Studies On The Chemical Constituents And Quality Control Of Euphorbia Humifusa

Euphorbia humifusa Willd is a traditional Chinese medicine and 16 samples were colleted from different habitats and batches. The chemical constituents of E. humifusa and its quality control method, as well as the HPLC fingerprint of this crude drug were investigated in detail.The chemical constituents of E. humifusa were studied and 8 compounds were isolated by a combination of silica gel, polyamide and Sephadex LH-20 column chromatography. Their structures were identified as apigenin-7-O-β-D-glucoside （E1）, luteolin-7-O-β-D-glucoside （E2）, kaempferol-3-O-β-D-glucoside （E3）, quercetin （E4）, brevofolin （E5）, quercetin-7-O-β-D-glucoside（E6）, astragalin-6"-O-gallate （E7） and quercetin-3-O-α-L-rhamnopyranosyl （1→6）-β-D-galactopyranoside （E8）. Compounds E3, E6, E7 and E8 are isolated from E. humifusa for the first time.The HPLC quantitative analysis of quercetin, luteolin and kaempferol in E. humifusa was performed on a Kromasil C₁₈ column （250mm×4.6mm, 5μm） with the mobile phase of methanol-0.1ï¼…phosphoric acid （52:48, v/v） and detective wavelength of 360nm. The linear ranges of quercetin, luteolin and kaempferol were 4.000～80.00μg·mL^-1（r=0.9999）, 0.848～16.96μg·mL^-1（r=0.9998） and 2.720～54.40μg·mL^-1（r=0.9999）, respectively. The recoveries of the three components were 98.2％（RSD=1.4％）, 100.3％（RSD=2.3％） and 101.1％（RSD=1.6％）, respectively. A HPLC quantitative analysis of gallic acid in E. humifusa was performed on a Diamonsil C₁₈ column （200mm×4.6mm, 5μm） with the mobile phase of methanol-0.1ï¼…phosphoric acid （10:90, v/v） and detective wavelength of 272nm. The linear range of gallic acid was 1.75～35.0μg·mL^-1（r=0.9996） and the recovery was 99.9％（RSD=1.8％）. A HPLC method was developed for the simultaneous determination of six flavonoids. The separation was performed on a Kromasil C₁₈ column （250mm×4.6mm, 5μm） with the mobile phase of methanol-acetonitrile-tetrahydrofuran-0.1ï¼…phosphoric acid （22:6:1:71, v/v/v/v） and detective wavelength of 330nm. The linear ranges of E6, E2, E8, E1, E7 and E3 were 1.440～28.80μg·mL^-1（r=0.9999）, 5.000～100.0μg·mL^-1（r=0.9999）, 5.500～110.0μg·mL^-1（r=0.9999）, 16.20～324.0μg·mL^-1（r=0.9998）, 15.00～300.0μg·mL^-1（r =0.9997） and 5.000～100.0μg·mL^-1（r=0.9997）, respectively. The recoveries of six flavonoids were 100.5％（RSD=2.2％）, 102.0％（RSD=1.8％）, 98.0％（RSD=2.0％）, 100.7％（RSD=2.2％）, 101.2％（RSD=2.0％） and 100.8％（RSD=3.0％）, respectively.The HPLC fingerprint of E. humifusa was established with Diamonsil-C₁₈ column as the stationary phase and acetonitrile-0.1ï¼…phosphoric acid as mobile phase, with UV detection at 330nm and the method was validated. The 16 batches of E. humifusa from different origins were analyzed and 16 co-peaks were obtained. All samples were identified with hierarchical clustering analysis and divided into three types. The type I was selected to develop the common recognition pattern. The similarities analysis method for quality control of E. humifusa was built by cosine, correlation and Euclidean.HPLC methods were developed for the quality control of Dijincao tablets. The linear ranges of quercetin, luteolin and kaempferol were 3.00～60.0μg·mL^-1（r=0.9999）, 4.48～89.6μg·mL^-1（r=0.9998） and 0.44～8.80μg·mL^-1（r=0.9998）, respectively. The recoveries of the three components were 100.1％（RSD=1.0％）, 100.5％（RSD=1.1％） and 98.8％（RSD=1.5％）, respectively. The linear range of gallic acid was 1.75～35.0μg·mL^-1（r=0.9996）. The recovery of gallic acid was 100.3％（RSD=2.2％）.