Efficient isolation and purification of five products from microbial biotransformation of cinobufagin by high‐speed counter‐current chromatography

Abstract

An efficient separation method of using high-speed counter-current chromatography was successfully established to directly purify cytotoxic transformed products of cinobufagin by Cordyceps militaris. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:6:3:4, v/v) was used in high-speed counter-current chromatography. A total of 9 mg of 4beta,12alpha-dihydroxyl-cinobufagin (1), 15 mg of 12beta-hydroxyl-cinobufagin (2), 8 mg of 5beta-hydroxyl-cinobufagin (3), 12 mg of deacetylcinobufagin (4) and 6 mg of 3-keto-cinobufagin (5) were obtained in a one-step separation from 400 mg of the crude extract with purity of 98.7, 97.2, 90.6, 99.1 and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified on the basis of (1)H-NMR and (13)C-NMR technology. All products (1-5) showed the potent activities against human carcinoma cervicis (Hela) and malignant melanoma (A375) cells in vitro.

Separation of bufadienolides from Helleborus thibetanus Franch. by a combination approach involving macroporous resin column chromatography and gradient countercurrent chromatography

“…Five bufadienolides were isolated from the microbial biotransformation of cinobufagin by Ma et al, utilizing a solvent system consisting of nhexane, ethyl acetate, methanol, and water (Wat) (4:6:3:4, v/v). The purity of these compounds exceeded 90% [17]. Li et al developed a stepwise elution method using a two-phase solvent system consisting of n-hexane/ethyl acetate/methanol/Wat at the ratios of 4:6:2:4 (v/v), 4:6:2.5:4 (v/v), and 4:6:3.2:4 (v/v) for the purification of bufadienolides from ChanSu by CCC.…”

“…Additionally, the utilization of CCC also exhibits economic advantages and facilitates high product purity [14,15]. Furthermore, the successful separation of bufadienolides from ChanSu was achieved through a one-step CCC [16][17][18]. Five bufadienolides were isolated from the microbial biotransformation of cinobufagin by Ma et al, utilizing a solvent system consisting of nhexane, ethyl acetate, methanol, and water (Wat) (4:6:3:4, v/v).…”

“…Five bufadienolides were isolated from the microbial biotransformation of cinobufagin by Ma et al., utilizing a solvent system consisting of n‐hexane, ethyl acetate, methanol, and water (Wat) (4:6:3:4, v/v). The purity of these compounds exceeded 90% [17]. Li et al.…”

Gamabufotalin, a major derivative of bufadienolide, inhibits VEGF-induced angiogenesis by suppressing VEGFR-2 signaling pathway

“…In recent years, some studies have revealed that natural products which exist in the traditional Chinese medicines [ 26 ], have the potential properties in treating different types of cancers. Toad venom (called “ Chansu ” in China) is a product of the skin gland of toads such as Bufo bufo gargarizans etc [ 27 , 28 ], which has been widely used as a cardiotonic, diuretic, anodyne and hemostatic agent [ 29 ]. Chansu has been used as significant anticancer agents, amending the life quality of cancer patients [ 30 ].…”