Skin anti-photoaging properties of ginsenoside Rh2 epimers in UV-B-irradiated human keratinocyte cells

Ginseng, one of the most widely used herbal medicines, has a wide range of therapeutic and pharmacological applications. Ginsenosides are the major bioactive ingredients of ginseng, which are responsible for various pharmacological activities of ginseng. Ginsenoside Rh2, known as an antitumour ginsenoside, exists as two different stereoisomeric forms, 20(S)-ginsenoside Rh2 [20(S)-Rh2] and 20(R)-ginsenoside Rh2 [20(R)-Rh2]. This work aimed to assess and compare skin anti-photoaging activities of 20(S)-Rh2 and 20(R)-Rh2 in UV-B-irradiated HaCat cells. 20(S)-Rh2, but not 20(R)-Rh2, was able to suppress UV-B-induced ROS production in HaCat cells. Both stereoisomeric forms could not modulate cellular survival and NO level in UV-B-irradiated HaCat cells. Both 20(S)-Rh2 and 20(R)-Rh2 exhibited suppressive effects on UV-B-induced MMP-2 activity and expression in HaCat cells. In brief, the two stereoisomers of ginsenoside Rh2, 20(S)-Rh2 and 20(R)-Rh2, possess skin anti-photoaging effects but possibly in different fashions.     

20(R)-Ginsenoside Rh2, not 20(S), is a selective osteoclastgenesis inhibitor without any cytotoxicity

Increased osteoclastic bone resorption plays a central role in the pathogenesis of many bone diseases, and osteoclast inhibitors are the most widely used treatments for these diseases. Ginsenosides, the main component of ginseng, have been known for their medicinal effects such as anti-inflammatory and anti-proliferative activities. In this study, we investigated the inhibitory effects of ginsenosides (ginsenoside 20(R)-Rh2 and ginsenoside 20(S)-Rh2) on osteoclastgenesis using RAW264 cells in vitro. Only ginsenoside 20(R)-Rh2 showed selective osteoclastgenesis inhibitory activity without any cytotoxicity up to 100 μM. These results implied that the stereochemistry of the hydroxyl group at C-20 may play an important role in selective osteoclastgenesis inhibitory activity.     

Transport characteristics of peptides and peptidomimetics: II. Hydroxyethylamine bioisostere-containing peptidomimetics as substrates for the oligopeptide transporter and P-glycoprotein in the intestinal mucosa.

Peptide bond bioisosteres, such as hydroxyethylamine (Hea), have frequently been used to stabilize metabolically labile peptide bonds in peptidomimetic drug design in an effort to increase the oral bioavailability of drug candidates. However, the impact of the peptide bond bioisosteres on the cell permeation characteristics of peptidomimetics is not well understood, particularly with respect to the effects on the substrate activity for proteins that can restrict (e.g. P-glycoprotein, P-gp) or facilitate (e.g. the oligopeptide transporter, OPT) intestinal mucosal permeation of peptidomimetics. In this study, terminally free and terminally modified (N-acetylated and C-amidated) peptidomimetics of H-Ala-Phe-OH and H-Ala-Phe-Ala-OH with the Ala-Phe peptide bonds replaced by Hea bioisosteres were synthesized. Transport characteristics of these peptidomimetics were investigated using Caco-2 cell monolayers as an in vitro model of the intestinal mucosa. The study showed that the Hea bioisostere stabilized the peptidomimetics to protease metabolism in Caco-2 cells. All terminally free peptidomimetics showed significant affinity and substrate activity for OPT. The affinity and substrate activity for OPT were stereoselective for peptidomimetics containing an S,S-configuration for the two adjacent chiral centers related to the Hea bioisostere. Three of the four terminally modified peptidomimetics showed significant substrate activity for P-gp and, interestingly, the substrate activity for P-gp was also stereoselective; however, it was in favor of an R,R-configuration for the two adjacent chiral centers related to the Hea bioisostere.     

Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells

The (−)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (−)CLA (1 or 5 μmol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (−)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 μmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (−)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 μmol/L had significantly lower intracellular uptake than (−)CLA with a ratio[(−)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 μmol/L. Besides, in the non-induced KB cells, (+)CLA(5 μmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (−)CLA(5 μmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (−)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs.     

Stereoselective determination of the epimer mixtures of itraconazole in human blood plasma using HPLC and fluorescence detection

Itraconazole is an antifungal drug widely used in a variety of fungal infections, which have become a significant public-health problem in recent decades. Itraconazole is a chiral drug consisting of two diastereoisomeric racemates, i.e., four stereoisomers. Data in the literature suggests that stereochemistry may play a significant role in the action and disposition of the drug and therefore stereoselective analytical methods for the determination of the drug in biological fluids are needed for the elucidation of that role. We report a stereoselective HPLC method that incorporates solvent extraction, the use of an internal standard, two chiral stationary phases in series, and fluorescence detection. The procedure is enantioselective and partially diastereoselective and provides the concentrations in blood plasma of the two epimer mixtures 2R,4S,2'R/2R,4S2'S and 2S,4R,2'R/2S,4R,2'S, respectively, each of which is a combination of the two epimers that differ in the configuration at the sec-butyl group. The analytical method has suitable sensitivity, recovery, precision, and accuracy. Analysis of the plasma of a human subject six hours after the oral administration of a single 200-mg dose of itraconazole showed a 3.4-fold difference between the concentrations of the epimer mixtures. The method has certain advantages over the published alternative procedure that uses LC-MS.     

Real-time analysis of P-glycoprotein-mediated drug transport across primary intestinal epithelium three-dimensionally cultured in vitro

P-glycoprotein (P-gp) is an efflux transporter that regulates bioavailability of orally administered drugs at the intestinal epithelium. To develop an in vitro experimental model that mimics P-gp-mediated intestinal drug transport in vivo, we employed normal intestinal epithelium three-dimensionally cultured. Physiological expression of P-gp mRNA and the expression of its protein at the apical membrane were observed in the small intestinal epithelium grown as cystic organoids. Rhodamine123 (Rh123), a substrate for P-gp, was actively transported in the basoapical direction and accumulated in the luminal space, while the epithelial integrity was kept intact. Furthermore, we were able to monitor the whole process of Rh123 transport and its inhibition by verapamil in real-time, from which kinetic parameters for Rh123 transport could be estimated by a mathematical modeling. The method here described to evaluate the dynamics of P-gp-mediated transport in primary intestinal epithelial cells would be instrumental in investigating the physiological function of P-gp and its inhibitors/inducers in vitro.     

Intestinal absorption and metabolism of chlorpheniramine enantiomers in rat

Chlorpheniramine (CPAM) is a chiral antihistaminic drug commercialized as a racemic mixture. The intestinal absorption and metabolism of CPAM have been investigated in rat using in vivo (oral and IV administration), in situ (intestinal loop model), and in vitro (everted sac model) experiments. Oral and IV administrations of 20 mg/kg of the racemic mixture show that the pharmacokinetics of CPAM are stereoselective, with higher AUCs for the (+)-S-enantiomer compared to its antipode. The monodesmethyl metabolite (DCPM) was quantifiable in blood and its pharmacokinetics are stereoselective after oral but not after IV administration. Experiments using intestinal loops and everted sacs showed that the absorption is not stereoselective and that in vivo stereoselective formation of DCPM is presumably due to stereoselective hepatic metabolism. Moreover, the in vitro and in situ absorption of CPAM are not modified by modulators of P-glycoprotein and cytochromes P450 (cyclosporin A, ketoconazole).     

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.     

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.     

Secondary phosphine oxides: tautomerism and chiral recognition monitored by multinuclear NMR spectroscopy of their Rh2[(R)-MTPA]4 adducts

Six secondary phosphine oxides and their tautomeric equilibria as free ligands and in the presence of an equimolar amount of the chiral dirhodium complex Rh* are described and discussed. Discrimination of enantiomers is easily possible by inspecting the (31)P NMR resonances; some (1)H and (13)C NMR resonances are useful as well. H/D exchange of the acidic protons in the phosphine oxides takes place with acetone-d(6), the solvent additive, after some hours but does not obscure the chiral recognition experiment. (103)Rh,(31)P coupling constants are discussed briefly. Decomposition of ligand molecules in 1:1-Rh*-adducts occurs slowly but completely.     

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.     

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.     

Methods of analysis and separation of chiral flavonoids

Although the analysis of the enantiomers and epimers of chiral flavanones has been carried out for over 20 years, there often remains a deficit within the pharmaceutical, agricultural, and medical sciences to address this issue. Hence, despite increased interest in the potential therapeutic uses, plant physiology roles, and health-benefits of chiral flavanones, the importance of stereoselectivity in agricultural, nutrition, pharmacokinetic, pharmacodynamic, pharmacological activity and disposition has often been ignored. This review presents both the general principles that allow separation of chiral flavanones, and discusses both the advantages and disadvantages of the available chromatographic assay methods and procedures used to separately quantify flavanone enantiomers and epimers in biological matrices.     

Lethal quercetin-digoxin interaction in pigs

Digoxin is a popular cardiac glycoside with very narrow therapeutic range. Quercetin is an ubiquitous antioxidant flavonoid. Digoxin is a substrate of P-glycoprotein (P-gp), a multi-drug efflux transporter, and quercetin was reported to be a modulator of P-gp. The aim of this study was to investigate the effect of quercetin on the absorption and disposition of digoxin in pigs. Pigs were orally given digoxin (0.02 mg/kg) with and without quercetin in crossover designs. The blood was collected via jugular vein and fluorescence polarization immunoassay was used to determine the serum concentration of digoxin. The pharmacokinetic parameters were calculated using WINNONLIN. The paired Student's t-test was used for statistical comparison. The coadministration of 50 mg/kg quercetin unexpectedly resulted in sudden death of two among three pigs within 30 min after digoxin administration. The coadministration of 40 mg/kg quercetin significantly elevated the C_max of digoxin by 413% and increased the AUC_0-t by 170%. The results indicated that a very serious pharmacokinetic interaction occurred between quercetin and digoxin. The concomitant administration of digoxin and quercetin or quercetin-containing herbs and dietary supplement should be avoided.     

Enzymatic preparation of 20(S, R)-protopanaxadiol by transformation of 20(S, R)-Rg3 from black ginseng

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S) → Rh2(S) → PPD(S) and Rg3(R) → Rh2(R) → PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.     

20(S)-protopanaxadiol and the ginsenoside Rh2 inhibit Na+ channel-activated depolarization and Na+ channel-dependent amino acid neurotransmitter release in synaptic fractions isolated from mammalian brain

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.     

Ginsenoside Rh2 is one of the active principles of Panax ginseng root to improve insulin sensitivity in fructose-rich chow-fed rats.

Ginsenoside Rh2, one of the ginsenosides contained in the Panax ginseng root, was employed to screen the effect on insulin resistance of rats induced by a diet containing 60% fructose. Single intravenous injection of ginsenoside Rh2 decreased the plasma glucose concentrations in 60 minutes in a dose-dependent manner from 0.1 mg/kg to 1 mg/kg in rats with insulin resistance induced by fructose-rich chow. Repeated intravenous injection of ginsenoside Rh2 (1 mg/kg per injection, 3 times daily) into rats which received fructose-rich chow for 3 consecutive days decreased the value of glucose-insulin index, the product of the areas under the curve of glucose and insulin during the intraperitoneal (i.p.) glucose tolerance test. This means that ginsenoside Rh2 has an ability to improve insulin action on glucose disposal. The plasma glucose lowering action of tolbutamide, induced by the secretion of endogenous insulin, is widely used to characterize the formation of insulin resistance. Time for the loss of plasma glucose lowering response to tolbutamide (10 mg/kg, i.p.) in rats during insulin resistance induction by fructose-rich chow was also markedly delayed by the repeated treatment of ginsenoside Rh2, as compared to the vehicle-treated control. Thus, the repeated treatment of ginsenoside Rh2 delayed the development of insulin resistance in high fructose feeding rats. Increase of insulin sensitivity by ginsenoside Rh2 was further identified using the plasma glucose lowering action of exogenous insulin in streptozotocin-induced diabetic rats (STZ-diabetic rats). Repeated injection of ginsenoside Rh2 at the same dosing (1 mg/kg, 3 times daily) into STZ-diabetic rats for 10 days made an increase of the responses to exogenous insulin. Taken together, it can be concluded that ginsenoside Rh2 has an ability to improve insulin sensitivity and it seems suitable to use ginsenoside Rh2 as an adjuvant for diabetic patients and/or the subjects wishing to increase insulin sensitivity.     

Immunological-adjuvant saponins from the roots of Panax notoginseng

The total saponin extract from the dried roots of Panax notoginseng (Burk.) F. H. Chen possesses immunological-adjuvant activities. Guided by in vivo immunological tests, further study on this fraction afforded three active dammarane-type saponins. Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods, including 1D- and 2D-NMR. The novel compound (20S)-protopanaxatriol 20-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside (1), and the two known compounds ginsenoside Rh4 (2) and notoginsenoside K (3) exhibited immunological-adjuvant activities on the humoral immune responses of ICR mice against ovalbumin (OVA).     

In vitro assessment of stereoselective hepatic metabolism of disopyramide in humans: comparison with in vivo data.

Metabolism of disopyramide (DP) enantiomers has been investigated in primary cultures of adult human hepatocytes. Results were compared with in vivo data obtained from a previous pharmacokinetic study (Le Corre et al. Drug Metab. Dispos. 16:858-864 1988). Metabolism of DP enantiomers as a function of incubation time showed constant velocity over time. The intracellular/extracellular distribution of both DP and mono-N-desisopropyldisopyramide did not appear to be stereoselective. Metabolism of DP enantiomers as a function of substrate concentration followed a first order kinetics. The average fractions of (-)-(R)-DP and (+)-(S)-DP metabolized in vitro (4.7 +/- 2.7 and 7.1 +/- 4.2%, respectively, n = 4) were about 5-fold lower than the fractions metabolized in vivo (26.0 +/- 6.0 and 40.2 +/- 8.8%, respectively, n = 6). The stereoselective index [(+)-(S)/(-)-(R)] of the N-dealkylation pathway obtained in vitro (1.51 +/- 0.11, n = 4) was very close to the one obtained in vivo (1.55 +/- 0.10, n = 6). These results highlight the interest of hepatocyte cultures in the evaluation of drug metabolism and especially in the assessment of stereoselectivity.