In Vitro Induction of Differentiation by Ginsenosides in F9 Teratocarcinoma Cells

Lee, Y.-N.,Lee, H.-Y.,Chung, H.-Y.,Kim, S.-I.,Lee, S.-K.,Park, B.-C.,Kim, K.-W. 부산대학교 유전공학연구소 1996 분자생물학 연구보 Vol.12 No.-

The aim of this study was to determine the ability of the ginsenosides, extracts of Panax ginseng C.A. Meyer, to cause differentiation of F9 teratocarcinoma stem cells as a model system. F9 stem cells cultured in the presence of the ginsenosides together with dibutyryl cyclic AMP (dbcAMP) became parietal endoderm-like cells. Moreover, the expression of differentiation marker genes, such as laminin B1 and type IV collagen, was increased after treatment with the ginsenosides. Among the various purified ginsenosides, Rh_1, and Rh_2, were the most effective at causing differentiation of F9 cells. Since ginsenosides and glucocorticoid hormone have similar chemical structures, we examined the possibility of the involvement of a glucocoticoid receptor (GR) in the differentiation process induced by the ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarded as a GR was detected. In F9 cells cultured in the medium containing the ginsenosides Rh_1, or Rh_2. In addition, F9 stem cells treated with the ginsenosides Rh_1, or Rh_2 and with RU486, a glucocorticoid antagonist with a high affinity for the GR, did not differentiate into endoderm cells morphologically, and the expression of laminin B1 gene was not induced in these cells. In a gel mobility shift assay, protein factors capable of binding to the glucocorticoid responsive element (GRE) specifically were detected in nuclear extracts of the ginsenoside-treated F9 cells. Moreover, overexpression of GR by cotransfection of GR expression vector and GRE-luciferase vector enhanced the transactivation activity of GRE promoter in the presence of ginsenosides Rh_1 or Rh_2 and was further augmented by dbcAMP. In addition, ginsenosides Rh_1 and Rh_2 bound to a GR assessed by whole-cell binding assay, even though the specific binding affinity was weaker compared to dexamethasone. Based on these data, we suggest that the ginsenosides Rh_1 and Rh_2 cause the differentiation of F9 cells and the effects of ginsenosides might be exerted bia binding with a GR or its analogous nuclear receptor. Copyright ⓒ 1966 Elsevier Science Ltd.

An optimized microwave-assisted extraction method for increasing yields of rare ginsenosides from Panax quinquefolius L.

Yao, Hua,Li, Xuwen,Liu, Ying,Wu, Qian,Jin, Yongri The Korean Society of Ginseng 2016 Journal of Ginseng Research Vol.40 No.4

Background: Rare ginsenosides in Panax quinquefolius L. have strong bioactivities. The fact that it is hard to obtain large amounts of rare ginsenosides seriously restricts further research on these compounds. An easy, fast, and efficient method to obtain different kinds of rare ginsenosides simultaneously and to quantify each one precisely is urgently needed. Methods: Microwave-assisted extraction (MAE) was used to extract nine kinds of rare ginsenosides from P. quinquefolius L. In this article, rare ginsenosides [20(S)-Rh1, 20(R)-Rh1, Rg6, F4, Rk3, 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5] were identified by high performance liquid chromatography (HPLC)-electrospray ionization-mass spectrometry. The quantity information of rare ginsenosides was analyzed by HPLC-UV at 203 nm. Results: The optimal conditions for MAE were using water as solvent with the material ratio of 1:40 (w/v) at a temperature of $145^{\circ}C$, and extracting for 15 min under microwave power of 1,600 W. Seven kinds of rare ginsenosides [20(S)-Rh1, 20(R)-Rh1, Rg6, F4, Rk3, Rk1, and Rg5] had high extraction yields, but those of 20(S)-Rg3 and 20(R)-Rg3 were lower. Compared with the conventional method, the extraction yields of the nine rare ginsenosides were significantly increased. Conclusion: The results indicate that rare ginsenosides can be extracted effectively by MAE from P. quinquefolius L. in a short time. Microwave radiation plays an important role in MAE. The probable generation process of rare ginsenosides is also discussed in the article. It will be meaningful for further investigation or application of rare ginsenosides.

Efficient Production of Various Minor Ginsenosides from PPD- and PPT-type Major Ginsenosides Using a Single Recombinant BglFc Isolated from Flavobacterium chilense

주베르,Sofiane Medjebouri,Qinzhen Liu,박혜윤,김가련,임완택 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.2

Background: Rare ginsenosides (F2, Rg3, Gyp- XVII, and C-K) are pharmaceutically active components of Panax ginseng, which are derived from the conversion of major ginsenosides through various transformation methods. To date, most studies have failed to identify a competent bacterial strain and recombinant enzyme for converting protopanaxadiol (PPD)- and protopanaxatriol (PPT)-type ginsenosides to target minor ginsenosides. Method: Our study identified and employed nine sets of clones from different glycoside hydrolase bacterial strains for major ginsenoside bioconversion. Among these nine clones, only BglFc was selected based on its strong biotransformation ability and capacity to generate complete minor ginsenosides. bglFc was cloned and expressed in Escherichia coli using the pGEX-4T-1 vector system, and the recombinant enzyme was used for efficiently producing minor ginsenosides. Results: Recombinant BglFc is 2,394 bp and 798 amino acid residues long, with a predicted molecular mass of 78.8 kDa. BglFc belongs to the glycoside hydrolase family 3, and demonstrates a promising ability to convert major ginsenosides into minor ones. The Km and Vmax values of pNPG were 0.81 ± 0.06 and 4.0 ± 0.2 mM·min-1·mg-1 of protein, respectively. Under optimal conditions (37°C, pH 7.0), the ginsenoside transformation pathways for BglFc were as follows: Rb1→Rd→Rg3(S)→Rh2(S); GypXVII →GypLXXV→C-K; GypLXXV→C-K; F2→C-K; Rb2 →C-O→C-Y; Rb3→C-Mx1→C-Mx; Rc→C-Mc1→CMc; Re→Rg2(S); and Rg1→Rh1(S), respectively. Conclusion: These results suggest that recombinant BglFc demonstrates a strong transformation activity for both PPD- and PPTtype major ginsenosides. Therefore, we conclude that BglFc can be used for gram unit production of various minor ginsenosides. Significance and Impact of Study: Previously, researchers have used a combination of enzymes for the production of minor ginsenosides. However, in this study, we found a favorable enzyme that can be used alone for the production of a different type of minor ginsenoside using the proposed conversion pathway.

Microbial conversion of major ginsenosides in ginseng total saponins by Platycodon grandiflorum endophytes

Cui, Lei,Wu, Song-quan,Zhao, Cheng-ai,Yin, Cheng-ri The Korean Society of Ginseng 2016 Journal of Ginseng Research Vol.40 No.4

Background: In this study, we screened and identified an endophyte JG09 having strong biocatalytic activity for ginsenosides from Platycodon grandiflorum, converted ginseng total saponins and ginsenoside monomers, determined the source of minor ginsenosides and the transformation pathways, and calculated the maximum production of minor ginsenosides for the conversion of ginsenoside Rb1 to assess the transformation activity of endophyte JG09. Methods: The transformation of ginseng total saponins and ginsenoside monomers Rb1, Rb2, Rc, Rd, Rg1 into minor ginsenosides F2, C-K and Rh1 using endophyte JG09 isolated by an organizational separation method and Esculin-R2A agar assay, as well as the identification of transformed products via TLC and HPLC, were evaluated. Endophyte JG09 was identified through DNA sequencing and phylogenetic analysis. Results: A total of 32 ${\beta}$-glucosidase-producing endophytes were screened out among the isolated 69 endophytes from P. grandiflorum. An endophyte bacteria JG09 identified as Luteibacter sp. effectively converted protopanaxadiol-type ginsenosides Rb1, Rb2, Rc, Rd into minor ginsenosides F2 and C-K, and converted protopanaxatriol-type ginsenoside Rg1 into minor ginsenoside Rh1. The transformation pathways of major ginsenosides by endophyte JG09 were as follows: $Rb1{\rightarrow}Rd{\rightarrow}F2{\rightarrow}C-K$; $Rb2{\rightarrow}C-O{\rightarrow}C-Y{\rightarrow}C-K$; $Rc{\rightarrow}C-Mc1{\rightarrow}C-Mc{\rightarrow}C-K$; $Rg1{\rightarrow}Rh1$. The maximum production rate of ginsenosides F2 and C-K reached 94.53% and 66.34%, respectively. Conclusion: This is the first report about conversion of major ginsenosides into minor ginsenosides by fermentation with P. grandiflorum endophytes. The results of the study indicate endophyte JG09 would be a potential microbial source for obtaining minor ginsenosides.

Mass Spectrometry Based Profiling and Imaging of Various Ginsenosides from <i>Panax ginseng</i> Roots at Different Ages

Lee, Jae Won,Ji, Seung-Heon,Lee, Young-Seob,Choi, Doo Jin,Choi, Bo-Ram,Kim, Geum-Soog,Baek, Nam-In,Lee, Dae Young MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.6

<P>(1) Background: <I>Panax ginseng</I> root is one of the most important herbal products, and the profiling of ginsenosides is critical for the quality control of ginseng roots at different ages in the herbal markets. Furthermore, interest in assessing the contents as well as the localization of biological compounds has been growing. The objective of this study is to carry out the mass spectrometry (MS)-based profiling and imaging of ginsenosides to assess ginseng roots at different ages; (2) Methods: Optimal ultra performance liquid chromatography coupled to quadrupole time of flight/MS (UPLC-QTOF/MS) was used to profile various ginsenosides from <I>P. ginseng</I> roots. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF)/MS-based imaging was also optimized to visualize ginsenosides in ginseng roots; (3) Results: UPLC-QTOF/MS was used to profile 30 ginsenosides with high mass accuracy, with an in-house library constructed for the fast and exact identification of ginsenosides. Using this method, the levels of 14 ginsenosides were assessed in <I>P. ginseng</I> roots cultivated for 4, 5, and 6 years. The optimal MALDI-imaging MS (IMS) was also applied to visualize the 14 ginsenosides in ginseng roots. As a result, the MSI cross sections showed the localization of 4 ginsenoside ions ([M + K]<SUP>+</SUP>) in <I>P. ginseng</I> roots at different ages; (4) Conclusions: The contents and localization of various ginsenosides differ depending on the cultivation years of <I>P. ginseng</I> roots. Furthermore, this study demonstrated the utility of MS-based profiling and imaging of ginsenosides for the quality control of ginseng roots.</P>

Stem-leaves of Panax as a rich and sustainable source of less-polar ginsenosides: comparison of ginsenosides from Panax ginseng, American ginseng and Panax notoginseng prepared by heating and acid treatment

Fengxiang Zhang,Shaojian Tang,Lei Zhao,Xiushi Yang,Yang Yao,Zhaohua Hou,Peng Xue 고려인삼학회 2021 Journal of Ginseng Research Vol.45 No.1

Background: Ginsenosides, which have strong biological activities, can be divided into polar or less-polar ginsenosides. Methods: This study evaluated the phytochemical diversity of the saponins in Panax ginseng (PG) root, American ginseng (AG) root, and Panax notoginseng (NG) root; the stem-leaves from Panax ginseng (SPG) root, American ginseng (SAG) root, and Panax notoginseng (SNG) root as well as the saponins obtained following heating and acidification [transformed Panax ginseng (TPG), transformed American ginseng (TAG), transformed Panax notoginseng (TNG), transformed stem-leaves from Panax ginseng (TSPG), transformed stem-leaves from American ginseng (TSAG), and transformed stem-leaves from Panax notoginseng (TSNG)]. The diversity was determined through the simultaneous quantification of the 16 major ginsenosides. Results: The content of ginsenosides in NG was found to be higher than those in AG and PG, and the content in SPG was greater than those in SNG and SAG. After transformation, the contents of polar ginsenosides in the raw saponins decreased, and contents of less-polar compounds increased. TNG had the highest levels of ginsenosides, which is consistent with the transformation of ginseng root. The contents of saponins in the stem-leaves were higher than those in the roots. The transformation rate of SNG was higher than those of the other samples, and the loss ratios of total ginsenosides from NG (6%) and SNG (4%) were the lowest among the tested materials. In addition to the conversion temperature, time, and pH, the crude protein content also affects the conversion to rare saponins. The proteins in Panax notoginseng allowed the highest conversion rate. Conclusion: Thus, the industrial preparation of less-polar ginsenosides from SNG is more efficient and cheaper.

Effects of gut microbiota on the pharmacokinetics of protopanaxadiol ginsenosides Rd, Rg3, F2, and compound K in healthy volunteers treated orally with red ginseng

Kim, Jeon-Kyung,Choi, Min Sun,Jeung, Woonhee,Ra, Jehyeon,Yoo, Hye Hyun,Kim, Dong-Hyun The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.4

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<SUB>∞</SUB>) 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.

Additive interaction of intrathecal ginsenosides and neostigmine in the rat formalin test

박천희,Park-Ne Kim,Seong Heon Lee,윤명하 대한마취통증의학회 2013 Korean Journal of Anesthesiology Vol.64 No.2

Background: The authors evaluated the effect of intrathecal mixture of ginsenosides with neostigmine on formalin-induced nociception and made further clear the role of the spinal muscarinic (M) receptors on the activity of ginsenosides. Methods: A catheter was located in the intrathecal space of male Sprague-Dawley rats. Pain was evoked by injection of formalin solution (5%, 50 μl) to the hindpaw. Isobolographic analysis was done to characterize drug interaction between ginsenosides and neostigmine. The antagonism of ginsenosides-mediated antinociception was determined with M1 receptor antagonist (pirenzepine), M2 receptor antagonist (methoctramine), M3 receptor antagonist (4-DAMP), M4 receptor antagonist (tropicamide). The expression of muscarinic receptor subtypes was examined with RT-PCR. Results: Intrathecal ginsenosides and neostigmine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Isobolographic analysis revealed an additive interaction between ginsenosides and neostigmine in both phases. Intrathecal pirenzepine, methoctramine, 4-DAMP, and tropicamide reversed the antinociception of ginsenosides in both phases. M1-M4 receptors mRNA detected in spinal cord of naïve rats and the injection of formalin decreased the expression of M1 receptor mRNA, but it had no effect on the expression of other three muscarinic receptors mRNA. Intrathecal ginsenosides little affected the expression of all of muscarinic receptors mRNA in formalin-injected rats. Conclusions: Intrathecal ginsenosides additively interacted with neostigmine in the formalin test. Furthermore, M1-M4 receptors exist in the spinal cord, all of which contribute to the antinocieption of intrathecal ginsenosides.

생쥐의 골 암성통증 모형에서 ginsenosides의 효과

윤명하,이형곤,김웅모,주진,김여옥,최금화 대한마취통증의학회 2010 Anesthesia and pain medicine Vol.5 No.4

Background:Ginsenosides have been used for a long time as an oriental folk medicine. Although ginsenosides modulate the nociceptive transmission, the effect of ginsenosides on a bone cancer pain has not been elucidated. The authors examined the effect of ginsenosides in a mouse model of bone cancer pain. Methods:Bone cancer was induced by intramedullary injection of osteolytic sarcoma cells in to the femur in male C3H/HeJ mice. Mice showing mechanical allodynia after 14 days after cancer cells inoculation were included in this study. Mechanical allodynia was evaluated by measuring the withdrawal threshold to von Frey filament applying on the femoral cancer site. Effect of ginsenosides (30, 100, 300 mg/kg) was examined at 15, 30, 60, 90, 120 min after intraperitoneal administration of ginsenosides. Results:After cancer cells injection into the femur, bone cancer was developed in simple X-ray. A paw withdrawal threshold in a cancer site was significantly decreased. Intraperitoneal ginsenosides did not effectively alter the withdrawal threshold in the cancer site. Conclusions:Taken together, ginsenosides may not be effective to attenuate the bone cancer pain.

Memory Enhancing and Neuroprotective Effects of Selected Ginsenosides

Hai Ying Bao,Jing Zhang,여수정,Chang-Seon Myung,Hyang Mi Kim,Jong Moon Kim,박정일,조정숙,Jong Seong Kang 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.3

The effects of ginsenosides Rg3(R), Rg3(S) and Rg5/Rk1 (a mixture of Rg5 and Rk1, 1:1, w/w), which are components isolated from processed Panax ginseng C.A. Meyer (Araliaceae), on memory dysfunction were examined in mice using a passive avoidance test. The ginsenosides Rg3(R), Rg3(S) or Rg5/Rk1, when orally administered for 4 days, significantly ameliorated the memory impairment induced by the single oral administration of ethanol. The memory impairment induced by the intraperitoneal injection of scopolamine was also significantly recovered by ginsenosides Rg3(S) and Rg5/Rk1. Among the three ginsenosides tested in this study, Rg5/Rk1 enhanced the memory function of mice most effectively in both the ethanoland scopolamine-induced amnesia models. Moreover, the latency period of the Rg5/Rk1-treated mice was 1.2 times longer than that of the control (no amnesia) group in both models, implying that Rg5/Rk1 may also exert beneficial effects in the normal brain. We also evaluated the effects of these ginsenosides on the excitotoxic and oxidative stress-induced neuronal cell damage in primary cultured rat cortical cells. The excitotoxicity induced by glutamate or Nmethyl- D-aspartate (NMDA) was dramatically inhibited by the three ginsenosides. Rg3(S) and Rg5/Rk1 exhibited a more potent inhibition of excitotoxicity than did Rg3(R). In contrast, these ginsenosides were all ineffective against the H2O2- or xanthine/xanthine oxidase-induced oxidative neuronal damage. Taken together, these results indicate that ginsenosides Rg3(S) and Rg5/Rk1 significantly reversed the memory dysfunction induced by ethanol or scopolamine, and their neuroprotective actions against excitotoxicity may be attributed to their memory enhancing effects.