Isolation of Three New Substituted Disaccharides from Leaves and Stems of Phlox Drummondii

Huma Aslam Bhatti*, Aneela Naz, Rubina, Samina naz and Shaheen Faizi

H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan

Keywords: Phlox drummondii; Polemoniaceae; Xylopyranose; Rhamanose

Introduction

The genus Phlox belongs to American family Polemoniaceae [1], which has about 15 genera and 300 species [2]. The generic name of Phlox means flame, alluding to the general brilliance of flowers [3]. This genus comprises about 70 species of hardy herbaceous perennials and half- hardy shrubs and annuals distributed in North and South America [4], Europe and Asia. Phlox drummondii, an annual phlox is a 45 cm (11/2 foot) branching plant with usually reddish purple blooms. It has given rise to many cultivated forms with petals of two colors and a star-like shape. It originated in Texas, USA [5] but has now cultivated all over the world.

Careful sifting of literature revealed that very little phytochemical work had been done on the genus Phlox, while no pharmacological work has been reported on its various parts. Using different solvent systems and preparative chromatography techniques on the extract of leaves and stem of phlox drummondii leads to the isolation of three novel compounds: 2-O-(β-3′-O-acetyl,4′-O-isovaleroyl- rhamnopyranosyl)-1-O-hexyl,3-O-acetyl-β-xylopyranose (Compound 1), 2-O-(β-2′,3′-Di-O-acetyl,4′-O-isovaleroyl- rhamnopyranosyl)-1-O-hexyl,3,4-di-O-acetyl-β- xylopyranose (Compound 2) and 2-O-(β-3′-O-acetyl,4′-O- isovaleroyl-rhamnopyranosyl)-1-O-hexyl,3,4-di-O-acetyl- β-xylopyranose (Compound 3) along with one known constituents that is 4-hydroxy benzoic acid (Compound 4) (Figure 1). These compounds were characterized by different spectroscopic techniques including UV, IR, EI-MS, FAB, 1H NMR and 2D-NMR techniques for new compounds.

Click to enlarge

Results and Discussion

The molecular formula of compound 1 was established as C26H44O12 with the aid of FAB mass spectrometry (negative- ion mode, [M-1]+ at m/z 547), while HR EIMS had important mass fragment ions at m/z 273.1338 (d), 231.1261 (e) and 85.0653 in (Figure 2). In the UV spectrum shown maxima at λ = 203nm which indicated absence of chromophore and Its IR spectrum shown the presence of hydroxyl group (ν = 3455cm-1) and ester moiety (ν = 1738 and 1162-1021 cm-1). The 13C NMR and 1H NMR signals of compound 1 is represented in (Table 1). In the DEPT spectrum of 13C NMR shown 26- resonance, out of the six were CH3, seven CH2, ten CH and three quaternary Carbons. The 1H NMR showed signals of anomeric protons at δ 4.40 (d, J = 6.5 Hz) and δ 5.02 (d, J = 1.6 Hz) represented the xylose and rhamnose sugar moieties, and J-values of that sugars; supported its β- and α- orientations [6]. The methyl signal of rhamnose sugar was appeared at δ 1.16 (d, J = 6.2 Hz, H-6′). The downfield signals of H-3, H-3′ and H-4′at δ 4.87 (t, J = 8.3 Hz, H-3), 5.15 (dd, J = 3.0 Hz, 9.8 Hz, H-3′) and 5.02 (t, J = 9.8 Hz, H-4′) as compared to simple disaccharides. Slowing K, et al. [7] revealed the acylated nature of disaccharides, which was also confirmed by the presence of two acetoxy methyl groups as singlets at 2.12 (3-COCH3) and 2.01 (3′- COCH3) and a isovaleryl moiety at C-4′. Two methyl groups appeared as a doublet at δ 0.93 (J = 6.5 Hz), while CH2 shown multiplet at δ 2.14-2.15 and methine proton signal appear at δ 2.08 as multiplet. Triplet of doublet for proton H-1′′a give a signal at δ 3.81 (J = 7.1 Hz, 7.1 Hz and 9.3 Hz) and H-1′′ b at δ 3.49 (J = 6.6 Hz, 6.6 Hz and as 9.3 Hz) and these were assigned to be diastereotopic methylene proton of a hexyloxyl moiety and remaining proton appears as a multiplet of H-2′′ at δ1.58, H-3′′ at δ1.32 and H-4′′, H-5′′ at δ1.24 and one triplet signal is appearing for H-6′′ at δ 0.86 with J = 7.0 Hz.

Click to enlarge

Further structure of the compound was also supported by HMBC, HMQC, COSY, and NOESY spectra. Its COSY spectrum illustrates the 1H-1H correlation of H-1(δ 4.40) with H-2 (δ 3.67) and H-3 (δ 4.87) with H-2 (δ 3.67) and H-4 (δ 3.71), while H-4 (δ 3.71) shows a correlation with H-5a (4.08) and H-5b (δ 3.31) for xylose sugar and rhamnose sugar; The connectivity’s of H-1′ (δ 5.02) with H-2′ (δ 3.96), H-3′ (δ 5.15) with H-2′ (δ 3.96) and H-4 (δ 5.08), H-4′ with H-5′ (δ 4.17), and H-5′ with H-6′ (1.16) are shown. HMBC correlation is represented in Figure 3, where an Anomeric proton of the xylose sugar is linked with the hexyl group predicted by its up field signal and is also confirmed by 3J-correlation of H-1 (δ4.40) with C-1′′ (δ 69.89) and H-1′′ (δ 3.81, 3.49) with C-1 (δ 101.52). Same while the anomeric proton of rhamnose sugar showed 3J-correlation of H-1′ (δ5.02) with C-2 (δ74.75) of the xylose sugar. Whereas H-2 (δ 3.67) of the xylose moiety is also linked with C-1′ (δ 99.56) of rhamnose sugar. Quaternary carbon of ester moiety of C-3 (δ171.99) show 2J-correlation with its methylene proton (δ 2.12), and also 3J-correlation with H-3 (δ4.87) at xylose sugar. Same connectivity was observed in rhamnose sugar, here quaternary carbon of ester moiety at position C-3′′ show 2J-connectivity with methylene proton and 3J-correlation with proton at postion H-3′′. At the C-4′ position of rhamnose sugar, isovaleryl group is attached and its connectivity is also confirmed by HMBC correlation. Here H-4′ (δ 5.08) show 3J-connectivity with C-1‴ (δ172.11), H-2‴ (δ 2.14-2.15) show 2J-connectivity with also C-1‴ (δ172.11) and H-4‴ (δ 0.93) with C-2‴ (δ43.40) show 2J HMBC correlation.

Click to enlarge

Compound 2 was obtained as a white powder. FAB (-ve) gave its molecular ion peak at 631 and revealed its molecular formula as C30H48014. EIMS show base peak at m/z 315 (fragment a) and other fragments ion peak shown in (Figure 3). The UV spectrum shown maxima at λ = 203nm, indicated absence of chromophore in this compound. Its IR spectrum gives several peak at the range of 1755-1715, indicated presence of ester moiety in that compound. 13C NMR and 1H NMR (Table 1) spectra were closely similar to the spectra of compound 1 except for some chemical shift values, such as H-4 and H-2′ which gives downfield signals as H-4 (δ 4.83, dt, J = 8.9 Hz, 5.6 Hz) and H-2′ (5.00, dd, J = 1.6 Hz, 3.2 Hz) because of the presence of ester group rather than OH in compound 1. Four acetoxy methyl proton appear at δ 2.08, δ 2.04, δ 1.98, and 1.93 ppm [8, 9, 10]. Compound 3 was obtained as solid wax. FAB (-ve) gives molecular ion peak at m/z 589 [M-H+], was indicated its molecular formula as C28H46O13, confirmed by its fragments ion peak m/z 273.1334 (C13H21O16) and m/z 231.1260 (C11H19O5) in HR-EIMS. The UV spectrum shown maximum absorbance at λ = 203nm, indicated absence of chromophore in structure. 1HNMR is closely similar to Compound 1 (Table 2). Except only shifting of downfield signals at position 4 due to presence of acetoxy group.

Acknowledgement

One of the authors, Aneela Naz thankfully acknowledges the Higher Education Commission (HEC) of Pakistan for awarding her HEC fellowship under the scheme of HEC-Merit Scholarship.