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OBJECTIVE To extract and isolate the four chemical components of Yao medicine Ventilago leiocarpa， and to conduct identification and content determination for them. METHODS The chemical components of V. leiocarpa were separated and purified by solvent extraction， extraction， silica gel column chromatography and preparative liquid chromatography； then the chemical structures of four isolated compounds were identified based on their spectral data. The contents of four components were determined by high performance liquid chromatography（HPLC）-quantitative analysis of multi-components by single-marker （QAMS） method， with the following chromatographic conditions： chromatographic column was Echway GowonTM C18 （250 mm× 4.6 mm， 5 μm）. The mobile phase was acetonitrile-0.1% phosphoric acid for gradient elution； the detection wavelength was 269 nm， and the column temperature was 25 ℃ . Using emodin as internal reference， the relative correction factors （fi/s） between emodin and the other 3 components were established and used to calculate the content. At the same time， the content of each component was calculated with the external standard method （ESM）， and the differences between these two methods were compared. RESULTS Four compounds were isolated from V. leiocarpa， and they were identified as emodin， frangulin A， pleuropyrone A， emodin-8-O-β-D-glucoside. The result of HPLC-QAMS showed that the fi/s of pleuropyrone A， emodin-8-O-β-D- glucoside and frangulin A were 1.147 2， 0.874 7 and 0.644 4， respectively. The content of these four components was measured as a good linearity （r≥0.999 6）； relative standard deviation （RSD） of precision， stability and reproducibility tests were all lower than 2.00%， and average recoveries were E-mail：dearhuangjianyou@126.com 99.41%-100.46%（RSD≤2.05%）. There was no significant difference between QAMS method and ESM （RSD＜3.00%）. CONCLUSIONS Emodin， frangulin A， pleuropyrone A and emodin- 8-O-β-D-glucoside are isolated from V. leiocarpa； among them， the last three components are all isolated from for the first time. The established HPLC-QAMS method is accurate and reliable for the determination of 4 components in V. leiocarpa， and can used for quality control of V. leiocarpa.
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Objective:To investigate the relationship between caspase recruitment domain protein 9 (CARD9) level and inflammatory response in cerebral tissue of ischemic brain injury mice.Methods:Totally 24 SPF BALB/c male mice were randomly(random number) divided into 4 groups: sham operated group, ischemia 3 h group, ischemia 6 h group, and ischemia 12 h group, 6 mice in each group. The permanant middle cerebral artery occlusion (pMCAO) model in the ischemia groups was established by using line embolism to block blood flow. Mice in each group were sacrificed at the predetermined time point after operation. CARD9 and p-p65NF-κB levels were detected by Western blot, and the inflammatory factors mRNA and protein including TNF-ɑ, IL-lβ and IL-6 were detected by RT-PCR and ELISA, respectively. The data were analyzed by SPSS 21.0 software, the comparison of measurement data between each two groups was analyzed by independent sample t test, and the correlations between CARD9 and inflammatory factors were analyzed by Pearson analysis. Results:Compared with the sham operated group, the CARD9 levels in the ischemia 3 h, 6 h and 12 h groups were increased significantly [(0.325±0.011) vs. (0.462±0.019), P=0.036; (0.735±0.036), P=0.003; (0.903±0.024), P=0.001], the p-p65NF-κB levels in the ischemia 3 h, 6 h and 12 h groups were increased significantly [(0.227±0.016) vs. (0.316±0.017), P=0.041; (0.445±0.021), P=0.016; (0.671±0.039), P=0.008], the TNF-ɑ levels in the ischemia 3 h, 6 h and 12 h groups were significantly increased [(0.53±0.06) vs. (1.06±0.10), P=0.009; (1.47±0.15), P=0.004; (2.78±0.18), P=0.001], the IL-lβ levels in the ischemia 3 h, 6 h and 12 h groups were significantly increased [(0.55±0.07) vs. (1.01±0.11), P=0.009; (2.13±0.16), P=0.003; (3.09±0.18), P=0.001], and the IL-6 levels in the ischemia 3 h, 6 h and 12 h groups were significantly increased [(1.99±0.18) vs. (4.10±0.41), P=0.006; (8.54±0.84), P=0.002; (11.56±0.96), P=0.001]. Pearson analysis showed that CARD9 was positively correlated with the p-p65NF-κB and TNF-ɑ, IL-lβ, IL-6 ( r=0.894, P=0.001; r=0.747, P=0.008; r=0.810, P=0.001; r=0.773, P=0.007). Conclusions:A positive correlation exists between CARD9 and inflammatory responses in the early stage of ischemic brain injury in mice
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OBJECTIVE：To establi sh the quality standard of Yao medicine Pileostegia tomentella，and to provide reference for its quality control. METHODS ：Totally 10 batches of P. tomentella were collected from Guangxi area. The properties of P. tomentella were observed and microscopic identification was conducted for the transverse section of its stems ，roots and powder. TLC method was established by using 7-hydroxycoumarin as reference. The contents of water ，total ash ，acid-insoluble ash and alcohol-soluble extract in P. tomentella were determined. The contents of skimmin ，7-hydroxycoumarin and 7-hydroxy-8- methoxycoumarin in P. tomentella were determined by HPLC. RESULTS ：The root and stem surface of P. tomentella possessed obvious wrinkles ，hard texture ，slight smell and bitter taste ，and there were secretory cells and needle crystal cells in the cross section. The medicinal powder contained calcium oxalate needle crystal ，calcium oxalate square crystal ，sclerotic cell and starch granules. The results of TLC showed that the spots of the same color were found in the corresponding positions of chromatograms of test samples and 7-hydroxycoumarin control. In the 10 batch of P. tomentella ，the contents of water were 9.1％-11.8％；those of total ash were 3.6％-6.7％；those of acid-insoluble ash were 0.10％-0.44％；those of alcohol-soluble extract were 8.9％-14.0％.The linear range of skimmin ，7-hydroxycoumarin and 7-hydroxy-8-methoxycoumarin were 6.088-182.640，1.568-47.040 and 1.096- 32.880 μg/mL（all r not less than 0.999 7）. The average recoveries were 99.47％，101.07％ and 99.89％（RSD were all less than 2％）. RSDs of precision ，stability（24 h），reproducibility and durability tests were all lower than 2％ . The contents of 3 components such as skimmin were 1.337-9.534，0.348-2.236， 0.083-1.088 mg/g，respectively. CONCLUSIONS ：It is preli- 201705） minarily proposed that the content of water in P. tomentella is not more than 12.0％ ；that of total ash is not more than 7.0％；that of alcohol-soluble extract is not less than 8.0％； E-mail：luguoshou@foxmail.com that of ski mmin is not less than 1.30 mg/g；that of 7-hydroxy- coumarin is not less than 0.30 mg/g；that of 7-hydroxy-8-methoxycoumarin is not less than 0.06 mg/g.
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OBJECTIVE：To study the effects of citrusinol on proliferation ，migration and the expression of skeleton-related proteins of human hepatocellular cells HepG 2，and to investigate its interaction mode with skeleton-related proteins. METHODS ： CCK-8 assay was used to detect the effects of different concentrations （12.5，25，50，100，200 μmol/L）of citrusinol on the proliferation of HepG 2 cells for 24 h. HepG 2 cells were divided into negative control group ，citrusinol low-concentration and high-concentration groups （50，100 μmol/L citrusinol）. After treated for 24 h，the migration of HepG 2 cells was detected by cell scratch test ；cell migration rate was calculated. mRNA and protein expression of F-actin ，β-tubulin and Ezrin in HepG 2 cells were determined by RT-PCR and Western blotting assay. Molecular docking software Schrodinger 2015 was used to analyze the interaction mode between citrusinol and above 3 kinds of proteins. RESULTS ：Citrusinol showed significant inhibition effect on the proliferation of HepG 2 cells （P＜0.05 or P＜0.01），in dose-dependent trend. Compared with negative control group ，cell migration， mRNA and protein expression levels of F-actin ， β-tubulin， Ezrin were decreased significantly in citrusinol low-concentration and high-concentration groups （P＜0.05 or P＜0.01）. Molecular docking results showed that the citrusinol could form hydrogen bond and hydrophobic bond with the above 3 skeleton-related proteins. CONCLUSIONS ：Citrusinol can inhibit the proliferation and migration of HepG 2 cells，the mechanism may be associated with the down-regulation of mRNA and protein expression of F-actin ，β-tubulin and Ezrin. The mode of its interaction with skeleton-related proteins may be the formation of hydrogen bond or hydrophobic bond.
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OBJECTIVE：To optimize ultrasonic-assisted ethanol-（NH4）2SO4 aqueous two-phase extraction technology of citru- sinol from Desmodium caudatum . METHODS ：Using the content of citrusinol as indexes ，with ethanol volume fraction ， solid-liquid ratio ，（NH4）2SO4 addition amount ，ultrasonic time and ultrasonic temperature as factors ，based on the single factor tests，Box-Behnken design-response surface methodology was used to optimize the extraction technology of citrusinol. RESULTS ： The optimized extraction technology of citrusinol included that ethanol volume fraction was 95.35％，the solid-liquid ratio was 1∶50.35 （g/mL），（NH4）2SO4 addition amount was 4.49 g，ultrasonic time was 48.7 min，ultrasonic temperature was 57.6 ℃. In 3 times of validation tests ，the extraction rates of citrusinol were 0.637 8，0.638 4，0.625 4 mg/g，respectively，which was close to predicted value（0.630 5 mg/g）. CONCLUSIONS ：The optimized ultrasonic-assisted ethanol- （NH4）2SO4 aqueous two-phase extraction technology is stable and feasible ，and can be used for the extraction of citrusinol from D. caudatum .