Triterpenoid glycosides, i.e., ginsenosides, are the main active principles of Panax ginseng C. A. Meyer and are responsible to the various pharmacological properties of this unique plant [1]. One of the most active minor constituents of P. ginseng is ginsenoside 20(S)-Rg3, which possesses a broad spectrum of biological activity, in particular, growth inhibition of malignant A549 lung cancer cells, U937 lymphoma, LNCaP prostate carcinoma, and SK-HEP-1 hepatoma. It is viewed as a potential anticancer drug among P. ginseng saponins [2]. 20(S)-Rg3 differs from the principal ginseng glycosides Rb1, Rb2, Rc, and Rd, which have protopanaxadiol as the aglycon, only by the lack of a carbohydrate group on C-20 [1]. This enables it to be prepared by selective incomplete deglycosylation of these saponins, including the use of various microorganisms [3].

We communicated earlier the isolation from soil samples taken from a ginseng field of several bacteria with β-glucosidase activity [4] and the use of several of them to convert ginsenosides Rb1 into Rd [5] and Rd into the minor glycoside F-2 and compound K [6]. In continuation of these studies, we present data on the biotransformation of Rd (1), which has β-sophorose and β-D-glucose residues in the C-3 and C-20 positions, respectively, of 20(S)-protopanaxadiol, into ginsenoside 20(S)-Rg3 (2) using the bacterium Flavobacterium sp. BGS36.

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Standard ginsenosides Rd (1) and 20(S)-Rg3 were purchased (Faces Biochemical Co., Wuhan, PRC). The preparation of Rd for deglycosylation experiments; the analysis of its biotransformation products using TLC and CHCl3:MeOH:H2O (65:35:10, lower phase) and HPLC on a Zorbax Eclipse XDB-C18 reversed-phase column, and the recording of mass spectra and PMR and 13C NMR spectra were carried out as previously described [5].

The bacterium Flavobacterium sp. BGS36 was cultivated in Ehrlenmeyer flasks in R2A liquid medium (200 mL) for 24 h at 30°C with constant stirring. Then, bacterial suspension (100 mL) with cell concentration 6 × 106 CFU/mL (colonyforming units/mL) was mixed with an aqueous solution of Rd (100 mL, 1 mM). The mixture was incubated for 24 h at 30°C with constant stirring. Analytical samples were taken every 3 h. When the incubation was finished, the mixture was extracted with water-saturated BuOH (2 × 200 mL). The resulting extract was evaporated in vacuo. The residue was used to isolate the biotransformation product. TLC and HPLC showed the presence in the collected samples of starting Rd (R f 0.38) and its transformation product 2 (R f 0.57). Thus, the concentration of the first glycoside decreased whereas that of the second increased in proportion to the incubation time.

Compound 2 corresponded to standard ginsenoside 20(S)-Rg3 according to TLC Rf value and HPLC retention time. Compound 2 was isolated from the obtained extract by preparative HPLC over an OptimaPak C18 column (250 × 10 mm, particle size 10 μm) and identified using mass and NMR spectroscopy in order to confirm this.

The difference in the m/z values of the [M + Na]+ molecular ions of starting Rd (m/z 969) [5] and its transformation product 2 (m/z 807) corresponded to a glucose residue. This indicated that it was cleaved from Rd during the biotransformation. A comparison of the PMR and 13C NMR spectra of these compounds provided evidence that this cleavage occurred at the C-20 glycoside of Rd. In fact, the 13C NMR spectrum of 2 contained resonances for only two anomeric C atoms (105.1 and 106.1 ppm), the chemical shifts of which were similar to those for the C-3 β-sophorose of Rd (Table 1). Also, C-20 underwent a strong-field shift in 2 (73.0 ppm) compared with that in Rd (83.6 ppm), indicating that C-20 in 2 lacked a carbohydrate component. The presence in the PMR spectrum of 2 of resonances for only two anomeric protons at 4.90 and 5.35 ppm and their agreement with those of the β-sophorose residue in Rd [5] also confirmed the aforementioned conclusion. Therefore, the Rd biotransformation product was the known triterpenoid glycoside Rg3, which had a β-sophorose residue in the C-3 position, like Rd, but differed from it by the lack of a glucose on C-20 [1]. This ginsenoside is known to be capable of existing as two different stereoisomers, 20(S)-Rg3 and 20(R)-Rg3, the 13C NMR spectra of which, despite their similarity, have noticeable differences in the chemical shifts of the C-17, C-21, and C-22 resonances [7, 8].

Table 1 13C NMR Spectra of Ginsenosides Rd (1) [5], 2, and 20(S)-Rg3 [7], δ, ppm*
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Table 1 shows that these C atoms in the 13C NMR spectrum of 2 had chemical shifts of 54.9, 28.0, and 36.1 ppm, respectively, which indicated unambiguously that it was identical to 20(S)-Rg3. Obviously, incomplete deglycosylation of 1 by bacterium Flavobacterium sp. BGS36 occurred stereospecifically to cleave the β-D-glucose on C-20, leading to the formation of only one stereoisomer, 20(S)-Rg3. An analogous phenomenon was observed during enzymatic transformation of ginsenosides Rb1 and Rd into 20(S)-Rg3 through the action of recombinant β-glucosidase from Microbacterium esteraromaticum [3].

The results indicated that Flavobacterium sp. BGS36 can be used for biotransformation of one of the principal ginseng glycosides Rd into the biologically more active minor ginsenoside 20(S)-Rg3.

3- O -[ β -D-Glucopyranosyl(1→2)- β -D-glucopyranosyl]-3 β ,12 β ,20 β -trihydroxydammar-24-ene (2). C42H72O13. Yield 84%, white powder, mp 292–294°C. 1H NMR (500 MHz, Py-d5, δ, ppm, J/Hz): 0.79 (3H, s, CH3-19), 0.94 (3H, s,CH3-30), 0.95 (3H, s, CH3-18), 1.08 (3H, s, CH3-29), 1.27 (3H, s, CH3-28), 1.42 (3H, s, CH3-21), 1.61 (3H, s, CH3-27), 1.64 (3H, s, CH3-26), 4.90 (1H, d, J = 7.0, H-1′), 5.35 (1H, d, J = 7.4, H-1″) (only characteristic proton resonances are given). Mass spectrum: FAB, m/z 807 [M + Na]+.