https://doi.org/10.5812/jjnpp.63754

In Vitro Antileishmanial Activity of Falcaria vulgaris Fractions on Leishmania major

authors:

avatar Abbas Ali Eskandarian 1 , avatar Hamed Jafari 2 , avatar Gholamreza Asghari 2 , avatar Mohammad Ali Mohaghegh 3 , avatar Mustafa Ghanadian 4 , avatar Hosein Ali Yousefi 1 , avatar Roghiyeh Faridnia 5 , avatar Hamed Kalani 1 , *

Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran
Department of Pharmacognosy, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, IR Iran
Department of Laboratory Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, IR Iran
Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, IR Iran
Student Research Committee, Department of Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, IR Iran
Jundishapur Journal of Natural Pharmaceutical Products: Vol.12, issue 3; e63754
published online: August 31, 2017
article type: Research Article
received: November 23, 2015
revised: December 20, 2016
accepted: January 7, 2017

How To Cite Eskandarian A A, Jafari H, Asghari G, Mohaghegh M A, Ghanadian M, et al. In Vitro Antileishmanial Activity of Falcaria vulgaris Fractions on Leishmania major. Jundishapur J Nat Pharm Prod. 2017;12(3):e63754. https://doi.org/10.5812/jjnpp.63754.

Abstract

Background:

In recent years, many reports have been published regarding the increasing frequency of drug resistance in Leishmania species to antimonial compounds. Accordingly, many researchers have tried to develop new medicines for the treatment of cutaneous leishmaniasis.

Objectives:

The aim of this study was to evaluate the in vitro antileishmanial activity of aqueous, ethyl acetate, and butanol fractions of Falcaria vulgaris against Leishmania major promastigotes.

Methods:

The total extract was obtained using the percolation method. Subsequently, chlorophyll and oil were eliminated from the total extract, using medium pressure liquid chromatography (MPLC), followed by liquid-liquid extraction (LLE) for fractionation of the total extract. The antileishmanial activity of each fraction was examined at 6 concentrations (40, 80, 160, 320, 640, and 1280 µg/mL) and 4 intervals (6, 24, 48, and 72 hours).

Results:

The results of repeated measures ANOVA showed P values of 0.16, 0.22, and 0.04 for the aqueous, ethyl acetate, and butanol fractions, respectively. Furthermore, one-way ANOVA test revealed a significant difference between these fractions at 48 and 72 hours (P = 0.04 for both). In addition, Tukey’s HSD test showed a significant difference between the butanol fraction and other fractions at 48 and 72 hours (P < 0.05). The greatest antileishmanial effect was observed in the butanol fraction with a half-maximal inhibitory concentration (IC50) of 343.8 µg/mL at 48 hours.

Conclusions:

As Falcaria vulgaris showed acceptable antileishmanial activity with no side effects in this study, it can be used as an effective complementary or alternative treatment for skin lesions of cutaneous leishmaniasis. However, further in vivo studies are required in the future.

1. Background

Cutaneous leishmaniasis (CL) is an important zoonotic disease worldwide (1, 2). Currently, pentavalent antimonials, such as meglumine antimoniate (Glucantime®), are used as first-line treatment for CL (3). However, these medicines pose some health risks due to their inherent toxicity (4), especially when systemic therapy is necessary for CL (5).

In recent years, many reports have been published regarding the increasing frequency of drug resistance in Leishmania species to antimonial compounds (6). Accordingly, many researchers have tried to develop new agents for the treatment of CL (7). One of the major approaches to the treatment of various types of leishmaniasis is the use of medicinal plants (8). The present study aimed to examine the effects of various fractions of Falcaria vulgaris on Leishmania major.

The genus Falcaria, which belongs to the family Apiaceae, is an herbaceous plant with 2 species endemic to Iran, i.e., F. vulgaris and F. falcarides. Spathulenol and carvacrol are 2 essential oils found at high concentrations in F. vulgaris extracts. Additionally, germacrene B is the most frequently found essential oil in F. falcarioides extracts (9). A previous study revealed that some essential oil compounds of this plant have antileishmanial activities (10).

The use of F. vulgaris in the treatment of skin wounds dates back to several decades ago. In a study conducted by Khazaei and Salehi, the effects of F. vulgaris extract on gastric ulcer were evaluated in animal models; the results showed that the ethanolic extract could significantly reduce the ulcer index (11). Likewise, another study confirmed the valuable effect of this plant on gastric ulcers (12). The therapeutic effects of F. vulgaris on skin wounds have been also discussed in the literature (13). In this regard, a previous study confirmed the antibacterial effects of this plant (14). However, no study has yet examined the effects of F. vulgaris extract on L. major, as the causative agent of skin wounds.

2. Objectives

We aimed to evaluate the in vitro antileishmanial activities of aqueous, ethyl acetate, and butanol fractions of F. vulgaris on L. major promastigotes.

3. Methods

3.1. Parasites

The standard strain of L. major (MRHO/IR/75/ER) was provided by the department of parasitology, Isfahan University of Medical Sciences, Isfahan, Iran. The parasites were maintained under laboratory conditions, allowing the repeated passage of BALB/c mice. Amastigotes were isolated from the infected mice and transformed into promastigotes in Novy-MacNeal-Nicolle (NNN) medium. The promastigotes, obtained from the third passage in the NNN medium, were gradually added to RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 µg/mL of streptomycin, and 100 IU/mL of penicillin at 25°C.

3.2. Plants

The aerial parts of F. vulgaris were collected in the spring from Khomein, Markazi Province, Iran. Subsequently, the collected plants were authenticated at the department of pharmacognosy, Isfahan University of Medical Sciences, Isfahan, Iran and stored in the herbarium (No., 2955).

3.3. Total Extract

The collected plants were left in darkness at room temperature to dry and were then ground to pass through a sieve. The powder was extracted using the percolation method (15), and the total extract was concentrated by a rotary evaporator.

3.4. Chlorophyll and Oil Elimination

Chlorophyll and oil were eliminated from the total extract via medium pressure liquid chromatography (MPLC), using petroleum ether and acetone (16).

3.5. Total Extract Fractionation

Fractionation was performed using the liquid-liquid extraction (LLE) method (17). Equal volumes of ethyl acetate and distilled water were mixed. The total extract was added to a separatory funnel, stirred vigorously, and allowed to form 2 separate phases. Subsequently, ethyl acetate was separated from the aqueous phase. Afterwards, butanol was added to the remaining aqueous phase at the same volume as the aqueous phase, stirred vigorously, and allowed to form 2 phases; then, the butanol phase was separated from the aqueous phase; the remaining aqueous phase was also collected. The fractions were lyophilized and kept at -20°C until further use.

3.6. Experimentation

Initially, butanol and ethyl acetate fractions were dissolved in isotonic saline, containing dimethyl sulfoxide (DMSO); it should be noted that the final volume of DMSO in the testing medium was approximately 0.2%. DMSO was not added to the aqueous fraction. Each fraction was studied at 6 concentrations (40, 80, 160, 320, 640, and 1280 µg/mL) in the RPMI medium, supplemented with FBS (10%), 100 µg/mL of streptomycin, 100 IU/mL of penicillin, and 1 × 106 exponentially growing promastigotes.

The single-blind experiments were performed in triplicate for each concentration at each interval (6, 24, 48, and 72 hours). It should be noted that a positive control containing amphotericin B (1 µg/mL) and the parasite, as well as 2 negative controls (1 with 0.2% DMSO and parasite and 1 with only the medium and parasite), was considered each time.

The negative control with DMSO was used for the ethyl acetate and butanol fractions, while the control without DMSO was applied for the aqueous fraction. At each interval (6, 24, 48, and 72 hours), 10 µL of each sample was gently mixed with 10 µL of 0.4% trypan blue on a glass slide. The mortality of parasites was determined by counting the number of alive (not stained) and dead (stained) promastigotes according to the following formula (1).

Equation 1.

Accordingly, at least 50 microscopic fields were examined for each sample.

3.7. Data Analysis

The normal distribution of data was evaluated using nonparametric Kolmogorov-Smirnov test. Interactions between concentration and time were separately analyzed by repeated measures analysis of variance (ANOVA) for each fraction. The groups were compared using one-way ANOVA test, followed by Tukey’s honest significant difference (HSD) test at P value below 0.05. The Microsoft Excel software (Microsoft Inc.) was used to determine the lethal dose 50 (LD50) by plotting the percentage of death versus different concentrations of each fraction at a given time.

Considering the nonlinearity of the effect of each fraction at different concentrations, type II regression analysis was performed; by putting Y = 50 in the equation, X values were calculated. The half-maximal inhibitory concentration (IC50) was calculated for only the butanol fraction at a concentration of 1280 µg/mL within 48- and 72-hour intervals, which produced the greatest effects, respectively. The statistical analyses were performed using SPSS version 16.

4. Results

The results of repeated measures ANOVA showed no significant differences for the aqueous (P = 0.16) and ethyl acetate (P = 0.22) fractions, whereas a significant difference was observed for the butanol fraction (P = 0.04). One-way ANOVA revealed a significant difference among fractions at 48 and 72 hours (P = 0.04 for both). Moreover, Tukey’s HSD test showed a significant difference between the butanol fraction and the other 2 fractions (P < 0.05) at 48 and 72 hours.

In addition, the IC50 values for the butanol fraction were 343.8 and 639.1 µg/mL at 48 and 72 hours, respectively. The data regarding the antileishmanial effects of fractions are presented in Figures 1 - 3. Moreover, the effects of F. vulgaris fractions on L. major promastigotes at 48 hours are compared in Figure 4. The butanol extract showed maximum antileishmanial effects in comparison with other fractions.

The Effects of Different Concentrations of Aqueous Fraction of F. Vulgaris on Leishmania Major at Different Intervals
The Effects of Different Concentrations of Aqueous Fraction of F. Vulgaris on Leishmania Major at Different Intervals
Figure 1.

The Effects of Different Concentrations of Aqueous Fraction of F. Vulgaris on Leishmania Major at Different Intervals

The Effects of Different Concentrations of Ethyl Acetate Fraction of F. Vulgaris on Leishmania Major at Different Intervals
The Effects of Different Concentrations of Ethyl Acetate Fraction of F. Vulgaris on Leishmania Major at Different Intervals
Figure 2.

The Effects of Different Concentrations of Ethyl Acetate Fraction of F. Vulgaris on Leishmania Major at Different Intervals

The Effects of Different Concentrations of Butanol Fraction of F. Vulgaris on Leishmania Major at Different Intervals
The Effects of Different Concentrations of Butanol Fraction of F. Vulgaris on Leishmania Major at Different Intervals
Figure 3.

The Effects of Different Concentrations of Butanol Fraction of F. Vulgaris on Leishmania Major at Different Intervals

The Effects of Different Concentrations of F. Vulgaris Fractions on Leishmania Major at 48 Hours
The Effects of Different Concentrations of F. Vulgaris Fractions on Leishmania Major at 48 Hours
Figure 4.

The Effects of Different Concentrations of F. Vulgaris Fractions on Leishmania Major at 48 Hours

5. Discussion

Considering its efficacy in the treatment of wounds, F. vulgaris was examined in the present study (18). Previous studies have shown that beta-caryophyllene, alpha-pinene, and limonene are compounds with antileishmanial activities (19-21). In this regard, one previous study showed that F. vulgaris is rich in these compounds (10). In addition, saponin is another compound in this plant with antileishmanial effects (22, 23).

According to some research, a few plants show mild to strong activities against Leishmania species (24-27). Asadi et al. (28) showed that the hydroalcoholic extracts of Stachys lavandulifolia and Mespilus germanica have antileishmanial activities. Some studies have also shown that these extracts are suitable adjuvants in vaccination against Leishmania species (29, 30). Recently, invivo studies have revealed that alcoholic extract of Hedera helix and ethanol extract of Peganum harmala exert antileishmanial effects on L. major (31, 32). Furthermore, another study showed that the aqueous extract of Artemisia sieberi has more antileishmanial activities than artemisinin (33).

Moreover, the mucilage extract of Cordia myxa has shown antileishmanial activities against L. major and L. infantum (34). The results of a previous study on the effects of 6 plants showed that Allium hirtifolium and Ziziphus spina-christi had greater inhibitory effects on parasites (35). Another study showed that the extracts of Stachys lavandulifolia Vahl. and Mespilus germanica L. leaves could inhibit the proliferation of L. major promastigotes.

In the present study, the aqueous fraction of F. vulgaris showed the greatest effect on parasite death during the first 6 hours (Figure 1). After this interval, changes reduced at all concentrations, and the difference was almost insignificant among different concentrations within 48 and 72 hours. As most solute compounds in the aqueous fraction are proteins and carbohydrates, they may act as a source of energy for parasite survival and proliferation; this may be the reason for the reduced death of parasites in this interval.

Likewise, the effects of ethyl acetate fraction on parasite mortality were almost similar to the aqueous fraction. In addition, the greatest effect of ethyl acetate fraction was observed during the first 6 hours. The changes were almost similar for all the fractions during the first 24 hours. In this study, the highest antileishmanial effect was observed for the butanol fraction with an IC50 of 343.8 µg/mL at 48 hours. As presented in Figure 3, the antileishmanial effect of butanol fraction was less significant than amphotericin B; in fact, amphotericin B destroyed 100% of parasites within 6 hours. Nevertheless, the butanol fraction showed a reliable and dose-dependent antileishmanial effect at 48 hours (Figure 3).

So far, researchers have introduced various antileishmanial compounds (36). Accordingly, the world health organization (WHO) has recommended the use of medicinal plants in the treatment of leishmaniasis (34). Based on the present findings, F. vulgaris has acceptable antileishmanial activity and can act as an effective agent in the treatment of wounds without any side effects. Therefore, it may be used as a useful complementary or alternative treatment for skin lesions of cutaneous leishmaniasis. However, further invivo studies are necessary in the future.

References

  • 1.

    Abdolmajid F, Ghodratollah SS, Hushang R, Mojtaba MB, Ali MM, Abdolghayoum M. Identification of Leishmania species by kinetoplast DNA-polymerase chain reaction for the first time in Khaf district, Khorasan-e-Razavi province, Iran. Trop Parasitol. 2015;5(1):50-4. [PubMed ID: 25709953]. https://doi.org/10.4103/2229-5070.145587.

  • 2.

    Mohaghegh M, Fata A, Salehi G, Berenji F, Bazzaz MM, Rafatpanah H, et al. Molecular identification of leishmania species using samples obtained from negative stained smears. Iran J Parasitol. 2013;8(2):337-41. [PubMed ID: 23914250].

  • 3.

    Oliveira LF, Schubach AO, Martins MM, Passos SL, Oliveira RV, Marzochi MC, et al. Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the New World. Acta Trop. 2011;118(2):87-96. [PubMed ID: 21420925]. https://doi.org/10.1016/j.actatropica.2011.02.007.

  • 4.

    Sundar S, Chakravarty J. Antimony toxicity. Int J Environ Res Public Health. 2010;7(12):4267-77. [PubMed ID: 21318007]. https://doi.org/10.3390/ijerph7124267.

  • 5.

    Palumbo E. Current treatment for cutaneous leishmaniasis: a review. Am J Ther. 2009;16(2):178-82. [PubMed ID: 19300044]. https://doi.org/10.1097/MJT.0b013e3181822e90.

  • 6.

    Ait-Oudhia K, Gazanion E, Vergnes B, Oury B, Sereno D. Leishmania antimony resistance: what we know what we can learn from the field. Parasitol Res. 2011;109(5):1225-32. [PubMed ID: 21800124]. https://doi.org/10.1007/s00436-011-2555-5.

  • 7.

    Monge-Maillo B, Lopez-Velez R. Therapeutic options for old world cutaneous leishmaniasis and new world cutaneous and mucocutaneous leishmaniasis. Drugs. 2013;73(17):1889-920. [PubMed ID: 24170665]. https://doi.org/10.1007/s40265-013-0132-1.

  • 8.

    Sen R, Chatterjee M. Plant derived therapeutics for the treatment of Leishmaniasis. Phytomedicine. 2011;18(12):1056-69. [PubMed ID: 21596544]. https://doi.org/10.1016/j.phymed.2011.03.004.

  • 9.

    Masoudi S, Ameri N, Rustaiyan A, Moradalizadeh M, Aberoomand Azar P. Volatile Constituents of Three Umbelliferae Herbs:Azilia eryngioedes(Pau) Hedge et Lamond,Laser trilobum(L.) Borkh. andFalcaria falcarioides(Bornm. et Wolff) Growing Wild in Iran. J Essent Oil Res. 2005;17(1):98-100. https://doi.org/10.1080/10412905.2005.9698843.

  • 10.

    Shafaghat A. Free radical scavenging and antibacterial activities, and GC/MS analysis of essential oils from different parts of Falcaria vulgaris from two regions. Nat Prod Commun. 2010;5(6):981-4. [PubMed ID: 20614839].

  • 11.

    Khazaei M, Salehi H. Protective effect of Falcaria vulgaris extract on ethanol induced gastric ulcer in rat. Iran J Pharmacol Ther. 2006;5(1):43-6.

  • 12.

    Khazaei M, Ghorbani R. Survey of protective effect of hydroalcoholic extract of Falcaria vulgaris on aspirin-induced gastric ulcer in rat [In Persian]. Sci J Kurdistan Univ Med Sci. 2006;11(1):16-25.

  • 13.

    Shakibaei D, Khoushbou S, Kaboodi B. The effect of the "Falcaria vulgaris" on deep skin wound remodeling time and skin tension power in rats [In Persian]. J Kermanshah Univ Med Sci. 2006;10(3):187-94.

  • 14.

    Moshafi MH, Mehrabani M, Mahdikhani S, Saffari F. Antibacterial effects of different fractions of extract of Falcaria vulgaris bernh fruits and bioautography of its effective fraction. Med J Tabriz Univ Med Sci. 2015;36(3):74-9.

  • 15.

    Seidel V. Initial and bulk extraction of natural products isolation. Methods Mol Biol. 2012;864:27-41. [PubMed ID: 22367892]. https://doi.org/10.1007/978-1-61779-624-1_2.

  • 16.

    Eskins K, Scholfield CR, Dutton HJ. High-performance liquid chromatography of plant pigments. J Chromatogr A. 1977;135(1):217-20. https://doi.org/10.1016/s0021-9673(00)86323-2.

  • 17.

    Tang B, Bi W, Tian M, Row KH. Application of ionic liquid for extraction and separation of bioactive compounds from plants. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;904:1-21. [PubMed ID: 22877739]. https://doi.org/10.1016/j.jchromb.2012.07.020.

  • 18.

    Masoumi SM, Kahrizi D, Rostami-Ahmadvandi H, Soorni J, Kiani S, Mostafaie A, et al. Genetic diversity study of some medicinal plant accessions belong to Apiaceae family based on seed storage proteins patterns. Mol Biol Rep. 2012;39(12):10361-5. [PubMed ID: 23086265]. https://doi.org/10.1007/s11033-012-1914-3.

  • 19.

    Ogeto TK, Odhiambo R. A, Shivairo RS, Muleke C. I, Osero B. O, Anjili C, et al. Antileishmanial activity of Aloe secundiflora plant extracts against Leishmania Major. Adv Life Sci Tech. 2013;13:9-17.

  • 20.

    Britta EA, Scariot DB, Falzirolli H, Ueda-Nakamura T, Silva CC, Filho BP, et al. Cell death and ultrastructural alterations in Leishmania amazonensis caused by new compound 4-Nitrobenzaldehyde thiosemicarbazone derived from S-limonene. BMC Microbiol. 2014;14:236. [PubMed ID: 25253283]. https://doi.org/10.1186/s12866-014-0236-0.

  • 21.

    Soares DC, Portella NA, Ramos MF, Siani AC, Saraiva EM. Trans- beta -Caryophyllene: An Effective Antileishmanial Compound Found in Commercial Copaiba Oil (Copaifera spp.). Evid Based Complement Alternat Med. 2013;2013:761323. [PubMed ID: 23864897]. https://doi.org/10.1155/2013/761323.

  • 22.

    Fazly Bazzaz BS, Haririzadeh G, Imami SA, Rashed MH. Survey of Iranian plants for alkaloids, flavonoids, saponins, and tannins [Khorasan Province]. Pharm Biol. 1997;35(1):17-30. https://doi.org/10.1076/phbi.35.1.17.13275.

  • 23.

    Germonprez N, Maes L, Van Puyvelde L, Van Tri M, Tuan DA, De Kimpe N. In vitro and in vivo anti-leishmanial activity of triterpenoid saponins isolated from Maesa balansae and some chemical derivatives. J Med Chem. 2005;48(1):32-7. [PubMed ID: 15633999]. https://doi.org/10.1021/jm031150y.

  • 24.

    Dutta A, Ghoshal A, Mandal D, Mondal NB, Banerjee S, Sahu NP, et al. Racemoside A, an anti-leishmanial, water-soluble, natural steroidal saponin, induces programmed cell death in Leishmania donovani. J Med Microbiol. 2007;56(Pt 9):1196-204. [PubMed ID: 17761483]. https://doi.org/10.1099/jmm.0.47114-0.

  • 25.

    Cruz Ede M, da Silva ER, Maquiaveli Cdo C, Alves ES, Lucon JJ, dos Reis MB, et al. Leishmanicidal activity of Cecropia pachystachya flavonoids: arginase inhibition and altered mitochondrial DNA arrangement. Phytochemistry. 2013;89:71-7. [PubMed ID: 23453911]. https://doi.org/10.1016/j.phytochem.2013.01.014.

  • 26.

    Rodrigues IA, Azevedo MM, Chaves FC, Alviano CS, Alviano DS, Vermelho AB. Arrabidaea chica hexanic extract induces mitochondrion damage and peptidase inhibition on Leishmania spp. Biomed Res Int. 2014;2014:985171. [PubMed ID: 24818162]. https://doi.org/10.1155/2014/985171.

  • 27.

    Yousefi E, Eskandari A, Gharavi MJ, Khademvatan S. In vitro activity and cytotoxicity of Crocus sativus extract against leihmania major (MRHO/IR/75/ER). Infect Disord Drug Targets. 2014;14(1):56-60. [PubMed ID: 25159304]. https://doi.org/10.2174/1871526514666140827101901.

  • 28.

    Asadi M, Bahrami S, Ansari Samani R. The effect of Stachys lavandulifolia vahl and Mespilus germanica L. leaves hydroalcoholic extracts on Leishmania major (MRHO/IR/75/ER) in vitro. Jundishapur J Nat Pharm Prod. 2010;(1):39-43.

  • 29.

    Santos WR, Bernardo RR, Pecanha LM, Palatnik M, Parente JP, Palatnik de Sousa CB. Haemolytic activities of plant saponins and adjuvants. Effect of Periandra mediterranea saponin on the humoral response to the FML antigen of Leishmania donovani. Vaccine. 1997;15(9):1024-9. [PubMed ID: 9261952]. https://doi.org/10.1016/S0264-410X(96)00292-7.

  • 30.

    Kaur A, Kaur PK, Singh S, Singh IP. Antileishmanial compounds from Moringa oleifera Lam. Z Naturforsch C. 2014;69(3-4):110-6. [PubMed ID: 24873031]. https://doi.org/10.5560/znc.2013-0159.

  • 31.

    Hooshyar H, Talari S, Feyzi F. Therapeutic Effect of Hedera helix Alcoholic Extract Against Cutaneous Leishmaniasis Caused by Leishmania major in Balb/c Mice. Jundishapur J Microbiol. 2014;7(4). e9432. [PubMed ID: 25147703]. https://doi.org/10.5812/jjm.9432.

  • 32.

    Khoshzaban F, Ghaffarifar F, Jamshidi Koohsari HR. Peganum harmala Aqueous and Ethanol Extracts Effects on Lesions Caused by Leishmania major (MRHO/IR/75/ER) in BALB/c Mice. Jundishapur J Microbiol. 2014;7(7). e10992. [PubMed ID: 25368792]. https://doi.org/10.5812/jjm.10992.

  • 33.

    Esavand Heydari F, Ghaffarifar F, Soflaei S, Dalimi A. Comparison Between in Vitro Effects of Aqueous Extract of Artemisia seiberi and Artemisinin on Leishmania major. Jundishapur J Nat Pharm Prod. 2013;8(2):70-5. [PubMed ID: 24624191]. https://doi.org/10.5812/jjnpp.9513.

  • 34.

    Saki J, Khademvatan S, Pazyar N, Eskandari A, Tamoradi A, Nazari P. In Vitro Activity of Cordia myxa Mucilage Extract Against Leishmania major and L. infantum Promastigotes. Jundishapur J Microbiol. 2015;8(3). e19640. [PubMed ID: 25861436]. https://doi.org/10.5812/jjm.19640.

  • 35.

    Feiz Haddad MH, Khodkar I, Samie M. In Vitro Anti-leishmanial Effects of Hydroalcoholic Extracts From Six Iranian Medicinal Herbs on Leishmania major (MRHO/IR/75/ER) Promastigotes. Jentashapir J Health Res. 2016;7(3). https://doi.org/10.17795/jjhr-33465.

  • 36.

    Hussain H, Al-Harrasi A, Al-Rawahi A, Green IR, Gibbons S. Fruitful decade for antileishmanial compounds from 2002 to late 2011. Chem Rev. 2014;114(20):10369-428. [PubMed ID: 25253511]. https://doi.org/10.1021/cr400552x.