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The Hypolipidemic Effect of Aqueous Extract of Hibiscus sabdariffa on Paracetamol-induced Hepatotoxicity in Albino Wistar Rats

Aim of the Study: This study was undertaken to ascertain if Hibiscus sabdariffa extract can affect the lipid profile (Total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL), very low density lipoprotein (VLDL), and low density lipoprotein (LDL)) levels in a paracetamolinduced hepatotoxicity using albino Wistar rat as a model. Materials and Methods: Thirty (30) rats used for this study were divided into three groups. Group A (n=10) served as control. Group B (n=10) was administered paracetamol only at a dose of 750 mg/kg body weight. Group C (n=10) was administered paracetamol (dose 750 mg/kg body weight) and aqueous extract of H. sabdariffa (dose 10 ml/kg body weight) of the animal for 3 weeks. All animals were allowed free access to clean drinking water and normal rat chow. Results: Results of the study revealed that TC was significantly lower (p<0.05) in the paracetamol + H. sabdariffa-treated group as compared to paracetamol-treated group and control respectively. Original Research Article Umoren et al.; EJMP, 31(7): 54-60, 2020; Article no.EJMP.55530 55 Similar trend was observed with TG, VLDL-c, LDL-c and HDL-c. However, the decrease in HDL-c was not statistically significant when compared to control. Conclusion: The presence of bioactive constituents vis; anthocyanins, flavonoids, polyvenols and free radical scavenging properties in H. sabdariffa enabled a hypolipidemic effect on the animals by lowering the levels of serum TG, VLDL-c, LDL-c despite challenge on the liver. However, it was unable to produce significant effect on HDL concentration -very important cholesterol required in high level to maintain homeostasis inside the body. This may be due to the challenge on the liver as a result of the paracetamol abuse.

Similar trend was observed with TG, VLDL-c, LDL-c and HDL-c. However, the decrease in HDL-c was not statistically significant when compared to control. Conclusion: The presence of bioactive constituents vis; anthocyanins, flavonoids, polyvenols and free radical scavenging properties in H. sabdariffa enabled a hypolipidemic effect on the animals by lowering the levels of serum TG, VLDL-c, LDL-c despite challenge on the liver. However, it was unable to produce significant effect on HDL concentration -very important cholesterol required in high level to maintain homeostasis inside the body. This may be due to the challenge on the liver as a result of the paracetamol abuse.

INTRODUCTION
Hibiscus sabdariffa L. also known as Roselle is an ideal crop for developing countries, and it is widely grown in Central and West Africa and South East Asia [1]. The calyxes and dried leaves of H. sabdariffa L. can be used for various purposes in different countries and some of the uses include: culinary use -in the preparation of herbal drinks/ tea, fermented drinks, wine, flavoring agents, chocolates, jam and cake. In Nigeria and Sudan, the dried calyxes are boiled with sugar to make a popular drink known as "Zobo" or "Kakade" [2].
Several studies, both in vitro [3,4] and in vivo [5,6] have shown that the extract of H. sabdariffa L. has a potent antioxidant effect. The antioxidant activity of the extract is due to its strong scavenging effect on reactive oxygen and free radicals [6,7].
The aqueous extract of the red and green H. sabdariffa has also been reported to cause significant decrease in the LDL-c levels, while no significant effect was observed in HDL-c and Triglyceride levels [8].
Recently, our laboratory studies have shown that despite the free radical scavenging property and presence of natural antioxidants in H. sabdariffa, these effects could not ameliorate the challenge on the liver due to paracetamol abuse in rats [13]. Based on this finding, it became expedient to investigate further to know if the aqueous extract of H. sabdariffa could positively affect lipid profile in a paracetamol-induced hepatotoxicity, hence this study.

Plant Materials
Fresh leaves of H. sabdariffa were purchased from Watt market in Calabar South, Cross River State, Nigeria. The leaves were authenticated by the Chief Botanist, Department of Botany, University of Calabar, Cross River State, Nigeria. 20 grams of the H. sabdariffa was weighed and grounded in an electric mill to obtain particles less than 2 mm. It was used to make an infusion by adding 1 liter of clean water and allowed to stand for 48 hours. The solution was filtered using Whatman's No. 1 filter paper. The filtrate was stored in clean plastic containers and refrigerated. The extract was brought out of the refrigerator 2 hours to oral administration. The extraction was carried out according to the method of [14] with little modification.

Experimental Animals
Thirty (30) albino Wistar rats of both sexes weighing between 100-200 g from the start of the experiment were used for this study to determine the following: Total cholesterol concentration, LDL-c, HDL-c, TG, VLDL-c concentrations. They were maintained in the animal facility of the Department of Physiology, University of Calabar, Nigeria, at a temperature of 30 ± 2°C and 12 h light/dark cycles. The rats were kept singly in improvised plastic metabolic cages with wire net covers. The rats were randomly assigned into three groups (A = control, B = paracetamoltreated group and C = paracetamol + aqueous leaf extract of H. sabdariffa-treated group). Each group consisted of ten rats. They were allowed free access to normal rat chow and clean drinking water. The paracetamol-treated group received intraperitoneal inducement of paracetamol (750 mg/kg body weight [15]. The paracetamol + extract of H. sabdariffa treated group received intraperitoneal inducement of paracetamol (750 mg/kg body weight) [15] and oral administration of aqueous leaf extract of H. sabdariffa (10 ml/kg body weight of rats). The animals in the control group were fed with only normal rodent chow and clean drinking water. Drug administration was done once daily for 3 weeks after which the blood samples were collected for analyses.

Preparation of Paracetamol Sample
The stock concentration of paracetamol was prepared by dissolving 750 mg of standard drug in 5 ml of distilled water bringing the stock concentration to 75 mg m/L. The dose used was 750 mg/kg body weight [15].

Preparation of animals for collection of blood samples
This was done by the method of [16] and used by [17].

VLDL-cholesterol concentration
The concentration of very low density lipoprotein cholesterol was determined using lipoprotein electrophoresis and ultracentrifugation [19].

Statistical Analysis
All results are presented as mean ± standard error of mean (SEM). The data were analyzed using a one-way analysis of variance (ANOVA) and p<0.05 was considered statistically significant [20].

Acknowledgement
All biochemical assays were carried out by Medichecks Diagnostic and Research Laboratory, Calabar, Cross River State, Nigeria.

RESULTS
Based on laboratory findings, the results of this research are expressed below.

Effect of Paracetamol and H. sabdariffa on TC Concentration
TC concentration in the control, paracetamoltreated and paracetamol + H. sabdariffa-treated groups are illustrated in Table 1. There was a significant decrease (p<0.05) in TC concentration in the paracetamol + H. sabdariffa-treated group as compared to control and paracetamol-treated groups respectively.

Effect of Paracetamol and H. sabdariffa on HDL-c Concentration
The HDL-c concentration in the control, paracetamol-treated and paracetamol + H. sabdariffa-treated groups are illustrated in Table  2. There was a significant decrease (p<0.05) in HDL-c in the paracetamol + H. sabdariffa-treated group as compared to control.

Effect of Paracetamol and H. sabdariffa on TG Concentration
TG concentration in the control, paracetamoltreated and paracetamol + H. sabdariffa-treated groups are illustrated in Table 3. There was a significant decrease (p<0.05) in TG concentration in the paracetamol + H. sabdariffatreated group as compared to control and paracetamol-treated groups respectively.

Effect of Paracetamol and H. sabdariffa on VLD-c Concentration
The VLDL-c concentration in the control, paracetamol-treated and paracetamol + H. sabdariffa-treated groups are illustrated in Table  4. There was a significant decrease (p<0.05) in VLDL-c concentration in the paracetamol + H. sabdariffa-treated group as compared to control and paracetamol-treated groups respectively.

Effect of Paracetamol and H. sabdariffa on LDL-c
The LDL-c concentration in the control, paracetamol-treated and paracetamol + H. sabdariffa-treated groups are illustrated in Table  5. There was a significant decrease (p<0.05) in LDL-c concentration in the paracetamol + H. sabdariffa-treated group as compared to control and paracetamol-treated groups respectively.

DISCUSSION
Lipid profile/ lipid panel is a panel of blood tests [21,22] that serves as an initial screening tool for abnormalities in lipids [23] such as cholesterol [24] and triglycerides [25]. The results of this test can identify certain genetic diseases and can determine approximate risks for cardiovascular disease [26] certain forms of pancreatitis [27,28] and other diseases.
H. sabdariffa has been reported to possess lipid lowering activity that could prevent diseases like hyper-lipidemia and cardiovascular diseases [29,30,9]. In 2003, [31] had reported the anticholesterol action of H. sabdariffa in reducing serum concentration of TGs, TC and LDL-C. These reports corroborate our result findings.
The main constituents of H. sabdariffa L. relevant for its pharmacological study are organic acid, anthocyanins and flavonoids [10,32]. These properties highlight the antioxidant activity of H. sabdariffa extract.
In the present study, H. sabdariffa revealed hypolipidemic activity in paracetamol-induced hepatotoxicity. 750 mg/kg-1 body weight of paracetamol and 10 ml/kg body weight of H. sabdariffa extract administered on rats caused significant reduction (P< 0.05) in serum cholesterol level (Table 1), serum TG (Table 3), serum VLDL-C (Table 4), and serum LDL-C (Table 5) respectively as compared to paracetamol-treated group and control respectively. There was however, no significant decrease in the serum HDL-C ( However, [37] had reported increased serum levels of HDL-c with H. sabdariffa extract; this is contrary to the findings of the present study. The HDL-c showed no significant change in the paracetamol + H. sabdariffa-treated group when compared to control. This may be specie specific, and also may depend on the dosage of substance abused. A high level of HDL-c is good because it helps remove other forms of cholesterol from bloodstream by scrubbing and keeping clean the endothelium of blood vessels. Conversely, a low HDL concentration as seen in this study predisposes the heart to attack and other forms of diseases.
Recent studies have shown that H. sabdariffa may not be able to ameliorate the challenge on liver due to paracetamol abuse in rats despite its free radical scavenging property and presence of natural antioxidants [13]. It is however not surprising that the aqueous extract of H. sabdariffa could not also exert its antioxidant property on the liver to boast the level of HDL concentration -very important cholesterol required in high level to maintain homeostasis inside the body. This implies that when paracetamol is abused by users, the effect can be detrimental to cells, tissues, and organs of the body. This should be seriously warned against.

CONCLUSION
The effect of H. sabdariffa on lipid profile level in a paracetamol-induced hepatotoxicity was investigated. Results from the findings showed that the aqueous extract of H. sabdariffa reduced levels of serum TG, VLDL-c, LDL-c despite challenge on the liver of cholesterol in the animal model. However, it was unable to produce significant effect on HDL concentration -very important cholesterol required in high level to maintain homeostasis inside the body this may be due to the challenge on the liver as a result of the paracetamol abuse. Conclusively, it may be adduced from the recent that the presence of bioactive constituents viz; anthocyanins, flavonoids, polyvenols and free radical scavenging properties in H. sabdariffa enabled a hypolipidemic effect on the animals despite challenge on the liver.
However, it is recommended that future studies be undertaken to ascertain the mechanism of action of this extract as the scope of this study was limited to lipid profile level estimation.

CONSENT
It is not applicable.

ETHICAL APPROVAL
Permission was sought for, and obtained from the Faculty Animal Research Ethics Committee of the Faculty of Basic Medical Sciences for the study. The ethical approval number: FAREC-FBMS/20/2018