Protective effect of Catharanthus roseus plant extracts against endosulfan and its isomers induced impacts on non-targeted insect model, Drosophila melanogaster and live brain cell imaging

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Highlights

  • Among the endosulfan and its isomers, alpha endosulfan is more toxic than total endosulfan and beta endosulfan.

  • 1/50th EC50 concentration of endosulfan and its isomers alters the various life-stage activities of Drosophila melanogaster; such as hatchability, pupation, emergence, body weight, survival rate, and reproductive performance.

  • Catharanthus roseus aqueous and methanolic plant extracts showed high levels of DPPH free radical scavenging activity and Fe3+ reducing power.

  • Life stage performance and protein profile recovery were observed by co-administration of 1/50th EC50 concentration of C. roseus with 1/5th EC50 concentration of endosulfan and its isomers.

  • The protective effects of C. roseus were confirmed by live brain cell imaging using TMRM (mitochondrial stain) and Hoechst (nuclear stain) stains.

Abstract

Endosulfan has been recognized as a highly controversial pesticide due to its acute toxicity, potential bioaccumulation, persistency, and long-range atmospheric transport. Several plant extracts act as antioxidant agents against wide-range of pesticide toxicity hazards through the free radicals scavenging properties. Plants' secondary metabolites are considered as efficient protective agents against various cellular toxic injuries. Understanding these properties of botanicals, several researchers currently focused on the detoxification and ameliorative potency of plant extracts against highly toxic chemicals. In our studies, we focused on the endosulfan total and its isomers (alpha and beta) induced changes on Drosophila melanogaster and their ameliorative effects by co-administrated with methanolic and aqueous extracts of Catharanthus roseus whole plant. We selected the 1/5th EC50 concentration of alpha-endosulfan, beta-endosulfan, and endosulfan (total) and co-administrated with 1/50th EC50 concentration of aqueous and methanolic extracts and evaluated their ameliorative effects, in terms of verifying the life stage activities, protein profiling and also by using live brain cells imaging. We finally concluded that, the methanolic and aqueous extracts inhibit the toxic impacts caused by endosulfan and its isomers and also increasing the survival rate of the test organism.

Introduction

The USEPA classifies endosulfan as category I (highly acutely toxic) pesticide and WHO considers endosulfan as moderately hazardous (class II) to various organisms (Siddique et al., 2003). Due to the low water solubility, chemical stability, high lipid solubility, and slow rate of biotransformation, the endosulfan is considered as a potential environmental pollutant. Several experimental studies suggest that endosulfan has endocrine-disrupting potential, causes Parkinson's disease, behavioral effects, reproductive disorders, neuronal effects, premature aging as well as cancer (Ahmed and Ahmad, 1993; ATSDR, 1993; Dalsenter et al., 1999; Bisson and Hontela, 2002; Hiremath and Kaliwal, 2002; Wang, 2006).

Several researchers are focused on the inhibiting or ameliorating effect of various plant extracts against the damage caused by narrow spectrum pesticides. Plant compounds mediated toxicity reduction studies of xenobiotic like malathion, maneb, arsenic, diazinon, rotenone, etc. have been done extensively (Venkatesan et al., 2003; Anilakumar et al., 2009; Ahmed et al., 2008, Ahmed et al., 2010; Abdallah et al., 2012; Manfo et al., 2014; Banaee et al., 2015). However, the literature and understanding of the botanicals mediated minimization of endosulfan toxicity is presently limited. Although Catharanthus roseus is a well-studied medicinal plant with high antioxidant properties, only a few studies have addressed the toxicity reduction by using C. roseus extracts. All parts of the plant exhibit a high degree of therapeutical properties, mainly high antioxidative potency (Djeriidane et al., 2006; Mustafa and Verpoorte, 2007; Ferreres et al., 2008; Pereira et al., 2010; Ferrers et al., 2011). Besides antioxidant and anti-cancerous activity, the plant compounds exhibit anti-diabetic, anti-allergic, anti-inflammatory, anti-microbial, anti-helminthic, anti-ulcer, anti-diarrheal, wound healing, hypotensive, hypolipidemic, memory enhancement, cardioprotective and vasodilator activities (Pillay et al., 1959; Chattopadhyay et al., 1991; Sekar, 1996: Nayak et al., 2007; Patil and Ghosh, 2010; Rajput et al., 2011; Patel et al., 2011). The plant's secondary compounds are incorporated into a wide range of commercial and industrial applications, served as resources for flavors, aromas and fragrance, bio-based fuels and plastics, enzymes, preservatives, cosmetics, natural pigments, and bio-active compounds. The extracts of C. roseus act as a very strong inhibitor for mild steel corrosion (Shahba et al., 2016; Rana et al., 2017).

D. melanogaster, an insect model with well-documented genetics and developmental biology, is the closet invertebrate to humans and has been used for toxicological studies as well as for studying human diseases (Jeibmann and Paulus, 2009). Therefore, the present study aimed to investigate the protective effect of C. roseus extracts (aqueous and methanolic extracts) against endosulfan and its isomers induced impacts on non-targeted insect model D. melanogaster with live cell brain imaging. We address the following topics in the paper, such as, (1) which endosulfan (endosulfan total, alpha-endosulfan and beta-endosulfan) is highly toxic and how it alters the various developmental stages of D. melanogaster – hatchability, pupation, emergence pattern, body weight, reproduction, and survival rate; (2) bioactive properties of C. roseus – polyphenol content, Fe3+ reducing power, DPPH free radical scavenging efficacy, organic and inorganic content of extracts (aqueous and methanol); (3) toxicity of methanolic and aqueous plant extracts (eclosion inhibition concentration used as an index for estimating the toxicity of plant extracts); (4) protective effect of C. roseus extracts against 1/5th EC50 concentration of endosulfan and its isomers – evaluated by monitoring the life stage performance recovery and protein profile variations (5) protective effects of C. roseus extracts validated by using live brain cell imaging of D. melanogaster using mitochondrial (TMRM) and nuclear stain (Hoechst).

Section snippets

Endosulfan total and its isomers

Endosulfan (C9H6C16O3S) is a polycyclic chlorinated hydrocarbon insecticide, it is a mixture of two stereoisomers, alpha-endosulfan (Endosulfan I; exo-configuration), and beta-endosulfan (Endosulfan II; endo-configuration). The technical grade of Endosulfan total and its isomers (α and β endosulfan) were procured from Sigma-Aldrich C. USA (Endosulfan 32015, Lot #SZBC166XV, Alpha-endosulfan 45468, Lot #SZBD205XV and Beta-endosulfan 33385, Lot #SZBD247XV).

Determination of effective concentration (EC50) of endosulfan and its isomers

The culture media prepared according to

EC50 concentration of endosulfan and its isomers

From the definitive test we arrived at the five different concentration of endosulfan (total) and alpha-endosulfan (2.28 ppm, 3.08 ppm, 4 ppm, 5.28 ppm, and 7 ppm) and beta endosulfan (2.5 ppm, 3.52 ppm, 5 ppm, 7 ppm, and 10 ppm). Toxicity response, EC50 in D. melanogaster against alpha-endosulfan, beta-endosulfan and endosulfan (total) was performed; 95% of confidence limits were obtained by strait line graphic interpolation methods of Litchfield and Wilcoxon (1949). The EC50 value of

Discussion

Several studies have emphasized that endosulfan shows acute and chronic toxicity in insects and mammals including human beings. The short-term toxicity of endosulfan can cause nervous system dysfunction, hyperactivity, decreased respiration, anemia, etc. it also causes vomiting, seizures, convulsions, and loss of consciousness, leading to death (Smith, 1991). The long-term exposure of endosulfan could be harmful to the central nervous system, immune system, and reproductive system. Endosulfan

Conclusion

The present study has undertaken to investigate the toxic effect of endosulfan and ameliorating effects of aqueous and methanol extracts of C. roseus. Endosulfan induced toxicity impact on D. melanogaster changed the C. roseus extract by increasing reduced body weight, increasing reduced life span, and overcome the developmental delay and also the reappearance of normal protein profile. Several studies have proved that the secondary metabolites of plants can cause a reduction of toxicity

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the Department of Zoology, University of Calicut for providing the infrastructural facility. And also, Dr. T. R. Santhosh Kumar, Scientist G, Rajiv Gandhi Centre for Biotechnology (RGCB) for providing valuable suggestions and laboratory facilities for doing live cell imaging. The first author is thankful to the Drosophila stock center, Department of Zoology, Mysore University for providing the model organism (D. melanogaster). This study financially supported by UGC – SAP, Government

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