1. Introduction
In today’s modern era, we are dependent on prescribed drugs, which can result in severe diseases. Drug-induced liver injury (DILI) is an uncommon, but potentially fatal, cause of liver disease that is associated with suggested medications, non-prescription drugs, and dietary supplements. According to published numbers, DILI is estimated to have an annual incidence of 10 to 15 per 10,000 to 100,000 persons exposed to prescription medications. About 44,000 people in the United States will experience DILI annually [
1]. Medications account for >50% of these, with 37% of cases attributable to APAP [
2]. Thus acetaminophen (APAP) is a major cause of abrupt liver failure.
Owing to the adverse effects of prescribed drugs [
3], natural herbal remedies are gaining more popularity in treating various diseases as they have the ability to prevent and minimize the danger of a variety of types of oxidative damage with few or no complications. The plant which yields the maximum amount of phytochemicals could be a good contender for liver injury treatment [
4].
Acetaminophen is metabolized by conjugation with sulfate and glucuronate, which are inert and excreted in the urine. However, when the activity of normal metabolic pathways is suppressed, excess APAP is metabolized by cytochrome P450 (CYP) proteins (particularly CYP2E1) and forms the highly reactive & highly toxic intermediate N-acetyl benzoquinone imine (NAPQI), which produces oxygen species (ROS) [
5,
6]. Neutralization and metabolism of NAPQI to the safer mercapturic acid is attained with the instant release of glutathione. In any case, once GSH is exhausted, the residual NAPQI causes hepatocyte damage due to an increased content of mitochondrial protein adducts, and the consequent oxidative stress caused by peroxynitrite, lipid peroxidation, and superoxide radicals [
7,
8]. In addition, an inflammatory response leads to programmed injury by the activation of c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) [
9,
10]. It has been proven that in APAP-induced liver injury, hepatic injury occurs when both the inflammatory response [
9] and oxidative stress [
7] are involved.
Curcuma longa and
Cinnamomum zeylanicum, commonly known as turmeric and cinnamon, are widely used as both herbs and spices [
11,
12]. Phytochemical constituents isolated from
C. longa and
C. zeylanicum include curcumin, dimethoxy curcumin, bisdemethoxycurcumin, curcumenol, ar-turmerone, vanillic acid, beta-sesquiphellandrene, 1-(4-hydroxy-3,5-dimethoxyphenyl), cinnamate, quercetin, trans-2-hydroxycinnamic acid, sinapic acid, gallic acid, cinnamic acid, and cinnamyl acetate [
13,
14]. The latest pharmacological studies suggest that cinnamon and turmeric possess antidiabetic, antimicrobial, anticancer, and anti-inflammatory properties due to the presence of natural bioactive compounds, making them valuable and adaptable plants with a wide range of therapeutic qualities [
12,
15].
Numerous research studies have examined curcumin’s bioactivity and health benefits, including its effects on the immune system, cancer prevention, hepatoprotection, neuroprotection, cardiovascular protection, and antioxidative and anti-inflammatory properties [
16]. Quercetin is recognized as a chemical with anti-inflammatory, anti-obesity, cardioprotective, and antioxidative properties. It is believed to be helpful in preventing neurological disorders, cancer, diabetes, heart disease, obesity, allergic asthma, and atopic illnesses [
17].
It is highly challenging to explain the molecular basis and mechanism of action of medicinal and edible plants using conventional pharmacological approaches due to the presence of numerous bioactive chemicals that can exert pharmacological effects through a variety of routes and targets [
18]. With the development of bioinformatics and molecular docking tools, it has become easier to screen for new drugs. Many reported receptors are significant in generating inflammatory responses and, thus, are potential targets for drug-induced liver injury. Molecular docking has been employed for more than three decades, resulting in the discovery and development of numerous drugs [
19]. The first stage in drug research and development is usually to find active compounds from existing substances. Several pharmaceutical corporations have libraries with hundreds of chemicals, but maintaining the library and executing high-throughput screening are both costly. Virtual screening provides a faster way to test millions of chemicals in several days. Molecular docking is one of the most-often utilized virtual screening procedures when the target protein’s 3D structure is available and used as a receptor [
20].
Recent research has focused on finding polypharmacological drugs that treat complicated (multifactorial) illnesses like cancer, neurological disease, and specific infections by acting on multiple targets. Molecular docking is a computer-aided drug design technique [
21] that is one of the computational methods employed in the hunt for multifunctional medications.
The use of bioactive compounds to reform medicines for curing multiple diseases in the future have shown encouraging results [
22]. In the current study, several antioxidative tests were performed, and the compounds were screened through an in silico analysis involving molecular docking. The compounds were docked with liver-injury protein receptors as mentioned above. The chosen multitarget compounds from the in silico study were then evaluated by an anticancer analysis, with a final examination in a liver-injury mouse model. In this model, we investigated the individual and combined degrees of protection of pure constituents against the hepatotoxicity of acetaminophen in mice. AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALP (alkaline phosphatase) as well as bilirubin (markers of liver function) were tested in the serum of mice [
23]. To check the hepatoprotective effect, histopathological analysis was also carried out.
3. Discussion
The current project was designed to screen for multitarget hepatoprotective pure compounds from
C. longa and
C. zeylanicum [
15] and to check their activity using in vitro and in vivo models for liver injury. Other linked assays like antioxidative and cell viability assays were also performed to demonstrate their potential as good therapeutic agents.
The liver is the main site of metabolism for most xenobiotics, so the generation of free radicals in the liver is higher than in other organs [
35]. ROS generated during the metabolism process of xenobiotics [
36] cause hepatic cell injury. Thus, antioxidants play a role in protecting from injury by scavenging free radicals. Phenolics were found to be high in
C. zeylanicum (117.5 ± 0.39 mg of Gallic Acid E/g of Extract), and a high content of flavonoids was found in
C. longa (98.37 ± 0.27 mg QE/g of Extract). The percentage inhibition of DPPH and the reducing power of individual and combined extracts were determined but there was no synergistic activity seen.
C. zeylanicum showed higher antioxidative activity (45.16 ± 0.66%). The results demonstrated that a high content of phenolics contributed more to the antioxidative activity than did flavonoids. The results were confirmed by previously available data [
37,
38,
39,
40,
41].
The major goals in finding a new drug are potency and protection, as all drugs can help to treat diseases as well as produce adverse effects. The basic screening of compounds was performed by MOE analysis of a library of 40 compounds based on their S-score. Only two compounds (i.e., curcumin and quercetin) were shortlisted from the MOE analysis, which was followed by Lipinski’s rule of five (Ro5) and an analysis of associated ADMET properties by the admetSAR and SwissADME online tool. The associated ADMET properties of potential compounds for different models, such as P-glycoprotein substrates, BBB penetration, gastrointestinal absorption, metabolism (including of cytochrome inhibitors), mutagenicity, and carcinogenicity showed positive results that strongly support the compounds’ suitability as drug candidates. A drug for therapeutic purposes can be selected by considering its metabolism. Cytochrome p450 is very important in drug metabolism. Various cytochromes are present, but CYP1A2, CYP2C9, CYP2D6, CYP3A4 and CYP2C19 are the important ones in drug metabolism. The clearance of compounds by these parameters indicate the safety of the compound. In our current findings, quercetin and curcumin are safe and tolerable. Our finding are supported by reported pharmacokinetics analyses of quercetin [
42] and curcumin [
43] compounds. The computational biology approaches have certain drawbacks and, therefore, one cannot fully depend on the results without the addition of wet-lab investigation and validation.
As reported in the literature, the antioxidative activity of plants is due to the presence of multiple bioactive compounds. Docking analysis specifically selected the multitargeting compounds [
13,
14]. Binding to more than one of the receptors/enzymes CYP2E1, MAPK, and TLR4 proved the multitargeting ability of the compounds. Our compounds specifically showed high interaction with cytochrome P450 enzymes (CYP2E1 and CYP2C9). Cytochromes are mainly involved in the metabolism of exogenous and endogenous compounds and drugs, especially for the metabolism of acetaminophen. Hepatic CYP2E1 plays a major role in APAP-induced liver injury [
44]. Compared with wild-type mice given acetaminophen, CYP2E1-knockout mice had a significantly higher resistance to liver damage [
45]. Weber et al. reported that TLR4 deficiency protects against hepatic injury in preclinical mice models [
46]. TLR4 is directly involved in hepatic inflammation and fibrosis, whereas upregulation of inflammatory factors like NF-κB by TLR4 is also important in hepatocarcinogenesis [
47]. Our docking analysis showing that the candidate drugs inhibit the TLR4 cell-surface receptor, because its activation causes the release of cytotoxic mediators (TNF-α and IL-6) along with the activation of the pro-apoptotic protein kinase JNK and NF-κB, which are cell-injury mediators [
48]. A previous study in which compounds of
C. longa and
C. zeylanicum were shown to be involved in the management and cure of liver injury [
41,
49] supports our finding. The scoring system confirms the correctness of docking by examining the lowest possible energy orientations. The docking results were justified by the in vivo results, as these receptors were dysregulated and caused oxidative stress in the case of injury, while oxidative stress was abated in the treatment groups.
Liver injury in later stages turns into liver cancer. So, it would be great if a hepatoprotective drug has the potential to treat cancer.
C. longa and
C. zeylanicum pure compounds have been tested against a variety of cell lines, including human cancer cell lines [
50,
51]. Cells were treated for 72 h, and the decreased viability of Huh7 and HepG2 cell lines and the lack of effect on the normal cell line Vero by quercetin and curcumin proved their anticancer activity, as mentioned above in
Figure 3. Compounds showed significant results individually as well as in combination; a finding that makes them very useful for future use. Although both candidate drugs had the same possible target, they did not enhance the effect of each other.
Increased levels of the serum biomarkers ALT, ALP, and AST in APAP-treated mice are expressed in liver injury, and in the case of damage, these liver enzymes are discharged into the blood, which indicate extent of the liver injury. The elevated levels of these markers in the APAP-treated group showed the extent of the hepatic cell injury, and a noteworthy decrease in the levels of these markers (AST, ALT, and ALP) in the pretreatment groups in comparison with the vehicle APAP-treated group confirmed their hepatoprotective potential. Liver injury causes a decrease in the level of TAC but pretreatment with the candidate drugs showed the recovered potential of antioxidants in the serum. The antagonistic effect of TAC and TOS proved the protective effect of quercetin and curcumin. No such significant differences were observed on the liver function tests (
Figure 8). The hepatoprotective effect of the screened compounds and their combination was comparable with that of silymarin, which we used as a standard drug. Our results are compatible with previous reports [
52,
53] stating that
C. longa,
C. zeylanicum are well known for their hepatoprotective abilities, as their pure compounds quercetin and curcumin have the potential to deal with oxidative stress.
The histopathological analysis also justified our in vitro results as this showed a clear effect of the different treatments on liver tissue. The APAP-treated group showed severe hepatocyte damage, inflammation, and disrupted architecture showing hepatotoxicity caused by the overdose. Pretreatment groups of quercetin and curcumin showed milder histological changes compared with APAP, suggesting a potential hepatoprotective effect. The combination-treated group showed moderate protection in the histological analysis, suggesting an additive or synergistic effect of the combined treatment. Small changes were observed in the silymarin group, suggesting a possible protective effect against liver damage. These findings suggest that quercetin, curcumin, and silymarin may have hepatoprotective properties, with silymarin having the mildest effect. Further studies are warranted to elucidate the underlying mechanisms and to determine the optimal doses and treatment durations for potential therapeutic applications.
From our observations, we conclude that the result of these treatments for liver injury follows the same trend as anticancer treatment results, proving that these are strongly associated studies. Both drugs, although having the same targets, did not contribute synergistically.