[Study on metabolites in vivo of Dangefentong Capsules based on UHPLC-Q/Orbitrap-MS/MS].

Dangefentong Capsules is a new traditional Chinese medicine preparation for the treatment of diabetic peripheral neuropathy. It is based on the Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix herb pair with salvianolic acids, tanshinones and pueraria flavonoids as main components. Studying the chemical composition in vivo of Dangefentong Capsules and its metabolites is of great significance for making clear its pharmacodynamic material basis and the action mechanism. The UHPLC-Q/Orbitrap-MS/MS was applied to rapidly analyze the metabolites and metabolic pathways of Dangefentong Capsules in Beagle dogs after gavage. Eclipse plus C_(18) column(2.1 mm×50 mm, 1.8 µm) was used, and gradient elution was performed with 0.1% formic acid aqueous solution(A)-formic acid acetonitrile solution(B). A heated electrospray ion source(HESI) was employed. The scanning mode was set as the positive and negative ion mode, and the mass scanning range was m/z 100-1 000. The plasma, urine and feces samples were collected after male Beagle dogs were administered with Dangefentong Capsules. The prototype components and metabolites were identified by UHPLC-Q/Orbitrap-MS/MS analysis combined with reference substances and references. The results showed that 107 chemical components were identified, including 58 prototype components and 49 metabolites. The identified prototype components included 42 components from Salviae Miltiorrhizae Radix et Rhizoma and 16 components from Puerariae Lobatae Radix. The metabolites consist of 21 and 28 metabolites of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix, respectively. They are mainly derived from the methylation, hydroxylation, sulfation and glucuronidation of salvianolic acids, tanshinones and pueraria flavonoids. This research rapi-dly analyzes the chemical components in vivo of Beagle dogs administered with Dangefentong Capsules, laying a basis for illustrating the pharmacodynamic material basis and mechanism of Dangefentong Capsules.

[Effects of different field processing methods on volatile components of Chuanxiong Rhizoma: an exploration based on headspace gas chromatography-mass spectrometry].

The volatile oil of Chuanxiong Rhizoma(CX) is known as an effective fraction. In order to seek a suitable method for processing CX and its decoction pieces, this study selected 16 volatile components as indices to investigate how different processing methods such as washing/without washing, sun-drying, baking, oven-drying and far-infrared drying at different temperatures affected the quality of CX and its decoction pieces(fresh CX was partially dried, cut into pieces, and then dried) by headspace gas chromatography-mass spectrometry(GC-MS), cluster analysis, principal component analysis and comprehensive weighted scoring. The results showed that the rapid washing before processing did not deteriorate the volatile components of CX. Considering the practical condition of production area, oven-drying was believed to be more suitable than sun-drying, baking, and far-infrared drying. The CX decoction pieces with a thickness of 0.3-0.4 cm were recommended to be oven-dried at 50 ℃. The integrated processing(partial drying, cutting into pieces, and drying) did not cause a significant loss of volatile components. For the fresh CX, the oven-drying at 60 ℃ is preferred. The temperature should not exceed 60 ℃, and drying below 60 ℃ will prolong the processing time, which will produce an unfavorable effect on volatile components. This study has provided the scientific evidence for field processing of CX, which is conducive to realizing the normalization and standardization of CX processing in the production area and stabilizing the quality of CX and its decoction pieces.

[Content determination of phenolic acids in fresh Salvia miltiorrhiza and effect of polyphenol oxidase on content].

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].

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].

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].

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.

[Comparative Study on Volatile Oil Composition of Chuanxiong Rhizoma, Angelicae Sinensis Radix and Ligustici Rhizoma et Radix].

OBJECTIVE: To identify Chuanxiong Rhizoma,Angelicae Sinensis Radix and Ligustici Rhizoma et Radix by establishing the HPLC specific chromatograms of their volatile oil and to compare their specific peaks. METHODS: The HPLC method used methanol-water as mobile phase. Their specific peaks were analysed by HPLC-MS. RESULTS: Under the selected spectrum condition, their HPLC specific chromatograms were established. Senkyunolide A, butylphalide, coniferylferulate, E-ligustilide, Z-ligustilide, neocnidilide and E-butylidenephthalide were identified as specific peaks in chromatograms based on their MS data. CONCLUSION: This method is simple, accurate and available to identify Chuanxiong Rhizoma, Angelicae Sinensis Radix and Ligustici Rhizoma et Radix. It provides reference for quality control of their medicinal materials and Chinese Patent Medicine.

[Optimization of processing technology for xanthii fructus by UPLC fingerprint technique and contents of toxicity ingredient].

The experiment's aim was to optimize the processing technology of Xanthii Fructus which through comparing the difference of UPLC fingerprint and contents of toxicity ingredient in water extract of 16 batches of processed sample. The determination condition of UPLC chromatographic and contents of toxicity ingredient were as follows. UPLC chromatographic: ACQUITY BEH C18 column (2.1 mm x 100 mm, 1.7 microm) eluted with the mobile phases of acetonitrile and 0.1% phosphoric acidwater in gradient mode, the flow rate was 0.25 mL x min(-1) and the detection wavelength was set at 327 nm. Contents of toxicity ingredient: Agilent TC-C18 column (4.6 mm x 250 mm, 5 microm), mobile phase was methanol-0.01 mol x L(-1) sodium dihydrogen phosphate (35: 65), flow rate was 1.0 mL x min(-1), and detection wavelength was 203 nm. The chromatographic fingerprints 16 batches of samples were analyzed in using the similarity evaluation system of chromatographic, fingerprint of traditional Chinese medicine, SPSS16.0 and SIMCA13.0 software, respectively. The similarity degrees of the 16 batches samples were more than 0.97, all the samples were classified into four categories, and the PCA showed that the peak area of chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeic acid were significantly effect index in fingerprint of processed Xanthii Fructus sample. The outcome of determination showed that the toxicity ingredient contents of all samples reduced significantly after processing. This method can be used in optimizing the processing technology of Xanthii Fructus.

[Effects of steaming and baking on content of alkaloids in Aconite Lateralis Radix (Fuzi)].

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.

[Studies on the chemical constituents of Pseudotsuga sinensis].

AIM: To study the chemical constituents of Pseudotsuga sinensis Dode (Pinaceae). METHODS: To separate the constituents of P. sinensis by using various kinds of chromatography and identify their structures on the basis of spectral analysis. RESULTS: Six compounds were isolated from P. sinensis. Their structures were established as 5,7,4'-trihydroxy-6-methylflavanone (poriol, I), 3,5,7,3',4'- pentahydroxyflavonone (quercetin, II), 5,7,3',5'-tetrahydroxy-6- methylflavanone (III), 5,7,3',5'-tetrahydroxyflavanone (IV), 3,5,7,3',5'-pentahydroxyflavanone (V) and 5-hydroxy-6-methylchromone-7-O-beta-D-glucopyranoside (VI) based on the analysis of spectral data of IR, UV, MS, 1D and 2D-NMR. CONCLUSION: Compounds III and VI are new compounds. All of six compounds were isolated from this plant for the first time.