In Vitro Antimicrobial Screening of Alkaloid Fractions from Strychnos potatorum

: Alkaloid fractions isolated from Strychnos potatorum L.f. (Loganiaceae) seed were tested for their antimicrobial properties against some pathogenic gram positive, gram negative and acid-fast bacteria and fungi. These fractions have shown considerable antimicrobial activity against both bacteria and fungi at the tested concentrations (100 & 200 µg/mL). Further, the growth of Proteus vulgaris , Staphylococcus aureus, Salmonella typhimurium, Vibrio cholerae, Mycobacterium tuberculosis, Aspergillus niger and Candida albicans were significantly inhibited. These findings have confirmed the use of this plant in treating of several microbial infections both in traditional and folk medicine in India.


Introduction
Plants contain numerous biologically active compounds, many of which shown to have antimicrobial activities 1 .Plant derived medicines have been a part of traditional healthcare in most parts of the world for thousands of years and there is increasing interest in plants as sources of agents to fight microbial diseases.Given the alarming incidence of antibiotic resistance of pathogenic microbes in particular, there is a constant need for discovering new and effective therapeutic agents 2 .
Strychnos potatorum L.f. is a medium sized (~ 15 m) glabrous deciduous tree occurs both in the tropic and sub-tropics of north and south-east parts of Africa and Indian peninsula, Sri Lanka and Myanmar of Asia.The seed, besides its bark and root, is primarily used in the Indian traditional systems of medicine for treating various diseases including microbial infections.It is used in Ayurveda for treating the eye and urinary tract infections 3 , gonorrhoea and kidney troubles in Unani and for the leucorrhoea, tuberculosis, venereal diseases and acute diarrhoea in Siddha medicine 4,5 .Alkaloids, the prime source of secondary metabolites isolated from several Strychnos species are known for their therapeutic importance.Therefore, an attempt has been made in order to give the experimental basis for its wide therapeutic use in traditional medicine.

Experimental
The seed material of Strychnos potatorum were collected from the Karpakpalli forest, Bidar district of Karnataka, India in December 2002.The plant was identified by Prof. Y.N.Seetharam and a herbarium specimen is deposited in the department of Botany Herbarium, Gulbarga University, Gulbarga with a voucher number HGUG-214 6 .

Extraction and fractionation of alkaloids
The pulverized powdered seed wetted with aqueous NH 4 OH and lixiviated overnight with (2.5 L) ethyl acetate at room temperature.The filtered organic phase was separated and basified with NH 4 OH (pH 11-12).This is extracted with (2.5 L) chloroform (3X) and passed through anhydrous Na 2 SO 4 and evaporated to yield 14.3 g of a brown sticky substance.

Biological activity
Escherichia coli (clinical isolate), Klebsiella sp., Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae (clinical isolate), Mycobacterium tuberculosis (clinical isolate), Mycobacterium tuberculosis H37Ra, Mycobacterium smegmatis, Aspergillus niger, Aspergillus fumigatus, Candida albicans and Microsporum gypseum were used.Agar-well diffusion assay for both gram positive and gram negative bacteria and fungi 7 and minimum inhibitory concentration assay for acid-fast bacilli were adopted 8 .For gram bacteria Müeller-Hinton agar, Rosebengal agar for Aspergillus sp. and for C. albicans and M. gypseum Sabauroud agar plates were used.While, in vitro antimycobacterial activity of alkaloid fractions (≤100 µg/mL) were determined using twofold serial broth dilution technique.Streptomycin, nystatin, rifampicin and ethambutol were used as standard antibiotics and brucine, an alkaloid for comparison.

Statistical analysis
Values are expressed for the zone of growth inhibition diameter of gram positive and negative bacteria and fungi as mean of triplicates ± SE.The significance of difference of data obtained statistically from student's paired t-test (P ≤ 0. 05) and was evaluated using Duncan's multiple range test 9 .

Results and Discussion
Seven alkaloid fractions (PB-I to PB-VII) were isolated from Strychnos potatorum seed and their UV, FT-IR, 1 H NMR and FAB-MS spectra have revealed that these are indole type of alkaloids with the basic strychnine skeleton, the prime type of alkaloid of Strychnos species.Further, from co-TLC with an authentic sample (kindly provided by Dr. Michel Frederich, Liege University, Belgium) and the literature available [10][11][12][13] fraction PB-IV was identified as diaboline (1-acetyl-19,20-didehydro-17,18-epoxycuron-17-ol), the most abundant alkaloid of S. potatorum (Figure 1).However, the structures of remaining alkaloids are to be established.These alkaloid fractions have exhibited considerable antimicrobial activity against the tested bacteria and fungi.Marked zone of growth inhibition of gram positive bacterium S. aureus and gram negative bacteria P. vulgaris, S. typhimurium, and V. cholerae by the fractions including diaboline (PB-IV) is observed (Table 1).Further, they have exhibited significant (P ≤ 0. 05) antifungal activity at higher concentration (200 µg/mL) especially against A. niger and C. albicans (Table 2).Furthermore, these alkaloid fractions showed considerable minimum inhibitory concentrations (IC 50 ) against M. tuberculosis (clinical isolate) at ≤100 µg/mL concentration (Table 3).Diaboline (PB-IV) and fraction PB-V were proved to be more effective against M. tuberculosis (12.5 µg/mL).On the other hand, the total alkaloid extract exhibited variable effect on the tested microorganisms.It may be because of intrinsic synergistic and/or antagonistic behaviour of these fractions when they are in a mixture.

Conclusion
It is evident from literature that alkaloids isolated from plants have considerable biological activity 1,2 .Similar observations were made by Caron et al. 14 of indole alkaloids for their antimicrobial properties 14 and antifungal properties 15 , isolated from various Strychnos species.Further, dolichantoside and palicoside, the glucoindole alkaloids of S. melladora have shown significant antimycotic activity against Candida species 16 .The mode of action of indole alkaloids is understood to some extent, and has studied the action of cryptolepine, serpentine and matadine, which stimulate topoisomerase II mediated DNA cleavage, where these alkaloids tightly bind to DNA specifically at GC-rich sequences and behave as typical intercalating agents 17,18 .These findings have further consolidated the wide use of S. potatorum seed in treating of various microbial infections both in traditional (Ayurveda, Unani and Siddha) and folk medicine.From the results obtained, it may be concluded that the antimicrobial activity of these alkaloid fractions has a broad spectrum and is relatively species specific.

Figure 1 .
Figure 1.The structure of Diaboline.However, the structures of remaining alkaloids are to be established.These alkaloid fractions have exhibited considerable antimicrobial activity against the tested bacteria and fungi.Marked zone of growth inhibition of gram positive bacterium S. aureus and gram negative bacteria P. vulgaris, S. typhimurium, and V. cholerae by the fractions including diaboline (PB-IV) is observed (Table1).Further, they have exhibited significant (P ≤ 0. 05) antifungal activity at higher concentration (200 µg/mL) especially against A. niger and C. albicans (Table2).Furthermore, these alkaloid fractions showed considerable minimum inhibitory concentrations (IC 50 ) against M. tuberculosis (clinical isolate) at ≤100 µg/mL concentration (Table3).Table1.Antibacterial activity of Strychnos potatorum alkaloid fractions.Zone of inhibition diameter (mm) of bacteria Drug, 100 µg/mL E. coli

Table 1 .
Antibacterial Values are the mean of three replicates (± SE) at 100 µg/mL concentration.Means within the column followed by the same letter do not differ significantly as determined by Duncan's multiple range test (P<0.05)among the treatments activity of Strychnos potatorum alkaloid fractions.Zone of inhibition diameter (mm) of bacteria *