Phenolic Compounds and Flavonoids in Various Extracts of <i>Ferula tadshikorum</i> and <i>Ferula sumbul</i> Grown in vitro Conditions

Background: Flavonoids, which are bioactive compounds found in medicinal plant extracts, are recognized for their significant therapeutic properties. To efficiently isolate these compounds from fresh plant materials, it is essential to optimize extraction methods. The most widely used approach involves solvent extraction using solvents such as methanol, ethanol, acetone, and hexane. This study aimed to determine the optimal extraction methods for isolating phenolic compounds and flavonoids from callus and regenerating plants of two medicinal species of the genus Ferula cultivated in vitro with or without indole-3-butyric acid (IBA) and 6-benzylaminopurine (BAP). This study focuses on Ferula tadshikorum and Ferula sumbul, with extracts analyzed quantitatively using high performance liquid chromatography (HPLC). The results revealed the presence of flavonoids such as kaempferol, quercetin, p-coumaric acid, and cinnamic acid at varying concentrations across different solvents. Notably, extracts from F. sumbul plants grown with Murashige and Skoog (MS) medium containing IBA and BAP exhibited higher levels of p-coumaric acid (9.588 µg/mL) in methanol, and kaempferol (9.595 µg/mL) in hexane. For F. tadshikorum, ethanol extraction yielded a significant amount of p-coumaric acid (29.9 µg/mL). Quercetin and cinnamic acid were detected in minimal quantities in both extracts, whereas kaempferol was notably higher (2.61 µg/mL) than in the methanol extract (0.617 µg/mL). Objectives: The present study represents the first attempt to determine the phenolic compounds in regenerating plants obtained through in vitro methods. Methods: In vitro regenerated plants of F. tadshikorum and F. sumbul, cultured on MS basal medium supplemented with IBA (0.5 mg/mL) and BAP (0.5 mg/mL), were ground into a fine powder in liquid nitrogen and then dried in a drying oven at 50°C before chemical analyses. The bioactive compounds were extracted by soaking the powders in methanol, ethanol, and hexane overnight. The extracts were filtered and evaporated under a vacuum at 35 - 36°C. The resultant slurry was partitioned with equal volumes of ethyl acetate (EA) and water to remove excess polar compounds. The EA fraction was vacuum-dried, and the mass of the crude extract was recorded. The extract was subsequently dissolved in methanol, ethanol, and hexane to achieve a concentration of 1 mg/mL. Finally, the solution was filtered through a 0.45 µm PTFE filter (German Acrodisc 13 CR) before analysis by HPLC. Results: We then analyzed methanol, ethanol, and hexane extracts from in vitro F. tadshikorum and F. sumbul plants grown under various combinations of hormones and hormone-free conditions using HPLC. When comparing extracts prepared with 3 solvents from 2 different samples of the F. sumbul, plants obtained in a combination of IBA and BAP hormones showed a higher content of p-coumaric acid in methanol extraction (9.58 µg/mL) and a higher content of kaempferol in the hexane extraction (9.59 µg/mL). During the extraction of callus and plant regenerants from in vitro F. tadshikorum plants using methanol and ethanol, a higher content of p-coumaric acid (29.9 µg/mL) was observed in the ethanol extract than in methanol. Quercetin and cinnamic acid were detected in very low quantities in both the extracts. Notably, kaempferol content was higher (2.61 µg/mL) than in the methanol extract (0.617 µg/mL). Conclusions: This study focused on the declining wild medicinal herbs in natural populations, providing an opportunity for systematic in vitro propagation year-round. This approach aims to establish these herbs as alternative sources for extracting biologically active secondary metabolites such as flavonoids.

Ҳали таржима қилинмаган