Transformation of Ginsenosides by A. niger and A. oryzae
PPT type ginsenoside Re is the most plentiful ginsenoside in unfermented KGB. Ginsenoside Rb1, Rb2, and Rd are also contained as the main PPD type ginsenosides of unfermented KGB. After fermentation, the ginsenoside profile was analyzed by TLC (Fig. 1) and HPLC (Fig. 2). The percentage weight of various ginsenosides in the n-butanol extracts was determined by HPLC (Fig. 3).
|Fig. 1. TLC Profiles of Ginsenosides in Unfermented and Fermented KGB Extracts|
Butanol extracts of unfermented KGB, niger-fermented KGB and oryzae-fermented KGB are presented as control, niger and oryzae, respectively.
|Fig. 2. HPLC Chromatograms of Ginsenosides Detected from (A) Unfermented KGB, (B) niger-Fermented KGB, (C) oryzae-Fermented KGB|
|Fig. 3. Ginsenoside Contents in Butanol Extract of Unfermented and Fermented KGB Measured by HPLC|
Percentage concentration=(weight of ginsenoside/weight of sample)×100%.
In terms of TLC results (Fig. 1), bands of ginsenosides on the plate went up after fermentation. Furthermore, bands of cK and Rh1 markedly appeared on the top of columns of niger and oryzae-fermented KGB, respectively. These show that the major ginsenosides have been transformed to their less polar forms.
With respect to HPLC results (Fig. 3), after fermentation by A. niger, for the main PPT type ginsenosides, Re decreased from 17.8 to 0.2%, Rg2 increased from 1 to 6.5% and Rh1 increased from 0.1 to 3.3%. As for PPD type ginsenosides, Rb1, Rb2 and Rd decreased to 0% and cK increased from 0 to 10.3%. Thus it can be seen that A. niger mainly transformed PPT type ginsenoside Re to Rg2 and to a lesser extent, to Rh1. Most PPD type ginsenosides such as Rb1, Rb2, and Rd were transformed to cK in the end. In our previous research,15) ginsenoside Rb1 was transformed to cK via Rd and F2 by crude enzyme extracted from various microorganisms, which is consistent with the present study. Ginsenoside Rg3 was also produced when KGB was sterilized by autoclave before fermentation and partly transformed to Rh2 by fermentation. These show that A. niger is more inclined to transform PPD type ginsenosides with a notably high cK yield while the PPT type ginsenoside transformation was stopped at ginsenoside Rg2, a less deglycosylated form.
After fermentation by A. oryzae, for the main PPT type ginsenosides, Re decreased from 17.8 to 0%, Rg1 increased from 2.4 to 9.3% and Rh1 increased from 0.1 to 6.8%. As for PPD type ginsenosides, Rb1 decreased from 1.2 to 0.5%, Rb2 decreased from 2.3 to 1.7%, and Rd decreased from 4.4 to 1.3%; F2 increased from 0 to 7.3%, and cK increased from 0 to 3.4% (Fig. 3). Thus it can be seen that A. oryzae transformed PPD type ginsenosides mainly to F2 and slightly to cK. Furthermore, ginsenoside Rg3 was also produced during sterilization. As for PPT type ginsenosides, Re was largely transformed to Rh1 via Rg1. Thus, it is apparent that A. oryzae may be more prone to transform PPT type ginsenosides with an efficient ginsenoside Rh1 production while the PPD type ginsenoside transformation was stopped at ginsenoside F2, a less deglycosylated form (Fig. 4).
|Fig. 4. Proposed Main Transformation Pathways of Ginsenoside Re by Mycotoxin Non-producing A. niger and A. oryzae|
Taken together, ginsenosides in ginseng berries were efficiently transformed into their nonpolar forms. KGB fermented by A. niger showed extremely high cK content while KGB fermented by A. oryzae contained abundant Rh1. This indicated that the A. niger tended to transform PPD type ginsenosides to cK and the A. oryzae tended to transform PPT type ginsenoside to Rh1. Different properties of ginsenoside transformation were showed between A. niger and A. oryzae, which may be explained by substrate specificity of glycosidase. Ginsenoside cK was reputed to exert various activities including anti-diabetic1,21) and anti-tumor22–25) effects. Rh1 was also reported to show anti-cancer,26) anti-allergy,27) anti-obesity28) and central nervous system protective activities.29)
Cytotoxicity of KGB on Cancer Cell Lines
The cytotoxicity of KGB and mycotoxin non-producing A. niger and A. oryzae fermented KGB on the human adenocarcinoma HT-29 cell line and hepatocellular carcinoma HepG2 cell line was investigated using the MTT assay.
Both A. niger and A. oryzae fermented KGB showed high inhibitory effects on the HT-29 cell line while unfermented KGB showed no cytotoxicity at the tested concentration (Fig. 5A). The minimum effective concentration of niger-fermented KGB was less than 2.5 µg/mL while that of oryzae-fermented KGB ranged from 2.5 to 5 µg/mL. The IC50 of niger-fermented KGB was between 10 and 20 µg/mL while the oryzae-fermented KGB was between 5 and 10 µg/mL.
|Fig. 5. Effects of Korean Ginseng Berry Extracts on Proliferation of HT-29 (A) and HepG2 (B) Cell Lines|
Antiproliferative effects of various PPD type and PPT type ginsenosides on HepG2 cell line (C). Proliferation of control group was set as 100%. Proliferation after treatment=(Abs after treatment/Abs of control)×100%. (*) Significant different with control group (p<0.05) when compared by Duncan’s multiple range tests. Values were expressed as the mean±standard deviation bars (n=4).
As for the HepG2 cell line, mold-fermented KGB also showed outstanding anti-proliferative activity while unfermented KGB only had a slight effect at concentrations larger than 20 µg/mL (Fig. 5B). The minimum effective concentration of niger-fermented KGB was less than 2.5 µg/mL while that of oryzae-fermented KGB ranged from 2.5 to 5 µg/mL. The IC50 value of niger-fermented KGB was about 10 µg/mL while that of oryzae-fermented KGB was almost 20 µg/mL.
To figure out which ginsenosides show cytotoxicity, PPD type ginsenoside Rb1, Rg3, cK, and PPT type ginsenoside Re and Rh1 were also tested by MTT assay (Fig. 5C). Rb1 showed no cytotoxicity at tested concentrations while its metabolized form, Rg3, showed slight cytotoxicity. Ginsenoside cK, the further deglycosylated form of Rb1, exhibited the most efficient cytotoxicity. However, PPT type ginsenosides showed no cytotoxicity. As A. niger is more inclined to transform PPD type ginsenosides, niger-fermented KGB showed stronger anti-proliferative activity than oryzae-fermented KGB.
Additionally, according to the previous study,30) cytotoxic activity is inversely proportional to the numbers of sugars linked to the aglycone of ginsenosides. Steamed AGB was reported to exhibit enhanced cytotoxicity as a result of that its main PPD type ginsenosides were transformed to Rg3.6) However, the niger-fermented KGB in our research showed a more outstanding effect than steamed AGB as its main PPD type ginsenosides were more completely transformed to cK.
Ginseng has been documented to prevent various degenerative diseases including cancer.31–34) Case control35,36) and cohort studies37) have reported that ingesting ginseng could significantly decrease the mortality from cancer to some extent and a clinical trial study indicated that Korean red ginseng powder might improve postoperative survival as well as restore immunity in patients with advanced gastric cancer.38)
The anti-cancer activity can be mainly ascribed to ginsenosides.34) Deglycosylated forms of PPD type ginsenosides such as cK have potent cytotoxicity and inhibit tumor cell invasion.39) Although PPT type ginsenosides have barely any cytotoxicity, they may also show curative effects against cancers. Protopanaxatriol was reported to inhibit the matrix metallopeptidase (MMP)-9 expression in the HT1080 human fibrosarcoma cell line and reduce the tumor cell invasion.40) It has also been observed that the fatty acid conjugate form of panaxatriol activated natural killer (NK) cells so as to kill cancer cells in an indirect way.41) The PPT type ginsenosides have been documented to suppress glycoprotein dependent multidrug resistance and increase the sensitivity of cancer cells to anti-cancer drugs.42) In addition, PPT type ginsenoside metabolites have been shown to increase the effects of anti-cancer drug by inhibiting breast cancer resistance protein.43)
Fermentation by A. niger and A. oryzae dramatically increased cytotoxicity of KGB on the HT-29 and HepG2 cell lines, which in turn confirmed that ginsenosides in KGB were highly transformed. The minimum effective concentration was about 2 µg/mL, which can be easily reached in the gastrointestinal tract. Therefore, fermented KGB may show anti-proliferative effect on colon cancer cells, like HT-29 cells. However, according to the previous research,9,44) only nanogram levels of deglycosylated forms of ginsenosides were detected in the plasma after oral intake of ginseng products, indicating that it is hard to expect any cytotoxicity on cancer cells located beyond the gastrointestinal tract, like HepG2 cells.
In summary, our findings indicated that A. niger tended to transform PPD type ginsenosides and A. oryzae tended to transform PPT type ginsenosides. Contents of ginsenoside cK and Rh1 were increased by approximately 10 and 7% in A. niger and A. oryzae fermented KGB extracts, respectively. Moreover, after fermentation by these mycotoxin free molds, anti-proliferative activity of KGB was dramatically enhanced. As PPD type ginsenosides exhibit stronger cytotoxicity and A. niger are more prone to transform PPD type ginsenosides, A. niger fermented KGB showed more remarkable anti-proliferative effect. However, as the most ginsenosides in KGB are of PPT type, fermentation with A. oryzae may be more meaningful. Since berry and root have the different profiles of ginsenoside, they may exhibit distinct activities. It is reported that Rh1 and cK act as novel agonists of estrogen and glucocorticoid receptor, respectively.45,46) Dey et al.47) compared the anti-hyperglycemic effects between berry and root, and found that ginseng berry exhibited more potent anti-hyperglycemic activity, and only ginseng berry showed marked anti-obesity effects in ob/ob mice administrated intraperitoneally. More studies are needed to figure out the distinct activities between ginseng berry and root.