OBJECTIVE:To study the effects of celastrol on the proliferation and apoptosis of human hepatoma HepG2 cells, and investigate its mechanism. METHODS:CCK-8 method was used to determine the cell activity 24,48,72 h after treated by 2, 5,10 μmol/L celastrol,and the proliferation inhibition rate and half inhibitory concentration(IC50)were calculated;flow cytome-try was conducted to detect the cell apoptosis rate and cycle change 24 h after treated by 2,5,10μmol/L celastrol,and the DMSO was used as negative control;rhodamine 123 staining method was used to determine the mitochondrial membrane potential 48 h af-ter treated by 2,5,10 μmol/L celastrol,and the DMSO was used as negative control;Western blot was adopted to detect the pro-apoptotic related genes Bax and B lymphoma 2(Bcl-2)protein expressions 0,12,24,36 h after treated by 5 μmol/L celastrol. RESULTS:2,5,10 μmol/L celastrol can inhibit cell proliferation,IC50 was 5.834 μmol/L. 2,5,10 μmol/L celastrol can induce apoptosis;5,10 μmol/L celastrol can block cell in G0/G1,S phases,compared with negative control group,with significant differ-ences(P<0.05 or P<0.01),and the above effects all showing certain concentration-dependent manner. 5 μmol/L celastrol can in-crease Bax protein expression and decrease Bcl-2 protein expression after cultured for 12,24,36 h,showing certain time-depen-dent manner;compared with 0 h,there was significant difference(P<0.05 or P<0.01). CONCLUSIONS:Celastrol can obvious-ly inhibit the proliferation of human hepatoma HepG2 cells and induce their apoptosis,and the mechanism may be related with strengthening mitochondrial permeability and promoting the release of apoptosis-inducing factor.

Objective To establish a method for the content determination of paeoniflorin in Gujing Pill. Methods Orthogonal test was used to optimize the condition of sample extraction and paeoniflorin content was detected by HPLC. HPLC was performed with Merck- LiChrospher RP- C18 column(4.6? 250 mm,5 ? m); methanol- water (25 ∶ 75) served as mobile phase and the detection wavelength was at 230 nm, the flow rate being 0.8 mL/min. Results Paeoniflorin has a good linearity within 0.27～ 1.35? g(r=0.9998) and the average recovery was 97.08 % with RSD 2.02 % (n=10). Conclusion This method is simple, sensitive, specific and can be used for the quality control of Gujing Pill.

Objective To optimize the solvent extraction of Rhubarbs. Methods With different concentrations of alcohol as the solvent, the amount of solvent, the extracting time and the extracting frequencies were assayed by orthogonal test to detect their influences on the extraction rate of rhubarb anthraquinone. And the extracts were analyzed and their pharmacodymamic effects were studied. Results Indicated by the content of total anthraquinones, the best conditions was: reflux extraction with 5- fold 50 % alcohol for 3 times, 0.5 h per time. The purgative activity of the extracts was stronger than that in the positive control group. Conclusion Optimization of the extracting conditions can promote the purgative activity of the extracts. 

Objective To establish a HPLC fingerprint analysis method of salt-prepared Cortex Phellodendri. Methods C18 column was used, with gradient elution by the mobile phase consisted of acetonitrile-0. 1% phosphoric acid (contained 0. 2 % triethylamine). The detection wavelength was at 230 nm, and the flow rate was at 0. 8 mL/min. Results Seventeen char-acteristic peaks were selected and the fingerprints on HPLC was set up. Conclusion HPLC fingerprint method is repro-ducible, accurate and stable, and can be used for the quality control of salt-prepared Cortex PheUodendri.

Objective To establish a HPLC fingerprint analysis method of salt-prepared Cortex Phellodendri. Methods C18 column was used, with gradient elution by the mobile phase consisted of acetonitrile-0.1 % phosphoric acid (contained 0.2 % triethylamine). The detection wavelength was at 230 nm, and the flow rate was at 0.8 mL/min. Results Seventeen characteristic peaks were selected and the fingerprints on HPLC was set up. Conclusion HPLC fingerprint method is reproducible, accurate and stable, and can be used for the quality control of salt-prepared Cortex Phellodendri.

Objective To establish a new method for the identification of Cortex Phellodendri. Methods A HPLC fingerprint at 220 nm has been established for identification of Cortex Phellodendri, using a Merck-Lichrospher RP-C_(18) column(4.6?250mm, 5?m) and ACN(A) with the buffer of 0.3% H_3PO_4-NH(CH_2CH_3)_2 (B) in gradient as the mobile phase. The results were compared with the chromatograms of three species of Rbizoma Coptidis under the same conditions. Results HPLC fingerprint of Cortex Phellodendri at 220nm consists of 14 specific peaks. The steady appearance of the peaks and their relative contents were considered as important signs for the identification of this crude drug. The chromatograms of Rhizoma Coptidis were obviously different from the fingerprint of Cortex Phellodendri. Conclusion This method has been proven to be feasible for the identification of Cortex Phellodendri.

Objective To establish a method for determination of Berberine and Phellodendrine in Cortex Phellodendri(CP).Methods Reverse-phase HPLC method was used for determination of Berberine and Phellodendrine in different batches of CP.The chromatographic conditions were:Merck-lichrospher RP-C18 column(4.6?250 mm,5 ?m),flow rate being 0.8 mL/min,column temperature at 25 ℃,detection wavelength at 284nm for Phellodendrine and 245nm for Berberine.Results The content of Berberine was 4 times and that of Phellodendrine in Chuan CP 2～3 times as much as that of Guan CP.Conclusion The method is proved to be feasible for quality assessment of Cortex Phellodendri.Limited contents of Berberine and Phellodendrine should be laid out for the great difference in Chuan CP and Guan CP.

Objective To evaluate the role of spinal sigma-1 receptors in the maintenance of bone cancer pain (BCP) in rats and the relationship with extracellular signal-regulated kinase (ERK).Methods Part Ⅰ Twenty-four female Sprague-Dawley rats,weighing 180-220 g,were randomized into 2 groups using a random number table:sham operation group (S group,n =4) and BCP group (n =20).BCP was induced by inoculating Walker 256 mammary gland carcinoma cells into the medullary cavity of the right tibia.Four rats were sacrificed on day 10 after inoculation in S group or on day 3,5,7,10 and 14 after inoculation in BCP group,and the L4-6 segments of the spinal cord were removed to measure the expression of sigma-1 receptors by Western blot.Part Ⅱ Forty female Sprague-Dawley rats,weighing 180-220 g,were randomized into 4 groups (n =10 each) using a random number table:sham operation group (S group),sigma-1 receptor inhibitor BD1047 group (BD group),BCP group,and BCP + BD1047 group (BCP + BD group).On day 10 to 14 after inoculation,normal saline 20 μl was injected intrathecally once a day in S and BCP groups,or BD1047 120 nmol/20μl was injected intrathecally once a day in BD and BCP + BD groups.Mechanical paw withdrawal threshold (MWT) to yon Frey filament stimulation was measured one day before inoculation,on day 3,5 and 7 after inoculation,and on day 10,12 and 14 after administration.After measurement of MWT on day 14 after inoculation,the rats were sacrificed and the L4-6 segments of the spinal cord were removed to determine the expression of phosphorylated ERK (p-ERK) by Western blot.Results Part Ⅰ Compared with group S,the expression of sigma-1 was significantly up-regulated and peaked on day 10 after operation in group BCP.Part Ⅱ Compared with S group,no significant changes were found in MWT and p-ERK expression at each time point in BD group,and MWT was decreased and p-ERK expression was up-regulated in BCP and BCP + BD groups.Compared with group BCP,after intrathecal injection of BD1047,MWT was significantly increased and the expression of p-ERK was down-regulated in BCP + BD group.Conclusion Spinal sigma-1 receptors are involved in the maintenance of BCP in rats possibly through promoting phosphorylation of ERK.

A new triterpenoid saponin and fourteen known triterpenoids were isolated from the methanol extract of the stems and leaves of Callicarpa integerrima Champ, which is used in Chinese folk medicine for stopping bleeding, expelling the wind, dissipating stagnation, and treating scrofula, by using various chromatographies, such as silica gel, Sephadex LH-20 and RP-C18 column chromatography. Their structures were identified as a new compound 2alpha, 3beta, 19alpha, 23-tetrahydroxy-olean-12-en-28-oic acid-28-O-beta-D-glucopyranosyl-(1 --> 4)-beta-D-glucopyranoside (1), together with fourteen known compounds: oleanolic acid (2), 3-acetyl oleanolic acid (3), 3beta-O-acetyl ursolic acid (4), 2alpha-hydroxy-ursolic acid (5), 2alpha, 3beta, 19alpha, 23-tetrahydroxy-urs-12-en-28-oic acid (6), alpha-amyrin-3-O-beta-D-glucopyranoside (7), pomolic acid (8), betulinic acid (9), ursolic acid (10), 2alpha, 3beta, 19alpha, 23-tetrahydroxy-olean-12-en-28-oic acid (arjungenin) (11), 2alpha-hydroxy-oleanolic acid (12), hederagenin (13), 2alpha, 19alpha-dihydroxy-ursolic acid (14) and pruvuloside A (15), by the spectroscopic techniques of NMR, HMBC, IR and MS, separately. All these compounds were obtained from this plant for the first time, and compounds 3, 4 and 15 were isolated from genus Callicarpa L. for the first time.

The study is to develop an HPLC method for simultaneous determination of rhamnazin (1), rhamnocitrin (2), rhamnetin (3), rhamnazin-3-O-beta-D-glucopyranoside (4), rhamnazin-3-O-beta-D-xylopyranosyl-(1-->4)-beta-D-glucopyranoside (5), rhamnazin-3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside (6), and rhamnocitrin-3-O-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside (7) in Nervilia fordii. The separation was performed on a Kromasil C18 column (250 mm x 4.6 mm, 5 microm) with 0.4% phosphoric acid-acetonitrile as the mobile phase in a gradient elution at a flow rate of 1.0 mL x min(-1). The detect wavelength was set at 256 nm, and the column temperature was set at 40 degrees C. There were good linear relationships between the logarithm values of concentrations and those of the peak areas of seven flavonoids (1-7) in the range of 0.55-70.00 microg x mL(-1) (r = 0.9997), 0.86-110.00 microg x mL(-1) (r = 0.9997), 0.39-50.00 microg x mL(-1) (r = 0.999 7), 0.55-70.00 microg x mL(-1) (r = 0.999 5), 1.33-170.00 microg x mL(-1) (r = 0.9998), 1.33-170.00 microg x mL(-1) (r = 0.9998), 0.16-20.00 microg x mL(-1) (r = 0.9995), respectively. The recoveries of the seven flavonoids were between 97.19%-99.45%, the relative standard deviations (RSDs) were between 0.91%-2.69%. The established method is rapid, accurate with high repeatability, which could provide scientific evidence for the quality control of Nervilia fordii.