HPLC Fingerprint Analysis of Benchmark Sample of Yanghetang / 中国实验方剂学杂志

ObjectiveTo establish a high performance liquid chromatography（HPLC） fingerprint of Yanghetang benchmark sample， and evaluate its quality with chemometric methods， so as to provide a reference for the quality control of this benchmark sample. MethodHPLC was used to establish the fingerprint of Yanghetang benchmark sample with ZORBAX SB-C18 column（4.6 mm×250 mm， 5 μm）， the mobile phase was consisted of acetonitrile（A） -0.05% phosphoric acid aqueous solution （containing 0.05% triethylamine solution）（B） for gradient elution（0-5 min， 2%-3%A； 5-15 min， 3%-5%A； 15-65 min， 5%-30%A； 65-90 min， 30%-70%A）， the flow rate was 1.0 mL·min-1， the column temperature was 35 ℃， and the detection wavelength was 210， 260 nm. Traditional Chinese Medicine（TCM） Chromatographic Fingerprint Similarity Evaluation System （2012 edition） combined with cluster analysis， principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to evaluate the quality differences between different batches of Yanghetang benchmark samples， and to find the main chemical components responsible for the quality differences. ResultHPLC fingerprint of Yanghetang benchmark sample was established， 13 common peaks were identified and attributed to each common peak， including peaks 2 and 8 from Rehmanniae Radix Praeparata， peaks 10 and 11 from Cinnamomi Cortex， peaks 1， 3-6 from fried Sinapis Semen， peak 13 from Ephedrae Herba， and peaks 7， 9， 12 from Glycyrrhizae Radix et Rhizoma. Eight of them were identified by comparing with control substance， which were 5-hydroxymethylfurfural（peak 2）， sinapine thiocyanate（peak 4）， glycyrrhizin（peak 7）， verbascoside（peak 8）， cinnamic acid（peak 10）， cinnamaldehyde（peak 11）， glycyrrhizic acid（peak 12） and ephedrine hydrochloride（peak 13）. The similarities of the HPLC fingerprints of 15 batches of Yanghetang benchmark samples with the control fingerprint were all greater than 0.80. The three chemometric methods could classify the samples into two categories. Eight differential components were screened out， among which 5-hydroxymethylfurfural， sinapine thiocyanate， verbascoside and ephedrine hydrochloride were identified. ConclusionThe established fingerprint analysis method is accurate， stable and reproducible， which basically reflects the overall chemical composition of Yanghetang benchmark sample， and can provide a basis for establishment of quality standards for compound preparations of this famous classical formula.

The physiological and biochemical responses of a medicinal plant (Salvia miltiorrhiza L.) to stress caused by various concentrations of NaCl.

Salvia miltiorrhiza, which is commonly known as Danshen, is a traditional Chinese herbal medicine. To illustrate its physiological and biochemical responses to salt stress and to evaluate the feasibility of cultivating this plant in saline coastal soils, a factorial experiment under hydroponic conditions was arranged on the basis of a completely randomised design with three replications. Five salinity treatments (0, 25, 50, 75 and 100 mM NaCl) were employed in this experiment. The results showed that salinity treatments of <100 mM NaCl did not affect the growth of Salvia miltiorrhiza in a morphological sense, but significantly inhibit the accumulation of dry matter. Salinity treatments significantly decreased the Chl-b content but caused a negligible change in the Chl-a content, leading to a conspicuous overall decrease in the T-Chl content. The Na(+) content significantly increased with increasing hydroponic salinity but the K(+) and Ca(2+) contents were reversed, indicating that a high level of external Na(+) resulted in a decrease in both K(+) and Ca(2+) concentrations in the organs of Salvia miltiorrhiza. Salt stress significantly decreased the superoxide dismutase (SOD) activity of Salvia miltiorrhiza leaves in comparison with that of the control. On the contrary, the catalase (CAT) activity in the leaves markedly increased with the increasing salinity of the hydroponic solution. Moreover, the soluble sugar and protein contents in Salvia miltiorrhiza leaves dramatically increased with the increasing salinity of the hydroponic solution. These results suggested that antioxidant enzymes and osmolytes are partially involved in the adaptive response to salt stress in Salvia miltiorrhiza, thereby maintaining better plant growth under saline conditions.

Protective effects of luteolin-7-glucoside against liver injury caused by carbon tetrachloride in rats.

Ixeris chinensis (Thunb.) Nakai has been used as a Chinese folk medicine; the information on the physiological and biochemical functions of the compounds extracted from I. chinensis is still scanty. We investigated the effects of luteolin -7-glucoside (LUTG) isolated from I. chinensis against liver injury caused by carbon tetrachloride (CCl4). CCl4 significantly increased the enzyme activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) in blood serum, as well as the level of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) in liver tissue, and decreased the levels of reduced glutathione (GSH). Pretreatment with LUTG was not only able to suppress the elevation of GPT, GOT, MDA and 8-OHdG, and inhibit the reduction of GSH in a dose-dependent manner in vivo, but also reduce the damage of hepatocytes in vitro. On the other hand, we also found LUTG has strong antioxidant activity against reactive oxygen species (ROS) in vitro in a concentration-dependent manner. The hepatoprotective activity of LUTG was possibly due to its antioxidant properties, acting as scavengers of ROS. These results obtained in vivo and in vitro suggest that LUTG had protective effects against hepatic oxidative injury induced by chemicals. Further studies on the pharmaceutical functions and immunological responses of LUTG may help in the development of a clinical application.