A New Flavonoid Glycoside from Salix denticulata Aerial Parts

Abstract

A new flavonoid glycoside (1) has been isolated from the aerial parts of Salix denticulata (Salicaceae) together with five known compounds, β-sitosterol, 2,6-dihydroxy-4-methoxy acetophenone, eugenol-1-O-β-d-glucopyranoside, 1-O-β-d-(3’-benzoyl) salicyl alcohol and luteolin-7-O-β-d-glucopyranosyl-(1-6)-glucopyranoside. The structure of 1 was elucidated as 2’,5-dihydroxy-3’-methoxyflavone-7-O-β-d-glucopyranoside by means of chemical and spectral data including 2D NMR studies.

1. Introduction

Salix denticulata which belongs to the Salicaceae family is a deciduous shrub indigenous to Central Himalayas (3000 meter) of India and is well known for its medicinal importance [1]. Previous studies on the plants of this genus led to isolation and elucidation of different compounds such as terpenoids [2], catechins [3], lignans [4], flavones [5,6] and other phenolic compounds [7]. This paper illustrates the isolation and structure revelation of a novel flavonoid glycoside (1) from the aereal parts of S. denticulata with the help of modern spectroscopic methods.

2. Results and discussion

Compound 1 was isolated as yellow crystalline solid, m.p. 210-212 °C, deduced molecular formula C₂₂H₂₂O₁₁ from its FAB-MS. It gave positive Molisch test, Shinoda test and blue color with FeCl₃, characteristic for flavone glycosides. The IR spectrum showed characteristic absorption bands for hydroxy (3350 cm^-1) and carbonyl (1460 cm^-1) functions. The ¹H NMR spectrum showed doublets at δ 6.46 (J = 1.8 Hz, H-6) and δ 6.76 (J = 1.8 Hz, H-8), indicating a tetrasubstituted aromatic ring. Other doublets at δ 7.12 (J = 3.4 Hz, H-3’), 6.92 (J = 8.4 Hz, H-6’) and 7.44 (J = 3.4, 8.4 Hz, H-5’) revealed the trisubstituted aromatic ring. The position of two singlets at δ 12.9 and 9.5 indicated two hydroxy groups, in which former is chelated with a carbonyl function and assigned at position OH-5. A sharp singlet at δ 3.61 correlated to C-4’ (δ 145.81), indicating OCH₃-4’. A doublet at δ 5.08 (J = 7.2 Hz) indicated anomeric signal with other signals in the range of δ 3.2-4.6 for a β linked sugar. In the ¹³C NMR spectrum, the downfield signal at δ 181.9 indicated a carbonyl group. The positions of substituted groups were confirmed by ¹H-¹³C correlation in HMBC (Figure 2) and HSQC. The correlation of H-3’ (δ 7.12) to C-4’ (δ 145.81) and C-5’ (δ 116.02); H-5’ (δ 7.44) to C-3’ (δ 113.5), C-4’ (δ 145.81) and C-6’ (δ 119.21) and H-6’ (δ 6.92) to C-1’ (δ 121.42) and C-4’ (δ 145.81) reveled the substitution at C-2’ (OH) and C-4’ in ring B. The correlation between the anomeric proton (δ 5.09) and C-7 (δ 162.98) indicated position of sugar at C-7. The sugar was identified as d-glucose by hydrolysis and direct comparison (co-PC) with authentic sugar. The chemical structure of compound 1 is given in Figure 1.

3. Experimental Section

3.1. General

Melting points were recorded on a Perfit melting point apparatus. UV spectra were measured on a Perkin-Elmer Lambda-25 spectrophotometer in methanol. IR spectra recorded on a Perkin-Elmer Spectrum RX1 FT-IR spectrometer (KBr discs). NMR spectra were obtained on Bruker Avance 300 and 500 spectrometers (300 MHz for ¹H and 125 MHz for ¹³C, CDCl₃ as solvent, TMS as internal standard). MS were recorded on Qualtro II-EIMS and Jeol SX-102 (FAB) mass spectrometer. Column chromatography was performed on silica gel (Merck 60-120 mesh, 15 × 100 cm). TLC was carried out on silica gel (Merck 10-40 μ) precoated plates, spots were visualized by spraying with 7% H₂SO₄.

3.2. Plant material

Aerial parts of S. denticulata were collected from Tungnath, Chamoli during the month of May and identified from Taxonomy Laboratory, Department of Botany, H.N.B. Garhwal University Srinagar. A voucher specimen (GUH-8036) of the plant has been kept in the Departmental Herbarium for future records.

3.3. Extraction and isolation

The shade dried aerial parts of S. denticulata (6 kg) were powdered and extracted exhaustively with 95% ethanol (3 times) to yield a black brown extract, which was concentrated under reduced pressure and defatted with n-hexane. The extract (380 g) was pre-adsorbed with silica gel and applied on the top of a column prepared by silica gel (500 g) in CHCl₃. The elution was first started with CHCl₃ and then CHCl₃ with increasing amounts of MeOH (0-30%). Elution with CHCl₃:MeOH = 22:3 afforded compound 1, whereas 9:1, 43:7, 41:9, 8:2 and 21:4 furnished β-sitosterol [8], 2,6-dihydroxy-4-methoxyacetophenone [9], eugenol-1-O-β-d-glucopyranoside, 1-O-β-d-(3’-benzoyl)-salicyl alcohol [10] and luteolin-7-O-β-d-glucopyranosyl-(1→6)-glucopyranoside [11,12], respectively.

3.4. 5-Hydroxy-2-(2-hydroxy-4-methoxyphenyl)-4-oxo-4H-chromen-7-yl β-d-glucopyranoside (1)

Yellow amorphous solid (60 mg); m.p. 210-212 °C (uncorr.); UV: ${\mathsf{\lambda }}_{\max }^{MeOH}$: 253, 278 and 353 nm; IR: ${\mathsf{\upsilon }}_{\max }^{KBr}$: 3373, 2907, 1703, 1293 cm^-1; NMR data: see

Table 1. ¹³C, ¹H-NMR, HSQC and HMBC data of compound 1 in CDCl₃.

Position	δC ppm	δH ppm (J Hz)	HSQC	HMBC
2	161.16	-	-C-	-
3	103.20	6.79, s	-CH-	4, 4a, 1’
4	181.93	-	-C-	-
4a	105.37	-	-C-	-
5	164.50	-	-C-	-
6	99.57	6.46 (d, 1.8)	-CH-	4a, 5, 7, 8
7	162.98	-	-C-	-
8	97.76	6.76 (d, 1.8)	-CH-	6, 7, 4a, 8a
8a	156.98	-	-C-	-
1’	121.42	-	-C-	-
2’	149.95	-	-C-	-
3’	113.59	7.12 (d, 3.4)	-CH-	4’, 5’
4’	145.81	-	-C-	-
5’	116.02	7.44 (dd, 3.4, 8.4)	-CH-	1’, 3’, 4’, 6’
6’	119.21	6.92 (d, 8.4)	-CH-	1’, 2’, 4’, 5’
1”	99.92	5.09 (d, 7.2)	-CH-	7
2”	70.36	3.29 (d, 8.8)	-C-	-
3”	73.15	3.34 (t, 8.8)	-CH-	-
4”	76.42	3.32, m	-CH-	-
5”	77.19	3.49, m	-CH-	-
6”	60.65	3.73, m	-CH2-	-
OCH3-4’	55.82	3.61, s	-CH3	4’
OH-5	-	12.9	-	-
OH-2’	-	9.5	-	-

; FAB-MS (m/z): 462 [M]⁺, 300 [M-glu]⁺ 149 [C₉H₉O₂]⁺; calcd. C 57.14, H 4.80; found C 57.86, H 4.37.