Journal of Ethnopharmacology 139 (2012) 712–720 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology jo urnal homepage: www.elsevier.com/locate/jethpharm Pharmacological, genotoxic and phytochemical properties of selected South African medicinal plants used in treating stomach-related ailments ∗ A. Okem, J.F. Finnie, J. Van Staden Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa a r t i c l e i n f o a b s t r a c t Article history: Ethnopharmacological relevance: The evaluated medicinal plants are used in South African traditional Received 3 August 2011 medicine in treating stomach-related ailments. Received in revised form Aims of the study: The study aimed at evaluating the pharmacological, genotoxic and phytochemical 10 November 2011 properties of the seven selected medicinal plants used for treating stomach-related ailments. Accepted 18 November 2011 Materials and methods: : Ethyl acetate (EtOAc), ethanol (EtOH) 70% and water extracts of the selected Available online 3 December 2011 plant parts were evaluated for their antimicrobial and anthelmintic activities using microdilution assays. Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), Gram-negative bac- Keywords: Anthelmintic terium (Escherichia coli) and Candida albicans were used for antimicrobial assays. Caenorhabditis elegans Antimicrobial was used for the anthelmintic assay. Plant extracts were also assayed for their cyclooxygenase-inhibitory Cyclooxygenase activity against cyclooxygenase-1 and -2 enzymes. The Ames test was used to evaluate the genotoxicity of Genotoxicity the plant extracts. A spectrophotometric method was used to determine the total phenolics, gallotannins, Medicinal plants flavonoids and saponins. Phenolics, Saponin Results: Twelve extracts exhibited minimum inhibitory concentration (MIC) <1 mg/mL against the bacte- rial test strains, and five extracts exhibited MIC <1 mg/mL against Candida albicans. The EtOAc extract of Tetradenia riparia had the best minimum lethal concentration (MLC) value (0.004 mg/mL) against Caenorhabditis elegans. All the EtOAc extracts exhibited percentage inhibition in the range of 50.7–94.7% ␮ against COX-1 and -2 enzymes at 250 g/mL. All the plant extracts were non-mutagenic towards Salmonella typhimurium tester strains TA98, TA100 and TA1537 without metabolic activation. Phyto- chemical analysis revealed relatively high amounts of total phenolics, gallotannins and flavonoids in the evaluated plant extracts. Conclusions: The general pharmacological activities exhibited by some of the plant extracts in this study support the traditional uses of the selected plants in treating stomach-related ailments. The Ames test showed that all the plant extracts were non-mutagenic but cytotoxicity tests are needed to ascertain the safety for long-term consumption. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction patients suffering from different kinds of infectious diseases such as persistence diarrhoea, gastroenteritis, and candidemia caused by In traditional medicine, healers often treat symptoms for stom- opportunistic etiologic organisms such as Candida spp and Entero- ach ailments and these encompass a broad spectrum of disorders coccus faecalis (Drouhent and Dupont, 1988; Hobson, 2003; Kayser, involving the lower and upper abdominal cavity. In developing 2003). Gastroenteritis is the inflammation of the digestive tract, countries there is a high rate of morbidity and mortality result- involving both the stomach and the small intestines which are char- ing from co-existing conditions of infectious and parasitic diseases. acterized by symptoms such as stomach pain, diarrhoea, dysentery, This is attributed to several conditions such as poor hygiene and vomiting, fever, inflammatory infections of the colon and abdom- lack of clean water that makes individuals vulnerable to infections inal cramp (WHO, 2003). Bacteria, viruses and parasitic organisms (Bi et al., 2004). The recent increase in the number of immuno- were identified long ago as the major etiologic agents of infectious suppressed and/or debilitated patients in South Africa resulting diseases that plague man. Escherichia coli and Staphylococcus aureus from the high rate of HIV/AIDS epidemic, have led to a number of are mostly associated with food poisoning. Escherichia coli is known to produce enterotoxins that induce watery diarrhoea and abdom- inal tissue damage resulting in acute or chronic abdominal pains ∗ and cramps (Kloos and Schleifer, 1981; Sleisenger and Fordtrand, Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897. E-mail address: [email protected] (J. Van Staden). 1993). The menace of diarrhoea and cholera caused by some of 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.11.034 A. Okem et al. / Journal of Ethnopharmacology 139 (2012) 712–720 713 these etiologic agents in tropical and subtropical countries have 2.2. Pharmacological screening been reported as one of the worst scenarios of disease outbreaks, as these have claimed lives of millions of people especially children 2.2.1. Antibacterial microdilution assay and infants (Cohen and Tartasky, 1997; Sarkar et al., 2007). Antibacterial activity of the plant extracts was determined using The use of plant-based medicine in treating infectious diseases the minimum inhibitory concentration (MIC) technique described has been in existence for thousands of years and will continue to by Eloff (1998) and as detailed by Ndhlala et al. (2009). Overnight provide mankind with new remedies (Cragg and Newman, 2007). cultures of a Gram-negative (Escherichia coli ATCC 11775) and two In South Africa, around 80% of the population relies mainly on tradi- Gram-positive (Staphylococcus aureus ATCC 12600 and Enterococcus tional medicine for their primary health care needs, largely because faecalis ATCC 19433) bacterial strains were used. Neomycin was of the high cost of Western medicine, availability of natural prod- used as the positive control and 70% EtOH was used as the negative ucts and the cultural beliefs of the people (Dauskardt, 1990; Cocks control. The MIC and minimum bactericidal concentration (MBC) and Møller, 2002). Medicinal plants are a reservoir of important were recorded as the concentration of the last well in which there biologically active compounds. Some of these have demonstrated was no bacterial growth. The assay was repeated twice in duplicate potent antimicrobial activities against an array of bacterial, fungal for each extract. and protozoan organisms, as well as anti-inflammatory and anti- cancer activities (Hernändez et al., 2000; Polya, 2003). Others have 2.2.2. Antifungal microdilution assay been reported to have antispasmodic effects, delay gastrointesti- The antifungal activity of the plant extracts was evaluated using nal transit, suppress gut motility, stimulate water adsorption or the microdilution assay described by Eloff (1998) and modified reduce electrolyte secretion (Palombo, 2006). Biologically active for fungi by Masoko et al. (2007). An overnight culture of Candida compounds from medicinal plants have now become the major albicans (ATCC 10231) was used. Amphotericin B (Sigma–Aldrich) focus for developing new and effective pharmaceuticals. This is as a (0.25 mg/mL) was used as the positive control. The MIC and result of the side effects and the resistance that pathogenic organ- minimum fungicidal concentration (MFC) were recorded as the isms develop against the antibiotic agents currently used (Essawi concentration of the last well in which there was no fungal growth. and Srour, 2000). The screening was done in duplicate and repeated twice for each In spite of the great advancement in instrumentation and extract. achievement in the pharmaceutical industries in the search for drug lead compounds from plants, much of the plant biodiver- 2.2.3. Anthelmintic colorimetric assay sity still remains unexplored as sources of novel principles (Cragg An in vitro determination of free-living nematode larvae via- and Newman, 2007). Growing evidence has shown that some of bility, as described by James and Davey (2007) with modification the plant’s secondary metabolites are toxic and/or carcinogenic, as outlined by Aremu et al. (2010), was used to evaluate the MLC which can induce adverse effects leading to mutation and/or degen- values of the plant extracts. A 3-day-old culture of Caenorhabditis erative diseases (Popat et al., 2001). Hence, evaluating natural elegans var. Bristol (N2) was used. Levamisole was used as the pos- products for their efficacy and toxic or genotoxic potential before itive control and 70% EtOH was used as the negative control. The applying them as therapeutic agents is becoming increasingly assay was done in duplicate and repeated twice for each extract. important. The present study evaluates the efficacy of seven South African 2.2.4. Anti-inflammatory assay medicinal plants used in traditional medicine for the treatment Both the cyclooxygenase-1 and -2 assays were conducted as of stomach-related ailments. The plant extracts were screened described by Jäger et al. (1996) and as outlined by Eldeen and for antibacterial and antifungal activities against some of the Van Staden (2008). The stock solution of COX-1 and -2 enzymes ◦ ␮ − pathogenic agents known to cause stomach-related ailments in (60 L) (Sigma–Aldrich Germany) stored