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

OBJECTIVETo evaluate the effects of aloin, cinnamic acid and 15 other kinds of natural chemicals on the activity of tyrosinase, in order to provide lightening agents in the treatment of hyperpigmentation disorders and cosmetic additives.

METHODSTyrosinase activity was estimated by measuring the oxidation rate of L-dopa. Inhibition of the enzyme was deduced according to the Lineweaver-Burk plots compared to the control.

RESULTSCadabine, paeonal, farrerol, evodin, cinnamic acid, aloin and sophorcarpidine had different levels of inhibition of tyrosinase. The inhibitory rates of cinnamic acid (2 mmol/L, 0.5 mmol/L), aloin (2 mmol/L) and the rest were significantly higher than that of hydroquinone (0.5 mmol/L) (P < 0.05).

CONCLUSIONSTyrosinase activity can be greatly inhibited by cinnamic acid, aloin and sophorcarpidine, of which sophorcarpidine functions as an uncompetitive inhibitor, compared to aloin and cinnamic acid, which are mixed-type inhibitors.

Cinnamates ; pharmacology ; Cosmetics ; pharmacology ; Emodin ; analogs & derivatives ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Humans ; Hyperpigmentation ; drug therapy ; Monophenol Monooxygenase ; antagonists & inhibitors ; Plant Preparations ; pharmacology

To generate transgenic mice in which both hygromycin (hyg) and neomycin (neo) resistance genes are expressed in murine fibroblast cells (MEFs), which are required for conditional gene knock-out and screening of drug resistant ES cell clones. To construct HygR-neoR expression vector, pTK-hygR-pA and PGK-neoR-pA were cloned into pBluescript vector. DNA fragments of tandem genes ( 4245bp ) were prepared by Kpn I and Xba I digestion and transgene was microinjected into pronucleus of zygotes to generate transgenic mice. Transgenic mice were identified by PCR and Southern blot; expression of hygR and neoR gene transcripts were detected by RT-PCR. 7 founder mice carrying hyg-neo resistant genes were obtained and 6 transgenic mouse lines were successfully established. The hygR and neoR gene transcripts were detected in the liver and/or ovary of transgenic mice from hn30, hn33, hn66 and hn67 mouse lines. In MEFs isolated from the mice of line hn66 and hn30, expression of hyg and neo resistant genes was also detectable. Transgenic mouse lines expressing two anti-drug genes have been established. The hyg and neo resistant gene transcripts were detected in the MEFs of two transgenic mouse lines.
Animals ; Cinnamates ; pharmacology ; Drug Resistance, Multiple ; genetics ; Fibroblasts ; metabolism ; Hygromycin B ; analogs & derivatives ; pharmacology ; Mice ; Mice, Transgenic ; Neomycin ; pharmacology ; Transgenes ; genetics

A significant number of organic carboxylic acids have been shown to influence the absorption and distribution of drugs mediated by organic anion transporters (OATs). In this study, uptake experiments were performed to assess the inhibitory effects of cinnamic acid, ferulic acid, oleanolic acid, deoxycholic acid, and cynarin on hOAT1, hOAT3, hOATP1B1, and hOATP2B1. After a drug-drug interaction (DDI) investigation, cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were found and validated to inhibit hOAT1 in a competitive manner, and deoxycholic acid was found to be an inhibitor of all four transporters. The apparent 50% inhibitory concentrations of cinnamic acid, ferulic acid, deoxycholic acid, and cynarin were estimated to be 133.87, 3.69, 90.03 and 6.03 μmol·L(-1) for hOAT1, respectively. The apparent 50% inhibitory concentrations of deoxycholic acid were estimated to be 9.57 μmol·L(-1) for hOAT3, 70.54 μmol·L(-1) for hOATP1B1, and 168.27 μmol·L(-1) for hOATP2B1. Because cinnamic acid, ferulic acid, and cynarin are ingredients of food or food additives, the present study suggests there are new food-drug interactions to be disclosed. In addition, deoxycholic acid may be used as a probe for studying the correlation of OATs and OATPs.
Carboxylic Acids ; pharmacology ; Cinnamates ; pharmacology ; Coumaric Acids ; pharmacology ; Deoxycholic Acid ; pharmacology ; Diet ; Drug Interactions ; HEK293 Cells ; Humans ; Organic Anion Transport Protein 1 ; antagonists & inhibitors ; Organic Anion Transporters ; antagonists & inhibitors ; Plant Extracts ; pharmacology ; Plants, Medicinal ; chemistry

Rosmarinic acid (RA), a phenolic acid, is one of the important secondary metabolites produced in Salvia miltiorrhiza. To observe the influence of salicylic acid (SA), an elicitor, on the synthesis of RA and related enzymes, we treated the cell suspension cultures of S. miltiorrhiza with SA and L-a-aminooxy-beta-phenylpropionic acid (AOPP), a competitive inhibitor of tyrosine aminotransferase (TAT). Under this condition, the activities of related enzymes, such as phenylalanine ammonia-lyase and TAT were traced and assayed; the accumulative amount of RA was measured. The results showed that the PAL activity reached the peak at 4 h, 124% higher than that of the control, and the content of RA reached its maximum ((5.914 +/- 0.296) mg/g dry weight) at 8 h, after treated by 6.25 mg/L SA on day 6 of the suspension culture. The results of treatment with 0.1 micromol/L AOPP showed that AOPP affected little on the TAT activity, while the PAL activity was significantly influenced, with 44% lower than that of the control at 6 h. Meanwhile, the reduced accumulation of RA ((4.709 +/- 0.204) mg/g dry weight) paralleled with the decrease in PAL activity. The co-treatment by 0.1 micromol/L AOPP and 6.25 mg/L SA relieved the restriction imposed by AOPP on PAL, and made the cell cultures accumulate more RA than sole treatment with AOPP, indicated that SA induced the accumulation of RA in suspension cell culture of S. miltiorrhiza, and the rate-limiting effect of PAL was stronger than TAT.
Cell Culture Techniques ; methods ; Cinnamates ; metabolism ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Cells ; metabolism ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; cytology ; growth & development ; metabolism ; Suspensions ; Tyrosine Transaminase ; metabolism

We studied the influence of the concentration of Ca2+ (0-50 mmol/L) in culture medium on the synthesis of rosmarinic acid (RA) and related enzymes in Salvia miltiorrhiza suspension cultures. Using verpamil (VP, a calcium channel antagonist) and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2+ in the culture medium. The synthesis of intracellular RA in 6-day incubation was significantly dependent on the medium Ca2+ concentration. At the optimal Ca2+ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2+ concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2+ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2+ in the culture medium was accomplished by the intracellular Ca2+, and the flow of Ca2+ from the extracellular to the intracellular environment could also participate in this process.
Calcium ; pharmacology ; Cinnamates ; metabolism ; Culture Media ; Culture Techniques ; methods ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Salvia miltiorrhiza ; chemistry ; enzymology ; growth & development ; Tyrosine Transaminase ; metabolism

Danshen (Salvia miltiorrhiza Bunge) hairy roots were obtained by infecting Danshen leaves with Agrobacterium rhizogenes 9402. Besides rosmarinic acid (RA) and salvianolic acid B (SAB), the hairy root could also produce salvianolic acid K (SAK), salvianolic acid L, ethyl salvianolic acid B (ESAB), methyl salvianolic acid B (MSAB), and a compound with a molecular weight of 538 (compound 538) identified by using LC-MS. Effects of methyl jasmonate (MeJA) and yeast elicitor (YE) on the accumulation of these compounds had been investigated. MeJA increased the accumulation of SAB, RA, SAK, and compound 538 from 4.21%, 2.48%, 0.29%, and 0.01% of dry weight to 7.11%, 3.38%, 0.68%, and 0.04%, respectively. YE stimulated the biosynthesis of RA from 2.83% to 5.71%, but depressed the synthesis of SAB, SAK and compound 538. It was indicated in all the results that these Danshen hairy roots could be used as alternative resources to produce salvianolic acids. Analysis of the content variation of these compounds after elicitation suggested that SAK and compound 538 might be the intermediates in the biosynthesis from RA to SAB in Danshen hairy roots.
Acetates ; pharmacology ; Alkenes ; analysis ; Benzofurans ; analysis ; Cinnamates ; analysis ; Cyclopentanes ; pharmacology ; Depsides ; analysis ; Oxylipins ; pharmacology ; Phenylpropionates ; analysis ; Plant Growth Regulators ; pharmacology ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Polyphenols ; analysis ; biosynthesis ; Salvia miltiorrhiza ; chemistry ; Yeasts ; chemistry

OBJECTIVETo investigate the effects of salvianolic acids on human umbilical vein endothelial cells (HUVEC) against damage induced by cholestane-3beta-5alpha-6beta-triol (chol-triol).

METHODSThe viability of HUVEC was measured by MTT method. The apoptosis of HUVEC induced by chol-triol was detected by flow cytometry and TUNEL assay. The production of malondialdehyd (MDA) in HUVEC was tested by thiobarbaturic acid (TBA) assay.

RESULTSThe viability of HUVEC treated with chol-triol 100 micro mol/L decreased by 39.8% while salvianolic acids 100 micro g/ml increased by 27.9%. The apoptotic rate of HUVEC measured by PI staining increased from 6% - 8% to 17% - 20% after chol-triol treatment for 12 h. Salvianolic acids 100 micro g/ml reduced the apoptotic rate to 10% - 14% after treatment HUVEC for 1 h prior to chol-triol treatment. In another experiment, chol-triol increased the number of TUNEL-positive cells 5 times, but salvianolic acids 10 micro g/ml and 100 micro g/ml reduced the number of TUNEL-positive cells by 36.9% and 61.2%, respectively. The production of MDA in HUVEC increased by 120.7% after chol-triol treatment for 12 h. Salvianolic acids 10 micro g/ml and 100 micro g/ml also decreased the concentration of MDA by 28.7% and 39.8%, respectively.

CONCLUSIONSalvianolic acids has protective effect on endothelial cells against damage induced by chol-triol.

Apoptosis ; drug effects ; Benzofurans ; pharmacology ; Caffeic Acids ; pharmacology ; Cell Survival ; drug effects ; Cells, Cultured ; Cholestanols ; toxicity ; Cinnamates ; pharmacology ; Depsides ; Endothelium, Vascular ; cytology ; drug effects ; Humans ; Lactates ; pharmacology ; Malondialdehyde ; metabolism

A new flavonone, named as (2R, 3S)-pinobanksin-3-cinnamate(1), together with six known compounds, pinocem-brin (2), pinobanksin (3), 3-O-acetylpinobanksin (4), galangin (5), kumatakenin(6), and 3-methylkaempferol (7), were isolated from a 95% ethanol extract of seeds of Alpinia katsumadai through a combination of various chromatographic techniques, including silica gel and Sephadex LH-20. The structure of compound 1 was elucidated by spectroscopic data analysis. Compound 1 exhibits a potent neuroprotective effect against the corticosterone-damaged PC12 cells, which may be underlying the effect by scavenging intracellular ROS.
Alpinia ; chemistry ; Animals ; Cell Death ; drug effects ; Cholestenones ; chemistry ; isolation & purification ; pharmacology ; Cinnamates ; chemistry ; isolation & purification ; pharmacology ; DNA Fragmentation ; drug effects ; Neuroprotective Agents ; chemistry ; isolation & purification ; pharmacology ; Oxidative Stress ; drug effects ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; Seeds ; chemistry

OBJECTIVETo explore the neuroprotective effect and mechanism of picroside II on extracellular regulated protein kinases1/2 (ERK1/2) signal transduction pathway in cerebral ischemia injuryrats. METHODS The middle cerebral artery occlusion (MCAO) model was established by inserting a monofilament into middle cerebral artery. Totally 96 successfully modeled Wistar rats were divided into the modelgroup, the treatment (picroside II) group, the Lipopolysachcaride (LPS) group, and the U0126 group according to random digit table. Each group was further divided into 3 subgroups, i.e. 6, 12, and 24 h sub-groups. Picroside II (20 mg/kg) was peritoneally injected to rats in the treatment group 2 h after ischemia.LPS (20 mg/kg) and Picroside II (20 mg/kg) were peritoneally injected to rats in the LPS group 2 h after ischemia. U0126-EtOH (20 mg/kg)and Picroside II (20 mg/kg) were peritoneally injected to rats in the U0126group 2 h after ischemia. Equal volume of normal saline was peritoneally injected to rats in the control groupand the model group. The neurobehavioral function was evaluated by modified neurological severity score(mNSS) test. The structure of neurons was observed using hematoxylin-eosinstaining (HE) staining. Theapoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of phosphorylated extracellular signal-regulated protein kinase1,2 (pERK1,2) in cortex was detected using immunohistochemistry (IHC) and Western blot.

RESULTSAfter cerebral ischemia injury neurological impairment score increased, the damage of neuron in the cortical area was aggravated, apoptotic cells increased in the model group as time went by. The expression of pERK1/2 increased more significantly in the model group than in the control group (P <0.05). The damage of neuron in the cortical area was milder, while apoptotic cells decreased, the expression of pERK1f2 obviously decreased more in the treatment group and the U0126 group (P < 0.05). The early damage of neuron in the cortical area was more severe, apoptotic cells and the expression of pERK12 were comparatively higher in early stage of the LPS group, but the expression of pERK1/2 was somewhat decreased in late stage.

CONCLUSIONSActivating ERK12 pathway could mediate apoptosis and inflammatory reactions of neurons after cerebral ischemia injury. Picroside II could protect the nerve system possibly through reducing activation of ERKI2 pathway, inhibiting apoptosis of neurons and inflammation reaction.

Animals ; Apoptosis ; Brain Ischemia ; drug therapy ; Cinnamates ; pharmacology ; Infarction, Middle Cerebral Artery ; drug therapy ; Iridoid Glucosides ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Neurons ; pathology ; Neuroprotective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar