Evaluation of Hepato-protective and Nephron- Protective Potential of Euphorbia nivulia Buch.-Ham. Against Carbon Tetrachloride-induced Toxicity in Sprague Dawley Rats

1 Department of Pharmacognosy, Faculty of Pharmacy & Pharmaceutical Sciences, University of Karachi, Karachi 75270, Pakistan. 2 Department of Pharmacognosy, Faculty of Pharmacy, Islamia University of Bahawalpur, Bahawalpur, Pakistan. 3 University College of Conventional Medicine, Islamia University of Bahawalpur, Bahawalpur, Pakistan. 4 Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan. 5 Faculty of Pharmacy, Islamia University of Bahawalpur, Bahawalpur, Pakistan. 6 Department of Pharmacognosy, Faculty of Pharmacy, Gomal University, Dera Ismail Khan, Pakistan. 7 Department of Pharmacy, Benazir Bhutto Shaheed University, Lyari, Karachi, Pakistan.


INTRODUCTION
Hepatic and kidney infections are the major health concerns in the world due to medications, toxic chemicals and reactive oxygen species (ROS) related oxidative stress [1]. Management of these life-threatening liver and kidney diseases is a major challenge to modern medicine [2]. Corticosteroids and immunosuppressive agents that are the only available drugs, exert several adverse effects. Several researches have proved that antioxidants can inhibit hepato-nephro toxicity by countering ROS [3][4] and offer protection against chemical induced renal failure and mitochondrial function maintenance [5] and may be useful as liver protective agents. Moreover, these antioxidants can search reactive oxygen species (ROS) that are the most usual cause of hepatic disorders [6], singlet oxygen [1] and superoxide anion [7]. Similarly, CCl 4 may be a reason for increasing the production of free radicals within liver including H 2 O 2 , which requires ferritin and hemoglobin for its conversion into OH ions. Decreased Hb level endorses excessive consumption of ferric ions for conversion of H 2 O 2 into OH ions. Increased WBC count and ESR is a highly frequent finding in inflammation due to oxidative stress [8][9]. Systemically administered CCl 4 in rats is distributed at higher concentrations in the kidney than in the liver [10]. Kidney has high affinity for CCl 4 [11] and contains cytochrome P450 predominantly in the cortex [12][13], thus CCl 4 is extensively metabolized in the kidney generating more reactive species. CCl 4 exposure causes damage to the kidney due to enhanced production of reactive oxygen species. It has also been reported that chronic administration of CCl 4 (0.15 ml/kg, sc, in olive oil) three times a week for seven weeks in rats caused various degrees of tubular and glomerular changes, interstitial mononuclear cell proliferation and fibrosis in the kidney [14]. Silymarin is a natural flavonoid complex having potent anti-oxidative as well as anti-inflammatory effects. It could be used as standard nephroprotective drug for kidney diseases [15]. Many studies have shown the efficacy of silymarin for drug/chemical nephrotoxicity [16] and diabetic nephropathy [17]. Silymarin is found to be effective as a complementary treatment for inflammatory conditions of liver as well [18]. Many synthetic anti-oxidants are being used in drugs but are carcinogenic. Thus, potent antioxidants and antihepato-nephrotoxic drugs of natural origin against liver and renal diseases are needed. This has led to increased dependency on alternative especially plant-based medicines [19]. Various medicinal plants/herbs belonging to Euphorbiaceae family have been reported to possess antioxidant bioactive molecules like triterpenes and flavonoids, both of which are reported to have hepato-protective activity [20]. Euphorbia nivulia-Buch. -Ham.is one of the members of Euphorbiaceae family that is rich in phytoconstituents including flavonoids and polyphenolics but no scientific validation has been done on its hepatic-nephroprotective potential. The current study was aimed to evaluate Euphorbia nivulia-Buch. -Ham's hepato-nephroprotective potentials against CCl 4 induced toxicity in Sprague Dawley rats. The collected parts of plant were cut into pieces, spread on filter paper at room temperature and dried under shade. Dried parts were then converted into fine particles by mean of electric grinder, and No.60 mesh was used to sieve. The powder was kept in closed amber colored glass bottle till used for the extraction.

Preliminary Phytochemical Analysis
Different phyto-constituents like flavonoids, alkaloids, glycosides, tannins, saponins and phenols, etc., were determined and results have been published in our recent article [22].

Antioxidant activity
By diphenyl-1-picrylhydrazyl (DPPH) reagent method [23] anti-oxidant activity of the extract was seen. Different concentrations (25-250 mg/ml) were used for IC 50 calculations. The total inhibition percentage of DPPH radicals was calculated by: Using EZ-Fit Enzyme Kinetics Software (Pirelli Scientific Inc. Amherst, (USA) IC 50 values were calculated.

Total assay antioxidant capacity / power reducing assay
Fe-reducing potency of plant extract was checked by the technique used by Nile and Park with slight modifications [24]. The calibration curve was created using Troop (100-2000 elm) and the effects were conveyed as 3M Troop equivalent (TE)/g fresh mass.

Total Phenolic content
Total phenolic content (TPC) was calculated by doing the slight modifications in Folin & Ciocalteu′s colorimetric methods [25]. TPC was designed using the usual calibration curve (ranging from 0-200 µg/mL) and data was conveyed as milligram Gallic acid equivalent per gram of dry extract (mg of GAE/g of DE).

Total flavonoid content
Total flavonoid content (TFC) was determined by modified colorimetric method [26]. TFC was designed by the calibration curve equation and stated as milligram Quercetin equivalent per gram of dried extract (mg of QE/g of DE).

HPLC Analysis of phenolic compounds
HPLC of En was done in Central Hi-Tech Laboratory, University of Agriculture, Faisalabad, Pakistan as defined before [27].

Acute toxicity assessment (In vivo)
Acute toxicity of En (70% aq. ethanol) extract was examined on Sprague-Dawley rats. There were six rats in each group (4 male, 2 females); (and these rats were orally controlled during morning (12 hrs fasting) with the extract at 150, 300, 500, 1000, 2000 mg/kg doses). Extracts of 150, 300, 500, 1000, 2000 mg/kg doses were orally administered to the rats. Advices of Organisation for Economic Cooperation and Development (OECD) 425 were monitored to perform toxicity studies.

Experimental Study Design
12-24 weeks old (adults) 48 male Sprague-Dawley rats weighing 180g to 200g were used for the study. Strategies of National Institute of Health, Islamabad were firmly monitored to conduct the trial. The following eight experimental groups, each with 6 animals, were studied.
Group-I: Control (Untreated; only standard food supply was offered) Group-II: Vehicle (10% DMSO in olive oil 1 ml/kg body weight) Group-III: Disease model (1 ml/kg 30% CCl 4 (in olive oil)) Group-IV: Drug control (CCl 4 + silymarin 50 mg/kg) Group-V: CCl 4 + En (150 mg/Kg) Group-VI: CCl 4 + En (300 mg/Kg) Group-VII: En (150 mg/Kg) Group-VIII: En (300 mg/Kg) CCl 4 was inserted intraperitoneally (ip), while vehicle and Silymarin were administered using gastric cannula. Doses of extract and positive standard were administered orally through gastric tube 30 minutes after the administration of CCl 4 . The treatments of CCl 4 and En (samples) were given in the early morning on alternate days (from day 1), thrice a week for 4 weeks.

Collection of blood sample and organs for biochemical investigation
After the final treatment, rats were fasted for 24 hours. Blood samples were collected after sedation with chloroform for hematological studies and serum analysis. Small portion of excised hepatic cells was stored in 10% formalin solution for tissue pathological studies.

Hematological studies and serum analysis
Neubauer hemocytometer (Feinoptik, Germany) used to estimate platelets, red blood corpuscles and white blood cells; Sahli's haemoglobin meter was used to count hemoglobin content. Modified Westergren method was used to measure erythrocyte sedimentation rate. Serum replicates analysis was done for alkaline phosphatase, alanine transaminase, aspartate transaminase, albumin and bilirubin by means of AMP diagnostic kits (Graz, Austria). Bradford method was used to regulate protein concentration [28].

Renal function tests and serum analysis
Serum albumin, urea and creatinine levels were estimated calorimetrically using commercial diagnostic kits. Then, creatinine/albumin ratio (C/A) was measured. After decapitation, kidneys were excised. Small portion of excised kidney was used for histopathological and biochemical studies.

Histopathological studies of liver/kidney
Specimen were fixed in paraffin and saved on hard blocks and divided in thin layers of 3-4μm, followed by staining with hematoxylin and eosin. Then light microscope (DIALUX 20 EB) at 40× power was used to examine these slides and photographed using HDCE-50B camera. All tissue pathological abnormalities were classified by using altered signs showing marked changes.

Statistical analysis
Tukey's multiple assessment tests were used to evaluate important changes among in vivo treatment groups using computer software Statistix 8.1. Statistical significance for demeanors was done at p < 0.05. Graph was created using software GraphPad5.

Extraction
% age yield of En extract was 7.14.

Preliminary Phytochemical Analysis
The results for various phytochemicals confirmed presence of alkaloids, flavonoids, glycosides, phenols, tannins and saponins in En, and have been shown in Table 1.

HPLC analysis
Quantitative sketching and chromatographic finger printing confirmed presence of polyphenols: gallic acid, quercetin,caffeic acid, benzoic acid, vanillic acid, chlorogenic acid, ferulic acid and syringic acid in the extract. These polyphenols were present in quantities of 1

Acute toxicity assessment (In vivo)
In acute toxicity assessment, all animals were observed carefully for development of any toxic signs or symptoms at different time intervals of 0, 30 min, 1, 2, 4, 6, 8, 12 h. and then daily for a period of 3 days. There was no toxic signs like lacrimation, salivation, piloerection, drowsiness, tremors, convulsions. Moreover, body weight, food consumption and water consumption was normal, and no mortality was observed in clinical parameters during acute study. So, it indicates that the LD50 of En extract is greater than 2000 mg/kg/day BW.

Hepato-/Nephro-protective activity
The current study demonstrated hepatonephroprotective role of En in CCl 4 induced hepatic-nephrotoxicity at different doses (150 mg and 300 mg/kg BW).

Hematological parameters
Effects of En and CCl 4 treatments are shown in Table 3 )/μl WBCs. But, no major difference in blood profile observed when En (150 and 300 mg/kg: groups VII and VIII) was given alone; thereby, validating the safety profile of the extract.    Fig. 3). CCl 4 administration in rats produced a major increase (p < 0.05) in the level of ALP = 378 ±2.58 u/L, AST = 98±1.15 u/L ± and ALT = 107 ±1.8 u/L as compared to the ALP = 144 ±1.29 u/L, AST = 43.0 ±2.08 u/L and ALT = 44 ±1.3 u/L in the control group. High and low doses of En extract (groups V-VI) markedly restored theCCl 4 produced raised levels of hepatic stress biomarkers. Strangely high level of serum markers was significantly decreased (p < 0.05) by co-administration of En (150 mg/kg) which tends to regulate these elevated levels (p < 0.05) as compared to control group. Still, coadministration of the high dose of En (300 mg/kg) significantly decreased the level of these enzymes, and the levels acquired were as: ALP = 150 ±2.30 u/L, AST = 42 ±1.39 u/L and ALT = 48 ±2.70 u/L in serum. High and low doses of En alone did not induce any change (p > 0.05) in the serum level of ALP, AST and ALT.

Defensive role of En on hepatic biochemical parameters (serum)
Serum albumin and bilirubin level were examined. The albumin level reduced (p < 0.05) while bilirubin level increased in CCl 4 intoxicated rats in contrast to the control group (

Effect of En on hepatic histoarchitecture
Histopathological investigations endorsed protective effects of En against CCl 4 induced hepatic damage (Fig. 2). Liver section of control group displayed smooth architecture and normal morphology with typical central vein, kupfer cells, hepatocytes and sinusoids as shown in Fig. 2a. Histopathological examination of CCl 4 treated animals (group III) caused noticeable elevation in fatty changes, inflammatory cells infiltrations (hepatic cytoplasm inflammation), cellular hypertrophy, centrilobular necrosis, vacuolization, ballooning and dilation of central vein as shown in Fig. 2b. Co-administration of high dose En (300 mg/kg) extract exhibited protective effects by restoring hepatic inflammation towards normal (Fig. 2d), which were comparable with shielding effects of Silymarin (group IV; Fig. 2c) with mild destruction of hepatocytes induced by CCl 4 . Administration of low dose En (150 mg/kg) had lessened injuries of the hepatic necrotic cells. Administration of En alone depicted the normal histoarchitecture of the liver samples, showing the safe and nontoxic behavior of the extracts. Pathohistological changes are summarized in Table 5 using scoring method.

Effect of En on renal biochemical parameters (serum)
Administration of CCl 4 to rats (Group III) significantly increases serum creatinine, serum urea and creratinine/ albumin ratio and decrease serum albumin level (P<0.05, P<0.05, P<0.05 & P< 0.001 respectively) in contrast to control group (Group I) (p<0.001) ( Table 6). A 4 weeks pre-treatment with En at 150 and 300 mg/kg dose (Groups V and VI) after CCl 4 intoxication showed renal safety in relations to serum urea, creatinine, and creatinine: albumin proportion levels in contrast to the toxic control group (Group III) (p<0.001). Associated administration of En to CCl 4 intoxicated rats caused a significant decrease in serum creatinine level and in creatinine/albumin ratio (P< 0.01) and significant increase in serum albumin level (P< 0.001) compared to CCl 4 group ( Table 6; Fig. 4).

Effect of en on renal histoarchitecture
Histopathological investigations endorsed protective effects of En against CCl 4 induced renal damage (Fig. 3). Kidney section of the control group displayed smooth architecture and normal morphology with normal structural and architectural integrity. The control group kidney showed normal proximal and distal tubules and normal and intact glomeruli (Fig. 3a). Four week CCl 4 chronic administration caused significant renal morphological damage, especially in the renal cortex. However, CCl 4 effect on the medulla was limited. CCl 4 -treated kidneys exhibited different forms of degeneration in affected glomeruli. Some showed mild dilatation of Bowman's space along with glomerular atrophy, while others exhibited congestion in the capillary loops. Proximal and distal tubules showed histological changes with inflammation, dilation and degeneration with preliminary signs of acute tubular necrosis. Due to sporadic congestion there was sporadic haemorrhage that results in renal tubular degeneration and detachment of tubules, and vacuolation of epithelial cells (Fig. 3b). Vacuolations or fatty changes in the renal cortex were clearly apparent after CCl 4 treatment. As an indicator of fibrosis and considerable congestion in the blood vessels, there was an evident increase in the connective tissue cells in these regions of infiltration. Rats treated with En alone (150 and 300 mg/kg) depicted the normal histoarchitecture of the kidney samples having no histological changes in kidney tissues, thus showing the safety and non-toxic behavior of the extract. CCl 4-induced abnormal histopathological changes significantly reduced in the En-treated groups. High dose En (300 mg/kg) coadministration exhibited protective effects towards normal by decreasing renal inflammation (Fig. 3e), these effects were comparable with shielding effects of silymarin (group IV; Fig. 3b). In the CCl 4 +ENE groups (V and VI), ENE markedly prevented congestion in glomeruli and vessels and other alterations, and the glomeruli and tubules were normal in their histology. Any increase in the connective tissue cells was not observed as well (Fig. 5e). Low dose En (150 mg/kg) administration lessened injuries of renal necrotic cells. Histopathological changes induced by CCl 4 and En extract in renal tissues (Quantification scores) is summarized in Table 7.

DISCUSSION
Traditional medicinal practices designed the base of clinical, pharmacological and chemical studies [29]. Plants have been the essential component of human culture due to their medicinal uses since time immemorial. In early drug discovery, plants as initial source of medicines have been used in ethnopharmacological stuff [30][31]. Herbs as a source of medicine are popular in almost every culture of the world because of their easy availability, effectiveness, economy and compatibility to human physiology.
DPPH analysis is well known to measure the scavenging potential of antioxidants appearing deep purple in color. In this analysis there is reduction of DPPH to DPPH 2 wherein the scavenger antioxidant molecule donates the proton. Reagent color modified from purple to yellow at 515 nm [32]. In short, antioxidant ability may be characteristics to phenolic as well as flavonoids contents present in En. Acute toxicity investigation confirmed the non-toxic behavior of the extract where during two weeks no death was noticed. In vivo studies the initial safety of the plant was analyzed. So the powerful antioxidant ability and non-toxic nature of the extract was acceptable reason for further assessment of its hepato-nephron protective potential against CCl 4 induced toxicity.
Determination of serum AST, ALT and ALP enzyme activity is an important indicator for hepatic membrane functional integrity. Elevated ALP, AST and ALT serum levels are measured as hepatotoxicity markers, these enzymes ooze out into the plasma when hepatic injury disturbs cellular membrane probity. In the current investigation, increased levels of hepatic serum markers (AST, ALT and ALP) might reverse hepatic damage due to CCl 4 intoxication. Increased ROS generation in hepatocytes leads to hepatic damaging action and cellular death because of protein oxidation, lipid peroxidation, and DNA damage [33]. Similarly, increased bilirubin serum level clearly indicates blockage in bile elimination due to hepatic cells' damage. Hepatocellular injury can be determined by assessment of serum bilirubin and hepatic biomarkers [34][35]. In case of decreased albumin level inflammation occurs due to oxidative stress [8]. In the current investigation, polyphenol enriched En (  Fig. 2 it is observed that co-administration of low and high dose En as well as silymarin ameliorated and decreased the CCl 4 induced toxic effects and damages. It is suggested that En Protective effects may be due to the phytoconstituents like polyphenols, sterols, tannins, terpenoids and flavonoids as suggested by Nabavi et al. [37]. En may be a strong hepatoprotective agent with free radical scavenging ability in hepatotoxicity. These investigations suggest a direct means of assessing the hepatic-protective effects against CCl 4 induced damages in liver Fig. 2. Liver section of the control group displayed smooth architecture and normal morphology with normal structural and architectural integrity Fig. 2a. Histopathological examination of CCl 4 treated animals (group III) show noticeable abnormal changes, like elevation in fats, hepatic cytoplasm inflammation, cellular hypertrophy, necrosis, ballooning, vacuolization, and central vein dilatation as shown in Fig. 2b. High dose En (300 mg/kg) co-administration showed protective effects towards normal by decreasing hepatic cells inflammation Fig. 2d, these effects were similar with protective effects of silymarin (group IV: Fig. 2c). Administration of low dose En (150 mg/kg) reduced the injuries of necrotic cells of the liver. Administration of En alone depicted the normal histoarchitecture of the liver, proving the safety and non-toxic behavior of the extract. Pathohistological changes are concised in Table  6 (scoring method was used). Histopathological investigations recognized protective effects of En against CCl 4 induced renal damage Fig. 3. Kidney section of the control group displayed smooth architecture and normal morphology with normal structural and architectural integrity. Moreover, the control group kidney showed normal proximal and distal tubules and normal, and intact glomeruli Fig. 3a. The CCl 4 chronic administration caused significant renal morphological damage, especially in the renal cortex. However, CCl 4 effect on the medulla was found limited. Due to sporadic congestion there was sporadic hemorrhage that results in renal tubular degeneration and detachment of tubules,and vacuolation of epithelial cells Fig. 3b. Vacuolations or fatty changes in the renal cortex, fibrosis and considerable profusion in the blood vessels were clearly apparent after CCl 4 treatment Fig. 3.
Rats treated with En alone (150 and 300 mg/Kg) depicted the normal histoarchitecture of the kidney samples having no histological changes in kidney tissues, thus showing the safety and nontoxic behavior of the extract. CCl 4-induced abnormal histopathological changes significantly reduced in the En -treated groups. High dose En (300 mg/kg) co-administration exhibited protective effects towards normal by decreasing renal inflammation Fig. 3e, these effects were analogous with protecting effects of silymarin (group IV: Fig. 3). In the CCl 4 + En groups (V and VI), En obviously prevented profusion in glomeruli and vessels and other changes, and histologically glomeruli and tubules were normal. Any increase in the connective tissue cells was not observed as well Fig. 3. Low dose En (150 mg/kg) administration lessened injuries of renal necrotic cells. Pathohistological changes are summarized in Table 4 (scoring method was used).
Results are in agreement with Moneim and El-Debi, 2012, who demonstrated that CCl 4 made a significant increase in kidney weight and comparative kidney weight due to a significant decrease in body weight and kidney swelling of rats [38]. Abnormal increased level of urea, and serum creatinine are possible gages of kidney injuries induced through CCl 4 treatment [39], also accompanied by histological changes such as severe proximal renal tubular necrosis followed by renal failure [40][41]. Serum creatinine level does not rise until at least half of the kidney nephrons are destroyed or damaged. After CCl 4 intoxication, rats developed urea elevation and chronic renal injury [42]. Level of proteinuria is high in renal injuries. Moreover, there is reduction in serum albumin in CCl 4 -treated rats due to glomeruli and tubules, which resulted in remarkable leakage. Results of the current study reveals that En significantly recovered renal injuries induced through CCl 4 intoxication in rats. Hematological investigation is used for assessment of hepatic disorders like inflammatory diseases. CCl 4 may be a reason for enhancing the production of free radicals including H 2 O 2 , which needs ferritin and hemoglobin for OH ions conversion. Lower Hb level,and increased WBC count is a highly frequent indicator in oxidative stress inflammation [8][9]. In this investigation, CCl 4 intoxicated rats exhibited increased WBC levels and decreased Hb, RBCs and platelets levels. The extract significantly restored CCl 4 mediated hematological abnormalities towards normal level. This restorative and protective property may be due to the protective potential of the polyphenols present in extract.

CONCLUSION
The current study recommends that Euphorbia nivulia Buch.-Ham (70% hydro alcoholic) extract possesses the possibility to improve the histopathological injuries triggered by CCl 4 and has the ability for restoration of normal hepatic and renal histoarchitecture. All this justifies its protective and shielding capacity to revoke the hepatic and renal damage caused by oxidative stress due to free radicals/ROS. Protective properties of the extract might probably be due to its various phytochemical constituents and antioxidant potential. Hepato and nephronprotective role of Euphorbia nivulia extract was comparable with silymarin which may be attributed to the presence of polyphenols and flavonoids. En allows us to conclude that extract may further be subjected to bio-assay guided isolation to identify the active component(s). The extract may be a good candidate as antioxidant and hepato-nephron-protective agent for developing new formulation(s).

ETHICAL APPROVAL
Strategies of National Institute of Health, Islamabad were firmly monitored to conduct the trial. The designed protocol was approved (Bch#0265) by the Ethical Committee of Quaid-i-Azam University, Islamabad, Pakistan.