Protective Effect of Momordica Charantia Against Hepatic Ischemic Reperfusion Injury Model in Rats

Parikh MP¹*, Patel AM¹, Patel KV² and Gandhi TR¹

¹Department of Pharmacology, Anand Pharmacy College, Anand, India

²Department of Pharmacy, Faculty of Technology and Engineering, M S University, India

*Corresponding author: : Parikh MP, Department of Pharmacology, Anand Pharmacy College, Anand-388001, Gujarat, India

Received: November 13, 2014; Accepted: January 26, 2015; Published: January 29, 2015

Abstract

Protection from the effects of ischemia and reperfusion injury (I/R) posesa serious problem in liver transplantation and resection surgery. Liver ischemia is a common cause of frequent clinical complications i.e.hepatocytes death, liver failure, and liver graft rejection. Oxidative stress playsa key role in hepatic I/R. Momordica charantia L. (Cucurbitaceae) is a strong antioxidant countering oxidative stress. This study was carried out to evaluate the effect of Momordica Charantia (MC) fruit extract on hepatic ischemic reperfusion injury in rats.Adult Wistar albino rats underwent 45 min of ischemia followed by 3 h of reperfusion by ligation of hepatic artery and left portal vein. Momodica charantia fruit extract was administered once daily for 4 days at a dose of 200mg/kg. Allopurinol (50mg/kg) was used as standard drug. After four days of drug administration, surgery was done on the fifth day and serum and liver samples were collected from all experimental animals after surgery. Biochemical parameters like SGOT (serum glutamic oxaloacetic transaminase), SGPT (serum glutamic-pyruvic transaminase), ALP (alkaline phosphatase), LDH (lactate dehydrogenase) and CRP (C-reactive protein) and various antioxidant parameters were measured from serum sample and liver homogenate, respectively. MC significantly (p<0.05) reversed I/R induced changes in liver damage marker -SGOT, SGPT and ALP, tissue damage marker LDH and inflammatory marker CRP. Changes in level of antioxidant parameters were also reversed by MC.MC possess protective effect against ischemic reperfusion injury that can be attributed to its antioxidant activity.

Keywords: Liver; Allopurinol; Oxidative stress; Inflammation; Liver markers

Introduction

The largest gland in the body, the liver, weighs around 1-2.3 kg. The cardinal physiological roles of liver include carbohydrate, fat and protein metabolism, breakdown of erythrocytes, defense against microbes, detoxification of drugs and noxious substances, inactivation of hormones, synthesis of vitamin A from carotene and secretion of bile [1].

Several diseases associated with liver are cholestatic liver diseases, primary biliary cirrhosis, primary sclerosing cholangitis, acute fulminant liver failure, alcoholic liver disease, autoimmune chronic hepatitis, biliary atresia (paediatric), chronic viral hepatitis, cryptogenic cirrhosis, hepatic malignancy, and metabolic disease [2]. Medical treatment of liver diseases and liver damage is always of prime importance. When drug therapy can no longer support life, a liver transplant is the ultimate option for end stage liver disease. Chronic viral hepatitis, alcoholic cirrhosis, and chronic cholestatic diseases are the most common liver diseases that require transplantation.

Liver Ischemia-Reperfusion (I/R) injury is a significant cause of morbidity and mortality in two principal settings. Firstly, it occurs in major liver resections [3] and transplantation [4,5] where anoxic or ischemic liver injury takes place. Secondly, it happens as a consequence of systemic hypoxia or with conditions that cause low blood flow to the liver resulting in insufficient perfusion. The latter occurs in hemorrhagic, cardiogenic, or septic shock with subsequent fluid resuscitation [6], in cardiovascular surgery with extracorporeal circulation [7], in laparoscopic surgery [8,9] and in abdominal compartment syndromes [10]. In the field of liver transplantation, I/R injury is closely related to the development of primary graft non-function (occurs in 5% of grafts) and primary graft dysfunction (occurs in 10-30% of grafts) [11]. Both conditions are associated with high rates of morbidity and mortality. I/R injury increase the incidence of subsequent graft rejection [12].

Liver injury induced by I/R is caused, by Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). There is evidence that during hepatic I/R there is generation and release of ROS and RNS with concomitant consumption of endogenous antioxidants and apoptotic or necrotic cell death [13-17]. Although the exact sources of ROS generation in liver I/R are still under investigation, the nicotinamide adenine dinucleotide phosphate oxidase (NADP), the xanthine/ xanthine oxido reductase system, and the mitochondria have been suggested to play key roles [18]. Administration of free radical scavenging agents that enhance the endogenous antioxidant system could reduce the post-ischemic tissue injury and so it can be useful in clinical settings against hepatic I/R damage [19].

The antioxidants with beneficial effect in hepatic ischemic reperfusion injury are: a-tocopherol [16], a-tocopherol/ascorbate [20], GSH [21], SOD derivatives [22], lipoic acid [23,24] and carvacrol [25]. Preconditioning is also another approach used in the patient prior to liver transplantation and resection. Ischemic preconditioning involves ischemia and reperfusion for a short period of time before exposure to prolonged ischemia and reperfusion.

Momordica charantia as an antioxidant is a strong free radical scavenger and also has hepatoprotective property [26]. Therefore, this study was carried out to evaluate the effect of Momordica charantia in hepatic ischemic reperfusion injury.

Materials and Methods

Collection of plant material

Fresh unripe fruits of Momordica charantiaL. (Cucurbitaceae) were collected in the morning from the local market of Anand, Gujarat, India and were then authenticated by Dr. A. S. Reddy, B. R. Doshi School of Biosciences, Sardar Patel University, Bakrol, Anand, Gujarat, India. (Herbarium specimen no: APCH34).

Extraction procedure

The fruits of Momordica charantia were air-dried and fine powdered, then extracted by repeated percolation which lasted about 6-8 h with hexane under reflux in Soxhlet extraction apparatus [27]. At the end of extraction process, the solvent was then evaporated and the remaining mass was measured. Extracted mass was stored at room temperature and labeled as MC. Extraction yield was 12.5%w/w. Pure powder of Allopurinol was obtained from Zydus Cadila Healthcare Ltd., Ahmedabad, Gujarat, India. Drug solutions were prepared freshly every day by suspending the drug in 0.5% Carboxymethyl Cellulose (CMC) suspension. All the chemicals used were of analytical grade and were obtained from Astron chemicals, Ahmedabad and SD fine chemicals, Mumbai. All the biochemical tests were performed using the standard reagent kits purchased from Coral Clinical Systems, Goa, India.

Experimental procedure

Wistar rats of either sex 200-250g obtained from Animal house of Anand Pharmacy College, Anand, were used as experimental animals for the study. The animals were housed in a group of 3 rats per cage under well-controlled conditions of temperature (22 ± 2°C), humidity (55 ± 5%) and 12hrs/12hrs light-dark cycle. Animals had free access to conventional laboratory diet (Pranav Agro Industries, Delhi, India) and R.O. water ad libitum. The protocol-APC/2012-IAEC/1211 of the experiment was approved by Institutional Animal Ethics Committee (IAEC) as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Animals were randomly allocated in six groups, with n=6 animals in each group, as follows: Group I Normal Control, Group II Model Control, Group III Sham operated, Group IV Vehicle control, Group V Standard Control, Group VI Momordica charantia 200 mg/kg (MC 200). Surgery was performed in Group II-VI. In Group III Sham surgery was performed in the absence of clamping of the vessels. Group IV received 0.5% CMC suspension (1ml/kg, i.p.) for 4 days, then surgery was performed on the fifth day. Group V and VI received Allopurinol 50mg/kg and Momordica charantia 200mg/ kg respectively for 4 days then surgery was performed on fifth day. For induction of I/R animals were fasted for 12 h before experiments. Anesthesia was induced with phenobarbitone (10mg/kg; i.p) and body temperature was maintained throughout surgery at 37±0.5°C with a lamp. The left branches of portal vein and hepaticartery were clamped by vascular clamp to induce complete ischemia of the median and left hepatic lobes. Sham surgery did not involve clamping of the vessels. After 45 min of ischemia, reperfusion was allowed for 3 h by removing the clamp. Blood samples were collected after anesthesia just before sacrificing and liver was rapidly removed for further analysis.Serum samples were separated by centrifugation (Plastocrafts Rota 4R-V/FA) at 3000 rpm for 15 min and stored at -20°C until the analysis was carried out.

SGOT, SGPT, ALP, LDH and CRP were estimated by spectrophotometry method by using standard kit.Antioxidant parameters- Malondialdehyde (MDA), total Glutathione (GSH), Superoxide dismutase (SOD) and Catalase (CAT) were measured in liver homogenate.

Statistical Analysis

Results represented are mean ± SEM. Statistical difference between the means of the various groups were analyzed using one way analysis of variance (ANOVA) followed by Dunnett's post hoc test with p value <0.05. Statistical analysis was done using GraphPad Prism software, version 5.03.

Results

Levels of SGOT, SGPT, ALP,LDH and CRP were found to be significantly (P<0.01) higher in model control animals as compared to normal control animals.Treatment with Allopurinol (50mg/kg) significantly (P<0.01) prevented the rise in SGOT, SGPT, ALP,LDH and CRP levels as compared tomodel controlgroup. Similarly, Momordica charantia (200mg/kg) significantly (P<0.01) prevented the rise in SGOT, SGPT, ALP, LDH and CRP levels as compared to model control animals as shown in Table 1.

Table 1: Effect of Momordica charantia on Liver injury maker and CRP levels.




  
    Groups 
    SGOT 
      (U/L) 
    SGPT 
      (U/L) 
    ALP 
      (U/L) 
    LDH 
      (U/L) 
    CRP 
      (mg/dl) 
  
  
    I 
    50.34±0.03
    38.02±0.22
    42.38±0.88
    254.54±0.11
    0.35±0.21
  
  
    II 
    353.08±0.07^#
    343.18±0.08^#
    257.34±0.09^#
    764.07±0.05^#
    8±0.33^#
  
  
    III 
    64.40±0.1
    52.96±0.31
    70.53±0.25
    288.72±0.12
    0.6±0.89
  
  
    IV 
    333.09±0.03
    331.06±0.09
    244.8±0.05
    750.58±0.55
    7.2±0.34
  
  
    V 
    97.87±0.12**
    82.54±0.01**
    78.18±0.22**
    327.84±0.41**
    4.4±0.78*
  
  
    VI 
    121.05±0.16**
    105.82±0.13**
    94.40±0.03**
    353.03±0.37**
    2.15±0.91**
  
  
    * significantly different from model    at p <0.05
      ** significantly different from model    at p <0.01
      # significantly different from normal    at p <0.01



Table 1:  Effect of Momordica charantia on Liver injury maker and CRP levels.

MDA level in liver homogenate of model control animals was significantly higher as compared to normal control animals. Treatment with Allopurinol (50mg/kg) and Momordica charantia (200mg/kg) significantly (P<0.05) prevented the rise in MDA levels as compared to model control group as shown in Figure 1.

Figure 1: Effect of Momordica charantia extract on MDA level in I/R. values represented are mean±SEM. Statistical significance was set at p<0.05.
#significantly different from normal group at p<0.05
*significantly different from model group at p<0.05

    
    
    Figure 1:  Effect of Momordica charantia extract on MDA level in I/R.
values represented are mean±SEM. Statistical significance was set at p<0.05.

#significantly different from normal group at p<0.05

*significantly different from model group at p<0.05

Decreased levels of CAT, SOD and GSHas depicted by the model control group, were significantly (P<0.05) increased in treatment control group compared to the model group as shown in Figure 2, 3 and 4 respectively.