ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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 .
ABSTRACT
A hybridoma cell line(B10)was established by fusing spleen cells of BALB/c mou- se immunized with human thyroglobulin(HTG) with SP2/0 myeloma cells. An averagefusing rate of 6.3% and antibody-secreting positive well rate of 58.3% were obtained.During the first two months, the supernatant of B10 culture had a titer of 1/128 to1/2048 measured by hemagglutination method, and the ascites was positive at 1/64000-128000 and 1/320000 respectively as measured by hemagglutination and radioimmunoass-ay.The B10 cell line is very stable and has very high activity to produce anti-HTGmonoclonal antibodies. After several times of preservation in liquid nitrogen andpassage in culture for one year,a recent determination shows that cell culture super-natant and ascites still have very high titer,being 1/4096 and 1/1048576 respectively asmeasured by hemagglutination method. The chromosome number of the B10 hybridomacell is 99.5?7.4.The success of the establishment of this cell line is briefly discussed.Attempt to establish diagnostic kit with this monoclonal antibody is being undertaken.