The liver is a dynamic organ important for a multitude of functions, namely the assimilation and conversion of nutrient materials absorbed from the gastrointestinal tract to a more useful form of energy, and the neutralization and removal of a number of drugs, xenobiotics and various other substances from the body Nadeem et al., 1997. The liver is a vibrant organ in the human body, playing an indispensable job of detoxification of countless endogenous and exogenous harmful substances Yang et al., 2010. As the liver is consistently exposed to environmental poisonous elements (such as drugs), various hepatic disorders can result; liver damage can lead to hepatic failure and eventual death Mukherjee et al., 2006.

Modern drugs are not suitable for liver diseases due to the high rate of undesirable effects. It is, therefore, necessary to search for alternative remedies Kang, 2002. There are some natural plants which show excellence at preventing, treating and curing liver diseases, while imposing only a few side effects. The hepatoprotective class of therapeutic regimens possesses the competence to protect liver damage and revitalize hepatocytes. International agencies, such as the World Health Organization, have insisted on the evaluation of herbs and herbal products by advanced scientific standards Neraliya and Gaur, 2004. About 600 herbal drugs are being marketed all over the world for hepatoprotective effects Sharma et al., 1991. However, only a limited number of formulations have been scientifically qualified for their prescribed use.

Examples of the compound formulations which have been scientifically validated for their intended use in liver diseases are: Amalkadi Ghrit, an herbal formulation Achliya et al., 2004, Liv-52 Syrup & Livomyn Suspension Sapakal et al., 2008, Liv-52, Livergen, Livokin, Octogen, Stimuliv and Tefroliv liquid formulations Girish et al., 2009, Livergen (a polyherbal liquid formulation) Arsul et al., 2011, Khamira Gaozaban Ambri Jadwar Ood Saleeb Wala Akhtar et al., 2013, Herbal drug Feroz and Khan, 2013, Herbal preparations F-I, F-II & F-III Sivakumar et al., 2014, and Virgoliv Syrup Ingawale et al., 2015.

Paracetamol (ParCM) is an excellent analgesic and antipyretic. The toxicity of ParCM is related to its biotransformation which mostly follows three steps: sulfation, glucuronidation and oxidation Mitchell et al., 1974. Oxidation is the most crucial step as far as hepatic toxicity is concerned Jollow et al., 1973. A small quantity of ParCM undergoes oxidation by cytochrome P450 2E1 to produce an extremely reactive component, N-acetyl-P benzoquinoneimine (NAPQI) Vermeulen et al., 1992. When ParCM is used in therapeutic doses, NAPQI is conjugated by hepatic glutathione (GSH) to produce a water-soluble product called mercapturic acid Lee et al., 1996. The rate of production of GSH counterbalances the toxicity of NAPQI synthesized as a consequence of ParCM oxidation so there is no damage to the hepatocytes so long as this balance is maintained Sabina et al., 2009. As higher doses of ParCM are administered, generation of GSH in the liver cells is diminished after a few hours and hepatic intoxication is evident when the level of GSH in liver is less than 30% of the normal value Makin and Williams, 1997. Uninhibited NAPQI becomes noxious by binding to macromolecules, including cellular proteins Vermeulen et al., 1992. Ascorbic acid (AsAc) is a popular antioxidant that can act as pro-oxidant under certain conditions Duarte and Lunec, 2005.

Polyherbal formulation (PoHF) has been claimed to be effective in liver inflammation and other liver aliments. The composition of PoHF consists of: Zingiber officinale (Ginger), Peganum harmala (Wild Rue), Cassia angustifolia (Senna), and Operculina turpethum (Turpeth). PoHF contains flavonoids & phenolic compounds (present in Zingiber officinale) Ghasemzadeh et al., 2010, alkaloids (present in Peganum harmala) Monsef et al., 2004, and glycosidic resins and beta-sitosterol (present in Operculina turpethum) Iweala et al., 2011.

The aim of this study was to validate the hepatoprotective effects of PoHF, which is currently being used without any scientific authentication. In our study, PoHF was used alone and in combination with ascorbic acid (AsAc) in ParCM-induced hepatic damage in rabbits. PoHF was evaluated on the basis of various biochemical and histopathological parameters.

ParCM-induced hepatotoxicity is an authentic method for evaluation of hepatoprotective effects of various medicinal plants and herbal formulations. Numerous studies have demonstrated that at high doses, ParCM produces hepatic harm or necrosis Vermeulen et al., 1992. Bonkovsky et al. (1994) reported that ParCM, in a single overdose or continual low doses, causes hepatic damage Bonkovsky et al., 1994. The extent of liver damage can be accessed from the level of enzymes like ALT, AST and ALP in the serum, as indicators of the physiological status of the liver. Liver enzymes residing in hepatic cells are released in the bloodstream after cellular insult by hepatotoxic dosage of ParCM Parmar et al., 2010. When hepatocytes are injured, the permeability of the cell membrane and transport mechanisms are altered, resulting in seepage of enzymes from the cells leading to an increase in serum levels of these biomarkers Jain and Singhai, 2012Raja et al., 2007.

In our study, elevated levels of all the aforementioned bio-indicators were observed in hepatic damage. The measure of ALT, AST, ALP and total bilirubin in ParCM intoxicated group B was significantly (P<0.05) higher than in the untreated group A ( Table 1 , Figure 1-4 ), demonstrating the damaging effects of ParCM on hepatic cell membranes from enzymes residing within the liver cells and leaking out of cell boundaries in the bloodstream, as reported by Raja et al. (2007) Raja et al., 2007 and Jain & Singha (2012) Jain and Singhai, 2012. Our results show similar liver damaging effects of ParCM, as which are in accordance with studies by Ahmad et al. (2010), Rehman et al. (2013), Naveed and Ibrar (2014), Rehman et al. (2015), and Mumtaz et al. (2015) Ahmad et al., 2010Mumtaz et al., 2015Naveed and Ibrar, 2014Rehman et al., 2015Rehman et al., 2013. The hepatotoxic effects of ParCM were further verified by histopathological analyses. Our findings revealed a severe congestion of the central vein and sinusoids, infiltration of inflammatory cells, and periportal fibrosis in liver tissues samples of group B ( Figure 5-8 ). Similar histopathological observations with high dose ParCM have also been reported Naveed and Ibrar, 2014Rehman et al., 2015Rehman et al., 2013.

The PoHF used in the study for evaluation of hepatoprotective effects caused a significant decline in serum levels of liver enzymes and total bilirubin, compared to levels for group B, indicating that the herbal product positively prevented the deteriorating effects of ParCM on the liver ( Table 1 , Figure 1-4 ).

Histopathological outcomes also reinforced the results of the biochemical analyses; central vein & sinusoids were mildly congested, infiltration of inflammatory cells was also of mild nature and no phenomenon of fibrosis was observed in group C ( Figure 5-8 ). The composition of PoHF includes different parts of natural plants and the beneficial effects achieved in our study may be attributed to the various constituents contained in the herbs of herbal formulation.

In group D (AsAc treatment), the level of liver biomarkers was significantly (P < 0.05) greater than that for group B and for group C, indicating severe disturbance in the transport mechanisms and permeability of the liver cell membranes ( Table 1 , Figure 1-4 ). The biochemical analysis data was fully supported by the histopathological results, as witnessed by the severe congestion of central vein & sinusoids, severe infiltration of inflammatory cells and periportal fibrosis in group D ( Figure 5-8 ). Indeed, Richardson and Ponka (1997) found that in spite of its role as a prototypical antioxidant, AsAc also exhibited pro-oxidant characteristics in different situations Richardson and Ponka, 1997. Halliwell and Gutteridge (1986) reported that the pro-oxidant action of AsAc depends on the presence of catalytic forms of metals Halliwell and Gutteridge, 1986. It was explained by Halliwell (1996) Halliwell, 1996 and Buettner and Jurkiewicz (1996) Buettner and Jurkiewicz, 1996 that if catalytic metal ions are available in excess as is the case in pathological conditions, ascorbic acid reduces the transition metal ions. This results in excessive production of ferrous ions which by interacting with hydrogen peroxide in Fenton reaction creates more hydroxyl radicals, leading to damaging effects.

Bauer et al. (2000) found that in ParCM-poisoned liver, heme oxygenase, an enzyme responsible for breakdown of heme from hemoglobin and ultimately liberation of more free iron, is upregulated Bauer et al., 2000. In light of this finding, it may be postulated that in our study due to intoxication with high dose of ParCM in group D, the activity of heme oxygenase might have accelerated resulting in frequent availability of free transition iron and presence of AsAc, causing reduction of transition metal ions, boost of the Fenton reaction, and detrimental effects on the liver cells. Wang et al. (2007), Park and Lee (2008), Kamel et al. (2010), and Marim et al. (2015) have all reported the damaging effects of AsAc in their studies Kamel et al., 2010Marim et al., 2015Park and Lee, 2008Wang et al., 2007.

The values of ALT, AST, ALP and total bilirubin in the blood serum of the animals of group E were significantly greater than group C ( Table 1 , Figure 1-4 ).

Microscopy of liver sections of group E showed moderate congestion of central vein & sinusoids, infiltration of inflammatory cells and severe fibrosis ( Figure 5-8 ), thereby corroborating the results obtained in biochemical analyses of blood serum. Although PoHF alone showed hepatoprotective effects in group C, here in group E (where PoHF & AsAc were co-administered) liver protection was not achieved. AsAc exhibited liver-damaging effects in group D; similar effects seen in group E may be ascribed to AsAc in ParCM poisoned animals.

Overall, administration of PoHF alone exhibited a remarkable hepatic cell protection in ParCM- intoxicated animals. Oral administration of AsAc, in a ParCM poisoned rabbit animal model, surprisingly resulted in more malicious effects. Co-administration of PoHF and AsAc (ideally to achieve synergistic effects to counter ParCM intoxication) was not achieved. This may be due to the possibility that AsAc transitions from an antioxidant to pro-oxidant in ParCM-intoxicated liver, thereby turning the AsAc from a beneficial to a noxious element.

PoHF is a successful hepatoprotective regimen in animals, even those administered with high dose ParCM. In our study, AsAc failed to generate hepatic cell protective effects and concomitant administration of PoHF with AsAc showed no benefit for ParCM-poisoned rabbits. Further studies of other hepatotoxicity inducers (besides ParCM) are warranted. Moreover, dose studies of AsAc and ParCM using a larger sample size and in other animal models are warranted.

As-Ac: Ascorbic acid; Par-CM: Paracetamol; Po-HF: Polyherbal formulation; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; CMC: Carboxy Methyl Cellulose; H & E: Hematoxylin & Eosin

Muhammad Fiaz designed the study, collected the literature, executed the experimental work and wrote the manuscript. Waseem Mehmood biochemically analyzed the blood serum. Ghulam Mustafa and Abdul Rauf performed the processing of the liver tissues, examined the slides of liver samples under microscope and concluded the findings of histopathology. Komal Najam reviewed the final manuscript. Naghma Fiaz statistically analyzed the data. Lubna Shakir and Alamgeer supervised the study.