Turk J Pharm Sci 10(3), 463-472, 2013 Original article DETERMINATION OF ISOFLAVONE CONTENT BY HPLC-UV METHOD AND IN VITRO ANTIOXIDANT ACTIVITY OF RED CLOVER {TRIFOLIUM PRATENSE L.) Nurgiin KU-CÜKBOYACI1'*, Onur KADIOĞLU1, Nezaket ADIGÜZEL2, Uğur TAMER3, Ayşegiil GÜVENÇ4, Bans BANİ5 1 Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330 Ankara, TURKEY Gazi University, Faculty of Science, Department of Biology, 06500 Ankara, TURKEY 3Gazi University, Faculty of Pharmacy, Department of Analytical Chemistry, 06330 Ankara, TURKEY 4Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, 06100 Ankara, TURKEY 5 Kastamonu University, Faculty of Arts and Science, Department of Biology, 37200 Kastamonu, TURKEY Abstract In this study, the methanol and acid hydrolyzed methanol extracts prepared from the aerial parts of two varieties of Trifolium pratense (red clover) collected from different parts of Turkey, namely T. pratense L. var. pratense and T. pratense L. var. sativum Schreb., have been analyzed to determine the concentrations of four isoflavones, daidzein (D), genistein (G), formononetin (F) and biochanin A (B). A RP-HPLC-UV method for the determination of these isoflavones in red clover was developed. Chromatographic separation was carried out by using a gradient system consisting of 0.3 % acetic acid and methanol on a reversed phase column, SupelcosilTM LC-18 (15 cm x 4.6 mm id, 5µm). The highest content of total D, G, F, and B were found as 0.0447, 0.0115, 0.0251 and 0.0039 % in T. pratense var. pratense, respectively. Antioxidant capacity of the methanol extracts of aerial parts of plant samples was also studied. Studied extracts were found to possess some antioxidant activities, as determined by the DPPH free radical scavenging test, thiobarbituric acid and trolox-equivalent antioxidant capacity assays. The phenolic contents of the samples were determined using Folin-Ciocalteu's reagent. Key words: Trifolium pratense, Fabaceae, Isoflavones, HPLC, Antioxidant activity. Kırmızı yonca (Trifolium pratense L.)'nın İzoflavon Iceriğinin YPSK-UV Metodu ile Tayini ve In vitro Antioksidan Aktivitesi Bu çalismada, Türkiye ’nin farkh yerlerinden toplanan Trifolium pratense ’nin (kırmızı yonca) iki varyetesinin, T pratense L. var. pratense and T pratense L. var. sativum Schreb., toprak üstü kısımlarından hazırlanan metanol ve asitle hidrolize edilmiş metanol ekstrelerinde dört izoflavonun, daidzein (D), genistein (G), formononetin (F) ve biokanin A (B), konsantrasyonları tayin edilmiştir. Kırmızı yonca içerisindeki bu izoflavonların tayini için bir ters faz YPSK-UV metodu geliştirildi. Kromatografik ayırim % 0.3 asetik asit ve metanol içeren bir gradient sistemin kullanıldigi ters faz bir kolonda, SupelcosilTM LC-18 (15 cm x 4.6 mm id, 5µm) gerçekleştirilmiştir. En yuksek total D, G, F ve B miktarları T. pratense var. pratense içerisinde sırasıyla % 0.0447, % 0.0115, % 0.0251 ve % 0.0039 olarak bulunmuştur. Bitki örneklerinin toprak üstti kısımlarının metanol ekstreleri antioksidan aktiviteleri bakımından da incelenmiştir. İncelenen ekstreler DPPH serbest radikal süpürücü, tiyobarbitürik asit ve troloks’a eşdeğer antioksidan kapasite tayinlerinde bazı antioksidan aktivitelere sahip bulunmuştur. Örneklerin fenolik madde içerikleri Folin-Ciocalteu reajanı kullanılarak belirlenmiştir. Anahtar kelimeler: Trifolium pratense, Fabaceae, İzoflavonlar, YPSK, Antioksidan aktivite. Correspondence: E-mail: [email protected]; Tel: +90 312 202 31 77. ^his paper is a part of Onur Kadıoğlu's dissertation for M. Sc. degree in Phtotherapy Program of Pharmacognosy at the Institute of Health Sciences, Gazi University, Ankara. 463 Nurgun KUCUKBOYACI, Onur KADIOĞLU, Nezaket ADIGÜZEL, Uğur TAMER, Ay§egul GÜVENQ, Ban§BANİ INTRODUCTION Trifolium pratense L. (Leguminosae), also known as red clover, is a perennial plant with trifoliate leaves, obovate to broadly elliptic, inflorescence globose to ovoid, and corolla reddish- purple to pink, rarely whitish. T. pratense is a very variable species. It has three varieties in Turkey, namely Trifolium pratense L. var. pratense, Trifolium pratense L. var. sativum Schreb., and Trifolium pratense L. var. americanum Harz. All varieties are widely cultivated as a fodder crop throughout the North temperate region (1). Red clover is traditionally used in the treatment of chronic skin diseases such eczema and psoriasis and whooping cough (2,3). Recently, it has been used for many human health benefits due to phytoestrogen isoflavones (4). Red clover contains a great number of flavone derivative compounds, principally isoflavones. Many flavonoids have been identified as glycoside and glycoside malonate derivatives of flavonoids in red clover. The main bioactive isoflavones are formononetin (F) and biochanin A (B), with smaller concentrations of daidzein (D) and genistein (G) (4). Therefore, it has received considerable interest as a rich source of estrogenic isoflavones. Phytoestrogens reported to have protective effects on estrogen-related conditions such as menopausal symptoms and estrogen-related diseases, such as several cancers, prostate, breast, etc., osteoporosis and cardiovascular diseases (4-6). Therefore, red clover preparations have recently suggested for alternative hormone replacement therapy (HRT) as selective estrogen receptor modulators and in the management of menopause symptoms (5). According to the substituents in the main bioactive isoflavone structure in red clover, the molecular structures of D, G, F and B are presented in Figure 1. H(X ^Q 0R2 Substituents No Isoflavones R1 R2 1 Daidzein H H 2 Formononetin H CH3 3 Geni stein OH H 4 Biochanin A OH CH3 Figure 1. Chemical structures of major isoflavones in Trifolium pratense L. The Genus Trifolium L. comprises 128 taxa, in which 11 of them are endemic to Turkey (1,7-10). A number of analytical methods have been reported for the detection and the quantification of the isoflavones in red clover (11-20). In our previous study, we applied new HPLC-chemometric approaches for the simultaneous chromatographic quantification of daidzein, genistein, formononetin, and biochanin A in the samples consisting of the aerial parts of Trifolium lucanicum Gasp. In addition, conventional HPLC was used for the determination of each compound in the extracts of T. lucanicum (21). As a part of our ongoing research on Trifolium species growing in Turkey, we investigated isoflavone contents of the aerial parts of two Trifolium pratense varieties. Additionaly, we determined total polyphenol contents and in vitro antioxidant potentials of methanolic extracts of these samples. In the present study, our aim is (i) to quantify principally bioactive isoflavones (daidzein, genistein, formononetin and biochanin A) in the extracted samples of aerial parts of red clover; 464 Turk J Pharm Sci 10(3), 463-472, 2013 (ii) to determine the content of total polyphenols in methanol extracts of the aerial parts of red clover; (iii) to evaluate the antioxidant activity and free radical scavenging activity of methanol extract prepared from the aerial parts of T. pratense varieties growing in Turkey. EXPERIMENTAL Chemicals In our study, methanol used in the HPLC analysis was of chromatographic grade (Merck, Darmstadt, Germany). Acetic acid (Merck, Darmstadt, Germany) was also used to prepare the mobile phase. In the extraction procedure, methanol and trifluoroacetic acid (TFA) were of analytical grade (Merck, Darmstadt, Germany). Isoflavone standards; daidzein (D), genistein (G), formononetin (F) and biochanin A (B) were purchased from Fluka (Buchs, Switzerland). Dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) was used by dissolving the isoflavone standards. All other chemicals were analytical reagent grade. Plant materials Plant materials were collected in flowering periods from different localities in Turkey. The plant samples were collected and identified by N. Adıgilzel and B. Bani. Authenticated voucher specimens were deposited in the Herbarium of GAZI, Ankara, Turkey. Their collecting locations are given below: T. pratense var. pratense: B6 Adana: Tufanbeyli, Yamanh village 1550 m, 20.05.2006 (B.B. 2774). T. pratense var. sativum: C9 Van: Çatak 1500 m, 16.6.2006 (B.B. 1836). HPLC Analysis oflsoflavones Sample preparation Two-hundred miligrams powdered materials were extracted with 80 % methanol for 15 min under reflux at 85 °C. This procedure was repeated two times. After cooling, the solution was filtered and the residue washed with 5 mL of 80 % methanol. The combined extract was evaporated under reduced pressure. The extract was dissolved in methanol and then aliquots of 3 mL were applied onto pre-conditioned SPE cartridges (Sep-Pak C18, Waters). Isoflavones were eluted with 80 % methanol and elution solvent was distilled in vacuo. This extracts (E1) were analysed for free isoflavones. Two-hundred miligrams powdered materials were extracted by using 20 mL of 80 % methanol (acidified to pH 3 with TFA) for 15 min under reflux at 85 °C. This procedure was repeated to times as a sequence treatment. After filtering the collected extract samples, 1 mL filtrate was diluted with 9 mL water and loaded on to a Sep-Pak C18 cartridge (Waters). Isoflavones were retained on the Sep-Pak C18 cartridge which was then washed with 10 mL of water twice and eluted with 80 % methanol. This extracts (E2) were analyzed for total isoflavones. Four isoflavones, D, G, F and B in the both final samples were determined by using the proposed conventional HPLC method. All samples