Qualitative and quantitative analysis of the chemical components in Yuquan capsules by using ultra-performance liquid chromatography-mass spectrometry.

The Yuquan capsules is a commonly used traditional Chinese Patent Medicine used for the treatment of diabetes mellitus. In this study, a high-throughput analytical method for identifying the chemical composition of Yuquan capsules was established for the first time by using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry. The data obtained were subjected to fragment analysis and this was combined with UNIFI processing of natural products. One-hundred sixteen compounds were characterized from Yuquan capsules. Twelve of the bioactive compounds were quantitatively analyzed by ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry. This study was undertaken to obtain a comprehensive chemical profile analysis as well as to evaluate the overall quality of Yuquan capsules. The results will provide a reference for the quality evaluation of different Yuquan preparations. In addition, the data will enable basic pharmacodynamic research into these extensively used capsules.

Quality Markers' Discovery and Quality Evaluation of Jigucao Capsule Using UPLC-MS/MS Method.

Jigucao capsules (JGCC) have the effects of soothing the liver and gallbladder and clearing heat and detoxification. It is a good medicine for treating acute and chronic hepatitis cholecystitis with damp heat of the liver and gallbladder. However, the existing quality standard of JGCC does not have content determination items, which is not conducive to quality control. In this study, serum pharmacochemistry technology and UNIFI data processing software were used to identify the blood prototype components and metabolites under the condition of the obvious drug effects of JGCC, and the referenced literature reports and the results from in vitro analysis of JGCC in the early stage revealed a total of 43 prototype blood components and 33 metabolites in JGCC. Quality markers (Q-markers) were discovered, such as abrine, trigonelline, hypaphorine and isoschaftoside. In addition, ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS) was used to determine the active ingredients in JGCC. The components of quantitative analysis have good correlation in the linear range with R2 ≥ 0.9993. The recovery rate is 93.15%~108.92% and the relative standard deviation (RSD) is less than 9.48%. The established UPLC-MS/MS quantitative analysis method has high sensitivity and accuracy, and can be used for the quality evaluation of JGCC.

An ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry identification and characterization of the active constituents from Abrus mollis Hance.

Abrus mollis Hance is a traditional Chinese medicine that is widely used to treat acute and chronic hepatitis, steatosis, and fibrosis. Its therapeutic qualities of it have long been acknowledged, although the active ingredients responsible for its efficacy and the mechanisms of its action are unknown. In this study, the chemical constituents absorbed into the blood from Abrus mollis Hance were assessed by using liquid chromatography-quadrupole-time-of-flight mass spectrometry and the data was analyzed with the UNIFI screening platform. The results obtained were compared to existing chromatographic-mass spectrometry information, including retention times and molecular weights as well as known reference compounds. 41 chemical constituents were found in Abrus mollis Hance, and these included 16 flavonoids, 13 triterpenoids, five organic acids, and two alkaloids. Experimentally it was found that Abrus mollis Hance had a therapeutic benefit when treating α-naphthalene isothiocyanate-induced acute liver injury in rats. In addition, 11 blood prototypical constituents, including six flavonoids, three triterpenoids, and two alkaloids, were found in serum samples following intragastric administration of Abrus mollis Hance extracts to rats. This novel study can be used for the quality control and pharmacodynamic assessment of Abrus mollis Hance in order to assess its efficacy in the therapeutic treatment of patients.

[Q-markers of Yuquan Capsules based on serum pharmacochemistry of Chinese medicine].

This study analyzed the quality markers(Q-markers) of Yuquan Capsules(YQC) based on serum pharmacochemistry of Chinese medicine and detected the components and metabolites of YQC absorbed into the blood by UPLC-Q-TOF-MS and UNIFI systems. As a result, 32 components of YQC were detected, including 17 prototype components and 15 metabolized components. Among them, 12 prototype components(ginsenoside Rh_2, genistein, formononetin, puerarin, daidzein, schizandrin A, schizandrin B, schizandrin C, schizandrol A, schizandrol B, gomisin D, and ononin) and 12 metabolized components(ginsenoside Rg_1, ginsenoside Rg_2, ginsenoside Rg_3, ginsenoside Ro, 3'-methoxypuerarin, daidzin, astragaloside Ⅱ, astragaloside Ⅳ, glycyrrhizic acid, liquiritigenin, isoliquiritin, and verbascoside) showed inhibitory effects and pharmacological activities against diabetes, and these 24 blood-entering components against diabetes were identified as Q-markers of YQC.

High throughput metabolomics explores the mechanism of Jigucao capsules in treating Yanghuang syndrome rats using ultra-performance liquid chromatography quadrupole time of flight coupled with mass spectrometry.

To explore the potential mechanism of the Chinese patent medicine Jigucao capsule in treating the serum metabolic profile of rats with Yanghuang syndrome, zingiber officinale Rosc. and ethanol simulates the syndrome background of traditional Chinese medicine and uses α-naphthyl isothiocyanate to induce liver damage in rats to prepare a Yanghuang syndrome model. The histopathological observation and the determination of biochemical indexes evaluate the therapeutic effect of the Jigucao capsule, and the metabolomic method analyzes the mechanism of the Jigucao capsule against Yanghuang syndrome. Jigucao capsule reduces the number of inflammatory cells, inhibits the proliferation of bile duct epithelial cells and hepatocyte necrosis. Compared with Yanghuang syndrome rats, the levels of alanine aminotransferase, alkaline phosphatase, and total bile acid were significantly reduced (P < 0.05). Furthermore, Jigucao capsule significantly reversed the abnormal levels of glucose 1-phosphate, phenylalanyl-cysteine, taurodeoxycholic acid, lysoPC (22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z), lysoPC (15:0), lysoPC (P-18:0), 7alpha-hydroxy-3-oxo-4-cholestenoate and 15(S)-hydroxyeicosatrieic acid and regulated part of the lipid metabolism and carbohydrate metabolism, Jigucao capsule has a therapeutic effect on Yanghuang syndrome rats. In short, this study sets for the first time elaborated on the underlying mechanism of Jigucao capsule resistance to Yanghuang syndrome rats from a metabolomics perspective, providing the basic data for the pharmacodynamic studies of the Jigucao capsule.

Rapid characterization of the constituents in Jigucao capsule using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

Jigucao capsule is a well-known Chinese patent medicine for the treatment of acute and chronic hepatitis and cholecystitis. The chemical components of Jigucao capsule were not clear resulting from the paucity of relevant studies, which hindered the research of the pharmacological mechanism, the comprehensive development, and utilization of Jigucao capsule in clinical studies. By establishing a high-throughput ultra-performance liquid chromatography quadrupole time of flight mass spectrometry in combination with intelligent UNIFI software data processing platform to automatically characterize and identify the chemical profile of Jigucao capsule, 144 compounds were determined rapidly, including 34 terpenoids, 25 flavonoids, 22 steroids, 21 phenylpropanoids, 10 glycosides, six alkaloids, 13 organic acids, and other 13 components. These compounds may be the active components of Jigucao capsule. In this study, a rapid and robust method for comprehensively analyzing the chemical composition of Jigucao capsule was described and established for the first time. The results will provide a reference for the quality control of Jigucao capsule and the establishment of a higher quality standard, as well as for the pharmacodynamic material basis research.

Effect of Yifei Huoxue Granule on the proliferation of rat pulmonary artery smooth muscle cells upon exposure to chronic hypoxic conditions in vitro.

OBJECTIVE: To investigate the inhibitory effect of Yifei Huoxue Granule (, YFHXG) on the hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and its mechanism of decreasing pulmonary arterial pressure. METHODS: Twenty male Sprague-Dawley (SD) rats were randomly divided into four groups: saline, and 0.66, 3.30 and 16.50 g/kg of YFHXG groups, the saline and different concentrations of YFHXG were given twice daily for 7 days, respectively. Serum-pharmacology method was used in the preparation of YFHXG serum. Tissue block anchorage was employed in the primary culture of rat PASMCs. The PASMCs were randomly divided into normoxia group, hypoxia group, and hypoxia+YFHXG group (0.66, 3.30 and 16.50 g/kg doses of YFHXG-treated serum groups, exposed to hypoxic condition). PASMCs in normoxia and hypoxia group were cultured with saline serum, hypoxia+YFHXG groups were cultured with different concentrations of YFHXG serum. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle was analyzed using flow cytometry. In addition, hypoxia inducible factor-1-alpha (HIF-1α) protein expression was evaluated by immunocytochemistry analysis, the concentration of intracellular reactive oxygen species (ROS) and Ca(2+) were determined by laser scanning confocal microscopy (LSCM). RESULTS: MTT assay and flow cytometry showed that hypoxia could directly activate the proliferation of PASMCs, while YFHXG dose-dependently inhibited hypoxia-induced proliferation of rat PASMCs. Immunocytochemistry showed that hypoxia enhanced HIF-1α protein expression, and LSCM showed that hypoxia significantly increased intracellular ROS and Ca(2+), while YFHXG decreased the expression of HIF- 1α and attenuated the hypoxia-induced increase in intracellular concentration of ROS and Ca(2+). CONCLUSIONS: YFHXG could inhibit hypoxia-induced proliferation of rat PASMCs, which may decrease pulmonary arterial pressure and vascular remodeling. The anti-hypoxia effect of YFHXG may be explained by its regulation of HIF-1α expression and of the levels of intracellular ROS and Ca(2+).

[Effect of yifei huoxue granule on the proliferation and the TGF-beta activity of rat pulmonary artery smooth muscle cells induced by platelet-derived growth factor BB].

OBJECTIVE: To explore the effects of Yifei Huoxue Granule (YFHXG) on the proliferation and the transforming growth factor-beta (TGF-beta) activity of rat pulmonary artery smooth muscle cells (PASMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: Using tissue block adhering wall method, the primary rat PASMCs were cultured. PASMCs at the log phase growth were randomly divided into the control group, the PDGF-BB group, the PDGF-BB + high YFHXG group (at the final concentration of 7.5 mg/mL), the PDGF-BB + middle YFHXG group (at the final concentration of 1.5 mg/mL), and the PDGF-BB + low YFHXG group (at the final concentration of 0.3 mg/mL), respectively. MTT assay were employed to determine the cell proliferation rate of each group. Flow cytometric analyses were used to detect the cell cycle constituent ratio and the proliferation index (PI). In addition, TGF-beta protein's expression was determined by immunocytochemical assay (SP method). RESULTS: Compared with the control group, the proliferation of PASMCs in the PDGF-BB group was obviously active (P<0.01). But when compared with the PDGF-BB group, along with the increased concentration of YFHXG, the growth of PASMCs was obviously inhibited, the cell ratio of G0/G1, phase obviously increased, the cell ratio of S + G2/M phase significantly decreased, and PI significantly decreased. Besides, the expression of TGF-beta protein decreased in a dose-dependent manner (P<0.05, P<0.01). CONCLUSIONS: PDGF-BB could directly stimulate the proliferation of PASMCs. YFHXG had a significant inhibition on the proliferation of rat PASMCs induced by PDGF-BB and could regulate the expression of TGF-beta.

[Effects of Herba Ephedra Sinicae and Fructus Schisandrae Chinensis on pathology of rats with bleomycin A(5)-induced idiopathic pulmonary fibrosis].

OBJECTIVE: To observe the different effects between Mahuang (Herba Ephedra Sinicae) and Wuweizi (Fructus Schisandrae Chinensis) on the pathological changes of rats with bleomycin A(5)-induced pulmonary fibrosis. METHODS: Ninety Wistar rats were randomly divided into sham operation group, model group, hydrocortisone group, Herba Ephedra Sinicae group, Fructus Schisandrae Chinensis group and Herba Ephedra Sinicae plus Fructus Schisandrae Chinensis group. There were 16 rats in each group except the sham operation group (10 rats). Pulmonary fibrosis was induced by a single intratracheal injection of bleomycin A5. Hematoxylin and eosin straining and immunohistochemical method were used after 7- and 28-day treatment to observe the pathology of lung injury, measure the inner diameter of pulmonary arterioles and the density of nuclear membrane. RESULTS: Compared with the sham operation group at 7 and 28 d, alveolar inflammation level was significantly increased in the model group (P<0.01). Alveolar inflammation level was decreased obviously in the hydrocortisone group (P<0.05) after 7- and 28-day treatment as compared with the model group, and that in Herba Ephedra Sinicae plus Fructus Schisandrae Chinensis group was also decreased obviously (P<0.05) at 28 d. Compared with the sham operation group, nuclear density of the model group was increased, while its inner diameter was decreased (P<0.05). In the Fructus Schisandrae Chinensis group, Herba Ephedra Sinicae plus Fructus Schisandrae Chinensis group and hydrocortisone group, the nuclear density was decreased (P<0.05) as compared with the model group. Inner diameter in the Herba Ephedra Sinicae group, Herba Ephedra Sinicae plus Fructus Schisandrae Chinensis group and hydrocortisone group was higher than that in the model group (P<0.05). Microvessel density of the model group was obviously higher than that of the sham operation group (P<0.05). Compared with the model group, Herba Ephedra Sinicae plus Fructus Schisandrae Chinensis group and hydrocortisone group had lower microvessel density (P<0.05). CONCLUSION: Herba Ephedra Sinicae combined with Fructus Schisandrae Chinensis can restrain pulmonary artery injury. The nuclear density and microvessel density can be reduced by Fructus Schisandrae Chinensis, while Herba Ephedra Sinicae can increase the inner diameter.

[Effect of serum containing tengcha total flavonoid and dihydromyricetin on proliferation and apoptosis of HepG2 cells].

OBJECTIVE: To observe the effect of serum containing Tengcha total flavonoid and dihydromyricetin on proliferation and apoptosis of HepG2 cells. METHOD: Serum containing respectively Tengcha total flavonid, dihydromyricetins and CTX and control serum were prepared by serological pharmacology method. MTT assay was used to observe the proliferation inhibition rate of HepG2 cells after incubated with different kinds of serum. Inverted microscope was utilized to observe the morphological changes after HepG2 cells were treated with different serum. AnnexinV/7AAD double label method was used to detect earlier period apoptosis cells. RESULT: Both serum containing 20% Tengcha total flavonid and serum containing 20% dihydromyricetin could restrain the HepG2 cells proliferation at different levels and the morpholological changes of apoptosis were observed. AnnexinV/7AAD double label method showed that the earlier period apoptosis cells rates were increased by serum containing 20% Tengcha total flavonoid, but serum containing 20% dihydromyricetin did not show influence on the earlier period apoptosis cells. CONCLUSION: Tengcha total flavonoid can restrain the HepG2 cells proliferation and induce earlier period apoptosis cells.

[An experimental study(II) on the inhibition of prostatic hyperplasia by extract of seeds of Brassica alba].

OBJECTIVE: To study the active components and their functionary mechanism of the extract of Brassica alba seeds, which inhibits experimental mice prostatic hyperplasia. METHOD: Prostatic hyperplasia of castrated male mice induced by testosterone propionate, the penetrability of capillary vessel of mice skin induced by histamine and the endermic flesh bud of rat induced by filter paper were used as experimental models. Sinalbin and beta-sitosterol separated from seeds of Brassica alba were used to test the activities. RESULT: Sinalbin and beta-sitosterol(16.0 mg.kg-1.d-1 and 8.0 mg.kg-1.d-1) could significantly inhibit mice prostatic hyperplasia induced by testosterone propionate and activity of serum acid phosphatase(P < 0.01 or P < 0.05), Sinalbin(16.0 mg.kg-1.d-1)could significantly inhibit the hyperplasia of endermic flesh bud in rat induced by filter paper(P < 0.05), beta-sitosterol(16.0 mg.kg-1.d-1 and 8.0 mg.kg-1.d-1) could significantly decrease the penetrability of capillary vessel of mice skin induced by histamine. CONCLUSION: Sinalbin and beta-sitosterol have anti-androgen and anti-inflammation activities.

[An experimental study(I) on the inhibition of prostatic hyperplasia with extract of seeds of Brassica alba].

OBJECTIVE: To study the effective fraction of the extract of seeds of Brassica alba, which inhibits experimental mice prostatic hyperplasia. METHOD: An experimental model of prostatic hyperplasia of castrated male mice induced by testosterone propionate was made. Fractions I, II and III were prepared by extracting the seeds of Brassica alba successively with ether, ethanol and water under reflux. Total extract was prepared by extracting the seeds of Brassica alba with 60% ethanol under reflux. The total extract and the three fractions were used to test the activities. RESULT: Total extract, fractions I and II could not only significantly inhibit mice prostatic hyperplasia induced by testosterone propionate and activity of serum acid phosphatase, but also decrease wet weight of preputial glands, while fraction III is inactive. CONCLUSION: Extract from seeds of Brassica alba can significantly inhibit mice prostatic hyperplasia induced by exterior hormone, possessing an activity of anti-androgen. Fractions I and II show an equivalent activity of total extract, which indicate that these fractions contain active components of seeds of Brassica alba which can inhibit prostatic hyperplasia.