Background
Despite its vital role in various physiological processes including maintenance and regulation of homeostasis, and detoxification of drugs and xenobiotics in the body, the liver is widely exposed to injury or toxicity due to direct exposure to various intoxicants such as like chemotherapeutic agents, chronic alcohol consumption and pathogenic microbes [
1]. One of the toxicants continuously associated with liver injury is paracetamol (PCM), which is an effective drug for the treatment of pain, inflammatory and fever. At its normal therapeutic doses, PCM can be obtain as an over-the-counter medicine since it is a safe drug for all age groups. However, the intentional or unintentional overdoses of PCM might cause acute damage to the liver, which may lead to fatal liver necrosis if not properly treated. Unfortunately, there are inadequate treatment selections to treat liver necrosis [
2].
At therapeutic dose, PCM undergo sulfation and glucuronidation in the liver wherein between 5 and 10% of it is rapidly metabolize into
N-acetyl-
p-benzoquinone imine (NAPQI) by the hepatic cytochrome P450 (CYP450) system, mainly CYP2E1. Being a reactive metabolite, NAPQI will form covalent binding with various cellular nucleophiles (e.g. RNA, DNA and proteins), which may result in cell death. Moreover, CYP2E1 also take part in the NADPH oxidase activity leading to the continuous production of free radicals that may result in hepatic injury. Concurrent to this, the glutathione redox system inactivates the formed NAPQI via glutathione conjugation resulting in the diminution of cellular GSH supplies and creation of protein adducts [
3,
4].
Excessive stress in the mitochondria resulting from extreme oxidative and nitrosative processes result in the initiation of enzymes of signaling cascade, which disrupts and destabilizes the membrane potential, causing the mitochondria to swell until its membrane rupture. The rupture of mitochondrial membrane will finally lead to the initiation of nuclear DNA fragmentation [
5]. Collectively, fragmentation of DNA together with impairment of mitochondrial play major role in hepatocyte necrosis seen in PCM-induced liver injury (PILI). Excessive formation of metabolites reduces GSH level in the liver, thus, PCM intoxication is currently treated using
N-acetylcysteine (NAC), a glutathione precursor, with aim of replenishing the GSH supplies in the liver. Despite being the only drug permitted to be used a cure for PILI, NAC is only effective following the oral or intravenous administration within 10 h of PCM overdose [
6].
To avoid depending on NAC as the only source of antidote for PILI, researchers have increase their effort to search for alternative sources of agents to treat PILI and natural products have been a good source of leads for treating various diseases including liver injury. Natural products, especially plants, have been playing key role in drug discovery in various traditional cultures due to their wide range of diversity of active bioactive molecules [
7]. Due to disease-inhibiting capabilities, these compounds are extremely useful as natural drugs that are less toxic, more effective and provide a venue for the development of natural products into the potent drug. For example, polyphenolic compounds have an imperative role in stabilizing lipid oxidation and are associated with antioxidant activity [
8].
Since PILI involved oxidative stress and inflammation, it is widely claimed that any compounds that can prevent oxidation and inflammation activities could also exert liver protective action [
9]. In this case, medicinal plants have been widely known as the major source of bioactive compounds with antioxidant and anti-inflammatory activities. One of the plants that have been actively studied for its medicinal potentials in our laboratory is
Dicranopteris linearis L. (Family
Gleicheniaceae), a type of fern known to the Malay as
‘Resam’. Other than that, it is important to highlight that
D. linearis is traditionally used to control fever, to get rid of intestinal worms, to treat boils, ulcers, and wounds [
10] whereas, scientifically, the plant has been proved to exert various pharmacological activities including anti-inflammatory [
11,
12], hepatoprotective [
13] and antioxidant [
14] to name a few. The hepatoprotective activity, in particular, was observed using the plant’s methanol extract (MEDL), which has been acknowledged to extract a mixture of polar, non-polar and intermediate polar bioactive compounds. Since MEDL contains mixture of bioactive compounds with different polarities, it is difficult to attribute the hepatoprotective activity to any particular group of bioactive compounds according to their polarity. Taking into consideration that the chloroform extract of
D. linearis (CEDL) demonstrated anti-inflammatory and antioxidant activities [
11,
15], wherein these activities have been reported to play role in the mechanisms of hepatoprotection, the present study was proposed to evaluate the hepatoprotective potential of this hydrophobic/lipid-soluble phytoconstituents (CEDL) of
D. linearis leaves using the in vivo PILI model in rats.
Discussion
CEDL ability to exert hepatoprotective activity against PILI in rats were investigated in this study. Liver function test, liver antioxidant enzyme levels and histological studies were performed to assess hepatoprotective properties of this plant. The results obtained show that overdose PCM administration altered various liver parameters by increasing the: i) LW and LW/BW ratio, and; ii) serum level of ALT and AST while decreasing the activity of several endogenous antioxidant enzymes, namely CAT and SOD. These findings were supported by histopathological observations, which show that PCM-induced severe degrees of histological damage to the liver tissue architecture due to the presence of necrosis, hemorrhage and inflammation.
It is widely known that PCM, at high doses, produces acute toxic effects that can result in PILI. This condition is attributed to PCM bioactivation to a toxic electrophile,
N-acetyl
p-benzoquinoneimine (NAPQI), which binds covalently to tissue macromolecules possibly triggering the oxidation of lipids or the perilous sulfhydryl groups (protein thiols) leading to the alteration in calcium homeostasis [
23]. Immense generation of reactive species might cause a diminution of protective physiological moieties, thus, initiating injury to the macromolecules in vital biomembranes that leads to liver injury [
24].
Liver enzymes (e.g. ALT and AST) and several other molecules, which are originally present in the cytoplasm of liver cells, leak into the bloodstream during liver injury resulting in the elevation of their level during blood test; thus, can and assist as an indicator for the liver injury [
25]. Unusually high levels of serum ALT and AST observed in the PCM-intoxicated group seen in the present study indicate PCM-induced liver dysfunction and denote injury to the hepatocytes. Interestingly, pretreatment with CEDL reversed the increased serum enzymes in the PILI group suggesting the extract potential to prevent the intracellular enzymes leakage through its membrane-stabilizing activity. Histopathological findings corroborate well with the biochemical results suggesting that CEDL significantly contribute to the reduction of degree of injury induced by PCM.
Antioxidant enzymes reduced the toxic effect caused by superoxide anion by scavenging those free radicals to form hydrogen peroxide. Two of the vital enzymes in the enzymatic antioxidant defense system are CAT and SOD [
26] and reduction in their activities have been reported to lead to several toxic effects. In the present study, it was observed that PILI caused a marked decrease in the activity of both enzymes whereas pretreatment with CEDL increased the hepatic SOD and CAT activities in PILI rats. This finding suggested that CEDL contained bioactive compounds that can modulate the endogenous enzymatic antioxidant enzymes and help prevents liver injury.
Other than that, CEDL has also been found in the present study to exert a high antioxidant capacity as proven using the ORAC assay despite its low TPC value and low free radical scavenging effects as shown using the DPPH and SOA assays. The contradictory findings between the ORAC assay against the DPPH- and SOA-assay could be partly attributed to the various mode of antioxidant actions reported elsewhere. Other than acting as a radical scavenger, other bioactive compounds have also been reported to exert antioxidant action by acting as electron donor, hydrogen donor, singlet oxygen quencher, peroxide decomposer, an enzyme inhibitor, metal-chelating agents, and synergist [
27]. Concerning the mechanisms of action involved, Lobo et al. [
27] have also cited two principal mechanisms via which antioxidants work, namely: i) a chain-breaking mechanism that involves donation of an electron to the free radical existing in the systems by primary antioxidant, and; ii) a mechanism that involves a quenching chain-initiating catalyst removing the ROS/reactive nitrogen species initiators (secondary antioxidants). Besides, some other bioactive compounds also can stimulate the synthesis of endogenous antioxidant molecules in the cell [
28]. The presence of various modes of antioxidant actions might, therefore, explained the extract’s high antioxidant capacity that helps to reduce oxidative damage to the tissues as well as improving the activity of hepatic antioxidant enzymes. On the other part, the low free radical scavenging activity of CEDL could be attributed to the presence of triterpenes as its major constituents. Interestingly, Arora et al. [
29] have earlier reported that
Centella asiatica’s extract enriched with triterpenes resulted in a loss of free radical scavenging potential, thus, could be used to justify the low free radical scavenging potential of CEDL. Concomitantly, Castellano et al. [
30] also reported that the data available currently concerning the antioxidant activity of triterpenes are rather inconsistent with various reports on the ability of triterpenes to react with diverse radical species resulted in a collection of varied results [
31‐
34].
Although phenolic compounds are present in CEDL, the number of phenolic compounds detected was low based on the TPC value recorded. The low TPC value in CEDL might be attributed to the occurrence of only hydrophobic flavonoids in the extract with the absence of tannins. Moreover, reports have shown the link between the level of TPC and the intensity of free radical scavenging activity [
35]. Phenolic compounds have been widely known to possess redox properties due to the existence of hydroxyl groups. Being good electron donors, these hydroxyl groups are responsible for facilitating free radical scavenging action [
36]. Concurrent with the link described above, the low free radical scavenging activity of CEDL could, therefore, be attributed to the low TPC value.
The presence of triterpenes followed by flavonoids as major constituents of CEDL might strongly contribute to the strong antioxidant capacity but low free radical scavenging activity, activation of the endogenous enzymatic antioxidant system and protection against PILI. The ability of triterpenes: i) to exert hepatoprotective against PILI [
37,
38], ii) to show antioxidant capacity via ORAC with low free radical scavenging activity [
30,
39], and; iii) to activate the endogenous enzymatic antioxidant system [
40] as seen with CEDL have been previously established. With regard to the antioxidant potential of CEDL, the antioxidant power of triterpenes is subject to debate since it is evidently affected by the singularities of the experimental systems engaged in its evaluation. For examples, oleanolic acid was found to capture ABTS+ radicals in a moderate-, dose-dependent-manner but cannot scavenge DPPH species. The latter observation is in agreement with the present study that shows CEDL lack of scavenging effect against the DPPH species. On the other hand, Castellano et al. [
30] also reported on the ability of triterpenes like oleanolic acid to moderately captures the peroxyl radicals generated in the ORAC assay, which is also seen in the present study. Other than that, flavonoids have also been reported: i) to ameliorate PILI [
41,
42], ii) to exert free radicals scavenging activity and marked antioxidant capacity [
43,
44], and; iii) to improve the endogenous enzymatic antioxidant system, particularly CAT and SOD [
45,
46].
Concerning the different in the antioxidant effect of flavonoids when measured against the DPPH radical scavenging assay or ORAC assay, Roy et al. [
43] reported that certain flavonoids such as epicatechin showed a higher ORAC reading than epigallocatechin gallate but, on the contrary, epigallocatechin gallate demonstrated a stronger DPPH radical scavenging activity than epicatechin. These differences were further explained through the structure-activity relationship investigation, which shows that the lower ORAC value of epigallocatechin and epigallocatechin gallate is attributed to the OH replacement at the 3′ position in pyrogallol moieties when compared to their non-3′-OH counterparts (such as epicatechin and epicatechin gallate) [
43]. However, the number of OH replacements is suggested to poorly correlate with the recorded ORAC value, but significantly affected the DPPH radical scavenging activity. Thus, the presence of flavonoids with non-3′-OH replacement in pyrogallol moieties in CEDL is postulated to contribute to the high ORAC value but low DPPH radical scavenging activity.
Preliminary qualitative phytochemical screening of CEDL demonstrated the presence of flavonoids. HPLC analysis of CEDL revealed at least two peaks showed the UV spectral that were characteristic of flavonoid-based bioactive compounds while comparison between the chromatogram of CEDL and several pure flavonoid standards revealed the presence of only hesperetin in the extract. Further analysis of CEDL using the UHPLC-HRMS procedure revealed the presence of 30 polyphenols, which were identified to belongs to the hydroxybenzoic acids, hydroxycinammates and flavonoid groups. Despite their presence in CEDL, these polyphenols contribute to low TPC content in CEDL and lack of free radical scavenging activity of CEDL and the latter effect of flavonoids has been discussed earlier. However, their presence and synergistic action also could be used to explain the significant antioxidant activity of CEDL as measured using the ORAC assay and hepatoprotective activity of CEDL as observed in the in vivo study.
Other than that, Vijayakumari and Leon Stephen Raj [
47] have recently performed the GC-MS analysis on CEDL collected from Marthandam, Kanyakumari district, India in which the GC-MS spectrum of CEDL demonstrated the presence of 9 different major peaks representing 1H-inden-1-ol, 2,3-dihydro, phenol 2,5-bis(1,1-dimethylethyl)-, heptacosane, di-n-decylsulfone, ethanone, 2-(2- benzothiazolylthio)-1-(3,5-dimethylpyrazolyl)-, 1-bromoeicosane, methoxyacetic acid, 2-tridecyl ester, octadecane, 3-ethyl-5-(2-ethylbutyl)- and 1,2,4-benzene tricarboxylic acid, 4-dodecyl dimethyl ester. Interestingly, some of these compounds, namely phenol,2,5-bis(1,1-dimethylethyl)-, ethanone,2-(2-benzothiazolylthio) -1-(3,5-dimethyl pyrazolyl)-, and octadecane,3-ethyl-5-(2-ethyl butyl)-1,2,4-benzene tricarboxylic acid, 4-dodecyl dimethyl ester, have been reported to exert antioxidant activity [
47]. It is believed that these compounds together with the triterpenes and flavonoids act synergistically to modulate the antioxidant properties of CEDL. The high antioxidant capacity of CEDL may help explain the significant hepatoprotective activity of the extract by counteracting the redox state precipitated intracellularly, hence, ensure hepatoprotection against PILI.
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