Antioxidant and Anticancer Activities of Some Maggots Methanol Extracts

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 12(1) pp: 111119 (2019) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Despite considerable progress in medical research, cancer is still the second most common cause of death according to the World Health Organization 2018 factsheet report and by 2020 it will have to cause the deaths of more than 10 million people.Surgical management of tumors is a widely accepted cancer treatment for many tumor types, and there have also been advances in the development of targeted therapies for cancer.However, surgery, chemotherapy and radiation treatment have serious side effects; therefore, there is a need for new, powerful, and highly effective anticancer agents.
Oxidative response plays a vital role in human health.An antioxidant is a molecule capable of slow/prevent the oxidation of other molecules and so prevent such pathogenic changes, free radicals are ubiquitous in our body and are generated by some physiological processes to eliminate invading these pathogenic microorganisms.Many diseases, including neurodegenerative diseases, autoimmune skin diseases, cardiovascular diseases, and chronic renal failure (Zhu et al., 2013), Cancer (Roy et al., 2015) are due to the oxidative stress activation and the deficiency of intracellular antioxidant defenses.Therefore, unstable and reactive free radicals can be reduced by antioxidants that protect cells from free radical attack (Souri et al., 2008).
One source of novel natural compounds that may have therapeutic utility in cancer and other diseases are insects.Most available antioxidant and antitumor agents are derived from plant, microbial and animal secondary metabolites (Oliver et al., 1994;Cragg et al., 1997), however, few data are available on insect-derived molecules.Insects comprise approximately 55% of total biodiversity and approximately 85% of all animal diversity (Chernysh et al., 2002).Insects are present in many types of ecological systems, from waterways to septic environments, stimulating scientists to look for a cheap and abundant supply of therapeutics in this arthropod class (Roy et al., 2015).In this study, the therapeutic potential of maggot extracts was examined as previously there have been few studies investigating the utility of maggot-derived compounds as cancer therapeutics.
Since few data are available regarding the antioxidant and anticancer activities of insect-derived materials, especially flies' maggots, the objectives of this study were to investigate some maggots crude extract from three representative species, M. domestica, L. sericata and C. albiceps, as a potential source for novel cancer agents as well as giving protection and preventing from diseases.

Tested Species:
Musca domestica (Diptera: Muscidae), Lucilia sericata and Chrysomya albiceps (Diptera: Calliphoridae) larvae were obtained from Medical Entomology Insectary, Animal House, Department of Zoology, Faculty of Science, Al-Azhar University and reared under controlled conditions of temperature, relative humidity and photoperiods.A standard rearing procedure (Busvine, 1962;Queiroz and Milwardde-Azevedo, 1991) was applied to provide maggots needed for the bioassay.

Preparation of Crude Maggots Extract:
Crude maggots extract was prepared according to Hassan et al., (2018) as the following; 500 of M. domestica, L. sericata and C. albiceps third instar maggots were washed with ethanol 70% as a disinfectant then with deionized water; the excess of water removed using filter paper.Each maggot species was completely homogenized in 50ml of 40 mM tris-HCl (pH 7.4).The homogenate was centrifuged at 10,000 rpm for 30 min at 4°C and the supernatant was used as buffer extract.Pure methanol was added to the residue and centrifuged again.The supernatant was taken and considered as methanolic extract.Extracts from tested species were dried and kept in the freezer at -4°C until needed for treatments.Antioxidant Assays :

DPPH Radical Scavenging Activity:
The free radical scavenging activity of the extracts was assayed using the stable free radical diphenyl-2-picryl hydrazyl (DPPH) 0.1 mM -DPPH solution, was obtained from Sigma Aldrich GmbH, Sternheim, Germany, it was added to 3 ml of each maggot extract.The mixture was shaken vigorously and incubated at room temperature for 30 min.The absorbance was measured at 517 nm using a Bausch and Lomb spectrophotometer 710 (Widowati et al., 2011).The IC 50 values were calculated using a log dose inhibition curve, and serial concentrations were prepared for each extract.A lower reaction absorbance indicates higher free radical activity (Choudary et al., 2006).The experiment was achieved in triplicates.The DPPH scavenging activity was calculated from this equation: DPPH scavenging (%) = (A 0 -A 1 )/A 0 ×100, where A 0 is the control absorbance and A 1 is the sample absorbance.The antioxidant and free radical scavenger eugenol was used as a positive control at concentration 50 μg/ml (Shekhar and Anju, 2014).

Total Antioxidant Capacity:
The total antioxidant capacity of the tested extracts was determined by Abdel-Hady et al. (2018).An aliquot of 0.1 mL of sample (200 μg/mL) solution was mixed with 1 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).Blank was contained 1 mL of the reagent solution and nearly volume of the same solvent used for the samples.The tubes were capped and incubated at 95°C for 90 min.After the samples had cooled to 25-27°C, the absorbance of the mixture was measured at 695 nm versus the blank and ascorbic acid was used as a standard.The experiment was repeated for 3 times.The antioxidant activity of the extracts was expressed as mg AAE eq./g extract.Cytotoxic Assay: 1-Cell Culture: Colon cancer (Caco-2) cell lines were obtained were obtained from the VACSERA-Cell Culture Unit, Cairo, Egypt.These cell lines were originally obtained from the American Type Culture Collection.

2-In Vitro Cytotoxicity by MTT Assay:
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay is based on the conversion of MTT into formazan crystals by living cell, which determines mitochondrial activity (Hansen et al., 1989).The tested extracts were tested at concentrations ranged from 25 to 0.048 μg/mL.Colon cancer (Caco-2) cell lines were cultured in RPMI-1640 medium (Sigma Co., St Louis, USA) supplemented with 10% inactivated fetal bovine serum (FBS; Gibco, UK), 100 units/ml penicillin and 100 µg/ml streptomycin.The cells were seeded in a 96-well plate at a density of 1.0x10 4 cells/well at 37 °C for 48 h in a 5% CO 2 incubator and humidified atmosphere in a sterile environment until half-confluent monolayer formed.The 200 μL of treatment medium (serum-free medium) was added in an independent manner to the cells.100 μL of MTT dye was added to each well then incubated for 2 hrs.Cells were washed by 100 μL of PBS and 150 μL of MTT destaining solution was added on microtiter shaker for at least 10 min or until all MTT dye that has been extracted formed the homogeneous solution.The optical density of each well was measured at 570 nm with an ELISA microplate reader (EXL 800 USA).The relative cell viability percentage was calculated by (A570 of treated samples/A570 of the untreated sample) X100, (Skehan et al., 2003).The results represent IC 50 is the percentage of inhibitory concentration of cell viability.

Statistical Analysis:
Statistical analysis was performed per the method described by (Armitage, 1974;Lentner et al., 1982).The analysis was done, and graphics were constructed using SigmaPlot.Data were assessed by calculating the mean (M), standard deviation (SD) and standard error (SE).

Antioxidant Activity of Crude Maggots Extract by DPPH:
DPPH is commonly used to test the ability of compounds as free-radical scavengers or hydrogen donors.Results of antioxidant activity using DPPH proved that the methanolic extracts of C. albiceps has the highest antioxidant activity than other extracts (37.18 ± 1.06 μg/mL) followed by L. sericata (75.28 ± 1.64 μg/mL) finally M. domestica extract has the lowest activity (103.13 ± 0.97 μg/mL) compared to 4.05 mg/ml for eugenol (Table 1).

2-MTT Assay for Cytotoxicity with Caco-2 Cells:
The activity of M. domestica, L. sericata and C. albiceps maggots' methanolic extract on tumor cell viability was evaluated by treating Caco-2 colon cancer cells with tested extracts using MTT assay.Data given in (Table 3, showed that, at the highest concentration (25 mg/ml), the lowest percentage of cell viability (2.41±0.01)recorded from treatment with the C. albiceps extract followed by L. sericata (3.64±0.03),and the least potent extract was from M. domestica (13.86±0.07).The IC 50 value of Doxorubicin (positive control) was 0.14±0.03.A concentration-dependent effect of tested extracts was observed.At a concentration of 0.195 mg/ml, cell viability percentages were (51.52±0.06;70.11±0.02for C. albiceps and L. sericata, respectively, while it recorded (100.49±0.04)for M. domestica.Overall, tested extracts possess significant cytotoxic activity against Caco-2 cell line.Median inhibitory concentrations of M. domestica, L. sericata and C. albiceps crude maggots extract are also summarized in table (3).The IC 50 values ranged from 0.27 to 1.91 mg/ml.The highest anticancer activity was obtained from the C. albiceps extract, which was more potent after the anticancer agent Doxorubicin.
The morphological changes in Caco-2 cell line exposed to various concentrations of different crude maggots extract are shown in (Figs.3-5).Results showed that Caco-2 cell lines exposed to 25, 12.5, 6.25, 3.12 and 1.56 mg/ml concentrations reduced the normal morphology of the cells and cell adhesion capacity of different tested extracts as compared to the control.Most of the cells at concentration 25 mg/ml lost their typical morphology and appeared smaller in size, shrunken and rounded.However, at concentration 0.78 mg/ml and lower, extracts did not cause any effects on the morphology of the cells (Fig. 3).Comparing the results, the cell viability was found to be concentrationdependent and it was highly affected by the crude extracts tested.The highest anticancer activity was recorded by C. albiceps extract, followed by L. sericata and M. domestica.

DISCUSSION
The present study reports the capability of maggots' whole body extracts as antioxidant and anticancer agents.Interestingly, these extracts were found to have therapeutics properties especially C. albiceps extract.Insects have been providing for years with many natural products, including (silk, honey, beeswax, propolis and royal jelly).Also, insects extract traditionally used in many diseases treatments, including arthritis treatment with pseudomyrmex ant venom and diabetics (Lockhart, 2007), larval grounds up bodies have been also used in folklore medicine in many parts of the world e.g.(Srivastava et al., 2009;Dossey, 2010).Dipterous flies are important insects, and their larvae are a source of protein, polyunsaturated fats, vitamins, minerals and other nutrients (Hwangbo et al., 2009).Despite the fact that only few insects have undergone clinical trials to provide their efficacy, scientists recently developing new potential medicines for treatment of many diseases such as cancer (Ratcliffe et al., 2014;Shehata et al., 2016), in addition to the use of maggots to advance the human health through its application in pharmacotherapy (Sherman and Cooper 2018).
Free radicals such as hydroxyl groups, peroxyl radicals, and single oxygen have a harmful effect due to their ability to oxidize cell components and causing different diseases (Bozin et al., 2008).The antioxidant property plays an important role in reducing chronic diseases like cancer and cardiovascular diseases by scavenging free radicals.Several synthetic antioxidants have been used to reduce the biological toxicity by exerting several deleterious effects.Therefore, there is a great demand of replacing synthetic antioxidants with natural oxidizing agents.Naturally, origin substances such as ground-up bodies have been widely used in medical industries and generally considered safe to use due to the presence of many compounds with antioxidant and anticancer activities (Suh et al., 2010).The extracts tested in this study revealed that C. albiceps extract displayed more potent scavenging ability, while scavenging ability of M. domestica was weaker than L. sericata.
Results obtained in this study showed also that Caco-2 cell exposed to different concentrations of tested extracts reduced the normal morphology of cells and cell adhesion capacity with preference to C. albiceps crude maggots extract that decreased the cell viability in a concentration-dependent manner, which may indicate the same principles involved in other anticancer agents.These results support those of previous studies, which established that M. domestica maggots could be used clinically to alleviate gastric cancer (Hou et al., 2007), and that alloferon -a peptide from Calliphora vicina-had cytotoxic effects on cancer cells at a dose of 25μg (Chernysh et al., 2002).However, the relationship between the order of potency with reference to antioxidant effects, and the order of potency against Caco-2 cells still unknown.

Conclusion
It could be concluded that the tested extracts showed relatively high concentration-dependent antioxidant effects, and C. albiceps extract recorded the highest levels of DPPH scavenging activity, followed by L. sericata and M. domestica.The different tested extracts inhibited the proliferation of tumor cells and the highest anticancer activity was recorded by C. albiceps crude maggots extract followed by L. sericata and M. domestica, and these results could be the first demonstration that larvae from these three particular insect species may have antioxidant and anticancer activities.

Table ( 1
): IC 50 values of the DPPH scavenging activity of the crude maggots extract.

Table ( 2
): Total antioxidant capacity of different tested extracts.