EFFECT OF ANTIOXIDANTS AND HEPATOPROTECTIVE ACTIVITIES OF METHANOL EXTRACT OF BEET ROOT (BETA VULGARIS L.) AGAINST CARBON TETRACHLORIDE INDUCED HEPATOTOXICITY IN RAT MODELSHTML Full Text
EFFECT OF ANTIOXIDANTS AND HEPATOPROTECTIVE ACTIVITIES OF METHANOL EXTRACT OF BEET ROOT (BETA VULGARIS L.) AGAINST CARBON TETRACHLORIDE INDUCED HEPATOTOXICITY IN RAT MODELS
Maximas H. Rose 1, P.N. Sudha*2 and K. Sudhakar 3
M.S University 1, Trinelveli, Tamil Nadu, India
Research Department of Chemistry, D.K.M. College for Women 2, Vellore, Tamil Nadu, India
Department of Zoology, Government Arts College 3, Thiruvannamalai, Tamil Nadu, India
ABSTRACT: A study on the effect of methanol extract of red beetroot (MEBV) against CCl4 induced hepatotoxicity in male albino rats of Wistar strain (150 to 180g) was done. Five groups with six rats each were used as positive control, negative control and experimental with an oral dose of CCl4 throughintra-peritoneal (IP) route and MEBV at 100µgm/ml, 200µgm/ml & 300µgm/ml per os, respectively, for 14 days. After 14 days, animals were sacrificed; blood samples were obtained and analyzed. The results showed that CCl4 administration was associated with triple fold increases in the activities of alanine amino transferase (ALT), aspartate amino transferase (AST) and Bilirubin (P<0.05) compared with the respective mean control values. The enzymatic and non-enzymatic antioxidants such as, serum catalase, GSH, Vit. E, Vit.C, & SOD, were lower than the respective mean values of the normal control. The blood parameters have got reduced. The hapatochords and hepatocytes architecture was also observed to be disturbed. However, treatment with MEBV restored the enzyme activities of the liver ALT, AST, Bilirubin, WBC, RBC, PCV and hepatic lobule architecture, to a very near normal level. This work showed that methanol extract of Beta vulgaris was successful at 300µgm/ml, in counteracting CCl4 induced hepatotoxicity and amelioration of the same (hepatic remediation) to near normalcy level in rat liver functions and amelioration may be due to the total phenolics, flavonoids and other antioxidants composition in the plant extract.
Antioxidants, Alanine amino-transferases, Aspartate transaminases, Glutathione, Super Oxide Dismutase, Catalase, Hepatotoxicity, Beta vulgaris
INTRODUCTION:The largest exocrine gland of our body, the liver, plays vital functions in association with homeostasis of the body.
Anabolic and catabolic pathways of nutrients that we consume and de-toxification of ingested food based chemicals are taken care by our liver. The variety of ingested chemicals induces liver injury by mostly causing oxidative stress in hepatic tissue and accounts for numerous diseases, including cancer.
Considering the fact of widespread and casual abuse of liver, like environmental toxins, prescription and over-the-counter drug use, which lead to hepatitis, cirrhosis and liver disease, more research light is thrown in the use of antioxidants for prevention and/or amelioration of hepatic injury 1. This amelioration process is often referred to as “The chemoprevention”, and a large body of evidence from various experiments has supported its efficiency 2.
Carbon tetrachloride (CCl4) is one of the most potent hepatotoxins and is widely used in scientific research to evaluate hepatoprotective agents 3. The hepatotoxic effect of CCl4 is largely due to its active metabolite, trichloromethyl free radical (CCl3 – and / or CCl3OO–) by chronic or acute vehicles 4. The regular exposure to hepatotoxic substances causes hepatocellular lipid accumulation (steatosis), hepato-cellular necrosis or hepatobiliary dysfunction5. Cirrhotic or neoplastic changes are usually considered as a result of chronic exposures. Peroxidation was first proposed as mechanism of carbon tetrachloride induced liver injury.
The CCl4 is shown to induce hepatocellular carcinomas in rodents by oral, inhalation, and parenteral exposure 3. The hepatotoxic effect of CCl4 is a result of its reductive dehalogenation by CYP2E1 enzyme into the highly reactive trichloromethyl and trichloromethyl peroxy free radicals. Both radicals form adducts with cellular macromolecules and abstract hydrogen from different molecules, thus initiating oxidation of lipids, proteins and DNA. These observations are consistent with the hypothesis that toxicants-induced hepatocellular injury results in the influx of Ca2+ (calcium ion) into the cell, initiating a series of cyto-toxic events common to various hepato-toxicants and resulting in cell death.
Several drugs and chemicals inhibit hepatocyte antioxidant defines against lipids peroxidation, thus increasing the severity and duration of peroxide damage 6.
Enzyme substances of liver like ALT (SGPT), AST (SGOT), and Bilirubin are also present in blood serum in very low concentration. These enzymes provide information about hepatic health 7. Liver diseases were among the first disorders to which serum test were applied and have proved to be useful in diagnostic purposes.
The red beetroot, (Beta vulgaris L.), a flowering plant of Amaranthaceae family, is widely available vegetable and is commonly consumed. It is reported to have water-soluble, non-phenolics antioxidants, the betalins, red betacyanins (principally betanin) and yellow betaxanthines, in its consumable leaves and roots 8. The antioxidant effects of betalains have been demonstrated mainly in various in vitro experiments. As very few investigations on beetroot activity have been performed in vivo, the present study is aimed at an evaluation of methanolic extract of Beta vulgaris (MEBV) and its anti-oxidants in ameliorating the induced hepatotoxicity to CCl4 exposure and circulating liver marker enzyme levels and proteins using Wistar albino rat models.
MATERIALS AND METHODS:
Chemicals and reagents: CCl4 , 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), 2,2-diphenyl-1picrylhydrazyl (DPPH), Anthrone Reagent, Folin-Ciocalteau Reagent, Ninhydrin, thiobarbituric acid (TBA) and 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), Methanol, 2,4- dinitrophenyl hydrazine-thiourea-CuSO4 reagent, and isobutyl alcohol were procured from Sigma-Aldrich Chemicals (Bangalore). All other chemicals and estimation kits used were of analytical grade and purchased from commercial sources.
Plant Material: Beetroot (Beta vulgaris L.) were purchased from a local market, Vellore, Tamil Nadu, India and the plants were authenticated by the Botany Department, Govt. Arts and Science College, Thiruvannamalai (Thiruvalluvar University), Tamil Nadu, India. The plant materials were washed in tap water, shade dried and powdered.
Extraction and fractionation: The powdered root material (800 g) was successively extracted with 95% methanol using Soxhlet extractor. The marc left after the methanol extraction was macerated with distilled water for 24 h. The solvents were distilled off under reduced pressure below 45ºC to afford Beta vulgaris methanol extract (MEBV, 12.7% w/w).
Preliminary phytochemicals screening: Preliminary phytochemicals analysis was performed to identify the nature of phyto-constituents in the methanol extract of beetroot (Beta vulgaris L.) 9.
- Antioxidant enzymes assay (DPPH scavenging assay): The free radical scavenging activity of methanol extract of Beta vulgaris (MEBV)was measured in vitro by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method 10. The extract of the plant was prepared at various concentrations (0.8, 4, 20 and 100 µg/ml) and added to a solution of 1.5 x 10-4m DPPH (sigma, Bangalore) in methanol and the reaction mixture was shaken vigorously. The amount of DPPH remaining was determined at 520nm and the radical scavenging activity was obtained from the equation. The absorbance (Abs) was taken at 520 nm. Quercetin, a known antioxidant was used as positive control. The amount of DPPH remaining was determined at 520nm and the radical scavenging activity was obtained from the equation.
| OD control – OD sample
= x 100
Radical Scavenging activity (%)
- Determination of total phenolics and flavonoids content: Total phenolics assay by Folin-Ciocalteu reagent method is routinely employed in studying phenolics antioxidants 11. The total phenolics and flavonoids content of plant extracts were determined as per Folin-Ciocalteau assay method 12. The total phenolics content was expressed as milligrams of gallic acid equivalents/g extract (mg GAE/g of dry mass) and the total flavonoids content was expressed in milligrams of quercetin equivalents/g of extract (mg QE/g of dry mass).
Experimental animals: The experimental animals used for the study were male Wistar rats Rattus norvegicus, weighing (160–180 g) and procured from Kings Institute of Preventive Medicine, Guindy, Chennai, and fed with standard rat chow (Amrut Laboratory Animal Feed, Bangalore, India) and water ad libitum. Forty eight animals were used for the experiment and maintained as per the Committee for the purpose of control and supervision of Experiments on Animals (CPCSEA) guidelines.
Animals were acclimatized for one week prior to experiment. The animals were maintained under standard laboratory conditions of temperature (25 ± 2%) and humidity (55 ± 5%) with 12 h light- dark cycle.
- Determination of LD50 of CCl4 for Male Wistar rats: The acute oral toxicity studies of Beta vulgaris extract was carried out as per OECD guidelines. The rats were orally fed with different doses of CCl4and the LD50 value was calculated as per the method of OECD – 423 guidelines and was found to be 2400ml/kg body weight for a period of 14 days (OECD – 423, 2001). The rats were treated with 1.9ml/kg of CCl4 daily intra-peritoneal (IP) to induce hepatotoxicity 13.
In vivo hepatoprotective activity: The rats were randomly divided into five groups, each consisting of five rats and treated as follows:
- Group I fed with normal diet and distilled water (served as positive control).
- Group II received CCl4 100mM (IP), (served as negative control)
- Group III received CCl4 100mM (IP) and 100μgm/ml (MEBV) p.o.,
- Group IV received CCl4 100mM (IP) and 200μgm/ml (MEBV) p.o.,
- Group V received CCl4 100mM (IP) and 300μgm/ml (MEBV) p.o.,
The rats were maintained in the above condition for 14 days and on the 15th day, the rats were anesthetized and sacrificed. Then the blood and liver samples were collected, processed, analyzed for various biochemical factors and antioxidant enzymes in the blood samples and they were duly reported.
Liver marker enzymes and Blood sampling assay: Activities of Alanine transaminase (ALT) or Serum Glutamic Pyruvic Transaminase (SGPT) and Aspartate aminotrasferase (AST) or Serum Glutamic Oxaloacetic Transaminase (SGOT) and Bilirubin levels were determined in serum proteins for the study 7.
However, the level of tissue toxicity is tested as Level of lipid peroxidation (LPO) and expressed in terms of GPx (Glutathione peroxidase), Glutathione (GSH) Superoxide dismutase (SOD) and catalase (CAT) 14. They were determined by the standard methods to assess oxidative stress, for the ‘in vivo’ subjects.
The blood samples were also analyzed for the red blood cells count, white blood cells count, packed cell volume (PCV %) also known Hematocrit (HCT) as per standard methods 14. The whole blood samples were collected into heparinized capillary tubes, filled up to 2/3 the length, sealed and centrifuged at 3,000rpm in a haematocrit centrifuge for 10 minutes. Packed cell volume was determined using a haematocrit reader and PCV was expressed as percentage erythrocytes blood contained and the haemoglobin concentration was estimated as per using the formula.
Hb concentration = ⅓ x PCV concentration
Histopathological Examination: Following standard procedure, the histopathological studies were done 15. For the observations at the light microscopic level, fresh tissue pieces of liver were fixed in Bouin’s fluid. Following two days of fixation, the specimens were washed and dehydrated through an ascending series of ethanol (70 – 100%).
Then, they were cleaned with xylene and embedded in paraffin wax, then sectioned at 5μm thickness using a rotary microtome. Sections were rehydrated in distilled water and stained with Hematoxylin-Eosin (H&E) and then examined under light microscopy.
Statistical analysis: The results were expressed as mean ± SE (n=6) and analyzed by one-way analysis variance (ANOVA) 16. The significant was considered to be as, p < 0.05 vs control.
Preliminary phytochemicals screening: Screening plant extracts for their antioxidant potency in order to identify their ability in scavenging free radicals (ROS) and binding with metal ions of oxidative reactions is considered as an important step prior to the isolation of antioxidant phytochemicals 17.
In the preliminary phytochemicals analysis, methanol extract of Beta vulgaris showed the presence of various phytoconstituents (Table 1 & Fig. 1). It was observed that, carbohydrate, protein, fat, total amino acid content and proline content are moderately high. The carbohydrates were 41.3g/ 100g, protein was 14.3g/100g, fat was 1.79g/100g and total amino acid content was (15.9 g/100g), indicating the phytoconstituents were moderately available in beetroot.
TABLE 1: BIOCHEMICAL FACTORS OF BETA VULGARIS (g/100g)
|Carbohydrates||41.3 (g/100g )|
|Protein||14.3 (g/100g )|
|Fat||1.79 (g/100g )|
|Total amino acid||15.9 (g/100g )|
FIG. 1: LIGHT MICROGRAPH OF A HE STAINED LIVER OF A POSITIVE CONTROL RAT ×300
Table 2 represents the Vitamins and Minerals Factors of the leafy parts of the plant Beta vulgaris. All the non – enzymatic antioxidants such as Total phenolics, b- Carotene, Ascorbic acid, Thiamine and Flavonoids were found to increase in high content.
Vit. C is measured to be at 441mg/100g, sodium at 454mg/100gm, calcium at 385mg/100g, Iron 11.6mg/100g, total ash at 4.8g/100g, b- Carotene and Thiamine at 32.6 and 0.28 µg/100g, respectively. The metal elements and vitamins have impact on various physiological routes involving non-enzymatic antioxidants 18.
Effect of MEBV on Antioxidant Activity (DPPH assay): The DPPH assay is the widely used screening method for antioxidant activity of plant extracts 19. DPPH is relatively stable, nitrogen-centered free radical which produces violet color in ethanol solution. It will be reduced to a yellow colored product, diphenylpicryl hydrazine, with the addition of the plant extracts in a concentration dependent manner. The reduction in the number of DPPH molecules can be correlated with the number of available hydroxyl groups. The Detoxification of excess Reactive Oxygen Species (ROS) produced during stress is important as it may induce membrane lipid peroxidation, enzyme inhibition and nucleic acid damage 20.
TABLE 2: VITAMINS AND MINERALS FACTORS OF BETA VULGARIS
|beta Carotene||32.6 (µg/100g)|
|Ascorbic acid||441 (mg/100g)|
|Total Ash||4.8 (g/100g)|
Table 3 shows the antioxidant properties which include the (1, 1-diphenyl-2- picrylhydrazyl) DPPH assay, the antioxidant enzymes such as catalase and superoxide dismutase (SOD) and the non-enzymatic antioxidant glutathione. The DPPH assay shows that MEBV has a 67% antioxidant activity. Similarly the other antioxidant enzymes such as SOD, GSH and Catalase showed decent levels of content [MEBV = SOD - 214mgm/ minute/ mg protein; the catalase activity was 56.9µmol/min/g; and Glutathione of MEBV was 56 mg/g;] the extract showed significant inhibition percentage (stronger hydrogen donating ability) and may be positively correlated with total phenolics content (TPC) 10.
TABLE 3: ANTIOXIDANTS ACTIVITY, TOTAL PHENOLICS AND FLAVONOIDS CONTENTS OF METHANOL EXTRACT OF B. VULGARIS
|SOD||214± 1.01 (mgm / minute / mg protein)|
|Catalase||56.9± 1.01 ( µ/mol/min/g)|
|GSH||56± 1.01 (mg/g)|
|Total phenolics Content||26.47 ± 1.05 (mg GAE/g dry extract)|
|Total flavonoids content||15.7 ± 0.01 (mg QE/g dry extract)|
GAE = Gallic acid equivalent; QE = Quercetin equivalent
Effect of MEBV on total phenolics (TPC) and flavonoids contents (TFC): The phytochemicals analysis showed the presence of total phenolics content (TPC) as 26.47 mg GAE/g and total flavonoids content (TFC) as 15.7 QE/g in MEBV. Flavonoids are natural polyphenolic molecules common to most flowering plants. They include flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Although not considered vitamins and flavonoids have a number of nutritional functions have been described as biological response modifiers. Most of them act as antioxidants and some have anti-inflammatory properties 21.
(Table 3) Antioxidants activity, total phenolics and flavonoids contents of methanol extract of B. vulgaris
Effect of MEBV on the liver function analysis: It was observed that, the level of ALT (SGPT) by CCl4 was at 101 ±1.11 IU/L is considerably reduced to 41 ±1.02 IU/L at 300μgm/ml MEBV, which is close to control value of 39.5 ±1.11 IU/L. (Table 4). Similarly the elevated level of AST (SGOT) by CCl4 (1288±1.02 IU/L) is effectively reduced to 409 ±1.02 IU/L by MEBV, at 300μgm/ml, thus reducing the liver marker enzymes near normal to the control 359.5 ±1.01 IU/L.
The elevated bilirubin level of 0.94mg/dL due to CCl4 toxicity is reportedly reduced by MEBV at 300μgm/ml, to 0.58mg/dL which is almost close to the normal control value of 0.48mg/dL.
TABLE 4: EFFECT OF METHANOL EXTRACT OF B. VULGARIS (L.) ROOT (MEBV) ON ALT, GOT AND BILIRUBIN IN CCl4-INDUCED HEPATOTOXICITY IN RATS
|Groups||ALT (SGPT)||AST (SGOT)||Bilirubin|
|Group-I||39.5 ±1.11 (IU/L)||359 ±1.01 (IU/L)||0.48 ±1.01 (mg/dL)|
|Group-II||101 ±1.11 (IU/L)||1288 ±1.02 (IU/L)||0.94 ±1.01 (mg/dL)|
|Group-III||81 ±1.02 (IU/L)||855 ±1.02 (IU/L)||0.87 ±1.02 (mg/dL)|
|Group-IV||51 ±1.02 (IU/L)||521 ±1.02 (IU/L)||0.64 ±1.02 (mg/dL)|
|Group-V||41±1.02 (IU/L)||409±1.02 (IU/L)||0.58±1.02 (mg/dL)|
Each value represents the mean ± SE (n = 6); Significance p≤0.05 vs. Control
Effect of MEBV on the level of non-enzymatic antioxidants: The level of non-enzymatic antioxidants (SOD, CAT and GPx, GSH, respectively) as observed in CCl4 control group rats (Table 5), were significantly (p<0.05) low, indicating the rise of ROS group and significant damage to the antioxidant system. However, the MEBV treated groups, recorded with rising values close to the normal control levels, indicating remarkable antioxidant effect. Inactivation, detoxification, removal of ROS and other free radicals depend on enzymatic and non-enzymatic antioxidants. The important enzymatic antioxidants in the tissues were SOD, CAT and glutathione peroxidase (GPx). These antioxidants together with GSH act to prevent the formation of free radicals and thereby prevent oxidative stress 22.
TABLE 5: EFFECT OF METHANOL EXTRACT OF B. VULGARIS (L.) ROOT (MEBV) ON ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANTS IN CCl4-INDUCED HEPATOTOXICITY IN RATS
|Group-I||5.01±1.02 (μgm/min/mg)||4.55±0.11 (U/min/mg)||7.88±1.05 (U/min/mg)||90.5±1.05 (U/min/mg)|
|Group-II||2.08±1.02 (μgm/min/mg)||1.42±1.05 (U/min/mg)||4.1±1.01 (U/min/mg)||42±1.04 (U/min/mg)|
|Group-III||3.6±1.02 (μgm/min/mg)||2.9±0.11 (U/min/mg)||6.4±1.01 (U/min/mg)||59±1.02 (U/min/mg)|
|Group-IV||4.45±1.11 (μgm/min/mg)||3.55±1.05 (U/min/mg)||6.9±1.02 (U/min/mg)||79.02±1.06 (U/min/mg)|
|Group-V||5.32±1.11 (μgm/min/mg)||4.7±1.05 (U/min/mg)||7.47±1.04 (U/min/mg)||84.72±1.08 (U/min/mg)|
Each value represents the mean ± SE (n = 6); Significance p≤0.05 vs. Control
Reduction in the enzyme activities towards the respective normal values by MEBV at different dose levels (100, 200 and 300 mg/kg) is an indication of stabilization of plasma membranes and repair of liver tissue damage. The first line of defense against ROS is, displayed by the antioxidative enzymes, such as SOD and CAT. The GSH, a non-enzymatic antioxidant, plays a pivotal role in protecting against organ toxicity. These antioxidants mainly convert active oxygen molecules into non-toxic compounds. It was observed that, Glutathione peroxidase (Gpx), Glutathione (GSH), Superoxide dismutase (SOD) and Catalase (CAT) levels of liver protein, which was low in the CCl4 diseased rats. And it may be due to the accumulation of superoxide radicals and hydrogen peroxide 23. Upon exposure to methanol extract of Beta vulgaris (MEBV), in this study, they have got improved to near normal levels as shown by the control. (The values at 300μgm/ml are given as per below;) theGPx of MEBV was 5.32±1.11, against CCl4 impacted value of 2.08±1.02μgm/ml; and the values were close to the control value of 5.01±1.02 μgm/ml. The GSH of MEBV = 4.7±1.05mg/mg, against CCl4 =1.42±1.05mg/mg; and the value was near the normal value of 4.55±0.11mg/mg; The value for SOD of MEBV was 7.47±1.04u/min/mg, as against CCl4 4.1±1.01u/min/mg; whereas the value of control was 7.88±1.02μgm/ml; and the Catalase (CAT) levels of MEBV at 84.72±1.08 micromol/mg, as against CCl4 exposed rats level of 42±1.04 micromol /mg liver protein and the value of the control group was 90.5±1.05 micromol/mg.
Glutathione is a tripeptide composed of three different amino acids, glutamate, cysteine and glycine that has numerous important functions within cells. Glutathione contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione is involved in various liver detoxification processes. Glutathione serves as a reductant is conjugated to drugs to make them more water soluble is involved in amino acid transport across cell membranes (the g-glutamyl cycle) is a part of the peptidoleukotrienes, serves as a cofactor for some enzymatic reactions and as an aid in the rearrangement of protein disulfide bonds 24.
Effect of MEBV on the blood parameters: As summarized in table 6, injection of CCl4 in rats with significant (P≤0.05) reduction of WBC, RBCs counts, Hb content as compared with control. Oral administration of MEBV for 14 days along with CCl4 ameliorated the suppressive effect of CCl4 in group III, IV and V at 300μgm/ml when compared with 100μgm/ml and 200μgm/ml. The amelioration by MEBV is reported to the near normal values that were exhibited in control group. The WBC count of 12.2 thousand in a unit blood was raised to the level of 21.1 thousands which was very close to the level of control (group-I) at 22.1 thousands per unit. Similarly the RBC count is raised from 5.7 million to 8.3 million which is close to the control values of 8.7 million per unit blood. The haemoglobin content in group-V was raised to 13.1 g/dL from 8.2 g/dL of CCl4 group-II which was close to 14.2 g/dL the value of the control. The packed cell volume too have bettered in group-V (43.1%) when compared with group-II (30%) which is near normal value of 47% in group-I.
TABLE 6: EFFECT OF METHANOL EXTRACT OF B. VULGARIS L ROOT (MEBV) ON THE BLOOD PARAMETERS OF THE CCl4 INDUCED HEPATOTOXIC LIVER OF THE RAT
|Group I||22.1±1.02 (*103 μL)||8.7±1.01 (*106 μL)||14.2±0.22 (g/dL)||47±1.12 (%)|
|Group II||12.2±1.02 (*103 μL)||5.7±1.01 (*106 μL)||8.2±0.22 (g/dL)||30±1.01 (%)|
|Group III||15.9±1.02 (*103 μL)||7.2±1.01 (*106 μL)||10.6±0.21 (g/dL)||37.5±1.02 (%)|
|Group IV||17.2±1.02 (*103 μL)||7.8±1.02 (*106 μL)||12.0±1.21 (g/dL)||39.7±1.02 (%)|
|Group V||21.1±1.12 (*103 μL)||8.3±1.02 (*106 μL)||13.1±1.21 (g/dL)||43.1±1.11 (%)|
Each value represents the mean ± SE (n = 6); Significance p≤0.05 vs. Control
Histopathological findings: Oral adminstration of methanol extract of Beta vulgaris for 14 days in addition to 1.9ml/kg b.w of CCl4 intraperitoneal route did offer remarkable hepatic remediation at 300μgm/ml level and restored the hepatic architecture close to the positive control group of rats 14. However, the CCl4 injection to negative control group revealed that, big areas of hepatic necrosis more or less in centro-lobular position and showed inflammation towards peripheral area (Fig. 2).
FIG. 2: LIGHT MICROGRAPH OF A HE STAINED LIVER OF A POSITIVE CONTROL RAT × 100
FIG. 3: LIGHT MICROGRAPH OF A HE STAINED LIVER OF CCL4 INTOXICATED RAT (NEGATIVE CONTROL)
Fig. 2 shows hexagonal or pentagonal lobules with central veins and peripheral hepatic triads or tetrads embedded in connective tissue. Hepatocytes are regular and contain a large spheroidal nucleus with a distinctly marked nucleolus and peripheral chromatin distribution. Fig. 3 show most hepatic lobules, the trabecular structure is lightly blurred and, in the remaining lobules, distinctly blurred. The cytoplasm of some cells shows rare empty vacuole-type spaces. ×300.
Also there was dilation of blood sinusoids, single cell necrosis which affected most of the cell cord.
The above results support the view on stabilization of plasma membranes of hepatocytes and thus reduced ROS levels by the activity of MEBV. Previous phytochemicals investigations on Beta vulgaris reported the presence of flavonoids and phenolics18, 25. The antioxidant activity, flavonoids and phenolics may be responsible for hepatoprotective activity 26.
DISCUSSION: The CCl4, a well-known hepato-toxin, has been widely used to screen new hepatoprotective agents. At cellular metabolism, this xenobiotic chemical is rapidly transformed by cytochrome P450 2E1 into a trichloromethyl radical which is converted into a peroxyl radical (ROO*) in the presence of oxygen. Similarly, superoxide radical (SO') and hydroxyl radicals (OH") are also produced as a part of normal metabolic processes. These radicals may interact with cellular macromolecules and initiate the peroxidative degradation of lipid membranes. Enhanced production of reactive oxygen species (ROS) and pro-inflammatory cytokines by activated Kupffer cells is also involved in liver damage initiated by CCl4-derived radicals. As CCl4 intoxication is known for the release of ROS and thus oxidative stress it leads to diverse pathological issues 27.
Generally the oxidative stress and cell damages are correlated with increased level of thiobarbituric reactive substances (TBA) and decrease in packed cell volume (PCV) due to destruction of erythrocytes (RBCs) 14. The packed cell volume (PCV) and haemoglobin concentration of CCl4 control group was reportedly decreased significantly (p<0.05), indicating that there might have been possible destruction of erythrocytes by CCl4 28. This observation agrees with the report that CCl4 toxicity can lead to destruction of hepatocytes and erythrocytes 14.
The elevated levels of ALT (SGPT), AST (SGOT) and Bilirubin due to CCl4 intoxication were moderately (p<0.05) reduced with MEBV treatment when compared with CCl4 control rats. Maximum activity was found with 300μgm/ml dose of MEBV.
Aminotransferases (AST or SGOT) are concentrated mainly in the liver, and their leaching into the circulation implicate that the integrity of the hepatocytes is disturbed. In this study, there is significant increase in the levels of ALT (SGPT), AST (SGOT) and bilirubin in the CCl4 treated group. Generally, bilirubin conjugates with glucoronic acid in the liver and is later excreted through bile. The presence of higher proportion of bilirubin in serum indicates that the process of conjugation is hampered, reflecting loss of hepatic function 29.
However treatments with MEBV has alleviated this situation and restored RBCs, WBCs levels to near normal values thus suggested MEBV has protective effect. The histopathological studies too support the hepatoprotective role of MEBV and its ability to suppress the oxidative stress caused by CCl4. This may be due to improved levels of SOD, GSH, Catalase enzymes and phytochemicals that are present already in the plant part as per preliminary screening and these may offer antioxidant activity such as phenolics, flavonoids, Vit. E, Vit. C, b- Carotene, Thiamine etc.
Thus, enzymatic scavenging of ROS could be efficiently achieved through the complex but elaborate coordination among the enzymes involved. TheHigher levels of antioxidant system enable the plant extract to act as a very good free radical scavenger and help to prevent diseases caused by oxidative damage 30.
The phenolics are ubiquitous secondary metabolites having wide therapeutic values like antioxidant, anti-carcinogenic and free radical scavenging activities. The scavenging ability of the phenolics is mainly due to the presence of hydroxyl groups. Flavonoids are a group of polyphenolic compounds that exhibit anti-hepatotoxic, anti-inflammatory activities. They inhibit enzymes such as aldose reductase and xanthine oxidase is capable of scavenging the ROS due to their phenolics hydroxyl groups and potent antioxidants. The presence of Flavonoids and Phenolics that are reported in the MEBV may reveal a positive correlation between phenolics content and antioxidant activity, suggesting phenolics and flavonoids might be the active phytochemicals in Beta vulgaris.
It may be recalled that the phenolics and flavonoids contents were found moderately in methanol extract of Beta vulgaris (MEBV).
CONCLUSION: In thisstudy, male wistar rats were exposed to CCl4 and methanol extract of Beta vulgaris, at 100, 200 and 300µgm/ml respectively. The rats, during the in vivo study, developed a significant hepatic damage and oxidative stress, as evidenced by the massive elevation in the liver marker enzyme activities of ALT (SGPT), AST (SGOT) and bilirubin as compared with normal control rats. The blood parameters and hepatic cellular architecture were also under disturbance. This may be an indication of cellular leakage and loss of functional integrity of hepatocytes cell membrane. The phytochemicals analysis revealed the high content of phenolics and flavonoids in Beta vulgaris; upon exposure to MEBV it was observed and recorded that the reversal of hepatotoxic effects and hepato-remediation do occur.
Hence based on the preliminary findings on antioxidant and hepatoprotective activities here reported, it may be concluded that, methanol extract of Beta vulgaris (MEBV) possess significant protection against CCl4-induced hepatotoxicity in the in vivo studies. This study also delivers the scope to continue study, identify and characterize the active principle(s) and the mechanism involved in the field of Hepatoprotection and Chemo-prevention of Hepatotoxicity offered by the antioxidants fraternity of Beta vulgaris.
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How to cite this article:
Rose MH, Sudhaa PN and Sudhakar K: Effect of antioxidants and hepatoprotective activities of methanol extract of beet root (Beta vulgaris L.) against carbon tetrachloride induced hepatotoxicity in rat models. Int J Pharm Sci Res2014; 5(6): 2546-55.doi: 10.13040/IJPSR.0975-8232.5(6).2536-45
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Maximas H. Rose , P.N. Sudha* and K. Sudhakar
Associate Professor, Research Department of Chemistry, D.K.M. College for Women, Vellore, Tamil Nadu, India
16 December, 2013
15 February, 2014
25 April, 2014
01, June 2014