In vitro antileishmanial and antimalarial activity of selected plants of Nepal.

BACKGROUND: Nepal is very rich in biodiversity, and no extensive effort has yet been carried out to screen plants that are used by traditional healers against parasitic diseases. The aim of this study was to evaluate the in vitro antileishmanial and antimalarial activity of crude methanolic or ethanolic extracts of 29 plant species that are currently used by local people of Nepal for treating different ailments.
METHODS: Crude extracts of leaves, twigs, aerial parts, and/or roots of the selected plants were evaluated for in vitro inhibitory activity against intracellular amastigotes of Leishmania infantum and against erythrocytic stages of Plasmodium falciparum. To determine the selectivity index (SI), cytotoxicity was assessed on MRC-5 cells in parallel.
RESULTS: Three plant species, namely Phragmites vallatoria and Ampelocissus tomentosa, for which no antiprotozoal activity has previously been reported, and Terminalia chebula revealed antiprotozoal activity. The extract of A. tomentosa exhibited moderate activity against L. infantum with an inhibitory concentration 50% (IC50) of 13.2 ± 4.3 µg/ml and SI >3, while T. chebula exhibited fairly good antiplasmodial activity with IC50 values of 4.5 ± 2.4 µg/ml and SI values >5.
CONCLUSION: In countries like Nepal, where the current health system is unable to combat the burden of endemic parasitic diseases, evaluation of local plants as a potential source of the drug can help in expanding the treatment options. The extent of untapped resources available in these countries provides an opportunity for future bioprospecting.

INTRODUCTION

Leishmaniasis and malaria represent major public health problems with significant morbidity and mortality in Asia, Africa, and Latin America [1,2]. Lack of vaccines, emergence of drug resistance, and expensive chemotherapeutics are some of the major challenges for the control of these vector-borne diseases, in addition to disadvantages including hospitalization for (parenteral) treatment, occurrence of adverse effects, long-term therapy leading to poor compliance and poor availability of drugs, especially to economically weak populations residing in rural areas [3-6].

Given the limited number of novel drugs in the pipeline and the expanding resistance against current drugs, it remains imperative to explore alternative ways to find new drugs. Plants contain a broad diversity of secondary metabolites such as alkaloids, flavonoids, and phenolic derivatives that may have therapeutic value, and hence may represent an attractive source for novel drugs [7]. However, screening of each and every individual plant parts against wide range of pathogens is virtually impossible and plant selection based on ethnobotany and traditional practices, such as Ayurveda [8], Unani, Siddha, traditional Chinese medicine, and Japanese Kampo medicine increases the probability of finding “hit” molecules that can be subsequently developed toward “lead” development [9,10].

In Nepal, there is a huge variation in the number of medicinal and aromatic plants (MAP) [11,12]. For example, compilation of the MAP database has listed 1624 medicinal plants in 2000 [13], rising to 1950 species in 2008 [14] clearly indicating that further exploration of the phytochemical and pharmacological properties of medicinal plants in Nepal should be continued. Up till now, very few indigenous Nepalese plants have been explored for their therapeutic potential against leishmaniasis and malaria. Starting from ethnobotanical literature and traditional use, the present study assessed the in vitro inhibitory activity potential of crude extracts of 29 selected Nepalese plants [Table 1], hence contributing to the medicinal knowledge of the local plant biodiversity.

Table 1

List of the selected plants for this study, their phytoconstituents, and traditional uses

MATERIALS AND METHODS

Plant Material

Leaves, twigs, aerial parts, and roots [Table 1] of selected plants were collected from different regions in Nepal [Figure 1] from December 2013 to April 2014. All the collected plant materials were identified in the Department of Plant Resources, Nepal, and Voucher specimens are deposited in Pharmacognosy Unit of Department of Plant Resources, Thapathali, Kathmandu, Nepal (http://www.dpr.gov.np).

Figure 1

Sampling site in Nepal for the collection of plant species

Extraction

The plant materials were washed thoroughly with water and shade dried at room temperature. Dried samples were crushed into powder by electric blending and subjected to Soxhlet extraction using polar solvents (ethanol and methanol). The extracts were evaporated on a rotary evaporator under vacuum till a solid mass was obtained. The extracts were kept at 4°C until analysis. All the extracts were kept in sealed vials, labeled properly, and transported to the Laboratory of Microbiology Parasitology and Hygiene, University of Antwerp, for integrated in vitro screening.

Parasites and Cell Culture

Standard techniques were used as previously described [9]. Briefly, ex vivo amastigotes of Leishmania infantum (MHOM/MA(BE)/67) were used for the in vitro antileishmanial assay. The strain was routinely passed in Syrian Golden hamsters every 6-10 weeks. The chloroquine (CQ)-resistant Plasmodium falciparum (K1 strain) was used for in vitro antiplasmodial activity testing. The human lung fibroblast cell line MRC-5 was cultured in minimum essential medium supplemented with 20 mM L-glutamine, 16.5 mM NaHCO3, and 5% fetal calf serum.

Biological In Vitro Assays

The integrated panel of microbial screens and standard screening methodologies were adopted as previously described [9]. Plant extracts were tested at dilutions ranging from 128 to 0.25 µg/mL using automated robotics with a 10-fold serial dilution strategy. Initially, 2-fold serial dilutions were made in 100% dimethyl sulfoxide (DMSO) to ascertain complete solubility during the dilution process. An immediate dilution step was performed in Milli-Q water before transferring the respective compound dilutions to the test plates (1/20 dilution: 10 µL compound solution +190 µL cell medium and test system) so that the final in-test concentration of DMSO did not exceed 1%.

Antileishmanial Activity

Mouse macrophages were stimulated by intraperitoneal injection of starch. 2 days after injection, macrophages were collected and seeded in each well (3 × 104) of a 96-well plate. The plates were incubated at 37°C and 5% CO2. After 2 days of outgrowth, ex vivo amastigotes were used to infect primary peritoneal mouse macrophages at a 10:1 infection ratio. The plates were further incubated for 2 h before the compound dilutions were added. After 5 days of incubation, cells were dried, fixed with methanol, and stained with 20% Giemsa to assess total intracellular amastigote burdens through microscopic reading. The results are expressed as the percentage reduction of amastigote burden compared to untreated control cultures and inhibitory concentration 50% (IC50)-values were calculated.

Antiplasmodial Assay

CQ-resistant P. falciparum 2/K 1-strain was cultured in human erythrocytes O+ at 37°C under microaerophilic atmosphere (3% O2, 4% CO2, and 93% N2) in RPMI-1640 supplemented with 10% human serum. 200 µL of infected red blood cells (1% parasitemia and 2% hematocrit) was added in each well of a 96 well plate containing prediluted extract. The test plates were kept in the modular incubator chamber for 72 h at 37°C, and subsequently, put at −20°C to lyse the red cells upon thawing. Next, 100 µL of Malstat™ reagent was put in new microtiter plate to which 20 µL of hemolyzed parasite suspension was added. After 15 min incubation at room temperature, 20 µl of nitro blue tetrazolium/polyethersulfone solution was added. The plate was incubated in the dark for another 2 h at room temperature and spectrophotometrically read at 655 nm. The IC50 was calculated from the drug concentration - response curves. According to the WHO guidelines ([45]), antiplasmodial activity is very good with IC50 <1 µg/ml; good to moderate if IC50 of 1-10 µg/ml; weak if 15-50 µg/ml, and inactive if IC50 >50 µg/ml, always taking into account a selectivity index (SI) higher than 10.

RESULTS

Only one plant extract (Ampelocissus tomentosa) exhibited moderate activity against L. infantum with an IC50 value of 13.2 ± 4.3 µg/ml and an SI value >3. Paris polyphylla also showed inhibitory activity but was also cytotoxic [Table 2].

Table 2

Antiprotozoal activity of extract of selected plants of Nepal and their cytotoxicity against MRC-5 cell lines

Antiplasmodial Activity

Three plant species, Phragmites vallatoria, A. tomentosa, and Terminalia chebula showed schizonticidal activity. Among them, T. chebula exhibited the best activity with IC50 values of 4.5 ± 2.4 µg/ml and SI values >5.

Cytotoxicity

Kalanchoe pinnata, P. polyphylla, and Pedilanthus tithymaloides were toxic to the MRC-5 cell line. K. pinnata was most toxic with cytotoxic concentration 50% value of 4.7 ± 1.8 µg/ml.

DISCUSSION AND CONCLUSION

Leishmaniasis and malaria continue to be major public health problems, and the available drugs are generally expensive and not devoid of toxic side effects. Associated with poor compliance, the threat of drug resistance is also an emerging issue. Despite different strategies such as drug repurposing, identifying new therapeutic targets by chemoinformatics or screening diverse libraries of natural products, no new drugs have reached the market during the last decade. The present study was carried out to explore the potential of Nepalese medicinal plants that are used as part of traditional medicine. Nepal is very rich in biodiversity, which has not yet been explored satisfactorily due to the geopolitical situation, the lack of sophisticated labs, and the availability of trained manpower in industry and academics. The selected medicinal plants were screened against protozoal diseases using a “whole-cell based” approach, which can be considered more valid than enzyme-based subcellular approaches [9].

In the present study, A. tomentosa showed selective antileishmanial (IC50 13.2 ± 4.3 µg/ml) and antimalarial (11.7 ± 3.5 µg/ml) activity. To our knowledge, the antiprotozoal activity of this plant has never been investigated, and no active constituents have been documented in the literature. Further studies on bioassay-guided fractionation to identify the putative active constituents and to better understand the therapeutic targets will be necessary, including a screening of other species of Ampelocissus genus.

Likewise, good antimalarial activity was found for T. chebula and P. vallatoria with an IC50 of 4.5 ± 2.4 and 12.0 ± 7.5, respectively, and SI of >5. This is the first observation that P. vallatoria showed potential activity against Plasmodium. The antiplasmodial activity of T. chebula has already been reported [22] with an IC50 = 4.76 µg/mL against the CQ-sensitive (3D7) strain of P. falciparum, hence supporting its use in traditional medicine.

P. tithymaloides was also found to be active against Leishmania but was not totally devoid of cytotoxicity. In traditional medicine, P. tithymaloides is been used in treating multiple diseases (from antimicrobial to anticancer) related to the diverse phytoconstituents [Table 1]. The antiprotozoal activity of this plant might be due to the presence of a diterpene, as species belonging to the family Euphorbiaceae are rich in diterpenoids and triterpenoids [46]. In previous studies, various poly-O-acylated jatrophane diterpenoids have shown in vitro antiplasmodial activity with IC50 values of 3.4-4.4 µg/ml, which has been confirmed in vivo, with 76% suppression of parasitemia in P. berghei infected mice [47,48]. Likewise, diterpenes such as jatrogrossidione and jatrophone have been found to have toxic effects against promastigotes of L. braziliensis, L. amazonensis, and L. chagasi with IC50 in the range of 0.75-5 µg/ml [49]. The moderate cytotoxic nature of P. tithymaloides might be due to the presence of pedilstatin or eurphorbol, which have already been established as irritants and carcinogens [50].

Non-selective antileishmanial activity was shown for P. polyphylla and K. pinnata. P. polyphylla is known as “satuwa” and is traditionally used as anthelmintic and for reducing fever in the Himalayan region of Nepal. Our findings on cell toxicity of some plant extracts (IC50 15 µg/ml) warrants for some vigilance as sometimes misleading information like “natural products are always safe” could eventually lead to deleterious health if high doses of these plants are consumed for a long time. Quite a lot of published literature indeed lacks parallel cytotoxicity evaluation. For example, P. polyphylla diosgenin-type saponins revealed antileishmanial activity (IC50 1.6 µg/ml) but without parallel cytotoxicity evaluation [42]. In our study, K. pinnata was highly cytotoxic (4.7 ± 1.8 µg/ml) while published data support that K. pinnata may possess immunosuppressive effects and inhibit disease progression in L. amazonensis-infected individuals [31,51,52]. The same research group more recently reported that this plant possessed immunomodulatory activity and highlighted that oral dose of K. pinnata extract (400 mg/kg) is comparable to Pentostam® (72 mg/kg) in reducing the hepatic and splenic parasitic burden [53].

Further research on these plants should now focus on the structural elucidation of the putative “active constituents,” in vitro evaluation using preset IC50 and SI cut-offs and in vivo evaluation in murine pharmacology models for pharmacokinetic and dynamic profiling.