Clinical pharmacokinetics of Ginkgolides Dropping Pills in healthy subjects / 中草药

Objective: In order to describe the pharmacokinetic profiles of two effective constituents ginkgolide A and ginkgolide B in healthy subjects, and to provide supports for setting out the clinical application of Ginkgolides Dropping Pills. Methods: Ten healthy subjects were enrolled in a randomized and open experimental design. Following a single oral administration of Ginkgolides Dropping Pills, blood samples which were anticoagulated by heparin sodium were collected at predetermined time, and then centrifuged to separate plasma samples. The total concentration of ginkgolide A and ginkgolide B in plasma samples and the lactone concentration of ginkgolide A and ginkgolide B were determined by a verified LC-MS/MS method, the pharmacokinetic parameters were calculated by WinNonlin 6.3 with non-compartment model. Results: After a single oral administration of Ginkgolides Dropping Pills, the tmax of lactone, total concentration of ginkgolide A respectively were (3.05 ± 1.40), (3.40 ± 1.22) h, the Cmax were (84.3 ± 32.8), (92.2 ± 35.0) ng/mL, respectively, and its Cmax ratio was 91.4%. The AUC0-t were (636 ± 183), (753 ± 205) ng∙h/mL, respectively, and its AUC0-t ratio was 84.5%, half-life time (t1/2) were (13.0 ± 10.3), (12.9 ± 8.49) h, respectively. The Tmax of lactone, total concentration of ginkgolide B were (3.15 ± 1.42), (3.35 ± 1.25) h, The Cmax were (74.1 ± 31.5), (148 ± 60.1) ng/mL, respectively, and its Cmax ratio was 50.1%.The AUC0-t were (627 ± 202), (1410 ± 431) ng∙h/mL, respectively, and its AUC0-t ratio was 44.5%, t1/2 were (13.2 ± 5.83), (13.7 ± 5.83) h, respectively. Conclusion: The results demonstrated that ginkgolide A and ginkgolide B were both at a moderate absorption and elimination rate, ginkgolide A mainly existed in human plasma upon lactone, while ginkgolide B presented as hydrolyzed forms with one or two lactone groups hydrolyzed and lactone, and the two forms of ginkgolide B were at equal exposure level after single oral administration of Ginkgolides Dropping Pills.

Ginkgolide A reduces airway inflammation in a neutrophil-predominant murine model of asthma via suppression of immune response of helper T cell 17 / 中国中西医结合急救杂志

Objective To determine the effects of ginkgolide A (GA) in a neutrophil-predominant murine model of asthma and explore underlying mechanisms. Methods Thirty-five female BALB/c mice were randomly divided into sham operation group (Sham group), asthma group, dexamethasone intervention control group (DEX group), low dose GA intervention group (L-GA group) and high dose GA intervention group (H-GA group), with 7 mice in each group. The asthma model was induced by intraperitoneal injection of 20 μg ovalbumin (OVA) and 75 μL Fluorine complete adjuvant (FCA) on day 0, 14 and 21, and challenged 30 minutes with 5% OVA atomization on days 22-24 consecutively; phosphate buffer (PBS) was sensitized and stimulated in Sham group. The mice in L-GA group and H-GA group were intraperitoneally injected with GA of 40 mg/kg and 80 mg/kg at 1 hour before each challenge, while the mice in DEX group were intraperitoneally injected with dexamethasone of 1 mg/kg. After 24 hours of the last OVA stimulation, the airway resistance was measured at the time of 0, 3, 6, 12, 25, 50 g/L acetylmethacholine aerosol stimulation. The total number of cells and cell classification in bronchoalveolar lavage fluid (BALF) were counted. The transforming growth factor-β1 (TGF-β1) and interleukin-17 (IL-17) in BALF were detected by enzyme linked immunosorbent assay (ELISA). The proportion of helper T cell 17 (Th17) to CD4+ T cell in lung tissue was detected by flow cytometry, and the pathological characteristics of lung tissue were evaluated. Results Compared with the Sham group, the airway hyper responsiveness (AHR), the total cells, the neutrophil counts, the levels of TGF-β1, IL-17 in BALF, and the proportion of Th17 cells in the lung tissue in the asthma group were significantly increased, obvious inflammatory cell infiltration and collagen fiber deposition around airway were observed, and airway inflammation score and mucus score were significantly increased. Compared with the asthma group, low and high doses of GA significantly reduced AHR, and there was a significant difference in airway resistance at the time of 50 g/L acetylmethacholine stimulation (cmH2O·s-1·mL-1: 5.29±0.40, 3.99±0.57 vs. 7.34±0.77, both P < 0.05); the total cells, neutrophil counts, and levels of TGF-β1, IL-17 in BALF, and the proportion of Th17 cells in lung tissue were significantly decreased [total cells count (×104/L): 21.00±1.00, 17.00±1.02 vs. 27.50±2.50; neutrophil count (×104/L): 12.600±0.600, 10.610±0.210 vs. 16.875±1.125; TGF-β1 (ng/L): 371.40±107.80, 289.60±70.76 vs. 551.90±68.34; IL-17 (ng/L): 60.75±11.79, 44.77±7.09 vs. 122.50±38.87; the proportion of Th17 cells: (5.53±0.40)%, (3.76±1.10)% vs. (8.30±1.19)%, all P < 0.05]; inflammatory cell infiltration around the airway and mucus secretion was significantly reduced, airway inflammation score and mucus score were significantly decreased (2.16±0.28, 1.16±0.28 vs. 3.77±0.25; 1.33±0.58, 1.17±0.29 vs. 3.67±0.58, all P < 0.05). The AHR, total cells, neutrophil counts, and IL-17 level in BALF, the proportion of Th17 cells in lung tissue and airway inflammation score decreased more obviously with the increase of GA dosage (all P < 0.05). For index mentioned above, no significant differences were observed between DEX group and asthma group. Conclusion GA treatment was effective in a murine model of neutrophil-predominant asthma via inhibiting response in the immune cells Th17.

Applying risk management to analytical methods for the desorbing process of ginkgo diterpene lactone meglumine injection / 中国天然药物

Analysis errors can occur in the desorbing process of ginkgo diterpene lactone meglumine injection (GDMI) by a conventional analysis method, due to several factors, such as easily crystallized samples, solvent volatility, time-consuming sample pre-processing, fixed method, and offline analysis. Based on risk management, near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were introduced to solve the above problems with the advantage of timely analysis and non-destructive nature towards samples. The objective of the present study was to identify the feasibility of using NIR or MIR spectroscopy techniques to increase the analysis accuracy of samples from the desorbing process of GDMI. Quantitative models of NIR and MIR were established based on partial least square method and the performances were calculated. Compared to NIR model, MIR model showed greater accuracy and applicability for the analysis of the GDMI desorbing solutions. The relative errors of the concentrations of Ginkgolide A (GA) and Ginkgolide B (GB) were 2.40% and 2.89%, respectively, which were less than 5.00%. The research demonstrated the potential of the MIR spectroscopy technique for the rapid and non-destructive quantitative analysis of the concentrations of GA and GB.

Relative bioavailability of terpene lactones in Beagle dogs after ig administration of domestic and imported Ginkgo Leaf Tablets / 中草药

Objective To compare the pharmacokinetic parameters and bioavailability of terpene lactones in Beagle dogs between domestic and imported Ginkgo Leaf Tablets. Methods Beagle dogs were ig administrated demestic and imported Ginkgo Leaf Tablets, and then the plasma of Beagle dogs were detected. LC-MS was used to determine the contents of terpene lactones (including ginkgolide A, ginkgolide B, and ginkgo lactone) in plasma of Beagle dogs. Plasma concentration-time curves were drawn and analyzed by DAS software to obtain pharmacokinetics parameter. Results The area under curve (AUC0-t) of GA, GB, and BB in Beagle dogs after ig administration domestic Ginkgo Leaf Tablets was 51.64, 19.86, and 72.90 ng∙h/mL, while it was 69.98, 24.35, and 169.60 ng∙h/mL after ig administration imported G. biloba leaf extract tablets, respectively. According to the contents of three components in two preparations, the relative bioavailability of GA, GB, and BB of domestic Ginkgo Leaf Tablets respectively was 37.77%, 33.70%, and 95.98%. Conclusion The oral bioavailability of the terpene lactones in imported Ginkgo Leaf Tablets was significantly higher than that of domestic tablets.

Effect of compatibility of ginkgolide A, ginkgolide B and ginkgolide K / 中国中药杂志

To investigate the best active compatibility of ginkgolide A, B and K (GA，GB，GK). The effects of GA, GB, GK alone, combinations of each two of them, and combinations of these three components on platelet-activating factor (PAF)-induced platelet aggregation activity and rat cerebral ischemia reperfusion model (tMCAO) were compared in this study. Different compatibilities of GA, GB and GK could significantly reduce the maximum aggregation rate of PAF-induced platelet aggregation, and the effect was most obvious in combination of the three. Different compatibilities of GA, GB and GK could alleviate the neural function, cerebral infarction volume and cerebral edema in the tMCAO model of rats to different degrees, and the effect of combinations of the three was stronger than those of combinations of two and single use. The combination of all of GA, GB and GK had the strongest effect on nerve injury caused by anti-platelet aggregation in tMCAO rats.

Applying risk management to analytical methods for the desorbing process of ginkgo diterpene lactone meglumine injection / 中国天然药物

Analysis errors can occur in the desorbing process of ginkgo diterpene lactone meglumine injection (GDMI) by a conventional analysis method, due to several factors, such as easily crystallized samples, solvent volatility, time-consuming sample pre-processing, fixed method, and offline analysis. Based on risk management, near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were introduced to solve the above problems with the advantage of timely analysis and non-destructive nature towards samples. The objective of the present study was to identify the feasibility of using NIR or MIR spectroscopy techniques to increase the analysis accuracy of samples from the desorbing process of GDMI. Quantitative models of NIR and MIR were established based on partial least square method and the performances were calculated. Compared to NIR model, MIR model showed greater accuracy and applicability for the analysis of the GDMI desorbing solutions. The relative errors of the concentrations of Ginkgolide A (GA) and Ginkgolide B (GB) were 2.40% and 2.89%, respectively, which were less than 5.00%. The research demonstrated the potential of the MIR spectroscopy technique for the rapid and non-destructive quantitative analysis of the concentrations of GA and GB.

Effects of ginkgolide A, B and K on platelet aggregation / 中国中药杂志

To investigate the effects of ginkgolide A (GA), ginkgolide B (GB) and ginkgolide K (GK) on platelet aggregation in rabbits, and compare the similarities and differences among these three components. The effects of different doses of ginkgolide A, B and K on platelet aggregation induced by platelet activating factor (PAF) were observed by using in vitro experiment. The results showed that three compounds could inhibit platelet aggregation induced by PAF in vitro, and the intensity was GK> GB> GA. It was further found that all of them can mobilize [Ca2+]i and enhance intracellular c-AMP level in a dose-dependent manner, which was consistent to the ability to antagonize PAF receptor. These findings indicated that GK was highly selective for PAF receptor, and may inhibit platelet aggregation by activating cAMP signaling pathway and inhibiting intracellular [Ca2+]i mobilization; GB and GA also had strong antagonism to PAF receptor, but the effect was weaker than that of GK.

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.

PXR Mediated Protection against Liver Inflammation by Ginkgolide A in Tetrachloromethane Treated Mice

The pregnane X receptor (PXR), a liver and intestine specific receptor,, has been reported to be related with the repression of inflammation as well as activation of cytochromosome P450 3A (CYP3A) expression. We examined the effect of PXR on tetrachloromethane (CCl4)-induced mouse liver inflammation in this work. Ginkgolide A, one main component of Ginkgo biloba extracts (GBE), activated PXR and enhanced PXR expression level, displayed both significant therapeutic effect and preventive effect against CCl4-induced mouse hepatitis. siRNA-mediated decrease of PXR expression significantly reduced the efficacy of Ginkgolide A in treating CCl4-induced inflammation in mice. Flavonoids, another important components of GBE, were shown anti-inflammatory effect in a different way from Ginkgolide A which might be independent on PXR because flavonoids significantly inhibited CYP3A11 activities in mice. The results indicated that anti-inflammatory effect of PXR might be mediated by enhancing transcription level of IkappaBalpha through binding of IkappaBalpha. Inhibition of NF-kappaB activity by NF-kappaB-specific suppressor IkappaBalpha is one of the potential mechanisms of Ginkgolide A against CCl4-induced liver inflammation.

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.

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.

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.