Primary research on components with anticomplement and antitussive activities from leave extracts of Chimonanthus nitens / 中草药

Objective: To investigate and analyze anticomplement and antitussive activities and the active ingredients of the extract of Chimonanthus nitens leaf. Methods: The classical anti-complement pathway and the concentrated ammonia-induced cough model was used to compare the activity of the different polar parts of C. nitens leaf, and the polar parts with anti-complement and antitussive activity were determined. A preliminary analysis of the chemical composition in activity extract was identified by high performance liquid chromatography-mass spectrometry. The main chemical components in the C. nitens leaf of antitussive and antibody activity were also evaluated. Results: The ethyl acetate extract of C. nitens leaf had both anti-complement and antitussive effects. A total of 28 compounds were initially identified through mass spectrometry analysis. Kaempferol-3-O-rutinoside and kaempferol had both antitussive and anti-complement activities. Conclusion: The ethyl acetate extract of C. nitens leaf has good anti-complement and antitussive activities, and the mainly active ingredients in it were kaempferol-3-O-rutinoside and kaempferol that could be used as quality-controlling chemical markers.

Simultaneous Determination of 6 Flavonoids in the Roots of Tetrastigma hemsleyanum by HPLC / 中国药房

OBJECTIVE： To establish a method for simultaneous determination of rutin， isoquercitrin， kaempferol-3-O- rutinoside， daunorubicin， quercetin and kaempferol in the roots of Tetrastigma hemsleyanum.  METHODS： HPLC method was adopted. The determination was performed on Alliance SilGreen C18 column with mobile phase consisted of 0.2％ phosphoric acid solution-acetonitrile solution （gradient elution） at the flow rate of 1.0 mL/min. The detection wavelength was 360 nm and the column temperature was at 35 ℃. The sample size was 15 μL. RESULTS： The linear range of rutin， isoquercitrin， kaempferol-3- O-rutinoside， daunorubicin， quercetin and kaempferol were 21.77-217.77， 12.37-123.75， 13.23-132.31， 4.63-46.30， 5.75-57.50， 3.36-33.66 μg/mL （all r＝0.999 9）， respectively. limit of detection of them were 0.217 8， 0.123 8， 0.066 2， 0.046 3， 0.191 7， 0.112 3 μg/mL， respectively. limit of quantitation of them were 0.435 6， 0.247 5， 0.165 4， 0.154 3， 0.575 0， 0.421 2 μg/mL， respectively. RSDs of precision （n＝6）， stability （24 h， n＝7） and reproducibility tests （n＝6） were lower than 3.20％. The average recoveries of them were 96.23％， 86.88％， 97.51％， 97.67％， 97.50％， 87.46％， RSDs were 1.85％， 1.90％， 1.84％， 1.87％， 1.25％， 2.01％ （n＝9）， respectively. CONCLUSIONS： The method is fast and simple， and could be applied for simultaneous determination of rutin， isoquercitrin， kaempferol-3-O-rutinoside， daunorubicin， quercetin and kaempferol in the roots of T. hemsleyanum.  

Quantitative Determination of Two Flavonoids and Four Phenoquinones of Huoxue Cuyu Capsules by HPLC / 中国药房

OBJECTIVE：To establish a method for simultaneous determination of two flavonoids （rutin and kaempferol-3-O- rutinoside） and four phenoquinones （dihydrotanshinone Ⅰ，cryptotanshinone，tanshinone Ⅰ and tanshinoneⅡA） in Huoxue cuyu capsules. METHODS：HPLC method was adopted. The determination was performed on Welch Ultimate XB-C18 column with mobile phase consisted of acetonitrile-0.1％phosphoric acid solution（gradient elution）at the flow rate of 1.0 mL/min. The column temperature was set at 20 ℃，and detection wavelength was set at 270 nm. The sample size was 10 μL. RESULTS：The linear range of rutin，kaempferol-3-O-rutinoside，dihydrotanshinone Ⅰ，cryptotanshinone，tanshinone Ⅰ and tanshinoneⅡA were 172.13-860.66 μg/mL（r＝0.999 7），15.33-76.66 μg/mL（r＝0.999 8），12.81-64.06 μg/mL（r＝0.999 3），5.90-29.52 μg/mL（r＝0.999 3），5.12-25.60 μg/mL（r＝0.999 2），6.71-33.57 μg/mL（r＝0.999 7），respectively. The limits of detection were 0.08，0.01，0.01，0.01，0.01，0.01 μg/mL. The limits of quantitation were 0.27，0.02，0.03，0.03，0.03，0.03 μg/mL，respectively. RSDs of precision，stability test and repetition tests were all lower than 2.0％（n＝6）. The recoveries were 97.54％-100.25％（RSD＝1.07％，n＝6），96.90％-101.91％（RSD＝1.73％，n＝6），96.24％-102.89％（RSD＝2.32％，n＝6），97.04％-102.18％（RSD＝1.82％，n＝6），95.06％-97.73％（RSD＝1.18％，n＝6），95.59％-101.40％（RSD＝2.29％，n＝6），respectively. CONCLUSIONS：The method is sensitive，rapid，simple and has good reproducibility. It can be used for simultaneous determination of rutin， kaempferol-3-O-rutinoside， dihydrotanshinone Ⅰ， cryptotanshinone， tanshinone Ⅰ， tanshinoneⅡA in Huoxue cuyu capsules.

Investigation and predictive analysis on Q-marker of Ziziphi Spinosae Semen based on human intestinal microbiota transformation / 中草药

Objective: The aim of this work was to investigate the quality markers (Q-markers) of Ziziphi Spinosae Semen (ZSS) based on biotransformation by human intestinal microbiota. Methods: In this study, in vitro biotransformation of ZSS aqueous extract by normal human intestinal microbiota was analyzed using UPLC-Q-Orbitrap-MS. Furthermore, the time course of the biotransformation was studied to probe into the biotransformation mechanism of compounds in ZSS by human intestinal flora. The change rules of flavonoids, saponins and alkaloids in the incubation solution at different time points were plotted based on the percentage of peak area of compounds. Results: A total of 31 original ingredients and four metabolites were characterized in transformed ZSS aqueous extract by human intestinal microbiota. No obvious degradation was observed for benzylisoquinoline alkaloids within 24 h. As far as flavones concerned, a wide range of metabolic reactions as well as significant reaction were shown. Meanwhile, these flavonoids were completely degraded during 24 h. In addition, both jujuboside A and jujuboside B were metabolized to their saponins by deglycosylation reactions. Thus, coclaurine, zizyphusine, kaempferol-3-O-rutinoside, spinosin, vicenin II, jujuboside A, and jujuboside B were referred as prospective Q-markers. Conclusion: The results indicated that the chemical compounds in ZSS were obviously affected by transformation. Intestinal transformation studies play an important role for the elucidation of therapeutic material basis of ZSS and it should be taken into account during the process of the investigation of Q-marker.

Effects of kaempferol-3-O-rutinoside on proliferation,migration and TG-FBR1 signaling pathway activation in vascular smooth muscle cells / 中国病理生理杂志

AIM:To investigate the effects of kaempferol-3-O-rutinoside(KR)on the proliferation,migration of vascular smooth muscle cells(VSMC)and the activation of transforming growth factor βreceptor 1(TGFBR1)signaling pathway in the cells.METHODS: The viability of VSMC was detected by MTT assay.The proliferation of VSMC was measured by EdU staining.The migration ability of VSMC was examined by Transwell assay.The protein levels of the mi-gration-associated proteins matrix metalloproteinase 2(MMP2)and matrix metalloproteinase 9(MMP9)were detected by Western blot.Molecular docking study was conducted to explore the interaction between KR and TGFBR 1.The protein le-vels of the phosphorylated TGFBR1,Smad2 and Smad3 were determined by Western blot.RESULTS: KR inhibited the viability of VSMC in a dose-and time-dependent manner.KR reduced the ratio of EdU-positive cells in a dose-dependent manner.KR dose-dependently suppressed the migration ability of VSMC and decreased the protein levels of MMP 2 and MMP9(P<0.05).KR docked into TGFBR1 with the binding energy of -9.804 kcal/mol by forming hydrogen bonds with SER-280,ARG-215,ASP-290 and LYS-335 of TGBFR1.KR dose-dependently suppressed the activation of TGFBR 1 and its downstream proteins Smad2 and Smad3(P<0.05).CONCLUSION: KR inhibits the proliferation and migration of VSMC,possibly via blocking the TGFBR1 signaling pathway.

Study on dynamic change of flavonoids in traditional SHE medicine-Shiliang Tea by quantitative analysis of multi-components by single-marker / 中草药

Objective: To establish an assay method of flavonoids (four components) in traditional SHE medicine-Shiliang Tea by quantitative analysis of multi-components by single-marker (QAMS), and to analyze the dynamic change of flavonoids at different harvest time and different collection places. Methods: With rutin as the internal reference substance, the relative correction factor (RCF) of kaempferol-3-O-rutinoside, quercetin, and kaempferol was calculated. Then the contents of four components were calculated, and the accuracy and feasibility of method was evaluated through external standard method. Results: The RCF of rutin to kaempferol-3-O-rutinoside, quercetin, and kaempferol were 1.158 with RSD 0.73%, 0.475 6 with RSD 1.55%, 0.431 9 with RSD 1.58%, respectively. There was no significant difference of assay results between QAMS method and external standard method. While the differences of content between different harvest months and two different species were significant. Conclusion: The QAMS method with rutin as internal reference substance can be used for quantitative analysis of four flavonoids in Shiliang Tea. It is suggested that the best harvest time of Shiliang Tea for flavonoids is in July and August.

Application of QAMS in quality control of Wuzi Yanzong Pills / 中草药

Objective To establish a quantitative analysis of multi-component with a single-marker (QAMS) method for the quality control of Wuzi Yanzong Pills (WYP). Methods Six main effective components (schisandrin, hyperin, quercitrin, kaempferol 3-O-rutinoside, deoxyschizandrin, and γ-schizandrin) of WYP were simultaneously separated on a reversed-phase column (Ultimate LP-C18) with high-resolution of each chromatographic peak by high performance liquid chromatography (HPLC). Schisandrin was selected as the internal reference, and the relative correlation factors (RCFs) of other five components were calculated to achieve QAMS. The ruggedness of RCFs was tested on different instruments and columns. Moreover, results of the QAMS were compared with the external standard method. Results Within a certain linear range, the RCFs of hyperin, quercitrin, kaempferol 3-rutinoside, deoxyschizandrin, and γ-schizandrin were 0.36, 4.86, 0.88, 7.34, and 6.35, respectively. The repeatability was good under different experimental conditions. There were no significant differences between the calculated value and estimated value on QAMS and external standard method. Conclusion The QAMS method can be used to assay the content of six components of WYP simultaneously and control the quality of WYP simplely, reliably, and accurately.

Multiple fingerprints and multi-comonent quantitative analysis of Danhong Injection / 中草药

Objective: To establish the multiple fingerprints of Danhong Injection (DI) using HPLC-UV-MS method and to simultaneously determine nine kinds of the medicinal components in DI. Methods: Separation was performed on Atiantis T3 analytical column (250 mm × 4.6 mm, 5 μm) with the mobile phase of 0.05% formic acid-50% acetonitrile by gradient elution, and negative-ion SIM mode was selected for mass spectrometric detection. Results: The multiple fingerprints reflected the chemical information of Salvia miltiorrhiza and safflower in DI. The similarity of the fingerprints was higher than 0.988 in all 11 batches of DI. 5-Hydroxymethyl furfural, sodium danshensu, protocatechuic aldehyde, coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid B, salvianolic acid A, and kaempferol-3-O-rutinoside showed the good linearity in their respective ranges of concentration, r ≥ 0.999 0.The average recovery of low-, mid-, and high-dose medicinal components (n = 9) was (99.0 ± 1.4)%, (102.0 ± 1.7)%, (99.3 ± 1.6)%, (97.6 ± 1.6)%, (100.0 ± 1.8)%, (97.9 ± 1.6)%, (100.5 ± 4.4)%, (100.6 ± 2.0)%, and (106.0 ± 4.7)%, respectively. The relative standard deviation for the method reproducibility was lower than 1.45%. The total contents of nine medicinal components in the 11 batches of DI were 2.61-3.06 mg/mL. Conclusion: This method is simple, accurate, and with good reproducibility, and the multiple fingerprints combined with the quantitative analysis could reflect the quality of DI better, which could be used to control the quality of DI.

Determination of Gallic Acid and Kaempferol-3-O-Rutinoside Contents inNymphaea Candida Presl. by HPLC / 中国中医药信息杂志

Objective To establish an HPLC method for the content determination of gallic acid and kaempferol-3-O-rutinoside inNymphaea candida Presl..MethodsThe separation was carried out on a Wondasil C18 column (250 mm×4.6 mm, 5μm). The mobile phase was methanol-0.1% acetic acid solution, with gradient elution;the flow rate was 1.0 mL/min;the detection wavelength was set at 270 nm;the column temperature was 30℃.Results The linear ranges of gallic acid and kaempferol-3-O-rutinoside were 0.065-0.585μg (r=0.999 1), 0.133-1.197μg (r=0.999 5), respectively. The average recoveries of gallic acid and kaempferol-3-O-rutinoside were 102.71%, 102.08%, with RSD of 1.97%, 0.46%, respectively.Conclusion This method was rapid, accurate, and reliable. It can be used for the determination of allic acid and kaempferol-3-O-rutinoside in Nymphaea candidaPresl..