Bioactive flavanoids from Glycosmis arborea

Background Glycosmis is a genus of evergreen glabrous shrub and distributed all over India. It possesses various medicinal properties and is used in indigenous medicine for cough, rheumatism, anemia, and jaundice. Glycosmis arborea is a rich source of alkaloids, terpenoids, coumarins, as well as flavonoids. Results The chemical investigation of methanol fraction of the leaves of G. arborea led to the isolation of one new flavone C-glycoside along with three known flavanoids, named as 5,7-dihydroxy-2-[4-hydroxy-3-(methoxy methyl) phenyl]-6-C-β-d-glucopyranosyl flavone (4), 5,7,4′-trihydroxy-3′-methoxy flavone (1), 5,4′-dihydroxy-3′-methoxy-7-O-β-d-glucupyranosyl flavanone (2), and 5,4′-dihydroxy-3′-methoxy-7-O-(α-l-rhamnosyl-(1‴→6‴)-β-d-glucopyranosyl) flavanone (3), respectively. The structures of all compounds were elucidated with the help of nuclear magnetic resonance spectrometry. Pure compounds and fractions were evaluated for pest antifeedant and antimicrobial activity. Conclusion Four compounds were isolated from the leaves of G. arborea. Among them, compound 4 showed significant antimicrobial activity.


Antifeedant assay
The antifeedant activity of the extracts against the polyphagous pest Spodoptera litura was tested using the leaf dip method [7]. Five percent concentrations of each extract were prepared by dissolving extracts in a small quantity of ethanol and diluting in water containing 0.05% Triton X-100. The leaf discs of about 5 cm 2 were prepared out of castor leaf (Ricinus communis L.) and were dipped for 30 s in an extract or compound separately. The leaf discs dipped only in water containing 0.05% Triton X-100 were used as controls. The leaf discs were air dried, and on each treated leaf disc, 10 larvae of S. litura (1 day old) were released. Three replications were maintained for each extract. Larval weight was taken after 4 days of treatment. Antifeedant activity of fractions and the purified compounds were tested against the polyphagous crop pest S. litura (Table 2).

Antibacterial assay
The in vitro antibacterial activity was tested by the disc diffusion method [8] using pathogenic strains of Agrobacterium tumifaciens, Pseudomonas syringae, and Pectobacterium. carotovorum. Concentrations of 200 and 500 μg/disc of compounds were impregnated on the discs. These discs were placed on the surface of the agar plates already inoculated with pathogenic bacteria. The plates were incubated at 37°C and examined at 48 h for zone of inhibition, if any, around the discs. Gentamicin was used in the assay as a standard control drug. An additional control disc without any sample but impregnated with an equivalent amount of solvent (DMSO) was also used in the assay. The result of antibacterial activity indicated that methanol fraction and compound 4 exhibited a mild to moderate activity (Table 3).

Chemistry
The repeated chromatography of methanol fraction of the leaves of G. arborea led to the isolation of four flavonoids by gradient elution with the CHCl 3 /MeOH mixture of increasing polarity. Compound 4 was isolated as a yellow solid which was further crystallized in acetone.   The exact proton and carbon assignments were made by a combination of 2D NMR experiments such as COSY, heteronuclear single-quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC). The position of sugar was confirmed by HMBC long-range correlation in which anomeric H-1″ showed long-range coupling with 162.97 (C-5), 104.97 (C-6), and 156.3 (C-7), suggesting the position of sugar at C-6 of aromatic ring B (Figure 2). The double bond position was confirmed by coupling of a proton singlet (δ 6.77) with 182.47 (C-4) and 121.98 (C-1 0 ), suggesting that it must be placed at C-3 and assigned as H-3. The 5-hydroxy flavone skeleton was assigned on the basis of 13 C NMR data which showed C-4 resonance at δ 182.47, characteristic of 5-hydroxy flavone [10,11]. The signal resonating at δ 56.8 was due to methoxy carbon. The upfield appearance of anomeric carbon (73.74) and proton (4.65) as compared to those of aromatic O-glycoside data and anomeric proton correlation with C-5, C-6, and C-7 in the long-range HMBC experiment exhibited its C-glycosidic nature which was confirmed by its resistance to acidic hydrolysis [12,13]. The remaining HMBC correlations are given in Table 1.
The structure was supported by the mass spectroscopic studies, which showed molecular ion peak at 478

Biological studies Antifeedant activity
All the fractions and compounds were tested for antifeedant activity. Among them, the hexane and methanol fractions showed significant antifeedant action against S. litura L. In a dual-choice leaf disc method, hexane and methanol fractions were tested for pesticidal potential.