Mechanism of bilobalide promoting neuroprotection of macrophages / 中国中药杂志

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 μg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 μg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 μg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.

An UHPLC-MS/MS method for simultaneous determination of quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-rutinoside, bilobalide and ligustrazine in rat plasma, and its application to pharmacokinetic study of Xingxiong injection / 中国天然药物

The present study was designed to develop and validate a rapid, sensitive, and reliable ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of five major active constituents in the traditional Chinese medicinal preparation Xingxiong injection (XXI) in rat plasma, including quercetin 3-O-rutinoside (QCR), kaempferol 3-O-rutinoside (KFR), isorhamnetin 3-O-rutinoside (ISR), bilobalide (BB), and ligustrazine (LGT). The plasma samples were pretreated by protein precipitation with acetonitrile. The chromatographic separation was achieved on a Waters Symmetry C analytical column (2.1 mm × 100 mm, 3.5 μm) with a mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B). Quantitation of the five bioactive constituents was achieved. Naringin was used as the internal standard (IS). All the calibration curves showed good linearity (r > 0.996) over the concentration range, with the lowest limit of quantification (LLOQ) between 2-18 ng·mL. The intra- and inter-day accuracy and precision of the analytes were both within acceptable limits. Moreover, satisfactory extraction recoveries (90.92%-104.03%) were obtained by protein precipitation. The validated method was successfully applied to a pharmacokinetic study of XXI in rats after intravenous administration at three doses. The pharmacokinetic parameters of the five compounds varied in a dose-dependent manner within the tested dosage range. The present study was the first report of pharmacokinetic study for XXI.

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.

Studies on the cell growth, differentiation and terpene lactone accumulation in Ginkgo biloba cell suspension cultures / 生物工程学报

To provide supports for Ginkgo biloba cell engineering for production of Terpene lactones (Ginkgolides and bilobalide), the cell suspension were established from calli induced from zygote embryos and stems of 30-day-old seedlings respectively. The relationship between cell growth, differentiation and the terpene lactone accumulation in these suspension cultures were investigated. HPLC determination indicated that, the ginkgolide B was found in the embryo derived cell suspension cultures at 0.044% of cell dry weight, and this result was the first time reported in this study. The accumulation of terpene lactone in the suspension cultures derived from both the embryo and seedling stems are effected by the level of the cell differentiation. The ginkgolide B was only found in small cell aggregates in the size smaller than 2mm, and the highest level of ginkgolide B was accumulated in cell aggregates in the size smaller than 1mm; however, the cell aggregates in the size bigger than 3mm could only produced bilobalide and ginkgolide A. In the same size aggregates of the suspension cultures the terpene lactone accumulation is strongly effected by the source of the explant. When the size of cell aggregates was in less than 1mm, the concentration of bilobalide, ginkgolide A and B in the cell suspension cultures derived from the embryos was 2, 1.4 and 0.56-fold, respectively, higher than that of cell cultures derived from seedling stems.