Protopanaxatriol Ginsenosides Research Articles

Effects of Different Culture Times Genes Expression on Ginsenoside Biosynthesis of the Ginseng Adventitious Roots in Panax ginseng

Panax ginseng is an ancient and very potent herb, which has a long history of medicinal use, and recent studies have shown that ginsenosides are the main active substances in its pharmacological effects. However, the saponin content of wild ginseng and cultivated ginseng can hardly meet the market supply, and the ginseng adventitious root suspension culture technology can produce ginsenosides in a targeted manner. The length of culture time is an important factor affecting the growth and development of plants and the accumulation of secondary metabolites. After transcriptome sequencing of ginseng adventitious root material at different culture times, the results showed that a total of 5784 differentially expressed genes were screened, which contained 239 transcription factors. KEGG analysis showed that these differentially expressed genes were mainly enriched in metabolic pathways and biosynthesis of secondary metabolites. A proposed temporal analysis of differentially expressed genes among the two groups distributed the differentially expressed genes under nine clusters, and the differentially expressed genes under different clusters had the same expression trends, indicating that these genes can be jointly involved in specific biological processes. Extraction of ginsenosides from ginseng adventitious roots using water-saturated n-butanol and detection of ginsenoside content by high-performance liquid chromatography revealed a significant increase in total saponins and protopanaxadiol ginsenosides (particularly significant for ginsenosides Rd and Rb1), an increase in bioaccumulation of some protopanaxatriol ginsenosides, and a decrease in some protopanaxatriol ginsenosides (S-Rh1, R-Rg3, and Rf) saponin content decreased. We also found seven genes involved in ginsenoside biosynthesis and that the changes in these genes’ expression may be related to the accumulation of ginsenosides.

반응표면분석법을 이용한 초산침지처리 태극삼의 진세노사이드 성분과 함량 모니터링

The study aimed to use response surface methodology (RSM) to monitor changes in the composition and contents of ginsenosides as a result of soaking fresh ginseng in an acetic acid solution during the manufacturing process of taegeuk ginseng, resulting in investigating the dynamic changes in protopanaxadiol and protopanaxatriol ginsenosides. The R2 of ginsenoside Rb1, Rb2, Re, Rf, Rg1, and total model formulas were 0.8420 (p<0.1), 0.8422 (p<0.1), 0.9183 (p<0.05), 0.9237 (p<0.05), 0.9007 (p<0.05), and 0.9236 (p<0.05), respectively. Prolonged soaking of protopanaxadiol ginsenoside Rb1, Rb2, Rb3, and Rc in high acetic acid concentrations released all sugars from the OH at position 20 of aglycone, resulting in relatively large amount of Rg3 being formed. Under similar conditions, protopanaxatriol ginsenoside Re and Rf were also converted to Rg1, Rg2, and Rh1 due to sugars released from OH at positions 6 and 20 of aglycone. Rg2 increased on conversion from Re; however, at high acetic acid concentrations, it was immediately converted to Rh1, resulting in a relative decrease. The total ginsenoside contents decreased with increase in the acetic acid solution soaking time. Thus, RSM confirmed that specific ginsenosides were reduced and other ginsenosides formed during the manufacturing process of taegeuk ginseng.

Protopanaxadiol and Protopanaxatriol Ginsenosides Can Protect Against Aconitine-induced Injury in H9c2 Cells by Maintaining Calcium Homeostasis and Activating the AKT Pathway.

This study aimed to investigate the effects of protopanaxadiol and protopanaxatriol ginsenosides on aconitine-induced cardiomyocyte injury and their regulatory mechanisms. The effects of ginsenosides on aconitine-induced cardiomyocyte damage were initially evaluated using H9c2 cells, and the molecular mechanisms were elucidated using molecular docking and western blotting. The changes in enzyme content, reactive oxygen species (ROS), calcium (Ca2+) concentration, and apoptosis were determined. Furthermore, an aconitine-induced cardiac injury rat model was established, the cardiac injury and serum physiological and biochemical indexes were measured, and the effects of ginsenoside were observed. The results showed that ginsenoside Rb1 significantly increased aconitine-induced cell viability, and its binding conformation with protein kinase B (AKT) protein was the most significant. In vitro and in vivo, Rb1 protects cardiomyocytes from aconitine-induced injury by regulating oxidative stress levels and maintaining Ca2+ concentration homeostasis. Moreover, Rb1 activated the PI3K/AKT pathway, downregulated Cleaved caspase-3 and Bax, and upregulated Bcl-2 expression. In conclusion, Rb1 protected H9c2 cells from aconitine-induced injury by maintaining Ca2+ homeostasis and activating the PI3K/AKT pathway to induce a cascade response of downstream proteins, thereby protecting cardiomyocytes from damage. These results suggested that ginsenoside Rb1 may be a potential cardiac protective drug.

Exogenous Putrescine Enhances Salt Tolerance and Ginsenosides Content in Korean Ginseng (Panax ginseng Meyer) Sprouts.

The effect of exogenously applied putrescine (Put) on salt stress tolerance was investigated in Panax ginseng. Thirty-day-old ginseng sprouts were grown in salinized nutrient solution (150 mM NaCl) for five days, while the control sprouts were grown in nutrients solution. Putrescine (0.3, 0.6, and 0.9 mM) was sprayed on the plants once at the onset of salinity treatment, whereas control plants were sprayed with water only. Ginseng seedlings tested under salinity exhibited reduced plant growth and biomass production, which was directly interlinked with reduced chlorophyll and chlorophyll fluorescence due to higher reactive oxygen species (hydrogen peroxide; H2O2) and lipid peroxidation (malondialdehyde; MDA) production. Application of Put enhanced accumulation of proline, total soluble carbohydrate, total soluble sugar and total soluble protein. At the same time, activities of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase in leaves, stems, and roots of ginseng seedlings were increased. Such modulation of physio-biochemical processes reduced the level of H2O2 and MDA, which indicates a successful adaptation of ginseng seedlings to salinity stress. Moreover, protopanaxadiol (PPD) ginsenosides enhanced by both salinity stress and exogenous Put treatment. On the other hand, protopanaxatriol (PPT) ginsenosides enhanced in roots and reduced in leaves and stems under salinity stress condition. In contrast, they enhanced by exogenous Put application in all parts of the plants for most cases, also evidenced by principal component analysis. Collectively, our findings provide an important prospect for the use of Put in modulating salinity tolerance and ginsenosides content in ginseng sprouts.

Analysis of chemical constituents in Shenmai Injection by LC-Q-TOF-MS and LC-IT-MS

The chemical constituents in Shenmai Injection(SMI) were qualitatively analyzed by using liquid chromatography/quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS) and liquid chromatography-ion trap-mass spectrometry(LC-IT-MS). The analysis was performed on an Agilent Zorbax SB-C_(18)(4.6 mm×250 mm, 5 μm) and gradient elution was carried out with 0.05% formic acid solution-acetonitrile as mobile phase at a flow rate of 0.6 mL·min~(-1) and a column temperature of 30 ℃. Mass spectrometry data of the components in SMI were collected in negative ion mode. The structures of components were speculated and identified by analyzing mass spectrometry data, comparing with standards, and referring to related literature. A total of 64 components in SMI were estimated, and the structures were confirmed in 16 of them by comparison with standards. Fifty-six compounds derived from Ginseng Radix et Rhizoma Rubra included 34 protopanaxadiol ginsenosides, 19 protopanaxatriol ginsenosides, 1 oleanane ginsenosides and 2 other glycosides. Eight compounds derived from Ophiopogonis Radix included 7 steroidal saponins, and 1 monoterpene glycoside. The results of this study would provide an important theoretical basis for the improvement of the quality control standards and the discovery of effective constituents in SMI.

Ginsenoside Rg1 promotes sleep in rats by modulating the noradrenergic system in the locus coeruleus and serotonergic system in the dorsal raphe nucleus

Panax ginseng Mayer has been used as tranquilizer to improve sleep disorder, but its active component is not defined. This study investigated the effects of the most abundant constituents of P. ginseng—protopanaxatriol ginsenoside Rg1 and protopanaxadiol ginsenoside Rb1—on sleep in rats. Male Sprague-Dawley rats received intragastrical injections of Rg1 and Rb1 for 3 days (5, 10, and 20 mg/kg/day). Sleep parameters were analyzed using electroencephalogram and electromyogram. Neuronal activation and monoaminergic neurotransmitters were evaluated using immunohistochemical fluorescence staining and HPLC, respectively. Rg1 treatment significantly increased the duration of total sleep, rapid eye movement sleep (REMS) and Non-REMS at the dose of 5, 10 and 20 mg/kg/day, and also prolonged the proportion of slow-wave sleep in the total sleep. The Non-REMS episodes were increased and the mean duration of each wakefulness episode was depressed by Rg1 treatment. Rb1 had no effect on sleep parameters. Rg1 treatment decreased the activity of noradrenergic neurons in locus coeruleus (LC) and increased the activity of serotonergic neurons in the dorsal raphe nucleus (DRN). Besides, Rg1 depressed extracellular norepinephrine concentrations in both LC and DRN and in other sleep-regulating brain regions of which functions can be modulated by monoaminergic neurotransmitters discharged from projecting noradrenergic and serotonergic neurons. In conclusion, Rg1 might be the sleep-promoting component in P. ginseng and its mechanism may be related to the modulation of noradrenergic and serotonergic systems. Our findings also highlight functional differences between Rg1 and Rb1.

Efficient separation determination of protopanaxatriol ginsenosides Rg1, Re, Rf, Rh1, Rg2 by HPLC

An efficient and flexible protocol was developed for simultaneous detection of the ginsenosides Rg1, Re, Rf, Rh1, and Rg2 in ginseng extract. In the analysis of white ginseng that contains no ginsenoside Rf, separation of the remaining ginsenosides was achieved within just 10 min under isocratic chromatographic conditions. For the analysis of red ginseng that contains ginsenoside Rf as a characteristic constituent, the gradient elution conditions were optimized. The method is based on high-pressure liquid chromatography employing a Shiseido UG 80 Capcell Pak NH2(4.6 mm I.D. × 250 mm, 5 μm)column and isocratic elution using acetonitrile (A) and water(B) in a ratio of 76: 24 (v/v), The optimal gradient elution conditions are as follows: 0–3 min, 89% A, 3–25 min, 89–84% A, 25–30 min, 84–82% A, 30–35 min, 82-76% A, then returning to 89% solvent A in 5 min. The flow rate was 0.80 mL min−1. The column temperature was set at 25℃ and the detection wavelength was at 203 nm. The working concentration ranges for ginsenoside Re, Rh1, Rg2, Rg1, and Rf were 0.23−1450.0 mg• L−1, 0.05−1130.0 mg• L−1, 0.11−687.0 mg• L−1, 0.051−1325 mg• L−1, and 0.55−800.0 mg• L−1, respectively. The method was validated for linearity, precision, and accuracy. And the further confirmation of five ginsenosides was conducted by QTOF-MS. Analysis of raw extracts of ginseng, white ginseng, and red ginseng for the five components showed satisfactory recovery.

Enhanced expression of ginsenoside biosynthetic genes and in vitro ginsenoside production in elicited Panax sikkimensis (Ban) cell suspensions.

Dual metabolite, i.e., ginsenoside and anthocyanin, co-accumulating cell suspensions of Panax sikkimensis were subjected to elicitation with culture filtrates of Serratia marcescens (SD 21), Bacillus subtilis (FL11), Trichoderma atroviridae (TA), and T. harzianum (TH) at 1.25% and 2.5% v/v for 1- and 3-week duration. The fungal-derived elicitors (TA and TH) did not significantly affect biomass accumulation; however, bacterial elicitors (SD 21 and FL11), especially SD 21, led to comparable loss in biomass growth. In terms of ginsenoside content, differential responses were observed. A maximum of 3.2-fold increase (222.2mg/L) in total ginsenoside content was observed with the use of 2.5% v/v TH culture filtrate for 1week. Similar ginsenoside accumulation was observed with the use of 1-week treatment with 2.5% v/v SD 21 culture filtrate (189.3mg/L) with a 10-fold increase in intracellular Rg2 biosynthesis (31mg/L). Real-time PCR analysis of key ginsenoside biosynthesis genes, i.e., FPS, SQS, DDS, PPDS, and PPTS, revealed prominent upregulation of particularly PPTS expression (20-23-fold), accounting for the observed enhancement in protopanaxatriol ginsenosides. However, none of the elicitors led to successful enhancement in in vitro anthocyanin accumulation as compared to control values.

Complete conversion of all typical glycosylated protopanaxatriol ginsenosides to aglycon protopanaxatriol by combined bacterial \u03b2-glycosidases

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as anti-inflammatory and anti-stress activities. However, the complete conversion of all typical glycosylated protopanaxatriol ginsenosides to APPT has not been achieved to date. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues at C-6 and the inner glucose at C-20 in protopanaxatriol (PPT), but not the outer rhamnose residues at C-6. In contrast, β-glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer rhamnose residue at C-6 but not the inner glucose residues at C-6 and C-20 in PPT. Thus, the combined use of DT-bgl and PF-bgl resulted in the complete the conversion of all typical glycosylated PPT ginsenosides, including R1, R2, Re, Rg1, Rg2, Rh1, Rf, F1, F3, and F5, to APPT. DT-bgl combined with PF-bgl completely hydrolyzed 1.0 mg ml−1 R1 and 1.0 mg ml−1 total PPT-type ginsenosides in Panax notoginseng root extract to 0.5 and 0.63 mg ml−1 APPT for 4 and 3 h, with molar conversions of 100% and productivities of 125 and 210 mg l−1 h−1, respectively. To the best of our knowledge, this is the first report of the complete conversion of all typical glycosylated PPT ginsenosides to APPT and the highest productivity of APPT obtained from ginseng extract achieved to date.

Ginsenosides and ginsenosidases in the pathobiology of ginseng-Cylindrocarpon destructans (Zinss) Scholten

To investigate the role that ginsenosides (and some of their metabolites) play in interactions between plants and phytopathogenic fungi (e.g. Cylindrocarpon destructans (Zinss) Scholten), we systematically determined the anti-fungal activities of six major ginsenosides (Rb1, Rb2, Rc, Rd, Re and Rg1), along with the metabolites of ginsenoside Rb1 (Gypenoside XVII (G-XVII) and F2), against the ginseng root pathogen C. destructans (Zinss) Scholten and non-ginseng pathogens Fusarium graminearum Schw., Exserohilum turcicum (Pass.) Leonard et Suggs, Phytophthora megasperma Drech. and Pyricularia oryzae Cav. Our results showed that the growth of both ginseng pathogens and non-pathogens could be inhibited by using the proto-panaxatriol (PPT) ginsenosides Re and Rg1. In addition, the growth of the non-pathogens could also be inhibited by using proto-panaxadiol (PPD) ginsenosides Rb1, Rb2, Rc and Rd, whereas the growth of ginseng pathogen C. destructans (Zinss) Scholten was enhanced by ginsenosides Rb1 and Rb2. In contrast, ginsenoside G-XVII and F2 strongly inhibited the hyphal growth of both C. destructans (Zinss) Scholten and the non-pathogens tested. Furthermore, addition of sucrose to the media increased the growth of C. destructans (Zinss) Scholten, whereas glucose did not affect the growth. Moreover, C. destructans (Zinss) Scholten and all four non-pathogens were able to deglycosylate PPD ginsenosides using a similar transformation pathway, albeit with different sensitivities. We also discussed the anti-fungal structure-activity relationships of the ginsenosides. Our results suggest that the pathogenicity of C. destructans (Zinss) Scholten against ginseng root is independent of its ability to deglycosylate ginsenosides.

The structure-activity relationship of ginsenosides on hypoxia-reoxygenation induced apoptosis of cardiomyocytes

Ginsenosides have been studied extensively in recent years due to their therapeutic effects in cardiovascular diseases. While most studies examined the different ginsenosides individually, few studies compare the therapeutic effects among the different types. This study examined how effective protopanaxadiol, protopanaxatriol ginsenosides Rh2, Rg3, Rh1, and Rg2 of the ginsenoside family are in protecting H9c2 cardiomyocytes from damage caused by hypoxia/reoxygenation. In the current study, a model of myocardial ischemia and reperfusion was induced in H9c2 cardiomyocytes by oxygen deprivation via a hypoxia chamber followed by reoxygenation. Our data show that structures similar to that of protopanaxadiol, which lacked the hydroxide group at C6, were more effective in lowering apoptosis than structures similar to protopanaxatriol with a hydroxide group at C6. As the compounds increased in size and complexity, the cardioprotective effects diminished. In addition, the S enantiomer proved to be more effective in cardioprotection than the R enantiomer. Furthermore, the immunoblotting analysis demonstrated that ginsenosides activate AMPK but suppress JNK signaling pathways during hypoxia/reoxygenation. Thus, ginsenosides treatment attenuated hypoxia/reoxygenation-induced apoptosis via modulating cardioprotective AMPK and inflammation-related JNK signaling pathways.

Effects of Panax ginseng extracts prepared at different steaming times on thermogenesis in rats

BackgroundPanax ginseng (PG) has a long history of use in Asian medicine because of its multiple pharmacological activities. It has been considered that PG in a type of white ginseng may induce undesirable thermogenic effects, but not in a type of red ginseng. However, there is a lack of evidence about the correlation between ginsenoside and thermogenesis. MethodsWe investigated the effects of PG with different ginsenoside compositions on body temperature, blood pressure, and thermogenesis-related factors in rats. ResultsWith increasing steaming time (0 h, 3 h, 6 h, and 9 h), the production of protopanaxadiol ginsenosides increased, whereas protopanaxatriol ginsenosides decreased in white ginseng. In both short- and long-term studies, administration of four ginseng extracts prepared at different steaming times did not induce significant changes in body temperature (skin, tail, and rectum) and blood pressure of rats compared to saline control. In addition, there were no significant differences in the molecular markers related to thermogenesis (p > 0.05), mRNA expressions of peroxisome proliferator-activated receptor-gamma coactivator-1α and uncoupling protein 1 in brown adipose tissue, as well as the serum levels of interleukin-6, inducible nitric oxide synthase, and nitrite among the treatment groups. ConclusionThese observations indicate that the potential undesirable effects of PG on body temperature could not be explained by the difference in ginsenoside composition.

Tartaric acid induced conversion of protopanaxadiol to ginsenosides Rg3 and Rg5 and their in situ recoveries by integrated expanded bed adsorption chromatography.

Panax ginseng has been applied in traditional Chinese medicine for over 2000 years. It is still one of the most popular herbs in recent decades. The prescribed ginseng-containing medicines consist of protopanaxadiol and protopanaxatriol ginsenosides, which are the major constituents of the herb. Minor ginsenosides at low levels in the herb, such as Rg3 and Rg5 , have attracted more rising attention than the major ones. The existing approaches to prepare Rg3 and Rg5 usually rely on either steamed red ginseng as the source or chemical/enzymatic conversion of protopanaxadiol to the targets. It is still highly desirable to effectively achieve such minor components. In this paper, a method integrated extraction of protopanaxadiol and conversion of it to Rg3 and Rg5 has been proposed. Protopanaxadiol was extracted and simultaneously converted to Rg3 and Rg5 by d,l-tartaric acid. The targets were absorbed by resins on expanded bed adsorption chromatography and were then separated from other ginsenosides in different stages. Compared with conventional methods, the developed process has advantages in shortening time consumption and improving the conversion ratio of protopanaxadiol, which is promising in directly achieving Rg3 and Rg5 from P. ginseng.

Protopanaxatriol Ginsenoside Rh1 Upregulates Phase II Antioxidant Enzyme Gene Expression in Rat Primary Astrocytes: Involvement of MAP Kinases and Nrf2/ARE Signaling.

Oxidative stress activates several intracellular signaling cascades that may have deleterious effects on neuronal cell survival. Thus, controlling oxidative stress has been suggested as an important strategy for prevention and/or treatment of neurodegenerative diseases. In this study, we found that ginsenoside Rh1 inhibited hydrogen peroxide-induced reactive oxygen species generation and subsequent cell death in rat primary astrocytes. Rh1 increased the expression of phase II antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, superoxide dismutase-2, and catalase, that are under the control of Nrf2/ARE signaling pathways. Further mechanistic studies showed that Rh1 increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to the antioxidant response element (ARE), and increased the ARE-mediated transcription activities in rat primary astrocytes. Analysis of signaling pathways revealed that MAP kinases are important in HO-1 expression, and act by modulating ARE-mediated transcriptional activity. Therefore, the upregulation of antioxidant enzymes by Rh1 may provide preventive therapeutic potential for various neurodegenerative diseases that are associated with oxidative stress.

Effects of steaming on saponin compositions and antiproliferative activity of Vietnamese ginseng

BackgroundSteaming of ginseng is known to change its chemical composition and biological activity. This study was carried out to investigate the effect of different steaming time-scales on chemical constituents and antiproliferative activity of Vietnamese ginseng (VG). MethodsVG was steamed at 105°C for 2–20h. Its saponin constituents and antiproliferative activity were studied. The similarity of chemical compositions between steamed samples at 105°C and 120°C were compared. ResultsMost protopanaxadiol and protopanaxatriol ginsenosides lost the sugar moiety at the C-20 position with 10–14h steaming at 105°C and changed to their less polar analogues. However, ocotillol (OCT) ginsenosides were reasonably stable to steaming process. Antiproliferative activity against A549 lung cancer cells was increased on steaming and reached its plateau after 12h steaming. ConclusionSteaming VG at 105°C showed a similar tendency of chemical degradation to the steaming VG at 120°C except the slower rate of reaction. Its rate was about one-third of the steaming at 120°C.

In silico screening of ginsenoside Rh1 with PPARγ and in vitro analysis on 3T3-L1 cell line

Peroxisome proliferator-activated receptor gamma (PPARγ) is a major transcription factor for the process of adipogenesis. Overexpression of PPARγ stimulates other adipogenic-specific proteins, which lead to several disease conditions including obesity. Natural products are of great choice to treat obesity, and Panax ginseng Meyer has been an important option. Ginsenoside Rh1, a protopanaxatriol ginsenoside, has shown to be effective in various diseases including obesity in high fat diet induced mice model. Therefore, considering Rh1 as an important compound, we performed structural analysis and its impact on important genes related to obesity. Initially, molecular interaction was assessed on Rh1 and PPARγ using docking method. The results showed Rh1 had strong hydrogen bond interaction with PPARγ active site. Furthermore, the stability of this complex structure was verified by molecular dynamic simulation. The pharmacokinetic properties of ginsenoside Rh1 were identified using the ADMET and PASS methods. Next, cell cytotoxic assay was performed at different concentrations, and to support the in silico screening, gene expression levels of adipogenic proteins were evaluated. Our findings suggest that ginsenoside Rh1 has binding interaction with PPARγ and from the cell level studies it was shown to interrupt the process of adipogenesis that may lead to work as an anti-obese component.

Ginsenoside Rb1 eliminates HIV-1 (D3)-transduced cytoprotective human macrophages by inhibiting the AKT pathway.

Abstract Acquired immunodeficiency syndrome patients treated with red ginseng, which contains protopanxadiol and protopanaxatriol ginsenosides as its main constituents, have been reported to remain healthy for >20 years in the absence of highly active antiretroviral therapy. Of these ginsenosides, ginsenoside Rh1, a protopanaxatriol ginsenoside, is known to eliminate cytoprotective HIV-1-infected macrophages by inhibiting pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK-1) phosphorylation. In this study, we investigated the capacity of ginsenoside Rb1, a protopanaxadiol ginsenoside, to eliminate cytoprotective primary human macrophages. We found that ginsenoside Rb1 could also eliminate cytoprotective primary human macrophages infected with HIV-1 D3. Ginsenoside Rb1 inhibited lipopolysaccharide/cycloheximide-induced AKT and glycogen synthase kinase-3β phosphorylation in the D3-transduced macrophages, but not the phosphorylation of PDK-1 and phosphoinositide-3-kinase (PI3K). Furthermore, we also observed that a combined treatment with ginsenoside Rb1 and miltefosine synergistically abolished the cytoprotective CHME5 cells expressing HIV-1 tat. Based on these findings, we can conclude that ginsenoside Rb1 can eliminate cytoprotective macrophages infected with HIV-1 by inhibiting the AKT pathway.

β-Glycosidase-assisted bioconversion of ginsenosides in purified crude saponin and extracts from red ginseng (Panax ginseng C. A. Meyer)

Major ginsenosides in ginseng (Panax ginseng) and its products are highly glycosylated, hence poorly absorbed in the gastrointestinal tract. β-Glycosidase-assisted deglycosylation of pure ginsenosides was peformed to study bioconversion mechanisms. Ginsenoside standard compounds, crude saponin, and red ginseng extracts were incubated with β-glycosidase (0.05% w/v, 55°C). β-Glycosidase has a broad specificity for β-glycosidic bonds, hydrolyzing the β-(1→6), α-(1→6), and α-(1→2) glycosidic linkages. The final metabolite of protopanaxadiol ginsenosides was Rg3 while the metabolite of protopanaxatriol ginsenosides was Rh1. β-Glycosidase treatment of red ginseng extracts resulted in a decrease in the amounts of Rb1, Rc, Re, and Rg2 after 24 h, whereas levels of the less glycosylated Rd, Rb1, Rg, Rg3, Rg1, and Rh1 forms increased. When crude saponin was incubated with β-glycosidase for 24 h, levels of Rb1, Rc, Re, and Rg1 decreased while levels of Rd, Rg3, and Rh1 increased as deglycosylated ginsenosides.

Protopanaxatriol ginsenoside Rh1 inhibits the expression of matrix metalloproteinases and the in vitro invasion/migration of human astroglioma cells

Malignant gliomas are the most common and fatal brain tumors in adults. In particular, the strong invasiveness of glioma cells into the normal brain tissue makes eradication of glioma very difficult. Matrix metalloproteinases (MMPs) play a pivotal role in glioma invasion, and thus controlling MMP expression has been suggested as an important therapeutic target for brain tumors. In the present study, we investigated the effect of protopanaxatriol ginsenoside Rh1 on MMP expressions in human astroglioma U87MG and U373MG cells. RT-PCR analysis showed that Rh1 inhibits the mRNA expressions of MMP-1, -3, and -9 in PMA-stimulated U87MG and U373MG cells. Rh1 also suppressed the promoter activities of MMP-1, -3 and -9. The ELISA, Western blot, and zymographic analyses revealed that Rh1 inhibits the protein expression and/or enzymatic activity of MMP-1, -3 and -9. In accordance with the strong inhibitory effects of Rh1 on MMPs, Rh1 efficiently inhibited the invasion and migration of U87MG and U373MG glioma cells as demonstrated by Matrigel invasion assay and wound healing assay. Further mechanistic studies revealed that Rh1 inhibits MAPK and PI3K/Akt signaling pathways and downstream transcription factors such as NF-κB and AP-1, which play an important role in MMP gene expressions. The data collectively suggest that ginsenoside Rh1 may have a therapeutic potential for malignant gliomas.

Characterizing a full spectrum of physico-chemical properties of (20S)-and (20R)-ginsenoside Rg3 to be proposed as standard reference materials

The authentication of the physico-chemical properties of ginsenosides reference materials as well as qualitative and quantitative batch analytical data based on validated analytical procedures is a prerequisite for certifying good manufacturing practice (GMP). Ginsenoside Rb1 and Rg1, representing protopanaxadiol and protopanaxatriol ginsenosides, respectively, are accepted as marker substances in quality control standards worldwide. However, the current analytical methods for these two compounds recommended by Korean, Chinese, European, and Japanese pharmacopoeia do not apply to red ginseng preparations, particularly the extract, because of the relatively low content of the two agents in red ginseng compared to white ginseng. In manufacturing fresh ginseng into red ginseng products, ginseng roots are exposed to a high temperature for many hours, and the naturally occurring ginsenoside Rb1 and Rg1 are converted to artifact ginsenosides such as Rg3, Rg5, Rh1, and Rh2 during the heating process. The analysis of ginsenosides in commercially available ginseng products in Korea led us to propose the inclusion of the (20S)- and (20R)-ginsenoside Rg3, including ginsenoside Rb1 and Rg1, as additional reference materials for ginseng preparations. (20S)- and (20R)-ginsenoside Rg3 were isolated by Diaion HP-20 adsorption chromatography, silica gel flash chromatography, recrystallization, and preparative HPLC. HPLC fractions corresponding to those two ginsenosides were recrystallized in appropriate solvents for the analysis of physico-chemical properties. Documentation of those isolated ginsenosides was achieved according to the method proposed by Gaedcke and Steinhoff. The ginsenosides were subjected to analyses of their general characteristics, identification, purity, content quantification, and mass balance tests. The isolated ginsenosides showed 100% purity when determined by the three HPLC systems. Also, the water content was found to be 0.534% for (20S)-Rg3 and 0.920% for (20R)-Rg3, meaning that the net mass balances for (20S)-Rg3 and (20R)-Rg3 were 99.466% and 99.080%, respectively. From these results, we could assess and propose a full spectrum of physico-chemical properties of (20S)- and (20R)-ginsenoside Rg3 as standard reference materials for GMP-based quality control.