|Year : 2012 | Volume
| Issue : 4 | Page : 307-312
Antioxidant and hepatoprotective effects of flower extract of Millingtonia hortensis Linn. on carbon tetrachloride induced hepatotoxicity
S Babitha1, David Banji2, Otilia J. F. Banji2
1 Research Scholar, R and D Cell, JNTU, Hyderabad, India
2 Department of Pharmacology, Nalanda College of Pharmacy, Nalgonda, AP, India
|Date of Submission||17-Oct-2011|
|Date of Decision||01-Nov-2011|
|Date of Acceptance||02-May-2012|
|Date of Web Publication||07-Nov-2012|
Department of Pharmacology, Nalanda College of Pharmacy, Nalgonda, AP
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Millingtonia hortensis Linn is an abundant resource of flavonoids, which might be beneficial in protecting liver tissue from injury. The hepatoprotective and antioxidant potential of ethanolic extract of M. hortensis on carbon tetrachloride (CCl 4 ) induced hepatotoxicity and the possible mechanism involved therein were investigated in rats. Materials and Methods: Preliminary phytochemical studies were carried out to determine the total phenol and flavonoid contents. 30 adult Wistar rats were allocated into 5 groups. Control group received vehicle, group-2 received CCl 4 alone (1 ml/kg body weight, intraperitonially), groups 3 - 5 received the ethanolic flower extract in 2 dose levels (200 and 400 mg/kg) and Curcumin (100 mg/kg) as a standard for 8 days orally, followed by CCl 4 as a single dose on the 8 th day. 48 hours later, blood was withdrawn, serum was subjected to biochemical assessments, and liver homogenate was examined for lipid peroxides, glutathione, superoxide dismutase, catalase and total protein levels. Furthermore, hepatic tissues were subjected to histopatological studies. Results: CCl 4 treatment produced a profound increase in the levels of malondialdehyde, hepatic marker enzymes and bilirubin content compared with the control (P < 0.05). Pre-treatment with the flower extract of M. hortensis significantly enhanced the levels of endogenous antioxidants and reduced the levels of hepatic marker enzymes in relation to the CCl 4 treated group (P < 0.05). Balloning degeneration and fatty changes in hepatocytes was prevented by pre-treatment with the flower extract. Conclusion: The antioxidant nature of the flower extract of M. hortensis could be responsible for averting damage to the liver.
Keywords: Antioxidant, curcumin, hepatoprotective, M. hortensis
|How to cite this article:|
Babitha S, Banji D, Banji OJ. Antioxidant and hepatoprotective effects of flower extract of Millingtonia hortensis Linn. on carbon tetrachloride induced hepatotoxicity. J Pharm Bioall Sci 2012;4:307-12
|How to cite this URL:|
Babitha S, Banji D, Banji OJ. Antioxidant and hepatoprotective effects of flower extract of Millingtonia hortensis Linn. on carbon tetrachloride induced hepatotoxicity. J Pharm Bioall Sci [serial online] 2012 [cited 2020 Feb 28];4:307-12. Available from: http://www.jpbsonline.org/text.asp?2012/4/4/307/103258
Liver is a predominant organ, playing a pivotal role in regulating various physiological processes in the body. It controls metabolism, secretion and storage. It has a capacity to detoxify substances and synthesize useful principles. The liver has an increased chance of being exposed to drugs, chemicals and xenobiotics leading to toxicity. This can have grave consequences on the functional capacity of the hepatocytes. ,, One of the principal causes of CCl 4 induced liver injury is lipid peroxidation by free radical derivatives of CCl 4 .  Antioxidative effect plays an important function in protection against CCl 4 induced liver injury. 
In spite of major advances in modern medicine, there are no effective drugs available that stimulate liver functions, protect liver damage or help regenerate hepatic cells.  On the contrary, the Indian system of medicine has proposed a number of medicinal preparations for the treatment of liver disorders.  Millingtonia hortensis Linn. (Syn Bignonia suberosa Roxb) is an important medicinal plant in Southern Asia, commonly known as the Indian cork tree. An applicability of this herb is wide with the decoction of the root being used to control food poisoning, reduce fever, as a lung tonic and a bronchodilator. Leaves of M. hortensis are used as antipyretic, remedy for asthma, in sinusitis, as a cholagogue and tonic in folklore medicine. The dried flowers are used as a treatment for asthma. , The flowers were reported to contain hispidulin (6 methoxy-5, 7, 4'-trihydroxyflavone), scutellarein, and scutellarein-5-glucuronide, respectively. The flowers also contain a flavonoid, cirsimaritin (hortensin; 5,4'-dihydroxy-6,7-dimethoxyflavone). 
Hispidulin, isolated from the chloroform extract of the flowers of M. hortensis, exhibit bronchodilator effect on rat's trachea.  Hispidulin, a bioactive flavonoid isolated from the flowers, was reported to exert an inhibitory activity on the 5- lipoxygenase pathway.  Essential oil from the flowers exhibited anti-microbial activities.  The methanol and aqueous extracts of the flowers were capable of inhibiting fungal growth.  Based on the presence of vital flavonoids in the flowers of M. hortensis, we explored the role of the flower extract in reversing damage induced by CCl 4 on the liver cells.
| Materials and Methods|| |
Glutathione, curcumin and thiobarbituric acid were procured from Sigma Aldrich, USA. 5, 5'-dithio bis-2-nitrobenzoic acid (DTNB, Ellman's reagent), and Trichloroacetic acid (TCA) was procured from Sisco Research Labs, Mumbai. Biochemical estimation kits were purchased from Erba Diagnostics, Transasia Biomedicals, India. All other reagents and chemicals were of analytical grade.
The flowers of M. hortensis were collected from Tumkur, Karnataka, India in the month of November 2009 and authenticated by Prof. Siddappa, H.O.D, Sree Siddaganga Boys College, Tumkur, Karnataka, India.
Preparation of the extract
Shade dried flowers of M. hortensis were powdered (60 g) and extracted with 70% alcohol under Soxlet extraction for a period of 8 hours to obtain the ethanolic extract. The extract was concentrated to dryness under reduced pressure and controlled temperature (40-50°C) in a rotavapor and stored in a refrigerator at 4°C till further use.
Preliminary phytochemical analysis
The extract was subjected to preliminary phytochemical studies to identify the presence of tannins, flavonoids and alkaloids. 
Determination of total phenolics
The total phenolic content of the extracts were determined by the Folin-Ciocalteau method with some modifications.  0.5 ml of the sample was added to 2.5 ml of 0.2 N Folin-Ciocalteau reagent and allowed to stand for 5 min. Then 2 ml of 75 g/L of Na 2 CO 3 was added, and the total volume was made up to 25 ml using distilled water. The above solution was then kept for incubation at room temperature for 2 h. Absorbance was measured at 760 nm on a spectrophotometer. Tannic acid (0 - 800 mg/L) was used to produce the standard calibration curve. The total phenolic content was expressed in mg of tannic acid equivalents per g of extract and all samples were analyzed in triplicate.
Estimation of total flavonoids
The total flavonoid content was determined using the Dowd method.  5 ml of 2% aluminium trichloride in methanol was mixed with the same volume of the extract solution (0.4 mg/ mL). Absorbance was recorded after 10 min at 415 nm, using a spectrophotometer against a blank sample. The total flavonoid content was determined using catechin (0 - 100 mg/L) as the standard. Total flavonoid content was expressed as mg of catechin equivalents per g of extract.
Albino rats of Wistar strain of either sex, weighing 180 - 220 g were procured and maintained on a 12 ± 1 h day and night schedule. The animals were fed with standard diet and water ad libitum. The study was approved by the Institutional Animal Ethical Committee, and experiments were conducted as per the guidelines of CPCSEA.
Oral toxicity in rats was carried out as per OECD-423 guidelines. 4 doses (5, 50, 300, 2000 mg/kg body weight) of ethanolic flower extracts were administered orally to groups containing 3 animals. The animals were regularly monitored for mortality, clinical signs and body weight changes daily for 3 days, and observation continued for the period of 14 days. At the end of the study period, all the animals were subjected to gross necropsy.
Carbon tetrachloride (CCl 4 ) induced hepatotoxicity
All animals except the control received Curcumin or extract followed by CCl 4 diluted with liquid paraffin oil (1:1 v/v, 1.0 ml/ kg, intraperitonially).  CCl 4 was used to inflict hepatic injury.
The rats were divided into 5 groups with 6 in each group. Group 1 served as normal control and received 2% acacia suspension orally; group 2 received CCl 4 in liquid paraffin (1: 1), 1 ml/kg b.w and served as the inducer control. Group 3 was treated with standard drug Curcumin (100 mg/kg), group 4 and 5 animals were treated with the extract (200 and 400 mg/kg), 2 % acacia served as the vehicle. All the treatments were given orally for 8 days. On the last day of treatment, all the animals except the normal group received a single dose of CCl 4 in liquid paraffin (1: 1), 1 ml/kg b.w intraperitonially (i.p.) after 1 h of the vehicle, extract or standard treatments. 48 h later , blood was withdrawn from the retro-orbital plexus under enflurane anesthesia and subjected to biochemical estimations. The animals were sacrificed , the liver was removed, weighed, and a portion was used for an estimation of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and total protein (TP).
Estimation of hepatoprotective parameters
The following biochemical estimations in serum were carried out using Erba diagnostic kits. Serum glutamate pyruvate transaminase (SGPT), Serum glutamate oxaloacetate transaminase (SGOT), Serum alkaline phosphatase (ALP), Total bilirubin (TB) and direct bilirubin (DB) were estimated on an autoanalyzer (Erba Chem V Plus).
At the end of the study, liver of different study animals were immediately fixed in 10% buffered neutral formalin solution. After fixation, tissues were embedded in paraffin, serial sections were cut, and each section was stained with hematoxylin and eosin. The slides were examined under light microscope, and photographs were taken. 
Estimation of antioxidant parameters
Estimation of SOD
Assay was based on the ability of SOD to inhibit the spontaneous oxidation of adrenaline to adrenochrome. The reaction mixture contained 3 × 10 -4 M adrenaline and 0.05 M carbonate buffer (pH 10.2). Decrease in absorbance of adrenochrome was measured at 480 nm. 1 unit of SOD activity is equal to the amount of enzyme, required to inhibit 50% auto-oxidation of adrenaline at 25°C. 
Estimation of GSH
To measure the reduced glutathione (GSH) level, the tissue homogenate (in 0.1 M phosphate buffer pH 7.4) was added to equal volume of 20% TCA containing 1 mM EDTA to precipitate the tissue proteins. The mixture was allowed to stand for 5 min prior to centrifugation for 10 min at 2000 rpm. The supernatant was then transferred to a new set of test tubes and 1.8 ml of the Ellman's reagent (DTNB), 0.1 mM) prepared in 0.3 M phosphate buffer with 1% of sodium citrate solution was added. The volume was made up to 2 ml in all the tubes. The absorbance was measured at 412 nm against blank.  Absorbance values were determined from a standard curve of GSH.
Estimation of CAT
The assay was based on the ability of CAT to scavenge hydrogen peroxide. The reaction mixture contained 30 mM of hydrogen peroxide and 0.05 M phosphate buffer (pH 7). Decrease in absorbance of hydrogen peroxide was measured at 240 nm. 1 unit of CAT is equal to the number of μmole of hydrogen peroxide decomposed per min at 25°C. 
Estimation of MDA
MDA was estimated in the liver homogenate by the method of Ohkawa et al. 1979.  1.5 ml acetic acid (20% v/v) pH 3.5, 1.5 ml thiobarbituric acid (0.8% w/v) and 0.2 ml sodium dodecyl sulfate (8.1% w/v) were added to 0.1 ml of processed tissue samples, and then heated at 95°C for 60 min. The mixture was cooled under tap water, and 5 ml of n-butanol:pyridine (15 : 1) 1 ml of distilled water was added. The mixture was vortexed vigorously. The samples were then centrifuged at 4000 rpm for 10 min. The organic layer was separated, and the absorbance was measured at 532 nm using a spectrophotometer. 1, 1, 3, 3 tetra ethoxy propane was taken as a standard.
Estimation of total protein
Total protein in the tissue homogenate was estimated by the modified Lowry's method.  This assay is based on the principle that proteins react with Folin's reagent to give a colored complex. To a 0.5 ml of liver homogenate, 1 N sodium hydroxide was added and made up to 5 ml. To 1 ml of this solution, 1 ml 10% TCA was added, allowed to stand for 10 min at 4°C and centrifuged for 10 min at 4°C at 4000 rpm. The supernatant was discarded, and the precipitate was dissolved in 1 ml 0.5 N sodium hydroxide. Alkaline copper tartarate reagent (4 ml) was added to the above solution and allowed to stand for 10 min at room temperature. A blank was prepared in the same manner, but without the liver homogenate. Finally, 0.5 ml of Folin's reagent was added, and the absorbance was recorded against the blank at 540 nm.
All values are expressed as mean ± S.E.M. Total variation present in a set of data was estimated by one-way analysis of variance (ANOVA) followed by Dunnet's post-hoc test. P < 0.05 was considered significant.
| Results|| |
The percentage yield of ethanolic extract was found to be 14.05%. Preliminary phytochemical investigation has revealed the presence of various phytoconstituents like flavonoids, phenolics, proteins and amino acids in ethanolic flower extract.
Total phenolic content and flavonoid content
The total phenolic content of the extract of flowers of M. hortensis was found to be 241.2 ± 3.0 mg tannic acid equivalent per g of extract. The total flavonoid content was 58.5 ± 2.06 mg equivalent of catechin per g of extract [Table 1].
|Table 1: Total phenolic and flavonoid content of M. hortensis flower extract|
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No mortality was observed up to a dose level of 2000 mg/kg body weight with the ethanolic extract of the flowers of M. hortensis. Therefore, a dose of 200 and 400 mg/kg was selected for the study.
The activities of various biochemical parameters in normal, inducer control and treated groups are represented in [Figure 1] and [Figure 2]. The activities of SGOT, SGPT, ALP, direct bilirubin and total bilirubin were significantly (P < 0.05) increased with a significant decrease in total protein levels in CCl 4 inducer control compared to normal control. Pre-treatment with the ethanolic flower extract of M. hortensis showed a significant decrease in the levels of the above markers and significant increase in tissue total protein levels compared with the inducer control (P < 0.05). Protection was observed maximally with the high dose of the extract.
|Figure 1: Effect of ethanolic flower extract of M. hortensis on SGOT, SGPT and ALP in CCl4 induced hepatotoxicity in rats. Values are mean ± SEM, N = 6. *P < 0.05 compared with the control group, #P < 0.05 compared with the CCl4 treated group, ‡P < 0.05 compared with the 200 mg/kg (low dose). Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT), Serum alkaline phosphatase (ALP)|
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|Figure 2: Effect of ethanolic flower extract of M. hortensis on TB, DB, TP and LW in CCl4 induced hepatotoxicity in rats. Values are mean ± SEM, N = 6. *P < 0.05 compared with the control group, #P < 0.05 compared with the CCl4 treated group, ‡P < 0.05 compared with the 200 mg/kg (low dose). Direct bilirubin (DB), Total protein (TP), Liver weight (LW)|
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The histopathological studies of the liver showed fatty changes, swelling, necrosis, inflammatory infiltration with loss of hepatocytes in CCl 4 control rats in comparison with normal control. The liver sections of rats treated with the higher dose of the extract showed regeneration of hepatocytes, normalization of fatty changes and decrease in necrosis of the liver. Curcumin treated group showed normalization of fatty accumulation and necrosis [Figure 3].
|Figure 3: Hepatoprotective action of ethanolic extract of M. hortensis against CCl4 induced hepatotoxicity: liver histopathology; (a) Normal control; (b) CCl4 - Paraffin oil (1:1, 1 ml/kg); (c) Standard curcumin (100 mg/kg) + CCl4; (d) MH (200 mg/kg) + CCl4; (e) MH (400 mg/kg) + CCl4|
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In vivo antioxidant assays
Antioxidants and MDA levels
[Figure 4] depicts the oxidative changes produced by CCl 4 and reversal following treatment with the extract. CCl 4 produced significant reduction in CAT, SOD and GSH activities and increases in the MDA levels compared to the control group (P < 0.05) in rat liver tissue. Pre-treatment of rats with M. hortensis flower extract (200, and 400 mg/kg) followed by CCl 4 significantly (P < 0.05) increased the levels of enzyme and non-enzymatic antioxidants compared to the CCl 4 -intoxicated group. MDA levels significantly declined following treatment with the extract compared to the CCl 4 control group (P < 0.05). Curcumin also produced significant (P < 0.05) increase in the antioxidant levels and decrease in the MDA levels compared with the CCl 4 treated group.
|Figure 4: Effect of ethanolic flower extract of M. hortensis on SOD, CAT, MDA and GSH in CCl4 induced hepatotoxicity in rats. Values are mean ± SEM, N = 6. *P < 0.05 compared with the control group, #P < 0.05 compared with the CCl4 treated group, ‡P < 0.05 compared with the 200 mg/kg (low dose). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione (GSH)|
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| Discussion|| |
The protective effect of M. hortensis on hepatic tissue in CCl 4 induced damage has been elucidated in this study. The metabolism of CCl 4 by cytochrome P 450 in the liver endoplasmic reticulum to a highly reactive CCl 3 radical is largely responsible for the toxicity produced by CCl 4 . The greater propensity to induce hepatic injury is due to the ability of CCl 4 to promote generation of free radical derivatives leading to lipid peroxidation. , The formed free radicals can inflict injury to the hepatic cell membrane, causing an alteration in the functioning of hepatic cells. Generally, a fine balance is maintained intracellularly between the proxidant and antioxidant levels. Exposure to hepatotoxins can produce an overwhelming increase in the levels of these reactive species. Overproduction can disturb the integrity of the hepatic cells. Aminotransferases are concentrated mainly in the liver, and their leaching into the circulation implicate that the integrity of the hepatocytes is disturbed. Our studies revealed a significant increase in the levels of SGOT, SGPT and ALP in the CCl 4 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.  In contrast, treatment with high dose of the extract produced a definite decline in the maker enzymes, signifying its role in protecting hepatic cells from injury. Further, an increase in the total protein content in hepatic tissues suggests that cellular functioning of hepatic tissues is restored.
The first lines of defense against ROS in an organism are displayed by the antioxidative enzymes, such as SOD, and CAT. GSH, a non-enzymatic antioxidant, also plays a pivotal role in protecting against organ toxicity. These antioxidants mainly convert active oxygen molecules into non-toxic compounds.  Reduced activities of SOD, CAT, and GSH in the liver and kidney have been observed after an injection with CCl 4, which may result in a number of deleterious effects due to accumulation of superoxide radicals and hydrogen peroxide.  An increased level of MDA indicated that more lipid peroxidation was induced by CCl 4 .  CCl 4 treated rats exhibited significantly increased MDA levels. Pre-treatment of the rats with M. hortensis caused significant and dose dependent reduction in the elevated levels of MDA which could be related to its antioxidant ability.
SOD has been reported as one of the most important enzyme in the enzymatic antioxidant defense system. It scavenges the superoxide anion to form hydrogen peroxide. , In the present study, it was observed that the ethanolic extract of flowers of M. hortensis caused a significant and dose-dependent increase in the hepatic SOD activity in the CCl 4 intoxicated rats. Catalase is an enzymatic antioxidant widely distributed in all animal tissues, and the highest activity is found in the red cells and in the liver. Catalase decomposes hydrogen peroxide and protects the tissue from highly reactive hydroxyl radicals.  We observed from our study that pre-treatment of the rats with ethanolic extract of M. hortensis significantly increased the hepatic catalase activity of the CCl 4 intoxicated rats.
Glutathione, a tripeptide, is one of the most abundant biological antioxidant present in the liver. It partakes in the removal of free radical species and maintains the thiol proteins. It also serves as a substrate for glutathione peroxidase and glutathione transferase (GST).  In the present study, decreased level of GSH was associated with an enhanced lipid peroxidation in CCl 4 treated rats. Treatment of rats with ethanolic extract of M. hortensis significantly increased the level of hepatic glutathione.
| Conclusion|| |
In the present study, the ethanolic extract of M hortensis exhibited significant hepatoprotective effect against CCl 4 induced acute hepatotoxicity in rats. The presence of abundant flavonoids in the ethanolic extract could be responsible for the beneficial response. The decline in the levels of marker enzymes and elevation in the levels of antioxidants can contribute to the hepatoprotective effect of the flower extract of M. hortensis.
| Acknowledgements|| |
Approval of ethical committees for conducting Animal studies (in vivo): The study was approved by the Institutional Animal Ethical Committee and experiments were conducted as per the guidelines of CPCSEA. (NCOP/IAEC/approval/03/2009).
The manuscript was not presented as part at a meeting, the organization in part or full. The manuscript has been read and approved by all the authors that the requirements for authorship document have been met, and each author believes that the manuscript represents honest work and authors alone content and writing of the paper.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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