Biotransformation of ginsenoside Rb₁ into F-2 and compound K by bacterium Sphingomonas sp. BG 25

Triterpenoid saponins and ginsenosides, the majority of which contain protopanaxadiol or protopanaxatriol as the aglycon, are important biologically active compounds of Panax ginseng C. A. Meyer. One of the principal ginsenosides, Rb₁ (1), has β-sophorose and β-gentiobiose residues in the C-3 and C-20 positions, respectively, of 20(S)-protopanaxadiol. The minor ginsenoside F-2 (2) with glucose residues on C-3 and C-20 and compound K (3) with only one glucose on C-20 have structures close to that of Rb₁ and are practically absent in ginseng roots [1]. Saponins 2 and 3 have broader spectra of biological activity than Rb₁. For example, compound K inhibits growth of cultivated melanoma B16-B6, hepatocellular carcinoma Hep-G2, and lung carcinoma 95-D cells whereas F-2 induces apoptosis of breast cancer stem cells [1,2].

Therefore, biotransformation of ginsenoside Rb₁ into F-2 and compound K, in particular using microorganisms, seemed of practical importance [3]. Microorganisms with amylase, xylanase, and cellulase activity were isolated earlier from soil samples taken from a ginseng field using specially synthesized substrates [4,5]. Among these, strains showing high β-glucosidase activity in cultivation on agar media with added esculin were identified. Herein we communicate data on the transformation of ginsenoside Rb₁ into F-2 and compound K through the action of one of these strains, Sphingomonas sp. BG 25.

Standard ginsenosides Rb₁, Rd, F-2, and compound K were purchased from Faces Biochemical Co., Ltd. (Wuhan, PRC). The isolation of 1 from ginseng roots, analysis of biotransformation products, and their preparative isolation and identification were carried out as before [6] with the exception that PMR and ¹³C NMR spectra were recorded on a Varian Unity Inova AS 400 spectrometer at operating frequencies of 400 and 100 MHz, respectively.

Bacterium Sphingomonas sp. BG 25 was cultivated in Ehrlenmeyer flasks in Luria–Bertani liquid medium (500 mL) for 48 h at 30°C with constant stirring. Then, bacterial suspension (125 mL) of concentration 8∙10⁶ CFU/mL (colony-forming units/mL) was placed into three sterile flasks and treated with an aqueous solution (125 mL, 1 mM) of Rb₁ ginsenoside. The first mixture was incubated for 24 h; the second, 72 h; the third, 96 h at 30°C with constant stirring. Analytical samples were taken every 6 h. After incubation, each mixture was extracted with water-saturated BuOH (250 mL). The resulting extract was evaporated in vacuo. The residue was analyzed. TLC and HPLC showed the presence in the first mixture of starting Rb₁ and its transformation product 4, the spectral characteristics of which agreed fully with those of ginsenoside Rd that were published by us [6]. The main constituents in the second and third mixtures were two other Rb₁ biotransformation products, 2 and 3, which had the same R _f values on TLC and HPLC retention times as standard ginsenoside F-2 and compound K. Both compounds were isolated from the obtained extracts by preparative HPLC over an OptimaPak C18 column (250 × 10 mm, 10 μm) and identified by spectral methods in order to confirm this.

A comparison of the [M + Na]⁺ ions in mass spectra of Rd (4) [6] and 2 showed that one glucose was cleaved from 4. Comparison of the PMR and ¹³C NMR spectra of these compounds indicated that this cleavage occurred from the β-sophorose residue in the C-3 position of Rd. In fact, the ¹³C NMR spectrum of 2 contained resonances for only two anomeric C atoms (106.7 and 98.1 ppm). The C-2′ resonance in the carbohydrate residue on C-3 (75.6 ppm) underwent a strong-field shift compared with that in the β-sophorose residue of 4 (83.7 ppm) (Table 1). Resonances of anomeric C atoms (106.7 and 98.1 ppm) in 2 were analogous to those of C-1′ (105.3 ppm) and C-1′″ (98.5 ppm) of 4, which indicated the presence in 2 of glucose residues in the C-3 and C-20 positions and; correspondingly, that it was identical to the known ginsenoside F-2 [1,2]. A comparison of the same spectral data of F-2 and 3 indicated that the latter contained only one glucose in the C-20 position because a strong-field shift of C-3 (78.2 ppm) in 3 was observed compared with that in 2 (88.7 ppm). Hence, 3 was the known compound K [2]. This was also confirmed by the fact that PMR and ¹³C NMR spectra of 2 and 3 were identical to those of F-2 and compound K, respectively, that were published before [7].

Table 1 ¹³C NMR Spectral Data for Rd (4), F-2 (2), and Compound K (3) (δ, ppm)

An analysis of the concentration change dynamics of these compounds in the incubation medium showed that Rb₁ was biotransformed by Sphingomonas sp. BG 25 as follows: Rb₁ → Rd → F-2 → compound K. Limiting the incubation time to 72–78 h enabled 2 and 3 to be obtained simultaneously. The main product was 3 if it was increased. The results indicated that Sphingomonas sp. BG 25 could be used in addition to other microorganisms [3,7] for biotransformation of Rb₁ into biologically more active ginsenoside F-2 and compound K.

3- O -( β -D-Glucopyranosyl)-20- O -( β -D-glucopyranosyl)-3 β ,12 β ,20 β -trihydroxydammar-24-ene (2), white powder, mp 183–185°C. ¹H NMR spectrum (400 MHz, Py-d₅, δ, ppm, J/Hz): 0.81 (3H, s, CH3-19), 0.95 (3H, s, CH₃-18), 0.96 (3H, s, CH₃-30), 1.00 (3H, s, CH₃-29), 1.30 (3H, s, CH₃-28), 1.59 (6H, s, CH₃-26, 27), 1.62 (3H, s, CH₃-21), 4.93 (1H, d, J = 7.6, H-1′), 5.18 (1H, d, J = 7.6, H-1′″) (only characteristic proton resonances are given). Mass spectrum: FAB, m/z 807 [M + Na]⁺. C₄₂H₇₂O₁₃.

20- O - β -D-Glucopyranosyl-3 β ,12 β ,20 β -trihydroxydammar-24-ene (3), white powder, mp 176–178°C. ¹H NMR spectrum (400 MHz, Py-d₅, δ, ppm, J/Hz): 0.85 (3H, s, CH₃-19), 0.92 (3H, s, CH₃-18), 0.96 (3H, s, CH₃-30), 1.01 (3H, s, CH₃-29), 1.21 (3H, s, CH₃-28), 1.58 (6H, s, CH₃-26, 27), 1.60 (3H, s, CH₃-21), 5.17 (1H, d, J = 7.6, H-1′″). Mass spectrum: FAB, m/z 645 [M + Na]⁺. C₃₆H₆₂O₈.