Natural product; Endogenous metabolite

To our knowledge, the present investigation describes the first ginkgolide, 4, having the general structure of naturally occurring ginkgolides, and which is not a PAF inhibitor at pharmacological-relevant concentrations. Using the standard test to determine PAF antagonism properties, the response yielded by 4 was at the noise level, whereas Ginkgolide J, a minor constituent of EGb 761, has an IC50 range of 12–54 µM [2].

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A new Ginkgo biloba extract P8A (TTL), 70% enriched with terpene trilactones, prevents Aβ1-42 induced inhibition of long-term potentiation in the CA1 region of mouse hippocampal slices. This neuroprotective effect is attributed in large part to Ginkgolide J that completely replicates the effect of the extract. Ginkgolide J is also capable of inhibiting cell death of rodent hippocampal neurons caused by Aβ1-42. This beneficial and multi-faceted mode of action of the ginkgolide makes it a new and promising lead in designing therapies against Alzheimer’s disease [2].

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A new Ginkgo biloba extract P8A (TTL), 70% enriched with terpene trilactones, prevents A beta(1-42) induced inhibition of long-term potentiation in the CA1 region of mouse hippocampal slices. This neuroprotective effect is attributed in large part to Ginkgolide J that completely replicates the effect of the extract. Ginkgolide J is also capable of inhibiting cell death of rodent hippocampal neurons caused by A beta(1-42). This beneficial and multi-faceted mode of action of the ginkgolide makes it a new and promising lead in designing therapies against Alzheimer's disease [2].

In cultured chicken embryonic neurons, ginkgolide J (100 μM) administration decreases apoptotic damage produced by serum deprivation (24 hours) or Staurosporine (200 nM, 24 hours) treatment [1].

Production of β-amyloid oligomers [2]
Oligomeric Aβ1-42 was prepared according to the methods of Stine et al. Lyophilized Aβ1-42 was allowed to equilibrate at room temperature for 30 min to avoid condensation upon opening the vial. The lyophilized peptide was resuspended in 1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP) to 1 mM using a glass gas-tight Hamilton syringe with a Teflon plunger. HFIP was allowed to evaporate in a fume hood and the resulting clear peptide film was dried under vacuum (6.7 mTorr) in a SpeedVac. We stored the dessicated pellet at −20°C. Immediately prior to use, the aliquots were resuspended to 5 mM in dimethylsulfoxide (DMSO) by pipette mixing followed by bath sonication for 10 minutes at 4°C. 5 mM Aβ1-42 in DMSO was diluted to 100 μM in ice-cold cell culture media, immediately vortexed for 30 seconds and incubated at 4°C for 24 hours. The concentration of Aβ was determined based on the amounts of total Aβ content in our preparation including different forms of oligomeric Aβ.

Electrophysiological Recordings [2]
fEPSPs were recorded from the CA1 region of the hippocampus by placing both the stimulating and the recording electrodes in CA1 stratum radiatum. BST was assayed by plotting the stimulus voltage (V) against slopes of fEPSP to generate input-output relations. For LTP experiments, baseline stimulation was delivered every minute at an intensity that evoked a response approximately 35% of the maximum evoked response. Baseline response was recorded for 15 minutes prior to beginning the experiment to assure stability of the response. LTP was induced using theta-burst stimulation (4 pulses at 100 Hz, with the bursts repeated at 5 Hz and each tetanus including 3 ten-burst trains separated by 15 seconds). P8A, GA, GB, GC, GJ, BB, GA-triether and vehicle in 0.1% DMSO were individually added to the bath solution for 20 min at the same time as Aβ1-42 before inducing LTP.

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Hippocampal neuronal cultures [2]
Hippocampal cell cultures were prepared according to the method previously described. Briefly, fetuses at embryonic day 18 (E18) from timed pregnant Sprague-Dawley rats were sacrificed and the hippocampi removed. Neurons were then dissociated, plated at a density of 106 cell/well on 6 well-plates coated with poly-L-lysine and maintained in a defined serum-free medium (95% neurobasal, 2% B-27 supplement, 0.5 mM L-glutamine, 0.6% glucose, 1% penicillin/streptomycin). These cultures are enriched in large pyramidal neurons that constitute the main initial target in AD pathogenesis. Cultures at 5–6 days in vitro (DIV) cells were used for the experiments.

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Neuronal cell death assay [2]
Hippocampal cultures were treated by adding 10 μM Aβ1-42 in its oligomeric form with or without P8A, or alternatively with and without each of the individual ginkgolides and bilobalide (GA, GB, GC GJ, BB). Similar to the LTP experiments, these compounds were dissolved in DMSO and added to the culture medium at a ratio of 1:1000 (v/v), yielding a 0.1% DMSO solution. After 24 hrs the number of viable cells was assessed by nuclear counting [49]. In a separate set of experiments viable cells were counted by using the ethidium homodimer/calcein AM combination of vital dyes (LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells), according to the manufacturer instructions. Values represent mean ± SEM of three consecutive experiments. Each experiment was performed in triplicate.

Slice preparation [2]
C57BL/6 mice (3–4 months of age) were decapitated, and their hippocampi were removed. Transverse hippocampal slices with a thickness of 400 μm were maintained in an interface chamber at 29°C, as previously described. They were perfused with perfusion buffer (124.0mM NaCl, 4.4mM KCl, 1.0mM Na2HPO4, 25.0mM NaHCO3, 2.0mM CaCl2, 2.0mM MgSO4, 10mM glucose) continuously bubbled with 95% O2 and 5% CO2 (flow rate 1ml/min in a chamber of 1 ml volume). Slices were permitted to recover for at least 90 minutes before recording.

[1]. Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer's disease. Pharmacopsychiatry. 2003 Jun; 36 Suppl 1:S8-14.

[2]. Synthesis and Biological Studies of a New Ginkgolide C Derivative: Evidence That the Cardioprotective Effect of Ginkgolides Is Unrelated to PAF Inhibition. DRUG DEVELOPMENT RESEARCH. 2001.54:191-201.

[3]. Protection against beta-amyloid induced abnormal synaptic function and cell death by Ginkgolide J. Neurobiol Aging. 2009;30(2):257-265.

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.[1]

C20H24O10

424.4

424.136

C, 56.60; H, 5.70; O, 37.70

107438-79-9

163776

White to off-white solid powder

1.6±0.1 g/cm3

760.4±60.0 °C at 760 mmHg

41-42 °C

273.6±26.4 °C

0.0±5.8 mmHg at 25°C

1.651

Ginkgolide-J has been reported in Ginkgo biloba

-0.68

3

10

1

30

925

0

C[C@@H]1C(=O)O[C@@H]2[C@]1([C@@]34C(=O)O[C@H]5[C@]3(C2)[C@@]6([C@@H]([C@H]5O)C(C)(C)C)[C@H](C(=O)O[C@H]6O4)O)O

LMEHVEUFNRJAAV-UHFFFAOYSA-N

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

8-tert-butyl-6,9,17-trihydroxy-16-methyl-2,4,14,19-tetraoxahexacyclo[8.7.2.01,11.03,7.07,11.013,17]nonadecane-5,15,18-trione

Ginkgolide J; 107438-79-9; 7beta-Hydroxyginkgolide A; BN-52024; UNII-M5297RI2UE; M5297RI2UE; BN 52024; Ginkgolide A, 7-hydroxy-, (7beta)-;

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 (~589.07 mM)

Solubility in Formulation 1: ≥ 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 2: ≥ 2.08 mg/mL (4.90 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 (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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 (Please use freshly prepared in vivo formulations for optimal results.)