Carbon tetrachloride (CCL4), isopropanol, chloroform, ethanol, hematoxylin, phosphomolybdic acid and acetic acid were purchased from Sigma. All the solvents used were of analytical grade.

P. plebeium R.Br. (local name: Gorakh pan; plant official name reaffirmed from www.plantlist.org site on 18 Oct 2016) is widely distributed in Pakistan, India, Bangladesh, Sri Lanka and Madagascar [9] ( Figure 1 ). It was collected from a local herbal practitioner’s shop in Lahore and was identified from a herbarium at Punjab University; a voucher number was issued (Specimen #LAH672015). The whole P. plebeium plant was washed and air dried until it was moisture free, and then grinded in a mechanical grinder. Next, 850 g of the powdered plant was macerated with 5.5 L of ethanol: water (70:30) mixture, followed by filtration through vacuum suction pump. The solvent was separated using a rotary evaporator at 125 rpm and 37° until a sticky oily mass was separated from the mixture. The waxy material (PGOE) was filter separated and the filtrate (PGAE) was evaporated to semisolid consistency.

The organic layer (PGOE) was removed and dried, an emulsion was made using tragacanth and distilled water, and the emulsion was stored at 4oC. The aqueous layer and organic emulsion were further diluted with distilled water to form a working dilution of final concentration of 25 mg/mL.

The animal experimental protocol was approved by the Animal Research Ethics Committee of the Pharmacy Department of the University of Lahore (ref# IAEC2014003). Wistar rats (n=24) of both sexes weighing from 150-250 g were obtained from the Animal House of the University of Lahore and were kept under standard laboratory conditions (i.e. 25° and humidity of 45-55%). The animals were provided with food & water ad-libitum.

Liver fibrosis was induced by CCl4 according to an established protocol [21]. Briefly, CCl4 was injected intraperitoneally (i.p.) at a dose of 0.25 µL per gram of rat body weight twice a week during the 1st week, 0.50 µL/g twice a week for the 2nd week, and 1 µL/g body weight twice a week from weeks 3 to 6. Treatment with P. plebeium extract was started from week 3 with concomitant i.p. administration of CCl4. Wistar rats (of both sexes) were randomly divided into the following groups. Notably, Group I (n=4) was the control group (i.e. received

i.p. administration of corn oil as vehicle control), Group II (n=4) was the CCL4 group (i.e. administered i.p. with CCl4 as mentioned above), Group III (n=4) consisted of rats bearing CCl4−induced liver toxicity that were treated with aqueous extract of P. plebeium (250 mg/kg), Group IV (n=4) consisted of rats bearing CCl4−induced liver toxicity that were treated with organic extract of P. plebeium (250 mg/kg), Group V (n=4) consisted of the control group treated with corn oil and the aqueous extract of P. plebeium (250 mg/kg), and Group VI (n=4) was the control group treated with corn oil and the organic extract of P. plebeium (250mg/kg). Corn oil and CCl4 were given i.p. and the extracts were given orally using oral gavage. Since most studies used 100-500 mg extract per kg of body weight [22]–[24], the dose of 250 mg/kg was, therefore, selected for this study. The body weights (in grams) of the rats were recorded biweekly for six weeks of the experiment.

At the end of the experiment, the rats were euthanized by chloroform anesthesia. Blood samples were collected by cardiac puncture and centrifuged immediately for serum separation at 4000 rpm for 5 min, and for other blood analyses. Liver damage was assessed by estimation of serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma- glutamyl transpeptidase (γGT) using commercially available test kits and the kinetic IFCC method (NDI Europa GmbH, Germany). The results were expressed as international units/liter (IU/L).

Liver sections were collected and fixed with 4% formalin at room temperature. Four µm thick sections were cut for Masson’s Trichrome staining and Hematoxylin & Eosin (H&E) staining according to previous protocols [25][26]. The liver sections were evaluated histologically with a camera attached to a light microscope.

Large lobules of liver samples were collected and stored at -80°C for PCR analysis. Total RNA was extracted from a small piece of frozen rat liver using WizolTM reagent, followed by cDNA synthesis using M-MLV cDNA synthesis kit (Enzynomics, Inc., Korea). Real-time PCR was conducted using TOPrealTM qPCR 2X PreMIX (SYBR Green with low ROX) by Enzynomics using primer sets and the methods previously described [21]. Real-time PCR data was analyzed by measuring relative expression and standard deviation as previously described [27].

The results are shown as mean with standard deviation. GraphPad Prism (version 5) was used to analyze the data. One-way analysis of variance with post hoc Bonferroni correction test was applied to compare the mean of groups; p<0.05 was considered significant.

Table 1 shows the weight variation (in grams) of animals from each group measured bi-weekly before dosing of animals. There was a non-significant increase in weight up to 3.8% from day one in the control group. In contrast, there was a non-significant decrease in weight to -18.1% compared to day 1 for the CCL4 treated group. The weight variation for all other groups also did not show any significant change.

Table 2 shows the results of the liver assays. Elevated levels of serum aminotransferases (ALT and AST) and γGT are the major indicators of liver injury. Liver markers were significantly higher (P<0.05) in the CCl4 treated group than the control group. The average values (mean±stdev; in IU/L) for the control group were 59.0±15.0, 56.2±1.0 and 9.3±7.2 for AST, ALT and γGT, respectively. On the contrary, the mean values (in IU/L) for the CCl4 treated group increased to 322.3±53.3, 191.82±21.5 and 16.79±1.58 for AST, ALT and γGT, respectively. The average values (in IUL/L) of AST, ALT and γGT in Group III were 104.2±7.3, 106.50±11.7 and 11.69±1.9, respectively. In Group IV, the average values (in IU/L) for AST, ALT and γGT were 164.04±19.8, 111.66±10.4 and 7.07±3.57, respectively. The levels of AST, ALT and γGT in group V and VI were not much different from the control (Group 1).

Table 3 shows the complete blood count (mean values) from each group. Count of white blood cells, red blood cells and platelets were not changed among the groups. Similarly, hemoglobin and hematocrit values remained the same among the groups.

Gene expression of αSMA, TGF-β and collagen was assessed for all groups ( Figure 2 ). CCl4 treatment in Group II animals caused a significant increase in mRNA level of αSMA (p<0.05), TGF-β (p<0.0001) and collagen (p<0.0001), compared to Group I. Treatment with P. plebeium aqueous extract (PPAE; Group III), after three weeks of CCl4, significantly prevented mRNA expression levels of αSMA (p<0.05), TGF-β (p<0.001) and collagen (p<0.001). Likewise, in animals treated with P. plebeium organic extract (PPOE; Group IV), the mRNA levels of TGF-β (p<0.0001) and collagen (p<0.0001) remained lower compared with the levels in Group II (CCl4 treated group). However, no repression was observed in αSMA expression following treatment with PPOE Figure 2 .

Gross and microscopic histopathological features of the liver in the various treatment groups are shown in Figure 3 . The top panel shows the liver images after excision from the animals.

Control (Group I) showed healthy liver with soft, smooth and shiny surface texture. However, liver of animals in the CCl_4-treated group (Group II) showed a nodular appearance with rough, hard patchy surface and no shine. The faint red color indicated decreased blood flow through the liver. PPAE (Group III) and PPOE (Group IV) treated rats were comparatively healthy; the livers from these groups were similar in color but with a smoother surface than those in the CCl_4- treated group (Group II). The livers of rats in the group treated with corn oil (control instead of CCL4) and P. plebeium aqueous extract (Group V) and Group IV rats were comparatively healthier, with bright, shiny and smooth surfaces. The middle panel shows H&E staining of the livers, where blue staining represents nuclei, pink represents cytoplasm, and deep pink or red represents muscle fibers. The central portal vein is shown by a one-sided black arrow and cellular necrosis is indicated by double-headed arrows. The liver section of the control group and the extract only group showed normal central vein and radiating hepatocytes with no histopathological changes. Liver sections from the CCl_4-treated rat show vacuolization of hepatocytes, inflammatory cell infiltration and hepatocyte necrosis. Liver sections treated with aqueous extract + CCl4 showed inflammatory cell infiltration with normal hepatocytes. Liver sections treated with organic extract + CCl4 showed inflammatory cell infiltration and fat deposition with dispersed hepatocytes.

The lower panel shows the Masson’s trichrome staining of the liver sections. Blue stain indicates fibrous tissue. The degree of fibrosis tissue (collagen) accumulation is directly related to intensity of the blue stain. Red indicates hepatocytes and dark red to black represents nuclei. The central portal vein is shown by a one-sided black arrow and collagen accumulation or fibrosis is indicated by double-headed arrows. The liver sections of control and extract only-treated rats showed normal liver morphology (i.e. central vein and radiating hepatocytes with no collagen accumulation). Liver sections from the CCl_4-treated rats showed extensive fibrosis and fibrous bands, indicating very high collagen accumulation leading to nodule formation. On the contrary, the liver tissue of animals treated with aqueous extract showed hepatic plates which were more than 1-cell thick, indicating regeneration of hepatocytes. The liver sections of animals treated with organic extract showed portal inflammation with no bile duct injury.