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Longxue Tongluo Capsules(LTC) has good efficacy against blood stasis syndrome during the recovery period of ischemic stroke. Its main active ingredient is the phenolic extract of Chinese dragon's blood. In our previous study, the primary mass fragmentation pathways of phenolic derivatives from LTC were clarified. Herein, the metabolites in rat plasma were characterized following the oral administration of loureirin A and loureirin C using liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectro-metry(LC-IT-TOF-MS), with 18 and 55 metabolites identified, respectively. On this basis, with the help of the obtained accurate molecular weight, characteristic fragment ions, reference comparison, combined with LTC database and natural products database self-created in our group, 18 prototypes and 106 metabolites were tentatively identified in rat plasma after oral gavage of LTC at a dose of 500 mg·kg~(-1). Glucuronidation, sulfonation, and methylation were major biotransformation pathways of LTC. This study preliminarily clarified the LTC constituents absorbed into blood and laid the foundation for clarifying the effective substances of LTC.
Subject(s)
Animals , Rats , Administration, Oral , Capsules , Chromatography, High Pressure Liquid , Chromatography, Liquid , Drugs, Chinese Herbal , Gas Chromatography-Mass SpectrometryABSTRACT
OBJECTIVE: To explore the new quality problems of Draconis Sanguis imported from Indonesia. METHODS: According to the quality standard of imported medicinal materials and Ch.P method, exploratory analysis was carried out on 10 batches of imported Draconis Sanguis and three batches of Draconis Sanguis fruits. RESULTS: The dracohodin content in three batches of Draconis Sanguis fruits was 1.2%-2.4%. Three batches of imported Draconis Sanguis were obtained by directly pulverization of Draconis Sanguis fruits, and loureirin A and loureirin B were detected in one batch of Draconis Sanguis, suggesting that dracaena cochinchinensis was added. CONCLUSION: Among the 10 batches of imported Draconis Sanguis, three batches are determined to be unqualified due to character inconformity, and the other batch is judged to be unqualified due to the addition of dracaena cochinchinensis.
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Loureirin A is a major active component of Draconis sanguis, a traditional Chinese medicine. This work aimed to investigate the activity of loureirin A against Candida albicans biofilms. 2, 3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)reduction assay and scanning electron microscopy were used to investigate the anti-biofilm effect. Minimal inhibitory concentration testing and time-kill curve assay were used to evaluate fungicidal activity. Cell surface hydrophobicity (CSH) assay and hyphal formation experiment were respectively carried out to investigate adhesion and morphological transition, two virulence traits of C. albicans. Real-time RT-PCR was used to investigate gene expression. Galleria mellonella-C. albicans and Caenorhabditis elegans-C. albicans infection models were used to evaluate the in-vivo antifungal effect. Human umbilical vein endothelial cells and C. elegans nematodes were used to evaluate the toxicity ofloureirin A. Our data indicated that loureirin A had a significant effect on inhibiting C. albicans biofilms, decreasing CSH, and suppressing hyphal formation. Consistently, loureirin A down-regulated the expression of some adhesion-related genes and hypha/biofilm-related genes. Moreover, loureirin A prolonged the survival of Galleria mellonella and Caenorhabditis elegans in C. albicans infection models and exhibited low toxicity. Collectively, loureirin A inhibits fungal biofilms, and this effect may be associated with the suppression of pathogenic traits, adhesion and hyphal formation.
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Objective To establish a quantitative analysis of multi-components by single marker (QAMS) for determination of five active components in Draconis Resina and discuss application of QAMS in quality control of ethnic medicines. Methods Using the method of HPLC, the Fortis Xi C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was composed of acetonitrile (A)-1.0% acetic acid (B) with gradient elution (0-10 min, A: 25%→30%; 10-60 min, A: 30%→50%) at the flow rate of 1.0 mL/min. The detection wavelength was 278 nm, the column temperature was 30 ℃ and the sample size was 10 μL. Pterostilbene was selected as an internal standard to establish the relative correction factors (RCFs) of 7, 4’-dihydroxyflavone, resveratrol, loureirin A, and loureirin B with reference to pterostilbene so as to achieve simultaneous determination of multi-indexed components. The contents of five active components were determined by both external standard method (ESM) and QAMS. Meanwhile, relative error (RE) between QAMS and ESM was analyzed to evaluate QAMS method. Results There were good linearities in the range of 10.23-102.27 μg/mL for 7,4’-dihydroxyflavone, 11.01-110.14 μg/mL for resveratrol, 9.47-94.72 μg/mL for loureirin A, 11.59-115.90 μg/mL for loureirin B and 24.35-243.52 μg/mL for pterostilbene, RCFs of 7,4’-dihydroxyflavone, resveratrol, loureirin A and loureirin B with reference to pterostilbene were 0.626, 1.064, 1.154, and 0.837 respectively, and repeatability was good in different experimental conditions (RSD < 3.0%).There were no significant difference between the quantitative results of the two methods. Conclusion QAMS method is feasible, credible, and can be used to determine multiple components in Draconis Resina. QAMS can be adopted as a novel strategy for quality control of ethnic medicines.
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Objective To investigate the molecular mechanism of Loureirin A mediated anti-hepatic fibrosis by evaluting its effects on proliferation , secretion ofα-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1), and expression of rat hepatic stellate cells in vitro . Methods Primary hepatic stellate cells were isolated and cultured from Sprague-Dawley rats. After activating and inducing primary hepatic stellate cells from qHSC to aHSC, the activated hepatic stellate cells model in vitro was established. Then we observed the morphological changes of static hepatic stellate cells and activated hepatic stellate cells with inverted phase contrast microscope. Cultured hepatic stellate cells were treated with different concentrations of loureirin A and the inhibitory rate of HSCs proliferation was measured by MTT assay. The expression of Frizzled-4 was measured by western blot analysis. The content ofα-SMA and TGF-β1 in the cultured HSCs'supernatant were measured by enzyme-linked immunosorbent assay (ELISA) . Results Loureirin A the proliferation of inhibited activated hepatic stellate cells in a time-dose-dependent manner compared with the control group,IC50=0.30 μg/μL. After loureirinA treatment of the HSCs, western blot analysis showed that Frizzled-4 expression level was obviously lower than control group. Loureirin A also inhibitedα-SMA and TGFβ1 (P<0.05) secretion in the cultured HSCs'supernatant in different degree by the assay of ELISA. Conclusions The molecular mechanism of Loureirin A and Wnt signaling pathway mediated anti-hepatic fibrosis and anti-angiogenesis may involve down-regulation the expression of Frizzled-4, inhibiting the synthesis and secretion ofα-SMA,TGF-β1and the proliferation of HSCs.
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AIM@#To analyze the composition of the Chinese herbal medicine Sanjie Zhentong Capsule (SJZTC) and test the therapeutic efficacy of each component in a rat model of endometriosis.@*METHODS@#A rapid resolution liquid chromatography (RRLC) method coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS) has been developed for the analysis of SJZTC. Two main ingredients, Drac(h)onis sanguis and saponin, were tested in the endometriosis model. Sixty Lewis female rats were in the estrous cycle stage when endometriosis was experimentally initiated by peritoneal implantation of endometrial tissue. Four weeks later, a second laparotomy was performed and implant volumes were measured. After that, the implanted rats were randomized into five study groups: control group (treatment with saline), anastrozole group (treatment with anastrole, 18 μg per day), loureirin A group (treated with loureirin A, 97.2 mg), ginsenoside Re group (treated with ginsenoside Re, 64.8 mg), and SJZTC groups (treated with SJZTC, 86.4 mg) administered once a day for 4 weeks via gastric gavage. After four weeks of treatment, a third laparotomy was performed, implant volumes were re-measured, and the levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-α) were tested.@*RESULTS@#A total of 38 compounds including, both the target and unknown compounds, were rapidly predicted in the capsule extract by the developed method. Compared with the control group, the anastrozole group, loureirin A group, ginsenoside Re group, and SJZTC treated group showed smaller implant volumes, as well as lower levels of VEGF and TNF-α in the peritoneal focus (P < 0.01 for all comparisons). Furthermore, parameters in the groups treated with SJZTC, loureirin A and ginsenoside Re were significantly better than the control group and the anastrozole group. These results indicate that SJZTC and its two main components are effective in reducing the development of endometriosis.
Subject(s)
Animals , Female , Humans , Rats , Capsules , Chemistry , Disease Models, Animal , Drugs, Chinese Herbal , Chemistry , Endometriosis , Drug Therapy , Rats, Inbred LewABSTRACT
Objective: To determine loureirin A and B in different brands of home made resina draconis,providing basis for new quality control method.Methods: A high pressure liquid chromatography(HPLC) method has been developed. UV detector wavelength was set at 270 nm.The mobile phase was acetonitrile acetic acid solution (40∶60).The flow rate was 1.0 ml/min and the column was at room temperature.Results: A good linear correlation was found in the range of 4 to 24 ?g/ml of loureirin A.The regression equation obtained was Y =855.8+803 7.1 X ( r =0.999 9); A good linear correlation was found in the range of 20 to 120 ?g/ml of loureirin B.The regression equation obtained was Y =219.3+808 1.8 X ( r =0.999 9).Conclusion: The quantities of loureirin B in all brands are lower than the limit of quality standard.The quantities of loureirin A are higher than that of loureirin B in the same sample.
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Objective: To establish HPLC method for the determination of loureirin A and loureirin B in Sanguis Draxonis Capsules.Methods: HPLC system included C 18 reverse phase column and acetonitrile 1% acetic acid (34∶66) as mobile phase, detection at 280nm and external standard method.Results:The standard curves of loureirin A was linear in the concentration range of 98～490ng, r =0.9996.The average recovery was 97.45%, RSD was 1.82%.The standard curves of loureirin B was linear in the concentration range of 43.2～259.2, r = 0.9993 .The average recovery was 96.92%, RSD was 1.57%.The contents of loureirin A and loureirin B in Sanguis Draxonis from heating free technology were higher than traditional technology.Conclusion: This heating free technology is a new technique that is a creation with higher extract rate,this method is worth populariging.