In vitro antimalarial activity of selected medicinal plants native to Tigray region of Ethiopia

Various medicinal plants are used as alternative remedies for the management of malaria, an important infectious disease responsible for around 228 million cases and 405,000 deaths worldwide in 2018. The worsening of the disease is highly associated to an emergence of drug-resistant parasites. Hence, the search of new alternative antimalarial agents from traditionally used medicinal plants is the most viable approach. The current study was aimed to evaluate the in vitro antiplasmodial property of Aloe elegans , Aloe monticola , Capparis tomentosa, Hygrophila schulli and Tephrosia gracilipes extracts. Leaf latexes were collected from Aloe elegans and Aloe monticola , whereas cold maceration with 70% ethanol was used to prepare extracts from roots of Capparis tomentosa and Tephrosia gracilipes , and leaves of Hygrophila schulli . The antiplasmodial activity of the extracts against asexual and gametocyte stages was tested using parasite lactate dehydrogenase assay and luciferase assay, respectively. Cell cytotoxicity was assessed on human microvascular endothelial cells by the MTT assay


Methods
Leaf latexes were collected from Aloe elegans and Aloe monticola, whereas cold maceration with 70% ethanol was used to prepare extracts from roots of Capparis tomentosa and Tephrosia gracilipes , and leaves of Hygrophila schulli . The antiplasmodial activity of the extracts against asexual and gametocyte stages was tested using parasite lactate dehydrogenase assay and luciferase assay, respectively. Cell cytotoxicity was assessed on human microvascular endothelial cells by the MTT assay

Results
Of the five selected medicinal plants, leaf latex of Aloe monticola showed the best activity against both asexual stages and stage V gametocytes of Plasmodium falciparum . The leaf latex of Aloe elegans and ethanolic extract of Hygrophila schulli leaves also showed antiplasmodial property against asexual stages. On the contrary, the roots of Capparis tomentosa and Tephrosa gracilipes were inactive. 3

Conclusion
Findings of this study may partly support the acclaimed traditional use of the leaves latexes of both Aloe elegans and Aloe monticola and the ethanolic extract of Hygrophila schulli leaves for the management of mild to moderate malaria.

Background
Beyond 80% of the global population are heavily using traditional medicines, commonly plant-based medicines, for management of numerous types of illnesses including malaria [1]. Malaria is an infectious disease caused by the parasites belonging to Plasmodium genus with P. falciparum causing the most life-threatening type of malaria [2]. Even though there is a progress in decreasing malaria cases and deaths, the global malaria burden seems to have reached a plateau and above 50% of the global communities are still at risk of malaria. Based on the World Health Organization (WHO) malaria report 2019, 228 million cases and 405,000 deaths from malaria were estimated worldwide in 2018. Beyond 90% of these cases and deaths occur in Sub-Saharan Africa and about 70% of the global deaths from malaria were in children under-five [3]. Malaria has also a great impact on the socio-economic growth of developing countries, costing to Africa more than 10-12 billion US$ per year and slowing down the economic growth by as much as 1.3% annually [4]. Plasmodium species (P. knowlesi) [5]. These hitches strengthened the imperative need to search new inexpensive and harmless antimalarial remedies which have 4 become a critical priority on the global malaria research agenda. Thus, as history had taught us, the traditional medicinal plants can represent a source of alternative remedies for malaria [6][7][8].
People believe that traditional medicines are easily accessible, inexpensive, and sometimes efficacious compared to the conventional medicines [9,10] [11,12]. However, the traditional use of these medicinal plants for malaria treatment has so far not been scientifically validated. The current research was conducted to appraise the antiplasmodial property of A. elegans, A. monticola, C. tomentosa, H. schulli, and T. gracilipes extracts on the basis of the traditional use.

Plant materials collection
The leaves latexes of A. elegans and A. monticola were collected from Axum and Hgumburda, Tigray regional state, Ethiopia, respectively. The root part of C. tomentosa and T. gracilipes were collected from the locality where the Kunama ethnic group has resided. The leaves of H. schulli were collected from Grakahsu, Tigray regional state, Ethiopia. All the samples were collected in November 2018.
Each plant was authenticated and specimens were deposited in the National 5 Herbarium, Addis Ababa University, Ethiopia.

Preparation of the extracts
The leaf latexes of both A. elegans and A. monticola were collected and dried as per the method described and were considered as an extract [1,13]. The leaf of H.
schulli and the roots of C. tomentosa and T. gracilipes were dried under shaded open air. These plant materials were pounded separately using grinding mill. The powdered plant materials were then macerated using 70% ethanol for three days separately and filtered using What man's filter paper. The marcs were re-macerated two times and filtered after 72 hours. The filtrates of each plant were combined separately and dried in oven at 40 ºC. The dried latexes and ethanolic extracts were stored in a refrigerator at -4 ºC until used for further investigation.
Antiplasmodial activity P. falciparum cultures were prepared based on the method described by Trager and Jensen [14] with minor modifications. The CQ-sensitive D10 and CQ-resistant W2 strains were preserved at 5% hematocrit (human type A+ erythrocytes) in RPMI 1640 medium (EuroClone, Celbio) supplemented with 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mM Hepes, and 2 mM glutamine. All the cultures were maintained at 37 °C in a standard gas mixture consisting of 1% O 2 , 5% CO 2 , 6 and 94% N 2 . Test samples were dissolved in DMSO and then diluted with medium to achieve the required concentrations (final DMSO concentration <1%, non-toxic to the parasite). Drugs were placed in 96-well flat-bottomed microplates and serial dilutions made. Asynchronous cultures with parasitaemia 1-1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 hours at 37 °C.
Parasite growth was examined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of the method of Makler [15,16]. The antiplasmodial activity is expressed as 50% inhibitory concentrations (IC 50 ) as mean ± standard deviation of at least three separate experiments performed in duplicate.
Antimalarial activity against stage IV-V P. falciparum gametocytes The 3D7elo1-pfs16-CBG99 transgenic strain expressing the CBG99 luciferase under the pfs16 gametocyte specific promoter was used and gametocytes cultures were conducted as described [17,18]. Methylene blue was used as positive control.
To trigger gametocytogenesis, asexual parasites cultures were diluted to 0.5% parasitaemia and medium was changed daily, to obtain a parasitaemia higher than 5% when the cultures were treated for 48-72h with N-acetylglucosamine (NAG) (Sigma-Aldrich) to clear residual asexual parasites and to obtain virtually pure gametocytes cultures. Stage IV-V gametocytes were obtained and used for the experiments after 12-14 days from the addition of NAG to the culture. The luciferase activity was taken as measure of gametocytes viability [18]. Briefly, 100 μL of culture medium were removed from each well to increase haematocrit; 70μL of resuspended culture were transferred to a black 96-well plate; 70μL of D-luciferin (1mM in citrate buffer 0·1 M, pH 5·5) were added. Luminescence measurements 7 were performed after 10 min with 500 ms integration time using a microplate reader Synergy4 (BioTek). The results are expressed as 50% inhibitory concentrations (IC 50 ), extrapolated from the non-linear regression analysis of the concentrationresponse curve. Each IC 50 value is the mean ± standard deviation of at least three separate experiments performed in duplicate.

Cell cytotoxicity assays
The long-term human microvascular endothelial cell line (HMEC-1) immortalized by SV 40 large T Antigen [19] was maintained in MCDB 131 medium (Invitrogen, Milan, Italy) supplemented with 10% fetal calf serum (HyClone, Celbio, Milan, Italy), 10 ng/ml of epidermal growth factor, 1 µg/mL of hydrocortisone, 2 mM glutamine and 20 mM Hepes buffer (EuroClone). For the cytotoxicity assays, cells were treated with serial dilutions of test compounds and cell proliferation evaluated using the MTT assay already described [20]. Plates were incubated for 72 h at 37°C in 5% CO 2 , post-hoc test was also carried out to compare mean of groups to each other. P<0.05 was considered as a significant.

Results
The antiplasmodial property of the tested medicinal plants' extracts against the CQsensitive D10 and CQ-resistant W2 strains of P. falciparum is presented in Fig. 1 and were not active with an IC 50 value >200 µg/mL against both D10 and W2 strains.
Plant extracts were then tested against stage V P. falciparum gametocytes in order to investigate their transmission blocking potential. Only A. monticola extract showed activity against gametocytes with an IC 50 of 93.92 ± 14.18 µg/mL. None of the extracts was toxic, until 500 µg/mL, when cytotoxicity was assessed against human microvascular endothelial cells (HMEC-1). The selectivity index (SI = IC 50 on HMEC-1/IC 50 on P. falciparum) of A. monticola was > 25 and >5 for asexual and sexual stages, respectively.

Discussion
History has taught us searching effective antimalarial agents from traditional medicines particularly medicinal plants [6,7]. In the current study, the promising in vitro antiplasmodial property [21,27]. Additionally, the methanolic extract of roots of C. tomentosa exerted a statistically significant (P<0.05) antimalarial activity against P. berghei in mice [28]. These controversies needs further study to explore the effectiveness of the root extracts of C. tomentosa using different chemical and bio-assays.
Various studies on the antimalarial activity of the genus Tephrosa have been reported. In one such study, the dicloromethane:methanol (1:1) roots extract of Tephrosia villosa, and stem extract of T. purpurea showed good to moderate antiplasmodial activity against the CQ-sensitive (D6) and CQ-resistant (W2) strains of P. falciparum [29,30]. Similarly, the flavonoid compounds and crude extract obtained from T. elata seedpods have been shown to exhibit good antiplasmodial activities (IC 50 = 2.8 -9.6 µg/mL) [31]. Another study on the dicloromethane:methanol (50:50) extract of the stem of T. purpurea, the aerial parts of T. subtriflora, and isolated flavonoids showed in vitro chemo-suppressive effect against the CQ-sensitive strains of P. falciparum (D6) [32,33]. Conversely, in this study, the ethanolic root extract of T. gracilipes was inactive (IC 50 >200 µg/mL), which could be due to interspecies, environmental and/or growth condition differences, though the plant is on use as a traditional medicine. Also T. gracilipes Plot of percentage growth of the CQ-resistant P. falciparum W2 strain versus dose of the plan