Ginsenoside F2 Research Articles

Ginsenosidases and the pathogenicity of Pythium irregulare

American ginseng (Panax quinquefolius L.) produces triterpenoid saponins, ginsenosides, that possess mild fungitoxic activity toward some common ginseng leaf pathogens. However, numerous oomycete root pathogens of ginseng, most notably Pythium irregulare Buisman, are able to partially deglycosylate 20 (S)-protopanaxadiol ginsenosides Rb1, Rd and gypenoside XVII via extracellular glycosidases, leading to a common product, ginsenoside F2. Conversion of the common 20 (S)-protopanaxadiols into F2 requires both β (1→6) and β (1→2) glucosidase activity. In the present study, the ability of nine distinct isolates of P. irregulare, as well as a P. ultimum Trow isolate and two isolates of Trichoderma hamatum (Bonord.) Bainier, to deglycosylate 20 (S)-protopanaxadiols, in vitro was examined. The pathogenicity of each isolate was also examined by scoring the severity of disease symptoms caused by each in separate inoculations of one- and two-year old ginseng seedlings. Disease severity was scored using a disease severity index, as well as by taking Fv/Fm measurements of leaves during a 14-day infection period. Based on these measurements, it was concluded that (1) the use of direct Fv/Fm measurements correlates strongly with observations of disease severity (R2=0.79), and that (2) the pathogenicity of P. irregulare isolates correlates with their ability to deglycosylate ginsenosides (R2=0.57). These results further support the hypothesis that the pathogenicity of P. irregulare on ginseng roots is dependent, in part, on the ability of this organism to deglycosylate ginsenosides.

Co-transformation of Panax major ginsenosides Rb1 and Rg1 to minor ginsenosides C–K and F1 by Cladosporium cladosporioides

Rb₁ and Rg₁ are the major ginsenosides in protopanaxadiol and protopanaxatriol. Their content in ginsenosides was 23.8 and 17.6%, respectively. A total of 22 isolates of β-glucosidase producing microorganisms were isolated from the soil of a ginseng field using Esculin-R2A agar. Among these isolates, the strain GH21 showed the strongest activities to convert ginsenoside Rb₁ and Rg₁ to minor ginsenosides compound-K and F₁, respectively. Ginsenosides Rb₁ and Rg₁ bioconversion rates were 74.2 and 89.3%, respectively. Meanwhile, the results demonstrated that the ginsenoside Rg₁ could change the biotransformation pathway of ginsenoside Rb₁ by inhibiting the formation of the intermediate metabolite gypenoside-XVII. GH21 was identified as a Cladosporium cladosporioides species based on the internal transcribed spacers (ITS) ITS1-5.8S-ITS2 rRNA gene sequences constructed phylogenetic trees.

Fungal sensitivity to and enzymatic deglycosylation of ginsenosides

A ginseng pathogen, Cylindrocarpon destructans, and five nonpathogens were tested for their sensitivity to a total ginsenoside fraction (T-GF), a protopanaxadiol-type ginsenoside fraction (PPD-GF) and a protopanaxatriol-type ginsenoside fraction (PPT-GF) from the roots of Panax ginseng C.A. Meyer. The results showed that T-GF inhibited growth of the five ginseng nonpathogens, while it promoted growth of the ginseng pathogen C. destructans. PPT-GF and PPD-GF both inhibited the growth of the five ginseng nonpathogens, although the activity of PPT-GF was higher than that of PPD-GF. PPT-GF and PPD-GF exhibited different activities on C. destructans: PPT-GF inhibited its growth, whereas PPD-GF significantly enhanced its growth. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can consecutively hydrolyze the terminal monosaccharide units from the sugar chains attached at C3 and C20 in PPD-type ginsenosides by extracellular glycosidase activity to yield four major products, gypenoside XVII (G-XVII), compound O, compound Mb and the ginsenoside F2. By contrast, the ginseng nonpathogens Aspergillus nidulans and Cladosporium fulvum have no extracellular glycosidase activity toward sugar chains attached to C3 in PPD-type ginsenosides. These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi might counter their toxicity by converting PPD-type ginsenosides into growth or host recognition factors. The ability of ginseng root pathogens to deglycosylate PPD-type ginsenosides may be a pathogenicity factor.

Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells

Ginsenoside F2 (F2) was assessed for its antiproliferative activity against breast cancer stem cells (CSCs). F2 induced apoptosis in breast CSCs by activating the intrinsic apoptotic pathway and mitochondrial dysfunction. Concomitantly, F2 induced the formation of acidic vesicular organelles, recruitment of GFP-LC3-II to autophagosomes, and elevation of Atg-7 levels, suggesting that F2 initiates an autophagic progression in breast CSCs. Treatment with an inhibitor of autophagy enhanced F2-induced cell death. Our findings provide new insights into the anti-cancer activity of F2 and may contribute to the rational use and pharmacological study of F2.

Changes in the Contents of Prosapogenin in the Red Ginseng (Panax ginseng) Depending on Steaming Batches

This study compared the contents of ginsenosides depending on steaming conditions of red ginsengs to provide basic information for developing functional foods using red ginsengs. The red ginseng steamed eight times at 98℃ ranked atop the amounts of prosapogenins ever detected in red ginsengs (ginsenoside Rg2, Rg3, Rg5, Rg6, Rh1, Rh4, Rk1, Rk3, F1, F4, 1.15%) among red ginsengs steamed more than twice. When steamed eight times at 98℃, 2.7 times as much prosapogenins such as ginsenosides Rg2, Rg3, Rg5, Rg6, Rh1, Rh4, Rk1, Rk3, F1, and F4 as those steamed just once at 98℃ was collected. In addition, the red ginsengs steamed eight times at 98℃ contained more amounting ginsenoside Rg3 (0.28%) than that in the red ginseng steamed several times at random. Accordingly, it is recommendable that red ginsengs steamed 8 times, which proved to be the optimal steaming condition, be used rather than those steamed 9 times (black ginsengs), in order to develop red ginseng products of high prosapogenin concentration and high functions.

Anti-Cancer Effect of Ginsenoside F2against Glioblastoma Multiforme in Xenograft Model in SD Rats

The glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Despite combination treatments of radiation and chemotherapy, the survival periods are very short. Therefore, this study was conducted to assess the potential of ginsenoside F2 (F2) to treat GBM. In in vitro experiments with glioblastoma cells U373MG, F2 showed the cytotoxic effect with IC50 of 50 μg/mL through apoptosis, confirmed by DNA condensation and fragmentation. The cell population of cell cycle sub-G1 as indicative of apoptosis was also increased. In xenograft model in SD rats, F2 at dosage of 35 mg/kg weight was intravenously injected every two days. This reduced the tumor growth in magnetic resonance imaging images. The immunohistochemistry revealed that the anticancer activity might be mediated through inhibition of proliferation judged by Ki67 and apoptosis induced by activation of caspase-3 and -8. And the lowered expression of CD31 showed the reduction in blood vessel densities. The expression of matrix metalloproteinase-9 for invasion of cancer was also inhibited. The cell populations with cancer stem cell markers of CD133 and nestin were reduced. The results of this study suggested that F2 could be a new potential chemotherapeutic drug for GBM treatment by inhibiting the growth and invasion of cancer.

Utilization of hydrolytic enzymes for the extraction of ginsenosides from Korean ginseng leaves

This study was performed to measure the amount of ginsenoside content obtained from dried ginseng leaf powder treated with various enzymes, including Ultraflo L. Ultraflo L showed superior results to other enzymes and untreated ginseng leaf in catalyzing the liberation of biological compounds. The total ginsenoside content and metabolites showed the highest levels (406.1μg/mg and 93.1μg/mg, respectively) in ginseng leaf extract treated with Ultraflo L (UTGL). In particular, the content of ginsenoside F2 in UTGL was 8.5-fold greater compared to that found in the roots. UTGL showed significantly higher DPPH and ABTS antioxidant activities (2.77mg/mL and 1.57mg/mL of IC50 value, respectively) than untreated ginseng leaf. Ginseng leaf treated with Ultraflo L has advantages over untreated ginseng leaf in terms of cost and source availability. These data also suggest that the leaves and stems of ginseng may be used as food resources, functional food materials, and feedstuffs.

Biotransformation of Ginsenoside Rb1to Prosapogenins, Gypenoside XVII, Ginsenoside Rd, Ginsenoside F2, and Compound K by Leuconostoc mesenteroides DC102

Ginsenoside Rb1is the main component in ginsenosides. It is a protopanaxadiol-type ginsenoside that has a dammarane-type triterpenoid as an aglycone. In this study, ginsenoside Rb1 was transformed into gypenoside XVII, ginsenoside Rd, ginsenoside F2 and compound K by glycosidase from Leuconostoc mesenteroides DC102. The optimum time for the conversion was about 72 h at a constant pH of 6.0 to 8.0 and the optimum temperature was about 30℃. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 h post-reaction (99%). The enzymatic reaction was analyzed by highperformance liquid chromatography, suggesting the transformation pathway: ginsenoside Rb1→ gypenoside XVII and ginsenoside Rd→ginsenoside F2→compound K.

Ginsenoside F1 production from ginsenoside Rg1 by a purified β-glucosidase from Fusarium moniliforme var. subglutinans

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F(1) from ginsenoside Rg(1). The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg(-1). It hydrolysed glucose-linked ginsenosides Rb(1), Rd and Rg(1) but not for other monosaccharide-linked ginsenosides, Rb(2), Rc, R(1), and Re. Under the optimum conditions of pH 6.0, 50 °C, 30 U l(-1) of enzyme, and 5 mg Rg(1) ml(-1), 4 mg F(1) ml(-1) was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l(-1) h(-1). This represents the highest productivity and conversion yield of F(1) yet reported.

Intestinal bacterial hydrolysis is required for the appearance of compound K in rat plasma after oral administration of ginsenoside Rb1 from Panax ginseng.

Ginsenoside Rb1 from Panax ginseng root is transformed into compound K via ginsenosides Rd and F2 by intestinal bacterial flora. Among 31 defined intestinal strains from man, only Eubacterium sp. A-44 transformed ginsenoside Rb1 into compound K via ginsenoside Rd. The ginsenoside Rb1-hydrolysing enzyme isolated from Eubacterium sp. A-44 was identical to a previously purified geniposide-hydrolysing beta-D-glucosidase. When ginsenoside Rb1 (200 mg kg-1) was administered orally to germ-free rats, neither compound K nor any other metabolite was detected in the plasma, intestinal tract or cumulative faeces 7 or 15 h after administration. Most of the ginsenoside Rb1 administered was recovered from the intestinal tract, especially the caeca, and cumulative faeces indicating poor absorption of ginsenoside Rb1. When ginsenoside Rb1 was administered orally to gnotobiote rats mono-associated with Eubacterium sp. A-44, a significant amount of compound K was detected in the plasma and considerable amounts were found in the caecal contents and cumulative faeces 7 and 15 h after administration. A small amount of ginsenoside Rb1 was detected in the caecal contents only 7 h after administration. These results indicate that orally administered ginsenoside Rb1 is poorly absorbed from the gut but that its metabolite compound K, produced by ginsenoside Rb1-hydrolysing bacteria such as Eubacterium sp. A-44 in the lower part of intestine, is absorbed.

Anti-complementary Ginsenosides Isolated from Processed Ginseng

As part of an ongoing search for immunomodulatory components aimed at the anti-complementary effect, ginsenosides isolated from processed ginseng were found to have inhibitory activity on complement activation through classical pathways. Activity-guided fractionation was used to isolate four ginsenosides, namely ginsenoside Rg₆, F₄, Rk₃, and Rh₄. Ginsenoside Rk₃ and Rh₄ had a 3 fold higher inhibition activity than rosmarinic acid which was used as a positive control while ginsenoside Rg₆ and F₄ showed only mild effects similar to that of the positive control. The results suggest that the activity of the corresponding ginsenosides may be increased by the glycosyl moiety at the C₆ position rather than the double bond conformation at C₂₀, and ginsenoside Rk₃ and Rh₄ could have a role in treating inflammatory diseases.

Bioconversion of ginsenoside rb1 into compound k by Leuconostoc citreum LH1 isolated from kimchi.

About 40 different types of ginsenoside (ginseng saponin), a major pharmacological component of ginseng, have been identified along with their physiological activities. Among these, compound K has been reported to prevent the development of and the metastasis of cancer by blocking the formation of tumors and suppressing the invasion of cancerous cells. In this study, ginsenoside Rb1 was converted into compound K via interaction with the enzyme secreted by β-glucosidase active bacteria, Leuconostoc citreum LH1, extracted from kimchi. The optimum time for the conversion of Rb1 to compound K was about 72 hrs at a constant pH of 6.0 and an optimum temperature of about 30°C. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 hrs post-reaction (99%). Both TLC and HPLC were used to analyze the enzymatic reaction. Ginsenoside Rb1 was consecutively converted to ginsenoside Rd, F2, and compound K via the hydrolyses of 20-C β-(1 → 6)-glucoside, 3-C β-(1 → 2)-glucoside, and 3-C β-glucose of ginsenoside Rb1.

Bioconversion of Ginsenoside Rd into Compound K by Lactobacillus pentosus DC101 Isolated from Kimchi

Ginsenosides are the principal components responsible for the pharmacological and biological activities of ginseng. Ginsenoside Rd was transformed into compound K using cell-free extracts of food microorganisms, with Lactobacillus pentosus DC101 isolated from kimchi (traditional Korean fermented food) used for this conversion. The optimum time for the conversion was about 72 h at a constant pH of 7.0 and an optimum temperature of about <TEX>$30^{\circ}C$</TEX>. The transformation products were identified by thin-layer chromatography and high-performance liquid chromatography, and their structures were assigned using nuclear magnetic resonance analysis. Generally, ginsenoside Rd was converted into ginsenoside F2 by 36 h post-reaction. Consequently, over 97% of ginsenoside Rd was decomposed and converted into compound K by 72 h post-reaction. The bioconversion pathway to produce compound K is as follows: ginsenoside Rd<TEX>$\rightarrow$</TEX>ginsenoside F2<TEX>$\rightarrow$</TEX>compound K.

Solirubrobacter ginsenosidimutans sp. nov., isolated from soil of a ginseng field

A Gram-reaction-positive, non-motile, non-spore-forming, aerobic rod, designated BXN5-15(T), was isolated from the soil of a ginseng field on Baekdu Mountain in China. Strain BXN5-15(T) grew optimally at 30 °C and pH 6.0-7.0 in the absence of NaCl on R2A agar. Strain BXN5-15(T) displayed β-glucosidase activity, which allowed it to transform ginsenoside Rb(1) (one of the dominant active components of ginseng) via Rd to minor ginsenoside F(2). On the basis of 16S rRNA gene sequence analysis, strain BXN5-15(T) was shown to belong to the genus Solirubrobacter. The closest phylogenetic relatives were Solirubrobacter soli Gsoil 355(T) (98.4 % 16S rRNA gene sequence similarity) and Solirubrobacter pauli B33D1(T) (96.4 %). Lower sequence similarities (<96.0 %) were found with all of the other recognized members of the order Solirubrobacterales. The predominant quinone was MK-7(H(4)). The major fatty acids (>10 %) were C(18 : 1)ω9c, iso-C(16 : 0) and C(18 : 3)ω6,9,12c. The G+C content of the genomic DNA was 70.6 mol%. DNA-DNA relatedness between strain BXN5-15(T) and S. soli KCTC 12628(T) was 23.3 %. On the basis of genotypic, phenotypic and chemotaxonomic data, strain BXN5-15(T) represents a novel species within the genus Solirubrobacter, for which the name Solirubrobacter ginsenosidimutans sp. nov. is proposed. The type strain is BXN5-15(T) ( = KACC 20671(T) = LMG 24459(T)).

Three new dammarane-type triterpene saponins from the leaves of Panax ginseng C.A. Meyer

Three new dammarane-type triterpene ginsenosides, together with six known ginsenosides, were isolated from the leaves of Panax ginseng C.A. Meyer. The new saponins were named as ginsenoside Rh11, ginsenoside Rh12, and ginsenoside Rh13. Their structures were elucidated as (20S)-3β,6α,12β,20-tetrahydroxydammara-25-ene-24-one 20-O-β-d-glucopyranoside (1), (20S)-3β,12β,20,24,25-pentahydroxydammarane 20-O-β-d-glucopyranoside (2), and (20S,23E)-3β,12β,20,25-tetrahydroxydammara-23-ene 20-O-β-d-glucopyranoside (3) on the basis of 1D and 2D NMR experiments and mass spectra. The known ginsenosides were identified as ginsenoside M7cd, ginsenoside Rg6, ginsenoside Rb3, gypenoside XVII, gypenoside IX, and 20-(E)-ginsenoside F4.

Changes in ginsenosides and antioxidant activity of Korean ginseng (Panax ginseng C.A. Meyer) with Heating Temperature and Pressure

This study was carried out to investigate the changes of ginsenoside compositions and antioxidant activity of fresh ginseng induced by thermal processing at different temperatures (25, 100, 121, and 150°C), pressure (0.1, 10, 20, and 30 MPa), and soaking solvents (water and ethanol). The levels of ginsenosides were similar trend with the pressure of 0.1–30 MPa, while there were significantly differences in heated ginseng with heating temperature and soaking solvent. When water and ethanol was used, the ginsenoside compositions significantly changed at 100 and 121°C, respectively, and it was rapidly decreased at 150°C. After heating, the level of 3 ginsenosides (Re, Rf, and Rg1) decreased and that of 5 other ginsenosides [Rb1, Rb2, Rb3, Rc, and Rg2(S)] increased up to 121°C compare to raw ginseng. Ginsenoside F2, F4, Rg2(R), Rk3, Rh4, Rg3(S), Rg3(R), Rk1, and Rg5, which was absent in raw ginseng, was detected in heated ginseng. Especially, ginsenoside Rg3(S), Rg3(R), Rk1, and Rg5 were remarkably produced after thermal processing. After heating, the phenolic compounds (1.43–11.62 mg/g), 50% inhibition concentration (IC50) value (1.48–3.11 mg/g), and ABTS radical scavenging activity (0.66–9.09 mg AA eq/g) of heated ginseng were increased.

Biotransformation pathways of ginsenoside Rb1 to compound K by β-glucosidases in fungus Paecilomyces Bainier sp. 229

Ginsenoside Rb1 is the most abundant ginsenoside in Panax (ginseng). The hydrolysis of this ginsenoside produces compound K, the biologically active ginsenoside of ginseng. We previously identified a fungus Paecilomyces Bainier sp. 229 (sp. 229), which can efficiently convert ginsenoside Rb1 to compound K. In this report, the ginsenoside hydrolyzing β-glucosidases were isolated from sp. 229 and the pathway of the biotransformation of ginsenoside Rb1 to compound K by sp. 229 was investigated. Based on reverse-phase HPLC and TLC analysis, we found the main metabolic pathway is as follows: ginsenoside Rb1 → ginsenoside Rd → ginsenoside F2 → compound K. Moreover, the results showed that there were other metabolic pathways: ginsenoside Rb1 → ginsenoside XVII → ginsenoside F2 → compound K and ginsenoside Rb1 → ginsenoside Rg3 → ginsenoside Rh2. These processes would allow the specific bioconversion of ginsenoside Rb1 to various ginsenosides using an appropriate combination of specific microbial enzymes.

Partial purification and characterization of three ginsenoside-metabolizing β-glucosidases from Pythium irregulare

The ginseng pathogen Pythium irregulare is able to selectively metabolize the 20( S) protopanaxadiol ginsenosides Rb1, Rb2, Rc, Rd, and gypenoside XVII via extracellular glycosidases, leading to the formation and partial assimilation of ginsenoside F2. Herein we have partially purified three ginsenoside-deglycosylating enzymes from P. irregulare culture filtrates, and provide preliminary characterization. A protocol involving acetone precipitation, chromatofocusing on PBE 94, gel filtration on Sephacryl S-200 HR and ion-exchange on Q Sepharose ™ Fast Flow resulted in a 13–25-fold purification. The three enzymes were induced in cultures grown in the presence of ginsenosides, and found to be acidic proteins (pI of 4.5–5.0), consisting of an apparent high molecular weight (∼160 kDa) homodimer of 78 kDa subunits, with β(1 → 6) activity, and two monomeric enzymes of 61 and 57 kDa, with β(1 → 2) activity. Primary sequence analysis identified them as β-glucosidases, with no homology to other saponin-deglycosylating enzymes. These are the first glycosidases purified from a Pythium species. We speculate that their role is likely to help Pythium find its host, and/or obtain nutrients/growth factors from its environment.

Dammarane-type glycosides and long chain sesquiterpene glycosides from Gynostemma yixingense

A new dammarane-type glycoside and a new long chain sesquiterpene glycoside, along with nine known compounds 20(S)-ginsenoside Rh1 ( 3 ), 20(R)-ginsenoside Rh1 ( 4 ), ginsenoside F1 ( 5 ), amarantholidoside IV ( 6 ), ginsenoside Rc ( 7 ), 20(S)-ginsenoside Rg2 ( 8 ), 20(R)-ginsenoside Rg2 ( 9 ), ginsenoside Rd ( 10 ) and gypenoside XLVI ( 11 ) were isolated from Gynostemma yixingense. The structures of the new compounds were determined on the basis of spectroscopic analysis, including 1D-, 2D-NMR and ESI-MS techniques as well as by comparison of the spectral data with those of related compounds as 2 α,3 β,20(S)-trihydroxydammar-24-ene-3-O-[ β- d-glucopyranosyl((1 → 2)- β- d-glucopyranosyl]-20-O-[ β- d-xylopyranosyl((1 → 6)- β- d-glucopyranoside] ( 1 ) (2E,6E)-10- β- d-glucopyranosyl-1,10,11-trihydroxy-3,7,11-trimethyldodeca-2,6-diene ( 2 ).

Quantitative Determination of Eight Major Constituents in the Traditional Chinese Medicinal Yi-Qi-Fu-Mai Preparation by LC

A high-performance liquid chromatography-diode array detection method was developed and validated for the simultaneous determination of eight marker components, ginsenoside Rf, Rb1, Rb2, Rb3, Rd, Rg3, 20(S)-ginsenoside F2 and schizandrin, present in traditional Chinese medicinal Yi-Qi-Fu-Mai preparations. Samples were prepared using a solid phase extraction procedure. Chromatographic separation was carried out on a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) by stepwise gradient elution with water (0.05% phosphoric acid, v/v) and acetonitrile as the mobile phase. Good linear relationships between were observed between peak areas and concentrations with r 2 values above 0.9990 for all the analytes. Average recoveries for the eight markers ranged from 97.7 to 100.4%. This method was found to be rapid, sensitive, accurate, and could be readily applied to the quality assessment of Yi-Qi-Fu-Mai preparations.