Flavone Glycosides from Calycotome Villosa Subsp. Intermedia

Abstract

The known flavonoid chrysin-7-O-(β-D-glycopyranoside) (chrysin glucoside, 1) as a major fraction and a new glycoside flavone, chrysin-7-O-β-D-[(6”- acetyl)glycopyranoside] (2) were isolated from the flowers and leaves of Calycotome Villosa Subsp. Intermedia, They were identified by UV-Vis, IR, ¹H-, ¹³C-NMR and ESI-MS.

Introduction

Calycotome villosa (Poiret) Link Subsp. Intermedia (C.Presl) belongs to the Papillionacea family. It is a 30-150 cm spiny shrub displaying yellow flowers during the spring that grows especially in the north of Africa and Spain [1]. Calycotome Villosa Subsp. Intermedia is not used in the Moroccan folk medicine and to the best of our knowledge, no phytochemical study of this plant has been reported. The first chemical investigation of this species has led to the isolation and structural elucidation of two flavonoid glycosides from the flowers and leaves. Flavonoids are a group of polyphenolic compounds widely distributed throughout the plant kingdom [2,3]. They exhibit varied biological effects and have been identified as antitumor agents, antioxidants, and free radical scavengers [4,5,6,7,8]. Flavonoids occur in nature as aglycones, glucosides and methylated derivatives [9]. In the glucoside form, a least one of the OH groups of the aglycone is glycosylated by one or more saccharides. The glycosidic linkage is normally located in the 3 or 7 position and the carbohydrate can be D-glucose, D-galactose, L-rhamnose and D-xylose.

Results and discussion

Two flavone glycosides were isolated from the methanolic extract of Calycotome Villosa Subsp. Intermedia. Compound (1) was obtained as a yellow powder from the cooled methanolic extract. Compound (2) was isolated as a pale yellow powder by repeated column chromatography. Their structures (Scheme 1) were identified as follows.

Scheme 1.

Scheme 1.

ESI-MS of compound (1), which gave a quasi-molecular ion peak at m/z 417 [M+H]⁺, and HRMS ESI (m/z 417.1179; calc.: 417.1185), as well as the elemental analysis (found: C 60.56%, H 4.79%; required: C 60.58%, H 4.81%) all indicated that the molecular formula should be C₂₁H₂₀O₉. The IR spectrum (KBr) indicated the presence of hydroxyl (3403 cm^-1) and carbonyl (1655 cm^-1) groups. Bands in the 1650-1050 cm^-1 range are typical of a flavone skeleton. The UV spectrum exhibited absorption maxima at 268 nm (band II) and 306 nm (band I) that are characteristic absorption bands of a flavone skeleton [10]. No shift in band I of compound 1 was observed with the addition of AlCl₃ and AlCl₃/HCl, suggesting formation of a hydroxyl-keto complex at the 5-OH and the absence of an o-dihydroxyl grouping in the B ring [10,11]. As indicated, the ESI mass spectrum of 1 gave a molecular ion peak at m/z 417 [M+H]⁺. A fragment at m/z 255 [M + H]⁺ was observed for the aglycone. A loss of 162 mass units from the molecular ion and a signal at δ 60.57 ppm, shown by DEPT to represent a CH₂ group, suggested glucose or galactose. The carbohydrate moiety was confirmed to be glucose by acid hydrolysis and TLC comparison with an authentic sample.

The ¹H-NMR spectrum exhibited a flavonoid pattern and showed signals at δ 7.04 (1H, s) 6.96 (1H, s) and 6.46 ppm (1H, s) typical of protons at C-3, C-8 and C-6 of a flavone skeleton. Chemical shifts of 8.08 (d, H2’, H6’) and 7.59 (m, H3’, H4’, H5’) suggested that there is no substitution in the B ring of the flavonoid. Thus, the NMR, IR and ESI-MS all suggested that 1 has a chrysin aglycone [12]. The signal at δ 12.79 ppm was assigned to the C-5 hydroxyl. This functionality was confirmed by a batochromic shift in the UV-visible spectrum of 1 after addition of AlCl₃. ¹H-NMR resonances at δ 3.20 to 3.70 ppm and signals in the ¹³C-NMR spectrum just below δ 77 ppm indicated the presence of a glucose moiety [13]. The signal at δ 5.13 ppm was assigned to the anomeric proton (H-1”) with a coupling constant (J 6.7 Hz) indicating a β-configuration [11].

Comparison of the ¹³C-NMR spectrum of compound 1 with that of chrysin aglycone showed that the signal of C-7 was observed to shift upfield slightly (-1.18 ppm), whereas the signals of C-3, C-6 and C-8 were displaced downfield by 2.56, 1.63 and 0.93 ppm respectively. However, the signals of C-4 and C-5 remained unaffected. This indicated that the glucosyl unit was attached to C-7 of the aglycone. DEPT experiments indicated the presence of one methylene carbon (δ 60.58 ppm) and seven quaternary carbons. Based on the above evidence and comparison with the values in literature for analogous compounds [12,14,15], the structure of 1 was established as a known flavonoid 7-O-(β-D-glucopyranosyl)chrysin. Its characteristics are in good agreement with those reported in the literature on a sample isolated from the flowers of Spartium Junceum [16] and a sample synthesised by Dangles et al. [17].

Compound 2 has a molecular formula, C₂₃H₂₂O₁₀, based on the HRMS-ESI (m/z 459.1276; calc.: 459.1291) and elemental analysis (found: C 60.24%, H 4.81%; required: C 60.26%, H 4.80%). Compound 2 also showed characteristic IR absorptions of a flavone skeleton. An absorption band at 1724 cm^-1 indicated that compound 2 had an acetyl group. The ¹H- and ¹³C-NMR data of 2 were quite similar to those of 1, except for some differences in the sugar moiety. These NMR data suggested that flavonoid 2 also has a chrysin aglycone. The ¹H-NMR spectrum exhibited a flavonoid pattern and showed two meta-coupled doublets at δ 6.50 and 6.85 ppm (1H, J = 1.6 Hz each) represented H-6 and H-8 respectively, and a signal at δ 6.98 ppm (1H, s) typical of the proton at C-3 of a flavone skeleton. The signal at δ 12.74 ppm (1H, s) is characteristic of a C-5 hydroxyl. This functionality was further confirmed by a batochromic shift in the UV spectrum in the presence of AlCl₃, as performed for 1 [11]. Furthermore the signals of a glycosidic moiety were visible (3.25 to 4.41 ppm), in particular the anomeric proton, which appeared at δ 5.12 ppm as a doublet with a coupling constant of J = 7.3 Hz indicating a β-linkage of the sugar unit to the aglycone [11]. The ¹³C-NMR spectrum confirms that 2 is indeed a glycoside of chrysin. The location at C-7 of the sugar moiety was established on the basis of the typical glycosylation shifts that occured with respect to chrysin, particulary the C-7 upfield shift (-1.48 ppm) and the downfield of C-6 (+1.53 ppm) and C-8 (+0.93 ppm) [12]. Morever, the cross peak from H-1” (δ 5.12) to C-7 (δ 162.9) in the HMBC spectrum confirmed that the glucosylation takes place in the 7 position. The UV spectral data of 2 with diagnostic shift reagents confirmed the susbstitution of the sugar unit at position C-7. The chemical shifts of the sugar unit carbons were similar to those of 1. However, the presence of the methyl signal at δ 20.6 ppm, together with the presence of a carbonyl signal at δ 170.2 ppm were observed in 2, indicating the presence of β-D-(6”-acetyl)glucose moiety. The location of the acetyl group on position 6” was established by the downfield shift of C-6” (+2.8 ppm), and the upfield shift of C-5” (-1.0 ppm) with respect to 1. The ¹H-COSY and HMBC spectrum confirmed the above assignments. Moreover, the ESI-MS exhibited the fragment ion characteristic of the chrysin aglycone unit at m/z 255. A loss of 204 mass units (C₈H₁₄O₆) from the molecular ion in the MS confirmed the structure of the sugar moiety. From these results, flavonoid 2 was identified as 7-O-β-D-[(6”-acetyl)glycopyranosyl]chrysin.

Conclusions

The isolation and spectroscopic characterisation of two flavonoids from the leaves and flowers of Calycotome Villosa Subsp. Intermedia was described. Isolation and identification of alkaloids from this plant species are in progress.