The efficacy and safety of nine South African medicinal plants in controlling Bacillus anthracis Sterne vaccine strain

Research article

• Ishaku Leo Elisha,
• Jean-Paul Dzoyem,
• Francien S. Botha and
• Jacobus Nicolaas EloffEmail author

BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201616:5

DOI: 10.1186/s12906-015-0980-1

©  Elisha et al. 2016

Received: 12 July 2015

Accepted: 23 December 2015

Published: 8 January 2016

Abstract

Background

Anthrax is a zoonotic disease caused by Bacillus anthracis, a Gram-positive spore-forming bacterium. The presence of the bacteria and the toxins in the blood of infected hosts trigger a cascade of pathological events leading to death. Nine medicinal plants with good activities against other bacteria were selected to determine their in vitro antibacterial activity against Bacillus anthracis Sterne strain. The cytotoxicity of the extracts on Vero kidney cells was also determined.

Results

The minimum inhibitory concentration (MIC) values of the extracts against Bacillus anthracis Sterne strain ranged from 0.02 to 0.31 mg/ml. Excellent MIC values were observed for the following plant species: Maesa lanceolata (0.02 mg/ml), Bolusanthus speciosus, Hypericum roeperianum, Morus mesozygia (0.04 mg/ml) and Pittosporum viridiflorum (0.08 mg/ml). The total antibacterial activity of the extracts ranged from 92 to 5562 ml/g. Total activity presents the volume to which the extract from 1 g of plant material can be diluted and still inhibit microbial growth. Maesa lanceolata and Hypericum roeperianum had the highest total activity with values of 5562 and 2999 ml/g respectively. The extracts of Calpurnia aurea had the lowest total activity (92 ml/g). The cytotoxicity determined on Vero cells indicated that most of the extracts were relatively non-toxic compared to doxorubicin (LC₅₀ 8.3 ± 1.76 μg/ml), except for the extracts of Maesa lanceolata, Elaeodendron croceum and Calpurnia aurea with LC₅₀ values at 2.38 ± 0.25, 5.20 ± 0.24 and 13 ± 2.26 μg/ml respectively. The selectivity index (SI) ranged from 0.02 to 1.66. Hypericum roeperianum had the best selectivity index, (SI = 1.66) and Elaeodendron croceum had lowest value (SI = 0.02).

Conclusions

The crude acetone extracts of the selected plant species had promising antibacterial activity against Bacillus anthracis. Maesa lanceolata extracts could be useful as a disinfectant and Hypericum roeperianum could be useful to protect animals based on its high total activity and selectivity index. Further investigation of these plant extracts may lead to the development of new therapeutic agents to protect humans or animals against anthrax.

Keywords

Bacillus anthracis Medicinal plants Antibacterial activity Total activity Cytotoxicity Selectivity index

Background

Anthrax is a serious zoonotic disease of great economic and social importance [1]. It is caused by Bacillus anthracis, a Gram-positive spore-forming bacterium [2]. The disease can affect most mammals and several species of birds, but is particularly important in herbivores, such as cattle, sheep and goats [1, 2]. Humans contract anthrax by handling infected animals or products of animals that have died from the disease [3]. Once infection sets in, spores germinate into capsulated bacilli capable of producing toxins. The presence of the bacteria and their toxins in the blood of the infected host triggers a cascade of pathological events leading to death [3]. B. anthracis has always been high on the list of agents that could be used in biological warfare and bioterrorism [4]. The disease is still endemic in many countries of Africa and Asia [3].

The major virulence factors of B.anthracis are encoded on two virulence plasmids pXO1 and pXO2 [5]. The tri-toxin bearing plasmid pXO1 codes for three toxins, which cause haemorrhage, oedema, and necrosis [6]. They comprise the lethal factor, oedema factor, and the protective antigen, in the host cell receptor component. The exotoxins are binary, with the protective antigen acting as the binding domain that allows entry of the toxin into the host cell. The smaller capsule bearing plasmid pXO2 encodes three genes (cap B, cap C, and cap A) involved in the synthesis of the poly-D- glutamyl capsule that inhibits host phagocytosis of the vegetative form of B. anthracis. Both plasmids are necessary for full virulence; loss of either results in an attenuated strain. Sterne B. anthracis strain, carries pXO1 and therefore can synthesise exotoxin, but does not have a capsule [5]. Microbiology laboratories use B. anthracis Sterne strain for accurate identification and diagnosis of anthrax, and occasionally for anthrax research [7].

B. anthracis spores have the capacity to contaminate a given area for a long time, because of their intrinsic ability to survive different environmental conditions and chemical disinfectants [2, 8]. Anthrax spores have been isolated after 60 years from contaminated sites [8, 9]. B. anthracis has been recovered from animal bones estimated to be over 200 years old in the Kruger National Park in South Africa [10].

Control measures employed during anthrax outbreaks include vaccination of livestock using the avirulent B. anthracis Sterne strain [7], burial or burning of dead animals and calcium oxide (lime) application on burial sites [2, 11]. Anthrax infections can be treated effectively with antibiotics, provided treatment is started early. Penicillin has been the antibiotic of choice for many years [1]. Where the use of penicillin is contraindicated or ineffective, ciprofloxacin and doxycycline are good alternatives [4]. B. anthracis natural or acquired resistance to broad spectrum antibiotics [12–14], as well as poor penetration of doxycycline into the central nervous system of infected individuals, necessitate the search for new antimicrobials that could offer effective and potent alternatives in face of bioterrorist attack or anthrax epidemics [4].

Plants have always played a central part in combating ailments in humans and livestock in many indigenous communities [15]. Herbal extracts or decoctions from different plant parts have been used in both ethnoveterinary and ethnomedicinal practices to treat anthrax in animals and humans [16–19]. Traditional use of the following plants have been documented in the treatment of anthrax in livestock and humans, Senna italica, Teucrium africanum, Ptaeroxylon obliquum, Achyrospermum schimperi, Teucrium polium [16, 17, 20].

The Phytomedicine Programme, University of Pretoria, has screened over 700 acetone leaf extracts of more than 530 tree species for their antimicrobial properties beneficial to man and animals [21]. Nine plant species with high antibacterial activities were selected from the database and tested against Bacillus anthracis Sterne strain. The cytotoxicity was also determined using the MTT assay against Vero kidney cells.