The chemical constituents from the stems and leaves of Clausena excavata were isolated and purified by column chromatography with silica gel, ODS, Sephadex LH-20 and RP-HPLC. The chemical structures of the isolated compounds were identified on the basis of physicochemical properties, spectroscopic analysis, as well as the comparisons with the data reported in literature. Nineteen compounds were isolated from the 90% ethanol extract of the stems and leaves of C. excavata, which were identified as methyl orsellinate(1), syringaresinol(2), lenisin A(3), scopoletin(4), osthenol(5), N-benzoyltyrarnine methyl ether(6), N-p-coumaroyltyramine(7), aurantiamide acetate(8), 1H-indole-3-carboxaldehyde(9), furostifoline(10), clausenalansine E(11), 3-formylcarbazole(12), clausine L(13), clausine E(14), methyl carbazole-3-carboxylate(15), glycosinin(16), murrayafoline A(17), clausine H(18) and 2,7-dihydroxy-3-formyl-1-(3'-methyl-2'-butenyl)carbazole(19). Among these isolated compounds, compounds 1-11 were isolated from C. excavata for the first time, and compounds 1, 2 and 10 were isolated from the genus Clausena for the first time. In addition, this study evaluated the anti-rheumatoid arthritis activities of compounds 1-19 by measuring their anti-proliferative effects on synoviocytes in vitro according to MTS method. Compounds 10-19 displayed remarkable anti-rheumatoid arthritis activities, which exhibited the inhibitory effects on the proliferation of MH7 A synovial fibroblast cells with the IC_(50) values ranging from(27.63±0.18) to(235.67±2.16) μmol·L~(-1).
Cell Proliferation ; Chromatography, Reverse-Phase ; Clausena ; Plant Leaves ; Synoviocytes

OBJECTIVETo develop a reversed-phase HPLC method for simultaneous determination of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde in Rhizoma Gastrodia.

METHODA Kromasil C18 column (4.6 mm x 250 mm, 5 microm) was used with a methanol-water-0.1% acetic acid gradient elution system. The eluates were detected at 270 nm, the flow rate was 1.0 mL x min(-1) and the column temperature was 35 degrees C.

RESULTThe linear range of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde were 19.1-383 (r = 0.999 9), 0.620-12.4 (r = 0.999 9), 2.45-49.0 (r = 0.999 9), 0.280-5.63 mg x L(-1) (r = 0.999 6), respectively. The average recoveries (n = 9) of the four components were 96.7% -97.7%, RSD < 1.6%.

CONCLUSIONThe method is accurate, sensitive and reliable for determination of gastrodin, adenosin, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde in Rhizoma Gastrodia.

OBJECTIVETo establish a RP-HPLC method for simultaneously determination of swertiamarin, gentiopicroside, sweroside, isoorientin in Gentiana lawrencei from Qinghai province.

METHODThe RP-HPLC method was used. Chromatographic column was the Kromasil C18 column (4.6 mm x 250 mm, 5 microm). The gradient elution solvent system was composed of acetonitrile (A) and (0.1% H3PO4) water (B). The ratio of acetonitrile was as follows: 0.00-27.00 min, 10% -17% (A); 27.00-45.00 min, 17% -33% (A); 45.01-55.00 min, 100%-100% (A). The detective wavelength was 240 nm; the flow rate was 1 mL x min(-1); column temperature was set at 25 degrees C.

RESULTSwertiamarin, gentiopicroside, sweroside and isoorientin were base-isolated. The method had good linearity within the ranges of 2.12-10.6 mg x mL(-1) for swertiamarin (r = 0.999 4), 2.46-12.3 g x L(-1) for gentiopicroside (r = 0.999 7), 2.47-12.4 g x L(-1) for sweroside (r = 0.999 2) and 0.309-1.54 mg x mL(-1) for isoorientin (r = 0.999 5).

CONCLUSIONThe method is rapid, precise and repeatable, can be applied to control the quality of G. lawrencei.

To develop a timesaving and easy operating Reverse Phase (RP) chromatography method, we adopted Thermo Pierce RP C18 Tip to separate small amount hippocampus peptide mixtures and to compare with high performance liquid chromatography (HPLC). According to the separation performance of 4 ACN gradient optimization methods, we determined the best ACN concentration gradient. The results showed that, the experiment took only 10 min by separating with eight ACN concentration gradient, which accounted 1/4 for HPLC. But as for the identified proteins, RP C18 Tip accounted 85.5% for HPLC. ACN gradient of 5%, 15%, 20% and 90% had best repeatability (P = 0.429) and result for separating 30 μg peptides. This method is easy to operate, timesaving and has low cost. It could be used into pretreatment of small amount complex proteomic samples.
Chromatography, High Pressure Liquid ; Chromatography, Reverse-Phase ; methods ; Peptides ; Proteins ; isolation & purification ; Proteomics ; methods

A precise and selective reversed phase high-performance liquid chromatographic method (HPLC) was used to quantify the levels of huperzine A in samples of three Huperzia serrata populations with a total of 73 individuals located in Zhejiang, Guangxi, Chongqing, respectively, as well as in one-to-one samples of these 73 individuals introduced in same site after one year. Huperzine A content variation both among and within populations, and the dynamic change of this alkaloid occurring in same population after one year introduction, were analyzed using SPSS 13.0 software (Coefficient of variation, One-way ANOVA analysis, Paired-samples T tests). The results indicated that huperzine A content varied significantly by geographical locations, especially change with longitude, i. e., the order of the huperzine A content was CQ population > GX population > ZJ population. The coefficients of variation (CV) were as follows: 0.36 (CQ), 0.44 (GX) and 0.40 (ZJ). This indicated that there was plentiful diversity concerned with huperzine A content among individuals within population. Moreover, this high diversity was still maintained after one year introduction. ANOVA analysis showed that there was significant difference among populations in huperzine A content. Finally, the significant change of huperzine A content was not observed in all three populations after one year introduction. The results presented in this study could provide evidence that the huperzine A content variation of H. serrata is the results of an interaction between genes and the environment, by comparison, is mainly controlled by genetic factor.
Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; methods ; Drugs, Chinese Herbal ; Huperzia ; chemistry

OBJECTIVETo develop an HPLC method for the determination of luteolin-7-O-glycoside, apigenin-7-O-glycoside, linarin, luteolin, apigenin and acacetin in Flos Chrysanthemi Indici simultaneously.

METHODThe reversed phase HPLC system consisting of a C18 column (4.6 mm x 250 mm, 5 microm) and a mixture of acetonitrile and 0.05% phosphate acid with gradient elution as the mobile phase was adopted. The absorbance was monitored at 326 nm.

RESULTThe linear response range was 1.08-21.5 mg x L(-1) (r = 0.9990), 0.278-5.57 mg x L(-1) (r = 0.9994), 5.58-112 mg x L(-1) (r = 0.9999), 0.521-10.4 mg x L(-1) (r = 0.9995), 0.162-3.25 mg x L(-1) (r = 0.9999) and 0.288-5.76 mg x L(-1) (r = 0.9998), respectively (n = 6). The average recoveries (n = 9) of six flavonoids were 96.34%-104.0%. All of RSD of precision (n = 6) and repeatability (n = 6) were less than 1.2% and 2.6%.

CONCLUSIONThe validation data demonstrated that the method was accurate and repeatable, and can be ase to measure the six flavonoids in Flos Chrysanthemi Indici.

OBJECTIVETo develop an HPLC method for determination of tomatine in the dried Solanum cathayanum of China Hubei Enshi.

METHODThe analysis was performed on a YMC-Pack ODS-AA column (4. 6 mm x 150 mm, 5 microm) eluted with acetonitrile and water in gradient mode. The concentration of acetonitrile in the mobile phase changes from 20% to 100% within 60 minutes. The detection wavelength was set 203 nm. The flow rate was 1.0 mL x min(-1) and column temperature was set at 30 degrees C.

RESULTThe linear relationship of tomatine was determined within the range from 0.1-0.6 g x L(-1) (r = 0.999 7). The average recovery as 98.93% with RSD 1.2%.

CONCLUSIONA convenient and reliable method was developed to determine the content of tomatine in the dried S. cathayanum.

OBJECTIVETo establish an RP-HPLC method for determination of atractylodinol in Ateractylodes lancea and compare the contents of atractylodinol in the herbs of different origins.

METHODThe samples were separated on an Agilent TC-C18 (4.6 mm x 250 mm, 5 microm) column with the mobile phase of acetonitrile-water (49:51). Flow rate was 1.0 mL x min(-1). The detection wave length was set at 337 nm. Column temperature was 30 degrees C.

RESULTThe linear range of atractylodinol was 9.12 x 10(-2) -9.12 mg x L(-1) (r = 0.999 9), the average recovery was 97.15%, RSD was 1.5% (n = 5). The contents of atractylodinol were in the range of 0.268-1.213 mg x g(-1) in the samples from different orgins. The contents of atractylodinol in samples growing in Dabieshan mountain were higher than those in Jiangsu province (P < 0.001).

CONCLUSIONThe established method for determination of atractylodinol is accurate and reliable, which can be used to evaluate the quality of A. lancea, the contents of atractylodinol in the sample was related with its morphological characteristic and geographic orgin.

OBJECTIVETo establish a reversed-phase high-performance liquid chromatographic (RP-HPLC) method for determination of phloridzin content.

METHODSA RP-HPLC method was established for determination of phloridzin using an Inertsil ODS-3 (4.6×150 mm, 5 µm) column with the detection wavelength of 288 nm, flow rate of 1.0 ml/min, and column temperature of 25 degrees celsius;.

RESULTSThe result showed that the phloridzin had a good linear relationship when its concentration ranged between 0.5988 and 89.72 µg/ml. The regression equation was Y=46.370 X-0.6728 (r=0.9999, n=3). The average recovery of phloridzin was 99.40% with the relative standard deviations (RSD) of 0.67%.

CONCLUSIONThis method is simple, quick and accurate for determination of phloridzin content.

In recent years, more and more researchers focus on the selectivity of RP-HPLC columns. Among these columns, C18 column is the most commonly used the column. Yet even the same filler can not give us the same separation effect with the columns of different brands and different types, therefore, it is important to choose the suitable HPLC column for carrying out an experiment. After study in recent years, some column classification, selection methods (e.g.: column parameter method, F value method) have been achieved and contributed to the finding of suitable columns and reducing the blindness of column selection. This paper summarizes the advantages of these methods and their limitations in the application process, and gives some advice and expectations to pharmaceutical analysis works based on the works of other researchers.
Chromatography, High Pressure Liquid ; methods ; Chromatography, Reverse-Phase ; instrumentation ; methods ; Drug Contamination ; Pharmaceutical Preparations ; isolation & purification