CHEMIJA. 2012. Vol. 23. No. 4. P. 306–311 © Lietuvos mokslų akademija, 2012 Distribution of formononetin, daidzein and genistein in Trifolium species and their aerial plant parts Giedrė Dabkevičienė1, Isoflavones are phenolic phytochemicals of major interest for health. Clovers are one of their alternative sources. The current study was designed to quantify three isoflavones in vari- Bronislava Butkutė1*, ous clover species and their aerial plant parts by a high-performance liquid chromatogra- phy and to identify the most promising source for medicinal use. Isoflavone contents were Nijolė Lemežienė1, quantified in the plants of 11 perennial and 4 annual species of genus Trifolium. Subject to aerial part and perennial clover species, the concentrations of formononetin, daidzein Valdas Jakštas2, and genistein varied in the wide ranges: <Limit of detection (LOD) – 2.19, <LOD – 1.42 and <LOD – 2.898 mg g–1, respectively. According to the sum of three isoflavones quanti- 1 Egidijus Vilčinskas , fied in leaves–stems–flowers, perennial clover species were arranged as follows:T. medium (6.01–3.50–2.93 mg g–1) > T. pratense > T. fragiferum > T. alpestre > T. rubens > T. och- 2 Valdimaras Janulis roleucum > T. pannonicum > T. hybridum > T. ambiguum > T. repens > T. montanum (0.092–0.022–0.090 mg g–1). Regarding the total isoflavone content, the annual clover spe- 1 Institute of Agriculture, cies were ranked in the sequence: T. incarnatum (3.27 mg g–1) > T. campestre > T. resupina- Lithuanian Research Centre tum > T. alexandrinum (0.517 mg g–1). for Agriculture and Forestry, LT-58344 Akademija, Key words: Trifolium spp., isoflavone, formononetin, daidzein, genistein, HPLC Kėdainiai District, Lithuania 2 Department of Pharmacognosy of Lithuanian University of Health Sciences, A. Mickevičiaus Str. 9, LT-44307 Kaunas, Lithuania INTRODUCTION 3-(4-methoxyphenyl)chromen-4-one] and biochanin A [5,7-dihydroxy-3-(4’-methoxyphenyl) chromen-4-one], re- Isoflavones are phenolic compounds, belonging to a wide- spectively, are present in red clover (Trifolium pratense L.) spread group of natural products flavonoids, which are [1, 2, 3] as well as in other Trifolium species [4–6]. Epide- found in a number of plant species, though predominately miological studies show that due to their phytoestrogenic in legumes. Isoflavones such as genistein [5, 7-dihydroxy- effect isoflavones exert a positive influence on hormonal 3-(4’-hydroxyphenyl)chromen-4-one] and daidzein [7-hy- functions and can prevent osteoporosis [7, 8], Alzheimer droxy-3-(4-hydroxyphenyl)chromen-4-one] are common disease [9], cancer [3], vascular pathologies and alleviate phytoestrogens of soy products. Daidzein, genistein and menopausal symptoms [2, 10]. Isoflavones are structural their 4’-methylated derivatives, formononetin [7-hydroxy- mimics of endogenous 17β-estradiol [11] and structures of the three isoflavones discussed in the current work are * Corresponding author. E-mail: [email protected] shown in Fig. 1. Distribution of formononetin, daidzein and genistein in Trifolium species and their aerial plant parts 307 out the HPLC experiment. Standards of daidzein (purity 99.9%) and genistein (purity 99.3%) were obtained from ChromaDex (Irvine, CA, USA) and formononetin (purity ≥99%) from Fluka-Sigma-Aldrich (Steinheim, Germany). The standard solutions were prepared by dissolving standard compounds in methanol. Sample preparation procedure included acid hydrolysis of glycoside forms and was performed under conditions described in the USP monograph [16]. The analyses were Daidzein R1 = H, R2 = OH carried out with the Waters 2695 Alliance HPLC system Formononetin R1 = H, R2 = OCH3 (Milford, MA, USA), equipped with a Waters 2487 UV/Vis Genistein R1 = OH, R2 = OH detector and a Waters XTerra RP18 150 × 3.9 mm column with a guard co lumn. The dual λ absorbance detector allowed monitoring of two wavelengths, 258 nm (for genistein ana- Fig. 1. The molecular structures of isoflavones daidzein, formononetin and lyte) and 301 nm (for daidzein and formononetin analytes). genistein A Waters 996 PDA detector was used for peak spectral iden- tification. Chromatographic and PDA data were recorded Currently, the main source of isoflavones used in human and peak area responses were integrated by the software Wa- nutrition is soy [10, 12]. However, some people are allergic ters Empower2 (Milford, MA, USA). The gradient chromato- to soy products. As a result, researchers are looking for alter- graphic elution was carried out according to Raudonis et al. native sources of isoflavones. Most research has focused on [17]. The linear through zero type calibration curves were the quantification of isoflavones in red clover and its separate generated using external standard solutions, prepared by dis- parts [4, 13–15]. Increasingly more countries are joining the solving standard compounds in methanol (0.2–30 μg mL–1). search for clover species that could be applied to meet spe- Value of the coefficient of determination for the curves was cific needs, including their use as a valuable source of phy- ≥0.9998 and the equation was y = 160329691 · x for ge- toestrogens. nistein, y = 55761134 · x for daidzein and y = 55059861 · x Our study was intended to quantify the content of for- for formononetin. Isoflavone concentrations were expressed mononetin, daidzein, genistein in various clover species and as mg g–1 of sample dry matter (DM). Limit of detection aerial plant parts by a high-performance liquid chromato- (LOD), i. e. the lowest quantity of isoflavone that can be re- graphy and to identify the most promising sources of isofla- liably detected by the method used, was determined with the vones. standard solution at a signal-to-noise ratio of three and was <0.100 μg mL–1. That corresponds to the isoflavone content EXPERIMENTAL <0.004 mg in 1 g DM of the tested material. Statistical data analysis was done using the software Plant material. Isoflavone studies were done on the plants ANOVA from the package SELEKCIJA [18]. of the 11 perennial and 4 annual clover species: T. alpes- tre L., T. ambiguum Bieb., T. fragiferum L., T. hybridum L., RESULTS AND DISCUSSION T. medium L., T. montanum L., T. ochroleucum Huds., T. pannonicum Jacq., T. pratense L., T. repens L., T. rubens L., Isoflavone amounts in the aerial parts of perennial Trifo- T. alexandrinum L., T. campestre Schreb., T. incarnatum L., lium species T. resupinatum L. In 2007 and 2008, genetic collections of Isoflavones were quantified separately in leaves, stems and clover species were established in a trial field using seed- flowers. The clover species and their aerial plant parts dif- lings grown in a greenhouse. Thirty plants per accession fered in the concentrations of the isoflavones tested. (2 replications, 15 plants per replication) were planted at The highest concentration of formononetin was de- 50 × 50 cm distances. The plants were cut at full flower- termined in aerial parts of T. fragiferum, T. medium and ing stage. Fresh samples were chopped, fixed at 105 °C for T. pratense (Table): in leaves 1.83, 1.69 and 1.51 mg g–1, 20 min, dried at (65 ± 5) °C, ground in a cyclonic mill with a in stems 1.98, 1.86 and 2.19 mg g–1, in flowers 0.763, 1.13 1 mm sieve and assayed for the following isoflavones: daid- and 1.66 mg g–1, respectively. For other species tested, the zein, formononetin and genistein. Isoflavones were quanti- levels of formononetin in various plant parts were consi- fied by a reversed-phase high-performance liquid chroma- derably lower. The contents of this isoflavone in aerial parts tography (RP-HPLC). of T. rubens ranged from 0.395 to 0.644 mg g–1. T. repens Isoflavone quantification. An HPLC grade acetonitrile plants accumulated even less formononetin – from 0.129 and trifluoracetic acid were purchased from Sigma-Aldrich to 0.155 mg g–1. In leaves and stems of T. pannonicum, for- (Steinheim, Germany), ethanol from Stumbras AB (Kaunas, mononetin was found in traceable concentration only, i. e. Lithuania). Ultra pure water (>18 MΩ cm) was used through- below the limit of detection (<LOD); however, in flowers 308 Giedrė Dabkevičienė, Bronislava Butkutė, Nijolė Lemežienė, Valdas Jakštas, Egidijus Vilčinskas, Valdimaras Janulis Table. The concentration of formononetin, daidzein and genistein in different aerial parts of perennial Trifolium species Formononetin Daidzein Genistein Leaves Stems Flowers Leaves Stems Flowers Leaves Stems Flowers T. rubens 0.395 0.644 0.401 0.308 0.253 0.065 0.058 0.143 0.129 T. repens 0.135 0.155 0.129 0.054 0.037 0.041 <LOD <LOD <LOD T. pratense 1.51 2.19 1.66 0.786 0.947 0.324 0.452 0.181 0.231 T. pannonicum <LOD <LOD 0.752 0.092 0.040 0.267 0.526 0.277 0.129 T. ochroleucum <LOD <LOD <LOD 0.044 0.011 0.026 0.970 0.682 0.498 T. montanum 0.028 <LOD 0.040 0.064 0.022 0.050 <LOD <LOD <LOD T. medium 1.69 1.86 1.13 1.42 0.917 0.961 2.90 0.719 0.841 T. hybridum 0.126 0.257 0.031 0.205 0.326 0.116 0.011 0.015 <LOD T. fragiferum 1.83 1.98 0.763 0.636 0.422 0.268 0.290 0.203 0.302 T. ambiguum 0.297 0.095 0.096 <LOD 0.018 0.038 <LOD <LOD <LOD T. alpestre 0.076 0.504 0.197 0.052 0.444 0.097 1.17 0.388 0.543 Average 0.553 0.699 0.473 0.333 0.312 0.205 0.580 0.237 0.243 MSE* 0.246 0.322 0.167 0.143 0.107 0.084 0.328 0.092 0.099 * Mean squared error. its content amounted to 0.752 mg g–1.