Co-delivery Nano System of MS-275 and V-9302 Induces Pyroptosis and Enhances Anti-Tumor Immunity Against Uveal Melanoma.

In the treatment of uveal melanoma (UVM), histone deacetylase inhibitors (HDACi) have emerged as a promising epigenetic therapy. However, their clinical efficacy is hindered by the suboptimal pharmacokinetics and the strong self-rescue of tumor cells. To overcome these limitations, reactive oxygen species (ROS)-responsive nanoparticles (NPs) are designed that encapsulate HDACi MS-275 and the glutamine metabolism inhibitor V-9302. Upon reaching the tumor microenvironment, these NPs can disintegrate, thereby releasing MS-275 to increase the level of ROS and V-9302 to reduce the production of glutathione (GSH) related to self-rescue. These synergistic effects lead to a lethal ROS storm and induce cell pyroptosis. When combined with programmed cell death protein 1 monoclonal antibodies (α-PD-1), these NPs facilitate immune cell infiltration, improving anti-tumor immunity, converting "immune-cold" tumors into "immune-hot" tumors, and enhancing immune memory in mice. The findings present a nano-delivery strategy for the co-delivery of epigenetic therapeutics and metabolic inhibitors, which induces pyroptosis in tumors cells and improves the effectiveness of chemotherapy and immunotherapy.

Metabolome-microbiome insights into therapeutic impact of 8-O-acetylharpagide against breast cancer in a murine model.

Iridoid glycosides extract, which is the main active extract of Ajuga decumbens Thunb, has been proved to have anti-breast cancer activity in previous studies. However, it is still unknown whether 8-O-acetylharpagide, a main active compound in the extract, has anti-breast cancer activity. In this study, 4 T1 breast cancer mice model was first successfully established. Then the anti-breast cancer effect of 8-O-acetylharpagide was systematically investigated. Feces were collected for metabolomics and 16S rRNA analysis to assess the potential mechanism. The results showed that 8-O-acetylharpagide was effective in reducing 4 T1 mouse tumor volume and weight compared with the model group. Metabolome analysis revealed 12 potential metabolite biomarkers in feces, mainly involved in primary bile acid biosynthesis and arachidonic acid metabolism. The 16S rRNA sequencing results demonstrated that 8-O-acetylharpagide modulated the abundance of the intestinal flora in 4 T1 mice. Spearman correlation analysis showed that calcitriol and prostaglandin G2 strongly correlated with Akkermansia, Firmicutes and Muribaculum. Overall, the active compound 8-O-acetylharpagide could inhibit significantly breast cancer growth in 4 T1 breast cancer model mice. The mechanism of the anti-breast cancer effect of 8-O-acetylharpagide may be related to the regulation of primary bile acid biosynthesis and arachidonic acid metabolism and modulation of the abundance of Akkermansia and Firmicutes.

Aging and brain free cholesterol concentration on amyloid-ß peptide accumulation in guinea pigs.

Alzheimer's disease is a complex multifactorial neurodegenerative disorder wherein age is a major risk factor. The appropriateness of the Hartley guinea pig (GP), which displays high sequence homologies of its amyloid-ß (Aß40 and Aß42) peptides, Mdr1 and APP (amyloid precursor protein) and similarity in lipid handling to humans, was appraised among 9-40 weeks old guinea pigs. Protein expression levels of P-gp (Abcb1) and Cyp46a1 (24(S)-hydroxylase) for Aß40, and Aß42 efflux and cholesterol metabolism, respectively, were decreased with age, whereas those for Lrp1 (low-density lipoprotein receptor related protein 1), Rage (receptor for advanced glycation endproducts) for Aß efflux and influx, respectively, and Abca1 (the ATP binding cassette subfamily A member 1) for cholesterol efflux, were unchanged among the ages examined. There was a strong, negative correlation of the brain Aß peptide concentrations and Abca1 protein expression levels with free cholesterol. The correlation of Aß peptide concentrations with Cyp46a1 was, however, not significant, and concentrations of the 24(S)-hydroxycholesterol metabolite revealed a decreasing trend from 20 weeks old toward 40 weeks old guinea pigs. The composite data suggest a role for free cholesterol on brain Aß accumulation. The decreases in P-gp and Lrp1 protein levels should further exacerbate the accumulation of Aß peptides in guinea pig brain.

Comparative Genomic Analysis of Shrimp-Pathogenic Vibrio parahaemolyticus LC and Intraspecific Strains with Emphasis on Virulent Factors of Mobile Genetic Elements.

Vibrio parahaemolyticus exhibits severe pathogenicity in humans and animals worldwide. In this study, genome sequencing and comparative analyses were conducted for in-depth characterization of the virulence factor (VF) repertoire of V. parahaemolyticus strain LC, which presented significant virulence to shrimp Litopenaeus vannamei. Strain LC, harboring two circular chromosomes and three linear plasmids, demonstrated ≥98.14% average nucleotide identities with 31 publicly available V. parahaemolyticus genomes, including 13, 11, and 7 shrimp-, human-, and non-pathogenic strains, respectively. Phylogeny analysis based on dispensable genes of pan-genome clustered 11 out of 14 shrimp-pathogenic strains and 7 out of 11 clinical strains into two distinct clades, indicating the close association between host-specific pathogenicity and accessory genes. The VFDB database revealed that 150 VFs of LC were mainly associated with the secretion system, adherence, antiphagocytosis, chemotaxis, motility, and iron uptake, whereas no homologs of the typical pathogenic genes pirA, pirB, tdh, and trh were detected. Four genes, mshB, wbfT, wbfU, and wbtI, were identified in both types of pathogenic strains but were absent in non-pathogens. Notably, a unique cluster similar to Yen-Tc, which encodes an insecticidal toxin complex, and diverse toxin-antitoxin (TA) systems, were identified on the mobile genetic elements (MGEs) of LC. Conclusively, in addition to the common VFs, various unique MGE-borne VFs, including the Yen-Tc cluster, TA components, and multiple chromosome-encoded chitinase genes, may contribute to the full spectrum of LC virulence. Moreover, V. parahaemolyticus demonstrates host-specific virulence, which potentially drives the origin and spread of pathogenic factors.

Metabolic profiling of emodin drug-induced liver injury and silybin treatment in rats using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry: A metabolomic and mechanistic approach.

Silybin, an active component in the plant Silybum marianum (L.) Gaertn., is commonly used to protect against liver disease. We investigated silybin's protective potential in rat liver against emodin-induced liver injury 4 weeks. It was found that aspartate aminotransferase and direct bilirubin serum biomarkers for liver toxicity significantly increased, and liver histopathology revealed cholestasis and necrosis in rats administered emodin alone, whereas aspartate aminotransferase and total bile acid levels in rats administered emodin and silybin simultaneously were changed compared to rats administered emodin alone. Liver mRNA and protein levels of Cyp7a1-which plays roles in cholesterol metabolism and bile acid synthesis-and Abcb11 (Bsep)-which facilitates bile salt secretion in hepatocyte canaliculi-were significantly altered with emodin, whereas cotreatment with silybin attenuated emodin's adverse effect. Metabolomic analysis using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry determined eight potential metabolite biomarkers in serum, urine, and liver tissue. Network analysis was conducted to conceptualize the interplay of genes, metabolites, and metabolic pathways for cholesterol metabolism and bile acid synthesis for liver injury. Overall, rats administered only emodin were shown to be a sound model to investigate fat-associated drug-induced hepatoxicity or liver injury and cotreatment of emodin with silybin prevents fatty liver injury. This metabolomic study revealed that emodin-induced fatty liver injury disrupted bile acid synthesis, vitamin B6 , and glycerophospholipid metabolism pathways and that silybin ameliorates liver injury on these compromised pathways.

UPLC-Q-TOF/MS-Based Metabolomics Approach to Reveal the Hepatotoxicity of Emodin and Detoxification of Dihydromyricetin.

Dihydromyricetin (DMY), an important flavanone found in Ampelopsis grossedentata, plays a protective role in liver injury. Our previous research found that DMY protected L02 cells against hepatotoxicity caused by emodin. In this study, serum, urine, and liver samples from rats were systematically used for biochemical analysis, pathological observation, and nontargeted metabolomics to evaluate the toxicity of emodin and DMY intervention. After oral administration of DMY, DMY may alleviate liver injury by improving liver metabolism. Approximately, 8 of 15 metabolites in rat urine and serum were significantly regulated by DMY. Metabolic pathway analysis showed that glutathione metabolism, pyrimidine metabolism, and tryptophan metabolism were the most affected pathways, and 18 proteins were predicted to be potential targets of DMY during the alleviation of liver injury induced by emodin. This research is of great significance in confirming the liver-protective effect of DMY, especially during acute liver injury caused by traditional Chinese medicine.

Identification of the active ingredients from Guangtongxiao decoction in rat bile based on ultra-performance liquid chromatography/Synapt G2 quadrupole time-of-flight tandem mass spectrometry.

OBJECTIVE: To identify the active ingredients and metabolites in rat bile after Guangtongxiao decoction (GTX) had been administered via the rectal route. METHODS: Drug-containing bile samples were collected via a catheter in the bile duct and could be used 5 h after rectal administration. The main active components and their metabolites in rat bile following rectal administration of GTX were identified and analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. RESULTS: Positive and negative modes were applied to analyze and identify the chemical ingredients in the bioactive fractions of GTX. Eight peaks were identified by comparison with the standard compounds: berberine hydrochloride, dehydrocorydaline, tetrahydropalmatine, corydaline, magnoflorine, magnolol, obacunone and albiflorin. Furthermore, 60 metabolites were detected in rat bile based on mass-fragmentation behaviors, and 21 metabolites were reported for the first time. CONCLUSION: Our findings provide a solid basis for further pharmacologic and pharmacokinetic studies of GTX.

UPLC/MS/MS-Based Metabolomics Study of the Hepatotoxicity and Nephrotoxicity in Rats Induced by Polygonum multiflorum Thunb.

Polygonum multiflorum Thunb. (PM) is one of the most frequently used natural products in China. Its hepatotoxicity has been proven and reported. However, chronic PM toxicity is a dynamic process, and a few studies have reported the long-term hepatotoxic mechanism of PM or its nephrotoxicity. To elucidate the mechanism of hepatotoxicity and nephrotoxicity induced by PM after different administration times, different samples from rats were systematically investigated by traditional biochemical analysis, histopathological observation, and nontargeted metabolomics. The concentrations of direct bilirubin (DBIL) at 4 weeks and total bile acid, DBIL, uric acid, and blood urea nitrogen at 8 weeks were significantly increased in the treatment group compared with those in the control group. Approximately, 12 metabolites and 24 proteins were considered as unique toxic biomarkers and targets. Metabolic pathway analysis showed that the primary pathways disrupted by PM were phenylalanine and tyrosine metabolism, which resulted in liver injury, accompanied by chronic kidney injury. As the administration time increased, the toxicity of PM gradually affected vitamin B6, bile acid, and bilirubin metabolism, leading to aggravated liver injury, abnormal biochemical indicators, and marked nephrotoxicity. Our results suggest that the hepatotoxicity and nephrotoxicity caused by PM are both dynamic processes that affect different metabolic pathways at different administration times, which indicated that PM-induced liver and kidney injury should be treated differently in the clinic according to the degree of injury.

UPLC-Q-TOF/MS-Based Serum Metabolomics Reveals the Anti-Ischemic Stroke Mechanism of Nuciferine in MCAO Rats.

Nuciferine is an aporphine alkaloid monomer that is extracted from the leaves of the lotus species Nymphaea caerulea and Nelumbo nucifera Gaertn. Nuciferine was reported to treat cerebrovascular diseases. However, the potential mechanism of the neuroprotective effects of nuciferine at the metabolomics level is still not unclear. The present research used neurological score, infarct volume, cerebral water content, and ultraperformance liquid chromatography to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS)-based serum metabolomics to elucidate the anti-ischemic stroke effect and mechanisms of nuciferine. The results showed that nuciferine significantly improved neurological deficit scores and ameliorated cerebral edema and infarction. Multivariate data analysis methods were used to examine the differences in serum endogenous metabolism between groups, and the biomarkers of nuciferine on ischemic stroke were identified. Approximately 19 metabolites and 7 metabolic pathways associated with nuciferine on treatment of stroke were found, which indicated that nuciferine exerted a positive therapeutic action on cerebral ischemic by regulating metabolism. These results provided some data support for the study of anti-stroke effect of nuciferine from the perspective of metabolomics.

Design, synthesis, and antitumor activity of desmosdumotin C analogues.

Desmosdumotin C (Des C), a natural product isolated from the roots of Desmos dumosus, has shown good antitumor activity. A three dimensional quantitative structure-activity relationship (QSAR) study using the comparative molecular field analysis (CoMFA) method was performed on 32 Des C analogues. Based on the QSAR, 18 new Des C analogues were designed and synthesized. An efficient three-step synthetic strategy toward Des C and its analogues was developed from commercial available 2, 4, 6-trihydroxyacetophenone. All synthesized compounds were evaluated against a panel of human cancer cell lines and showed ED50 values ranging from 1.1 to 25.1 µΜ.

Phytochemicals protect L02 cells against hepatotoxicity induced by emodin via the Nrf2 signaling pathway.

Dihydromyricetin (DMY), hyperoside and silybin are phytochemicals that belong to a class called flavonoids, and they have been used in liver protection pharmaceutical preparations, but the specific mechanism of these chemicals is still unclarified. This study aims to investigate the hepatoprotective effects and potential mechanism of these phytochemicals. The immortalized human hepatocyte cell line L02 was treated with 200 µM emodin for 48 h, and this was used as a hepatocyte injury model. The L02 cells were treated with both 200 µM emodin and different concentrations of DMY/hyperoside/silybin for 48 h to investigate the protective effects of these phytochemicals. The CCK-8 assay was used to detect cell viability. RT-qPCR and western blotting were performed to examine the mRNA and protein expression, respectively, of the classic bile acid synthetic pathway gene CYP7A1, the bile acid efflux transporter bile salt export pump (BSEP), the nuclear factor erythroid-2-related factor 2 (Nrf2) and the drug processing gene CYP1A2. DMY, hyperoside and silybin prevented the impairment of cell viability that was caused by emodin-induced hepatotoxicity in a dose-dependent manner, and at a low concentration (10 µM), the protective effect followed the order hyperoside > DMY > silybin, while at a high concentration (160 µM), the protective effect followed the order DMY > hyperoside > silybin. These phytochemicals reduced the expression of CYP7A1 at both the mRNA and protein levels. BSEP was not influenced by the phytochemical intervention. When 200 µM emodin was used for 48 h with the addition of the phytochemicals at 200 µM, the nuclear protein expression of Nrf2 significantly increased and CYP1A2 expression decreased. DMY, hyperoside and silybin prevented the hepatotoxicity induced by emodin in the L02 cells, potentially, via the Nrf2 signaling pathway.

The Pathogenesis of Diabetes Mellitus by Oxidative Stress and Inflammation: Its Inhibition by Berberine.

A substantial knowledge on the pathogenesis of diabetes mellitus (DM) by oxidative stress and inflammation is available. Berberine is a biologically active botanical that can combat oxidative stress and inflammation and thus ameliorate DM, especially type 2 DM. This article describes the potential of berberine against oxidative stress and inflammation with special emphasis on its mechanistic aspects. In diabetic animal studies, the modified levels of proinflammatory cytokines and oxidative stress markers were observed after administering berberine. In renal, fat, hepatic, pancreatic and several others tissues, berberine-mediated suppression of oxidative stress and inflammation was noted. Berberine acted against oxidative stress and inflammation through a very complex mechanism consisting of several kinases and signaling pathways involving various factors, including NF-κB (nuclear factor-κB) and AMPK (AMP-activated protein kinases). Moreover, MAPKs (mitogen-activated protein kinases) and Nrf2 (nuclear factor erythroid-2 related factor 2) also have mechanistic involvement in oxidative stress and inflammation. In spite of above advancements, the mechanistic aspects of the inhibitory role of berberine against oxidative stress and inflammation in diabetes mellitus still necessitate additional molecular studies. These studies will be useful to examine the new prospects of natural moieties against DM.

Mechanism of Chinese Medicine Herbs Effects on Chronic Heart Failure Based on Metabolic Profiling.

Chronic heart failure (CHF) is a major public health problem in huge population worldwide. The detailed understanding of CHF mechanism is still limited. Zheng (syndrome) is the criterion of diagnosis and therapeutic in Traditional Chinese Medicine (TCM). Syndrome prediction may be a better approach for understanding of CHF mechanism basis and its treatment. The authors studied disturbed metabolic biomarkers to construct a predicting mode to assess the diagnostic value of different syndrome of CHF and explore the Chinese herbal medicine (CHM) efficacy on CHF patients. A cohort of 110 patients from 11 independent centers was studied and all patients were divided into 3 groups according to TCM syndrome differentiation: group of Qi deficiency syndrome, group of Qi deficiency and Blood stasis syndrome, and group of Qi deficiency and Blood stasis and Water retention syndrome. Plasma metabolomic profiles were determined by UPLC-TOF/MS and analyzed by multivariate statistics. About 6 representative metabolites were highly possible to be associated with CHF, 4, 7, and 5 metabolites with Qi deficiency syndrome, Qi deficiency and Blood stasis syndrome, and Qi deficiency and Blood stasis and Water retention syndrome (VIP > 1, p < 0.05). The diagnostic model was further constructed based on the metabolites to diagnose other CHF patients with satisfying sensitivity and specificity (sensitivity and specificity are 97.1 and 80.6% for CHF group vs. NH group; 97.1 and 80.0% for QD group vs. NH group; 97.1 and 79.5% for QB group vs. NH group; 97.1 and 88.9% for QBW group vs. NH group), validating the robustness of plasma metabolic profiling to diagnostic strategy. By comparison of the metabolic profiles, 9 biomarkers, 2-arachidonoylglycerophosphocholine, LysoPE 16:0, PS 21:0, LysoPE 20:4, LysoPE 18:0, linoleic acid, LysoPE 18:2, 4-hydroxybenzenesulfonic acid, and LysoPE 22:6, may be especially for the effect of CHM granules. A predicting model was attempted to construct and predict patient based on the related symptoms of CHF and the potential biomarkers regulated by CHM were explored. This trial was registered with NCT01939236 (https://clinicaltrials.gov/).

Identification and characterization of the structure-activity relationships involved in UGT1A1 inhibition by anthraquinone and dianthrone constituents of Polygonum multiflorum.

The adverse effects of Polygonum (P.) multiflorum, including abnormal bilirubin metabolism, are a serious public health issue. As uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) is the only enzyme responsible for bilirubin metabolism, we investigated the inhibitory effect of a P. multiflorum extract and 10 anthraquinone and dianthrone compounds on UGT1A1 in rat liver microsomes in vitro. The P. multiflorum extract exhibited the strongest inhibitory effect on UGT1A1 activity (inhibition constant [Ki] = 0.3257 µM, 1422 µg of material/mL), followed by cis-emodin dianthrones (Ki = 0.8630 µM), trans-emodin dianthrones (Ki = 1.083 µM), emodin-8-O-glc (Ki = 3.425 µM), and polygonumnolide C2 (Ki = 4.291 µM). Analysis of the structure-activity relationships of these compounds suggested that the spatial orientation of the molecules and the presence of particular functional groups affect UGT1A1 inhibition. A mechanistic analysis showed that all the tested compounds docked into two of the nine active sites of UGT1A1 and suggested that hydrophobic interactions and hydrogen bonds are important for the affinity of the tested compounds for UGT1A1; moreover, their interaction energies were generally in agreement with the Ki values. These findings provide insight into adverse reactions to P. multiflorum and identify the pharmacophores involved in inhibition of UGT1A1.

Estimating the Differences of UGT1A1 Activity in Recombinant UGT1A1 Enzyme, Human Liver Microsomes and Rat Liver Microsome Incubation Systems in Vitro.

Estimating the extent to which drugs inhibit uridine 5'-diphosphate-glucuronosyltransferases1A1 (UGT1A1) enzyme activity is important for predicting hepatotoxicity and neurotoxicity. UGT1A1 enzyme activity is commonly evaluated by detecting the elimination of bilirubin substrate or the generation of bilirubin glucuronides. However, the present methods are inadequate for accurately assessing bilirubin metabolism, selecting incubation conditions, and comparing different systems. Therefore, in our study, we first established a ultra-performance liquid chromatography (UPLC)-MS/MS method to identify bilirubin and bilirubin glucuronides. To ensure the reaction was linear, we performed assays to optimize the protein concentration and incubation time. Finally, we measured UGT1A1 activity using three different systems. Data revealed the optimum incubation conditions were 10 min with 0.5 mg/mL human liver microsomes (HLM), recombinant human UGT1A1 (rUGT1A1), and rat liver microsomes (RLM). Bilirubin glucuronidation obeyed Michaelis-Menten kinetics in all three systems. The diversity in bilirubin metabolism among species was revealed. rUGT1A1 had the strongest binding affinity for bilirubin, but the lowest metabolism velocity. Compared with the other systems, RLM exhibited a significant difference. It has the lowest CLint and the highest Km. The difference in parameters between three systems may be attributable to the species differences. In conclusion, these in vitro studies provide useful information regarding drug interactions and the prediction of toxicity for future studies.

[Pharmacokinetics of 8-O-acetylharpagide and harpagide after oral administration of Ajuga decumbens extract in beagle dog].

8-O-acetylharpagide and harpagide are two kinds of effective component of Ajuga decumbens extract. A sensitive LC-MS/MS method has been established for pharmacokinetics of 8-O-acetylharpagide and harpagide in beagle dog after oral administration of from A. decumbens extract. Female beagle dogs received orally 12.9, 25.7 mg x kg(-1) p. o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartment analysis. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), which was run at a flow rate of 0.3 mL x min(-1). Chromatographic separation was achieved on an Agilent ZORBAX XDB-C18 column (2.1 mm x 50 mm, 3.5 microm) using a gradient elution of 5% B at 0-2 min, 95% B at 2. 1-5 min and 5% B at 5. 1-10 min. All analytes, including the IS, were monitored under positive ionization conditions and quantified in MRM mode with transitions of m/z 429.2-369.2 for 8-O-acetylharpagide, m/z 387.2-207.2 for harpagide, and m/z 149.2-103.1 for IS. High purity nitrogen was employed as both the nebulizing and drying gas. Other parameters of the mass spectrometer were optimized as follows: drying gas flow 10.0 L x min(-1); drying gas temperature 300 degrees C; capillary voltage 4 000 V. Results showed that 8-O-acetylharpagide and harpagide showed a dose-dependence profile. T(max) of 8-O-acetylharpagide is 1.7 h, and T(max) of harpagide is 1.57 h, which was higher than T(max) of 8-O-acetylharpagide and harpagide after oral administration of from A. decumbens extract in rats. The different pharmacokinetic parameters may be due to the species differences of rat and beagle dog.

Pharmacokinetics of 8-O-acetylharpagide and harpagide after oral administration of Ajuga decumbens Thunb extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga decumbens Thunb is a medicinal plant native to China popularly used to treat chronic pelvic inflammation and hysteromyoma. Its main bioactive components are iridoid glycosides, such as 8-O-acetylharpagide and harpagide that had presented antibacterial, anti-inflammatory, and antiviral activities. AIM OF THE STUDY: To establish a sensitive LC-MS/MS method and compare the pharmacokinetics of 8-O-acetylharpagide and harpagide in rats after oral administration of their pure forms and from compounds obtained from Ajuga decumbens extract. MATERIALS AND METHODS: Rats received orally 15 mg/kg (equivalent of 6 mg/kg 8-O-acetylharpagide and 1.5mg/kg harpagide), 30 mg/kg and 60 mg/kg of Ajuga decumbens Thunb extract and were compared to animals that received 12 mg/kg of 8-O-acetylharpagide or 3mg/kg of harpagide p.o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartmental analysis. RESULTS: Results showed that the iridoid glycosides were quickly absorbed by oral route and showed a dose-dependence profile. Pharmacokinetic parameters of both glycosides were essentially the same except Tmax when dosed as the extract or pure forms. CONCLUSION: 8-O-acetylharpagide was metabolized to harpagide, which affected the pharmacokinetic profiles of harpagide when dosed as the extract. This pharmacokinetic study seems to be useful for a further clinical study of Ajuga decumbens Thunb extract.

Mechanisms of inhibiting proliferation of vascular smooth muscle cells by serum of rats treated with Dahuang Zhechong pill.

UNLABELLED: Dahuang Zhechong pill (DHZCP), a famous and classical Chinese herbal prescription, consists of twelve traditional Chinese drugs: Eupolyphaga sinensis Walker., Rheum officinale Baill., Scutellaria baicalensis Georgi., Glycyrrhiza uralensis Fisch., Prunus persica Batsch., Prunus armeniaca L., Paeonia lactiflora Pall., Rehmannia glutinosa Libosch., Toxicodendron vernicifluum F.A. Barkl., Tabanus bivittatus Mats., Hirudo nipponica Whitman. and Holotrichia diomphalia Bates., and is clinically used to treat hepatic diseases, gynecopathy and atherosclerosis in China. Our previous studies confirm that DHZCP is able to significantly inhibit proliferation of vascular smooth muscle cells (VSMCs) in vivo and in vitro. AIM OF THE STUDY: To investigate the mechanisms of inhibition of VSMCs proliferation by DHZCP with the method of Serum Pharmacology. MATERIALS AND METHODS: VSMCs proliferation of rat was assayed by measuring the cell viability with the MTT method, and platelet-derived growth factor (PDGF) expression in VSMCs was examined by the immunocytochemical method. Cycle and apoptosis of VSMCs were evaluated with flow cytometry. RESULTS: The serum of DHZCP-treated rats not only inhibited endothelin-1 (ET-1) stimulated cell proliferation and PDGF expression in VSMCs, but also promoted apoptosis of the proliferated VSMCs. Meanwhile, the serum of rats containing DHZCP interfered with the cycle of PDGF-stimulated VSMCs, increasing proportion of the cells in G(0)/G(1) phases and decreasing proportion of the cells in S and G(2)/M phases. CONCLUSION: These suggest that the inhibitory effect of DHZCP on VSMCs proliferation is partially attributed to depressing PDGF expression in VSMCs, retarding the cell cycle and to promoting apoptosis of VSMCs.

Fluorenone alkaloid from Caulophyllum robustum Maxim. with anti-myocardial ischemia activity.

A new fluorenone alkaloid (caulophine) was isolated from the radix of Caulophyllum robustum Maxim. (collected from the Qinling mountains) using cell membrane chromatography as the screening method. Caulophine was identified as 3-(2-(dimethylamino)ethyl)-4,5-dihydroxy-1,6-dimethoxy-9H-fluoren-9-one based on physicochemic and spectroscopic analyses, particularly by NMR spectroscopic data (i.e., COSY, HMQC, HMBC, NOESY). Caulophine possessed anti-myocardial ischemia activity.

[Metabolic kinetic of puerarin in beagle liver microsomal by HPLC-ESI-MS].

OBJECTIVE: To develop a HPLC-ESI-MS method for the determination of puerarin and its metabolite and study the metabolic kinetics in beagle dog liver microsomes. METHOD: Beagle dog liver microsomes were prepared by using ultracentrifugation method. Chromatography was performed on a Shimadzu C18 column (2.0 mm x 150 mm, 5 microm). Amethanol-water gradient system was used. ESI interface was applied in the positive, and SIM m/z 417 was puerarin and m/z 531 was daidzein. RESULT: The puerarin was metabolized by NADPH regenerating system in beagle dog microsomes. The Michaelis-Menten parameters Km and Vmax in beagle dog microsomes were initially estimated by analyzing Lineweave-Brurk plot. The Vmax Km of puerarin were (0.047 +/- 0.006) mg x min(-1) x g(-1), (1.22 +/- 0.53) mg x L(-1). CONCLUSION: The puerarin and daidzein can be rapidly determined by HPLC-MS in beagle dog microsomes and the puerarin was metabolized to daidzein by CY P450. The study can give help for Baige capsule.