Curtisia dentata leaves were collected from the Buffelskloof Nature Reserve situated in Mpumalanga Province, South Africa. The plant was authenticated and identified by Mr John Burrows, Botanist and Reserve Manager, Buffelskloof Private Nature Reserve and a voucher (specimen No: B.C.Turpin-2062) has been deposited in the Herbarium of the Buffelskloof Private Nature Reserve. The leaves were then washed with distilled water, dried on a laboratory bench for five weeks and ground into thin powder (2 mm mesh) using Scientec Hammer Mill, Germany. Powders were kept in plastic bottles at room temperature until required.

Acetone, chloroform, ethyl acetate, ethanol (all were AR grade from Merck, South Africa), DPPH, (Sigma, Germany), Muller Hinton broth, Muller Hinton agar (Oxoid) ADC Middlebrook supplement, Middlebrook 7H9 broth (Fluka), glycerol, Iodo-nitro-terazolium chloride (Fluka), Mycoplasma agar, Mycoplasma broth, Yeast malt broth and Yeast Malt agar (Oxoid).

About 10 g of the ground leaves were extracted with 40 ml acetone, ethanol, chloroform and ethyl acetate three times separately using a sonicator at 15 °C. The extracts were then filtered through Whatman's No.1 paper and then concentrated using Buchi Rotary evaporator. The residues were then weighed and kept in a refrigerator until needed.

Shortly, the cytotoxicity studies were carried out using MTT Cell Proliferation Assay as described by Mosman (1983[23]). The Human embryonic kidney (HEK293) and Human hepatocellular carcinoma (HepG2) cells were all grown to confluences in 25 cm³ flasks. This was then trypsinized and plated into 96 well plates at specific seeding densities (5x10⁴ cells per well). Cells were incubated overnight at 37 °C. Medium was then removed and fresh medium (MEM + Glutmax + antibiotics) was added. Isolated compounds (50-300 μg/ml) were then added in triplicate and incubated for 4 h. Antibiotics used were 100 µg/ml of both pencicllin G and streptomycin. Thereafter medium was removed and replaced by complete medium (MEM + Glutmax + antibiotics + 10 % Fetal bovine serum). After 48 h cells were subjected to the MTT assay and the results for different concentrations were reported as mean±SE, using Microplate reader (Meter tech. Σ 960, USA) at 570 nm. The wells with cells only were used as control. The percentages of inhibition were then calculated using

Percentage cell inhibition = 100 -Abs (Sample) / Abs (Control) x 100

(Sreejaya and Santhy, 2013[43]), while the IC₅₀ were obtained from the logarithmic curve of % inhibition v/s concentrations of the isolated compounds. The Selectivity index was calculated as follows: SI=LD₅₀ in mg/ml/ MIC in mg/ml (Bagla et al., 2014[1]).

The microbial resistance of various strains against common antibiotics, especially in developing countries, has resulted in tremendous selective pressure on antibiotics. These have prompted the need to investigate the antimicrobial potential of traditional medicinal plants against pathogenic strains of both bacterial and fungal strains. A variety of extracts, essential oils and compounds from South African medicinal plants have been investigated for antimicrobial activity against variety of etiologic agents of devastating human infections (Buwa and Van Staden, 2006[3] ; Tshikalange et al., 2008[47]; van Vuuren, 2008[48]; Mulaudzi et al., 2015[25]) with the intention of finding new and alternative medicines.

The antimicrobial properties of Curtisia dentata leaf extracts and isolated compounds are presented in Table 1(Tab. 1). The acetone extract exhibited lowest MIC of 0.01 mg/ml against Candida albicans compared to other extracts while ethanol and chloroform extracts exhibited MIC of 0.10 mg/ml against Mycoplasma hominis. Contrarily, Shai et al. (2009[39]), reported MIC values of leaves and stem bark acetone extracts of 0.11 and 0.61 mg/ml respectively. Recently, Nielsen et al. (2012[31]), reported the MIC values of the methanol extracts of the stem bark of 156.25 µg/ml against β-lactamase Escherichia coli, ampicillin-resitant Klebsiella pneumoniae, chloramphenicol resistant Citrobacter, methicilin-resistant Staphylococcus aureus (MRSA) and carbennicillin-resistant Pseudomonas aeruginosa (CRCF), while the leaves exhibited MIC values of 78.12 and 625 µg/ml against MRSA and CRCF respectively. Although these results are not comparable to the data presented in the current paper due to differences in type and origin of the microbial strains used and other environmental conditions, including collection times, they may well suggest that most of the biological active secondary metabolites may be embedded into the leaves than the stem bark.

The ethyl acetate and ethanol extracts exhibited potent MBC of 0.20 against Mycoplasma hominis while chloroform extract exhibited similar MBC against C. albicans. According to Chitemerere and Mukanganyama (2011[4]), MIC values of 0.06 to 0.5 mg/ml are referred to as potent when using similar method as the one in our study. Earlier, the diethyl ether extract exhibited moderate MIC of 3.13 mg/ml against Proteus mirabilis (Fadipe et al., 2014[11], 2015[12]). Betulinic acid revealed most potent antimicrobial activity compared to other isolated compounds. In other studies, betulinic acid isolated from different plants was found to possess variety of biological activity, including anti-HIV, anti-inflammatory, anti-malarial, anti-feedant, anti-parasitic, catalytic activity of Topo II, anticancer and actinociceptive activity (Theo et al., 2009[45], Moghaddam et al., 2012[19]).

Interestingly, the organic extracts from C. dentata revealed most potent MIC values against common pathogenic sexually transmitted infecting organisms and those isolated from HIV patient in our current study. These results in a way, validates the use of the plant species in the treatment of such infections. Besides being recognised as the genital and neonatal pathogen, M. hominis may inhabit the immune systems of both immune-compromised and immune-competent individuals, resulting in serious extra-genital infections such as pneumonia, mediastinitis, pericarditis, endocarditis, osteitis, arthritis and wound infections (Pascual et al., 2010[36]). Moreover it may cohabit with other organisms which may include C. albicans which causes vaginitis (Njunda et al., 2011[32]). Besides exhibiting bactericidal/fungicidal activity, the ethanol extract exhibited the best total activity of 2400 mL/g, suggesting that one gram may be diluted with 2400 ml of a solvent and still inhibit the growth of the organism. Interestingly, bactericidal antibiotics are recommended in the treatment of severely ill and immunosuppressed patients (Nemeth et al., 2015[29]). Although the results obtained in the current work validates the use of the plant in the treatment of variety of infections, the mode of action of these extracts and some isolated compounds remains unknown. Although both the extracts and isolated compounds exhibited potent antimicrobial activity against Candida albicans and Mycoplasma hominis, which are implicated as causative agents of sexually transmitted infections (STIs), there is a need to explore the biological activity of the plant species against other organisms belonging to the similar sphere like Neisseria gonorrhoea, Trichomonas vaginalis and other organisms causing several related infections.

According to Doughari et al. (2011[7]), the rigorous toxicity studies of C. dentata needs to be carried out to enable identification of new biologically active compounds and cautions to be issued on dangerous practices or toxicity effects, if any, as the species is extensively used in the treatment various human infections. The results for the cytotoxicity investigations are shown below (Table 2(Tab. 2)) and suggest that the isolated compounds inhibit the growth of selected human cells in a dose dependent manner. Earlier, the acetone and dichloromethane extracts of C. dentata reportedly exhibited LC₅₀ of 0.007 and 0.024 µg/ml respectively in an MTT assay against Monkey kidney cells (Shai et al., 2008[41]). The higher LD₅₀ implies that it would take a large quantity of the extract to cause a toxic response, while small LD₅₀ values are highly toxic and could be dangerous (Okeleye et al., 2013[33]). In our current study, both betulinic acid and β-sitosterol exhibited LD₅₀ of > 300 µg/ml against the selected cell lines, suggesting that the compounds are less toxic. Ursolic acid exhibited the lowest LD₅₀ of 122.4 µg/ml against HEK 293 cell line while lupeol exhibited LD₅₀ of 278.8 and 289.4 4 µg/ml against HEK 293 and Hep G2 respectively. However, according to Sahranavard et al. (2009[38]), the isolated compounds are referred to as toxic when exhibiting LD₅₀ of about 100 µg/ml or less. Some isolated compounds and plant extracts from plant materials reportedly exhibited very low IC₅₀, hence toxic (Mthethwa et al., 2014[24]; Behzad et al., 2014[2]; Tiwary et al., 2015[46]).

The β-sitosterol and betulinic acid revealed percentage inhibition of 25.0 to 41. 30 %. According to El-Sharkawy et al. (2013[10]), the percentage of inhibition ranging from 25 to 50 is referred to as normal. Elsewhere, β-sitosterol exhibited LD50 of 21.9 and 21.6 against normal (HFB4) and cancer (MCF7) cells respectively (Singab et al., 2012[42]). Generally, the isolated compounds are less toxic hence relevant to use in pharmacological setups.

The selectivity index (SI) of ursolic acid and lupeol against HEK 293 and HepG2 are reported in Table 3(Tab. 3). SI indicates the cytotoxic selectivity or safety of the crude extract or isolated compound against the selected cell lines (Machana et al., 2011[16]). The compounds exhibited low selectivity index values, indicating the similar cytotoxicity and antimicrobial activity. According to Makhafola et al. (2014[17]), the in-vitro toxicity does not equate to in vivo toxicity because of difference in physiological microenvironment in live animal and tissue culture. Moreover, other factors relating to chemical kinetics which may include absorption, biotransformation, distribution and excretion, which influence the exposure at the level of target cells in vivo, cannot be adequately simulated in vitro.

The results for the antioxidant activity of both acetone and ethyl acetate are shown in Table 4(Tab. 4). The acetone extract exhibited better inhibition of DPPH than ethyl acetate extract, while ascorbic acid completely inhibits DPPH at lower concentrations. Acetone extract inhibits DPPH at a comparable state at 0.08 mg/100 ml. According to Mongalo et al. (2012[21]), the extract is a good inhibitor of DPPH if it gives inhibition of about 75 % at 0.75 mg/100ml, suggesting that the acetone extract of the plant species in the current study exhibited moderate free radical scavenging activity while ethyl acetate was relatively poor.

However, there is a need to explore the antioxidant properties of the extracts against other forms of radicals. The antioxidant activity may be of greater important in preventing oxidative stress which may be involved in many fatal infections and diseases.

The newly isolated compound (1) was isolated and identified as β-Sitosterol (NMR data not shown), while the other three compounds (2, 3 and 4) were identified earlier (Fadipe et al., 2014[11]) (Figure 1(Fig. 1)).