Six new (1-6) and seven known depsidones (7-13) were isolated from the culture of an ant (Monomorium chinensis)-derived fungus Spiromastix sp. MY-1. Their structures were elucidated by extensive spectroscopic analysis including high resolution MS, 1D and 2D NMR data. The new bromide depsidones were obtained through supplementing potassium bromide in the fermentation medium of Spiromastix sp. MY-1. All isolated compounds showed various bioactivities against the tested phytopathogenic bacteria. Particularly, new bromide compound 4, named spiromastixone S, exhibited the strongest activity against Xanthomonas oryzae pv. oryzae with a MIC value of 5.2 μmol·^-1.
Animals ; Anti-Bacterial Agents ; Ants ; Bromides ; Depsides ; Fungi ; Lactones ; Microbial Sensitivity Tests ; Molecular Structure

Salvianolic acid B(Sal B), tanshinone Ⅱ_A(TSN Ⅱ_A), and glycyrrhetinic acid(GA) lipid emulsion(GTS-LE) was prepared by the high-speed dispersion method combined with ultrasonic emulsification.The preparation process of the emulsion was optimized by single-factor method and D-optimal method with appearance, centrifugal stability, and particle size of the emulsion as evalua-tion indexes, followed by verification.In vitro release of Sal B, TSN Ⅱ_A, and GA in GTS-LE was performed by reverse dialysis.In vivo pharmacokinetic evaluation was carried out in mice.The acute liver injury model was induced by acetaminophen.The effect of oral GTS-LE on the acute liver injury was investigated by serum liver function indexes and pathological changes in liver tissues of mice.The results showed that under the optimal preparation process, the average particle size of GTS-LE was(145.4±9.25) nm and the Zeta potential was(-33.6±1.45) mV.The drug-loading efficiencies of Sal B, TSN Ⅱ_A, and GA in GTS-LE were above 95%, and the drug release in vitro conformed to the Higuchi equation.The pharmacokinetic results showed that the C_(max) of Sal B, TSN Ⅱ_A, and GA in GTS-LE was 3.128, 2.7, and 2.85 times that of the GTS-S group, and AUC_(0-t) of Sal B, TSN Ⅱ_A, and GA in GTS-LE was 3.09, 2.23, and 1.9 times that of the GTS-S group.After intragastric administration of GTS-LE, the activities of alanine aminotransferase and aspartate aminotransferase were significantly inhibited, the content of malondialdehyde was reduced, and the structure of hepatocytes recovered to normal.In conclusion, GTS-LE can delay the release of Sal B and promote the release of TSN Ⅱ_A and GA.The encapsulation of three drug components in the emulsion can improve the oral bioavailability to varying degrees and can effectively prevent the acute liver injury caused by acetaminophen.
Abietanes/therapeutic use* ; Acetaminophen/therapeutic use* ; Alanine Transaminase/metabolism* ; Animals ; Antipyretics/therapeutic use* ; Aspartate Aminotransferases/metabolism* ; Benzofurans/therapeutic use* ; Chemical and Drug Induced Liver Injury/prevention & control* ; Depsides/therapeutic use* ; Emulsions ; Glycyrrhetinic Acid/therapeutic use* ; Liver/drug effects* ; Malondialdehyde ; Mice

Caffeic acid and its oligomers are the main water-soluble active constituents of the traditional Chinese medicine(TCM) Arnebiae Radix. These compounds possess multiple biological activities such as antimicrobial, antioxidant, cardiovascular protective, liver protective, anti-liver fibrosis, antiviral and anticancer activities. The phenylpropanoid pathway in plants is responsible for the biosynthesis of caffeic acid and its oligomers. Glycosylation can change phenylpropanoid solubility, stability and toxic potential, as well as influencing compartmentalization and biological activity. In view of the important role played by de-glycosylation in the regulation of phenylpropanoid homeostasis, the biosynthesis of caffeic acid and its oligomers are supposed to be under the control of relative UDP-glycosyltransferases(UGTs). Through the data mining of Arnebia euchroma transcriptome, we cloned 15 full-length putative UGT genes. After recombinant expression using the prokaryotic system, the crude enzyme solution of the putative UGTs was examined for the glycosylation activities towards caffeic acid and rosmarinic acid in vitro. AeUGT_01, AeUGT_02, AeUGT_03, AeUGT_04 and AeUGT_10 were able to glycosylate caffeic acid and/or rosmarinic acid resulting in different mono-and/or di-glycosylated products in the UPLC-MS analyses. The characterized UGTs were distantly related to each other and divided into different clades of the phylogenetic tree. Based on the observation that each characterized UGT exhibited substrate or catalytic similarity with the members in their own clade, we supposed the glycosylation abilities towards caffeic acid and/or rosmarinic acid were evolved independently in different clades. The identification of caffeic acid and rosmarinic acid UGTs from A. euchroma could lead to deeper understanding of the caffeic acid oligomers biosynthesis and its regulation. Furthermore, these UGTs might be used for regiospecific glycosylation of caffeic acid and rosmarinic acid to produce bioactive compounds for potential therapeutic applications.
Boraginaceae/genetics* ; Caffeic Acids ; Chromatography, Liquid ; Cinnamates ; Cloning, Molecular ; Depsides ; Glycosyltransferases/genetics* ; Phylogeny ; Tandem Mass Spectrometry

OBJECTIVE: To evaluate the effect of rosmarinic acid (RA) on mitophagy and hypertrophy of cardiomyocytes exposed to high glucose (HG). METHODS: Rat cardiomyocytes (H9c2) exposed to HG (25 mmol/L) were treated with 50 μmol/L RA or with both RA treatment and Parkin siRNA transfection, with the cells cultured in normal glucose (5.5 mmol/L) and HG as the controls. The expressions of PINK1, Parkin and LC3II/LC3I in the cells were detected by Western blotting. The formation of mitochondrial autophagosomes was observed by transmission electron microscope. Flow cytometry was employed to detect the level of reactive oxygen species (ROS) and apoptotic rate of the cells. The activities of respiratory chain complex enzymes were measured by spectrophotometry. Fluorescence enzyme labeling and RESULTS: RA treatment significantly increased the expression levels of PINK1, Parkin and LC3-II/I ( CONCLUSIONS RA can protect rat cardiomyocytes against oxidative stress injury and cardiomyocyte hypertrophy induced by HG by activating Parkin-mediated mitophagy.
Animals ; Cinnamates ; Depsides ; Glucose ; Hypertrophy ; Mitophagy ; Myocytes, Cardiac ; Protein Kinases ; Rats ; Reactive Oxygen Species ; Ubiquitin-Protein Ligases/genetics*

Rosmarinic acid,a hydrosoluble polyphenolic hydroxyl compound,is the active ingredient in such traditional Chinese medicines as Menthae Haplocalycis Herba,Salviae Miltiorrhizae Radix et Rhizoma,Rosemary,Perillae Folium. Because of its good anti-inflammatory,anti-oxidant and anti-tumor effects,it is widely used in food,medicine and other fields. However,the metabolic process and metabolites of rosmarinic acid in vivo have not been completely defined. In this study,an efficient method of ultra-high performance liquid chromatography combined with linear ion trap-Orbitrap(UHPLC-LTQ-Orbitrap) mass spectrometer was used to analyze the metabolites in vivo of rosmarinic acid in rats. Plasma,urine and feces samples were collected after oral administration of rosmarinic acid. After biological samples were processed by solid phase extraction,Acquity UPLC  BEH C18 column(2. 1 mm × 100 mm,1. 7 μm) was used with 0. 1% formic acid(A)-acetonitrile(B) solution as the mobile phase at the speed of 0. 30 m L·min-1 and temperature of 35 ℃ under gradient conditions. The plasma,urine,feces and the blank samples were then analyzed by ESI-LTQ-Orbitrap under both negative and positive ion modes. Based on the accurate mass measurement(<5),MS/MS fragmentation patterns,standards and literatures,a total of 36 metabolites were screened out and identified in the biological samples collected from rats after intragastric administration. Three were identified 3 from rat plasma,31 from urine,and 7 from feces. The main metabolic pathways of rosmarinic acid in rats can be divided into five parts. Rosmarinic acid were first decomposed into small molecules,such as trans-caffeic acid,coumaric acid,m-hydroxybenzoic acid and Danshensu,which were followed by sulfation,methylation,glucuronic acid conjugation and glucose conjugation. The results showed that UHPLC-LTQ-Orbitrap mass spectrometer could be used to analyze the metabolism of rosmarinic acid in rats,and provide reference for further studies on toxicology,pharmacodynamics and secondary development of Chinese medicine.
Animals ; Chromatography, High Pressure Liquid ; Cinnamates/metabolism* ; Depsides/metabolism* ; Drugs, Chinese Herbal/metabolism* ; Rats ; Tandem Mass Spectrometry ; Rosmarinic Acid

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA, MeJA and Ag, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.
Amino Acid Sequence ; Benzofurans ; Biosynthetic Pathways ; genetics ; Cinnamates ; Depsides ; Gene Expression Regulation, Plant ; genetics ; Oxidoreductases ; genetics ; Phenylpropionates ; metabolism ; Phenylpyruvic Acids ; metabolism ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; chemistry ; enzymology ; genetics ; metabolism ; Plants, Genetically Modified ; Recombinant Proteins ; analysis ; biosynthesis ; Salvia miltiorrhiza ; chemistry ; enzymology ; genetics ; metabolism ; Sequence Alignment

The present study was designed to analyze the major constituents in Prunellae Spica and establish a method for simultaneous determination of two constituents contained in Prunellae Spica. High performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-QTOF-MS/MS) technique was used to identify the constituents in the extractive of Prunellae Spica. High performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was used to simultaneously quantify two kinds of constituents contained in Prunellae Spica. Principal component analysis (PCA) was applied to compare the similarity and difference among samples from different regions of China. In the present study, 22 compounds were identified and some new fragmental pathways of triterpenic acids were discovered. An accurate and reliable HPLC-ELSD method was developed and validated for the first time to simultaneously quantify multiple constituents, including rosmarinic acid, maslinic acid, corosolic acid, betulin, oleanolic acid, and ursolic acid in the extract of Prunellae Spica. (PCA) revealed some similarities and differences among different samples from different regions of China. In conclusion, our results from this study would be helpful in establishing a scientific and rational quality control method for Prunellae Spica.
China ; Chromatography, High Pressure Liquid ; methods ; Cinnamates ; chemistry ; Depsides ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Molecular Structure ; Prunella ; chemistry ; Tandem Mass Spectrometry ; methods ; Triterpenes ; chemistry

The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.
Animals ; Benzofurans ; chemistry ; pharmacology ; Cinnamates ; chemistry ; pharmacology ; Depsides ; chemistry ; pharmacology ; Lactates ; chemistry ; pharmacology ; Male ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Solubility

OBJECTIVE: To exlpore the eff ect of depsides salts from Salvia miltiorrhiza on human hepatoma cell line SMMC-7721 xenograft tumors and the possible mechanisms. METHODS: A total of 36 nude mice were divided into 6 groups: A model group, a negative control group, a positive control group, and 3 treatment groups at low, middle or high dose (n=6). The tumor model of nude mice was given depsides salts at a dose of 10, 20 or 50 mg/kg every 3 day for 16 days. Then samples of subcutaneous tumors in nude mice were collected. The morphological changes of tumor samples were observed by HE staining and the expression of vascular endothelial growth factor (VEGF) and the tumor antigen Ki67 was detected by immunohistochemical method. RESULTS: The tumor growth was inhibited by all doses of depsides salts. The morphology of tumors was shrinkage, broken and irregularly arranged compared with the tumors in the model group and the negative control group. Morphological changes were more obvious in tumors with treatment at high dose. Expression of VEGF and Ki67 in treatment groups and the positive control group were lower than that in the model group and the negative control group, with a significant difference (P<0.05). CONCLUSION Depsides salts from Salvia miltiorrhiza can inhibit the growth of human hepatoma cell line SMMC-7721 tumor in nude mice, which is related to the inhibition of Ki67 and VEGF.
Animals ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; drug effects ; Depsides ; pharmacology ; Humans ; Ki-67 Antigen ; metabolism ; Liver Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Salts ; Salvia miltiorrhiza ; chemistry ; Vascular Endothelial Growth Factor A ; metabolism ; Xenograft Model Antitumor Assays

To establish a method for the determination of astilbin, peoniflorin, rasmarinci acid, isofraxidin and liquiritin contained in Shaolin Xiaoyin tablets, in order to lay a foundation for designing late-stage dosage forms and clinical medication schemes. In this paper, efforts were made to establish a method for the determination of the blood concentration of the five components and study the in vivo pharmacokinetics in rats. The blood concentration was determined by HPLC. Phenomenex C18 column (4.6 mm x 250 mm, 5 microm) was adopted and eluted with methanol-acetonitrile-0.05% formic acid, the flow rate was 0.8 mL x min(-1), and the wavelength was 275 nm. The samples were processed by the solid phase extraction method. After oral administration of Shaoling Xiaoyin tablets, the rat bloods were collected at different time points to determine the blood concentrations. The experimental results showed that the baseline separation could be adopted for the five components, and astilbin, peoniflorin, rasmarinci acid, isofraxidin and liquiritin showed good linear relations within ranges of 2.48-248, 0.213 6-21.36, 0.531-53.1, 0.704-70.4, 0.253-25.3 mg x L(-1). All the five components could be absorbed in blood and excreted quickly. The method established in this paper is rapid and accurate, and could be used for in vivo analysis on preparations containing similar components. The main components in Shaoling Xiaoyin tablets could be absorbed and excreted quickly, and thus suitable to be made into sustained release tablets. Common preparations are required to be taken for 4-6 times a day.
Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; Cinnamates ; blood ; pharmacokinetics ; Coumarins ; administration & dosage ; blood ; pharmacokinetics ; Depsides ; blood ; pharmacokinetics ; Drugs, Chinese Herbal ; analysis ; pharmacokinetics ; Flavanones ; administration & dosage ; blood ; pharmacokinetics ; Flavonols ; administration & dosage ; blood ; pharmacokinetics ; Glucosides ; administration & dosage ; blood ; pharmacokinetics ; Male ; Monoterpenes ; administration & dosage ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley