Evaluation of the antibacterial and anticancer activities of some South African medicinal plants

Background Several herbs are traditionally used in the treatment of a variety of ailments particularly in the rural areas of South Africa where herbal medicine is mainly the source of health care system. Many of these herbs have not been assessed for safety or toxicity to tissue or organs of the mammalian recipients. Methods This study evaluated the cytotoxicity of some medicinal plants used, inter alia, in the treatment of diarrhoea, and stomach disorders. Six selected medicinal plants were assessed for their antibacterial activities against ampicillin-resistant and kanamycin-resistant strains of Escherichia coli by the broth micro-dilution methods. The cytotoxicities of methanol extracts and fractions of the six selected plants were determined using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay). Results The average minimum inhibitory concentration (MIC) values of the plants extracts ranged from 0.027 mg/mℓ to 2.5 mg/mℓ after 24 h of incubation. Eucomis autumnalis and Cyathula uncinulata had the most significant biological activity with the least MIC values. The in vitro cytotoxicity assay on human hepatocarcinoma cell line (Huh-7) revealed that the methanol extract of E. autumnalis had the strongest cytotoxicity with IC50 of 7.8 μg/mℓ. Ethyl acetate and butanol fractions of C. uncinulata, Hypoxis latifolia, E. autumnalis and Lantana camara had lower cytotoxic effects on the cancer cell lines tested with IC50 values ranging from 24.8 to 44.1 μg/mℓ; while all the fractions of Aloe arborescens and A. striatula had insignificant or no cytotoxic effects after 72 h of treatment. Conclusions Our results indicate that the methanol fraction of E. autumnalis had a profound cytotoxic effect even though it possessed very significant antibacterial activity. This puts a query on its safety and hence a call for caution in its usage, thus a product being natural is not tantamount to being entirely safe. However, the antibacterial activities and non-cytotoxic effects of A. arborescens and A. striatula validates their continuous usage in ethnomedicine.


Background
Various plants are used in the treatment of gastrointestinal related diseases. Several studies have documented reports on some herbs used in ethnotherapy of diarrhea, dysentery, vomiting, stomach cramps and other associated ailments [1][2][3]. Contrary to the belief of a large proportion of the populace that anything natural is safe, many commonly used herbs cause acute toxicity effects and in the long term may be toxic. The toxic effects may range from diarrhoea, hypersensitivity reactions, nausea or vomiting, to organ-targeted toxicity; immunotoxicity, embryo/foetal and prenatal toxicity, mutagenicity/genotoxicity, hepatotoxicity, nephrotoxicity, presence of epileptogenic compounds, cardiac toxins, gastrointestinal toxins to carcinogenicity [4]. Other adverse effects of herbal medicines may include cardiovascular, neurological and dermatologic toxic effects. In the review by Luyckx and Naicker [5], it was stated that 'drug-induced nephrotoxicity reportedly contributes to up to 26% of cases of hospital-acquired acute kidney injury (AKI) and 18% of cases of global community-acquired AKI ...' The review [5] further revealed that folk remedies account for up to 35% of cases of AKI in the developing world.
Many of the plants widely acclaimed to be of therapeutic values have not enjoyed vigorous assessments to gauge their safety. A number of cases of complications arising after the administration of medicinal herbs have been reported. Foyaca-Sibat and co-investigators described the case reports of two patients with neuromyotonia not associated with malignancies. The patients were reported to have developed acute renal failure while under treatment with herbal medications by their traditional healer in the former Transkei region of South Africa [6].
Other researchers have also identified medicinal plants with potential toxicity such as the extracts of Athrixia phylicoides DC. (Bush tea) [7]; and a flavonol glycoside from Bauhinia galpinii [8]. Genotoxicity and mutagenic effects in the Salmonella microsome assay have been reported in Crinum macowanii, Chaetacme aristata Planch. (Celastraceae), Plumbago auriculata Lam. (Plumbaginaceae), Catharanthus roseus (L.) G.Don. (Apocynaceae) and Ziziphus mucronata Willd. (Rhamnaceae) [9]. Additionally, Michellamine B-an alkaloid dimmers isolated from Ancistrocladus korupensis was inhibitory to several laboratory and clinical strains of HIV-1, including the AZT resistant strain G910-6 and the pyridinone-resistant strain A17; as well as strains of HIV-2. However, the high toxicity of this compound to several human cell lines prevented its further evaluation [10]. Data on the cytotoxic assessments of herbs are very few compared with the huge number of plants acclaimed to have therapeutic values [11,12]. This study investigated the cytotoxic effects of Aloe arborescens, A. striatula, Cyathula uncinulata, Eucomis autumnalis, Hypoxis latifolia and Lantana camara commonly used in the treatment of gastrointestinal infections in the Oliver R. Tambo District Municipality (ORTDM), Eastern Cape Province, South Africa.

Plant material, extraction and fractionation
Fresh plant parts were collected in ORTDM, Eastern Cape Province, South Africa between July 2008 and February 2010. The plants were identified in the Kei herbarium, Walter Sisulu University, South Africa where voucher specimens have been deposited. Information on the selected herbs are presented in Table 1 [13][14][15][16][17][18][19]. The air-dried plant parts were extracted three times with methanol (Merck, Japan) and filtered using a Buchner funnel and Whatman No. 1 filter paper. The extracts were concentrated under reduced pressure at a temperature of 40°C using a rotating evaporator to yield methanol extract. The methanol extract was then suspended in deionised water and partitioned sequentially with Ethyl acetate and n-butanol. The fractions were concentrated under reduced pressure to yield the corresponding fractions and the remaining water fraction.

Antibacterial assay
The antibacterial assay was by the determination of minimum inhibitory concentration values of plant extracts and fractions against Gram negative bacteria strains. The broth dilution method was carried out in 96-well microtitre plates using ampicillin-resistant and kanamycin-resistant strains of Escherichia coli as the test organisms. A McFarland No1 standard suspension of bacteria inoculum was prepared in sterile Mueller Hinton Broth. Triplicate tests were performed in a series of two-fold dilutions of extract (10 mg/mℓ) as previously described [20]. Kanamycin was used as the positive control for ampicillin-resistant E. coli strain while ampicillin was used in the case of kanamycin-resistant E. coli strain. Plates were incubated at 37°C for 18 h and an hour before the end of incubation, 40 μℓ of 0.2 mg/ml INT (p-iodonitrotetrazolium salt) solution was added to each well. The lowest concentration indicating inhibition of growth was recorded as the MIC. This was indicated by the clear well after further incubation with INT as opposed to the pinkish colouration in growth wells.  [18], treatment of wound [19].

Cytotoxicity assays
The in vitro cytotoxicities of the selected herbs and solvent-solvent fractions on a human hepatoma cell line, (Huh-7), which was established from a hepatocellular carcinoma were examined using a modified MTT assay [21]. The Huh-7 was maintained at -80°C in Dulbecco's Modified Eagle Medium (DMEM) and recovered from preservative by centrifugation. The pellet was re-suspended in fresh DMEM and cultured in a humidified atmosphere at 37°C using RPMI 1640 supplemented with 10% foetal bovine serum, 100 U/mℓ penicillin G and 100 ug/ml streptomycin and L-glutamine (Gibco BRL) in 5% CO 2 incubator (Thermo Fischer Scientific, Wakenyaku Co. Ltd, Japan). Cells were sub-cultured every 2 days after confluent growth was observed. The MTT assay was carried out as follows. Briefly, the cells at a density of 1 × 10 4 per mℓ were seeded in each well of a flat-bottom 96-well plate containing 100 μℓ of the growth medium. Cells were permitted to adhere for 24 h, and then treated with various fractions at concentrations 0, 1, 10 and 100 μg/mℓ for 72 h. After that, 20 μℓ of 5 mg/mℓ MTT in phosphate buffered saline (PBS) was added to each well and the plate was incubated for an additional 2 h. The medium was discarded and the formazan blue, which formed in the cells, was dissolved with 100 μℓ MTT stop solution (Triton-X100-20 mℓ; Isopropyl alcohol-500 mℓ; HCl-2 mℓ). After incubation at 37°C for 10 min, the absorbance of the dissolved solutions was detected at 490 nm on a microplate ELISA reader (Thermo Labsystems, Japan). Cytotoxicity was expressed as the concentration of extracts or fractions inhibiting cell growth by 50% (IC 50 ). All tests and analyses were run in triplicate. Statistical analyses were carried out using MS Excel 2007.

Antibacterial activities
Active antibacterial extracts were revealed as clear spots of inhibition of the growth of test organisms in microtitre wells. The pinkish contrasting wells of bacterial growth was indicative of non-cleavage of the tetrazolium salt to yield the pinkish or purplish formazan products [22]. The average MIC values of the plants extracts ranged from 0.27 mg/mℓ and 2.5 mg/mℓ after 24 h of incubation ( Figure 1). The Ethyl acetate fraction of each type of plant was the most active fraction against the test bacteria. E. autumnalis had the least MIC (0.27 mg/mℓ) followed by C. uncinulata (0.39 mg/mℓ) and thus demonstrated good antibacterial activities. The methanol extract of H. latifolia had the highest value of MIC (2.5 mg/mℓ).

Cytotoxicities of methanol extract of plants extract and fractions
The results of cytotoxicity assays of the extracts and fractions of the investigated plants on human hepatoma cell line (Huh-7) are shown in Table 2. The methanol extract of E. autumnalis (IC 50 7.8 μg/mℓ) was more cytotoxic than the control toxic substance, berberine (IC 50 9.8 μg/mℓ), other plant extracts and solvent fractions. Of the solvent fractions, the Ethyl acetate and butanol fractions of C. uncinulata, H. latifolia, E. autumnalis and L. camara had moderate cytotoxic effects on the cancer cell lines tested with IC 50 values ranging from 24.8 to 44.1 μg/mℓ while all the fractions of A. arborescens and A. striatula had insignificant or no cytotoxic effects after 72 h of treatment (IC 50 1000 to >1000 μg/mℓ).

Discussion
This study evaluated the cytotoxicity and antibacterial activities of methanol extracts and solvent fractions of A. arborescens, A. striatula, C. uncinulata, E. autumnalis, H. latifolia and L. camara. Among all the samples, the methanol extract of E. autumnalis exhibited greatest cytotoxicity on the cell line tested. However, the water fraction of E. autumnalis and that of other plants showed insignificant cytotoxicity on the cell line, compared with the polar solvent fractions ( Table 2).
The Ethyl acetate fraction of E. autumnalis had the least MIC (0.27 mg/mℓ) followed by C. uncinulata (0.39 mg/mℓ) against the test bacteria and thus demonstrated  good antibacterial activities. Various biological activities of Eucomis were reported [23,24]. E. autumnalis is known for its anti-inflammatory and antispasmodic activities and these have been attributed to components such as homoisoflavones and flavonoids [25]. E. autumnalis also contains some steroidal triterpenoids which are known to be beneficial in wound therapy [25]. However, the bulb was reported to be toxic [25] agreeing very much with our findings.
Eucosterol glycoside, a lanosterol oligosaccharides isolated from E. bicolor demonstrated antitumor activity by causing 44% inhibition of TPA-stimulated 32 P incorporation into phospholipids of HeLa. This activity has been suggested to probably relate to the use of bulb decoctions of E. autumnalis to relieve abdominal distensions and abdominal pain by the Tswana and Pedi tribes of South Africa [26]. According to Koorbanally et al. [27], sheep drenched with fresh bulbs of E. autumnalis in an animal feeding trial presented with listlessness, anorexia, foaming at the mouth, tympanites, an inactive rumen and a strong pulse leading eventually to death within twentyfour hours. Despite these submissions Eucomis is one of the most traded genera of plants in South Africa [28], hence the need for caution in their usage.

Conclusions
In conclusion, the results obtained indicated that the methanol extract of E. autumnalis exhibited much greater cytotoxicity than the methanol extract and solvent fractions of all other plants investigated even though it had strong antibacterial activities. E. autumnalis showed selective anticancer activity against the human hepatoma cell line, whereas the two Aloe spp. were non toxic on the cell line. In addition, the study showed that Aloe arborescens, A. striatula and C. uncinulata may be candidate plants for eventual drug design. Medicinal plants are natural products and may have therapeutic potentials; however, being natural does not make them automatically safe.