In vitro isoflavonoid production and analysis in natural tetraploid Trifolium pratense (red clover) calluses

Abstract

Isoflavones are polyphenolic phytoestrogens, predominantly found in leguminous plants. Trifolium pratense L., Fabaceae (red clover), is rich in isoflavones that possess estrogenic activity due to their similar molecular structure and effectiveness in preventing health conditions such as menopause, osteoporosis, cardiovascular disease, hypertension and hormone-dependent cancers. In this study, presence and amount of various phytoestrogens in the tetraploid plant and in the calluses derived from the plants were investigated. Calluses were generated from explants obtained from natural tetraploid T. pratense seedlings. The best callus formation was obtained from hypocotyl explants cultured in Phillips Collins and Gamborg B5 media containing different plant growth regulators. Flowers of plants and calluses were analysed for formononetin, biochanin A, genistein and daidzein contents using HPLC. In HPLC analysis, high levels of formononetin (0.249 µg/mg) were determined in natural tetraploid T. pratense flowers in addition to genistein and biochanin A. In calluses, highest isoflavone content (1.15 µg/mg formononetin) was observed in modified Gamborg B5 medium. Biochanin A content of calluses and the plant were found to be nearly the same. But formononetin and genistein contents of the calluses in this medium were found to be respectively 4.62 and 21.39 folds higher than the tetraploid plant.

Comprehensive evaluation of phytoestrogen accumulation in plants and in vitro cultures of Medicago sativa L. ‘Elçi’ and natural tetraploid Trifolium pratense L.

“…Kasparová et al (2006) used Gamborg medium for callus and suspension cultures in 4 different diploid varieties of T. pratense L. As a result, TLC and HPLC only detected formononetin. Our results were in accord with the previous study (Ercetin et al, 2012) in terms of formononetin and genistein production in callus culture with Gamborg B5 medium. In conclusion, a cell suspension culture method was developed for M. sativa L. 'Elçi' , in which daidzein and formononetin were produced in as little as 18 days.…”

Identification and Qualitative Analysis of β-Sitosterol and Some Phytoestrogens in In Vivo and In-Vitro Samples of Lepidium sativum: A Semi-Arid Bone Healing Plant

“…In the present investigation Chloroform: methanol (8:2) combination showed best results and exhibited coherence with the results of [13,14]. HPTLC technique with other biochemical assays supplemented evidence to confirm the presence of some bioactive compounds, which have provided the plants an important position in folkloric usage [15] In the present study, quantitative and qualitative biochemical methods can be employed as a stability indicator [16] that can be used for the routine quality control analysis and quantitative determination of β-sitosterol from Lepidium sativum.…”

Phytochemical Composition of Different Red Clover Genotypes Based on Plant Part and Genetic Traits

“…The content of these compounds varies greatly depending on different factors. However, the majority of studies found that formononetin and biochanin A are the dominant isoflavones in red clover [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. In this study, concentrations of formononetin were in ranges of 0.22–4.68 mg/g, 0–0.95 mg/g and 0.10–1.54 mg/g in leaves, flowers and stems, respectively, while these values for biochanin A were 0.59–5.94 mg/g, 0.17–1.52 mg/g and 0.08–0.56 mg/g ( Table 2 ).…”