Iran J Parasitol: Vol. 14, No. 4, Oct-Dec 2019, pp.521-533 Iran J Parasitol Tehran University of Medical Open access Journal at Iranian Society of Parasitology Sciences Public a tion http://ijpa.tums.ac.ir http://isp.tums.ac.ir http://tums.ac.ir Original Article Anti-Leishmanial and Immunomodulatory Effects of Epigallo- catechin 3-O-Gallate on Leishmania tropica: Apoptosis and Gene Expression Profiling Morteza SADUQI 1, *Iraj SHARIFI 2, Zahra BABAEI 2, Alireza KEYHANI 2, Mahshid MOSTAFAVI 2, Maryam HAKIMI PARIZI 2, Mahdiyeh GHASEMIAN 2, Mehdi BAMOROVAT 2, Fatemeh SHARIFI 3, Mohammad Reza AFLATOONIAN 4, Fariba SHARIFIFAR 5, Pooya GHASEMI NEJAD 2, Ahmad KHOSRAVI 2, Ehsan SALARKIA 2, Rajender S. VARMA 6 1. Department of Medical Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran 2. Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran 3. Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran 4. Research Center of Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran 5. Herbal and Traditional Medicines Research Center, Department of Pharmacognosy, Kerman University of Medical Sciences, Kerman, Iran 6. Regional Center of Advanced Technologies and Materials, Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, 783 71 Olomouc, Czech Republic Received 08 Jan 2019 Abstract Accepted 11 Mar 2019 Background: Pentavalent antimonials such as meglumine antimoniate (MA, Glucantime), are the first-line treatment against leishmaniasis, but at present, they have basically lost their efficacy. This study was aimed to explore epigallocatechin 3-O-gallate (EGCG), alone or in combination with MA against Leishmania tropica stages. Keywords: Methods: All experiments were carried out in triplicate using colorimetric assay, macrophage Epigallocatechin 3-O-gallate; model, flow cytometry and quantitative real-time PCR. This experimental study was carried out Leishmania tropica; in 2017 in Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Immunomodulatory role; Iran. Apoptosis; Results: Promastigotes and amastigotes were more susceptible to EGCG than MA alone, but Gene expression the effect was more profound when used in combination. EGCG exhibited high antioxidant level with a remarkable potential to induce apoptosis. Furthermore, the results showed that the level of gene expression pertaining to Th-1 was significantly up-regulated (P<0.001). Conclusion: EGCG demonstrated a potent anti-leishmanial effect alone and more enhanced *Correspondence Email: lethal activity in combination. The principal mode of action entails the stimulation of a synergis- [email protected] tic response and up-regulation of the immunomodulatory role towards Th-1 response against L. tropica. 521 Available at: http://ijpa.tums.ac.ir Saduqi et al.: Anti-Leishmanial and Immunomodulatory Effects of Epigallocatechin … Introduction eishmaniasis is a neglected tropical and crobial (against bacteria, viruses and fungi) subtropical complex disease caused by and antiparasitic activities with diverse ranges Lover 20 species of the genus Leishmania of therapeutic potentials (8). (1). Cutaneous leishmaniasis is the most fre- The existence of the basic effector Th-1 and quent type which consists of approximately Th-2 subtypes of T-lymphocytes (CD4) is 70%-75% of the overall reported cases (2). now well accepted, and is being used to design The magnitude of the disease burden due to therapeutics (drugs) and prophylactic (vaccine) the chronic conflict in the Middle East coun- strategies. Although the killing mechanism of tries has significantly been increased by a fac- the host cells is so complex and multifactorial, tor of 6 to 10. Consequently, CL represents a the functions of Th-1 and Th-2 cells correlate major and large-scale global health challenge well with their distinctive cytokine pattern (9). (3). Th-1 is basically involved in cell-mediated de- Biological control measures of numerous layed-type hypersensitivity (DTH) response vectors and reservoirs are impossible and and provides killing mechanism against the there is neither efficacious vaccine nor effec- leishmanial stages which ultimately can be- tive drugs against all forms of leishmaniasis come suppression. On the other hand, Th-2 available universally. Pentavalent antimony cytokines encourage antibody production, are compounds (SbV) such as meglumine anti- commonly found in association with strong moniate (Glucantime) are the first-line of antibody and allergic reactions and eventually therapy and are widely used drugs over the last are responsible for the progression of parasite seven decades, which have significantly lost infection (10). their efficacy. Moreover, the choice of other So far, no study has been carried out to ex- chemical alternatives for the same reasons is plore the effect of the major component of extremely limited. In fact, all of these synthetic green tea, epigallocatechin 3- O-gallate chemicals have insufficient action and emer- (EGCG) on the etiological agents of CL in the gence of disease is a common phenomenon Old World. EGCG is the most abundant, (4,5). widely used and the most effective constituent Antioxidants are collectively a diverse group of green tea with great anticancer properties of compounds, including natural products, (11,12) and potent leishmanicidal activities which possess beneficial health effects. Limi- against leishmanianiasis in the New World tation for insufficient action of SbV empha- species (13–16) which possesses a broad range sizes an urgent need for new additive natural of antimicrobial effects (17). products, combinatory medicines or new In addition, this study was primarily de- treatment modalities to be used as alternative signed to evaluate the leishmaniacidal effect of medicines against leishmaniasis (6,7). Medici- EGCG and its antioxidant level, cytotoxic in- nal plants, vegetables, fruits, polyphenols and dex, apoptotic values and potentials to express many other plant extracts possess antioxidant distinct gene profiling, alone or in combina- properties that are effective against the infec- tion with Glucantime, the drug of choice tious agents, degenerative diseases, cancer, against various types of leishmaniasis, includ- neurologic and cardiovascular disorders, and ing the mechanism of action. We believe such reactive oxygen species (ROS) generated as a broad range of experimental sets performed the result of oxidative stress in normal meta- here are unique and able to elucidate the po- bolic processes. Phenolic flavonoids such as tential effects of the active constituent of green tea consist a wide spectrum of antimi- green tea, EGCG on the L. tropica pro- Available at: http://ijpa.tums.ac.ir 522 Iran J Parasitol: Vol. 14, No. 4, Oct-Dec 2019, pp.521-533 mastigote and amastigote stages. This investi- The parasite was diluted in complete medi- gation could also be used as a model for the um and counted by a Neubauer chamber. Mu- Leishmania species in the Old World. rine macrophage cell-line (J774-A1; ECACC no. 91051511) was prepared from the Pasteur Materials and Methods Institute of Iran (Tehran). Parasite and macrophage cell-line Reagents This experimental study was carried out in Rosewell Park Memorial Institute medium 2017 in Leishmaniasis Research Center, Ker- (RPMI 1640), FCS, Pen/Strep, MTT (3-(4,5- man University of Medical Sciences. The dimethylthiazol-2-yl)-2,5-diphenyltetrazolium standard strain of L. tropica promastigotes bromide, a tetrazole), DPPH (zinc sufate, 2,2- (MHOM/IR/75/Mash2) were seeded in diphenyl-1-picrylhydrozy), BHA (butylated RPMI 1640 medium, supplemented with 100 hydroxyanisole) and epigallocatechin 3-O- IU penicillin/mL, 100 µg streptomycin/mL, gallate (EGCG) (Fig.1) were purchased from 10%(v/v) fetal calf serum (FCS) at 56 °C for Sigma-Aldrich, France. Glucantime (meglu- 30 min and allowed to grow at 24 °C±1 °C mine antimoniate) was kindly provided by the and pH 7.2 (all were purchased from Sigma, Provincial Health authorities in Kerman Uni- Aldrich, France). versity of Medical Sciences originally pur- chased from Sanofi-Aventis, France. Fig. 1: Epigallocatechin-3-O-Gallate Structure, the main component of green tea Anti-promastigote assay nation of EGCG plus Glucantime in RPMI The activity of EGCG, Glucantime alone or 1640 (pH 7.2) in 96-well microplate, were in combination, by a standard colorimetric cell prepared. Logarithmic-phase promastigotes viability MTT assay for determining the sus- (2x106) were harvested from the culture medi- ceptibility of promastigotes was evaluated um and incubated at 24 °C±1 °C for a stand- (18). Various dilutions of EGCG or Glucan- ard time of 72 h. Promastigotes were cultured time (25, 50, 100, 200, 400 and 500µg/mL) in complete medium with no EGCG or Glu- and the same serial concentrations for combi- cantime used as the untreated control group 523 Available at: http://ijpa.tums.ac.ir Saduqi et al.: Anti-Leishmanial and Immunomodulatory Effects of Epigallocatechin … and complete medium with no parasites as the with FITC annexin V and 7AAD, for flow blank. All experiments were repeated thrice cytometry to promastigotes, was evaluated. (19). The experimental reactions were stopped The serial dilutions of EGCG and Glucantime by isopropanol alcohol and read by spectro- were prepared on the day of experimentation photometer at 492 nm (BioTek- ELx800 as previously mentioned. The anti- Winooski,