Abstract

The root bark of Anisophyllea dichostyla R. Br. is traditionally used in the Democratic Republic Congo for the treatment of several conditions such as anorexia, fatigue and intestinal infections. We have identified and quantitated several polyphenol antioxidants in the methanol extract of the root bark (120 g). The polyphenol content (3.32 g/kg) was predominantly ellagitannins (25%) and polyhydroxyflavan-3-ols (catechins and procyanidins, 75%) with 3′-O-methyl-3,4-methylenedioxo ellagic acid 4′-O-β-d-glucopyranoside and (–)-epicatechin as the major species in each class. These two compounds and the following species were identified unequivocally by NMR spectroscopy: (+)-catechin, (−)-epicatechin 3-O-gallate, 3-O-methyl ellagic acid, 3,3′-di-O-methyl ellagic acid, 3′-O-methyl-3,4-methylenedioxo ellagic acid, 3′-O-methyl-3,4-methylenedioxo ellagic acid 4′-O-β-d-glucopyranoside, and 3′-O-methyl ellagic acid 4-O-β-d-xylopyranoside. The following additional compounds were purified by semi-preparative HPLC and tentatively identified on the basis of UV spectra, HPLC–ESI-MS and nano-ESI-MS–MS: (+)-catechin-3-O-β-d-glucopyranoside, epicatechin-(4β → 8)-catechin (procyanidin B₁), epicatechin-(4β → 8)-epicatechin (procyanidin B₂), an (epi)catechin trimer, 3-O-methyl ellagic acid 4-O-β-d-glucopyranoside, (–)-epicatechin 3-O-vanillate, 3,4-methylenedioxo ellagic acid 4′-O- β-d-glucopyranoside, and 3,3′-di-O-methyl ellagic acid 4-O-β-d-xylopyranoside. Fractionation of the raw extract by column chromatography on silicic acid yielded 10 fractions. In the hypoxanthine/xanthine oxidase antioxidant assay system, CC-9 which contained a range of polyphenols dominated by (−)-epicatechin-O-gallate proved to be the most potent antioxidant fraction (IC₅₀ = 52 μg/mL) in terms of ROS scavenging. In terms of XO inhibition CC-8, dominated by (epi)catechin trimer and which also contained appreciable amounts of 3′-O-methyl ellagic acid 4′-O-β-d-xylopyranoside, as well as the catechins (+)-catechin-3-O-β-d-glucopyranoside, epicatechin-(4β → 8)-catechin (procyanidin B₁), and (−)-epicatechin 3-O-gallate, proved to be the most potent (IC₅₀ = 36 μg/mL).

Keywords: Anisophyllea dichostyla; Antioxidant capacity; Chemoprevention; Ellagitannins; HPLC; HPLC–ESI-MS; NMR; Procyanidins; Xanthine oxidase

Abbreviations: BSTFA, N-methyl-N-(trimethylsilyl)-trifluoroacetamide; CC, Column chromatography; COLOC, Correlation spectroscopy for long-range couplings; COSY, Correlation spectroscopy; DCM, Dichloromethane; DEPT, Distortionless enhancement polarization transfer; DMSO, Dimethyl sulphoxide; D₂O, Deuterium oxide; nano-ESI-MS, Nano-electrospray mass spectrometry; NOE, Nuclear Overhauser effect; HPLC, High-performance liquid chromatography; HPLC–ESI-MS, High-performance liquid chromatography electrospray ionization mass spectrometry; NMR, Nuclear magnetic resonance; ROESY, Rotating-frame Overhauser-effect spectroscopy; ROS, Reactive oxygen species; TFA, Trifluoroacetic acid; TMS, Tetramethylsilane; XO, Xanthine oxidase

Article Outline

1. Introduction

2. Materials and methods

2.1. Chemicals and reagents

2.2. Plant material

2.3. Extraction protocol

2.4. Column chromatography on silicic acid

2.5. Analytical HPLC

2.6. HPLC–ESI-MS

2.7. Semi-preparative HPLC and fraction collection

2.8. Nano-ESI-MS

2.9. Acid hydrolyses

2.10. NMR spectroscopy

2.11. Measurement of antioxidant capacity

2.12. Statistics

3. Results

3.1. Isolation and identification of polyphenols

3.2. Quantitation of polyphenols

3.3. Antioxidant capacity

3.4. Structure elucidation and NMR signal assignments

3.4.1. Catechin and epicatechin

3.4.2. Epicatechin 3-O-gallate

3.4.3. Ellagitannins

3.4.4. 3-O-methyl ellagic acid

3.4.5. 3,3′-di-O-methyl ellagic acid

3.4.6. 3′-O-methyl-3,4-methylenedioxo ellagic acid

3.4.7. 3′-O-methyl-3,4-methylenedioxy ellagic acid 4′-O-β-d-glucopyranoside

3.4.8. 3′-O-methyl ellagic acid 4-O-β-d-xylopyranoside

4. Discussion

Acknowledgements

References