Transformation of Ginsenoside Rc into (20S)-Rg₃ by the Bacterium Leuconostoc sp. BG78

Ginsenoside (20S)-Rg₃ is one of the most active minor dammarane glycosides of Panax ginseng C. A. Meyer and exhibits a broad spectrum of biological activity including hepatoprotective, immunostimulating, neuroprotective, and antitumor. It inhibits cell growth of malignant A549 lung carcinoma, U937 lymphoma, LNCaP prostate carcinoma, and SK-HEP-1 hepatoma and is of interest in medicine as a potential antitumor agent [1]. One factor limiting the application of ginsenoside (20S)-Rg₃ is its low content in the total saponins of Panax ginseng. (20S)-Rg₃ differs from the principal dammarane glycosides Rb₁, Rb₂, Rc (1), and Rd (2) that have the aglycon protopanaxadiol only by the lack of a carbohydrate on C-20 [2]. This enables it to be produced by selective incomplete deglycosylation of these saponins, including by using various microorganisms [3, 4].

We reported earlier the use of several bacteria with β-glucosidase activity for transformation of ginsenoside Rb₁ into the minor glycoside F-2 and compound K [5] and Rd into the aforementioned ginsenoside (20S)-Rg₃ [6]. Herein we communicate data on the transformation of another principal glycoside, Rc (1) with a C-3 β-sophorose and a C-20 α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside, into (20S)-Rg₃ by the bacterium Leuconostoc sp. BG78.

Standard ginsenosides Rc, Rd, and (20S)-Rg₃ were purchased (Faces Biochemical Co., Wuhan, PRC). Glycoside 1 was isolated from six-year P. ginseng roots as described before [7] using in the final step preparative HPLC over an OptimaPak C18 column (250 × 10 mm, 10 μm). PMR, ¹³C NMR, and mass spectra of isolated Rc agreed with those published for this compound [4, 7]. The Rc biotransformation products were analyzed using TLC and solvent system CHCl₃–MeOH–H₂O (65:35:10, lower phase) and HPLC over a Zorbax Eclipse XDB-C18 reversed-phase column. Mass spectra and NMR spectra (¹H and ¹³C) were recorded as before [8].

Bacterium Leuconostoc sp. BG78 was cultivated in 200-mL Ehrlenmeyer flasks in MRS liquid growth medium for 24 h at 37°C with constant stirring. Then, cells were harvested by centrifugation (3000 g, 15 min, 4°C), rinsed twice with phosphate buffer (20 mM, pH 7.0), and suspended in the same buffer (20 mL). The resulting bacterium suspension (10 mL each) was placed into two sterile flasks and treated with freshly prepared MRS medium (40 mL) and an aqueous solution of ginsenoside Rc (50 mL, 1 mM). The first mixture was incubated for 48 h; the second, for 96 h at 37°C with constant stirring. Analytical samples were taken every 6 h. After the incubation was finished, each mixture was extracted twice with watersaturated BuOH (100 mL). The resulting extract was evaporated in vacuo. The residue was used to isolate the biotransformation products.

TLC and HPLC showed that the samples of the first mixture contained starting Rc (R _f 0.27) and two transformation products, 2 (R _f 0.38) and 3 (R _f 0.57). The Rc concentration decreased in proportion to the incubation time. The concentration of 2 increased, reaching a maximum after 36 h, and then decreased gradually. This was accompanied by a proportional increase in the concentration of 3. This was clearly visible in the analyses of samples taken from the second mixture after incubation for 36 h. The R _f values on TLC and retention times in HPLC of compounds 2 and 3 corresponded to those of standard ginsenosides Rd and (20S)-Rg₃. Compounds 2 and 3 were isolated from the extracts obtained from the first and second mixtures, respectively, using preparative HPLC over an OptimaPak C18 column. They were identified by mass and NMR spectroscopy. The spectral properties of 2 agreed fully with those of ginsenoside Rd reported by us [8]; of 3, with those of ginsenoside (20S)-Rg₃ described by us [6].

The difference in the m/z values for the [M + Na]⁺ molecular ions of starting ginsenoside Rc (1101) and its transformation product 2 (m/z 969) [8] corresponded to an α-L-arabinofuranose residue. This indicated that it was cleaved from Rc during the biotransformation. A comparison of the ¹³C NMR spectra of these compounds confirmed this conclusion. In particular, the ¹³C NMR spectrum of 2 (Table 1) contained resonances for only three anomeric C atoms (98.5, 105.3, and 106.3 ppm). The resonance of C-6″′ in the C-20 carbohydrate of 2-R₂ (63.0 ppm) underwent a strong-field shift relative to that in the 1-R₂ residue of 1 (68.5 ppm). An analogous comparison of the spectral characteristics of 2 and 3 [6] indicated that (20S)-Rg₃ formed as a result of the cleavage from Rd of the C-20 glucose.

Table 1 ¹³C NMR Spectra of Ginsenosides 1, 2, and 3 (125 MHz, δ, ppm)

Thus, incomplete deglycosylation of 1 by Leuconostoc sp. BG78 involved formation of Rd and its subsequent transformation into (20S)-Rg₃ (70–75% yield). Rd was formed more rapidly from Rc than its transformation into (20S)-Rg₃. This was probably related to the more active α-L-arabinofuranosidase, which catalyzes the first step, than β-glucosidase, which is responsible for the biotransformation of Rd into (20S)-Rg₃.

The results indicated that Leuconostoc sp. BG78 could be used to transform principal glycoside Rc into the biologically more active minor ginsenoside (20S)-Rg₃. This distinguished it from other Leuconostoc bacterial strains that transform Rc into other minor dammarane glycosides [3].

3- O -[ β -D-Glucopyranosyl-(1→2)- β -D-glucopyranosyl]-20- O -[ α -L-arabinofuranosyl-(1→6)- β -Dglucopyranosyl]-3 β ,12 β ,20 β -trihydroxydammar-24-ene (1). C₅₃H₉₀O₂₂. White powder, mp 198–201°C. ¹H NMR spectrum (500 MHz, Py-d₅, δ, ppm, J/Hz): 0.79 (3H, s, CH₃-19), 0.93 (6H, s, CH₃-18, 30), 1.07 (3H, s, CH₃-29), 1.25 (3H, s, CH₃-28), 1.59 (3H, s, CH₃-26), 1.61 (3H, s, CH₃-21), 1.64 (3H, s, CH₃-27), 4.90 (1H, d, J = 7.5, H-1′), 5.11 (1H, d, J = 7.5, H-1″″), 5.35 (1H, d, J = 7.5, H-1″), 5.62 (1H, d, J = 7.2, H-1″″) (only characteristic proton resonances are given). FAB mass spectrum: m/z 1101 [M + Na]⁺.