Isolation of a flavone glucoside from Glycosmis mauritiana (Rutaceae)

1 Introduction

Glycosmis mauritiana (syn. Limonia pentaphylla Auct; Glycosmis pentaphylla Auct. pl.; Limonia mauritiana Lam.), belongs to the family Rutaceae, commonly known as Ash-sheora, Orange berry, Rum Berry and Gin Berry. According to Rastogi et al. (1980) there is some perplexity in the name of Glycosmis mauritiana. Glycosmis mauritiana is native to India, Malaysia, China, Sri Lanka, Myanmar, Thailand, Indonesia and Malaya. Glycosmis mauritiana is a small tree or shrub. Fruits have spicy taste. Which ove widely described in old traditional Indian medicines (G.R.I.N. Taxonomy for Plants, 2009; TopTropicals.com-rare plants for home and garden, 2009; Rastogi et al., 1980; Stone, 1985).

As part of our investigations on the chemical investigations of medicinal plants of Rutaceae family found in Shahjahanpur district (Intekhab et al., 2008a,b; Intekhab and Aslam, 2008, 2009), here we are reporting the isolation and characterization of flavone glucoside from ethyl acetate extract of the roots of Glycosmis mauritiana.

2 Experimental

2.4

2.4 Compound

Orientin (Luteolin-8-C-glucoside; Lutexin) C₂₁H₂₀O₁₁: yellow powder, UV λ_nm: (MeOH) 350, 269, 257, (MeOH–NaOMe) 268, 277, 405, (MeOH–NaOAc) 272, 277, 327, 393), (MeOH–NaOAc–H₃BO₃) 267, 303, 375, 422, (MeOH–AlCl₃) 274, 333, 418, (MeOH–AlCl₃–HCl) 267, 276, 313, 357, 384; IR (KBr) ν_max: 3410 (–OH), 1655 (α, β-unsaturated carbonyl group), 1613 (aromatic C⚌C) cm⁻¹; ESIMS: 448 [M]⁺, 430 [M-H₂O]⁺, 314 [M-C₈H₆O₂]⁺, 299 [M-aglycone-CH₂]⁺, 286 [M-162]⁺, 134 [M-314]⁺, 69 [M-379]⁺; ¹H NMR (300 MHz, DMSO-d₆) δ: 13.15 (1H, s, 5-OH), 10.82-9.15 (3H, s, 3′, 4′, 7′-OH), 7.52 (1H, dd, J = 2.2, 8.3 Hz, H-6′), 7.45 (1H, d, J = 2.2 Hz, H-2′), 6.90 (1H, d, J = 8.3 Hz, H-5′), 6.65 (1H, s, H-3), 6.56 (1H, s, H-6), 5.05 (1H, d, J = 7.0 Hz), 3.30–3.90 (sugar protons, m). ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.16 (C-2), 102.41 (C-3), 182.03 (C-4), 160.47 (C-5), 98.33 (C-6), 162.80 (C-7), 104.65 (C-8), 156.00 (C-9), 104.00 (C-10), 121.97 (C-1′), 114.07 (C-2′), 145.95 (C-3′),149.90 (C-4′), 115.78 (C-5′), 119.45 (C-6′), 73.50 (C-1″), 70.90 (C-2″), 78.88 (C-3″), 70.83 (C-4″), 82.04 (C-5″), 61.76 (C-6″); Rf: 0.58: EtOAc–HOAc–HCO₂H–H₂O (10:1:1:2.5), 0.67: EtOAc–butanone–HCO₂H–H₂O (5:3:1:1); elemental analysis: Calcd. %C = 54.82, %H = 4.55, %O = 43.64. Found: %C = 54.96, %H = 4.48, %O = 43.75.

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3 Results and discussion

Compound, a yellow powder, exhibited a molecular ion peak at m/z 448 [M + H]⁺ in its electro spray mass spectrum corresponded to the molecular formula C₂₁H₂₀O₁₁. The compound showed positive ferric chloride and Shinoda tests for flavonoids, suggested that the compound may be a flavonoid (Geissman, 1962; Markham, 1982). These results also suggested that compound is a flavonoid derivative with a free hydroxyl group at C-5 Geissman, 1962.

The UV spectrum in MeOH gave maxima at 269 and 350 nm, respectively, indicating that the compound belongs to the flavone groups. 3′, 4′- or 3′, 4′, 5′-oxygenated flavones usually exhibit two absorption peaks (or one maximum with a shoulder) between 250 and 275 nm, while the 4′-oxygenated equivalents have only one peak in this range (Markham and Mabry, 1994). The methanol UV spectra of this compound showed two peaks at 257 and 269 nm, respectively, indicating that the B-ring contain 3′, 4′- or 3′, 4′, 5′-OH groups. The NaOMe spectrum of the compound was stable for 5 min confirming the absence of 3-OH group. The NaOAc/H₃BO₃ spectrum also indicated for the presence or absence of an ortho-dihydroxy group at all locations of the flavonoid nucleus (Markham and Mabry, 1975; Harborne and Baxter, 1999; Porter, 1988; Mabry and Markham, 1975).

Flavones containing ortho-dihydroxy group in ring – B shows a consistent 12–30 nm bathochromic shift of band I in the presence of NaOAc/H₃BO₃, diagnostic for the presence of an ortho-dihydroxy group (at C-6, 7 or C-7, 8) in the A-ring. Since this compound produced a significant bathochromic shift (350–375 nm) is diagnostic for the presence of an ortho-dihydroxy group (at C-3′, 4′ or C-4′, 5′) in the B-ring.

The presence of an ortho-dihydroxy group in the B-ring of flavones can be detected by comparison of the spectrum of the flavonoid in the presence of AlCl₃ with that obtained in AlCl₃/HCl. A hypsochromic shift of 30–40 nm was observed in band I of the AlCl₃ spectrum on the addition of acid concluded the presence ortho-dihydroxy group.

The bathochromic shift with AlCl₃, i.e. the band I shift from 350 nm, splitting into two bands with peaks at 418 nm 333 nm, indicated the presence of an OH-group in position 5 (Markham and Mabry, 1975; Harborne and Baxter, 1999). After adding the NaOAc, the band shift II by 12 nm (277–269 nm), indicated the presence of OH-group at position 7. After the addition of NaOAc + H₃BO₃ the shift of band I by 25 nm (375–350 nm), indicated the presence of OH-group in positions 3′ and 4′ (Markham and Mabry, 1975; Harborne and Baxter, 1999; Porter, 1988; Porter, 1994).

The IR spectra showed absorption bands at 3410 (–OH), 1655 (α, β-unsaturated carbonyl group), 1613 (aromatic C⚌C) cm⁻¹ functionalities. The ¹H NMR spectrum of the compound exhibited signal at δ 13.15 (1H, s) attributed a chelated hydroxyl group. Further four signals observed at δ 10.82–9.15 were due to a phenolic hydroxyl groups. The ¹H NMR also demonstrated two one proton doublets at δ 7.45 (1H, d, J = 2.2 Hz) and δ 6.90 (1H, d, J = 8.3 Hz) and one double doublet 7.52 (1H, dd, J = 2.2, 8.3 Hz) assignable to H-2′, H-5′ and H-6′ protons, respectively. The appearance of two doublets and their coupling constant values are further in agreement with the hydroxy groups at C-3′ and C-4′ i.e. a luteolin moiety as a basic skeleton (Porter, 1988, 1994; Mabry and Markham, 1975; Mabry et al., 1970; Ito et al., 1988).

The ¹H NMR displayed one proton singlet at δ 6.65 could be assigned to H-3 proton (Porter, 1994). In addition, the methine carbon signal at δ_c 102.41 was attributed to C-3 in the ¹³C NMR spectrum, indicating a 5, 7 dihydroxy-flavone. In ¹H NMR a one proton singlet appeared at δ 6.56 attributed for H-6 proton. The resonances of the anomeric proton observed in the low-field region in the ¹H NMR spectra at δ 5.05 (1H, d, J = 7.0 Hz) of the compound, implied that compound was luteolin glucoside.

No sugar was released when compound heated prolong with acid, confirmed the presence of C-glycosylation. The [M-18]⁺ peaks in mass spectrum also supported the presence of C-glycosylation (Mabry et al., 1970).

The structure was further supported by its ¹³C NMR spectrum (Ito et al., 1988; Agrawal, 1989), which demonstrated a downfield signal at δ 182.03 clearly assigned to carbonyl carbon C-4. The three downfield signals appeared at δ 145. 95, 149.90, 160.47 and 162.80 were assigned to C-3′, 4′, 5 and 7, bearing hydroxyl group. Further, a signal at δ 98.33 assigned to C-6 further supported that hydroxyl group present at C-5 and C-7. The anomeric carbon signaled at δ 73.50 in its ¹³C NMR spectrum indicating the 8-C-β-d-glucopyranoside structure of the compound (Tomczyk et al., 2002). The position of sugar was concluded to be at C-8-OH based on comparison of ¹H and ¹³C NMR spectra with those reported in the literature of known compound orientin (luteolin 8-C-β-d-glucopyranoside) (Tomczyk et al., 2002).

4 Conclusion

From the survey of the literature to the best of our knowledge luteolin 8-C-β-d-glucopyranoside was previously unknown from Glycosmis mauritiana and further examination of the constituents of this plant is currently in progress.