Effect of Punica granatum peel and Vitis vinifera seeds on Biochemical Indices in DEN induced Hepatocellular Carcinoma in Rats
Ashok Kumar K1*, Vijayalakshmi K2, Saravanan D1
1 Department of Biotechnology, School of Life Sciences, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai, Tamilnadu, India.
2Department of Biochemistry, Bharathi Women’s College, Chennai, Tamilnadu, India.
*Corresponding Author E-mail: ashokkumar_kh5@yahoo.com
ABSTRACT:
Plants are the richest source of traditional medicines in treatment against many diseases. Many of the plant materials wasted are the reservoir of phytochemicals possessing important biological activities. In this present study, the extracts of Punica granatum peel and Vitis vinifera a seeds were evaluated in rats against (diethylnitrosamine) DEN induced hepatocellular carcinoma for assessing the hepatoprotective potential. Rats were divided into four groups. The first group served as normal control group, the second group received DEN at a dose of 200 mg/kg body weight by Single intraperitoneal administration. The third one received DEN as in DEN-treated group and co-treated with 400 mg/kg Punica granatum peel extract (EPGP). The final group also received DEN and co-treated with 400 mg/kg Vitis vinifera seeds extract (EVVS). DEN administration to rats resulted in significantly elevated levels of serum AFP, bilirubin, triglycerides, cholesterol and urea. The levels of plasma total protein, albumin and blood glucose were significantly decreased in DEN treated rats. No significant changes were noted in creatinine levels of DEN induced rats. Co-treatment with the Punica granatum peel and Vitis vinifera seed extracts orally for 12 weeks significantly reversed the DEN induced alterations of above mentioned parameters in the serum. The present study indicates that both the selected extracts exhibits good hepato protectivity against DEN induced hepatocellular damage in rats.
KEYWORDS: Vitis vinifera, Punica granatum, Diethylnitrosamine, Alpha feto protein, Hepatocellular carcinoma
INTRODUCTION:
The causes of HCC are chronic viral hepatitis, alcohol, nitroso compounds toxins such as aflatoxin, hemochromatosis and non-alcoholic fatty liver disease5,6,7,8. The survival rate of Liver cancer patients can be improved if we perform early detection and treating them9. Since the liver is the major role in the metabolism of ingested materials, it is more often susceptible to carcinogenic insult. Moreover, due to the high tolerance of liver, HCC is detected at the latter stage and once detected in most cases treatment has a poor prognosis10. DEN is a powerful environmental hepatocarcinogen that has been used as an initiating agent for hepatocarcinogenic activities11 and has been shown to be metabolized to its active ethyl radical metabolite which interacts with DNA causing mutation, which would lead to carcinogenesis12,13. Liver damage can be cured by the use of allopathic medicine, but the disadvantage of allopathic medicine are their side effects, so one has to use the herbal medicine for the treatment purpose14. Hence, there is a great demand for the development of natural anticancer agents.
The pomegranate tree is useful in many ways and almost every part of it possesses medicinal properties which can cure broad classes of diseases. This plant different parts is used in folklore medicine for the treatment of various diseases such ashepatic damage, ulcer, snakebite, etc. The unripe fruit is a good appetizer and tonic, useful in vomiting, but causes biliousness15. Punicagranatum is the Fruit of Energy, Vitality and Medicinal Value. The fruitsmedicinal values have been known since ancient times16. The nature’s power fruit has strong antioxidant, anti-inflammatory, antibacterial properties, recent studies have shown some anticancer activities17. The pomegranate is a symbol of life, longevity, health, femininity, fecundity, knowledge, morality, immortality and spirituality18. The fruit is good for heart, stomach and enhances the production of hemoglobin. It is a good diuretic agent and gives strength and its pulp is a good antidiarrhoeal agents19,20. The dried fruit coat is grounded and mixed with water and taken internally to treat stomachache and diarrhea21. The peels of Punicagranatum are powerful astringent and cure for diarrhea and oral aphthae, and the juicea “refrigerant” 22 and “blood tonic”23. Modern uses of pomegranate derived products now include treatment of acquired immune deficiency syndrome (AIDS),24 and cardiovascular protection25, 26. Pomegranate peel aqueous extract can reduce blood sugar through regeneration of ß cells27. Pomegranate peel is a rich source of tannins, flavonoids, polyphenols and some anthocyanins as delphinidins, cyanidins, etc28.
Vitisvinifera is medicinal plants traditionally used in the treatment of many diseases and disorders in systems of medicine like Ayurveda, Unani, Siddha and Homeopathy 29. V.vinifera is common fruit used in conditions like burning sensations, hemorrhages, anemia, leprosy, skin diseases, syphilis, asthma, Jaundice and bronchitis30,31. The seeds of the grape are used in herbal medicine and as a dietary supplement32. Many literature has been reported that grape seed extract possess a broad spectrum of pharmacological and therapeutic effects such as anti-inflammatory, antioxidative, and antimicrobial activities, as well as having hepatoprotective, cardioprotective, and neuroprotective effects. Vitisvinifera possess potential anti stress activity and pretreatment with the extracts of its seeds extract have shown significant protective affect against cold water swimming stress33. Seedless grape varieties were developed and sold in markets to appeal customers, but research has proved that many of the bioactive properties of grapes may actually come from the seeds.
MATERIALS AND METHODS:
Plant material:
The Punica granatum and Vitis vinifera belongs to families Lythraceae and Vitaceae respectively. These fruits were collected from the Koyambedu market, Chennai, India from the same cultivar. The plants were identified and authenticated by Dr. P. Jayaraman, Director of National Institute of Herbal Science, Plant Anatomy Research Centre, Chennai. These Voucher specimens are maintained in plant anatomy research Centre, Chennai (PARC/2009/360 and PARC/2009/361).
Drugs and chemicals:
Diethylnitrosoamine, Phenobarbital, picric acid, Folinciocalteau’s reagent, bilirubin, sulphanilic acid, Bovine serum albumin, Cholesterol bromocresol green, and Diacetylmonoxime were obtained from Sigma chemicals and other used chemicals were of analytical grade.
Preparation of extract:
P. granatum peel and V. vinifera seeds were separated from the fruits and cleaned thoroughly using sterilized water. The selected plant materials were shade dried and pulverized to fine powder individually in a mechanical grinder. 100g of Course powder was weighed and extracted by keeping it for 24 hours in the orbital shaker maintaining constant RPM at room temperature 34. Both the extracts were collected and filtered using Whatman No.1 filter paper separately. The solvents were evaporated under reduced pressure in a rotary evaporator until solvents are completely evaporated from the extracts. The obtained ethanol extracts were used for further studies.
Selection of animal:
Male wistar rats (150-200 g) were obtained from Kings Institute, Chennai, India and used for the studies. The animals were housed in cages and maintained under standard conditions of humidity, temperature (25.2˚C) and light (12 h light/12 h dark) at BRULAC, Saveetha University, Chennai, India. They were fed with standard pelleted diet (M/s Pranav Agro Industries Ltd., India) and had free access to water. Experimental animals used for the studies were handled according to the instruction in the manual of the Institutional Legislation, regulated by the committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA) under the Ministry of Social Justice and Empowerment, Government of India (IAEC No. Bio chem BWC.004/2009).
Experimental design:
Hepatic cancer was induced in rats using DEN (200mg/kg body weight by i. p) and after two weeks the carcinogenic effect was promoted by 0.05% Phenobarbital, which was supplemented to the experimental animals through drinking water for 20 successive weeks.
The experimental animals were divided in to four groups of seven each.
Group I - Normal vehicle control.
Group II Hepatic cancer induced in rats using DEN (200mg/kg body weight by single i.p. injection). Phenobarbital, which was supplemented to the experimental animals through drinking water up to 20 successive weeks.
Group III Cotreatment with P. granatum peel extract (400 mg/Kg body weight) by oral gavage for 12 weeks.
Group IV Cotreatment with V. vinifera seeds extract (400 mg/Kg body weight) by oral gavage for 12 weeks.
Collection of blood:
The experimental animals used for the study were killed by cervical decapitation after the experimental period. The blood was collected with ethylene diamine tetra acetic acid (EDTA) and without EDTA for the separation of plasma and serum to determine the constituents of blood. The remaining packed cells after the removal of plasma were washed with isotonic saline to remove the buffy coat. 4 ml of packed cells were then washed thrice with isotonic Tris-HCl buffer 0.31 M pH 7.4. Haemolysis was performed by pipetting out the washed red blood cell suspension in to polypropylene centrifuge tubes, which contained hypotonic buffer (Tris-HCl buffer 0.015 M pH 7.2). Erythrocyte ghosts were sedimented using a high speed refrigerated centrifuge at 20,000xg for 40 minutes. The supernatant was aspirated carefully and used for further analysis.
Tumour marker:
The level of serum Alpha feto protein is estimated using ELISA kit supplied from Anogen (Mississauga, Ontario, Canada) following standard procedure provided in kit manual.
Biochemical parameters:
The blood glucose level was estimated by method of35. The level of urea in blood was assayed by the method of 36. Serum creatinine level was estimated by the method of37. Serum cholesterol estimated by Zak’s method38. Bilirubin in serum was estimated by van den bergh reaction39,40. The total protein levels in serum were determined using Folin- Ciocalteau reagent41,42. Serum albumin is estimated by dye binding method43. Triglycerides level in serum was estimated by the method of 44.
RESULTS AND DISCUSSION:
Serum alpha feto protein (AFP) is a useful tumour marker for the detection and monitoring of liver cancer. AFP is a serum protein that is elevated in conditions like hepatocellular carcinoma. Exposure to hepatocarcinogens or hepatotoxins is frequently associated with elevated serum concentration of AFP. This marker protein can be reactivated during liver regeneration and in hepatocellular carcinoma45. AFP has high specificity for hepatocarcinoma. The serum concentration of AFP can be used to confirm hepaticarcinoma and plays a major role in diagnosis of tumor response to therapy. More than 90% of patients with hepatocellular carcinoma have elevated serum AFP levels.
Figure 1 shows the concentration of serum AFP in the animals of different groups selected for study. The result shows elevated level (p<0.001) of AFP in DEN induced group when compared with control. There was significant decrease (p<0.001) in the serum level of AFP in the groups treated with selected extracts (group III and VI) when compared to group II. The genes that control AFP synthesis, is repressed in adult cells and are activated by unknown mechanisms in association with cancer. This phenomenon was designated as an oncofetal gene expression46. Possible explanations for the re-initiation of AFP synthesis by neoplastic hepatocytes include either increased transcription of AFP gene or post-translational modification affecting AFP production. The major control of AFP production is therefore probably at the level of gene transcription. Steady state AFP transcription may begin prior to the development of histologically obvious or symptomatic hepatocellular carcinoma47. AFP gene expression was increased in rat liver during feeding of fumonisin B1, a carcinogenic mycotoxin produced by Fusarium moniliforme.48
Figure 1. Effect of the EPGP and EVVS on the serum AFP in experimental animals Values are expressed as Mean ± SD; n= 7 animals. Comparisons are made between: a - (Control rats) group I; b – (DEN treated rats) group II:Statistical Significance:* p<0.001.
Figure 2. Effect of the EPGP and EVVS on the serum protein and albumin in experimental animals Values are expressed as Mean ± SD; n= 7 animals. Comparisons are made between: a - (Control rats) group I; b – (DEN treated rats) group II: Statistical Significance:* p<0.001.
Blueberries treatment lowers the level of serum AFP in DEN induced hepatocarcinogenesis which was at elevated level and shows chemo preventive activity 49. The AFP concentration was brought near the normal range in the animals treated with EPGP and EVVS extracts. The plasma proteins level may be decreased in certain conditions like liver diseases and its dysfunction. From the Figure 2 it was found that there was a significant decrease (p< 0.001) in total protein in DEN induced animals (Group II) when compared to normal animals (Group I). Group III and group IV, which are treated with selected fruit extracts showed significant increase (p< 0.001) in protein level when compared to group II. The total protein and albumin levels are decreased in hepatotoxic conditions due to disturbances in the carbohydrate, protein and lipid metabolisms. The marked reduction of the total protein synthesis in the HCC induced rats in this study is in agreement with the early observations of decreased ribosomal RNA in DEN induced animals. Since the decrease in RNA occurs in cells where RNA polymerases are activated, defective intranuclear RNA processing is the most likely explanation of the observed decrease 50. The V. vinifera fruit extract increases the level of total serum protein in H2O2 and Methionine induced liver damage where the level of protein in serum was lowered 51. The serum protein level decreases in the CCl4 induced hepatic damage in rats which were significantly increased on treatment with guava leaf52.
Albumin a serum protein transports essential fatty acids from adipose tissue to muscles. It is biosynthesized in the liver. Consequently, decreased albumin levels may be associated with liver disease 53. An albumin deficiency can lead to many medical problems. In this study there was a significant decrease (p< 0.001) in the level of serum albumin in the HCC induced group. The EPGP and EVVS supplemented groups showed significant improvement (p< 0.001) when compared to HCC induced group (Figure 2). Vitisvinifera root extract administration increased the level of albumin in CCl4 induced hepatotoxicity rats as V. vinifera root extract enhanced the synthesis of TP and albumin, which accelerates the regeneration process and the protection of liver cells 54.
Figure 3. Effect of the EPGP and EVVS on the serum bilirubin in experimental animals Values are expressed as Mean ± SD; n= 7 animals. Comparisons are made between: a - (Control rats) group I; b – (DEN treated rats) group II: Statistical Significance:* p<0.001.
The Liver function was assessed by estimating the level of bilirubin in serum. Figure 3 represent the level of bilirubin in serum of control and experimental animals. Bilirubin in group II animals were significantly increased (p<0.001) when compared to control rats (group I) and its level are significantly decreased in EPGP (p<0.001) and EVVS (p<0.001) treated groups when compared to group II. Bilirubin is a byproduct of the breakdown of red blood cells in the liver and is a good indicator of liver function. High levels of bilirubin in serum cause jaundice and are indicative of damage to the liver and bile duct 55. The elevated level of bilirubin in group II (HCC induced) animals in the present study corresponds to the extensive liver damage induced by carcinogen DEN. The bilirubin level significantly decreases in the CCl4 induced hepatotoxic rats on treatment with Cyphostemma glaucophilla leaves where the level of bilirubin was elevated56. Buteamono sperma bark shows hepatoprotective activity against paracetamol induced liver damage and significantly lowers the levels of bilirubin elevated in paracetamol toxicity 57. The reduced level of bilirubin as a result of selected extracts administration observed during the present study might probably be due to the presence of flavonoids and tannins which are documented for its hepatoprotective properties.
Figure 4. Effect of the EPGP and EVV Son the serum cholesterol and triglycerides in experimental animals Values are expressed as Mean ± SD; n= 7 animals. Comparisons are made between: a - (Control rats) group I; b – (DEN treated rats) group II: Statistical Significance:* p<0.001, ** p<0.01.
Lipid plays an important role in the pathogenesis of many complications involved with cancer. The elevated level of serum cholesterol and serum Triglycerides in DEN, poses to be a risk factor for developing micro vascular complication leading to cancer. In the present study the serum cholesterol level was elevated (p<0.001) in group II (Figure 4). The level of cholesterol was significantly decreased in group III and Group IV treated with EPGP (p<0.001) and EVVS (p<0.001) respectively. This clearly shows that the selected extracts reversed the elevated levels of cholesterol on treatment in rats. The level of serum cholesterol increases in Hydrogen peroxide and Methionine induced liver damage. This elevated level lowered significantly on treatment with alcoholic extract of V. vinifera fruit 51. The serum cholesterol level elevates in carbon tetra chloride induced liver damage and the level is significantly lowered by treatment with alkaloid fraction extracted from the seeds of LepidiumSativum58.In the present work, figure 4shows significant increase (p<0.001) in serum TG on DEN induced HCC group. The level of serum TG was lowered (p<0.001) in EPGP and EVVS supplemented groups when compared to DEN treated group. The aqueous Olive Leaf Extract lowers the level of TG in TCA-intoxicated rats that showed elevated serum TG level59. The mechanism of the TG lowering effect may be attributed to many factors such as reducing the oxidative stress by the antioxidant activity of its polyphenols.
Figure 5, 6 and 7 represents the changes of urea, creatinine and glucose respectively in blood samples of control and experimental animals. The urea level (p<0.001) was significantly increased in group II animals when compared to group I animals. The amount of urea excreted depends upon the glomerular filtration rate and when excretion fails the urea level in blood increases. The glucose level (p<0.001) was significantly reduced in group II animals when compared with group I animals. The groups treated with the selected extracts showed significant increase in glucose (p<0.001) and significant decrease in urea (p<0.01) when compared with group II. The cancer cells have an altered metabolism that is the higher rate of glycolysis60. In the present study the glucose levels in the blood of cancer bearing animals is reduced due to the increased uptake of glucose for energy production in the cancer cells because glycolysis is the only energy giving pathway in cancer cells. Increased glycolysis may lead to tumour progression 61.
Figure 5. Effect of the EPGP and EVVS on blood urea of experimental animals Values are expressed as Mean ± SD; n= 7 animals. Comparisons are made between: a - (Control rats) group I; b – (DEN treated rats) group II: Statistical Significance:
*p<0.001
Cancer cells need large amounts of glucose to progress because cancer cells undergo anerobic respiration. As the tumor progress and the cancer cell uses more and more glucose resulting in hypoglycemic conditions. The development of hypoglycemia due to increased glycolysis was observed in experimental animals with carcinoma 62,63,64.
Figure 6. Effect of EPGP and EVVS on blood glucose of experimental animals. Values are expressed as Mean ± SD; n= 7 animals. Comparisons are made between: a - (Control rats) group I, b – (DEN treated rats) group II: Statistical Significance:* p<0.001
The creatinine level is increased in DEN induced group when compared to control group but to non-significant level. The EPGP and EVVS treated groups showed slight decrease when compared to positive control. The increased levels of serum creatinine in DEN induced group may be due to insufficiency of glomerular to filter the proteins through its structure and increased muscle tissue catabolism or impairment in carbohydrate metabolism 65, 66. Treatment with the aqueous leaves extract of Cyphostemma glaucophilla against CCl4 induced liver damage in rats decreases the level of creatinine which was increased on CCl4 induction. 56
Figure 7. Effect of the EPGP and EVVS on serum creatinine of experimental animals Values are expressed as Mean ± SD, n= 7 animals. Comparisons are made between: a - (Control rats) group I, b – (DEN treated rats) group II: Statistical Significance: NS - Non-significant
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Received on 21.12.2018 Modified on 31.01.2019
Accepted on 23.02.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(6): 2758-2764.
DOI: 10.5958/0974-360X.2019.00462.1