Extraction process,enzymatic properties and practical application of glucuronic hydrolase in Scutellaria baicalensis stems and leaves / 中成药

AIM To study the extraction process,enzymatic properties and practical application of glucuronic hydrolase in Scutellaria baicalensis stems and leaves(sbsl GUS).METHODS With granularity,water consumption,extraction time and extraction frequency as influencing factors,enzymatic activity as an evaluation index,the extraction process was optimized by orthogonal test on the basis of single factor test.The relationship between substrate(baicalin)concentration and enzymolysis rate,after which Vmax and Km were calculated,the effects of pH value,temperature and metal ion on enzymatic activity were investigated,pH stability and heat stability were evaluated.sbsl GUS was adotped in the enzymolysis of baicalin to prepare baicalein,then the effects of pH value,temperature,reaction time,initial substrate concentration and enzyme addition on transfer rate were investigated.RESULTS The optimal extraction process was determined to be 40 mesh for granularity,10 times for water consumption,15 min for extraction time,and 3 times for extraction frequency.The enzymolysis accorded with the kinetics of enzymatic reaction,Km was 0.006 3 mol/L,Vmax was 70.42 μmol/h,the strongest enzymatic activity was found at the pH value of 6.0,temperature of 45℃and metal ion of 100 mmol/L Cu2+,sbsl GUS demonstrated good stability at the ranges of 4.0-7.0 for pH value and 4-30℃for temperature.The optimal preparation process was determined to be 6.0 for pH value,45℃for temperature,more than 12 h for reaction time,67.2 mmol/L for initial substrate concentration,and 1 mL/0.269 mmol baicalin for enzyme addition,the transfer rate was 97.83%.CONCLUSION sbsl GUS enzymolysis exhibits high efficiency and mild condition,which can provide a simple preparation method for obtaining baicalein,and expand the application path of Scutellaria baicalensis stems and leaves.

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.

Comparative study on free and bound phenolic acids before and after drying of Salvia miltiorrhiza / 中国中药杂志

There were significant differences in phenolic acid content between fresh and dried Salvia miltiorrhiza before and after drying. That is to say, the content of phenolic acid in S. miltiorrhiza significantly increased with the increase of dehydration during the drying process.In order to investigate the differences and transformation of free and bound phenolic acids before and after the drying process of S.miltiorrhiza, we studied hydrolysis method, hydrolysates and hydrolysis regularity of phenolic acids in S.miltiorrhiza. UPLC method was used to determine four main hydrolysates of bound phenolic acids, namely danshensu, caffeic acid dimer(SMND-309), caffeic acid, przewalskinic acid A(prolithosperic acid), and three main free phenolic acids in S.miltiorrhiza, namely rosmarinic acid, lithospermic acid, salvianolic acid B. The results of the acid-base hydrolysis experiment of salvianolic acid showed that the alkaline hydrolysis effect was significantly better than acid hydrolysis. The optimal alkaline hydrolysis condition was hydrolysis at 70 ℃ for 4 h with 2 mol·L~(-1) NaOH solution containing 1% ascorbic acid(Vit C). The hydrolysates of free phenolic acids were the same with the hydrolysates of bound phenolic acids. Fresh S.miltiorrhiza contains a low level of free phenolic acids and a high level of bound phenolic acids, which were exactly opposite to dried S.miltiorrhiza. It was suggested that a large amount of bound phenolic acids was accumulated during the growth of S.miltiorrhiza. These bound phenolic acids were coupled with polysaccharides on the cytoderm through ester bonds to form insoluble phenolic acids, which was not easy to be detected by conventional methods. However, during drying and dehydration processes, the bound phenolic acids were converted to a large amount of free phenolic acids under the action of the relevant enzyme.

Study on dynamic changes of chemical constituents during different drying processes of Salvia miltiorrhiza / 中国中药杂志

There is no consensus on the drying methods of Salvia miltiorrhiza in ancient and modern times,especially on the content of phenolic acid in fresh S. miltiorrhiza. In order to further explore the content of main components in fresh S. miltiorrhiza and study the dynamic changes during the drying process,the content of main components was used as the index in this study to evaluate the processing method,drying method,correlation between dehydration rate and component content for fresh S. miltiorrhiza. In addition,the sealed and unsealed parallel control groups were set to carry out verification test during the drying process. UPLC method was used for determination of seven main components including rosmarinic acid,lithosperic acid,salvianolic acid B,cryptotanshinone,tanshinoneⅠ,methylene salianolate and tanshinone ⅡAin S. miltiorrhiza. The results showed that the fresh S. miltiorrhiza contained low levels of phenolic acid,and the content of phenolic acid increased significantly with the increase of dehydration rate during drying process,while the change of tanshinone was not obvious. In the comparison of three drying methods,we found that drying at 50 ℃ was better than drying in the sun,and drying in the sun was superior to drying in the shade. So,drying at 50 ℃ was the best drying method. The correlation between dehydration and phenolic acid content of S. miltiorrhiza was analyzed by verification test and SPSS software,which further proved that the dehydration rate was significantly positively correlated with the content of phenolic acid components. This study provides reference for the production processing and drying methods of S. miltiorrhiza medicinal materials,which is of great significance for improving the quality of S. miltiorrhiza.

Effects of steaming and baking on content of alkaloids in Aconite Lateralis Radix (Fuzi) / 中国中药杂志

To study the effect of steaming and baking process on contents of alkaloids in Aconite Lateralis Radix (Fuzi), 13 alkaloids were analyzed by UPLC-MS/MS equipped with ESI ion source in MRM mode. In steaming process, the contents of diester-diterpenoid alkaloids decreased rapidly, the contents of monoester-diterpenoid alkaloids firstly increased, reached the peak at 40 min, and then deceased gradually. The contents of aconine alkaloids (mesaconine, aconine and hypaconine) increased all the time during processing, while the contents of fuziline, songorine, karacoline, salsolionl were stable or slightly decreased. In baking process, dynamic variations of alkaloids were different from that in the steaming process. Diester-diterpenoid alkaloids were degraded slightly slower than in steaming process. Monoester-diterpenoid alkaloids, aconine alkaloids and the total alkaloids had been destroyed at different degrees, their contents were significantly lower than the ones in steaming Fuzi at the same processing time. This experiment revealed the dynamic variations of alkaloids in the course of steaming and baking. Two processing methods which can both effectively remove the toxic ingredients and retain the active ingredients are simple and controllable, and are valuable for popularization and application.

Inhibitory effect of human umbilical cord-derived mesenchymal stem cells on interleukin-17 production in peripheral blood T cells from spondyloarthritis patients / 中国实验血液学杂志

In this study, the inhibitory effect of human umbilical cord-derived mesenchymal stem cells (hUCMSC) on interleukin-17 (IL-17) production in peripheral blood T cells from patients with spondyloarthritis (SpA) were investigated, in order to explore the therapeutic potential of hUCMSC in the SpA. Peripheral blood mononuclear cells (PBMNC) were isolated from patients with SpA (n = 12) and healthy subjects (n = 6). PBMNC were cultured in vitro with hUCMSC or alone. The expression of IL-17 in CD4(+) T cells or γ/δ T cells were determined in each subject group by flow cytometry. IL-17 concentrations in PBMNC culture supernatants were measured by ELISA. The results indicated that the proportion of IL-17-producing CD4(+) T cells and IL-17-producing γ/δ T cells of SpA patients were 4.5 folds and 5 folds of healthy controls [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (0.75 ± 0.25)%, P < 0.01; CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.06 ± 0.02)%, P < 0.01]. After co-culture of PBMNC in patients with hUCMSC, the increased proportions of CD3(+)CD4(+)IL-17(+) cells and CD3(+)γδTCR(+)IL-17(+) cells in SpA patients were inhibited significantly by hUCMSC [CD3(+)CD4(+)IL-17(+) cells (3.42 ± 0.82)% vs (1.81 ± 0.59)% (P < 0.01); CD3(+)γδTCR(+)IL-17(+) cells (0.30 ± 0.10)% vs (0.16 ± 0.06)% (P < 0.01]. In response to phytohemagglutinin (PHA, 1 µg/ml), PBMNC from SpA patients secreted more IL-17 than that from healthy control [(573.95 ± 171.68) pg/ml vs (115.53 ± 40.41) pg/ml (P < 0.01)]. In the presence of hUCMSC, PBMNC of SpA patients produced less amount of IL-17 [(573.95 ± 171.68) pg/ml vs (443.20 ± 147.94) pg/ml, (P < 0.01)]. It is concluded that the IL-17 production in peripheral blood T cells from SpA patients can be inhibited by hUCMSC, which have therapeutic potential for SpA.