The cytotoxicity and antimicrobial activities of harman alkaloids including harmane, harmine, harmalol and harmaline were investigated. The cytotoxicity was monitored by the brine shrimp lethality test and microdilution method was used to determine MIC and MBC of the compounds. Harmane showed the most cytotoxicity and the most antimicrobial activity.
PDF Abstract XML References Citation
How to cite this article
The harman alkaloids are a group of beta-carboline alkaloids that present in some medicinal plants such as Peganum harmala L. (syrial rue) and Banisteriopsis caapi L. (yage). These plants have been used for the treatment of various diseases including cancers and malaria in oriental traditional medicine for 2 millennia (Nadikarni, 1976). A number of pharmacological effects have been attributed to such alkaloids such as tremorogenesis (Louis et al., 2002), hypothermia (Fattah et al., 1995), hallucinogenesis (Grella et al., 1998) monoamine oxidase inhibition (May et al., 1991; Rommelspacher et al., 1994). They are also known to bind to receptors like 5-HT receptors and the benzodiazepine binding site of GABA receptors (Saano et al., 1982). The spasmolytic effects of harmane, harmine and harmaline have also been reported on guinea-pig isolated trachea (Shi et al., 2001). In addition, these compounds possess antioxidative and radical scavenging properties (Tse et al., 1991; Herraiz et al., 2002) inhibition of platelet aggregation (Saeed et al., 1993), cytotoxic effects on cancer cell lines and inhibition of DNA topoisomerase I (Sobhani et al., 2002 ). Reports are also available on cardiovascular actions of these alkaloids (Shi et al., 2000) and the vasorelaxant effects of harmane, harmine, harmaline and harmalol seem to contribute to their hypotensive effects (Shi et al., 2001). Anthelmentic and antiprotozoal activities have also been attributed to harman alkaloids (Ancolio et al., 2002).
In the present study, we evaluated the cytotoxicity and antimicrobial activity of harman alkaloids including harmane, harmine, harmalol and harmaline.
MATERIALS AND METHODS
Harman alkaloids including harmane, harmine, harmalol and harmaline were obtained from Fluka Chemical Co. and used without further purification.
The following strains were used as test organism: Staphylococcus aureus, Escherchia coli, Proteus vulgaris and Candida albicans. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of all alkaloids were determined by microdilution method in Mueller- Hinton broth (Difco) for bacteria and the Sabourad medium for Candida albicans. Inoculates were prepared in the same medium at a density adjusted to a 0.5 McFarland turbidity standard [108 colony-forming units (CFU) mL-1] and diluted 1:10 for the broth microdilution procedure. Microtiter plates were incubated at 37°C and the MICs were recorded after 24 h of incubation for bacteria and after 48 h of incubation for Candida albicans. The Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration of compounds at which the microorganism tested did not demonstrate visible growth. Minimum Bacteriostatic Concentration (MBC) was defined as the lowest concentration yielding negative subcultures or only one colony.
Brine Shrimp Bioassay
The brine shrimp lethality test was used. In brief, brine shrimp (Artemia salina) eggs were hatched in artificial seawater (38 g L-1 of sea salt). After 48 h of incubation, ten brine shrimps were transferred to each sample vial using a pasteur pipette and artificial sea water was added to make 5 mL. Sample vials were previously prepared by dissolving 10 mg of each compound in 1 mL of DMSO. From this solution 5, 10, 15, 30 and 50 μL was transferred to vials corresponding to 10, 20, 30, 60 and 100 μg mL-1. The solvent was then evaporated overnight. Survivors were counted after 24 h and the LC50 values, with 95% confidence intervals were determined using probit analysis. Control vials were prepared using DMSO only. Three replicates were prepared for each dose level.
The MIC and MBC values of these compounds show the following order of potency for Staphylococcus aureus, Escherchia coli and Proteus vulgaris inhibition: harmane>harmine>harmaline>harmalol and following order of potency for Candida albicans inhibition: harmane>harmaline>harmalol>harmine (Table 1 and 2).
It is revealed from the Table 3 that all the compounds tested in this study, caused brine shrimp death compared with other cytotoxic natural compounds for example berberin chloride (LC50 = 22.5 μg mL-1) and strychnine sulfate (LC50 = 77.2s μg mL-1) (Meyer et al., 1982).
|Table 1:||Minimum inhibitory concentration (MIC mg mL-1) of harman alkaloids|
|Table 2:||Minimum bactericidal concentration (MBC mg mL-1) of harman alkaloids|
|Table 3:||Cytotoxicity of harman alkaloids|
Harmine showed the most cytotoxicity (LC50 = 9.88 μg mL-1) followed by harmane (LC50 = 23 μg mL-1), harmaline (LC50 = 23.74 μg mL-1) and harmalol (LC50 = 50 μg mL-1).
The results of antimicrobial investigation confirmed the antibacterial and antifungal activity of harman alkaloids. Due to their strong toxicity and their possible genotoxicity in mammalian cells, harman alkaloids can not represent interesting therapeutic antibacterial and antifungal agents, however their presence in several medicinal plants might contribute to the prevention of bacterial and fungal infections in the populations using traditional medicine.
The cytotoxicity of plant materials is considered to be due to the presence of antitomour compounds. Harman alkaloids including compounds tested in this study are major compounds in some medicinal plants such as peganum harmala and the results of this study demonstrates that these compounds have probably defense role in this plants. They showed very good cytotoxicity and more study are needed to demonstrate usefulness of them. These compounds with their analogues can be new anticancer drugs in the future and harman alkaloids containing plants will be a good source for them.
- Ancolio, C., N. Azas, V. Mahiou, E. Ollivier and C. Di Giorgio et al., 2002. Antimalarial activity of extracts and alkaloids isolated from six plants used in traditional medicine in Mali and Sao Tome. Phytother. Res., 16: 646-649.
- Grella, B., M. Dukat, R. Young, M. Teitler, K. Herrick-Davis, C.B. Gauthier and R.A. Glennon, 1998. Investigation of hallucinogenic and related beta-carbolines. Drug Alcohol Depend., 50: 99-107.
- Herraiz, T. and J. Galisteo, 2002. Tetrahydro-beta-carboline alkaloids that occur in foods and biological products. Free Radic. Res., 36: 923-928.
- Louis, E.D., W. Zheng, E.C. Jurewicz, D. Watner, J. Chen, P. Factor-Litvak and M. Parides, 2002. Elevation of blood β-carboline alkaloids in essential tremor. Neurology, 59: 1940-1944.
- May, T., H. Rommelspacher and M. Pawlik, 1991. Harman binding experiments. I: A reversible and selective radioligand for monoamine oxidase subtype A in the CNS of the rat. J. Neurochem., 56: 490-499.
- Rommelspacher, H., T. May and B. Salewski, 1994. Harman (1-methyl-beta-carboline) is a natural inhibitor of monoamine oxidase type A in rats. Eur. J. Pharmacol., 252: 51-59.
- Shi, C.C., S.Y. Chen, G.J. Wang, J.F. Liao and C.F. Chen, 2000. Vasorelaxant effect of harman. Eur. J. Pharmacol., 390: 319-325.
- Shi, C.C., J.F. Liao and C.F. Chen, 2001. Comparative study on the vasorelaxant effects of three harmala alkaloids in vitro. Jpn. J. Pharmacol., 85: 299-305.
- Shi, C.C., J.F. Liao and C.F. Chen, 2001. Spasmolytic effects of three harmala alkaloids on guinea pig isolated trachea. Pharmacol. Toxicol., 89: 259-264.
- Sobhani, A.M., S.A. Ebrahimi and M. Mahmoudian, 2002. An in vitro evaluation of human DNA topoisomerase I inhibition by Peganum harmala L. seeds extract and its beta-carboline alkaloids. J. Pharm. Pharm. Sci., 5: 19-23.
- Tse, S.Y.H., I.T. Mak and B.F. Dickens, 1991. Antioxidative properties of harmane and β-carboline alkaloids. Biochem. Pharmacol., 42: 459-464.