Rapid Determination of 30 Polyphenols in Tongmai Formula, a Combination of Puerariae Lobatae Radix, Salviae Miltiorrhizae Radix et Rhizoma, and Chuanxiong Rhizoma, via Liquid Chromatography-Tandem Mass Spectrometry.

Tongmai formula (TMF) is a herbal preparation composed of three traditional Chinese medicinal materials: Puerariae Lobatae Radix (Gegen), Salviae Miltiorrhizae Radix et Rhizoma (Danshen) and Chuanxiong Rhizoma (Chuanxiong). It has been used to treat cardiovascular diseases for decades. To develop a reliable and convenient analytical method for a comprehensive determination of polyphenols in TMF and the ascertainment of their chemical correlations with its herbal components, a method combining high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was developed and validated for rapid determination of 30 polyphenols in TMF and its three herbal components. The chromatographic separation was carried out on a Chromolith Fastgradient RP-18 endcapped 50-2 column using an optimized gradient elution. Statistical analysis of obtained data demonstrated that the method had a desirable linearity, precision, and accuracy, as well as excellent sensitivity. The obtained results indicated that, among the 30 polyphenols in TMF, 22 originated from Gegen, 6 originated from Danshen, and 2 originated from Chuanxiong. The major polyphenols in TMF have been identified as puerarin, mirificin, salvianolic acid B, salvianic acid A, 3'-hydroxypuerarin, 3'-methoxypuerarin, and salvianolic acid A, with a combined contribution of 19.2% of the preparation. The development and validation of this method will greatly facilitate future pharmacological studies of TMF and its herbal components, as well as polyphenols in cardiovascular therapies.

Metabolism of 20(S)-Ginsenoside Rg2 by Rat Liver Microsomes: Bioactivation to SIRT1-Activating Metabolites.

20(S)-Ginsenoside Rg2 (1) has recently become a hot research topic due to its potent bioactivities and abundance in natural sources such as the roots, rhizomes and stems-leaves of Panax ginseng. However, due to the lack of studies on systematic metabolic profiles, the prospects for new drug development of 1 are still difficult to predict, which has become a huge obstacle for its safe clinical use. To solve this problem, investigation of the metabolic profiles of 1 in rat liver microsomes was first carried out. To identify metabolites, a strategy of combined analyses based on prepared metabolites by column chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was performed. As a result, four metabolites M1-M4, including a rare new compound named ginsenotransmetin A (M1), were isolated and the structures were confirmed by spectroscopic analyses. A series of metabolites of 1, MA-MG, were also tentatively identified by UPLC-Q-TOF/MS in rat liver microsomal incubate of 1. Partial metabolic pathways were proposed. Among them, 1 and its metabolites M1, M3 and M4 were discovered for the first time to be activators of SIRT1. The SIRT1 activating effects of the metabolite M1 was comparable to those of 1, while the most interesting SIRT1 activatory effects of M3 and M4 were higher than that of 1 and comparable with that of resveratrol, a positive SIRT1 activator. These results indicate that microsome-dependent metabolism may represent a bioactivation pathway for 1. This study is the first to report the metabolic profiles of 1 in vitro, and the results provide an experimental foundation to better understand the in vivo metabolic fate of 1.

High-Performance Liquid Chromatography with Diode Array Detector and Electrospray Ionization Ion Trap Time-of-Flight Tandem Mass Spectrometry to Evaluate Ginseng Roots and Rhizomes from Different Regions.

Ginseng, Panax ginseng C. A. Meyer, is an industrial crop in China and Korea. The functional components in ginseng roots and rhizomes are characteristic ginsenosides. This work developed a new high-performance liquid chromatography coupled with electrospray ionization ion trap time-of-flight multistage mass spectrometry (LC-ESI-IT-TOF-MS(n)) method to identify the triterpenoids. Sixty compounds (1-60) including 58 triterpenoids were identified from the ginseng cultivated in China. Substances 1, 2, 7, 15-20, 35, 39, 45-47, 49, 55-57, 59, and 60 were identified for the first time. To evaluate the quality of ginseng cultivated in Northeast China, this paper developed a practical liquid chromatography-diode array detection (LC-DAD) method to simultaneously quantify 14 interesting ginsenosides in ginseng collected from 66 different producing areas for the first time. The results showed the quality of ginseng roots and rhizomes from different sources was different due to growing environment, cultivation technology, and so on. The developed LC-ESI-IT-TOF-MS(n) method can be used to identify many more ginsenosides and the LC-DAD method can be used not only to assess the quality of ginseng, but also to optimize the cultivation conditions for the production of ginsenosides.

Liquid Chromatography with Tandem Mass Spectrometry: A Sensitive Method for the Determination of Dehydrodiisoeugenol in Rat Cerebral Nuclei.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is developed for the quantification of dehydrodiisoeugenol (DDIE) in rat cerebral nuclei after single intravenous administration. DDIE and daidzein (internal standard) were separated on a Diamonsil™ ODS C18 column with methanol-water containing 0.1% formic acid (81:19, v/v) as a mobile phase. Detection of DDIE was performed on a positive electrospray ionization source using a triple quadrupole mass spectrometer. DDIE and daidzein were monitored at m/z 327.2→188.0 and m/z 255.0→199.2, respectively, in multiple reaction monitoring mode. This method enabled quantification of DDIE in various brain areas, including, cortex, hippocampus, striatum, hypothalamus, cerebellum and brainstem, with high specificity, precision, accuracy, and recovery. The data herein demonstrate that our new LC-MS/MS method is highly sensitive and suitable for monitoring cerebral nuclei distribution of DDIE.

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.

[Study on material base of corydalis rhizoma].

Corydalis Rhizoma, the dried tuber of Corydalis yanhusuo (Papaveraceae) distributed traditionally mainly in south-eastern and now cultivated in northwestern and other district in China, is one of the commonly used and well-known traditional Chinese medicine. It has been widely used to treat spastic pain, abdominal pain, pain due to injury, and promote blood circulation. Its main chemical constituents were alkaloids, which were divided into the two types of protoberberines and aporphines. Among them, some alkaloids were found to elicit profound effects on the dopaminergic system in the central nervous system, which plays an important role in regulating nociception. In this article, the chemical composition and structure-types, new methods of qualitative and quantitative analysis as well as characteristics of biotransformation, absorption, distribution, metabolism, excretion, pharmacokinetic, and drug-drug interaction for the alkaloids were revealed. These results would greatly contribute to the establishment of bioactive material base of Corydalis Rhizoma.

Limonoid constituents of Euodia rutaecarpa var. bodinieri and their inhibition on NO production in lipopolysaccharide-activated RAW264.7 macrophages.

A new limonoid compound, named evorubodinin (1), was isolated from the dried and nearly ripe fruits of Euodia rutaecarpa (Juss.) Benth. var. bodinieri (Dode) Huang (family Rutaceae), together with two known limonoid compounds, limonin (2) and evolimorutanin (3). The chemical structure of 1 was elucidated by spectroscopic method and single-crystal X-ray diffraction. The inhibitory effects of the isolated compounds 1-3 and the structurally related compounds evodol (4), shihulimonin A1 (5), evodirutaenin (6), 12α-hydroxyrutaevin (7), and rutaevin (8) on nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 macrophages were also assayed. All compounds 1-8 showed the inhibitory activity, in which both 7 and 8 with the uncommon 5ß-H configuration more efficiently inhibited NO production. The results provided valuable information for further investigation of compounds 1-8 as anti-inflammatory agents or lead compounds.

Ginsenjilinol, a new protopanaxatriol-type saponin with inhibitory activity on LPS-activated NO production in macrophage RAW 264.7 cells from the roots and rhizomes of Panax ginseng.

One new dammarane triterpene saponin named ginsenjilinol (1) was isolated from the roots and rhizomes of Panax ginseng C.A. Mey., together with two known saponins ginsenoside Rf (2) and ginsenoside Re5 ( = panajaponol A, 3). Based on IR, HR-ESI-MS, and 1D as well as 2D NMR ((1)H-(1)H COSY, NOESY, HSQC, and HMBC) spectral data, the chemical structure of the new saponin was elucidated as 3ß,12ß,20S,26-tetrahydroxydammar-24E-en-6α-O-ß-d-glucopyranosyl-(1 â†’ 2)-O-ß-d-glucopyranoside. The ability of the isolated saponins to inhibit nitric oxide production by lipopolysaccharide-activated RAW 264.7 cells was also assayed. All of the isolated saponins exhibited the significant activity in a concentration-dependent manner at concentrations of 60-200 µM with the half maximal inhibitory concentration (IC50) values of 70.96 ± 2.05 µM for 1, 74.14 ± 2.65 µM for 2, and 79.83 ± 1.78 µM for 3, respectively, whereas indomethacin had an IC50 of 63.75 ± 3.33 µM as a positive control drug.

Intestinal bacterial transformation - a nonnegligible part of Chinese medicine research.

Intestinal bacteria play an essential part in the metabolism of the constituents of herbal drugs, and a lot of investigations have been done to unveil their functions and mechanisms in modification of these constituents and their effect. This review provides a progressive description of intestinal bacterial transformation with respect to properties, reactions, correlation with the effect of herbal drugs, research interests, and methodology. In addition, the problems encountered during the investigation are addressed and perspectives are proposed.

[Biotransformation by human intestinal flora and absorption-transportation characteristic in a model of Caco-2 cell monolayer of d-corydaline and tetrahydropalmatine].

OBJECTIVE: To study the biotransformation by human intestinal flora, and the absorption and transportation characteristic in a model of human colon adenocarcinoma cell lines (Caco-2 cell) monolayer of d-corydaline (CDL) and tetrahydropalmatine (THP). METHOD: CDL or THP was incubated with crude enzymes of human intestinal flora under the anaerobic environment and 37 degrees C conditions to transform CDL or THP. Caco-2 cell monolayer was used as an intestinal epithelial cell model for determination of the permeability of CDL or THP from apical side (AP side) to basolateral side (BL side) or from BL side to AP side. Transportation parameters and permeability coefficients (P(app)) were then calculated, and P(app) values were compared with the reported values for model compounds, propranolol as a well absorbed drug and atenolol as a poor absorbed drug. The concentration of CDL or THP was measured by HPLC coupled with photodiode array detector. RESULT: CDL or THP in the human intestinal flora incubation system did not happen biotransformation. In the Caco-2 cell monolayer model, the P(app) magnitudes of both CDL and THP were 1 x 10(-5) cm x s(-1) in the bi-directional transport, which were identical with propranolol. And their transports were concentration dependent between 0-180 min. CONCLUSION: Both CDL and THP may be stable in the human intestinal flora incubation system, and their absorption and transportation in the human Caco-2 cell monolayer model are mainly via passive diffusion mechanism.

[Chemical constituents from roots and rhizomes of Panax ginseng cultivated in Jilin province].

The chemical constituents of the roots and rhizomes of Panax ginseng were systematically investigated by various column chromatographic methods including Amberlite XAD-4 macroporous adsorptive resins and silica gel as well as high-performance liquid chromatography, and their chemical structures were identified by physico-chemical properties and spectral analyses. Twenty-eight compounds were isolated from the 70% ethanolic-aqueous extract and identified as koryoginsenoside R1 (1), ginsenoside Rg1 (2), ginsenoside Rf (3), notoginsenoside R2 (4), ginsenoside Rg2 (5), notoginsenoside Fe (6), ginsenjilinol (7), ginsenoside Re5 (8), noto-ginsenoside N (9), notoginsenoside R1 (10), ginsenoside Re2 (11), ginsenoside Re1 (12), ginsenoside Re (13), ginsenoside Rs2 (14), ginsenoside Ro methyl ester (15), ginsenoside Rd (16), ginsenoside Re3 (17), ginsenoside Re4 (18), 20-gluco-ginsenoside Rf (19), ginsenoside Ro (20), ginsenoside Rc (21), quinquenoside-R1 (22), ginsenoside Ra2 (23), ginsenoside Rb1 (24), ginsenoside Ra1 (25), ginsenoside Ra3 (26), ginsenoside Rb2 (27), and notoginsenoside R4 (28). All isolated compounds are 20 (S) -protopanaxadiol or protopanaxatriol type triterpenoid saponins. Compound 1 was isolated from the roots and rhizomes of P. ginseng cultivated in Jilin province for the first time and compound 6 was isolated from the roots and rhizomes of P. ginseng for the first time. The 1H-NMR data of compounds 6, 14 and 19 were assigned for the first time.

[Flavonoid glycosides from dried and nearly ripe fruits of Evodia rutaecarpa].

OBJECTIVE: To study the chemical constituents from the dried and nearly ripe fruits of Evodia (Euodia) rutaecarpa. METHOD: The compounds were separated and purified by solvent and chromatographic methods. Their structures were identified by spectroscopic techniques. RESULT: Fifteen compounds were separated from the normal butanol extracts of the 70% aqueous ethanol extract of the dried and nearly ripe fruits of E. rutaecarpa. Among of them, four compounds were reported in the essay and identified as diosmetin-7-O-beta-D-glucopyranoside (1), isorhamnetin-3-O-rutinoside (2), diosmin (3) and chrysoeriol-7-O-rutinoside (4). CONCLUSION: Compounds 1, 3 and 4 were separated from the dried and nearly ripe fruits of E. rutaecarpa for the first time.

Biotransformation products of phellopterin by rat liver microsomes and the inhibition on NO production in LPS-activated RAW264.7 cells.

Four new coumarins (2',3'-dihydroxyphellopterin, E-5-methoxytrichoclin acetate, Z-5-methoxytrichoclin acetate, and E-5-methoxytrichoclin) and three known coumarins (byakangelicol, byakangelicin, and Z-5-methoxytrichoclin) were produced by liver microsomes from rats pre-treated with sodium phenobarbital. The chemical structures were elucidated on the basis of their spectroscopic data. The inhibitory activities of nitric oxide (NO) production in lipopolysaccharide-activated macrophage-like cell line RAW264.7 were tested. The main biotransformation product, byakangelicin, showed inhibitory activities of NO production with the IC50 value of 217.83 µM, whereas the parent compound phellopterin showed cytotoxic effect on RAW264.7 cell at the concentration from 40 to 400 µM.

Aquisiflavoside, a new nitric oxide production inhibitor from the leaves of Aquilaria sinensis.

A new flavonoid glycoside, named aquisiflavoside, was isolated from the leaves of Aquilaria sinensis (Lour.) Gilg, and its structure was elucidated by spectroscopic analyses. Aquisiflavoside was evaluated for the inhibition of nitric oxide (NO) production induced by lipopolysaccharide in macrophage cell line RAW 264.7 and exhibited a potent inhibitory activity against NO production in a dose-dependent manner with an IC(50) value of 34.95 µM.

Biotransformation of prim-O-glucosylcimifugin by human intestinal flora and its inhibition on NO production and DPPH free radical.

prim-O-Glucosylcimifugin (PGCN), a highest content chromone in the roots of Saposhnikovia divaricata, was incubated with human intestinal flora (HIF), and two biotransformation products were obtained from the incubated solution by chromatographic methods. The chemical structures of the two biotransformation products were elucidated as cimifugin (CN) and 5-O-methylvisamminol (MVL), respectively, on the basis of NMR and MS data. The biotransformation product CN was formed through a deglucosylation of PGCN by ß-glucosidase secreted from the HIF, and then the hydroxymethyl group of CN was reduced to lead to occurrence of MVL. All of these compounds were evaluated for their effect on the inhibition of nitric oxide production induced by lipopolysaccharide in macrophage cell line RAW 264.7 and for 1,1-diphenyl-2-picrylhydrazyl free-radical scavenging activity in cell-free bioassay system.

New triterpenoids from the stems and leaves of Panax ginseng.

Three new dammarane-type triterpene saponins ginsenosides Rh(18) (1), Rh(19) (3) and Rh(20) (4), along with two new triterpene sapogenins 12ß,23(R)-epoxydammara-24-ene-3ß,6α,20(S)-triol (2) and dammara-(20E)22,25-diene-3ß,6α,12ß,24S-tetrol (5) were isolated from the stems and leaves of Panax ginseng C. A. Mey. Their structures were elucidated on the basis of spectral evidence and comparisons with literature data.

Three new isoflavone glycosides from Tongmai granules.

Three new isoflavone glycosides, 3'-methoxydaidzein-7,4'-di-O-ß-d-glucopyranoside (1), biochanin A-8-C-ß-d-apiofuranosyl-(1 â†’ 6)-O-ß-d-glucopyranoside (2), daidzein-7-O-ß-d-glucopyranosyl-(1 â†’ 4)-O-ß-d-glucopyranoside (3), and a new natural isoflavone glycoside, daidzein-7-O-α-d-glucopyranosyl-(1 â†’ 4)-O-ß-d-glucopyranoside (4) were isolated along with 18 known isoflavones from the EtOAc and n-BuOH fractions of the aqueous extraction of Tongmai granules. All the isoflavones were obtained and determined for the first time from Tongmai granules. The structures of these compounds were elucidated by spectral methods. It was confirmed that the compounds 1-4 were originally from Puerariae Lobatae Radix based on HPLC-DAD analysis of the crude drug extract. The isoflavones isolated were tested for their antioxidative activities by measuring the capacities of scavenging the 2,2'-diphenyl-1-picrylhydrazyl radical.

Intestinal permeability of the constituents from the roots of Saposhnikovia divaricata in the human Caco-2 cell monolayer model.

The bidirectional intestinal permeability of the active constituents from the roots of Saposhnikovia divaricata, including four coumarins, anomalin (1), 5-methoxy-7-(3,3-dimethylallyloxy)coumarin (2), decursin (3), and decursinol angelate (4), as well as four chromones, cimifugin (5), prim-O-glucosylcimifugin (6), 3'- O-angeloylhamaudol (7), and sec-O-glucosylhamaudol (8), was studied by using the Caco-2 cell monolayer. These compounds were assayed by HPLC, and their transport parameters, including apparent permeability coefficients (P(app)), were then calculated. The bidirectional P(app) values of the compounds were compared with those of the markers, propranolol and atenolol. Compounds 1-5 and 7 were assigned to well-absorbed compounds, while 6 and 8 were assigned to moderately absorbed compounds. The transport of 1-7 increased linearly as a function of time up to 180 min and concentration within the test range of 10-200 µM, thus their passive diffusion mechanism was proposed. The results provided some useful information for predicting the intestinal absorption in vivo of these compounds.