Comparative effects of ethanol leaf and stem bark extracts of Irvingia gabonensis (BUSH MANGO) on sodium arsenite-induced lipid profile perturbtions in wistar rats

Exposure to arsenic orchestrates a myriad of noxious health effects, including cancer. Different parts of Irvingia gabonensis are used as herbal remedies in traditional medicine. In this study, the comparative effects of the ethanol leaf (ELEIG) and stem bark extracts (ESEIG) of Irvingia gabonensis on sodium arsenite (SA)-induced lipid profile disturbances in Wistar rats were investigated. Fifty five Wistar rats weighing between 100 g and 179 g were distributed into eleven groups (n=5). Group 1 (control) received feed and water ad libitum. Group 2 received SA at a dose of 4.1 mg/kg body weight (kgbw) for 14 days. Groups 3–11 were treated with the extracts with or without SA. Treatment was done by oral intubation for 14 days. Serum concentrations of total cholesterol (TC), triacylglycerol (TAG), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), very low density lipoprotein cholesterol (VLDL-c), total lipids (TL) and atherogenic index of plasma (AIP) were used to determine the lipid profile effects of the extracts. Exposure to SA caused significant (p ˂ 0.05) increases in all assayed parameters, relative to control. Post-treatment and simultaneous treatment with ELEIG and ESEIG mitigated the effects of SA. In addition, ELEIG alone at various doses produced results comparable with control values. However, ESEIG alone caused significant (p ˂ 0.05) increases in all assayed parameters, relative to control. These results show that ELEIG and ESEIG ameliorate SA-induced lipid profile disturbances in Wistar rats. However, long-term administration of ESEIG alone may be discouraged.


Background
The environment plays a vital role in the wellbeing of humans and animals. However, a distortion in the environment by environmental pollutants can negatively impact the health of its occupants (humans and animals).
Several heavy metals (such as arsenic, cadmium and lead) are established environmental pollutants which have been linked to the etiologies of diverse disease conditions [1]. Arsenic occurs at high concentrations in ground water of different countries around the World, including Nigeria [2][3][4]. Reports also have it that an estimated 200 million people all over the World are at health risks associated with high arsenic concentrations in drinking water [5,6]. Epidemiological studies have also revealed that exposure to inorganic arsenicals (e.g. arsenite) majorly via occupational and environmental exposure, is detrimental to health [7,8].
As the popular saying goes "health is wealth". In the quest of ensuring a good health condition, many plants of acclaimed medicinal values have been used over the years by most locals and herbal medicine practitioners for therapeutic purposes especially those in low income countries. Irvingia gabonensis (also called bush mango or wild mango) is a plant used in traditional and modern medicine in treating various disease conditions [9,10]. The aqueous maceration of the leaves is used as antidote against poisonous substances. In Senegal, the decoction of the stem bark is used in treating gonorrhea, liver and gastrointestinal disorders [11]. Other medicinal properties have also been attributed to the stem bark [11][12][13]. Furthermore, the stem bark and leaf extracts have been reported to possess hematological, hepatoprotective, anti-diabetic and prophylactic effects in animal models [14][15][16][17]. Ewere and co-workers [18][19][20] have also documented the medicinal effect of leaf extracts of Irvingia gabonensis under conditions of induced toxicities in Wistar rats. However, there is a dearth of scientific information on the effect of leaf and stem bark extracts of Irvingia gabonensis on lipid profile in sodium arseniteintoxicated experimental animal models.

Chemicals and reagents
Assay kits for lipid profile (total cholesterol (TC), triacylglycerol (TAG), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), very low density lipoprotein cholesterol (VLDL-c) and total lipids (TL) concentrations) were purchased from Sunlong Biotech co. (Shangyi, Hangzhou, Zhejiang, China). Sodium arsenite was purchased from British Drug House (BDH) chemicals, Poole, England. Absolute ethanol was purchased from JHD, China. All other chemicals / reagents used in this study were of analytical grade.

Preparation of plant extracts
Fresh leaves and stem bark of I. gabonensis were sourced from a local community in Akwa Ibom State, Nigeria. The samples were authenticated (James Daniel UUH 042116 (Uyo)) by a taxonomist of the Department of Pharmacognosy and Herbal Medicine, University of Uyo, Nigeria. They were washed using clean water to eliminate contaminants, before air-drying for 1 week in Biochemistry laboratory, University of Uyo, Nigeria. They were then pulverized using a clean manual grinder (leaves), mortar and pestle (stem).
About 850 g and 2800 g of pulverized leaves and stem bark, respectively, were macerated in absolute ethanol for 72 h with intermittent stirring to ensure proper extraction of the bioactive agents. The samples were filtered repeatedly through a clean muslin cloth and the filtrates were concentrated using a water bath at 45°C. The paste-like gel extracts obtained after concentration were stored in a refrigerator at 4°C prior to use.

Experimental animals
Fifty-five (55) healthy, non-pregnant female Wistar rats (body weights between 100 g and 179 g) were obtained at the animal house facility of Faculty of Basic Medical Sciences, University of Uyo, Nigeria. They had free access to feed and water, and were acclimatized for seven (7) days in the same facility in a well ventilated room under standard conditions.

Experimental design
After acclimatization and just before treatment commenced, the experimental animals were randomly assigned to eleven groups (n=5) in standard experimental animal cages and weighed using a digital weighing balance (Camry electronic scale EK5350, China) after overnight fast to get their initial body weights. They were administered treatment as shown in Table 1.

Collection of blood samples for analysis
On termination of treatment, all experimental animals were subjected to overnight fast with access to water only. They were chloroform-anesthetized and sacrificed using lower abdominal incision about 24 h after the last treatment. Whole blood samples were obtained by cardiac puncture using sterile syringes and needles and collected in sterile plain sample bottles for biochemical analysis. Sera were obtained from clotted blood samples in the plain bottles after centrifugation using a table top centrifuge (Model 800-1, Zeny Inc. Salt Lake, USA) at 3000 rpm for 15 min. The separated sera were preserved in a refrigerator at 4°C prior to analyses.

Biochemical analyses
Serum total cholesterol (TC) concentration was determined according to the method of Beaumont et al. [21]. Serum triacylglycerol (TAG) concentration was determined according to the method of Fossati [22]. Serum HDL-c concentration was determined according to the method of Warnick et al. [23]. Serum LDL-c concentration was determined according to the method of Badimon et al. [24]. Serum VLDL-c concentration determination was done using the Friedewald equation (Friedewald et al. [25]. Serum total lipid concentration was determined according to the method of Frings and Dunn [26] and Atherogenic index of plasma (AIP) of the samples was calculated by using the formula:

Statistical analysis
Data obtained are presented as mean ± standard deviation (SD) and were analysed with one-way analysis of variance (ANOVA) for differences between groups using SPSS Software (IBM, version 20). P values ˂ 0.05 were considered statistically significant.

Effect of ELEIG on lipid profile of experimental rats
Results obtained revealed that intoxication with SA led to significant (p ˂ 0.05) increases in TC, TAG, HDL-c, LDL-c, VLDL-c, TL and AIP when compared with control. Post-treatment with ELEIG at various doses, produced significant (p ˂ 0.05) and non-significant (p ˃ 0.05) decreases in the concentrations of the assayed parameters, in dose-dependent and independent manner, relative to group 2 (administered SA only). Simultaneous treatment with ELEIG at various doses, took similar trend as the post-treatment. In addition, treatment with ELEIG alone at various doses, led to no-significant (p ˃ 0.05) differences in TC, HDL-c, VLDL-c (at doses of 200 mg/kgbw and 400 mg/kgbw), as well as TAG (at 200 mg/kgbw), LDL-c (at 100 mg/kgbw) and AIP (at doses of 100 mg/kgbw and 200 mg/kgbw) relative to control. Treatment with ELEIG alone also produced significant (p ˂ 0.05) decreases in LDL-c (at doses of 200 mg/kgbw and 400 mg/kgbw). It also produced significant (p ˂ 0.05) increases in TL at various doses, relative to control.

Effect of ESEIG on lipid profile of experimental rats
Post-treatment with ESEIG at various doses, produced significant (p ˂ 0.05) and non-significant (p ˃ 0.05) decreases in the concentrations of the assayed parameters, in dose-dependent manner, relative to group 2 (administered SA only), except AIP. Simultaneous treatment with ESEIG at various doses followed similar trend as the post-treatment, except AIP. However, treatment with ESEIG alone at various doses, led to significant increases in all the assayed parameters, relative to control. The detailed results are presented in Tables 2 and 3, respectively.

Discussion
Documented evidence have shown that environmental pollutants such as heavy metals play a key role in the development of dyslipidemia [27,28]. A prevalence of cardiovascular diseases such as hypertension, atherosclerosis, and heart diseases among others, has also been reported in arsenic-exposed populations [29,30]. Wade and co-workers [31] had reported an association between cardiovascular diseases and moderate arsenic exposure among subjects in China. Other epidemiological studies have also reported the association between arsenic and cardiovascular diseases [32][33][34]. It is therefore a possibility that arsenic poisoning may orchestrate the development of cardiovascular diseases via modification of lipid metabolism. A disturbance of the normal concentrations of key lipids: total cholesterol (TC), triacylglycerol (TAG), high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and total lipid, is reflective of ill health [20].
In this study, administration of sodium arsenite alone produced significant increases in serum concentrations of total cholesterol (TC), triacylglycerol (TAG), HDL-c, LDL-c, VLDL-c and total lipids, compared with control.   Data are expressed as mean ±SD (n=5); a p ˂ 0.05 compared with group 1; b p ˂ 0.05 compared with group 2 c p ˂ 0.05 compared with group 3; d p ˂ 0.05 compared with group 4; e p ˂ 0.05 compared with group 5; f p ˂ 0.05 compared with group 6; g p ˂ 0.05 compared with group 7; h p ˂ 0.05 compared with group 8; i p ˂ 0.05 compared with group 9; j p ˂ 0.05 compared with group 10; k p ˂ 0.05 compared with group 11; AIP= Attherogenic index of plasma; SA = Sodium arsenite Administration of sodium arsenite alone also produced significant and non-significant increases in atherogenic index of plasma (AIP) relative to control. Cardiovascular diseases may arise as a consequence of metabolic disorders such as dyslipidemia and diabetes [35]. Several documented evidence have linked elevation of serum cholesterol, LDL-c and triacylglycerol concentrations with the etiology of cardiovascular diseases [36][37][38][39][40][41]. Contrary to several documented reports stating decreases in HDL-c concentration in dyslipidemia, sodium arsenite intoxication produced significant increases in serum HDL-c concentration when compared with control. HDL-c is generally referred to as "good cholesterol" due to its function in reverse cholesterol transport, removing cholesterol from within the artery and transporting same back to the liver for onward excretion and utilization [42]. However, some recent studies have shown that too high HDL-c concentration may not necessarily be a good omen. There are different possible explanations to the significant increases in HDL-c observed in this study.
Firstly, HDL-c has been reported to possess antioxidant and anti-inflammatory properties [43]. It is involved in mopping up lipid hydroperoxides induced by oxidative stress. Sodium arsenite has been reported to induce oxidative stress as well as lipid peroxidation [44,45]. The observed significant increase in HDL-c concentration in this study may have arisen in an attempt by HDL-c to mop up lipid hydroperoxides generated by sodium arsenite. Secondly, documented evidence also has it that increased HDL-c levels may be due to the inefficient clearance of LDL-c (bad cholesterol) from circulation [46]. Thirdly, oxidative stress has the capacity to modify the protective effects of HDL-c by oxidizing it, thereby making it proatherogenic rather than antiinflammatory [47][48][49]. Finally, the increased HDL-c may be dysfunctional HDL-c which may increase risk of premature death [50]. Pathological processes such as inflammation can overwhelm the antioxidant and antiinflammatory functions of HDL-c, converting it to a 'dysfunctional' pro-inflammatory, pro-oxidant, prothrombotic, and proapoptotic particle which may impair reverse cholesterol transport and thus predispose to cardiovascular diseases [51,52].
The result obtained from this study is very vital because it contributes and agrees with a growing body of evidence that very high HDL-c levels may not be protective. According to various recent reports, very high levels of HDL-c may be associated with an increased risk of heart attack and death [50,53,54].
Treatment with ethanol leaf extract of Irvingia gabonensis (ELEIG) 2 weeks after (post-treatment) produced significant decreases in serum concentrations of TC, TAG, HDL-c and total lipid in dose-dependent manner while dose-independent significant decreases were observed for LDL-c and VLDL-c, compared with group 2, administered sodium arsenite only. In addition, simultaneous treatments with ELEIG produced significant decreases in serum concentrations of TAG, LDL-c, and total lipid in dose-dependent manner while doseindependent significant decreases were observed for TC, HDL-c and VLDL-c when compared with group 2, administered sodium arsenite only. Simultaneous treatment with ELEIG at doses of 200 mg/kgbw and 400 mg/ kgbw respectively, also produced significant decreases in AIP when compared with group 2, administered sodium arsenite only. The reversal of sodium arsenite-induced dyslipidemia by ELEIG may therefore be by its mitigation of oxidative stress and inflammation induced by sodium arsenite which have been widely reported to play a key role in the etiology of cardiovascular diseases. This is possibly due to the inherent antioxidant phytochemicals present in the extract [55]. Administration of ELEIG alone at different doses also produced comparable results with that of the control.
On the other hand, treatment with ethanol stem bark extract of Irvingia gabonensis (ESEIG) both simultaneously and 2 weeks after (post-treatment) produced significant and non-significant decreases in serum concentrations of the assayed lipid profile parameters when compared with group 2, administered sodium arsenite only except AIP of the treated groups, which were not significantly different. However, administration of ESEIG alone at various doses culminated in significant increases in all assayed lipid profile parameters relative to control. Since elevations in lipid profile parameters have been associated with cardiovascular diseases, ESEIG may therefore be slightly toxic when administered solely on a long-term basis. This is in tandem with previous findings of Ewere et al. [56].

Conclusion
The results obtained from this study indicate that ethanol leaf and stem bark extracts of Irvingia gabonensis mitigate sodium arsenite-induced lipid profile perturbations in Wistar rats. Ethanol leaf of Irvingia gabonensis extract may therefore be useful in combating arsenic poisoning. However, ethanol stem bark extract of Irvingia gabonensis may be slightly toxic when administered alone on a long-term basis. Thus, long-term administration of the stem bark extract may not be encouraged.