Impaired tricarboxylic acid cycle flux and mitochondrial aerobic respiration during isoproterenol induced myocardial ischemia is rescued by bilobalide / 药物分析学报

There is an urgent need to elucidate the pathogenesis of myocardial ischemia (MI) and potential drug treatments.Here,the anti-MI mechanism and material basis of Ginkgo biloba L.extract (GBE) were studied from the perspective of energy metabolism flux regulation.Metabolic flux analysis (MFA) was performed to investigate energy metabolism flux disorder and the regulatory nodes of GBE components in isoproterenol (ISO)-induced ischemia-like cardiomyocytes.It showed that[U-13C]glucose derived m+2 isotopologues from the upstream tricarboxylic acid (TCA) cycle metabolites were markedly accu-mulated in ISO-injured cardiomyocytes,but the opposite was seen for the downstream metabolites,while their total cellular concentrations were increased.This indicates a blockage of carbon flow from glycolysis and enhanced anaplerosis from other carbon sources.A Seahorse test was used to screen for GBE components with regulatory effects on mitochondrial aerobic respiratory dysfunction.It showed that bilobalide protected against impaired mitochondrial aerobic respiration.MFA also showed that bilobalide significantly modulated the TCA cycle flux,reduced abnormal metabolite accumulation,and balanced the demand of different carbon sources.Western blotting and PCR analysis showed that bilobalide decreased the enhanced expression of key metabolic enzymes in injured cells.Bilobalide's efficacy was verified by in vivo experiments in rats.This is the first report to show that bilobalide,the active ingredient of GBE,protects against MI by rescuing impaired TCA cycle flux.This provides a new mechanism and potential drug treatment for MI.It also shows the potential of MFA/Seahorse combi-nation as a powerful strategy for pharmacological research on herbal medicine.

Advances research on chemical constituents, preparations and adverse reactions of Ginkgo biloba / 药物评价研究

The leaves and fruits of Ginkgo biloba L.are embodied in Pharmacopoeia of People's Republic of China,mainly contain ginkgo flavonoids,terpene lactones,phenolic acids,isopentenyl alcohol,steroids and other chemical compositions.The extract and active ingredients can be made into tablets,capsules,granules,oral liquid,injection,dripping pills,syrup,tincture and other formulations,clinically used for cardiovascular and cerebrovascular disease treatment.The adverse reactions are allergies,diarrhea,bleeding,liver and kidney toxicity.This review contains the chemical composition,dosage form and clinical adverse reactions of Ginkgo biloba in recent years,for providing a reference of further development and rational utilization of this plant resource.

Study on blood-brain distribution of lactones in rats after oral administration of Ginkgo biloba extract by blood-brain synchronization microdialysis / 中草药

Objective: To study the blood and brain drug concentration and drug distribution coefficient of lactones in rats after oral administration of Ginkgo biloba extract (GBE). Methods: Blood-brain synchronization microdialysis was used in combination with HPLC-MS/MS inthis study. Probes were buried into rat brain and jugular vein to collect the blood and brain micro dialysis liquid in rats after oral GBE in different periods. The lactones in the dialysate were analyzed by HPLC-MS/MS, and the blood and brain drug concentration was depicted according to the results. Then the drug distribution coefficients of each component in blood and brain (AUCbrain/AUCblood) were calculated. Results: After GBE suspension (600 mg/kg) oral gavaging in the rats, the blood samples and brain samples were obtained and detected by HPLC/MS/MS. Ginkgolides A, B, and C were detected in both blood and in brain, but bilobalide could be detected just in blood. The drug distribution coefficients in the blood and brain (AUCbrain/AUCblood) were as follows: Ginkgolide A was 2.911%, ginkgolide B was 3.126%, and ginkgolide C was 0.337%. Conclusion: Simultaneous multiple microdialysis technique can be used to detect multiple components in different tissues simultaneously. It also has other characteristics, such as continuous sampling on living animals with small sample volume and little tissue damage, which can save animals. In all, simultaneous multiple microdialysis could be a good method for exploring the effect components in Chinese medicine and their distribution at effect sides.

Determination of Six Active Components in Dengyinnaotong Capsules by HPLC-MS/MS / 中国药学杂志

OBJECITVE: To establish an HPLC-MS/MS method for simultaneous determination of six active components in Dengyinnaotong capsules, ie, scutellarin, bilobalide, ginkgolide A, ginkgolide B, ginsenoside Rg1 and notoginsenoside R1. METHODS: The chromatographic separation was carried out at 30℃ on a Phenomenex Luna C18 (4.6 mm×150 mm, 5 μm) column eluted by gradient program. The flow rate was 0.3 mL·min-1. The six compounds were separated within 10.0 min. RESULTS: The regression curves for the six compounds showed good linearity in wide ranges. The recoveries were around from 94.8% to 108.5%. CONCLUSION: The established method is accurate, reliable, specific and reproducible, which can be used for the quality control of Dengyinnaotong Capsules.

Determination of content of Shuxuening Injection based on quantitative analysis of multi-components by single marker / 中草药

Objective: To establish a quantitative analysis method for determining the contents of artemisinin, quercetin, isorhamnetin, Ginkgo esters, and lactones a, b, c in Shuxuening Injection by using multi-components with a single marker (QAMS), and set up methodological evaluation model of the above experiment. Methods: With artemisinin and Ginkgo esters as indexes, the relative correction factor (fk/s) between artemisinin and the other two total flavonoids and the fk/s between Ginkgo esters and lactones a, b, c were established. The contents of artemisinin and Ginkgo esters in Shuxuening Injection were determined by external standard method, the contents of other five components in Shuxuening Injection were calculated by the relative correction factor. The results of the content of five batches of Shuxuening Injection determined by QAMS and tested by external standard method were carried out by t test. Results: The fk/s of quercetin and isorhamnetin with reference to artemisinin were 1.873 and 0.324, the fk/s of ginkgolide A, B, C with reference to Ginkgo esters were 2.280, 1.659, and 1.429. And the repeatability was good under different experimental conditions. There were no significant differences between the calculated value and estimated value on QAMS and external standard method of five batches of Shuxuening Injection, and the results showed that fk/s was authentic. Conclusion: The established correction factor has good repeatability, and could be used for quantitative analysis and quality evaluation of multiple components in Shuxuening Injection.

Correlation of in vitro release between total flavonoids and different ingredients in sustained-release pellets of Ginkgo biloba extract by f2 fit factor method / 中草药

Objective: To find a new method to evaluate the in vitro release of Ginkgo biloba extract (GBE) sustained-release pellets, f2 fit factor method was used to study the correlation of in vitro release between total flavonoids and different ingredients (including flavonoids and terpenoids). Methods: The release rates in vitro of total flavonoids and different ingredients (quercitrin, isorhamnetin, lutin, quercetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide) were detected by UV and HPLC-MS respectively, and then f2 fit factor was calculated between total flavonoids and different ingredients. Also the micro-structures of pellets before and after drug release were detected by scanning electron microscopy (SEM), which could explain the drug release mechanism combined with the fitted equation. Results: All f2 values were greater than 50 between the total flavonoids and different ingredients of the in vitro release from GBE pellets of optimized preparation, which indicated that there might be a good correlation between them. The drug release mechanism further verified the reliability of the results. Conclusion: The f2 fit factor method could be applied in the evaluation of in vitro release for multi-component sustained-release preparations of Chinese materica medicine.

Study on quantitative determination method of flavonoids and terpene lactone components in Dipyridamole Injection / 中草药

Objective: To establish a method for quantitative determination of Dipyridamole Injection. Methods: The flavonoids were determined using Agela C18 column (150 mm × 4.6 mm, 5μm), acetonitrile-0.4% phosphoric acid (gradient) as the mobile phase with flow rate 1.0 mL/min, detection wavelength of 360 nm, column temperature of 35℃. The lactones were determined using Agela C18 column (150 mm × 4.6 mm, 5 μm), methanol-tetrahydrofuran and water (10:75) solution (gradient) as the mobile phase with flow rate 0.8 mL/min, with evaporative light scattering detector, and column temperature of 30℃. Results: Respectively taking 10 batches of Dipyridamole Injection, the contents of seven major components such as quercetin, kaempferide, isorhamnetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide were determined as 12.4, 20.8, 5.5, 7.2, 2.6, 4.7, and 102.0 μg/mL. Conclusion: The established method is with good stability, repeatability, and precision, which conforms to the requirements and can be used for the quality control of Dipyridamole Injection.

Electrocardiographic profile of guinea pig heart submitted to Ginkgo biloba extract and its terpenoids / Perfil eletrocardiográfico do coração de cobaia submetido ao extrato de Ginkgo biloba e seus terpenóides

Electrocardiographic effects produced by Ginkgo biloba extract (EGb) and by ginkgolides A (GA) and B (GB), and bilobalide (BB) were investigated in guinea pig heart mounted in Langendorff apparatus (Tyrode, 34 ± 0.1 ºC, 95% O2, 5% CO2). Electrocardiographic parameters were evaluated in the conditions: 1) control with Tyrode and DMSO, 2) EGb (n=4), GA (n=5), GB (n=5) or BB (n=6), and 3) washout. The results showed that 0.1 and 1.0 mg/ml of EGb do not change the electrocardiographic parameters. However, 10 mg/ml of EGb increased the PR interval (PRi) at 21% (p<0.001). This increase was also observed for 50 mM GA (20%, p<0.001) and 70 mM BB (13%, p<0.001), which indicates Ca2+ channel block. However, the 50 mM GB reduced the PRi at 11 % (p<0.001). The GA (23%, p<0.001), GB (16%, p<0.001), and BB (40%, p<0.001) reduced the QT interval (QTi), which suggests the activation of the potassium channel. However, EGb increased QTi (6%, p<0.001). The EGb (28%, p<0.05) and GB (13%, p<0.05) reduced the heart rate. Atrioventricular (AV) block was observed with EGb, GA, and BB. We can conclude that EGb and its terpenoids alter the ECG parameters inducing AV block, which indicates possible arrhythmogenic potential.

Os efeitos eletrocardiográficos produzidos pelo extrato de Ginkgo biloba (EGb) e gingkolídeos A (GA) e B (GB), e bilobalide (BB) foram investigados em coração de cobaia montado sistema de Langendorff (Tyrode, 34 ± 0.1 ºC, 95% O2, 5% CO2). Os parâmetros do ECG foram avaliados nas condições: 1) Tyrode e DMSO, 2) EGb (n=4), GA (n=5), GB (n=5) ou BB (n=6) diluídos em DMSO e 3) washout. Os resultados demonstram que 0,1 e 1,0 mg/mL de EGb não alteraram os parâmetros eletrocardiográficos. Entretanto, 10 mg/ml de EGb aumentaram o intervalo PR (PRi) em 21% (p<0.001). Esse aumento também foi observado com GA a 50µM (20%, p<0,001) e BB a 70 mM (13%, p<0,001) indicando bloqueio de canais de cálcio. Por outro lado, GB reduziu o PRi (11%, p<0,001). O intervalo QT (QTi) foi reduzido por GA (23%, p<0,001), GB (16%, p<0,001) e BB (40%, p < 0.001) sugerindo uma ativação de canais de potássio. Entretanto, EGb aumentou o QTi (6%, p<0.001). A frequência cardíaca foi reduzida por EGb (28%, p<0.05) e GB (13%, p<0.05). Bloqueios átrio-ventriculares (BAV) foram observados com EGb, GA e BB. Podemos concluir que EGb e os terpenos alteram parâmetros eletrocardiográficos induzindo BAV e demonstrando possível potencial arritmogênico.

Study on the Protective Effect of Bilobalide on Ischemical Reperfusion Injury in Rats Undergoing Liver Transplantation / 中国药房

OBJECTIVE:To observe the effect of Bilobalide on ischemical reperfusion injury in orthotopic liver transplantation model rats and to discuss its protective effect on ischemical reperfusion injury of rats undergoing liver transplantation.METHODS:The model of orthotopic liver transplantation was established by in rats by setting up the bridge between portal vein and left renal vein and building blood bypass by inlaying of canal in the inferior vena,after modeling,the rats were treated with Bilobalide,then levels of NO(active medium of blood vessel)and ET1 before ischemia and at 10 min,30min and 2h after reperfusion were determined by nitratase reduction method;serum levels of ALT,AST,ALP,LDH enzymology,ATP and MDA in the liver tissues were determined as well.The hepatic tissue sample was taken at 2h and fixed with formalin to be made into specimen for observation of the ultrastructure of liver cells and hepatic lobules under electron microscope.RESULTS:After being treated by Bilobalide,serum NO level was elevated and the pathological elevated levels of ALT,AST and LDH were brought down,the microcirculation in ischemical reperfusion injury rats was improved and the extent of damage in the ultrastructure of liver cells was lessened in rats.CONCLUSION:Bypass of portal vein and inferior vena cava prior liver transplantation is helpful for the prevention of ischemical reperfusion injury of liver.The balance of NO/ET1 is possibly a factor influencing the blood flow in the microcirculation of transplanted liver.Bilobalide was proved to be of protective effect on ischemical reperfusion injury in rats.

Isolation and Purification of Ginkgolide A, B and Bilobalide / 中草药

A simple method for the isolation and purification of ginkgolide A, B and bilobalide(ginkgo terpene trialctone) was developed. A commercially available extract of leaves of Ginkgo biloba L.containing ＞6％ ginkgo terpene trilactone was used as the raw material. After partition in EtOAc, the en-riched extract was separated to give individual terpenes by preparative liquid chromatography. GinkgolideA, B and bilobalide could be isolated with high purity by recrystallizing in MeOH-H2O.

Quality and quantity analysis of terpene lactones from Ginkgo biloba leaves by GC-MS / 中草药

Object To develop a capillary GC-MS analytical method for identification and deter- mination of ginkgolide A, B, C and bilobalide (GA, GB, GC and BB) in Ginkgo biloba L. leaves. Methods The leave samples were extracted in ultrasonic bath with ethanol-water (20∶80). The extract was purified by liquid-liquid extraction with ethyl acetate followed by solid-phase extraction on a column mixed with acid Al 2O 3, active carbon and celite. The terpenes were trimethylsilylated by BSTFA (with 1% TMCS) for 60 min at 100 ℃ and determined by GC-MS with HP-5 MS capillary column in the selected-ion monitoring mode. The intense fragment ions were chosen as monitoring ions for quantitative analysis. Cholesterol was used as an internal standard. Column temperature gradient: initial temperature 180 ℃, maintained 1 min, and then increased at 20 ℃/min to 260 ℃, and finally at 2 ℃/min up to 300 ℃, maintained 2 min. Results The retention times of GA, GB, GC and BB were 13.7,14.3,15.3 and 6.8 min, the major fragmentation ions (monitoring) were at m/z 537, 625, 713 and 455 (299), the average recoveries of GA, GB, GC and BB were 102.0%, 99.4%, 96.0%, 96.3%, RSD were 0.54%, 2.40%, 1.98% and 2.43%, respectively. Conclusion This method is repeatable, specific, accurate and easy to operate. It is adoptable for quality and quantity analysis of terpene lactones from G. biloba leaves.

Pathological changes in lungs of rats infected with Pneumocystis carinii pneumonia after treatment with bilobalide / 重庆医科大学学报

Objective:To study the Pathological changes in lungs of rats infected with Pneumocystis carinii pneumonia after treatment with bilodalide(BL). Methods:PCP rat models were established of hypodermic injection of dexamethasone twice a week for 6 weeks. The PCP rats were treated with BL 30mg/(kg.d)?8days and killed to obtain lungs. Pathological changes in lung tissues were observed under light and electron microscope .Infected and normal rats were established as controls. Results: The number of Pc cysts was markedly reduced in the lung print smear and inflammation in the lung was lightened. The investigation of thinner lung tissue section by transmission electronic microscope showed that the Pneumocystis carinii treated with bilobalide had formation of vacuoles in trophozoites,swelling and destroying of cell organ, rupture of cytomembrane and some high density electronic granulae were investigated in the cytoplasm. Membrane of cyst was destroyed too. Conclusion:Bilodalide can kill Pc and alleviate inflammation in the lung tissues of PCP rats.