Acetylcholinesterase Inhibitory Activities of Flavonoids from the Leaves of Ginkgo biloba against Brown Planthopper

Ginkgo biloba is a traditional Chinese medicinal plant which has potent insecticidal activity against brown planthopper. The MeOH extract was tested in the acetylcholinesterase (AChE) inhibitory assay with IC 50 values of 252.1 μg/mL. Two ginkgolides and thirteen flavonoids were isolated from the leaves of Ginkgo biloba. Their structures were established on the basis of spectroscopic data interpretation. It revealed that the 13 isolated flavonoids were found to inhibit AChE with IC 50 values ranging from 57.8 to 133.1 μg/mL in the inhibitory assay. AChE was inhibited dose dependently by all tested flavonoids, and compound 6 displayed the highest inhibitory effect against AChE with IC 50 values of 57.8 μg/mL.


Introduction
Ginkgo biloba (Ginkgoaceae) is the oldest living tree, with a long history of use in traditional Chinese medicine [1].The extract of dried green leaves which was extracted with an acetone/water mixture has been standardized to contain 6% terpene trilactones (ginkgolides and bilobalide) and 24% flavonoids [2].Those flavonoids are almost flavonol-Oglycosides, a combination of aglycones kaempferol, quercetin, and isorhamnetin, with glucose or rhamnose or both linking to different positions of the flavonol moiety [3].Moreover, brown planthopper, Nilaparvata lugens (Homoptera: Delphacidae) is a major rice pest in many countries of Asia.And its infestation is initiated every year by a windborne from the tropics and subtropics.Acetylcholinesterase (AChE) is a substrate-specific enzyme that degrades the neurotransmitter acetylcholine in the nerve synapse [4].As reported, linarin was isolated from Buddleja davidii, with a detection limit of 10 ng in the bioautographic TLC assay, which is at the same level as the known active compound galantamine [5].
In this study, we describe the isolation and structures as well as acetylcholinesterase inhibitory activity of thirteen flavonoids together with two ginkgolides from the leaves of Ginkgo biloba (Figures 1 and 2).

Extraction and Isolation.
The air-dried leaves of Ginkgo biloba (5.0 kg) were powdered and extracted exhaustively by maceration with MeOH at room temperature.The extract solution was concentrated under diminished pressure to afford a residue, which was partitioned between ethyl acetate (5 × 1 L) and water (5 × 1 L) to give 25 g and 34 g of extracts from these two layers, respectively.The ethyl acetate extract was subjected to column chromatography over silica gel and eluted with a gradient CHCl 3 -MeOH (100 : 1-2 : 1) to afford eight fractions.Fr.  [7].A control reaction was carried out using phosphate buffer instead of test compounds and was considered as 100% activity.Inhibition, in %, was calculated in the following way: where  compound is the absorbance of the test compounds containing reaction and  control is the absorbance of the reaction control.IC 50 was obtained by plotting the inhibition percentage against using concentrations.

Results and Discussion
With the aim of researching active compounds by measuring acetylcholinesterase-inhibiting activity from the leaves of Ginkgo biloba, the MeOH extract and compounds 1-15 were tested in the acetylcholinesterase inhibitory assay.The MeOH extract proved to be active with IC 50 = 252.1 g/mL.In order to substantiate the result and find out which compound inhibited the enzyme, the extract was subjected to column chromatography over silica gel, RP-18, Sephadex LH-20, and semipreparation HPLC to yield the active compounds 1-13.[16], taxifolin (12), and genistein (13) [17]; two ginkgolides, namely, ginkgolide B ( 14) and ginkgolide C (15) [18] were obtained from the leaves of Ginkgo biloba.

Structural Elucidation of
The structure determination of compounds 1-15 was established using NMR spectral method, and their spectral data were compared to previous literature values.

AChE Inhibitory Activity.
The AChE inhibitory activity of the evaluated extract of Ginkgo biloba at different concentrations (31.25, 62.5, 125, 250, and 500 g/mL) showed that the extract has moderate inhibitory activity with the IC 50 = 252.1 g/mL (Table 1).Ginkgolides B and C were found to be inactive in the AChE inhibitory test, while their effect target seemed to be GABA receptor [19].
Flavonoids have previously been reported to be multipotent agents in combating Alzheimer's disease (AD) by enhancing acetylcholine levels.Among the 13 isolated flavonoids, they were found to inhibit AChE with IC 50 values ranging from 57.8 to 133.1 g/mL in the inhibitory assay.AChE was inhibited dose dependently by all tested flavonoids.As shown in Table 1, compound 6 exerted the most promising activity with IC 50 values of 57.8 g/mL following by the inhibitory property of compound 8 that exhibited an IC 50 value of 73.1 g/mL as compared with a positive control, chlorpyrifos (IC 50 = 12.4 g/mL).Compounds 4, 10, and 12 showed weak inhibitory activity, with IC 50 values of 110.9, 112.6, and 133.1 g/mL, respectively.

Conclusion
In conclusion, we isolated two ginkgolides and thirteen flavonoids from the leaves of Ginkgo biloba.The AChE inhibitory activity of test compounds revealed that flavonoids were found to inhibit AChE with IC 50 values ranging from 57.8 to 133.1 g/mL, and that ginkgolides were inactive.These inhibitors enhance the signal transmission in nerve synapses by prolonging the effect of acetylcholine, which is harmful to brown planthopper.The isolation and identification of AChE inhibitors of these compounds may be of interest to clarify the physiological role of this enzyme and to provide novel pesticide.

Table 1 :
AChE inhibitory activity of compounds 1-15.All compounds were examined in a set of experiments repeated three times; IC 50 values of compounds represent the concentration that caused 50% enzyme activity loss.b No inhibitory activity. a