Polyphenols from Ginkgo biloba

By Sephadex LH-20 gel chromatography of an extract from Gingko biloba leaves, polymeric proanthocyanidins were eluted after the fractions of flavonol glycosides and biflavone glycosides. A purified proanthocyanidin polymer accounted for 86.6% of the total proanthocyanidins, and for 37.7% of the total antioxidant activity of this leaf extract. For structure elucidation, the polymer was submitted to acidic depolymerization in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D-and reverse 2D-NMR (HSQC, HMBC) spectra of their peracetylated derivatives, MALDI-TOF-MS and CD-spectroscopy. The observations resulting from the degradation with phloroglucinol were confirmed by 13C-NMR spectroscopy of the polymer. The mean molecular weight of the polymeric fraction was estimated to be 9–10 flavan-3-ol-units.


Introduction
Extracts of Ginkgo biloba L. (Maidenhair tree, Ginkgoaceae) are widely used in herbal medicine for the treatment of mild to moderate cognitive disorders, concentration problems, tinnitus and dementia [1]. Ginkgo extracts are also used against Alzheimer's disease [2]. Most of the extracts are standardized to a typical composition of 6% terpene trilactones, 24% of flavonol glycosides and less than 5 ppm of ginkgolic acids [3,4]. Ginkgo leaves and standardized extracts contain large amounts of proanthocyanidins (PAC, 4-12% and 7% in leaves and extracts, respectively) [5,6]. In spite of their quantitative importance, relatively little is known about the composition of the PAC in Ginkgo. The only compounds identified to date are dimeric procyanidins and prodelphinidins [7]. The knowledge of the phytochemical composition of the polymeric proanthocyanidin fraction (acetone-water-eluted fraction, AWF) is, however, of importance for the understanding of the relation between the chemical structure and the antioxidant activity of Ginkgo extracts.
Our study focused on the isolation and characterization of a fraction containing polymeric proanthocyanidins (AWF) from Ginkgo biloba by degradation with phloroglucinol and identification of the break-down products

Results and Discussion
A defatted crude acetone/water (7:3) extract of Ginkgo biloba leaves was repeatedly extracted with ethyl acetate to remove the terpenoids, flavonoids, isoflavonoids, phenolic acids, alkyl phenols and the lower oligomeric proanthocyandins. The remaining watersoluble residue was further fractionated on an LH-20 Sephadex column with methanolwater (1:1) as a mobile phase to separate carbohydrates, relatively polar flavonoids and the higher oligomeric proanthocyanidns from polymeric condensed tannins [8]. The AWF fraction was then eluted with acetone-water (7:3).
In order to distinguish the compounds relevant for the antioxidant activity of Ginkgo biloba leaf extract, the ethyl acetate fraction (EAF), the methanol-water fraction (MWF) and the acetone-water eluted fraction (AWF) were investigated by ESR spectroscopy (Tab. 1). The antioxidant activity of the three fractions (EAF, MWF, AWF) was expressed as its ability to reduce a synthetic free radical species obtained from the reaction with Fremy's salt [9].
By HPLC and TLC-examinations we could demonstrate that the EAF contains mainly terpenoids, flavonoids, flavan-3-ols, lower oligomeric proanthocyanidins and other phenolic compounds. The MWF contains mainly carbohydrates, flavonol glycosides and higher oligomeric proanthocyanidins.
The EAF and the MWF possess relatively strong antioxidant properties (Tab. 1.), most likely related to the excellent radical scavenging properties of the ginkgolides, biflavones and the flavonol glycosides. 37.7 % of the total antioxidant activity of Ginkgo biloba crude extract was detected in the AWF -the fraction which also contained the highest proanthocyanidin concentration. Thus, our objective was to obtain detailed structural information on this proanthocyanidin fraction. The AWF fraction (for details see experimental part) showed an optical rotation of +81° (c 0.1, MeOH), which corresponds to a molar proportion of subunits with a relative 2,3-cis stereochemistry of 83.5 % [10]. By integration of the 13 C NMR-signals close to δ = 77 ppm and δ = 84 ppm (solvent: MeOHd 4 , 99 MHz), an approximately 4.5:1 ratio was obtained for cis : trans isomers [11,12]. The ratio of the signal intensities at (δ = 115-116 ppm) and at 107 ppm revealed that the AWF fraction is composed of approximately 15 % of procyanidin (PC) and 85 % of prodelphinidin (PD) [12]. The mean average molecular size of the polymeric proanthocyanidin in the AWF fraction was estimated to be 9-10 flavan-3-ol units by integration of the C-3 signals of the extender units at 73 ppm and the corresponding signal of the lower flavan-3-ols at 68 ppm [13].

Tab. 1.
Antioxidant activity and proanthocyanidin (PAC) content of the isolated fractions from Ginkgo biloba leaf crude extract. In order to elucidate the structure in more detail, the AWF fraction was degraded in the presence of phloroglucinol under acidic conditions at ambient temperature for 30 min [13,14]. The reaction resulted in the cleavage of the terminal flavanoid units, which were identified as epigallocatechin in relatively high amounts, catechin, gallocatechin, and relatively low quantities of epicatechin. Among the monomeric phloroglucinol-captured products, epigallocatechin-(4β→2)-phloroglucinol was isolated as the main monomeric adduct in addition to small amounts of epicatechin-(4β→2)-phloroglucinol and gallocatechin-(4α→2)-phloroglucinol. The overall ratio of monomers is consistent with the 13 C NMR data. The structures of the flavan-3-ols and the monomeric phloroglucinol adducts were identified on the basis of 1D-and 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives. Comparison of the data with authentic samples from earlier work and with published values allowed the identification of these compounds [16][17][18][19][20][21][22][23].
Compound 1 showed a prominent quasi-molecular ion peak at m/z 1384 [M+Na] + in the MALDI-TOF-MS of its peracetate (compound 1a), which suggests a B-type diflavanoid constitution composed of two (epi)gallocatechin units and one additional phloroglucinol ring. The 1 H-NMR spectrum at ambient temperature showed complex signal duplication due to rotational isomerism. The assignment of signals and the point of interflavan-linkage were achieved by extensive 2D-NMR experiments. The spectral pattern of the heterocyclic region of compound 1a was almost identical with the corresponding procyanidin derivative epicatechin-(4β→8)-epicatechin-(4β→2)-phloroglucinol [17]. The location of the interflavanoid linkages was recognized for 1a by long-range correlations (HMBC) of H-4 (C) with C-8a (D). This key correlation indicates that the flavan-3-ol units are C-4/C-8 linked [25]. Under the conditions employed, compound 1 gave phloroglucinol and epigallocatechin-(4β→2)-phloroglucinol as the main degradation products in the reaction with 0.1 M ethanolic HCl [27]. These degradation products were identified by co-chromatography in comparison with authentic compounds. The high amplitude positive cotton effect at 200-240 nm in the CD-spectrum of 1a confirmed the absolute configuration as 4R [25,26]. In conjunction with the optical rotation [α] 20 = +99.3° (c 0.10, MeOH), compound 1 was characterized as epigallocatechin-(4β→8)-epigallocatechin-(4β→2)-phloroglucinol.
To the best of our knowledge, compounds 1 and 2 as well as the NMR-data of their peracetate derivatives are described for the first time.
In conclusion, we found that the AWF fraction accounted for 37.7 % of the total antioxidant activity of Ginkgo biloba leaf extract.The flavan-3-ol units in the AWF fraction showed great similarity in the chemical structure to the dimeric procyanidins and prodelphinidins previously identified by paper chromatography and HPLC [7]. In addition, the 2,3-cisconfiguration and 3',4',5'-trihydroxylated B-rings were predominant in the isolated polymer, and 3-O-galloylated units were not observed.
Future investigations will deal with the pharmacological testing of the AWF fraction to corroborate their presumed contribution to antioxidant mechanisms and the overall clinical effects of Ginkgo extracts in the treatment of mild to moderate cognitive disorders, tinnitus, and dementia.

Plant material
Ginkgo biloba L. dried plant material (Ch-B.: 4407655) was obtained from Martin Bauer GmbH, Vestenbergsgreuth, Germany. Identification was performed by microscopic investigations. A voucher specimen is retained in the documentation file of the School of Pharmacy, Petra University, under the code Ginkgo 1.

Quantitative analysis of proanthocyanidins
The content of proanthocyanidins in the crude extract and the three fractions (EAF, MWF, AWF) was determined photometrically after acidic depolymerization to the corresponding anthocyanidins [9]. In the crude extract and all fractions, 1 mg of the dried sample was dissolved in 10 mL of a solution of concentrated hydrochloric acid in n-butanol (10:90, v/v). The closed vial containing the solution was mixed vigorously and heated for 90 min in a boiling water bath. After the solution was cooled to room temperature, the absorbance at 550 nm was measured using a Novaspec II spectrophotometer (Pharmacia LKB, Uppsala, Sweden). The content of PAC (calculated as mg cyanidin/L) was calculated by the molar extinction coefficient of cyanidin (ε = 17 360 L mol −1 cm −1 ).

Electron spin resonance (ESR) analysis
For measuring the antioxidant activity of the crude extract and the three fractions, 1 mg of the crude extract and each fraction was dissolved in 1 mL methanol. Aliquots (500 μL) were allowed to react with an equal volume of Fremy's salt (1 mM in phosphate buffer pH 7.4). The ESR spectrum of Fremy's radical was obtained after 20 min, by which time the reaction was completed. Signal intensity was obtained by integration. The antioxidant activity, expressed as molar equivalents of Fremy's salt reduced by one mol of antioxidant, was calculated by comparison with a control reaction with methanol. Spectra were obtained at 21 °C on a Miniscope MS 100 spectrometer (Magnettech). The microwave power and modulation amplitude were set to 10 dB and 1500 mG, respectively. For the measurement, 50 μL of the reaction mixture was added with a micropipette.

Extraction and preparation of the polymeric proanthocyanidin fraction
Air-dried material (5 kg) was exhaustively extracted with acetone/water (7: 3, 35 L). The combined extracts were evaporated in vacuo to a volume of 2 L, filtered to remove the precipitated chlorophyll, concentrated and defatted with petroleum benzene 30-50 °C (liquid crude extract). A part of the liquid crude extract (10 ml) was evaporated to dryness and freeze-dried to yield 2.2 g dried crude extract. Successive extractions of the remaining liquid crude extract with ethyl acetate (8 L) followed by evaporation of the solvent yielded 33.5 g of solid residue in the ethyl acetate fraction (EAF).

Degradation with phloroglucinol and isolation of proanthocyanidin cleavage products
An aliquot of the AWF fraction of Ginkgo biloba obtained as described above (18 g) was treated under shaking with phloroglucinol (13.5 g) in 1% HCl in EtOH (100 mL) for 30 min. at room temperature [14]. The solution was concentrated under reduced pressure to give 31.5 g of reaction products (PA degradation fraction).