ABSTRACT
Recently, the use of pentavalent antimony compounds for Leishmaniasis treatment has been associated with disease recurrence, drug resistance, and severe side effects. Therefore, there is a need to develop alternative treatment strategies. This study investigates the in vitro effects of Zingiber officinale on promastigotes and amastigotes of Leishmania major and Leishmania tropica.
Promastigotes and amastigotes of Leishmania major and Leishmania tropica were cultured and mass-produced in an RPMI1640 medium enriched with other necessary compounds. The MTT colorimetric method and calculating the IC50 value were used to evaluate the anti-leishmania activity of hydroalcoholic extract of Zingiber officinale.
The hydroalcoholic extract of Zingiber officinale inhibited the growth of Leishmania major and Leishmania tropica promastigotes in 24, 48, and 72 hours after in vitro incubation. The IC50 of hydroalcoholic extract of Zingiber officinale was 56 μg/mL for Leishmania major and 275 μg/mL for Leishmania tropica promastigotes after 72 hours. The IC50 of hydroalcoholic extract of Zingiber officinale was 75 μg/mL for Leishmania major and 325 μg/mL for Leishmania tropica amastigotes after 72 hours.
The results showed that hydroalcoholic extract of Zingiber officinale has cytotoxicity properties, and Leishmania tropica has a higher resistance to hydroalcoholic extract of Zingiber officinale than Leishmania major. Further research is recommended.
Keywords: Zingiber officinale, Z. officinale, gingers, Leishmania major, Leishmania tropica
INTRODUCTION
The protozoan parasite of Kinetoplastida called Leishmania causes Leishmaniasis, which has different forms of clinical presentation, including skin and mucosal lesions and visceral signs (1,2). Leishmania is an intracellular parasite that lives in the phagolysosome of vertebrate phagocytic cells. Phlebotomine sand flies play the role of the vector of this parasite (3-5). This tropical infection, commonly seen and ignored in developing countries, is an emerging public health problem in about 2 to 4 million new cases and almost leads to 70,000 deaths each year (6). According to the World Health Organization (WHO) report, around 13 million people have been affected, 350 million others are at the risk of exposure to this disease, and 2 million new patients are added to this number annually (7-10). Iran is one of 10 countries where 75% of global cutaneous leishmaniasis cases have been reported. In addition, it ranks first place in the Middle East in terms of reported cases of cutaneous leishmaniasis (7).
Today, with the increase in the number of patients with defective immune systems, opportunistic infections such as leishmaniasis are increasing. The treatment with pentavalent antimony drugs, which is the first preference for treating leishmaniasis, is limited due to its numerous side effects and drug resistance (11). Pentavalent antimony compounds, including meglumine antimoniate (glucantime), and sodium stibogluconate (pentostam), remain the first-line treatment for all clinical forms of Leishmaniasis. These compounds inhibit adenosine triphosphate production by interrupting the parasite phosphodiesterase enzyme (12-14). However, due to inherent toxicity and frequent infections, these drugs have side effects such as liver and heart disorders and biochemical changes (15). Considering the drug resistance and tremendous side effects of these compounds, researchers seek alternative forms of treatment that are more effective with fewer side effects (16).
It is essential to study medicinal plants to find a suitable drug against the Leishmania parasite and Leishmaniasis (17). Several herbal compounds have been used so far for the treatment of cutaneous leishmaniasis lesions. Various researchers have recently evaluated the in vivo and in vitro effects of the parasite’s essential oils and extracts of some local and native plants. One of these plants is Zingiber officinale, that antifungal, antibacterial, and even antiparasitic properties have been proven (17-21). Zingiber officinale is a yellow plant with purple petioles. Its main constituents include sugars (50-70%), fats (3-18%), oleoresins (4-5.5%) and spicy compounds (1-3%). The ingredients of Zingiber officinale include gingerol shogaols, zingerone, Zingiberene, and Zabolin, which make the odor and flavor of Zingiber officinale (22,23).
This study, among the MTT colorimetric, aims to determine the in vitro effects of the extract of Zingiber officinale on promastigotes and amastigotes of Leishmania major (MRHO/IR/75/ER) and Leishmania tropica (MHOM/IR/02/Mash 10).
METHODS
Cultivation of Promastigotes
The standard promastigotes of L. tropica (MHOM/IR/02/Mash 10) and L. major (MHOM/IR/75/ER) were prepared from Ahvaz Jundishapur University of Medical Sciences. Parasites were culture in RPMI1640 enriched with 10% fetal calf serum (FCS) and antibiotics (100 IU/mL penicillin and 100 IU/mL streptomycin) at 25±2 °C. The optimal 1×106 per mL parasite was obtained by counting promastigotes by a hemocytometer slide.
Cultivation of Amastigotes
To culture, the amastigotes forms of L. major and L. tropica, in the first step, the enriched RPMI1640 medium’s acidity with 10% FCS containing 1×106 mL of the parasite was reduced to pH=5-5.5. Culture flasks were kept at an incubator (25 °C & 5% CO2). The temperature was increased by 2-3 °C daily until the optimum temperature of 32°C was obtained. Within four days, the parasites were transformed from promastigotes to amastigotes (24).
Preparation of Zingiber officinale Extract
The Zingiber officinale rhizome was washed with water and dried, and then completely ground. Extraction was carried out using the 80% methanol percolation method and then evaporated at 40 °C and a low pressure using a rotary evaporator device, and the extract was condensed. The condensed extract was stored at 60 °C in a hot, dry oven and then was kept at 4 °C until use. The Zingiber officinale extracts were prepared at 600, 300, 150, 75, 37.5, 18.75, and 9.375 µg/mL concentrations.
Promastigotes were exposed to different concentrations of Zingiber officinale extract. 100 µL of the RPMI1640 medium enriched with 10% FCS containing at least 1×105 promastigotes were placed in 96-well plates, and 100 µL of the hydroalcoholic solution of Zingiber officinale extract was added to the wells at concentrations of 600, 300, 150, 75, 37.5, 18.75, and 9.375 µg/mL. The promastigotes were incubated for three days at 25±2°C. The RPMI1640 culture medium containing parasites without Zingiber officinale extract was used as a control. All tests were performed in triplicate. Amastigotes were exposed to different concentrations of Zingiber officinale extract. 100 µL of the RPMI1640 medium enriched with 10% FCS containing at least 1×105 amastigotes was placed in 96-well plates, and 100 µL of the Zingiber officinale extract solution was added to the wells at concentrations of 1.200, 600, 300, 150. 75, 37.5, and 18.75 µg/mL. The amastigotes were incubated for three days at 32 °C. The RPMI1640 culture medium containing parasites without Zingiber officinale extract was used as a control.
The parasites incubated with Zingiber officinale extract were evaluated using the methyl thiazolyl tetrazolium assay (MTT) method after 24, 48, and 72 hours. The MTT powder was prepared at a concentration of 5 mg/mL in PBS (Phosphate Buffered Saline), and 20 µL of it was added to each well so that the final concentration of MTT would reach 0.5 mg/mL. The plates were incubated at 25±2 °C for 2-5 hours. Subsequently, 100 µL of 1% dimethyl sulfoxide was added to each well and mixed well to help solve the insoluble crystalline formazan formed due to the reduction of tetrazolium by the succinate dehydrogenase enzyme. After 20 minutes of incubation, the wells’ optical density (OD) was read with The ELISA reader device at 570 nm. The cell viability percentage was calculated using the Excel software and the formula: [Viable cell %= (AT-AB/(AC-AB) ×100], where AB represents the optical density of the blank well, AC represents the optical density of the control well, and AT represents the optical density of the drug-treated cell. The results were calculated in an IC50 measurement by linear regression test (25).
Statistical Analysis
IC 50 values of promastigotes and intracellular amastigotes were calculated for the mean and standard deviation. We performed all tests in triplicate. The mean and standard deviation of the three trials were registered. The data were analyzed using SPSS 19. Statistical analysis of the differences among mean values gotten for the experimental groups was done by analyzing variance (ANOVA). Values less than 0.05 were considered significant.
RESULTS
The IC50 values of the Zingiber officinale extract for L. major after 24, 48, and 72 hours were 112, 90, and 56 µg/mL, respectively, while the IC50 values for L. tropica at those times were 600, 390, and 275 µg/mL respectively.
The IC50 values of the Zingiber officinale extract for amastigotes of L. major after 24, 48, and 72 hours were 130, 105, and 75 µg/mL, respectively, while the IC50 values for L. tropica at those times were 720, 430, and 325 µg/mL respectively. According to these results, Zingiber officinale extract showed a concentration and time-dependent cytotoxicity activity against promastigotes and amastigotes of L. major and L. tropica. However, the value of time for inhibitory effect was observed less than an increase in Zingiber concentrations. Over time, the cell viability increased but was still not significant for L. tropica promastigotes, L. major amastigotes, and L. tropica amastigotes between 24 and 48 hours. At the same time, cell viability was substantial between 24 and 72 hours (Table 1, Figures 1, 2, 3, 4).
DISCUSSION
Leishmaniasis is a public health concern throughout the world, especially in tropical and subtropical countries (9). There are various drugs for the treatment of Leishmaniasis, but their toxicity, side effects, and drug resistance are problems associated with their use (14,26). According to the WHO, almost 80% of people use traditional medicines to treat their illnesses (27). Today, herbal drugs for treating parasitic diseases, especially Leishmaniasis, have special significance, and essential oils and extracts of various herbs have been used in recent years to treat cutaneous Leishmaniasis. Since herbal medicines are more cost-effective and have fewer side effects than chemical medicines, they can be an excellent alternative. Many studies have been conducted on the effect of medicinal herbs such as Thyme, Yarrow, Propolis, Sideritis, Medlar tree leaf, and many other herbs on Leishmaniasis. These studies indicate that the medicinal herb extract has an inhibitory effect on parasite growth in some cases. The inhibitory effect of Zingiber officinale extract on many microorganisms has been reported in recent years (28-30).
A study conducted by Shoaie et al. (20) in 2012 on clinical species of Candida albicans showed that Zingiber officinale has an inhibitory effect on the growth of it. The minimum inhibitory concentration of Zingiber officinale extracts on Candida albicans was 62.25 µg/mL (20). Feizi et al. (31) investigated the effects of methanol extract of Zingiber officinale on protoscoleces at three concentrations of 25, 50, and 100 mg/mL at different times. This study showed that the ethanolic extract of Zingiber officinale in concentrations of 50, 100, and 150 mg/mL killed 100% of the protoscoleces after 60, 90, and 120 minutes (31). Duarte et al. (32), investigated the effect of Zingiber officinale extract and F10 fraction on promastigotes of L. amazonasis and obtained the IC50 values of 125.5 µg/mL for aqueous extract of Zingiber officinale and 49.8 µg/mL for the F10 fraction of Zingiber officinale F10.
In some studies, nanoemulsion preparation based on plant extracts for anti-Leishmania use has been very promising (33). One of these anti-Leishmania extracts is derived from Artemisia dracunculus (Tarragon). Some active ingredients of which are flavonoids, phenolic acids, coumarins, and Alkamides (33,34). Another herbal medicine that has been attributed to the anti-amastigote forms of L. tropica properties is Zataria multiflora (35), however, there are limited studies on L. tropica in the literature. Further, the analyses confirmed that the main components of essential oil were thymol (monoterpenoid phenol), carvacrol (phenolic monoterpenoid), and p-cymene (monoterpene), that the presence of all these active ingredients in Zingiber officinale has also been reported (35-37). The therapeutic activities of Zingiber are mainly attributed to its active plant compounds 6-gingerol, 6-shogaol, zingerone, and other phenolics and flavonoids. Gingerol has been reported as the most abundant bioactive compound in Zingiber with various medicinal effects, including antioxidant, analgesic, anti-inflammatory, and antipyretic properties (38,39). A study evaluating 6-gingerol alone in combination with amphotericin B on the L-major stage using experimental and in vivo rat models stated that the binding affinity of 6-gingerol and IFN-g (interferon-gamma) was the basis of the docking conformations (38).
This study investigated the inhibitory effects of Zingiber officinale extract on both promastigotes and amastigotes of L. major and L. tropica. The study showed an increase in the extract concentration and an increase in incubation time, decreasing the survival percentage of the parasite’s two strains. Besides, the Zingiber officinale extract has a more significant inhibitory effect on L. major than L. tropica. As a result, higher concentrations of Zingiber officinale are needed to kill the L. tropica parasite, and the Zingiber officinale extract had a more significant effect on the promastigotes of the two strains than on their amastigotes. However, the value of time for inhibitory effect in this study was less observed than an increase in Zingiber concentrations.
CONCLUSION
This study confirms the in vitro inhibitory effects of Zingiber officinale on the promastigotes and amastigotes of both L. major and L. tropica. Since the pathogens form of this parasite is intracellular, further experiments are necessary to evaluate the effect of the mentioned extract on the Leishmania parasite in animal models and human volunteers in later stages.
ACKNOWLEDGMENT
This study is financially supported by Grant (no: OG-94112) from the Research Deputy of Ahvaz Jundishapur University of Medical Sciences, Iran. The authors would like to kindly appreciate the cooperation of the parasitology department staff for the period of conduction. The Ethics Committee of Ahvaz Jundishapur University of Medical Sciences approved this experiment, and ethical approval no: IR.AJUMS.REC.1394.151.
*Ethics
Ethics Committee Approval: The Ethics Committee of Ahvaz Jundishapur University of Medical Sciences approved this experiment, and ethical approval no: IR.AJUMS.REC.1394.151.
Informed Consent: Given that this study was performed in vitro, there was no need for informed consent.
Peer-review: Externally and internally peer-reviewed.
*Authorship Contributions
Surgical and Medical Practices: J.S., E.B., R.A., Concept: J.S., R.A., Design: J.S., R.A., Data Collection or Processing: E.B., R.A., Analysis or Interpretation: J.S., R.A., Literature Search: J.S., E.B., R.A., Writing: J.S., E.B., R.A.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: This study is financially supported by Grant (no: OG-94112) from the Research Deputy of Ahvaz Jundishapur University of Medical Sciences, Iran.
