Introduction
Cutaneous leishmaniasis is a parasitic, zoonotic disease caused by various species of Leishmania parasites. The disease has several names in different parts of the world [1, 2].
The disease could be traced in ancient times. Potteries from the prehistoric period of Indians in Ecuador and Peru indicate images of skin lesions and malformations in the human face, which are a relatively accurate indication of cutaneous and cutaneous-mucosal lesions of leishmaniasis (Chiclero’s ulcer or Aleppo boil).
Other reports suggest the familiarity of Greek and Muslim Iranian physicians with the disease [3]. Cunningham in 1885 and Frith in 1891 were the first ones who observed Leishmania tropica. In 1898, Beruschi, a Russian military surgeon, discovered the protozoan nature of the disease with the study of cutaneous ulcers in Turkmenistan. In 1900, William Leishman isolated small elliptical bodies in the spleen of a patient, which was later called Leishman [4].
World Health Organization (WHO) reports suggest that about 350 million people in 88 countries around the world are currently at risk of developing this disease. The number of people infected with the disease is now estimated to be 12 million, and about 1-2 million new cases occur annually. About 90% of cases of cutaneous leishmaniasis have been reported in Afghanistan, Brazil, Iran, Peru, Saudi Arabia, and Syria [5]. The rate of contamination in Iran is about 16.000, but its actual rate is more than three times the reported statistics [6].
In 1902, Shalgin stated that the mosquito would transmit the disease. Wright from Boston was the first to describe the parasite in 1903 when he saw it in an American soldier and named it Helcosoma tropica. In 1904, Rojers managed to culture the parasite for the first time and observed its flagellate form in a medium containing rabbit blood, which is used nowadays in most research laboratories. In 1906, Lühe described Leishmania tropica as the organism responsible for causing oriental sore. The culture medium containing rabbit blood developed by Rojers was introduced and named by Navi, Nicole, and McNeil as the N.N.N culture medium, and it is used now in most research laboratories. In 1911, another scientist, Vianna, from South America called this parasite Leishmania braziliensis. In the same year, Vianna showed that the vector of the disease is Phlebotomus papatasi, and later on, Phlebotomus seregenti. The others also confirmed that result. In 1915, Gashe, a medical professor at Dar al-Fonoun, tested 21 stray dogs in Tehran. Of those, 15 dogs had leishmaniasis [6, 7, 8].
Cutaneous leishmaniasis has long been known in Iran, and nowadays, our country is considered as one of the most important endemic areas of the disease in the world [9]. In Iran, the anthroponotic type or the urban type of cutaneous leishmaniasis has been commonly found in cities of Tehran, Shiraz, Kerman, and Bam, as well as Khorasan Province, while the rural type has been observed in provinces of Isfahan, Golestan, Khuzestan, and Ilam as well as cities of Bushehr, Semnan, and Dehloran [10].
Cutaneous leishmaniasis in Iran has been clinically observed in rural (wet wounds) and urban (dry wounds) forms. Rural cutaneous leishmaniasis is a zoonosis disease and called ZCL (Zoonotic Cutaneous Leishmaniasis). The urban cutaneous leishmaniasis is known as anthroponotic and called Anthroponotic Cutaneous Leishmaniasis (ACL). Leishmania major is the causative agent of rural cutaneous leishmaniasis, and Leishmania tropica is the cause of urban cutaneous leishmaniasis. It should be noted that the ZCL type is dominant in most parts of Iran. The recorded statistics of the patients with cutaneous form in Iran are about 20000 each year, and some believe that the actual figures are about 4 to 5 times that number, and it is one of the most important parasitic diseases in Iran after malaria [11]. In the Middle East, 14 of the 22 countries are involved with leishmaniasis. Rural focal areas of cutaneous leishmaniasis are mostly found in Afghanistan, Egypt, Iran, Iraq, Jordan, Libya, Morocco, Tunisia, Palestine, Pakistan, Saudi Arabia, Syria, and Yemen, while the urban type is more seen in Afghanistan, Iran, Iraq, Morocco, Pakistan, Saudi Arabia, Syria, and Yemen. Leishmania parasite that causes visceral leishmaniasis in Iran is either Leishmania infantum or Leishmania donovani. Visceral leishmaniasis has been one of the most important endemic diseases in the northwestern part of Iran in the last 10 years [12, 13].
In areas where endemic leishmaniasis exists, chemical and physical treatment methods, including copper sulfate, acid, water, or hot metals, have also been used. Although these methods have been invasive and the scarring from them can be worse than the sore itself, amastigotes have been proven to be sensitive to heat. Therefore, if localized heat up to 40˚C or ultrasound waves is applied to the wound, the wound healing will be expedited. It has been even mentioned in some cases that the thermal treatment has a curable effect like glucantime [8, 14].
No proper vaccine or drug to prevent the parasite and no suitable chemical or physical method to fight with its carrier have been developed so far. The appropriate treatment for leishmaniasis depends on the infecting Leishmania species and its clinical syndrome. Pentavalent antimonials, sodium stibogluconate (pentostam) and meglumine antimoniate (glucantime) have been the main treatments in recent decades- but with increasing reports on their treatment failure. Amphotericin B deoxycholate is an effective but relatively toxic drug and has not been identified as a thoroughly satisfactory treatment so far.
The use of medicinal herbs in the treatment of parasitic diseases dates back to ancient times when Cinchona succirubra (Rubiaceae) was used as an antimalarial drug [15]. The treatment of cutaneous leishmaniasis has been documented in traditional medicine in various forms, such as oral remedies, ointment, and poultice. In this medicine, herbs have also been used for their topical and systemic effects [1]. In general, the use of medicinal plants for the treatment of diseases has a long history [16]. Recent research on natural plant compounds has revealed antileishmanial effects of quinoline, alkaloids (such as cupsarin and skimmianine), isoquinoline alkaloids (such as lymacine and isotetradine), flavonoids (such as luteolin and fistin), saponins (such as alpha-hederin), naphthoquinones (such as lapachol and plumbagin), terpenes and tetralenes on some species of Leishmania [1]. This article reviews several herbs that are used to treat cutaneous leishmaniasis in Iran and some countries around the world. We hope that herbal medicines become a proper approach to prevent drug resistance and the toxic effects of antileishmanial chemical drugs.

Materials and Methods
This research is a review study. It is based on valid articles published from 1983 to 2018 in this field. The databases used for this article were PubMed, Science Direct, and Google Scholar. The qualified papers contained the history of identifying Leishmania parasites, treatment of the disease using medicinal herbs as well as physical, chemical, biological therapeutic methods with topical and systematic approaches.

Results
The treatment of cutaneous leishmaniasis has been discussed since 1911. It consists of topical or systemic approaches in four physical, chemical, biological, and immunological modes [17]. The topical treatment of cutaneous leishmaniasis is a suitable method since it is easier to apply, absorbed at the site, and without the complications of systemic drugs. Mapacrine, emetine, and berberine acid sulfate can be used for injection into the lesion, which are all chemical treatments. Among the above methods, the use of emetine is highly recommended by the WHO in endemic areas [18]. According to the findings, the protocol for the treatment of cutaneous leishmaniasis is often recommended with the following drugs: glucantime, pentamidine isethionate, amphotericin B, fluconazole, itraconazole, ketoconazole, and allopurinol [19, 20].
One of the essential issues in the treatment of this disease is the side effects of chemical drugs. For example, the side effects of sodium stibogluconate include joint pain, muscular pain, increased liver enzymes, anemia, leukopenia, thrombocytopenia, and electrocardiogram changes in inverse T and QT forms, prolonged ST, and changes in clinical function tests [20, 21]. Before 1990, amphotericin B has been rarely used due to its side effects, including renal disorders, anorexia, lethargy, fever, chills, bone pain, changes in electrolytes, and occasionally heart attacks. The drug affects the metabolism of leishmaniasis and fungi sterols. This drug attaches to the ergosterol in the Leishmania membrane, causes cell permeability, and kills the parasite. Based on the WHO recommendation, amphotericin B is used in cases where patients are resistant to antimony compounds. This drug is taken through intravenous injection. The above complications have been observed in 25% of users [22].
The chemical pentamidine medicine has fewer clinical complications than sodium gluconate, but in 30%-50% of the subjects, it leads to complications such as tachycardia, headache, vomiting, muscular pains, hypoglycemia, severe low blood pressure, sudden death, thrombocytopenia, anemia, neutropenia, and increased levels of liver enzymes [23, 24]. Many studies have shown that some natural plant compounds have antileishmanial effects against some species of Leishmania [25, 26, 27, 28, 29, 30, 31, 32]. This review article examined and discussed a number of herbs used to treat cutaneous leishmaniasis since ancient times.
Herbal treatments of cutaneous leishmaniasis in ancient Iranian medicine
Several reports of this disease in ancient texts of traditional medicine have revealed that in the time of Zakariya al-Razi, the following procedure has been used to treat the disease ulcers in the hands and feet (Table 1):


They grinded the caraway (Cuminum cyminum L.) (Mosaddegh, 2010) and separated its shell. Then, they washed the powder thoroughly with warm water and rubbed over the wound overnight. On the next morning, they put the hand or foot in hot water and washed it with water. This process was repeated several times until full recovery [3].
In Khorasan Province, the natural extract of barberry (Berberis vulgaris) (Hashemi, 2012; Maspi, 2010) plant has been used in traditional treatment. Studies on the antileishmanial effect of barberry alcoholic extract have reported a significant leishmanicidal effect. The results show a significant decrease in the size of the ulcers in mice exposed to the extract compared to the control group after two weeks of starting the treatment. The sores of the control group that had been only in contact with ethanol were reported still growing and worsening. Various alkaloids are found in the barberry plant, including berberine, jatorrhizine, columbamine, palmatein, oxyacanthine, berbamine, bervulcine, isotetradine, volrysin, and magnoflorine. Berberine effectively eliminates the Leishmania major in macrophages in the peritoneal cavity of the mice, while its topical use has induced anti-leishmaniasis effects [10, 15].
Birthwort (Aristolochia maurorum L.) has been introduced in ancient Iranian traditional medicine as a plant with a warm and dry nature. This herb has been used as an antidote for plant and animal toxins and a lethal agent for the gastric worm. The poultice of this plant has been used to neutralize the lethality of scorpion venom and insects’ bites and other wounds and used with honey to treat fresh and chronic ulcers. It was also used with Lilium and honey to fill and heal the wounds. If rubbed with oil on the body, it will kill and repel the lice. With wound disinfecting properties, this herb has been used to heal wounds of cutaneous leishmaniasis [19].
Camphor (Cinnamomum camphora) (Lamidi, 2005; Dutta, 2007) with its cool and dry nature has been the antidote for venoms of scorpions and snakes, and the healing agent for obstinate wounds. Its 1%-3% solution was used as an antipruritic remedy. In non-toxic concentrations (less than 100 µg/mL), camphor has anti-inflammatory effects. According to the research results, the anti-inflammatory effects of camphor are related to its regulating effects on cytokines, nitric oxide, prostaglandin E2 production, and oxidative stress. This material was also used to heal cutaneous leishmaniasis ulcers [2, 4].
In the past, honey (Ramezani, 2012) has been used as a therapeutic agent in the treatment of infectious ulcers [9], including oriental sores. Honey has been placed as a coating on wounds, burns, and scratches to reduce edema, inflammation, and pain. By encoding the enzymes producing hydrogen peroxide and other substances, and bees give antibacterial properties to honey [1]. In a study, honey with glucantime has been used inside the lesion to treat cutaneous leishmaniasis. Then, it was put on the wound sufficiently to exacerbate its effect. It was shown in this study that the therapeutic effect of honey when used with glucantime decreases compared with using the glucantime alone. This study revealed that honey does not have anti-inflammatory properties but provides a nutritional source for damaged cells [33].
Boswellia (Boswellia carterii) (Verma, 2004; Torres, 1999) with a warm and dry nature has been used in traditional medicine to heal and repair deep wounds, especially fresh injuries, and stop their bleeding as well as to prevent the spread of obstinate wounds. It was also used to heal cutaneous leishmaniasis ulcers. The anti-inflammatory effects of boswellia have been demonstrated both in vitro and in vivo conditions. Having triterpenoids, especially beta-boswellic acid and its derivatives, boswellia is a specific inhibitor of 5-lipoxygenase 1 enzyme and inhibits and reduces leukotrienes. By reducing the number of white blood cells in the inflammation site, it will reduce inflammatory responses and accelerate the healing process. Antibacterial, antifungal, and anti-parasitic properties of boswellia have also been proven in this study [5, 34].
Lemon (Citrus limon) (Mohseni, 2012; Dutta, 2007) with a cold and dry nature has been used in ancient Iranian medicine to treat tiny, red, and burning papules of the skin and to eliminate the pus. This plant has been applied to heal cutaneous leishmaniasis as well [1, 4]. It has anti-fungal, antibacterial, immune system regulating, and anti-larval effects, and its cytotoxic effects on cancer cells of prostate, stomach, lung, and chest have been proven [13].
Herbal remedies for cutaneous leishmaniasis in modern medicine
During an experiment, Torres Santos et al. found out that 2’,6’-dihydroxy-4’-methoxychalcone extracted from Piper aduncum has a significant effect against the promastigotes and amastigotes of the Leishmania amazonensis so that the IC50 of this substance against promastigotes was 0.5 µg/mL and against the intracellular amastigotes was 24 µg/mL [34] (Table 2).




Discussion
Hojjati et al. stated that Aloe vera has multiple uses in medicine and examined the effect of the substance secreted from Aloe vera leaves on cutaneous leishmaniasis [43]. The promastigotes of the species causing cutaneous and visceral leishmaniasis were sensitive to Aloe vera leaf, and their IC50 ranged from 100-180 µg/mL. Such data revealed that Aloe vera leaf could improve the host macrophages function through direct anti-leishmaniasis activity and may be used as an effective antileishmanial agent in pharmaceutical research [43].
Soudi et al. examined the antileishmanial effect of the root extract of Echinacea purpurea cultivated in Iran. According to their results, the root extract of this plant had an irreversible leishmanicidal effect on Leishmania major promastigotes in vitro and could be used with a concentration of 50 mg/mL in clinical studies [25].
Taran et al. studied the effectiveness of Pistacia atlantica gum topically in the treatment of cutaneous leishmaniasis in an animal model (Balb/c mice). They did not provide the dilution. Their results indicated that the gum of this tree could be used to control rural cutaneous leishmaniasis and prevent the development of the sore [44].
Grecco et al. extracted two flavonoids, sakuranetin , and naringin, from the leaves of Bacchari sretusa plant and tested them in vitro against the promastigotes of various Leishmania species [45]. In the end, the second compound acted against Leishmania amazonensis, Leishmania braziliensis, Leishmania major, and Leishmania chagasi with an IC50 value between 43 and 52 mg/mL, while the first compound showed no anti-parasitic activity in spite of having a chemical structural similarity [45]. There is an antioxidant component in this plant: 2, 2’-diphenyl-1-picrylhydrazyl [46].
Sawadogo et al. chose five plants from the central and western parts of Burkina Faso [19]. These plants were traditionally used to treat parasitic diseases and cancers. In previous studies, the antioxidant and antiproliferative activities of these plants have been examined. In this study, the alcoholic extract of these plants was evaluated to show their possible antileishmanial and antitrypanosomal activity. They used colorimetric and spectrophotometric methods and finally concluded that the extract of Lantana ukambensis had an antileishmanial activity with an IC50 of 9.6 mg/mL [19].
Feily et al. examined the green tea (Camellia sinensis) extract activity against Leishmania major promastigotes in vitro compared with glucantime, in which, the parasite promastigotes were exposed to 6 different concentrations of the extract [26]. The positive control group was treated with 85 mg/mL glucantime, and the control group received no medication. The green tea extract showed significant antileishmanial activity against parasite promastigotes in different concentrations, and with increasing the extract dose, the anti-parasitic effect increased, too. The average live promastigotes at a concentration of 12 mg/mL of green tea were approximately equal to the 85 mg/mL concentration of glucantime, and higher concentrations of green tea were more effective than glucantime. This study was merely done qualitatively by counting the number of parasites and the mortality rate of the parasites [26].
Ogeto et al. studied the antileishmanial activity of the extracts of Aloe secundiflora against Leishmania major promastigotes in vitro [47]. Among the extracts of this plant, the aqueous and methanolic extracts had the highest inhibitory effect on the growth of promastigotes. The IC50 values for the aqueous and methanolic extracts were 279.488 and 42.824 μg/mL, respectively [47].
Rodrigues et al. studied the cell-killing effects of Croton cajucara essential oil and one of its essential compounds, 7-hydroxy-calamenene, against Leishmania chagasi [48]. The minimum inhibitory concentration of parasite growth was 250 μg/mL for the essential oil and 15.6 μg/mL for the 7-hydroxy-calamenene. Transmission electron microscopy studies revealed significant nuclear and kinetoplastid changes in the promastigotes of Leishmania chagasi. The promastigotes and macrophages treated with essential oil had reduced by 52.8%, while the production of nitric oxide by parasite-infected macrophages had decreased by 80% [48].
Saleheen et al. studied the effect of the aqueous extract of Allium cepa on the promastigotes of Leishmania major, Leishmania donovani, Leishmania tropica, and Leishmania mexicana [27]. The extract concentration range tested for antileishmanial activity (0.7-10 mg/mL) was reported to be lethal for all the mentioned Leishmania species, while the concentration of 0.376 mg/mL killed, on average, 50% of all examined parasites after 72 hours. These researchers identified sulfur compounds in the plant, such as S-alkyl-L-cysteine sulfoxide, responsible for the antileishmanial effect [27].
In an experimental study, Shariatifar et al. examined the effects of Cassia fistula (known as the golden rain tree) on the promastigotes of leishmaniasis, causing agents in the culture medium [49]. To do this research, the fruit of Cassia fistula was obtained from the market and powdered. The aqueous extracts of the plant were prepared with different concentrations and added to the culture medium of the promastigotes, and their effects were evaluated. This extract was added at different concentrations on three Leishmania-inoculated culture media [49].
Counting the number of promastigotes showed that the plant extract had prevented the growth of Leishmania major in the culture medium at concentrations of 0.1, 0.2, 0.3 to 0.9 mg/mL, while the organism had proliferated in control culture media. In the next step, concentrations of 1, 2, 3 to 9 mg/mL were prepared and added to the culture medium. Again, no growth was observed in the media containing the plant extract at this stage. In the third stage, the concentrations of one-thousandth of the Cassia fistula fruit extract were studied, which revealed a small growth rate of the parasites at the concentrations of 0.001 and 0.002 mg/mL. However, no growth was seen at concentrations of 0.003 to 0.009 mg/mL.
Regarding the effect of the extract of this plant on cutaneous leishmaniasis, it was proposed that this herbal drug will be examined on laboratory mice after identifying its organic compound, and if proven to be useful, it can be used to treat cutaneous leishmaniasis in the living tissue [50].
Amanzadeh et al. studied the inhibitory effect of aqueous and alcoholic extracts of Allium stipitatum on the growth of Leishmania infantum in laboratory conditions and reported that the concentration of 0.01 to 0.1 mg/mL of this extract on the third day and the concentration of 0.2 mg/mL on the first day had inhibited the parasite’s growth [50].
Sharif et al. examined the effect of methanolic extracts of tarragon (Artemisia dracunculus) and chamomile (Matricaria chamomilla) on Leishmania major under in vitro conditions. The mentioned extracts showed significant antileishmanial activity at different concentrations [28].
Mirzaii et al. evaluated the antileishmanial activity of Esfand or wild rue, with the scientific name of Peganum harmala, on the growth of promastigotes of the Leishmania major parasite in comparison with a trivalent antimony drug, potassium antimonyl tartrate, using MTT method in vitro. Both the extract and the antimony drug inhibited the growth of the parasite in the culture media after 72 hours. In other words, the power of both factors in controlling the growth was almost equal, so that their strength had increased by increasing the concentration. The IC50 values for the extract and antimycotic drug were 1832.65±89.72 μg/mL and 17.87±2.05 μg/mL, respectively. Regarding the side effects of antimony medications, the extract of this plant can be used as a drug against Leishmania major in laboratory conditions [4].
Ahmad et al. and Garcia et al. studied the effects of Bidens pilosa and pomegranate grains (Punica granatum) against promastigotes and amastigotes of Leishmania amazonensis and measured their toxicity against Balb/c mice peritoneal macrophages [51, 52]. These extracts showed considerable growth inhibitory effect against extracellular promastigotes and amastigotes. The IC50 values of these plants against extracellular amastigotes were calculated as 42.6 and 69.6 μg/mL, respectively. The antiparasitic activity of these plants has been reported against other parasitic agents, which is due to the presence of flavonoids (catechin and epicatechin) and epigallocatechin gallate. In various studies, epigallocatechin gallate has been introduced as a potent antioxidant, an anti-cancer, and an anti-parasitic agent. Flavonol glycoside (quercine) is another antioxidant found in these plants [51, 52].
Barati et al. examined antileishmanial effects of the extracts of Thymus vulgaris, Peganum harmala, and Myrtus communis and control drug, tartar emetic, by MTT method in vitro and the results were calculated as IC50 for each extract. The IC50s of Thymus vulgaris, Peganum harmala, and Myrtus communis extracts were obtained as 7.4 μg/mL, 7.2 μg/mL, and 5.8 μg/mL, respectively. The IC50 of tartar emetic was calculated to be 4.7 μg/mL. The extract of Myrtus communis with the lowest IC50 had a better effect than other extracts. All extracts showed significant antileishmanial impact [29].
Barati et al. studied the antileishmanial effects of the extracts of Artemisia aucheri Boiss, Ferula assa-foetida L., Gossypium hirsutum, and tartar emetic control drug on Leishmania major promastigotes by MTT method in vitro and the results were calculated as IC50 for each of the extracts individually. The IC50s of the extracts of Artemisia aucheri Boiss, Ferula assa-foetida L., and Gossypium hirsutum were estimated to be 5.9, 7.5, and 3.6 μg/mL, respectively. The IC50 of tartar emetic was calculated to be 4.7 μg/mL. Although the extract of Gossypium hirsutum showed a higher effect on the promastigotes than the other two extracts, all of these extracts had antileishmanial activity [53].
Maspi et al. sought to assess the lethal effects of alcoholic and aqueous extracts of Calendula officinalis on Leishmania major promastigotes (MRHO/IR/75/ER) under laboratory conditions [15]. They reported that the mentioned extracts killed all the parasites at a concentration of 500 μg/mL, and lower concentrations showed dose-dependent antileishmanial activity. The IC50 obtained from the alcoholic and aqueous extracts after 24 hours were 170 μg/mL and 215 μg/mL, respectively [15].
Asadi et al. evaluated the effect of hydroalcoholic extracts of Hypericum perforatum and Mespilus germanica on Leishmania major promastigotes under in vitro conditions. Different concentrations of the extracts were examined on the parasites. The results indicated that the number of promastigotes decreased with increasing the concentrations of the extracts [54].
Khademvatan et al. evaluated the cytotoxic effects of different concentrations of miltefosine on the promastigotes of Leishmania major and Leishmania tropica and observed the characteristics of programmed cell death or apoptosis [55]. They used the MTT colorimetric assay to determine the survival potential of these two parasites and reported the results as IC50. Miltefosine induced death with apoptotic properties in both species, and the IC50 values of these two parasites were determined after 48 hours of incubation as 22 and 11 µmole, respectively.
In the same study, Khademvatan et al. assessed the possibility of inducing apoptosis by the extract of Allium sativus on Leishmania major promastigotes. They cultured the promastigotes of this parasite in the RPMI-1640 medium and treated them by different concentrations of garlic and calculated IC50 using the MTT method. They found that garlic had a dose-dependent cytotoxic effect, with approximately 100% death at a concentration of 93 μg/mL [55].
Shariatifar et al. examined the effect of the ethanolic extract of Mespilus germanica on the cutaneous leishmaniasis in the BALB/c mice. The extract could reduce the diameter of the ulcer and the number of Leishmania major parasites in the wound. The extract succeeded in reducing the wound diameter up to 20% in 3 mice at a concentration of 60%. At the same concentration, it had reduced the number of parasites to 66.4% in 8 mice. At 40% concentration, the number of ulcer parasites decreased by 26.7% in 4 mice and decreased by 82% in 9 mice [49].
Fani et al. studied the macrophages infected with Leishmania major exposed to the aqueous extract of garlic (Allium sativum), after incubation time and performing MTT test. They found that the garlic extract had macrophages destroyed promastigotes at a garlic dose of 37 μg/mL for 48 hours [30].
Barazesh et al. evaluated the antileishmanial activity of curcumin and its derivatives (Curcumin, indium, Gallium curcuma, and Diacetyl curcumin) against Leishmania major promastigotes by MTT method in vitro [56]. Curcumin is an active ingredient in plant therapy and is responsible for many biological effects of Curcuma longa. It has potent antioxidant, anti-inflammatory, and anti-cancer properties. The IC50 values for Gallium curcuma, and Diacetyl curcumin, and amphotericin B (control drug) were calculated as 38, 32, 26, 52, and 20 μg/mL, respectively. Having lower IC50s than the Diacetyl curcumin analog, Gallium curcumin and Curcumin idinum were stronger agents against Leishmania major promastigotes [56].
Soozangar et al. studied the effects of Achillea millefolium on Leishmania major in vitro. All concentrations of this extract reduced the number of Leishmania parasites. They concluded that the extract of this plant could be used in further studies on Leishmania parasites and in developing herbal medicines [57].
Yektaian et al. designed and experimented to investigate the effect of hydroalcoholic extract of three herbs of Achillea millefolium, Artemisia absinthium L., and walnut (Juglans regia L.) leaves and evaluate the synergism effect of the three mentioned extracts on Leishmania major parasites in vitro [58]. Based on the study objective, the extracts of all three plants were prepared at concentrations of 25 μg/mL. These concentrations were then injected into the parasite containing media in a PBS buffer solvent plus 2% DMSO and were examined and analyzed at intervals of 0, 6, 24, 48, and 72 hours in terms of the number of parasites. It was found that the extracts of plants increased the immobilization of parasites. This lack of mobility has a direct relation with time. For example, glucantime after 24 hours and amphotericin B after 30 minutes destroyed the parasites compared with the effect of plant extracts after 24 hours. The results of this study showed that the combination of the three plants’ extracts has been effective on Leishmania parasite. However, further studies are needed to demonstrate this effect in laboratory animals and volunteer patients [59].
Naserifar et al. studied the effects of various concentrations of the aqueous extract of Scophularia straiata on the growth of Leishmania major in BALB/c mice peritoneal macrophages under in vitro conditions. The aqueous extract of this plant had a proper antileishmanial activity in eliminating this parasite in the macrophage and the culture medium. At the concentration of 25 μg/mL and on the third day, it removed the amastigotes within the macrophages, and it killed the promastigotes in the RPMI-1640 medium at the same concentration on the third day [16].
Artemisinin is one of the most prominent antileishmanial drugs. Artemisinin is a terpene lactone isolated from Artemisia annua, which is known as an anti-malarial and antileishmanial drug. In vitro, this drug had stopped the activity of the Leishmania major, strain Friedlin, changing the metabolites of various metabolic cycles, including galactose metabolism pathway, sphingolipid biosynthesis pathway as well as the biosynthesis pathways of valine, leucine, and isoleucine [60].
Yousefi et al. and Samarqandian and Borji studied the effects of Crocus sativus and its apoptotic activity against Leishmania major promastigotes in vitro. The chemical analysis of saffron extract showed that this plant contained antioxidant compounds such as anthocyanin, lycopene, flavonoids, carotenoids (crocin and crocetin), picocrocin, and safranal. The IC50 of saffron was 0.7 mg/mL after 48 hours of incubation, which indicated the high activity of this plant against the promastigotes of Leishmania major parasites [60, 61].
In an experimental study, Banyadian et al. first prepared the essential oil of the fresh plant of Lavendula officinalis, which was dried and powdered by the steam distillation method [31]. Then, the concentrations of 0.5%, 1.5%, 5%, 10%, 15%, and 20% of the essential oil and a concentration of 33.8% of the glucantime were separately added to the culture medium containing promastigotes of Leishmania major (106 parasites/mL). The number of live parasites after adding the essential oil and the drug were counted after 24, 48, and 72 hours by 10% trypan blue dye using the Neubauer chamber. The results showed that the promastigotes propagation rate significantly decreased after adding the plant essential oil compared with the control group. The lethal effect was also observed at a concentration of 10% and higher so that no alive parasites were seen in the groups after 72 hours. The results of this study indicated the inhibitory growth effect and the lethal impact of essential oil of lavender plant in laboratory conditions on the promastigote form of Leishmania major. Therefore, further studies are recommended to discover the antileishmanial effect of this plant on the amastigote form of the parasite .
Gharirvand-Eskandari and Doudi evaluated and analyzed the antileishmanial effects of the extract and essential oil of Medicago lupulina leaves on Leishmania major promastigotes (clinical isolate) by MTT method and reported that the IC50 of glucantime was equal to 19 μg/mL after 48 hours. The IC50 values for alcoholic extract and essential oil of this plant after 48 hours were equal to 130 and 340 μg/mL, respectively [62].
In another study, Gharirvand-Eskandari and Doudi examined the antileishmanial effect of alcoholic extract of Medicago lupulina leaves on the promastigotes of Leishmania major, the standard strain (MRHO/IR/75/ER) in vitro and obtained its IC50 after 48 hours as 98 μg/mL. The IC50 for glucantime was obtained 12 μg/mL in this study after 48 hours [62].
The results of Shahanipoor et al revealed that the glucose oxidase enzyme, together with ethanolic extract of propolis, not only had antimicrobial activity against gram-positive and gram-negative bacteria isolated from cutaneous leishmaniasis ulcers but also managed to heal the wounds [63].
The results of Eskandari et al. research in 2016 suggested that the extract of Portulaca oleracea L. was effective on the promastigotes of Leishmania major (clinical isolates). Using the MTT standard method, the IC50s for the alcoholic extract and essential oil of the plant leaves were obtained as 360 and 680 μg/mL, respectively, after 48 hours, while these values for glucantime were respectively 12 and 19 μg/mL after 48 hours [64].
The results of Shirazi and Doudi research demonstrated that applying the alcoholic extract of Crataegus microphylla leaves on the promastigotes of Leishmania major parasite (MRHO/IR/75/ER) by MTT method provided the glucantime IC50 value of 18.616 μg/mL, while the IC50 for alcoholic extract was obtained as 1094 μg/mL. Meanwhile, the results of the alcoholic extract of Cichorium intybus leaves revealed an IC50 of 1094 μg/mL [65].
The results of Albakhit et al. research showed that the IC50 of glucantime on the promastigotes of Leishmania major was equal to 21.96 μg/mL, while the IC50s of methanolic extracts of the kernel and date fruit of Phoenix dactylifera L. were respectively 23 μg/mL and 500 μg/mL. Also, the IC50s for the alcoholic and aqueous extracts of Ziziphus spina-christi were obtained as 60 and 80 μg/mL, respectively. Although glucantime was more effective than the extracts of the plants mentioned above, all extracts had significant effects on the promastigotes of Leishmania major [66].
Maroufi et al. investigated the effect of Juice (Crataegus aronia) on Leishmania major in vitro. The exposure was limited to 24, 48, and 72 hours by arranging (0/5-60 µg/mL), (20/94-20/11 µg/mL) and (55/14-35/19 µg mL) [67].

Conclusion
Medicinal plants have been the focus of attention since ancient times. The chemical drugs have many clinical complications, so medicinal plants can be considered as an alternative to synthetic drugs.

Ethical Considerations
Compliance with ethical guidelines
The study was reviewed by the Research Committee of Islamic Azad University of Falavarjan.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions
Conceptualization and writing-original draft preparation: Monir Doudi; Data curation and validation: Elham Gharirvand Eskandari; Data analysis: Mahbubeh Setork; Visualization, supervision, project administration: All authors.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The researchers of this study would like to thank the Deputy Director of Research and Technology at the Islamic Azad University of Falavarjan.


References