Protective effects of coconut water against the intraperitoneal infused carbon tetrachloride-induced toxicity—evaluations of biochemical, haematological and histopathological profiles in rats

Coconut water is a relished beverage traditionally used as a remedy for childhood diarrhoea and gastroenteritis. It can be given to an individual who has hangover or suspected to have ingested a toxic substance. In this study, we evaluated the protective effect of coconut water against carbon tetrachloride (CCl4)-induced toxicity in rats. Administration of coconut water decreased significantly albumin, aspartate transaminase (AST), alanine transaminase (ALT), urea, creatinine, bicarbonate (HCO3−), total cholesterol, triglycerides, low-density lipoprotein, very low-density lipoprotein and malondialdehyde (MDA) levels compared to the non-pretreated group. Furthermore, high-density lipoprotein, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) increased significantly (p < 0.05) in coconut water-pretreated groups compared to the negative control. There were no observed pathological changes in the coconut water-pretreated groups, but slight enlargement of the central veins and tubules was evident when compared to the negative control. Therefore, intake of coconut water may be protective against toxicity induced by CCl4 as its pretreatment elevated antioxidant parameters (GSH, SOD and CAT) and did not alter biochemical parameters in rats.


Background
Coconut water is a relished beverage gotten from coconuts (Cocos nucifera L) (Gordon and Jackson 2017). Previously, coconut water was mostly acquired as a fresh beverage in regions where it was accessible, by cutting open a green nut fresh off the tree; today, it is a $300 + million business (Gordon and Jackson 2017). It is a perennial plant that bears fruit 12-13 times a year for up to 60-70 years, providing between 30 and 75 fruits per year. The fruit, in the shape of a nut, grows on trees of different heights depending on the type of coconut and is an important source of money for many farmers and countries in the far East in particular, where it is utilized in a number of ways and exported to other countries across the world (Gordon and Jackson 2017). The fruit is a significant source of oilseeds, particularly in many developing nations such as the Philippines, Sri Lanka, India, Malaysia and portions of West Africa, Latin system's reductive dehalogenation to create trichloromethyl free radical, which rapidly interacts with molecular oxygen to produce trichloromethylperoxyl radicals (Li et al. 2004). Excessive formation of CCl 4 free radicals can result in membrane lipid oxidation, which can progress to hepatorenal injury (Li et al. 2004). In this study, we investigated the protective effects of coconut water against CCl 4 -induced toxicity in rats.

Collection and preparation of coconut water
Healthy tender coconuts were gathered from Ndi Ekpere Compound, Amaiyi Igbere in Bende Local Government Area of Abia State, Nigeria. The coconut was de-husked, and the epicarp was gently opened to avoid losing or contaminating the coconut water. Every day, new coconut water was collected and filtered in a sterile condition using a ready-to-use Whatman filter No 1.

Animal use and care
Healthy rats (male and female) (8 weeks old; weight 151.78 ± 19.45 g, n = 60) bred locally at the Department of Biochemistry, Faculty of Biological Sciences, Abia State University, Uturu, were used for the study. The rats were housed in a metal cage for at least a week before the commencement of the study to allow for acclimatization under natural atmospheric conditions. They were fed with standard rat chow (Vital Feed) and water ad libitum. The laboratory procedures and tests were carried out with the permission of the Abia State University Ethical Committee (ABSU/REC/BMR/0026) and in accordance with World Health Organization recommendations for 'good laboratory practice' .

Experimental procedure
Healthy Wistar rats (male, n = 30 and female, n = 30) were randomly divided into five groups. To prevent mating, the male and female rats were placed in separate cages. Groups 3-5 were treated with different doses of coconut water for 14 days. After the 14th day pretreatment with coconut water, groups 2-5 were given 1 mL/ kg of CCl 4 (Merck, UK) diluted in olive oil at a 1:1 ratio by intraperitoneal (i.p.) route. The procedure is shown as: The rats were sacrificed after 24 h of CCl 4 treatment. The rats were weighed and humanely sacrificed by cervical dislocation. The blood samples were collected through cardiac puncture and distributed into dry plain bottles for biochemical tests and ethylenediaminetetraacetic acid (EDTA) bottles for haematological investigations. The liver and kidney were harvested and used to assess their histopathology.

Antioxidant markers
Other researchers' methodologies were used to determine oxidative stress indicators. Techniques for measuring superoxide dismutase (SOD) were used as reported by Sun and Zigma (1978). Catalase was estimated as revealed by Aebi (1984). Malondialdehyde and glutathione were evaluated using the methods described by Buege and Aust (1978) and Sedlak and Lindsay (1968), respectively.

Histopathological assessments
The liver and kidneys were fixed in 10% v/v formalin before being encased in paraffin wax and sectioned. Each specimen was cut at 5 µm and stained with haematoxylin and eosin dyes. Using X40 magnification light microscope, the stained portions were examined for histological changes and photomicrographs were taken.

Statistical analysis
The generated data were represented as mean ± standard deviation. To determine the difference between the groups, statistical analysis was performed with R ™ statistical program (https:// cran.r-proje ct. org/ src/ base/ R3/), version 3.0.3. A significant relationship was determined using the Tukey test post hoc at a 95% level of confidence.

Results
Body weight of 14 days treatment of coconut water on CCl 4 -induced toxicity Figure 1 represents the protective effect of coconut water on body weight of CCl 4 -induced toxicity in both male and female rats. No significant changes (p > 0.05) in body weight were observed in both sexes in all the treatment groups.

Protective effect of coconut water on liver biomarkers of CCl 4 -induced toxicity
Intake of coconut water decreased the values of albumin, AST and ALT compared to the negative control. However, total protein and bilirubin levels had a dosedependent significant difference (p < 0.05) between the coconut water-pretreated groups (4 and 6 mL/kg) and the negative (CCl 4 ) control. ALP values did not show any significant difference in the male rats. In the female rats, there were significant differences (p < 0.05) in the values of albumin, AST, ALT, ALP and bilirubin among the coconut water-pretreated groups compared to the CCl 4 group ( Table 1). Table 2 shows the data set obtained from the protective effect of coconut water on kidney function of CCl 4 -induced toxicity in both sexes. There were significant differences observed in the values of urea, creatinine, Na + , K + and HCO 3 − in the coconut waterpretreated groups compared to the CCl 4 -treated group.

Protective effect of coconut water on haematological parameters of CCl 4 -induced toxicity
In the CCl 4 group, the values of RBC, PCV, HB, platelet, MCV and neutrophils were decreased compared to the coconut water-treated groups. Furthermore, the values of WBC and lymphocytes were increased in the negative control compared to coconut water-treated groups (Table 3). Table 4 shows the result obtained from the protective effect of coconut water on lipid profiles of carbon tetrachloride-induced toxicity in both male and female rats. Groups administered with coconut water had a significant decrease (p < 0.05) in the values of TC, triglycerides, LDL and VLDL. The values of the HDL decreased significantly in the CCl 4 group compared to the coconut watertreated groups.

Protective effect of coconut water on antioxidant enzymes of CCl 4 -induced toxicity
The result presented in Table 5 is the protective effect of coconut water on antioxidant enzymes of CCl 4 -induced toxicity in both male and female rats. Administration of coconut water caused a significant elevation in the values of GSH, SOD and CAT. The values of MDA decreased in the treatment group compared to its untreated counterparts in both male and female rats.

Protective effect of coconut water on histology section of liver and kidney
The liver and kidney histology sections of the male and female rats are presented in Figs. 2, 3, 4 and 5. The photomicrograph of the normal control showed a normal liver architectural design, but the treated rats showed a slight Table 1 Protective effect of coconut water on liver biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B) rats AST, aspartate aminotransferase; ALT, alanine transaminase; and ALP, alkaline phosphatase. Values are presented as mean ± SD, n = 6. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/ kg) compared to the negative control More so, the hepatocytes appeared a bit distorted and many microcysts within the stroma were found (A-J). In the kidney histology (K-T), no pathological change was observed in the normal control unlike the CCl 4 -treated groups. While the negative control had a slight dilated and shrunken glomerulus, a slight enlargement of the tubules was observed in the coconut water-treated group, although there were no pathological changes observed amongst the groups (Figs. 4, 5).

Discussion
In this study, we examined the protective effect of coconut water against CCl 4 -induced toxicity in rats. The findings of the study revealed no significant changes in body weight in the experimental rats. This might be attributable to the brief period of CCl 4 delivery following coconut water pretreatment. In other reports, body weight measurement has been described as plausible evidence in toxicological research ). Elevations of liver biomarkers; enzymes (AST, ALT and ALP), bilirubin, albumin and total proteins are strong indicators for liver insults (Orieke et al. 2019;Ugbogu et al. 2019;Emmanuel et al. 2021). When the liver is under severe attack, these enzymes are released into the blood and thus give insight on the hepatic status of the liver (Ugbogu et al. 2019). Dutta et al. (2018) reported that CCl 4 is hepatotoxic by producing ROS. Increased ROS has been associated with decreased antioxidant levels ). In the present study, the negative control had elevated levels of liver enzymes and administration of coconut water decreased the values of albumin, AST and ALT. Our findings correlate with the results of other researchers (Anurag and Rajamohan 2003). Anurag and Rajamohan (2003) showed that coconut water counteracted an increased level of liver enzymes caused by isoproterenol exposure. The reported hepatoprotective activities of coconut water might be linked to its antioxidant activities (Tsai et al. 2009). A functional renal system may be related to the kidney's filtration capacity, commonly known as the glomerular filtration rate (GFR). A reduction in GFR may produce an increase in urea and creatinine in the serum, indicating renal disease (Jose 2014). Additionally, in kidney test, a high serum urea level indicates renal failure, which usually progresses to severe kidney damage in long-term instances (Gowda et al. 2010). In our study, urea, creatinine and HCO 3 − significantly decreased in the coconut water-treated groups when compared to the negative control. Abnormal elevations of creatinine, urea and electrolytes levels are implicated in kidney insults (Ugbogu et al. 2019;Emmanuel et al. 2021).
Haematological parameters are those that relate to blood and blood-forming organs. The quantity and shape of RBC, WBC and platelets are important in disease diagnosis and monitoring (Owoeye et al. 2011). In this work, groups pretreated with coconut water prior to CCl 4 induction had a significant haematoprotective effect Table 2 Protective effect of coconut water on kidney biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B) rats Na + , sodium ion; Cl − , chloride ion; K + , potassium ion; and HCO 3 − , bicarbonate. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control in both male and female rats compared to the negative control. In the negative control, the levels of RBC, PCV and HB were decreased, and this may be associated with the toxic free radicals generated from CCl 4 which consequently affected haematopoiesis in the experimental animals as revealed in other studies (Rahmouni et al. 2011). Therefore, haematological parameters are very sensitive and are crucial for predicting the toxicological and pathological effect of potentially toxic substances in both human and animals (Kong et al. 2016). Previous study on the effects of CCl 4 on haematological parameters showed that acute CCl 4 toxicity led to transient decrease in the HB concentration and reticulocyte count as well as PCV and RBC counts by extension which is in consonant with this present study (Saba et al. 2010;Emmanuel et al. 2021). The rats administered with coconut water prior CCl 4 induction had decreased levels TC, triglycerides, LDL and VLDL, while HDL levels increased compared to the untreated group. This demonstrates that CCl 4 could disrupt lipid metabolism. Atherosclerosis and coronary heart disease have been linked to higher levels of cholesterol, triglycerides, LDL and VLDL (Sandhya and Rajamohan 2008;Emmanuel et al. 2021). Our findings confirmed the previously reported evidence that coconut water lowered the increased levels of TC, triglycerides, LDL and VLDL in rats induced by a high cholesterol fat feed (Sandhya and Rajamohan 2006). Table 3 Protective effect of coconut water on haematological parameters of carbon tetrachloride-induced toxicity in male (A) and female (B) rats RBC, Red blood cells; PCV, packed cell volume; HB, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; and WBC, white blood cell. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control Basophil (%) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Page 7 of 11 Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 CCl 4 has been found to be toxic by causing the production of free radicals capable of interacting with sulphur-containing proteins, which can impair antioxidant activity (Tsai et al. 2009). This study revealed that CCl 4 has a negative effect on antioxidants, as seen by a decrease in SOD, GSH and CAT and an increase in MDA. Our findings are consistent with the findings of Rahmouni et al. (2011) that discovered that CCl 4 may reduce erythrocyte GSH, SOD and CAT activities while increasing erythrocyte MDA levels.
In the histology of the liver and kidney, pathological changes were evident; however, there were slight enlargement of the central veins and tubules, respectively, compared to the negative control. Coconut water administration revealed a dose-dependent protection compared to the untreated group. Injuries in the liver included deformed hepatocytes, dilated sinusoids, microvascular steatosis and an enlarged central vein, while congested blood vessels and dilated tubules were detected in the kidney of the negative control. These findings support the Table 4 Protective effect of coconut water on lipid profiles of carbon tetrachloride-induced toxicity in male (A) and female (B) rats TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; and VLDL-C, very low-density lipoprotein cholesterol. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control  Table 5 Protective effect of coconut water on antioxidant enzymes of carbon tetrachloride-induced toxicity in male (A) and female (B) rats GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; and MDA, malondialdehyde. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control assertion that CCl 4 has haematotoxic, hepatotoxic and nephrotoxic potentials (Rahmouni et al. 2011;Elshater et al. 2013;Emmanuel et al. 2021).

Conclusions
Our findings show that coconut water protects rats from CCl 4 -induced toxicity. Its pretreatment for 14 days following CCl 4 toxicity elevated GSH, SOD, CAT levels and decreased MDA levels. These data show that coconut water may protect against lipid peroxidation by scavenging the harmful radicals produced by CCl 4 and thereby normalizing the quantity and distribution of lipids in rats' systemic circulation. The observed increase in these antioxidants may be related to coconut water's hepato/reno-protective potentials and reduced cellular oxidative damage. Notwithstanding the health benefits of coconut water, it can be noted that its contents may be influenced and varied by the type of cultivar, geographical location and the stage of maturation of the nut.