Phenolic Constituents from Fallopia multiflora (Thunberg) Haraldson

Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Institute of Marine Biochemistry, VAST, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Faculty of Chemical Technology, Hanoi University of Industry, 298-Cau Dien, Bac Tu Liem, Hanoi, Vietnam Center for Research and Technology Transfer, VAST, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam


General.
in layer chromatography was performed using precoated Kieselgel 60 F254 (Merck) and visualized by UV light 254 nm and 10% H 2 SO 4 reagent under heating.Column chromatography was performed using silica gel 60 (Merck, 70-230 mesh).Optical rotations were read on a JASCO P-2000 digital polarimeter.NMR experiments were performed on a Bruker AM500 FT-NMR spectrometer.e HR-ESI-MS data were obtained from a LTQ Orbitrap XL Mass Spectrometer ( ermo Scientific).GC analysis was carried out on an Agilent 7890B GC System using a column SPB-1 (0.25 mm × 30 m), detector FID, column temp.210 °C, injector temperature 270 °C, detector temperature 300 °C, and He carrier gas.

Plant Material.
e roots of F. multiflora were collected in Dong Van commune, Ha Giang province, Vietnam, on October 2014 and identified by Dr. Nguyen e Cuong, Institute of Ecology and Biological Resources, VAST.Voucher specimens (HaGiang-03) have been deposited at the herbarium of the Institute of Ecology and Biological Resources.

Extraction and Isolation.
e air-dried and powdered roots of F. multiflora (3.5 kg) were extracted with methanol (6 L × 3 times) in a sonic bath for 30 min at 40 °C.e combined extracts were evaporated under reduced pressure to give crude extract (430.0 g), which was then resuspended in water (3 L) and successively partitioned by n-hexane and ethyl acetate (each 1 L × 3 times) to obtain n-hexane (51.7 g) and ethyl acetate (214.5 g) residues, respectively.e aqueous solution was passed through a Diaion HP-20 column and eluted by 0, 50, and 100% methanol in water to give three fractions W1-3.Fraction W3 was chromatographed on a silica gel column using mobile phase of a gradient of 0-100% methanol in dichloromethane to obtain seven fractions F1-7.Fraction F4 was purified by a silica gel column chromatography eluting with dichloromethane-methanol 6 : 1 (v/v) to yield 9 (457.0mg).Fraction F5 was passed through a C-18 column using acetone-water 2 : 3 (v/v) as eluent to obtain 3 (8.0mg).Compounds 1 (12.8 mg), 2 (5.4 mg), and 7 (4.5 mg) were purified from F6 by a silica gel column eluted by dichloromethane-methanol 6 : 1 (v/v).Repeated column chromatography using C18 and Sephadex LH-20 stationary phases were applied for fraction F7 to afford compounds 6 (6.8 mg) and 10 (6.0 mg).

Acid Hydrolysis and Sugar Identification.
e compounds 1 and 3 (each 1 mg) were individually dissolved in 1 ml of dioxane/1 N HCl (1 : 1 v/v) and heated at 80 °C for 3 h.e acidic solution was neutralized with silver carbonate and extracted with CH 2 Cl 2 .e aqueous layer was concentrated to dryness using nitrogen gas.e residue was then dissolved 2 Journal of Chemistry in 0.1 mL of pyridine, followed by addition of 0.1 mL of 0.06 M L-cysteine methyl ester hydrochloride in pyridine.After heating at 60 °C for 2 h, 0.1 mL trimethylsilylimidazole was then added to the solution, followed by heating at 60 °C for another 1.5 h.e dried product was partitioned with nhexane and water (0.1 mL each), and the organic layer was analyzed by GC.D-glucose and D-apiose were detected at 14.15 and 4.61 min, respectively.
e coupling constants indicated the β (J � 7.5 Hz) and α (J � 2.5 Hz) conformations of the glycosyl units.Analysis of the 13 C NMR and HSQC spectra of 1 revealed the presence of an aromatic methyl at δ C 20.2 (3-CH 3 ), an acetyl group (δ C 208.2 and 32.6) and ten aromatic signals of a naphthalene skeleton (Table 1).In addition, an apiofuranosyl-(1→6)glucopyranoside moiety was recognized [5].Acid hydrolysis and GC analysis led to the identification of D-glucose and D-apiose.Comparison with reported data suggested the skeleton was 6-hydroxymusizin [6].
e 13 C NMR and HSQC spectra of 3 showed the signals characteristic for a methoxy, an aromatic methyl, a glucose moiety and a naphthalene skeleton.ese data were almost identical to that of torachrysone 8-O-β-D-glucopyranoside (2) except for the replacement of the acetyl group by a carboxylic group (δ C 171.0) at C-2 [7,8].e HMBC correlations confirmed the position of the methoxy and glucose groups at C-6 and C-8, respectively (Figure 2).Acid hydrolysis and GC analysis led to the identification of D-glucose.erefore, compound 3 was deduced as 6-methoxy-3-methyl-1,6,8-trihydroxy-2naphthoic acid 8-O-β-D-glucopyranoside.
By means of spectroscopic methods and in comparison with the reported data, the structure of the known compounds was identified as torachrysone 8-O-β-D-glucopyranoside (2),  9), benzyl gentiobioside (10), and catechin (11).Naphtolic derivatives including torachrysone 8-O-β-D-glucopyranoside (2) have been identified in the Polygonaceae [3,[8][9][10][11][12] and other families such as Fabaceae, Rhamnaceae, Araliaceae, and Euphorbiaceae [13][14][15][16].All those compounds possessed a hydroxyl group at C-1, an acetyl group at C-2, and a methyl at C-3 in the naphthalene skeleton.However, compound 3 bearing a carboxylic group instead of an acetyl group in the same skeleton has not been found previously from the natural source.us, compound 3 could be considered as a specific chemotaxonomic marker for F. multiflora.e anthraquinones and stilbenes including emodin (5), emodin 8-O-glucopyranoside (6) and 2,3,5,4′-tetrahydroxystilbene-2-O-β-glucoside ( 9) are main constituents of F. multiflora and have been listed as chemical markers for quality control of this plant material [2,17].e present study reports for the first time the occurrence of benzyl gentiobioside (10) in Polygonaceae, which can be used as a characteristic chemical constituent of F. multiflora in contrast to other species belonging to the genus Fallopia and family Polygonaceae.

Table 1 :
NMR data of compounds 1 and 3.