Yan-Fang Yang

New triterpene saponins from the roots of Glycyrrhiza yunnanensis and their rapid screening by LC/MS/MS

The roots of Glycyrrhiza yunnanensis Cheng f. et L. K. Dai ex P. C. Li are used as licorice in traditional medicine of Southwest China. Triterpene saponins are the major chemical constituents. In this study, one new oleanane-type triterpenoid, glyyunnansapogenin I (I), seven new triterpene saponins, yunganosides E3 (II), L (III), M (IV), N1 (V), O (VI), P (VII) and N2 (VIII), together with four known saponins (IX-XII) were isolated from the roots of G. yunnanensis by preparative chromatography. Their structures were identified by spectroscopic analysis including NMR and HR-MS. Based on (-)-ESI-MS/MS fragmentation behaviors of these reference standards, an LC/MS/MS method using neutral loss scan and precursor ion scan on a triple quadrupole mass spectrometer was established to rapidly and comprehensively analyze triterpene saponins in G. yunnanensis. Combined with high-accuracy qTOF mass spectrometry analysis, a total of 50 saponins were detected, and their structures were identified or tentatively characterized. This is the first systematic study on triterpene saponins in G. yunnanensis.

Simultaneous Determination of Eight Ginsenosides in Rat Plasma by Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry: Application to Their Pharmacokinetics

A high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was successfully developed and validated for the identification and determination of eight ginsenosides: ginsenoside Rg1 (1); 20(S)-ginsenoside Rh1 (2); 20(S)-ginsenoside Rg2 (3); 20(R)-ginsenoside Rh1 (4); 20(R)-ginsenoside Rg2 (5); ginsenoside Rd (6); 20(S)-ginsenoside Rg3 (7); and 20(R)-ginsenoside Rg3 (8) in rat plasma. The established rapid method had high linearity, selectivity, sensitivity, accuracy, and precision. The method has been used successfully to study the pharmacokinetics of abovementioned eight ginsenosides for the first time. After an oral administration of total saponins in the stems-leaves of Panax ginseng C. A. Meyer (GTSSL) at a dose of 400 mg/kg, the ginsenosides 6, 7, and 8, belonging to protopanaxadiol-type saponins, exhibited relatively long tmax values, suggesting that they were slowly absorbed, while the ginsenosides 1–5, belonging to protopanaxatriol-type saponins, had different tmax values, which should be due to their differences in the substituted groups. Compounds 2 and 4, 3 and 5, 7 and 8 were three pairs of R/S epimerics at C-20, which was interesting that the t1/2 of 20(S)-epimers were always longer than those of 20(R)-epimers. This pharmacokinetic identification of multiple ginsenosides of GTSSL in rat plasma provides a significant basis for better understanding the clinical application of GTSSL.

Transport of Twelve Coumarins from Angelicae Pubescentis Radix across a MDCK-pHaMDR Cell Monolayer—An in Vitro Model for Blood-Brain Barrier Permeability

Angelicae Pubescentis Radix (APR), a widely used traditional Chinese medicine, is reported to have central nervous system activities. The purpose of this study was to characterize the blood-brain barrier permeability of twelve coumarins from APR including umbelliferone (1), osthol (2), scopoletin (3), peucedanol (4), ulopterol (5), angepubebisin (6), psoralen (7), xanthotoxin (8), bergapten (9), isoimperatorin (10), columbianadin (11), and columbianetin acetate (12) with an in vitro model using a MDCK-pHaMDR cell monolayer. The cell monolayer was validated to be suitable for the permeation experiments. The samples’ transports were analyzed by high performance liquid chromatography and their apparent permeability coefficients (Papp) were calculated. According to the Papp value, most coumarins could be characterized as well-absorbed compounds except for 4, 10 and 11 which were moderately absorbed ones, in concentration-dependent and time-dependent manners. The results of P-glycoprotein (P-gp) inhibitor (verapamil) experiments showed that the transport of coumarin 4 was affected by the transport protein P-gp. Sigmoid functions between permeability log(Papp AP-BL*MW0.5) and log D (at pH 7.4) were established to analyze the structure-activity relationship of coumarins. The results provide useful information for discovering the substance basis for the central nervous system activities of APR, and predicting the permeability of other coumarins through BBB.

Pharmacokinetics Studies of 12 Alkaloids in Rat Plasma after Oral Administration of Zuojin and Fan-Zuojin Formulas

Zuojin formula (ZJ) is a traditional Chinese medicine (TCM) prescription consisted of Coptidis Rhizoma (CR) and Euodiae Fructus (EF), and has been used to treat gastrointestinal (GI) disease for more than 700 years. Fan-Zuojin formula (FZJ) is a related TCM prescription also consisted of CR and EF with the opposite proportion. In recent years, ZJ was getting more attention for its antitumor potential, but the indeterminate pharmacokinetic (PK) behavior restricted its clinical applications, and the PK differences between ZJ and FZJ were also largely unknown. Consequently it is necessary to carry out a full-scale PK study to demonstrate the physiological disposition of ZJ, as well as the comparative PK study between ZJ and FZJ to illustrate the compatibility dose effects. Therefore a liquid chromatographic–tandem mass spectrometry (LC–MS/MS) method was established and validated for the determinations of coptisine, epiberberine, palmatine, berberine, 8-oxocoptisine, 8-oxoepiberberine, noroxyhydrastinine, corydaldine, dehydroevodiamine, evodiamine, wuchuyuamide-I, and evocarpine in rat plasma. PK characteristics of 12 alkaloids after oral administration of ZJ and FZJ were compared, and the result was analyzed and discussed with the help of an in silico study. Then an integrated PK study was carried out with the AUC-based weighting method and the total drug concentration method. The established method has been successfully applied to reveal the PK profiles of the 12 alkaloids in rat plasma after oral administration of ZJ and FZJ. The results showed that: (1) double peaks were observed in the plasma concentration-time (C–T) curves of the alkaloids after ZJ administration; but the C–T curves approximately matched the two-compartment model after FZJ administration; (2) There were wide variations in the absorption levels of these alkaloids; and even for a certain alkaloid, the dose modified systemic exposure levels and elimination rate also varied significantly after administration of ZJ and FZJ extracts. The results could be interpreted as follows: firstly, inhibition effect on GI motility caused by the high content CR alkaloids (especially berberine) in ZJ could delay the Tmax, and increase the absorption and systemic exposure levels of the other alkaloids, and also lead to the double peak phenomenon of these alkaloids. However, for quaternary protoberberine alkaloids (QPA), double peaks were primarily caused by the different Ka value in two intestinal absorption sites; Secondly, absorption was the major obstacle to the systemic exposure level of the alkaloids from CR and EF. In silico and PK studies suggested that the absorption of these alkaloids, except QPAs, mainly depended on their solubility rather than permeability; Thirdly, EF could promote the absorption and accelerate the elimination of QPAs, and had a greater influence on the former than the latter. At last the integrated PK analysis suggested that berberine and dehydroevodiamine could be regarded as the representative components to reflect the PK behaviors of CR and EF alkaloids after administration of ZJ and FZJ. In conclusion, the absorption, elimination and systemic exposure level of these alkaloids were mainly influenced by the proportion of EF and CR, the pharmacological effect on GI motility, and the physicochemical property of these alkaloids. These findings would be helpful for a better understanding of the activities and clinical applications of ZJ, FZJ and other related TCM prescriptions.

Metabolites identification of bioactive licorice compounds in rats

Licorice (Glycyrrhiza uralensis Fisch.) is one of the most popular herbal medicines worldwide. This study aims to identify the metabolites of seven representative bioactive licorice compounds in rats. These compounds include 22β-acetoxyl glycyrrhizin (1), licoflavonol (2), licoricidin (3), licoisoflavanone (4), isoglycycoumarin (5), semilicoisoflavone B (6), and 3-methoxy-9-hydroxy-pterocarpan (7). After oral administration of 250mg/kg of 1 or 40mg/kg of 2-7 to rats, a total of 16, 43 and 31 metabolites were detected in the plasma, urine and fecal samples, respectively. The metabolites were characterized by HPLC/DAD/ESI-MS(n) and LC/IT-TOF-MS analyses. Particularly, two metabolites of 1 were unambiguously identified by comparing with reference standards, and 22β-acetoxyl glycyrrhizin-6″-methyl ester (1-M2) is a new compound. Compound 1 could be readily hydrolyzed to eliminate the glucuronic acid residue. The phenolic compounds (4-7) mainly undertook phase II metabolism (glucuronidation or sulfation). Most phenolic compounds with an isoprenyl group (chain or cyclized, 2-5) could also undertake hydroxylation reaction. This is the first study on in vivo metabolism of these licorice compounds.

Simultaneous quantification of twenty-one ginsenosides and their three aglycones in rat plasma by a developed UFLC–MS/MS assay: Application to a pharmacokinetic study of red ginseng

&NA; To track the pharmacokinetic features of red ginseng (RG), a rapid and sensitive ultra fast liquid chromatographic coupled with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC–MS/MS) method was developed for simultaneous quantification of twenty‐one ginsenosides and their three aglycones, including 18 prototype compounds (ginsenosides Rb1, Rb2, Rc, Rd, Re, Rg1, Rg5, Rh4, Rk1, Rk3, 20(S)‐Rf, 20(S)‐Rg2, 20(R)‐Rg2, 20(S)‐Rg3, 20(R)‐Rg3, 20(S)‐Rh1, 20(R)‐Rh1, 20(S)‐NG‐R2), and 6 metabolites (ginsenosides 20(S)‐Rh2 and Rh3, 20(S)‐protopanaxadiol (PPD), 20(S)‐protopanaxatriol (PPT), 20(R)‐PPT, ginseng saponin compound K) of RG in rat plasma after oral administration of RG water extract at a single dose of 4 g/kg body weight to rats. All analytes with internal standard (digoxin) were detected by multiple reaction monitoring in negative ionization mode and separated on an ACQUITY UPLC® BEH RP‐C18 column (1.7 &mgr;m, 100 × 2.1 mm). This established method was well validated in terms of linearity, sensitivity, intra‐ and inter‐day precisions, accuracy, recovery, matrix effect, stability, and had a lower limit of quantification at the concentration range of 0.12–8.12 ng/mL for all of analytes. This UFLC–MS/MS approach was successfully applied to the pharmacokinetic study for RG water extract in rats. We firstly proposed that Rb1, Rb2, Rc, Rd, Rg1, Rg5, 20(S)‐Rg3, 20(S)‐Rh2, and 20(S)‐PPD measured in rat plasma were suitable pharmacokinetic markers of RG extract in rats due to their high systemic exposure levels. Thus, this specific and reliable method will be useful for future applications to pharmacokinetic studies for various sources of ginsenoside samples and Panax herbs in vivo. Graphical abstract Figure. No caption available. HighlightsA UFLC–MS/MS method was established to quantify 24 triterpenoids in rat plasma.The validated method was successfully applied to pharmacokinetic study of RG.This study firstly indicated suitable pharmacokinetics markers of RG in vivo.

The Blood-Brain Barrier Permeability of Lignans and Malabaricones from the Seeds of Myristica fragrans in the MDCK-pHaMDR Cell Monolayer Model

The blood-brain barrier (BBB) permeability of twelve lignans and three phenolic malabaricones from the seeds of Myristica fragrans (nutmeg) were studied with the MDCK-pHaMDR cell monolayer model. The samples were measured by high-performance liquid chromatography and the apparent permeability coefficients (Papp) were calculated. Among the fifteen test compounds, benzonfuran-type, dibenzylbutane-type and arylnaphthalene-type lignans showed poor to moderate permeabilities with Papp values at 10−8–10−6 cm/s; those of 8-O-4′-neolignan and tetrahydrofuran-lignan were at 10−6–10−5 cm/s, meaning that their permeabilities are moderate to high; the permeabilities of malabaricones were poor as their Papp values were at 10−8–10−7 cm/s. To 5-methoxy-dehydrodiisoeugenol (2), erythro-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)-propan-1-ol acetate (6), verrucosin (8), and nectandrin B (9), an efflux way was involved and the main transporter for 6, 8 and 9 was demonstrated to be P-glycoprotein. The time and concentration dependency experiments indicated the main transport mechanism for neolignans dehydrodiisoeugenol (1), myrislignan (7) and 8 was passive diffusion. This study summarized the relationship between the BBB permeability and structure parameters of the test compounds, which could be used to preliminarily predict the transport of a compound through BBB. The results provide a significant molecular basis for better understanding the potential central nervous system effects of nutmeg.

Metabolites identification of glycyrin and glycyrol, bioactive coumarins from licorice.

Coumarins are an important group of bioactive constituents in licorice (Glycyrrhiza uralensis), a worldwide popular herbal medicine. This study aims to elucidate the metabolism of two major licorice coumarins, glycyrin and glycyrol in rats. After oral administration of 40mg/kg glycyrin, neither the parent compound nor its metabolites could be detected in rats plasma or urine samples, indicating that glycyrin had poor oral bioavailability. Two hydroxylated metabolites, 4″-hydroxyl glycyrin and 5″-hydroxyl glycyrin, were detected in rat liver microsome incubation system. Among them, the major metabolite 4″-hydroxyl glycyrin, which is a new compound, was obtained by microbial transformation of Syncephalastrum racemosum AS 3.264. Its structure was fully identified by 1D and 2D NMR. Meanwhile, glycyrol, together with three metabolites, were detected in rats urine and fecal samples after oral administration (40mg/kg). Their structures were tentatively characterized by LC/MS. Glycyrol mainly undertakes hydroxylation metabolism, accompanied by hydration and dehydrogenation as minor reactions. This is the first systematic study on metabolism of glycyrin and glycyrol. The results could be valuable to evaluate druggability of these bioactive natural products.

Plasma pharmacokinetics and cerebral nuclei distribution of major constituents of Psoraleae fructus in rats after oral administration

BACKGROUND The fruit of Psoralea corylifolia L., Psoraleae fructus (PF), is widely used in traditional Chinese medicine as a well-known herbal tonic. Previous studies have shown that PF and its major constituents may have potential values in the treatment of Parkinson and Alzheimer diseases, though their pharmacokinetics and brain distribution were largely unknown. PURPOSE To develop a liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for simultaneous studies of the plasma pharmacokinetics and cerebral nuclei (including cerebellum, thalamus, brainstem, hippocampus, corpus striatum and cortex) distribution in rats of eleven known PF compounds following as psoralen, isopsoralen, psoralidin, bavachin, bavachinin, isobavachin, isobavachalcone, bavachalcone, neobavaisoflavone, corylifol A, and corylin. METHODS Rats were orally administered via gavage at a single dose of PF extract at 1.2 g/kg. The eleven known PF compounds were extracted from rat plasma and cerebral nuclei at different time points, and then determined by the established LC-MS/MS method. Non-compartmental pharmacokinetic profiles were calculated, and the distribution in rat plasma and cerebral nuclei were compared. RESULTS The results showed that all the tested compounds were quickly absorbed into rat plasma and distributed almost evenly to the cerebral nuclei. The distribution concentrations at different nuclei varied at one determined time point, but the overall trends were basically similar to the plasma concentration-time results. Psoralen and isopsoralen, the two highest coumarins contained in PF, displayed far higher plasma concentrations (AUC0→∞, plasma≈53,884∼65,578 ng·h/ml) and central nervous system penetration (AUC0→∞, brain nuclei ≈44,659∼65,823 ng·h/g) than the prenylflavonoids (other compounds except psoralidin, AUC0→∞, plasma≈69∼324 ng·h/ml; AUC0→∞, brain nuclei ≈119∼3662 ng·h/g). However, the total brain-to-plasma ratios of the prenylflavonoids were higher than the coumarins, suggesting the prenylflavonoids can more readily enter the brain than the coumarins. CONCLUSION The established LC-MS/MS method is sensitive and specific for the simultaneous quantitation of the eleven PF compounds in rat plasma and cerebral nuclei. The results of plasma pharmacokinetics and cerebral nuclei distribution may reveal the possible substance basis for the CNS activities of PF, and highlight the application possibility of PF and its major constituents in the treatment of Parkinson and Alzheimer diseases.

Elucidation of Compatibility Interactions of Traditional Chinese Medicines: In Vitro Absorptions Across Caco‐2 Monolayer of Coptidis Rhizoma and Euodiae Fructus in Zuojin and Fanzuojin Formulas as A Case

Traditional Chinese medicines are often combined as formulae and interact with each other. As for Coptidis Rhizoma (CR) and Euodiae Fructus (EF), the most classical compatibilities were Zuojin (ZJF) and Fanzuojin formulas (FZJF) with reverse mixture ratios and opposite effects. To compare in vitro absorption interactions between CR and EF, bidirectional transports across Caco‐2 cell monolayer of extracts of two formulas and equivalent single herbs were studied. Eighteen alkaloids from CR and EF were determined by liquid chromatography coupled to tandem mass spectrometry. Parameter apparent permeability coefficient (Papp) and efflux rate (ER) values showed that most alkaloids were well or moderately absorbed and six quaternary protoberberine alkaloids from CR had obvious efflux. ZJF compatibilities reduced both Papp BL→AP and ER values of three indole alkaloids, and increased ER values of two quinolone alkaloids from EF. FZJF compatibilities obviously affected the bidirectional Papp values of CR alkaloids, weakened ERs of five protoberberines from CR and enlarged ERs of two quinolones from EF. Conclusions were drawn that different compatibility ratios of CR and EF led to different interactions on the in vitro absorption of alkaloids. The results may provide a good reference for interaction studies on the compatibilities of traditional Chinese medicines. Copyright