Natural Product Research Vol. 23, No.

8, 20 May 2009, 719723

Biological activity of alkaloids from Solanum dulcamara L.

Padma Kumar, Bindu Sharma* and Nidhi Bakshi
Department of Botany, Laboratory of Plant Tissue Culture and Secondary Metabolites, University of Rajasthan, Jaipur, India (Received 19 March 2008; final version received 26 June 2008) Alkaloids are well known for their antimicrobial activity. Though all natural alkaloids come from plants, not all plants produce alkaloids. Plants of the Solanaceae family are known for their high alkaloid content. Alkaloids are found in all plant parts like roots, stems, leaves, flowers, fruits and seeds. In the present study, those plant parts of Solanum dulcamara were selected which have been reported to produce a high content of a specific alkaloid: solanine (from unripe fruits), solasodine (from flowers) and -solamarine (from roots). These alkaloids were extracted from various parts of S. dulcamara by well-established methods and were screened for their antibacterial activity. Human pathogenic bacteria, viz., Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, were selected for the study. All three alkaloids inhibited the growth of E. coli and S. aureus. However, no significant activity was observed against E. aerogenes. Minimum inhibitory concentration and minimum bactericidal concentration were also evaluated. Keywords: antibacterial activity; solanine; Solanum dulcamara; solasodine; -solamarine

1. Introduction Plants of Solanaceae are known for their alkaloid content. For the present study, Solanum dulcamara Linn (Solanaceae), commonly known as bittersweet (a scrambling, shrubby plant), has been selected. Alkaloids in general are well known for the biological activities that make them very useful pharmaceutical agents. Solanum dulcamara is known for its diuretic, diaphoretic, and narcotic properties. It is commonly used in rheumatism and in skin disease. Although the plant has been screened for its antimicrobial activities, a systematic study of the antibacterial activity of alkaloids of S. dulcamara is lacking; solanine, solasodine and -solamarine have been extracted and screened for their antibacterial activity in the present study.

2. Materials and methods Solanum dulcamara plants were collected from Mount Abu (Rajasthan) in the month of November and were submitted to the Herbarium of the Botany Department, University of Rajasthan, Jaipur, India.
*Corresponding author. Email: bindushr111@gmail.com

ISSN 14786419 print/ISSN 10292349 online 2009 Taylor & Francis DOI: 10.1080/14786410802267692 http://www.informaworld.com

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2.1. Test bacteria Enterobacter aerogenes, Escherichia coli and Staphylococcus aureus were procured from Sawai Man Singh Medical College, Jaipur and were maintained on nutrient agar medium. A fresh suspension of the micro-organisms under investigation was prepared from a freshly grown culture on agar slant before every antibacterial assay. In the present study, those plant parts were selected which have been reported to produce a high content of a specific alkaloid. Solanine from unripe fruit (Khanna, Kumar, & Singhvi, 1988), solasodine from flowers (Kumar & Khanna, 1986) and -solamarine from roots (Kupchan, Stanley, Barboutti, John, & Cesar, 1965) were extracted by routine extraction procedures available in the literature.

2.2. Antibacterial susceptibility testing For antibacterial screening, the filter paper disc method (Gould & Bowie, 1952; Jain & Sharma, 2001) was followed. Sterilised filter paper discs (6.0 mm diameter) were saturated with 10 and 20 mL of 100 mg mL1 test solution, so that each disc contained 1 and 2 mg test compounds, respectively. Disc with extracts were then air dried to remove any residual solvent that might interfere with the determination. The discs were then placed on the surface of sterilised nutrient agar medium that had been inoculated with test bacterial suspension (1 108 CFU mL1 inoculum size) and air dried to remove the surface moisture. The thickness of the agar medium was kept equal in all the petri plates and a standard disc of streptomycin (1 mg disc1) was used in each plate for reference. Before incubation, the petri plates were placed for 1 h at 4 C to allow the diffusion of the compounds from the disc into the medium. Plates were then incubated at 37 C for 2024 h. Thereafter, the zone of inhibition or depressed growth was measured. All the experiments were done in five replicates and the activity index was calculated for each of them (Table 1). Activity index AI Inhibition zone of the sample : Inhibition zone of the standard

2.3. Determination of minimum inhibitory concentration Minimum inhibitory concentration (MIC) of the compounds for test organisms was determined using 2-fold serial microdilution method (Basri & Fan, 2005) and concentrations ranging from 5 to 0.0098 mg mL1 were prepared in 96-well microtitre plates. Thereafter, 100 mL of inoculum (1 108 CFU mL1 inoculum size) was added to each well. Each compound was assayed in triplicate. Bacterial suspensions were used as controls. The microplates were incubated at 37 C for 24 h. The MIC values were taken as the lowest concentration of the compounds in the wells of the microtitre plates that showed no turbidity after 24 h of incubation. The turbidity of the wells in the microtitre plates was interpreted as visible growth of the micro-organisms. The minimum bactericidal concentration (MBC) was determined by subculturing the contents of the wells of the microtitre plates on a sterile agar plate. The least concentration showing no visible growth on agar plates was taken as the MBC value (Table 2).

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Solanine, solasodine and -solamarine (1 and 2 mg disc1) were screened against the test bacteria (Table 1). The inhibition zones of the extracts were compared with the inhibition zone produced by the standard (streptomycin). Solanine, solasodine and -solamarine showed the best efficacy against S. aureus. In the case of solanine and -solamarine, inhibition zones were nearly double the standard. The inhibition zone produced by solasodine was almost equal to the standard. During the studies, it was observed that within 96120 h (34 days), the inhibition zone produced by standard against S. aureus disappeared, while even after 4 months the inhibition zones of solanine, solasodine and -solamarine remained unchanged. This proves that the duration of the activity of the extracts against bacterium was much more than streptomycin (standard drug). All three

Table 1. Antibacterial activity of solanine, solasodine and -solamarine. Test bacteria Test compound Solanine Solasodine -solamarine Streptomycin Concentration of content per disc 1 mg 2 mg 1 mg 2 mg 1 mg 2 mg 1 mg E. aerogenes IZ ve ve ve ve ve ve ve AI E. coli IZ ve 10 mm ve 08 mm ve 08 mm 10 mm AI S. aureus IZ 22 mm 38 mm 12 mm 25 mm 23 mm 24 mm 12 mm AI 1.8 1.0 1.9

Inhibition zone of the sample Notes: IZ Inhibition zone (include disc diameter). AI Activity index Inhibition zone of the standard :

Table 2. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of extracts of S. dulcamara. E. coli S. aureus

Solanine Solasodine -solamarine Solanine Solasodine -solamarine Test bacteria concentration (mg mL1) MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 5.0000 2.5000 1.250 0.6250 0.3125 0.1563 0.0781 0.0391 0.0195 0.0098

Notes: Absence of bacterial growth; Presence of bacterial growth.

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alkaloids (solanine, solasodine and -solamarine) showed a negative response against E. coli at 1 mg disc1, but at 2 mg disc1 the responses were positive. None of the extract showed a positive response against E. aerogenes. Results obtained for MIC are coinciding well with the results obtained by disc diffusion assay. MIC for solanine, solasodine and -solamarine were 1.25, 0.625 and 1.25 mg mL1, respectively against E. coli, while it was 0.625 mg mL1 against S. aureus for all three extracts tested (Table 2). The MIC and MBC values of solanine and -solamarine against E. coli were the same (1.25 mg mL1); similarly, against S. aureus, MIC and MBC values obtained were also same (0.625 mg mL1) for solanine, indicating their bactericidal activity. On the other hand, the higher MBC values recorded for solasodine (against E. coli and S. aureus) and -solamarine (against S. aureus) indicate their bacteriostatic effect. Earlier workers (Dhawan, Patnaik, Rastogi, Singh, & Tandon, 1977; Mathes, 1963, 1967; Mishra, Ulubelen, & Johansson, 1983; Samy, Ignacimuthu, & Sen, 1998) have observed the antibacterial activity of various medicinal plants. Digrak, Alma, Llcim, and Sen (1999) investigated the antibacterial activity of various commercial plant extracts and found that Mimosa bark extracts had the most pronounced antibacterial activity. Gopalkrishanan et al. (2000) determined the effect of petroleum ether, chloroform and methanolic extracts of dried leaves of Acalypha indica against six bacterial species (E. coli, S. aureus, Pseudomonas aeruginosa, Salmonella typhoza, Bacillus subtilis and Klebsiella pneumonia). Elizabeth (2001) tested antimicrobial activity of Allium sativum on three human pathogenic bacteria, viz. S. aureus, Salmonella typhimurium and Yersinia enterocolitica by disc diffusion method. In the present study, all three alkaloids, solanine, solasodine and -solamarine, showed better results than the standard (streptomycin) against S. aureus. Amongst the three, -solamarine showed best response against S. aureus. The duration of activity of the extracts against bacterium was found to be more when compared to the standard, although alkaloids in the study showed a negative response against E. coli at 1 mg but showed activity at 2 mg. Further values of MIC and MBC (Table 1), which are much less (52 mg mL1 in the case of E. coli and 51 mg mL1 in the case of S. aureus), prove that the compounds have good potential to resist E. coli and S. aureus, even at low concentration. The study opens the field of research for extracting and identifying compounds from new sources (plants) which might inhibit E. aerogenes, which showed great resistance against the test compounds when compared to S. aureus and E. coli. From the present study, it appears that alkaloids of S. dulcamara have strong biological activity, which can be exploited for future antibacterial drugs, specifically for diseases caused by the notorious E. coli and S. aureus.

Acknowledgement
The authors are thankful to the Head of the Botany Department, University of Rajasthan, Jaipur, India, for providing all facilities for the present investigation.

References
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