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Leishmaniasis is a major public health problem worldwide. The aim of the present study was to investigate medicinal plants with anti-Leishmania activity which used in Iran.
Data were systematically gathered from five English databases including Ebsco, Science Direct, PubMed, Google Scholar and Scopus, four Persian databases including Magiran, Iran doc, Iran medex and the Scientific Information Database (SID) from 1999 to April 2015. Information obtained included plant family, extraction method, concentrations of extracts, animal models and parasite strains.
A total of 68 articles including 188 experiments (140 in vitro and 48 in vivo) between 1999 and 2015, met our eligibility criteria. Thoroughly, 98 types of plants were examined against three genera of Leishmania spp. For the heterogeneity study conducted, it was showed that there was a great deal of variation among studies. Based on random effect, meta-analysis pooled mean of IC50 was obtained 456.64 (95% CI: 396.15, 517.12).
The most Iranian plants used as anti-leishmanial activity were Artemisia species, Allium sativum, Achilleamille folium, Peganum harmala and Thymus vulgaris. The present systematic and meta-analysis review provide valuable information about natural products with anti-Leishmania activity, which would be examined in the future experimental and clinical trials and herbal combination therapy.
Leishmaniasis is a parasitic disease caused by an obligate intracellular parasite of genus Leishmania, which is transmitted to human by the bite of a female sand fly . The disease has wide clinical spectrums from self-limiting cutaneous to fatal visceral form which depends on both host immune response and the species of Leishmania parasite. The World Health Organization (WHO) emphasizes on leishmaniasis as one of the seven important infections . Approximately, 350 million people in 98 countries are at the risk of infection. It is estimated that 12 million people are affected with the disease and about 1.5 million new cases of cutaneous leishmaniasis (CL) are reported annually. Approximately, 90% of the CL cases occur in eight countries of Afghanistan, Saudi Arabia, Syria, Iran, Algeria, Iraq, Brazil and Peru , . Pentavalent antimony is conventionally used from 1959 for leishmaniasis but it is toxic with side effects, which requires prolong injections. The efficacy of pentavalents has been decreased and the emergence of resistance limits their usage , . The first line drugs in leishmaniasis including meglumine antimoniate (Glucantime), pentamidine (Pentacarinat), and sodium stibogluconate (Pentostam) are not effective orally and require prolonged injections. The second line drugs such as amphotricine B and pentamidine are very toxic . In the absence of an effective vaccine, there is an urgent need for new and more effective drugs to replace or supplement those in current use. Plant derivatives or plant extracts are likely to provide a valuable source of new medicinal agents. The urgent need for substituting treatments has led to a program for screening natural products in leishmaniasis. Actually, the WHO recommended the use of traditional medicine in societies with poor health services. Moreover, the data obtained from reviewing would lead to the emergence of natural products with anti-leishmanial activity and would be the way for the production of new effective synthetic compounds. It has been estimated that there are about 250,000 medicinal plant species in the world. Nevertheless, the biological activities of only about 6% of them have been evaluated. Besides, only around less than 1% of medicinal plant compounds have been assessed in clinical trials , .
About 35% of approved drugs belong to natural products or semi synthetic derivatives, while 30% are synthetic molecules based on natural products or pharmacophore developed from natural compounds. It is noteworthy, out of 15 antiparasitic medications that have been approved by health authorities between January 1981 and June 2006, 65% are natural products or derivatives .
Medicinal plants are an effective source of pharmaceutical products in Iran , . A critical evaluation of the clinical data due to the adverse effects has shown that herbal medicine is generally accepted better than synthetic medications. However, potentially, serious adverse events including herbal drug interactions have been described. This suggests the need to be attentive when using herbal therapies, mainly in specific situations such as throughout pregnancy and in the children age group . About 820 forms of herbal drugs are produced in Iran , . However, in different cultures and countries, indigenous medicinal plants are used to treat parasitic diseases such as leishmaniasis. Hence, clinical trials and empirical studies have been carried out about medicinal plants in different parts of the world especially in Asian countries including Iran , . Our study attempts to provide an overview on the native medicinal plants, which was investigated against Leishmania parasite in Iran.
An exclusive search was performed through all scientific databases from April 1999 to August 2015 including five English databases (Science Direct, Scopus, Ebsco, Pub Med and Google Scholar), four Persian databases (Iran medex, Magiran, Iran doc) and the Scientific Information Database (SID). All articles which related to the medicinal plants and leishmaniasis were chosen (Fig. 1). Additionally, reference lists of all articles were reviewed for prevention of missing relevant data. The search terms were: “ Leishmania,” “plant extract,” “herbal extract,” “medicinal plants,” “traditional medicine,” and “ herbal medicine“ alone or in combination together. Furthermore, the synonyms of herbal medicines were considered as follow: herbal preparations, herbal medications, herbal products, herbal remedies, medicinal herbs and phytopharmaceuticals. Other relevant topics such as Leishmania parasite were also reviewed and included if the appropriate outcomes were retrieved. The search was performed both in English and Persian languages.
Papers selected for inclusion were studied carefully; repetitive papers, studies out of Iran and papers with poor methodology were excluded. (See Fig. 1). The following information was extracted: the year of publication, the first author, parasite species, herbal plant, type of extract, part of plant used for extraction, concentrations, exposure time, animal models, diameter of lesions and outcomes. Two reviewers independently screened studies identified for inclusion and determined study eligibility (Kapp index showed an agreement 89% between two reviewers). Disagreements were resolved by the third opinion.
In this meta-analysis, the mean and 95% confidence intervals of the half-maximal inhibitory concentration (IC50) values were calculated for each individual study in order to estimate the pooled mean of herbal extract effect on Leishmania spp. in Iran. The results were reported using a random-effect model with 95% confidence interval (CI). Heterogeneity among studies was evaluated by the Q-Cochran test (p < 0.1 indicate heterogeneity) and I-square statistic [low (25%–49%), moderate (50%–74%) and high (≥75%) . Subgroup analysis was performed to investigate potential sources of heterogeneity . Publication bias was evaluated using the funnel plots and Egger test . Statistical software Stata 11 (Stata Corp, College Station, TX, USA) was used to data analysis.
Out of 7500 articles of literature searched from 1999 to 2015, 68 articles with 188 experiments (140 in vitro and 48 in vivo), met our eligibility criteria and included the current systematic review and meta-analysis. Unpublished data, duplicated papers, congresses proceeding abstracts were excluded from our systematic review and meta-analysis. Totally, data extracted comprised of 98 types of plants, their families, extraction methods, animal models IC50 and Leishmania species. In the in vitro studies, all of plant extracts were tested on three genera of Iranian strains of Leishmania spp including L. tropica, L. major and L. infantom. In the in vivo studies, most of L. major strains were including (MHOM/64/IR/ER75), (MRHO/IR/75/ER), (MRHO/SU/59/P), (MRHO/IR/76/ER) and also most L. tropica strains were (MHOM/IR/2002/Mash2) and (MHOM/TN/80/IPI1), which were maintained in Balb/c mice and human. Therefore, we attempt to summarize a list of various studies in the list of herbs and natural products with antileishmanial activity (Table 1, Table 2). Briefly, most leishmanicidal agents studied from natural sources in Iran were Artemisia species, Allium sativum, Achillea millefolium (Yarrow), Peganum harmala and Thymus vulgaris. Heterogeneity test was conducted and Q statistic was very large (Q = 1945, df = 61, I-square = 100%, p < 0.001), showing that there was a great variation among studies. Based on random effect model, the pooled mean of IC 50 was obtained 456.64 (95% CI: 396.15, 517.12). Begg's test showed that there was no publication bias among all studies (t = 1.25, p = 0.215). Subgroup meta-analysis of characters such as stem bark, preparation, family and botanical name was carried out. The results showed that IC50 was significantly different among the parts used (or stem bark) (p < 0.001) and the “Aerial” and “Leaves or twigs” parts were most parts used. Also subgroup analysis revealed that there was a significant difference in preparing characters including: hexane, dichloromethane, hydroalcoholic, ethyl acetate with higher IC50 values and aqueous or methanolic with lower IC50 values (p < 0.001) (Table 3). The IC50 values for Allium spp, Alkanna spp and Artemisia spp showed the significant difference (p < 0.001), so that it could be with the highest value for Allium spp. For details on the models or mechanism-based bioassays utilized for selecting crude plant extracts, fractions and pure compounds against the Leishmania parasite, the original references should be consulted (Fig. 2).
Leishmaniasis is an important parasitic disease all around the world. For reducing the resistance in endemic areas, alternative strategies including the use of herbal plants are considered , . The present study showed a wide range of plant extracts with antileishmanial properties in vitro and in vivo experiments. Among all medicinal plants, the genus Artemisia (Astraceae) is a large, heterogeneous, and widely dispersed genus all over the world. These species are small shrubs biennial and perennial or annual herbs. The genus Artemisia has 30 species in Iran out of which two are endemic . Artemisia plants contain chemical components such as sesquiterpenes, monoterpenes lactones, flavonoides, coumarins, sterols and polyacetylenes . Artemisis species has cytotoxic and anti-inflammatory activity , , . The results of a study carried out by Niloofarzadeh et al. (2008), showed that hydroalcoholic extracts of propolis Thymus vulgaris and Achillea millefolium were significantly more effective than systemic glucantime or alcoholic extract for the treatment of dermal leishmaniasis in Balb/c mice. The highest efficacy was observed for propolis, followed by Achillea millefolium and then Thymus vulgaris . The efficacy of ethanol extract of the root leaves and stem of Berberis vulgaris were topically used on experimental dermal lesions of Balb/c mice. The result after two weeks statistically revealed a significant reduction of ulcer size in mice . Doroodgar et al. (2008) reported the effect of various concentrations of Artemisia essence in Balb/c mice. They showed that cutaneous lesions in mice inoculated by L. major were enlarged after the application of higher concentration of the Artemisia essence. As a result, the lesions did not heal, and their size increased. In addition, parasitologic examination also remained positive . The result showed that the size of lesion in mice received 40, 60, and 80% of Rubia. tinctorum extracts revealed no significant difference in comparison with the lesion size in control group . Seidlitzia rosmarinus (S. rosmarinus) has been traditionally used in Mashhad and its suburbs for the treatment of CL. Despite little available data about the possible efficacy of this plant against leishmaniasis, the efficacy of herbal extracts of S. rosmarinus against cutaneous leishmaniasis in Balb/c mice was examined in this study. The natives in Khorasan Province used pure dried leaves' powder of S rosmarinus leaves on their cutaneous lesions. Therefore, alcoholic extract of stem and leaves, which is almost similar to the pure powder, was used in this study. In this study, Eucerine was used as a base for the extracts; however, the results could be different if the researcher used vaseline or lanoline as a base for transdermal delivery of herbal extract . The ulcer size in Balb/c mice received Eucerine alone was significantly increased more than other groups which approved the previous suggestion.
The administration form of a drug is also important. In the present study, the extracts were topically used as an ointment, but the results would be different if the extracts were administered intralesionaly. Recent studies have shown that nanoparticles of anti-leishmanial drugs are highly effective to treat CL. The important advantages of such drugs are low dosage and minimum adverse reactions .
In the present systematic review and meta-analysis, the Begg's test showed no publication bias among all studies (t = 1.25, p = 0.215). In addition, subgroup analysis revealed that there was a significant difference in extracting preparation including hexane, dichloromethane, hydroalcoholic and ethyl acetate with higher IC50 values, and aqueous or methanolic with lower IC50 values. (p < 0.001) (Table 3).
However, several studies have demonstrated that the hexane and ether acetate extracts present low or no toxicity to host cells at the effective concentrations , . Ribeiro et al. (2014) evaluated anti-leishmania activity of 44 extracts and fractions derived from 16 Brazilian plant species against L. amazonensis. Among them, the most potent extracts were the hexanic extract .
In general, the ethanolic extracts were less effective and more toxic than the hexanoic extracts and buthanolic, dichloromethane ethyl acetate and hexanic fractions in the mammalian cells . Thus, the application of the hexanic extract against Leishmania parasites as a potent fraction is recommended in the in vivo experiments.
In the study of Hooshyar et al. (2014), a significant decrease was shown in the main lesion size, or the small lesions were not completely disappeared after treatment by Hedera helix (H.helix) alcoholic extract. Their results disagreed with those of Talari et al. who used, 100 and 50 mg/mL of H. helix extract and observed that all promastigotes of L. major were killed in vitro . This difference of findings may be due to different preparation methods and concentration of the plant extract was used in two studies. Different extract was gathered from eleven Iranian Artemisia species. Their leishmanicidal activities against the growth of L. major showed that ethanol extracts especially those taken from A. ciniformis, A. santolina and A. kulbadica had the strongest effects . In the present study, they demonstrated the inhibitory effect of different extracts from eleven Artemisia species on the growth of L. major promastigotes in vitro. It was previously reported that the aqueous extract and essential oil of A. herbaalba had antileishmanial activity against L. tropica and L. major promastigotes . In addition, the aqueous extract of leaves of A. indica exhibited leishmanicidal activity (IC50 = 430 μg/mL) . Here, some of tested Artemisia spp showed most strong antileishmanial activities. In this study, all tested extracts exhibited antileishmanial activity after incubation, however, ethanol extracts from A. ulbadica and A. ciniformis showed the stronger leishmanicidal activity at value of (IC50 = 25 μg/mL). Growth inhibitory effect of ethanol extract of other plants such as Haplo phylum myrtifolium against L. tropica promastigotes were previously reported (IC50 = 10.9 μg/mL) . Comparing the antileishmanial effect of non-polar extracts revealed that ethyl acetate extract of A. fragrans had less antileishmanial activity against L. major promastigotes. Ethyl acetate extracts of studied Artemisia species (except for A. turanica and A. fragrans) were also more active in comparison with their dichloromethane extract. In vitro antileishmanial activity of ethyl acetate and dichloromethane extracts of Ircinia spinosula (IC50 = 16.09, 47.38 μg/mL) were reported against L. major promastigotes . The lethal dose (LD50) of dichloromethane extract and hexane extract of Calophyllum brasiliense on L. amazonensis promastigotes were 40 mg/mL and 20 mg/mL, respectively . In comparison with other extracts, Artemisia studying species hexane extracts (except for A. fragrans) were less active than L. major. Hexane extracts of A. biennis, A. annua, A. turanica, A. fragrans and A. absinthium were less effective than other species. Other investigators have also reported lower activity of hexane extracts of plants than Leishmania species in comparison with other extracts. For example, ethanol extracts of Arbutus unedo significantly decreased L. tropica promastigotes counts .
Leishmanicidal activity of Allium sativum (garlic extract) has been established against infection with L. major, so that it can induce a Th1-type response, stimulate INF-γ and NO production in macrophage and thus prevent the progression of the infection , . To improve the therapeutic efficacy and reduce toxicity, above mentioned natural molecules can be applied as either scaffold for producing and exploring new immune drugs or natural immunomodulators in synergy and in combination with existing drugs , . Targeting anti-leishmanial drugs to macrophages with drug delivery systems reflects a hopeful strategy overcoming the problems associated with the current treatment protocols.
Another important issue is the safety of natural remedies. Although natural immune therapy in different generations has been tested and approved, it is necessary to prove the overall pharmacological safety of the correction. Chemical agents in Iranian drug market have disadvantages such as high cost and side effects. Considering the effectiveness of these plants would make them as a source of natural and safe agents for the treatment of leishmaniasis. However, anti -leishmanial drugs or natural compounds are safe when their selectivity index is more than 10 .
In conclusion, the present review showed that a range of plant extracts had effects on promastigote stage of Leishmania and interesting antileishmanial properties exhibited in vitro and in vivo. Therefore, it might be possible to use the extracts instead of chemical drugs. However, almost all of the authors claimed successful results about their investigated plants, but their studies really had limitations which affected with the accuracy of their results. Some of defects included in these studies are described in detail as lacking of randomized double blind clinical trials in all of human based studies. Also some of investigations were performed in vitro and were not performed in vivo , . The period of exposure of extracts was not enough in some of the studies  and at last in one study, the toxicity level of the plant investigated was very high for testing in volunteer patients . Most of data published were obtained from animal model and were not tested on human . According to all documented data, phytotherapy has provided a large and hopeful vision to new, safe, and effective leishmaniacidal agents. Nevertheless, it needs to generalize all results obtained from in vitro and in vivo studies on the efficacy of plant extracts, metabolites or formulations against different Leishmania species to validate their activities. We concluded that the mechanism of action was enhancing the hosts' cellular immunity.
The present systematic investigation on anti-leishmanial activity of the medicinal plants together with their toxicity, mechanism of action and chemical properties for improvement is the most favorable formulation urgently required to confirm their efficacy in the treatment of leishmaniasis.
As a whole, the present systematic review provide valuable information about the natural products with anti -leishmanial activity which would be very favorable for experimental and clinical trials and herbal combination therapy studies. Consequently, further clinical researches are needed to establish the effective and safe medicinal plants therapy. It is necessary to find their active components, and potential toxic effects would lead to producing the well-tolerated and safe drugs for leishmaniasis.
This project was funded by Mazandaran University of Medical Sciences (no:2025).
There has been no financial support for this work that could have influenced its outcome.
Study concept: Mahdi Fakhar.
Data collection: Masoud Soosaraei.
Data interpretation: Masoud Soosaraei, Mahdi Fakhar, Saeed HosseiniTeshnizi, Hajar Ziaei Hezarjaribi.
Writing the paper: Masoud Soosaraei, Mahdi Fakhar.
All authors declare that they have no conflicts of interest.
Research registry 2025.
The authors wish to acknowledge all researchers that their publications were used in our review. This project was funded by Mazandaran University of Medical Sciences (no:2025).