Though many experimental procedures have been investigated for Leishmania species, use of natural products remains stand-alone due to variety of chemical constituents present which work synergistically on multiple-target bringing about therapeutic effect (Bastos et al., 2016). This is collaborated by Mutoro et al. (2018b), in which the growth of Leishmania major parasites was inhibited by Plumbago capensis and Solanum nigrum extracts combined. Plants therefore may offer an alternative remedy as they are affordable, less toxic and easily available. The investigations of plants   belonging  to family of Leguminosae,   Rosaceae,   Aloaceae, Compositae, Myrtaceae, Barberidaceae, Boraginaceae, Lamiceae, Solaneceae, Juglandaceae, Asteraceae, Fabaceae etc have proven to be effective against Leishmaniasis (Da Silva et al., 2018; Bahmani et al., 2015; Serakta et al., 2013). Kinuthia et al. (2013) reported that Moringa stenopetala, Callistemon citrinus and Allium sativum plants are effective in elimination of L. major. More than twenty- six (26) Asteraceae plant families have demonstrated usefulness in allergies, burns; examples are Giardia lamblia and Leishmania species (Ivanescu et al., 2015).

The findings by Shamsi et al. (2018) implicated Matricaria chamomilla, Echinacea purpurea, Artemisia sieberi and Lavandula spica to be effective against Leishmaniasis. In Kenya, Warbugia ugandensis has been used by healers in management of Leishmaniasis (Ngure et al., 2009). These findings offer hope that natural products with antiparasitic activity could aid as templates for the manufacture of antileishmanial drugs. In the current work,  the  activity  of  B.  pilosa,  T.  diversifolia and S. oleraceus; methanol, hexane, dichloromethane, ethyl acetate extracts and aqueous phase on L. major parasites was investigated. The plant has been used ethnobotanically to treat Leishmaniasis but has not been scientifically rationalized.

Plant collection and extraction

Fresh aerial parts of S. oleraceus (Figure 1a), T. diversifolia (Figure 1b) and B. pilosa (Figure 1c) were collected from the Egerton University, Botanical garden (0°22'11.0"S, 35°55'58.0"E) with the help of a taxonomist for identification; they were assigned voucher number SK 214, SK 215 and SK 216 respectively . The botanical garden was chosen due to its richness in a wide variety of indigenous plant and its proximity to the laboratory where the analysis was performed. The aerial parts of S. oleraceus, T. diversifolia and B. pilosa were collected in September 2017 and processed as described by   King’ondu et al. (2009).

Briefly, the plant parts were chopped into pieces, dried under a shade for 21 days and finally ground to powder in an electric mill. Approximately, 500 g of powdered plant was soaked in methanol overnight at room temperature. The filtrate obtained through Whatman No.1 filter paper was evaporated in rotary evaporator (BUCHI R-200) at 30-35°C under reduced pressure. The methanol extract was mixed with water and partitioned sequentially with hexane, dichloromethane and ethyl acetate to obtain their respective extracts after evaporation.

Phytochemical analysis

Phytochemical screening for alkaloids, glycosides, saponins, tannins, flavonoids, terpenoids and anthraquinones was done employing the methods of Santhi and Sengottuvel (2016) and Verma et al. (2018).

Preparation of test compounds

Stock solutions of methanol, hexane, dichloromethane, ethyl acetate and aqueous phase of B. pilosa, T. diversifolia and S. oleraceus were made in 5 ml Schneider’s Drosophila insect medium in readiness for assays. Each stock solution was filtered through 0.22 µm filter flasks in a laminar flow hood. Some plant extracts that did not dissolve in the medium were dissolved in 1% dimethyl Sulfoxide (DMSO) to prevent solvent carry-over. All the prepared plant extracts were stored at 4°C and retrieved later for use.

Cytotoxicity on Vero cells

The cytotoxicity assay on Vero cells was conducted by the 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) (Sigma®) protocol (Wabwoba et al., 2010). The quantity of insoluble formazan obtained from reduction of MTT reagent was proportional to number of viable cells. Vero cells were cultured and maintained in Minimum Essential Medium (MEM) (ATCC® 30-2003™) and supplemented with 10% Fetal bovine serum (FBS). Vero cells were harvested by trypsinization and pooled in 50 ml vials. A hundred microliters (100 µl) of the cell suspension was placed into two wells of rows A-H in  a 96- flat-bottomed microtiter plate at a concentration  of 1×10⁶ cells per ml of the culture medium per well and incubated at 37 ^o C in 5% CO₂ to facilitate plating. MEM was gently aspirated off and 200 µl of concentration (10 mg/ml) of the extracts was placed into two wells before dilutions of 2.0×10³, 1.0×10³, 5.0×10², 2.5×10², 1.25×10², and 6.25×10¹ were carried out. The tests were carried out in duplicate. The Vero cells and test extracts were then incubated at 37°C for 48 h in 5% CO₂. Ten micro-litres (10 µl) of MTT was added to each well and further incubated for 3 h until a purple precipitate (formazan) was observed. The medium with MTT was aspirated off and the formazan solubilized with 100 μL of DMSO left to settle for 10 min. The absorbance reading for each well was measured using a micro-titer plate reader at wavelength of 562 nm. The number of viable cells was calculated at each concentration, according to Wang et al. (2006)’s formula: [(A/B) × 100], where A was the absorbance value for treated cells and B was the absorbance value for untreated cells. The extracts were considered cytotoxic if IC₅₀ was less than 30 µg/ml (IC ₅₀ ≤ 30 µg/ml) and non-cytotoxic if otherwise as stated by Sudha and Masilamani (2012).

Parasite preparation

The L. major strain (IDUB/KE/94=NLB-144) was sourced from Institute of Primate Research (IPR), Kenya. The parasites were grown to stationary phase at 25°C in Schneider’s Drosophila insect medium supplemented with 20% heat inactivated fetal bovine serum (FBS)-HYCLONE® USA), 100 µg/ml penicillin and 500 U/ml streptomycin (Hendricks and Wright, 1979), and 250 µg/ml 5-fluorocytosine arabinoside (Kimber et al., 1981). The stationary-phase meta-cyclic stage promastigote were obtained by centrifugation at 1500 × g and 4°C for 15 min. The meta-cyclic promastigote was used for analysis.

Anti-promastigote assays

The in vitro tests were done according to Mikus and Steverding (2000), using Alamar Blue bioassay. L. major meta-cyclic promastigote at the log phase (1×10⁶ parasites per ml) of the culture medium was grown in a flat bottomed 24-well plate at 25°C for 48 h. Aliquots (50 µl) of the promastigotes were transferred into two wells of rows A-H in a 96-flat bottomed microtiter plate and further incubated for 24 h at 27°C. Two hundred microliters (200 µl) of (10 mg/ml) the plant extracts were added before serial dilutions of 10×10³, 5×10³, 2.5×10², 1.25×10², and 6.25×10¹ were done. The control wells contained L. major promastigotes in medium whereas the blank wells had the medium alone and positive control contained pentostam (Glaxo Operations (UK) Limited, Barnard Castle, UK) at concentration of 100 ug/ml. Each crude extract was tested in duplicate. After incubation for 24 h, the plates were removed and 10 µl of MTT was added into each well and further incubated for 4 h period. The medium and MTT were aspirated off, then 100 µl of Dimethyl sulfoxide (DMSO) was added and the plates shaken for 5 min. Optical densities readings were measured at 562 nm using micro titer reader. A decrease in fluorescence representing inhibition was deduced as percent of control culture fluorescence. Optical densities reading was used to generate 50% inhibitory concentration (IC₅₀) for the different plants using the Alamar blue bioassay program.

Data analysis

The IC₅₀ values were determined using Alamar blue program V2. The data presented for activity were analyzed using SAS system. All  the  statistical  comparisons  were done  with Ryan –Einof-Gabriel-Wels multiple ranges tests and assigned at P≤ 0.05 level of significance.

Anti-leishmanial activity

Majority of world population depend solely on herbs for their remedies against numerous infectious diseases (WHO, 2017). Therefore, the advent of synthetic drugs led to neglect of natural products as source of antimicrobials (Ezatpour et al., 2015). However, the management of Leishmaniasis using these drugs have posed challenges such as adverse side effects on users, cost, requirement for long periods of administration and parasite resistance. Moreover, there are no effective vaccines available to cure the disease (Al-Hajj et al., 2018). In this work, antileishmanial activities of B. pilosa, T. diversifolia and S. oleraceus; methanol, hexane, dichloromethane, ethyl acetate extracts and aqueous phase was investigated on L. major. Consequently, the results showed the plants had significant antileishmanial activity and inhibited the growth of L. major parasites. The flavonoids and phenolic compounds present in B. pilosa confer the plant   with    pharmacological   activity   (Cortés-Rojas   et  al., 2013). Interestingly, the plant has been used traditionally to cure illness as pain, diabetes, edema and inflammation (Lima et al., 2011). Other studies conducted have implicated B. pilosa, 1-phenyl-hepta-1,3,5-triyne to have antimalarial activity and cancer inhibitor (Kumari et al., 2009). The presence of flavonoids, abundant class of compounds in B. pilosa (Lima et al., 2011), promotes formation of cleavable complex as well as induces apoptosis through arrest of G1 phase (Mittra et al., 2000). This in turn inhibits growth of Leishmania parasites and subsequent low IC₅₀ values.

According to Al-Hajj et al. (2018), flavonoids inhibit DNA topoisomerase I activity of L. donovani enzyme responsible for relegation step in parasite growth. In Leishmania donovani parasites, presence of flavonoids causes reversal of multi-drug resistance through binding to glycoprotein like transporter and inhibits synthesis of heat shock proteins responsible for evasion of host immunity (Hamarsheh et al., 2017). Therefore, a lower IC₅₀ for activity, a better antileishmanial activity for B. pilosa; hexane, dichloromethane, ethyl acetate extracts and aqueous phase. According to Makwali et al. (2012), presence of saponins in the B. pilosa extracts complemented the activity of the flavonoids by acting on the parasite membrane inducing a drop in membrane potential. Our study was further complemented by Delmas et al. (2000), in which alpha- Hederin and other saponins isolated had antileishmanial activity. Therefore, in contrast to a single therapy, the various bioactive compounds present in the extract led to a synergy (Makwali et al., 2012) as it brought concept of structure-function approach in killing the parasites. Moreover, studies by Mutoro et al. (2018b) demonstrated that combinational approach in treatment reduces toxicity, lowers parasite resistance and optimizes drug efficacies.

T. diversifolia extracts showed activity against Leishmania parasites, in which dichloromethane showed strongest antileishmanial activity. The report herein was corroborated    by    De   Toledo   et   al.  (2014),   in   which  dichloromethane extract from T. diversifolia had strong activity against Leishmania parasites. Notably, the dichloromethane extract becomes concentrated with large quantities of sesquiterpenes compounds from T. diversifolia glandular where they are manufactured and stored during successive partitioning (De Toledo et al., 2014). Additionally, T. diversifolia dichloromethane treated promastigotes lost distinct traits such as change in body morphology and loss of motility apparatus. Studies by Cortes-Selva et al. (2005), demonstrated the ability of sesquiterpenes to reverse multidrug resistance in Multi-Drug Resistance Leishmania tropica lines.

S. oleraceus has been used by different cultures to avert various diseases such as diarrhea, pneumonia and hepatitis (McDowell et al., 2011). Our study showed activity against Leishmania parasites thus validating the use of plant. Both dichloromethane and hexane extract had the highest activity. This could be attributed to polyphenols and flavonoids present (Table 1). Our study is in agreement with Jimoh et al. (2011) who showed that phenols were responsible for pharmacological activity.

On the other hand, activity decreased from ethyl acetate to aqueous phase, an indicator that bioactive compounds in S. oleraceus mainly remained in hexane and dichloromethane extracts, thus significant antileishmanial activity. The findings by Jimoh et al. (2011) revealed S. oleraceus methanol to have high flavonoid contents compared to aqueous phase. Moreover, the ethyl acetate extract registered a low activity. This could be as a result of low concentration of bioactive constituents. In summary, the activity of the various extracts on Leishmania parasites could be linked to the cell biology, where the chemical composition of the cell comprises ether lipids (LPGs, GPI-anchored metalloprotease, gp63, etc) which gives the parasite ability to evade host immunity and virulence (Weingartner, 2012). The availability of chemical constituents in the three plants leads to attachment of the corresponding groups in the parasite cell membrane. This attachment causes disruption of the Leishmania cell membrane and leakage of parasite contents leading to eventual death.

The findings of our study demonstrated that B. pilosa, T. diversifolia and S. oleraceus; methanol, hexane, dichloromethane, ethyl acetate extracts and aqueous phase had antileishmanial activity against L. major parasites. Therefore, with these promising results, the plants can be explored as alternative source of drugs for the management of Leishmaniasis as they are easily accessible and safe. Moreover, the results obtained provide basis for scientific rationalization of B. pilosa, T. diversifolia and S.  oleraceus  for  use  in  pharmaceutical drug developments.

The authors have not declared any conflict of interests.

The authors are grateful to Professor S. Kariuki, a taxonomist (Department of Biological Science, Egerton University) for identification of plant species and Kenya Medical Research Institute (KEMRI). This work was funded by ASAFEM Project.