Prenylated flavonoids from the stem wood of Commiphora opobalsamum (L.) Engl. (Burseraceae)

2.1 General

Proton and Carbon-13 nuclear magnetic resonance (NMR) were recorded at 200 and 50 MHz, respectively using a Mercury-200BB apparatus with tetramethylsilane (TMS) as internal reference. A low resolution mass spectrum (LR-MS) was produced on a Finnigan SSQ 700 mass spectrometer. Infrared (IR) spectra were recorded on FTIR-8400 spectrophotometer and UV spectra were measured on a Shimadzu UV-240, following Mabry et al. (1970). Column chromatography was carried out using Sephadex LH-20 and silica gel (230–400 mesh). Analytical TLC and PTLC were performed on precoated Merck F₂₅₄ silica gel plates and visualized first under vis–UV light (254 and 366 nm), then sprayed with vanillin–H₂SO₄ and heated at 105 °C (Harborne, 1998).

2.2 Plant material and sampling site

The North kordofan region lies between latitude 12° 43⁻ – 13° 42⁻ N and longitude 30° 14⁻ – 31° 55⁻ E. It is characterized by a dry, hot climate, typically tropical continental with a relatively short rainy season. Plant material was collected from Wad al Baga, a rainy forest about 15 km south of El-Obeid city, capital of the region (Fig. 1) between August and September 2009. Taxonomical identification was determined using the available relevant African Flora (El Amin, 1990; Maydell, 1990) and by means of a comparison with herbarium specimens conserved in the Herbaria of Soba Forests Research Centre, and voucher specimen was deposited in the Herbarium of Botany Department, University of Kordofan (voucher no. B01185).

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Figure 1

Location map of Northern kordofan, Sudan. 1: Khartoum state; 2: North Kordofan state; 3: Northern state; 6: Northern Darfur state; 8: South Kordofan state; 10: White Nile; 18: East Kordofan.

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2.3 Extraction, isolation and characterization

Shade dried stem wood was milled into powder (1300 g) and successively extracted in a Soxhlet with hexane, dichloromethane (DCM), acetone, and methanol (Harborne, 1998). Acetone extract was evaporated in a rotatory evaporator, dried (14 g) and subjected to chromatography on silica gel eluted with hexane–EtOAc and EtOAc–MeOH solvent systems. The 25 column fractions obtained were combined according to their TLC profile into 14 major fractions (A₁ to A₁₄). Further fractionation led to the isolation of two new prenylated flavonoids along with four known flavonoids. Their structures were determined by analysis of their spectroscopic data (UV, IR, MS, ¹H- and ¹³C-NMR) in comparison with those reported in the literature.

2.4 Compound 1 (6-(3,3-dimethylallyl)-2,3-diyhdrokaempferol)

Collected as colorless crystals (20 mg) by repeated crystallization of the residue obtained from fraction A₃; m.p. 200–202 °C. UV/VIS data are shown in Table 1. IR (KBr) cm⁻¹: 3552 (OH), 1616 (C⚌O), 1521. ¹H-NMR (Acetone-d₆), δ: 7.40 (2H, m, H-2′/6′), 6.89 (2H, m, H-3′/5′), 6.01 (1H, s, H-8), 4.64 (1H, d, J = 11.6 Hz, H-3), 5.06 (1H, d, J = 11.6 Hz, H-2), 5.23(1H, m), 3.26 (2H, d, J = 6.8 Hz), 1.75 and 1.64 (s each, all –CH₃). ¹³C-NMR (Acetone-d₆), δ: 83.7 (C-2), 72.5 (C-3), 161.3 (C-5), 108.7 (C-6), 164.6 (C-7), 94.8 (C-8), 158.2 (C-9), 130.8 (C-2′/6′), 115.2 (C-3′/5′), 158.1 (C-4′), 17.2(CH₃, C-4‴), 25.2 (CH₃, C-5‴), 122.7 (CH, C-2‴), 20.9 (CH_2, C-1‴), 130.7 (C-3‴). LR-MS (EI, 70 eV): m/z (%) = 356.14 (68), [M]^•+.

2.5 Compound 2 and 3

A precipitate filtered out from fractions (A₄ and A₅) was separated on a Sephadex LH-20 eluted with DCM/MeOH (9:1) and the obtained fractions were further purified by preparative TLC using DCM/EtOAc (7:3) as developing solvent to yield compound 2 and 3.

2.6 Compound 4 (quercetin)

Major fractions A₆, A₇ and A₈ were repeatedly chromatographed on Sephadex LH-20 eluted with DCM/MeOH (8:2 and 1:1) then purified by preparative TLC to give compound 4. This compound was obtained as a yellowish-green powder (15 mg); m.p. 310–312 °C. UV/VIS data are shown in Table 1. IR (KBr) cm⁻¹: 3394 (OH), 1654 (C⚌O), 1514, 1560. ¹H-NMR (Acetone-d₆), δ: 7.82 (1H, d, J = 2.1 Hz, H-2′), 7.70 (1H, dd, J = 2.1 and 8.4 Hz, H-6′), 6.99 (1H, d, J = 8.4 Hz, H-5′), 6.25 (1H, d, J = 2.1 Hz, H-6), 6.52 (1H, d, J = 2.1 Hz, H-8), 12.18 (s, 5-OH). LR-MS (EI, 70 eV): m/z (%) = 302.12 (100), [M]^•+.

2.7 Compounds 5 and 6

Major fractions A₉, A₁₀, A₁₁ and A₁₂ were re-grouped (750 mg) and subjected to a silica gel column eluted with gradient of hexane/EtOAc (7:3, 1:1, 3:7, 1:9 and 100% EtOAc) to afford sixty fractions. Fractions 33–38 and 41–55 were combined to give sub-fractions f₁ (120 mg) and f₂ (300 mg), respectively.

3.1 Compound 5

The UV spectrum of this amorphous yellow powder demonstrated a similar profile as compound 1, and indicated the presence of a flavanonol skeleton with 5,7-dihydroxy groups. The IR spectrum exhibited absorptions at 3419 cm⁻¹ (OH), 1635 cm⁻¹ (C⚌O), 1519 and 1579 cm⁻¹ for aromatic structure, 1444 cm⁻¹ for gem-dimethyl groups and broad bands at 2925 and 1070 cm⁻¹ demonstrated the glycosidic nature. The ¹H- and ¹³C-NMR spectra (Tables 2 and 3) showed a typical pattern to compound 1, except the position of H-3 (5.07 ppm) which indicated the acylation of C₃–OH and this was confirmed further from the absence of substantial chemical shift effects for the aromatic ring protons of compound 5 relative to 1 which according to Moco et al. (2006) suggested attachment of the glucose moiety to the OH group at C-3. The ¹³C-NMR signals at 101.2 (C-1″), 74.5 (C-2″), 77.8 (C-3″), 71.1 (C-4″), 77.9 (C-5″) and 62.4 (C-6″) along with resonances at 3.5–3.2 ppm and coupling constant 7.2 Hz of H-1‴ proton in ¹H-NMR confirmed the presence of the O-sugar moiety in β-configuration. The molecular ion peak at m/z 354.22 corresponding to M⁺–C₆H₁₁O₆ (glucose moiety) and the ¹³C-NMR, DEPT-135, HMBC and HSQC analysis confirmed the molecular formula C₂₆H₃₀O₁₂. Thus, compound 5 was identified as 6-(3,3-Dimethylallyl)-2,3-dihydrokaempferol-3-β-O-glycoside (Fig. 2) and it was the first report of its isolation from natural sources.

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Figure 2

Structures of prenylated flavonoids isolated from Commiphora opobalsamum.

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3.2 Compound 6

The UV spectrum (MeOH) of this new prenylated flavanone showed one major peak with a shoulder at 285 and 320 nm corresponding to flavanones (Mabry et al., 1970). The IR spectrum indicated the presence of OH (3438 cm⁻¹), C⚌O (1635 cm⁻¹), an aromatic structure (1519 cm⁻¹) and dimethyl groups (1438 cm⁻¹). The ¹H- and ¹³C-NMR (Tables 2 and 3) showed spectral data similar to those of compounds 1 and 5, except the higher shift of C-3 to 43.6 which indicated the absence of OH at that position. This was supported by both the higher shift up field of H-3 (2.75 ppm) relative to that of compound 5 and the presence of cross peak of C-3 with the protons at 2.75 and 3.21 ppm in the HSQC spectra. Low shift of C-7 to 164.3 (∼2.4 ppm) relative to that of compound 5 indicated the acylation of the C₇–OH by the glucose moiety which was further confirmed by downfield shift of H-8 to 6.29 ppm relative to 5.9–6.1 ppm, the typical position in 5,7-dihydroxyflavanons (Markham, 1982). The LR-MS analysis showed a base peak at m/z 340.27 (100%) corresponding to M⁺-glucose moiety and suggested the molecular formula C₂₆H₃₀O₁₁ which was confirmed from ¹³C-, ¹H-HSQC and HMBC spectra. Thus, compound 6 was identified as 6-(3,3-Dimethylallyl) naringenin-7-O-β-glucoside (Fig. 2) and to the best of our knowledge it was isolated for the first time from a natural source.

In conclusion, fractionation of the acetone extract of C. opobalsamum stem wood afforded six compounds, two new prenylated flavonoids along with known flavonols (kaempferol and quercetin) and flavanonols (6-[3,3-Dimethylallyl]-2,3-diyhdrokaempferol and aromadendrin). To the best of our knowledge all the known isolated flavonoids except quercetin (Abbas et al., 2007) are reported from this plant species for the first time.