Evaluation of the In Vitro Antiplasmodial, Antileishmanial, and Antitrypanosomal Activity of Medicinal Plants Used in Saudi and Yemeni Traditional Medicine

The antiplasmodial, antileishmanial, and antitrypanosomal activity of twenty-five medicinal plants distributed in Saudi Arabia and Yemen was evaluated. The plants were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi, and free trypomastigotes of T. brucei. To assess selectivity, cytotoxicity was determined on MRC-5 cells. Criteria for activity were an IC50 < 10 μg/mL and high selectivity (SI). Seven plants showed interesting antiprotozoal activity in one or more models. Extracts of Caralluma penicillata and Acalypha ciliata showed fairly good activity against P. falciparum with IC50 of 6.7 and 10.8 μg/mL and adequate selectivity (SI > 9.6 and >5.9). Interesting activity against L. infantum was obtained with Verbascum bottae (IC50 of 3.2 μg/mL, SI 10.2) and Solanum glabratum (IC50 8.1 μg/mL, SI 3.4). The extracts of C. penicillata, Leucas virgata, Loranthus regularis, and V. bottae exhibited moderate activity against T. brucei (IC50 8.5, 8.1, 8.3, and 2.3 μg/mL; SI > 7.6, 7.7, 4.3, and >14.1). These results partly support the traditional use of some of the selected medicinal plants and warrant further investigations into the putative active constituents.


Introduction
About one billion people are afflicted with what the World Health Organization classifies as neglected tropical diseases (NTDs). A subset of life-threatening NTDs includes Kala azar (visceral leishmaniasis), sleeping sickness (African trypanosomiasis), and Chagas disease (American trypanosomiasis) which all may lead to fatal complications but are restricted to limited geographical areas and specific groups. Most current chemotherapeutics are toxic and marginally effective, must be given by injection, and become compromised by the development of drug-resistance [1]. Despite the large numbers of people at risk and the substantial burden of disease, with few exceptions, no major interventions have been developed for generations [2]. Malaria is yet another even bigger threat in many parts of the world with resistance spreading to almost all classes of antimalarials [3]. Hence, safe, effective, and more affordable therapeutics are clearly needed, whereby the natural biodiversity with its numerous plants, microorganisms, and marine organisms constitutes a broad source of potentially new molecules with great variety of structures and pharmacological potential. In addition, it is estimated that two-thirds of the world population still rely on traditional medical remedies, mostly plants, because of limited availability or affordability of pharmaceutical medicines [4].
In the present study, we evaluated twenty-five medicinal plants used in traditional medicine in Yemen and Saudi Arabia for their in vitro antiplasmodial, antileishmanial, and antitrypanosomal potential.

Antitrypanosomal Activity. T. brucei
Squib-427 strain (suramin-sensitive) was cultured at 37 ∘ C and 5% CO 2 in Hirumi-9 medium [10] supplemented with 10% fetal calf serum (FCS). About 1.5 × 10 4 trypomastigotes were added to each well and parasite growth was assessed after 72 h at 37 ∘ C by adding resazurin [11]. For Chagas disease, T. cruzi Tulahuen CL2 (benznidazole-sensitive) was maintained on MRC-5 cells in minimal essential medium (MEM) supplemented with 20 mM L-glutamine, 16.5 mM sodium hydrogen carbonate, and 5% FCS. In the assay, 4 × 10 3 MRC-5 cells and 4 × 10 4 parasites were added to each well. After incubation at 37 ∘ C for 7 days, parasite growth was assessed by adding the -galactosidase substrate chlorophenol red -D-galactopyranoside [12]. The color reaction was read at 540 nm after 4 h and absorbance values were expressed as a percentage of the blank controls.

Cytotoxicity
Assay. MRC-5 SV2 cells were cultivated in MEM supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate, and 5% FCS. For the assay, 10 4 MRC-5 cells/well were seeded onto the test plates containing the prediluted extracts and incubated at 37 ∘ C and 5% CO 2 for 72 h. Cell viability was assessed fluorimetrically after 4 hours of addition of resazurin. Fluorescence was measured (excitation 550 nm, emission 590 nm) and the results are expressed as % reduction in cell viability compared to the blank controls.

Results
For the selection of relevant plant species for our screening program, different places in Yemen and Saudi Arabia were visited where elderly people with profound knowledge of folk medicine were interviewed. In total, 25 medicinal plants were collected (Table 1), extracted, and evaluated in the integrated in vitro screen for antileishmanial, antiplasmodial, and antitrypanosomal potential ( Table 2). Seven plants showed interesting activity (acceptable potency and selectivity) in one or more models (Table 2).

Cytotoxicity. The selectivity of the antiprotozoal activity was assessed on MRC-5 cells. Clear cytotoxicity was found for
Gomphocarpus fruticosus (IC 50 2.3 g/mL), Centaurothamus maximus (IC 50 9.3 g/mL), and Hypoestes forskalei (IC 50 11.0 g/mL). The observed antiprotozoal inhibition of these plant species is therefore considered as nonselective.

Discussion
In continuation of our search for substances of plant origin with pharmacological effects, 25 plants were collected from Saudi Arabia and Yemen and screened for their antiplasmodial, antileishmanial, and antitrypanosomal activity potential. It is important to mention that to the best of our knowledge, this study represents the first report on antiprotozoal evaluation for most of the investigated plants. Although some plants have already been partly investigated, knowledge remains very limited in many cases. Based on potency (IC 50 ) and selectivity, seven plant extracts are considered promising enough to pursue further purification and biological evaluation of individual constituents. The methanol extract of the C. penicillata (collected from Saudi Arabia) exhibited antiplasmodial activity with adequate selectivity (IC 50 6.7 g/mL, SI 9.6). Some side-activity was present against T. brucei (IC 50 8.5 g/mL, SI 7.6), which matches our previously published data on C. sinaica showing antileishmanial and antitrypanosomal activity. However, C. sinaica was inactive against P. falciparum [13]. Pregnane glycosides which represent the major compounds in Caralluma species are believed to be responsible for the observed effects. Isolation and characterization of some acylated pregnane glycosides revealed antiparasitic activity for C. tuberculata and C. penicillata. The pregnane glycosides penicilloside E isolated from C. penicillata and caratuberside C isolated from C. tuberculata exhibited a pronounced antitrypanosomal activity (IC 50 1.0 and 1.8 g/mL) [14,15].
Interesting antiplasmodial activity was obtained with A. ciliata that is traditionally used in the treatment of malaria. Our result is in agreement with literature data on other Acalypha species [16][17][18][19][20]. We previously reported interesting antiplasmodial activity for the methanol and aqueous extracts of A. fruticosa [16], whereby both extracts showed complete inhibition of schizont maturation at 7.8 g/mL. Furthermore, Bradacs et al. [17] reported that the leaf extracts of A. grandis significantly affected P. falciparum without showing obvious effects on other protozoa. Additionally, the extract and fractions of A. wilkesiana dose-dependently reduced parasitaemia induced by chloroquine-sensitive P. berghei infection in prophylactic, suppressive, and curative mouse models [18].
Interesting antileishmanial and antitrypanosomal activities were observed for S. glabratum. Antiprotozoal properties have indeed been reported for extracts from other Solanum species [21][22][23][24][25]. Abdel-Sattar et al. [21] demonstrated potent in vitro antitrypanosomal activity for the methanol extract of S. schimperianum (IC 50 0.061 g/mL). It is recently reported that the extract of S. torvum inhibited the proliferation of promastigotes of L. donovani [22]. The fruits of S. stramonifolium var. stramonifolium were shown to have marginal activity against amastigotes of L. amazonensis [23]. Although S. sisymbriifolium failed to inhibit promastigotes of L. amazonensis and L. brasiliensis (IC 50 of 33.8 and 20.5 g/mL), the steroid derivative Cilistol-A as the main active principle of the chloroform fraction exhibited significant activity against both Leishmania species (IC 50 6.6 and 3.1 g/mL) [24]. It seems that the antileishmanial as well as antitrypanosomal activities can be attributed to the steroidal alkaloids, which represent the major constituents in Solanum species. Abreu Miranda et al. [25] isolated solamargine and solasonine from the fruits of S. lycocarpum and showed in vitro leishmanicidal activity against promastigotes of L. amazonensis. Our failure to demonstrate activity against P. falciparum (IC 50 > 64 g/mL) is not in agreement with literature data on other Solanum species. For example, Chinchilla et al. [26] reported antimalarial effect for S. arboretum; Kamaraj et al. [27] found some effect for S. torvum against chloroquine-sensitive (3D7) and chloroquine-resistant strains of P. falciparum. Moreover, diosgenone which is a spirostan-type steroidal saponin isolated from S. nudum, showed a high activity against FCB-2 strain of P. falciparum [28]. These results showed that diosgenone could be a new therapeutic alternative for the treatment of malaria [28].
One of the more remarkable plants with antileishmanial and antitrypanosomal activities was V. bottae, which showed selective activity against L. infantum and T. brucei (IC 50 3.2 and 2.3 g/mL; SI 10.2 and 14.1). This result outperforms data reported on other Verbascum species. A very marginal antileishmanial activity for V. arcturus against L. donovani (IC 50 57 g/mL) was reported and no activity against P. falciparum was reported [29]. Manjili et al. [30] obtained an IC 50 of 451 g/mL for a V. thapsus extract against L. major promastigotes, illustrating inefficacy.
Another plant with antitrypanosomal activity was L. virgata (IC 50 8.8 g/mL against T. brucei), which is in agreement with literature data on other Leucas species [31][32][33][34]. Our results on the antiplasmodial inactivity of L. virgata are not in agreement with the effects noted for L. aspera and L. cephalotes. It is recently reported that the leaf ethyl acetate and methanol extracts of L. aspera had good antiplasmodial activity (IC 50 7.81 and 22.7 g/mL with SI values of 5.4 and 2.0) [32]. A similar study was conducted by Kamaraj et al. [33] who reported similar results for L. aspera (IC 50 12.5 g/mL). In addition, it is reported that L. cephalotes showed promising antiplasmodial activity (IC 50 < 5 g/mL) in addition to promising activities against L. donovani with IC 50 values of 3.61 g/mL (SI = 8) [34]. Apparently, these findings are mostly attributed to the presence of essential oil constituents as well as diterpenoids [35][36][37][38]. Triterpenoids, such as ursolic acid isolated from some Leucas species [39], showed significant antitrypanosomal activity: it inhibited all movement of T. cruzi epimastigotes at 40 g/mL after 48 h incubation [40].
Notably antitrypanosomal potencies against T. brucei were also displayed by the methanolic extract of L. regularis (IC 50 9.5 g/mL, SI 4.3). Based on the literature review and to the best of our knowledge this is the first report on antiprotozoal activity of the genus Loranthus.
The methanolic extract of R. officinalis inhibited both P. falciparum and T. cruzi (IC 50 11.4 and 8.8 g/mL; SI 1.9 and 2.5) which is in agreement with data reported previously [35,40]. The inhibitory effect was attributed to the presence of essential oils and triterpenoids, such as ursolic acid and oleanolic acid [40].

Conclusion
In conclusion, this preliminary study led to the identification of seven plant extracts, namely, A. ciliata, C. pencillata, L. virgata, L. regularis, R. officinalis, S. glabratum, and V. bottae exhibiting relevant antiplasmodial, antileishmanial, and antitrypanosomal activity in one or more models. The obtained results support to some extent the traditional uses of some plants for the treatment of parasitic diseases. Isolation, purification, and structure elucidation of constituents from some of these investigated plants are warranted to support discovery of novel antiplasmodial, antileishmanial, and antitrypanosomal compounds.