1. Introduction
Genetic selection and improvement of economically important traits in broiler production have been used for several years as vital strategies in the commercialization of the poultry industry to increase market body mass and growth rate. This technique has led to the production of fast-growing commercial broilers having altered physiological requirements and that are potentially susceptible to oxidative stress [
1]. Oxidative stress is caused by exposure to reactive oxygen intermediates, such as the superoxide anion, hydrogen peroxide, and hydroxyl radical, which can damage proteins, nucleic acids, and cell membranes [
2]. Consequently, oxidative stress leads to the initiation and progression of liver damage.
Carbon tetrachloride (CCl
4) intoxication has long been known as a model for the induction of toxicity and has been the subject of many toxicological studies in vitro and in vivo [
3,
4]. The liver is the main target organ for CCl
4 toxicity due to its high content of cytochrome
p-450 [
5]. It is well established that exposure to CCl
4 leads to necrosis and cirrhosis of liver tissue induced by the trichloromethyl radical (CCl
3) activated by cytochrome P450. Necrosis and cirrhosis are reflected by disruption of hepatic metabolic activities and leakage of liver enzymes into the bloodstream [
6]. In addition, deleterious effects of CCl
4 on hepatic antioxidant enzymes, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), have also been reported [
7]. Medicinal plants have been shown to be beneficial in ameliorating oxidative damage [
8], through elimination of hydrogen peroxide and the scavenging of other free radicals.
Vernonia amygdalina (VA) is a shrub of between 1 and 5 m in height, which grows throughout tropical Africa. The plant, generally known as bitter leaf due to the bitterness of its leaves, is used as the main vegetable or spice in the popular bitter-leaf soup [
9]. The wood, usually from the root, has been used as a chewing stick. It is much valued as a tooth cleaner, an appetizer, and for gastro-intestinal infection [
10]. Some wild chimpanzees have been observed to use this plant for the treatment of parasite-related diseases in Tanzania [
11]. The plant is used in traditional medicine for the treatment of a variety of ailments, including malaria [
12]. An antidiabetic effect of VA was reported by Erukainure et al. [
13], while Okunlola et al. [
14] gave a report of its antioxidant properties. Additionally, its anticancer property has been reported [
15]. Some bioactive constituents have been identified in the leaves of the plant, including steroid glycosides like vernonioside A1, A2, A3 [
16] and vernonioside B2, B3, B4 [
17]. Moreover, flavonoids, such as luteolin, luteolin 7-O- glycosides, and luteolin 7-O-glucuronide, as well as certain sesquiterpene lactones [
18] have also been reported.
Although there are substantial reports on the nutritional importance of VA [
9,
19] in poultry feed, there is a paucity of information on its hepatoprotective effects and mechanisms of action in this species. This study was therefore designed to investigate the potential protective effects of VALE on oxidative status and liver damage in broilers subjected to CCl
4-induced oxidative stress.
4. Discussion
This study evaluated the hepatoprotective effects of
Vernonia amygdalina in broilers subjected to oxidative stress. In the current study, VALE significantly improved growth performance in terms of BWG and FCR. Birds treated with VALE had better BWG and better FCR. The result from this study is consistent with the findings of Durunna et al. [
29] and Tokofai et al. [
19], who reported improved growth performance in VA-fed birds. The improvement in weight gain observed in the treated group resulted in a decrease in the feed conversion ratio. The improved performance of the birds may be attributed to the bioactive compounds of VALE. Indeed, it is reported to contain flavonoids, phenolics, alkaloids, terpenes, triterpenoids, steroidal glycosides, sesquiterpene lactones, vitamin A and C, etc. [
30,
31]. According to the findings of Fiesel et al. [
32], supplementation of polyphenol-rich plant products improved the gain:feed ratio in growing pigs. As gut morphology (villus height:crypt depth ratio) and apparent total tract digestibility of nutrients were not generally influenced, the improvement of the gain:feed ratio by the supplementation of the plant products was probably not due to enhanced nutrient digestibility. Similar to our findings, Olobatoke and Oloniruha [
33] reported that the inclusion of bitter leaf powder in cockerel diets significantly improved FCR. Adaramoye et al. [
34] indicated that the improvement observed may be associated with the beneficial effect of the bitter leaf in strengthening gastrointestinal enzymes, thereby improving digestion and nutrient uptake. Huffman et al. [
35] also reported that bitter leaf enhanced the production of gastrointestinal enzymes (chymotrypsin), which may not only improve feed utilization, but also aid digestion of sporozoites and other intestinal parasites affecting feed efficiency.
The liver, being the main target of reactive oxygen species (ROS), is also involved in the response to attacks induced by ROS. Any disturbance in the functioning of liver tissue is expressed through liver biomarkers and enzymatic functions. CCl
4 is converted to its toxic trichloromethyl free radical by cytochrome P450 2E1 [
3]. This free radical, with the potential of oxidative stress, causes destruction of DNA, and disruption of cell structure integrity and hepatic cell metabolism [
36]. The better total protein and globulin of the birds of VALE + CCl
4 indicates the ability of VALE to combat the stress caused by CCl
4 and modulate the production of proteins by the liver. This ability of VALE to reduce the deleterious effects of CCl
4 is ensured by its antioxidant compounds, such as phenols and vitamin C. Our observations of the hematochemical parameters in the present study are consistent with the results showing hypoproteinemia and albuminuria in broilers administered CCl
4 [
37], rats [
38], and Japanese quail [
39]. It is generally thought that oxidative stress is always accompanied by catabolism, as it promotes proteolysis, lipolysis, and glycogenolysis [
40]. The elevated serum total cholesterol and triglyceride values in CCl
4-treated birds in the present study are in agreement with previous studies indicating that oxidative stress reduced the rate of lipolysis and the activity of lipolytic enzymes [
41]. However, the cholesterol level in the VALE group was significantly reduced; this may be explained by the presence of cholesterol-lowering compounds in VALE. Saponins have been shown to reduce serum cholesterol levels [
42]. Two main mechanisms are involved in the reduction of serum cholesterol by saponins. In the first mechanism, saponins form insoluble complexes with cholesterol, thereby inhibiting its intestinal absorption. The second mechanism suggests that saponins form large aggregates with bile salts in the intestine and thus inhibit ileal reabsorption of bile salts. The latter effect triggers increased synthesis of bile salts from cholesterol in the liver, resulting in depletion of serum cholesterol. The CCl
4 group birds tended to have lower blood glucose levels compared to the control, contrary to the results of [
43]. The main metabolic pathway for the utilization of glucose by skeletal muscles under stress conditions is glycolysis [
21], and this can be reflected by the increased activity of LDH under this condition [
44]. The significantly lower LDL and significantly higher HDL in the VALE + CCl
4 group compared to the CCl
4 group reflects the ability of the bioactive compounds in VALE to mitigate the impact of CCl
4 stress. It is interesting to note that in addition to the fact that VALE in the present study normalized the HDL of birds subjected to the adverse effect of CCl
4, this index was even higher than that of the control group. This improvement can be attributed to the pharmacological properties of VALE through its flavonoids [
45].
Serum MDA, as well as TAC, ALP, AST, ALT, and GGT are used as indicators of oxidative stress and tissue damage. MDA as an indicator of lipid peroxidation and oxidative stress is used to assess the extent of oxidative degradation of lipids. Birds treated with CCl
4 in this study had a higher serum MDA content, which reflects lipid peroxidation [
46], a result of oxidative stress in biological systems [
47]. The binding of the toxic free radical of CCl
4 (trichloromethyl radical) to macromolecules induces peroxidative degradation of polyunsaturated fatty acids [
21]. This degradation leads to the formation of lipid peroxides, which in turn produce MDA with potentially harmful effects [
3]. Thus, a lower MDA content indicates a lower reaction of lipid peroxidation [
48]. The improved MDA of the birds of VALE + CCl
4 than those of CCl
4 in the present study indicates that the bioactive compounds of VALE enhanced the antioxidant status of the birds during exposure to CCl
4, suggesting its ability to scavenge peroxyl and hydroxyl radicals. Indeed, the antioxidant property of VALE has been attributed to the presence of their flavonoids. Our results differ from those of Moradi et al. [
39], who reported that there was no difference in the effect of CCl
4 on lipid peroxidation in Japanese quail. This discrepancy in the results may be due to the difference in poultry species investigated. The rapid growth rate of the commercial broiler chickens could be responsible for the susceptibility to oxidative stress conditions. Birds in the CCl
4 + VALE group showed a lower rate of tissue damage in this study. This result suggests the ability of VALE to attenuate the toxic effects of CCl
4 by reducing the increased activity of serum enzymes. The results obtained are in agreement with those showing a significant increase in serum AST and ALT levels in broilers treated with CCl
4 [
22] and Wistar rats [
49], AST in Japanese quails [
50], and ALP in Japanese quails [
51].
In this study, the elevated serum ALP and GGT concentrations in the CCl
4 group birds can be attributed to liver damage and biliary system damage. Adesanoye and Farombi [
52] reported the hepatoprotective and antioxidant potential of VA (500 mg/kg body weight) in rats. VA could therefore act by inhibiting the activation of CCl
4 by inactivating the cytochrome P450 system. As a potent antioxidant, it can also influence the enzymatic systems associated with glutathione and superoxide dismutase through the elimination of free radicals. Action on cytochrome P450 or inhibition of oxidative damage may be responsible for the protective effect against liver damage induced by oxidative stress, such as the findings observed in the present study. In agreement with our results, it was demonstrated that the negative effect of CCl
4 could be attenuated by
Zingiber officinale [
53] and green tea [
54] and
Tanacetum parthenium [
43].
Serum CAT and SOD concentrations were significantly lower in the CCl
4-treated groups compared to those in the VALE group. The enzymatic antioxidant system comprising SOD and CAT is the first line of antioxidant defense that converts harmful molecules, including hydrogen peroxides and hydroperoxides, into harmless molecules [
55]. Similarly, SOD catalyzes the excess superoxide radicals into hydrogen peroxide and oxygen; CAT catalyzes the decomposition of hydrogen peroxide into water and molecular oxygen [
55]. The better upregulation of SOD and CAT of the birds treated with CCl
4 and supplemented with VALE than the birds with CCl
4-induced hepatotoxicity without VALE in this study suggests that improvement in these endogenous enzymes is among the protective mechanisms of action of VALE. This improvement may be attributable to the polyphenols in VALE, which have the ability to reverse the oxidative stress-induced impairment. The results obtained in this study are in agreement with a report where a decrease in the concentration of SOD in broilers [
21] was reported. This decrease could be due to the inhibitory effect of MDA on antioxidant enzymes and its accelerating effect on oxidative damage on biomolecules.