Natural sesquiterpene trilactone from Ginkgo biloba; Endogenous Metabolite

Bilobalide (1-100 µM) completely suppresses the NMDA-evoked release of choline in a concentration-dependent manner with IC50 value of 2.3 µM[1].
Bilobalide (1, 5 and 10 μM) alone for 24 h does not affect cell viability of SH-SY5Y cells. The best protective effect was obtained at 10 μM[2], and pre-treating cells with bilobalide prevents Aβ 1-42, H2O2, and serum deprivation-induced decreases in cell viability.
Bilobalide (5 and 10 μM; 24 h) treatment dose-dependently raises levels of p-Akt (Ser473 and Thr308) in SH-SY5Y cells[2].

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Bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, has been proposed to exert protective and trophic effects on neurons. However, mechanisms underlying the protective effects of bilobalide remain unclear. Using human SH-SY5Y neuroblastoma cells and primary hippocampal neurons, this study investigated the neuroprotective effects of bilobalide. We mimicked aging-associated neuronal impairments by applying external factors (beta amyloid protein (Abeta) 1-42, H(2)O(2) and serum deprivation) consequently inducing cell apoptosis. As markers for apoptosis, cell viability, DNA fragmentation, mitochondrial membrane potential and levels of cleaved caspase 3 were measured. We found that, bilobalide prevented Abeta 1-42-, H(2)O(2)- and serum deprivation-induced apoptosis. To better understand the neuroprotective effects of Bilobalide, we also tested the ability of bilobalide to modulate pro-survival signaling pathways such as protein kinase C (PKC), extracellular-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. It was found that, bilobalide dose-dependently increased PI3K activity and levels of phosphorylated Akt (p-Akt Ser473 and Thr308), which could be maintained up to at least 2 h after bilobalide withdrawal in cells treated with or without Abeta 1-42, H(2)O(2) or serum-free medium. In addition, application of PI3K/Akt inhibitor LY294002 could abrogate both the protective effects of Bilobalide against Abeta 1-42-, H(2)O(2)- and serum deprivation-induced apoptotic cell damage and Bilobalide-induced increase in PI3K activity and levels of p-Akt (Ser473 and Thr308). In contrast, application of PKC inhibitor staurosporine (STS) did not affect the protective effects of bilobalide. Moreover, no change in levels of phosphorylated ERK1/2 (p-ERK1/2) was observed in bilobalide-treated cells. These results further suggested that the PI3K/Akt pathway might be involved in the protective effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration [2].

Bilobalide (20 mg/kg) completely suppresses the NMDA-induced release of choline in vivo while basal choline levels were not significantly affected. When NMDA is retrograde dialyzed into the hippocampus of freely moving rats, this results in the release of choline in vivo. The effect caused by NMDA is completely inhibited by Bilobalide (20 mg/kg intravenously)[1].

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In rat hippocampal slices superfused with magnesium-free buffer, glutamate (1 mM) caused the release of large amounts of choline due to phospholipid breakdown. This phenomenon was mimicked by N-methyl-D-aspartate (NMDA) in a calcium-sensitive manner and was blocked by NMDA receptor antagonists such as MK-801 and 7-chlorokynurenate. The NMDA-induced release of choline was not caused by activation of phospholipase D but was mediated by phospholipase A2 (PLA2) activation as the release of choline was accompanied by the formation of lyso-phosphatidylcholine (lyso-PC) and glycerophospho-choline (GPCh) and was blocked by 5-[2-(2-carboxyethyl)-4-dodecanoyl-3,5-dimethylpyrrol-1-yl]pentano ic acid, a PLA2 inhibitor. Bilobalide, a constituent of Ginkgo biloba, inhibited the NMDA-induced efflux of choline with an IC50 value of 2.3 microM and also prevented the formation of lyso-PC and GPCh. NMDA also caused a release of choline in vivo when infused into the hippocampus of freely moving rats by retrograde dialysis. Again, the effect was completely inhibited by Bilobalide which was administered systemically (20 mg/kg i.p.). Interestingly, convulsions which were observed in the NMDA-treated rats were almost totally suppressed by Bilobalide. We conclude that release of choline is a sensitive marker for NMDA-induced phospholipase A2 activation and phospholipid breakdown. Bilobalide inhibited the glutamatergic excitotoxic membrane breakdown both in vitro and in vivo, an effect which may be beneficial in the treatment of brain hypoxia and/or neuronal hyperactivity [1].

Cells (5–6 × 106) were treated with the indicated concentrations of Bilobalide for 24 h. The medium containing Bilobalide was then aspirated off. After washed twice with phosphate-buffered saline (PBS), cells were challenged with or without Aβ 1-42 (100 μg/ml), H2O2 (500 μM) or serum deprivation for the indicated periods of time. In some cases, to test if the protective effects of Bilobalide were associated with modulation of intracellular pro-survival pathways such as PKC and PI3K/Akt pathways, LY294002 (10 μM) or STS (500 nM) was also added to the medium 15 min before the addition of Bilobalide. The medium containing LY294002 was aspirated off 24 h after the addition of bilobalide while the medium containing STS was aspirated off immediately before the addition of bilobalide. In all experiments, an equivalent volume of vehicle was added to control cultures.

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MTT assay [2]
Cell viability was determined by a colourimetric 3-4,5-dimethyithiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay according to Kobayashi et al. Briefly, cells were plated into 96-well plates. After treated as described above, 10 μl 5 mg/ml MTT solution was added to each well of the 96-well plates and incubated for 4 h at 37°C. Then, the medium containing MTT was aspirated off and 200 μl of DMSO was added to each well. With DMSO as the blank, the absorbance was then read with the Victor-2 Multilabel counter at a wavelength of 490 nm.

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TUNEL apoptosis assay [2]
Oligonucleosomal DNA fragmentation in apoptotic cells was measured using the Cell Death Detection ELISAplus Kit (TUNEL Apoptosis Assay Kit) according to the manufacturer’s protocol. Cells were treated as previously described prior to ELISA detection, and spectrophotometric data were obtained using the same Victor-2 Multilabel counter at 405 nm against a reference wavelength of 490 nm.

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Flow cytometric analysis of mitochondrial membrane potential using JC-1 [2]
Mitochondrial membrane potential was analyzed using an aggregate-forming lipohilic dye 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanineiodide (JC-1). JC-1 was stocked as 1 mg/ml DMSO solution and freshly diluted with culture medium. Cells in different treatments were loaded with JC-1 (at the final concentration of 5 μg/ml) at 37°C for 20 min. After that, cells were collected by centrifugation at 400×g for 3 min. The pellets were re-suspended in 0.5 ml PBS. Flow cytometry analysis was then performed within 10 min by using a flow cytometer. Green and red fluorescence were analyzed on the PMT2 (525 nm BP) and PMT3 (575 nm BP) channels, respectively. The percentages of red and green fluorescence were quantified by Expo32TM software and mitochondrial membrane potential was expressed as red/green fluorescence ratio.

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Western blot assay of signaling proteins [2]
The expression levels of cleaved caspase 3, p-Akt (Ser473 and Thr308), total Akt, p-ERK1/2 (Thr202/Tyr204) and total ERK1/2 were examined by Western blot analysis. After treated as previously described, cells were trypsinized and collected by centrifugation at 400×g for 3 min. The pellets were lysed in lysis buffer (50 nM Tris–HCl, pH 7.4, 150 nM sodium chloride, 5 nM ethylene dinitrilotetra-acetic acid, 0.1% sodium dodecyl sulfate, 1% TritonX-100, 0.1 mg/ml Phenylmethylsulfonyl, 5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin), and the protein concentration was determined with a DC protein assay kit.

Microdialysis experiment. [2]
I-shaped, concentric probes were manufactured according to Santiago and Westerink (1990). The probes had an outside diameter of 0.24 mm and an exchange length of 4 mm and were equipped with a dialysis membrane with a molecular weight cutoff of 10,000 Da. For probe implantation, the rats were anaesthesised with pentobarbital (60–80 mg/kg i.p.) and placed in a stereotactic frame. The probe was implanted into the right ventral hippocampus using the following coordinates: AP +2.5 mm; L –5.8 mm; DV –7.5 mm (Paxinos and Watson 1986). The experiments were carried out on freely moving animals on the first and second day following surgery. The microdialysis probes were perfused at a constant rate of 2.0 ml/min with a low-magnesium artificial cerebrospinal fluid (concentrations in mM: NaCl 147; KCl 4; CaCl2 1.2; MgCl2 0.12) containing 10 mM neostigmine. After four samples had been taken to estimate the basal efflux of choline, the perfusion fluid was changed to a solution containing 1 mM NMDA. In some experiments, the rats were pretreated by i.p. injection with Bilobalide (20 mg/kg) 1 h prior to the start of NMDA infusion. Aliquots of the dialysis fluid were collected in 15-min intervals and analyzed for choline by HPLC.

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10-30 mg/kg; oral

Rats

[1]. Bilobalide, a constituent of Ginkgo biloba, inhibits NMDA-induced phospholipase A2 activation and phospholipid breakdown in rat hippocampus. Naunyn Schmiedebergs Arch Pharmacol. 1999 Dec;360(6):609-15.

[2]. Bilobalide prevents apoptosis through activation of the PI3K/Akt pathway in SH-SY5Y cells. Apoptosis. 2010 Jun;15(6):715-27.

Bilobalide is a terpenoid trilactone found in extracts of Ginkgo biloba.
Bilobalide has been reported in Ginkgo biloba with data available.
See also: Ginkgo (part of).

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In conclusion, our results demonstrate that bilobalide, a sesquiterpene lactone isolated from Ginkgo leaves, potently inhibits the NMDA-induced, PLA2-dependent release of choline from hippocampal phospholipids both in vitro and in vivo. This effect likely contributes to the apparent therapeutic potential of Ginkgo biloba extracts in neurodegenerative disorders.[1]

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In summary, this study mimicked aging-associated neuronal impairments by using external factors (Aβ 1-42, H2O2 and serum deprivation) consequently inducing cell apoptosis. The protective effects of bilobalide against cell apoptosis and the ability of bilobalide to modulate pro-survival signaling pathways such as PKC, ERK1/2 and PI3K/Akt pathways were investigated. We found that, bilobalide prevented Aβ 1-42-, H2O2- and serum deprivation-induced decrease of cell viability, DNA fragmentation, mitochondrial membrane potential depolarization, and cleavage of caspase 3. Further, we suggested that the PI3K/Akt pathway might be involved in the anti-apoptotic effects of bilobalide. Since modern technology allows production of purified bilobalide with high bioavailability, these results suggest that bilobalide may be useful in developing therapy for diseases involving age-associated neurodegeneration. [2]

C15H18O8

326.3

326.1

C, 55.21; H, 5.56; O, 39.23

33570-04-6

73581

White to off-white solid powder

1.6±0.1 g/cm3

651.7±55.0 °C at 760 mmHg

247.5±25.0 °C

0.0±4.4 mmHg at 25°C

1.606

-0.45

2

8

1

23

650

6

O=C(O1)C[C@@]2([C@]1([H])C[C@]3(C(C)(C)C)O)[C@@]34[C@](OC([C@@H]4O)=O)([H])OC2=O

MOLPUWBMSBJXER-YDGSQGCISA-N

InChI=1S/C15H18O8/c1-12(2,3)14(20)4-6-13(5-7(16)21-6)10(19)23-11-15(13,14)8(17)9(18)22-11/h6,8,11,17,20H,4-5H2,1-3H3/t6-,8-,11-,13-,14+,15+/m0/s1

(1S,4R,7R,8S,9R,11S)-9-tert-butyl-7,9-dihydroxy-3,5,12-trioxatetracyclo[6.6.0.01,11.04,8]tetradecane-2,6,13-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: ≥ 2.08 mg/mL (6.37 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 2: ≥ 2.08 mg/mL (6.37 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 20.8 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: ≥ 2.08 mg/mL (6.37 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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 (Please use freshly prepared in vivo formulations for optimal results.)