The protective effect of Hibiscus sabdariffa calyxes extract against cypermethrin induced oxidative stress in mice

Journal Pre-proof The protective effect of Hibiscus sabdariffa calyxes extract against cypermethrin induced oxidative stress in mice

Ali Mezni, Lazher Mhadhbi, Abdelhafidh Khazri, Badreddine Sellami, Mohamed Dellali, Ezzeddine Mahmoudi, Hamouda Beyrem PII:

S0048-3575(19)30455-9

DOI:

https://doi.org/10.1016/j.pestbp.2019.09.007

Reference:

YPEST 4463

To appear in:

Pesticide Biochemistry and Physiology

Received date:

26 June 2019

Revised date:

14 September 2019

Accepted date:

25 September 2019

Please cite this article as: A. Mezni, L. Mhadhbi, A. Khazri, et al., The protective effect of Hibiscus sabdariffa calyxes extract against cypermethrin induced oxidative stress in mice, Pesticide Biochemistry and Physiology (2019), https://doi.org/10.1016/ j.pestbp.2019.09.007

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© 2019 Published by Elsevier.

Journal Pre-proof The protective effect of Hibiscus Sabdariffa calyxes extract against Cypermethrin induced oxidative stress in mice Ali MEZNIa,* [email protected], Lazher MHADHBIa Abdelhafidh KHAZRIa, Badreddine SELLAMIb, Mohamed DELLALIa, Ezzeddine MAHMOUDIa, Hamouda BEYREMa a

University of Carthage - Environmental Biomonitoring Laboratory (LBE) - Faculty of

Sciences of Bizerte - Zarzouna 7021 Tunisia b

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Institut National des Sciences et Technologies de la Mer, Tabarka, Tunisia

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*

Corresponding author.

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Abstract

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Cypermethrin (Cyp) is a kind of pyrethroids compound that is broadly used against different species of insects and pests. Cyp can also elicit a range of neurotoxic, immunotoxic,

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genotoxic and reproductive toxic effects on various experimental organisms. The aim of this study was to evaluate the protective effects of Hibiscus sabdariffa against the toxicity damage

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induced by Cyp exposure. The Hibiscus sabdariffa calyxes extract was given to mice (200-

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500 mg/kg bw). The mice, which were treated with Cyp and Hibiscus sabdariffa, were divided into six groups of six mice each. Groups I, IV and VI were used as control and groups

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II CYP control (20mg/kg body weight)., groups III and V were treated with Hibiscus sabdariffa extract (200 and 500 mg/kg body weight) plus (20mg/kg body weight) for 21 days Furthermore, HPLC was used to identify the compound fraction. This result showed Cyp induced biochemical changes in all organs of mice. Cyp caused decreased CAT activity, inhibition of AChE activity and increased the levels of H2 O 2 and MDA in brain, heart, liver and kidney. Hibiscus sabdariffa exhibited antioxidant effect and significantly attenuated the neurotoxicity of Cyp. Hibiscus sabdariffa exhibits neuroprotective effects and can be an effective and novel alternative approach to reduce the risk caused by pyrethroid compound. Keywords: Pesticide, the Hibiscus sabdariffa calyxes extract, antioxidant, neurotoxicity Introduction Pesticides are used largely by man to combat plagues of the harvest and vectors of transmissible diseases. Pyrethroids have the widespread application to control plant pests in

Journal Pre-proof agriculture and health (Prusty et al., 2015). Utilization of these synthetic compounds has been a growing process compared to the other pesticides like organochlorines, due to their low toxic effects on mammals (Desai et al., 2016). Pyrethroids classified by the World Health Organization as moderately harmful, class II (WHO, 1995). Cypermethrin [RS-a-cyano-3phenoxybenzyl (1RS)-cis-, trans-3-(2,2,-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] is the highly active type II synthetic pyrethroid insecticide containing the αcyano group. Cypermethrin has been found to cause significant morphological and behavioral, biochemical and neurotoxic stress in freshwater fishes (Kumar et al., 2007). The toxicity of cypermethrin is based on its interaction with the sodium channels located in the nerve cells,

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where it is able to maintain the channel in the open configuration, thus generating repeated

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nerve impulses in the affected organs (Bradberry et al., 2005).

Cypermethrin also causes induction of DNA damage and micronucleus in vitro in human

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lymphocytes (Suman et al., 2005). Both in vitro and in vivo experiments with rat peripheral

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blood lymphocytes showed that cypermethrin severely damages DNA and causes imbalance in the prooxidant/antioxidant status in lymphocytes (Suja et al., 2004 ; Gabbianelli et al.,

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2004). In vivo studies have also shown that cypermethrin causes free radical-mediated tissue damages.

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In recent years, interest has increased in using natural products for pharmacological purposes, as a form of complementary or replacement therapy (Panda and Naik, 2009). Hibiscus

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sabdariffa L., also known as roselle, is an annual, herbaceous medicinal plant that belongs to the Malvaceae family. This species is usually cultivated for its fibers and calyces, and

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includes three different genotypes: green, red (which is the most common type used) and dark red (Barhé, and Tchouya, 2015). The importance of vegetables, fruits, legumes and berries as part of a healthy diet is generally accepted. One possible reason why these foods promote good health could be the presence of a range of antioxidants in edible plants, for example vitamins C and D, carotenes, selenium, folates and phenolic compounds, including flavonoids. Polyphenols with their antioxidant property, transfer an electron to the free radicals, which thus become stable as their electrons are paired (Fraga, 2007). Encouraged by the above mentioned properties of Hibiscus sabdariffa, the current study examines the ameliorative potential of this plant extract against the biochemical alterations induced by Cyp in mice. Clinical significance

Journal Pre-proof This work also aimed to investigate the protective effect of Hibiscus sabdariffa L against oxidative stress and neurotoxicity induced by CYP in brain, heart, liver and kidneys of mice. Materials and methods Chemical α Cypermethrin (purity 99.7%, Fluka, Sigma Aldrich, St. Louis, USA) were obtained from Sigma Aldrich.

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Plant material

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Hibiscus sabdariffa L calyxes were collected from Mednine, South of Tunisia, in October2014. In order to minimize the degradation of compounds, calyxes were dried in

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lyophilizer and stored at 4°C until use.

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Preparation of extract

The extraction of Hibiscus sabdariffa L. calyxes for their polyphenols analysis was performed

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as described previously by Wojdyło et al. (2013). Calyxes were dried and grounded with an electric mincer (FP3121 Moulinex) until a fine the powder was obtained. Powder was

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dissolved in 10% ethanol in the dark, vigorously vortexed for 25 min, centrifuged at 14,000g for 30 min at 4 °C for debris elimination and the supernatant containing soluble polyphenols

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was ready to use. Total phenolic content was determined by the Folin-Ciocalteau colorimetric method, flavonoids and condensed tannins according to Dewanto et al. 2002; Sun et al. 1998

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respectively. Extract 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 out on a C18 column (Zorbax Eclipse XDBC18, 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 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 by 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 Thirty-six male mouse mus musculus (32–38g) from the Pasteur Institute (Tunis) were used in agreement with the NIH guidelines (1985). They were provided with food and water ad

Journal Pre-proof libitum and maintained in animal house at controlled temperature 22±2°C with a 12 h light– dark cycle. Mice were randomly divided into six groups of six animals each and received daily intraperitoneal (IP) injection during 21 days as follows: — Group 1 (Control): mice administered with 10% ethanol during 21 days (n = 6). — Group 2 (Cyp): mice receiving a single dose of Cyp (20 mg/kg) at day21. (n = 6). — Group 3 (Cyp + HSE200): mice treated both with 200mg/kg bw of HSE during 21days and a single dose of Cyp on day 21 (n = 6). — Group 4 (HSE200): mice treated with HSE (200mg/kg bw) during 21days (n = 6). —Group 5(Cyp + HSE500): mice treated both with 500mg/kg bw of HSE during 21days and

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a single dose of Cyp on day 21 (n = 6).

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— Group 6 (HSE500): mice treated with HSE (500mg/kg bw) during 21days (n = 6). At the end of the exposure period, mice were killed by decapitation, their organs (brain, heart,

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liver and kidney) collected, weighed and homogenized in phosphate buffer saline pH 7.4 with

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an ultra-thurrax homogenizator. After centrifugation (10 min at 10.000g, 4 °C), the supernatant was used for evaluation of Cyp-induced oxidative stress status in mice.

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Biochemical analyses

Assay of lipid peroxidation Lipid peroxidation in the hepatic tissue was estimated

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colorimetrically by measuring thiobarbituric acid reactive substances (TBARS) which were

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expressed in terms of malondialdehyde (MDA) content according to Draper and Hadley (1990) method. Briefly, aliquots of liver homogenates were mixed with 1 mL of 5 % TCA

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and centrifuged at 4,000 g for 10 min. 1 ml of thiobarbituric acid reagent (TBA, 0.67 %) was added to 500 ml of supernatant and heated at 95 °C for 15 min. The mixture was then cooled and was measured for absorbance at 532 nm. The MDA values were calculated using 1, 1, 3, 3-tetraethoxypropane as the standard and expressed as nmoles of MDA/g of tissue. Catalase (CAT) was assayed by the decomposition of hydrogen peroxide according to the method of Aebi (1974). Decrease in absorbance due to H2 O 2 degradations was monitored at 240 nm for 1 min and the enzyme activity was expressed as μmol H2 O2 consumed/min/mg protein. H2 O 2 was determined enzymatically according to Kakinuma et al. (1979) using a commercially available kit from Biomaghreb. Briefly, in the presence of peroxidase, H2 O 2 reacts with 4-amino-antipyrine and phenol to give a red colored quinoneimine which was absorbed at 505 nm and results expressed as mmol H2 O2 /mg protein.

Journal Pre-proof Acetylcholinesterase activity (AChE; EC 3.1.1.7) 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 using One-way ANOVA using the software STATISTICAs 8.0. After testing ANOVA assumptions, statistical significance was evaluated through one-way ANOVA. Significant

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considered as significant (95% confidence interval).

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differences were assessed using the Tukey HSD test. A probability level of less than 0.05 was

The data analysis follows the methods of standard community analysis described by Clarke

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and Goreley(2005) using “PRIMER 6” (polysmouth routines in multivariate ecological

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research). The obtained data were transformed (logx+1) and subjected to Principal Component Analysis (PCA) ordination on the basis of Bray Curtis similarity to simplify its

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interpretation Results

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Hibiscus sabdariffa calyxes extract composition

The analysis of new cultivars from South of Tunisia Hibiscus sabdariffa L. calyxes revealed

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the presence of a wide range of flavonoid phenolics (Table 1). Concerning Hibiscus, different flavan-3-ols and flavonols (mainly quercetin derivatives), flavanone, and dihydrochalcone

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were found, as expected. Content of total phenolic compounds ranged between 1409.75 and 8432 mg/100 g dw, only Polymeric proanthocyanidins, Procyanidin B2, Quercetin, Epicatechin and Catechin were more abundant.

Effects of cypermethrin and HSE on the brain The present study showed (Figure1A) that Cyp administration displayed a significant (p<0.05) increase of brain Hydrogen peroxide (H2 O2 ) levels compared to normal control values. The co-treatment with HSE (200 and 500mg/kg) efficiently alleviated all the deleterious effects especially at 500mg/kg. CYP increased lipoperoxidation MDA (Figure1B) with significantly manner (p ≤ 0.001) compared to control. All dosage of HSE efficiently brought the MDA level to near control level.

Journal Pre-proof Figure1C showed that Cyp exposure resulted in a significant decrease in CAT activity in the brain and a simultaneous treatment with Cyp and HSE showed a significant restoration in CAT activity when compared to animals treated with Cyp alone. Brain AChE activity (Figure 1D) was significantly inhibited (p ≤ 0.05) in Cyp -treated mice compared to control. HSE efficiently protected from this alteration specially the highest dose of 500 mg/kg. The Principal Component Analysis (PCA) of the different biomarkers in the brain showed that Cyp induced an oxidative stress and Cyp –HSE 500 counterbalanced all alterations. The data plot given in the Fig 2A showed that Cyp effected H2 O2 and MDA with similar manner

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contrary for CAT and AChE. Fig 2B indicated the correlation between biomarkers, this result

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showed a positive correlation between MDA and H2 O2 and the other hand between CAT and

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Effects of cypermethrin and HSE on the heart

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AChE. Negative correlation between MDA, CAT and AChE, H2 O2 .

Figure 3 A showed that the MDA level in the heart was significantly higher (p≤ 0.001) in

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Cyp-treated mice compared to control. Only the highest dose of HSE (500mg/kg) efficiently brought the MDA level to near control level. No statistically significant changes were

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observed when control group was compared to mice receiving different doses of HSE. Cyp induced an increase of H2 O2 level in the heart +218, 51% (Figure 3B) the co-treatment

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reduced the level near to control by significant manner even at the lower dose of HSE 200mg/kg. Figure 3 C and D presented a significant inhibition of heart CAT and AChE

activity.

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activities induced by Cyp and the HSE treatment with different dose restored this decrease of

The PCA loading plots (Fig. 4A), showing the relationships between biomarkers in PC1 and PC2, depict positive associations as biomarkers clustered together (< 90° angle), with smaller angles representing stronger associations. Conversely, biomarkers on opposite sides of the origin (approximately 180°) are negatively associated. Biomarkers at a 90° angle to each other have no association. Overall, the results of the statistical method used to assess correlations between biomarkers strongly agreed. Fig 4B provides all correlation coefficients.

Effects of Cypermethrin and HSE on Liver MDA and H2 O2 in the liver were significantly increased (p ≤ 0.05) in Cyp-treated mice compared to control (Figure 5A, B) respectively +63.2% and +64.05%. MDA and H2 O2 levels decreased significantly in Cyp with HSE-treated groups with different doses (200 and

Journal Pre-proof 500mg/kg) compared with Cyp treated group.HSE restored all this changes the level near to control group. In Figure 5 C and D Cyp induced a significant inhibition of CAT and AChE activities in liver and HSE co-treatment exhibited the hepatoprotection property with different doses. The result of PCA was presented in Fig 6A to investigate the relationship between biomarkers. Fig 6B showed a positive correlation between H2 O 2 and MDA (r=0.95) and between CAT and AChE (r=0.967). Negative correlation between MDA and CAT (r= -0.941) and between H2O2 and AChE (r= -0.956).

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Effects of Cypermethrin and HSE on Kidney

Figure 7 showed that Cyp induced an oxidative stress on kidney which presented by a

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significant increase of MDA and H2 O 2 level (Figure 7A and B) and inhibition of CAT and

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AChE activities (Figure 7 C and D). The co-treatment with HSE at various doses (200 and 500mg/kg) protect the kidney against Cyp-induced oxidative stress.

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The PCA was presented in Fig 8 A investigate that Cyp induced similarly AChE and CAT, on the other hand MDA and H2 O2 . The Cyp- HSE 500 group rewarded the effect of Cyp. Fig 8B

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Discussion

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showed the correlation between biomarkers with different values.

Recently, there has been an explosion of interest in studying the involvement of free radicals

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in oxidative stress. Cyp is known to induce oxidative stress (khazri et al., 2015), which is regarded as the imbalance between reactive oxygen species (ROS) production and the ability of antioxidants to destroy the ROS, which results in toxic effect through the creation of peroxides and free radicals. To overcome this problem, every organism has unique enzyme system (Shi et al., 2011) which by facing ROS exhibits variations in their activities (Loro et al., 2012). Phenolic compounds, which are widely distributed in plants, were considered to play an important role as dietary anti-oxidants for the prevention of oxidative damage in living systems (Block, 1992; Hertog and Feskens, 1993).The present study was designed to evaluate whether pretreatment with HSE, would have protective influences on Cyp - induced toxicity in the brain, heart, liver and kidney of mice. The neurotoxicity induced by Cyp exposure was evaluated by the measurement of some oxidative marker in the brain of mice. Cyp provoked an increased oxidative stress status, evidenced by high lipoperoxidation (MDA), an increase of H2 O2 level and affected antioxidant enzyme activities as CAT. On the

Journal Pre-proof other hand, Cyp induced the inhibition of the AChE activity. The Cyp had been found accumulate in the brain (Tao et al 2008) caused an alteration of biochemical parameters in brain (Dahman et al 2011). The Cyp crosses the blood-brain barrier and induced neurotoxicity (Azeez and Al-Hussary 2012). Also, it inhibited AChE activity (Kumar et al., 2009). Furthermore, α-Cyp-mediated neurotoxicity is contributed by its ability to induce free radical generation (Giray et al., 2001). In addition, other studies demonstrate the similar results; oral administration of Cyp induced an oxidative stress in the brain, where it decreased the antioxidant enzyme activities and an increase of MDA level (Sharma et al 2014). In addition, our results showed that Cyp induced an oxidative damage in the heart; it was

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proved by an increase of H2 O2 and MDA level and an inhibition of CAT and AChE activities.

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The increase of MDA and H2 O2 levels induced the decrease of the antioxidant enzyme activities (Kucukkurt et al 2010). Our study is in agreement with other studies, Ince et al

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(2012) showed that Cyp induced an oxidative stress in heart presented by the increase of

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antioxidant biomarkers such as MDA and the decrease of the enzyme activities such as CAT and SOD this alteration was observed in histopathology study. This alteration in balance of

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oxidative and antioxidant can produced haemorrhages within myocardium, disruption of branching structure, and loss of striation of cardiac tissue (Grewal et al 2010). The changes

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observed in heart suggested mainly prolonged harmful effect of Cyp. Our work also demonstrate that Cyp induced an oxidative stress in liver and kidney, Cyp was considered one

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of the pyrethroid group were found to exert their damaging effect by the generation of free radicals in the target organs (Youssef et al 2003) this tow organs plays an important role in the

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detoxification process by the way of oxidative stress response (Abdou et al 2012). Our study is similar with the results of Sankar et al (2012) showed that Cyp induced an oxidative stress in liver and kidney by the increase of MDA level and decrease of antioxidant enzymes activities. It can be concluded that the oxidative damage may be the primary cause of tissue alteration induced by Cyp toxicity (Manna et al 2004). In the same way studies showed that Cyp has been determined to accelerate the generation of free radicals in organs such as liver and kidney (Eraslan et al 2008). Hibiscus sabdariffa extract exhibited protection by its antioxidant action, blocking mitochondrial dysfunction and apoptosis induced by Cyp. Based on our current findings, Hibiscus sabdariffa can be a novel natural therapy for reducing the endogenous toxins mediated neuronal insult. In addition, this study demonstrates that Hibiscus pigments exhibited an anticytotoxicity effect against the Cyp-induced cytotoxicity probably via their ability of quenching free radicals and decreasing MDA formation in all organs of mice.

Journal Pre-proof Previous work shows that Hibiscus sabdariffa play an essential role in its prophylactic effect against chronic diseases (Khaghani et al., 2011; Sindi et al., 2014). The nutrients can scavenge reactive oxygen and free radicals, inhibit the activity of xanthine oxidase, decrease the peroxidation of lipids and boost the antioxidant activities, decrease inflammation and increase the mitochondrial functions (Da-Costa-Rocha et al., 2014).Through their free radicals scavenging capacity, Hibiscus sabdariffa and its phenolic compounds have been shown to have protective effects against oxidative stress (Liu et al., 2002). Undoubtedly, the most result drawn from the present study is the protective effect offered by HSE against Cyp toxicity. This goal was achieved using HSE, and, interestingly, our data

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should be paralleled with a recent work of Perez-Torres et al 2019 that proved the

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cardioprotective effect of HSE against ischemia and reperfusion. Koch et al 2019, demonstrate that HSE prevent the neurotoxicity induced by amyloid-β and Seung et al 2018

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proved the neuroprotective property of this extract against streptozotocin toxicity. Other study

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showed the hepatoprotective effect of HSE (Famurewa et al 2019) and the protection of

Cadmium induced nephrotoxicity. CONCLUSIONS

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kidney. The study of Orororo et al 2018 showed curative and protective effect of HSE against

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In the present study, we could demonstrate that a single dose of Cypermethrin caused an oxidative stress in brain, heart, liver and kidney of mice. Hibiscus sabdariffa administration

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was able to protect mice against toxicity induced by Cyp. Our results have established a basis for more comprehensive investigations to evaluate potential benefits of this plant consumption

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and to assess the mode of action. Nevertheless, further studies are needed to decide if application of HSE as an adjuvant against pesticide toxicity can be considered.

Acknowledgments

The authors wish to thank the Ministry of High education, University of Carthage, Faculty of Sciences of Bizerte, Tunisia for financial support of this work. Pr. David Sheehan is gratefully acknowledged as a native English speaker for her helpful criticisms and for the English improvements. Disclosure statement No potential conflict of interest was claimed by the authors.

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Fig. 1. Effect of Cyp and Hibiscus sabdariffa calyxes extracts on MDA(A), H2 O 2 levels (B), CAT (C) and AChE activities (D) in Brain. Results are expressed as mean ± SD (N = 6), a, b,

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c, d: data not sharing a common letter are significantly different (p ≤ .05).

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Fig 2. Variables distribution plots assayed by principal component analysis (PCA) between all measured biomarkers in brain (A), Correlation matrixes between biomarker responses (B).

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Fig. 3. Effect of Cyp and Hibiscus sabdariffa calyxes on MDA (A) ,H2 O2 levels (B), CAT (C) and AChE activities (D) in Heart. Results are expressed as mean ± SD (N = 6), a, b, c, d:

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data not sharing a common letter are significantly different (p ≤ .05). Fig 4. Variables distribution plots assayed by principal component analysis (PCA) between all

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measured biomarkers in heart (A), Correlation matrixes between biomarker responses (B). Fig. 5. Effect of Cyp and Hibiscus sabdariffa calyxes on MDA (A) ,H2 O2 levels (B), CAT (C)

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and AChE activities (D) in Liver. Results are expressed as mean ± SD (N = 6), a, b, c,d: data not sharing a common letter are significantly different (p ≤ .05). Fig 6. Variables distribution plots assayed by principal component analysis (PCA) between all measured biomarkers in Liver (A), Correlation matrixes between biomarker responses (B). Fig. 7. Effect of Cyp and Hibiscus sabdariffa calyxes on MDA (A) ,H2 O2 levels (B), CAT (C) and AChE activities (D) in Kidney. Results are expressed as mean ± SD (N = 6), a, b, c, d: data not sharing a common letter are significantly different (p ≤ .05). Fig 8. Variables distribution plots assayed by principal component analysis (PCA) between all measured biomarkers in Kidney (A), Correlation matrixes between biomarker responses (B).

Table 1. Identification and quantification of phenolic compounds in Hibiscus sabdariffa L. extracts (mean ± SD).

Journal Pre-proof Pea

Rt

k

λ max

[M-

MS2 (m/z)

Tentative

mg/g

identification

extract

191(100),179(80),173(5),161(16),135(

3-O-Caffeoylquinic

2.6±0.1

20)

acid

109(100)

5-

-

(nm)

H]

(min

(m/z

)

)

Non-anthocyanine 1

2

4.89

5.23

328

353

285.330c

127

h

5.75±0.08

(Hydroxymethyl)furfr al

7.34

328

328

353

353

191(78), 179(34), 173(100), 161(5),

4-O-Caffeoylquinic

135(10)

acid

191(100),179(87),173 (3), 161(5), 135(12)

354

611

595

317(12)

301(100)

4 7

17.9

356

609

301(100)

5 8

19.1

350

463

pr

16.1

350

301(100)

e-

6

13.2

21.2

1.53±0.06

Myricetin-

0.961±0.00

pentosylhexoside

1

Quercetin-

1.031±0.00

pentosylhexoside

2

Quercetin-3-O-

1.07±0.01

rutinoside Quercetin-3-O-

tr

glucoside 347

285(100)

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rn

7

593

al

5 9

5-O-Caffeoylquinic

1.44±0.08

acid

Pr

5

f

4

6.9

oo

3

kaempferol-3-O-

tr

rutinoside Total phenolic acids

5.602±0.00

Total flavonoids non-

4

anthocyanine

4.007±0.00

Other compounds

3 5.75±0.08

Graphical Abstract Highlights 

CYP can also elicit a range of neurotoxic, immunotoxic and genotoxic effects and reproductive toxicity in various experimental organisms



CYP-induced biochemical changes in all organs of mice



Hibiscus sabdariffa exhibited antioxidant effect and significantly attenuated the neurotoxicity of Cyp