INTRODUCTION:

Liver is an important organ in human body responsible for metabolism and it is continuously exposed to different types of drugs, pollutants and xenobiotics¹. Globally liver diseases lay a huge burden on mankind and available drugs have serious side effects². Paracetamol is commonly used drug but it causes severe liver damage when used in overdoses^3,4. Drugs which are used to treat paracetamol induced toxicity are reported with severe side effects⁵.

Many plants are also reported to posses hepatoprotective activity against paracetamol induced toxicity like Aerva lanata Linn⁶ (manokaran, 2008), Madhuca longifolia⁷ (Umadevi, 2011), Butea monosperma⁸ (Pradeep, 2011). Hence there is an utmost requirement to discover a drug having Hepato-protective potential with no or fewer side effects.

Poloxamers are synthetic tri-block copolymers. They have hydrophobic polyoxypropylene chain centrally, which is flanked by two hydrophilic polyoxyethylene chains. 4:2:4 is the weight proportion of these three chains. These are amphiphilic surface copolymer whose hydrophilicity, molecular size and hydrophobicity can by shifted by changing the central and side chains of the molecule. In 1950’s an expansive group with various qualities were produced and made accessible commercially through the BASF Corporation conveying the proprietary name, Pluronic®. At the point when promoted as a Pluronic®, the poloxamer is given a particular designation containing a letter that shows the condition of the polymer at room temperature, with a "P" demonstrating a powder, "L" for liquid or "F" for flakes, after this letter there are two or three digit number representing to the molecular weight of the Pluronic®. Molecular weight of the polyoxypropylene in the polymer is determined by multiplying first or second digit by 300 and percentage of the polymer that is polyoxyethylene is determined by multiplying the last number by 10. Based on percentage of oxyethylene, molecular weight and many other parameters, different grades of poloxamer are identified as poloxamer (68, 88, 98, 108, 124, 188, 237, 338, and 407)⁹. Because of adsorption property, size and structure, poloxamers¹⁰ are used in cosmetics¹¹, emulsions¹², nanoparticle synthesis¹³, effective dispersants for inks/pigments¹⁴. Pharmaceutical applications of poloxamer include multi-drug resistance (MDR) reversal^15,16,17, oral chemotherapy¹⁸, gene delivery^19,20, lung cancer treatment²¹, ophthalmic diseases treatment²² and also to synthesize different drug delivery forms like cubosomes²³, thermosensitive gels^{24, 25}, micelles²⁶. P-407 and P-188, was used to enhance the dissolution rate of many poorly water soluble drugs like Bicalutamide²⁷, Pioglitazone²⁸, etoricoxib²⁹, Diacerein³⁰, Cefexime³¹.

PLURONIC F68 is a P-188, having a molecular weight of 8400 Daltons, is a copolymer linear and nonionic in nature. In humans, t1/2 of P188 is 18 hours and can be given safely up to 72 hours. It was approved as therapeutic reagent by FDA to decrease blood viscosity before transfusions³². P188 is well known for its surfactant properties so applied widely in industries, pharmaceutical companies. P 188 when used along with P 407 enhances the residence time of active component at the site of administration by increasing viscosity of formulation and decreasing temperature of sol-gel transition³³. P-407 in combination with Carbopol increased the drug retaining time of Diclofenac potassium ophthalmic preparation³⁴. P-188 also has suitable mucoadhesive force³⁵.Other reported applications of P-188 are decreases inflammation and tissue damage after experimental brain injury in rats³⁶ , useful excipient for intramuscular delivery of naked DNA³⁷, treatment of cardiovascular diseases³⁸, repair damaged cell membranes³⁹, facilitates the Repair of Alveolus Resident Cells in Ventilator-injured Lungs⁴⁰.

MATERIALS AND METHODS:

Drugs and Chemicals:

Silymarin purchased from Microlabs Pvt. Ltd. Tamil Naidu India. SGOT, SGPT and ALP kits were obtained from Span diagnostics, Surratt, India. Pentzocine from Dr. Reddy Labs Pvt. Ltd. Formaldehye, Paracetamol and all other chemicals used in the study are of analytical grade.

Animals:

Wistar albino rats, weighing 150-250gm were obtained from Synzyme Laboratories Shamshabad, Telangana India. Animals were housed in poly acrylic cages and maintained under standard laboratory conditions (temperature 25 ± 2⁰C) with dark and light cycle (12/12 h). Water ad-libtum and feed as pellets. All procedures described were reviewed and approved by the Institutional Animals Ethical Committee (1970/PO/Re/S/17/CPCSEA). Acute oral toxicity studies were carried according to OECD guideline-423.

Assesment of Hepato-protective Activity of P-188:

Animals were randomly divided into five groups of four animals in each. Group I served as Normal Control and fed orally with normal saline 5 ml/kg BW. Group II served as Negative Control received normal saline 5 ml/kg BW. Group-III, IV and V received Polaxamer-188-50mg/kg, Polaxamer-188-100mg/kg and Silymarin 25mg/kg BW⁴¹ respectively. All the treatments i.e., normal saline and Polaxamer-188 and silymarin are given to their respective groups for 7 days. On the seventh day, paracetemol suspension was given by oral route, in a dose of 750mg/kg BW to all rats except Group I.

Biochemical studies:

After last dose all the animals were kept on fasting for 18hrs. Blood was collected from all animals by puncturing retro-orbital plexus. Samples were allowed to clot for 30 min at room temperature and serum was separated by centrifugation at 2500rpm for 15 min and utilized for the estimation of various biochemical parameters namely SGOT, SGPT⁴², ALP⁴³. After collection of blood samples rats in different groups were sacrificed and their livers were excised immediately and keep it in 10% formalin.

Histopathological Studies:

Small pieces of liver tissues in each group were collected in 10% neutral buffered formalin for proper fixation. These tissues were processed and embedded in paraffin wax. Sections of 6µm in thickness were cut and stained with hematoxylin and eosin (H&E). These sections were examined photo microscopically for necrosis, statuses and fatty changes of hepatic cells⁴⁴.

RESULTS AND DISCUSSION:

Acute Toxicity Studies:

After observing mortalities and behavioral profile for the stipulated time, LD-50 of P-188 was found to be more than 2000 mg/kg. Further, in accordance with the OECD guidelines, very low doses i.e., 1/40^th (50mg/kg) and 1/20^th (100mg/kg) of LD-50 were taken as lower and higher dose of P-188 respectively for conducting Hepato-protective activity.

Assessment of Hepato-protective Activity of P-188:

Biochemical studies:

In negative control group there was a significant increase in the level of SGOT, SGPT and ALP levels as compared to Normal Control Group indicating the induction of toxicity. Whereas the animals in group III, IV and V showed significant decrease in the levels of enzymes as compared to negative control group (Table 1) indicating the efficacy of Hepato-protective role of P-188.

Histopathological Studies:

histopathological results of negative control animal showed inflammation with mild cholestasis in their liver tissues indicating the effect of paracetamol in inducing liver toxicity. Liver tissues of animals treated with P-188 (50mg/kg) showed mild or no inflammation, more number of normal hepatocytes, normal hepatic parenchyma, kupffer cell hyperplasia and no evidence of necrosis (Figure 1). P-188 (100 mg/kg) showed scattered lymphocytes among normal hepatocytes and kupffer cell hyperplasia, no incidence of inflammation and necrosis and silymarin 25mg/kg shows normal hepatic parenchyma with mild inflammation in portal area. Normal liver shows normal parenchyma with no sign of inflammation.

DISCUSSION:

Present study was conducted to evaluate Hepato-protective efficacy of P-188 against paracetamol challenged rats. It is one of the commonly used models for screening hepto-protective drugs by measuring the quantity of enzymes released in circulation. Overdose of paracetamol may cause hepatic injury, necrosis, liver failure and finally may lead to death of recipient⁴⁵. Cytochrome P-450 is responsible for the metabolism of paracetamol. After overdose metabolic pathways viz., sulphation and glucoronidation gets saturated and paracetamol is converted in to toxic metabolite N-acetyl-pbenzoquinoneimine (NAPQI)⁴⁶. NAPQI may bind covalently to unsaturated lipid membrane and causes liver injury majorly indicated by increase in the levels of SGOT, SGPT and ALP levels⁴⁷. Necrosis of liver cells increases the circulating levels of SGOT and SGPT⁴⁸. Injury to liver parenchymal cells also causes increased biliary pressure which in turn increases ALP levels of serum⁴⁹. Results of our study clearly reflects the hepatotoxic condition of negative control group animals (increase in the level of SGOT, SGPT and ALP) and P-188 as hepatoprotective drug by decrease in the level of these enzymes as compared to negative control group. Our results of biochemical studies are in acoordance with Venkatalakshmi P and Ragadevi M⁵⁰ where they reported Protective effect of Tinospora cordifolia linn. on Paracetamol and Alcohol Induced Hepatotoxicity in Albino Rats. Results of serum enzyme levels are in accordance with the results of histopathological evaluation. Features of hepatotoxicity include Inflammation with cholestasis, hepatic necrosis and decrease in the no. of normal hepatocytes which are clearly shown in the photomicrographs of liver tissue of negative control group animals whereas non-inflammed condition, distortion of liver cell architecture is decreased upto a great extent in P-188 and silymarin treated group animals. Moreover P-188 showed dose dependent protection in this study.

CONCLUSION:

P-188, a well known thermogelling polymer is available in the market since last 50 years, but from last one or two decades its therapeutic efficacy in different types of chronic diseases is also explored and reported. In current research, P-188 was found to produce highly significant (p<0.0001) decrease in SGOT, SGPT and ALP levels as compared to negative control group levels. Moreover these biochemical results also support the results of histopathological pictographs where P-188 was found effective in restoring the distorted architecture which was reported in the livers of negative control group animals. Based on these evaluated parameters, we can conclude that P-188 can be used as Hepato-protective agent though further studies are profoundly needed for complete assurance.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 17.08.2018 Modified on 16.09.2018

Research J. Pharm. and Tech 2019; 12(2):574-578.

DOI: 10.5958/0974-360X.2019.00102.1

Groups and treatment	SGPT (U/L)	SGOT(U/L)	ALP(U/L)
Group I-Normal Control (Normal Saline 5 ml/kg)	152.5±1.63****	142.5±1.74****	46.07±1.09****
Group II- Negative Control (Normal Saline 5 ml/kg)	212.7±3.71	231.7±3.06	96.25±0.65
Group III- Polaxamer-188 (50mg/kg)	185.8±3.48***	154.8±2.53****	56.02±1.39****
Group IV-Polaxamer-188 (100mg/kg)	144.0±2.33****	141.0±0.42****	51.12±0.67****
Group V- Standard (Silymarin 25mg/kg)	159.2±5.60****	150.3±1.15****	54.40±2.20****