In vitro antipromastigote activity of decoction and hydrodistillation extracts of Clematis hirsuta leaves against Leishmania donovani and Leishmania aethiopica

Abstract

Leishmaniasis remains a significant health concern in four eco-epidemiological regions of the world (WHO, 2024). Annually, there are thought to be 700,000 to 1 million new cases of leishmaniasis. Only 25–45% of the estimated 50–90,000 new cases of VL that are reported to WHO each year are actually seen worldwide. Only about 200,000 CL new cases are reported to WHO each year, out of an estimated 600,000 to 1 million worldwide (WHO, 2023). The current study was aimed at scientifically validating the antileishmanial potential of Clematis hirsuta by testing the in vitro antipromastigote activity of Clematis hirsuta decoction and hydro distilled leaves extracts and to test for cytotoxicity. The antipromastigote activities of the extracts of Clematis hirsuta against Leishmania aethiopica and Leishmania donovani at at 100 µg/ml and their cytotoxic effects on human red blood cells were evaluated. The decoction extracts of C. hirsuta inhibited the growth of L. aethiopica and L. donovani by 75.36 ± 1.47% and 87.37 ± 0.39%, respectively, at 100 µg/ml. The hydro distilled extract inhibited the growth of L. aethiopica and L. donovani by 97.22 ± 0.02% and 97.54 ± 0.07%, respectively. The IC₅₀ values of decocted extract were 0.01 µg/ml and 0.002 µg/ml against L. aethopica and L. donovani, respectively. The IC₅₀ values of the hydro distilled extracts were 0.39 µg/ml and 0.06 µg/ml against L. aethopica and L. donovani, respectively. In the hemolysis assay, the decoction extracts resulted in 18.18 ± 2.14% hemolysis on red blood cells. The hydro distilled extract caused 57.57 ± 4.28% hemolysis on red blood cells. The CC₅₀ values of decocted extract and hydro distilled extracts were > 1000 µg/ml and 881.0 µg/ml on red blood cells respectively. In terms of the selectivity index values, the decocted extract had an SI > 1000 for both Leishmania promastigotes. The hydro distilled extract had an SI of 2258.97 for L. aethiopica and 14,683.3 for L. donovani. In conclusion, the decocted extract showed greater than 75% activity, whereas the hydro distilled extract showed greater than 97% activity against both Leishmania promastigotes. The extracts were less toxic against RBC. Both extracts were selective against both Leishmania promastigotes but further studies are recommended.

Introduction

The protozoan parasite known as Leishmania, which belongs to the family Trypanosomatidae, causes all three types of leishmaniasis (cutaneous, mucocutaneous and visceral leishmaniasis) in both the Old and New Worlds. It can be found across Asia, Africa, the Middle East, and Central and South America. Over 90 countries that are poverty stricken are affected. It is a complicated disease caused by different species with different clinical symptoms and locations of occurrence, which causes confusion even among experts [1].

Leishmaniasis remains a significant health concern in four eco-epidemiological regions of the world: West and South-East Asia, East Africa, North Africa, and the Americas [2]. The occurrence of cutaneous leishmaniasis (CL) ranges from 600,000 to 1 million cases per year, 95 percent of these cases are reported from the Americas, Mediterranean basin, Middle East, and Central Asia. Yearly the occurrence of visceral leishmaniasis (VL) is currently estimated at 50,000 to 90,000 cases, with Brazil, East Africa and India accounting for majority of cases. Furthermore, mucocutaneous leishmaniasis (MCL) is estimated to account for 90% occurrence in Bolivia, Brazil, Ethiopia and Peru. Poverty, population migration, malnutrition, poor cleanliness, and an immunocompromised health status are all conditions that increase the likelihood for leishmaniasis [3]. In Ethiopia, where the disease is endemic and very frequent, people in the highlands are more likely to get cutaneous leishmaniasis than those in the lowlands, where visceral leishmaniasis is more common [4–8]. Estimated 4500–5000 new instances of VL are disclosed every year, where the disease burden is quite high and the nation is severely afflicted by both MCL and VL [9]. In addition, L. aethiopica, L. donovani, L. major, and L. tropica are all prevalent in the nation [10].

The leishmaniasis is transmitted by the insect vector known as the sand fly. Seventy distinct kinds of phlebotomine sand flies of the Diptera Family Psychodidae genera are subdivided into Phlebotomus in the Old World and Lutzomyia in the New World transmit the parasite Leishmania [11]. Owing to its wide range of species, leishmaniasis has been classified as either Old World or New World based on location. Asia, the Middle East, Africa, and Southern Europe are all part of the Old World, which refers to the Eastern Hemisphere. The New World, on the other hand, refers to the Western Hemisphere, specifically Mexico, Central America, South America, and the United States [1].

These flies can also transmit viruses such the Changuinola and Chandipura viruses, sand fly fever, and vesicular stomatitis. A bacterial infection called bartonellosis is also spread by a number of high Andean phlebotomines. Although research into phlebotomines in the Old World's more desert areas gave rise to the common name “sand fly,” the Americas contain an equally diversified species population as well [12].

Sand fly species, ecology and behavior heavily influence the epidemiology, also the accessibility of a diverse scope of non-human hosts, and the species and genetic variant of Leishmania parasites can determine distribution. Sand flies in some areas interact entirely among wild or tamed animals, with zero human interaction, whereas in others, livestock may be an important reservoir host of infection for humans. In India, without the presence of animals infections can be transmitted between people. As such leishmaniasis epidemiology is complicated [13].

Pentavalent antimonials have historically been employed to treat leishmaniasis; nevertheless, patients have experienced significant adverse effects, including arthralgias, myalgias, pancreatitis, leukopenia, and cardiotoxicity [14–16]. Despite their high cost, liposomal amphotericin B (AmpB) formulations are believed to be effective [17]. Perhaps the most well-known drug for leishmaniasis treatment is amphotericin B [18].

In an attempt to find new antileishmanial drugs, secondary metabolites isolated from plants have received a lot of interest in the past [15, 19–21]. Investigations using substances taken from plant tissue and/or pure molecules with antileishmanial activity have been conducted [22]. Economical and eco-friendly qualities of medicinal plants make them a more appealing alternative to chemotherapy than other treatments. Moreover, natural compounds made from plants are thought to be a secure and successful leishmaniasis treatment [23].

Clematis hirsuta belongs to the Ranunculaceae family. It is woody and perennial climber that can grow to be up to 4 m long [24]. According to Hao et al. [25], several medicinal chemicals found in Clematis species include glycosides, saponins, alkaloids, xanthones, and anthocyanidins. As a result, plants in the Clematis genus are utilized as medicines. It is used as an analgesic, anti-rheumatic, and anti-inflammatory in herbal medicine in Asia [26]. However, it’s widely prevalent over tropical Africa and areas with intermediate altitudes, such as Ethiopia. Known locally by some as “Hidda Feetii”, it is among the medicinal plants used in Ethiopia to treat a variety of illnesses [27]. It’s also locally known by the people of Debre Libanos monstery as “Azo Hareg” and its leaves and stem are used to treat leishmaniasis, while its leaves also treat ‘Yeshererit Beshita’ (Herpeszoster) and Haemorrhoids. When it comes to its veterinary use, its leaves are used to treat ‘Yeferes Ekeke’ (Lymphangitis) [28]. There are different uses for it in different places. To treat earaches for example, fresh leaves of C. hirsuta are smashed, compressed, and a tiny quantity of the fluid put via the ear canal over the course of two days [29].

According to Asmamaw Habtamu and Yalemtsehay Mekonnen [24] research, by using agar disk diffusion methods, the crude chloroform extract of C. hirsuta leaves at the concentration of 200 mg/ml on P. aeruginosa demonstrated the highest inhibition zone (12.33 ± 0.50 mm). In addition, C. hirsuta leaves were found to have antifungal activity in another research as well [30]. This shows the plant has been tested on anti parasitic and anti fungal activity.

Thus the aim of this study was to assess the in vitro antipromastigote activity of the decocted and hydro-distilled leaves extracts of Clematis hirsuta against Leishmania donovani and Leishmania aethiopica.

Material and methods

Test strains

The clinical isolates Leishmania donovani (VL: GR1140), Leishmania aethiopica (CL: 584/17) and human blood sample were obtained from the Leishmaniasis Research and Diagnostic Laboratory, Department of Immunology, Microbiology and Parasitology of School of Medicine, Addis Ababa University. The Leishmania aethiopica (CL: 584/17) strain was acquired from a patient from Gurage zone, Tiya woreda. The Leishmania donovani (VL: GR1140) strain was acquired from a patient from Amhara region, Gondar. A human blood sample was taken from a 24 year old healthy woman. All experiments were performed in accordance with relevant guidelines and regulations (protocol number 092/21/SOP).

Plant material collection and authentication

The Clematis hirsuta plant leaves were collected from the highlands of Ochollo village, Gamo Zone, South Ethiopia (520 km south west of Addis Ababa, Ethiopia, at latitude 6°25′09.0"N and longitude 37°48′00.8"E), which is located in the Rift Valley above the west shore of Lake Abaya. Mr. Melaku Wondeafrash a senior botanist at National Herbarium of Addis Ababa University is the one who authenticated the plant. The plant was collected from individual farm land and a verbal agreement was created before collecting the plant specimen. The research team collected the plant specimen under the guidance of the Department of Plant Biology and Biodiversity Management, College of Natural and Computational Sciences, Addis Ababa University (AAU), where a botanical specimen with the code BS006 was given and deposited for at the national herbarium future reference (Letter of approval REF:DPBBM/CNCS/85/17/2025).

Preparation of the extracts

Decoction extraction

Fresh leaves of C. hirsuta (600 g) were cleaned with water to remove dirt. The plant material was then socked in enough amount of distilled water and subjected to decoction on a hot plate for 1 h. After the decoction cooled, it was filtered first with sterile gauze and then with Whatman no. 1 filters paper. The filtrate was then centrifuged and lyophilized. The dried extract (0.44% yield) was finally labeled as CHD and transferred to an amber-coloured bottle and stored in a refrigerator at 4 °C until use as described previously by Zintchem et al. [31].

Hydro distillation extraction

Fresh leaves of C. hirsuta (600 g) were chopped into small pieces and subjected to hydro distillation (boiling the plant in distilled water) for 3 h using a distillation apparatus (Clevenger apparatus). The condensate was then collected and extracted with chloroform (3x) using a separatory funnel. The organic solvent was concentrated in a rotary evaporator at temperature not exceeding 35ºC. The oil (1.86% yield) was then transferred into vial and labeled as CHH and stored in a refrigerator at 4ºC for further experiment as described by Abdellatif and Hassani [32].

Leishmania promastigote culture

The promastigote, Leishmania donovani and Leishmania aethiopica, were grown in Novy–MacNeal–Nicolle (NNN) media supplemented with Locke’s solution. Once they were able to grow very well, they were transferred to 50 ml corning flask containing liquid media (complete liquid media), which was composed of 40 ml of RPMI medium, 2 ml of L-glutamine, 1 ml of pen strip (penicillin) and 10 ml of FBS (fetal bovine serum). A 3 × 10⁶ promastigotes/ml logarithmic growth phase was observed via a hemocytometer (counting chamber). It was checked for clear and non-cluster formations while it was observed under a microscope before proceeding.

Antipromastigote assay

The CL and VL assays were performed as described by Tewabe et al. [33] in triplicate with the following protocol. A uniform amount of 100 µl was added throughout the 96-well plates. In the first row, 120 µl of complete media was added, and 80 µl of extracts of CHD (after serial dilution the concentration will be from 100–0.78125 µg/ml), CHH (after serial dilution the concentration will be from 100–0.78125 µg/ml) and the positive control (amphotericin B) (after serial dilution the concentration will be from 10–0.078125 µg/ml) were added in triplicate. 100 µl was then serial diluted from these wells and discarded on the last row. Thus a two fold serial dilution was performed. 100 µl of selected parasite (584/17 for CL; GR 1140 for VL) was added to all wells except for the blank well which contained only the media. Thus, one column well in the plate then contained media and parasite which acted as a negative control. Finally, the cap of 96 well plates was cleaned using a guaze and closed. The plate was then covered with an aluminum foil and labeled and incubated for 72 h and on the 68 h, 20 µl alamar blue was added in each well. After 4 h it was read on FluoroskanAscent™, Thermo Scientific at an excitation wavelength of 544 nm and an emission wavelength of 590 nm (nano meter).

The median inhibitory concentration (IC₅₀) to parasites was obtained directly from linear equation of dose–response curves as well as the table format provided by Fluoroskan ascent readings. The IC₅₀ value was expressed as numbers. The antipromastigote activity of the extract was expressed as a percentage of inhibition following the formula of Tadele et al. [34]:

Hemolysis assay

Hemolysis assays were performed using the human blood sample provided by the laboratory as described by Abeje et al. [35]. Phosphate buffer saline (PBS) was made first by mixing 1000 ml of distilled water and one phosphate buffer powder packet in a beaker. Four milliliters of blood was taken from the EDTA tube, which was stored with a syringe. High-volume PBS and 2 ml of human blood were added to a 50 ml Falcon tube. A cold centrifuge at 4 °C was used to centrifuge the mixture at 3500 rpm for 10 min. The pellet was saved and the supernatant was discarded. A drop of PBS was added and the mixture was centrifuged at the same time. This process was repeated three times. The pellet volume reached 1 ml through this process after the third round of centrifugation. Forty-nine milliliters of PBS was added to a 1 ml pellet, which was subsequently mixed, resulting in a red blood cell suspension. This experiment was performed in duplicate. Like in the antipromastigote assay, 100 µl of the red blood cell suspension was mixed with 100 µl of the serially diluted extracts (1000–7.8125 µg/ml) in an Eppendorf tube. Triton X-114 (2%) and 1% DMSO were used as positive and negative controls, respectively. The Eppendorf tubes were incubated at 37 °C for 2 h, except for Triton, which was incubated for 30 min. The tubes were then centrifuged at 1000 rpm for 10 min. Seventy-five microliters of the supernatant was transferred to a 96-well plate. The data were then read spectrophotometrically at a wavelength of 530 nm via a Victor3 multilabel reader.

The median cytotoxic concentration (CC₅₀) to monocytes from human blood was obtained directly from the linear equation of the dose‒response curves.

The hemolytic effects are expressed as percentages following Zohra and Fawzia [36] formula:

Selectivity Index (SI)

The CC₅₀ against red blood cells and the corresponding IC₅₀ against Leishmania promastigote were used to determine the selectivity index (SI) of each extract. The following formula was used to calculate selectivity of the extracts and the standards of killing parasites as opposed to mammalian cells [37].

Ethical consideration

All research experiments performed in this study were approved by the Institutional Review Board, School of Pharmacy, College of Health Sciences, and Addis Ababa University, with protocol number 092/21/SOP dated January 2022. The review board reviews basic research involving patients and human volunteers. All experiments were performed in accordance with relevant guidelines and regulations. This study strongly adhered to the Declaration of Helsinki in the process of Ethics approval, consent to participate and data collection.

Statistical analysis

The IC₅₀ of the antipromastigote was calculated from sigmoidal dose response curve of the percentage of inhibition. The cytotoxicity was calculated from the sigmoidal dose‒response curve of the percentage of hemolysis. GraphPad Prism 8.4.2 (GraphPad Software, LLC, CA, USA) computer software was used for data analysis. Microsoft excel was used to express the values as the means ± standard errors of the means. The ratio of CC₅₀ to IC₅₀ was used to determine selectivity index (SI) [38].

Results

Growth inhibition screening

The ability of the plant extracts of C. hirsuta to inhibit Leishmania promastigotes at 100 µg/ml via the decoction extract of C. hirsuta was 75.36 ± 1.47% and 87.37 ± 0.39% growth inhibition of L. aethiopica and L. donovani, respectively. The hydro distilled extract (100 µg/ml) of C. hirsuta inhibited L. aethiopica and L. donovani growth by 97.22 ± 0.02% and 97.54 ± 0.07%, respectively. Compared with the decoction extract, the hydro distilled extract showed greater percentage inhibition in both cases. The positive control (Amp B) inhibited the growth of L. aethiopica and L. donovani by 98.73 ± 0.12% and 98.31 ± 0.17%, respectively (Table 1).

Table 1.

Percentage of inhibition of C. hirsuta extracts against Leishmania promastigotes (mean ± SEM)

Types of extracts and Concentration	% of inhibition of extracts
	Against L. aethiopica	Against L. donovani
CHD (100 µg/ml)	75.36 ± 1.47	87.37 ± 0.39
CHH (100 µg/ml)	97.22 ± 0.02	97.54 ± 0.07
Amp B (10 µg/ml)	98.73 ± 0.12	98.31 ± 0.17

CHD decoction extract of leaves of C. hirsute, CHH hydro distilled extract of leaves of C. hirsute, Amp B Amphotericin B (positive control);

Percentage of growth inhibition of extracts and standard drug

At different concentrations, each extract, along with Amp B, had an increasing sigmoid curve when tested against both Leishmania promastigotes (Fig. 1). As presented in Fig. 1, the decocted and hydro distilled extracts did not make an S-shaped graph. However, the uphill graph suggested that CHD and CHH had high percentages of inhibition at concentrations ranging from 100–0.78125 µg/ml. The figure shows that the curve at the highest peak is the highest for the inhibitor. The opposite is true for the lowest peak on the graph.

Fig. 1.

Percentage of growth inhibition of extracts and standard drug. A Extracts against L. aethiopica; (B) extracts against L. donovani; (C) Amp B against L. aethiopica; (D) Amp B against L. donovani; CHD: decoction extract of leaves of C. hirsuta; CHH: hydro distilled extract of leaves of C. hirsuta; Amp B: amphotericin B (positive control); Conc (µg/ml): Concentration was expressed/transformed to log ^x

Antipromastigote assay

The mean values of IC₅₀ of the extracts against Leishmania promastigotes are shown in Table 2. The IC₅₀ values of decocted extract were 0.01 µg/ml and 0.002 µg/ml against L. aethiopica and L. donovani respectively. The IC₅₀ values of hydro distilled extracts were 0.39 µg/ml and 0.06 µg/ml against L. aethiopica and L. donovani respectively. The IC₅₀ value of the positive control (Amp B) was 0.14 µg/ml and 0.01 µg/ml against L. aethiopica and L. donovani respectively. The IC₅₀ value of the decocted extract was lower than the hydro distilled extract of C. hirsuta in both cases (Table 2).

Table 2.

The mean value (IC₅₀) of the extracts against Leishmania promastigotes

Types of extracts	Against L. aethiopica		Against L. donovani
	IC50 (µg/ml) (95% CI)	R2	IC50 (µg/ml) (95% CI)	R2
CHD	0.01 (0.001—0.04)*	0.74	0.002 (0.0003–0.007)*	0.85
CHH	0.39 (0.23–0.56)*	0.87	0.06 (0.02–0.11)*	0.86
Amp B	0.14 (0.08–0.21)*	0.80	0.01 (0.008–0.02)*	0.908

The values are expressed as mean; Calculated at 95% CI; CHD decoction extract of leaves of C. hirsute, CHH hydro distilled extract of leaves of C. hirsute, Amp B Amphotericin B; R2 is measurement of fitness (regression coefficient); 95% CI: 95% confidence interval; Differences with P value < 0.05 (*) were considered significant

Hemolysis assay

The percentage of red blood cell destruction that the extracts can cause in an in vitro environment at specific concentrations was assessed. The decoction extract of C. hirsuta resulted in 18.18 ± 2.14% hemolysis of red blood cells. The hydro distilled extract of C. hirsuta caused 57.57 ± 4.28% hemolysis of red blood cells. Compared with the decoction extract, the hydro distilled extract resulted in a greater percentage of hemolysis. The positive control hemolysis percentage was 100% (Table 3).

Table 3.

Hemolysis percentage of extracts against human red blood cells

Types of extracts	% of hemolysis of extracts against human red blood cells
CHD (1000 µg/ml)	18.18 ± 2.14
CHH (1000 µg/ml)	57.57 ± 4.28
Triton X-114	100

The values are expressed as mean ± SEM; CHD: decoction extract of leaves of C. hirsuta; CHH: hydro distilled extract of leaves of C. hirsuta; Trtion X-114: positive control; 1000 μg/ml indicates the concentrations of the extracts

Hemolysis percentage curve of extracts against human red blood cells

At different concentration each extract had an increasing curve (Fig. 2).

Fig. 2.

Hemolysis percentage curve of extracts against human red blood cells. CHD: decoction extract of leaves of C. hirsuta; CHH: hydro distilled extract of leaves of C. hirsuta; Conc (µg/ml): Concentration was expressed/transformed to log ^x

As presented in Fig. 2, the decocted and hydro distilled extracts made an uphill graph. This graph suggests that CHH has higher percentages of hemolytic activity than CHD at concentrations ranging from 1000–7.8125 µg/ml. shows that the curve at the highest peak was the highest hemolytic, in this case, CHH, and the curve at the lowest peak was CHD.

Cytotoxic effect of Clematis hirsuta extracts

The concentration at which 50% of red blood cell destruction can occur in an in vitro environment was assessed and the median cytotoxic concentration (CC₅₀) against human red blood cells is shown in Table 4. The decocted extract had CC₅₀ value > 1000 µg/ml in red blood cells (erythrocyte). The CC₅₀ values of hydro distilled extract was 881.0 µg/ml in red blood cells. The CC₅₀ value of the hydro distilled extract was lower than the decocted extract of C. hirsuta (Table 4).

Table 4.

Median cytotoxic concentration (CC₅₀) result for human red blood cells

Types of extracts	Against red blood cells
	CC50 (µg/ml) (95% CI)	R2
CHD	> 1000*	-
CHH	881.0 (748.9–1118) *	0.92

The values are expressed as means; CHD decoction extract of leaves of C. hirsute, CHH hydro distilled extract of leaves of C. hirsute, R² is measurement of fitness (regression coefficient), 95% CI 95% confidence interval; Differences with P value < 0.05 (*) were considered significant

Selectivity index of Clematis hirsuta extracts

The selectivity index was determined to evaluate the ability of the extracts to selectively destroy either Leishmania promastigotes or the red blood cells. The selectivity index of the decocted extract showed a SI > 1000 on both Leishmania promastigotes. The hydro distilled extract had an SI of 2258.97 and 14,683.3 for L. aethiopica and L. donovani respectively. The SI value of the decocted extracts was much higher than the hydro distilled extracts (Table 5).

Table 5.

Selectivity index of plant extracts against red blood cells

Types of extracts	Against red blood cells (CC50 of cell/IC50 of parasite)
	L. aethiopica	L. donovani
CHD	> 1000	> 1000
CHH	2258.97	14683.3

The values are expressed as ratio of CC₅₀ of red blood cells and IC₅₀ of Leishmania promastigote; CHD: decoction extract of leaves of C. hirsuta; CHH: hydro distilled extract of leaves of C. hirsuta

Discussion

In this study, the plant C. hirsuta extracts (decocted and hydro-distilled extracts) were tested for their activity against L. aethiopica and L. donovani. This was accomplished by determining the percentage of inhibition. Following that, the 50% concentration at which the extracts can have an inhibition effect (IC₅₀) was determined. The hemolysis percentage and CC₅₀ were also calculated to test the toxicity of the extracts on red blood cells. Finally, selectivity index was determined.

The percentage of growth inhibition of the promastigotes L. aethiopica and L. donovani shown by the decocted extract was 75.36 ± 1.47% and 87.37 ± 0.39% respectively, at a concentration of 100 µg/ml. The percentage of L. aethiopica and L. donovani inhibited by the hydro distilled extract were 97.22 ± 0.02% and 97.54 ± 0.07% respectively, at a concentration of 100 µg/ml (Table 1). This showed that the hydro distilled extract had a greater inhibition than the decocted extract at a concentration of 100 µg/ml. In other study plant latex of Vernonia brachycalyx showed strong inhibitory activity on the flagellate and extracellular premature stages of L. aethiopica and L. donovani, with IC50 values of 6.82 ± 0.18 and 6.34 ± 0.20 respectively [39].

The results of the present study are in line with those of De Queiroz et al. [40] who identified several plants extracts that are active against Leishmania amazonensis in Brazil. The same authors reported that plant extracts from Chenopodium ambrosioides and Pfaffia glomerata showed direct efficacy against the parasite with 87.4% and 88.7% suppression of growth respectively. In addition, the Ruta graveolens extract dramatically reduced the quantity of the promastigotes, resulting in 74.4% suppression at 100 µg/mL [40]. This finding revealed that decocted and hydro distilled extracts are ideal inhibitor of the Leishmania promastigotes in this study compared with the Brazilian plants described in another study.

First, the hydro distilled extract had similar inhibitory effects on L. donovani and L. aethiopica. These findings led to the conclusion that CHH is a good inhibitor against L. donovani and L. aethiopica. Second, this extract showed a very high inhibition for both Leishmania promastigotes compared with the decocted extract and slightly lower inhibition compared with the positive control (Amp B). This finding suggested that CHH is a better inhibitor on both L. aethiopica and L. donovani than CHD.

Compared with the L. aethiopica, the decoction extract had better inhibitory effects on L. donovani. It confirms that CHD is more effective in L. donovani than in L. aethiopica. In contrast with the hydro distilled extract, the decocted extract also had the lowest percentage of inhibition. However, against L. donovani the decocted extract had a much closer inhibition percentage to that of CHH. These findings suggest that CHD is more or less a good inhibitor against L. donovani than CHH is. This confirms the activity of the plants leaves in the traditional treatment of leishmaniasis as it is known to be used by the people of Debre Libanos monstery [28].

The antipromastigote activity of the decocted extracts was lower than that of the positive control (Amp B), whereas, that of the hydro distilled extracts was greater than that of Amp B. The IC₅₀ value represents the concentration at which the extracts can destroy 50% of the test strains. Therefore, the lower the IC₅₀ value is the better activity of the extract. The IC₅₀ values of decocted extract were lower against both L. aethiopica and L. donovani than they were against CHH. Thus, at a very low concentration, it is very active against both of the test strains. The IC₅₀ values of hydro distilled extracts were also low but not as low as those of CHD against both L. aethiopica and L. donovani. The IC₅₀ value of the positive control (Amp B) was also low against L. aethiopica and L. donovani (Table 2). As reported by [41], Ferula communis and Otostegia integrifolia showed better activity with IC₅₀ value of 11.38 ± 0.55 and 13.03 ± 0.87 µg/mL against L. aethiopica, respectively. However, the same plant extracts exhibited lower activity against L. donovani with IC₅₀ values of 23.41 ± 2.32 and 17.24 ± 1.29 µg/mL, respectively.

The antipromastigote activity of the extracts was determined by screening (ranges). An IC₅₀ less than or equal to 5 μg/mL has strong activity; IC₅₀ between 5 and/or equal to 20 μg/mL has good activity; IC₅₀ between 20 and/or equal to 30 μg/mL has mild activity; IC₅₀ between 30 and/or equal to 64 μg/mL has poor activity; and IC₅₀ greater than 64 μg/mL has no activity [42]. Both decocted and hydro distilled extracts had strong activity against both L. aethiopica and L. donovani. This finding proves its activity against Leishmania promastigotes. When the IC₅₀ values of CHH and Amp B were compared, the IC₅₀ value was closer to that of L. donovani. This indicated that 0.06 μg/mL CHH destroyed 50% of L. donovani, whereas 0.01 μg/mL Amp B destroyed 50% of L. donovani. These findings suggested that CHH and Amp B had very strong activities against L. donovani. However, the activity of Amp B was greater than that of CHH. The IC₅₀ value of CHH and Amp B when tested against L. aethiopica were greater than those when they were tested on L. donovani. Hence, Amp B had much stronger activity than CHH. In the same genus of C. hirsuta a study conducted by [43], Clematis simensis possesses antileishmanial activity with IC₅₀ outcomes of 46.12 ± 0.03 and 8.18 ± 0.10 µg/mL on the promastigotes of L. aethiopica and L. donovani, respectively. However, E abyssinica showed stronger activity with IC₅₀ outcomes of 16.07 ± 0.05 µg/mL and 4.82 ± 0.07 µg/mL on L. aethiopica and L. donovani, respectively.

The IC₅₀ values of the decocted extracts were the lower in this study. When CHD was against L. donovani, it had a lower IC₅₀ value than when it was used against L. aethiopica. This meant that 0.002 μg/mL of CHD destroyed 50% of L. donovani. This suggests that it is more active against L. donovani. When the CHD and Amp B IC₅₀ value were compared, they had further values from each other. In both cases, when CHD was tested on L. aethiopica and L. donovani, it had a better activity than Amp B and CHH. However, in both the test strains, the activity was closer to that of Amp B than CHH, suggesting it had higher activity than CHH.

Other plant extracts, such as Brucea antidysenterica J.F. Mill Seeds and its solvent fractions were tested for antileishmanial activity; the ethyl acetate fraction exhibited high antipromastigote activity, with 4.14 ± 0.62 ≤ IC₅₀ ≤ 6.77 ± 0.47 μg/mL whereas the aqueous fractions presented the lowest activity, with 189.3 ± 8.70 ≤ IC₅₀ ≤ 208.9 ± 20.2 μg/mL. Its crude extract (80% methanol) showed 20.77 ± 1.55 ≤ IC₅₀ ≤ 27.83 ± 1.06 μg/mL of antipromastigote activity [38]. Compared with those of C. hirsuta, the antipromastigote activity of the crude leaf extracts of C. hirsuta was greater than that of the methanol extract and fractions of Brucea antidysenterica.

After the activity of the plants extracts was determined, the toxicity level of the extracts was subsequently determined. The decocted extract had 18.18 ± 2.14% hemolysis while hydro distilled extract had 57.57 ± 4.28% hemolysis at concentrations of 1000 µg/ml as compared to Triton X-114 which showed 100% hemolysis. Compared with the decocted extract, the hydro distilled extract had greater hemolytic activity, whereas the decocted extract had the lowest hemolytic activity. Both the decocted and hydro distilled extracts had less hemolytic activity than positive control. Thus the hydro distilled extract can destroy more than 50% of red blood cells, whereas Triton X-114 can assure 100% destruction. As reported by [44], most of the extracts showed low hemolytic activity and high anti-hemolytic activity as well as high selectivity indices (SI). Hemolysis studies are part of the preclinical evaluation of new herbal drugs, ensuring their safety before use in humans. A lower percentage of hemolysis in the presence of the natural product indicates a higher anti-hemolytic capacity [45].

The use of hemolysis percentage led to the determination of CC₅₀ values of the plant extracts. This was the determination of the half concentration at which hemolysis can occur by these plant extracts. The toxicity criteria fall within different ranges: < 10 μg/mL very strong cytotoxicity, 10–100 μg/mL strong cytotoxicity and 100–500 μg/mL moderate cytotoxicity [46].

The decocted extracts and hydro distilled extract had a CC₅₀ of > 1000 µg/ml and 881.0 µg/ml respectively (Tables 3 and 4). The CC₅₀ value of the decocted extract was greater than the hydro-distilled extract. This meant that the decocted extract and the hydro distilled extract destroyed 50% of red blood cells at concentrations > 1000 µg/ml and 881.0 µg/ml respectively with the range of concentration being 1000–7.8125 µg/ml. This finding showed that the hydro distilled extract was more moderately cytotoxic than the decocted extract, which was less toxic. Over all, the extracts were less cytotoxic than the other extracts on the basis of their range. Since Triton X-114 had 100% hemolysis, it was more cytotoxic than CHH and CHD were.

Once the CC₅₀ values and IC₅₀ values were determined, the selectivity index was calculated. Leishmania parasites are believed to be selectively destroyed by an extract with selective index (SI) > 1 whereas, mammalian host cells are believed to be selectively destroyed by a selective index (SI) < 1 [46]. The decocted extract had a SI > 1000 on for both Leishmania promastigotes species. While the hydro distilled extract had SI of 2258.97 on L. aethiopica and 14,683.3 on L. donovani (Table 5). Accordingly, both extracts are selective against both Leishmania promastigotes. Compared with SI, the hydro distilled extract of SI against L. aethiopica has less selectivity for the parasite against L. donovani. It also had less selectivity than the decocted extract. This finding shows that the hydro distilled extract had a small chance of selectivity against red blood cells compared with the decocted extract.

The limitation of this study was the difficulty in culturing the parasites strains to a certain volume for assay capabilities. They can be problematic if they are not monitored regularly and sub-cultured frequently. Even with proper maintenance some parasites strains were unable to grow as much as needed. Creating a proper environment along with proper selection of ideal test strains helped to overcomee this problem but even the ideal volume of strains for the assay was difficult to grow. The next steps would be, doing an antiamastigote assay and further understanding the plant extracts capabilities.

Conclusion

The results of the present study revealed that the leaves of C. hirsuta extracts (decocted and hydro-distilled) have different levels of antipromastigote activity at different concentrations against both L. aethiopica and L. donovani. Compared with the decocted extract, the hydro distilled extract was more active against both L. aethiopica and L. donovani. The hydro distilled extract of C. hirsuta was more active against both L. aethiopica and L. donovani. Notably, its effectiveness was comparable to Amp B, a standard drug for treating leishmaniasis, which highlights its potential for being an alternative. The extracts were also found less toxic against human red blood cells. This research shows that the plant C. hirsuta has antipromastigote activity, which opens a door to further studies. We suggest that drug manufacturing from this plant for the distant future may provide a better alternative to the current drugs for the treatment of leishmaniasis.

Abbreviations

Amp B

Amphotericin B

CHD

Clematis hirsuta Decoction extract

CHH

Clematis hirsuta Hydrodistilled extract

CL

Cutaneous leishmaniasis

FBS

Fetal bovine serum

FDA

Food and drug administration

L- Amp B

Liposomal amphotericin B

MCL

Mucocutaneousleishmaniasis

NNN

Novy–MacNeal–Nicolle(NNN) media

PBS

Phosphate buffer saline

PKDL

Post kala-azar dermal leishmaniasis

RPMI

Roswell Park Memorial Institute (RPMI-1640 medium)

Sb^v

Pentavalentantimonials

SSG

Sodium stibogluconate

VL

Visceral leishmaniasis

Authors’ contributions

Authors’ contributions Etsegenet Abebe, Yehenew Asmamaw, Gurja Belay, Tegenu Gelana, Helen Nigussie and Solomon Mequanente conceptualized the manuscript, participated in the data collection techniques, performed the data analysis, interpreted the data, and drafted the manuscript. Abebe Ejigu, Yehenew Asmamaw, Dawit Araya, Markos Tadel, Betelhem Tatek and Werissaw Hailesilassie participated in the revision of the study design, data collection techniques, and statistical analysis. Etsegenet Abebe, Gurja Belay, Tegenu Gelana and Helen Nigussie participated in the revision of the data analysis, revised the paper for intellectual content and participated in the drafting of the manuscript. All the authors reviewed and approved the final version.

Funding

No funding available.

Data availability

Data availability The raw data is available at our hand and the corresponding author of this manuscript can provide the information to access the research data supporting the results of our manuscript.

Declarations

Ethics approval and consent to participate

All research experiments performed in this study were approved by the Institutional Review Board, School of Pharmacy, College of Health Sciences, and Addis Ababa University, with protocol number 092/21/SOP dated January 2022. The review board reviews basic research involving patients and human volunteers. All experiments were performed in accordance with relevant guidelines and regulations. This study strongly adhered to the Declaration of Helsinki in the process of the Ethics approval, consent to participate and data collection.

Based on protocol number 092/21/SOP, informed consent was obtained from the individuals participated in this study after a necessary explanation about the purpose, benefit, and risk of the study.

Consent for publication

Consent for publication is not applicable for this research because the clinical isolates Leishmania donovani (VL: GR1140), Leishmania aethiopica (CL: 584/17) and human blood sample were obtained from the Leishmaniasis Research and Diagnostic Laboratory, Department of Immunology, Microbiology and Parasitology of School of Medicine, Addis Ababa University. Thus the individuals responsible for this laboratory are part of the researchers and the authors of this manuscript.

Competing interests

The authors declare no competing interests.