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Aim To investigate the protective effect of cyclovirobuxine D(CVB-D) on aldosterone (ALD)-induced primary neonatal rat cardiac myocytes (PNRC-Ms) injury and the possible mechanism. Methods PNRCMs were extracted by trypsin, and the PNRCMs injury model was established by ALD (10 μmol · L
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We have developed a new method using HPLC-CAD (charged aerosol detector) for the quantitative analysis of cyclovirobuxine D and related substances in the API of Huangyangning tablets. The related substances were further studied by HPLC-Q-Exactive coupled with hybrid quadrupole-orbitrap mass spectrometry. A HILIC column of XBridge Amide (4.6 mm×250 mm, 5 μm) was used, and the mobile phase was composed of acetonitrile and 100 mmol·L-1 ammonium formate (85∶15), which was adjusted to pH 2.8 with formic acid. Isocratic mode elution was adopted at a flow rate of 1.1 mL·min-1. The column temperature was set at 30 ℃. For CAD, the temperature of atomization and gas pressure were respectively set at 35 ℃ and 62.2 psi. This method detected and quantified five related substances to cyclovirobuxine D. The results showed that the LOD and LOQ of cyclovirobuxine D was 12.588 ng and 28.323 ng, respectively with an average recovery of 95.74% (RSD = 1.79%, n = 6). The content of cyclovirobuxine D in 12 batches of API samples provided by three manufacturers was from 79.94% to 88.49%, with an average value of 82.20%. The total content of the five related substances was from 15.99% to 22.15% with an average value of 20.10%, using an external standard method with cyclovirobuxine D as the reference and according to the CAD uniform response to non-volatile substances. The newly developed HPLC-CAD method has advantages in terms of the comprehensiveness of signals from Buxus alkaloids without UV absorption and with high sensitivity to its trace-related substances; the method yields good separation between the components and is compatible with mass spectrometry. It is applicable for the accurate quantitative analysis of main components and related substances in the API of Huangyangning tablets.
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Objective To study the in situ intestinal absorption characteristics of cyclovirobuxine D (CB) hydroxypropyl-β-cyclodextrin inclusion complex (CBHD) in rats, and to explore the effect of cytochrome P450 inhibitor ketoconazole (KET) on CB and CBHD in situ intestinal absorption. Methods Twenty-four male rats were randomized into CB, CBHD, KET+CB and KET+CBHD groups, with 6 rats in each group. In situ intestinal absorption was adopted in a rat model. One-way intestinal perfusion model was employed to investigate the absorption of CB and CBHD in the intestinal segments of rats and the effects of KET on CB and CBHD absorption. The concentration of CB was determined by highperformance liquid chromatography with fluorescence detector (HPLC/FLD; Lichrospher C18 column [250 mm×4.6 mm, 5 μm]). The mobile phase was methanol-water with volume ratio being 85: 15. The excitation wavelength was set at 231 nm, and emission wavelength was set at 385 nm. The column temperature was 25 °C, and flow rate was 1.0 mL/min. The injection volume was 20 μL. Results The specificity of HPLC/FLD method was good and the standard curve equation was A=106.7 C+41.861 (R2=0.999 08) based on the linear regression of CB concentration (C) with CB peak area (A), indicating that the CB mass concentration was linear in the range of 0.5 to 20.0 μg/mL. The intra-day precision of the 1.0, 5.0 and 10.0 μg/mL samples was 2.25%, 2.44% and 3.04%, and the inter-day precision was 4.22%, 2.00% and 2.50%, respectively. The precision was good and the method was in accordance with the requirements of methodology. The recovery rates of the 1.0, 5.0 and 10.0 μg/mL samples were 99.08%, 98.24% and 97.25%, respectively, which were also in accordance with the requirements of methodology. The intestinal absorption rate constant (Ka) values of CBHD with KET were 4.18, 5.05, 1.91 and 2.85 times those of CB, and the effective permeability (Peff) values were 4.92, 5.98, 2.19 and 3.24 times those of CB in the duodenum, jejunum, ileum and colon, respectively (all P<0.05). Conclusion KET can improve the intestinal absorption of CB and CBHD in rats.
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Objective To explore the release and percutaneous penetration of cyclovirobuxine D patches at different concentrations in vitro. Methods The release curves of cyclovirobuxine D patch in vitro were fitted by ritger-peppas mathematical model, and the patch release mechanism was discussed according to the fitting parameters. At the same time, compared the percutaneous permeability characteristics of 0.25,0.5,1.0,2.0 mg.( cm2 )-1 of cyclovirobuxine D patch by using a modified Franz diffusion cell, with isolated rat skin serving as transdermal barrier. Results Ritger-Peppas model fitting equation for cyclovirobuxine D patch [1.00 mg.(cm2)-1]was: Mt/M=0.964 6 t1.621 6.And the percutaneous penetration curve was best fitted to Higuchi kinetics equation.The drug release rate from the patch matrix was greater than the rate of penetration through the skin, indicating the patch at the time through rat′s skin was a passive diffusion process, and transdermal process was rate-limited by skin. Conclusion Kinetics equation fitting is an effective method for analyzing drug release and permeation behavior of cyclovirobuxine D patch in vitro.
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Objective To develop a pre-column derivatization HPLC/FLD method for determining the content of cyclovirobuxine D (CB) in rat plasma, and to evaluate the pharmacokinetics of cyclovirobuxine D phospholipid complex (CBPC) and CB in SD rats by oral administration. Methods The solvent evaporation method was used to prepare CBPC. The preparation protocol was optimized by central composite design, with the ratio of phospholipid and CB, concentration of principal agent as the independent variables, and with the compound rate as the response variable. Twelve male rats were evenly randomized into two groups with each containing 6 animals. Rats were orally given CBPC and CB (60 mg/kg, with CB count). Blood samples were collected from the retinal venous plexus of SD rats after oral administration of CBPC and CB at 15 min, 30 min, 1, 2, 3, 4, 5, 6, 8, 12 and 24 h time points, and the blood concentrations of CB was determined by HPLC/FLD. The HPLC method employed the C18 column (250 mm × 4.6 mm,5 μm) with a mobile phase of methanol-water (85:15) at a flow rate of 1.0 mL·min-1. The excitation wavelength was set at 231 nm, emission at wavelength 385 nm, and the column temperature was 25:. Results The pharmacokinetic parameters of CBPC and CB were calculated as follows: AUC0-t(1 703.81±549.38) μg·h·L-1 and (619.93±75.67) μg·h·L-1, Tmax (6.00±0) h and (4.33±0) h, Cmax(82.32±9.55) μg·L-1 and (69.27±8.66) μg·L-1. The relative bioavailability of CBPC was 274.84%. Conclusion Phospholipid complex can improve the oral bioavailability of CB in rats.
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Objective To exPlore the theraPeutic effect of cycloVirobuxine_D ( CVB_D) on heart failure by obserVing the influences of CVB_D on cardiac function,myocardial morPhology and structure in rats with heart failure. Methods The rat model of heart failure was established by ligating left anterior descending coronary artery,and then the rats were randomly diVided into 5 grouPs,Plus the sham grouP. EchocardiograPhy was used to assess cardiac function,including EF,LVSF,SV,HR,CO and so on. Myocardial tissue section was obtained. Change in the myocardial tissue was obserVed by HE staining. Myocardial ultra_structure was obserVed by using transmission electron microscoPe. Results As comPared with the model grouP,EF and LVSF were significantly increased in medium and high dose CVB_D grouPs (P<0. 05 or P<0. 01),SV was increased in low and medium dose CVB_D grouPs (P<0. 05),CO was increased in low,medium and high dose CVB_D grouPs (P<0. 05),LVIDs was decreased and LVPWs was increased in high dose CVB_D grouP (P<0. 05). Under light microscoPe,changes of myocardial structures were obserVed in different CVB_D dose grouPs: myocardial fiber gradually became tidy, striPes became clear, and contour became clear. The changes of myocardial ultra_structure were obserVed under transmission electron microscoPe: the myocardial fiber breakage was reduced and the number of mitochondria was increased,swelling degeneration was alleViated. Conclusion CVB_D significantly imProVed cardiac contraction and blood PumPing function of rats with heart failure, exerted mitigatiVe effect on damage of cardiac muscle fiber and mitochondria. It suggests that CVB_D may be effectiVe in the treatment of heart failure.
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Aim To investigate the cardioprotective effects of Cyclovirobuxine D on pure cultared neonatal rat cardiomyoeytes exposed to hypoxia-reoxygenation (H/R) injury and the mechanisms. Methods The hypoxia/reoxygenation(H/R) injury model of pure cultured neonatal rat cardiomyocytes was developed.The concentration of lactate dehydrogenase (LDH) and malonialdehvde (MDA), the activity of super-oxide dismutes (SOD) and caspase-3, the apoptosis rate of cardiomyocyte were measured.Results Compared with the control group, the model group showed lower activity of SOD and higher concentrations of LDH , MDA, the apoptosis rate and activity of caspase-3(P
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Objective To investigate the in-vitro transdermal permeability of cyclovirobuxine D ethosomes.Methods Ethosomes,liposomes,saturated aqueous solution and 35.5% saturated alcohol solution of cyclovirobuxine D were prepared.The in-vitro transdermal permeability of different kinds of preparations of cyclovirobuxine D was studied by in-vitro permeation experiments.Results Cyclovirobuxine D ethosomes enhanced transdermal flux rate of cyclovirobuxine D and the enhancement ratio was 8.85,superior to liposomes and saturate alcohol.The dermatic hold-up amount of cyclovirobuxine D in 35.5% saturated alcohol solution,liposomes and ethosomes was in a decreasing sequence.Conclusion Compared with the liposomes,the ethosomes can enhance the transdermal flux rate of cyclovirobuxine D,which could increase the systematic action of the drug.
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AIM: To investigate the electrophysiological changes of diabetic myocardium and effects of cyclovirobuxine D (CVB-D) on its electrophysiology. METHODS: Diabetes was induced in male SD rats, using a single injection of alloxan into tail vein. Untreated age-matched animals were used as controls. Animal electrocardiogram (ECG) was recorded by 2 weeks. Effects of CVB-D on isolated right ventricular papillary muscle from experimental diabetic rats and control group were observed by recording the transmembrane potentials with conventional glass microelectrodes. RESULTS: QT intervals in ECG and action potential duration (APD) at all levels were significantly lengthened in myocardium from week 2 of diabetes. Within the concentration of 13.3-63.3 ?mol?L~(-1), CVB-D prologated APD of diabetes in dose-dependent manner and more than that of control. Within the concentration of 33.3-63.3 ?mol?L~(-1), CVB-D depressed RP, APA, V_(max) and OS of diabetes in dose-dependent way and more than that of control. In addition, CVB-D at concentration of 20 ?mol?L~(-1) prologated APD in a time-dependent manner. The most prologation of APD was attained about 40 min in control, while more than 40 min in diabetes. CONCLUSION: The results show that QT intervals in ECG and APD at all levels are significantly lengthened in myocardium from week 2 of diabetes. CVB-D prolongates APD and inhibits RP, APA, OS and V_(max) more in diabetes than in control.
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Objective To isolate and elucidate the structures of alkaloids in Huangyangning. Methods Alkaloids of Huangyangning were separated with preparative HPLC. The molecular structures were elucidated on the basis of chemical evidences and spectral analyses (UV, IR, MS, 1H-NMR, 13C-NMR, COSY, DEPT, HMQC, and HMBC). Results Cyclovirobuxine D is the major component in Huangyangning and cyclobuxine D and cyclovirobuxine C are the two related alkaloids. Conclusion It is demonstrated that all the Huangyangning alkaloids have the same structural frame with only minor differences in substitution through chromatographic and spectral analyses. Therefore, it is not easy to purify cyclovirobuxine D by using usual column, re-crystallization, or chemical approaches for the existence of the related alkaloids.
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Objective To set up the reference standard of cyclovirobuxine D.Methods Thermal analysis,HPLC/MS,HPLC with terminal wavelength,HPLC with fluorescence derivation and with ultraviolet derivation,TLC and nonaqueous titration methods were applied to determine the content of cyclovirobuxine D control.Results Thermal analysis can not be used to analyse the purity of cyclovirobuxine D ,and HPLC/MS,HPLC with terminal wavelength,HPLC with fluorescence derivation and HPLC with ultraviolet derivation can obtain the same purity.Conclusion The methods used for the assay of cyclovirobuxine D control were practical.
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AIM: To study the effect of CVB-D on contraction of guinea pig Oddi's sphincter in vitro. METHODS: Within the range from 1?10 -7 M to 3?10 -6 M, CVB-D's effects on the smooth muscle under the following conditions were observed: 1) the two phases of the Oddi's sphincter contractive curve caused by 80 mM K+ and 5?10 -6 M Ach; 2)together with verapamil, the contraction of Oddi's sphincter induced by 80 mM K+ and 5?10 -6 M Ach respectively. RESULTS: The relation between the amount of CVB-D and rapid contraction of desived inihibitory response was possessed of a dose-effect one, as well as ratio of plateau and peak of contractions, and slightly elevated contraction in continuous phase. CVB-D kept rapid phase peak value and plateau/peak value decreased markedly that expressed a dose-effect relationship. Contrary to CVB-D. Ach caused rapid, continuous phase and plateau/peak value to fall according as CVB-D decreased. Combination of verapamin and CVB-D led rapid phase peak value induced by KCl to lessen, but do not influence verapamin plateau used only and vanish plateau of rapid phase induced by Ach. CONCLUSION: CVB-D's effects on the contraction of Oddi's sphincter are related to the agonists and contractive phases, which reflect its effects on Ca 2+ channels.