Berberine is a potential alternative for metformin with good regulatory effect on lipids in treating metabolic diseases.

Metformin (MTF) and berberine (BBR) share several therapeutic benefits in treating metabolic-related disorders. However, as the two agents have very different chemical structure and bioavailability in oral route, the goal of this study is to learn their characteristics in treating metabolic disorders. The therapeutic efficacy of BBR and MTF was systemically investigated in the high fat diet feeding hamsters and/or ApoE(-/-) mice; in parallel, gut microbiota related mechanisms were studied for both agents. We discovered that, although both two drugs had almost identical effects on reducing fatty liver, inflammation and atherosclerosis, BBR appeared to be superior over MTF in alleviating hyperlipidemia and obesity, but MTF was more effective than BBR for the control of blood glucose. Association analysis revealed that the modulation of intestinal microenvironment played a crucial role in the pharmacodynamics of both drugs, in which their respective superiority on the regulation of gut microbiota composition and intestinal bile acids might contribute to their own merits on lowering glucose or lipids. This study shows that BBR may be a good alternative for MTF in treating diabetic patients, especially for those complicated with dyslipidemia and obesity.

Microbiota-derived short-chain fatty acids mediate the effects of dengzhan shengmai in ameliorating cerebral ischemia via the gut-brain axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengzhan shengmai (DZSM) formula, composed of four herbal medicines (Erigeron breviscapus, Panax ginseng, Schisandra chinensis, and Ophiopogon japonicus), is widely used in the recovery period of ischemic cerebrovascular diseases; however, the associated molecular mechanism remains unclear. AIM OF THE STUDY: The purpose of this study was to uncover the links between the microbiota-gut-brain axis and the efficacy of DZSM in ameliorating cerebral ischemic diseases. MATERIALS AND METHODS: The effects of DZSM on the gut microbiota community and bacteria-derived short-chain fatty acid (SCFA) production were evaluated in vivo using a rat model of cerebral ischemia and in vitro through the anaerobic incubation with fresh feces derived from model animals. Subsequently, the mechanism underlying the role of SCFAs in the DZSM-mediated treatment of cerebral ischemia was explored. RESULTS: We found that DZSM treatment significantly altered the composition of the gut microbiota and markedly enhanced SCFA production. The consequent increase in SCFA levels led to the upregulation of the expression of monocarboxylate transporters and facilitated the transportation of intestinal SCFAs into the brain, thereby inhibiting the apoptosis of neurocytes via the regulation of the PI3K/AKT/caspase-3 pathway. The increased intestinal SCFA levels also contributed to the repair of the 2VO-induced disruption of gut barrier integrity and inhibited the translocation of lipopolysaccharide from the intestine to the brain, thus attenuating neuroinflammation. Consequently, cerebral neuropathy and oxidative stress were significantly improved in 2VO model rats, leading to the amelioration of cerebral ischemia-induced cognitive dysfunction. Finally, fecal microbiota transplantation could reproduce the beneficial effects of DZSM on SCFA production and cerebral ischemia. CONCLUSIONS: Our findings suggested that SCFAs mediate the effects of DZSM in ameliorating cerebral ischemia via the gut microbiota-gut-brain axis.

Relationship between Tissue Distributions of Modified Wuzi Yanzong Prescription () in Rats and Meridian Tropism Theory.

OBJECTIVE: To investigate the relationship between tissue distributions of modified Wuzi Yanzong prescription (, MWP) in rats and meridian tropism theory. METHODS: A high-performance liquid chromatography with Fourier transform-mass spectrometry (HPLC-FT) method was used to identify the metabolites of MWP in different tissues of rats after continued oral administration of MWP for 7 days. The relationship between MWP and meridian tropism theory was studied according to the tissue distributions of the metabolites of MWP in rats and the relevant literature. RESULTS: Nineteen metabolites, mainly flavanoid compounds, were detected in the different rat tissues and classified to each herb in MWP. Further, it was able to establish that the tissue distributions of the metabolites of MWP were consistent with the descriptions of meridian tropism of MWP available in literature, this result might be useful in clarifying the mechanism of MWP on meridian tropism. In the long run, these data might provide scientific evidence of the meridian tropism theory to further promote the reasonable, effective utilization, and modernization of Chinese medicine. CONCLUSION: The tissue distributions of MWP in vivo were consistent with the descriptions of meridian tropism of MWP.

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry.

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC-HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).

Mass spectral fragmentation analysis of triterpene saponins from Ardisia crenata Sims by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

We used the electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) technique to study the characteristic mass fragmentation patterns of eight triterpene saponins from Ardisia crenata Sims. Eight triterpene saponins were analyzed using parent mass list-triggered data-dependent multiple-stage accurate mass analysis at a resolving power of 100,000 in the external calibration mode. The chemical formula with unsaturation numbers was calculated from accurate m/z values of precursor, and product ions were obtained and used to assign the structures of eight triterpene saponins and two trace unknown compounds. The mass accuracies obtained for all full-scan MS and MS(n) spectra were within 7 ppm (< 5 ppm in most cases) in the ESI negative-ion mode. On FTICR-MS and FTICR-MS/MS, the eight triterpene saponins showed characteristic mass fragmentation patterns that facilitated the identification of their structural types, including the individual monosaccharide types, the monosaccharide numbers, and the sequences of the substituted saccharide groups. We proposed their fragmentation mechanisms. Based on their characteristic mass fragmentation patterns and fragmentation mechanisms, two unknown trace triterpene saponins were identified in the mixture.

Identification and characterization of flavonoids from semen zizyphi spinosae by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry.

Semen zizyphi spinosae (SZS) has been used to treat insomnia and anxiety for thousands of years. In this paper, a novel high-performance liquid chromatography coupled with the photodiode array detector/linear ion trap-MS(n) (HPLC-PDA/LTQ-MS(n)) method was established to separate and identify flavonoids from the extract of SZS. Separation was performed on an HYPERSIL C(18) column by gradient elution using CH(3)CN/H(2)O-CH(3)COOH as the mobile phase at a flow rate of 0.8 ml/min. UV spectral data, accurate molecular weights, and multi-stage MS/MS fragmentation information were obtained. Electrospray ionization/MS/MS fragmentation patterns were proposed. Nineteen flavonoid glycosides were identified or tentatively characterized based on their retention time, UV spectral data, accurate molecular weights, and mass fragmentation behavior. The method was useful for separation and identification of the flavonoid components from SZS and could be applied to other complex samples, especially for minor constituents.

A rapid resolution liquid chromatographic method for fingerprint analysis of raw and processed caowu (Aconitum kusnezoffii).

A sensitive and reliable rapid resolution liquid chromatographic (RRLC) method coupled with diode array detection has been developed for the fingerprint analysis of raw and processed caowu (Aconitum kusnezoffii). The RRLC fingerprints were established with a Zorbax Extend C18 analytical column (4.6 x 50 mm, 1.8 microm) and gradient elution. Analysis run time was <10 min. The method displayed good precision, stability, and repeatability in fingerprint analysis. Characteristic RRLC fingerprints of caowu were generated and used to assess the consistency and differences in the products. Raw and processed caowu from different sources were analyzed under the developed RRLC conditions, yielding contrasting RRLC fingerprints. Comparison of the RRLC fingerprints of processed and raw caowu indicated that the major constituents changed during processing. Meanwhile, a peak area ratio analysis method was applied to assess their chromatographic fingerprints. In characterizing the constituents of caowu, 11 major chromatographic peaks were identified by mass spectrometry and compared with reference standards and reference data. In summary, RRLC fingerprinting is a rapid and useful way to evaluate the quality of raw and processed caowu.

Fingerprinting analysis of Rhizoma chuanxiong of commercial types using 1H nuclear magnetic resonance spectroscopy and high performance liquid chromatography method.

The (1)H nuclear magnetic resonance ((1)H NMR) fingerprints of fractionated non-polar extracts (control substance for a plant drug (CSPD) A) from Rhizoma chuanxiong, the rhizomes of Ligusticum chuanxiong Hort., of seven specimens from different sources were measured on Fourier Transform (FT)-NMR spectrometer and assigned by comparing them with the (1)H NMR spectra of the isolated pure compounds. The (1)H NMR fingerprints showed exclusively characteristic resonance signals of the major special constituents of the plant. Although the differences in the relative intensity of the (1)H NMR signals due to a discrepancy in the ratio of the major constituents among these samples could be confirmed by high performance liquid chromatography analysis, the general features of the (1)H NMR fingerprint established for an authentic sample of the rhizomes of L. chuanxiong exhibited exclusive data from those special compounds and can be used for authenticating L. Chuanxiong species.

Steroidal glycosides from Dregea sinensis var. corrugata screened by liquid chromatography-electrospray ionization tandem mass spectrometry.

The MS fragmentation behavior of the C-21 steroidal glycosides from Dregea sinensis var. corrugata was investigated by positive and negative ion electrospray ionization MS using a multistage tandem mass spectrometer equipped with an ion trap analyzer. The mass fragmentation patterns of steroidal glycosides substituted with an orthoacetate group were summarized, and the fragmentation patterns were applied to the online structure identification of the steroidal glycosides in the extract. Eighteen new C-21 steroidal glycosides were identified by means of HPLC-HRESIMS and HPLC-DAD-ESIMS(n). Three new compounds (1, 4, and 7) were identified by HPLC-DAD-ESIMS(n), and their structures were elucidated by use of 1D and 2D NMR methods. The structures identified by MS are fully consistent with those elucidated by NMR data. The present study shows that HPLC-DAD-ESIMS(n) can be used as an effective tool to rapidly identify compounds and guide the isolation of target compounds from crude plant extracts.

Chemical constituents of the stems of Ephedra sinica.

A new naphthalene derivative, 1-methyl-2,3-methylenedioxy-6-naphthalenecarboxylic acid methyl ester (1), and a new alkaloid, ( +/- )-1-phenyl-2-imido-1-propanol (2), together with the four known compounds, ephedrine, pseudoephedrine, N-methylephedrine, and 6-methoxykynurenic acid, have been isolated from the stems of Ephedra sinica, a famous traditional Chinese herbal medicine. The structures of 1 and 2 were determined by spectroscopic methods, including 1D- and 2D-NMR experiments as well as HR-EI-MS analysis.

New spermidines from the florets of Carthamus tinctorius.

Two new spermidine compounds, namely safflospermidine A (1) and safflospermidine B (2), together with two known compounds, N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (3) and N(1),N(5),N(10)-(E)-tri-p-coumaroylspermidine (4), were isolated from the florets of Carthamus tinctorius L. Their structures were elucidated by spectroscopic means.

LC-MS determination and pharmacokinetic studies of paeonol in rat plasma after administration of different compatibility of Su-Xiao-Xin-Tong prescriptions.

A rapid and specific liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method has been developed and validated for the identification and quantification of paeonol in rat plasma. Paeonol and internal standard were isolated from plasma samples by liquid-liquid extraction with chloroform. The chromatographic separation was accomplished on a Zorbax-SB C18 column (100x2.1 mm, 3.5 microm). The mobile phase consisted of acetonitrile and 0.1% aqueous formic acid (64:36) was delivered at a flow rate of 0.2 mL/min. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring mode via electrospray ionization source. Linearity was established for the range of concentrations 0.0525-15.8 microg/mL with a coefficient correlation (r) of 0.9995. The intra- and inter-day precision (RSD%) was lower than 9.34% and accuracy ranged from 93.7 to 102.3%. The lower limit of quantification was 0.0525 microg/mL. The proposed method was used to determine the concentration of paeonol for pharmacokinetic studies. The pharmacokinetics of different compatibility prescriptions of Su-Xiao-Xin-Tong were studied and compared.

Structural characterization of constituents with molecular diversity in fractions from Lysidice brevicalyx by liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance.

A combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography (HPLC/ESI-MSn), and hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR), have been extensively utilized for on-line analysis of natural products, analyzing metabolite and drug impurity. In our last paper, we reported an on-line analytical method for structural identification of trace alkaloids in the same class. However, the structural types of the constituents in plants were various, such as flavanoids, terpenoids and steroids. It is important to establish an effective analytical method for on-line structural identification of constituents with molecular diversity in extracts of plants. So, in the present study, the fragmentation patterns of some isolated stilbenes, phloroglucinols and flavanoids from Lysidice rhodostegia were investigated by ESI-MSn. Their fragmentation rules and UV characteristics are summarized, and the relationship between the spectral characteristics, rules and the structures is described. According to the fragmentation rules, NMR and UV spectral characteristics, 24 constituents of different types in the fractions from L. brevicalyx of the same genus were structurally characterized on the basis of HPLC/HRMS, HPLC-UV/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Of these, six (10, 13, 14, 16, 17 and 23) are new compounds and all of them are reported from L. brevicalyx for the first time. The aim is to develop an effective analytical method for on-line structural identification of natural products with molecular diversity in plants, and to guide the rapid and direct isolation of novel compounds by chemical screening.

[Evaluation of the quality of Gastrodia elata Bl. by HPLC-DAD/MS].

The chromatographic fingerprint of Gastrodia elata Bl. (Tianma) was developed to compare the quality of Tianma samples from different habitats and processing methods. The above analysis method was established by HPLC-DAD technique. And an HPLC method was used to analysis the contents of gastrodin (GAS) and p-hydroxybenzyl alcohol (HBA) in Tianma from different habitats and processed methods. Experiments of chromatographic fingerprint analysis were carried out with a Zorbax XDB C18 column (250 mm x 4.6 mm, 5 microm). The mobile phase consisted of acetonitrile and 0.1% aqueous acetic acid in gradient elution mode. The column was maintained at 25 degrees C. Detection was set at 270 nm. The mass spectra were recorded using as ESI source in the negative mode with ion spray voltage at 3500 V, source temperature at 335 degrees C, gas spray at 8.3 kPa and gas flow rate at 9 L x min(-1). The HPLC methods of quantitative analysis were the same as those of chromatographic fingerprint analysis except the mobile phase, which consisted of acetonitrile and 0.1% aqueous acetic acid in isocratic elution mode with the ratio of 4.5 to 95.5 (v/v). Data of chromatographic fingerprint were analyzed by the "similarity evaluation system for chromatographic fingerprint of TCM (Version 2004 A)" software to compare the quality of Tianma. Samples from different habitats with the same processing method were of high similarity, though a few samples showed evident difference in fingerprint graphics. For Tianma samples with different processing methods, the contents of common peaks were different and the processing method of freezing to dry was better than others. With HPLC-MS technique, 8 major common peaks in the fingerprint of Tianma were identified by their MS spectra and comparison with the reference standards. The results of similarity analysis for chromatographic fingerprint were basically consistent with those of quantitative analysis. The established HPLC-DAD/MS methods can be used to evaluate the quality of Tianma.

Determination of hydroxysafflor yellow A in rat plasma and tissues by high-performance liquid chromatography after oral administration of safflower extract or safflor yellow.

A simple and reproducible HPLC method for quantification of hydroxysafflor yellow A (HSYA) in rat plasma and tissues after oral administration of safflower extract or safflor yellow (SY) was developed. Sample preparation was achieved by protein precipitation of plasma and tissue homogenates with three volumes of methanol. p-Hydroxybenzaldehyde was used as the internal standard (IS). HSYA and IS were separated on a Hypersil BDS-C(18) column with a gradient elution system composed of acetonitrile and aqueous acetic acid. UV detection was used at 320 nm. The calibration curves were linear in all matrices (r(2) > 0.999) in the concentration ranges 0.51-101.36 microg/mL for plasma, 12.27-2454.46 microg/g for intestines and 0.96-192.20 microg/g for lung. The intra-day and inter-day precision were all less than 12.5%, and the extract recovery was in the range 64.1-103.7% with RSD less than 15.6% for HSYA in all matrices. The method was used successfully to quantify HSYA in rat plasma and tissue samples to support a pharmacokinectic study.

[Quality analysis and evaluation of Rhizoma Coptidis under different cultivation conditions].

AIM: To develop methods for the fingerprint analysis of Rhizoma Coptidis and the determination of berberine, palmatine and jatrorrhizine in Rhizoma Coptidis, and analyze the contents of these three alkaloids in Rhizoma Coptidis under different cultivation conditions, from different areas and processed with different methods. METHODS: Two methods (HPLC-UV and HPLC-MS) have been developed and used in fingerprint analysis of Rhizoma Coptidis. An HPLC method was used to determine the contents of three alkaloids. RESULTS: With HPLC-MS techniques, seven major chromatographic peaks in the fingerprint analysis of Rhizoma Coptidis were identified by their MS spectra and compared with the reference standards. In different cultivation conditions, shading conditions and growing ages have obvious influence on the contents of three alkaloids in Rhizoma Coptidis, while planting density was not the major factor that influenced the contents of three alkaloids. The contents of three alkaloids of Coptidis samples were almost higher than those of Coptidis reference material. For Coptidis samples from different cultivation area, the contents of these three alkaloids were different greatly. For Coptidis samples processed with different methods, the contents of three alkaloids were not influenced obviously by processing methods. CONCLUSION: The results showed that the ecology cultivation method to replace the traditional shading method was feasible and provided the theoretical foundation for scientifically processing Rhizoma Coptidis.

High-performance liquid chromatographic assay for the active saponins from Panax notoginseng in rat tissues.

A reversed-phase liquid chromatographic method was used to determine the ginsenosides Rg1, Rb1 and Rd of Panax notoginseng in rat tissues (kidney, liver, heart, spleen and lung) after the administration of total saponins of P. notoginseng. The tissue samples were treated with solid-phase extraction prior to HPLC. The calibration curves for the three saponins were linear in the given concentration ranges. The intra-day and inter-day assay coefficients in tissues were between 76 and 120% respectively. The recoveries of all the tissues were higher than 70%. This method was applied to evaluate the distribution of the three major saponins of P. notoginseng in rat tissues.

Simultaneous quantification of six major active saponins of Panax notoginseng by high-performance liquid chromatography-UV method.

A simple, sensitive and specific high-performance liquid chromatography-UV (HPLC-UV) method has been developed for the first time to simultaneously quantify the six major active saponins of Panax notoginseng, namely notoginsenoside R1, ginsenoside Rg1, Rb1, Rg2, Rh1 and Rd. Astragaloside IV is used as the internal standard. This HPLC assay was performed on a reversed-phase C18 column with gradient elution of acetonitrile and 0.01% formic acid in 30 min. The method provided good reproducibility and sensitivity for the quantification of six saponins with overall intra- and inter-day precision and accuracy of less than 4.0% and higher than 90%, respectively. This assay is successfully applied to the determination of the six saponins in 23 notoginseng samples. The results indicated that the developed HPLC assay can be readily utilized as a quality control method for P. notoginseng.

Metabolism of ferulic acid in rats.

Ferulic acid is the major active constituent in many natural Chinese medicinal herbs. The metabolism of ferulic acid has been investigated using solid-phase extraction and HPLC-DAD methods that were established to separate and analyze the metabolites in urine, feces and bile. Three metabolites, identified by enzymatic hydrolysis, HPLC-DAD, HPLC-MS and MS/MS, are all glucuronic acid conjugates of ferulic acid. Ferulic acid conjugated with one glucuronic acid at different positions produces M1 and M3. Ferulic acid conjugated with two glucuronic acids produces M2, which is the main metabolite. A metabolic pathway is proposed.

Chemical fingerprint and metabolic fingerprint analysis of Danshen injection by HPLC-UV and HPLC-MS methods.

HPLC-UV and HPLC-MS techniques were used in fingerprint analysis of Danshen injection and its raw materials (roots and rhizoma of Salvia miltiorrhiza). HPLC profiles of Danshen injections from a Chinese pharmaceutical factory and their raw materials were established as their characteristic fingerprint and employed to assess their consistency and difference. To develop the representative fingerprint of Danshen injection, 10 batches of samples were analyzed under the same HPLC conditions. The results showed that 10 batches of Danshen injections had very similar HPLC fingerprints. To characterize the major constituents of Danshen injection for quality control, 11 major chromatographic peaks were characterized by their MS spectra and comparison with the reference standards. Through comparison of the HPLC profiles of Danshen injection with its raw material, it was found that they are greatly different, which indicated the changes of major constituents in the course of preparation procedure. In addition, the rat's plasma was analyzed by HPLC-MS technique after intravenous administration of Danshen injection at different time intervals to explore the in vivo metabolism of the major active constituents. Except for protocatechuic aldehyde, the major phenolic acids in Danshen injection appeared in rat's plasma after intravenous administration, but quantity of each phenolic acids was very different from that in Danshen injection. With the administration time prolonged danshensu and salvianolic acid B disappeared quickly, salvianolic D, lithospermic acid and salvianolic A slowly decreased and maintained relatively high concentration after 30 min of intravenous administration. This indicated that polyphenolic acids were significant for biological activity of Danshen injection. It might be concluded that chemical fingerprint combined with metabolic fingerprint is a useful means to control the quality and to clarify the possible mechanism of action of herbal products.