Hostname: page-component-586b7cd67f-t7czq Total loading time: 0 Render date: 2024-11-22T08:44:23.783Z Has data issue: false hasContentIssue false
  • English
  • Français

Antifilarial activity of azadirachtin fuelled through reactive oxygen species induced apoptosis: a thorough molecular study on Setaria cervi

Published online by Cambridge University Press:  23 July 2018

N. Mukherjee ,
N. Joardar  and
S.P. Sinha Babu Open the ORCID record for S.P. Sinha Babu [Opens in a new window]
N. Mukherjee
Affiliation:
Parasitology Laboratory, Department of Zoology (Centre for Advanced Studies), Visva-Bharati University, Santiniketan 731 235, West Bengal, India Cancer Biology & Inflammatory Disorder Division, CSIR - Indian Institute of Chemical Biology, 4- Raja S.C. Mullick Road, Kolkata 700 032, West Bengal, India
N. Joardar
Affiliation:
Parasitology Laboratory, Department of Zoology (Centre for Advanced Studies), Visva-Bharati University, Santiniketan 731 235, West Bengal, India
S.P. Sinha Babu*
Affiliation:
Parasitology Laboratory, Department of Zoology (Centre for Advanced Studies), Visva-Bharati University, Santiniketan 731 235, West Bengal, India
*
Author for correspondence: S.P Sinha Babu E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Efficacious therapeutic strategies against lymphatic filariasis are always sought after. However, natural products are a promising resource for developing effective antifilarial agents. Azadirachtin, a significant tetranortriterpenoid phytocompound found in Azadirachta indica, was evaluated in vitro for antifilarial potential against the filarial parasite Setaria cervi. Dye exclusion and MTT assay confirmed the antifilarial potential of azadirachtin against S. cervi with a median lethal dose (LC50) of 6.28 μg/ml for microfilariae (mf), and 9.55 μg/ml for adult parasites. Morphological aberrations were prominent in the histological sections of the azadirachtin-exposed parasites. Moreover, alterations in the reactive oxygen species (ROS) parameters in treated parasites were evident. Induction of apoptosis in treated parasites was confirmed by DNA laddering, acridine orange (AO)/ethidium bromide (EtBr) double staining and in situ DNA fragmentation. The downregulation of anti-apoptotic CED-9 and upregulation of proapoptotic EGL-1, CED-4 and CED-3 at both the transcription and translation levels confirmed apoptosis execution at the molecular level. Changes in the gene expressions of nuc-1, cps-6 and crn-1 further clarified the molecular cause of DNA degradation. Furthermore, azadirachtin was found to be non-toxic in both in vitro and in vivo toxicity analyses. Therefore, the experimental evidence detailed the pharmacological effectiveness of azadirachtin as a possible therapeutic agent against filariasis.

Keywords

anthelminticapoptosisazadirachtinlymphatic filariasisreactive oxygen speciesSetaria cervitoxicity
Type
Research Paper
Information
Journal of Helminthology , Volume 93 , Issue 5 , September 2019 , pp. 519 - 528
Copyright
Copyright © Cambridge University Press 2018 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

*

These authors contributed equally.

References

Beard, J (1989) Tree may hold the key to curbing Chagas’ parasite. New Scientist 124, 31.Google Scholar
Billker, O et al. (2002) Azadirachtin disrupts formation of organized microtubule arrays during microgametogenesis of Plasmodium berghei. Journal of Eukaryotic Microbiology 49, 489–497.Google Scholar
Biswas, K et al. (2002) Biological activities and medicinal properties of neem (Azadirachta indica). Current Science 82, 1336–1345.Google Scholar
Boeke, SJ et al. (2004) Safety evaluation of neem (Azadirachta indica) derived pesticides. Journal of Ethnopharmacology 94, 25–41.Google Scholar
Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.Google Scholar
Brahmachari, G (2004) Neem—an omnipotent plant: a retrospection. Chembiochem 5, 408–421.Google Scholar
Brophy, PM and Pritchard, DI (1992) Immunity to helminths: ready to tip biochemical balance. Parasitology Today 8, 419–422.Google Scholar
Cardiff, RD, Miller, CH and Munn, RJ (2014) Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harbour Protocols. doi: 10.1101/pdb.prot073411.Google Scholar
Circu, ML and Aw, TY (2010) Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biology & Medicine 48, 749–762.Google Scholar
Fischer, AH et al. (2008) Hematoxylin and eosin staining of tissue and cell sections. Cold Spring Harbour Protocols. doi:10.1101/pdb.prot4986.Google Scholar
Gorman, A, McGowan, A and Cotter, TG (1997) Role of peroxide and superoxide anion during tumour cell apoptosis. FEBS Letters 404, 27–33.Google Scholar
Gucchait, A et al. (2017) Development of novel anti-filarial agents using carbamo(dithioperoxo)thioate derivatives. European Journal of Medicinal Chemistry 148, 598–610.Google Scholar
Kaushal, NA, Kaushal, DC and Ghatak, S (1987) Identification of antigenic proteins of Setaria cervi by immunoblotting technique. Immunological Investigations 16, 139–149.Google Scholar
Lettre, G and Hengartner, MO (2006) Developmental apoptosis in C. elegans: a complex CEDnario. Nature Reviews Molecular Cell Biology 7, 97–108.Google Scholar
Lustigman, S and McCarter, JP (2007) Ivermectin resistance in Onchocerca volvulus: towards a genetic basis. PLoS Neglected Tropical Diseases 1, e76.Google Scholar
Mishra, V et al. (2005) Antifilarial activity of Azadirachta indica on cattle filarial parasite Setaria cervi. Fitoterapia 76, 54–61.Google Scholar
Mukherjee, N et al. (2014a) In vitro antifilarial activity of Azadirachta indica aqueous extract through reactive oxygen species enhancement. Asian Pacific Journal of Tropical Medicine 7, 841–848.Google Scholar
Mukherjee, N et al. (2014b) Antifilarial effects of polyphenol rich ethanolic extract from the leaves of Azadirachta indica through molecular and biochemical approaches describing reactive oxygen species (ROS) mediated apoptosis of Setaria cervi. Experimental Parasitology 136, 41–58.Google Scholar
Mukherjee, N et al. (2014c) Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria. Research in Veterinary Science 97, 310–318.Google Scholar
Mukherjee, N et al. (2016a) Oxidative stress plays major role in mediating apoptosis in filarial nematode Setaria cervi in the presence of trans-stilbene derivatives. Free Radical Biology & Medicine 93, 130–144.Google Scholar
Mukherjee, N et al. (2016b) Phenolics and terpenoids; the promising new search for anthelmintics: a critical review. Mini Reviews in Medicinal Chemistry 16, 1415–1441.Google Scholar
Mukherjee, S et al. (2015) Ginger extract ameliorates phosphamidon induced hepatotoxicity. Indian Journal of Experimental. Biology 53, 574–584.Google Scholar
Nayak, A et al. (2012) Molecular evidence of curcumin-induced apoptosis in the filarial worm Setaria cervi. Parasitology Research 111, 1173–1186.Google Scholar
OECD (Organization for Economic Cooperation and Development) (1995) Guidelines for testing chemicals. Repeated dose 28-d oral toxicity study in rodents, no. 407. Paris, France: OECD.Google Scholar
OECD (Organization for Economic Cooperation and Development) (2009) Chronic Toxicity Studies. OECD Guideline for the Testing of Chemicals. Section 4: Health Effects, No. 452. Adopted by the Council on 7th September 2009.Google Scholar
Parrish, JZ et al. (2003) CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptotic DNA degradation. EMBO Journal 22, 3451–3460.Google Scholar
Pessoa, LM (2001) Atividade ovicidal in vitro de plantas medicina medicinais contra Haemonchus contortus. MSc thesis. Universidade Estadual do ceara. Fortaleza, Brazil.Google Scholar
Pietrosemoli, S, Ovalez, R and Montilla, T (1999) Empleo, de hojas de nematodes gastrointestinales de bovines a pastoreo. Revista de la Facultad de Agronomia de la Universidad del Zulia 16, 220–225.Google Scholar
Radwan, MU et al. (2001) Residual activity of orally administrated pesticides used on fruits and vegetables on rat blood parameters behaviour. Annals of Agricultural Science Cairo 46, 365–382. (abstract)Google Scholar
Rahman, WA, Lee, R and Sulaiman, SF (2011) In vitro anthelmintic activity of neem plant (Azadirachta indica) extract against third-stage Haemonchus contortus larvae from goats. Global Veterinaria 7, 22–26.Google Scholar
Raizada, RB et al. (2001) Azadirachtin, a neem biopesticide: subchronic toxicity assessment in rats. Food and Chemical Toxicology 39, 477–483.Google Scholar
Rembold, H and Annadurai, RS (1993) Azadirachtin inhibits proliferation of sf-9 cells in monolayer-culture. Journal of Biosciences 48, 495–499.Google Scholar
Roy, B et al. (2014) Design and green synthesis of polymer inspired nanoparticles for the evaluation of their antimicrobial and antifilarial efficiency. RSC Advances 4, 34487.Google Scholar
Saini, P et al. (2012) Effect of ferulic acid from Hibiscus mutabilis on filarial parasite Setaria cervi: molecular and biochemical approaches. Parasitology International 61, 520–531.Google Scholar
Saxena, RC (2002) Pests of stored products. In Schmutterer, H (ed.), The Neem Tree. Weinheim: Wiley Online Library, pp. 524–537.Google Scholar
Schmutterer, H (2002) The Neem Tree Azadirachta indica A. Juss. and Other Meliaceous Plants. Sources of Unique Natural Products for Integrated Pest Management, Medicine, Industry and Other Purposes. 2nd edition. Mumbai: Neem Foundation.Google Scholar
Shenoy, RK (2008) Clinical and pathological aspects of filarial lymphedema and its management. Korean Journal of Parasitology 46, 119–125.Google Scholar
Simon, HU, Haj-Yehia, A and Levi-Schaffer, F (2000) Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415–418.Google Scholar
Syng-Ai, C, Kumari, AL and Khar, A (2004) Effect of curcumin on normal and tumor cells: role of glutathione and bcl-2. Molecular Cancer Therapeutics 3, 1101–1108.Google Scholar
Tripathi, A, Shrivastav, TG and Chaube, SK (2012) Aqueous extract of Azadirachta indica (neem) leaf induces generation of reactive oxygen species and mitochondria-mediated apoptosis in rat oocytes. Journal of Assisted Reproduction and Genetics 29, 15–23.Google Scholar
Van der Esch, SA, Carnevali, F and Amici, A (2009) Effect of neem derived products on gastrointestinal nematodes in vitro and in vivo in sheep. In Kleeberg, H and Strang, R (eds), Biological Control of Plant, Medical and Veterinary Pests. Proceedings of the 14th workshop, Wetzlar, Germany.Google Scholar
Verkerk, RHJ and Wright, DJ (1993) Biological activity of neem seed kernel extract and synthetic azadirachtin against larvae of Plutella xylostellal. Journal of Pesticide Science 37, 83–91.Google Scholar
WHO (World Health Organization) (2017) Lymphatic filariasis. http://www.who.int/mediacentre/factsheets/fs102/en/.Google Scholar
Yu, H et al. (2015) Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos. Bioscience Reports 35, 1–12.Google Scholar
Supplementary material: PDF

Mukherjee et al. supplementary material

Mukherjee et al. supplementary material 1

Download Mukherjee et al. supplementary material(PDF)
PDF 402 KB