Hindawi Publishing Corporation

BioMed Research International

Volume 2015, Article ID 938747, 6 pages
[Link]

Research Article
Evaluation of Antileishmanial Activity of Albaha Medicinal
Plants against Leishmania amazonensis

Saeed S. Al-Sokari,1 Nasser A. Awadh Ali,2 Lianet Monzote,3 and Mohamed A. Al-Fatimi4
1
Department of Biology, Faculty of Sciences, Al Baha University, Saudi Arabia
2
Pharmacognosy Department, Faculty of Clinical Pharmacy, Al Baha University, Saudi Arabia
3
Departamento de Parasitologı́a, Instituto de Medicina Tropical Pedro Kourı́, La Habana, Cuba
4
Pharmacognosy Department, Faculty of Pharmacy, Aden University, P.O. Box 5411, Ma’alla, Aden, Yemen

Correspondence should be addressed to Nasser A. Awadh Ali; alinasser9678@[Link]

Received 1 July 2015; Accepted 4 August 2015

Academic Editor: Yu-Ping Tang

Copyright © 2015 Saeed S. Al-Sokari et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Sixteen methanolic extracts obtained from thirteen plant species, selected either from ethnobotanical or chemotaxonomical
data, were screened for their antileishmanial activity against Leishmania amazonensis. The cytotoxic activity against normal
peritoneal macrophages from normal BALB/c mice was also determined. Eight extracts had IC50 values ranging from <12.5 to
37.8 𝜇g/mL against promastigotes. Achillea biebersteinii flower, Euphorbia helioscopia, and Solanum incanum leaf extracts showed
antileishmanial activities with IC50 between <12.5–26.9 𝜇g/mL and acceptable selectivity indices of 8–5. The other leishmanicidal
plant extracts, with IC50 ranging from 18.0 to 29.5 𝜇g/mL, exhibited low selectivity indices.

1. Introduction medicine knowledge can be useful to open new ways in the

search of new antileishmanial agents. A number of published
Leishmaniasis is distributed worldwide, especially in tropical studies have demonstrated that many plant extracts exhibited
and subtropical areas and more than 12 million people are activity against Leishmania parasites [5–8]. Some reports
currently infected. About 20 000 to 30 000 deaths occur were published about the antiprotozoal activity of Saudi
annually. Cutaneous leishmaniasis (CL) is the most common medicinal plants [5, 9–11]. In this study, the plant species were
form of leishmaniasis and causes skin lesions, mainly ulcers. selected either from ethnobotanical or chemotaxonomical
About 95% of CL cases occur in the Americas, the Mediter- data [12]. It is noteworthy to mention that to the best of
ranean basin, the Middle East, and Central Asia. Over two- our knowledge, this study represents the first report on
thirds of new CL cases occur in 6 countries: Afghanistan, antileishmanial activity for some of the tested plants. The aim
Algeria, Brazil, Colombia, Iran, and Syria. An estimated 0.7 of this study was to investigate the antileishmanial activity of
million to 1.3 million new cases occur worldwide annually the 16 methanol extracts from 13 selected plant species from
[1, 2]. Albaha region, against Leishmania amazonensis.
In Saudi Arabia, the disease was first described in 1973
by Morsy and Shoura [3]. Currently, CL is common in
the human population in different localities, including the 2. Material and Methods
Eastern Province of Saudi Arabia and in particular the Al-
Hassa Oasis that is a known endemic area for CL [4]. At 2.1. Plant Materials. The plant material was collected in
the moment, no good conventional treatments for CL in March-April 2014 from different locations in Albahah town
most countries are available. The conventional drugs are and outskirts of Albaha, except myrrh oleo gum resins that
very toxic and not 100% effective. Therefore, there is an were purchased from the herbal shops under the name
urgent need to discover new therapeutic agents. Traditional (Somali Myrrha) (Table 1). The plants were taxonomically
2 BioMed Research International

Table 1: Selected plants studied, ethnobotanical information and characteristics.

Plant family (voucher specimen no) Part tested𝑎 Local name

Species Traditional uses
(yield in %)
Asteraceae (CP-101) Thafra Antispasmodic and for1 kidney
Achillea biebersteinii Afan. Fl (1.5)
inflammation
(CP-101) Thafra As antispasmodic and for kidney
Achillea biebersteinii Afan. L (2.6)
inflammation
Calotropis procera (Aiton) Asclepiadaceae (CP-091) Alashur
L (4.2) For treating leprosy and filariasis
[Link]
Chenopodium murale L. Amaranthaceae (CP-081) F (7.3) Jkheara Leishmaniasis1
Burseraceae (CP-071) Somali mir Gum used for treating
Commiphora myrrha L. Resin (70)
leishmaniasis2
Sapindaceae (CP-061) Shath For treating chronic ulcer, burns,
Dodonaea viscosa Jacq. L (3.5)
leishmaniasis2
Euphorbia helioscopia L. Euphorbiaceae (CP-051) AP (4.2) Al-dehin Antiseptic
Lamiaceae (CP-041) Al-shiah As antispasmodic, antiseptic
Lavandula dentata L. AP (2.9)
when the leaves chewed1
Pulicaria crispa [Link] Asteraceae (CP-102) AP (3.1) Arararabi Antimalarial, stomach disorders2
Punica granatum L. Punicaceae (CP-011) Fl (2.5) Al-roman Anthelmintic, antiseptic2
Ruta chalepensis L. Rutaceae (CP-121) L (5.2) Al-shathab Antimicrobial2
Solanum incanum L. Solanaceae (CP-131) F (7.6) Al-hadak Antiseptic2
Leaves as dressing for healing
Solanum incanum L. Solanaceae (CP-132) L (3.9) Al-hadak wounds, paste of fruits for
treating leishmaniasis2
Verbesina encelioides (Cav.) Asteraceae (CP-021) L (3.7) Safeara Wounds, skin diseases2
Benth & Hook. f. ex A. Gray
Withania somnifera (L.) Solanaceae (CP-011) F (4.6) Alobeb Chronic dermatitis2
Dunal
Withania somnifera Solanaceae (CP-011) L (8.5) Alobeb
a
AP, aerial parts; F: fruits; L: leaves; Re: resins; Fl: flowers: 1 most information obtained from reference [12] and 2 interviewing with local people.

identified at the Faculty of Science, Department of Botany, of streptomycin/mL, 100 U penicillin/mL). Parasites were not
Aden University, Yemen. Voucher specimens of the plant used after the tenth passage to carry out in vitro experiments.
material are deposited at the Pharmacognosy Department,
Faculty of Clinical Pharmacy, Albaha University, Saudi Ara- 2.3.2. Antipromastigote Assay. Fifty microliters of Schneider’s
bia. medium with HFBS and antibiotics was distributed in each
well of a 96-well plate. In the first one, additional 48 𝜇L of
2.2. Preparation of Extracts. Air-dried and powdered plant medium was added as well as 2 𝜇L of extracts. Then, five
material (10 g) was extracted under shaking at room temper- dilutions 1 : 2 were carried out taking 50 𝜇L each time to test
ature with MeOH (4 × 100 mL). The obtained extracts were concentrations of the extracts ranging from 12.5 to 200 𝜇g/mL
filtered and evaporated to dryness in vacuo at 40∘ C. The yields and a final volume was completed to 100 𝜇L after addition of
of each dried extract were calculated in %. The resulting dried 50 𝜇L of parasites at 4 × 105 promastigotes/mL in logarithmic
crude extracts were stored at 4∘ C. For testing, the extracts phase. Also, parasites treated with DMSO or pentamidine
were dissolved in dimethylsulfoxide (DMSO) at 20 mg/mL. (Richet, Buenos Aires, Argentina) were also included as
controls. Plates were sealed with parafilm and incubated
2.3. Antileishmanial and Cytotoxicity Assays at 26∘ C during 72 hours. Then, 20 𝜇L of a solution of 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
2.3.1. Parasite Cultures. Strain MHOM/77BR/LTB0016 of (MTT) (SIGMA, St. Louis, MO, USA) at 5 mg/mL prepared
L. amazonensis, provided by Immunology Department at and filtered in the moment of use was added in each well.
Oswaldo Cruz Foundation (FIOCRUZ) from Brazil, was An additional incubation of 4 hours was performed, the
used. Parasites were routinely isolated from normal BALB/c supernatant was eliminated, and the formazan crystals were
mice lesions and maintained as promastigotes at 26∘ C in dissolved with 100 𝜇L of DMSO. The optical density was
Schneider’s medium (SIGMA, St. Louis, MO, USA) contain- determined using a spectrophotometer (Sirio S Reader, 2.4-
ing 10% heat-inactivated fetal bovine serum (HFBS) (SIGMA, 0, Italy), at a test wavelength of 560 nm, using 630 nm
St. Louis, MO, USA) and corresponding antibiotics (100 𝜇g as reference wavelength [13, 14]. The median inhibitory
BioMed Research International 3

concentration (IC50 ) was obtained from linear dose-response 3. Results and Discussion
curves fit to data by means of the linear equation model. The
evaluations were performed in triplicate and the results are Sixteen methanolic extracts of thirteen plant species belong-
expressed as mean ± standard deviation. ing to ten families gathered from Albaha region were inves-
tigated for their in vitro antileishmanial activity. Table 1 gives
the names of the plants, family, parts investigated, voucher
2.3.3. Cytotoxicity Assay. The cytotoxic median concentra- specimen numbers, local names, yields in %, and their
tion (CC50 ) of the extracts was also determined on normal traditional uses. Results of antileishmanial activity, cytotoxic
peritoneal macrophages from normal BALB/c mice [13]. effects on peritoneal macrophage from BALB/c mice, and
Resident macrophages were collected from peritoneal cavities selectivity of methanolic extracts studied are summarized
of animals in ice RPMI 1640 medium (SIGMA, St. Louis, Mo, in Table 2. Out of the 16 methanol extracts tested against
USA) supplemented with antibiotics. Suspension was seeded promastigote forms of L. amazonensis, three extracts showed
at 30000 cells/well and incubated for 2 hours at 37∘ C in 5% promising activity with potent leishmanicidal activities and
CO2 . Nonadherent cells were removed by washing and 50 𝜇L good selectivity indices: A. biebersteinii flower (IC50 =
of medium complemented with 10% of HFBS and antibiotics 26.9 𝜇g/mL; SI = 5), E. helioscopia leaf (IC50 < 12.5 𝜇g/mL;
was added. Additional 48 𝜇L was distributed in first well SI = 8), and S. incanum leaf (IC50 = 18.0 𝜇g/mL; SI = 8).
along with 2 𝜇L of tested extracts. Plant extracts were tested at In the case of extracts from Myrrh resin, W. somnifera and
5 concentrations (200, 100, 50, 25 and 12.5 𝜇g/mL) to establish aerial parts of P. crispa also showed good antileishmanial
a full dose-titration and determination of the IC50 (inhibitory activity but a low SI was observed. The rest of extracts tested
concentration 50%). The plate was incubated under the were either inactive or nonspecific due to high cytotoxicity
same conditions during 72 hours. Macrophages treated with against normal peritoneal macrophages from BALB/c mice
DMSO or pentamidine were included as controls. Cells (Table 2). The promising extracts were further tested against
viability was determined using the colorimetric assay with intracellular amastigotes of L. amazonensis, and four of these
extracts were active (Table 2); in particular the extract of
MTT as previously described, where 15 𝜇L was added to each
flowers from A. biebersteinii displayed the most promising
well, and CC50 was obtained from dose-response curves fit to
activity.
data by means of the linear equation model. The evaluations
Phytochemical studies of the genus Achillea have revealed
were performed in triplicate and the results are expressed as
the presence of a number of phytochemicals that exhibited
mean ± standard deviation.
antileishmanial activity. Santos et al. 2010 [17] reported the
Then, the selectivity index (SI) ratio was obtained from antileishmanial activity of an essential oil from the leaves
calculation: CC50 for macrophage/IC50 for promastigotes and flowers of A. millefolium. Therefore, the antileishmanial
[15]. Extracts with SI ≥ 5 or with IC50 < 12.5 𝜇g/mL against activity of A. biebersteinii may be attributed either to the com-
promastigotes were selected for further experiments. ponents of the essential oil [17] or alkamides that have been
isolated from Achillea species and possess antileishmanial
2.3.4. Antiamastigote Assay. The peritoneal macrophages activity [18].
were obtained as previously described and plated at 106 /mL However, E. helioscopia has been the subject of abundant
in 24-Well Lab-Tek (Costar, USA). Nonadherent cells were phytochemical and biological investigations, but there are
removed by washing after incubation of 2 hours at 37∘ C in 5% no reports regarding its antileishmanial activity [19]. The
CO2 . Macrophages were then infected with stationary-phase plant is used traditionally in Saudi Arabia for removing
of L. amazonensis promastigotes at 4 : 1 parasite/macrophage warts and orally to dispel worms [20]. Several compounds
ratio and incubated again during 4 hours at the same have been isolated from E. helioscopia including jatrophone
conditions. Free parasites were removed by washing and type diterpenoids and the flavones quercetin, and may be
1000 𝜇L of RPMI completed medium was added in each responsible for the leishmanicidal activity of the plant extract
well. In the first well, additional 990 𝜇L of medium and [21]. In agreement partially with our results, Duarte et al.
10 𝜇L of the tested extracts were added and four dilutions reported antileishmanial activity for the stilbene piceatannol
1 : 2 were carried out taking 1000 𝜇L each time, testing final isolated from Euphorbia lagascae against promastigotes of
concentrations between 12.5 and 100 𝜇g/mL. The plate was L. donovani, L. infantum, and L. major with moderate
incubated at the same conditions for 48 hours [16]. Then, activity [22]. Several triterpenoids isolated from Euphorbia
monolayer was fixed in absolute methanol, stained with resinifera and Euphorbia officinarum were found to possess
Giemsa, and examined under light microscopy. Number of antileishmanial activity [23].
intracellular amastigotes and percent of infected macrophage Interestingly, antileishmanial activity was observed for S.
were determined by counting 25 macrophages per sample, incanum leaves. A number of Solanum species with antileish-
and the results were expressed as percent of reduction of manial activity have been reported [24–28]. It was found that
the infection rate (obtained by multiplying the percentage the extract of S. torvum inhibited the proliferation of pro-
of infected macrophages by the number of amastigotes mastigotes of L. donovani [24]. The fruits of S. stramonifolium
per infected macrophages) in comparison to controls [15]. were shown to have marginal activity against amastigotes
IC50 value was determined from the concentration-response of L. amazonensis [25]. This activity may be attributed to
linear curves. The evaluations were performed in triplicate the steroid derivative such as cilistol-A or steroidal alkaloids
and the results were expressed as mean ± standard deviation. which form the main compounds in Solanum species [26, 27].
4 BioMed Research International

Table 2: Activity and cytotoxicity of plant extracts against L. amazonensis and peritoneal macrophage from BALB/c mice, respectively.

Species (part testeda ) IC50 b ± SDc (𝜇g/mL) CC50 d ± SD (𝜇g/mL) SIe IC50 ± SD (𝜇g/mL)
Promastigotes Macrophage Amastigotes
A. biebersteinii (Fl) 26.9 ± 2.9 131.3 ± 6.1 5 13.6 ± 0.6
A. biebersteinii (L) >200 — — —
C. procera (L) >200 — — —
C. murale (F) >200 — — —
Myrrha resin (Re) 17.5 ± 0.01 49.8 ± 2.0 3 —
D. viscosa (L) >200 — — —
E. helioscopia (AP) <12.5 82.5 ± 4.4 7 31.1 ± 3.9
L. dentata (AP) >200 — — —
P. crispa (AP) 23.5 ± 2.5 79.0 ± 7.3 3 —
P. granatum (Fl) >200 — — —
R. chalepensis (L) >200 — — —
S. incanum (F) 37.8 ± 2.7 174.6 ± 6.9 5 24.4 ± 3.0
S. incanum (L) 18.0 ± 2.8 149.2 ± 3.8 8 31.0 ± 3.8
V. encelioides (L) >200 — — —
W. somnifera (F) 29.5 ± 1.0 84.9 ± 4.7 3 —
W. somnifera (L) 20.3 ± 6.8 <12.5 0 —
a
AP, aerial parts; F: fruits; L: leaves; Re: resins; Fl: flowers.
b
IC50 : Concentration of extract causing 50% growth inhibition.
c
SD: Standard deviation.
d
CC50 : Concentration of extract causing 50% of mortality of peritoneal macrophage from BALB/c.
e
SI: Selectivity index. —: Not done.

The oleo gum resins (myrrh) of Commiphora species have that the leishmanicidal activity has been mostly related to the
long been used for health problems such as stomachache, terpenoid constituents of the plant [35].
colds, wounds, malaria, fever and as an antiseptic and against
skin infections [28]. Previous studies on the Commiphora 4. Conclusion
genus reported no antileishmanial activity of C. parvifolia
and C. ornifolia [29]. This is the first report on the antileish- In conclusion, the evaluation of antileishmanial activity of 16
manial activity of myrrh oleo gum resins. Volatile oil of extracts from 13 plants of Albaha region against L. amazo-
different Commiphora species was found to be rich in beta- nensis has been reported for the first time and revealed that
elemene, alpha-copaene, alpha-humulene, beta-selinene, and four extracts were found to have promising antileishmanial
germacrene B. Commiphora oil rich in alpha-humulene phytochemical constituents. In particular, the extract of A.
may be responsible for antileishmanial activity [28, 30]. biebersteinii was the most promising extract and should be
Antiplasmodial activities of C. opobalsamum and C. schimperi further investigated using bioactivity-guided isolation of the
were previously reported [31]. active constituents. Moreover, the evaluation of antileishma-
W. somnifera fruits and leaf (IC50 of 20.3 and 29.5 𝜇g/mL) nial activity confirms the ethnobotanical uses of certain plant
exhibited, in the present study, more remarkable but nonse- extracts such as myrrh oleo gum resins and S. incanum.
lective antileishmanial activity than that reported previously
and collected from India (IC50 : 63.0 𝜇g/mL) [8]. However,
similar activity with an extract collected from Oman was
Conflict of Interests
reported (IC50 : 22.1 𝜇g/mL) [32]. The difference in the activity The authors declare no conflict of interests.
may be explained in the light of the presence and/or quantities
of bioactive compounds in plants that are influenced by
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