Amelioration of isoniazid and rifampicin-induced liver toxicity by Amaranthus graecizans subsp . silvestris in rat BJP

Amaranthus graecizans subsp. silvestris, a folk medicine for the treatment of inflammation, was used to evaluate its hepatoprotective potential against rifampicin and isoniazid-induced liver damage. Wistar albino rats were divided into four groups: Group I served as control (distilled water treated), Group II served as hepatotoxic group (isoniazid 50 mg/kg and rifampicin 100 mg/kg, treated), Group III served as positive control (silymarin 100 mg/kg, treated) while Group IV served as A. graecizans subsp. silvestris extract (400 mg/kg) treated group. The results suggest that the liver markers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin) were significantly increased in the animals of Group II. The methanolic extract showed a significant decrease in the raised liver enzymes of Group IV and encountered the liver damage caused by isoniazid and rifampicin. Histopathological examination of liver also revealed the improved architecture in the extract-treated group. Thus, the methanolic extract has potential liver protective action due to its phytochemicals. Article Info Received: 18 June 2017 Accepted: 12 September 2017 Available Online: 30 September 2017 DOI: 10.3329/bjp.v12i3.32985 Cite this article: Ishtiaq S, Afridi MSK. Amelioration of isoniazid and rifampicin-induced liver toxicity by Amaranthus graecizans subsp. silvestris in rat. Bangladesh J Pharmacol. 2017; 12: 354-58. Amelioration of isoniazid and rifampicin-induced liver toxicity by Amaranthus graecizans subsp. silvestris in rat Saiqa Ishtiaq, Muhammad Shaharyar Khan Afridi and Nawal Masood Punjab University College of Pharmacy, University of the Punjab, Allama Iqbal Campus, Lahore 54000, Pakistan. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. and Nakalembe, 2016). In Pakistan, it is called ‘Phulari’ by the natives and it is predominantly northerly distributed. The leaves of the plant are used to treat inflammation, piles and gonorrhea (Hussain et al., 2010; Arshad et al., 2011; Nisar et al., 2011). The plant possesses remarkable anti-oxidant properties that make it a suitable candidate to counter oxidative stress (Ishtiaq et al., 2014). The methanolic extract of the herb is also reported to have significant analgesic and antiinflammatory action which ensures the promising antioxidant abilities. The plant has also shown anti-cholinesterase and anti-protease activity (Ishtiaq et al., 2017). Oxidative stress is one of the main reason behind multiple degenerative diseases including liver damage. Parenchymal cells are primary cells subjected to oxidative stress-induced injury in the liver (Li et al., 2015). As, it plays a core role in liver aberrations and their progression, the utilization of herbal drugs with anti-oxidant potentials have been suggested as healing agents, as well as adjuncts, to neutralize liver damage (Casas-Grajales and Muriel, 2015). With this view, the present project was designed to evaluate hepatoprotective potential of Amaranthus graecizans subsp. silvestris, against isoniazid and rifampicin-induced liver toxicity in Wistar rats. Materials and Methods


Introduction
Directly observed treatment short course (TB-DOTS) with isoniazid, rifampicin and pyrazinamide are highly effective treatment for the management of tuberculosis. Both isoniazid and rifampicin are well documented for their hepatotoxicity. Isoniazid produces liver injury by producing reactive toxic metabolites (isonicotinic acid, and hydrazine and acetylhydrazine free radical) that interacts with the liver proteins and results into anomaly of the liver anatomy. On the other hand, rifam -picin when given in combination with isoniazid causes cytochrome P450 enzyme-induction and results in the increased metabolism of isoniazid (Gangadharam, 1986;Wang et al., 2016;Pandit et al., 2012).
The animal model of hepatotoxicity is usually produced by either paracetamol or carbon tetrachloride. Then, different plants are examined to see their hepatoprotec-tive effects (Qadir and Ahmad, 2017). However, antitubercular drugs-induced hepatotoxicity and the effect of the plant to overcome it is observed only in a few plants such as Monotheca buxifolia (Ullah et al., 2016), Moringa oleifera (Pari and Kumar, 2004).
Amaranthus graecizans subsp. silvestris (Vill.) Brenan, also known as "Mediterranean amaranth", belongs to the family Amaranthaceae, occurs in the warmer parts of Europe to the cooler regions of Western Asia and North Western India, also found in tropical Africa (Flora of Pakistan, 1972). It is considered as a wild food plant, semi-cultivated in Africa and consumed as a vegetable (Tabuti, 2007). It is also used as a folk medicine. The young leaves and shoot of the plant either boiled or steamed are employed to treat sore throat, as an immune system booster and to relieve joint pain by the natives in Uganda. The adverse effect associated with its use is the throat irritation (Nabatanzi and Nakalembe, 2016). In Pakistan, it is called 'Phulari' by the natives and it is predominantly northerly distributed. The leaves of the plant are used to treat inflammation, piles and gonorrhea (Hussain et al., 2010;Arshad et al., 2011;Nisar et al., 2011). The plant possesses remarkable anti-oxidant properties that make it a suitable candidate to counter oxidative stress . The methanolic extract of the herb is also reported to have significant analgesic and antiinflammatory action which ensures the promising antioxidant abilities. The plant has also shown anti-cholinesterase and anti-protease activity (Ishtiaq et al., 2017).
Oxidative stress is one of the main reason behind multiple degenerative diseases including liver damage. Parenchymal cells are primary cells subjected to oxidative stress-induced injury in the liver (Li et al., 2015). As, it plays a core role in liver aberrations and their progression, the utilization of herbal drugs with anti-oxidant potentials have been suggested as healing agents, as well as adjuncts, to neutralize liver damage (Casas-Grajales and Muriel, 2015). With this view, the present project was designed to evaluate hepatoprotective potential of Amaranthus graecizans subsp. silvestris, against isoniazid and rifampicin-induced liver toxicity in Wistar rats.

Chemicals
Methanol, isoniazid and rifampicin were procured from the Pacific Pharmaceuticals Ltd. Lahore. Diagnostic kits of transaminases, alkaline phosphatase and bilirubin were obtained from the Global (UK). Silymarin was acquired from the Wilson's Healthcare, Pakistan. All the chemicals were of analytical grade and reagents were freshly prepared in the laboratory.

Plant material
Fresh plant parts were collected from the Chakwal and authenticated by Dr. Muhammad Ajaib, Department of Botany, Mirpur University of Sciences and Technology, Pakistan against voucher specimen of GC. Herb. 2329. The collected material was cleaned, dried and then powdered and preserved in amber colored bottles.

Animal
Wistar rats of either sex were purchased from the University of Veterinary and Animal Sciences, Lahore Pakistan. The animals were kept at room temperature 25ºC under 12 hours of dark and light cycles. The animals were fed with the standard pelleted diet with water ad libitum.

Preparation of methanolic extract
The powder (500 g) was macerated in 700 mL of 90% methanol for seven days. The macerated material was shaken periodically to facilitate extraction. After seven days, the sample solution was first filtered through Whatman No. 1 filter paper. The extract was evaporated using the rotary evaporator at a temperature lower than 40ºC. The extract was collected and stored in an airtight container which was placed in the refrigerator for further investigations (Miliauskas et al., 2004).

Phytochemical screening
The phytochemical analysis was carried out according to the standard procedures .

Study design
The study was conducted for 21 days. The weight of the rat was between 150-200 g. Twenty animals were randomly divided into four groups (n=5). Group I was given with distilled water as a control. Group II was administered with isoniazid (50 mg/kg) and rifampicin (100 mg/kg). Group III was treated with silymarin (100 mg/kg), isoniazid (50 mg/kg) and rifampicin (100 mg/ kg). Group IV was treated with A. graecizans subsp. silvestris (400 mg/kg), INH (50 mg/kg) and RMP (100 mg/kg).
All the treatment groups were administered orally in distilled water (10 mL/kg) for 21 days. At the end of the treatment, animals were anesthetized by i.p. administration of 5 mL/kg of a solution of 1% chloralose in 25% urethane (w/v). Blood samples of animals were collected by cardiac puncture in sterile heparinized tubes and allowed to clot for 30 min. Serum was separated and used for the assay of serum marker enzymes (Salama et al., 2013).

Histopathological examination
Fresh liver tissues (previously trimmed to 7 μm thick) were placed in plastic cassettes and immersed in neutral buffered formalin for 24 hours. Fixed tissues were processed routinely and then fixed in paraffin, sectioned, deparaffinized and rehydrated. The tissues were then stained with eosin and hematoxylin. The extent of combinational necrosis of isoniazid and rifampicin was evaluated by structural changes in liver sections stained with hematoxylin and eosin (Habbu et al., 2008).

Statistical analysis
The values are expressed as mean ± SEM. SPSS 21 was used for the statistical analysis. Statistical significance of the differences between control and treated groups was calculated using One-way ANOVA with post-hoc Student's t-test. p<0.05 was considered to be significant.

Phytochemical analysis
Phytochemical screening revealed the presence of alkaloids, carbohydrates, saponins, flavonoids, phenols, steroids and glycosides in the extract whereas proteins and triterpenoids were absent.

Hepatoprotective activity
The hepatoprotective activity of the extract showed significant p<0.05 reduction in aminotransferases, alkaline phosphatases and total bilirubin levels. Significant results were also obtained with silymarin but the more pronounced hepatoprotective effect was observed with    (Table I).

Histopathological examination
The histopathological examination also showed less damage to the hepatocytes and liver architecture in methanolic extract and silymarin treated groups when compared with isoniazid (50 mg/kg and rifampicin (100 mg/kg) treated group (Figure 1). The detailed histopathological observations were tabulated (Table  II).

Discussion
Methanolic extract of A. graecizans subsp. silvestris was evaluated for its hepatoprotective potentials against the combination of isoniazid and rifampicin. It was observed that the methanolic extract of A. graecizans subsp. silvestris, significantly (p<0.05) reduced the liver profile parameters (ALT, AST, ALP and total bilirubin) and showed the protection of hepatocytes against isoniazid-and rifampicin-induced liver injury when compared with the hepatotoxic group. The histopathological assessment also revealed the regeneration of hepatocytes against the destruction caused by these anti -tubercular agents.
The hepatoprotective activity of the plant extract could be due to the presence of the alkaloids, saponins, phenolic compounds, tannins, flavonoids and steroids (Agbafor et al., 2014).
The basic phytochemical group screening of the A. graecizans subsp. silvestris also unveiled the occurrence of alkaloids, carbohydrates, saponins, flavonoids, phenol, steroids, and glycosides in the sample whereas proteins and triterpenoids were absent.
This variety of phytochemicals in the extract may produce their hepatoprotective effect either by preventing or scavenging the free radicals generated as it holds appreciable anti-oxidant activity  or through an enhanced protein synthesis as observed in histopathological sections of the liver. The extract may also increase the excretion of isiniazid and rifampicin.
A number of plants, containing these phytochemicals demonstrated hepatoprotective effects in various researches when evaluated. Various plants i.e. Celosia cristata and Bupleurum scorzonerifolium are the rich source of saponins, has shown to exhibit hepatoprotective effect (Wang et al., 2010;Matsuda et al., 1997). Similarly, the plants enrich with flavonoids and phenolic acids i.e. Equisetum arvense and Laggera alata, also possess hepatoprotectant potentials (Wu et al., 2006;Oh et al., 2004). Quercetin is a flavonoid, when it's given to hepatocompromized rats, it results in the lowering of elevated hepatic enzyme levels (Janbaz et al., 2004). Certain plants i.e. Hyperricum erectum, Sarcandra glabra etc. with hepatoprotective glycosides are also reported in the literature (An et al., 2009;Li et al., 2006).

Conclusion
A. graecizans subsp. silvestris showed the presence of alkaloids, carbohydrates, saponins, flavonoids, phenol, steroids, and glycosides. The methanolic extract showed hepatoprotective action in rat model against isoniazid plus rifampicin-induced hepatotoxicity.

Financial Support
Punjab University College of Pharmacy, University of the Punjab

Ethical Issue
The protocols followed to carry out the project was approved by the Animal Ethical Committee of Punjab University College of Pharmacy, prepared by National Institute of Health (AEC/ PUCP/1041/4313).

Conflict of Interest
We declare that we have no conflict of interest.