Research Article Received: 28 October 2014
Revised: 19 April 2015
Accepted article published: 1 June 2015
Published online in Wiley Online Library: 1 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7281
Aqueous extract of tomato seeds attenuates rotenone-induced oxidative stress and neurotoxicity in Drosophila melanogaster† Gokul Krishna and Muralidhara * Abstract BACKGROUND: Tomato seeds, a major by-product from the food processing industry, constitute a rich source of bioactives and a large population consumes tomato (either in raw or cooked form). In the present study, initially we assessed the antioxidant activity of aqueous extract of tomato seeds (TSE) in selected chemical systems and further explored the neuroprotective effects of TSE utilising the rotenone (ROT) model of neurotoxicity in Drosophila. RESULTS: Adult male flies (Oregon K) were fed TSE-enriched medium (0.1–0.2%) with or without ROT (500 𝛍mol L−1 ) for 7 days. The propensity of TSE to protect flies against ROT-induced lethality, locomotor phenotype, oxidative stress and neurotoxicity was investigated. TSE offered marked protection against ROT-induced mortality, while survivors exhibited improved locomotor phenotype. TSE significantly attenuated ROT-induced oxidative stress, mitochondrial dysfunctions, protein carbonyls content, restored the cholinergic function and dopamine levels. CONCLUSION: We hypothesise that the efficacy of tomato seed extract to attenuate ROT-mediated neurotoxicity may be largely related to the combined antioxidant activity of bioactives resulting in abrogation of oxidative stress and mitochondrial dysfunction. More importantly, our approach provides an experimental paradigm to rapidly assess the potential neuroprotective effects of common dietary components employing Drosophila, since it corroborates previous evidence in a mouse model. © 2015 Society of Chemical Industry Keywords: tomato; seeds; Drosophila; rotenone; oxidative stress; neurotoxicity; protection
INTRODUCTION
J Sci Food Agric 2016; 96: 1745–1755
explored for possible health benefits. However, so far, application of these by-products has been limited to their use as additives, dietary supplements or as a food preservative.9 Previously, grape seed extract has been demonstrated to prevent amyloid-𝛽 deposition10 and glutamate-mediated excitotoxic injury.11 In a recent study, tomato seed extract was shown to exhibit anti-proliferative effect against rat basophile leukaemia.12 Further, an extract obtained from the mucilaginous myxotesta of tomato seed matrix was demonstrated to possess anti-platelet activity.13 Several lines of evidence implicate an important aetiological role for agrichemicals in triggering neurodegeneration.14,15 It has been reported that the dopaminergic neurons are especially vulnerable to the toxic effects of several chemicals suggesting an environmental basis for disease development.16 Oxidative processes are known to contribute to the neurodegeneration
∗
Correspondence to: Muralidhara, Biochemistry and Nutrition Department, CSIR – Central Food Technological Research Institute (CFTRI), Mysore 570020, India. E-mail: [email protected], [email protected] † This paper was presented, in part, at the XXI Indian Convention of Food Scientists and Technologists (ICFOST) on ‘Innovations in food science and technology to fuel the growth of the Indian food industry’ held during 20–21 January 2012, Pune, India.
Department of Biochemistry and Nutrition, CSIR – Central Food Technological Research Institute (CFTRI), Mysore 570020, India
www.soci.org
© 2015 Society of Chemical Industry
1745
Tomato, one of the most important economic crops in the world, continues to be a part of the human diet either as fresh in salads or processed industrially.1 Tomatoes accumulate a variety of secondary metabolites including phenolic compounds, phytoalexins and glycoalkaloids such as tomatine.2 Several clinical studies have demonstrated the protective effects of tomato with more frequent consumption associated with overall reduced incidences of several pathological conditions including cancer and cardiovascular diseases.3 One of the most important properties of tomato extract is their antioxidant capacity against radicals and other reactive species. Over-production of reactive species is associated with the pathogenesis of several disorders. Abundant reports have proven potential antioxidant activity of tomato in several free radical and experimental models4,5 which has been attributed to presence of carotenoids and phenolic compounds. Recently, studies in rats have shown that tomato powder effectively supressed hydrogen peroxide-induced lipid peroxidation6 and one of the bioactives, lycopene significantly abrogates A𝛽-mediated neuroinflammation.7 There is a constant need to reutilise by-products derived from agricultural, as well as food processing industries. Although these by-products pose increasing disposal problem from an environmental viewpoint, they constitute an important source of bioactive compounds.8 For this reason, by-products derived from agroindustries have been considered as valuable components and
www.soci.org evident in Parkinson’s disease (PD) or following exposure to pesticides such as rotenone (ROT). The neurodegenerative process induced by ROT involves the damage to the dopaminergic neurons through production of oxidative radicals.17 – 19 Specifically, ROT is a high-affinity inhibitor of mitochondrial complex I which can lead to neuronal death and cause similar pathology in animal models that is characteristic of PD.14 Drosophila represents a powerful model to obtain insights into the aetiology of various neurodegenerative diseases.18,20 Further, the fly system offers a convenient platform to test the efficacy of phytochemicals and pharmacological agents to abrogate neuropathological aberrations induced by chemical toxicants.21 – 24 In our laboratory, we have successfully utilised Drosophila to assess the neuroprotective efficacy of plant extracts employing model neurotoxicants such as rotenone.25,26 Despite the presence of several classes of antioxidant compounds, the neuromodulatory potential of aqueous extract of tomato seeds (TSE) has not been hitherto explored. Hence, based on our preliminary findings in vitro in chemical systems, we hypothesised that TSE may be a potential neuroprotective candidate and evaluated the same in a well-established ROT model of neurotoxicity in flies. Initially, we examined the ability of TSE alone to modulate endogenous levels of oxidative markers and antioxidant defence status in head/body regions of flies. Subsequently, we assessed its propensity to alleviate ROT-induced mortality, locomotor deficits, oxidative stress and neurotoxicity in flies employing a short-term co-treatment paradigm.
MATERIALS AND METHODS Chemicals Rotenone (purity ≥ 95%), thiobarbituric acid (TBA), 2′ ,7′ -dichloro fluorescein diacetate, acetylthiocholine iodide, thiazolyl blue tetrazolium bromide (MTT), dopamine were procured from Sigma–Aldrich (St Louis, MO, USA). All other chemicals used were of analytical grade.
1746
Preparation of tomato seed aqueous extract and HPLC analysis The aqueous extract of tomato seeds was prepared as previously described.12 Tomato seeds were procured from the local market of Mysore, Karnataka, India, and used without any pre-treatment. Briefly, seeds were cleaned to get rid of the unwanted exogenous matter. A 20% (w/v) solution of the powdered seed in Milli-Q water was boiled for 30 min. The extract was filtered through a Buchner funnel, frozen followed by lyophilisation (Labconco Corp., Kansas, MO, USA) under reduced pressure (
Revised: 19 April 2015
Accepted article published: 1 June 2015
Published online in Wiley Online Library: 1 July 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7281
Aqueous extract of tomato seeds attenuates rotenone-induced oxidative stress and neurotoxicity in Drosophila melanogaster† Gokul Krishna and Muralidhara * Abstract BACKGROUND: Tomato seeds, a major by-product from the food processing industry, constitute a rich source of bioactives and a large population consumes tomato (either in raw or cooked form). In the present study, initially we assessed the antioxidant activity of aqueous extract of tomato seeds (TSE) in selected chemical systems and further explored the neuroprotective effects of TSE utilising the rotenone (ROT) model of neurotoxicity in Drosophila. RESULTS: Adult male flies (Oregon K) were fed TSE-enriched medium (0.1–0.2%) with or without ROT (500 𝛍mol L−1 ) for 7 days. The propensity of TSE to protect flies against ROT-induced lethality, locomotor phenotype, oxidative stress and neurotoxicity was investigated. TSE offered marked protection against ROT-induced mortality, while survivors exhibited improved locomotor phenotype. TSE significantly attenuated ROT-induced oxidative stress, mitochondrial dysfunctions, protein carbonyls content, restored the cholinergic function and dopamine levels. CONCLUSION: We hypothesise that the efficacy of tomato seed extract to attenuate ROT-mediated neurotoxicity may be largely related to the combined antioxidant activity of bioactives resulting in abrogation of oxidative stress and mitochondrial dysfunction. More importantly, our approach provides an experimental paradigm to rapidly assess the potential neuroprotective effects of common dietary components employing Drosophila, since it corroborates previous evidence in a mouse model. © 2015 Society of Chemical Industry Keywords: tomato; seeds; Drosophila; rotenone; oxidative stress; neurotoxicity; protection
INTRODUCTION
J Sci Food Agric 2016; 96: 1745–1755
explored for possible health benefits. However, so far, application of these by-products has been limited to their use as additives, dietary supplements or as a food preservative.9 Previously, grape seed extract has been demonstrated to prevent amyloid-𝛽 deposition10 and glutamate-mediated excitotoxic injury.11 In a recent study, tomato seed extract was shown to exhibit anti-proliferative effect against rat basophile leukaemia.12 Further, an extract obtained from the mucilaginous myxotesta of tomato seed matrix was demonstrated to possess anti-platelet activity.13 Several lines of evidence implicate an important aetiological role for agrichemicals in triggering neurodegeneration.14,15 It has been reported that the dopaminergic neurons are especially vulnerable to the toxic effects of several chemicals suggesting an environmental basis for disease development.16 Oxidative processes are known to contribute to the neurodegeneration
∗
Correspondence to: Muralidhara, Biochemistry and Nutrition Department, CSIR – Central Food Technological Research Institute (CFTRI), Mysore 570020, India. E-mail: [email protected], [email protected] † This paper was presented, in part, at the XXI Indian Convention of Food Scientists and Technologists (ICFOST) on ‘Innovations in food science and technology to fuel the growth of the Indian food industry’ held during 20–21 January 2012, Pune, India.
Department of Biochemistry and Nutrition, CSIR – Central Food Technological Research Institute (CFTRI), Mysore 570020, India
www.soci.org
© 2015 Society of Chemical Industry
1745
Tomato, one of the most important economic crops in the world, continues to be a part of the human diet either as fresh in salads or processed industrially.1 Tomatoes accumulate a variety of secondary metabolites including phenolic compounds, phytoalexins and glycoalkaloids such as tomatine.2 Several clinical studies have demonstrated the protective effects of tomato with more frequent consumption associated with overall reduced incidences of several pathological conditions including cancer and cardiovascular diseases.3 One of the most important properties of tomato extract is their antioxidant capacity against radicals and other reactive species. Over-production of reactive species is associated with the pathogenesis of several disorders. Abundant reports have proven potential antioxidant activity of tomato in several free radical and experimental models4,5 which has been attributed to presence of carotenoids and phenolic compounds. Recently, studies in rats have shown that tomato powder effectively supressed hydrogen peroxide-induced lipid peroxidation6 and one of the bioactives, lycopene significantly abrogates A𝛽-mediated neuroinflammation.7 There is a constant need to reutilise by-products derived from agricultural, as well as food processing industries. Although these by-products pose increasing disposal problem from an environmental viewpoint, they constitute an important source of bioactive compounds.8 For this reason, by-products derived from agroindustries have been considered as valuable components and
www.soci.org evident in Parkinson’s disease (PD) or following exposure to pesticides such as rotenone (ROT). The neurodegenerative process induced by ROT involves the damage to the dopaminergic neurons through production of oxidative radicals.17 – 19 Specifically, ROT is a high-affinity inhibitor of mitochondrial complex I which can lead to neuronal death and cause similar pathology in animal models that is characteristic of PD.14 Drosophila represents a powerful model to obtain insights into the aetiology of various neurodegenerative diseases.18,20 Further, the fly system offers a convenient platform to test the efficacy of phytochemicals and pharmacological agents to abrogate neuropathological aberrations induced by chemical toxicants.21 – 24 In our laboratory, we have successfully utilised Drosophila to assess the neuroprotective efficacy of plant extracts employing model neurotoxicants such as rotenone.25,26 Despite the presence of several classes of antioxidant compounds, the neuromodulatory potential of aqueous extract of tomato seeds (TSE) has not been hitherto explored. Hence, based on our preliminary findings in vitro in chemical systems, we hypothesised that TSE may be a potential neuroprotective candidate and evaluated the same in a well-established ROT model of neurotoxicity in flies. Initially, we examined the ability of TSE alone to modulate endogenous levels of oxidative markers and antioxidant defence status in head/body regions of flies. Subsequently, we assessed its propensity to alleviate ROT-induced mortality, locomotor deficits, oxidative stress and neurotoxicity in flies employing a short-term co-treatment paradigm.
MATERIALS AND METHODS Chemicals Rotenone (purity ≥ 95%), thiobarbituric acid (TBA), 2′ ,7′ -dichloro fluorescein diacetate, acetylthiocholine iodide, thiazolyl blue tetrazolium bromide (MTT), dopamine were procured from Sigma–Aldrich (St Louis, MO, USA). All other chemicals used were of analytical grade.
1746
Preparation of tomato seed aqueous extract and HPLC analysis The aqueous extract of tomato seeds was prepared as previously described.12 Tomato seeds were procured from the local market of Mysore, Karnataka, India, and used without any pre-treatment. Briefly, seeds were cleaned to get rid of the unwanted exogenous matter. A 20% (w/v) solution of the powdered seed in Milli-Q water was boiled for 30 min. The extract was filtered through a Buchner funnel, frozen followed by lyophilisation (Labconco Corp., Kansas, MO, USA) under reduced pressure (
