Effects of ginsenosides on glycine receptor alpha1 channels expressed in Xenopus oocytes

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate the excitatory ligand-gated ion channel activity. Recent reports showed that ginsenosides attenuate nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides also affect the inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human glycine alpha1 receptor channel activity expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Treatment of ginsenoside Rf enhances glycine-induced inward peak current (IGly) with dose dependent and reversible manner but ginsenoside Rf itself did not elicit membrane currents. The half-stimulatory concentrations (EC50) of ginsenoside Rf was 49.8 +/- 8.9 microM. Glycine receptor antagonist strychnine completely blocked the inward current elicited by glycine plus ginsenoside Rf. Cl- channel blocker 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) also blocked the inward current elicited by glycine plus ginsenoside Rf. We also tested the effect of eight individual ginsenosides (i.e., Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Ro) in addition to ginsenoside Rf. We found that five of them significantly enhanced the inward current induced by glycine with the following order of potency: Rb1 > Rb2 > Rg2 > or = Rc > Rf > Rg1 > Re. These results indicate that ginsenosides might regulate gylcine receptor expressed in Xenopus oocytes and this regulation might be one of the pharmacological actions of Panax ginseng.     

Generation of ginsenosides Rg3 and Rh2 from North American ginseng

Rg3 and Rh2 ginsenosides are primarily found in Korean red ginseng root (Panax ginseng C.A. Meyer) and valued for their bioactive properties. We quantified both Rh2 and Rg3 ginseng leaf and Rg3 from root extracts derived from North American ginseng (Panax quinquefolius). Quantification was obtained by application of HPLC with ion fragments detected using ESI-MS. Ginseng leaf contained 11.3+/-0.5 mg/g Rh2 and 7.5+/-0.9 mg/g Rg3 in concentrated extracts compared to 10.6+/-0.4 mg/g Rg3 in ginseng root. No detectable Rh2 was found in root extracts by HPLC, although it was detectable by ESI-MS analysis. Ginsenosides Rg3 and Rh2 were detected following hot water reflux extraction, but not from tissues extracted with 80% aqueous ethanol at room temperature. Therefore ginsenosides Rg3 and Rh2 are not naturally present in North American ginseng, but are products of a thermal process. Using ESI-MS analysis, it was found that formation of Rg3 and Rh2, among other compounds, were a function of heating time and were breakdown products of the more abundant ginsenosides Rb1 and Rc. Our findings that heat processed North American ginseng leaf is an excellent source of Rh2 ginsenoside is an important discovery considering that ginseng leaf material is obtainable throughout the entire plant cycle for recovery of valuable ginsenosides for pharmaceutical use.     

Enzymatic preparation of genuine prosapogenin, 20(S)-ginsenoside Rh1, from ginsenosides Re and Rg1

It was found that a lactase preparation from Penicillium sp. nearly quantitatively hydrolyzed ginsenosides Re and Rg1, which are major saponins in roots of Panax ginseng, to a minor saponin, 20(S)-ginsenoside Rh1 [6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol]. This is the first report on the enzymatic preparation of ginsenoside Rh1 with a high efficiency. This enzyme also readily hydrolyzed ginsenoside Rg2 to ginsenoside Rh1.     

Effects of ginsenoside Rg2 on the 5-HT3A receptor-mediated ion current in Xenopus oocytes

Treatment with ginsenosides, major active ingredients of Panax ginseng, produces a variety of pharmacological or physiological responses with effects on the central and peripheral nervous systems. Recent reports showed that ginsenoside Rg2 inhibits nicotinic acetylcholine receptor-mediated Na+ influx and channel activity. In the present study, we investigated the effect of ginsenoside Rg2 on human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity, which is also one of the ligand-gated ion channel families. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. The ginsenoside Rg2 itself had no effect on the oocytes that were injected with H2O as well as on the oocytes that were injected with the 5-HT3A receptor cRNA. In the oocytes that were injected with the 5-HT3A receptor cRNA, the pretreatment of ginsenoside Rg2 inhibited the 5-HT-induced inward peak current (I5-HT) The inhibitory effect of ginsenoside Rg2 on I5-HT was dose dependent and reversible. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 was 22.3 +/- 4.6 microM. The inhibition of I5-HT by ginsenoside Rg2 was non-competitive and voltage-independent. These results indicate that ginsenoside Rg2 might regulate the 5-HT3A receptors that are expressed in Xenopus oocytes. Further, this regulation on the ligand-gated ion channel activity by ginsenosides might be one of the pharmacological actions of Panax ginseng.     

An application of β-glycosidase to transformation of ginsenosides for the effective production of specific ginsenosides with biological efficacy

Over the past several decades, the pharmacological effects of ginsenosides in Panax ginseng roots have been extensively investigated. Here, we developed a method for producing specific ginsenosides (F1 and F2) with good yields (F1:162 mg/g, F2:305 mg/g) using ??-glycosidase purified from Aspergillus niger. In addition, each ginsenoside (at least 25 species) was separated and purified by high performance liquid chromatography (HPLC) using five different types of solvents and different purification steps. In addition, the Rg3:Rh2 mixture (1:1, w/w) was shown to inhibit a specific lung cancer cell line (NCI-H232) in vivo, displaying an anticancer effect at a dose lower than achieved using treatments with single Rg3 or Rh2. This finding suggests that the combination of ginsenosides for targeting anticancer is more effective than the use of a single ginsenoside from ginseng or red ginseng.     

Evidence that the tertiary structure of 20(S)-ginsenoside Rg(3) with tight hydrophobic packing near the chiral center is important for Na(+) channel regulation

Ginsenosides are the active ingredients of Panax ginseng. Ginsenoside Rg(3) exists as two stereoisomers of carbon-20: 20-S-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(S)-Rg(3)) and 20-R-protopanaxatriol-3-[O-beta-d-glucopyranosyl (1-->2)-beta-glucopyranoside] (20(R)-Rg(3)). Recently, we reported that 20(S)-Rg(3) regulates voltage-dependent Ca(2+) channel activity and several types of ligand-gated ion channels, whereas 20(R)-Rg(3) does not have this activity. In this study, we investigated the structure-activity relationship of these two stereoisomers by NMR spectroscopy and by measurement of the current in Xenopus oocytes expressing the mouse cardiac voltage-dependent Na(+) channel (Na(v)1.5). We found that 20(S)-Rg(3) but not 20(R)-Rg(3) inhibited Na(+) channel current in a dose- and voltage-dependent manner. The difference between Rg(3) epimers in voltage-dependent ion channel regulation indicates that the structure of 20(S)-Rg(3) may be geometrically better aligned than that of 20(R)-Rg(3) for interaction with receptor regions in Na(+) channels. The (1)H and (13)C NMR chemical shifts, including all hydroxyl protons of 20(S)-Rg(3) and 20(R)-Rg(3), were completely assigned, and their tertiary structures were determined. 20(S)-Rg(3) has more tight hydrophobic packing near the chiral center than 20(R)-Rg(3). Tertiary structures and activities of 20(S)-Rg(3) and 20(R)-Rg(3) indicate that 20(S)-Rg(3) may have stronger interactions with the receptor region in ion channels than 20(R)-Rg(3). This may result in different stereoselectivity of Rg(3) stereoisomers in the regulation of voltage-dependent Na(+) channel activity. This is the first structural approach to ginsenoside action on ion channel.     

A role for the carbohydrate portion of ginsenoside Rg3 in Na+ channel inhibition

We showed recently that ginsenosides inhibit the activity of various types of ion channel. Here we have investigated the role of the carbohydrate component of ginsenoside Rg3 in the inhibition of Na+ channels. The channels were expressed in Xenopus oocytes by injecting cRNAs encoding rat brain Nav1.2 alpha and beta1 subunits, and analyzed by the two-electrode voltage clamp technique. Treatment with Rg3 reversibly inhibited the inward Na+ peak current (INa) with an IC50 of 32.2 +/- 4.5 microM, and the inhibition was voltage-dependent. To examine the role of the sugar moiety, we prepared a straight chain form of the second glucose and a conjugate of this glucose with 3-(4-hydroxyphenyl) propionic acid hydrazide (HPPH). Neither derivative inhibited INa. Treatment with the carbohydrate portion of ginsenoside Rg3, sophorose [beta-D-glucopyranosyl (1-->2)- beta-glucopyranoside], or the aglycone (protopanaxadiol), on their own or in combination had no effect on INa. These observations indicate that the carbohydrate portion of ginsenoside Rg3 plays an important role in its effect on the Na+ channel.     

Prevention of ginsenoside-induced desensitization of Ca2+-activated Cl- current by microinjection of inositol hexakisphosphate in Xenopus laevis oocytes: involvement of GRK2 and beta-arrestin I

We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca(2+)-activated Cl(-) currents via Galpha(q/11)-phospholipase C-beta3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl(-) channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl(-) channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl(-) channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP(6)), which is known to bind beta-arrestins and interfere with beta-arrestin-induced receptor down-regulation, and cRNAs coding beta-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 +/- 4.1, 9.2 +/- 2.3, and 2.6 +/- 2.2% of the first responses, respectively. Preintraoocyte injection of InsP(6) before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration-, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC(50) was 13.9 +/- 8.7 microM. Injection of cRNA coding beta-arrestin I but not beta-arrestin II blocked InsP(6) effect on prevention of ginsenoside-induced Cl(-) channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl(-) current. However, the GRK2-caused loss of ginsenoside effect on Cl(-) current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl(-) channel desensitization is mediated via activation of GRK2 and beta-arrestin I.     

Biotransformation of ginsenosides Re and Rg1 into ginsenosides Rg2 and Rh1 by recombinant β-glucosidase

Ginsenosides Re and Rg1 were transformed by recombinant β-glucosidase (Bgp1) to ginsenosides Rg2 and Rh1, respectively. The bgp1 gene consists of 2,496?bp encoding 831 amino acids which have homology to the glycosyl hydrolase families 3 protein domain. Using 0.1?mg enzyme ml(-1) in 20?mM sodium phosphate buffer at 37°C and pH 7.0, the glucose moiety attached to the C-20 position of ginsenosides Re and Rg1, was removed: 1?mg ginsenoside Re ml(-1) was transformed into 0.83?mg Rg2?ml(-1) (100% molar conversion) after 2.5?h and 1?mg ginsenoside Rg1?ml(-1) was transformed into 0.6?mg ginsenoside Rh1?ml(-1) (78% molar conversion) in 15?min. Using Bgp1 enzyme, almost all initial ginsenosides Re and Rg1 were converted completely to ginsenosides Rg2 and Rh1. This is the first report of the conversion of ginsenoside Re to ginsenoside Rg2 and ginsenoside Rg1 to ginsenoside Rh1 using the recombinant β-glucosidase.     

Characterizing the mechanism for ginsenoside-induced cytotoxicity in cultured leukemia (THP-1) cells

Pure ginsenoside standards (saponins Rh2, PD, and PT), along with an Rh2-enhanced North American ginseng (Panax quinquefolius) leaf extract (LFRh2), were tested for cytotoxic activity in cultured THP-1 leukemia cells. Thermal treatment of ginseng leaf resulted in production of both Rh2 and Rg3 content that was confirmed by liquid chromatography - mass spectrometry (LC-MS). Flow cytometry of cells stained with annexin V - fluorescein isothiocyanate and propidium iodide showed that the LFRh2 significantly (p < or = 0.05) increased apoptosis (18% +/- 0.4%) after 23 h at a concentration that inhibited cell viability by 50% (LC50 (72 h) = 52 microg/mL. In comparison, a similar significant (p < or = 0.05) increase in apoptotic cell numbers occurred at 41 h of exposure for pure ginsenoside standards, PD (LC50 (72 h) = 13 microg/mL), PT (LC50 (72 h) = 19 microg/mL), and Rh2 (LC50 (72 h) = 15 microg/mL). Although no further increase in apoptosis was observed in THP-1 cells after exposure to increasing concentrations of LFRh2 and pure Rh2, PD, and PT standards, a significant (p < 0.05) increase in the percentage of necrotic cells did occur after exposure of cells to different ginsenosides at elevated concentrations. THP-1 caspase-3 activity was greatest (p 相似文献   

Understory light and root ginsenosides in forest-grown Panax quinquefolius

The objective of this study was to determine the relationship between light levels in the understory of a broadleaf forest and the content of six ginsenosides (Rg(1), Re, Rb(1), Rc, Rb(2,) and Rd) in 1- and 2-year-old American ginseng (Panax quinquefolius L.) roots. Our results revealed that ginsenoside contents in 1- and 2 year-old roots collected in September were significantly related to direct and total light levels, and duration of sunflecks. At this time, the effect of light levels accounted for up to 48 and 62% of the variation in ginsenoside contents of 1- and 2-year-old American ginseng roots. Also, red (R) and far red (FR) light, and the R:FR ratio significantly affected Rd, Rc, and Rg(1) contents in 2-year-old roots, accounting for up to 40% of the variation in ginsenoside contents.     

Differential effect of ginsenoside metabolites on the 5-HT3A receptor-mediated ion current in Xenopus oocytes

Ginsenosides are major active ingredients of Panax ginseng. They have a number of pharmacological and physiological actions and are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is derived from protopanaxadiol (PD) ginsenosides, whereas M4 is derived from protopanaxatriol (PT) ginsenosides. Recent reports show that ginsenosides act as pro-drugs for these metabolites. In previous work we demonstrated that the ginsenoside Rg2 regulates human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity [Choi et al. (2003)]. In the present study, we investigated the effect of CK and M4 on the activity of the human 5-HT3A receptor channel. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. Treatment with CK or M4 had no effect on oocytes injected with 5-HT3A receptor cRNA. However pretreatment with M4 or CK followed by injection of 5-HT3A receptor cRNA led to reversible inhibition of the 5-HT-induced inward peak current (I(5-HT)). Half maximal inhibitory concentrations (IC50) of CK and M4 were 36.9 +/- 9.6 and 7.3 +/- 2.2 microM, respectively. Inhibition by M4 was non-competitive and voltage-independent. These results indicate that M4, a metabolite of PT ginsenosides, acts primarily on 5-HT3A receptors and further, that ginsenosides as well as ginsenoside metabolites can influence 5-HT3A receptor channel activity in Xenopus oocytes.     

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.     

Neuroprotective effect of individual ginsenosides on astrocytes primary culture

Most of the known pharmacological effects of Panax ginseng on the central nervous system are due to its major components - ginsenosides. Although the antioxidant ability of ginseng root has already been established, this activity has never been evaluated for isolated ginsenosides on astrocytes. The activity of protopanaxadiols Rb(1), Rb(2), Rc and Rd, and protopanaxatriols Re and Rg(1) was evaluated in vitro on astrocytes primary culture by means of an oxidative stress model with H(2)O(2). The viability of astrocytes was determined by the MTT reduction assay and by the LDH release into the incubation medium. The effects on the antioxidant enzymes catalase, superoxide dismutase (SOD), glutathione peroxidases (GPx) and glutathione reductase (GR) and on the intracellular reactive oxygen species (ROS) formation were also investigated. Exposure of astrocytes to H(2)O(2) decreased cell viability as well as the antioxidant enzymes activity and increased ROS formation. Oxidative stress produced significant cell death that was reduced by previous treatment with the tested ginsenosides. Ginsenosides Rb(1), Rb(2), Re and Rg(1) were effective in reducing astrocytic death, while Rb(1), Rb(2), Rd, Re and Rg(1) decreased ROS formation, ginsenoside Re being the most active. Ginsenosides from P. ginseng induce neuroprotection mainly through activation of antioxidant enzymes.     

Bifidus fermentation increases hypolipidemic and hypoglycemic effects of red ginseng

Antihyperlipidemic and antihyperglycemic effects of Red Ginseng (RG, steamed and dried root of Panax ginseng C. A. Meyer, family Araliaceae), major component of which is ginsenoside Rg3, and Bifidodoterium-fermented RG (FRG), major component of which is ginsenoside Rh2, were investigated. Orally administered RG and FRG potently reduced the serum triglyceride levels in corn-oil-induced hypertriglycemidemic mice as well as total cholesterol and triglyceride levels in Triton WR-1339-induced hyperlipidemic mice. Of the saponin and polysaccharide fractions of RG and FRG, the polysaccharide fraction inhibited postprandial blood glucose elevation of maltose- or starch-loaded mice and reduced the blood triglyceride levels in corn-oil-induced hypertriglycemidemic mice. The saponin fraction and its ginsenosides Rg3 and Rh2 reduced blood triglyceride and total cholesterol levels in Triton WR1339-induced hyperlipidemic mice. The inhibitory effect of FRG and its main constituents against hyperlipidemia and hyperglycemia in mice were more potent than those of RG. These findings suggest that hypolipidemic and hypoglycemic effects of RG can be enforced by Bifidus fermentation and FRG may improve hyperlipidemia and hyperglycemia.     

Can ginsenosides protect human erythrocytes against free-radical-induced hemolysis?

Many studies have focused on the free-radical-initiated peroxidation of membrane lipid, which is associated with a variety of pathological events. Panax ginseng is used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress. Many reports have been devoted to the effects of ginsenosides, the major active components in P. ginseng, on the lipid metabolism, immune function and cardiovascular system. The results, however, are usually contradictory since the usage of mixture of ginsenosides cannot identify the function of every individual ginsenosides on the experimental system. On the other hand, every individual ginsenosides is not compared under the same experimental condition. These facts motivate us to evaluate the antioxidant effect of various individual ginsenosides on the experimental system of free-radical-initiated peroxidation: the hemolysis of human erythrocyte induced thermally by water-soluble initiator, 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The inhibitory concentration of 50% inhibition (IC(50)) of AAPH-induced hemolysis of the erythrocyte has been studied firstly and found that the order of IC(50) is Rb3 - Rb1Rc>Re>Rh1>R1>Rg2>Rb3. Rg3, Rd and Rh2, however, act as synergistic prooxidants in the above experimental system. Rg1 does not show any synergistic antioxidative property. Although the antioxidative and prooxidative mechanism of various ginsenosides with or without TOH in AAPH-induced hemolysis of human erythrocytes will be further studied in detail, this information may be useful in the clinical usage of ginsenosides.     

Simultaneous Determination and Analysis of Major Ginsenosides in Wild American Ginseng Grown in Tennessee

Ginsenosides are the major constituent that is responsible for the health effects of American ginseng. The ginsenoside profile of wild American ginseng is ultimately the result of germplasm, climate, geography, vegetation species, water, and soil conditions. This is the first report to address the ginsenoside profile of wild American ginseng grown in Tennessee (TN), the third leading state for production of wild American ginseng. In the present study, ten major ginsenosides in wild American ginseng roots grown in TN, including Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rg3, were determined simultaneously. The chemotypic differences among TN wild ginseng, cultivated American ginseng, and Asian ginseng were assessed based on the widely used markers of ginsenoside profiling, including the top three ginsenosides, ratios of PPD/PPT, Rg1/Rb1, Rg1/Re, and Rb2/Rc. Our findings showed marked variation in ginsenoside profile for TN wild ginseng populations. Nevertheless, TN wild ginseng has significant higher ginsenoside content and more ginsenoside diversity than the cultivated ginseng. The total ginsenoside content in TN wild ginseng, as well as ginsenosides Rg1 and Re, increases with the age of the roots. Marked chemotypic differences between TN wild ginseng and cultivated American ginseng were observed based on the chemotypic markers. Surprisingly, we found that TN wild ginseng is close to Asian ginseng with regard to these characteristics in chemical composition. This study verified an accessible method to scientifically elucidate the difference in chemical constituents to distinguish wild from the cultivated American ginseng. This work is critical for the ecological and biological assessments of wild American ginseng so as to facilitate long‐term sustainability of the wild population.     

Molecular docking studies of anti-apoptotic BCL-2, BCL-XL, and MCL-1 proteins with ginsenosides from Panax ginseng

Anti-apoptotic proteins such as BCL-2, BCL-XL and MCL-1 bind with pro-apoptotic proteins to induce apoptosis mechanism. BCL-2 family proteins are key regulators of apoptosis process. Over expression of these anti-apoptotic proteins lead to several cancers by preventing apoptosis. A number of studies revealed that ginseng derivatives reduce tumor growth. Ginseng, the most valuable medicinal herb found in eastern Asia belongs to Araliaceae family. In this study, docking simulations were performed for anti-apoptotic proteins with several ginsenosides from Panax ginseng. Our finding shows ginsenosides Rf, Rg1, Rg3 and Rh2 have more binding affinity with BCL-2, BCL-XL and MCL-1 and other ginsenosides also interact with each anti-apoptotic proteins. Therefore, ginseng derivatives represent a novel class of potent inhibitors and could be used for cancer chemotherapy.     

High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.     

Ginsenosides regulate ligand-gated ion channels from the outside

Treatment with ginsenosides, the major active ingredients of Panax ginseng, produces a variety of physiological effects on the central and peripheral nervous systems. Ginsenosides inhibit various types of ligand-gated ion channel but it is not clear whether they act from within or outside the cell since they are somewhat membrane-permeable. In the present study, we used the Xenopus oocyte gene expression system to determine from which side of the cell membrane the ginsenoside Rg3 (Rg3), and M4, a ginsenoside metabolite, act to regulate ligand-gated ion channel activity. Ligand-gated ion currents were measured using the two-electrode voltage clamp technique. Rg3 and M4 inhibited 5-HT3A and a3b4 nACh receptor-mediated ion currents when present outside of the cell but not when injected intracellularly. We also examined the effect of these agents on oocytes expressing the gustatory cGMP-gated ion channel, which is known to have a cGMP binding site on the intracellular side of the plasma membrane and is only activated by cytosolic cGMP. Rg3 inhibited cGMP-gated ion currents when applied extracellularly or to an outside-out patch clamp, but not when injected into the cytosol or when using an excised inside-out patch clamp. These results indicate that Rg3 and M4 regulate ligand-gated ion channel activity from the extracellular side.