Background
The liver has a pivotal role in regulating many important functions, such as metabolism, secretion, storage; and plays a role in regulating various physiological processes [
1]. Furthermore, it is involved in detoxification of a variety of drugs and xenobiotics and thereforeat increased susceptible to the toxicity from these agents. It is frequently abused by poor drug habits and alcohol and widely exposed to environmental toxins, prescribed and over-the-counter drugs, which cause various liver diseases [
2]. Liver diseases have become aworldwide problem as a result of extremely poor prognosis and high mortality due to the lack of effective prevention or drug available that stimulates liver function, offer protection against liver damage, or help to regenerate hepatic cells [
3,
4]. Therefore, attempts are perpetually being made to discover new treatment for liver disease, and the discovering process have been paid great attention to the investigation of the efficacy of plant-based drugs used in traditional medicine as they are cheap and have little side effects. Besides, WHO reported that 80% of the world population relies mainly on plant-based drugs [
5].
In lieu of the aforementioned problem, patients suffering from liver diseases have turned to complementary and alternative medicine (CAM), which includes among others the use of herbal medicines as their sources of hepatoprotective agents. According to White et al. [
6], over 50% of patients that required health care used CAM either in conjunction with, or separate from, conventional health care. Despite its uses for treatment of various types of diseases, the use of CAM is also popular in patients with liver disease but is not well documented [
6]. According to Bhawna and Kumar [
7], hepatoprotective plants contain a variety of phytochemicals like phenols, coumarins, monoterpenes, glycosides, alkaloids, and xanthenes. In addition, hepatoprotective properties are related with phytoextracts or phytocompounds rich in natural antioxidants as reported from previous studies [
8‐
10]. One of the plants that have been studied extensively in our laboratory for its medicinal potentials is
Dicranopteris linearis (L. (Gleicheniaceae), locally known as “resam”, is common in secondary forests and grows well in poor clay soil [
11].
D. linearis has been used in Malay traditional medicine to reduce body temperature and to control fever [
12]. In addition, there are few reports of its traditional uses in other parts of the world, with only 2 reports describing its use to treat external wounds, ulcers, and boils by the people of Papua New Guinea, to eliminate intestinal worms by the people of Indochina, and to treat asthma and female sterility by the tribes living on an Indian mountain [
13]. Scientifically, the leaf extracts of
D. linearis have been reported to possess antinociceptive, anti-inflammatory and antipyretic [
12], gastroprotective [
14], antistaphylococcal [
15], antioxidant [
16], and anticancer activities [
17] properties.
The present study was performed based on three reasons, namely: i) the previous reports on the anti-inflammatory and antioxidant activities of
D. linearis leaves; ii) the reports linking the anti-inflammatory and antioxidant activities to the hepatoprotective mechanism [
3,
18,
19], and; iii) no scientific report to date to prove on the hepatoprotective potential of
D. linearis leaves. It is postulated that
D. linearis leaves will exert hepatoprotective activity that could be linked to its antioxidant activity. Therefore, the aim of the present study was to determine the hepatoprotective activity of methanol extract of
D. linearis (MEDL) using the carbon tetrachloride (CCl
4)-induced acute liver damage in rats model. In addition, the antioxidant activity, phytochemical content and HPLC profile of MEMM were also verified to support the hepatoprotective potential of MEDL. The hepatoprotective potential of the MEDL was compared with silymarin, a known, commercially available hepatoprotective agent.
Discussion
CCl
4 has been widely used in animal models to investigate chemical-induced liver injury. The CCl
4-induced experimental damage involves the formation of free radicals and the occurrence of lipid peroxidation in cellular and organelle membranes [
27]. CCl
4 will be metabolized in the liver to form highly reactive trichloromethyl free radicals (·CCl
3), which are responsible in triggering the toxicity processes in the liver. This free radicals are further converted to trichloromethyl peroxyl radical (CCl
3OO·) that act as the initiator of lipid peroxidation [
28‐
30]. The presence of excessive amount of free radicals accelerate peroxidative degradation of cellular membrane, which resulted in the breakdown of cell integrity and the leakages of ALT and AST, and, subsequently, lead to the elevation of serum ALT and AST levels. Moreover, several remarkable pathological characteristics associated with CCl
4-induced hepatotoxicity, namely fatty liver, cirrhosis and necrosis, could be seen and have been thought to result from the formation ad action of reactive intermediates (i.e trichlorometyl free radicals (CCl
3+) metabolized by the mixed function cytochrome P450 in the endoplasmic reticulum.
One of the ways to measure the extent of hepatic damage is through the determination of the level of cytoplasmic enzymes (i.e. ALT, AST and ALP) that leak into the blood circulation, which is associated with massive centrilobular necrosis, ballooning degeneration and cellular infiltration of the liver [
31]. Measurement of ALT is more liver-specific to determine hepatocellular damage [
32]. However, measurement of AST is still commonly used to assess liver function because it is a sensitive indicator of mitochondrial damage, particularly in centrilobular regions of the liver [
33]. According to Ahmed and Khater [
34], transaminase levels return to normal due to the healing of hepatic parenchyma and the regeneration of hepatocytes. In the present study, CCl
4–induced increase in the liver/body weight ratio indicating that the liver damage model had been successfully developed. This is further supported by the increase in the level of serum ALT, AST and ALP and histological scoring and microscopic findings. Interestingly, pretreatment with MEDL effectively protected the rodents against CCl
4-induced hepatic intoxication, which are evidenced by significant reduction in the liver/body weight ratio, levels of serum liver enzymes. Furthermore, it is well established that intoxication with CCl
4 leads to extensive necrosis in the liver centrilobular regions around the central veins [
9] and fatty infiltration [
35]. Interestingly, the microscopic examination also revealed the ability of pre-treated MEDL to reduce inflammation, steatosis and necrosis as indicated by decreased in histological scoring.
As described earlier, the basis of CCl
4-induced liver toxicity lies in its biotransformation to free radicals via the cytochrome P450 system [
36]. Since free radicals play important role in CCl
4-induced hepatotoxicity, it seems rational to suggest that compounds with capability to neutralize such free radicals might also possess a hepatoprotective activity. In fact, various natural products (e.g extract of
Artemisia campestris and pure compound like ginsenosides) with antioxidant potential have been reported to protect against CCl
4-induced hepatotoxicity [
36]. In the present study, MEDL was proven to exhibit radicals scavenging activities using various
in vitro antioxidant models suggesting that the extract perhaps protected the hepatocytes by ameliorating oxidative stress and inhibiting lipid peroxidation. In addition, it is also plausible to add anti-inflammatory activity as another possible mechanism through which MEDL exerts the hepatoprotective activity. This suggestion is based on previous reports that
D. linearis possess anti-inflammatory activity, which might explained the ability of MEDL to reduce inflammation associated with CCl
4 as observed during microscopic examination.
MEDL has also been shown to contain high TPC value, which have been associated with high antioxidant activity [
37,
38] as well as anti-inflammatory activity [
39,
40]. These claims are in line with previous findings by Wu et al. [
27] who reported that TPC-rich extract of
Laggera pterodonta exerts both hepatoprotective and antioxidant activities when assessed using the
in vitro primary cultured neonatal rat hepatocytes. Adetutu and Owoade [
41] also reported that TPC-rich extract of
Hisbiscus sabdariffa exhibits both hepatoprotective and antioxidant activities when assessed using
in vivo CCl
4-induced rat hepatotoxic model. The positive correlations between the high antioxidant activity and high TPC have been reported elsewhere [
27,
41] and are parallel with our findings. Thus, the antioxidant activity attributed by the presence of high concentration of TPC in MEDL could be suggested to play significant role in the observed hepatoprotective activity. Interestingly, one class of compounds that contributed to the high TPC reading is flavonoids, which have been known to play remarkable role in anti-inflammatory activity. Flavonoids have been detected in MEDL whereas MEDL has been shown to attenuate inflammation in CCl
4-induced liver injury. This seems to suggest the role of anti-inflammatory activity of
D. linearis as reported previously in enhancing the observed hepatoprotective activity.
Phytochemical analysis of MEDL demonstrated the presence of several phytocontituents (e.g. flavonoids, tannins, saponins and triterpenes) essentially associated with antioxidant and/or hepatoprotective effects. Earlier studies have claimed that phenolic compounds possess diverse pharmacological effects (i.e. antioxidant, anti-inflammatory, and hepatoprotective) [
27,
30,
42]. MEDL exerted prominent hepatoprotective effects against CCl
4-induced liver damage possibly via the antioxidant- and anti-inflammatory-modulated mechanisms, which is in compliance with the description on the pharmacological properties of phenolic compounds in the aforementioned reports. HPLC analysis of MEDL demonstrated the presence of four major peaks with their λ
max value that fall in the region of 192-348.2, 192-349.4, 192-349.4, and 264.9-347 nm, respectively. Based on report made by Tsimogiannis et al. (2007), each of the peaks represents flavonoid-based compounds. According to Tsimogiannis et al. (2007), flavonoids are divided into several subgroups, namely flavonols, flavones, dihydroflavonols, flavanonols and flavanones based on the UV-Vis spectra within which they fall. The UV-Vis spectra of flavonoids falls within two absorbance bands, labeled as Band A and Band B wherein Band A represents flavones or flavonols and lies in the range of 310–350 nm or 350–385 nm, respectively. Meanwhile Band B falls in the range of 250 and 290 nm and is much the same in all the aforementioned flavonoid subgroups. As for the flavanones and dihydroflavonols subgroup, Band A falls between 300–330 nm while Band B was detected in the range of 277–295 nm. Interestingly, the presence of low amount of flavonoids in MEDL is concurrent with the HPLC profile of MEDL that shows only four major peaks. Based on previous report, at least flavonoids such as rutin and quercitrin have been isolated from
D. linearis and, interestingly, rutin and quercitrin have also been reported to exert antioxidant activity [
43,
44] and hepatoprotective activity [
44,
45]. Hence, flavonoids, like rutin and quercitrin, in addition to other polyphenolic constituents may be the prominent bioactive compounds responsible for antioxidant and hepatoprotective activities of MEDL.
It is also noteworthy to highlight on the inconsistency with regards to the presence of low amount of flavonoids in MEDL as detected in the phytochemical screening and the high TPC value of MEDL, and to provide possible explanation for this inconsistency. It is plausible to suggest that the high TPC value is attributed to the presence of other types of flavonoids, particularly, the flavonol 3-
O- glycosides [
46]. This suggestion, which is supported by the UV spectra characteristics of major peaks as detected in the HPLC, is based on the fact that flavonoid glycosides are soluble in methanol:water and acetone:water solvent systems, but not very soluble in water alone [
47‐
49]. With the high presence of saponin in MEDL, the existence of flavonoids glycosides could be attributed to the tensioactive effect of saponins, which are present in relatively high quantities when compared to the flavonoids [
50].
Competing interest
The authors declare that there is no competing interest.
Authors’ contribution
FHK, FY, SSM and MFFK carried out the animal studies, phytochemical screening, HPLC analysis, biochemical analysis and draft the manuscript. NM involved in the macroscopic and microscopic analysis and helped to draft the manuscript. LKT and MZS involved in the antioxidant study and statistical analysis. MNS participated in the design of the study and involved in the statistical analysis. ZAZ conceived of the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.