|Year : 2014 | Volume
| Issue : 1 | Page : 59-63
Hepatoprotective activity of ethanolic extract of Leea indica (Burm.f.) Merr. (Leeaceae) stem bark against paracetamol induced liver toxicity in rats
Garima Mishra1, RL Khosa2, Pradeep Singh1, KK Jha1
1 Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India
2 Department of Pharmacy, Bharat Institute of Technology, Meerut, Uttar Pradesh, India
|Date of Web Publication||1-Jul-2014|
Teerthanker Mahaveer University, Moradabad - 244001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Liver diseases have become one of the major causes of morbidity and mortality all over the world. Among them, synthetic drug-induced liver injury is one of the most common causative factor that poses a major clinical and regulatory challenge. The herbal medicinal plants have pivotal role in management of various diseases including liver disorders. Therefore, it is inevitable to discover novel hepatoprotective agents from natural sources. Objective: To evaluate the hepatoprotective activity of ethanolic extract of Leea indica stem bark against paracetamol (PCM) induced hepatotoxicity in rats. Materials and Methods: Hepatotoxicity was induced by PCM (2 g/kg b.w., p.o.) and biochemical parameters such as serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), serum alkaline phosphatase (SALP), serum bilirubin (total and direct), and triglyceride level were estimated. Silymarin (100 mg/kg b.w.) was employed as standard hepatoprotective agent. Histopathological changes in liver were also studied. Results: The ethanolic extract (200 mg/kg and 400 mg/kg b.w.) treatment exhibited significant decrease in elevated level of serum marker enzymes, bilirubin (total and direct), and triglycerides when compared to positive control group. The ethanolic extract at dose of 400 mg/kg b.w. was found to be more potent than 200 mg/kg. Conclusion: The ethanolic extract of Leea indica bark seems to justify the promising hepatoprotective effect on PCM induced liver damage in rats.
Keywords: Bilirubin, hepatoprotective, Histopathology, Leea indica, paracetamol, SGOT, SGPT
|How to cite this article:|
Mishra G, Khosa R L, Singh P, Jha K K. Hepatoprotective activity of ethanolic extract of Leea indica (Burm.f.) Merr. (Leeaceae) stem bark against paracetamol induced liver toxicity in rats. Niger J Exp Clin Biosci 2014;2:59-63
|How to cite this URL:|
Mishra G, Khosa R L, Singh P, Jha K K. Hepatoprotective activity of ethanolic extract of Leea indica (Burm.f.) Merr. (Leeaceae) stem bark against paracetamol induced liver toxicity in rats. Niger J Exp Clin Biosci [serial online] 2014 [cited 2022 Jul 1];2:59-63. Available from: https://www.njecbonline.org/text.asp?2014/2/1/59/135732
| Introduction|| |
Irregular food habits, smoking, alcoholism, and over-consumption of chemical drugs are among the main causes of hepatic disorders. Surveys have shown that about 2-3% of the Indian population are carriers of hepatitis virus.  Liver is one of the largest organs in human body and the chief site for intense metabolism and excretion.  Thus, liver diseases remain one of the serious health problems and its disorders are numerous with no effective remedies. There are lots of drugs with hepatocurative activity described in classic Ayurvedic texts.  Apart from the drugs mentioned in these Ayurvedic texts, there are many other drugs such as Wedelia chinensis, Vitex negundo, Cassia fistula, Costus speciosus, Caeselpinia crista etc. used to treat liver disorders in India.
Leea indica (Burm.f.) Merr. (family-Leeaceae) commonly known as Kurkurjiwah in hindi is a large evergreen perennial shrub growing up to 2-3 m in height with stout, soft woody stems with numerous stilt roots.  Traditionally, the whole plant is used to relieve headache, body pain, and skin complaints. , The roots have been traditionally applied in the treatment of diarrhea, dysentery, spasm, and as sudorific. The decoction of root given in colic, is cooling and relieves thirst. The juice of young leaves is used as digestive agent whereas roasted leaves are applied to relieve vertigo. ,
A number of compounds have been reported from plants belonging to the genus Leea. The methanolic extract of Leea indica is reported to possess strong antioxidant activity.  Therefore, based on these findings, the present study was designed to investigate the hepatoprotective activity of ethanolic extract of Leea indica against paracetamol (PCM) induced liver damage in rats.
| Materials and methods|| |
Collection and Authentication of Plant Material
Leea indica stem bark was purchased from Khari Bawli, Delhi and authenticated by Dr. D.C. Saini, Senior Scientist, Palaeobotany, Birbal Sahni Institute of Palaeobotany (BSIP), Lucknow, India. A voucher specimen no. BSIP 07 was deposited in BSIP herbarium and crude drug sample is preserved in the Department of Pharmacognosy, Teerthanker Mahaveer College of Pharmacy, TMU, Moradabad.
Preparation of Plant Extract
The shade dried bark was mechanically reduced to moderately coarse powder. The dried powder was extracted with petroleum ether (60-80°C) and ethanol using a soxhlet apparatus successively. The ethanolic extract thus obtained was concentrated under reduced pressure by using rotary evaporator and completely dried in water bath. Further, the extracts were subjected to preliminary phytochemical investigation.
In order to determine the presence of alkaloids, carbohydrates, flavonoids, proteins, amino acids, phenols, tannins, glycosides, and steroids, a preliminary phytochemical study was performed with ethanolic extract. The standard methods for various phytoconstituents are as follows. ,,
Test for Alkaloids
2-3 ml test solution may give cream color precipitate with Mayer's reagent to ensure the presence of alkaloids.
Reddish brown colored precipitate may appear when 2-3 ml test solution was added to Dragendorff's reagent to show the presence of alkaloids.
2-3 ml test solution may give reddish brown precipitate with Wagner's reagent which may correspond to the presence of alkaloids.
Yellow colored precipitate may appear when 2-3 ml of test solution was mixed with Hager's reagent.
Tests for Carbohydrates
The test solution was treated with few drops of alcoholic α-naphthol solution. The solution was shaken and concentrated H 2 SO 4 was added gently through the sides of the test tube. A purple to violet color ring may form at the junction of the two liquids.
Tests for Flavonoids
Few fragments of magnesium ribbon and concentrated HCl when added drop wise to the test solution, pink to red colour may be observed.
Tests for Amino Acids
3 ml test solution was heated with 3 drops of 5% Ninhydrin solution in boiling water bath for 10 min. Purple or bluish color may indicates the presence of amino acids.
Tests for Proteins
3 ml of test solution was mixed with 5 ml of Millon's reagent, a white precipitate may appear which turn red upon gentle heating or precipitate may dissolve giving red color solution.
3 ml test solution was added to a mixture of 4% NaOH and few drops of 1% CuSO 4 . Violet or pink color may appears.
Tests for Tannins and Phenolic Compounds
5% Ferric chloride solution
Test solution may show deep blue-black color to confirm the presence of free tannins. A brownish green precipitate may appears if condensed tannins were present.
Lead acetate solution
Test solution may give white precipitate.
Red precipitate may appear.
Dilute NH 4 OH and potassium ferricyanide
Test solution may give red color when treated with dilute NH 4 OH and potassium ferrycyanide.
Tests for Glycosides
A thick section may indicate yellow to orange color with sodium picrate.
To the solutions of aqueous or alcoholic extract 1ml pyridine and 1ml alkaline sodium nitroprusside solution were added. Pink to red color may appear.
Keller Kiliani Test (test for deoxy-sugars)
2 ml extract of the drug was added to glacial acetic acid containing a trace amount of 0.5% ferric chloride. It was transferred to a small test tube; 0.5 ml of concentrated H 2 SO 4 was carefully added by the side of the test tube. Reddish brown colour if appear at the junction of the two liquids and upper layer appear bluish green, may indicates the presence of glycosides.
Tests for Steroids
A mixture of 2 ml chloroform and 2 ml conc. H 2 SO 4 was added to the test solution, shaken well and allowed to stand for some time, chloroform layer may appear red indicating the presence of sterols while acid layer may show greenish yellow colored fluorescence to reveal the presence of triterpenoids.
2 ml extract was mixed with chloroform and 1-2 ml acetic anhydride was added to it, the solution was boiled and cooled, conc. H 2 SO 4 was added from the side of the test tube which may show first red then blue and finally green color.
Standard kits for biochemical estimation of SGOT, SGPT, SALP, and Bilirubin were obtained from Span Diagnostics Ltd., India. PCM was procured from E. Merck (India) Ltd, Mumbai. All chemicals and solvents were of analytical grade and collected from Ranbaxy Fine Chemicals Ltd., Mumbai, India.
Preparation of the Drug Solution
Oral suspensions containing 200 and 400 mg/ml of ethanolic extract of Leea indica were prepared in normal saline.
Wistar albino rats (180-200 g) of either sex were used for the experiment. Animals were maintained under standard conditions (12 hrs light/dark cycle; 25 ± 2°C; 45-60% relative humidity) and were fed standard diet and water ad libitum. All the animals were acclimatized to laboratory conditions for a week before commencement of the experiment. All experimental protocols were approved by Institutional Animal Ethical Committee (IAEC) of Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad (Reg No. 1205/c/09/CPCSEA).
Acute Toxicity Study
The acute toxicity study was carried out as per the guidelines set by Organization for Economic Co-operation and Development (OECD-423), received from Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Social Justice and empowerment, Govt. of India.  The ethanolic extract was orally administered to adult Wistar albino rats. The groups were continuously observed for mortality and behavioral changes during first 24hrs and then daily for a fortnight. The oral LD 50 was found to be more than 3000 mg/kg.
Assessment of Hepatoprotective Activity
Wistar rats were divided into five groups having six animals in each (Control, Positive control, Standard, Test low and high doses). Group I (control) and group II (positive control) received saline 1 ml/kg for one week, group III (standard) received silymarin (100 mg/kg p.o.), group IV (test low doses), and group V (test high doses) received 200 and 400 mg/kg oral dose of ethanolic extract respectively once a day for one week. On the fifth day, after the administration of respective treatments, all the animals of groups II, III, IV & V were administered PCM at a dose of 2 g/kg p. o. , On the seventh day, that is after 48 hours of administration, blood was withdrawn by retro orbital puncture  for the estimation of biochemical parameters. After that, animals were sacrificed under ether anesthesia. The liver was collected, washed, and used for histopathological studies. 
Serum separated by centrifugation of blood sample at 7000 rpm for 10 minutes was used for estimation of serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT),  serum alkaline phosphatase (SALP),  serum bilirubin (total and direct) and triglyceride level. After collection of blood, the liver was immediately removed and rinsed in ice cold normal saline, blotted with filter paper and weighed.
The liver samples were fixed in 10% neutral formalin, dehydtrated in graded alcohol and then embedded in paraffin. Microtome sections (5 μm) were prepared from each liver sample and stained with hematoxylin-eosin dye  for microscopic observation of liver histoarchitecture.
The results were expressed as mean ± standard error mean (SEM). The statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Bonferroni's Multiple Comparison Test.
| Results|| |
Preliminary Phytochemical Screening
Preliminary phytochemical investigtion of ethanolic extract of Leea indica showed the presence of carbohydrate, alkaloids, glycosides, flavonoides, and phenolic compounds.
The extract was found to be safe in the doses used and no mortality up to a dose of 3000 mg/kg, b.w. for ethanolic extract. Hence, 200 and 400 mg/kg b.w. p.o. were selected for the study.
The results of hepatoprotective activity of ethanolic extract of Leea indica on PCM treated rats are summarized in [Table 1]. The hepatic enzymes SGPT, SGOT, ALP, bilirubin, and triglycerides in serum were significantly increased (P < 0.05) in PCM treated (positive control group) rats when compared with control. Silymarin (100 mg/kg) treated animals showed significant decrease (P < 0.001) in the levels of SGPT, SGOT, ALP, bilirubin, and triglycerides when compared with positive control group rats. The ethanolic extract significantly increased the levels of SGPT, SGOT, ALP, bilirubin, and triglycerides when compared with positive control group rats but effect was less than standard.
|Table 1: Effect of ethanolic extract of Leea indica bark on Paracetamol induced hepatotoxicity in rats|
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The histoarchitecture of the liver sections of control rats showed no histological changes in sinusoids and hepatocytes and no damage in central vein [Figure 1]a. The PCM treated group showed hepatic cell necrosis along with deterioration of central vein [Figure 1]b. In the group treated with silymarin, the section showed mild diffuse granular degeneration and necrosis in hepatic cells [Figure 1]c while the group treated with ethanolic extract (200 mg/kg and 400 mg/kg b.w.) revealed minimal necrosis and regeneration of hepatocytes [Figure 1]d and e.
|Figure 1: Histopathology of liver tissues at 10×. (a) Photomicrograph of liver from control group (group I) animal showing normal architecture and no necrosis and no cytoplasmic vacuolation (10×), (b) Photomicrograph of liver from animal of toxic (positive control, group II) group treated with 2 g/kg, p.o. of PCM showing marked vacuolation and portal inflammation, (c) Photomicrograph of liver from animal treated with PCM and silymarin (group III) showing normal architecture with regenerative activity with mild focal |
vacuolation, (d) Photomicrograph of liver from animal treated with PCM and low dose (200 mg/kg) (group IV) of ethanolic extract of Leea indica bark showing patchy hepatocyte vacuolation with regenerative
activity and area of normal hepatocytes, (e) Photomicrograph of liver from animal treated with PCM and high dose (400 mg/kg) (group V) of ethanolic extract of Leea indica bark showing patchy hepatocyte
vacuolation with regenerative activity and area of normal hepatocytes
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| Discussion|| |
The present study investigated the protective effect of ethanolic extract of Leea indica bark compared with the protective effect of silymarin on PCM-induced hepatotoxic rat model. PCM is an analgesic and antipyretic agent known to cause centrilobular hepatic necrosis at toxic doses. It is metabolically activated by cytochrome P-450 to form a reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) that is detoxified by glutathione (GSH) to form an acetaminophen-GSH conjugate. After a toxic dose of acetaminophen, total hepatic GSH is depleted by as much as 90%, and as a result, the metabolite covalently binds to cysteine groups on protein, forming acetaminophen-protein adducts.  Silymarin treatment normalized the elevated biochemical parameters, caused by PCM by its stabilizing action on plasma membrane. Silybin dihemisuccinate, a soluble form of flavonoid of silymarin, has protected rats against liver glutathione depletion and lipid peroxidation induced by acute PCM hepatotoxicity and showed potential benefits of silymarin as an antidote.  Assessment of liver function can be made by the estimation of serum levels of biochemical markers like SGOT, SGPT, SALP, total and direct bilirubin which are leaked out into systemic circulation during necrotic cell damage and hence are referred as sensitive indicators of liver injury. Pretreatment with silymarin and ethanolic extract of Leea indica stem bark at 200 mg/kg and 400 mg/kg b.w. significantly attenuated the increased level of these serum markers, in a dose dependent manner, as compared with positive control. The histological examination of the liver sections of silymarin (100 mg/kg b.w.) and ethanolic extract (200 mg/kg and 400 mg/kg b.w.) treated groups revealed that the normal cellular architecture was retained when compared with positive control group, thereby confirming the protective effect of the extract.
Preliminary phytochemical studies of ethanolic extract of Leea indica revealed the presence of carbohydrate, alkaloids, glycosides, flavonoides, and phenolic compounds. The presence of these constituents has been reported for antioxidant potential  therefore, they might be responsible for protective action against PCM-induced hepatic damage.
| Conclusion|| |
From the current investigation on hepatoprotective effect of Leea indica stem bark against PCM induced hepatotoxicity in rats, it can be concluded that the ethanolic extract has ability to reduce the elevated levels of various biomarker enzymes and lipid content suggesting that the plant has promising hepatoprotective potential.
Further research needs to be conducted in order to evaluate the possible mechanism by which Leea indica stem bark mediates the possible hepatoprotection.
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