Research Article - Der Pharma Chemica ( 2017) Volume 9, Issue 2
Healing Potency of Citrus, Hesperetin and Naringenin Loaded with Silicate Nanoparticles on Neurotoxicity Induced by Acrylamide Toxic DoseMaha Z. Rizk1, Abo-elmatty D.M2, Hanan F. Aly1, Howaida I. Abd-Alla3, Samy M. Saleh2 and Eman A.Younis1
2Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Egypt
3Chemistry of Natural Compounds Department, National Research Centre, Giza, Dokki 12622, Egypt
The current research demonstrated the effect of silica loaded nanoparticles (citrus, naringenin and hesperetin) on acrylamide (ACR) intoxicated rats, through measuring specific selected biomarkers on brain. Brain neurotransmitters noradrenaline (NA), adrenaline (A), dopamine (DA) and serotonine (5-HT), acetylcholine esterase (ACHE) andcaspase-3 enzyme activity and DNA degradation (Comet) were evaluated. ATPase enzyme activity and total protein content were also evaluated. Besides, the presented data showed significant deterioration in the all selected biomarkers investigation in aclylamide-intoxicated rats. It showed significant reduction in brain neurotransmitters (NAADA and 5-HT), ATPase enzyme activity and total protein content while significant elevation in AchE and Caspase-3 enzyme activity and DNA damage. Treatment of intoxicated rats with the three silica loaded nanoparticles indicated amelioration with different percentage of improvement in the all measured biomarkers as well as in rat’s brain architecture. Thus it could be concluded that, the using of natural product nanoparticles can up-regulate brain neurotransmitters, suppressing DNA damage, diminished brain damage that leads to delay disease development and/or its complication
Acrylamide, ATPase, Brain neurotransmitters, Comet assay
Acrylamide is a reactive monomer used in many technological applications, but it is the incidental formation during cooking of common starchy foods that leads to pervasive human exposure, typically in the range of 1 μg/Kg body weight (bw)/day (d) .
Acrylamide formation was found to occur during the browning process by the Maillard reaction of reducing sugars with the aminoacid asparagine at temperatures higher than 120ºC . Acrylamide, a human neurotoxicant and rat tumorigenic . Evidence has been reported that acrylamide is neurotoxic [4,5] among over-exposed humans and is amulti-site carcinogen in rats exposed by ingestion over a lifetime [6,7].
Exposure to neurotoxic agents is a common event in the workplace and in the general environment. The already large number of neurotoxic substances  is constantly increasing with newly generated compounds as they needed for a rapidly changing and growing market . Risk assessment is a fundamental process for prevention, especially for neurotoxicity . Acrylamide neurotoxicity in both laboratory animals and humans is characterized by ataxia and distal skeletal muscle weakness. In addition, acrylamide intoxication in rodent models is associated with selective nerve terminal damage in both central and peripheral nervous systems . A growing body of evidence indicated that the nerve terminal is a primary site of acrylamide action and that inhibition of corresponding membrane fusion processes impaired neurotransmitter release and promoted eventual degeneration . This might be attributed to, easily absorption by all routes of administration, and both the Central Nervous System (CNS) and Peripheral Nervous System (PNS) are the selective targets for its toxicity. Furthermore, acrylamide induced neurotoxic effects discernible in terms of diminished ATPase activity, enhanced activity of acetylcholine esterase and dopamine, noradrenaline, adrenaline, serotonine depletion. Marked enhanced DNA damage and caspase-3- activity [13,14]. During the past decade, there has been a rapid progress on research in the areas of nano-science and nanotechnology. Nanomaterial has specific properties, such as small size, large surface area, shape, and special structure . Because of the unique dimensional and morphological properties, nanomaterial can be physically and chemically manipulated and widely used in industrial and biomedical processes as well as organ or cell specific drug delivery . The rapid growth of nanotechnology industry has led to the wide-scale production and application of engineered NPs. Moreover, nano-sized crystalline SiO2 is commonly used in semiconductor manufacture .
Natural dietary components include vegetables, fruits and spices; they have drawn an immense attention due to their ability to suppress various phases of carcinogenesis and neurodegenerative intervention because of the presence of phytochemicals 
Natural flavonoids with more of a lipophilic chemical structure and antioxidant properties are promising candidates for neurodegenerative intervention . On the other hand, Citrus aurantium, a fruit commonly known as bitter orange, it belongs to the Rutaceae family. Citrus fruits are an abundant source of various flavonoids, which have been used as a traditional herbal medicine in Korea and China . Naringenin is an important natural flavanone. It is known to be a multi-functional agent, a powerful anti-oxidant, anti-inflammatory, antitumor, anti-depressant, hepatoprotective and neuroprotective compound . Also naringinen was known to exhibit free-radical scavenging activity . Moreover, Hesperetin (Hsp) flavanone is the major circulating aglycone metabolite of hesperidin, abundant in citrus fruit and drinks . Hsp has been shown to be a potential antioxidant, anti-inflammatory, neuroprotective agent . Hence, the present study is designed to examine total ethanol extract of Citrus aurantium beside two citrus flavonoids as naringenin and hesperetin isolated from Citrus aurantium (sour orange) family Rutaceae to ameliorate neurotoxicity induced by acrylamide in experimental rat’s model.
Materials and Methods
The fruits were obtained from Banha, Qalyubia governorate, Egypt. All the fruits were of eating quality, and without blemishes, or damage. The pericarp region (peel) is separated from the edible part. The white, spongy inner part of the peel, called the mesocarp, or albedo was separated, dried at room temperature and grinded.
All chemicals used in the present study were of high analytical grade, products of Sigma (USA), Merck (Germany), BDH (England), Riedel de Hàen (Germany) and Fluka (Switzerland), preparation of the nanomaterials based on Tetraethoxyorthosilicate (TEOS). Isolation of flavanones (naringinen and hesperetin) Naringenin  and hesperetin  were done on the basis of chromatographic properties, 1H and 13C NMR spectroscopic data with literature values.
Preparation of the nanomaterial based on Tetraethoxyorthosilicate (TEOS)
It is prepared according to Haron et al.,  unpublished data.
Male Wistar albino rats (100: 120 g) were selected for this study. They were obtained from the Animal House, National Research Center, Egypt. All animals were kept in controlled environment of air and temperature with access of water and diet.
Anesthetic procedures and handling with animals were complied with the ethical guidelines of Medical Ethical Committee of the National Research Centre in Egypt and performed for being sure that the animals not suffer at any stage of the experiment (Approval no: 13115).
Doses and route of administration
CR suspended in H2O and was injected intraperitoneal in a dose of 50 mg/kg body weight five times weekly for ten consecutive days . SOAE, hesperetin and naringenin nanoparticles were administered orally five times weekly for one month and half post ACR induction in a dose 100 mg/kg body weight [28,29]. Donepezil-HCl, a reference drug, was administered orally five times weekly for one month and half post ACR treatment in a dose 50 mg/kg body weight .
Ninety male Wister strain albino rats will be used in the present study. Animals will be divided randomly into 9 groups of ten rats each. Group 1: Will be normal healthy control rats. Groups 2-4: Will be normal healthy rats orally administrated with silica loaded nanoparticles: Citrus (SOAE), naringenin and hesperetin for one month and half, respectively. Group 5: Will be injected intrapretonial with toxic dose of ACR for 10 days and served as intoxicated group. Groups 6 and 8: Will be orally administrated with silica loaded nanoparticles: SOAE, naringenin and hesperetin post ACR administration respectively for one month and half. Group 9: Will be orally administrated with donepezil-HCl as reference drug post ACR administration for one month and half.
Determination of brain biomarkers
Estimation of brain neurotransmitters
Determination of adrenaline: Adrenaline activity was measured by a quantitative enzyme-linked immune sorbent assay (ELISA) technique according to the manufacturer’s instructions . Determination of noradrenaline, dopamine and serotonin: Brain NA, DA and 5-HT were determined using high performance liquid chromatography HPLC) technique according to Zagrodzka .)
Estimation of acetylcholine esterase activity
Serum acetyl cholinesterase was measured by a quantitative enzyme-linked immunosorbent assay (ELISA) technique according to Wen .
Estimation of brain caspase-3 activity
Brain caspase-3 activity was measured by a quantitative enzyme-linked immunosorbent assay (ELISA) technique according to the manufacturer’s instructions Savitha .
Estimation of DNA damage percent by Comet assay in brain tissues
Single cell gel electrophoresis assay (also known as comet assay) was performed as previously described by Singh .
Estimation of ATPase activity in brain
ATPase in brain was estimated according to the method of Matteucci .
Estimation of brain total protein content
Total protein content was estimated according to the method of Bradford .
The main toxic endpoints of ACR are known as neurotoxicity in humans and animals, developmental and reproductive toxicity in rodents, and nontoxicity and carcinogenicity in rodents. Neurotoxicity of ACR is known from accidental intoxications and from chronic occupational exposures . The present results clearly demonstrate significant reduction in brain neurotransmitter levels (adrenaline, noradrenaline, serotonine and dopamine), ATPase enzyme activity and total protein content while significant elevation in acetylcholine esterase enzyme activity, DNA damage and caspase-3 enzyme activity in ACR intoxicated rats. A body of evidence suggests that oxidative stress mechanisms plays a vital role in ACR-induced neurotoxicity . ACR is known to affect membranes and cytoskeletal protein via oxidative stress resulting in disrupted neurotransmission in rodents [40,41]. Goldstein and Lowndes  suggested that defective neurotransmission in ACR-intoxicated laboratory animal might be mediated by changes in transmitter synthesis, storage uptake and release [42,43]. In concomitant with the present results LoPachin et al. , showed that significant decrease in epinephrine, norepinephrine and dopamine contents in all tested brain areas at higher dose of acrylamide; this may be due to; axon and nerve terminal degeneration which caused changes in transmitter synthesis, storage uptake, release and reduction in synaptic vesicle as a result the content of neurotransmitters is decreased. With ACR exposure, dopaminergic neurotransmission appears to be largely affected as evident by significant depletion in DA levels. Previous workers have demonstrated that ACR reduces DA levels in brain and furthermore affects DA receptor density, DA release and uptake both in vivo and in vitro exposure . ACR affects both cholinergic and dopaminergic neurotransmission . It is well accepted that energy depletion and mitochondrial dysfunctions apart from oxidative stress are vital factors associated with most of the neurodegenerative mechanisms . In the present study, ACR markedly enhanced the activity levels of AchE, a significant biological component of the cholinergic function and membrane. AchE is known to contribute to membrane integrity and permeability occurring during synaptic transmission and conduction . Enhanced activation of AchE leads to faster acetylcholine degradation and a subsequent down stimulation of acetylcholine receptors which causes a reduction of cholinergic neurotransmission and related functions such as cell proliferation and promotes apoptosis . The Monoamine neuro-transmitter serotonin (5-HT) is concentrated in a cluster of neurons in the region of brain stem, involved in temperature regulation, learning, sensory perception and onset of sleep . It is known that transmission of nerve impulse involves several steps, including transmitter synthesis, storage, release, reaction with receptor and termination of transmitter actions . Evidence has been adduced to show that ACR interacts with tyrosine and tryptophan. Tyrosine is the precursor amino acid for synthesis of catecholamine’s such as DA and NA. It is likely that by complexing with tyrosine, ACR may decrease the critical concentration of tyrosine in brain leading to an inhibition of catecholamine synthesis. Reactivity of ACR with tryptophan may presumably result in an inhibition of biosynthesis f 5-HT which requires precursor amino acid, tryptophan . The current results clearly demonstrated significant increase in caspase enzyme activity in brain tissue of acrylamide intoxicated rats. The current results run in parallel with Sumathi et al.  demonstrated acrylamide toxicity preceded an elevation in DNA destruction as specified by the DNA fragmentation increment as well as the number of comets recorded. Fragmentation of DNA beside the elevation in the Comets appearance has also been declared by Bondy et al.  as a consequence of exposure to acrylamide. Further, acrylamide is recognized to enhance a reactive oxygen species level which is renowned, to elicit destruction for various macromolecules as well as to DNA. DNA injury is considered to be one of the biomarkers and identical discriminatory of apoptosis . Hence, the current study markedly investigated that, the toxicity in response to acrylamide can enhance apoptosis as represented in the comet micrographs which obviously detected cells derangement.
In addition the current research recorded significant reduction in the activity of ATPase in acrylamide intoxicated rats. This result is in concomitant with Martyniuk et al.  and Sumathi et al.  who found that, ACR was shown to influence energy output by preventing glycolytic pathway, so the low activity of ATPase would in turn particularly modified the ion exchange and cells neurotransmission.
It is considered that ATPase be one of the principal ingredient, of the ACR induced -neurotoxic effect which leads to distal and recessive nerve fiber degradation . Moreover, accumulation of Aβ as a consequence of acrylamide exposure causes hydrogen peroxide and hydroxyl radical production via specific chemical reactions. The output of these reactive oxygen species stimulated membrane lipid peroxidation, which can deteriorate the membrane function ion-motive ATPase (Na+/K+- and Ca+-ATPases) leading to depolarization of membrane as well as a reduction in ATP cellular levels .
With respect to total protein content, the current results demonstrated significant decrease in total protein content in the ACR induced rats. This observation is in accordance with the results of several authors [56,57], as they found that, brain toxicity is accompanied by a fall in whole-body protein turnover. The reduction in the total protein content in the brain injected with ACR may be due to the increase in amino acids deamination and impairment in cellular proteins construction. Sherlock and Dooley , ascertained perturbation in protein synthetic machinery in the brain. Furthermore, the lower protein level that is observed in ACR induced rats might be also related to formation of the toxic N-nitroso compounds lead to suppressing of oxidative phosphorylation as mentioned by Anthony. Also, the Nitroso-compounds interact with cellular DNA, RNA and protein resulted in biochemical and physical alterations of these macromolecules . Flavonoids are a group of naturally occurring substances, including flavones, flavanones, and isoflavones, having several beneficial biological activities of flavonoids, including antioxidant, antitumor, and antiinflammation properties [60,61]. Some of these flavonoids (SOAE, hesperetin and naringenin), due to their phenolic structures, have antioxidant effect and inhibit free radical-mediated processes . Naringenin was found to possess antitumor, anti-inflammatory and hepatoprotective effects . In addition Hsp has been shown to be a potential anti-oxidant, anti-inflammatory, neuroprotective agent [64,65]. Accordingly, nano-components (SOAE, hesperetin and naringenin) which possess antioxidant properties are possibly to be defensive against ACR-induced neurotoxic effects . In summary, the present data indicated that acrylamide induced brain damage which might be related to oxidative stress. Administration of the three nano-components lessened the negative effects of acrylamide on the brain by inhibiting free radical mediated process; an effect that could be attributed to the antioxidant property of three nano-components.
 L. Camachoa, J.R. Latendresseb, L. Muskhelishvilib, R. Pattonb, J.F. Bowyerc, M. Thomasc, D.R. Doerge, Toxicol. Lett., 2012, 211, 135-143
 A. Becalski, B.P.Y. Lau, D. Lewis, S.W. Seaman, J. Agr. Food. Chem., 2003, 51, 808-8021.
 R.G. Tardiff, M.L. Gargas, C.R. Kirman, M.L. Carson, L.M. Sweeney, Food Chem. Toxicol., 2010, 48, 658-667.
 World Health Organization (WHO), 2005.
 C.J. Calleman, Y. Wu, F. He, G. Tian, E. Bergmark, S. Zhang, H. Deng, Y. Wang, K.M. Crofton, T. Fennell, Toxicol. Appl. Pharmacol., 1994, 126, 361-371.
 G.E. Johnson, S.H. Doak, S.M. Griffiths, E.L. Quick, D.O. Skibinski, Z.M. Zair, G.J. Jenkins, Mutat. Res., 2009, 678, 95-100.
 M.A. Friedman, E. Zeiger, D.E. Marroni, D.W. Sickles, J. Agric. Food Chem., 2008, 56, 6024-6030.
 R. Lucchini, L. Benedetti, E. Albini, L. Alessio, Int. Arch. Occup. Environ. Health., 2005, 78, 427-437.
 Research and Consultancy Solutions (RNCOS), 2007.
 D.S. Rohlman, R. Lucchini, W.K. Anger, D.C. Bellinger, C. Van Thriel, Neurotoxicol., 2008, 556-567.
 R.M. LoPachin, Neurotoxicol., 2003, 25, 617-630.
 R.M. LoPachin, C.D. Balaban, J.F. Ross, Toxicol. Appl. Pharmacol., 2004, 188, 135-153.
 E.J. Lehning, R.M. Lopachin, J. Matthew, J. Eichberg, J. Toxicol. Environ. Health., 1994, 42, 331-342.
 M.I. Yousef, F.M. El-Demerdash, Toxicol., 2006, 133, 219-214.
 G. Oberdörster, E. Oberdörster, J. Oberdörster, Environ. Health. Perspect., 2005, 113, 823-839.
 L.L. Hsieh, H.J. Kang, H.L. Shyu, C. Chang, Water. Sci. Technol., 2009, 60, 1295-1301.
 J.J. Wang, B.J. Sanderson, H. Wang, Mutation res., 2007, 628, 99-106.
 Y.Chen, S.C. Shen, H.Y. Lin, Biochem. Pharm., 2003, 66, 1139-1150.
 S.L. Hwang, G.C. Yen, J. Agric. Food Chem., 2009, 57, 2576-2582.
 M.J. Jeff, Therapeutic Research Facility. Natural Medicines Comprehensive Database, 4th edn, 2002.
Y,  L.T. Yi, C.F. Li, X. Zhan, C.C. Cui, F. Xiao, L.P. Zhou, Y. Xie, Involvement Prog. Biol. Psychiatr., 2010, 34, 1223-1228.
 A. Russo, R. Acquaviva, A. Campisi, V.Sorrenti, C. Di Giacomo, G. Virgat, M.L. Barcellon A.Vanella, Cell Biol. Toxicol., 2000, 16, 91-98
 I. Erlund, M.L. Silaste, G. Alfthan, M. Rantala, Y.A. Kesaniemi, A. Aro, Eur. J. Clin. Nutr., 2002, 56, 891-898.
 E.J. Choi, W.S. Ahn, Arch. Pharm. Res., 2008, 31, 1457-1462.
 S.H. Jeon, W. Chun, Y.J. Choi, Y.S. Kwon, Arch. Pharm. Res., 2008, 31, 978-982.
 J.M. Vasconcelos, A.M.S. Silva, J.A.S Cavaleiro, Phytochem., 1998, 49, 1421-1424.
 A.A Haroun, A.M. Elnahrawy, H.I. Abd-Alla, 2016.
 K. Pradeep, S. Park, K. CheolKo, European J. Pharmacol., 2008, 587, 273-280
 A. Jain, A. Yadav, A.I. Bozhkov, V.I. Padalko, S.J.S. Flora, Ecotoxicol. Environ. Saf., 2011, 74, 607-614.
 S. Sonkusare, K. Srinivasan, C. Kaul, P. Ramarao, Life Sci., 2005, 77, 1-14.
 H.A. Fernando, Diabetes. Res., 2013, 95, 2013:1903.
 J. Zagrodzka, A. Romaniuk, M.Wieczorek, P. Boguszewski, Acta. Neurobiol. Exp., 2000, 60, 333-343.
 G. Wen, W. Hui, C. Dan, W. Xiao-Qiong, T. Jian-Bin, L. Chang-Qi, L. De-Liang, C. Wei-Jun, L. Zhi-Yuan, L. Xue-Gang, Acta. Histochem. Cytochem., 2009, 42, 137-142.
 B. Savitha, C.N. Naik, R. Guruprasad, S. Arjunp, S. Priyanka, S. Math, J. Inves. Clini., 2014, Dentistry.
 N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, Exp. Cell. Res., 1988, 175, 184-191.
 E. Matteucci, F. Cocci, L. Pellegrini, G. Gregori, O. Giampietro, Enz. Protein., 1994, 48(2), 105-119.
 M.M. Bradford, Anal. Biochem., 1976, 72, 248-254.
 P. Wolfram, Food. Chem. Toxicol., 2008, 46, 1360-1364.
 S. Mehri, K. Abnous, S.H. Mousavi, V.M. Shariaty, H. Hosseinzaddeh, Cell. Mol. Neurobiol., 2011, 30, 185-191.
 S. Yu, F. Son, J. Yu, X. Zhao, L. Yu, G. Li, Neurochem. Res., 2006, 31, 1197-2
 Y.J. Zhu, T. Zeng, Y.B. Zhu, S.F. Yu, Q.S. Wang, L.P. Zhang, Neurochem. Res., 2008, 33, 2310-2317.
 B.D. Goldstein, H.E. Lowndes, Neurotoxicol., 1986, 2, 297-312.
 D.W. Sickls, S.T. Brady, A. Testino, M.A. Friedmanand R.W. Wrenn, J. Neurosci. Rev., 1996, 46, 7-17.
 R. LoPachin, D. Barber, D. He, S. Das, Toxicological. Sci., 2006, 89(1), 224-234.
 H.H. Ahmed, G.A. Elmegeed, S.M. El-Sayeed, M.M. Abd-Elhalim, W. ShoushaGh, R.W. Shafic, Eur. J. Med. Chem., 2010,
 B. Ling, N. Authier, D. Balayssac, A. Eschalier, F. Coudore, 2005, Pain., 119, 104-112.
 M.T. Lin, M.F. Beal, 2006, Nature., 443, 787-795.
 R. Schmatz, C.M. Mazzanti, R. Spanevello, N. Stefanello, J. Gutierres, M. Correa, Eur. J. Pharmacol., 2009, 610, 8-42.
 Q.H. Jin, H.Y. He, Y.F. Shi, X.J. Ahang, Acta. Pharmacol., 2004, 25, 1013-1021.
 D.S. Strac, D. Muck-Seler, N. Pivac, Psychiatria. Danubina., 2015, 27(1), 14-24
 N.S. Prasad, Muralidhara, Neuro. Toxicol., 2012, 33, 1254-1264.
 M.E. Hidalgo, C. De la Rosa, Quim Nova., 2009, 70, 32-1467.
 T. Sumathi, C. Shobana, V. Mahalakshmi, R. Sureka, M. Subathra, A. Vishali, K. Rekha, Asian. J. Pharm. Clin. Res., 2013, 6, (3) 80-90.
 S.C. Bondy, D. Liu, S. Guo-Ross, Neurochem. Int., 1998, 33, 51-54.
 C.J. Martyniuk, B. Fang, J.M. Koomen, T. Gavin, L. Zhang, D.S. Barbert, Chem. Res. Toxicol., 2011, 24, 2302-2311.
 L. Wang, X. Yan, Z. Zeng, J. Lv, P. Liu, C. Liu, 2010, 35, 1740-1744.
 J. George, K.R. Rao, R. Stern, G. Chandrakasan, Toxicol., 2001, 156, 129-138.
 S. Sherlock, J. Dooley, Malden, MA: Blackwell Science 2002.
 M.L. Anthony, K.P. Gartland, C.R. Beddell J.K. Lindon, Arch.Topical., 1994, 68, 43-53.
 G. Ramakrishnan, H.R. Raghavendran, R. Vinodhkumar, T. Devaki, Chem. Biol. Interact., 2006, 161, 104-114.
 Y.C. Chen, S.C. Shen, W.R. Lee, W.C. Hou, L.L. Yang, T.J.F. Lee, J. Cell. Biochem., 2001, 82, 537-548.
 Y.C. Chen, S.C. Shen, W.R. Lee, H.Y. Lin, C.H. Ko, C.M. Shih, L.L. Yang, Arch. Toxicol., 2002, 76, 351-359.
 N.J. Montvale, PDR for Herbal Medicines. Medical Economics Company., 2000.
 A. Hirata, Y. Murakami, M. Shoji, Y. Kadoma, S. Fujisawa, Anticancer. Res., 2005, 25, 3367- 3374.
 E.J. Choi, Life. Sci., 2008, 82, 1059-1064.
 Q. Xie, Y. Liu, H. Sun, Y. Liu, X. Ding, D. Fu, J. Agr. Food. Chem., 2008, 56, 6054-6060.