In vitro study of the efficacy of Solanum nigrum against Leishmania major [version 1; peer review: 1 approved with reservations, 1 not approved]

Leishmania parasites (Kinetoplastida: Trypanosomatidae) are obligate intracellular parasites of macrophages that causes visceral and cutaneous leishmaniases. Currently, there is inadequate therapeutic interventions to manage this endemic tropical disease, transmitted mainly by phlebotomine sandflies hence there is need to develop affordable and effective therapeutic measures. This study determined the in vitro efficacy of Solanum nigrum methanolic and aqueous plant extracts on Leishmania major parasites.  Cytotoxic effects of the extracts were determined using vero cells and reported as percentage viability of the cells. The promastigote parasites of Leishmania major were cultured and grown for 3 days in different concentrations of extracts to determine the MIC and IC50 values. The in vitro antileishmanial efficacy was done on macrophages infected with L. major amastigote parasites and then treated with extracts in varying concentrations. The study revealed that all the test extracts had lower toxicity than control drugs, pentostam (IC50= 0.0 92 mg/ml) and amphotericin B (IC50=0.049 mg/ml).  The extracts tended to show a dose dependent cytotoxic effect which corresponded to high vero cells viability as their concentration increased.  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. High concentrations of the test extracts and control drugs resulted to low infectivity and multiplication of L. major amastigotes. Findings from this study demonstrate that S. nigrum extracts have potential antileishmanial activities however; further investigation needs to be done on pure compound isolation, in vivo assays and clinical trials so as to use the promising compounds as effective antileishmanial agents. Open Peer Review


Introduction
Leishmaniasis is a widespread disease caused Leishmania parasites which are transmitted by the sandfly. Desjeux (1998) reported that the disease occurred in different clinical forms, which ranged from cutaneous self-healing lesions to a fatal visceralizing form, and also included the metastasizing mucocutaneous and post-kala-azar dermal leishmaniasis. Leishmaniasis represents an important health and socioeconomic problem in 88 countries around the world, where this disease is endemic according to a study by Dujardin et al. (2008). Despite the death toll and disease burden of leishmaniasis, there is an acute lack of suitable therapies. Treatment of the disease depends on a limited number of drugs with limitations such as high cost, unacceptable host toxicity, poor efficacy, lack of availability, and acquired parasite drug resistance as reported in studies by Barrett & Fairlamb (1999);Fairlamb (2003) and Stuart et al. (2008). Therefore, the development of cheap, available, effective and less toxic drugs is of paramount importance. Medicinal plants are the best alternative since they possess natural active components that can be effective against parasitic infections or can be used in development of commercial drugs.
Several studies have reported that some plants are effective against Leishmania parasites both in vitro and in vivo. The findings of studies by Kinuthia et al. (2014); Ndeti et al. (2016) and Njau et al. (2016) revealed that plants such as Allium sativum, Callistemon citrinus, Moringa stenopetala and Aloe secundiflora were effective against Leishmania major parasites. A study by Wabwoba et al. (2010) indicated that a combination of M. stenopetala with Allium sativum induced apoptotic effect in Leishmania major promastigotes. Similar results were reported in studies by McClure et al. (1996) where the growth of Leishmania mexicana and Leishmania chagasii were inhibited by A. sativum, and by Khademvatan et al. (2011) where A. sativum extract induced apoptosis in Leishmania major parasites. Schlein et al. (2001) reported that Ricinus communis (Malpighiales: Euphorbiaceae) possessed antileishmanial effects both in Phlebotomus duboscqi (Diptera: Psychodidae), the vector for L. major in Israel, and in in vivo in BALB/c mice when used alone, as revealed in a study by Oketch et al. (2006), or in combination with Azadiracta indica (Sapindales: Meliaceae) as indicated by Jumba et al. (2015).
The findings of some studies have shown that Solanaceae family plants have medicinal effects against various parasitic infections. The findings of Laban et al. (2015) showed that Solanum aculeastrum was effective against Leishmania major parasites. Additionally, Mishra et al. (2013) reported that a prenyloxy-naphthoquinone obtained from roots of Plumbago zeylanica (Caryophllales: Plumbaginaceae) has anti-leishmanial activity against Leishmania donovani. There was a significant difference between the EC 50 for the isolated compound and miltefosine, the standard drug (P< 0.001) against L. donovani promastigotes and amastigotes. Studies on L. major in Kenya by Makwali et al. (2015) have shown that Plumbago capensis possess anti-leishmanial effects. The current study evaluated the in vitro anti-leishmanial activity of Solanum nigrum extracts on L. major parasites.

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) andCock (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.

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 IC 50 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 IC 50 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% CO 2 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 flatbottomed microtiter plate at a concentration of 1×10 6 cells per ml of the culture medium per well and incubated at 37°C in 5% CO 2 . 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% CO 2 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 IC 50 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 1×10 6 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 IC 50 and antipromastigote assay Metacyclic promastigotes at a concentration of 1×10 6 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 2.0×10 3 , 1.0×10 3 , 5.0×10 2 , 2.5×10 2 , 1.25×10 2 , and 6.25×10 1 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 IC 50 values for the different plant extracts using the Chemosen program v2.

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% CO 2 . 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)

Statistical analysis
The IC 50 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 IC 50 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 dosedependent cytotoxic effect which corresponded to low IC 50 values as their concentration increased. Methanolic extracts of S.nigrum from Bungoma and Kisii had IC 50 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).  (Table 3).
For extracts that shared the same MICs, higher IC 50 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).
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 dosedependent. The difference between the IRs of test extracts and the control drugs were statistically significant (P< 0.05).
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

Discussion
This study has shown that S. nigrum has anti-leishmanial activity against Leishmania parasites. The results indicated that the plant extracts of S. nigrum obtained from Kisii and Bungoma have the potential to inhibit L. major promastigotes in vitro.
The current study further established that the concentrations of the extracts that were effective (MIC) against L. major promastigotes in vitro were relatively high (>0.5 mg/ml), as compared to those of pentostam and amphotericin B, which both inhibited the promastigotes at 0.03125 mg/ml. The efficacy of methanolic extracts was better than their respective aqueous counterparts. Schneider's Drosophila medium was used as a negative control and supported maximum survival of the L. major promastigotes in vitro. This was expected because this medium supports the growth of Leishmania promastigotes and amastigotes, as described by Hendricks & Wright, 1979. The efficacy of test extracts was higher than that of Schneider's Drosophila medium. The slight differences that have been noted between the two allopatric plants could be due to factors such as difference in the presence and composition of the phytochemicals; a study by Aritho et al. (2017) on T. vogelii also revealed such differences.
A study by Son et al. (2003) showed that extracts from S. nigrum leaves had the potential to be used in treatment of tumors, especially liver cancer, and also used for treatment of lung cancer, bladder and gastric carcinoma as indicated by stud-  Estevez et al. (2007) showed that the extracts of S. stramonifolium had activity against L.amazonensis amastigotes. This activity was attributed to steroid derivatives which include cilistol A and steroidal alkaloids, which form the main components in Solanum species (Abreu Miranda et al., 2013;Filho et al., 2013).
Cytotoxic assays using Vero cells showed that the test extracts were less toxic compared to the standard antileishmanial drugs. Generally, the increase in the dose of the extracts led to a higher cytotoxic effect on L. major promastigotes, resulting in inhibition of the growth of the parasites. Many drugs used for treatment of leishmniasis are highly toxic (Santos et al., 2008) and this study confirmed that pentostam and amphotericin B are toxic compared with the extracts tested. The continued use of the contemporary leishmaniasis drugs despite their toxicity is mainly due to lack of an alternative. The use of herbal medicine can be a cheaper and available alternative. The aqueous extracts of both S. nigrum from Bungoma and Kisii (IC 50 , 0.76 mg/ml and 0.64 mg/ml, respectively) were less toxic than methanolic extracts (IC 50 of 0.57 mg/ml and 0.50 mg/ml, respectively).
The lower the toxicity of the test extracts, the higher the viability of Vero cells after exposure to extracts and vice versa. According to Das et al. (2007) the Solanaceae family plants have been reported to be poisonous both to humans and livestock. Their toxicity has been attributed to the presence of tropane alkaloids, which when ingested in large quantities, causes anticholinergic effects. Another study by Glossman-Mitnik (2007) reported that the toxicity of S. nigrum which is edible is due to solanine, a glycoalkaloid which causes toxicity as the concentration increases.  et al. (2010), who observed that the IRs of amphotericin B and pentostam at 100 µg/ml were 9.0% and 11%, respectively. In this study, however, although the difference between MI for amphotericin B and pentostam at 50 µg/ml was not statistically significant, the in vitro efficacy of amphotericin B in suppressing the amastigotes multiplication was higher than that of pentostam.

Conclusion
The findings of this study have justified the claimed medicinal importance of Solanum nigrum as a remedy for various infections. It can be concluded that the crude extracts of S. nigrum possess considerable anti-leishmanial activity, especially against Leishmania major, which were used in this study. The plant may contain potent anti-parasitic compounds, effective in the treatment of Leishmania infections. However, further investigation needs to be conducted on pure compound isolation, toxicological studies and clinical trials so as to use the promising compounds as effective antileishmanial agents.  .

Grant information
The author(s) declared that no grants were involved in supporting this work.
In the beginning of the manuscript, the authors mention volume of the suspension of cells from which 100ul of cells were directly used and finally declared it as 1x10 6 cells per ml. To our minds the centrifugation and counting of the cells with its procedure is extremely important to claim any activity of anti leishmanial compounds. Otherwise results are difficult to be reproduced.
It is also not clear, how the authors could test 6 dilutions of 4 extracts?. As per the description of plate designing the cells were plated only in two wells of A-H row? (as mentioned in line no 11 in the above subheading). This totals to only 16. It is serious concern for this reviewer.
Again authors have not mentioned the time duration of incubation before aspirating out the MEM medium. It is the required time for the adhering to the solid phase. Otherwise cell will also be aspirated off with the medium, and making the study claims untenable.
Again the same section authors have no-where mentioned the standard drugs and concentrations thereof, if at all used for the assay. But in the result section the results of the standard drugs are mentioned on the top. How come?
Authors should also mention the exact timing of incubation performed by them after MTT addition , not 2-4 hrs, which is very arbitrary and not acceptable.

Evaluation of MIC & Evaluation of IC 50 and anti-promastigote assay.
A big question is, if the authors performed two different tests to evaluate MIC and IC 50 value individually? If not then the heading can be merged as Antipromastigote assay to evaluate the IC 50 and MIC of the Test extracts. Evaluation of IC 50 and antipromastigote assay.
What does authors mean by serial dilutions of 2.0×10 3 , 1.0×10 3 , 5.0×10 2 , 2.5×10 2,1.25×10 2, and 6.25×10 1 ? The dilutions are written in different manner, either as log or serial, but not like this. if these are extract's dilutions than there should be uniformity in the unit representation of the concentration of the drugs for all assays.
Somewhere it is mg/ml and somewhere its ug/ml for the same drugs.
Again there is no discussion of standard drugs under this subheading.
Authors should mention method of visual reading by microscopic observation (if done) before mentioning the survival comparison.
Anti-amastigote assay: The amount of sterile cold PBS injected in the peritoneum seems very low in volume.10 ul will go no where and no one can withdraw it back. write carefully.
What was the ratio of macrophage and promastigotes used for this assay. No counting and ratio is mentioned.
Were 4 hrs sufficient for the infection incubation as the promastigotes takes at least 8-12 hrs for engulfment to Maximum 24 hrs. in the manuscript again the plate was incubated for 24 hrs but after the washing step. After reading their protocol, we really cannot believe the results, if these are genuine. And what was the need of second incubation. In this section the concentration of plant extracts used are missing.
Authors must have mentioned the magnification (x) used for the microscopic observation.
Amastigotes are only possible to visualize at 1000 x magnification with oil which is not possible in 24 well plate directly. We can't understand how authors could count the amastigotes.
Statistical analysis: All the experiments could have been performed in duplicate wells in the same assay. And also could have been repeated thrice for mean and SD Value, which is not done in cytotoxic and antipromastigote assays mentioned in this manuscript. Culture was done in duplicate only in antiamastigote assay, for which p value can be calculated by simple way. ANOVA and Tukey's Test is not needed.

Results:
As there are so many flaws in methodology section there is no meaning of evaluating-the results. At every step the results must match with the methodology used. This is missing in this otherwise poorly written paper. Even the concentration unit is not uniform. The IC 50 value of standard drugs are not even appropriate . There is need for the authors to demonstrate the need for investigating the plants they used given that a number of studies show promising results. What was different with the plant?

Methods
The mice were handled in accordance with the regulations set by the Animal Care and Use Committee at KEMRI.