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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 149-154

In vitro anti-leishmanial activity of Satureja khuzestanica jamzad and Oliveria decumbens vent. Extracts on Leishmania major and Leishmania infantum promastigotes


1 Department of Myco-Parasitology, Urmia University of Medical Sciences, Urmia, Iran
2 Department of Medicinal Chemistry, Faculty of Pharmacy, Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran
3 Abadan School of Medical Sciences, Abadan, Iran
4 Department of Biology, Facultyof Science, University of Zabol, Zabol, Iran

Date of Web Publication30-Oct-2019

Correspondence Address:
Dr. Batool Sadeghi Nejad
Abadan School of Medical Sciences, Abadan.
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrptps.JRPTPS_39_18

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  Abstract 

Background and Objectives: Leishmaniasis is the main health problem and affects millions of people, especially in developing countries. On the other hand, there is no immunoprophylaxis (vaccination) accessible for the treatment of Leishmania infections and commercial drugs are unsatisfactory. Therefore, there is an effort to find alternative herbal remedies. The objective of the present study was to state the antileishmanial activity of two herbal medicines such as Satureja khuzestanica Jamzad and Oliveria decumbens Vent. leaf extracts on the promastigotes of Leishmania major and Leishmania infantum. Materials and Methods: The hydroethanolic extracts of each plant were extracted and their antileishmanial effects evaluated in different concentrations (0–156 μg/ml) and at various hours (24, 48, and 72h) using colorimetric (3[4, 5dimethylthiazol2yl]2, 5 diphenyltetrazolium bromide) assay. The concentrationresponse curves of tested extracts and glucantime solutions as a reference were designed, and 50% of inhibitory concentration (IC50) values were recorded. Results: Antileishminal activity of S. khuzestanica, O. decumbens, and glucantime drug on L. major and L. infantum promastigotes were revealed with IC50 values of 4.3 and 5.5 μg/ml for S. khuzestanica, 0.85 and 0.23 μg/ml for O. decumbens, and 40.2 and 18.5 μg/ml for glucantime after 72h incubation. Conclusion: These results revealed that compounds from S. khuzestanica and O. decumbens have antileishmania properties that necessary to survey the effects of these extracts on leishmania genus in animal models in the future.

Keywords: Antileishmanial, Leishmania infantum, Leishmania major, Oliveria decumbens, promastigote, Satureja khuzestanica


How to cite this article:
Khademvatan S, Eskandari A, Nejad BS, Najafi S. In vitro anti-leishmanial activity of Satureja khuzestanica jamzad and Oliveria decumbens vent. Extracts on Leishmania major and Leishmania infantum promastigotes. J Rep Pharma Sci 2019;8:149-54

How to cite this URL:
Khademvatan S, Eskandari A, Nejad BS, Najafi S. In vitro anti-leishmanial activity of Satureja khuzestanica jamzad and Oliveria decumbens vent. Extracts on Leishmania major and Leishmania infantum promastigotes. J Rep Pharma Sci [serial online] 2019 [cited 2019 Nov 16];8:149-54. Available from: http://www.jrpsjournal.com/text.asp?2019/8/2/149/269939




  Introduction Top


Leishmaniasis is a chronic disease caused by protozoan parasites belonging to the genus Leishmania transmitted by sandflies. Leishmaniasis is an important disease with three main clinical manifestations of leishmaniasis are recognized as follows: cutaneous leishmaniasis (CL), muco-CL, and visceral leishmaniasis.[1],[2] Antileishmanial drugs such as pentavalent antimonial compounds, meglumine antimoniate (glucantime), sodium stibogluconate (Pentostam), amphotericin B, pentamidine, and paromomycin have been used against human leishmaniasis.[3],[4] The mentioned drugs have limitations for being used due to the prolonged term of treatment, high cost, high toxicity, painful implementation, the absence of oral formulation (e.g., amphotericin B can be used only intravenously), or important side effects and high-resistant parasites against these drugs.[4],[5] In addition, other probable complications of these drugs are damage to the heart, liver, pancreas, hematopoietic tissues, and renal failure.[5] Other drugs include oral drug miltefosine have been used for the treatment of human visceral Leishmania infections, and fluconazole has also been effective against CL.[6] Since medicinal plants contain valuable components, easily accessible and cheap, the use of such native plants could be surveyed as wealthy sources of antileishmanial compounds.[7],[8],[9],[10]Satureja khuzestanica Jamzad leaf extracts (Marzeh Khuzestani in Persian), family of (Lamiaceae) is an endemic plant that it grows in the Southern and Western parts of Iran, especially Khuzestan and Lorestan provinces.[11] The extract of S. khuzestanica has biological properties such as antibacterial,[12] antifungal,[13] antiviral,[14] and antileishmanial activity.[10] Other plant, Oliveria decumbens Vent., family of (Umbelliferae) is an ancient herbal medicine which has been used in Behbahan (Khuzestan, South West of Iran). The essential oil of aerial parts of O. decumbens was analyzed, and antimicrobial properties of this plant carried out against Gram-positive bacteria,  Escherichia More Details coli, Aspergillus niger, and Candida albicans.[12] The present study aimed to evaluate the in vitro antileishmanial activity of two hydroalcoholic extracts of S. khuzestanica and O. decumbens in different concentrations (0–156 µg/ml) and at various hours (24, 48, and 72h) on L. major and Linfantum promastigotes by (3- [4, 5-dimethylthiazol-2-yl]-2, 5 dipheny ltetrazolium bromide) (MTT) colorimetric assay.


  Materials and Methods Top


Ethics statement

The protocol of this study was approved by the Ethics Committee of Abadan School of Medical Sciences, Abadan, Iran. The University Ethics Committee code number was IR.SBMURECH.1395: No. 93U-029.

Plant materials and extract

The medicinal plants of S. khuzestanica and O. decumbens were collected from Shiyon Dezful district in Khuzestan province, and the specimens were identified by agricultural and natural resources center (ANRC), Ahvaz, Iran and deposited at the Herbarium of ANRC with voucher numbers of 7936 and 5288 for S. khuzestanica and O. decumbens, respectively. For the preparation of hydroethanolic extracts, 10g of shade-dried and powdered plant material was macerated in 100mL ethanol with 85% of (Merck) solution on a rotary shaker for 72h.[15] The plant extracts were filtered through Whatman No. 1 and the filtrated hydroethanolic extracts were evaporated and dried at the room temperature. Dried extracts were obtained and stored at 4°C for further assay.

Parasite culture

Parasites-Leishmania (L.) major (MRHO/IR/75/ER) and Leishmania (L.) infantum (MCAN/IR/96/LONDON 49) promastigotes strains were obtained from the School of health, Tehran University of Medical Sciences, Iran and were maintained in RPMI-1640 (Sigma, Chemical company) medium and supplemented with 10% of fetal calf serum sigma, 100 IU/mL penicillin and 100 µg/ml streptomycin. All promastigotes (106 parasites/ml) were incubated at 26°C for 24, 48, and 72h in fresh RPMI-1640.[16]

Promastigote viability using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide assay

MTT is colorimetric assay measured reduction of MTT dye (tetrazolium) into formazan by mitochondrial enzymes in viable cells. To prepare the stoke solution MTT (Sigma Chemical Co., St. Louis, Mo.), 5mg from yellow powder was soluble in 1ml phosphate-buffered saline and kept in −20°C. At working, a freshly solution was prepared with mixing one volume of MTT stock solution in 9ml of RPMI-1640 medium containing 2.5% of colony-stimulating factor, the final concentration of MTT reaches to 0.5mg/ml.[17]

Add promastigotes, selected plants extract and medicine

Aliquot 100 µl L. major and L. infantum promastigotes (106 parasites/ml) added to per well of 96-well microplate and incubated for 6h. Afterwards, 10 µl the two-fold dilutions of selected plant extracts ranged from 0 to 156 µg/ml and 20 µl working MTT reagent were added to per well of 96-well microplate. Negative control well was contained only 100 µl culture medium plus 20 µl working MTT reagent and glucantime drug was used as positive control. After 3–4h incubation and centrifugation, separated the supernatant solution and added 100 µl pure dimethyl sulfoxide to per well for dissolving of purple formosan crystals. After 15min of shaking, optical density (OD) by ELISA reader in wavelength of 570nm and reference wavelength of 630nm.[16],[18]

Statistical analysis

Statistical analyses of the differences in mean values between different experimental groups were performed with the two-tailed Student’s t-test. The significance of difference was measured using analysis of variance and with a confidence interval of 95%, P = 0.05 or less were considered as statistically significant.


  Results Top


In this study, the antileishmania activity of hydroethanolic extracts of selected herbals was determined using MTT assay. Inhibitory concentration (IC50) values were for the hydroethanolic extract of S. khuzestanica at 24, 48, and 72h for L. major 41.2, 13.3, and 4.3 µg/ml and for Linfantum promastigote 42.1, 30.2, and 5.5 µg/ml, respectively, whereas these values were for O. decumbens at 24, 48, and 72h for L. major promastigote 22.3, 2.7, and 0.85 µg/ml and for Linfantum promastigote 7.1, 1.13, and 0.23 µg/ml, respectively. The findings demonstrated that the hydroethanolic extracts of S. khuzestanica and O. decumbens had potent antileishmanial activity against the forms of L. major and Linfantum promastigotes in vitro after 24, 48, and 72h of incubation (P < 0.05). These results also revealed that the hydroethanolic extract of O. decumbens leaf in comparison with the hydroethanolic extract of S. khuzestanica leaf had significantly (P < 0.05) higher leishmanicidal effect on the promastigotes of L. major and Linfantum since it exhibited lower IC50 values for the tested promastigotes. The effect of the antileishmanial activity of the S. khuzestanica leaf extract against the forms of L. major and Linfantum promastigotes in various concentrations and at different hours (24, 48 and 72h) are shown in [Table 1] and [Figure 1]. Furthermore, antileishmanial activity of the O. decumbens leaf extract on L. major and Linfantum promastigotes in different concentrations and at various hours (24, 48, and 72h) are shown in [Table 1] and [Figure 2]. Glucantime drug also revealed IC50 values of 40.2 and 18.5 µg/ml for L. major and Linfantum promastigotes after 72h incubation, respectively. The comparison of means of antileishmanial activity of S. khuzestanica, O. decumbens, and glucantime drug against the forms of L. major and Linfantum promastigotes after 72h revealed statistically significant (P < 0.05).
Table 1: IC50 of S. khuzestanica, O. decumbens leaf extracts and glucantime against L. major and L. infantum promastigotes after 24, 48 and 72h incubation

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,
Figure 1: a) Viability of L. major promastigotes and b) Viability of L. infantum promastigotes in the various concentrations of S. khuzestanica after 24,48,72 h

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,
Figure 2: a) Viability of L. major promastigotes and b) Viability of L. infantum promastigotes in the various concentrations of O. decumbens after 24,48,72 h

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IC50 value of S. khuzestanica, O. decumbens, and glucantime at 72h after treatment were 50.62 ± 30.31 µg/ml, 34.43 ± 28.79 µg/ml, and 60.15 ± 34.36 µg/ml, respectively. The P values for each plant and drug are presented in [Table 2].
Table 2: Results of comparison of paired-samples t-test for each plants and drug against tested promastigotes after 72 h

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  Discussion Top


The CL is also a major health problem in Iran, especially in Mashhad, Northeast of Iran.[19] Natural products, such as herbal extracts, have been used as pure compounds for the treatment of various diseases such as infectious diseases. The use of herbal medicines in Asia for the remedy of skin diseases returns back to the past. One of the important points is that the side effects of herb medicines are less than that of the chemical drugs.[20] Until now, several studies have been demonstrated the potential antileishmania effects of various herbal extracts on parasites-leishmania in the past.[21],[22],[23],[24],[25],[26] These studies showed that a number of medicinal plants have moderate-to-strong antileishmanial activity. However, to the best of our knowledge, based on the previous literature, no studies have been carried out on the effects of S. khuzestanica and O. decumbens plants extract on the in vitro growth of L. major and L. infantum promastigotes by MMT assay. In the present study, the hydroethanolic extracts of S. khuzestanica and O. decumbens significantly (P < 0.05) inhibited the growth rate of promastigotes forms of L. major and L. infantum. These results also revealed that promastigotes forms of L. infantum were more susceptible to the hydroethanolic extract of O. decumbens and glucantime drug while the hydroethanolic extract of S. khuzestanica was more sensitive to L. major than L. infantum promastigote after 72h of incubation. Allahdin et al. studied the leishmanicidal activity of Camellia sinensis against promastigotes forms of L. major and Linfantum. The results of the mentioned study revealed that C. sinensis had IC50s of 19 µg/ml, 12 µg/ml and glucantime had IC50s of 21.8 µg/ml, and 10.16 µg/ml for L. major and Linfantum promastigotes after 72h of incubation, respectively. These results were found to be C. sinensis was more sensitive to Linfantum promastigotes than L. major promastigotes.[27] There is the consistency between the results of the aforementioned study and those of our research. In general, based on our findings, antileishmanial efficacy of O. decumbens extract was significantly higher than S. khuzestanica extract. In the present study, the results showed that by increasing the concentrations of tested plant extracts or drug, while the inhibitory effect on the growth of L. major and Linfantum promastigotes will be increased, OD decreases. The reason for the reduction of OD is the furosian reduction, which is produced by the action of the mitochondria dehydrogenases of the active metabolic cells and is shown to be linked to the number of live cells.[28] In addition, previous phytochemical studies have also reported that the S. khuzestanica contained saponins, reducing sugars, flavonoids, tannins, phenols, proteins, and triterpenes.[29] Furthermore, the essential oil of aerial parts of O. decumbens contained γ-terpinene, myristicin, thymol, ρ-cymene, and carvacrol.[30] Moreover, in several studies, strong antibacterial, antifungal, and anti-parasitic activities of these compounds and their derivations such as α-pinene, 1, 8-cineole, limonene, thymol, and carvacrol against some pathogenic microorganisms have been confirmed.[31],[32],[33],[34] Therefore, phytocompounds in these plants can be responsible for their antileishmanial efficacy, while the precise mechanism of their activity is unknown. However, Sikkema et al. investigated the antimicrobial mechanisms of some terpenoid compounds, such as monoterposes[35] and reported that terpenoid compounds damage the structures of the organs of microorganism’s body. On the other hand, in other studies, indicated that antimicrobial activity of plants according to the ability of the terpenes, not only affect penetrability but also other cell membrane functions. These compounds may pass through the cell membrane, therefore, penetrating into the cell and interacting with vital intracellular sites.[36],[37]S. khuzestanica leaf extracts contain active compounds, which could be used as suitable herbal drug in the treatment of experimental CL in vivo[38] and used as leishmaniacidal agent in vitro.[10],[39] Kheirandish et al. studied the cytotoxic and antileishmanial activity of S. khuzestanica essential oil (SKEO) by the MTT method. The present research revealed the potent antileishmanial activity of SKEO and this plant had no toxic effect on mammalian cells.[40] Furthermore, Moghaddas et al. studied Iranian native plants, including S. khuzestanica on the treatment of CL.[19] The findings of this study reported that carvacrol-rich essential oil of S. khuzistanica showed strong antibacterial activity. Given that the strong antimicrobial properties of carvacrol contain in S. khuzestanica has already been reported,[12] the antileishmanial properties of this plant may be due to this compound.


  Conclusions Top


According to mentioned results, the antileishmanial effects of S. khuzestanica and O. decumbens extracts may be recommend in the treatment of CL as a complement or substitute treatment. However, further studies are necessary and should be investigated in cell culture and in vivo assays in animal and human models to emphasis it.

Acknowledgments

The authors would like to thank and appreciate all who have cooperated with this research. This study was financially supported by Deputy of Research, Abadan School of Medical Sciences (Grant No. 93u-029).

Financial support and sponsorship

This study was financially supported by grant (No: 93u-29) from the Research Deputy of Abadan School of Medical Sciences, Abadan, Iran.

Conflicts of interest

There are no conflicts of interest.



 
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