Study on metabolites of Laportea bulbifera extract in rat feces based on UHPLC-Q-TOF-MS~E technique / 中国中药杂志

This project is to study the metabolites of Laportea bulbifera extract in rat feces. After the SD rats were gavaged with the extract(136 g·kg~(-1), according to the crude drug dose), the metabolites in their feces were detected by UHPLC-Q-TOF-MS~E technique, and the obtained mass spectrometry data was combined with UNIFI software for prediction. The prototype components and metabolites in rat feces were identified with reference materials and related literature. A total of 43 metabolites were identified(including 8 prototype components and 35 metabolites). The metabolic pathways mainly include monocaffeoylquinic acid(hydrogenation reduction, ring-opening cracking, sulfation, hydroxylation, glucuronidation), quercetin(O-C2 bond ring-opening cleavage, C2-C3 double bond reduction, rutin carbonylation) and so on. The metabolites and metabolic process of L. bulbifera extract in rat feces were clarified, which provided a basis for the study of the active substances and its mechanism of action.

Spectrum-effect Relationship of Anti-inflammatory Active Components from Nonvolatile Fraction of Blumea balsamifera Based on Gray Correlation Analysis / 中国实验方剂学杂志

Objective: To study the correlation between UPLC fingerprint of anti-inflammatory effect of active components from nonvolatile fraction of Blumea balsamifera, and to provide the basis for clarifying the anti-inflammatory material basis of B. balsamifera. Method: UPLC was used to establish fingerprint of nonvolatile fraction of 12 batches of B. balsamifera and their common fingerprint peaks were identified by UPLC-Q-TOF-MS.The corresponding pharmacodynamic data were obtained by auricle swelling and inflammation model mice induced by xylene, and spectrum-effect relationship was established by gray correlation analysis. Result: A total of 14 common peaks in nonvolatile fraction of B. balsamifera were established by UPLC fingerprint and 9 common peaks of them were identified.The correlation between UPLC fingerprint and the anti-inflammatory activity was from 0.717 1 to 0.550 5.The contribution of chemical compositions represented by each characteristic peak to the anti-inflammatory efficacy was in the order of peak 3 > peak 9 > peak 4 > peak 11 > peak 2 > peak 1 > peak 14 > peak 7 > peak 6 > peak 5 > peak 12 > peak 8 > peak 10 > peak 13, and the top two peaks with strong contribution to anti-inflammatory effect were peak 3 and peak 9, they were 3-O-caffeoylquinic acid and 3, 5-di-O-caffeoylquinic acid identified by contrast reference substances, respectively. Conclusion: The active substances in nonvolatile fraction of B. balsamifera are obtained through the study on the relationship between spectrum and efficiency, and the anti-inflammatory efficacy of the nonvolatile fraction is the result of combination of various components.It is clear that the caffeoylquinic acid derivates act as predominant anti-inflammatory active substance of nonvolatile fraction of B. balsamifera.

Effect of Inula cappa on CYP450 Enzyme in Rats by Cocktail Probe Drugs / 中国实验方剂学杂志

Objective:To study on the effect of Inula cappa extract on the activities of six cytochrome P450(CYP450) enzymes in rats by Cocktail probe method. Method:Rats in the I. cappa high and low dose groups were given oral administration of active fractions of I. cappa at a dose of 8.127,2.709 g·kg-1·d-1 of the material for 7,14 d,repectively.Probe drugs(caffeine,midazolam,tolbutamide,omeprazole,metoprolol,chlorzoxazone) were simultaneously injected to rats after administration.Plasma was collected at different time after the administration of probe drugs.The plasma concentrations of these six probes were measured by UPLC-MS and their corresponding pharmacokinetic parameters were calculated with DAS 2.0. Result:Compared with the control group,only the apparent volume of distribution(V) of midazolam was increased;area under the curve(AUC0-t and AUC0-∞)and half-life period(T1/2) of caffeine,midazolam,tolbutamide and omeprazole were increased and the clearance rate(CL) of them was decreased in rats of I. cappa groups.But there were no differences in pharmacokinetic parameters of chlorzoxazone and metoprolol. Conclusion:I. cappa can reduce the enzymatic activities of CYP3A,CYP1A2,CYP2C9 and CYP2C19 in rats at different degree,among which CYP3A is the strongest.

Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis / 中国中药杂志

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

Protective effect of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of HUVEC cells / 中国中药杂志

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H₂O₂ was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 μmol·L⁻¹ H₂O₂ for 0.5 h. Treatment of H₂O₂ also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H₂O₂ model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H₂O₂-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.

Absorption of Inula cappa extract based on everted intestinal sac method / 中国中药杂志

To investigate the absorptive characteristics of Inula cappa extract based on the rat everted intestinal sac method . Nine representative ingredients in I. cappa extract were selected as the study objects. An UPLC-MS/MS method was established to determine and detect their cumulative absorption amount for expounding the absorptive characteristics of ingredients in different intestinal sections. According to the results， the transport mechanism of 8 compounds showed passive diffusion by the reverted gut sac method. And scopolin was actively transported in the intestine. The best absorption site of chlorogenic acid was duodenum. The best absorption site of cryptochlorogenic acid， 1，3--dicaffeoylquinic acid， luteolin-7-glucoside and 3，4--dicaffeoylquinic acid were jejunum. The best absorption site of neochlorogenic acid， scopolin， 4，5--dicaffeoylquinic acid and 3，5--dicaffeoylquinic acid was ileum. The absorption of all the compounds was affected by pH and bile. All of the nine ingredients in I. cappa extract could be absorbed in intestines， but with differences in the absorption rate， the best absorptive site and mechanism， indicating that the intestinal absorption of I. cappa extract was selective.

Metabolism of Inula cappa extract by rat intestinal bacteria in vitro / 中国中药杂志

To investigate the metabolism of major components in Inula cappa by rat intestinal bacteria in vitro. I. cappa extract was incubated for 24 h with rat intestinal bacteria under anaerobic environment. After the samples were precipitated by n-butanol, UPLC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites, combined with data software such as Metabolite Tools, Data Analysis and so on. The potential metabolites in rat intestinal bacteria were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results injected that fourteen metabolites were detected in rat intestinal flora. Various types of metabolic reactions happen to caffeoylquinic acid in intestinal flora, including isomerization, hydrolyzation, there were also methylation, hydrogenation and acetylation of caffeic acid. At the same time, a methylate of dicaffeoylquinic acid was also detected. Presumably, caffeoylquinic acids were gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.

Researching on fingerprint of Inulacappa by HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>This study is to establish the fingerprint and find out the common chromatographic peaks of Inula cappa by HPLC.</p><p><b>METHOD</b>The HPLC analysis was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 150 mm, 1.8 μm) with 0.1% fomic acid aqueous solution-0.1% fomic acid acetonitrile solution as mobile phase at a flow rate of 0.3 · mL(-1) · min(-1); The detective wavelength is 325 nm; The column temperature is 45 °C.</p><p><b>RESULT</b>The results indicated that 5 of 17 common peaks were identified . The similarity about 10 groups of Inulacappais is over 0.95.</p><p><b>CONCLUSION</b>This method is able to be a scientific basis of quality assessment according to its convenient and reliable.</p>

Structures and hepatocytotoxicity of co-occurring substances in oleanolic acid tablets

Tablets of oleanolic acid [OA] have been approved by SFDA in China as an adjuvant therapy for acute and chronic hepatitis. Co-occurring substances present in the tablets of OA and their hepatocytotoxicity have not yet been reported. In the current investigation, the crude OA drug was separated by repeated column chromatography. The structures of the isolated compounds were characterized by spectral analysis and the cytotoxicity of each compound was evaluated in vitro against the human normal liver cell L02 at concentrations from 0.125 to 1000 micromol/L using the MTT method. As a result, OA and its 11 co-occurring trace compounds including one new triterpenoid, 3-O- [4-oxopentanoyl]- olean-12- en-28-oic acid, were isolated and structurally characterized. Cytotoxicity tests indicated that these compounds were all non-toxic at concentrations up to 50micromol/L. Clear structure-activity relationship [SAR] was also observed. The results suggested that OA tablets of similar origin might not cause obvious cytotoxicity to the normal liver cell. The work may facilitate further SAR studies of OA-type triterpenoids.

Design, synthesis and anti-oxidative evaluation of L-amino acid prodrugs of scutellarein / 药学学报

To design and synthesize a series of novel scutellarein 4'-L-amino acid prodrugs with more potent anti-oxidative activity and improved physicochemical properties. Scutellarein was used as lead compound, according to successful experience of improving bioavailability of oral administration drugs by active transport mechanism, principle of hybridization was used to introducing L-amino acid structural fragments at 4'-position of scutellarein to design and synthesize target scutellarein 4'-L-amino acid prodrugs. The synthetic compounds were tested on their physicochemical properties and in vitro anti-oxidative activity against H202 induced oxidative damage in PC12 cells. Five compounds were found to have more potent anti-oxidative activity than positive control VE. Moreover the physicochemical properties of synthesized compounds were evaluated, and the results revealed that L-amino acid ether derivatives are more stable (t1/2 9-92 h) than their corresponding ester derivatives (t1/2 0.5 h). Water solubility of scutellarein 4'-L-amino acid ester and ether derivatives were 1 796-4 100 microg.mL-1 and 27.7-81.1 microg.mL-1 respectively, in comparison with scutellarin, the solubility of compounds 18, 19 and 22, 24-27 increased about 120-280 fold and 2-6 fold respectively. All these results suggested that L-amino acid prodrug strategy has significant potential in scutellarein prodrug design.

Design, synthesis and anti-hBV evaluation of adefovir mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs / 药学学报

A series of adefovir mono-L-amino acid esters, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs with more potent anti-HBV activity and lower nephrotoxicity were designed and synthesized. Adefovir bis (L-amino acid) ester was used as lead compound, according to pathological and pharmacological findings that non-steroidal anti-inflammatory drugs can effectively inhibit the organic anion transporter 1 (hOAT1)-mediated adefovir phosphonic acid pairs of anion transport across tubular basement membrane thereby reducing the nephrotoxicity of adefovir. Flatten design principle was used to introducing non-steroidal anti-inflammatory drugs structural fragments to design and synthesize target adefovir mixture ester prodrugs. HepG2 2.2.15 cell line was used as in vitro anti-HBV activity evaluation model. Five compounds exhibited antiviral activity, and compound 18 showed the most potent anti-HBV activity and relatively high selective index (EC50 3.92 micromol L(-1), SI 9.97). HK-2 cell line was used as in vitro model to evaluate nephrotoxicity. Results suggested the target compounds have lower cytotoxicity than the positive control. Moreover, by analyzing the primary structure and activity relationship of these compounds, it could suggest that mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs strategy has significant potential in the acyclic nucleoside phosphonates prodrug design.

Separation and purification technology of main active composition of Hongye Xingtong soft capsules with macroporous resin / 中国中药杂志

<p><b>OBJECTIVE</b>To study the technical conditions of the extraction and purification of the active composition from Polygonum orientale and Crataegus pinnatifida Bge. in Hongye Xingtong soft capsules with the macroporous resin.</p><p><b>METHOD</b>The orientm, isorientm and hyperoside were used as index to screen the five kinds of resins. And the technical conditions of the enrichment and purification of D101 resin selected out of above were all-round studied.</p><p><b>RESULT</b>The D101 was fit for adsorbing orientm, isorientm and hyperoside. Under the optimal conditions, the transfer rate of orientm, isorientm and hyperoside was above 91%, and the total solid was cut down by more than 60%.</p><p><b>CONCLUSION</b>The D101 is greatly effective for the enrichment and purification of the active composition of P. orientale and C. pinnatifida Bge.</p>

Technology of eliminating tannin with polyamide-adsorption for preparing Xinshao lyophilization / 中国中药杂志

<p><b>OBJECTIVE</b>To study the process of eliminating tannin with polyamide-adsorption for preparing Xinshao lyophilyzation.</p><p><b>METHOD</b>The transfer rate of paeoniflorin, tannin examination, the test of the local stimulus and abnormal toxicity on mice were adopted as indexes to optimize the process of tannin adsorption and resolution of polyamide-adsorption, and the effect of eliminating tannin of polyamide-adsorption was compared with traditional methods.</p><p><b>RESULT</b>The effect of eliminating tannin with polyamide-adsorption was good, and the transfer rate of paeoniflorin with polyamide-adsorption was superior to the other methods. Furthermore, the abnormal toxicity decreased evidently and the security of the preparation was ensured.</p><p><b>CONCLUSION</b>The method of polyamide-adsorption provides a useful reference to the technique of eliminating tannin in the traditional Chinese medicine injections.</p>

Study on RP-HPLC fingerprint of xinshao injection / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the fingerprint detecting standard of xinshao Injection by reversed phase high performance liquid chromatography.</p><p><b>METHOD</b>The chromatographic conditions were as follows: an Diamonsil C18 column was used, the mobile phase was composed of 80% acetontrile and 0.1% phosphoric acid with gradient elution, the flow rate 1.0 mL min(-1), column temperature was 40 degrees and the UV absorbance detection was set at 230 nm.</p><p><b>RESULT</b>A standard HPLC fingerprint procedure was developed for xinshao Injection, with 8 common peaks. Under the same selected chromatographic conditions, the HPLC fingerprints of raw drugs and their intermediate, finished products were obtained with good separation and correlation (no less than 0.97) according to the technical requirements of fingerprint on Injection of Chinese traditional medicine.</p><p><b>CONCLUSION</b>This method was accurate, repeatable and can be used as the important parameters of the quality control for xinshao injection.</p>

Study on RP-HPLC fingerprint of compound hongcao injection / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the fingerprint detecting standard of Compound Hongcao Injection by reversed phase high performance liquid chromatography.</p><p><b>METHOD</b>The chromatographic conditions were as follows: an Inersil-ODS-3 column was used; the mobile phases (acetontrile and 0.1% phosphoric acid) with gradient elution; the flow rate 1.0 mL x min(-1) and the UV absorbance detection at 300 nm.</p><p><b>RESULT</b>Under the same selected chromatographic conditions, the HPLC fingerprints of raw drugs and their intermediate, finished products were obtained with good separation and correlation according to the technical requirements of fingerprint on Injection of Chinese traditional medicine.</p><p><b>CONCLUSION</b>17 common peaks as well as their retention times and peak area ratios on HPLC fingerprint can be used as the important parameters of the quality control for Compound Hongcao Injection.</p>

Studies on chemical constituents in herb of Polygonum orientale / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of the essential substance from Polygonum oriental.</p><p><b>METHOD</b>Compounds were isolated with silica gel and polyamide chromatography and their structures were determined by spectral analysis and chemical evidence.</p><p><b>RESULT</b>Six compounds were obtained and identified as myricitrin, luteolin, gallic acid, catechin, protocatechuic acid, p-hydroxycinnamic acid.</p><p><b>CONCLUSION</b>Six compounds were isolated from P. oriental for the first time.</p>

Determination of gallic acid in Melastoma dodecandrum by RP-HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a RP-HPLC method for the determination of gallic acid in Chinese herb Melastoma dodecandrum, and to evaluate the quality of the herb.</p><p><b>METHOD</b>The RP-HPLC analysis was achieved by using a Polaris C18 column and tetrahydrofuran-methanol-0.2% phosphoric acid (0.5:0.5:99) as the mobile phase, with a flow rate of 1.0 mL x min(-1), and detected by UV at 274 nm.</p><p><b>RESULT</b>The content of gallic acid was from 0.020% to 0.081%, in ten groups of M. dodecandrum collected from different locations.</p><p><b>CONCLUSION</b>The method is a simple, convenient and rapid. It can be used for quality evaluation of M. dodecandrum.</p>

Qualitative and quantitative studies on qianliebeixi capsules / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method of quality control on qianliebeixi capsules.</p><p><b>METHOD</b>To clarify semen vaccarine by TLC, to determinate the contents of quertin and kaempferol in prepations, by HPLC. Chromatographic conditions: C18 column with METH-0.2%H3PO4(47:53) as mobile phase; the temperature of column was 40 degrees C, and quercetin was detected at 370 nm with flowing rate 1 ml/min.</p><p><b>RESULT</b>The spots of semen vaccarine were clear in TLC, no interference in negative contrast. The average recovery rate of quertin was 98.9% and the RSD was 2.2%; the average recovery rate in kaempferol was 100.4% and the RSD was 1.7%.</p><p><b>CONCLUSION</b>The resolution of spots were perfect in TLC, the spots were easy to distinguish, The method is simple, rapid as well as reproducible and can control the quality of qianliebeixi capsules effectively.</p>