PAFR ( IC50 = 3.6 μM )

In vitro activity: Ginkgolide B potently exhibits a platelet-activating factor (PAF) receptor.[1] Ginkgolide B (0.5 μM–12 μM) treatment of PMN causes a rapid and weak production of reactive oxygen species, as shown by chemiluminescence. Ginkgolide B potentiates the CL response induced by fMet-Leu-Phe and zymosan. Ras/MAPK signaling pathway is altered and cyst cell differentiation is induced by ginkgolide B.[2] Ginkgolide B encourages endothelial gene expression and proliferation, and it significantly improves the ability of endothelial cells to integrate into vascular networks and the migration response triggered by vascular endothelial growth factor. Ginkgolide B shields EPCs from cell death caused by H₂O₂. The phosphorylation of eNOS, Akt, and p38 is induced by ginkgolide B, and this promotes cell proliferation and function.[3]

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Ginkgolide B inhibits MDCK cyst formation and growth. [2]
An MDCK cyst model was used to evaluate the effects of ginkgolide B on cyst formation. MDCK cells did not form cysts but grew into colonies in the absence of forskolin without or with ginkgolide B (Fig. 1A, top two panels). In the presence of 10 μM forskolin, however, cysts were seen on day 4 and progressively expanded over the next 8 days (Fig. 1A, third panel). When MDCK cells were incubated with ginkgolide B (at 0.125, 0.5, or 2 μM) in the presence of 10 μM forskolin for 6 days, cyst formation was significantly inhibited (Fig. 1A, bottom panel), and this effect of ginkgolide B was dose dependent with an inhibition by up to 69% at 2 μM ginkgolide B (Fig. 1B). The numbers of total colonies (cysts with diameter >50 μm plus noncyst colonies) were similar to the original seeded cell numbers in all groups, indicating that ginkgolide B was not toxic to MDCK cells.
To determine the inhibition of ginkgolide B on cyst enlargement, the established cysts (on day 4 cultured with forskolin) were exposed to 0.125, 0.5, or 2 μM ginkgolide B in the presence of 10 μM forskolin. Cysts continuously enlarged with forskolin stimulation (Fig. 2A, top). Ginkgolide B remarkably inhibited cyst enlargement (Fig. 2A, middle, and B). This inhibition was abolished when ginkgolide B was removed after 4-day treatment (Fig. 2A, bottom, and C).

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Ginkgolide B retards cyst development in embryonic kidney cyst model. [2]
To evaluate the effect of ginkgolide B on renal cyst formation and growth in whole kidney organs, mouse embryonic kidneys at E13.5 were cultured on Transwell filters in the absence or presence of 100 μM 8-Br-cAMP as described previously. In the absence of 8-Br-cAMP, kidneys grew without cyst formation over 4 days, whereas numerous cystic structures were seen in the presence of 8-Br-cAMP as previously described (Fig. 3A, top) (37, 45). Ginkgolide B significantly inhibited cyst formation and growth in the embryonic kidneys (Fig. 3A, middle). Cysts grew and enlarged again following ginkgolide B washout after 2-day treatment, which suggests that the cyst inhibition of ginkgolide B was reversible (Fig. 3A, bottom). Moreover, the inhibition of ginkgolide B on cyst formation and growth was dose dependent (Fig. 3B), as shown by quantitative image analysis (Fig. 3C). Kidney growth with or without 8-Br-cAMP incubation was not affected by ginkgolide B (data not shown).

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In this study, we investigated the effects of Ginkgolide B (GB) on proliferation and differentiation of EPCs, and the involved signaling pathway in vitro. EPC proliferation, migration, adhesion and angiogenesis activities were assessed with the WST-8 assay, Transwell chamber assay, cell counting and angiogenesis kit, respectively. Apoptosis was detected with annexin V and propidium iodide staining. The protein expression of angiogenesis-related makers was detected by Western blot, and related gene expression was determined by real-time polymerase chain reaction (RT-PCR). The results showed that GB promoted the proliferation and endothelial gene expression, and markedly enhanced vascular endothelial growth factor-induced migration response and the capability to incorporate into the vascular networks in EPCs. GB protected EPCs from H2O2-induced cell death. GB induced the phosphorylation of eNOS, Akt and p38, which in turn promoted cell proliferation and function. In conclusion, the present study demonstrates that GB, at a near medical applied dose, increases the number and functional activities of EPCs with involvement of Akt/endothelial nitric oxide synthase and mitogen-activated protein kinase (MAPK)/p38 signal pathways. These findings raise the intriguing possibility that GB may play an important role in the protection and revascularization of blood vessels [3].

Ginkgolide B (2 μM) significantly inhibits the formation of MDCK cysts in a dose-dependent manner, with up to 69% reduction. Ginkgolide B also significantly inhibits cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model.[2] Ginkgolide B (50 mg/kg p.o.) applied prior to ischemia significantly lessens neuronal damage.[4] In the mouse model of focal ischemia, 30 minutes of pretreatment with Ginkgolide B (100 mg/kg, s.c.) decreases the infarct area. Ginkgolide B (1 μM) shields hippocampal neurons and astrocytes from neonatal rats' primary cultures from glutamate-induced neuronal damage. Ginkgolide B (100 μM) reduces apoptotic damage induced by staurosporine.[5] Ginkgolide B (1 mg/kg i.v. or 10 mg/kg p.o.) inhibits bronchoconstriction, the hematocrit increase, and the concomitant thrombopenia and leukopenia induced by PAF-acether (33 ng/kg–100 ng/kg) in animals anesthetized with pentobarbitone or ethyl carbamate. Aerosolized PAF-acether-induced bronchoconstriction is lessened by ginkgolide B at a dose of 3 mg/kg. At a dosage of 300 μM, ginkgolide B also prevents PAF-acether-stimulated alveolar macrophages from producing superoxide. Injecting 100 ng of PAF-acether into a perfused lung causes thromboxane to form, but ginkgolide B prevents this from happening. Pretreating parenchyma lung strips with Ginkgolide B (100 μM) reduces the release of thromboxane and partially inhibits the contraction caused by PAF-acether (0.1 μM).[6] Ginkgolide B prevents ischemia injury from developing.[7] Ginkgolide B treatment shows a significant decrease in brain edema, neurological deficits, and the volume of the infarction. Moreover, ischemia/reperfusion (I/R)-induced NF-κB, microglia activation, and pro-inflammatory cytokine production are inhibited by ginkgolide B. In post-ischemic brains, ginkgolide B raises Bcl-2 protein levels while lowering Bax protein levels.[8] Ginkgolide B reduces platelet aggregation and prevents Akt phosphorylation and phosphatidylinositol 3 kinase (PI3K) activation in platelets activated by collagen and thrombin. Ginkgolide B reduces plasma PF4 and RANTES levels in ApoE^−/− mice.Ginkgolide B reduces P-selectin, PF4, RANTES, and CD40L expression in aortic plaque in ApoE^−/− mice.Furthermore, ginkgolide B inhibits macrophage and vascular cell adhesion protein 1 (VCAM-1) expression in aorta lesions in ApoE^−/− mice.[9]

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Ginkgolide B inhibits renal cyst development in PKD mice. [2]
Kidney-specific Pkd1 knockout mice (Pkd1flox/−;Ksp-Cre) were used to determine whether ginkgolide B has any cyst inhibition activity in vivo. Ginkgolide B was subcutaneously injected into mice every 12 h at 16 mg·kg−1·day−1 from day 1 to day 4 of age. During the treatment period, wild-type mice and Pkd1flox/−;Ksp-Cre mice, with or without ginkgolide B treatment, were indistinguishable in their activity and behavior. After the treatment, there was no difference in body weight between the mouse groups (data not shown). Kidney sizes and weights in Pkd1flox/−;Ksp-Cre mice were greater than those in wild-type mice. However, ginkgolide B significantly reduced the kidney sizes and weights in Pkd1flox/−;Ksp-Cre mice (Fig. 4A). Ginkgolide B did not affect the liver sizes and weights in all groups (Fig. 4B). Figure 4C shows representative central coronal kidney sections. Kidney sections from ginkgolide B-treated Pkd1flox/−;Ksp-Cre mice showed fewer cysts of all sizes than those in untreated PKD mice. Image analysis of hematoxylin- and eosin-stained sections showed remarkably smaller fractional cyst areas per kidney in ginkgolide B-treated PKD mice compared with untreated PKD mice (Fig. 4D).

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We investigated the effect of the known antagonist of platelet-activating factor (PAF), Ginkgolide B, on postischemic neuronal damage in the rat. Neuronal necroses were evaluated in the hippocampus 7 days after a 10-min forebrain ischemia. Preischemic application of ginkgolide B (50 mg/kg p.o.) significantly reduced neuronal damage. It is suggested that the antagonism of PAF is responsible for this beneficial effect of ginkgolide B.[4]

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The standardized Ginkgo biloba extract EGb 761(definition see editorial) has been shown to produce neuroprotective effects in different in vivo and in vitro models. Since EGb 761 is a complex mixture containing flavonoid glycosides, terpene lactones (non-flavone fraction) and various other constituents, the question arises as to which of these compounds mediates the protective activity of EGb 761. Previous studies have demonstrated that the non-flavone fraction was responsible for the antihypoxic activity of EGb 761. Thus, we examined the neuroprotective and anti-apoptotic ability of the main constituents of the non-flavone fraction, the ginkgolides A, B, C, J and bilobalide. In focal cerebral ischemia models, the administration of bilobalide (5-20 mg/kg, s. c.) 60 min before ischemia dose-dependently reduced the infarct area in mouse brain and the infarct volume in rat brain 2 days after the onset of the injury. 30 minutes of pretreatment with ginkgolide A (50 mg/kg, s. c.) and ginkgolide B (100 mg/kg, s. c.) reduced the infarct area in the mouse model of focal ischemia. In primary cultures of hippocampal neurons and astrocytes from neonatal rats, ginkgolide B (1 microM) and bilobalide (10 microM) protected the neurons against damage caused by glutamate (1 mM, 1 h) as evaluated by trypan blue staining. In addition, bilobalide (0.1 microM) was able to increase the viability of cultured neurons from chick embryo telencepalon when exposed to cyanide (1 mM, 1h). Furthermore, we attempted to find out whether ginkgolides A, B, and J and bilobalide were also able to inhibit neuronal apoptosis (determined by nuclear staining with Hoechst 33 258 and TUNEL-staining). Ginkgolide B (10 microM), ginkgolide J (100 microM) and bilobalide (1 microM) reduced the apoptotic damage induced by serum deprivation (24h) or treatment with staurosporine (200 nM, 24h) in cultured chick embryonic neurons. Bilobalide (100 microM) rescued cultured rat hippocampal neurons from apoptosis caused by serum deprivation (24h), whereas Ginkgolide B (100 microM) and bilobalide (100 microM) reduced apoptotic damage induced by staurosporine (300 nM, 24h). Ginkgolide A failed to affect apoptotic damage neither in serum-deprived nor in staurosporine-treated neurons. The results suggest that some of the constituents of the non-flavone fraction of EGb 761 possess neuroprotective and anti-apoptotic capacity, and that bilobalide is the most potent one. In contrast, ginkgolic acids (100-500 microM) induced neuronal death, which showed features of apoptosis as well as of necrosis, but these constituents were removed from EGb 761 below an amount of 0.0005 %. Taking together, there is experimental evidence for a neuroprotective effect of EGb 761 that agrees with clinical studies showing the efficacy of an oral treatment in patients with mild and moderate dementia [5].

MDCK cyst model.[2]
Type I MDCK cells (ATCC no. CCL-34) were cultured in an atmosphere of 5% CO2-95% air at 37°C in a 1:1 mixture of DMEM and Ham's F-12 nutrient medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. For cyst generation, 400 MDCK cells were suspended in 0.4 ml of ice-cold MEM containing 2.9 mg/ml collagen, 10 mM HEPES, 27 mM NaHCO3, 100 U/ml penicillin, and 100 μg/ml streptomycin (pH 7.4). The cell suspension was plated onto 24-well plates. After incubation for ∼90 min at 37°C, collagen gel solutions were freeze-dried. Then, 1.5 ml of MDCK cell medium containing 10 μM forskolin was added to each well, and plates were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C.
To evaluate the inhibition of Ginkgolide B on cyst formation using the MDCK cyst model, 0.125, 0.5, and 2 μM Ginkgolide B were added, respectively, in the culture medium with 10% fetal bovine serum in the presence of 10 μM forskolin from day 0 to day 6. The medium containing forskolin and ginkgolide B was changed every 12 h. On day 6, cysts (with diameters >50 μm) and noncyst cell colonies were counted by phase-contrast light microscopy. To study the effect of ginkgolide B on MDCK cyst growth, the established cysts (on day 4 cultured with forskolin) were exposed to different concentrations of ginkgolide B in the presence of forskolin from day 4 to day 12. The medium with 10% fetal bovine serum containing forskolin and ginkgolide B was changed every 12 h for 8 days. Furthermore, to test the reversibility of the inhibition on cyst growth by ginkgolide B, the established cysts were exposed to forskolin and ginkgolide B from day 4 to day 8, and then ginkgolide B was removed from day 8 to day 12. Micrographs showing the same cysts in collagen gels (identified by markings on plates) were obtained every 2 days. Cyst diameters were measured using Image-Pro Plus 6.0 to determine the growth rate of cysts. At least 10 cysts/well and 3 wells/group were measured for each condition.

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Embryonic kidney cyst model.[2]
Mouse embryonic kidneys at embryonic day 13.5 (E13.5) were dissected and placed on transparent Falcon 0.4-μM diameter porous cell culture inserts as described previously. The lower chambers were filled with a 1:1 mixture of DMEM/Ham's F-12 nutrient medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 10 mM HEPES, 5 μg/ml insulin, 5 μg/ml transferrin, 2.8 nM selenium, 25 ng/ml prostaglandin E, 32 pg/ml T3, 250 U/ml penicillin, and 250 μg/ml streptomycin. Medium was replaced every 12 h. As indicated, 100 μM 8-Br-cAMP and Ginkgolide B were added. Kidneys were photographed using a Nikon inverted microscope (Nikon TE 2000-S) equipped with ×2 objective lens, 520-nm band-pass filter, and high-resolution PixeLINK color CCD camera. Cyst area was calculated by dividing the total cyst area by total kidney area.

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MDCK tubule model.[2]
To determine whether Ginkgolide B promotes MDCK cell to form tubules, MDCK cells were cultured in 3T3 conditioned medium (3T3 CM) with increasing doses of ginkgolide B (at 0.125, 0.5, or 2 μM) for 12 days. The medium containing ginkgolide B was changed every 12 h. On day 12, the numbers of cells forming tubule-like structures were counted at 20–35 random sites in each culture dish using phase-contrast light microscopy. Furthermore, to examine whether ginkgolide B induces tubulogenesis from MDCK cysts formed within three-dimensional collagen gels, the cysts were established by exposing them to forskolin for 4 days as mentioned above. Then, the medium was replaced with fresh 3T3 CM with or without different concentrations of ginkgolide B over the next 8 days. Tubules were monitored and photographed every 2 days. On day 12, the length of the longest tubule from each cyst was measured by Image-Pro Plus 6.0.

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Cytotoxicity and apoptosis.[2]
An MTT assay was used to assess Ginkgolide B cytotoxicity. MDCK cells in a 96-well plate were exposed to ginkgolide B at 0, 0.1, 1, 10, or 100 μM for 24 h. Twenty microliters of MTT solution (5 mg/ml) was added and incubated for 4 h at 37°C. The medium was then removed, and 150 μl DMSO was added. The absorbance at 490 nm was measured. Cell viability was shown as the OD490 value.

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The in situ cell death detection kit (Roche Diagnostics, Indianapolis, IN) was used to measure apoptosis. MDCK cells were seeded on eight-chamber polystyrene tissue culture-treated glass slides and incubated with Ginkgolide B at 0.125, 0.5, or 2 μM. Gentamycin (2 mM) was used as a positive control. Three days later, the assay was performed according to the manufacturer's instructions. Five microscopic fields were analyzed per condition. The apoptotic index was calculated as follows: apoptotic index (%) = (apoptotic cell number/total cell number) × 100.

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Cell proliferation.[2]
Cell Counting Kit-8 (CCK-8) was used to assay the proliferation of MDCK cells incubated with or without Ginkgolide B in the absence and presence of 10 μM forskolin at indicated time points (12, 24, 36, 48, 72, and 96 h). MDCK cells (1,000 cells·100 μl−1·well−1) in a 96-well plate were cultured in a 37°C humidified 5% CO2 incubator. Each well was used with 10 μl CCK-8 solution and was incubated for 1 h. The absorbance at 450 nm was measured. The cell proliferation rate was expressed as the OD450 value.

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cAMP measurement.[2]
MDCK cells cultured in six-well plates were exposed to Ginkgolide B (at 0.125, 0.5, or 2 μM) for 30 min with or without 10 μM forskolin stimulation. Intracellular cAMP content was determined by RIA following the procedure recommended in the cAMP RIA kit.

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CFTR function assay.[2]
Fischer rat thyroid (FRT) epithelial cells stably coexpressing human CFTR and the high-sensitivity I−-sensing green fluorescent protein YFP-H148Q/I152L were used as described previously. At the time of the assay, cells were washed with PBS and then incubated with PBS containing forskolin (20 μM) and Ginkgolide B (at 0.01, 0.1, 1, or 10 μM) for 20 min. Measurements were carried out using FLUOstar fluorescence plate readers, equipped with 500 ± 10-nm excitation and 535 ± 15-nm emission filters. Each well was assayed individually for I− influx by recording fluorescence continuously (200 ms/point) for 2 s (baseline) and then for 12 s after rapid (<1 s) addition of 165 μl PBS in which 137 mM Cl− was replaced by I−. The I− influx rate was computed by fitting the final 11.5 s of the data to an exponential for extrapolation of initial slope and normalizing for background-subtracted initial fluorescence. All experiments contained negative control (DMSO vehicle) and positive control CFTRinh172.

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Short-circuit current measurements.[2]
MDCK cells in Snapwell inserts (transepithelial resistance 1,000–2,000 Ω) were cultured in media containing Ginkgolide B (at 0.1, 1, or 10 μM) for 1 or 48 h. Ginkgolide B was washed out with medium 1 h before short-circuit current measurements. Snapwell inserts containing MDCK cells were mounted on a standard Ussing chamber system. Permeabilization reagent (250 μg/ml amphotericin B) was added on the basolateral membrane of the insert. The hemichambers were filled with 5 ml of 65 mM NaCl, 65 mM Na-gluconate, 2.7 mM KCl, 1.5 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, Na-HEPES, and 10 mM glucose (apical), and 130 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, Na-HEPES, and 10 mM glucose (basolateral) (pH 7.3). Short-circuit current was recorded continuously using a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, FL) with Ag/AgCl electrodes and 1 M KCl agar bridges.

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Western blotting.[2]
MDCK cells were seeded in 6-well plates in a 1:1 mixture of DMEM and Ham's F-12 nutrient medium containing 10% FBS for 2 h, followed by serum starvation for 24 h. Then, MDCK cells were exposed to medium containing Ginkgolide B (at 0.125, 0.5, or 2 μM) for 1 h with or without 10 μM forskolin stimulation. Western blot analysis was performed as described in the manufacturer's instructions.

Pkd1/Ksp-Cre mouse model of ADPKD. [2]
Pkd1flox mice and Ksp-Cre transgenic mice in a C57BL/6 background were generated as described previously. Ksp-Cre mice express Cre recombinase under the control of the Ksp-cadherin promoter. Pkd1−/−;Ksp-Cre mice were generated by cross-breeding Pkd1flox/flox mice with Pkd1+/−:Ksp-Cre mice. Neonatal mice (age 1 day) were genotyped by genomic PCR. Ginkgolide B (16 mg·kg−1·day−1) or a saline DMSO vehicle control (0.05 ml/injection) was administrated by subcutaneous injection on the back of neonatal mice two times a day using a 1-ml insulin syringe, beginning at age 1 day (5 mice/group). Pkd1flox/+;Ksp-Cre or Pkd1flox/− mice from the same litter were used as wild-type. Body weight was measured at the age of 4 days. Kidneys were removed and weighed and fixed for histological examination. Protocols were approved by the Peking University Health Center Committee on Animal Research.

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Dissolved in PBS; 0.6 mg/day; Intragastric administration

Eight-week-old male ApoE / mice

[1]. Biochem Pharmacol . 1987 Sep 1;36(17):2749-52.

[2]. Am J Physiol Renal Physiol . 2012 May 15;302(10):F1234-42.

[3]. Eur Cell Mater . 2011 May 28:21:459-69.

[4]. J Cereb Blood Flow Metab . 1990 Jan;10(1):133-5.

[5]. Pharmacopsychiatry . 2003 Jun:36 Suppl 1:S8-14.

[6]. Eur J Pharmacol . 1986 Aug 7;127(1-2):83-95.

[7]. J Neurochem . 1988 Dec;51(6):1900-5.

[8]. Eur J Pharm Sci . 2012 Nov 20;47(4):652-60.

[9]. PLoS One . 2012;7(5):e36237.

Ginkgolide B has been reported in Ginkgo and Ginkgo biloba with data available.
See also: Ginkgolide B (annotation moved to).

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In summary, our study demonstrated for the first time that ginkgolide B significantly inhibited cyst development. Ginkgolide B induced no apparent toxicity and apoptosis, but inhibited forskolin-induced cyst epithelial cell proliferation and promoted epithelial cell differentiation. The cyst-inhibitory activity of ginkgolide B may be mediated through the Ras/MAPK signaling pathway. Further evaluation is needed for ginkgolide B as a novel promising candidate drug for ADPKD.[1] Ginkgolide B does not affect chloride transporter CFTR function.[2]
To determine the effect of ginkgolide B on CFTR function, CFTR activity in FRT cells expressing CFTR was determined by an I−-sensitive fluorescence assay and in MDCK cells by an Ussing chamber assay. There was no difference in CFTR-mediated I− secretion between ginkgolide B-treated and untreated FRT cells (Fig. 7A). Figure 7B shows that ginkgolide B did not reduce the short-circuit current in MDCK cells with forskolin stimulation. Ginkgolide B did not alter CFTR function as seen by short-circuit current in MDCK cells after 1- vs. 48-h incubation with 0.1, 1, or 10 μM ginkgolide B followed by washout. The representative curves are showed in Fig. 7C.

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Ginkgolide B does not affect intracellular cAMP content.[2]
Figure 8 shows the intracellular cAMP levels in MDCK cells incubated with forskolin in the presence or absence of ginkgolide B (at 0.125, 0.5, or 2 μM). Intracellular cAMP content was significantly increased in MDCK cells by forskolin. There was no difference in intracellular cAMP concentration between ginkgolide B-treated and untreated MDCK cells with forskolin stimulation.

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Ginkgolide B regulates intracellular signaling pathways.[2]
To study the mechanism by which ginkgolide B inhibits cysts, the signaling proteins involved in the Ras/MAPK pathway were analyzed by Western blotting. The highest p-ERK expression levels were found in serum-starved MDCK cells exposed to 10 μM forskolin for 60 min (data not shown). Based on this time course, serum-starved MDCK cells were treated with ginkgolide B at 0.125, 0.5, and 2 μM for 60 min in the presence of 10 μM forskolin. Ginkgolide B significantly decreased levels of Ras, B-raf, p-MEK, and p-ERK but increased Raf-1 levels in MDCK cells with forskolin stimulation. Ginkgolide B did not affect the expression of Egr-1. We also examined the effect of ginkgolide B on CREB phosphorylation in MDCK cells cotreated with 10 μM forskolin for 60 min. Forskolin-mediated CREB activation was significantly inhibited by 0.5 and 2 μM ginkgolide B, whereas total CREB content was not affected (Fig. 9).

C20H24O10

424.4

424.136

C, 56.60; H, 5.70; O, 37.70

15291-77-7

65243

White to off-white solid powder

1.6±0.1 g/cm3

762.4±60.0 °C at 760 mmHg

280°C (dec.)

274.3±26.4 °C

0.0±5.8 mmHg at 25°C

1.651

0.52

3

10

1

30

925

0

O[C@H]1[C@]2([H])OC([C@@H](C)[C@]2(O)C23C(=O)O[C@@]4([H])C12C1([C@H](C(O[C@@]1([H])O3)=O)O)[C@@H](C4)C(C)(C)C)=O

SQOJOAFXDQDRGF-UHFFFAOYSA-N

InChI=1S/C20H24O10/c1-6-12(23)28-11-9(21)18-8-5-7(16(2,3)4)17(18)10(22)13(24)29-15(17)30-20(18,14(25)27-8)19(6,11)26/h6-11,15,21-22,26H,5H2,1-4H3

8-tert-butyl-6,12,17-trihydroxy-16-methyl-2,4,14,19-tetraoxahexacyclo[8.7.2.01,11.03,7.07,11.013,17]nonadecane-5,15,18-trione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.89 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.90 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.90 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 10mg/mL

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Solubility in Formulation 5: 5 mg/mL (11.78 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 44°C.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)