Biomarkers
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Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice Khazri Abdelhafidh, Lazher Mhadhbi, Ali Mezni, Sellami Badreddine, Hamouda Beyrem & Ezzeddine Mahmoudi To cite this article: Khazri Abdelhafidh, Lazher Mhadhbi, Ali Mezni, Sellami Badreddine, Hamouda Beyrem & Ezzeddine Mahmoudi (2017): Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice, Biomarkers, DOI: 10.1080/1354750X.2017.1390609 To link to this article: http://dx.doi.org/10.1080/1354750X.2017.1390609
Accepted author version posted online: 11 Oct 2017.
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Author Information ------------------------------------------------Dr abdelhafidh khazri (Corresponding Author) Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Dr , Lazher MHADHBI Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Dr Ali Mezni Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Professor Badreddine Sellami Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Professor Hamouda Beyrem Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Professor Ezzeddine Mahmoudi Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia
Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice Abdelhafidh KHAZRIa, Lazher MHADHBIa, Ali MEZNIa, Badreddine SELLAMIb , Hamouda BEYREMa, Ezzeddine MAHMOUDIa
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E-mail address: [email protected]
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Phone: + 216 502275 38
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Unité d’Ecotoxicologie et d’Ecologie Côtière (GREEC),
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Faculté des Sciences de Bizerte, 7021 Zarzouna–Bizerte.
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* Corresponding Author: Abdelhafidh KHAZRI
Abstract Context: Cypermethrin (CYP) is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control. The toxicity of CYP is well studied in many organisms Objective: The aim of present study was to investigate the protective effect of Zizyphus lotus (Zizyp) fruit against neurotoxicity and oxidative stress induced by CYP in mice.
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used as control and CYP control (20mg/kg body weight). While, groups III was orally treated with Zizyphus lotus fruit (5 g/kg body weight) plus CYP (20mg/kg body weight) for 18 days.
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Furthermore, HPLC-ESI-MS-MS (Q-Tof) and GC-MS were used to identify the compounds
Results:
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fraction.
Antioxidant enzyme catalase (CAT), neurotoxicity enzyme acetylcholinesterase
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(AChE) activities and hydrogen peroxide (H2O2), malondialdehyde (MDA) levels were determined in the liver, kidney and heart. CYP caused decreased CAT activity, inhibition of
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AChE activity and increased the levels of H2O2 and MDA in heart, liver and kidney. Conclusion: Our results indicate that, Zizyp fruit is markedly effective in protecting mice against
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CYP-induced biochemical changes. This protection may be due to its antioxidant property and
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scavenging ability against active free radicals.
Keywords: Cypermethrin, Zizyphus lotus jujube fruits, HPLC-ESI-MS-MS, Mice, Oxidative stress,
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Materials and methods: Mice were divided into four groups of six each: groups I and II were
Introduction CYP, type II pyrethroid, has potent insecticidal property. It plays a role in agriculture, protection of food stuff, disease vector control and home pest control (Sankar et al. 2011). CYP can be found in trace amounts or at higher concentrations in fruits, vegetables, cow’s milk and bread (Sankar et al. 2010). The toxicity of pyrethroid insecticides to mammalian animals has received much attention in recent years because animals exposed to these insecticides exhibited changes in
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and genotoxic effects and reproductive toxicity in various experimental systems (Yousef et al. 2003; Sood 2006). This compound is a hydrophobic molecule which can easily pass through the
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cell membrane and disturb its structure and cause leakage of cytoplasmic enzymes (Manna et al.
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2004; Hussien et al. 2013). Currently, oxidative stress are among the most important subjects in pesticide toxicology (López et al. 2007; Nakamura et al. 2007) as pesticides including CYP may
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be directly involved in these processes. CYP may be responsible for the induction of oxidative stress by interfering with different pathways in some organisms. For example, khazri et al. 2015, indicated that CAT activity as well as MDA and H2O2 levels were induced significantly in the
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gills of freshwater mussels. Giray et al. (2001) reported that single (170 mg /kg ) or repeated (75 mg/ kg per day for 5 days) oral administration of CYP was found to produce significant oxidative
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stress in cerebral and hepatic tissues in rats.
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Nowadays, there is an increased demand for using plants in therapy; the ‘‘back to nature” slogan, instead of using synthetic drugs which might have adverse effects and consequently might be
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more dangerous than the disease itself (Mady et al. 2001). The genus Zizyp belonging to the family Rhamnaceae is widespread in tropical and subtropical regions. In Africa, Zizyp jujube is widely distributed in Mediterranean region, like Tunisia, Algeria, Morocco, and Libya (Richardson et al. 2004). Recently, several pharmacological researches (Borgi et al. 2007; Borgi
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their physiological activities (Sakr 2003). CYP can also elicit a range of neurotoxic, immunotoxic
et Chouchane 2009) have been reported on the various species of Zizyp (Rhamnaceae) particularly Zizyp which is commonly known as ”sedra” and the edible fruit called ”nbeg”. This plant is used in folklore medicine for the treatment of various diseases such as diabetes, bronchitis, diarrhea and abscess (Le-Floc’h 1983).
Clinical significance Hence, the present study was undertaken to explore the toxic effect of the insecticide CYP on mice. This work also aimed to investigate the protective effect of Zizyp fruits against oxidative stress and neurotoxicity induced by CYP in heart, liver and kidneys of mice.
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Plant material
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Composition of Zizyphus lotus jujube fruits extract
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Zizyp fruits were collected from Jendouba, North of Tunisia, in August 2016. In order to minimize the degradation of compounds, fruits were dried in lyophilizer and stored at 4°C until use.
The extraction of Zizyp jujube fruits for their polyphenols analysis was performed as described
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previously by Wojdyło et al. (2013). Fruits were dried and grounded with an electric mincer (FP3121 Moulinex) until a fine powder was obtained. Powder was dissolved in 10% ethanol in
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the dark, vigorously vortexed for 25 min, centrifuged at 14,000g for 30 min at 4 °C for debris elimination and supernatant containing soluble polyphenols was ready to use. Total phenolic
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content was determined by the Folin-Ciocalteau colorimetric method, flavonoids and condensed
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tannins according to Dewanto et al. 2002; Sun et al. 1998 respectively. Fruits composition was established by HPLC–MS/MS analysis. Briefly liquid chromatography was performed using a Perkin Elmer system series 200 equipped with a binary micro-pump. The analyses were carried
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out on a C18 column (Zorbax Eclipse XDB-C18, 4.6 ×150 mm, particle size 5 mm). The mobile phase A was 0.1% formic acid in water and the mobile phase B was 0.1% formic acid in acetonitrile. Elution was performed at a flow rate of 1 mL min-1 and an injection volume of 20
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Materials and methods
mL. Tandem mass spectrometry (MS/MS) was carried out using a 3200 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex Forster city USA) equipped with an electrospray ionization (ESI) interface. Data were acquired and processed with Analyst 1.5.1 software. The detector was set in the negative ion mode. The ion trap mass spectrometer was operating in the m/z 50–1700 mass range.
Animals and experimental design. Twenty-four male mice (195–200 g) from Pasteur Institute (Tunis) were used in agreement with the ethic committee of Tunis University. They were provided with food and water add libitum and maintained in animal house at fixed temperature of 22 ± 2 °C with a 12 h light–dark cycle. Mice were randomly divided into four groups of six animals each that were daily treated by intraperitoneal (ip) route.
Group II: Zizyp: mice receiving 5 g/kg Zizyp fruits for 18 days
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Group III: CYP: mice receiving a single dose of CYP (20 mg/kg) at days 18.
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Group IV: CYP + Zizyp fruits: mice treated both with Zizyp fruits and a single dose of CYP at days 18
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At the end of the treatment, mice were anesthetized with urethane (40 mg/mL), sacrificed. Heart, Liver and Kidney were isolated, homogenized in PBS buffer pH 7.4 with an ultrathurax T25 homogenizator,
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centrifuged (30 min at 9,000g, 4°C) and the resulting supernatant was used for heart , liver and kidney lipid peroxidation, neurotoxicity and oxidative stress analyses.
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Biochemical analyses
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CAT activity was measured by the decrease in absorbance at 240 nm due to H2O2 consumption according to method of Aebi (1974). The reaction volume and reaction time were 1 mL and 1 min, respectively. The
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reaction solution contained 80 mM phosphate buffer, pH 6.5 and 50 mM H2O2 (Ni et al., 1990). Specific CAT activity was given as nmol/min/ mg protein.
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MDA, a marker of lipid peroxidation was determined according to Draper and Hadley, 1965. An aliquot of the homogenate was mixed with butylated hydroxy toluene/trichloroacetic acid (BHT/TCA) solution containing 1% (w/v) BHT dissolved in 20% TCA (w/v) and centrifuged at 4000g for 15 min at 4 °C.
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Group I: control: mice receiving 10% ethanol for 18 days
Supernatant was blended with 0.6 N HCl and 120 mmol /L. MDA, a marker of lipid peroxidation was determined according to Draper and Hadley . An aliquot of the homogenate was mixed with butylated hydroxy toluene/trichloroacetic acid (BHT/TCA) solution containing 1% (w/v) BHT dissolved in 20% TCA (w/v) and centrifuged at 4000g for 15 min at 4 °C. Supernatant was blended with 0.6 N HCl and 120 mmol /L.
Heart, Liver and Kidney AChE activity was determined according to the method of Ellman et al. 1961, using acetylcholine as substrate. The reaction mixture containing sample and 0.33 mM
DTNB into 100 mM phosphate buffer pH 7.4 was started by the addition of substrate and incubated at 37°C. Absorbance was followed at 412 nm every 30 s for 5min and AChE activity expressed as nmol/min/mg protein. Statistical analysis Data are expressed as mean ± SEM. for all the experiments. Biochemical data were analyzed
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assumptions, statistical significance was evaluated through one-way ANOVA. Significant
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differences were assessed using the Tukey HSD test. A probability level of less than 0.05 was
Results Zizyphus lotus jujube fruits extract composition
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considered as significant (95% confidence interval).
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The analysis of new cultivars from North Tunisia Zizyp jujube fruits revealed the presence of a wide range of flavonoid phenolics (Table 1). Concerning jujubes, different flavan-3-ols and
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flavonols (mainly quercetin derivatives), flavanone, and dihydrochalcone were found, as expected. Content of total phenolic compounds ranged between 1409.75 and 8432 mg/100 g dw,
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only Polymeric proanthocyanidins, Procyanidin B2, Quercetin, Epicatechin and Catechin were
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more abundant.
Effects of Cypermethrin and Zizyphus lotus jujube on oxidative markers
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The present study showed that CYP administration displayed a significant (P < 0.05) increase of heart, liver and kidney Hydrogen peroxide (H2O2) levels compared to normal control and Zizyp values after 18 days. Simultaneous treatment with CYP and Zizyp showed significant restoration
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using One-way ANOVA using the software STATISTICAs 8.0. After testing ANOVA
in H2O2 levels in heart, liver and kidney tissues (Figure 1). CYP treatment significantly attenuated the CAT activity in heart, liver, and kidney tissues and Zizyp treatment significantly restored the enzyme to near normal status in all the tissues. CYP exposure resulted in significant decrease in CAT activity in heart, liver and kidney (Figure 2). Simultaneous treatment with CYP and Zizyp showed significant restoration in CAT activity in heart, liver and kidney tissues when compared to animals treated with CYP alone.
Effects of Cypermethrin and Zizyphus lotus on lipid peroxidation MDA levels in heart, liver and kidney tissues were significantly higher (p ≤ 0.001) in CYPtreated mice compared to control (Figure 3). MDA levels decreased significantly in CYP with Zizyp-treated group compared with cypermethrin treated group. No statistically significant changes were observed when control group was compared to mice receiving Zizyp alone or in
Effects of Cypermethrin and Zizyphus lotus on neurotoxicity
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AChE activity in heart, liver and kidney tissues were significantly inhibited (p ≤ 0.05) in cypermethrin-treated mice compared to control (Figure 4). AChE activity increased significantly
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in cypermethrin with Zizyp-treated group compared with CYP treated group. No statistically
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significant changes were observed when control group was compared to mice receiving Zizyp alone or in combination with CYP.
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Discussion
The mode of action of CYP can be expected to have two ways: it may accumulate in cell
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membrane due to the hydrophobic nature and small molecular size, CYP passes through the cell and disturb membrane structure (Saxena et al. 2005). CYP has also been shown to induce
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oxidative stress and generation of reactive oxygen species (ROS) in experimental systems. It has
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been demonstrated that ROS may cause DNA damage, which could lead to single-strand breaks and mutation (Woo et al. 2009; Jin et al. 2011). For protecting themselves against the damaging effects of activated free radicals, like O2.-(main form of reactive species of oxygen), various
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organisms can stimulate their antioxidant enzyme activities, protecting molecular targets against oxidative injury (Bouayed et Bohn 2010). The overall results of this study showed that oral exposure to cypermethrin introduces significant oxidative stress in heart, liver and kidney tissues
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combination with CYP.
of mice as was evident by the elevation of the level of H2O2. Several studies have demonstrated that pesticides exposure induced increased generation of reactive oxygen species (ROS) leading to oxidative stress and alterations in the cellular antioxidant defense system (Mansour et Mossa 2009). Decreased of CAT activity also indicated the occurrence of an oxidative insult. CAT activity is considered to be a good indicator of antioxidant status or oxidative stress. Significant decrease in CAT activity in this study may be either due to the excessive of H 2O2 synthesis indicating towards CYP induced oxidative stress in these organs. The depression of this enzyme
activity as observed in the present study reflects perturbations in normal oxidative mechanisms during CYP toxicity. CYP has been demonstrated to cause a significant decrease in the CAT activity in the rats (Sankar et al. 2012). Similar result has been found in animal exposed to different pyrethroid compounds (Fetoui et al. 2010). CYP may be a potential indicator for fatty acid metabolism, and implicitly of a possible membrane lipid peroxidation. To disclose the matter, MDA tissular quantification was tested in heart, liver and kidney tissues. Our results
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to CYP. Our data are in accordance with previous results, which present a decrease in membrane
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fluidity in CYP exposed and an increase on MDA content (Sankar et al. 2012). The results of the
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present study also indicate that CYP caused significant inhibition of AChE activity in mice after exposure to 150 μg/L of CYP. This decrease of AChE acivity has been showed in all study
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organs when compared to the control mice. Similar patterns were observed in rats, Daphnia magna and fish exposed to pyrethroid under laboratory conditions (Mokhtar et al. 2006; Héla et
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al. 2015).
Results obtained in the present study indicated that extract Zizyp fruits (5g/kg) successfully
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maintained normal biochemical parameters against the toxicity induced by CYP in mice. The administration of Zizyp fruits markedly elevated the level CAT in the heart, liver and kidney,
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which nearly approached to normal level of untreated mice. It seems that Zizyp fruits extract
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(5mg/kg) could protect the heart, liver and kidney by improving enzymatic antioxidant defense system to effectively alleviate the generation of in vivo free radicals activated by CYP. Interestingly, extract Zizyp fruits (5mg/kg) was able to inhibited lipid peroxidation at higher level
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after CYP treatment. These results have provided the evidence for the pharmacological effect of Zizyp fruits in CYP induced oxidative stress. This finding is in agreement with observation of (Chang et al. 2010). In this study, in vitro antioxidant assays indicate that plant extracts showing
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showed an important leveling up of MDA, indicating lipid peroxidation resulting from exposure
higher antioxidant activity and higher phenol and flavonoid contents could be significant source of natural antioxidant
Conclusion It can be concluded that chronic exposure to CYP causes heart, hepatic and renal toxicities. The mechanism of such pathological facts may be prompted by the free radical release and the lipid peroxidation that it induces. The use of Zizyp fruit was ascertained to reduce the harmful effects of CYP in the mentioned parameters. In addition to post-harvest investigations, further studies
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should be strongly considered for functional and nutritive applications.
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found in Zizyp fruits. The results of our study suggest that the antioxidant potential of Zizyp fruits
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Acknowledgments
The authors wish to thank the Ministry of High education, University of Carthage, Faculty of
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Sciences of Bizerte, Tunisia for financial support of this project. Pr. David Sheehan is gratefully acknowledged as a native English speaker for her helpful criticisms and for the English
Declaration of interest
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improvements.
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Authors do not have any potential conflict of interest
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may determine the bioavailability and the physiological relevance of the elucidated constituents
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Figure and table legends Fig. 1. Effect of CYP and Zizyp fruits on H2O2 levels, Heart (A), Liver (B) and Kidney (C). Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant: Fig. 2. Effect of CYP and Zizyp fruits on CAT activity, Heart (A), Liver (B) and Kidney (C).
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Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant:
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Fig. 3. Effect of CYP and Zizyp fruits on MDA levels, Heart (A), Liver (B) and Kidney (C).
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Fig. 4. Effect of CYP and Zizyp fruits on AChE activity, Heart (A), Liver (B) and Kidney (C).
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Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant:
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Table 1. LC–MS/MS data of some phenolic compounds found in Zizyp lotus fruits
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Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant:
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ISSN: 1354-750X (Print) 1366-5804 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmk20
Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice Khazri Abdelhafidh, Lazher Mhadhbi, Ali Mezni, Sellami Badreddine, Hamouda Beyrem & Ezzeddine Mahmoudi To cite this article: Khazri Abdelhafidh, Lazher Mhadhbi, Ali Mezni, Sellami Badreddine, Hamouda Beyrem & Ezzeddine Mahmoudi (2017): Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice, Biomarkers, DOI: 10.1080/1354750X.2017.1390609 To link to this article: http://dx.doi.org/10.1080/1354750X.2017.1390609
Accepted author version posted online: 11 Oct 2017.
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Author Information ------------------------------------------------Dr abdelhafidh khazri (Corresponding Author) Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Dr , Lazher MHADHBI Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Dr Ali Mezni Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Professor Badreddine Sellami Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Professor Hamouda Beyrem Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia ------------------------------------------------Professor Ezzeddine Mahmoudi Email: [email protected] Affiliation 1: Universite de Carthage Faculte des Sciences de Bizerte, Bizerte, 7021 Tunisia
Protective effect of Zizyphus lotus jujube fruits against cypermethrin-induced oxidative stress and neurotoxicity in mice Abdelhafidh KHAZRIa, Lazher MHADHBIa, Ali MEZNIa, Badreddine SELLAMIb , Hamouda BEYREMa, Ezzeddine MAHMOUDIa
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E-mail address: [email protected]
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Phone: + 216 502275 38
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Unité d’Ecotoxicologie et d’Ecologie Côtière (GREEC),
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Faculté des Sciences de Bizerte, 7021 Zarzouna–Bizerte.
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* Corresponding Author: Abdelhafidh KHAZRI
Abstract Context: Cypermethrin (CYP) is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control. The toxicity of CYP is well studied in many organisms Objective: The aim of present study was to investigate the protective effect of Zizyphus lotus (Zizyp) fruit against neurotoxicity and oxidative stress induced by CYP in mice.
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used as control and CYP control (20mg/kg body weight). While, groups III was orally treated with Zizyphus lotus fruit (5 g/kg body weight) plus CYP (20mg/kg body weight) for 18 days.
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Furthermore, HPLC-ESI-MS-MS (Q-Tof) and GC-MS were used to identify the compounds
Results:
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fraction.
Antioxidant enzyme catalase (CAT), neurotoxicity enzyme acetylcholinesterase
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(AChE) activities and hydrogen peroxide (H2O2), malondialdehyde (MDA) levels were determined in the liver, kidney and heart. CYP caused decreased CAT activity, inhibition of
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AChE activity and increased the levels of H2O2 and MDA in heart, liver and kidney. Conclusion: Our results indicate that, Zizyp fruit is markedly effective in protecting mice against
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CYP-induced biochemical changes. This protection may be due to its antioxidant property and
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scavenging ability against active free radicals.
Keywords: Cypermethrin, Zizyphus lotus jujube fruits, HPLC-ESI-MS-MS, Mice, Oxidative stress,
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Materials and methods: Mice were divided into four groups of six each: groups I and II were
Introduction CYP, type II pyrethroid, has potent insecticidal property. It plays a role in agriculture, protection of food stuff, disease vector control and home pest control (Sankar et al. 2011). CYP can be found in trace amounts or at higher concentrations in fruits, vegetables, cow’s milk and bread (Sankar et al. 2010). The toxicity of pyrethroid insecticides to mammalian animals has received much attention in recent years because animals exposed to these insecticides exhibited changes in
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and genotoxic effects and reproductive toxicity in various experimental systems (Yousef et al. 2003; Sood 2006). This compound is a hydrophobic molecule which can easily pass through the
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cell membrane and disturb its structure and cause leakage of cytoplasmic enzymes (Manna et al.
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2004; Hussien et al. 2013). Currently, oxidative stress are among the most important subjects in pesticide toxicology (López et al. 2007; Nakamura et al. 2007) as pesticides including CYP may
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be directly involved in these processes. CYP may be responsible for the induction of oxidative stress by interfering with different pathways in some organisms. For example, khazri et al. 2015, indicated that CAT activity as well as MDA and H2O2 levels were induced significantly in the
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gills of freshwater mussels. Giray et al. (2001) reported that single (170 mg /kg ) or repeated (75 mg/ kg per day for 5 days) oral administration of CYP was found to produce significant oxidative
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stress in cerebral and hepatic tissues in rats.
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Nowadays, there is an increased demand for using plants in therapy; the ‘‘back to nature” slogan, instead of using synthetic drugs which might have adverse effects and consequently might be
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more dangerous than the disease itself (Mady et al. 2001). The genus Zizyp belonging to the family Rhamnaceae is widespread in tropical and subtropical regions. In Africa, Zizyp jujube is widely distributed in Mediterranean region, like Tunisia, Algeria, Morocco, and Libya (Richardson et al. 2004). Recently, several pharmacological researches (Borgi et al. 2007; Borgi
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their physiological activities (Sakr 2003). CYP can also elicit a range of neurotoxic, immunotoxic
et Chouchane 2009) have been reported on the various species of Zizyp (Rhamnaceae) particularly Zizyp which is commonly known as ”sedra” and the edible fruit called ”nbeg”. This plant is used in folklore medicine for the treatment of various diseases such as diabetes, bronchitis, diarrhea and abscess (Le-Floc’h 1983).
Clinical significance Hence, the present study was undertaken to explore the toxic effect of the insecticide CYP on mice. This work also aimed to investigate the protective effect of Zizyp fruits against oxidative stress and neurotoxicity induced by CYP in heart, liver and kidneys of mice.
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Plant material
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Composition of Zizyphus lotus jujube fruits extract
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Zizyp fruits were collected from Jendouba, North of Tunisia, in August 2016. In order to minimize the degradation of compounds, fruits were dried in lyophilizer and stored at 4°C until use.
The extraction of Zizyp jujube fruits for their polyphenols analysis was performed as described
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previously by Wojdyło et al. (2013). Fruits were dried and grounded with an electric mincer (FP3121 Moulinex) until a fine powder was obtained. Powder was dissolved in 10% ethanol in
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the dark, vigorously vortexed for 25 min, centrifuged at 14,000g for 30 min at 4 °C for debris elimination and supernatant containing soluble polyphenols was ready to use. Total phenolic
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content was determined by the Folin-Ciocalteau colorimetric method, flavonoids and condensed
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tannins according to Dewanto et al. 2002; Sun et al. 1998 respectively. Fruits composition was established by HPLC–MS/MS analysis. Briefly liquid chromatography was performed using a Perkin Elmer system series 200 equipped with a binary micro-pump. The analyses were carried
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out on a C18 column (Zorbax Eclipse XDB-C18, 4.6 ×150 mm, particle size 5 mm). The mobile phase A was 0.1% formic acid in water and the mobile phase B was 0.1% formic acid in acetonitrile. Elution was performed at a flow rate of 1 mL min-1 and an injection volume of 20
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Materials and methods
mL. Tandem mass spectrometry (MS/MS) was carried out using a 3200 QTRAP mass spectrometer (Applied Biosystems/MDS Sciex Forster city USA) equipped with an electrospray ionization (ESI) interface. Data were acquired and processed with Analyst 1.5.1 software. The detector was set in the negative ion mode. The ion trap mass spectrometer was operating in the m/z 50–1700 mass range.
Animals and experimental design. Twenty-four male mice (195–200 g) from Pasteur Institute (Tunis) were used in agreement with the ethic committee of Tunis University. They were provided with food and water add libitum and maintained in animal house at fixed temperature of 22 ± 2 °C with a 12 h light–dark cycle. Mice were randomly divided into four groups of six animals each that were daily treated by intraperitoneal (ip) route.
Group II: Zizyp: mice receiving 5 g/kg Zizyp fruits for 18 days
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Group III: CYP: mice receiving a single dose of CYP (20 mg/kg) at days 18.
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Group IV: CYP + Zizyp fruits: mice treated both with Zizyp fruits and a single dose of CYP at days 18
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At the end of the treatment, mice were anesthetized with urethane (40 mg/mL), sacrificed. Heart, Liver and Kidney were isolated, homogenized in PBS buffer pH 7.4 with an ultrathurax T25 homogenizator,
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centrifuged (30 min at 9,000g, 4°C) and the resulting supernatant was used for heart , liver and kidney lipid peroxidation, neurotoxicity and oxidative stress analyses.
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Biochemical analyses
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CAT activity was measured by the decrease in absorbance at 240 nm due to H2O2 consumption according to method of Aebi (1974). The reaction volume and reaction time were 1 mL and 1 min, respectively. The
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reaction solution contained 80 mM phosphate buffer, pH 6.5 and 50 mM H2O2 (Ni et al., 1990). Specific CAT activity was given as nmol/min/ mg protein.
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MDA, a marker of lipid peroxidation was determined according to Draper and Hadley, 1965. An aliquot of the homogenate was mixed with butylated hydroxy toluene/trichloroacetic acid (BHT/TCA) solution containing 1% (w/v) BHT dissolved in 20% TCA (w/v) and centrifuged at 4000g for 15 min at 4 °C.
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Group I: control: mice receiving 10% ethanol for 18 days
Supernatant was blended with 0.6 N HCl and 120 mmol /L. MDA, a marker of lipid peroxidation was determined according to Draper and Hadley . An aliquot of the homogenate was mixed with butylated hydroxy toluene/trichloroacetic acid (BHT/TCA) solution containing 1% (w/v) BHT dissolved in 20% TCA (w/v) and centrifuged at 4000g for 15 min at 4 °C. Supernatant was blended with 0.6 N HCl and 120 mmol /L.
Heart, Liver and Kidney AChE activity was determined according to the method of Ellman et al. 1961, using acetylcholine as substrate. The reaction mixture containing sample and 0.33 mM
DTNB into 100 mM phosphate buffer pH 7.4 was started by the addition of substrate and incubated at 37°C. Absorbance was followed at 412 nm every 30 s for 5min and AChE activity expressed as nmol/min/mg protein. Statistical analysis Data are expressed as mean ± SEM. for all the experiments. Biochemical data were analyzed
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assumptions, statistical significance was evaluated through one-way ANOVA. Significant
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differences were assessed using the Tukey HSD test. A probability level of less than 0.05 was
Results Zizyphus lotus jujube fruits extract composition
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considered as significant (95% confidence interval).
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The analysis of new cultivars from North Tunisia Zizyp jujube fruits revealed the presence of a wide range of flavonoid phenolics (Table 1). Concerning jujubes, different flavan-3-ols and
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flavonols (mainly quercetin derivatives), flavanone, and dihydrochalcone were found, as expected. Content of total phenolic compounds ranged between 1409.75 and 8432 mg/100 g dw,
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only Polymeric proanthocyanidins, Procyanidin B2, Quercetin, Epicatechin and Catechin were
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more abundant.
Effects of Cypermethrin and Zizyphus lotus jujube on oxidative markers
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The present study showed that CYP administration displayed a significant (P < 0.05) increase of heart, liver and kidney Hydrogen peroxide (H2O2) levels compared to normal control and Zizyp values after 18 days. Simultaneous treatment with CYP and Zizyp showed significant restoration
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using One-way ANOVA using the software STATISTICAs 8.0. After testing ANOVA
in H2O2 levels in heart, liver and kidney tissues (Figure 1). CYP treatment significantly attenuated the CAT activity in heart, liver, and kidney tissues and Zizyp treatment significantly restored the enzyme to near normal status in all the tissues. CYP exposure resulted in significant decrease in CAT activity in heart, liver and kidney (Figure 2). Simultaneous treatment with CYP and Zizyp showed significant restoration in CAT activity in heart, liver and kidney tissues when compared to animals treated with CYP alone.
Effects of Cypermethrin and Zizyphus lotus on lipid peroxidation MDA levels in heart, liver and kidney tissues were significantly higher (p ≤ 0.001) in CYPtreated mice compared to control (Figure 3). MDA levels decreased significantly in CYP with Zizyp-treated group compared with cypermethrin treated group. No statistically significant changes were observed when control group was compared to mice receiving Zizyp alone or in
Effects of Cypermethrin and Zizyphus lotus on neurotoxicity
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AChE activity in heart, liver and kidney tissues were significantly inhibited (p ≤ 0.05) in cypermethrin-treated mice compared to control (Figure 4). AChE activity increased significantly
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in cypermethrin with Zizyp-treated group compared with CYP treated group. No statistically
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significant changes were observed when control group was compared to mice receiving Zizyp alone or in combination with CYP.
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Discussion
The mode of action of CYP can be expected to have two ways: it may accumulate in cell
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membrane due to the hydrophobic nature and small molecular size, CYP passes through the cell and disturb membrane structure (Saxena et al. 2005). CYP has also been shown to induce
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oxidative stress and generation of reactive oxygen species (ROS) in experimental systems. It has
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been demonstrated that ROS may cause DNA damage, which could lead to single-strand breaks and mutation (Woo et al. 2009; Jin et al. 2011). For protecting themselves against the damaging effects of activated free radicals, like O2.-(main form of reactive species of oxygen), various
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organisms can stimulate their antioxidant enzyme activities, protecting molecular targets against oxidative injury (Bouayed et Bohn 2010). The overall results of this study showed that oral exposure to cypermethrin introduces significant oxidative stress in heart, liver and kidney tissues
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combination with CYP.
of mice as was evident by the elevation of the level of H2O2. Several studies have demonstrated that pesticides exposure induced increased generation of reactive oxygen species (ROS) leading to oxidative stress and alterations in the cellular antioxidant defense system (Mansour et Mossa 2009). Decreased of CAT activity also indicated the occurrence of an oxidative insult. CAT activity is considered to be a good indicator of antioxidant status or oxidative stress. Significant decrease in CAT activity in this study may be either due to the excessive of H 2O2 synthesis indicating towards CYP induced oxidative stress in these organs. The depression of this enzyme
activity as observed in the present study reflects perturbations in normal oxidative mechanisms during CYP toxicity. CYP has been demonstrated to cause a significant decrease in the CAT activity in the rats (Sankar et al. 2012). Similar result has been found in animal exposed to different pyrethroid compounds (Fetoui et al. 2010). CYP may be a potential indicator for fatty acid metabolism, and implicitly of a possible membrane lipid peroxidation. To disclose the matter, MDA tissular quantification was tested in heart, liver and kidney tissues. Our results
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to CYP. Our data are in accordance with previous results, which present a decrease in membrane
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fluidity in CYP exposed and an increase on MDA content (Sankar et al. 2012). The results of the
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present study also indicate that CYP caused significant inhibition of AChE activity in mice after exposure to 150 μg/L of CYP. This decrease of AChE acivity has been showed in all study
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organs when compared to the control mice. Similar patterns were observed in rats, Daphnia magna and fish exposed to pyrethroid under laboratory conditions (Mokhtar et al. 2006; Héla et
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al. 2015).
Results obtained in the present study indicated that extract Zizyp fruits (5g/kg) successfully
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maintained normal biochemical parameters against the toxicity induced by CYP in mice. The administration of Zizyp fruits markedly elevated the level CAT in the heart, liver and kidney,
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which nearly approached to normal level of untreated mice. It seems that Zizyp fruits extract
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(5mg/kg) could protect the heart, liver and kidney by improving enzymatic antioxidant defense system to effectively alleviate the generation of in vivo free radicals activated by CYP. Interestingly, extract Zizyp fruits (5mg/kg) was able to inhibited lipid peroxidation at higher level
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after CYP treatment. These results have provided the evidence for the pharmacological effect of Zizyp fruits in CYP induced oxidative stress. This finding is in agreement with observation of (Chang et al. 2010). In this study, in vitro antioxidant assays indicate that plant extracts showing
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showed an important leveling up of MDA, indicating lipid peroxidation resulting from exposure
higher antioxidant activity and higher phenol and flavonoid contents could be significant source of natural antioxidant
Conclusion It can be concluded that chronic exposure to CYP causes heart, hepatic and renal toxicities. The mechanism of such pathological facts may be prompted by the free radical release and the lipid peroxidation that it induces. The use of Zizyp fruit was ascertained to reduce the harmful effects of CYP in the mentioned parameters. In addition to post-harvest investigations, further studies
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should be strongly considered for functional and nutritive applications.
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found in Zizyp fruits. The results of our study suggest that the antioxidant potential of Zizyp fruits
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Acknowledgments
The authors wish to thank the Ministry of High education, University of Carthage, Faculty of
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Sciences of Bizerte, Tunisia for financial support of this project. Pr. David Sheehan is gratefully acknowledged as a native English speaker for her helpful criticisms and for the English
Declaration of interest
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improvements.
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Authors do not have any potential conflict of interest
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may determine the bioavailability and the physiological relevance of the elucidated constituents
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Figure and table legends Fig. 1. Effect of CYP and Zizyp fruits on H2O2 levels, Heart (A), Liver (B) and Kidney (C). Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant: Fig. 2. Effect of CYP and Zizyp fruits on CAT activity, Heart (A), Liver (B) and Kidney (C).
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Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant:
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Fig. 3. Effect of CYP and Zizyp fruits on MDA levels, Heart (A), Liver (B) and Kidney (C).
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Fig. 4. Effect of CYP and Zizyp fruits on AChE activity, Heart (A), Liver (B) and Kidney (C).
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Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant:
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Table 1. LC–MS/MS data of some phenolic compounds found in Zizyp lotus fruits
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Results are expressed as mean ± SD (N = 6), p < 0.05 was considered significant:
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