Medicinal plants with hepatoprotective potentials against carbon tetrachloride-induced toxicity: a review

Carbon tetrachloride (CCl4) is a well-characterized hepatotoxic agent. With rising cases of liver diseases, the identification, assessment, and development of hepatoprotective agents from plants source has become imperative. With arrays of literature on plants with hepatoprotective potentials, this review sourced published literatures between 1998 and 2020 and systematically highlighted about 92 medicinal plants that have been reported to protect against CCl4-induced liver injury in animal models. The results show that herbal plants provide protection for the liver against CCl4 by downregulation of the liver marker enzymes and activation of antioxidant capacity of the liver cells with the restoration of liver architecture. We also provided the traditional and accompanying pharmacological uses of the plants. A variety of phytochemicals mostly flavonoids and polyphenols compounds were suggested to offer protection against liver injuries. It can be concluded that there are a variety of phytochemicals in plant products with hepatoprotective activity against CCl4-induced toxicity in animal models.


Background
The liver being an important organ is often exposed to array of threats [1]. Injury to the liver can lead to deterioration of its functions and may culminate in organ failure [2]. The likely risk factors for the development of the liver diseases have been suggested to include pathogenic microorganisms and viruses, hepatotoxins, overdose and duration of drugs, obesity and malnutrition, alcohol, autoimmune disorders, type-2 diabetes, and genetic factors [1]. The diseases of the liver are of public health concern because orthodox remedies for liver diseases produce limited results with attendant side effects. As such, utilization of complementary and alternative herbal medicine has attracted research interest for novel plausible hepatoprotective agents capable of ameliorating or reversing liver injury with little side effects [3,4]. Over the years, this search has gained impetus with many studies focusing on hepatoprotective potentials of plant drugs.
Carbon tetrachloride (CCl 4 ) is a known hepatotoxicant in humans and animal models [5]. It has been successfully used in hepatotoxicity research as a model and to appraise hepatoprotective agents [6,7]. With reports on the rise of liver diseases and numerous literature reports on plants with potential hepatoprotective activity, this review highlighted the mechanism of CCl 4 toxicity, the significance, effectiveness, and underlying mechanisms of herbal plant extracts on CCl 4 -induced toxicity in experimental animal models. Ugwu and Suru Egyptian Liver Journal (2021) 11:88 Main text

Insight on the mechanism of carbon tetrachloride hepatotoxicity
Prior to the Montreal Protocol, CCl 4 was formerly and widely used as a fire suppressant, as a precursor to refrigerants, propellants for aerosol cans, as a cleaning agent, a widely used solvent in organic chemistry, as a pesticide, and anesthetics [8,9]. However, it is rarely used today because of adverse health effects and environmental safety concerns. Symptoms associated with acute inhalation of low-medium doses include headache, weakness, lethargy/general anesthesia, nausea, vomiting, and respiratory arrest. For medium to high oral exposure, the liver is known to be the primary site of CCl 4 -induced toxicity beginning with acute but progressive centrilobular injury that may culminate in cell death [10].

Experimental deductions
Due to the complex nature of CCl 4 -induced liver damage, there have emerged several independent mechanisms to explain each of the facets of the associated changes. The interrelationship among diverse mechanisms proposed for each of these associated changes has not been wellestablished/outlined. This is primarily because early and later changes associated with the hepatotoxic development have been mixed up. As a result, a harmonized understanding of the intricate mechanisms involved in hepatic damage has become partly elusive. However, this has not obscured the following experimental deductions ( Fig. 1): • Changes in endoplasmic reticulum (ER) function due to decrease in glucose-6 phosphatase [11], which may not be unconnected with CCl 4 -induced glycogen depletion and attendant protection from carbohydrate-rich diets [12,13]. Besides, CCl 4 -induced disruption and disassociation of polyribosomes from ER alters its anabolic function as manifested in decreased incorporation of amino acids into proteins such as albumin and fibrinogen [14]. Additionally, CCl 4 -induced hypomethylation of 2′-O-ribose moieties in rRNA might have resulted from transient increase in cytosolic Ca 2+ . This increase may activate the selective destruction of rRNA methylases via Fig. 1 Hepatotoxic mechanism of CCl 4 the action of demethylases or proteases. Overall, the protein synthetic function of ER in the centrilobular region may be hampered with an attendant defects in the ability of the liver to effectively respond to additional insults [10]. • Calcium homeostasis underlies some aspects of CCl 4 hepatotoxicity (plasma membrane blebbing and fatty accumulation-steatosis); CCl 4 may elicit dramatic redistribution of intracellular Ca 2+ stores, albeit no total cellular change [10]. Calcium ion (Ca 2+ ) homeostasis is maintained by 3 mechanisms: (i) Ca 2+ extrusion by plasma membrane ATPase, (ii) Ca 2+ sequestration by mitochondria, and (iii) Ca 2+ sequestration by liver ER. So, CCl 4 may cause decreased Ca 2+ sequestration by ER and mitochondria, decreased extrusion by plasma membrane ATPase, as well as blockage of gap junctional intercellular communication may favor increase cytosolic Ca 2+ . An ATP-dependent Ca 2+ sequestration by hepatic ER has been shown to be disrupted by CCl 4 [15]. Endoplasmic reticulum membrane permeability may also be altered, being one indicator of impending cell death [16]. • Rapid destruction/decrease in cytochrome P 450 in centrilobular regions (suggesting that CCl 4 was metabolized by ER mixed-function oxidase system), which is orchestrated by low levels of reduced glutathione (GSH) and low oxygen tension. In turn, low level oxygen tension may limit competition between O 2 and CCl 4 for cytochrome P 450 binding (i.e., CCl 4 may readily bind to cytochrome P 450 ). • Metabolic products [trichloromethyl (CCl 3 * ) or peroxytrichloromethyl (CCl 3 -OO * ) free radical] elicit damage: lipid peroxidation of vulnerable unsaturated fatty acids in membrane phospholipids and destruction of haem moiety of cytochrome P 450 . • Blockage of gap junctional communication by CCl 4 thereby shutting down intercellular communication. • Changes in mitochondrial function: disruption of oxidative phosphorylation due partly to chelation of calcium [17].

Making sense out of experimental deductions
The hepatic biotransformation of CCl 4 primarily involves metabolic activation to transient reactive intermediates. Under low oxygen partial pressure, cytochrome P 450 catalyzes the reductive de-halogenation of CCl 4 resulting in predominant formation of CCl 3 * and CHCl * radicals [18,19]. These reactive intermediates may bind covalently to cellular components (membranes, microsomes) and impinge on mostly lipid metabolism (increased synthesis, decreased transport out of the hepatocyte) thereby culminating in hepatic steatosis (fatty liver) [20,21].
Dianzani [22] reported that covalent modification of lipoproteins occurs prior to their decreased transport out of hepatocytes. Intracellular maturation of lipoproteins in the Golgi apparatus is dependent on galactosylation which is catalyzed by glucosyl-and galactosyltransferases [23]. The CCl 4 -induced damage of Golgi apparatus and eventual reduction in the activities of these enzymes may explain the observed decrease in lipoprotein secretion associated with CCl 4 intoxication. Thus, CCl 4 -induced inhibition of lipoprotein secretion, and its attendant hepatic steatosis mainly result from covalent binding of CCl 4 metabolites to cell constituents, but not due to lipid peroxidation.
Under high oxygen partial pressure, however, CCl 3 * may interact with oxygen to form CCl 3 -OO * . The peroxy radicals may elicit the peroxidation of unsaturated fatty acids especially in membrane phospholipids of intracellular and plasma membranes [24]. Some of the lipid peroxidative products may inflict further damage leading to increased membrane permeability and a comprehensive loss in membrane integrity [25]. Thus, both covalent binding of CCl 4 metabolites and lipid peroxidation work in tandem to elicit the hallmark of damage seen in CCl 4 -induced hepatotoxicity. The consequences of loss of membrane integrity are enormous and may lead to cascade of events culminating in liver necrosis. These events may include disturbed Ca 2+ homeostasis/dramatic redistribution of Ca 2+ in hepatocytes, leakage/efflux of K + , and influx of Na + [10,26].
Beside the peroxidative action, CCl 4 -derived free radicals and their attendant oxidative stress have been shown to enhance NF-kB expression, which in turn initiates the synthesis of cytotoxic cytokines, which may be partly responsible for liver injury [27]. Tumor necrosis alpha (TNF-α) has been implicated in CCl 4 -induced hepatocellular damage [28]. At lower doses of CCl 4 , inflammatory responses prevail. Healthy hepatocytes are insensitive to tissue necrosis factor alpha (TNF-α) action, but become sensitive once protein and RNA synthesis are inhibited [29].
Summarily, CCl 4 hepatotoxicity may be due to a combination of factors such as the thorough inhibition of protein synthesis, the severe derailment of intracellular Ca 2+ sequestration, and the effect on membrane integrity. These factors may result and progress through a series of steps that contribute to various extents to the ultimate damage: reductive dehalogenation, covalent binding of resulting radicals; inhibition of protein synthesis (in particular, apolipoprotein synthesis), assembly, packaging and release of VLDL and HDL, fat accumulation;      Active components.

Traditional plants with anti-hepatotoxic potential
In this review, numerous experimental studies on the medicinal plants effectiveness to ameliorate CCl 4 -induced hepatotoxicity in animal models were presented. The botanical names, ethnopharmacological and pharmacological uses of plants traditionally used to treat liver-related diseases were presented in Table 1. The comprehensive details on in vivo studies of medicinal plants with hepatoprotection against CCl 4 -induced hepatotoxicity alongside the active phytochemicals and their probable mechanisms of action are presented in Table 2.

Discussion
For about three decades, extracts from different natural products have been identified to be hepatoprotective at varied doses against CCl 4 -induced toxicity by reducing oxidative stress on liver enzymes. The findings from this review show that only few studies tested these natural products on hepatic cell lines ( Table 2). Without separating the whole extract to identify the active components, a large number of hepatoprotective products will increase without corresponding clinical relativity [123]. There is an urgent need to study individual components of the plant extract especially in experimental animal models. The major drawback of herbal medicine is its potential hepatotoxicity in man which could cause acute to chronic liver injury with underlining mechanism of toxicity not clearly understood due to factors such as the synergistic and multi-organ targeted nature of the various components [124][125][126][127].
The protection provided by herbal plants against CCl 4 -induced hepatotoxicity is basically due to the inhibitory nature of the phytochemicals present in them [70,101]. These phytochemicals are able to inhibit the microsomal enzymes to restrict the generation of free radicals and stop lipid peroxidation through its antioxidant ability [66]. They can also enhance the regeneration of liver cells, radical scavenging, and stimulation of the antiinflammatory ability of the liver cells against the inflammation induced by CCl 4 [102].
The treatment of the animal models with these herbal extracts showed beneficial effects through several biochemical and histological results. From the results in Table 2, it is clear that these plants extract downregulated serum liver marker enzymes like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin, and malondialdehyde (MDA) while upregulating the activity of antioxidant enzymes and total protein. The medicinal plants also downregulated the inflammatory markers expression in the hepatic cells. Some of these reported studies confirmed the hepatoprotective effectiveness of these medicinal plant products through histological reports [43,54]. This review also reported numerous phytochemicals with possible hepatoprotective potentials ranging from flavonoids (quercetin, kaempferol), phenols, sobatum, courmarins, gallic acid, rutin, alkaloids, saponins, vitamin C, caffeic acid, etc. This review presented a number of plant species with ethnopharmacological relevance in the treatment of liver injury and their medicinal/pharmacological uses from literature.

Conclusion
We, therefore, conclude that there are a variety of phytochemicals in plant products with hepatoprotective activity against CCl 4 -induced toxicity by downregulation of liver marker enzymes, and activation of antioxidative capacity of the liver cells that leads to the restoration of the liver architecture.

Future perspectives
There is need to validate the efficacy of some of the reported active components which can be likely candidate for therapeutic purposes. Research should move from whole plant extract experiment to isolation of bioactive components and testing the extract on culture cell lines.