Content determination of phenolic acids in fresh Salvia miltiorrhiza and effect of polyphenol oxidase on content / 中国中药杂志

There is no consensus on the content, accumulation, transformation and content determination methods of phenolic acids in fresh Salvia miltiorrhiza. In order to find out the true content of phenolic acids in fresh S. miltiorrhiza, a variety of treatment me-thods were used in this study to prepare sample solution. The content changes of phenolic acids in S. miltiorrhiza samples with different dehydration rates were investigated during drying and shade drying processes. Polyphenol oxidase(PPO) of S. miltiorrhiza was extracted and purified by ammonium sulfate precipitation and dialysis to investigate the enzymatic properties. The content of rosmarinic acid, lithosperic acid and S. nolic acid B in S. miltiorrhiza was determined by UPLC. The results showed that the content of phenolic acids in fresh S. miltiorrhiza was highest when it was homogenized with 1 mol·L~(-1) HCl solution or 1 mol·L~(-1) HCl methanol solution. There was no significant difference in the content of phenolic acids in S. miltiorrhiza with different dehydration rates, indicating that there was no correlation between phenolic acid content and dehydration rate. The optimum pH of S. miltiorrhiza PPO was 7.6 and the optimum temperature was 40 ℃. With catechol as substrate, S. miltiorrhiza PPO had the enzymatic browning reaction which was in compliance with Michaelis equation, with Michaelis constant K_m of 0.12 mol·L~(-1) and V_(max) of 588.23 U·min~(-1). The inhibitory effect of citric acid, disodium ethylenediamine tetraacetate, ascorbic acid and sodium sulfite on S. miltiorrhiza PPO increased with the increase of inhibitor concentration, and sodium sulfite showed the strongest inhibitory effect. The present study proved that there were a large number of phenolic acids in fresh S. miltiorrhiza, which were the secondary metabolite of primitive accumulation during the growth of S. miltiorrhiza, rather than the induced product of postharvest drying and dehydration stress. This study has reference value and significance for the cultivation, harvest and processing of S. miltiorrhiza.

Pharmacokinetics of eight active ingredients of Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix combination in rats / 中国中药杂志

To reveal the rationality of compatibility of Salviae Miltiorrhizae Radix et Rhizoma(SMRR) and Puerariae Lobatae Radix(PLR) from the perspective of pharmacokinetics, this study established a UPLC-MS/MS method for quantitative determination of PLR flavonoids(3'-hydroxy puerarin, puerarin, puerarin 6″-O-xyloside, 3'-methoxy puerarin, puerarin apioside) and salvianolic acids and tanshinones(salvianolic acid B, cryptotanshinone, and tanshinone Ⅱ_A) in plasma of rats. Rats were given SMRR extract, PLR extract, and SMRR-PLR extract by gavage and then plasma was collected at different time. UPLC separation was performed under the following conditions: Eclipse C_(18) column(2.1 mm×50 mm, 1.8 μm), 0.1% formic acid in water(A)-0.1% formic acid in acetonitrile(B) as mobile phase for gradient elution. Conditions for MS are as below: multiple reaction monitoring(MRM), ESI~(+/-). Comprehensive validation of the UPLC-MS/MS method(specifically, from the aspects of calibration curve, precision, accuracy, repeatability, stability, matrix effect, extract recovery) was performed and the result demonstrated that it complied with quantitative analysis requirements for biological samples. Compared with SMRR extract alone or PLR extract alone, SMRR-PLR extract significantly increased the AUC and C_(max) of PLR flavonoids and tanshinones in rat plasma, suggesting that the combination of SMRR and PLR promoted the absorption of the above components. The underlying mechanism needs to be further studied.

Effect of Paiteling on HeLa Cell Proliferation and Metastasis Ability and PI3K/Akt Signal Transduction Pathway / 中国实验方剂学杂志

Objective:To explore the effect of Paiteling on the proliferation，metastasis and invasion of HeLa cells and relevant proteins of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Method:① HeLa cells were divided into blank group and Paiteling concentration gradient groups (3.906，2.604，1.953，1.563，1.302，1.116，0.977 g·L-1). After drug intervention for 24 h，the cell morphological changes were observed under microscope. The cell viability was measured by thiazole blue (MTT) colorimetry，and the half inhibitory concentration (IC50) of Paiteling on HeLa cells was calculated. ② HeLa cells were divided into blank group，cisplatin group (0.01 g·L-1)，Paiteling high-dose group (2.974 g·L-1)，Paiteling medium-dose group (1.487 g·L-1) and Paiteling low-dose group (0.991 g·L-1). Cell proliferation and toxicity test (CCK-8) method was used to detect the effect of Paiteling on the proliferation ability of HeLa cells，scratch test was used to detect cell migration，and invasion test (Transwell) was used to detect changes in cell invasion ability. ③ Inhibitor LY294002 group (0.006 g·L-1) was added. Western blot (WB) was used to detect the expressions of Paiteling on PI3K，Akt，recombinant human B-cell lymphoma factor-xl (Bcl-xl)，and B-cell lymphoma/leukemia associated D protein (Bad). Result:① Compared with the blank group，microscopic observation showed that the number of cells in the treatment group was significantly reduced, and the cell morphology was incomplete. MTT experiments showed that Paiteling has a significantly inhibitory effect on HeLa cell proliferation (P<0.01). The IC50 of Paiteling on HeLa cells was calculated as 2.974 g·L-1. ② The CCK-8 experiment showed that compared with the blank group，all the drug-treated groups had an inhibitory effect on HeLa cell proliferation at 24，36，48 h (P<0.01), compared with the cisplatin group，middle and low-dose Paiteling groups showed a reduced inhibitory effect on HeLa cell proliferation at each time point (P<0.01). The scratch test showed that，compared with the blank group，each drug-added group could inhibit the migration ability of HeLa cells (P<0.01)，and the cell migration rate of the high-dose Paiteling group was lower than that of the cisplatin group (P<0.05). Transwell experiments showed that compared with the blank group，the number of membranes permeated by HeLa cells in each drug-treated group was decreased (P<0.01)，and the number of membranes permeated in the middle and low-dose Paiteling groups was increased compared with the cisplatin group (P<0.01). ③ Western blot showed that compared with the blank group，the expression levels of PI3K，Bcl-xl，and Akt in the high，medium，and low-dose Paiteling groups and the LY294002 group decreased (P<0.05，P<0.01)，while the expression of Bad increased (P<0.01). Compared with the high-dose Paiteling group，the PI3K，Akt，and Bcl-xl protein expressions were increased in the low-dose Paiteling group (P<0.01)，whereas Bad expression was decreased (P<0.01). Conclusion:Paiteling can inhibit HeLa cell proliferation，metastasis and invasion ability in a dose-dependent and time-dependent manner，which may be related to its effect on the expressions of PI3K/Akt signaling pathway-related proteins.