Methanol extract of Dicranopteris linearis L. leaves impedes acetaminophen-induced liver intoxication partly by enhancing the endogenous antioxidant system

Medicinal plants have been the subjects of man’s interest since a long time ago with almost every culture has a history of plants’ use to treat various diseases [1]. Approximately 80% of the people in the developing countries still depend on medicinal plants as their main source of health care needs. The revival of public awareness in plant-based medicine combined with rapid expansion of pharmaceutical industries in the recent couple of years has imposed an increase demand for medicinal plants [2]. This is further supported by the fact that approximately 80% of claimed traditional medicine involves the use of plant extracts, which suggests that the medicinal plants still continue to occupy an important niche in modern medicine and may play a leading role in the introduction of new therapeutic agents [3]. A number of medicinally-effective compounds currently used as modern drugs are derived from medicinal plants, which may consist of complex structures. Thus, synthesizing these bioactive compounds preferably in minimal chemical steps and at low budget is sometimes not accessible. Therefore, the use of plant-based extracts, either as whole or semi-purified for the treatment of diseases as applied in the traditional medicine practices can be seen as a reasonable and practical solution to overcome the issue. One of the medicinal fields where the application of medicinal plants as an alternative medicine has gained remarkable attention is related to the treatment of liver diseases or damages [4].

Liver is important to the body as it helps to regulate astounding range of imperative physiological functions that help in the maintenance, performance and regulating homeostasis of the body. Being involved in the metabolism processes, the liver is usually exposed to various types of endogenous and exogenous substances including those with toxic potentials. Normally the liver will detoxify and excrete these substances from the body. However, over or continuous exposure of the liver to toxic chemicals can inflict damages to the liver because of the release of free radicals (i.e. reactive oxygen species (ROS) or reactive nitrogen species (RNS)) resulting from the metabolisms of the toxic chemicals. The ROS or RNS, on the other hand, act by causing damage to the membrane of hepatic cells found mainly in the liver parenchyma cells resulting in several repercussions on human health. Liver damage is any circumstance that may cause liver inflammation or tissue injury and affects liver function. Liver disease badly affects over 10% of the world population [3].

Drugs have been one of the important causes of liver injury. In the United States, approximately 2000 cases of acute liver failure occur annually and drugs account for over 50% of them (37% are due to acetaminophen, 13% are idiosyncratic reactions due to other medications). Meanwhile, drugs also account for 2–5% of cases of patients hospitalized with jaundice and approximately 10% of all cases of acute hepatitis. Of these, acetaminophen (APAP) has been widely associated with liver intoxication. It is a commonly used drug to treat pain and fever relief [5]. APAP is commonly considered as a “safe drug” when taken within the suggested therapeutic dose because it can be purchased easily without prescriptions. However, it can be hepatotoxic when an overdose is administered. Accidental or intentional overdose with acetaminophen (APAP) mainly cause for the acute liver failure case. Overdose of APAP cause the generation of free radicals results in the depletion of glutathione and also cause dose-related hepatocellular necrosis [6].

Although a number of alternative treatments are available to cure most kind of the liver damages cases, several types remain untreatable and the emergence of drug resistance is persistent [1, 3]. Hence, novel treatment approaches are crucial to improve therapeutic outcome. Almost half of the agents currently used in liver therapy derived from either natural products or derivatives of natural products with plants being the main contributor [3]. One of the plants that are currently being investigated for its hepatoprotective potential is Dicranopteris linearis L. This plant belongs to the family Gleicheniaceae and, despite its limited medicinal uses among the Malays [7], it has been scientifically proven to exert antinociceptive, anti-inflammatory and antipyretic [8, 9], antiproliferative and antioxidant [10], and chemopreventive [11] activities. We have recently reported on the hepatoprotective activity of methanol extract of D. linearis against the CCl₄-induced liver injury model [12].

Taking into account the ability of MEDL to exert antioxidant and anti-inflammatory activities [8‐10] which are important in the augmentation of liver protection against damages [13], and the fact that CCl₄–induced a different mechanisms of liver intoxication in comparison to APAP, the present study was proposed to establish the hepatoprotective activity of MEDL against APAP-induced liver damage model. In addition, we also determined the involvement of some endogenous enzymatic antioxidant system, namely catalase (CAT) and superoxide dismutase (SOD), in the attenuation of APAP-induced hepatotoxicity by MEDL and analysed the phytoconstituents of MEDL using the ESI-UHPLC and GCMS methods.

Acetaminophen is metabolically activated to form a reactive metabolite by several types of cytochrome P450 such as cytochromes 2E1, 1A2, 3A4, and 2A6 via a direct two-electron oxidation. This reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI1), covalently binds to protein to form protein adducts. At therapeutic doses, NAPQI1 is detoxified by glutathione (GSH) to form an acetaminophen-GSH conjugate. However, at a toxic dose of APAP, total hepatic GSH is remarkably depleted resulting in the metabolite binding covalently to cysteine groups on protein to form APAP-protein adducts. Events that generate hepatocellular death following the formation of APAP-protein adducts are poorly understood. However, it is suggested that covalent binding to critical cellular proteins results in subsequent loss of activity or function and eventually cell death and lysis [18]. Mitochondrial proteins as well as proteins involved in cellular ion control have been postulated to be the primary cellular targets, which upon binding with the reactive metabolites, results in the loss of energy production. In addition, the formation of protein adducts also lead to alterations of plasma membrane ATPase activity. A number of these cellular proteins that bound to intoxicated APAP have been isolated and identified such as glutamate dehydrogenase, glutathione peroxidise, carbonic anhydrase III etc. [19]. McGill et al. [19], themselves, have reported among others the involvement of mitochondrial damage and nuclear DNA fragmentation in the APAP induced liver damage.

Other than Kamiyama et al. [20] who demonstrated that lipid peroxidation play minimal role in APAP-induced liver damage via in vitro techniques, Jaeschke et al. [21] used the in vivo model to show the less critical function of lipid peroxidation in APAP-induced liver damage. The lack of lipid peroxidation involvement in APAP-induced model might be attributed to the role played by peroxynitrite, which is a highly reactive nitrating and oxidizing species. In overdose, APAP is metabolized by cytochrome P450 to form a reactive metabolites, NAPQI, which then reacts with hepatic glutathione (GSH) causing the latter depletion by approximately 90%. In addition NAPQI binds covalently to proteins as described earlier to form APAP-cysteine adducts, which then leads to the centrilobular hepatic necrosis. Further study showed that nitrated tyrosine, protein adducts formed as a result of nitration of tyrosine by peroxynitrite, occurs in hepatic centrilobular cells wherein they co-localized in cells containing the APAP-cysteine adducts. This nitrated tyrosine are formed from the action of peroxynitrite, which is a highly reactive nitrating and oxidizing species formed by the rapid reaction of nitric oxide (NO) and superoxide. It has been reported that the activated Kupffer cells are mechanistically vital in NO and superoxide formation. However, other cellular sources of NO and superoxide may also be important. Hepatocytes and stellate cells express inducible nitric oxide synthase (iNOS) while endothelial cells constitutively express eNOS. On the other hand, various sources produce superoxide, including damaged mitochondria. It is noteworthy to highlight that intoxication with hepatotoxin that caused hepatic GSH depletion (i.e. APAP, chloroform, bromobenzene, and allyl alcohol) leads to peroxynitrite formation, which then promotes toxicity.

Although the role of free radicals remains vital in the development of APAP-induced hepatotoxicity, the pathway taken by the free radicals to induce liver cells damage is different from the one used by CCl_4. Instead of lipid peroxidation, APAP-induced liver damage uses peroxynitrite as a mediator of hepatotoxicity. Based on this fact, it is reasonable to propose that for any compounds/extracts to be considered as suitable candidates to be developed into hepatoprotective drugs, they should possess significant free radical scavenging and antioxidant effects [22]. This proposed statement is in line with the previous study that demonstrated the ameliorative effect of aqueous extract of Dicranopteris linearis leaves against CCl₄- and APAP-induced liver injury, which are believed to be dependent on the extract’s antioxidant status [23]. With regard to MEDL, it has been shown to exert remarkable free radical scavenging and antioxidant activities as confirmed by several tests [12]. The antioxidant property of MEDL could be associated with the presence of several flavonoids that have been identified as part of the phytoconstituents of MEDL, such as rutin and quercitrin [10, 12]. Previously, rutin has been reported to attenuate APAP- induced hepatotoxicity in rats [24]. Albeit no data has been found on the hepatoprotective activity of quercitrin, there is a report on the ability of isoquercitrin, which is an isomer of quercetin, to prevent APAP-induced liver damage [25]. Recent UHPLC analysis of MEDL revelaed the presence of several other bioactive compounds  that exert hepatoprotective activity in addition to rutin such as catechin [26] and gallic acid [27]. Catechin and gallic acid were also reported to possess antioxidant and anti-inflammatory activities [28, 29] and, thus, are believed to synergistically act with rutin to exert the hepatoprotective effect. In addition, several volatile compounds with reported anti-inflammatory and antioxidant activities have also been identified using the GCMS equipment in present study. These compounds, namely phenol, 2,6-bis(1,1-dimethylethyl), 2-hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-, methyl palmitate, methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionate, gibberellin A3, N-methyl-1-adamantaneacetamide and shikimic acid, are thought to synergistically contribute to the attenuation of APAP-induced liver intoxication [30‐39].

The involvement of endogenous antioxidant defence mechanism in the protection of liver against oxidative stress has been greatly acknowledged [40]. As described earlier, parenchymal cells are the main site where oxidative stress took place, which if not attenuated can lead to liver injury. As part of the endogenous defence mechanisms, the liver develops its own complicated antioxidant system consisting of a number of enzymatic and non-enzymatic antioxidants that serves to protect the liver from ROS/RNS-induced damage and, at the same time, provide maintenance to the liver’s redox homeostasis. Nevertheless, the presence of too much ROS/RNS can disturb the liver’s homeostasis, which can trigger oxidative stress that can lead to liver injury [41]. The involvement of some of the members of endogenous antioxidant system, namely SOD, CAT and GSH, in the modulation of hepatoprotective activity of MEDL against APAP were analysed in the present study. From the results obtained, the function of SOD, CAT and GSH in protecting the liver against the action of ROS/RNS was reduced following intoxication by APAP. The observed reduction in function of some of the endogenous antioxidant members as seen with SOD, CAT and GSH suggested the development of liver injury. This finding was further supported by the increase in level of serum liver enzymes, particularly of ALT and AST, as well as the microscopic observation that shows destruction in the liver architecture in the group intoxicated with APAP. Concurrent with the above observations, the present study also demonstrates considerable increase in the rat’s liver weight as well as liver/body weight ratio following the liver intoxication with APAP, which might also supported the condition of liver damage. In comparison, pre-treatment with MEDL caused remarkable attenuation of toxic effect triggered by APAP administration indicated by the increase in SOD and GSH level, and CAT activity in liver homogenates. In addition to the increase in the level or activity of several enzymatic and non-enzymatic components of endogenous antioxidant system, the recovery of APAP intoxicated liver can be indicated by the reduction in the serum ALT and AST level, the rat’s liver weight as well as liver/body weight ratio and progressive recovery of the liver tissue at microscopic level towards normal architecture in a dose-dependent manner. Thus, it is plausible to suggest that the ability of MEDL to exhibit hepatoprotective activity against APAP intoxication could be attributed, in part, to its capability to modulate the action of endogenous antioxidant system, in particular, the activation of SOD, CAT and GSH [40, 41].

The anti-inflammatory potential of MEDL in vitro was tested using the LOX and XO assays. LOXs are enzymes that play role in the metabolism of leukotrienes (LTs) and are susceptible to the action of antioxidants with most of their action could be associated with the inhibition of lipid hydroperoxide formation via the scavenging of lipidoxy or lipid peroxy radicals formed in the course of enzyme peroxidation [42]. On the other hand, XO function as a main biological source of oxygen-derived free radicals that contribute to the oxidative damage of living tissues such as in the medical condition known as gout, which is distinguished by hyperuricemia that initiates uric acid deposition in the joints resulting in painful inflammation [43]. Despite the fact that the chloroform and aqueous extracts of D. linearis have been reported to demonstrate anti-inflammatory activities when assessed using the various animal models [8, 9], MEDL showed low inhibitory effect against LOX activity (<20% inhibition) with no activity detected against XO. These discrepancies in reports could plausibly suggest that MEDL exerts the anti-inflammatory activity via mechanisms that did not involve inhibition of LOX and XO. The extracts of D. linearis were earlier shown to inhibit inflammation when assessed using the carrageenan-induced paw edema assay and cotton pellet-induced granuloma assay. The former assay, in particular, signifies a conventional model of edema formation and hyperalgesia, and has been comprehensively used in the development of non-steroidal anti-inflammatory drugs as well as selective COX1–2 inhibitors. COX-2 is quickly induced in the spinal cord and other regions of the CNS following carrageenan injection in the paw [44]. According to these reports, the COX-2-mediated rise in prostaglandin (PG) E2 synthesis at the central level contributes to the severity of the inflammatory and pain responses in this model. Therefore, it can be suggested that D. linearis extracts exhibited anti-inflammatory activity mainly via modulation of the COX-dependent pathway.

Phytochemicals analysis of MEDL using the GCMS or UHPLC-ESI/HRMS revealed the presence of several volatile (methyl palmitate [33, 34] and shikimic acid [38, 39]) and non-volatile bioactive compounds (gallic acid [28] and catechin [29]) with reported antioxidant and/or anti-inflammatory activities. In addition, several of these compounds such as methyl palmitate [45], quercetin [46], rutin [47] and gallic acid [48] have been reported to exert hepatoprotective activity and are believed to synergistically act together in MEDL to produce the observed liver protective effect against APAP intoxication.

In conclusion, MEDL exerts hepatoprotective activity against APAP-induced intoxication possibly via its ability to partly activate the endogenous antioxidant system such as SOD, CAT and GSH. The hepatoprotective potential of MEDL could also be attributed to its potential antioxidant and anti-inflammatory activities and the presence of various non-volatile bioactive compounds that might act synergistically to enhance the hepatoprotective effect.