|Year : 2018 | Volume
| Issue : 1 | Page : 83-88
Hepatoprotective effect of beetroot juice on liver injury in male Sprague–Dawley rats
Fidelis E Olumese, HA Oboh
Department of Medical Biochemistry, School of Basic Medical Sciences, University of Benin, College of Medical Sciences, Benin City, Edo State, Nigeria
|Date of Web Publication||11-Jun-2018|
Dr. Fidelis E Olumese
Department of Medical Biochemistry, School of Basic Medical Sciences, University of Benin, College of Medical Sciences, Benin City, Edo State
Source of Support: None, Conflict of Interest: None
Background: The ability of plant extract to improve injury in the liver has gained interest in recent times. This could be due to the side effects and expense of modern medicines that are used to manage hepatic diseases; hence beetroot juice as a potential hepatoprotective agent was evaluated. Materials and Methods: Thirty Sprague–Dawley rats were assigned by weight into six groups average body weight (160.20 ± 2.54 g). Group I: rats plus distilled water (Normal control) 2 ml/kg; Group II: rats received olive oil (2 ml/kg); Group III: rats received carbon tetrachloride (CCl4) suspended in olive oil (2 ml/kg) and 250 mg/kg of beetroot extract. Group IV: rats received CCl4suspended in olive oil (2 ml/kg) and 500 mg/kg of beetroot extract. Group V: rats received olive oil (2 ml/kg) and 100 mg/kg of silymarin; Group VI: rats received (2 ml/kg)CCl4suspended in olive oil. Liver injury was induced by oral administration of CCl4using gastric gavage at 2 ml/kg every 48 h for 14 days, followed by treatment with beetroot extract and silymarin. Animals were euthanized by decapitation, blood and liver tissue harvested for biochemical and histopathological evaluations. Results: Alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels were significantly (P < 0.05) increased in the groups treated with extract and silymarin when compared to the animals administered only CCl4, whereas malondialdehyde level was significantly (P < 0.05) increased in the group administered only CCl4when compared to other groups. Histopathologically, the animals treated with 500 mg/kg of extract showed a normal hepatocyte and mild portal congestion. Conclusion: Beetroot juice has potential hepatoprotective effects on the liver in a dose-dependent manner.
Keywords: Beetroot juice, carbon tetrachloride, hepatoprotective, liver injury
|How to cite this article:|
Olumese FE, Oboh H A. Hepatoprotective effect of beetroot juice on liver injury in male Sprague–Dawley rats. Ann Trop Pathol 2018;9:83-8
| Introduction|| |
The well-documented health benefits of a diet high in fruit and vegetables have led to a growing interest in functional foods and their application in health and disease. Herbal medicine is gaining popularity in developing countries as it has been estimated that 80% of the world population still depend mainly on traditional medicine and traditional treatment involving the use of plant extract. Nigeria is richly endowed with indigenous plants which are used in herbal medicine to cure diseases and heal injuries. Some of the plants are used as food or medicine. These plants exhibit a wide range of biological and pharmacological activities such as anti-cancer, anti-inflammatory, diuretic, oxytocic, laxative, antispasmodic, antihypertensive, antidiabetic, and antimicrobial functions.
Cultivars of Beta vulgaris are grown throughout Europe and North America and in some part of West Africa. Beetroots are grown mainly in Jos, Plateau State of Northern Nigeria. This could be probably because beets develop best under cool conditions. Beets are grown primarily for the enlarged bulbous root, which forms near or just above the soil surface. The plant is naturally a biennial, producing a rosette of leaves and a bulbous root one year, and a seed stalks the following year. Beets and their relatives are grown throughout the world for human and stock food. Sugar beets and Chard are among the more familiar types.
Beetroot is a rich source of phytochemical compounds that includes ascorbic acid, carotenoids, phenolic acids, and flavonoids.,, Beetroot is also one of the few vegetables that contain a group of highly bioactive pigments known as betalains., A number of investigations have reported betalains to have anti-inflammatory and antioxidant capabilities in vitro and a variety of in vivo animal models.,, This has sparked interest in a possible role for beetroot in clinical pathologies characterized by oxidative stress and chronic inflammation such as liver disease,, arthritis, and cancer.,,, Beetroot's effect on the vascular endothelium of the blood vessels increases the blood flow, and this is largely attributed to its high inorganic nitrate, betaine and NO contents. Nitrate is not the only constituent of beetroot proposed to have beneficial effects in health and disease.
The liver as a vital organ in the body is primarily responsible for the metabolism of endogenous and exogenous agents. It plays an important role in drug elimination and detoxification and liver damage may be caused by xenobiotics, alcohol consumption, malnutrition, infection, anemia, and medications. Hepatotoxic agents can react with the basic cellular components and consequently induce almost all types of liver lesions. Chemical toxins (including acetaminophen, carbon tetrachloride (CCl4), galactosamine, and thioacetamide) are often used as the model substances causing experimental hepatocyte injury in both in vivo and in vitro conditions., CCl4 treatment results in centrilobular steatosis, inflammation, apoptosis, and necrosis.,, If the damage exceeds the repair capacity of the liver, it will progress to fibrosis and cirrhosis.,
A number of studies have shown that plant extracts having antioxidant activity protect against CCl4 hepatotoxicity by inhibiting lipid peroxidation and enhancing antioxidant enzyme activity. This study is to determine the hepatoprotective effect of aqueous extract of Beta vulgaris on Sprague–Dawley rats.
| Materials and Methods|| |
Plant material and extraction
Beetroot (Beta vulgaris) was obtained from a local vendor in a vegetable market on Airport Road, Benin City, Nigeria. The plant was identified by a Taxonomist at the Department of Plant Biology and Biotechnology at the University of Benin and a voucher number UBHB374 was deposited in the Herbarium. The beetroots were rinsed with clean water, peeled, and blended. The juice was extracted from the beetroot using a muslin cloth and distilled water and the shaft discarded. The juice obtained was stored in an airtight container (10 kg of beetroot yielded approximately 3 L of juice after extraction) and allowed to settle for 20–30 min. The juice obtained was freeze dried [Armfield vacuum freeze dryer Model FT 33, England] and ready for use. The freeze-dried sample is composed of a 10% beetroot juice containing 9808.0 mg GAE/100 ml polyphenols and 8334.0 mg QE/100 ml flavonoids. The freeze-dried sample was stored at 0°C until ready for use.
The freeze-dried beetroot was screened for the presence of saponins (foam test), alkaloids (Dragendorff's reagent), tannins (gelatin test), phenols (ferric chloride test), flavonoids (lead acetate test), and phytosterols (Keller–Killani and the Salkowski test)., A 10 g freeze-dried beetroot sample was dissolved in 100 ml distilled water, methanol, ethanol, and chloroform, respectively. Thereafter, they were homogenized to a uniform consistency with an Ultra-Turrax homogenizer (USA). The filtrate obtained from the filtered extract was used for the screening.
Thirty healthy Sprague–Dawley rats, male and female of average body weight (160.20 ± 2.54 g) were grouped according to sex and weight into six groups of five animals each. They were obtained from the animal house in the Department of Anatomy, University of Benin, Benin City, Nigeria. The animals were housed in spacious plastic cages with animal beds in the animal house of the Department of Medical Biochemistry, University of Benin, Nigeria. The animals were acclimatized for two weeks and maintained under standard conditions of temperature (23°C ± 2°C) and (12 h light/dark cycle). The rats were fed standard pelleted feed (Top Feeds; Premier Feed Mills Co. Ltd., Ibadan, Nigeria) for two weeks. Before the study, food and water were given ad libitum. Approval for the study was obtained after a review of the protocol by the Ethical Committee of the College of Medical Sciences, University of Benin, Benin City, Nigeria, with number CMS/REC/2016/002.
Thirty healthy Sprague–Dawley male rats were assigned by weight into six groups (five rats per group, average body weight [160.20 ± 2.54 g]). The six groups are composed as follows: Group I: Healthy rats that received 2 ml/kg distilled water (normal control); Group II: Healthy rats that received 2 ml/kg olive oil (normal plus, olive oil); Group III: Healthy rats which received 2 ml/kg CCl4 in olive oil (negative control); Group IV: Healthy rats that received 2 ml/kg CCl4 in olive oil and 250 mg/kg body weight of beetroot extract/day. Group V: Healthy rats which received 2 ml/kg CCl4 in olive oil and 500 mg/kg body weight of beetroot extract/day. Group VI: Healthy rats that received 100 mg/kg body weight of silymarin/day and 2 ml/kg CCl4 in olive oil (positive control). Liver injury was induced by the oral administration of 40% concentration of CCl4 in olive oil (GOYA Extra Virgin Olive oil, Manufactured by Goya En Espana, S. A. U. Sevilla, Spain) which served as a vehicle at a dose of 2 ml/kg body weight every 48 h (between 8.00 am and 9.00 am) for 14 days using gastric gavage which was modified. Animals were fasted overnight and euthanized by decapitation. Blood was collected for biochemical evaluation, while the liver was dissected, freed of adherent tissues, and weighed.
Determination of serum markers in liver damage
The serum from each group was used to determine the levels of alanine aminotransferase (ALT: Randox, UK. Cat. No AL 100), aspartate aminotransferase (AST: Randox, UK. Cat. No AS 101), alkaline phosphatase (ALP: Teco Diagnostics, USA. A506), and total protein (Randox, UK. Cat. No TP 245) using commercial kits according to the manufacturer's direction.
Measurement of lipid peroxidation in liver tissue
The extent of lipid peroxidation was determined by measuring thiobarbituric acid (TBA) reactive substances, in terms of malondialdehyde (MDA) formation. To 0.6 ml of the liver homogenate sample in test tubes, 0.3 ml of trichloroacetic acid (TCA)-TBA-HCl mixture was added and incubated in laboratory water bath (SurgiFriend Medicals, England) at boiling temperature for 15 min. The content of each test tube was allowed to cool and then centrifuged for 10 min to remove flocculent precipitates (Centrifuge Model 80-2; Uniscope SM 801 A at 2130 ×g, Max speed: 4000 r/min, CHINA). The absorbance of each supernatant was read at 532 nm against a blank which contained 0.3 ml of TCA-TBA-HCl solution dissolved in 0.6 ml of distilled water. The concentration of MDA was determined.
The liver harvested was trimmed of any adherent tissue and placed in sterile containers, containing 10% buffered formalin. The tissues were cut so that they can enter the cassettes and the areas of interest exposed for processing. The processing was done by removing the tissue from buffered formalin and dehydrated by passing it through graded concentration of alcohol starting from the lowest (70%, 90%, 95%, and absolute). The alcohol in the tissues was cleared with xylene and the latter was replaced with paraffin wax by impregnation. Staining was done with hematoxylin and eosin, microtome was used to section at 5 μ and trimmed 10 microns. The processed tissues on the slides were analyzed microscopically.
Biochemical data were expressed as mean ± standard error of the mean. The difference between the groups was tested using ANOVA. Duncan's multiple range test was used to test for significant difference among the means (P < 0.05).
| Results|| |
The phytochemical screening of beetroot revealed the presence of phenols, alkaloids, tannins, flavonoids, saponins, phytosterols, and glycosides. The phytochemical determination of beetroot as assessed is contained in [Table 1]. The weight of liver was significantly (P < 0.05) reduced in the group of animals administered only olive oil when compared to other groups [Table 2]. The ALT level was significantly (P < 0.05) increased in the group of animals administered 250 mg of beetroot extract and 100 mg/kg body weight of silymarin when compared to the animals that were treated with only CCl4[Figure 1]. There was a significant (P < 0.05) increase in AST level on administering 250 and 500 mg/kg body weight of extract when compared to the group that was treated with only CCl4 [Figure 1]. The level of ALP was significantly (P < 0.05) reduced at 250 mg/kg of extract, while at 100 mg/kg of silymarin it was significantly (P < 0.05) increased when compared to negative control [Figure 1].
|Table 2: Effect of beetroot extract on relative liver weight in Sprague-Dawley rats|
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|Figure 1: Effect of oral administration of different doses of beetroot extract and silymarin on serum liver enzymes in Sprague–Dawley rats. Serum concentrations of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in the liver were expressed as mean ± standard error of the mean. Values with different superscripts are significantly different when P < 0.05. Group 1 (normal rats + distilled water); Group II (normal rats + olive oil); Group III (normal rats + CCl4); Group IV (normal rats + 250 mg extract + CCl4); Group V (normal rats + 500 mg extract + CCl4); Group VI (normal rats + 100 mg silymarin + CCl4)|
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The concentration of total protein was significantly (P < 0.05) reduced in the group of animals administered 500 mg of beetroot extract when compared to the other groups [Figure 2]. Nevertheless, the level of MDA was significantly (P < 0.05) increased in the group of animals that were administered only CCl4 when compared to the tested doses of extract and silymarin [Figure 3].
|Figure 2: Effect of oral administration of different doses of beetroot extract and silymarin on serum protein in Sprague–Dawley rats. Serum concentration of protein was expressed as mean ± standard error of the mean. Values with different superscripts are significantly different when P < 0.05. Group 1 (normal rats + distilled water); Group II (normal rats + olive oil); Group III (normal rats + CCl4); Group IV (normal rats + 250 mg extract + CCl4); Group V (normal rats + 500 mg extract + CCl4); Group VI (normal rats + 100 mg silymarin + CCl4)|
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|Figure 3: Effect of oral administration of different doses of beetroot extract and silymarin on malondialdehyde concentration in the liver homogenate of Sprague–Dawley rats. Homogenate concentration of in the liver was expressed as mean ± standard error of the mean. Values with different superscripts are significantly different when P < 0.05. Group 1 (normal rats + distilled water); Group II (normal rats + olive oil); Group III (normal rats + CCl4); Group IV (normal rats + 250 mg extract + CCl4); Group V (normal rats + 500 mg extract + CCl4); Group VI (normal rats + 100 mg silymarin + CCl4)|
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The histological examination of the liver of rats treated with aqueous extract of beetroot showed a well-formed normal hepatocyte and a mild portal congestion at 500 mg/kg body weight compared to the controls (Plates 1, 2, and 3) [Figure 4].
|Figure 4: Photomicrograph of sections of the rat liver after 14 days of treatment with silymarin, 250 mg and 500 mg of extract (plates 4, 5, and 6, respectively) compared to control (normal, olive oil and CCl4;Plates 1, 2, and 3, respectively). Plates 1 and 2: A-hepatocyte, B-sinusoids, C-central vein. Plate 3: A-macrovesicular steatosis, B-moderate vascular congestion, C-mild periportal infiltrates of inflammatory cells. Plate 4: A-patchy macrovesicular steatosis (100 mg silymarin). Plate 5: A-patchy macrovesicular steatosis (250 mg of extract). Plate 6: A-normal hepatocyte and B-mild portal congestion (500 mg of extract). Sections were stained with H and E ×400|
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| Discussion|| |
To ascertain whether the freeze-dried beetroot juice extract would ameliorate liver damage, rats were treated with 250 and 500 mg of the beetroot extract and 100 mg silymarin. CCl4 intoxication is used for modeling liver injury in rats. Hepatotoxicity of CCl4 is the result of cytochrome P-450 dependent reductive dehalogenation to form a highly reactive trichloromethyl free radical CCl3. CCl4-induced damage is characterized by hepatocyte membrane damage, caused by lipid peroxidation, increased plasma levels of hepatic enzymes, fatty degeneration (steatosis, i.e. accumulation of triglycerides in the liver), reduced β-oxidation of fatty acids, and necrosis.,
MDA is a reactive aldehyde that could be cytotoxic. Since it is formed as a by-product of lipid peroxidation, it can also be used as an indicator of the amount of lipid peroxidation. The free radicals produced by the metabolism of CCl4 attack polyunsaturated fatty acids in the cellular membrane, initiates lipid peroxidation, and ultimately resulting in a loss in membrane integrity., In this study, we found that the organ weight ratio were significantly (P < 0.05) increased in negative, positive, and treated groups when compared to the control groups; this may be due to the highly reactive trichloromethyl free radical generated from CCl4 which had predilection for liver membrane, thereby eliciting inflammatory processes.
A single dose of CCl4 leads to centrizonal necrosis and steatosis, while prolonged administration leads to liver failure, cirrhosis, and hepatocellular carcinoma. Overproduction of trichloromethyl-free radicals is considered the initial step in a chain of events that eventually lead to membrane lipid peroxidation and finally to cell apoptosis and necrosis., The evidence accumulating presently suggests that free radicals can oxidatively damage mitochondria, regulate gene expression, thereby contributing to the development of fibrosis and can perpetuate chronic inflammatory processes. In chronic hepatocyte injury, mainly in cirrhosis, ALT is more commonly elevated than AST, however, as fibrosis progresses, ALT activities typically decline, and the ratio of AST to ALT gradually increases, so that by the time cirrhosis is present, AST is often higher than ALT., Increases in serum AST, ALT, and ALP levels by CCl4 have been attributed to hepatic structural damage because these enzymes are normally localized to the cytoplasm and are released into the circulation after cellular damage has occurred. Cellular membranes that are weak allow sufficient leakage of calcium into the cytosol which disrupts intracellular calcium homeostasis. High calcium levels in the cytosol activate calcium-dependent proteases and phospholipases that can increase the breakdown of membranes. This ultimately increases intracellular calcium, which activates endonucleases that can cause chromosomal damage and also contribute to cell death. In this study, the level of AST was significantly (P < 0.05) reduced in the group administered only CCl4 when compared to the groups treated with beetroot juice extract and silymarin. This reduction is probably not unconnected with the necrosis and steatosis processes that may have occurred in the liver, however, on the administration of the tested doses of extract and silymarin, there was an upregulation of the enzyme activities. Similarly, the leakage of ALT from a compromised liver that has undergone chemical assault will have its concentration highly elevated in the serum. In end-stage cirrhosis, the levels of ALT and AST enzymes are generally not elevated and may be low due to massive tissues destruction. Interestingly, this study showed that the activity of ALT was significantly (P < 0.05) reduced in the group of rats that had only CCl4. However, the group of animals treated with 250 mg/kg body weight of beetroot extract/day for 14 days showed a significant (P < 0.05) increase in ALT when compared to the negative control. It was also observed that at 500 mg of extract and 100 mg/kg body weight of silymarin, there was upregulation of ALT concentration. It is proposed that these increases may be due to the extract's ability to regenerate liver tissue by interrupting necrosis or fibrosis processes, thereby enhancing the production of new cells. The observed elevation of ALP in the groups treated with CCl4 may be due to the stimulation of its production from other sources other than the liver. Total protein is a quantitative measurement of the concentration of all proteins present in serum which excludes clotting factors. It suggests the presence of an autoimmune component to chronic liver disease. Total protein could be elevated in a liver that has been assaulted with CCl4. An increase in protein breakdown with an overall fall in whole-body protein turnover has been reported in patients with alcoholic cirrhosis. When there is an elevated total proteins, it is an indication of increased catabolism of other proteins, in addition to collagen. This catabolism that is enhanced may be due to the extreme centrilobular necrosis of the liver tissue during hepatic fibrosis. This study showed that at 500 mg/kg body weight of beetroot extract/day, there was a significant (P < 0.05) reduction in the serum total protein when compared to the group of animals that were treated with 250 mg of extract and silymarin. This healing effect of the tested dose (500 mg) on the liver may be that the extract was able to prevent lipid peroxidation and the accumulation of MDA which enhances collagen synthesis in hepatic fibrosis.
The elevation of MDA level in rats treated with CCl4 is attributed to enhanced lipid peroxidation, leading to tissue damage and failure of antioxidant defense mechanisms to prevent the formation of excessive free radicals. In this study, the level of MDA was significantly (P < 0.05) reduced in the group of rats administered beetroot extract or silymarin when compared to the negative control. The overwhelming significant (P < 0.05) reduction in MDA on administering 500 mg/kg body weight of beetroot to the rats may be attributed in part to its antioxidant and free radical scavenging abilities. This is evidenced in our histopathological evaluation which revealed that at a dose of 500 mg/kg body weight, there was a normal hepatocyte when compared to the 250 mg of extract and positive control (silymarin-treated group).
| Conclusion|| |
The results obtained from this study suggest that oral administration of 250 or 500 mg/kg of beetroot extract can ameliorate rat liver injury through possibly the interruption of apoptosis machinery or secondary necrosis. Further studies will be required to isolate its active ingredients and determine the mechanism of action.
We thank Tetfund for supporting us with a grant that enabled this research and Dr. G. I Eze of Anatomy Department, University of Benin, for expert assistance in the preparation of the slides, color photomicrographs, and its interpretation.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]