Compound K Research Articles

Intestinal Metabolite Compound K of Ginseng Saponin Potently Attenuates Metastatic Growth of Hepatocellular Carcinoma by Augmenting Apoptosis via a Bid-Mediated Mitochondrial Pathway

It was recently shown that compound K (CK), an intestinal bacterial metabolite of ginseng saponin, exhibits antihepatocellular carcinoma (HCC) activity, and Bid is a potential drug target for HCC therapy. This paper reports a novel mechanism of CK-induced apoptosis of HCC cells via Bid-mediated mitochondrial pathway. CK dramatically inhibited HCC cells growth in concentration- and time-dependent manners, and a high dose of CK could induce HCC cell apoptotic cell death. Furthermore, the effective dose of CK potently attenuated the subcutaneous tumor growth and spontaneous HCC metastasis in vivo. At the molecular level, immunohistochemical staining revealed that Bid expression in subcutaneous tumor and liver metastasis tissues decreased dramatically in CK-treated groups compared to untreated controls, which also implies that Bid may play a critical role in the growth and progression of HCC. Further study shows that translocation of full-length Bid to the mitochondria from nuclei during cytotoxic apoptosis was associated with the release of cytochrome c from mitochondria, indicating that full-length Bid is sufficient for the activation of mitochondrial cell death pathways in response to CK treatment in HCC cells. Taken together, the results not only reveal a Bid-mediated mitochondrial pathway in HCC cells induced by CK but also suggest that CK may become a potential cytotoxic drug targeting Bid in the prevention and treatment of HCC.

Ginsenoside Compound K Attenuates Metastatic Growth of Hepatocellular Carcinoma, which Is Associated with the Translocation of Nuclear Factor-κB p65 and Reduction of Matrix Metalloproteinase-2/9

The intestinal metabolite of ginseng saponin, compound K (CK), has various chemopreventive and chemotherapeutic activities, including anti-tumor activity. However, the functional mechanisms through which CK attenuates metastatic growth in hepatocellular carcinoma (HCC) remain unclear. Here, using multiple IN VITRO and IN VIVO models, we reported that CK strongly attenuated colony formation, adhesion, and invasion of HCC cells IN VITRO and dramatically inhibited spontaneous HCC metastatic growth IN VIVO. At the molecular level, immunofluorescence and Western blotting analysis confirmed that inhibition of metastatic growth of HCC induced by CK treatment caused a time-dependent decrease in nuclear NF- κB p65 and a concomitant increase in cytosolic NF- κB p65, indicating that CK suppressed the activation of the NF- κB pathway. Meanwhile, our study showed that the inhibition of matrix metalloproteinase2/9 (MMP2/9) expression caused by CK treatment was associated with NF- κB p65 nuclear export. Taken together, our results not only revealed that NF- κB p65 nuclear export and the reduction of MMP2/9 expression were associated with the metastatic inhibition induced by CK, but also suggested that CK may become a potential cytotoxic drug in the prevention and treatment of HCC.

Effect and Mechanism of Ginsenosides CK and Rg1 on Stimulation of Glucose Uptake in 3T3-L1 Adipocytes

The glucoregulatory activities of ginsenosides compound K (CK) and Rg1 were investigated in 3T3-L1 adipocytes. Both compounds significantly enhanced glucose uptake in 3T3-L1 adipocytes in a dose-response manner, which is correlated with increased GLUT4 translocation from intracellular vesicles to the plasma membrane in adipocytes. The stimulating effects of CK and Rg1 on glucose uptake and GLUT4 translocation are associated with activation of AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways; both are key pathways in mediating glucose uptake in animal cells. In addition to the acute stimulus effect of glucose uptake, prolonged incubation of CK and Rg1 significantly induced GLUT4, but not GLUT1, expression at both the mRNA and protein levels in 3T3-L1 adipocytes. Further studies showed that CK inhibited and Rg1 enhanced triglyceride accumulation in adipocytes, suggesting that the mechanism of action of these ginsenosides may not be completely identical. In summary, this study demonstrated insulin-like activities of CK and Rg1 in adipocytes. These findings are important in understanding the hypoglycemic properties and potential applications of ginseng and ginsenosides.

Changes of ginsenosides in Korean red ginseng ( Panax ginseng) fermented by Lactobacillus plantarum M1

To obtain microorganisms for the microbial conversion of ginsenosides in red ginseng powder (RGP), Lactobacillus species (M1–M4 and P1–P4) were isolated from commercial ginseng products. Strain M1 was determined to be L. plantarum by 16S rRNA sequencing. Red ginseng powder (RGP) fermented by L. plantarum M1 had a high total content of ginsenosides (142.4 mg/g) as compared to the control (121.8 mg/g). In particular, the ginsenoside metabolites Rg3, Rg5, Rk1, Compound K (CK), Rh1, and Rg2 showed a high level in the fermented RGP (65.5 mg/g) compared to the control (32.7 mg/g). During fermentation for 7 days, total sugar content decreased from 8.55 mg/g to 4 mg/g, uronic acid content reached its maximum (53.43 μg/g) at 3 days, and total ginsenoside content increased to 176.8 mg/g at 4 days. In addition, ginsenoside metabolites increased from 38.0 mg/g to 83.4 mg/g at 4 days of fermentation. Using everted instestinal sacs of rats, the fermented red ginseng showed a high transport level (10.3 mg of polyphenols/g sac) compared to non-fermented red ginseng (6.67 mg of polyphenols/g sac) after 1 h. These results confirm that fermentation with L. plantarum M1 is very useful for preparing minor ginsenoside metabolites while being safe for foods.

Myocardial Protection of Contractile Function After Global Ischemia by Compound K in the Isolated Heart

Ginsenosides are among the most well-known traditional herbal medicines frequently used for the treatment of cardiovascular symptoms in South Korea. The anti-ischemic effects of compound K (CK), a metabolite of ginsenoside Rb1, on ischemia-induced isolated rat hearts were investigated through the analyses of the changes in the hemodynamics (blood pressure, aortic flow, coronary flow, and cardiac output) and the measurement of the infarct region. The subjects in this study were divided into four groups: the normal control, the CK-alone group, the ischemia-induced group without any treatment, and the ischemia-induced group treated with CK. No significant differences in perfusion pressure, aortic flow, coronary flow, and cardiac output were found between the groups before ischemia was induced. The oxygen and buffer supply was stopped for 30 min to induce ischemia 60 min after reperfusion in the isolated rat hearts, and the CK was administered 5 min before ischemia induction. The CK treatment significantly prevented decreases in perfusion pressure, aortic flow, coronary flow, and cardiac output under ischemic conditions. In addition, the hemodynamics (except for the heart rate) of the group treated with CK significantly recovered 60 min after reperfusion, unlike in the control group. CK significantly limited the infarct. These results suggest that CK treatment has distinct anti-ischemic effects in an exvivo model of an ischemia-reperfusion-induced rat heart.

Compound K Induces Apoptosis via CAMK-IV/AMPK Pathways in HT-29 Colon Cancer Cells

Although compound K (CK), an intestinal metabolite of ginseng protopanaxadiol saponins, has been known to induce apoptosis in various cancer cells, association of AMP-activated protein kinase (AMPK) with apoptosis in HT-29 colon cancer cells remains unclear. We hypothesized that CK may exert an anticancer activity through modulating the AMPK pathway in HT-29 cells. CK-induced apoptosis was associated with the disruption of the mitochondrial membrane potential, release of apoptogenic factors (cytochrome c and apoptosis-inducing factor) from mitochondria, and cleavage of caspase-9, caspase-3, caspase-8, Bid, and PARP proteins. This apoptotic effect of CK on colon cancer cells was found to be initiated by AMPK activation, and AMPK was activated through phosphorylation by Ca2+/calmodulin-activated protein kinase-IV (CAMK-IV). Treatment of HT-29 cells with compound C (AMPK inhibitor) or siRNA for AMPK completely abolished the CK-induced apoptosis. STO-609, CAMKs inhibitor, also attenuated CK-induced AMPK activation and apoptosis. In conclusion, the present study demonstrates that CK-mediated cell death of HT-29 colon cancer cells is regulated by CAMK-IV/AMPK pathways, and these findings provide a molecular basis for the anticancer effect of CK.

STRUCTURE‐FUNCTION ANALYSIS OF TRANSFROMED PHYTOCHEMICAL GLYCOSIDES

Glycosides are important food components which can confer beneficial functions for human health. Transformation, especially hydrolysis, of the sugar moieties tends to improve the bioavailability of the administered glycosides in vivo. Selected strains of Bifidobacterium possessed potent ginsenoside‐transforming activities. Final major products such as compound K, Rh2, and Rh1 were differentially obtained depending on the microorganisms. When compared between various ginsenosides, compound K (CK) showed anti‐tumor activities and Rh1 showed strong anti‐allergic effect. Bifidobacterium sp. Int‐57 efficiently hydrolyzed genistin to genistein and daidzin to daidzein, respectively, in Puerariae radix (PR) and soybean. Transformation of the platycodin saponins from Platycodon grandiflorum A. DC reduced the cytotoxicity on V79‐4 (Chinese hamster lung fibroblasts) and the hemolytic toxicity, while the sensory scores responsible for the pungency, bitterness and after‐tastes were improved. Additionally, transformation of gastrodin from Gastrodia elata Blume to 4‐hydroxybenzyl alcohol, furostanol diosgenin saponins from Trignoella foenum‐graecum to spirostanol forms, and baicalin and wogonoside from Scutellariae radix to baicalein and wogonin, respectively, were carried out by selected strains of Bifidobacterium and Lactobacillus. In conclusion, proper transformation may improve the functionality and the sensory value and reduce the cyto‐toxicity of the functional glycosides present in various functional food raw materials.

Compound K, Intestinal Metabolite of Ginsenoside, Attenuates Hepatic Lipid Accumulation via AMPK Activation in Human Hepatoma Cells

Compound K (CK) is a major intestinal metabolite of ginsenosides derived from ginseng radix. Although antidiabetic and antihyperlipidemic activities of CK have been investigated in recent years, action mechanism of CK remains poorly understood. Therefore, we examined whether CK affects the lipid metabolism in insulin-resistant HepG2 human hepatoma cells. In this study, a significant increase in AMP-activated protein kinase (AMPK) was observed when the cells were treated with CK. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase (ACC), a substrate of AMPK. CK attenuated gene expression of sterol regulatory element-binding protein 1c (SREBP1c) in time- and dose-dependent manners. Genes for fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1), well-known target molecules of SREBP1c, were also suppressed. In contrast, gene expressions of peroxisome proliferator-activated receptor alpha (PPAR-alpha) and CD36 were increased. These effects were reversed by treatment of compound C, an AMPK inhibitor. However, there were no differences in gene expressions of SREBP2, hydroxymethyl glutaryl CoA reductase (HMGR), and low-density-lipoprotein receptor (LDLR). Taken together, AMPK mediates CK induced suppression and activation of SREBP1c and PPAR-alpha, respectively, and these effects seem to be one of antidiabetic and/or antihyperlipidemic mechanisms of CK in insulin-resistant HepG2 human hepatoma cells.

Studies on absorption, distribution and metabolism of ginseng in humans after oral administration

Aim of the study This study was aimed to characterize the absorption, distribution and metabolism of ginseng in human subjects using pharmacokinetic experiments based on the metabolism by microflora obtained from the feces. Materials and methods We examined how intestinal microflora transforms ginsenoside-Rb1 (G-Rb1) and ginseng extracts into Compound k (C-K) using in vitro experiments with microbial flora obtained from the feces of 32 male subjects. Subsequently, Cmax, Tmax and the area under the blood concentration curve (AUC) for C-K in plasma were estimated by quantitative analysis using LC–MS/MS. The correlation between C-K transforming activity and the AUC of C-K in plasma and other properties were analyzed. Results C-K was absorbed into the blood 24 h after oral administration of ginseng, with average values for Tmax, Cmax, and AUC as follows: 10.76 ± 2.07 h, 27.89 ± 24.46 (ng/ml), and 221.98 ± 221.42 (μg h/ml), respectively. There was a correlation between the C-K transforming activity of G-Rb1 and the C-K transforming activity of ginseng extract by intestinal microflora (Spearman's correlation coefficient = 0.402, p < 0.05). Conclusion The absorption of the final metabolites ginseng is independent of the metabolite transforming activity of intestinal microflora, but the Tmax, Cmax and AUC of the transformed metabolites are dependent on the activity of each individual's microbial flora.

고려홍삼으로부터 분리한 compound K 함유분획에 의한 대식세포의 toll-like receptor-의존성 신호전달로 활성조절 분석

본 연구에서는 고려홍삼으로부터 새로 분리한 CK 함유분획을 이용하여 마우스 대식세포에 대한 선천면역반응 조절에 미치는 영향을 조사하였다. 본 연구에서 사용된 농도의 CK 함유분획에서는 세포독성 효과가 관찰되지 않았으며 CK함유 분획의 전처리에 의하여 그람음성세균의 LPS, 또는 CpG-ODN에 의해 유도되는 NF-<TEX>${\kappa}B$</TEX>와 MAPK 활성 및 전염증성 사이토카인, NO의 분비가 TLR4 및 TLR9 특이적으로 억제되었다. 이와 같은 결과는 CK 함유분획이 TLR4을 매개로하는 염증반응뿐만 아니라 TLR9을 통한 염증반응에도 영향을 미치는 것으로 해석된다. 따라서 앞으로 CK 함유 분획에 포함된 개별 사포닌 등 시료 성분에 대한 면밀한 분석, 그리고 이들 개별 물질이 각각의 신호전달 체계에 미치는 영향과 그 기작에 대한 연구가 더욱 필요할 것으로 사료되며 염증억제제로서의 개발 가능성을 탐구하기 위한 생체 내에서의 효능 및 작용기전 분석이 요구된다. Compound K (CK), a protopanaxadiol ginsenoside metabolite, was previously shown to have immunomodulatory effects. In this study, we isolated the CK rich fractions (CKRF) from Korean Red Ginseng and investigated the regulation of CKRF-mediated inflammatory signaling during Toll-like receptor (TLR)-mediated cellular activation. Among various TLR ligands, CKRF considerably abrogated TLR4- or TLR9-induced inflammatory signaling. Both LPS and CpG-containing oligodeoxynucleotides (CpG-ODN) stimulation rapidly activates mitogen-activated protein kinases [MAPKs; extracellular signal-regulated kinases 1/2 and p38], NF-<TEX>${\kappa}B$</TEX>, and expression of pro-inflammatory cytokines tumor necrosis factor-<TEX>${\alpha}$</TEX>, and interleukin-6 in murine bone marrow-derived macrophages (BMDMs) in a time- and dose-dependent manner. Of interest, pre-treatment of CKRF in either LPS/TLR4- or CpG-ODN/TLR9-stimulated macrophages substantially attenuated the LPS-induced inflammatory cytokine production and mRNA expressions, as well as MAPK and NF-<TEX>${\kappa}B$</TEX> activation. To our knowledge, this is the first description of the inhibitory roles for CKRF in TLR4- or TLR9-associated signaling in BMDMs. Collectively, these results demonstrate that CKRF specifically modulates distinct TLR4 and TLR9-mediated inflammatory responses, and further studies are urgently needed for their in vivo roles for potential therapeutic uses, such as in systemic inflammatory syndromes.

The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor, induces tolerance to endotoxin‐induced lethal shock

Compound K (C-K), a protopanaxadiol ginsenoside metabolite, was previously shown to have immunomodulatory effects. Here, we describe a novel therapeutic role for C-K in the treatment of lethal sepsis through the modulation of Toll-like receptor (TLR) 4-associated signalling via glucocorticoid receptor (GR) binding. In mononuclear phagocytes, C-K significantly repressed the activation of TLR4/lipopolysaccharide (LPS)-induced NF-κB and mitogen-activated protein kinases (MAPKs), as well as the secretion of pro-inflammatory cytokines. However C-K did not affect the TLR3-mediated expression of interferon-β or the nuclear translocation of IRF-3. C-K competed with the synthetic glucocorticoid dexamethasone for binding to GR and activated glucocorticoid responsive element (GRE)-containing reporter plasmids in a dose-dependent manner. In addition, the blockade of GR with either the GR antagonist RU486 or a siRNA against GR substantially reversed the anti-inflammatory effects of C-K. Furthermore, TLR4-dependent repression of inflammatory response genes by C-K was mediated through the disruption of p65/interferon regulatory factor complexes. Importantly, pre- or post-treatment with C-K significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock by lowering their systemic inflammatory cytokine levels and by reversing the lethal sequelae of sepsis. Collectively, these results demonstrate that C-K, as a functional ligand of GR, regulates distinct TLR4-mediated inflammatory responses, and suggest a novel therapy for Gram-negative septic shock.

Compound K Enhances Insulin Secretion with Beneficial Metabolic Effects in db/db Mice

Compound K (CK) is a final metabolite of panaxadiol ginsenosides. Although Panax ginseng is known to have antidiabetic activity, the active ingredient is not yet fully identified. In our preliminary studies, panaxadiol ginsenosides showed insulin secretion stimulating activity. Therefore, it would be interesting to know whether and how CK has antidiabetic activity. In in vitro studies using HIT-T15 cells and primary cultured islets, CK enhanced the insulin secretion in a concentration-dependent manner. This effect, however, was completely abolished in the presence of diazoxide (K+ channel opener) or nifedipine (Ca2+ channel blocker). Insulin secretion stimulating activity of a single oral CK administration was also confirmed with an oral glucose tolerance test (OGTT) using ICR mice. From these studies, we may conclude that CK lowered the plasma glucose level by stimulating insulin secretion and this action was presumably associated with an ATP-sensitive K+ channel. In a long-term study using C57BL/KsJ db/ db mice, CK treatment significantly decreased the fasting blood glucose levels in a dose-dependent fashion. OGTT revealed that CK improved glucose tolerance with increased insulin levels 30 min after the glucose challenge. Concurrently, CK treatment prevented the destruction of islets and preserved more insulin. Next, to gain insight into the extra-pancreatic molecular mechanism of CK, we performed a global gene expression profiling study in the liver and adipose tissues. According to DNA microarray analysis, CK shifted glucose metabolism from hepatic glucose production to hepatic glucose utilization in the liver and improved insulin sensitivity through enhancing plasma adiponectin levels, resulting in overexpression of genes for adipogenesis and glucose transporter in the adipose tissue. Taken together, we may suggest that CK could be developed as a therapeutic tool in type 2 diabetic patients with disability of insulin secretion and/or insulin resistance.

Transformation of Bioactive Compounds by Fusarium sacchari Fungus Isolated from the Soil-Cultivated Ginseng

Ginsenoside bioactive compounds, namely, compound K (C-K), compound Mx (C-Mx), and ginsenoside Mc (G-Mc), were the metabolites of ginsenosides Rb 1, Rb 2, Rb 3, and Rc by intestinal microflora of humans or rats, microorganisms, and enzymes, and C-K showed beneficial effects in vitro and in vivo as an antitumoral agent. The objective of this work was to explore an efficient procedure for biotransformation of these bioactive compounds. Thus, a filamentous fungus, Fusarium sacchari, was first obtained from the soil-cultivated ginseng, which was verified to possess a potent capacity of transformation of C-K, C-Mx, and G-Mc. The optimal biotransformation conditions of F. sacchari with C-K, C-Mx, and G-Mc were obtained as follows: transforming temperature, 30 degrees C; transforming time, 6 days; rotary speed, 160 rpm; pH of the medium, 5.5. HPLC analysis indicated that these three bioactive compounds were key metabolites and their structures were confirmed by (1)H and (13)C NMR analysis. Moreover, the in vitro antitumor activities of C-K, C-Mx, and G-Mc and the in vivo antitumor activities of the transformed product mainly containing these compounds were also evaluated. Among C-K, C-Mx, and G-Mc, C-K exhibited the most potent antitumor activities. The in vivo study showed that the transformed products by F. sacchari had much more antitumor activity than those of commonly used ginsenoside Rg3 and Paclitaxel.

Compound K의 인슐린분비 및 탄수화물 대사에 미치는 영향

진세노사이드의 인슐린 분비 활성을 비교해 본 결과 PPD 계열 진세노사이드가 인슐린의 분비를 촉진하는 경향을 보였으며, 그 중에서도 CK의 인슐린 분비 촉진 효과가 가장 뛰어났다. CK는 RIN-m5F cell line과 일차 배양한 췌장 소도 세포에서 용량 의존적으로 인슐린의 분비를 촉진하였고 이러한 CK의 인슐린 분비 촉진 기전은 ATP-sensitive <TEX>$K^+$</TEX> 채널의 봉쇄에 의한 것임을 확인하였다. H4IIE cell line에서 간 세포내 당신생과 관련된 효소의 발현을 측정한 결과 CK는 dexamethasone/cAMP에 의한 PEPCK 와 G6Pase의 발현을 억제하였다. 이로 미루어 볼 때, CK는 간에서 당의 신생을 억제하여 공복 시 혈당을 감소시킬 수 있음을 시사하였다. 또한 3T3-L1 cell line에서 TG의 함량과 <TEX>$PPAR-{\gamma}$</TEX> 유전자의 발현에 미치는 영향을 살펴본 결과 CK는 <TEX>$PPAR-{\gamma}$</TEX>의 발현을 억제하여 결과 지방세포의 분화를 억제하였다. 결론적으로 CK는 췌장에서 ATP-sensitive <TEX>$K^+$</TEX> channel을 봉쇄함으로 인슐린 분비를 촉진시키고 또한 간세포에서 당 신생을 억제함으로 식후 및 공복 시 혈당을 감소시킬 것으로 기대된다. Compound K (CK) is a final metabolite of panaxadiol ginsenosides. Although panax ginseng is known to have anti-diabetic activity, the active ingredient is not yet fully identified. Therefore, it would be interesting to know whether and how CK has an anti-diabetic activity. First, insulin secretion-stimulating activity of CK was examined using RIN-m5F cell line and primary cultured islets. CK enhanced the insulin secretion in a concentration dependent manner. This effect, however, was almost completely abolished in the presence of diazoxide, <TEX>$K^+$</TEX> channel opener, indicating that the insulin secretion-stimulating activity of CK is presumably due to blockade of ATP sensitive <TEX>$K^+$</TEX> channel. In addition, effects of CK on gene expressions of hepatic enzymes (phosphoenolpyruvate carboxykinase[PEPCK], glucose-6-phos-phatase[G6Pase]) and on adipocyte differentiation in H4IIE and 3T3-Ll cells, respectively, were examined. CK suppressed the induction of PEPCK and G6Pase mRNA expressions under the dexamethasone/cAMP stimulation condition. CK also reduced the <TEX>$PPAR-{\gamma}$</TEX> mRNA expression and triglyceride accumulation in a dose dependent manner as compared to the control. The present study suggests that CK deserves to examine whether it shows an anti-diabetic activity in animal and human studies.

Ginsenosides compound K and Rh2 inhibit tumor necrosis factor-α-induced activation of the NF-κB and JNK pathways in human astroglial cells

Ginsenosides, the main component of Panax ginseng, have been known for the anti-inflammatory and anti-proliferative activities. In this study, we investigated the molecular mechanisms responsible for the anti-inflammatory effects of ginsenosides on activated astroglial cells. Among 13 different ginsenosides, intestinal bacterial metabolites Rh(2) and compound K (C-K) showed a significant inhibitory effect on tumor necrosis factor-alpha (TNF-alpha)-induced expression of intercellular adhesion molecule-1 in human astroglial cells. Pretreatment with C-K or Rh(2) suppressed TNF-alpha-induced phosphorylation of IkappaBalpha kinase and the subsequent phosphorylation and degradation of IkappaBalpha. Additionally, the same treatment inhibited TNF-alpha-induced phosphorylation of MKK4 and the subsequent activation of the JNK-AP-1 pathway. The inhibitory effect of ginsenosides on TNF-alpha-induced activation of the NF-kappaB and JNK pathways was not observed in human monocytic U937 cells. These results collectively indicate that ginsenoside metabolites C-K and Rh(2) exert anti-inflammatory effects by the inhibition of both NF-kappaB and JNK pathways in a cell-specific manner.

Effect of Ginsenosides on Glucose Uptake in Human Caco-2 Cells Is Mediated through Altered Na+/Glucose Cotransporter 1 Expression

In this study, we measured the effect of ginsenosides on glucose uptake using the Caco-2 cell system. At submicromolar concentrations, these compounds exhibited marked effects on the rate of glucose transport across the differentiated Caco-2 cell monolayer. Compound K (CK), the main intestinal bacterial metabolite of the protopanaxadiol ginsenosides, significantly enhanced the steady-state glucose transport rate to about 50% of the control sample rate (from 1.54 +/- 0.09 to 2.25 +/- 0.15 nmol/min). Conversely, the protopanaxatriol ginsenoside Rg1 inhibited glucose transport to about 70% of the original rate (from 1.54 +/- 0.09 to 1.02 +/- 0.05 nmol/min). Consistent with the effect on glucose uptake rate, CK and Rg1 conferred a significant and paralleled alteration on both the protein and mRNA expression levels of the Na+/glucose cotransporter 1 (SGLT1) gene. Unlike SGLT1, there is no significant alteration on the protein or mRNA levels of GLUTs in CK- or Rg1-treated cells. Taken together, our results demonstrate that ginsenosides CK and Rg1 elicited potent enhancing and suppressing effects, respectively, on glucose uptake across human intestinal Caco-2 monolayer through modulation of SGLT1 expression.

Anti-diabetic Effects of Compound K versus Metformin versus Compound K-Metformin Combination Therapy in Diabetic db/db Mice

Compound K (CK) is a major intestinal metabolite of ginsenosides derived from ginseng radix. In our preliminary studies, CK has shown to exhibit anti-hyperglycemic effect through its insulin-secreting action, similar to that of insulin secretagogue sulfonylureas. Metformin, a biguanide, improves insulin resistance by reducing gluconeogenesis and enhancing peripheral glucose uptake, promoting reduction of the plasma glucose level. The aim of this study was to compare the anti-diabetic effects of CK and metformin due to differences in their mechanisms of action and also to investigate whether treatment of CK and metformin in combination show synergistic or additive effects compared to each drug alone. Seven week-old male db/db mice were treated for 8 weeks. CK was given at a dose of 10 mg/kg, metformin at 150 mg/kg and the same dosage of each drug was applied to CK plus metformin combination group. Significant improvements were observed in plasma glucose and insulin levels, homeostasis model assessment-insulin resistance (HOMA-IR) index and in hematoxylin and eosin-stained liver tissues in combination group. Although further studies to elucidate the benefits of co-administration of CK and metformin are needed, our findings may provide basis to the discovery of a new combination therapy on diabetes control in type 2 diabetics.

Purification and Characterization of New Special Ginsenosidase Hydrolyzing Multi-Glycisides of Protopanaxadiol Ginsenosides, Ginsenosidase Type I

In this paper, the new type ginsenosidase which hydrolyzing multi-glycosides of ginsenoside, named ginsenoside type I from Aspergillus sp.g48p strain was isolated, characterized and generally described. The enzyme molecular weight was about 80 kDa. Ginsenosidase type I can hydrolyze different glycoside of protopanaxadiol type ginsenosides (PPD); i.e., can hydrolyze the 3(carbon)-O-beta-glucoside of Rb1, Rb2, Rb3, Rc, Rd; can hydrolyze 20(carbon)-O-beta-glucoside of Rb1, 20-O-beta-xyloside of Rb3, 20-O-alpha-arabinoside(p) of Rb2 and 20-O-alpha-arabinoside(f) of Rc to produce mainly F2, compound-K (C-K) and small Rh2, but can not hydrolyze the glycosides of protopanaxatriol type ginsenoside (PPT) such as Re, Rf, Rg1. So, when the ginsenosidase type I hydrolyzed ginsenosides, the enzyme selected ginsenoside-aglycone type, can hydrolyze different glycosides of PPD type ginsenoside; however no selected glycoside type, can hydrolyze multi-glycosides of PPD type ginsenosides. These properties were novel properties, and differentiated with the other previously described glycosidases.

The Inhibitory Effects of Korean Red Ginseng Saponins on 5- HT3AReceptor Channel Activity Are Coupled to Anti-Nausea and Anti-Vomiting Action

We performed in vitro and in vivo studies to know whether the inhibitory effects of ginsenosides on <TEX>$5-HT_{3A}$</TEX> receptor channel acctivity are coupled to anti-nausea and anti-vomiting action. In vitro study, we investigated the effect of compound K (CK) and M4, which are ginsenoside metabolites, on human <TEX>$5-HT_{3A}$</TEX> receptor channel activity expressed in Xenopus oocytes using two-electrode voltage clamp technique. Treatment of CK or M4 themselves had no effect in both oocytes injected with <TEX>$H_2O\;and\;5-HT_{3A}$</TEX> receptor cRNA. In oocytes injected with <TEX>$5- HT_{3A}$</TEX> receptor cRNA, M4 treatment inhibited more potently 5-HT-induced inward peak current <TEX>$(I_{5-HT})$</TEX> than CK with dose-dependent and reversible manner. The half-inhibitory concentrations <TEX>$(IC_{50})$</TEX> of CK and M4 were <TEX>$36.9\;\pm\;10.1\;and\;7.3\;\pm\;2.2\;{\mu}M$</TEX>, respectively. The inhibition of <TEX>$I_{5-HT}$</TEX> by M4 was non-competitive and voltage-independent. These results indicate that M4 might regulate <TEX>$5-HT_{3A}$</TEX> receptors. In vivo experiments, injection of cisplatin (7.5 mg/kg, i.v.) induced both nausea and vomiting with 1 h latency. These episodes reached to peak after 2 h and persisted for 4 h. Pre-treatment of GTS (500 mg/kg, p.o.) significantly reduced cisplatin-induced nausea and vomiting by <TEX>$51\;\pm\;8.4\;and\;48.8\;\pm\;6.4\%$</TEX> during 4 h compared to GIS­untreated group, respectively. These results show the possibility that in vitro inhibition of <TEX>$5-HT_{3A}$</TEX> receptor channel activity by ginsenosides might be coupled to in vivo anti-emetic activity.

Differential effect of bovine serum albumin on ginsenoside metabolite-induced inhibition of alpha3beta4 nicotinic acetylcholine receptor expressed in Xenopus oocytes.

Ginsenosides, major active ingredients of Panax ginseng, that exhibit various pharmacological and physiological actions are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. Recent reports shows that ginsenosides might play a role as pro-drugs for these metabolites. In present study, we investigated the effect of bovine serum albumin (BSA), which is one of major binding proteins on various neurotransmitters, hormones, and other pharmacological agents, on ginsenoside Rg2-, CK-, or M4-induced regulation of alpha3beta4 nicotinic acetylcholine (ACh) receptor channel activity expressed in Xenopus oocytes. In the absence of BSA, treatment of ACh elicited inward peak current (I(ACh)) in oocytes expressing alpha3beta4 nicotinic ACh receptor. Co-treatment of ginsenoside Rg2, CK, or M4 with ACh inhibited I(ACh) in oocytes expressing (alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. In the presence of 1% BSA, treatment of ACh still elicited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and co-treatment of ginsenoside Rg2 or M4 but not CK with ACh inhibited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. These results show that BSA interferes the action of CK rather than M4 on the inhibitory effect of I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and further suggest that BSA exhibits a differential interaction on ginsenoside metabolites.