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

Authors: Ramzi A. Mothana; Nawal M. Al-Musayeib; Mohamed F. Al-Ajmi; Paul Cos; Louis Maes
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

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 screenedin 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.


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## Body

## 1. 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 theirin vitro antiplasmodial, antileishmanial, and antitrypanosomal potential.


## 2. Materials and Methods

### 2.1. Plant Material

Twenty-five plants were selected randomly from different areas of Yemen and Saudi Arabia in March and June 2008 and were identified at the Pharmacognosy Departments, Colleges of Pharmacy, King Saud and Sana’a Universities, Saudi Arabia and Yemen. The plants were chosen mainly on the basis of their local medicinal knowledge. Voucher specimens were deposited at the departments. The botanical names, plant parts used, and the traditional uses in the collected areas are presented in Table1.Table 1
List of selected plants and their use in traditional medicine.
Plant species
Family
Part used
Traditional uses
Acalypha ciliataForssk.
Euphorbiaceae
L
Malaria, anthelmintic, and scabies (a, d)
Acalypha fruticosaForssk.
Euphorbiaceae
L
Skin diseases, malaria, and wounds (a, b, d)
Amaranthus viridis L.
Amaranthaceae
L
Anthelmintic, cancer, and impotence (a, b)
Barleria trispinosa(Forssk.) Vahl
Acanthaceae
L, S
Warts (a, d)
Caralluma penicillata(Deflers) N.E.Br.
Asclepiadaceae
L
Diabetes, stomach ulcer, and smallpox (a, c)
Centaurothamus maximus(Forssk.)
Asteraceae
L
Wounds (a)
Wagenitz & Dittrich
Cissus rotundifolia(Forssk.) Vahl
Vitaceae
L
Malaria, liver disease, and otitis (a, b, c, d)
Coccinia grandis(L.) Voigt
Cucurbitaceae
L, T
Anthelmintic, diuretic,  and pneumonia (a)
Dichrocephala integrifolia(L.f.) O.
Asteraceae
L, S
Wounds (a)
Kuntze
Fagonia indica Burm. f.
Zygophyllaceae
L, T
Diabetes, diuretic, and headache (a, b, c)
Forsskaolea tenacissimaL.
Urticaceae
L, S
Diuretic, kidney disease (a)
Gomphocarpus fruticosus (L.) Ait. f.
Asclepiadaceae
L
Tumors, skin disease, scabies, and itching (a, b)
Hypoestes forskalei (Vahl) R. Br.
Acanthaceae
L
Fungal skin disease, scabies, itching, and warts (a)
Kedrostis foetidissima(Jacq.) Cogn.
Cucurbitaceae
L, S
Warts (a)
Kleinia pendula(Forssk.) DC.
Asteraceae
R
Otitis (a)
Leucas virgata
Labiatae
L, T
Heartburn, indigestion, and stomach problems (a)
Loranthus regularis Steud. ex Sprague
Loranthaceae
R
Diabetes, kidney disease (a)
Ochradenus baccatusDel.
Resedaceae
L, F
Diuretic, antiseptic, cough, and itching  (a, b)
Otostegia fruticosa (Forssk.) Briq.
Labiatae
L, F
Antiparalytic, eye diseases (a)
Oxalis corniculata L.
Oxalidaceae
L, F
Antiparasitic, antivertigo, and mouth inflammation (a, b, c)
Rosmarinus officinalis L.
Labiatae
L, S
Antiseptic, cholagogue (a, d)
Solanum glabratum Dunal
Solanaceae
L, T
Diuretic, scabies, syphilis, cough, hemorrhoids (a, b, c, d)
Taraxacum officinale F.H. Wigg
Asteraceae
L
Gastrointestinal troubles (a)
Tecoma stans (L.) H.B.K.
Bignoniaceae
L, S
Diabetes (a, b)
Verbascum bottae (Deflers) Huber-Mor.
Scrophulariaceae
L, F
Cough, skin disease, and rheumatism (a, b)
F: flower, L: leaves, R: roots or rhizomes, S: stems, and T: fruits. a: information has been taken from native people; b: Al-Dubai and Al-Khulaidi (1996) [5]; c: Fleurentin and Pelt (1982) [6]; d: Schopen (1983) [7].


### 2.2. Extraction of Plant Materials

The air-dried and powdered plant material (50 g) was extracted with 500 mL methanol (CH3OH) using a Soxhlet apparatus for 8 hours. The obtained methanolic extract was filtered and evaporated in a rotator evaporator and freeze dryer. The dried extracts were stored at −20°C until used. Stock solutions were prepared in 100% DMSO at 20 mg/mL just prior toin vitro evaluation.


### 2.3. Reference Drugs

For the different tests, appropriate reference drugs were used as positive control: tamoxifen for MRC-5, chloroquine forPlasmodium falciparum, miltefosine forLeishmania infantum, benznidazole forTrypanosoma cruzi, and suramin forTrypanosoma brucei. All reference drugs were obtained either from the fine chemical supplier Sigma or from WHO-TDR.


### 2.4. Biological Assays

The integrated panel of microbial screens and standard screening methodologies were adopted as previously described [8]. All assays were performed at the Laboratory of Microbiology, Parasitology and Hygiene at the University of Antwerp, Belgium. Plant extracts were tested in triplicate at 5 concentrations (64, 16, 4, 1, and 0.25 μg/mL) to establish a full dose-titration and determination of the IC50 (inhibitory concentration 50%). The in-test concentration of DMSO did not exceed 0.5%.


#### 2.4.1. Antileishmanial Activity

L. infantum MHOM/MA (BE)/67 amastigotes were collected from the spleen of an infected donor hamster and used to infect primary peritoneal mouse macrophages. Macrophages (3×104) were seeded in each well of a 96-well plate. After 2 days of outgrowth, 5×105 amastigotes were added to each well and incubated for 2 h at 37°C. The prediluted extracts were subsequently added and the plates were further incubated for 5 days at 37°C and 5% CO2. Intracellular amastigotes burdens were microscopically assessed after Giemsa staining and expressed as a percentage of the burdens in the blank controls.


#### 2.4.2. Antiplasmodial Activity

Chloroquine-resistantP. falciparum K1-strain was grown in human erythrocytes O+ at 37°C under a low oxygen atmosphere (3% O2, 4% CO2, and 93% N2) in RPMI-1640 medium supplemented with 10% human serum. Infected human red blood cells (200 μL, 1% parasitaemia and 2% hematocrit) were added to each well containing the prediluted extracts and incubated for 72 h. After incubation, test plates were frozen at −20°C and parasite multiplication was measured by the Malstat method [9].


#### 2.4.3. Antitrypanosomal Activity

T. brucei Squib-427 strain (suramin-sensitive) was cultured at 37°C and 5% CO2 in Hirumi-9 medium [10] supplemented with 10% fetal calf serum (FCS). About 1.5 × 104 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 × 103 MRC-5 cells and 4 × 104 parasites were added to each well. After incubation at 37°C for 7 days, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-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.


#### 2.4.4. Cytotoxicity Assay

MRC-5SV2 cells were cultivated in MEM supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate, and 5% FCS. For the assay, 104 MRC-5 cells/well were seeded onto the test plates containing the prediluted extracts and incubated at 37°C and 5% CO2 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.


## 2.1. Plant Material

Twenty-five plants were selected randomly from different areas of Yemen and Saudi Arabia in March and June 2008 and were identified at the Pharmacognosy Departments, Colleges of Pharmacy, King Saud and Sana’a Universities, Saudi Arabia and Yemen. The plants were chosen mainly on the basis of their local medicinal knowledge. Voucher specimens were deposited at the departments. The botanical names, plant parts used, and the traditional uses in the collected areas are presented in Table1.Table 1
List of selected plants and their use in traditional medicine.
Plant species
Family
Part used
Traditional uses
Acalypha ciliataForssk.
Euphorbiaceae
L
Malaria, anthelmintic, and scabies (a, d)
Acalypha fruticosaForssk.
Euphorbiaceae
L
Skin diseases, malaria, and wounds (a, b, d)
Amaranthus viridis L.
Amaranthaceae
L
Anthelmintic, cancer, and impotence (a, b)
Barleria trispinosa(Forssk.) Vahl
Acanthaceae
L, S
Warts (a, d)
Caralluma penicillata(Deflers) N.E.Br.
Asclepiadaceae
L
Diabetes, stomach ulcer, and smallpox (a, c)
Centaurothamus maximus(Forssk.)
Asteraceae
L
Wounds (a)
Wagenitz & Dittrich
Cissus rotundifolia(Forssk.) Vahl
Vitaceae
L
Malaria, liver disease, and otitis (a, b, c, d)
Coccinia grandis(L.) Voigt
Cucurbitaceae
L, T
Anthelmintic, diuretic,  and pneumonia (a)
Dichrocephala integrifolia(L.f.) O.
Asteraceae
L, S
Wounds (a)
Kuntze
Fagonia indica Burm. f.
Zygophyllaceae
L, T
Diabetes, diuretic, and headache (a, b, c)
Forsskaolea tenacissimaL.
Urticaceae
L, S
Diuretic, kidney disease (a)
Gomphocarpus fruticosus (L.) Ait. f.
Asclepiadaceae
L
Tumors, skin disease, scabies, and itching (a, b)
Hypoestes forskalei (Vahl) R. Br.
Acanthaceae
L
Fungal skin disease, scabies, itching, and warts (a)
Kedrostis foetidissima(Jacq.) Cogn.
Cucurbitaceae
L, S
Warts (a)
Kleinia pendula(Forssk.) DC.
Asteraceae
R
Otitis (a)
Leucas virgata
Labiatae
L, T
Heartburn, indigestion, and stomach problems (a)
Loranthus regularis Steud. ex Sprague
Loranthaceae
R
Diabetes, kidney disease (a)
Ochradenus baccatusDel.
Resedaceae
L, F
Diuretic, antiseptic, cough, and itching  (a, b)
Otostegia fruticosa (Forssk.) Briq.
Labiatae
L, F
Antiparalytic, eye diseases (a)
Oxalis corniculata L.
Oxalidaceae
L, F
Antiparasitic, antivertigo, and mouth inflammation (a, b, c)
Rosmarinus officinalis L.
Labiatae
L, S
Antiseptic, cholagogue (a, d)
Solanum glabratum Dunal
Solanaceae
L, T
Diuretic, scabies, syphilis, cough, hemorrhoids (a, b, c, d)
Taraxacum officinale F.H. Wigg
Asteraceae
L
Gastrointestinal troubles (a)
Tecoma stans (L.) H.B.K.
Bignoniaceae
L, S
Diabetes (a, b)
Verbascum bottae (Deflers) Huber-Mor.
Scrophulariaceae
L, F
Cough, skin disease, and rheumatism (a, b)
F: flower, L: leaves, R: roots or rhizomes, S: stems, and T: fruits. a: information has been taken from native people; b: Al-Dubai and Al-Khulaidi (1996) [5]; c: Fleurentin and Pelt (1982) [6]; d: Schopen (1983) [7].


## 2.2. Extraction of Plant Materials

The air-dried and powdered plant material (50 g) was extracted with 500 mL methanol (CH3OH) using a Soxhlet apparatus for 8 hours. The obtained methanolic extract was filtered and evaporated in a rotator evaporator and freeze dryer. The dried extracts were stored at −20°C until used. Stock solutions were prepared in 100% DMSO at 20 mg/mL just prior toin vitro evaluation.


## 2.3. Reference Drugs

For the different tests, appropriate reference drugs were used as positive control: tamoxifen for MRC-5, chloroquine forPlasmodium falciparum, miltefosine forLeishmania infantum, benznidazole forTrypanosoma cruzi, and suramin forTrypanosoma brucei. All reference drugs were obtained either from the fine chemical supplier Sigma or from WHO-TDR.


## 2.4. Biological Assays

The integrated panel of microbial screens and standard screening methodologies were adopted as previously described [8]. All assays were performed at the Laboratory of Microbiology, Parasitology and Hygiene at the University of Antwerp, Belgium. Plant extracts were tested in triplicate at 5 concentrations (64, 16, 4, 1, and 0.25 μg/mL) to establish a full dose-titration and determination of the IC50 (inhibitory concentration 50%). The in-test concentration of DMSO did not exceed 0.5%.


### 2.4.1. Antileishmanial Activity

L. infantum MHOM/MA (BE)/67 amastigotes were collected from the spleen of an infected donor hamster and used to infect primary peritoneal mouse macrophages. Macrophages (3×104) were seeded in each well of a 96-well plate. After 2 days of outgrowth, 5×105 amastigotes were added to each well and incubated for 2 h at 37°C. The prediluted extracts were subsequently added and the plates were further incubated for 5 days at 37°C and 5% CO2. Intracellular amastigotes burdens were microscopically assessed after Giemsa staining and expressed as a percentage of the burdens in the blank controls.


### 2.4.2. Antiplasmodial Activity

Chloroquine-resistantP. falciparum K1-strain was grown in human erythrocytes O+ at 37°C under a low oxygen atmosphere (3% O2, 4% CO2, and 93% N2) in RPMI-1640 medium supplemented with 10% human serum. Infected human red blood cells (200 μL, 1% parasitaemia and 2% hematocrit) were added to each well containing the prediluted extracts and incubated for 72 h. After incubation, test plates were frozen at −20°C and parasite multiplication was measured by the Malstat method [9].


### 2.4.3. Antitrypanosomal Activity

T. brucei Squib-427 strain (suramin-sensitive) was cultured at 37°C and 5% CO2 in Hirumi-9 medium [10] supplemented with 10% fetal calf serum (FCS). About 1.5 × 104 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 × 103 MRC-5 cells and 4 × 104 parasites were added to each well. After incubation at 37°C for 7 days, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-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.


### 2.4.4. Cytotoxicity Assay

MRC-5SV2 cells were cultivated in MEM supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate, and 5% FCS. For the assay, 104 MRC-5 cells/well were seeded onto the test plates containing the prediluted extracts and incubated at 37°C and 5% CO2 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.


## 2.4.1. Antileishmanial Activity

L. infantum MHOM/MA (BE)/67 amastigotes were collected from the spleen of an infected donor hamster and used to infect primary peritoneal mouse macrophages. Macrophages (3×104) were seeded in each well of a 96-well plate. After 2 days of outgrowth, 5×105 amastigotes were added to each well and incubated for 2 h at 37°C. The prediluted extracts were subsequently added and the plates were further incubated for 5 days at 37°C and 5% CO2. Intracellular amastigotes burdens were microscopically assessed after Giemsa staining and expressed as a percentage of the burdens in the blank controls.


## 2.4.2. Antiplasmodial Activity

Chloroquine-resistantP. falciparum K1-strain was grown in human erythrocytes O+ at 37°C under a low oxygen atmosphere (3% O2, 4% CO2, and 93% N2) in RPMI-1640 medium supplemented with 10% human serum. Infected human red blood cells (200 μL, 1% parasitaemia and 2% hematocrit) were added to each well containing the prediluted extracts and incubated for 72 h. After incubation, test plates were frozen at −20°C and parasite multiplication was measured by the Malstat method [9].


## 2.4.3. Antitrypanosomal Activity

T. brucei Squib-427 strain (suramin-sensitive) was cultured at 37°C and 5% CO2 in Hirumi-9 medium [10] supplemented with 10% fetal calf serum (FCS). About 1.5 × 104 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 × 103 MRC-5 cells and 4 × 104 parasites were added to each well. After incubation at 37°C for 7 days, parasite growth was assessed by adding the β-galactosidase substrate chlorophenol red β-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.


## 2.4.4. Cytotoxicity Assay

MRC-5SV2 cells were cultivated in MEM supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate, and 5% FCS. For the assay, 104 MRC-5 cells/well were seeded onto the test plates containing the prediluted extracts and incubated at 37°C and 5% CO2 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.


## 3. 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 (Table1), extracted, and evaluated in the integratedin 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).Table 2
Antiprotozoal activity and cytotoxicity(IC50—μg/mL) of the methanolic extracts of the investigated plants.
Plant species
P. falciparum
L. infantum
T. cruzi
T. brucei
MRC-5
IC50
SI
IC50
SI
IC50
SI
IC50
SI
IC50
Acalypha ciliata
10.8 ± 1.3
>5.9
>64.0
34.4 ± 7.3
>1.9
32.7 ± 3.7
>2.0
>64.0
Acalypha fruticosa
27.1 ± 6.2
>2.4
>64.0
35.7 ± 5.8
>1.8
32.9 ± 4.1
>2.0
>64.0
Amaranthus polygamus
>64.0
>1
>64.0
>64.0
>64.0
>64.0
Barleria trispinosa
59.8 ± 5.6
>1.1
>64.0
>64.0
>64.0
>64.0
Caralluma penicillata
6.7 ± 0.9
>9.6
34.6 ± 4.5
>1.9
28.0 ± 1.7
>2.3
8.5 ± 1.5
>7.6
>64.0
Centaurothamus maximus
12.1 ± 3.4
<1
32.5 ± 0.5
<1
8.3 ± 0.7
1.1
35.8 ± 2.3
9.3 ± 1.8
Cissus rotundifolia
>64.0
>
>64.0
>64.0
>64.0
>64.0
Coccinia grandis-fruits
>64.0
>
>64.0
>64.0
>64.0
>64.0
Coccinia grandis-leaves
38.6 ± 7.3
>1.7
43.1 ± 4.8
>1.5
36.9 ± 2.4
>1.7
32.7 ± 4.3
>2
>64.0
Dichrocephala integrifolia
10.2 ± 2.5
2.4
20.3 ± 2.6
1.2
6.6 ± 0.5
3.7
37.6 ± 4.9
24.5 ± 3.7
Fagonia indica
>64.0
>64.0
>64.0
>64.0
>64.0
Forsskaoleatenacissima
>64.0
>64.0
>64.0
>64.0
>64.0
Gomphocarpus fruticosus
>64.0
>64.0
2.6 ± 0.5
1
>64.0
2.3 ± 0.4
Hypoestes forskalei
8.8 ± 2.0
1.3
8.1 ± 1.3
1.4
9.1 ± 1.0
>1.2
8.1 ± 0.5
>1.4
11.0 ± 2.1
Kedrostis foetidissima
62.2 ± 8.3
>1
>64.0
60.2 ± 3.5
>1.1
>64.0
>64.0
Kleinia pendula
38.9 ± 6.4
>1.6
>64.0
32.3 ± 3.7
>2
32.7 ± 4.0
>2
>64.0
Leucas virgata
>64.0
>64.0
>64.0
8.3 ± 0.9
>7.7
>64.0
Loranthus regularis
>64.0
32.5 ± 0.5
1.3
33.6 ± 2.5
1.2
9.5 ± 2.1
4.3
40.6 ± 5.5
Ochradenus baccatus
>64.0
>64.0
>64.0
>64.0
>64.0
Otostegia fruticosa
34 ± 5.2
>1.9
>64.0
36.6 ± 4.9
>1.8
35.2 ± 3.7
>1.8
>64.0
Oxalis corniculata
>64.0
>64.0
54.7 ± 7.6
>1.2
34.7 ± 5.2
>1.8
>64.0
Rosmarinus officinalis
11.4 ± 2.7
1.9
32.5 ± 0.5
8.8 ± 1.2
2.5
13.2 ± 2.0
1.7
22.1 ± 1.7
Solanum glabratum
>64.0
8.1 ± 1.3
3.4
8.5 ± 0.9
3.3
30.0 ± 4.8
27.9 ± 3.4
Taraxacum officinale
>64.0
>64.0
45.6 ± 7.3
>1.4
>64.0
>64.0
Tecoma stans
36.3 ± 5.8
>1.8
38.1 ± 4.7
>1.7
31.7 ± 3.7
>2.0
36.9 ± 6.3
>1.7
>64.0
Verbascum bottae
29.9 ± 4.5
1.1
3.2 ± 0.3
10.2
27.0 ± 4.2
1.2
2.3 ± 0.9
14.1
32.5 ± 5.9
Chloroquine
0.3 ± 0.1
Miltefosine
3.32 ± 0.7
Benznidazole
2.2 ± 0.5
Suramin
0.03 ± 0.02
Tamoxifen
—
11.0 ± 2.3
IC50 values of reference drugs are expressed in μM concentrations.


### 3.1. Antiplasmodial Activity

The methanolic extracts ofCaralluma penicillata andAcalypha ciliata showed relevant activity againstP. falciparum with IC50 of 6.7 and 10.8 μg/mL, respectively, and acceptable selectivity (SI > 9.6 and > 5.9). The extracts ofDichrocephala integrifolia andRosmarinus officinalis showed similar activity with IC50 values of 10.2 and 11.4 but with low selectivity (SI = 2.4 and 1.9).Hypoestes forskalei demonstrated nonselective inhibition (IC50 8.8 μg/mL; SI 1.3).


### 3.2. Antileishmanial Activity

The extract ofVerbascum bottae showed promising and selective activity againstL. infantum (IC50 3.2 μg/mL; SI 10.2);Solanum glabratum exhibited moderate antileishmanial activity (IC50 8.1 μg/mL; SI 3.4).H. forskalei demonstrated nonselective inhibition (IC50 8.1 μg/mL; SI 1.4).


### 3.3. Antitrypanosomal Activity

AgainstT. brucei, the extracts ofC. penicillata,Leucas virgata,Loranthus regularis, andV. bottae exhibited the most remarkable activity with IC50 values of 8.5, 8.1, 8.3, and 2.3 μg/mL, respectively, and acceptable selectivity (SI > 7.6, 7.7, 4.3, and >14.1). AgainstT. cruzi, some inhibitory potential was shown forD. integrifolia,R. officinalis, andS. glabratum, however, with lower selectivity (IC50 6.6, 8.8, and 8.5 μg/mL, resp., with SI < 4).


### 3.4. Cytotoxicity

The selectivity of the antiprotozoal activity was assessed on MRC-5 cells. Clear cytotoxicity was found forGomphocarpus fruticosus (IC50 2.3 μg/mL),Centaurothamus maximus (IC50 9.3 μg/mL), andHypoestes forskalei(IC50 11.0 μg/mL). The observed antiprotozoal inhibition of these plant species is therefore considered as nonselective.


## 3.1. Antiplasmodial Activity

The methanolic extracts ofCaralluma penicillata andAcalypha ciliata showed relevant activity againstP. falciparum with IC50 of 6.7 and 10.8 μg/mL, respectively, and acceptable selectivity (SI > 9.6 and > 5.9). The extracts ofDichrocephala integrifolia andRosmarinus officinalis showed similar activity with IC50 values of 10.2 and 11.4 but with low selectivity (SI = 2.4 and 1.9).Hypoestes forskalei demonstrated nonselective inhibition (IC50 8.8 μg/mL; SI 1.3).


## 3.2. Antileishmanial Activity

The extract ofVerbascum bottae showed promising and selective activity againstL. infantum (IC50 3.2 μg/mL; SI 10.2);Solanum glabratum exhibited moderate antileishmanial activity (IC50 8.1 μg/mL; SI 3.4).H. forskalei demonstrated nonselective inhibition (IC50 8.1 μg/mL; SI 1.4).


## 3.3. Antitrypanosomal Activity

AgainstT. brucei, the extracts ofC. penicillata,Leucas virgata,Loranthus regularis, andV. bottae exhibited the most remarkable activity with IC50 values of 8.5, 8.1, 8.3, and 2.3 μg/mL, respectively, and acceptable selectivity (SI > 7.6, 7.7, 4.3, and >14.1). AgainstT. cruzi, some inhibitory potential was shown forD. integrifolia,R. officinalis, andS. glabratum, however, with lower selectivity (IC50 6.6, 8.8, and 8.5 μg/mL, resp., with SI < 4).


## 3.4. Cytotoxicity

The selectivity of the antiprotozoal activity was assessed on MRC-5 cells. Clear cytotoxicity was found forGomphocarpus fruticosus (IC50 2.3 μg/mL),Centaurothamus maximus (IC50 9.3 μg/mL), andHypoestes forskalei(IC50 11.0 μg/mL). The observed antiprotozoal inhibition of these plant species is therefore considered as nonselective.


## 4. 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 (IC50) and selectivity, seven plant extracts are considered promising enough to pursue further purification and biological evaluation of individual constituents.The methanol extract of theC. penicillata (collected from Saudi Arabia) exhibited antiplasmodial activity with adequate selectivity (IC50 6.7 μg/mL, SI 9.6). Some side-activity was present againstT. brucei (IC50 8.5 μg/mL, SI 7.6), which matches our previously published data onC. sinaica showing antileishmanial and antitrypanosomal activity. However,C. sinaica was inactive againstP. falciparum [13]. Pregnane glycosides which represent the major compounds inCaralluma species are believed to be responsible for the observed effects. Isolation and characterization of some acylated pregnane glycosides revealed antiparasitic activity forC. tuberculata andC. penicillata. The pregnane glycosides penicilloside E isolated fromC. penicillata and caratuberside C isolated fromC. tuberculata exhibited a pronounced antitrypanosomal activity (IC50 1.0 and 1.8 μg/mL) [14, 15].Interesting antiplasmodial activity was obtained withA. ciliata that is traditionally used in the treatment of malaria. Our result is in agreement with literature data on otherAcalypha species [16–20]. We previously reported interesting antiplasmodial activity for the methanol and aqueous extracts ofA. fruticosa [16], whereby both extracts showed complete inhibition of schizont maturation at 7.8 μg/mL. Furthermore, Bradacs et al. [17] reported that the leaf extracts ofA. grandis significantly affectedP. falciparum without showing obvious effects on other protozoa. Additionally, the extract and fractions ofA. wilkesiana dose-dependently reduced parasitaemia induced by chloroquine-sensitiveP. berghei infection in prophylactic, suppressive, and curative mouse models [18].Interesting antileishmanial and antitrypanosomal activities were observed forS. glabratum. Antiprotozoal properties have indeed been reported for extracts from otherSolanum species [21–25]. Abdel-Sattar et al. [21] demonstrated potentin vitro antitrypanosomal activity for the methanol extract ofS. schimperianum (IC50 0.061 μg/mL). It is recently reported that the extract ofS. torvum inhibited the proliferation of promastigotes ofL. donovani [22]. The fruits ofS. stramonifolium var.stramonifolium were shown to have marginal activity against amastigotes ofL. amazonensis [23]. AlthoughS. sisymbriifolium failed to inhibit promastigotes ofL. amazonensis andL. brasiliensis (IC50 of 33.8 and 20.5 μg/mL), the steroid derivative Cilistol-A as the main active principle of the chloroform fraction exhibited significant activity against bothLeishmania species (IC50 6.6 and 3.1 μg/mL) [24]. It seems that the antileishmanial as well as antitrypanosomal activities can be attributed to the steroidal alkaloids, which represent the major constituents inSolanum species. Abreu Miranda et al. [25] isolated solamargine and solasonine from the fruits ofS. lycocarpum and showedin vitro leishmanicidal activity against promastigotes ofL. amazonensis. Our failure to demonstrate activity againstP. falciparum (IC50 > 64 μg/mL) is not in agreement with literature data on otherSolanum species. For example, Chinchilla et al. [26] reported antimalarial effect forS. arboretum; Kamaraj et al. [27] found some effect forS. torvum against chloroquine-sensitive (3D7) and chloroquine-resistant strains ofP. falciparum. Moreover, diosgenone which is a spirostan-type steroidal saponin isolated fromS. nudum, showed a high activity against FCB-2 strain ofP. 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 wasV. bottae, which showed selective activity againstL. infantum andT. brucei (IC50 3.2 and 2.3 μg/mL; SI 10.2 and 14.1). This result outperforms data reported on otherVerbascum species. A very marginal antileishmanial activity forV. arcturus againstL. donovani (IC50 57 μg/mL) was reported and no activity againstP. falciparum was reported [29]. Manjili et al. [30] obtained an IC50 of 451 μg/mL for aV. thapsus extract againstL. major promastigotes, illustrating inefficacy.Another plant with antitrypanosomal activity wasL. virgata (IC50 8.8 μg/mL againstT. brucei), which is in agreement with literature data on otherLeucas species [31–34]. Our results on the antiplasmodial inactivity ofL. virgata are not in agreement with the effects noted forL. aspera andL. cephalotes. It is recently reported that the leaf ethyl acetate and methanol extracts ofL. aspera had good antiplasmodial activity (IC50  7.81 and 22.7 μg/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 forL. aspera (IC50 12.5 μg/mL). In addition, it is reported thatL. cephalotes showed promising antiplasmodial activity (IC50 < 5 μg/mL) in addition to promising activities againstL. donovani with IC50 values of 3.61 μg/mL (SI = 8) [34]. Apparently, these findings are mostly attributed to the presence of essential oil constituents as well as diterpenoids [35–38]. Triterpenoids, such as ursolic acid isolated from someLeucas species [39], showed significant antitrypanosomal activity: it inhibited all movement ofT. cruzi epimastigotes at 40 μg/mL after 48 h incubation [40].Notably antitrypanosomal potencies againstT. brucei were also displayed by the methanolic extract ofL. regularis (IC50 9.5 μg/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 genusLoranthus.The methanolic extract ofR. officinalis inhibited bothP. falciparum andT. cruzi (IC50 11.4 and 8.8 μg/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].


## 5. 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, andV. 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.



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*Source: 905639-2014-05-21.xml*