Efficacy of the combination of crude extracts of Solanum nigrum and Plumbago capensis on 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 sagatal 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 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 from Kisii and Bungoma, Kenya (0° 40' 49.7352'' S and 34° 46' 37.4196'' E), where the plant is abundant. Plumbago capensis whose activity has been established (Makwali et al., 2015) was collected from Upper Hill area of Nairobi County, Kenya (1°17'59.0"S, 36°48'58.0"E) The plants were transferred to the Center of Traditional Medicine and Drug Research (CTMDR) at KEMRI (Nairobi, Kenya) and dried at 25°C in the shade until they became brittle and attained a constant weight. The dried plants were separately ground using an electric mill (Christy & Norris Ltd., Chelmford, England, model 8) into powder followed by extraction using water and analytical grade methanol (Sigma, 82762). The methanolic extracts were prepared as described by Mutoro et al. (2018a). 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 (Sigma, Z240079) and concentrated using a rotary evaporator (Cole-Parmer - Stuart - RE400) to obtain dry methanolic extracts. The extracts were coded as A, B and C for methanolic extracts of S. nigrum (Bungoma), S. nigrum (Kisii) and P. capensis, respectively. The aqueous extracts were also prepared as described by Mutoro et al. (2018a). 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 was frozen, dried and weighed. The extracts were coded as D, E and F for P. capensis, 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 six 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 (Orchid scientific, SMP 01) in the animal housing kept at 23–25°C under twelve hours in light and twelve hours in dark 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 and quantification of nitric oxide produced by macrophages treated with blends of extracts.

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 (Fisher Scientific, 21720024) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Hyclone^® USA, SH30071031H), 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 (Thermo Fisher Scientific 75004061 mySPIN 6 Mini Centrifuge, 1189M94EA). 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 as described by Mutoro et al. (2018a). Briefly, plant extracts were made in Schneider’s Drosophila culture medium (Fisher Scientific, 21720024) 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.

Evaluation of minimum inhibitory concentration (MIC) and active ratios of test extracts and blends on L. major promastigotes

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 and aqueous test extracts whose concentrations were 2000 µg/ml, 1000 µg/ml, 500 µg/ml and 250 µg/ml. Similarly, the promastigotes were treated with combined extracts in fixed ratios of 2000:250, 1000:500, 500:1000 and 250:2000. The L. major promastigotes treated with the single extracts and the blends were stained with 100 µL of trypan blue dye while on a microscope slide and observed under the light microscope (XSZ-107 Series Biological Microscope, Sam-Tech Diagnostics) to check their motility and viability compared to the Schneider’s Drosophila medium as the negative control. The lowest concentration of the individual test plant extracts and the blends in which no live promastigotes were observed was the MIC and active ratio for the individual test extracts and the blends respectively.

Anti-amastigote assay

The anti-amastigote assay was carried out as described by Mutoro et al. (2018a). The peritoneal macrophages were obtained from 4 clean BALB/c mice. The mice were anaesthetized using 100µl pentobarbitone sodium (Sagatal®; Sigma, P3761). The body surface of the mouse was disinfected with 70% ethanol after which it was torn dorso-ventrally to expose the peritoneum. 10µl of sterile cold phosphate buffered saline (PBS) was injected into the peritoneum. After injection, the peritoneum was gently massaged for 2 minutes to dislodge and release macrophages into the PBS. The peritoneal macrophages were then harvested by withdrawing the PBS. The PBS containing the macrophages was washed through centrifugation at 2,000g for 10 minutes and the pellet obtained was re-suspended in RPMI-1640 culture medium. The macrophages were adsorbed in 24-well plates for 4 hours at 37°C in 5% CO₂. Non-adherent cells were washed with cold sterile PBS and the adherent macrophages were incubated overnight in RPMI culture medium. Adherent macrophages were then infected with L. major promastigotes and were further incubated at 37°C in 5% CO₂ for 4 hours 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 hours in RPMI-1640 culture medium. The infected macrophages were then treated with combinations of both aqueous and methanolic extracts at fixed ratios of 500:125, 125:125 and 125:500. Pentostam and liposomal amphotericin B were used as positive control drugs to compare the parasite inhibition with that of blends of plant extracts. The medium and blends of 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 (Thermo Scientific™, 9990715). The number of amastigotes were determined by counting microscopically at least 100 infected macrophages in triplicate cultures, and the count was expressed as infection rate (IR) and multiplication index (MI) as described by Berman & Lee, (1984) in the formulas below;

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

        MI (%) = $\frac{Numberofamastigotesinexperimentalculture/100macrophages}{Numberofamastigotesincontrolculture/100macrophages}\times 100$

Determination of nitric oxide production

Measurement of nitric oxide (NO) production was carried out as described by Gamboa-Leon et al. (2007). BALB/c mice peritoneal macrophages at a concentration of 1×10⁵ cells per culture medium were placed in each well in 96-well microtiter plates and allowed to adhere at 37°C in 5% CO₂ humidified atmosphere. 2 hours later, the peritoneal macrophages were incubated further in RPMI -1640 medium with 10% FBS for 48 hours in presence of blends of aqueous and methanolic test extracts and the controls. At least 100 µl of macrophage culture supernatants were collected and frozen until when they were required for NO measurement. NO was measured using the Greiss reaction for nitrites (NO₂) as described by Hollzmuller et al., 2002. NO₂ is one of the products released when the breakdown of NO occurs in the macrophages. NO in the collected supernatants is therefore estimated by quantifying the NO₂ content. A nitrite standard reference curve was prepared by dispensing 50 µl of RPMI-1640 with 10% FBS into wells in rows B-H of the first 3 columns in a 96 well plate. 100µM sodium nitrite solution were added to the remaining 3 wells in row A of a 96-well micro titer plate, and immediately a six serial two fold dilutions (50 µl/well) were performed in triplicate columns 1, 2 and 3 down the plate to generate a curve that corresponded to the concentrations 100, 50, 25, 12.5, 6.25, 3.125 and 1.563 µM.

Secondly, 50 µl of the sample supernatant from the wells with macrophages treated with blends of test extracts were added into the wells in triplicate at fixed ratio of 125:125. Greiss reagent A (Fisher Scientific, G7921) were dispensed to all the experimental samples and into the wells containing sodium nitrite solution. Following an incubation of 5 minutes at room temperature, 50 µl of Greiss reagent B (Fisher Scientific, G7921) in water were dispensed into all the wells and incubated for a further 5 minutes at room temperature before measuring the optical density (OD) of the purple/magenta azo compound at 520 nm using micro titer reader (PerkinElmer). The values for the standard nitrite for each of the blend of extracts were read from the standard curve plotted, by reading the NO concentration that corresponds to the absorbance values of the samples.

Statistical analysis

The 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.

Minimum inhibitory concentrations (MIC) of individual test extracts on L. major promastigotes

MICs for single extracts and active ratios for the blends of the extracts were detected by looking at the motility and viability of the parasites in the wells as compared to the Schneider’s Drosophila medium (SIM) as the negative control (Table 1 and Table 2).

The MICs of the individual methanolic extracts, S. nigrum from Bungoma (A), S. nigrum from Kisii (B) and P.capensis (C) against L. major promastigotes were 1mg/ml, 0.5 mg/ml and 0.25 mg/ml respectively. The MICs of the individual aqueous extracts, P.capensis (D), S. nigrum from Kisii (E) and S. nigrum from Bungoma (F) were 0.5 mg/ml, 2 mg/ml and 2 mg/ml respectively. In comparison, the MICs of pentostam and amphotericin B against L. major promastigotes were both 0.03125 mg/ml. The Schneider’s Drosophila medium supported the survival of L. major promastigotes to the maximum (Table 3).

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

The survival levels of promastigotes after treatment with blends of extracts were determined by comparing with survival in both the positive controls (Pentostam and Amphotericin B) and negative control (Schneider’s Drosophila medium). A blend of S. nigrum (Bungoma) and P. capensis (AC) and S. nigrum (Kisii) and P. capensis (BC) methanolic extracts in a ratio 250:2000 inhibited the L. major promastigotes in vitro 100% while ratios 500:1000 and 1000:500 decreased the promastigotes to minimum survival (25%) levels (+) and moderate (50%) levels (++) respectively. Ratio 2000:250 supported the survival of promastigotes to highly moderate (75%) levels (+++) when compared with the controls. A blend of S. nigrum from Bungoma and Kisii (AB) methanolic extracts was efficacious in inhibiting the parasites growth to minimum levels (+) at ratio 1:8 and there was moderate growth at ratios 1000:500 and 500:1000 (Table 4).

A blend of the aqueous extracts of P. capensis and S. nigrum from Kisii (DE) at ratio of 2000:250 led to complete inhibition of parasites growth while ratios 1000:500, 500:1000 and 250:2000 supported growth of the parasites. A blend of aqueous extracts of P. capensis and S. nigrum from Bungoma (DF) at ratio 2000:250 inhibited the growth of the parasites to minimum levels (+) and a blend of S. nigrum from Kisii and Bungoma (EF) supported growth of the parasites at all ratios (Table 4).

Both pentostam and amphotericin B drugs were able to inhibit the growth of L. major promastigotes in vitro at a concentration of 31.25 µg/ml. The Schneider’s Drosophila medium, on the other hand, supported the maximum (100%) growth survival of L. major promastigotes as indicated by four pluses (++++) when compared to the positive controls, pentostam and amphotericin B (Table 4).

Anti-amastigote assay (Macrophage assay)

When the methanolic extracts were combined in ratios of 500:125, 125:125 and 125:500, the blend of S. nigrum from Kisii and P. capensis (BC) had the lowest infection rate of 46.7% at a concentration ratio of 125:500. Similarly, the methanolic extracts combinations of S. nigrum from Bungoma and Kisii (AB) and S. nigrum from Kisii and P. capensis (AC) resulted to infection rate (IR) of 61.7% at the same ratio (Table 5).

Combinations of the aqueous extracts of P. capensis and S. nigrum from Kisii (DE), P. capensis and S. nigrum from Bungoma (DF) and S. nigrum from Kisii and Bungoma (EF) at the ratio of 125:500 resulted to infection rates of 70.0%, 78.0% and 78.7% respectively. The efficacy of combined aqueous extracts S. nigrum from Kisii and Bungoma (EF) in the ratio of 125:500, in inhibiting the infectivity and multiplication of L. major amastigotes in BALB/c peritoneal macrophages in vitro was lower than all the other blends of extracts (Figure 1 and Table 5). The blend of methanolic extract of S. nigrum from Kisii and P. capensis (BC) performed the best with the multiplication index (MI) of 50.6% at the ratio of 125:500.

Figure 1. In vitro activities of blends of extracts at ratios of 500:125, 125:125 and 125:500 as represented by infection rates of Leishmania major amastigotes in treated BALB/c mice peritoneal macrophages.

RPMI 1640 medium which had no drug incorporated supported the growth of L. major amastigotes in peritoneal macrophages (Figure 2) more effectively and this was indicated by a high infection rate (IR) of 89.7 % (Table 5). In contrast, the leishmaniasis drugs, pentostam and liposomal amphotericin B inhibited the in vitro survival of L. major amastigotes more effectively and this corresponded to low infection rates of 26.3% and 21.0 % respectively at a concentration of 50 µg/ml (Table 4). The IRs and MIs associated with all the combined extracts were significantly different (P< 0.05) from those of pentostam and amphotericin B at all the concentrations studied.

Figure 2. In vitro activities for controls (pentostam and amphotericin B) as represented by infection rates of Leishmania major amastigotes in treated BALB/c mice peritoneal macrophages.

Determination of nitric oxide production

Nitric oxide (NO) plays a key role as a leishmanicidal effector molecule in host macrophages (Gamboa-Leon et al., 2007). Therefore the effect of the blends of test extracts on NO production was evaluated in vitro. BALB/c mice peritoneal macrophages were incubated in RPMI-1640 medium for 48 hours in the presence or in absence of blends of test extracts. In order to determine the amount of NO triggered by the combined extracts, their optical densities (absorbencies) were determined using Griess Reagent system. All the absorbencies for the combined extracts ranged between 0.034 to 0.041 (Table 6). These OD corresponded to < 5 µM of NO in the standard nitrite curve for the blends of both the methanolic and aqueous extracts. RPMI-1640 medium produced similar negligible levels of NO (Table 7).