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In Vitro Pharmacological Evaluation and Phenolic Content of Ten South African Medicinal Plants Used As Anthelmintics ⁎ A.O

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In Vitro Pharmacological Evaluation and Phenolic Content of Ten South African Medicinal Plants Used As Anthelmintics ⁎ A.O Available online at www.sciencedirect.com South African Journal of Botany 76 (2010) 558–566 www.elsevier.com/locate/sajb In vitro pharmacological evaluation and phenolic content of ten South African medicinal plants used as anthelmintics ⁎ A.O. Aremu, A.R. Ndhlala, O.A. Fawole, M.E. Light, J.F. Finnie, J. Van Staden Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Received 12 January 2010; received in revised form 13 April 2010; accepted 19 April 2010 Abstract Helminth infection is regarded as one of the neglected tropical diseases (NTDs). Although the disease is common in rural areas, information on the pharmacology of South African medicinal plants used against this disease is limited. We investigated the efficacy of ten South African medicinal plants against Caenorhabditis elegans. Because of the increased susceptibility of a host to microbial infections and other inflammatory responses associated with helminth infections, the antimicrobial and cyclooxygenase (COX) inhibitory activities of the plants were also investigated. Phenolics including flavonoids, condensed tannins and gallotannins have been linked to many pharmacological activities. Thus, the phenolic content of the plant extracts were quantitatively evaluated. In the three bioassays, organic solvent extracts from Cyathea dregei (roots and leaves), Felicia erigeroides (leaves and stems), Hypoxis colchicifolia (leaves) and Senna petersiana (leaves) exhibited noteworthy pharmacological activities while Acokanthera oppositifolia (leaves) had good COX inhibitory activity. The concentration of phenolics ranged from 56.7 to 1.7 mg GAE/g dry matter in Ocimum basilicum and Cotyledon orbiculata var. dactylopsis, respectively. Flavonoids, condensed tannin and gallotannin content also varied greatly among the plant extracts investigated. © 2010 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Anthelmintic; Antimicrobial; Cyclooxygenase inhibition; Phenolic content; South African medicinal plants; Tannin 1. Introduction ignorance are the factors that sustain the parasite life cycle and favour the proliferation of the disease vectors (Brooker et al., A large number of the South African population still depend on 2006). In South Africa, the prevalence of helminth infections has traditional medicine (TM) for their health care needs due to its been reported in provinces such as KwaZulu-Natal and affordability, accessibility and cultural importance (Light et al., Mpumalanga. Helminth infections have remained relatively 2005). Among the variety of diseases treated by traditional healers high over time, and the most vulnerable groups are women and in TM are helminth infections. These infections are regarded as children (Jinabhai et al., 2001). one of the neglected tropical diseases (NTDs) due to their high Globally, a number of medicinal plants have been used for the prevalence among the economically disadvantaged people in the treatment of helminth infections by the local people (Waller et al., rural areas in different regions of the world. In most developing 2001). South Africa is endowed with more than 24000 indigenous countries, helminth infections are considered a major health plants, representing about 10% of all higher plants that occur concern because the factors that predispose humans to these globally. Approximately 3000 plant species are used as medicines infections abound in these regions (Ijagbone and Olagunju, by an estimated 200000 traditional healers in South Africa (Van 2006). Poor sanitation, poverty, unsafe water, malnutrition and Wyk et al., 1997). Although the plant kingdom has remained poorly explored, natural products isolated from plants have been postulated to remain an essential part of the search for novel ⁎ Corresponding author. Tel.: +27 33 2605130; fax: +27 33 2605897. medicines against human diseases (Balunas and Kinghorn, 2005). E-mail address: [email protected] (J. Van Staden). There is a higher potential of discovery and development of more 0254-6299/$ - see front matter © 2010 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2010.04.009 A.O. Aremu et al. / South African Journal of Botany 76 (2010) 558–566 559 potent and efficient drugs from plants because a large number of extracts were concentrated in vacuo using a rotary evaporator medicinal plants have remained understudied for their pharmaco- (Büchi, Germany) at 30 °C. The concentrates were transferred to logical and phytochemical properties (Gurib-Fakim, 2006). pre-weighed glass vials and completely dried under a stream of Although helminth infections do not always cause obvious air. Water extracts were collected into pre-weighed glass jars and diseases, the host's health integrity could be compromised due to freeze-dried. The percentage yield (Table 3) of each dried extract the effect of the metabolic activities of the parasites on nutrition as in terms of the starting plant material was determined after which well as the toxic products of excretion (Deacon, 2006). This it was stored in the dark at 10 °C until required for analysis. probably accounts for the increased susceptibility and enhances Phenolic compounds were extracted from dry plant material as the progression of bacterial diseases such as tuberculosis and described by Makkar (2000). Ground plant samples (1 g) were other opportunistic infections among helminth hosts (Bentwich extracted with 10 ml of 50% methanol (MeOH) by sonication in et al., 1995; Brooker et al., 2004). In addition, microorganisms cold water for 20 min. The extracts were then filtered in vacuo such as bacteria (Escherichia coli, Klebsiella pneumoniae and through Whatman No.1 filter paper. Staphylococcus aureus) and fungi (Candida albicans) have been implicated in the increased infection rate in immune-compro- 2.2. Pharmacological evaluation mised patients worldwide (Sleigh and Timburg, 1998). Globally, the increase in helminth infection and their growing resistance to 2.2.1. Anthelmintic assay most chemotherapeutics is a major problem facing human health An in vitro colourimetric assay used in the determination of free- (James and Davey, 2009). In addition, research on the discovery living nematode larvae viability, as described by James and Davey of new chemotherapies for helminth infections has been neglected (2007) with slight modifications, was used to evaluate the by governments, researchers and the pharmaceutical industries minimum lethal concentration (MLC) values of the extracts. A 3- due to the poor economic prospects and the presumed low priority day-old Caenorhabditis elegans var. Bristol (N2) culture was of the diseases that go with it (Geary et al., 1999). prepared by subculturing a stock culture seeded with autoclaved E. The importance of pharmacological screening of plant extracts coli. The subcultured C. elegans was washed with 5 ml of M9 for multiple biological activities has been extensively documen- buffer (Brenner, 1974) into a sterile McCartney bottle and, using a ted (Houghton et al., 2005).Theimportancehasbeen UV–visible spectrophotometer (Varian Cary 50, Australia), the demonstrated for diseases such as Alzheimer's and diabetes. optical density (OD) at 530 nm was measured. Thereafter, 5 ml of The use of multiple bioassays gives a clearer indication of the M9 buffer (sufficient for inoculating one microtitre plate) were effect of the extracts in relation to the disease state (Houghton adjusted with appropriate volumes of the prepared stock of C. et al., 2007). This study was aimed at screening extracts of elegans culture to obtain a culture mixture in the OD530 range of medicinal plants to determine their efficacy as anthelmintics. 0.04–0.06 (approximately 100 worms per 50 µl). Organic solvent Besides the anthelmintic investigation, antimicrobial activity and extracts were redissolved in DMSO (25 mg/ml), and water extracts cyclooxygenase (COX) enzymes inhibition of the plant extracts in sterile water (50 mg/ml). Each redissolved extract (100 µl) was were carried out as microbial infection and pain are often two-fold serially diluted with 100 µl sterile distilled water down a associated with helminth diseases. The plant extracts were also 96-well microtitre plate. A similar two-fold serial dilution of 100 µl investigated for phenolic composition, to provide a clue on of 1 mg/ml levamisole (Sigma-Aldrich, Germany) was used as a different phenolic compounds that could be responsible for any reference drug. The prepared C. elegans culture (50 µl) containing biological activity observed. approximately 100 worms (C. elegans) were added to each well of the microtitre plate. C. elegans culture, DMSO and water were 2. Materials and methods included as controls. The microtitre plates were covered with parafilm and incubated at 20 °C for 48 h in the dark. Thereafter, 2.1. Plant collection and extract preparation 50 µl of 1.25 mg/ml p-iodonitrotetrazolium chloride (INT) (Sigma-Aldrich, Germany) was added to the wells of the Plant material of ten species (Table 1) was collected from the microtitre plate and further incubated at 20 °C for 24 h. Active University of KwaZulu-Natal (UKZN) Botanical Garden, Pieter- organisms biologically reduce the colourless INT to a pink– maritzburg and Mount Gilboa (29° 15.450′ S, 30° 20.289′ E) red colour (McGaw et al., 2007). The concentration of the between February and April 2009. Voucher specimens of the plants lowest clear well was
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