AMPK; MMP-9; Sirtuin

After a 24-hour treatment, 3T3-L1 cells' adipogenesis was inhibited by ginkgolide C (3-100 μM), but its viability was unaffected. When compared to the control group, ginkgolide C (10-100 μM) dramatically reduced lipid accumulation and increased glycerol release in 3T3-L1 adipocytes. In differentiated 3T3-L1 adipocytes, ginkgolide C inhibits the expression of PPAR-α and PPAR-γ and decreases the expression of C/EBPα, C/EBPβ, and SREBP-1c. Furthermore, in differentiated 3T3-L1 adipocytes, Ginkgolide C (3-100 μM) dose-dependently inhibits the expression of mRNA and adipogenesis-related proteins (FAS, LPL, and aP2). Additionally, in a concentration-dependent manner, ginkgolide C (3-100 μM) increases the phosphorylation of AMPKα and ACC-1 and significantly promotes the production of Sirt1 [1]. In a dose-dependent manner, ginkgolide C (1, 10, 50, 100, and 500 mM) dramatically inhibited the rat platelet aggregation induced by collagen (10 mg/mL). In collagen-stimulated platelets, ginkgolide C (50, 100 mM) induces the formation of activated MMP-9 (86 kDa) from MMP-9 precursor (92 kDa) [2].

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Ginkgolide C, isolated from Ginkgo biloba leaves, is a diterpene lactone derivative [corrected] reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK), resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C), ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway.[1]

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In this report, we investigated the effect of Ginkgolide C (GC) from Ginkgo biloba leaves in collagen (10 mug/ml)-stimulated platelet aggregation. It has been known that matrix metalloproteinase-9 (MMP-9) is released from human platelets, and that it significantly inhibited platelet aggregation stimulated by collagen. Zymographic analysis confirmed that pro-MMP-9 (92-kDa) was activated by GC to form an activated MMP-9 (86-kDa) on gelatinolytic activities. And then, GC dose-dependently inhibited platelet aggregation, intracellular Ca(2+) mobilization, and thromboxane A(2) (TXA(2)) formation in collagen-stimulated platelets. In addition, GC significantly increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. Therefore, we demonstrate that the inhibitory effect of GC on platelet aggregation might be involved into the following pathways. GC may increase intracellular cAMP and cGMP production and MMP-9 activity, inhibit intracellular Ca(2+) mobilization and TXA(2) production, thereby leading to inhibition of platelet aggregation. These results strongly indicate that GC is a potent inhibitor of collagen-stimulated platelet aggregation. It may be a suitable tool for a negative regulator during platelet activation [2].

GGC/Ginkgolide C exerts anti-tumour effects in preclinical model [3]
We examined the impact of GGC/Ginkgolide C to modulate tumour growth in xenograft model as per the protocol specified in Figure 5A. The results suggested that in control group I, tumour volume sharply increased but markedly decreased in groups II and III (Figure 5C). Moreover, tumour size and weight were attenuated in groups II and III as compared with control (Figure 5B and D), without altering the body weight (Figure 5E).

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GGC/Ginkgolide C alters levels of various oncogenic markers [3]
First as measured by immunohistochemical analysis, it found that GGC/Ginkgolide C treatment caused a marked down-regulation in the expression of p-STAT3, Ki-67 and VEGF proteins (Figure 6A). Thereafter, as shown in Figure 6B, GGC increased PTPε protein level but decreased the levels of phopho-STAT3 and different phosphorylated kinases as measured by western blotting. Next, cleaved caspase-3 and PARP levels were significantly elevated in tissues harvested from GCC exposed mice (Figure 6D). Finally, GGC reduced the expression of diverse oncogenic proteins that can regulate tumourigenesis in the NSCLC model (Figure 5E).

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In this study, we investigated whether Ginkgolide C (GC), a potent anti-inflammatory flavone, extenuated MI/R injury through inhibition of inflammation. In vivo, rats with the occlusion of the left anterior descending (LAD) coronary artery were applied to mimic MI/R injury. In vitro, primary cultured neonatal ventricular myocytes exposed to hypoxia/reoxygenation (H/R) were applied to further discuss the anti-H/R injury property of Ginkgolide C/GC. The results revealed that GC significantly improved the symptoms of MI/R injury, as evidenced by reducing infarct size, preventing myofibrillar degeneration and reversing the mitochondria dysfunction. Moreover, histological analysis and Myeloperoxidase (MPO) activity measurement showed that GC remarkably suppressed Polymorphonuclears (PMNs) infiltration and ameliorated the histopathological damage. Furthermore, GC pretreatment was shown to improve H/R-induced ventricular myocytes viability and enhance tolerance of inflammatory insult, as evidenced by suppressing expression of CD40, translocation of NF-κB p65 subunit, phosphorylation of IκB-α, as well as the activity of IKK-β. In addition, downstream inflammatory cytokines modulated by NF-κB signaling were effectively down-regulated both in vivo and in vitro, as determined by immunohistochemistry and ELISA. In conclusion, these results indicate that GC possesses a beneficial effect against MI/R injury via inflammation inhibition that may involve suppression of CD40-NF-κB signal pathway and downstream inflammatory cytokines expression, which may offer an alternative medication for MI/R diseases [4].

Cell Viability Assay [1]
3T3-L1 cells were treated with various concentrations of Ginkgolide C in 96-well plates for 24 h. Cell viability was analyzed by the MTT assay as previously described. The culture medium was removed, and the cells were incubated with 100 μL MTT solution (5 mg/mL) for 4 h at 37°C. After plates were washed, isopropanol was added to dissolve formazone crystals, followed by absorbance detection with a spectrophotometer at 570 nm.

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Adipocyte Differentiation [1]
3T3-L1 cells (104/mL) were seeded in 6-well plates and adipocyte differentiation was induced as previously described. Briefly, 3T3-L1 cells were cultured in DMEM containing 10% fetal bovine serum and stimulated with 1 μM dexamethasone, 0.5 mM 1-isobutyl-3-methylxanthine, and 10 μg/mL insulin for 2 days. Two days later, DMEM supplemented with 10 μg/mL insulin was used as differentiation medium for 2 days, with changes every 2 days. On day 8, the differentiated adipocytes were treated with Ginkgolide C (3–100 μM).

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Oil Red O Staining [1]
Differentiated adipocytes were treated with Ginkgolide C on a 3 cm plate for 24 h, and cells were fixed with 10% formalin for 30 min. Next, cells were washed with phosphate-buffered saline and stained with oil red O for 1 h, followed by observation of the oil droplets under microscopy. Plates were washed and treated with isopropanol and the lipid accumulation was quantified by measuring absorbance at an optical density of 490 nm with a microplate reader.

Experimental protocol [3]
A549 xenograft model was established, and athymic nu/nu female mice were randomized into the following four different treatment groups (n = 6/group). Tumour volumes were measured by Digimatic calliper every 5 days, and mice body weight was measured at about 2- or 3-day intervals. Mice were killed 5 days later after the last therapy and tumour tissues were further processed as described before.

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Western blot analysis of tumour tissues [3]
Lung tumour tissues from control and experimental mice were minced and lysis in T-PER Tissue Protein Extraction Reagent. Equal amount of lysates resolved in a 10–15% SDS-polyacrylamide gel and electrotransferred to nitrocellulose membranes. Then, the membranes were blotted with various indicated antibodies.

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Immunohistochemical study [3]
Solid tumours from all experimental mice were fixed with 10% neutral buffered formalin, processed and embedded in paraffin. Sections were cut and deparaffinized in xylene and dehydrated in graded alcohol and finally hydrated in water. Antigen retrieval was performed by boiling the slide in 10 mM sodium citrate (pH 6.0) for 20 min. Immunohistochemistry was performed following the manufacturer’s instructions. Briefly, endogenous peroxidases were quenched with 3% hydrogen peroxide. Non-specific binding was blocked by incubation in the blocking reagent in the ImmPRESS Reagent Kit according to the manufacturer’s instructions. Sections were incubated overnight with primary antibodies: p-STAT3, anti-Ki-67 and anti-VEGF (at 1 : 100 dilutions). Slides were subsequently washed several times in 1× PBS and were incubated with ImmPRESS reagent according to the manufacturer’s instructions. Immunoreactive species were detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB) as a substrate. Sections were counterstained with Gill’s haematoxylin and mounted under glass coverslips. Images were taken using a Nikon ECLIPSE Ts2 (magnification, ×20). Positive cells (brown) were quantitated using the iSolution Lite x64.

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In Vivo I/R Procedure to Induce MI/R Injury in Rats [4]
MI/R surgery was precisely implemented according to the procedure in Figure 2A. The rats were anesthetized with 3% sodium pentobarbital (40 mg/kg, i.p.) and mechanically ventilated the lung with a rodent respirator. Holter monitoring electrocardiogram was continuously used to monitor the changes of S-T segment in order to estimate the success of surgery. After a left thoracotomy, a suture tied with a plastic tube was twined round the LAD coronary artery to form a snare for reversible LAD occlusion. According to procedure, transient regional myocardial ischemia for 40 min was realized by straining the suture, and reperfusion for 120 min was initiated by releasing the suture and removing the tension. The blood samples were collected before the rats were sacrificed.
Rats were randomly divided into 6 groups as follows (n = 8 per group): Control group, non-I/R rats received saline; I/R group, I/R rats received saline; Aspirin group, I/R rats received 16 mg/kg of Aspirin; 8, 16, 32 mg/kg Ginkgolide C/GC group, I/R rats received 8, 16, 32 mg/kg of Ginkgolide C/GC. Saline, Aspirin and Ginkgolide C/GC were administered intraperitoneally for 7 days before I/R procedure.[4]

[1]. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway. Evid Based Complement Alternat Med. 2015;2015:298635.

[2]. Ginkgolide C inhibits platelet aggregation in cAMP- and cGMP-dependent manner by activating MMP-9. Biol Pharm Bull. 2007 Dec;30(12):2340-4.

[3]. Abrogation of STAT3 activation cascade by Ginkgolide C mitigates tumourigenesis in lung cancer preclinical model. J Pharm Pharmacol. 2021 Dec 7;73(12):1630-1642.

[4]. Ginkgolide C Alleviates Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Inhibition of CD40-NF-κB Pathway. Front Pharmacol. 2018 Feb 21:9:109.

ginkgolide-C has been reported in Ginkgo biloba with data available.
See also: Ginkgo (part of).

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Overweight and obesity are not only the result of adipocyte proliferation but also the result of excess lipid accumulation in liver and adipocyte tissue. Obesity leads to metabolic syndrome and insulin resistance and causes type II diabetes, hypertension, and high cholesterol. We confirmed that ginkgolide C significantly inhibited transcription factors and enzymes associated with adipogenesis and promoted Sirt1/AMPK activity to increase lipolysis in differentiated adipocytes. We suggest that ginkgolide C holds potential for improving metabolic syndrome and insulin resistance.[1]

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In traditional Chinese medicine, G. biloba is widely used to treat cardiovascular disease, and G. biloba extract (Egb761) is also reported to have neuroprotective effects. Several compounds have been isolated, and ginkgolides A and B have multiple pharmacological activities, including improved platelet aggregation and neuroprotection against cerebral ischemia. In this study, we investigated the antiobesity effect of ginkgolide C in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes that were treated with ginkgolide C showed a reduced accumulation of droplets, a decrease in adipogenesis-related transcription factors, and downregulated expression of fatty acid synthesis enzymes. We also found that ginkgolide C promoted production of lipolysis-related enzymes and regulated AMPK pathway activity.[1]

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Objectives: Ginkgolide C (GGC) isolated from Ginkgo biloba (Ginkgoaceae) leaf can demonstrate pleiotropic pharmacological actions. However, its anti-oncogenic impact in non-small cell lung cancer (NSCLC) model has not been reconnoitered. As signal transducer and activator of transcription 3 (STAT3) cascade can promote tumour growth and survival, we contemplated that GGC may interrupt this signalling cascade to expend its anti-cancer actions in NSCLC.
Methods: The effect of GGC on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation and apoptosis was examined. The in-vivo effect of GGC on the growth of human NSCLC xenograft tumours in athymic nu/nu female mice was also investigated.
Key findings: GGC attenuated the phosphorylation of STAT3 and STAT3 upstream kinases effectively. Exposure to pervanadate modulated GGC-induced down-regulation of STAT3 activation and promoted an elevation in the level of PTPε protein. Indeed, silencing of the PTPε gene reversed the GGC-promoted abrogation of STAT3 activation and apoptosis. Moreover, GGC exposure significantly reduced NSCLC tumour growth without demonstrating significant adverse effects via decreasing levels of p-STAT3 in mice tissues.
Conclusions: Overall, the findings support that GGC may exhibit anti-neoplastic actions by mitigation of STAT3 signalling cascade in NSCLC.[3]

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GC can exhibit significant cardioprotective effects through reducing infarct size, inhibiting inflammatory response, improving myocardial histological structure and alleviating PMNs infiltration during I/R injury. Inhibition of excessive inflammation via suppressing CD40/NF-κB signal pathway should be the key mechanism of GC in the protective of MI/R injury. Thus, GC will be a prospective and preventive agent in the management of MI/R injury. [4]

C20H24O11

440.4

440.131

C, 54.55; H, 5.49; O, 39.96

15291-76-6

15291-76-6

9867869

White to off-white solid powder

1.7±0.1 g/cm3

813.8±65.0 °C at 760 mmHg

>3000ºC

291.4±27.8 °C

0.0±6.6 mmHg at 25°C

1.671

0.16

4

11

1

31

957

12

C[C@@H]1C(=O)O[C@@H]2[C@]1([C@@]34C(=O)O[C@H]5[C@]3([C@H]2O)[C@@]6([C@@H]([C@H]5O)C(C)(C)C)[C@H](C(=O)O[C@H]6O4)O)O

AMOGMTLMADGEOQ-DTDWCABLSA-N

InChI=1S/C20H24O11/c1-5-12(24)28-11-8(22)18-10-6(21)7(16(2,3)4)17(18)9(23)13(25)30-15(17)31-20(18,14(26)29-10)19(5,11)27/h5-11,15,21-23,27H,1-4H3/t5-,6-,7+,8+,9+,10-,11+,15+,17+,18-,19-,20-/m1/s1; SMILES: C[C@@H]1C(O[C@H]2[C@@H]([C@]34[C@@H]5OC([C@@]3([C@@]12O)O[C@@H]6OC([C@@H]([C@]46[C@H](C(C)(C)C)[C@H]5O)O)=O)=O)O)=O

Ginkgolide A, 1,7-dihydroxy-, (1alpha,7beta)-

BN-52022; BN52022; BN-52,022; UNII-32ZQ957R4A; 15291-76-6; 32ZQ957R4A; 1alpha,7beta-Dihydroxyginkgolide A; BN 52,022; Ginkgolide A, 1,7-dihydroxy-, (1alpha,7beta)-; BN 52022; Ginkgolide C

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)

DMSO : ~250 mg/mL (~567.67 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.68 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 25.0 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 2: ≥ 2.5 mg/mL (5.68 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 3: ≥ 2.5 mg/mL (5.68 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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