In vitro study of the efficacy of Solanum nigrum against Leishmania major

Ethical considerations

The proposal for this research work was submitted to the KEMRI Scientific Steering Committee (SSC), for approval and was given ethical clearance (Number: KEMRI/SSC-2028) on the use of the mice as the animal model by the Ethical Review Committee (ERC). All experimental animals at the end of the experiment were sacrificed by injection of 100 µl sodium pentobarbital and disposed of according to the regulations of Animal Care and Use (ACUC) through incineration.

Experimental design

The in vitro studies were carried out using a comparative study design. Pentostam (Glaxo Operations (UK) Limited, Barnard Castle, UK) and amphotericin B (AmBisome^®; Gilead, Foster City, CA, USA) were used as the standard drugs to compare their efficacy and toxicity with those of the test extracts. RPMI-1640 and Schneider’s Drosophila media (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used as the control in in vitro experimental chemotherapeutic studies.

Plant collection and preparation of the extracts

Fresh leaves of Solanum nigrum were collected form Kisii and Bungoma, Kenya, where the plant is abundant. The plants were transferred to the Center of Traditional Medicine and Drug Research (CTMDR) at KEMRI (Nairobi, Kenya) and dried at 25°C until they became brittle and attained a constant weight. The dried plants were separately ground using an electric mill (Christy& Norris Ltd., Chelmford, England) into powder followed by extraction using water and analytical grade methanol. The methanolic extracts were prepared as described by Mekonnen et al. (1999) and Cock (2012). Immediately, 100 g of ground plant material was soaked in 500 ml of analytical grade methanol for 72 h at room temperature with gentle shaking. The mixture was filtered using Whatman No.1 filter papers and concentrated using a rotary evaporator to obtain dry methanolic extracts. The extracts were coded as A and B for methanolic extracts of S. nigrum (Bungoma) and S. nigrum (Kisii), respectively. The aqueous extracts were prepared as described by Delahaye et al. (2009). Briefly, 100 g of the dried ground plant material in 600 ml of distilled water was placed in a water bath at 70°C for 1.5 h. The mixture was filtered using Whatman No.1 filter papers and then the filtrate frozen, dried and weighed. The extracts were coded as C and D for S. nigrum (Kisii) and S. nigrum (Bungoma), respectively. The extracts were then stored at 4°C until required for bioassays.

Mice and Leishmania parasites

A total of four 8-week-old male inbred BALB/c mice with weights that ranged between 25 and 29 g were obtained from KEMRI. There were eight BALB/c mice per cage in the animal house kept at 23–25°C under a 12/12 h light/dark cycle and were fed on standard diet in the form of mouse pellets and given tap water ad libitum. The mice were handled in accordance with the regulations set by the Animal Care and Use Committee at KEMRI. The mice were used for extraction of peritoneal macrophages that were used for anti-amastigote assay.

The Leishmania major strain (IDUB/KE/94=NLB-144) which was originally isolated in 1983 from a female Phlebotomus dubosqi collected from Marigat, Baringo County in Kenya were used. The parasites were grown to stationary phase at 25°C in Schneider’s Drosophila medium supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Hyclone^® USA), 100 U/ml penicillin and 500 µg/ml streptomycin (Hendricks & Wright, 1979), and 250 µg/ml 5-fluorocytosine arabinoside (Kimber et al., 1981). The stationary-phase metacyclic stage promastigotes were then harvested by centrifugation at 1500g for 15 min at 4°C. The metacyclic promastigotes were then used for the in vitro assays.

Preparation of the stock solutions of test extracts

Stock solutions of the crude plant extracts were made in Schneider’s Drosophila culture medium for anti-leishmanial assays and filtered through 0.22-µm filter flasks in a laminar flow hood (Biological Safety Cabinet). The stock solutions were then stored at 4°C and retrieved later for both in vitro bioassays.

Cytotoxicity assay using Vero cells to determine IC₅₀ values

This assay was used to test the cytotoxicity of individual extracts against Vero cells (Thermo Fischer Scientific, Waltham, Massachusetts, USA) and the results were presented as IC₅₀ values. The assay was carried out as described by Wabwoba et al. (2010). Vero cells were grown in minimum essential medium (MEM) (ATCC® 30-2003™) supplemented with 10% FBS, penicillin (100 IU/ml) and streptomycin (100 µg/ml) in 25 ml cell culture flasks incubated at 37°C in a humidified 5% CO₂ for 24 h. The Vero cells were harvested by trypsinization and pooled in 50-ml centrifuge tubes from which 100 µl of cell suspension was moved into two wells of rows A-H in a 96-well flat-bottomed microtiter plate at a concentration of 1×10⁶ cells per ml of the culture medium per well and incubated at 37°C in 5% CO₂. The MEM was gently aspirated off and 150 µl of the test extracts (A, B, C and D) were added at concentrations of 1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml and 31.25 µg/ml in the micro titer plates. The plates containing the Vero cells and test extracts were further incubated at 37°C for 48 h in a humidified 5% CO₂ atmosphere. The controls wells comprised of Vero cells and medium while the blank wells had medium alone. A total of 10 µl of (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent were added into each well and incubated further for 2–4 h until a purple precipitate (formazan) was visible under the microscope. The media together with MTT reagent was gently be aspirated off, after which 100 µl of dimethyl sulfoxide (DMSO) was added, and vigorously shaken for 5 minutes in order to dissolve formazan. The absorbance (optical density) was measured for each well plate using a microtiter plate reader at wavelength of 570 nm. The IC₅₀ values of the extracts were determined automatically using the Chemosen program 2.

Evaluation of minimum inhibitory concentration (MIC)

The MICs were determined as described by Wabwoba et al. (2010). Briefly, the L. major metacyclic promastigotes at concentration of 1×10⁶ promastigotes per ml of the culture medium were treated with individual methanolic test extracts A and B whose concentrations were 2000 µg/ml, 1000 µg/ml, 500 µg/ml and 250 µg/ml. These test procedures were repeated for aqueous extracts C and D. The lowest concentration of the individual test extracts in which no live promastigotes were observed was the MIC.

Evaluation of IC₅₀ and antipromastigote assay

Metacyclic promastigotes at a concentration of 1×10⁶ promastigotes per ml of the culture medium were grown for 48 h in 24-well microtiter plates at 25°C. Aliquots of the promastigotes were transferred into 96 well microtiter plates and incubated further at 27°C for 24 h after which 200 µl of the highest concentrations (2 mg/ml) of the individual test extracts were added before serial 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 control wells contained L. major promastigotes in culture medium alone whereas the blank wells had the culture medium alone. The plates were incubated further at 27°C for 48 h and 10 µl of MTT reagent was into each well and incubated further for 4 h. The medium and MTT reagent were aspirated off the wells. Next, in each well, 100 µl of DMSO was added and the plates shaken for 5 minutes. Absorbances were read at 562 nm using a microtiter reader. The absorbance readings were used to generate IC₅₀ values for the different plant extracts using the Chemosen program v2.

Survival of L. major promastigotes was stratified as follows: ++++, 75–100% survival compared with control; +++, 50–<75% survival compared with control; ++, 25–<50% survival compared with control; +, <25% survival compared with control; -, absence of live promastigotes.

Anti-amastigote assay

The anti-amastigote assay was carried out as described by Delorenzi et al. (2001). The peritoneal macrophages were obtained from four clean BALB/c mice. The mice were anaesthetized using 100 µl pentobarbital sodium (Sagatal®). The body surface of the mouse was disinfected with 70% ethanol after which it was opened dorso-ventrally to expose the peritoneum. A total of 10 µl sterile cold PBS was injected into the peritoneum. After injection, the peritoneum was gently massaged for 2 min to dislodge and release macrophages into the PBS. The peritoneal macrophages were then be harvested by withdrawing the PBS. The PBS containing the macrophages was washed through centrifugation at 2,000g for 10 min and the pellet obtained was re-suspended in RPMI-1640 culture medium. The macrophages were adsorbed in 24-well plates for 4 h at 37°C in 5% CO₂. Non-adherent cells were washed away with cold sterile PBS and the adherent macrophages were incubated overnight in RPMI-1640 culture medium. Adherent macrophages were then infected with L. major promastigotes and were further incubated at 37°C in 5% for 4 h, after which they were washed with sterile PBS to remove the free promastigotes, which were not engulfed by the macrophages. This was followed by incubation of the preparation for 24 h in RPMI-1640 culture medium.

Pentostam and liposomal amphotericin B at concentrations of 125 µg/ml, 250 µg/ml and 500 µg/ml were used as positive control drugs to compare the parasite inhibition with that by plant extracts. The medium and test extracts or drug was replenished daily for 3 days. After 5 days, the macrophages were washed with sterile PBS at 37°C, fixed in methanol and stained with 10% Giemsa. The number of amastigotes was determined by counting at least 100 macrophages in duplicate cultures, and the count was expressed as infection rate (IR) and multiplication index (MI) as described by Berman & Lee (1984) in the calculations below:

                                                                                                             IR (%) = Number of infected macrophages per 100 macrophages

$$MI(\%)=\frac{Numberofamastigotesinexperimentalcultureper100macrophages}{Numberofamastigotesincontrolcultureper100macrophages}\times 100$$

Statistical analysis

The IC₅₀ values were determined using Chemosen program v2. Data for infection rates and multiplication indices were saved as percentages and then were analyzed using SPSS 13.0 programme. The results were expressed as mean values ± standard deviation (SD). Statistical analysis were done using one way ANOVA and Tukey’s post hoc test and p values < 0.05 were considered significant.

Cytotoxicity assays using Vero cells

Generally, all the test extracts studied were less toxic (i.e. higher IC₅₀ values) to Vero cells when compared to the control drugs, pentostam (0.03 mg/ml) and amphotericin B (0.01 mg/ml) (Table 1).

The methanolic and aqueous extracts tended to show a dose-dependent cytotoxic effect which corresponded to low IC₅₀ values as their concentration increased. Methanolic extracts of S.nigrum from Bungoma and Kisii had IC₅₀ values of 0.57 mg/ml and 0.50 mg/ml while those their aqueous counterparts were 0.76 mg/ml and 0.64 mg/ml (Table 1).

The survival of L. major promastigotes after exposure to extracts and controls

Preliminary studies involved exposure of the L. major promastigotes to extracts and control drugs at varying concentrations in vitro. The L. major parasites cultured in Schneider’s Drosophila medium were taken as the negative control because the parasites continued to multiply. Both pentostam and amphotericin B used were able to inhibit the Leishmania major promastigotes growth at an MIC of 31.25 µg/ml. The Schneider’s Drosophila medium, on the other hand, led to maximum survival of L. major promastigotes (++++) (Table 2).

Both aqueous extracts of S. nigrum from Bungoma and Kisii (C and D) inhibited the survival of Leishmania major promastigotes at an MIC of 2000 µg/ml. S. nigrum methanolic extracts from Bungoma (A) lowered L. major parasites at a concentration of 1000 µg/ml, while that from Kisii (B) inhibited L. major multiplication at an MIC of 500 µg/ml.

The concentrations of the extracts that were effective against L. major promastigotes in vitro were high (>0.5 mg/ml) compared to those of the standard drugs (0.03125 mg/ml). The efficacy of methanolic extracts was better than their respective aqueous counter parts (Table 2).

Minimum inhibitory concentrations (MIC) and IC₅₀ values of the extracts on L. major promastigotes

The MICs of S. nigrum from Bungoma (A) and S. nigrum from Kisii (B) methanolic extracts were 1000 µg/ml and 500 µg/ml, respectively. Both aqueous extracts of S. nigrum from Kisii (C) and S. nigrum from Bungoma (D) had MIC values of 2000 µg/ml. The standard drugs had lower MICs (31.25 µg/ml) against L. major promastigotes compared to the test extracts. Schneider’s Drosophila medium, which was the negative control, supported maximum survival of Leishmania major promastigotes (Table 3).

IC₅₀ values determined indicated the effectiveness of the test extracts or the controls in inhibiting the promastigotes by 50%. Pentostam and amphotericin B had IC₅₀ values of 0.08 mg/ml and 0.04 mg/ml, respectively, while S. nigrum from Bungoma (A) and S. nigrum from Kisii (B) methanolic extracts had IC₅₀ values of 1.81 mg/ml and 1.28 mg/ml, respectively. Similarly, aqueous extracts of S. nigrum from Kisii (C) and S. nigrum from Bungoma (D) had IC₅₀ values of 1.18 mg/ml and 1.84 mg/ml, respectively. Based on the IC₅₀ values, the aqueous extract of S. nigrum from Kisii (C) was the most effective of the extracts (Table 3).

For extracts that shared the same MICs, higher IC₅₀ values tended to correspond to increased in vitro survival of promastigotes of Leishmania. The methanolic extract of S. nigrum from Kisii seemed to be more efficacious in vitro since it knocked out the promastigotes at a lower MIC level (0.5 mg/ml) when compared to all other extracts whose effective MIC level was ≥1 mg/ml (Table 3).

Anti-amastigote assay (macrophage assay)

RPMI-1640 medium with no drug had an infection rate of 96.7% (Table 4), which implied that it supported maximum growth of Leishmania major amastigotes in peritoneal macrophages (Figure 1). The leishmaniasis drugs pentostam and liposomal amphotericin B inhibited the in vitro survival of L. major amastigotes, corresponding to low infection rates of 26.3% and 21.0%, respectively, at a concentration of 50 µg/ml (Table 4).

Figure 1. Showing a BALB/c mice peritoneal macrophage having engulfed L. major amastigotes at 5 days post infection in RPMI-1640 medium culture.

Am= amastigote, Nuc= Nucleus, and Cyt= Cytoplasm.

At a concentration of 125 µg/ml, the methanolic extracts of S. nigrum from Bungoma (A) and S. nigrum from Kisii (B) had infection rates of 71.0±2.3% and 68.0±2.7%, respectively. Similarly, the infection rates of aqueous extracts, S. nigrum from Kisii (C) and S. nigrum from Bungoma (D) were 78.0±2.5% and 85.3±1.2% (Table 4).

The methanolic extract of S. nigrum from Kisii (B) inhibited the survival of L. major amastigotes better than the other extracts in all the concentrations studied (Figure 2). High concentrations of the test extracts and control drugs resulted in low IRs and MIs of L. major amastigotes. The efficacies were dose-dependent. The difference between the IRs of test extracts and the control drugs were statistically significant (P< 0.05).

Figure 2. The in vitro activities of the test extracts and controls against L. major amastigotes in BALB/c peritoneal macrophages.

When the MIs of amastigotes in peritoneal macrophages treated with 125 µg/ml of methanolic test extracts (A and B) were compared with those treated with 50 µg/ml of amphotericin or pentostam, using one-way ANOVA, there was a statistically significant difference (P<0.001). A Tukey post hoc test revealed that the MI of methanolic extracts of S. nigrum from Kisii (A and B) at 500 µg/ml were statistically significant from that of pentostam and amphotericin B (P= 0.001).

When the infection rates of methanolic extracts of 500 µg/ml A and B were compared with those of amphotericin B using Tukey’s post hoc test, the difference in each case was statistically significant, (P<0.001 and P=0.001, respectively). Comparisons of the IRs for extracts C and D with those of amphotericin B or pentostam followed a similar trend, where Tukey’s post hoc test indicated a significant difference (P<0.05) for each comparison. The MIs of pentostam and amphotericin B were not statistically different (P≥ 0.05) at a concentration of 25 µg/ml.