Extraction and isolation of lithospermic acid B from Salvia miltiorrhiza Bunge using aqueous two-phase extraction followed by high-performance liquid chromatography.

A rapid and effective method integrating separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge was developed by combining an aqueous two-phase system extraction with preparative chromatography. An aqueous two-phase system of n-butyl alcohol/KH2 PO4 was chosen from seven systems. The influence of parameters including concentration of KH2 PO4 , n-butyl alcohol concentration, pH, and the ratio of an aqueous two-phase system to crude extract were investigated using a single factor design. Response surface methodology was subsequently used to find the optimal compositions of an aqueous two-phase system. Keeping a solvent-to-solid ratio of 10, the final optimized composition of an aqueous two-phase system was 39.1% w/w n-butyl alcohol and 22.6% w/w KH2 PO4 . Under these conditions a recovery yield of 99.8% and a high partition coefficient of 310.4 were obtained. In a pilot-scale experiment using optimized conditions, 18.79 g of lithospermic acid B with a purity of 70.5% and in a yield of 99.8% was separated from 0.5 kg of crude extract. Subsequently, 9.94 g lithospermic acid B with a purity of 99.3% and recovery yield of 70.3% was obtained with a preparative chromatographic process, and the two-step total recovery was 70.1%.

Expression of gap junctional proteins Cx37 mRNA in cerebral arterial endothelium induced by soluble eggs antigen of Schistosome / 中国地方病学杂志

Objective To explore the effects of gap junctional(GJ)proteins in pathogenesis of cerebral schistosomiasis, through observing the expression of gap junctional proteins Cx37 mRNA in cultured cerebral arterial endothelium incubated with soluble eggs antigen(SEA). MethodsCerebral artery endothelial cells of rabbits were incubated with SEA, and the experiments were divided into control group and SEA 1 - 5 groups (SEA concentrations were 10.0％ ,5.0％ ,3.3％ ,2.5％,2.0％, respectively), reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of Cx37 mRNA and protein in cerebral artery endothelial cells of rabbits, respectively. Results Cx37 mRNA levels of control and SEA 1 - 5 groups were 0.239 ± 0.037, 0.260 ± 0.043, 0.218 ± 0.310, 0.647 ± 0.040, 0.419 ± 0.036, and 0.513 ± 0.038, respectively;SEA 3 - 5 groups were higher than control group of mRNA levels(all P＜ 0.05). Cx37 protein levels of control and SEA 1 - 5 group were 0.401 ± 0.045, 0.485 ± 0.048, 0.749 ± 0.052, 1.119 ± 0.063, 1.015 ± 0.057 and 0.605 ±0.047, respectively, of which SEA 2 - 5 groups were higher than control group(all P ＜ 0.05). ConclusionsExpression levels of Cx37 mRNA and protein in cultured cerebral artery endothelial cells incubated with SEA are higher than those of control cerebral artery endothelial cells, which suggests that the gap junction proteins may play an important role in pathogenesis of cerebral schistosomiasis through SEA and its secretion in infiltration of brain tissue and deposition in the cerebral arteries.

Hydrolytic kinetics of lithospermic acid B extracted from roots of Salvia miltiorrhiza.

The hydrolytic kinetics of lithospermic acid B (LAB) extracted from the roots of Salvia miltiorrhiza (Chinese herb: danshen) was investigated by using reversed-phase high-performance liquid chromatography (HPLC) with UV-vis detection. The influences of initial drug concentration, pH and temperature on hydrolysis of LAB were studied in aqueous solutions. The results showed that initial concentration of LAB has no effect on the degradation rate at pH 2.0. The hydrolysis followed pseudo-first-order kinetics at 90 degrees C. The log k(obs)-pH profile indicated that the optimal stability range was at pH 2.0-5.0. The rate constant of overall hydrolysis as a function of temperature under the given conditions obeyed the Arrhenius equation. Analysis of the acid-induced degraded solution of LAB by liquid chromatography-mass spectrometry (LC-MS) revealed at least four degradation products [M-H](-) ion at m/z 197, 137, 537 and 537, respectively. Three of these degradation products, i.e. danshensu (DSU), protocatechuic aldehyde (PRO), and lithospermic acid, were further identified by comparing the retention times with standard samples. According to the structure of LAB and its hydrolysis behavior in solution, the other product was proposed to be the isomer of lithospermic acid.

Kinetics and mechanism of degradation of lithospermic acid B in aqueous solution.

The degradation of lithospermic acid B (LAB) was investigated as a function of buffer concentration, pH and temperature. Stability tests were performed using a stability-indicating high-performance liquid chromatography (HPLC) with UV-vis detection. The degradation followed pseudo-first-order kinetics under all experimental conditions. The maximum stability of LAB was observed at pH 2.0. The logk(pH)-pH profile described by specific acid-base catalysis and water molecules agreed with the experimental results. The overall degradation rate constant as a function of the temperature under the given conditions obeyed the Arrhenius equation. The chemical fate of LAB in mild acidic solution was investigated, and nine degradation products were detected and tentatively identified by LC-MS analysis. The primary degradation pathway involving the cleavage of ester bond and ring-opened of benzofuran in the LAB are proposed.