 1 and Perera C.O. 2

Transcription

1 ISOFLAVONE CHARACTERIZATION FROM LEGUMES OTHER THAN SOYBEANS Koh L.W. 1 and Perera C.O. 2 Food Science and Technology, Department of Chemistry, Faculty of Science, National University of Singapore, 10 Kent Ridge Road, Singapore ABSTRACT Isoflavones are sub-class of phytoestrogens. They are natural compound in plants that are believed to be beneficial to health, particularly in reducing the risk of hormone-associated health problems in humans. Phytoestrogens are found abundantly in legumes. Isoflavones characterization from other plants includes arrowroot, and chickpea was investigated. Lotus root, a member of the Nelumbonaceae family was also included in this study. Their isoflavones content was compared to soybean. Isoflavones were extracted with 80% methanol. The extracted materials were saponified to convert malonyl and acetyl isoflavones esters to the six types of isoflavones that were considered in this research namely daidzin, daidzein, glycitin, glycitein, genistin, and genistein. HPLC was used to characterize the extracted isoflavones. Isoflavone content was expressed in terms of mg/g of dry matter. These legumes seem to have same similar types of isoflavones (daidzin, daidzein and glycitin). Lotus root was suspected to contain daidzin and glycitin. Soybean appeared to have the highest amount of isoflavones (aglycone + aglycone 1 equivalent). It was followed by arrowroot, chickpea, and lotus root. INTRODUCTION Previous researches had suggested that dietary phytoestrogens have a high potential in the prevention of Western diseases such as atherosclerosis, osteoporosis, cardiovascular diseases, several kinds of cancer and postmenopausal complaints (Krenn et al., 2002). Being a sub class of phytoestrogens, isoflavone has similar health benefits. It is largely found in the plant family of Leguminosae. Most of the research done on isoflavones concentrated on soybeans and its products. The aim of this research was to characterize the isoflavones in other plant and legumes available in the market and compare them to soybean. Three main isoflavones (aglycone) found in soybean are daidzein, glycitein and genistein. The isoflavones also appear as the glucosides conjugates (daidzin, glycine and genistin) and the glucoside esters of the parent isoflavones. Acetyl and malonyl isoflavone glucoside esters are the most abundant forms of isoflavones found in soy bean. (Klump et al., 2001a,b,c). This study concentrated on three legumes which were arrowroot (Pueraria lobata), chickpea (Cicer arietinum), and soybean (Glycine max). Isoflavones determined in arrowroot and chickpea were compared to those in soybean. The pharmacological studies of Pueraria lobata showed that it contains four types of isoflavones which were daidzein, daidzin, puerarin and daidzein~4,7-diglucoside (Fang, 1980). Puerarin is the most abundant isoflavone in arrowroot (Guerra, 2000). Chickpea contains the isoflavone formononetin, biochanin A, daidzein and genistein (Koster et al., 1983). Soybean is one of the legumes that had the most

2 attention in the field of isoflavone studies. It had been reported that soybean contains daidzein, genistein, glycitien and their glucosides and glucoside esters (Yang et al., 2001). Lotus root (Nelumbo nucifera) was studied as well. The determinations of isoflavones in these samples were done by using HPLC with a C18 column. MATERIALS AND METHODS The determination of isoflavones in soybean, arrowroot, chickpea and lotus root was carried out by using AOAC approved method The samples were dried and defatted (only for chickpea and soybean because the rest have low % of fats) using HPLC graded hexane. Isoflavones were extracted at 60 C water bath for 2 hours in 80% HPLC graded methanol. The extracts were cooled to room temperature and saponified with 2M NaOH solution to convert isoflavone glucoside ester forms to isoflavone aglycone forms leaving the three major isoflavones intact. Next, the extracts were acidified with acetic acid glacial and then, diluted with 80% methanol. The extracts were centrifuged at 4500 rpm for 15 minutes and were filtered with PTFE filter before injecting into the HPLC C 18 column. The HPLC used was manufactured by Waters (515 HPLC, 2 pump & in-line Degasser AF) with a photo-diode array detector (Waters 2996). Isoflavones were separated with mobile phase A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile) in the C 18 column (YMC ODS-AM 303) and the peaks were detected by UV detection at 260nm. The flow rate was set at 0.8 ml/min. The peaks of the separated isoflavones were compared with those in the standard curve prepared by running the standard isoflavones (only daidzin, daidzein, genistin, genistein, glycitin and glycitein were run) at different concentrations, depending on the type of isoflavones being analysed. The concentrations of isoflavones in the samples were computed in terms of mg/g of dried matter. RESULTS Table 1: Isoflavones (Glucoside ) content in the respective legumes in terms of dry matter Samples Glucoside forms (mg of isoflavone / g dry matter) Daidzin Genistin Glycitin Total glucoside Arrowroot Chickpea tr Soybean Lotus root * * * tr, present in trace amount, less the detection threshold *These compounds are only predicted that they were the respective isoflavones. Table 2: Isoflavones (Aglycone) content in the respective legumes in terms of dry matter Sample Aglycone conjugates form (mg of isoflavone / g dry matter) Daidzein Genistein Glycitein Total aglycone Arrowroot Chickpea Soybean Lotus root

3 Notes: All the values calculated were based on the concentration (ppm) of isoflavones in 1ml of diluted extracts computed by the computer. The computation was done in accordance to the standard curve by comparing the area of the sample peaks with those of the standards. This concentration was multiplied by the dilution factor and expressed in term of mg/g of dry matter. Table 3: Samples and their total aglycones plus aglycones equivalent. Sample Initial aglycone Aglycone Total aglycone (mg/g) (mg/g) equivalent (mg/g) Arrowroot Chickpea Soybean Lotus root * *These compounds are only predicted that they were the respective isoflavones. Note: According to (Klump et al., 2001) Glucosides can be converted to aglycone equivalent using the following formula: Aglycone equivalent = (0.625 x glucoside of genistein) + (0.637 x glucoside of glycitein) (1) + (0.611 x glucoside of daidzein) DISCUSSION Based on the results, soybean had the highest content of the six isoflavones of interest with the total amount of mg/g of glucosides and mg/g of aglycones. In soybeans and foods derived from soy, isoflavones are found in concentrations ranging from 0.1 to 5 mg/g (Coward et al., 1993).The results obtained in this studies fall within this range. Results show that arrowroot did not contain genistein and glycitein and had less isoflavone than soybean. Puerarin and daidzin are two major isoflavones in arrowroot. (Chen, 2001). Based on the results, genistin and daidzin had the highest value in arrowroot. Genistin and puerarin might have similar retention time and resulting in the larger peaks area of genistin and hence causing the higher value of genistin. Some literatures stated that arrowroot had higher content of isoflavone than soy bean. The highest total concentration of isoflavones, with regard to edible seeds, was found in arrowroot (over 200,000 µg/100g), followed by soybeans (ranged from 37,300 µg/100g to 140,300 µg/100g) and chickpea (1,150 to 3,600 µg/100g) (Mazur, 1998). Arrowroot might contain isoflavone other than the six types of isoflavone that considered here. Therefore, arrowroot appeared to have less isoflavones then soy beans. However, different research might come out with different results because different analyte was measured. For instance, Mazur s (1998) result of soybeans contradicts with Coward et al.(1993). Chickpea had all the six isoflavones studied. Genistin only contained in trace amounts and was not able to be detected by the HPLC systems because the value did not reach the threshold level. Chickpea indeed had less isoflavone compare to soybean according to Mazur (1998). The concentration of isoflavone obtained by Mazur (1998) was mg/g. This figure was different with the result obtained here because Mazur (1998) investigated different isoflavones compare to the six in this study. Chickpea was ranked the third after soybean according to Mazur (1998). The major isoflavone in chickpea were formononetin and biochanin A (both were not included in this research). Results achieved in this study might contradict with those obtained by others because the samples that we used were not from the same source. Plants were not uniform in every country

4 due to many reasons such as climate, soil type and cultivars variation. Secondly, the techniques that we used were not the same. Determination by HPLC and GC-MS will not give the same results because they have different sensitivity. Thirdly, different HPLC systems and columns will have different sensitivity and might separate the desired compound at different retention time. Fourthly, HPLC was not sufficiently sensitive for those legumes with low concentration of the analyte. Finally, this research was just a preliminary study comparing isoflavone in soybean with other legumes. Only the six aglycones and glucosides were taken into account. In conclusion, the major isoflavones found in soybean was daidzin and genistin. Chickpea contains all the six isoflavones in soybean but with different concentration. Arrowroot did not contain genistein and glycitein and most of the isoflavones separated were daidzin and genistin. Lotus root, might have daidzin and glycitin. However, both separated compound might be something else which had the same retention as the analyte. Soybean had the highest amount of the total isoflavones of interest. This was followed by arrowroot, chickpea and lotus root in this research. Isoflavone is one of the most interesting research areas. Today, many studies on soybean had been done. In the future, more studies should be done on the isoflavone content and their types in other legumes especially arrowroot and chickpea. They were among the member of the Leguminosae family that had the highest concentration of isoflavone. Perhaps, an acceptable range of isoflavone content in these legumes should be obtained such as those obtained by Coward et al (1993) on soybean. Lotus root, a non Leguminosae member, might be another good target. In nature, both chickpea and arrowroot were relatively cheap plant produce. However, isoflavones extracted and purified from them, could be very expensive compounds that can be used as supplements in the medical field as well as for food supplementation. ACKNOWLEDGEMENTS I am very grateful for the huge support and guidance that were given to me by various people during my research. There were so many of them that if I were to name each and everyone, there will be no room for my report. However, I must express my deepest gratitude to NUS for this opportunity, to my supervisor, Assoc. Prof. Perera for his endless guidance, to post graduate, Miss Molamma P. Prabhakaran for mentoring me throughout this study, to all FST lab technicians and seniors for their advices, as well as to my parents for their precious moral support. REFERENCE Chen, G., Zhang, J., and Ye, J. (2001). Determination of puerarin, daidzin and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection. In Journal of Chromatography A, vol. 923, Coward, L., Barnes, N. C., Setchell, K. D. R., and Barnes, S. (1993). Genistein, daidzein and their β-glycoside conjugated: Antitumor isofolavones in soybean Foods from American and Asian diets. In Journal of Agricultural and Food Chemistry, vol. 41, Fang, Q. (1980). Some current study and research approaches relating to the use of plants in the traditional Chinese medicine. In Journal of Ethnopharmacology, vol. 2, Guerra, M.C., Speroni, E., Broccoli, M., Cangini, M., Pasini, P., Minghetti, A., Crespi-Perellino, N., Mirasoli, M., Cantelli, G., and Paolini, M. (2000). Comparison between Chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin. Antioxidant properties and effects on rat liver CYP-catalysed drug metabolism. In Life Sciences, vol. 67,

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