Abstract
Context: Ginkgo biloba L (Ginkgoaceae) is a traditional herbal medicinal plant for the treatment of mild to moderate cognitive disorders, tinnitus, and dementia. These uses may be correlated with the presence of radical scavenging compounds.
Objective: The chemical composition and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of the flavan-3-ols and proanthocyanidins from G. biloba were studied.
Material and methods: The compounds have been isolated using column chromatography on Sephadex LH-20 and MCI gel and the structures were determined on the basis of 1D- and 2D-NMR (HSQC, HMBC) experiments of their peracetylated derivatives, MALDI-TOF-MS and by acid-catalyzed degradation with phloroglucinol. The DPPH radical scavenging activities of the compounds were investigated.
Results: The new trimeric prodelphinidin, epigallocatechin-(4β→8)-epigallocatechin-(4β→8)-catechin (compound 7), has been isolated from the air-dried leaves of the title plant, in addition to catechin, epigallocatechin, gallocatechin, and three dimeric proanthocyanidins. The dimeric prodelphinidin epigallocatechin-(4β→8)-epigallocatechin (compound 6) showed the strongest DPPH radical scavenging activity, with IC50 1.7 μg/mL, 10 times more active than the positive control, BHT (IC50 17.3 µg/mL), followed by the new trimeric proanthocyanidin epigallocatechin-(4β→8)-epigallocatechin-(4β→8)-catechin with IC50 2.1 µg/mL. The crude extract exhibited high DPPH radical scavenging activity with IC50 15.5 µg/mL comparable with that of BHT.
Discussion and conclusion: The results showed that all the isolated compounds from the tannin fraction exhibited potent free radical scavenging activities, which were higher than that of BHT, suggesting that the condensed tannins from G. biloba leaves strongly contribute to the overall antioxidant effects.
Introduction
Herbal medicinal products, based on Ginkgo extracts, are among the top-selling phytopharmaceuticals. The leaves of Ginkgo biloba L. (Ginkgoaceae), Maidenhair tree, are the starting material of pharmaceutical preparations for the treatment of insufficient central and peripheral blood flow (CitationOberpichler-Schwenk & Kriegelstein, 1992). Ginkgo extracts are also used as powerful antioxidants (CitationSiddique et al., 2000). The characteristic ingredients to which the beneficial effects are attributed are ginkgolides, bilobalide, and biflavonoids (CitationSingh et al., 2008; Citationvan Beek & Montoro, 2009).
Large amounts of proanthocyanidins (4–12%) (CitationLang & Wilhelm, 1996) are found in Ginkgo leaves and standardized extracts contain 7% (CitationSchenen, 1988). In spite of their importance, relatively little is known about the composition of the proanthocyanidins in the title plant. The compounds that were identified using paper chromatography and high-performance liquid chromatography (HPLC) were dimeric procyanidins and prodelphinidins (CitationStafford et al., 1986). Many researchers have reported that flavan-3-ols and proanthocyanidins had antioxidant and radical scavenging activities (CitationRösch et al., 2003; CitationQa’dan et al., 2006). The knowledge of the chemical structure of the proanthocyanidin fraction in Ginkgo leaves is of great importance for better understanding of their role as powerful radical scavengers, antioxidative agents and for the determination of the contribution of this group of compounds to the total radical scavenger activity of the crude extract.
During our phytochemical investigations of G. biloba, one new trimeric prodelphinidin, epigallocatechin-(4β→8)-epigallocatechin-(4β→8)-catechin (compound 7), in addition to catechin (compound 1), epigallocatechin (compound 2), gallocatechin (compound 3), and three known dimeric proanthocyanidins (compounds 4–6) were isolated from the leaves of this plant. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of all purified compounds was evaluated ().
Figure 1. Chemical structures of the isolated compounds from Ginkgo biloba leaves.
Materials and methods
General experimental procedures
Butylated hydroxytoluene (BHT, ≥99.0%) was obtained from Sigma (St. Louis, MO). DPPH was obtained from Fluka (Munich, Germany). All other reagents used were of analytical grade.
1H-NMR spectra were recorded in CDCl3 on a Bruker AM 600 (600 MHz) relative to CHCl3. 13C-NMR spectra were recorded at 150 MHz. CD data were obtained in MeOH on a Jasco J 600. MALDI-TOF mass spectrometer: LAZARUS II (home-built), N2-laser (LSI VSL337ND) 337 nm, 3 ns puls width, focus diameter 0.1 mm, 16 kV acceleration voltage, 1 m drift length, data logging with LeCroy9450A, 2.5 ns sampling time and expected mass accuracy ±0.1%, sample preparation: acetylated compounds were deposited from a solution in CHCl3 on a thin layer of 2,5-dihydroxybenzoic acid (DHB) crystals.
Analytical TLC was carried out on aluminum sheets (Kieselgel 60 F254, 0.2 mm, Merck, Munich, Germany) using acetone–toluene–formic acid (60:30:10) as mobile phase. Compounds were visualized by spraying with vanillin–HCl reagent and 1% methanol FeCl3 solution. Acetylations were performed in pyridine–acetic acid anhydride (1:1.2) at ambient temperature for 24 h.
Plant material
G. biloba L. (Ginkgoaceae) 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.
Extraction and isolation
Air-dried material (5 kg) was exhaustively extracted with acetone/water (7:3, 35 L) and the combined extracts evaporated in vacuo to 2 L, filtered to remove the precipitated chlorophyll, concentrated and defatted with petroleum benzene 30–50°C. Successive extractions with ethyl acetate (8 L) followed by evaporation of solvent yielded 33.5 g ethyl acetate fraction (EAF).
The EAF (30.0 g) were subjected to CC on Sephadex LH-20 (5.5 × 68 cm) with EtOH–H2O (6 L), EtOH–MeOH 1:1 (7 L), MeOH (3 L), and acetone–H2O 7:3 (4 L) to give 10 fractions. Fraction 3 (3800–4200 mL, 1.3 g) was subjected to chromatography on MCI gel CHP 20 P (25 × 250 mm) with a 10–80% MeOH linear gradient (17 mL/fraction) to afford catechin (compound 1) (subfractions 29–41, 23 mg). Fraction 4 (4200–4900 mL, 1.1 g) was separated by MCI gel CHP 20 P (25 × 250 mm) with a 10–80% MeOH linear gradient (17 mL/fraction) to afford epigallocatechin (compound 2) (subfractions 21–27, 219 mg) and gallocatechin (compound 3) (subfractions 69–89, 46 mg). Fraction 4 (4900–5770 mL, 2.2 g) was separated on MCI gel with the same gradient as above to afford epigallocatechin-(4β→6)-catechin (compound 5) (subfractions 30–34, 7 mg) and epigallocatechin-(4β→8)-catechin (compound 4) (subfractions 45–54, 19 mg).
Epigallocatechin-(4β→8)-epigallocatechin (compound 6) was achieved from fraction 8 (9200–9700 mL, 1.1 g) followed by MCI gel chromatography (subfractions 41–49, 71 mg). All compounds were identified after acetylation by their physical data (NMR, MS, CD) and by comparison with authentic samples and published values (CitationDe Mello et al., 1996; CitationPloss et al., 2001; CitationQa’dan et al., 2003).
The remaining water phase was evaporated to dryness (450 g). A portion (250 g) of the water phase was successively applied to CC on Sephadex LH-20 (55 × 900 mm) with 5 L EtOH–H2O and 20 L MeOH–H2O (1:1) to afford eight fractions.
Epigallocatechin(4β→8)-epigallocatechin-(4β→8)-catechin (compound 7)
Fraction 5 (4750–5100 mL, 156 mg) achieved from Sephadex LH-20 column was subjected to chromatography on MCI gel CHP 20P (25 × 450 mm) with a 20–60% MeOH linear gradient (17 mL/subfraction) to afford an amorphous powder (subfraction 14–23, 31 mg). Compound 7: [α]20 = +107.6° (c = 1.65, MeOH). Eighteen milligrams were acetylated to give compound 7a: MALDI-TOF-MS: [M+Na]+ m/z 1637. 1H-NMR (CDCl3, 600 MHz; duplication due to the dynamic rotational isomerism; two sets of signals in the ratio ca. 2:1; signal set of the minor rotamer was not determined): δ 1.44–2.39 (m, OAc), δ 2.70 [dd, J = 8.3/16.8 Hz, H-4a (I)], δ 3.16 [dd, J = 5.5/16.8 Hz, H-4b (I)], δ 2.61 [m, H-4 (I)], δ 4.62 [brs, H-4 (F)], δ 4.71 [brs, H-4 (C)], δ 5.09 [d, J = 7.8 Hz, H-2 (I)], δ 5.18 [m, H-3 (I)], δ 5.36 [brs, H-2 (C)], δ 5.39 [m, H-3 (F)], δ 5.44 [m, H-3 (C) and H-2 (F)], δ 6.64 [d, J = 2.3 Hz, H-6 (A)], δ 6.69 [s, H-6 (G)], δ 6.75 [s, H-6 (D)], δ 6.77 [d, J = 2.3 Hz, H-8 (A)], δ 6.85 [d, J = 2.1 Hz, H-2′ (H)], δ 6.88 [brs, H-2′/H-6′ (B)], δ 6.95 [brs, H-2′/H-6′ (E)], δ 7.08 [d, J =8.3 Hz, H-5′ (H)]. 13C-NMR (CDCl3, 150 MHz): δ 25.6 [CR-4 (I)], δ 25.8 [C-4 (I)], δ 33.9 [C-4 (C)], δ 35.0 [C-4 (F)], 62.5 [CR-3 (I)], 67.5 [C-3 (I)], δ 71.1 [C-3 (C)], δ 73.3 [C-2 (F)], δ 74.3 [C-2 (C)], 78.9 [CR-2 (I)], δ 79.3 [C-2 (I)], δ 108.7 [C-6 (A)], δ 109.5 [C-8 (A)], δ 110.8 [C-4a (G)], δ 111.2 [C-4a (A)], δ 112.8 [C-4a (D)], δ 111.3 [C-6 (D)], δ 116.5 [C-8 (G)], δ 117.1 [C-8 (D)], δ 118.3 [brs. [C-2′(H), C-2′ (E), and C-6′ (E)]], 119.3 [C-2′ (B) and C-6′ (B)], δ 121.9 [C-6′ (H)], δ 123.3 [C-5′ (H)], δ 133.5 [C-1′ (E)], δ 134.2 [C-4′ (E)], δ 135,0 [C-1′ (H)], δ 135.1 [C-4′ (B)], 135.2 [C-1′ (B)], 141.2 [C-4′ (H)], δ 142.0 [C-3′ (H)], δ 142.1 [C-3′ (E) and C-5′ (E)], δ 147.1 [C-7 (D)], δ 148.1 [C-5 (G)], δ 149.0 [C-7 (A)], δ 151.6 [C-8a (G)], δ 152.1 [C-8a (D)], δ 154.7 [C-8a (A)].
Purified proanthocyanidin (10 mg, compound 7) was reacted with phloroglucinol (10 mg) in 1% HCl in EtOH (1 mL) for 15 min at room temperature with continuous shaking (CitationFoo & Karchesy, 1989). The solution was then concentrated under a stream of N2 to dryness and purified on preparative TLC. The main phloroglucinol adduct was further purified on prep. TLC on cellulose (t-BuOH–CH3COOH–H2O; 60:20:20) to yield epigallocatechin-(4β→2)-phloroglucinol (6.7 mg).
DPPH free radical scavenging activity assay
The free radical scavenging activity was measured according to the method of CitationBlois (1958). A 1.5 mL aliquot of 0.25 mM DPPH solution in ethanol and 1.5 mL of the crude extract and isolated compounds were mixed. The mixture was shaken vigorously and allowed to reach steady state at room temperature for 30 min. Decolorization of DPPH was determined by measuring the absorbance at 517 nm with a Varian spectrophotometer. The DPPH radical scavenging activity was calculated according to the following equation:
where A0 is the absorbance of the control (blank, without isolated compounds) and A1 is the absorbance in the presence of the isolated compound or standard sample.
Sample solution (100 μL) at different concentration (10–500 μg/mL) was added to 3 mL of DPPH solution (0.1 mM methanol solution).
Results and discussion
Phytochemical analysis
The EAF obtained from the aqueous acetone extract of the leaves of G. biloba was chromatographed on Sephadex LH-20 and MCI gel chromatography, etc (s. Exp.). Three known dimeric prodelphinidins, epigallocatechin-(4β→8)-catechin (compound 4), epigallocatechin-(4β→6)-catechin (compound 5), and epigallocatechin-(4β→8)-epigallocatechin (compound 6), have been isolated in addition to the flavanols catechin (compound 1), epigallocatechin (compound 2), and gallocatechin (compound 3). The identity of all compounds was established by physical properties (1D- and 2D-NMR, circular dichroism (CD), optical rotation [α], and MALDI-TOF-MS) of the corresponding derivatives obtained after peracetylation, compared with authentic samples and published data (CitationDe Mello et al., 1996; CitationPloss et al., 2001; CitationQa’dan et al., 2003).
The remaining aqueous phase (s. Exp.) was further fractionated on Sephadex LH-20 and MCI gel to give compound 7.
The structure of compound 7 was determined on the basis of its 1D- and 2D-NMR (HSQC, HMBC), CD, and [α] data of its peracetylated derivative.
Compound 7 showed a prominent quasimolecular ion peak at m/z 1637 [M+Na]+ in the MALDI-TOF-MS of its peracetate (compound 7a), indicative of a trimeric proanthocyanidin composed of two (epi)gallocatechin units and one (epicatechin) moiety. The 1H-NMR of compound 7a showed close structural resemblance to that of the analogous trimeric prodelphinidin epigallocatechin-(4β→8)-gallocatechin-(4α→8)-catechin (CitationQa’dan et al., 2003), except for the different stereochemistry of one of the extender units. The pyrogallol type B- and E-rings of the extender units could be determined with the long-range coupling (4J) between the H-2 (C) and H-2 (F) proton signals through the respective C-2′ and C-6′ carbons. The small heterocyclic coupling constants of J2,3(C) and J2,3(F) confirmed the relative 2,3-cis-stereochemistry of both extender units corresponding to two epigallocatechin units (CitationFletcher et al., 1977). The C-4/C-8 bonding position of the interflavanoid linkages likewise was recognized by 1H-13C long-range correlations (HMBC) of the H-4 (C) with the C-8a (D) and the H-4 (F) with the C-8a (G) (CitationBalas & Vercauteren, 1994). The structure elucidation of compound 7 was corroborated by acid-catalyzed reaction in the presence of phloroglucinol (CitationFoo & Karchesy, 1989). Compound 7 gave phloroglucinol and epigallocatechin-(4β→2)-phloroglucinol, and a small amount of epigallocatechin-(4β→8)-epigallocatechin-(4β→2)-phloroglucinol as degradation products, and catechin as releasing terminal flavan-3-ol. These degradation products were identified by co-chromatography in comparison with authentic compounds (CitationQa’dan et al., 2006). The high amplitude positive cotton effect at low wavelengths (210–240 nm) in the CD spectrum of compound 7a confirmed the absolute configuration of the extender units as 4R (CitationBarrett et al., 1979; CitationBotha et al., 1981). In conjunction with the optical rotation [α]20 = +107.6° (c = 1.65, MeOH), compound 7 was characterized as epigallocatechin-(4β→8)-epigallocatechin-(4β→8)-catechin.
To the best of our knowledge, compound 7 and the NMR data of its peracetate derivative have not been described previously. In conclusion, the isolation and characterization of the three dimeric proanthocyanidins, in addition to the trimer epigallocatechin-(4β→8)-epigallocatechin-(4β→8)-catechin, reflect the relative frequency of the type of the interflavanoid linkages in the lower oligomeric fraction. Interflavanoid linkages seem to be predominantly 4→8 bonds. In addition, the 2,3-cis-configuration and the 3′,4′,5′-trihydroxylated B-rings were predominant in the isolated flavan-3-ol units and proanthocyanidins and the absence of 3-O-galloylated units was observed.
The ability of the crude extract and the isolated compounds to reduce the free radical DPPH to yellow-colored DPPH was determined by the DPPH assay test at 517 nm (CitationBlois, 1958). The free radical scavenging activity increased with increasing concentrations of the crude extract, flavan-3-ols, proanthocyanidins, and butylated hydroxytoluene (BHA). Based on these data, it can be concluded that the isolated flavan-3-ols (compounds 1–3) and proanthocyanidins (compounds 4–7) exhibited potent DPPH radical scavenging activity, with IC50 values between 6.3 µg/mL (compound 1) and 1.7 µg/mL (compound 6), 3–10 times more active than the positive control, BHT. The crude extract was found to have relatively higher DPPH radical scavenging activity with IC50 15.5 μg/mL. The latter could be due to the proanthocyanidins, biflavonoids, flavonol derivatives, and ginkgolides, which are present in the crude extract and are known to have powerful antioxidant and DPPH radical scavenging activities (CitationGuang-Hua et al., 2009). The results showed that the antioxidant activity of the plant extract and isolated compounds have the following order: epigallocatechin-(4β→8)-epigallocatechin (compound 6) ≥ epigallocatechin-(4β→8)-epigallocatechin-(4β→8)-catechin (compound 7) > gallocatechin (compound 3) ≈ epigallocatechin (compound 2) > epigallocatechin-(4β→6)-catechin (compound 5) > epigallocatechin-(4β→8)-catechin (compound 4) > crude extract (CE) (). Our results confirmed the antioxidant potential of flavan-3-ols and proanthocyanidins (CitationRösch et al., 2003; CitationQa’dan et al., 2006). As expected, the monomeric compounds 2 and 3 showed higher radical scavenging activity than catechin (compound 1) (CitationXia et al., 2003). Although the new trimer (compound 7) has numerous hydroxyl groups, epigallocatechin-(4β→8)-epigallocatechin (compound 6) was slightly more effective than compound 7. Similarly, the monomeric compounds 2 and 3 were more effective than the dimeric compounds 4 and 5. Therefore, the number of hydroxyl groups did not always seem to be important for the DPPH radical scavenging activities.
Table 1. Radical scavenging activities of the crude extract, the positive control [butylated hydroxytoluene (BHT)] and the isolated compounds 1–7 against DPPH.
Conclusion
The results showed that the flavan-3-ols and proanthocyanidin fraction exhibited potent free radical scavenging activities, which were higher than that of BHT, suggesting that the condensed tannins from G. biloba leaves strongly contribute to the overall antioxidant effects. Future investigations will include pharmacological testing of individual compounds to demonstrate the contribution of these antioxidants to the medicinal uses for the treatment of mild to moderate cognitive disorders, tinnitus, and dementia.
Acknowledgements
We wish to acknowledge the help of Prof. Dr. R. Anwander (Inst. f. anorganische Chemie, Tuebingen) for the NMR spectra, Dr. H. Luftmann (Inst. f. organische Chemie, Muenster) for the MALDI-MS-spectra and Prof. Dr. V. Buß (Theoretische Chemie, Duisburg) for the CD spectra.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing the paper.
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