Effect of Rhizophagus intraradices on growth of Salvia miltiorrhiza / 中国中药杂志

The study aimed to explore the effects of inoculation of Rhizophagus intraradices on the biomass, effective component content, and endogenous hormone content of Salvia miltiorrhiza through pot experiments. The number of leaves, plant height, dry weight of aboveground and underground parts, branch number, root number, root length, root diameter, and other biomass were mea-sured by weighing and counting methods. The content of salvianolic acid B, caffeic acid, rosmarinic acid, tanshinone Ⅰ, tanshinone Ⅱ_A, cryptotanshinone, and other effective components was determined by ultra-high performance liquid chromatography. The content of ABA and GA_3 was determined by triple quadrupole mass spectrometry. The correlations between biomass and effective components and between effective components and plant hormones ABA and GA_3 were analyzed. The results showed that R. intraradices significan-tly increased the aboveground dry weight, leaf number, and root number of S. miltiorrhiza by 0.24-0.65 times, respectively. The content of salvianolic acid B and rosmarinic acid in the aboveground part and the content of salvianolic acid B, caffeic acid, rosmarinic acid, tanshinone Ⅰ, and tanshinone Ⅱ_A in the underground part were significantly increased by 0.44-1.78 times, respectively. R. intraradices infection significantly increased the GA_3/ABA values of aboveground and underground parts by 3.82 and 76.47 times, respectively. The correlation analysis showed that caffeic acid, the effective component of the aboveground part, was significantly positively correlated with plant height, tanshinone Ⅱ_A, the effective component of the underground part, was significantly positively correlated with biomass root number, cryptotanshinone, and dry weight, while rosmarinic acid was significantly negatively correlated with dry weight. There were significant positive correlations between cryptotanshinone and ABA, tanshinone Ⅱ_A and ABA and GA_3, and caffeic acid and GA_3. In conclusion, R. intraradices can promote the accumulation of biomass and secondary metabolites of S. miltiorrhiza and regulate the balance between plant hormones ABA and GA_3, thereby promoting the growth of S. miltiorrhiza.

Modification of C20 oxidase in tanshinone biosynthesis pathway / 中国中药杂志

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Tanshinone IIA ameliorates myocardial ischemia/reperfusion injury in rats by regulation of NLRP3 inflammasome activation and Th17 cells differentiation

Purpose: Tanshinone IIA is a well-known lipophilic active constituent refined from traditional Chinese medicines, danshen. It has been previously demonstrated to possess various biological properties, including anti-inflammatory, antioxidant, promoting angiogenesis effect and so on. However, the mechanism of tanshinone IIA on myocardial ischemia-reperfusion injury (MI/RI) remains unclear. In this study, we investigated the effect of tanshinone IIA on MI/RI. Methods: MI/RI rat models were set up. Echocardiographic evaluation and hematoxylin and eosin staining were performed to analyze the cardiac function and morphology of MI/RI. Western blot was conducted for the detection of protein expression of pyrin domain containing 3 (NLRP3) and caspase-1 in heart tissues. Flow cytometry and real-time polymerase chain reaction were used for the detection of proinflammatory cytokines and Th17 cells differentiation. Results: We found that tanshinone IIA alleviated the myocardial damage of MI/RI, ameliorated the overall and local inflammatory reaction, and produced a cardioprotective effect by inhibiting of NLRP3 inflammasome activation and Th17/Treg cells differentiation. Conclusions: Our results highlighted the cardio-protective effect of tanshinone IIA on MI/RI and uncovered its underlying mechanism related to the NLRP3 inflammasome inhibition and the modulation of Th17/Treg cells differentiation.

Research on batch-to-batch quality control of compound Danshen extract based on physical characterization and multivariate statistical analysis / 中国中药杂志

Physical attributes of Chinese herbal extracts are determined by their chemical components, and the physical and chemical attributes jointly affect the preparation process performance and the final product quality. Therefore, in order to improve the quality control of Chinese herbal extracts, we should comprehensively study the batch-to-batch consistency of physical and chemical attributes as well as the correlations between them. This paper first explored the physical attributes affecting the preparation process performance of the compound Danshen extract and developed a method for characterizing the texture attributes. With such main chemical components as water, phenolic acids, saponins, and saccharides and texture, rheology, and other physical attributes taken into consideration, the batch-to-batch quality fluctuation of products from different production lines and time was analyzed by principal components analysis(PCA). Finally, the correlation and partial least squares(PLS) analysis was conducted, and the regression equation was established. The fitting result of the PLS model for dynamic viscosity was satisfying(R~2Y=0.857, Q~2=0.793), suggesting that the chemical components could be adjusted by the component transfer rate in the extraction process, the impurity removal rate in the alcohol precipitation process, and the water retention rate of the concentration process to meet the control of the extract dynamic viscosity. This study clarified the correlations between physical and chemical attributes of the compound Danshen extract and established a method for controlling its physical attributes based on process regulation, which would provide reference for improving the quality control of Chinese herbal extracts.

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.

Cryptotanshinone but not tanshinone IIA inhibits angiogenesisin vitro

In the course of screening of angiogenesis inhibitor from natural products, cryptotanshinone from Salvia miltiorrhiza was isolated as a potent small molecule inhibitor of angiogenesis. Cryptotanshinone inhibits bFGF-induced angiogenesis of BAECs at ten micromolar ranges in vitro without cytotoxicity. Tanshinone IIA, another tanshinone isolated from S. miltiorrhiza, which is structurally very similar to cryptotanshinone except C-15 position of dihydrofuran ring does not inhibit angiogenesis induced by bFGF. These results demonstrate that cryptotanshinone is a new anti-angiogenic agent and double bond at C-15 position of the dihydrofuran ring plays a crucial role in the activity.