• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.410-418
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• 2019

To investigate a novel ${\beta}$-glucosidase from Bifidobacterium breve ATCC 15700 (BbBgl) to produce compound K (CK) via ginsenoside $F_2$ by highly selective and efficient hydrolysis of the C-3 glycoside from ginsenoside Rd, the BbBgl gene was cloned and expressed in E. coli BL21. The recombinant BbBgl was purified by Ni-NTA magnetic beads to obtain an enzyme with specific activity of 37 U/mg protein using pNP-Glc as substrate. The enzyme activity was optimized at pH 5.0, $35^{\circ}C$, 2 or 6 U/ml, and its activity was enhanced by $Mn^{2+}$ significantly. Under the optimal conditions, the half-life of the BbBgl is 180 h, much longer than the characterized ${\beta}$-glycosidases, and the $K_m$ and $V_{max}$ values are 2.7 mM and $39.8{\mu}mol/mg/min$ for ginsenoside Rd. Moreover, the enzyme exhibits strong tolerance against high substrate concentration (up to 40 g/l ginsenoside Rd) with a molar biotransformation rate of 96% within 12 h. The good enzymatic properties and gram-scale conversion capacity of BbBgl provide an attractive method for large-scale production of rare ginsenoside CK using a single enzyme or a combination of enzymes.

• Journal of Ginseng Research
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• 제45권2호
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• pp.334-343
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• 2021

Background: Gut microbiota mainly function in the biotransformation of primary ginsenosides into bioactive metabolites. Herein, we investigated the effects of three prebiotic fibers by targeting gut microbiota on the metabolism of ginsenoside Rb1 in vivo. Methods: Sprague Dawley rats were administered with ginsenoside Rb1 after a two-week prebiotic intervention of fructooligosaccharide, galactooligosaccharide, and fibersol-2, respectively. Pharmacokinetic analysis of ginsenoside Rb1 and its metabolites was performed, whilst the microbial composition and metabolic function of gut microbiota were examined by 16S rRNA gene amplicon and metagenomic shotgun sequencing. Results: The results showed that peak plasma concentration and area under concentration time curve of ginsenoside Rb1 and its intermediate metabolites, ginsenoside Rd, F2, and compound K (CK), in the prebiotic intervention groups were increased at various degrees compared with those in the control group. Gut microbiota dramatically responded to the prebiotic treatment at both taxonomical and functional levels. The abundance of Prevotella, which possesses potential function to hydrolyze ginsenoside Rb1 into CK, was significantly elevated in the three prebiotic groups (P < 0.05). The gut metagenomic analysis also revealed the functional gene enrichment for terpenoid/polyketide metabolism, glycolysis, gluconeogenesis, propanoate metabolism, etc. Conclusion: These findings imply that prebiotics may selectively promote the proliferation of certain bacterial stains with glycoside hydrolysis capacity, thereby, subsequently improving the biotransformation and bioavailability of primary ginsenosides in vivo.

• Journal of Ginseng Research
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• 제47권3호
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• pp.408-419
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• 2023

Background: Ginsenoside compound K (CK), the main active metabolite in Panax ginseng, has shown good safety and bioavailability in clinical trials and exerts neuroprotective effects in cerebral ischemic stroke. However, its potential role in the prevention of cerebral ischemia/reperfusion (I/R) injury remains unclear. Our study aimed to investigate the molecular mechanism of ginsenoside CK against cerebral I/R injury. Methods: We used a combination of in vitro and in vivo models, including oxygen and glucose deprivation/reperfusion induced PC12 cell model and middle cerebral artery occlusion/reperfusion induced rat model, to mimic I/R injury. Intracellular oxygen consumption and extracellular acidification rate were analyzed by Seahorse multifunctional energy metabolism system; ATP production was detected by luciferase method. The number and size of mitochondria were analyzed by transmission electron microscopy and MitoTracker probe combined with confocal laser microscopy. The potential mechanisms of ginsenoside CK on mitochondrial dynamics and bioenergy were evaluated by RNA interference, pharmacological antagonism combined with co-immunoprecipitation analysis and phenotypic analysis. Results: Ginsenoside CK pretreatment could attenuate mitochondrial translocation of DRP1, mitophagy, mitochondrial apoptosis, and neuronal bioenergy imbalance against cerebral I/R injury in both in vitro and in vivo models. Our data also confirmed that ginsenoside CK administration could reduce the binding affinity of Mul1 and Mfn2 to inhibit the ubiquitination and degradation of Mfn2, thereby elevating the protein level of Mfn2 in cerebral I/R injury. Conclusion: These data provide evidence that ginsenoside CK may be a promising therapeutic agent against cerebral I/R injury via Mul1/Mfn2 mediated mitochondrial dynamics and bioenergy.

• Journal of Ginseng Research
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• 제40권4호
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• pp.351-358
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• 2016

Background: This study was designed to investigate whether ginsenoside Rb1 (Rb1) and compound K (CK) ameliorated insulin resistance by suppressing endoplasmic reticulum (ER) stress-induced inflammation in adipose tissue. Methods: To induce ER stress, epididymal adipose tissue from mice or differentiated 3T3 adipocytes were exposed to high glucose. The effects of Rb1 and CK on reactive oxygen species production, ER stress, TXNIP/NLRP3 inflammasome activation, inflammation, insulin signaling activation, and glucose uptake were detected by western blot, emzyme-linked immunosorbent assay, or fluorometry. Results: Rb1 and CK suppressed ER stress by dephosphorylation of $IRE1{\alpha}$ and PERK, thereby reducing TXNIP-associated NLRP3 inflammasome activation in adipose tissue. As a result, Rb1 and CK inhibited IL-$1{\beta}$ maturation and downstream inflammatory factor IL-6 secretion. Inflammatory molecules induced insulin resistance by upregulating phosphorylation of insulin receptor substrate-1 at serine residues and impairing insulin PI3K/Akt signaling, leading to decreased glucose uptake by adipocytes. Rb1 and CK reversed these changes by inhibiting ER stress-induced inflammation and ameliorating insulin resistance, thereby improving the insulin IRS-1/PI3K/Akt-signaling pathway in adipose tissue. Conclusion: Rb1 and CK inhibited inflammation and improved insulin signaling in adipose tissue by suppressing ER stress-associated NLRP3 inflammation activation. These findings offered novel insight into the mechanism by which Rb1 and CK ameliorate insulin resistance in adipose tissue.

• Journal of Ginseng Research
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• 제35권2호
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• pp.191-199
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• 2011

The human ether-a-go-go-related gene (HERG) cardiac $K^+$ channels are one of the representative pharmacological targets for development of drugs against cardiovascular diseases such as arrhythmia. Panax ginseng has been known to exhibit cardioprotective effects. In a previous report we demonstrated that ginsenoside $Rg_3$ regulates HERG $K^+$ channels by decelerating deactivation. However, little is known about how ginsenoside metabolites regulate HERG $K^+$ channel activity. In the present study, we examined the effects of ginsenoside metabolites such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) on HERG $K^+$ channel activity by expressing human a subunits in Xenopus oocytes. CK induced a large persistent deactivatingtail current ($I_{deactivating-tail}$) and significantly decelerated deactivating current decay in a concentration-dependent manner. The $EC_{50}$ for persistent $I_{deactivating-tail}$ was $16.6{\pm}1.3$ ${\mu}M$. In contrast to CK, PPT accelerated deactivating-tail current deactivation. PPD itself had no effects on deactivating-tail currents, whereas PPD inhibited ginsenoside $Rg_3$-induced persistent $I_{deactivating-tail}$ and accelerated HERG $K^+$ channel deactivation in a concentration-dependent manner. These results indicate that ginsenoside metabolites exhibit differential regulation on Ideactivating-tail of HERG $K^+$ channel.

• Journal of Ginseng Research
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• 제33권4호
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• pp.268-277
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• 2009

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.

• Journal of Ginseng Research
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• 제44권4호
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• pp.611-618
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• 2020

Background: It is well recognized that gut microbiota is involved in the biotransformation of ginsenosides by converting the polar ginsenosides to nonpolar bioactive ginsenosides. However, the roles of the gut microbiota on the pharmacokinetics of ginsenosides in humans have not yet been fully elucidated. Methods: Red ginseng (RG) or fermented red ginseng was orally administered to 34 healthy Korean volunteers, and the serum concentrations of the ginsenosides were determined using liquid chromatography-tandem mass spectrometry. In addition, the fecal ginsenoside Rd- and compound K (CK)eforming activities were measured. Then, the correlations between the pharmacokinetic profiles of the ginsenosides and the fecal ginsenoside-metabolizing activities were investigated. Results: For the RG group, the area under the serum concentratione-time curve values of ginsenosides Rd, F2, Rg3, and CK were 8.20 ± 11.95 ng·h/mL, 4.54 ± 3.70 ng·h/mL, 36.40 ± 19.68 ng·h/mL, and 40.30 ± 29.83 ng·h/mL, respectively. For the fermented red ginseng group, the the area under curve from zero to infinity (AUC_∞) values of ginsenosides Rd, F2, Rg3, and CK were 187.90 ± 95.87 ng·h/mL, 30.24 ± 41.87 ng·h/mL, 28.68 ± 14.27 ng·h/mL, and 137.01 ± 96.16 ng·h/mL, respectively. The fecal CK-forming activities of the healthy volunteers were generally proportional to their ginsenoside Rd-eforming activities. The area under the serum concentration-time curve value of CK exhibited an obvious positive correlation (r = 0.566, p < 0.01) with the fecal CK-forming activity. Conclusion: The gut microbiota may play an important role in the bioavailability of the nonpolar RG ginsenosides by affecting the biotransformation of the ginsenosides.

• The Korean Journal of Physiology and Pharmacology
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• 제16권2호
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• pp.113-118
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• 2012

Ginsenosides are low molecular weight glycosides found in ginseng that exhibit neuroprotective effects through inhibition of $N$-methyl-D-aspartic acid (NMDA) receptor channel activity. Ginsenosides, like other natural compounds, are metabolized by gastric juices and intestinal microorganisms to produce ginsenoside metabolites. However, little is known about how ginsenoside metabolites regulate NMDA receptor channel activity. In the present study, we investigated the effects of ginsenoside metabolites, such as compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT), on oocytes that heterologously express the rat NMDA receptor. NMDA receptor-mediated ion current ($I_{NMDA}$) was measured using the 2-electrode voltage clamp technique. In oocytes injected with cRNAs encoding NMDA receptor subunits, PPT, but not CK or PPD, reversibly inhibited $I_{NMDA}$ in a concentration-dependent manner. The $IC_{50}$ for PPT on $I_{NMDA}$ was $48.1{\pm}4.6\;{\mu}M$, was non-competitive with NMDA, and was independent of the membrane holding potential. These results demonstrate the possibility that PPT interacts with the NMDA receptor, although not at the NMDA binding site, and that the inhibitory effects of PPT on $I_{NMDA}$ could be related to ginseng-mediated neuroprotection.

• Journal of Ginseng Research
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• 제44권3호
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• pp.461-474
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• 2020

Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown. Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues. Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.

• 한국식품위생안전성학회지
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• 제36권5호
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• pp.418-429
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• 2021

본 연구에서는 백삼 및 홍삼 추출물과 그 효소 가수 분해물을 제조하고 이를 첨가하여 제조된 양갱의 항산화 활성 및 품질 특성을 조사하였다. 백삼과 홍삼 추출물의 효소 가수분해를 위해서는 Rapidase C80 max, Pyr-flo, Ultimase MFC를 선정하였다. Rapidase C80 max, Pyr-flo, Ultimase MFC로 가수분해 한 백삼과 홍삼에서는 효소반응 전에는 검출되지 않았던 ginsenoside F2와 Compound K (CK)가 검출되었으며 특히 홍삼의 2차 효소 반응군에서 CK의 함량이 가장 높았다. 효소 가수분해 전 또는 후의 백삼 및 홍삼을 함유한 양갱을 제조한 후 폴리페놀 함량, 항산화 능력을 분석한 결과 백삼 및 홍삼을 함유하지 않은 양갱(대조군)에 비해 총 폴리페놀 함량, DPPH 라디칼 소거능, ABTS 라디칼 소거능, FRAP 분석 결과 모두 우수하였으며, 첨가한 백삼 및 홍삼에 효소반응을 진행할수록 유의적으로 항산화 활성이 증가하였다(P<0.05). 밝기(L^*)는 무첨가 양갱(대조군)이, 적색도(a^*)는 홍삼양갱(RG)이, 황색도(b^*)는 백삼양갱(WG)이 높게 나타났고, 조직감은 홍삼 첨가 후 2차 가수분해까지 진행한 양갱(RG-T2)이 강도(hardness), 탄력성(springiness), 씹힘성(chewiness), 응집성(cohesiveness), 검성(gumminess) 모두 유의하게 높은 결과를 나타내었다(P<0.05). 결론적으로, 백삼 또는 홍삼 추출물에 Rapidase C80 max, Pyr-flo, Ultimase MFC를 처리하면 진세노사이드 탈당화에 매우 유용하여 생리활성이 우수한 CK를 생산할 수 있으며, 효소로 가수분해 된 백삼 및 홍삼을 첨가한 양갱은 대조군(인삼 무첨가 양갱)에 비해 총 폴리페놀과 항산화 활성을 유의하게 증가시킨다는 것을 알 수 있었다. 이러한 결과는 향후 기능성 양갱 제조에 우수한 기초자료로 활용될 수 있을 것으로 보인다.