AU2014100873A4 - Quantitative Analysis of the Flavonoid Glycosides and Terpene Trilactones in the Extract of Ginkgo biloba and Evaluation of their Inhibitory Activity towards ß-Amyloid Peptide Aggregation - Google Patents

Abstract

Quantitative Analysis of the Flavonoid Glycosides and Terpene Trilactones in the Extract of Ginkgo biloba and Evaluation of their Inhibitory Activity towards p Amyloid Peptide Aggregation The standard extract of Ginkgo biloba leaves (EGb761) is used clinically in Europe for the symptomatic treatment of impaired cerebral function in primary degenerative dementia syndromes. In this invention, seven flavonoid glycosides were isolated from the extract of Ginkgo biloba leaves and characterized by spectroscopic analysis. Furthermore, an UIPL chromatography method was established for the simultaneous quantification of these seven flavonoids. The inhibitory activities of these flavonoids (compounds 1-7), as well as four TTLs, i.e. ginkgolides A, B, and C and bilobalide (compounds 8-11), were evaluated towards A342 aggregation using a thioflavin T fluorescence assay. It was found that three flavonoids (compounds 1, 3 and 4) exhibited moderate inhibitory activities, whereas the other four flavonoids (compounds 2, 5, 6 and 7), as well as the four terpene trilactones, showed poor activity. This is the first report of the inhibition of AD aggregation of two characteristic acylated flavonoid glycosides (compounds 6, 7) in Ginkgo leaves, on the basis of which the structure-activity relationship of these flavonoids (compounds 1-7) was discussed. <DocRef#00128414-CX > M006.037.DRF 22 Spec draft final

Description

Quantitative Analysis of the Flavonoid Glycosides and Terpene Trilactones in the Extract of Ginkgo biloba and Evaluation of their Inhibitory Activity towards p Amyloid Peptide Aggregation 5 CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application having Serial No. 61/943,499 filed 24 Feb 2014, which is hereby incorporated by reference herein in its entirety. 10 FIELD OF INVENTION [0002] This invention relates to a method of screening a compound for treating Alzheimer disease. In particular, the compound is isolated from Ginkgo biloba. 15 BACKGROUND OF INVENTION [0003] Alzheimer disease (AD) is the main type of senile dementia, and is characterized by a range of pathological features, including the formation of senile plaques and neurofibrillary tangles. In recent years, there has been an increase in the number of reports identifying amyloid 0 peptide (AD) as a primary pathological factor in the occurrence of 20 AD, with gradual changes in the balance between the state of AD production and clearance leading to the accumulation of aggregated AD. This accumulation of AD then triggers a series of complex reactions that ultimately leads to neuronal death and cognitive dysfunction 1. [0004] The abnormal production and aggregation of AD and the deposition of fibrils in 25 the brain are regarded as key steps in the onset of AD, and the development of therapeutic strategies is aimed at preventing the aggregation of AD or promoting the destabilization of the preformed fibrils. Therefore these methods represent viable approaches for the prevention and treatment of AD . M006.037.DRF 1 Spec draft final [0005] EGb761 is the standard extract of the leaves of Ginkgo biloba which is currently 30 manufactured by several companies in Germany and France. On the grounds of proven clinical efficacy, EGb761 has been approved for the symptomatic treatment of impaired cerebral function in dementia syndromes by the German Federal Health Authority and for cerebral circulatory insufficiency and AD in France. The main indications for EGb761 are primary degenerative dementia, vascular dementia and mixed forms of dementia, which 35 are characterized by several major symptoms, including memory loss, poor concentration, depression, vertigo, tinnitus, and headache. The approved daily dose of EGb761 is 120 to 240 mg in 2 or 3 doses and is typically recommended for at least 8 weeks. The results of a large number of in vivo and in vitro studies have provided strong evidence in support of the clinical use of EGb761 . 40 SUMMARY OF INVENTION [0006] It is therefore an object of the present invention to provide a method of screening a compound for treating Alzheimer disease. It is another object of the present invention to provide a method of extracting and isolating bioactive compounds from Ginkgo biloba. 45 [0007] Accordingly, the present invention, in one aspect, provides a method of screening a therapeutic agent as a drug candidate for treating Alzheimer disease, comprising: [0008] a) exposing said agent to a thioflavin T fluorescence assay; [0009] b) determining the inhibitory activity of said agent towards A342 aggregation; [0010] c) comparing the inhibitory activity of said agent with the inhibitory activity of a 50 control compound; and [0011] d) identifying a drug candidate that has a stronger inhibitory activity of step a) than that of said control compound. [0012] In an exemplary embodiment of the present invention, said control compound is selected from the group consisting of quercetin 3-0--D-rutinoside, quercetin 3-0-a-L M006.037.DRF 2 Spec draft final 55 (-D-glucopyranosyl)-(1,2)- rhamnopyranoside and quercetin 3-0-a-(6"'-p-coumaroyl glucopyranosyl-3-1,2-rhamnopyranoside). [0013] In another aspect, the present invention provides a method of extracting and isolating a compound from EGb761 of Ginkgo biloba leaves, comprising: [0014] a) extracting EGb761 powder with methanol in an ultrasonic water bath to obtain 60 an extract; [0015] b) filtering and concentrating said extract of step (a) to obtain a residue; [0016] c) eluting said residue of step (b) with a gradient of methanol in water by a Diaion HP-20 adsorbent resin column chromatography to obtain fractions 1-12; [0017] d) eluting the fraction 3 with methanol in water and storing the resulting solution 65 in 4 'C to obtain a precipitate; and collecting said precipitate by filtration and re crystallization to yield quercetin 3-0--D-rutinoside (compound 1); [0018] e) eluting the fraction 7 with methanol in water to obtain eluate; [0019] f) purifying said eluate of step (d) by column chromatography over an ODS column with a gradient of methanol in water to obtain 12 sub-fractions; 70 [0020] g) eluting the sub-fractions 11 and 13 with a gradient of methanol in water on TSK Toyopearl HW-40 gel to yield quercetin 3-0-a-(6"'-p-coumaroyl glucopyranosyl-3 1,2-rhamnopyranoside) (compound 6), and kaempferol 3-0-a-(6"'-p-coumaroyl glucopyranosyl-3-1,2-rhamnopyranoside) (compound 7), respectively; [0021] h) eluting the sub-fraction 10 with a gradient of methanol in water on TSK 75 Toyopearl HW-40 gel to yield kaempferol 3-0--D-rutinoside (compound 3), isorhamnetin 3-0--D-rutinoside (compound 4), kaempferol3-0-a-L-(3-D glucopyranosyl)-(1,2)-rhamnopyranoside (compound 5), respectively; and M006.037.DRF 3 Spec draft final [0022] i) obtaining quercetin 3-0-a-L-( -D-glucopyranosyl)-(1,2)- rhamnopyranoside (compound 2) by the alkaline hydrolysis of quercetin 3-0-a-(6"'-p-coumaroyl 80 glucopyranosyl-0-1,2-rhamnopyranoside); [0023] In a further exemplary embodiment of the present invention, the fraction 3 was eluted with 30% methanol in water in the step (d). [0024] In a further exemplary embodiment of the present invention, the fraction 7 was eluted with 70% methanol in water in the step (e). 85 BRIEF DESCRIPTION OF FIGURES [0025] Fig. 1 shows HPLC fingerprint of the total extract of the leaves of Ginkgo biloba [0026] Fig. 2 shows the structures of compounds 1-7 90 M006.037.DRF 4 Spec draft final DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0027] As used herein and in the claims, "comprising" means including the 95 following elements but not excluding others. [0028] Flavonoid glycosides (FGs) and terpene trilactones (TTLs) are the two main groups of constituents in EGb761 which represent 24% and 6% of the total weight of EGb761, respectively. To identify the bioactive compounds in EGb761, individual components of FGs from EGb761 were isolated and their inhibitory activities as well as 100 single compounds of TTLs towards AJ42 aggregation were evaluated using a thioflavin T (ThT) fluorescence assay . Using a variety of different chromatographic purification techniqueswe seven of the main FGs (compounds 1-7) from EGb761 were isolated and their structures were elucidated based on their HR-MS, 1 H-NMR and 13 C-NMR spectral data. 105 [0029] An ultra-high performance liquid chromatography (UPLC)-UV method was also developed for the quantitative analysis of these compounds, which allowed for the quantification of these seven FGs as well as the four main TTL components (compounds 8-11) in the extracts and preparations of Ginkgo biloba leaves. Finally, the bioactivities of these flavonoids and terpene lactones were also investigated, on the basis of which the 110 structure-activity relationship of these flavonoids (1-7) was discussed. [0030] Reagents and Materials [0031] Ginkgolides A, B, and C, and bilobalide (8-11) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) (purity>98%). Compounds 1-7 were isolated in our laboratory and characterized by MS 115 and NM'IR spectroscopy (purity> 95%). [0032] The A342 peptide was produced by Bachem AG (Bubendrof, Switzerland) with purity greater than 95%, as determined by HPLC. [0033] Thioflavin T (ThT), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). M006.037.DRF 5 Spec draft final 120 Trifluoroacetic acid (TFA) was purchased from International Laboratory (South San Francisco, CA, USA). [0034] Tablets made from the extract of Ginkgo biloba leaves for isolation (distributed by General Nutrition Corporation Pittsburgh, PA, USA) were purchased from the Manning pharmacy in Hong Kong. 125 [0035] Purifications by column chromatography were performed over Diaion HP-20 adsorbent resin (Mitsubishi Chemical Co., Japan), ODS (Davisil, 35-60 pim, Grace, Columbia, MD, USA) or Toyopearl TSK HW-40 gel (Tosoh Corporation, Japan). Silica gel 60 F 2 54 HPTLC aluminium sheets (Merck, Darmstadt, Germany) were used for analysis by TLC. 130 Example 1 Isolation, Preparation and Structural Elucidation of Acylated and Non Acylated FGs from EGb761 [0036] The EGb761 tablets were ground into powders, which were extracted with methanol for 1 h in an ultrasonic water bath, and this process was repeated for three times. The combined extracts were filtered and concentrated under reduced pressure to give a 135 residue, which was subjected to a Diaion HP-20 adsorbent resin column chromatography eluting with a gradient of methanol in water to result in 12 fractions. [0037] Fraction 3 (1.2 g), which was eluted in 30% methanol in water (v/v), was stored in a refrigerator at 4'C and gave a yellow precipitate. The precipitate was collected by filtration and re-crystallized from methanol to give compound 1 (10 mg) as an amorphous 140 yellow powder. [0038] Fraction 7 (2.3 g), which was eluted with 70% methanol in water (v/v), was purified by column chromatography over an ODS column eluting with a gradient of methanol in water to result in 12 sub-fractions. Sub-fractions 11 (0.1 g) and 13 (0.26 g) were then chromatographed over TSK Toyopearl HW-40 gel eluting with a gradient of 145 methanol in water to yield compounds 6 (22 mg) and 7 (52 mg), respectively. Sub fraction 10 (0.49 g) was subjected to the same procedure to yield compounds 3 (25 mg), 4 (43 mg) and 5 (36 mg). M006.037.DRF 6 Spec draft final [0039] Compound 2 was prepared by the alkaline hydrolysis of compound 6 according to the procedure described below [5]. Compound 6 (15mg) was dissolved in 0.4 mM solution 150 of NaOMe in MeOH (0.5 mL), and the resulting solution was stirred in an ice bath for 3 h. A residue was resulted from subsequent acidification with 0.3 N HCl and concentration of the solution, which was extracted with EtOAc. The EtOAc extract was then purified by column chromatography over ODS eluting with a gradient of methanol in water to give compound 2 (5 mg) as a yellow powder. 155 [0040] High resolution ESI-MS analysis was conducted on an Agilent 6230 time-of-flight mass spectrometer (Agilent, Santa Clara, CA, USA) in the positive ion mode. H and "C NMR spectra were recorded on a Bruker CyroFIT NMR Spectrometer (Bruker Daltonics, MA, USA) at 600 and 150 MHz, respectively. Example 2 Quantitative analysis of FGs by UPLC-UV 160 [0041] Samples of Ginkgo extract (5 mg) were accurately weighed and transferred into a 2 mL tube followed by a solution of 60% methanol in water (v/v), and the resulting mixture was briefly shaken for mixing. The mixture was then sonicated for 10 min before being filtered through a 0.22 ptm PTFE filter for analysis. [0042] The reference compounds were accurately weighed and dissolved in a mixture of 165 60% methanol in water (v/v) to prepare a standard mixed stock solution containing compounds 1 (300.0 ptg/mL), 2 (144.0 ptg/mL), 3 (150.0 ptg/mL), 4 (150.0 p.g/mL), 5 (150.0 ptg/mL), 6 (150.0 ptg/mL) and 7 (150.0 ptg/mL). Working standard solutions for the calibration curves were prepared by diluting the mixed standard solutions with a mixture of 60% methanol in water (v/v) to the appropriate concentrations. The standard solutions 170 were then stored at 4'C prior to being analyzed. [0043] UPLC was performed on an Acquity UPLC system (Waters Corp, Milford, MA, USA) equipped with a binary solvent delivery system, auto-sampler unit, and diode array detector (DAD). All the operations, including the acquisition and analysis of data, were controlled by using Hystar software (Bruker). The chromatography was performed on an 175 Acquity BEH C18 column (2.1 x 100 mm, 1.7 ptm). The mobile phases consisted of 0.1% formic acid in water (A) and 0.l1% formic acid in acetonitrile (B). M006.037.DRF 7 Spec draft final [0044] The following gradient elution procedure was used for the analysis of the sample: 0-2 min, 5-15% B; 2.1-20 min, 15-22% B; 20.1-23 min, 100% B; 23.1-25 min, 5% B. For quantitative analysis, the flow rate was set at 0.35 mL/min, the column temperature 180 was kept at 40'C, the detector signal was set at 260 nm (260 nm refers to the set wavelength of the UV detector), and the injection volume was set at 2 piL. [0045] A calibration curve was established for the quantitative analysis of the flavonoids by plotting the peak areas of analytes against the concentrations of the standard solutions, and then the amounts of the different FGs in the samples were then calculated using the 185 calibration curve. Example 3 Quantitative analysis of TTLs by a HPLC-ELSD Method. [0046] Standard and test solutions were prepared and analyzed according to the procedure described for USP28-NF23 [6] [0047] The standard solutions were prepared as follows. Accurately weighed ginkgolides 190 A, B and C and bilobalide were dissolved in methanol, and the resulting solutions was sonicated for 5 min before being diluted with methanol to obtain solutions of different concentrations, including 0.1, 0.5, 1.0, 2.0, 4.0, and 8.0 mg/mL. The solutions were then passed through a 0.45 ptm filter. [0048] The test solutions were prepared as follows. An accurately weighed portion of the 195 Ginkgo sample was placed in a 30 mL glass tube with screw cap and PTFE gasket, followed by addition of 10.0 mL of 90% methanol solution. The tube was then sealed and the mixture was heated with stirring at 90'C for 30 min. The hot suspension was then mixed on a vortex mixer for 5 min before being heated at 90'C for 30 min. The mixture was then cooled to room temperature, and passed through a 0.45 pim filter. 200 [0049] The mixtures were analyzed by HPLC using a Waters 2695 HPLC system equipped with a Waters 2420 evaporative light scattering detector (ELSD) (Waters Corp., Milford, MA, USA). A Phenomenex C18 column (250 x 4.60 mm, 5 tm) was used for the chromatography eluting with mobile phases consisting of water (A) and MeOH (B). M006.037.DRF 8 Spec draft final [0050] The Following gradient elution procedure was used for the analysis: 0-23 min, 205 25-48% B; 23-25 min, 48-75% B; 25-30 min, 75% B; 30-35 min, 75-90% B; 35-40 min, 90-25% B; 40-50 min, 25% B. [0051] The HPLC system was operated with a flow rate of 1. OmL/min with column and drift tube temperatures of 30 and 55 0 C, respectively. The nebulizer temperature was set at 36'C and the gas pressure was 25 psi. The injection volume for the quantitative analysis 210 was set to 10 pL. Example 4 Preparation of AD Stock Solutions [0052] The AD stock solutions were prepared according to a simple TFA pre-treatment protocol [. The A342 peptide was suspended in TFA at a concentration of 2 mg/mL, and the resulting mixture was sonicated for 20 min at room temperature to allow for 215 facilitating complete dissolution. A gentle stream of nitrogen gas was passed over the mixture to remove the TFA, and the resulting residue was treated with HFIP to give a suspension, which was cooled on ice without agitation until the peptides were dissolved completely. The HFIP was then removed by evaporation with a gentle stream of nitrogen gas. The resulting peptide was lyophilized overnight on a freeze drying system 220 (Labconco, Kansas City, MO, USA) to remove any trace amounts of TFA and HFIP, and then dissolved in cold 0.02% ammonia solution to a final concentration of 250 [iM and stored at -80'C before assay. Example 5 Polymerization Assay [0053] A polymerization assay was performed in the current study using a previously 225 described procedure [8- 9]. A reaction mixture containing 25 ptM A342 was mixed with different amounts (i.e., 0.3, 1.0, 3.0, 10, 30 and 50 pM) of compounds 1, 2 and 6, as well as 1% DMSO, 50 mM phosphate buffer (pH 7.5) and 100 mM NaCl. All of the compounds for testing were initially dissolved in DMSO at concentrations of 30, 100, and 300 pM, and 1, 3 and 5 mM, and then added to the reaction mixture to final 230 concentrations of 0.3, 1.0, 3.0, 10, 30 and 50 [tM, respectively. Compounds 3, 4, 5, 7, 8, 9, M006.037.DRF 9 Spec draft final 10 and 11 showed very low levels of activity in the preliminary activity test and were subsequently tested at a final concentration of 100 [tM in the full assay. [0054] Twenty microliter aliquots of the mixtures were injected into PCR tubes, which were then placed into the PCR system (Gene Amp PCR System 9700, Applied 235 Biosystems, Foster City, CA, USA) and incubated of 24h without agitation. The reactions were stopped by adding a ThT solution. Fluorescence measurements were conducted in triplicate from every tube with the mean values being reported. The concentrations of test compounds in the testing solutions were diluted to 1/150 of those in the reaction solution to minimize any quenching effects. 240 Example 6 Fluorescence Spectroscopy [0055] Fluorescence spectroscopy was conducted on an Envision 2104 Multilabel Reader (Perkin Elmer, Waltham, MA, USA)), with optimized excitation and emission wavelengths of 430 (BW 24 nm) and 470 nm (BW 24 nm), respectively. The reaction mixtures contained 5 ptM ThT and 50 mM of glycine-NaOH buffer (pH 8.5). IC 50 values 245 were calculated using the GraphPad Prism 5 software. [0056] Results and Discussion [0057] 1. Isolation and Structural Elucidation of the FGs [0058] Seven flavonoids (compounds 1-7) were isolated from the total extract of the leaves of Ginkgo biloba. HPLC fingerprint of the total extract showed that these seven 250 compounds accounted for 52 % of the total peak area of the 39 peaks observed in the HPLC chromatogram. [0059] All these seven compounds were obtained as yellow amorphous powders and their structures were determined based on a comparison of their MS and NIR spectral data with those in the literature [10, 11, 12, 13]. Compounds 1-7 were identified as quercetin 3-0 255 0-D-rutinoside (compound 1), quercetin 3-0-a-L-( -D-glucopyranosyl)-(1,2) rhamnopyranoside (compound 2), kaempferol 3-0-p-D-rutinoside (compound 3), isorhamnetin 3-0--D-rutinoside (compound 4), kaempferol3-0-a-L-(3-D M006.037.DRF 10 Spec draft final glucopyranosyl)-(1,2)-rhamnopyranoside (compound 5), quercetin 3-O-U-(6"'-p coumaroyl glucopyranosyl-3-1,2-rhamnopyranoside) (compound 6), and kaempferol 3-0 260 u-(6"'-p-coumaroyl glucopyranosyl-0-1,2-rhamnopyranoside) (compound 7), respectively. The structures of these seven compounds were shown in Fig. 2. [0060] 2. Quantification of the FGs in Extracts and Preparations of Ginkgo biloba leaves [0061] 2.1 Method Validation [0062] The regression equations, linearity, intra- and inter-day precision properties, and 265 LOD and LOQ values of the seven analytes were determined using our UPLC-UV method. As shown in Table 1, satisfactory linearity levels were observed for compounds 1-7 (R>0.999). The LODs (S/N = 3) and LOQs (S/N = 10) of these analytes were less than 7.40 ptg/mL and 24.75 ptg/mL, respectively. The results listed in Table 2 showed that the intra- and inter-day variations were less than 1.63% (n = 5) and 3.65% (n = 5) for all 270 of the analytes, respectively, demonstrating that this method exhibited a high level of precision for all of the analytes tested. The repeatability values (RSDs, n = 5) of compounds 1-7 were determined to be less than 1.94%, and the results of the recovery study showed that the average recovery rates of all the seven compounds were in the range of 95.07-104.73% with average RSDs of 1.52% (n = 6). Taken together, these 275 results demonstrated that UPLC-UV method used in this study was suitable for the subsequent quantitative analysis of the seven flavonoid glycosides. Table 1. Linearitvx and sensitivit ofr cojnds 1-7 Linear range LOD LOQ Compound Regression Equation R2 (pg/mL (pg/mL) (pg/mL) 1 Y = 9.1207x-3.07 25.0-300.0 0.9999 2.33 8.09 2 Y= 6.4290x+6.08 24.0-144.0 0.9995 4.44 14.66 3 Y= 11.370x-4.72 12.5-150.0 0.9999 2.74 9.10 4 Y =7.9820x-17.96 25.0-150.0 0.9996 4.02 13.46 5 Y = 9.1149x-5.52 25.0-150.0 0.9997 4.30 14.33 M006.037.DRF 11 Spec draft final 6 Y = 6.6509x+4.18 25.0-150.0 0.9998 7.17 23.89 7 Y = 7.583 1x-3.76 25.0-150.0 0.9999 7.40 24.75 Table 2. Precision, repeatability and recovery of compounds 1-7 Precision (n = - Repeatabilityn ---- -- ) Rcovery (n =6) Compound Intra-day Inter-day Concentration

RSD/%

0 Mean RSD/% 0 1 0.53 1.29 6.92 1.67 99.8 0.68 2 1.63 3.65 3.33 1.05 100.6 1.23 3 0.67 1.78 3.74 1.02 99.3 2.08 4 0.65 1.3 4.75 1.94 99.9 1.52 5 0.42 1.09 2.61 1.11 100.3 1.61 6 0.36 1.15 6.31 1.45 102.8 1.65 7 0.54 0.39 4.78 1.15 98.1 1.90 280 [0063] 2.2 Quantitative Analysis of Compounds 1-7 in the Extracts and Tablets of Ginkgo biloba [0064] In this study, a total of 11 samples for UPLC-UV analysis was collected, including the extracts and botanical products of Ginkgo biloba. The details of these samples were shown in Table 3. 285 Table 3. Details of the samples subjected to quantitative analysis Sample Description Sl Extract, extracted from tanakan* tablets purchased from Watsons, HK S2 Extract, extracted from tanakan* tablets purchased from Tianjin, China S3 Extract, extracted from Ginaton* tablets purchased from Tianjin, China S4 Extract, extracted from Ginaton* tablets purchased from Tianjin, China S5 Extract, extracted from the leaves of Ginkgo biloba (purchased from Lotus Pond Market (Sichuan Province, China) and grown in Shandong Province, China) S6 Extract, extracted from the leaves of Ginkgo biloba (purchased from Lotus Pond Market (Sichuan Province, China) and grown in Hunan Province, China) S7 Extract, purchased from Ningbo Traditional Chinese Phannaceutical CO., Ltd, Zhejiang Province, China M006.037.DRF 12 Spec draft final S8 Tanakan* tablet purchased from Watsons, HK S9 Tanakan* tablet purchased from Tianjin, China S1O Ginaton@ tablet purchased from Tianjin, China Si1 Ginaton@ tablet purchased from Tianjin, China [0065] Samples Si-i1 were subjected to UPLC-UV analysis to allow for compounds 1-7 in these samples to be quantified. The results of this analysis, as shown in Table 4, revealed that similar levels of the seven FGs were present in the two commercial Ginkgo 290 tablets tanakan* and Ginaton®. The levels of the seven FGs in tanakan* were found to be a little higher than those in Ginaton®, tanakan® and Ginaton®, and showed very small batch-to-batch variation, which reflected a high level of quality control during the manufacture of EGb761 and the tablets. Table 4. FGs in the extracts and preparations of Ginkgo biloba (n = 2) Contents (pg/mg) Contents ()a) Si S2 S3 S4 S5 S6 S7 S8 S9 S10 Sll 1 6.95 6.24 5.83 6.46 1.88 1.75 29.54 2.18 2.03 1.77 1.78 2 3.68 3.16 2.99 3.33 2.00 1.46 16.37 1.18 1.00 0.91 0.94 3 3.84 3.70 3.33 3.62 1.22 1.15 18.58 1.18 1.18 0.99 1.00 4 5.77 5.19 5.04 4.74 2.18 2.11 17.45 1.73 1.67 1.55 1.49 5 3.37 3.00 2.90 2.66 1.79 1.25 26.83 1.07 0.97 0.89 0.85 6 6.02 6.52 5.70 6.53 2.67 2.73 32.53 2.19 1.92 1.76 1.82 7 4.47 4.76 4.33 4.70 1.64 1.53 25.27 1.57 1.46 1.34 1.33 Sum 34.10 32.58 30.12 32.03 13.39 11.98 166.57 11.10 10.19 9.20 9.21 295 aaaon [0066] 3. Determination of the Different TTLs using the HPLC-ELSD Method [0067] 3.1 Calibration Curve [0068] The linearity values of the TTLs (compounds 8-11) were determined by analyzing the curves of the four TTL standards at five different concentrations. The results were 300 expressed as correlation coefficients (R2) and summarized in Table 5. Table 5. Linearity of the TTLs M006.037.DRF 13 Spec draft final Compound Linearity (pig/mL) Regression Equation R2 8 36.725-734.50 logA = 1.58 logC+1.44 0.9993 9 28.225-1129.0 logA = 1.51 logC+1.54 0.9991 10 39.625-792.50 logA = 1.57logC+1.38 0.9994 11 30.275-605.50 logA = 1.55 logC+1.46 0.9996 [0069] 3.2 Quantitative Analysis of the Extracts and Preparations of Ginkgo biloba [0070] The results of the quantitative analysis using USP protocol [6] indicated that there were specific differences between the two commercial Ginkgo tablets, tanakan* and 305 Ginaton , in terms of the amounts of the TTLs that they contained. For example, there were more of the four TTLs in tanakan* than there were in Ginaton*, although both products showed good batch-to-batch repeatability in their TTL contents as shown in Table 6. 310 Table 6. TTLs in the extracts and preparations of Ginkgo biloba (n = 2) Compound Contents (pg/mg) Contents (0%)a) Si S2 S3 S4 S5 S6 S7 S8 S9 S10 Sl 8 5.99 6.23 4.94 5.52 5.02 3.51 42.04 2.17 2.11 1.78 2.04 9 14.57 14.78 11.73 11.84 5.59 4.59 53.23 1.00 1.09 0.82 1.00 10 6.00 6.21 6.09 6.08 6.14 4.10 13.57 2.15 2.14 1.63 1.70 11 14.73 13.3 10.94 10.88 5.37 4.18 32.98 3.23 3.22 2.63 2.72 Sum 41.28 40.52 33.69 34.31 22.11 16.36 141.82 8.55 8.55 6.86 7.46 a: The amount of EGb761 per tablet was considered to be 40 mg based on the label for this preparation. [0071] 4. Bioactivity Assay of the Main FGs and TTLs [0072] The inhibitory activities of compounds 1-11 were evaluated using a ThT assay. Among the main FGs, three compounds (compounds 1, 2, and 6) showed moderate and 315 dose-dependent inhibitory activities towards A342 aggregation, with IC 50 values in the range of 33 to 67 ptM as shown in Table 7. The other four FGs (compounds 3, 4, 5, and M006.037.DRF 14 Spec draft final 7), however, showed weak effect as shown in Table 8. Furthermore, even at very high concentrations (i.e., 100 M), the four main TTLs (compounds 8-11) exhibited very little inhibitory activity towards AJ42 aggregation (Table 8). 320 Table 7. IC 50 values of FGs 1, 2 and 6 towards A042 aggregation Compounds IC 50 on Aggregation (pM) 1 33.02±4.84 2 67.12 11.66 6 32.56 4.83 Table 8. Inhibition rates (IRs) of compounds 3-5 and 7-11 towards A042 aggregation Compounds* IR on Aggregation (%) 3 30.89 ± 3.90 4 23.62± 1.39 5 30.49 ± 1.36 7 34.98 ± 1.38 8 21.10 ±2.96 9 13.56± 1.31 10 13.92 ±2.35 11 14.84 ± 2.36 *All of the compounds were tested at a concentration of 100 RM. 325 [0073] Although the results of this invention for ginkgolides A, B, and C (8-10) were consistent with those reported in previous studies' 15, the results of this invention for bilobalide (11) contrasted significantly with that reported by Luo et al. [15, who found that bilobalide exhibited significant in vitro activity towards the inhibition of A340 aggregation. The significant difference probably attributed to the different sequence 330 between A340 and A042. [0074] Conclusions [0075] Seven major FGs including two characteristic components (compounds 6, 7) were isolated from the extract of Ginkgo biloba leaves and their structures were identified on M006.037.DRF 15 Spec draft final the basis of MS and NNIR spectral evidences. A UPLC-UV method was developed for 335 the simultaneous determination of these flavonoids in Ginkgo extract and products. The linearity, LOD, LOQ, precision, repeatability and recovery characteristics of this method revealed that it was suitable for the quantitative analysis of the FGs in EGb761. Regarding the inhibitory activity of the pure compounds towards AD fibril formation, significant differences were observed between these seven compounds. For example, 340 compounds 1, 2 and 6 exhibited moderate inhibitory activities towards AJ42 aggregation, whereas compounds 3, 4, 5 and 7 showed barely any effect thereon. The four TTLs were also shown not to exhibit any inhibitory activity towards AJ42 aggregation. This study therefore represents the first reported example of the isolation of acylated flavonoid glycosides (6, 7) from EGb761 with inhibitory activity against AD aggregation. 345 [0076] Furthermore, the results of the current study have provided some structure-activity relationships for the FGs in terms of inhibitory activity towards AJ42 aggregation. Of the seven FGs evaluated in the current study, three FGs (compounds 1, 2, 6) with the aglycone of quercetin showed much stronger activity than those with the aglycone of kaempferol or isorhamnetin, indicating that the observed activity was closely associated 350 with the aglycone moiety, and that the number and position of the hydroxyl groups could have a significant impact on the activity. The difference observed between the activities of compounds 1 and 2 was attributed to the linkage of the sugar moiety. It is noteworthy that the acylated compound 6 showed much stronger inhibitory activity than that of compound 2. This difference in activity was attributed to the three-dimensional 355 conformation formed as a consequence of the coumaroyl group in 6. This conformation is essential for the compound to form a non-covalent interaction with the p-sheet structure of the amyloid peptide [13, 16] [0077] In conclusion, it has shown that the seven FGs from the extract of the leaves of Ginkgo biloba showed moderate to weak inhibitory activity towards AJ42 aggregation, 360 whereas four TTLs had little effect on AJ42 aggregation. [0078] The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled M006.037.DRF 16 Spec draft final in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set 365 forth herein. [0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred 370 methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and disclose specific information for which the reference was cited in connection with. [0080] All references cited above and in the following description are incorporated by reference herein. The practice of the invention is exemplified in the following non 375 limiting examples. The scope of the invention is defined solely by the appended claims, which are in no way limited by the content or scope of the examples. [0081] References 1. Cavallucci, V.; D'Amelio, M.; Cecconi, F. AD Toxicity in Alzheimer's Disease. Mol. Neurobiol. 2012, 45, 366-378. 380 2. Savonenko, A.V.; Melnikoval, T.; Hiat, A.et al. Alzheimer's Therapeutics: Translation of Preclinical Science to Clinical Drug Development. Neuropsychopharmaco. 2012, 37, 261-277. 3. Cott, J.M.; Raman, A. Clinical Efficacy and Pharmacodynamics. In American herbal pharmacopoeia and therapeutic compendium: Ginkgo leaf, Ginkgo leaf 385 dry extract, Ginkgo biloba L.; Herabalist R.U.; Graff, A. American Herbal pharmacopoeia: Scotts Valley, CA, USA, 2003; pp. 26-27. 4. Levine, H III. Thioflavine T interaction with synthetic Alzheimer's disease p amyloid pepetides: Detection of amyloid aggregation in solution. Prot. Sci. 1993, 2, 404-410. M006.037.DRF 17 Spec draft final 390 5. Jiang, Z.-H.; Tanaka, T.; Kouno, I. Chilianthins A-F, six triterpene esters having dimeric structures from Rhoitelea chiliantha DIELS et HAND.-MAZZ. Chem. Pharm. Bull. 1996, 44, 1669-1675. 6. The United States Pharmacopeial Convention. The United States Pharmacopeia, 28th revision/National Formulary, 2 3 rd Edition; The United State Pharmacopeial 395 Convention: Rochville, United States of America, 2003; pp. 2094-2095. 7. Jao, S.C.; Ma, K.; Talafous, J.; Orlando, R.; Zagorski, M.G. Trifluoroacetic acid pretreatment reproducibly disaggregates the amyloid p-peptide. Amyloid Int. J. Exp. Clin. Invest. 1997, 4, 240-252. 8. Naiki, H.; Hasegawa, K.; Yamaguchi, I.; Nakamura, H.; Gejyo, F.; Nakakuki, K. 400 Apolipoprotein E and antioxidants have different mechanisms of inhibiting Alzheimer's beta-amyloid fibril in vitro. Biochem. 1998, 37, 17882-17889. 9. Ono, K.; Hasegawa, K.; Yamada, K.; Naiki, H. Nicotine breaks down performed Alzheimer's beta-amyloid fibril in vitro. Biol. Psychiatry. 2002a, 52, 880-886. 10. Nasr, C.; Lobstein-Guth, A.; Haag-Berrurier R.A. Quercetin coumaroyl 405 glucorhamnoside from Ginkgo biloba. Phytochem. 1987, 26, 2869-2870. 11. Nasr, C.; Haag-Berrurier R.A.; Lobstein-Guth, A.; Anton, R. Kaempferol coumaroyl glucorhamnoside from Ginkgo biloba. Phytochem. 1986, 25, 770-771. 12. Tang, Y.P.; Wang, Y.; Lou, F.C.; Li, Y.F.; Wang, J.H. Flavonol glycosides from the leaves of Ginkgo biloba. Acta Pharm. Sin. 2000, 35, 363-3 66. 410 13. Gao, J.H.; Shi, F.G.; Song, G.Q. Further NMR investigation and conformational analysis of an acylated flavonol glucorhamanoside. Magn. Reson. Chem. 1996, 34, 249-254. 14. Ramassamy, C.; Christen, Y.; Poirier, J. Ginkgo biloba extract (EGb761), 0 amyloid peptide and apolipoprotein E in Alzheimer's disease. In Advances in 415 Ginkgo biloba extract (EGb761) as a neuroprotective agent: from basic studies to clinical trial; Christen Y. Solal: Marseille, France, 2001; Volume 8, pp. 71-89. 15. Luo, Y.; Smith J.V.; Paramasivam V.; Paramasivam V.; Burdick A.; Curry, K.J.; Buford J.P.; Khan, I.; Netzer W.J.; Xu, H.X.; Butko P. Inhibition of amyloid-p M006.037.DRF 18 Spec draft final aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761. Proc. 420 Natl. Acad. Sci. 2002, 99, 12197-12202. 16. Porat, Y.; Abramowitz, A.; Gazit, E. Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem. Biol. Drug. Des.2006, 67, 27-37. 425 <DocRef#00128414-CX > 430 M006.037.DRF 19 Spec draft final

Claims (5)

1. A method of screening a therapeutic agent as a drug candidate for treating Alzheimer disease, comprising: a) exposing said agent to a thioflavin T fluorescence assay; b) determining the inhibitory activity of said agent towards A342 aggregation; c) comparing the inhibitory activity of said agent with the inhibitory activity of a control compound; and d) identifying a drug candidate that has a stronger inhibitory activity of step a) than that of said control compound.

2. The method of claim 1, wherein said control compound is selected from the group consisting of quercetin 3-0--D-rutinoside, quercetin 3-0-a-L-(-D-glucopyranosyl) (1,2)- rhamnopyranoside and quercetin 3-0-a-(6'"-p-coumaroyl glucopyranosyl-p 1,2-rhamnopyranoside) .

3. A method of extracting and isolating a compound from EGb761 of Ginkgo biloba leaves, comprising: a) extracting EGb761 powder with methanol in an ultrasonic water bath to obtain an extract; b) filtering and concentrating said extract of step (a) to obtain a residue; c) eluting said residue of step (b) with a gradient of methanol in water by a Diaion HP-20 adsorbent resin column chromatography to obtain fractions 1-12; d) eluting the fraction 3 with methanol in water and storing the resulting solution in 4 'C to obtain a precipitate; and collecting said precipitate by filtration and re crystallization to yield quercetin 3-0--D-rutinoside; e) eluting the fraction 7 with methanol in water to obtain eluate; f) purifying said eluate of step (d) by column chromatography over an ODS column with a gradient of methanol in water to obtain 12 sub-fractions; g) eluting the sub-fractions 11 and 13 with a gradient of methanol in water on TSK Toyopearl HW-40 gel to yield quercetin 3-0-a-(6"'-p-coumaroyl glucopyranosyl 3-1,2-rhamnopyranoside), and kaempferol 3-0-a-(6"'-p-coumaroyl glucopyranosyl-3-1,2-rhamnopyranoside), respectively; M006.037.DRF 20 Spec draft final h) eluting the sub-fraction 10 with a gradient of methanol in water on TSK Toyopearl HW-40 gel to yield kaempferol 3-0--D-rutinoside, isorhamnetin 3-0 -D-rutinoside, kaempferol3-0-a-L-( -D-glucopyranosyl)-(1,2) rhamnopyranoside, respectively; and i) obtaining quercetin 3-0-a-L-( -D-glucopyranosyl)-(1,2)- rhamnopyranoside by the alkaline hydrolysis of quercetin 3-0-a-(6'"-p-coumaroyl glucopyranosyl-p 1,2-rhamnopyranoside);

4. The method of claim 3, wherein the fraction 3 was eluted with 30% methanol in water in the step (d).

5. The method of claim 3, wherein the fraction 7 was eluted with 70% methanol in water in the step (e). <DocRef#00128414-CX > M006.037.DRF 21 Spec draft final