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Year : 2014  |  Volume : 33  |  Issue : 4  |  Page : 216-221

Protective effect of extracts of Baliospermum montanum (Willd.) Muell.-Arg. against paracetamol-induced hepatotoxicity-an in vivoand in vitrostudy

1 Department of Pharmacognosy and Phytochemistry, Creative Educational Society's College of Pharmacy, Chinnatekur, Kurnool, Andhra Pradesh, India
2 Department of Pharmacy, The MS University of Baroda, Vadodara, Gujarat, India

Date of Web Publication19-Dec-2014

Correspondence Address:
S V Suresh Kumar
Creative Educational Society's College of pharmacy, Chinnatekur, Kurnool, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0257-7941.147427

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Aim: Evaluation of hepatoprotective effect of ethyl methyl ketone and methanol sub-fractions obtained from methanol fraction of total methanol extract (ME) was carried out both in vivo and in vitro using paracetamol-induced toxicity.
Settings and Design: Hepatoprotective activity in vivo was assessed by determining the serum levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase, total bilirubin, total cholesterol, total protein (TPTN), and albumin in serum. The studies were supported by histopathological examination of liver sections. In vitro activity was assessed by determining the change in hepatocyte viability and other parameters such as GOT, GPT and TPTN.
Materials and Methods: The ME of the roots of Baliospermum montanum was prepared and fractionated with chloroform and methanol to get activity guided fraction. The bio-active guided methanol fraction was further fractionated with ethyl methyl ketone and methanol. Both the fractions were evaluated for hepatoprotective activity against paracetamol-induced toxicity.
Statistical Analysis: For determining the significant intergroup difference each parameter was analyzed separately, and one-way analysis of variance was carried out and the individual comparisons of the group mean values were done using Dunnet's test.
Results: Methanol sub-fraction prevented hepatic damage in vivo, induced by paracetamol, whereas both the sub-fractions showed hepatoprotective effect by restoring altered parameters in the selected in vitro model. The results were comparable with the standard hepatoprotective drug silymarin.
Conclusions: This study underlines the therapeutic potential of B. montanum as per claims in Ayurveda in liver disorders.

Keywords: Baliospermum montanum, methanol sub-fraction, paracetamol, silymarin

How to cite this article:
Suresh Kumar S V, Mishra S H. Protective effect of extracts of Baliospermum montanum (Willd.) Muell.-Arg. against paracetamol-induced hepatotoxicity-an in vivoand in vitrostudy. Ancient Sci Life 2014;33:216-21

How to cite this URL:
Suresh Kumar S V, Mishra S H. Protective effect of extracts of Baliospermum montanum (Willd.) Muell.-Arg. against paracetamol-induced hepatotoxicity-an in vivoand in vitrostudy. Ancient Sci Life [serial online] 2014 [cited 2022 Nov 29];33:216-21. Available from: https://www.ancientscienceoflife.org/text.asp?2014/33/4/216/147427

  Introduction Top

Baliospermum montanum (Willd.) Muell.-Arg. (Euphorbiaceae) commonly known with the name of Danthi in India is a stout, monoecious undershrub with many shoots arising from the base. The roots are described as purgative, anthelmintic and diuretic, and also used to treat pains, enlarged spleen, inflammations and leukoderma, [1] abdominal tumors and cancer. [2] The Ayurveda claims the usefulness of roots in the treatment of jaundice. [1] Presence of steroids, terpenoids and flavonoids; [3] phorbol esters [4] has been reported in the plant. Our preliminary studies on roots of B. montanum showed significant hepatoprotective activity of total methanol extract (ME) at a dose level of 200 mg/kg, against carbon tetrachloride-induced hepatotoxicity (unpublished data). In the present study, the bio-active total ME has been further fractionated in an attempt to enrich hepatoprotective activity and to identify active constituents. The hepatoprotective activity of ethyl methyl ketone and methanol sub-fraction at various dose levels was assessed using paracetamol as hepatotoxin both in vivo and in vitro. Silymarin was used as a positive control.

  Materials and methods Top

Plant material

The roots of the B. montanum were procured from center for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal, Kerala state, and their identity was confirmed. The voucher specimen (HDT/CMPR/04/SVSK/05-06) was deposited in the herbarium of the Institute.

Preparation of extracts

450 g of powdered roots of the plant was completely extracted with methanol using soxhlet apparatus. The ME concentrated in vacuum yielded solid mass (6.25%; w/w). The ME (25 g) was fractionated on silica gel (60-120 mesh) using chloroform fraction of ME (CFME) and methanol fraction of ME (MFME). The recovered fractions were finally evaporated to give yields of 12.06% and 68.86% of solid, respectively. Further, 40 g of the methanol fraction was fractionated on silica gel using ethyl methyl ketone MFME (EMKMFME) and methanol fraction MFME (MFMFME) with resultant yields of 8.47 and 73.44%, respectively. The thin layer chromatographic studies of ME revealed the presence of flavonoids, terpenoids, and steroids; [5] the CFME showed the terpenoids and steroids with trace quantities of flavonoids; while MFME flavonoids with trace quantities of terpenoids. The sub-fractions EMKMFME showed flavonoids, while the MFMFME, flavonoids and traces of terpenoids. The sub-fractions, EMKMFME and MFMFME were selected at dose levels of 50, 100 and 150 mg/kg for in vivo and; at dose levels of 100, 500 and 1000 μg/ml for in vitro studies. The results were compared with a standard hepatoprotective drug silymarin (100 mg/kg in vivo and 100 μg/ml in vitro). All the test substances were suspended in the vehicle, that is, 5% acacia mucilage for in vivo and 30% dimethyl sulfoxide (DMSO) for in vitro.


Wistar albino rats of either sex weighing 175-225 g, maintained under standard husbandry conditions were used. Animals were allowed to take standard laboratory feed and water ad libitum. The experiments were performed after the experimental protocol was approved by the Institutional Animal Ethics Committee.

Hepatoprotective activity in vivo

Toxicity studies

Acute toxicity studies were performed for EMKMFME and MFMFME according to the acute toxic classic method [6] described by OECD. Female albino rats were used for acute toxicity study. The animals were kept fasting for overnight providing only water. The rats were divided into two groups of 3 animals each. The groups of rats were administered orally with appropriate extracts of B. montanum at a dose of 300 mg/kg. The animals were observed continuously after dosing during first 30 min, periodically for first 24 h with special attention given during first 4 h and daily thereafter, for a total of 14 days. As there was no mortality seen at this dose level, the procedure was repeated with further dose (2000 mg/kg) using fresh animals.

Hepatotoxins and test substances

Test substances including silymarin were suspended in the vehicle, that is, 5% acacia mucilage for administration. Paracetamol at a dose level of 3 g/kg in 5% acacia mucilage was administered orally.

Paracetamol-induced hepatotoxicity

Rats were divided into 9 groups of 6 each, control, paracetamol, silymarin, and test groups. The rats of control and paracetamol group received four doses of 5% acacia mucilage (1 ml/kg, p.o.) at 24 h intervals (0 h, 24 h, 48 h, and 72 h). The rats of paracetamol group received a single dose of paracetamol (3 g/kg p.o.) 30 min after the administration of the third dose of vehicle. The animals in silymarin and test groups received three doses of respective test substances (silymarin 100 mg/kg; EMKMFME and MFMFME 50, 100, and 150 mg/kg p.o.) at 0 h, 24 h, and 48 h. Paracetamol (3 g/kg p.o.) was administered 30 min after the third dose of respective test substances. After 48 h of paracetamol treatment, blood was collected, and serum was separated for estimation of biochemical parameters. [7]

Assessment of liver function

Glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) were estimated by a UV-kinetic method based on the reference method of the international federation of clinical chemistry. [8] Alkaline phosphatase (ALKP), [9] total bilirubin (TBL), [10] total cholesterol (CHL), [11] total protein (TPTN), [12] albumin (ALB) [13] were estimated. All the estimations were carried out using standard kits on auto analyzer.

Histopathological studies

Animals were sacrificed to remove the liver. The liver was fixed in Bouin's solution for 12 h, and then embedded in paraffin using conventional methods, [14] cut into 5 μm thick sections and stained using hematoxylin-eosin dye. The sections were then observed for histopathological changes.

Hepatoprotective activity in vitro

Hepatotoxins and test substances

For in vitro studies paracetamol (300 μg/ml), was used to produce a submaximal toxicity in isolated rat hepatocytes. The test solutions are tested at dose levels of 100, 500, and 1000 μg/ml. Silymarin at a dose level of 100 μg/ml was used as a positive control. All the substances are dissolved in 30% DMSO. [15]

Isolation of rat hepatocytes

The method developed by Sarkar et al., [16] was used for the isolation of hepatocytes with slight modifications. The livers were isolated under aseptic conditions and placed in N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES) buffer I containing HEPES (0.01 M), NaCl (0.142 M), and KCl (0.0067 M), pH 7.4. The livers were cut into small pieces and then incubated with a HEPES buffer II, containing HEPES (0.1 M), NaCl (0.0667 M), KCl (0.0067 M), and 0.5% collagenase type IV, pH 7.6 for about 45 min at 37°C in an incubator with constant shaking. Hepatocytes were obtained after filtration and cold centrifugation (4°C, 200 rpm/min for 2 min, and 3 times) and suspended in HEPES buffer I. The viability of the hepatocytes was assessed by trypan blue (0.2%) exclusion method.

Primary cultures of rat hepatocytes

The method of Tingstrφm and Obrink [17] with slight modifications was used for the primary culturing of rat hepatocytes. The freshly isolated viable hepatocytes were suspended in culture medium RPMI-1640 supplemented with calf serum (10%), HEPES and gentamicin (1 μg/ml). These cells (approximately 1-1.2 × 10 6 /ml) were then seeded into culture bottles and incubated at 37°C in the atmosphere of 5% CO 2 in carbon dioxide incubator. Upon incubation for 24 h, the hepatocytes formed a monolayer. The newly formed cells were round, and most appeared as individual cells. These cells were 95-96% viable as confirmed by trypan blue exclusion test.

Hepatic cytotoxicity testing

The EMKMFME and MFMFME were tested for their hepatic cytotoxicity at dose levels of 500, 1000, and 1500 μg/ml on isolated rat primary cultured hepatocytes. The hepatocytes suspensions were incubated with respective sub-fractions for 24 h in CO 2 incubator at a temperature of 37°C. The hepatic cytotoxicity was assessed after 24 h of incubation with these sub-fractions, by calculating the percentage viability [18] and also by estimating TPTN content [12] in the surrounding medium. The test substances were dissolved in 30% DMSO [15] and used for the activity.

Assessment of hepatoprotective activity

Twenty hour after the establishment of the monolayers of hepatocytes, the medium was decanted and the culture was washed with HEPES buffer I and finally, the hepatocytes were suspended in 5 ml of HEPES buffer I. Hepatocyte suspensions (0.1 ml) in triplicate were distributed into various culture tubes labeled as control, toxicant, standard (silymarin + toxicant) and test (test sample + toxicant). The control group received 0.1 ml of vehicle (30% DMSO) and toxicant groups received 0.1 ml of paracetamol dissolved in 30% DMSO, while the test groups received 0.1 ml of respective test solutions (100, 500, and 1000 μg/ml of extracts dissolved in 30% DMSO) followed by 0.1 ml of paracetamol dissolved in 30% DMSO. The standard groups received 0.1 ml of silymarin solution (100 μg/ml) followed by paracetamol. The contents of all culture tubes were made up to 1 ml with HEPES buffer I, mixed well and incubated in a CO 2 incubator for 24 h at 37°C. In test and standard groups the hepatocytes were incubated with respective solutions for 30 min and then exposed to the hepatotoxin. After incubation, hepatocyte suspensions were collected to assess cell damage. Cell viability was evaluated by trypan blue exclusion method. [19] Hepatocytes suspensions were centrifuged at 200 rpm and the leakage of the enzymes GOT, GPT, and TPTNs secreted outside the cells were determined from the supernatant. [19]

Assessment of hepatoprotective activity

The effect of different extracts in liver protection was determined by measuring the increase in the percentage of viable cells in that group of cells incubated with extracts, compared with the control and toxicant groups. Reversal of toxin-induced elevations in the levels of enzymes such as GOT [8] and GOT; [8] toxin-induced reductions in levels of proteins were also considered to be an important criterion of hepatoprotective activity.

Statistical analysis

The mean values ± standard error of the mean are calculated for each parameter. Percentage reduction against the hepatotoxin by the test samples was calculated by considering the difference in enzyme level between the hepatotoxin-treated group and the control group as 100% reduction. For determining the significant intergroup difference, each parameter was analyzed separately, and one-way analysis of variance [20] was carried out and the individual comparisons of the group mean values were done using Dunnet's test. [21]

  Results Top

Hepatoprotective activity in vivo

Toxicity studies

Ethyl methyl ketone methanol fraction of methanol extract and MFMFME from B. montanum did not show mortality even at the dose level of 2000 mg/kg and therefore considered safe.

Assessment of hepatoprotective activity

Significant (P < 0.05) increase in the serum levels of GOT, GPT, ALKP, TBL, and CHL; decrease in the levels of TPTN and ALB occurred in normal rats upon treatment with paracetamol. MFMFME at dose levels of 150 mg/kg, exhibited a significant decrease (P < 0.05) in all the elevated biochemical levels and significant (P < 0.05) increase in depleted TPTN and ALB levels similar to that observed in silymarin-treated group. EMKMFME at a dose level of 150 mg/kg also restored the altered biochemical parameters, due to paracetamol treatment significantly (P < 0.05) as observed with MFMFME. The activities exhibited by EMKMFME and silymarin are not similar statistically (P < 0.05). The results obtained are shown in [Table 1].
Table 1: Effect of EMKMFME and MFMFME of Baliospermum montanum on paracetamol-induced hepatotoxicity in rats-in vivo

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Histological examination of liver sections of control group revealed normal cellular architecture [Figure 1]a which was disturbed by paracetamol treatment as evidenced by disarrangement and degeneration of hepatic cells with centrilobular and bridged necrosis [Figure 1]b. Treatment with MFMFME 150 mg/kg, followed by paracetamol resulted the absence of necrosis and less disarrangement and degeneration of hepatocytes [Figure 1]d indicating marked protective activity similar to that observed in silymarin treated rat liver sections [Figure 1]c]. Liver sections of the rats treated with EMKMFME 150 mg/kg followed by paracetamol [Figure 1]e also showed less pathological changes as evidenced by disarrangement and degeneration of hepatocytes with necrosis.
Figure 1: Photomicrographs representing effect of ethyl methyl ketone methanol fraction methanol extract (EMKMFME) and methanol fraction methanol fraction methanol extract (MFMFME) against paracetamol-induced hepatotoxicity in rats. (a) Normal rat liver section; (b) liver section of the rat intoxicated with paracetamol; (c) liver section of the rat treated with silymarin and intoxicated with paracetamol; (d) liver section of the rat treated with MFMFME 150 mg/kg and intoxicated with paracetamol; (e) liver sections of the rat treated with EMKMFME 150 mg/kg and intoxicated with paracetamol. H and E, ×400. cv: central vein, vc: vacuole, ss: sinusoidal spaces, hc: hepatocytes

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Hepatoprotective activity in vitro

Hepatic cytotoxicity testing

When normal hepatocytes were treated with the extracts under test, there are no alterations in the values of % viable cells and TPTN content as compared to the control at the dose level up to 1500 μg/ml indicating that the extracts were not toxic to the cells.

Assessment of hepatoprotective activity

Incubation of hepatocytes with 100 μg/ml solution of paracetamol resulted in the induction of significant (P < 0.05) submaximal toxicity which was indicated by 70.43% and 49.51% depletion in viability and TPTN content of hepatocytes, respectively. Similarly, an elevation about 138.51% and 120.58% of GOT and GPT levels were observed, respectively, upon treatment with paracetamol.

Hepatocytes treated with MFMFME in concentrations of 100-1000 μg/ml showed a concentration dependent significant (P < 0.05) protective effect by restoring the viability of hepatocytes (34.58-77.35%), TPTN content (31.03-68.47%), GOT (53.67-83.81%), and GPT (70.45-83.19%). EMKMFME at concentrations of 500 and 1000 μg/ml also showed significant (P < 0.05) protective effect by restoring the viability of cells (20.54-33.78%), TPTN (45.81-57.14%), GOT (05.84-19.16%), and GPT (19.02-58.12%). Results are shown in [Table 2]. The maximum protection was obtained with MFMFME 1000 μg/ml, and the activity was statistically similar (P < 0.05) to the silymarin activity. Similarly, the activity obtained with EMKMFME 1000 mg/kg was not similar (P < 0.05) to the activity of silymarin.
Table 2: Effect of Baliospermum montanum against paracetamol-induced toxicity on rat hepatocytes (in vitro)

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  Discussion Top

The present study reveals the hepatoprotective activity of ethyl methyl ketone and methanol sub-fractions of methanol fraction obtained from total ME of B. montanum against paracetamol-induced toxicity in both in vivo and in vitro.

Paracetamol, a common antipyretic agent, is safe in therapeutic doses but can produce fatal hepatic necrosis in man, rats, and mice with toxic doses. [22] When the liver cell plasma membrane is damaged, a variety of enzymes normally located in the cytosol are released into the blood stream. Their estimation in the serum is a useful marker for the extent and type of hepatocellular damage. [23] The MFMFME of B. montanum used in the study preserved the structural integrity of the hepatocellular membrane in a dose-dependent manner as evident from the protection provided as compared to the enzyme level in the hepatotoxin treated rats.

From [Table 1], it is evident that the MFMFME was able to reduce all the elevated biochemical parameters due to the hepatotoxin intoxication in vivo. The levels of TPTNs and ALB were reduced due to the hepatotoxin intoxication. The reduction is attributed to the damage produced and localized in the endoplasmic reticulum which results in the loss of P 450 leading to its functional failure with a decrease in protein synthesis. Increased protein and ALB levels suggest the stabilization of the endoplasmic reticulum leading to protein synthesis. Suppression of cholesterol levels suggests that the extract reversed the inhibition of the synthesis of bile acids from cholesterol. Reduction in the levels of serum GOT and serum GPT toward the normal value is an indication of plasma membrane stabilization as well as repair of hepatic tissue damages caused by paracetamol. Reduction of ALKP levels with concurrent depletion of raised bilirubin level suggests the stability of the biliary function during injury with paracetamol. The protective effect exhibited by MFMFME is similar to silymarin treatment.

The histological examination of the liver sections showed that the normal liver architecture was disturbed by hepatotoxin treatment with resultant centrilobular and bridged necrosis. In the sections obtained from the rats treated with MFMFME and treatment with paracetamol, the normal cellular architecture was retained as observed with silymarin, thereby confirming the protective effect of the extract. Although the less visible changes are observed in the sections of the rats treated with EMKMFME and intoxicated with paracetamol, the intensity was less compared to MFMFME treated rat sections.

Leakage of cytosolic enzymes out of the cells occurred due to increase in cell permeability, membrane damage, and cell necrosis caused by the paracetamol. In the present study, the hepatotoxin employed reduced cell viability possibly due to injury of plasma membrane of hepatocytes resulting in the leakage of cellular enzymes. Incubation of hepatocytes with extracts significantly restored their viability as well as altered biochemical parameters induced by hepatotoxin [Table 2]. Though both the extracts were able to restore the changes caused by hepatotoxin significantly, the maximum restoration was exhibited by MFMFME at dose levels of 1000 μg/kg. A possible mode of action of these extracts may be due to the stimulation of hepatic cell regeneration, stabilization of plasma membrane, and activation of the reticuloendothelial system.

Hepatoprotective activity of various flavonoid compounds was reported in the literature by many authors. Galisteo et al., [24] reported the hepatoprotective activity of flavonoids of Rosmarinus tomentosus. The hepatoprotective effect of quercetin was reported by Janbaz et al., [25] Protective effect of rutin was reported by Janbaz et al., [26] Silymarin obtained from Silybum marianum is a good hepatoprotective agent. [27] Flavonoids are also reported [3] in B. montanum and were also confirmed in our studies. In accordance with these results, it may be hypothesized that flavonoids, which are present in MFMFME and EMKMFME, could be considered responsible for the hepatoprotective activity. Further work is in progress to isolate the bioactive principle the plant. In conclusion, this study underlines the therapeutic claims of B. montanum.

  References Top

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  [Figure 1]

  [Table 1], [Table 2]

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