Phytochemical investigation and antimicrobial activity of Derris scandens

2.1 General experimental procedure

UV (MeOH) and IR (KBr) spectra were measured on Hitachi U-3200 and Shimadzu-8900 spectrophotometers, respectively. EI-MS and HR-EI-MS were carried out using MAT 8200 and Micromass LCT mass spectrometers, in m/z. The ¹H NMR spectra were recorded on a Bruker AMX-500 spectrometer using TMS as an internal reference. The chemical shifts are reported in ppm (δ) while the coupling constants (J) are in Hertz. The ¹³C NMR spectra were recorded at 125 MHz on the same instrument. Column chromatography (CC) was carried out using silica gel (70–230 and 230–400 mesh; E-Merck, Darmstadt, Germany) and Aluminum sheets precoated with silica gel 60 F 254 (0.2 mm thick; E-Merck) were used for TLC to check the purity of the compounds and were visualized under UV light (254 and 366 nm) followed by ceric sulfate as the spray reagent.

2.2 Plant material

D. scandens plants were collected from Karachi, Pakistan, and identified through comparison with earlier identified herbarium specimen (voucher specimen, No. ICP-201) present in Herbarium, Department of Botany, University of Peshawar, Pakistan.

2.3 Compound isolation and identification

The dried and powdered roots and stem of D. scandens were extracted with methanol at r.t. for two weeks. The crude extract of roots (6.5 g) and stem (5.3 g) of D. scandens was subjected to column chromatography (n-hexane, n-hexane/CH₂Cl₂ and CH₂Cl₂/MeOH, in the order of increasing polarity) to separate different fractions. Two coumarins viz., scandenin (1, 5.4 mg) (Pelter and Stainton, 1964) and scandenin A (2, 5.7 mg) (Rao et al., 2007) were purified from fraction DSR-3 by silica gel column chromatography (CC) and with n-hexane/ethyl acetate (10:1) as the eluent. Fraction DSR-1 was further separated by silica gel column chromatography and eluted with n-hexane–EtOAc (9:1) to give triterpene named betulinic acid (3, 2.2 mg) (Ikuta et al., 1995). Similarly, another triterpene viz., lupeol (4, 3.0 mg) (Carvalho et al., 2001) was also isolated from fraction DSR-1, after elution with a mixture of n-hexane–EtOAc (8.5:1.5). Repeated column chromatography of fraction DSS-1 using n-hexane–acetone (9.5:0.5) as the eluent afforded β-amyran-3-one (5, 2.5 mg) (Krishnaveni and Rao, 2000). Moreover fraction DSR-4 gave one triterpene named β-amyrin (6, 8.0 mg) (Heupel, 1985) upon elution with n-hexane–EtOAc (9:1) and one steroid viz., ß-sitosterol (7, 3.7 mg) (Rubinstein et al., 1976) was isolated from fraction DSS-4 with n-hexane–EtOAc (8:2) as eluent. Finally DSS-7 gave ß-sitosterol glucopyranoside (8, 7.8 mg) (Seo et al., 1978) using n-hexane–EtOAc (2.5:7.5) as the eluent. All the above compounds (Fig. 1) were identified by an intensive comparison of their NMR spectral data reported in the literature.

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Figure 1

Structure of different compounds isolated from D. scandens.

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2.4 Bioactivity test – agar diffusion test

Tests for antifungal, antibacterial, and antialgal activities were performed as previously described (Schulz et al., 1995; Höller et al., 2000). The test organisms for the agar diffusion and screening tests were the bacteria Bacillus megaterium de Bary (Gram positive) and Escherichia coli (Migula) Castellani & Chambers (gram negative), the fungi Microbotryum violaceum [formerly known as Ustilago violacea (Pers.) Roussel (Ustomycetes), Mycotypha microspora Fenner (Zygomycetes)], and the alga Chlorella fusca Shih Krauss (Chlorophyceae). The crude extract of roots and stem of plant D. scandens was subjected to column chromatography and resulted in 12 fractions (DSR 1–12) for roots and 10 fractions (DSS1–10) for stems tested in an agar diffusion assay for their antifungal, antibacterial, and algicidal properties. Reference compounds were penicillin, nystatin, actidione, and tetracycline.

The extracted compounds were dissolved in acetone at a concentration of 1 mg/mL. Fifty microliters of the solutions (50 μg) was pipetted onto a sterile filter disk (Schleicher & Schuell, 9 mm), which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organism (Schulz et al., 1995).