Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model

Jila Sherafati; Mohammad Saaid Dayer; Fatemeh Ghaffarifar; Kamran Akbarzadeh; Majid Pirestani

doi:10.1371/journal.pone.0283355

. 2023 Aug 3;18(8):e0283355. doi: 10.1371/journal.pone.0283355

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model

Jila Sherafati ^1,², Mohammad Saaid Dayer ^1,^*, Fatemeh Ghaffarifar ¹, Kamran Akbarzadeh ³, Majid Pirestani ¹

Editor: Alireza Badirzadeh⁴

PMCID: PMC10399734 PMID: 37535629

Abstract

Leishmaniasis is a zoonotic disease caused by an intracellular parasite from the genus Leishmania. Lack of safe and effective drugs has increasingly promoted researches into new drugs of natural origin to cure the disease. The study, therefore, aimed to investigate the anti-leishmanial effects of Lucilia sericata larval excretion/secretion (ES) in combination with Apis mellifera honey as a synergist on Leishmania major using an in vitro model. Various concentrations of honey and larval ES fractions were tested against promastigotes and intracellular amastigotes of L. major using macrophage J774A.1 cell line. The inhibitory effects and cytotoxicity of ES plus honey were evaluated using direct counting method and MTT assay. To assess the effects of larval ES plus honey on the amastigote form, the rate of macrophage infection and the number of amastigotes per infected macrophage cell were estimated. The 50% inhibitory concentration (IC₅₀) values were 21.66 μg/ml, 43.25 60 μg/ml, 52.58 μg/ml, and 70.38 μg/ml for crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey alone, respectively. The IC₅₀ for positive control (glucantime) was 27.03 μg/ml. There was a significant difference between viability percentages of promastigotes exposed to different doses of applied treatments compared to the negative control (p≤ 0.0001). Microscopic examination of amastigote forms revealed that dosages applied at 150 to 300 μg/ml significantly reduced the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Different doses of larval products plus honey did not show a significant toxic effect agaist macrophage J774 cells. The larval ES fractions of L. sericata in combination with A. mellifera honey acted synergistically against L. major.

Introduction

Leishmaniasis is a complex of vector-borne diseases caused by different species of an obligate intracellular protozoa of the genus Leishmania [1, 2]. These zoonotic diseases manifest in three main forms including cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL). Species such as Leishmania major, L. tropica, L. infantum and L. aethopica in the Old World and L. perivuana, L. braziliensis and L. mexicana in the New World are transmitted by infected bites of different sand-fly species to cause CL in humans [3]. The increasing prevalence of CL in new geographical areas has been attributed to poverty, immune-related diseases, migration, disasters, and drug resistance [4, 5]. Despite application of many control and prevention programs, CL is still persistent in some areas due to widespread insect vectors, poor health, income level, and drug resistance (CDC ref). About 95% of CL cases occur in the Americas, the Mediterranean basin, the Middle East, and Central Asia. The new CL cases are estimated at between 600,000 and 1 million worldwide annually [6].

CL is more likely to cause dry (urban) and wet (rural) wounds leaving ugly scars in exposed areas of the body such as the hands and the face, imposing economic and health burdens [7, 8]. The routine therapies of CL include using pentavalent antimony compounds (Sb), thermotherapy (hot and/or cryo), and topical and intralesional administration of Paromomycin, Amphotericin B and Pentamidine. The former being the first line therapy in many countries. However, the antimonial drugs are associated with clinical side effects such as cardiac toxicity, clinical pancreatitis, arthralgia, myalgia, nausea/vomiting, liver transaminase abnormalities, pancytopenia, and renal toxicity. This is why many studies are underway to find alternative natural compounds [9–11]. Nowadays, researchers have presented evidence that natural compounds including insect products could be promising candidates for development of new anti-Leishmania agents [12, 13].

The larvae of Lucilia sericata (Diptera: Calliphoridae) are used for maggot therapy of drug-resistant chronic wounds [14]. The maggot therapy results in removal of necrotic tissue, stimulation of granulation and production of antiseptics against pathogenic microorganisms such as gram-positive and negative bacteria, fungi, and parasites, particularly Leishmania [15–17]. In addition, larval excretion/secretion (ES) of L. sericata involves of proteolytic enzymes (trypsin-like and chymotrypsin-like serine proteases, metalloproteinase and aspartyl proteinases) which are able to digest bacteria and necrotic tissue [18, 19].

On the other hand, honey produced by Apis mellifera (Hymenoptera: Apoidea) has strong antibacterial activity and helps clearing the infection [20, 21]. Clinical and laboratory evidences showed that honey, also, has antimicrobial properties against a wide range of viruses, fungi and protozoa [22–24]. The antibacterial activity of honey has been attributed to its natural hydrogen peroxide content [25]. The production of hydrogen peroxide by glucose oxidase in honey does not cause any tissue damage but prevents microbial invasion [25–27]. Therefore, honey has been effective in healing CL lesions [28].

In the present study, anti-leishmanial activity of honey in combination with crude and fractionated ES of L. sericata larvae was investigated in an in vitro model.

Materials and methods

Ethics statement

This study did not involve animals and was performed in vitro. However, the Ethics committee of Tarbiat Modares University approved all protocols described in the current study (Approval No. IR.MODARES.REC.1399.124).

Insect rearing and larval ES collection

To obtain larval ES, about 100 stage II and III larvae of L. sericata were selected from previously established colonies and disinfected as described by Cruz-Saavedra et al [29, 30]. The ES was collected by centrifugation at 4000 g for 10 minutes [17]. Finally, the upper phase was removed and used for experimentation.

Fractionation of the crude ES

The fractionation of the crude ES into two fractions of >10 and <10 kDa was performed through amicon ultra_4 fillers by centrifugation at 7500 g for 40 minutes following the manufacturer’s instructions. For sterilization, both the crude ES and its fractions were passed through 0.22 μm syringe filter. The sterilized products were cultured on agar blood medium and incubated at 35°C for 48 h to ensure no microbial growth happens. The crude ES and its fractions were then stored at -20°C until use.

Bradford protein assay

The amount of protein in solution was determined using a standard curve obtained by ploting OD readings against a serial dilution of Bovine serum albumin (BSA) concentrations (0, 31.25, 62.5, 125, 250, 500, 1000 μg/ml) tittered by the macro assay (No.DB9684-50ml) kit, following the manufacturer’s instructions. The reactions took palce in 8-well plates upon incubation at 25°C for 5 minutes. The asorbance was measured using an ELISA reader (Model 680, BIORAD) at 570 nm [31].

Analysis of larval ES products by SDS-PAGE

The protein profile of the crude and fractionated larval ES (>10 and <10 kDa) of L. sericata were analyzed using 1-mm-thick 12.5% Sodium dodecyl sulfate–polyacrylamide gel at a 110-V constant voltage using “Mini-Protein III” system (Bio-Rad) under reduction conditions. The gels were then stained with a solution containing Coomassie brilliant blue G250 and methanol. The molecular weights of the protein bands were estimated in comparison with a prestained protein ladder (PAGEmark, 786–418). About 20–25 protein bands were observed for crude ES, which corresponded to bands in both the >10 kDa and <10 kDa fractions. The protein profiles of larval ES and its fractions were presented in our previous study [30]. The profile obtained from ES separation showed that the separation was done with high accuracy.

Honey preparation as a synergist

The synergistic effects of honey in combination with either the crude ES or ES fractions were tested using honey purchased from a beekeeper (bees fed on Astragalus spp) from Saqqez City in Kurdistan Province. The honey was purely natural as no sucrose was used to feed the bees. The honey solution was first filter sterilized and then tested for any microbial contamination on blood agar medium by incubation at 35°C [32]. In the laboratory, honey solutions were prepared by dilution with distilled water to obtain the following concentrations: 350, 300, 250, 200, 150, 100, 50 and 25 μg / ml [33, 34]. Finally, the solutions were stored at -20°C until used [33].

Parasite cultivation

L. major strain MRHO/IR/75/ER was obtained from Pasteur Institute of Iran. The promastigotes were incubated in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, USA) and 100 μg/mL penicillin-streptomycin (Thermo Fisher Scientific, USA) at 26°C.

Culture of the murine macrophage cell line

The mouse macrophage cell line J774A.1 was purchased from Pasteur Institute of Iran (https://fa.pasteur.ac.ir/) registered under the Cell Bank Code: C483. The cell line J774A.1 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% heat-inactivated FBS (Gibco, USA) and 100 μg/mL penicillin-streptomycin (Thermo Fisher Scientific, USA). The cell suspension was incubated in 25 ml cell culture flasks (Jet Biofil) at 37°C under a humidified atmosphere of 5% CO₂.

Promastigote assay

Promastigotes of L. major were cultured in RPMI medium supplemented with 20% FBS in 96-well plates (SPL, Korea) at a concentration of 1× 10⁶ cells/mL in the presence of honey, the crude ES + honey, ES >10 kDa + honey, ES<10 kDa + honey and incubated at 26°C. A serial dilution was performed from the crude ES and its products plus honey with initial concentration of 350 μg/ml. Glucantime was added to the promastigote culture in the positive control, whereas in the negative control, the cell culture was left without treatment. All experiments were performed in triplicate. In order to determine the parasite viability, the proliferation of promastigotes was determined by cell counting using a hemocytometer (Neubauer chamber) after 24, 48 and 72-h incubation [35].

Macrophage cytotoxicity test

Mice macrophage cell monolayers (cell line J774A.1) were washed with phosphate buffered saline (PBS) for 5 minutes at 37°C followed by washing with DMEM medium containing 10% FBS, then centrifuged at 200 g for 10 minutes at 4°C. The cell suspension was cultured in a 96-well plate (SPL, Korea) at 1×10⁶ cells/mL concentration for 24 h. The cultured cells were then exposed to different concentrations of compounds including honey, honey + crude ES, honey + ES >10 kDa, honey + ES <10 kDa, glucantime (positive control) and PBS (negative control). Then, the plates were incubated at 37°C for 24 h under 5% CO₂ atmosphere. Then, 20 μl of MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at final concentration of 5 mg/ml was added to each well. The experimental plates were again incubated at 37°C for 4 h, followed by removal of the top liquid from the wells. Subsequently, 100 μl of dimethyl sulfoxide (DMSO) was added to each well. The optical absorbance was read after 15 minutes using an ELISA reader (Model 680, BIORAD Co.) at a wavelength of 570 nm [36].

Amastigotes assay

Macrophage cells (J774A.1) were seeded at 2 × 10⁶ cells/mL on glass coverslips in 12-well culture plates (SPL, Korea) and incubated at 37°C under 5% CO₂ atmosphere for 24 h [6]. Non-adherent cells were washed with PBS. Adhered macrophages were exposed to 1 × 10⁵ stationary phase Leishmania promastigotes/well and incubated at 37°C under 5% CO₂ atmosphere for 24 h. Extracellular promastigotes in the wells were removed by washing with PBS. Subsequently, test compounds were added to the experimental plates at different concentrations based on IC₅₀ values obtained in promastigote assay. The compounds included 1) honey (350 and 150 μg/ml); 2) honey + crude ES (350 and 150 μg/ml); 3) honey + ES >10 kDa (350 and 150 μg/ml); 4) honey + ES <10 kDa (350 and 150 μg/ml); and 5) glucantime (100 and 50 μg/ml). The experiments were done in triplicate. The negative control did not receive any treatment. After 72 hours, the glass coverslips were fixed with methanol, stained with 10% Giemsa and examined by light microscopy using immersion oil. The number of infected macrophages and the average number of parasites per macrophage in 100 cells were counted randomly in ten fields under a light microscope.

Statistical analyses

The standard curve and its equation were obtained through online software at (https://www.aatbio.com)) and the graph was replotted using GraphPad Prism in the Saturation Binding Data Graph section. The statistical analyses of results were done using GraphPad Prism version 6.07 by one-sample t-test and ANOVA. Data were presented as the means ± standard deviation (SD). The independence of the two categorical variables was determined using chi-square and / or Fisher’s exact tests. The dose-response curve was plotted to determine the IC₅₀ of promastigote assay using nonlinear regression. Amastigote infection data was evaluated using the equations presented in [37]. Statistical significance was assigned at the level of (P<0.05).

Results

Bradford protein assay

Bradford protein assay was performed to calculate the protein concentration of L. sericata larval products. The average net absorbance at 595 nm for seven standard dilutions from low to high concentrated were as follows; 0.72, 0.785, 0.825, 0.901, 1.01, 1.25, and 1.84. Average net absorbance at 595 nm for the crude ES, ES >10 kDa, and ES <10 kDa were 1.62, 1.48 and 1.16 respectively. The average protein concentrations for the crude ES, ES >10 kDa, and ES <10 kDa were 809.8, 680.6, and 385.3 μg/ml respectively (Fig 1). Consequently, the maximum concentration used in the current study was 350 μg/ml.

Synergistic effects of ES products plus honey on promastigotes growth

The IC₅₀ values and Area Under the Curve (AUC) of the crude ES + honey, ES >10 kDa + honey, ES <10 kDa + honey, honey, and glucantime were evaluated against promastigotes at 24, 48, and 72 h. The lowest IC₅₀ values for the crude ES + honey, ES >10 kDa + honey, ES <10 kDa + honey, honey, and glucantime after 72 h exposure time were 21.66 μg/ml (log = 1.336), 43.25 μg/ml (log = 1.636), 52.58 μg/ml (log = 1.721), 70.38 μg/ml (log = 1.847), and 27.03 μg/ml (log = 1.423) respectively (Fig 2). Regardless of the applied doses, the viability percentages of promastigotes in treated groups was significantly lower than that in the negative control (p ≤ 0.0001). On the other hand, there were no statistically significant differences in the viability percentages of promastigotes treated with the crude ES + honey, ES >10 kDa + honey, and ES <10 kDa + honey compared to glucantime with P values equal to 0.4029, 0.9011, and 0.6972 respectively, though the difference was significant between honey and glucantime (P = 0.0118). The crude ES + honey showed higher toxicity at low concentrations compared with glucantime. Fig 3 shows that longer exposure time and higher concentration of ES plus honey lead to greater toxicity to promastigotes (Figs 2 and 3).

Fig 2 — (a) crude ES + honey. (b) ES >10 kDa + honey. (c) ES <10 kDa + honey. (d) Honey. (f) Glucantime at 24, 48 and 72 h. intervals.

Fig 3 — Mean (±SD) numbers of viable promastigotes exposed to various combinations of ES products at different doses and time intervals: (a) 24 h. (b) 48 h. (c) 72 h. Treatment groups significantly differed from the control group at P < 0.05.

Synergistic cytotoxicity of ES products plus honey to macrophages by MTT

MTT test showed that ES products plus honey exerted synergistic effects on the macrophage cell line J774A.1 after 48 and 72 hours in a dose-dependent manner. The crude ES + honey, ES >10 kDa + honey, ES <10 kDa + honey induced the same effects on the viability of macrophage cells as glucantime did with no significant difference between them (P > 0/05). The difference between honey and glucantime treatment was significant, in other words, honey showed less toxicity (P = 0.0141). However, all treatment groups as well as glucantime were significantly different from the negative control (P < 0/0001) (Fig 4).

Susceptibility of amastigotes to ES products plus honey as a synergist

After 72 hours’ exposure time, honey alone and honey plus larval ES products significantly reduced the infection rate of amastigotes and inhibited their growth compared to the control group (P = 0.004 and P = 0.0002, respectively). There was a statistically significant difference between ES <10 kDa plus honey compared to glucantime against amastigotes in terms of infection rate (P = 0.048 and P = 0.039 respectively) and percentage of growth inhibition (P = 0.033 and P = 0.004 respectively). There was no significant difference between infection rates of amastigotes exposed to the crude ES plus honey and ES >10 kDa plus honey compared with those exposed to glucantime (P = 0.559 and P = 0.184). The former treatments inhibited amastigotes growth rates in macrophages in the same manner compared with glucantime at P = 0.221 and P = 0.453 respectively. The effect of ES <10 kDa plus honey was significantly less that glucantime in term of reduction of infection rate and growth inhibition (P = 0.041 and P = 0.003 respectively) (Fig 5 and Table 1).

Table 1. Parameters indicating susceptibility of L. major amastigotes to L. sericata larval ES products with honey as a synergist.

Treatment	Dosages (μg/ml)	Infected cells %	Parasite load	Survival index
No treatment control	0	85.37 ± 3.575	3.894 ± 0.091	273.97 ± 1.151
Crude ES + honey	150	40 ± 2.828	1.33 ± 0.087	53.45 ± 6.187
	300	20.67 ± 3.535	1.081 ± 0.155	22.09 ± 1.661
ES >10 kDa + honey	150	49 ± 2	1.868 ± 0.051	91.6 ± 6.233
	300	43.564 ± 2.516	1.535 ± 0.046	66.908 ± 8.11
ES <10 kDa + honey	150	58.34 ± 2.517	2.217 ± 0.06	129.426 ± 6.118
	300	49.01 ± 1.91	2.1 ± 0.014	103.04 ± 3.12
Honey	150	66.33 ± 2.515	2.517 ± 0.151	166.932 ± 4.323
	300	57.3 ± 1.527	2.273 ± 0.61	130.38 ± 3.994
Glucantime	50	39.66 ± 2.081	1.96 ± 0.038	77.89 ± 1.52
	100	30.23 ± 1.527	1.68 ± 0.061	51.957 ± 3.56

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For the negative control group, the parasite load was 3.894 ± 0.091 amastigotes per macrophage, whereas those for groups treated with the crude ES plus honey and ES >10 kDa plus honey at 300 μg/ml concentration were 1.081 ± 0.155 and 1.535 ± 0.046 amastigotes/macrophage cells respectively. The latter treatments both resulted in lower amastigotes/macrophage compared with glucantime applied at 100 μg/ml concentration (1.68 ± 0.061 amastigotes/macrophage). However, treatment with ES <10 kDa plus honey and honey at 300 μg/ml resulted in 2.1 ± 0.014 and 2.273 ± 0.61 amastigotes/macrophage respectively. These were less effective in comparison with glucantime. The survival indexes of amastigotes in groups treated with glucantime and the negative control were 51.927 ± 3.56 and 273.97 ± 1.151 respectively. The survival indexes in groups treated with the crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey at 300 μg/ml concentrations were 22.09 ± 1.66, 66.908 ± 8.11, 103.39 ± 3.12, and 130.38 ± 3.994 respectively (Table 1).

Discussion

Being a zoonotic diseases, the cutaneous leishmaniasis presents a major public health problem worldwide, especially in the tropics and subtropics [38, 39]. Due to high toxicity, cost, side effects and drug resistance incurred by pentavalent antimonials, researches are increasingly focused on finding cheaper drugs with minimal or no side effects [40, 41]. Insect-derived products including larval peptides and honey have been widely used to treat chronic wounds since ancient times [42]. The current study aimed to investigate leishmanicidal effects of L. sericata derived larval ES in combination with honey as a synergist on L. major promastigotes, the causative agent of cutaneous leishmaniasis (CL). In addition, the effects of two ES fractions together with honey were studied against the macrophage cell line J774A.1 and intracellular amastigotes.

Recent findings revealed that honey is a potent agent for cleaning infected wounds and has antimicrobial properties against a wide range of microbes, fungi and protozoa [33, 43, 44]. Its effectiveness on Leishmania parasite has been also reported [45]. In the present study, honey showed antileishmanial activity against L. major cells in accordance with others’ findings [46]. Consistent with our study, honey incorporated with hydroalcoholic extract of Nigella sativa was found effective against Leishmania parasite [24]. Other studies reported the same results as in our study [28, 33, 47].

In agreement with previous reports, our results indicated the effects of larval ES plus honey on L. major promastigotes [37, 48, 49]. Comparison of IC₅₀ values of different combinations showed that larval ES plus honey exerted a higher toxic effect on promastigotes than each of the components as stand-alone treatment, though all treatments showed significant differences with the negative control (p < 0.0001). The same results were reported by other researchers [37, 49]. They even showed that highly concentrated larval ES exerted stronger lethal effects on exposed promastigotes than macrophage cells [37, 50].

A study involving cytotoxicity tests of larval ES products of Calliphora vicinia and L. sericata against macrophage (J774A.1) and L. major cells resulted in more than 40% toxicity to cells. This toxicity rate was higher than that reported in our study, although the ambiguity over the exact concentration used in aforementioned study makes the comparison difficult [51]. The report by Pinilla et al on the toxic effects of Sarconesiopsis magellanica larval ES on fibroblasts using the human lung-derived MRC5 cell line contradicted our reports. The authors showed that the larval ES was lethal to MRC5 cells at low concentrations [52]. They also indicated that the survival rate of the cells did not change when exposed to larval ES products of L. sericata and S. magellanica at concentrations below 10 μg/ml, yet, the products became effective as the concentrations approached 20 μg/ml. This discrepancy with our results may be attributed to different insect strains, cell lines and laboratory conditions. However, the study by [37] who examined the effects of L. sericata larval products on peritoneal macrophages and J774A.1 cells, and that by [49] who examined ES products of L. sericata and S. magellanica on U937 cell line, were consistent with our results.

The larval products of L. sericata with honey had strong anti-Leishmania effects against intracellular amastigotes of L. major. All combinations reduced the infection rate and parasite load of infected macrophages, but the efficacy was even stronger when applying the crude ES plus honey and ES> 10 kDa plus honey compared with glucantime at 100 μg/mL. We found synergism between larval products and honey especially for the crude ES and ES> 10 kDa at sub-lethal concentrations against L. major infecting macrophage cell line J774A.1. The reduction in infection rate was more than 50% with the application of the crude ES plus honey and ES> 10 kDa plus honey at 150 μg/ml concentration and ES <10 kDa plus honey at 300 μg/ml concentration. However, these treatments were less toxic to the used macrophage cell line. The cell line maintained more than 88% viability when treated with the crude ES plus honey, ES> 10 kDa plus honey and ES< 10 kDa plus honey at 300 μg/ml concentrations.

Our finding was consistent with the report [37] in which the larval saliva and hemolymph of L. sericata larvae were tested on macrophage cell line J774A.1 infected with the L. tropica parasite. A similar result was obtained by examination of larval ES of L. sericata and S. magellanica on U937 cell line infected with Leishmania panamensis parasite [49]. The larval ES produced sharper decrease in parasite survival index at higher concentrations. This was consistent with the study by Rahimi et al [37], but contrasted earlier studies in which susceptibility of J774A.1. cell line infected with L. major amastigotes and U937 human cell line infected with L. panamensis amastigotes was evaluated [49, 51].

The parasite load and survival index values of macrophage cells infected with L. major were lower upon treatment with the crude ES plus honey and ES> 10 kDa plus honey compared with ES< 10 kDa plus honey or honey alone. Furthermore, significant decreases in parasite loads and survival indexes were observed in treated groups compared to the untreated control (P = 0.05), which was consistent with findings of Rahimi et al [37]. Our study confirmed the result of Sherafati et al that honey acts as a synergist with ES products [30]. Indeed, leishmanicidal effects of larval ES products in combination with honey to intracellular amastigotes provide a promising basis for in vivo experimentation in future.

A number of studies have shown anti-leishmanial effects of larval products on Leishmania species such as L. amazonensis [53], L. tropica [48], L. major [54], and L. panamensis [29] under in vitro and in vivo conditions. Our study provides a new evidence on leishmaniacidal and potential therapeutic effects of larval ES products of L. sericata combined with honey as a synergist to L. major promastigotes and intracellular amastigotes.

Conclusions

To our knowledge, this is the first study dealing with the effects of L. sericata larval-ES products in combination with A. mellifera honey on different infective stages of L. major. The study followed a quantitative approach to test the cytotoxicity of ES products plus honey on mice macrophage cell line J774A.1. It should be noted that the crude ES plus honey at a concentration of 300 μg/ml was more effective than glutamine as a standard drug at a concentration of 100 μg/ml. Also, there was no significant difference between toxicity of ES> 10 kDa plus honey with that of glucantime. Our results showed that there was a synergistic effect between honey and larval ES products for the inhibition of intracellular and extracellular cells of L. major. The larval excretion/excretion product contains a mixture of proteolytic, glycolytic, lipolytic, as well as nucleoid enzymes which are released on wound surfaces for maggot therapy to take place using L. sericata larvae. The following activation of collagenases, gelatinases, sterolisins, MMPs (MMP-2 and MMP-9) enables many biological and pathological processes to accelerate wound healing [55]. However, the inhibitory action of ES components of Lucilia sericata in combination with honey or its derivatives such as royal jelly or wax in against Leishmania major needs further investigation both under in vitro and in vivo conditions.

Supporting information

S1 File

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Acknowledgments

The authors would like to thank the Department of Parasitology and Medical Entomology and the Student Research Committee of Tarbiat Modares University for their assistance in this project.

Abbreviations

CL: Cutaneous leishmaniasis
ES: Excretion and secretion
%I: Infection percentage
%DI: Decreased infection percentage
SVI: Survival index
PL: Parasite load. PBS: Phosphate buffered saline
BSA: Bovine serum albumin
NNN: Novy-Macneal-Nicolle
FBS: Fetal bovine serum
RPMI: Roswell Park Memorial Institute
DMEM: Dulbecco’s Modified Eagle Medium
MTT: 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
DMSO: Dimethyl sulfoxide
IC50: Inhibitory concentration for 50% of parasite
SDS: PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
AUC: Area Under Curve
MMPs: Matrix metalloproteinases

Data Availability

All relevant data are within the manuscript and its Supporting Information file.

Funding Statement

This study was financially supported by the Student Research Committee, the Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. (Grant no: Med/8288)”. We also declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0283355.r001

Decision Letter 0

Alireza Badirzadeh

21 Sep 2022

PONE-D-22-17517Evaluating leishmanicidal activity of Lucilia sericata larval products in combination with Apis mellifera honey using an in vitro modelPLOS ONE

Dear Dr. Dayer,

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear authors;

First of all, I congratulate you on carrying out this valuable study. But in my opinion, some improvements are necessary to enrich the manuscript. So, I have listed my comments.

1. The title should be more impressive. Therefore, I suggest “Leishmaniacidal potential of Lucilia sericata products and Apis mellifera honey combination: an in vitro evaluation.”

2. Language editing in the text of the manuscript is required.

3. Please employ MeSh to check your keywords; I think J744 is not an appropriate keyword.

4. In the introduction, you mentioned the existence of three forms of the disease, but it is better to say that there are three main forms of the disease.

5. According to your manuscript, new cases of CL have been estimated at 1.2 million. Still, the website of WHO says,” It is estimated that between 600 000 to 1 million new cases occur worldwide annually”.

6. Although in lines 210 and 216, you indicate the treatment of honey plus Glucantime, I could not find more information in “Material and Method” and table 1. Please clarify the treatment.

7. Despite only one table in the manuscript, you named it “Table 2”! Please correct it.

8. You have mentioned further studies are needed to detect the active components of larval ES and honey. Please mention probable active ingredients, and discuss them in brief.

Best wishes

Reviewer #2: The manuscript entitled " Evaluating leishmanicidal activity of Lucilia sericata larval products in combination with Apis mellifera honey using an in vitro model" describes the effects of larval products of L. sericata mixed with honey on Leishmania major promastigotes and infected macrophages. The following contains some point that should be addressed by the authors.

The Leishmania species (major) used in this study should be included in the title.

The authors should describe the experimental processes to check the purity of the

two fractions of ES (>10 and <10 kDa).

Figure is not necessary.

The viable amastigotes were not evaluated, only % of infected macrophages and number of intracellular amastigotes in J774 macrophage cultures.

A screening for lipopolysaccharide (LPS/endotoxin) contamination in larval products and honey should be performed

Images of treated and untreated promastigotes and infected macrophages could be included in the manuscript.

The authors suggested further studies to detect the active components of larval ES and honey as well as the mechanism of inhibition on the Leishmania parasite (line 314). Some suggestion that these issues could be discussed in the manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Aug 3;18(8):e0283355. doi: 10.1371/journal.pone.0283355.r002

Author response to Decision Letter 0

21 Dec 2022

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Our response:

We did the required modification according to PLOS ONE style patterns. Thanks.

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Our response:

Our study did not involve human or animal specimens. However, the undertaken methodologies of the study were approved by the Ethics Committee of Tarbiat Modares University.

3. Thank you for stating the following financial disclosure:

“MSD has received a grant (Grant no: Med/8288) from Tarbiat Modares University, Tehran, Iran https://www.modares.ac.ir/en No” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Our response:

Thanks for your suggestion. The sentence was edited, and included in the cover letter as instructed.

4. Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“MSD has received a grant (Grant no: Med/8288) from Tarbiat Modares University, Tehran, Iran https://www.modares.ac.ir/en

No” Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Our response:

Thanks for your assistance. The statement was included in the cover letter as required.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter.

Our response:

The supporting data was provided as a separate file and uploaded.

6. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical.

Our response:

Sure. We have done so.

Our response:

Thanks for your attention. We included a reference for the sole table in the main text as required.

8. Please include a copy of Table 1 which you refer to in your text on page 11. [Note: HTML markup is below. Please do not edit.]

Our response:

Sure. We did so.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

-------------------------

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

-------------------------

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy [1] requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

-------------------------

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

5. Review Comments to the Author

Reviewer #1: Dear authors;

First of all, I congratulate you on carrying out this valuable study. But in my opinion, some improvements are necessary to enrich the manuscript. So, I have listed my comments.

1. The title should be more impressive. Therefore, I suggest “Leishmaniacidal potential of Lucilia sericata products and Apis mellifera honey combination: an in vitro evaluation.”

Our response:

Thanks. We agree. The title was changed as kindly suggested.

2. Language editing in the text of the manuscript is required.

Our response:

The whole manuscript has now been thoroughly revised for English style. Thank you.

3. Please employ MeSh to check your keywords; I think J744 is not an appropriate keyword.

Our response:

Thank you for your attention. We substituted it with the word “macrophage”.

4. In the introduction, you mentioned the existence of three forms of the disease, but it is better to say that there are three main forms of the disease.

Our response:

Thank you. Corrected as kindly suggested.

Our response:

Thanks for your attention. The correction was made and a new reference added.

6. Although in lines 210 and 216, you indicate the treatment of honey plus Glucantime, I could not find more information in “Material and Method” and table 1. Please clarify the treatment.

Our response:

Thanks for your attention. We are sorry for this mistake. In fact, we did not have honey + glucantime treatment, but used glucantime as the first line treatment to compare with. So, we have corrected this mistake in the main text.

7. Despite only one table in the manuscript, you named it “Table 2”! Please correct it.

Our response:

Thanks. It has been corrected.

8. You have mentioned further studies are needed to detect the active components of larval ES and honey. Please mention probable active ingredients, and discuss them in brief.

Best wishes

Our response:

Thanks. We have briefly discussed it the text.

Reviewer #2:

The manuscript entitled " Evaluating leishmanicidal activity of Lucilia sericata larval products in combination with Apis mellifera honey using an in vitro model" describes the effects of larval products of L. sericata mixed with honey on Leishmania major promastigotes and infected macrophages. The following contains some point that should be addressed by the authors.

The Leishmania species (major) used in this study should be included in the title.

Our response:

Thanks for your suggestion. We added the species name in the title.

The authors should describe the experimental processes to check the purity of the two fractions of ES (>10 and <10 kDa). Figure is not necessary.

Our response:

As kindly suggested, we have added a section to describe the evaluation method for protein band purity in the main text.

The viable amastigotes were not evaluated, only % of infected macrophages and number of intracellular amastigotes in J774 macrophage cultures.

Our response:

Yes. We did not check the amastigote viability as correctly noticed. But we checked the percentage of growth inhibition, and the infection rate which presented in both Table 1 and Figure 5. So, the necessary corrections have now been introduced.

A screening for lipopolysaccharide (LPS/endotoxin) contamination in larval products and honey should be performed Images of treated and untreated promastigotes and infected macrophages could be included in the manuscript.

Our response:

Thanks for the suggestion. As the ES products and honey, used in this study, were freshly prepared and immediately applied to perform the experimentations, we did not attempt to test LPS contamination. In fact, such tests had not been undertaken by many references which were examined for this paper such as no. 37 and 49. In addition the recent profiling of ES proteins and peptides of Lucilia sericata did not report any LPS trace as an integral component, rather they proved the suppressive action of ES to bacterial LPS. The same was reported by many recent researches for honey being protective against LPS. However, as the respected reviewer has rightly mentioned, the LPS role as an exogenous contaminant may not be ruled out. This is particularly important when any ES products are to be developed as prescription drugs. We will surely consider the suggestion in our future researches. Many thanks.

Our response:

Many thanks. Some explanations were added in the discussion section.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? [2]). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy [3].

Reviewer #1: No

Reviewer #2: No

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PLoS One. doi: 10.1371/journal.pone.0283355.r003

Decision Letter 1

Alireza Badirzadeh

30 Jan 2023

PONE-D-22-17517R1Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro modelPLOS ONE

Dear Dr. Saaid Dayer,

Please submit your revised manuscript by 3/28/2023. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Dear authors;

First, I congratulate you due to carrying out this valuable rearch, and thank you to apply all comments.

Sincerely yours

Reviewer #2: There is no satisfactory section to describe the evaluation method for protein band purity in the revised text.

In addition, figure 1 show only a bovine serum albumin standard curve not a concentration of L. sericata larval products curve. The authors describe in the text: the average net absorbance at 595 nm for seven standard dilutions from low to high…. Average protein concentrations for the crude ES, ES >10 kDa, and ES <10 kDa were 809.8, 680.6, and 385.3 µg/ml …(Figure 1) (lines 175-180).

Please rewrite this section and consider do not show a BSA standard curve to describe protein concentrations of larval products. A table or just the description in the text are good options.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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PLoS One. 2023 Aug 3;18(8):e0283355. doi: 10.1371/journal.pone.0283355.r004

Author response to Decision Letter 1

21 Feb 2023

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

6. Review Comments to the Author

Reviewer #1: Dear authors;

First, I congratulate you due to carrying out this valuable rearch, and thank you to apply all comments.

Sincerely yours

Our response:

Thanks you so much.

Reviewer #2: There is no satisfactory section to describe the evaluation method for protein band purity in the revised text.

Our response:

Thank you for the comment. We have added a section describing the evaluation method of protein purity as we did in our earlier study.

Please rewrite this section and consider do not show a BSA standard curve to describe protein concentrations of larval products. A table or just the description in the text are good options.

Our response:

Thank you for your attention

We have now introduced the required changes in Fig. 1. The previous graph was drawn using online site (at https://www.aatbio.com), which unfortunately did not have the ability to accommodate the results of larval products. Therefore. we have used Prism software, Saturation Binding Data Graph section, to combine and redraw the corresponding data. As shown, the graph now indicates the standard curve as well as the OD’s obtained from crude and fractionated larval extracts.The data analysis part of this point was also added.

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PLoS One. doi: 10.1371/journal.pone.0283355.r005

Decision Letter 2

Alireza Badirzadeh

7 Mar 2023

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model

PONE-D-22-17517R2

Dear Dr. Mohammad Saaid Dayer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Alireza Badirzadeh

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Dear authors;

I believe we must try to develop natural medicines, so your valuable manuscript could get my interest, and you adopted all of my primary comments. Therefore, the current version is completely eligible to be published in the journal.

Sincerely yours

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

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PLoS One. doi: 10.1371/journal.pone.0283355.r006

Acceptance letter

Alireza Badirzadeh

15 Mar 2023

PONE-D-22-17517R2

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model

Dear Dr. Dayer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alireza Badirzadeh

Academic Editor

PLOS ONE

Associated Data

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Supplementary Materials

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information file.

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PERMALINK

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model

Jila Sherafati

Mohammad Saaid Dayer

Fatemeh Ghaffarifar

Kamran Akbarzadeh

Majid Pirestani

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Insect rearing and larval ES collection

Fractionation of the crude ES

Bradford protein assay

Analysis of larval ES products by SDS-PAGE

Honey preparation as a synergist

Parasite cultivation

Culture of the murine macrophage cell line

Promastigote assay

Macrophage cytotoxicity test

Amastigotes assay

Statistical analyses

Results

Bradford protein assay

Fig 1. Bovine serum albumin (BSA) standard curve and OD’s obtained for crude and fractionated larval extracts.

Synergistic effects of ES products plus honey on promastigotes growth

Fig 2. Dose-response curves and Areas Under Curves related to ES products with honey as a synergist on promastigotes.

Fig 3.

Synergistic cytotoxicity of ES products plus honey to macrophages by MTT

Fig 4. Viability of macrophage cells exposed to different concentrations of larval products + honey as a synergist.

Susceptibility of amastigotes to ES products plus honey as a synergist

Fig 5. Susceptibility of L. major amastigote grown in macrophages to L. sericata larval products plus honey as a synergist compared with glutamine at 72 h. post-treatment (level of significance * P < 0.05).

Table 1. Parameters indicating susceptibility of L. major amastigotes to L. sericata larval ES products with honey as a synergist.

Discussion

Conclusions

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Alireza Badirzadeh

Roles

Author response to Decision Letter 0

Decision Letter 1

Alireza Badirzadeh

Roles

Author response to Decision Letter 1

Decision Letter 2

Alireza Badirzadeh

Roles

Acceptance letter

Alireza Badirzadeh

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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