INTRODUCTION:

Liver is the largest glandular and major organ for metabolism. The liver reacts with different types of responses to injury in response to variety of metabolic, toxic, microbial, circulation and neoplastic insults¹. Hepatic injury leads to disturbances in transport function of hepatocytes resulting in leakage of plasma membrane thereby causing an increased enzyme level in serum.

The plant selected for the present study is Actinodaphne madrasapatana which belongs to Lauraceae family. It is endemic, occasionally found in peninsular India. Common on rocky hill slopes at higher elevations. The leaves and flowers of this plant constitute the drug. Leaves are used for curing diabetes and wound healing². Flowers are used to cure mania.

The aerial parts of this plant are reported to have large amount of flavonoids. Flavonoids are reported to be majorily involved in treating liver disorders³. Hence the leaves of A. madarasapatana are selected for assessing hepatoprotective activity in wistar albino rats against carbon tetrachloride induced hepatotoxicity.

MATERIALS:

Plant material:

The leaves of Actinodaphne madraspatana were collected from native species growing in deciduous forests of Tirumala region, Andhra Pradesh, India. The leaves have been identified taxonomically and authenticated by Dr. S. Madhava Chetty, Associate Professor, Department of Botany, Sri Venkateswara University, Tirupathi, Andhra Pradesh, India.

Preparation of the plant extract:

The leaves of Actinodaphne madraspatana were subjected to soxhlet extraction with methanol. The extract was concentrated by vacuum distillation. The extract was subjected to phytochemical screening⁴.

Experimental animals:

Male Wistar Albino rats (150-200 g) were procured from Albino Research Center. They were randomly housed in standard polypropylene cages and maintained under the standard conditions: room temperature (25± 3)⁰C, humidity 45 % - 55 %, 12/12 h light/dark cycle. They were fed with commercially available pellet diet obtained from Amruth foods, Pranav Agro Industries, Sangli, India and water was allowed ad libitum. The animals were acclimatized to the laboratory conditions minimum one week prior to the behavioural experiments. Animals used in this study were treated and cared for in accordance with the guidelines recommended by CPCSEA (Reg.No: MRCP/IAEC/ CPCSEA/1217/a/2). The study was approved by Institutional Animal Ethics Committee of Malla Reddy College of Pharmacy, Secundrabad (MRCP/IAEC/ CPCSEA/MPCOL/2011-12/5).

METHODS:

Acute toxicity studies:

The overnight fasted rats were divided into 4 groups, each group consisting of 3 female animals. The extracts were given in various doses (5, 50, 300, and 2000 mg/kg) by oral route. After administration of the extract, the animal were observed continuously for the first 2 hours and at 24 hrs to detect changes in behavioral responses and also for tremors, convulsion, salivation, diarrhoea, lethargy, sleep, and coma and also were monitored up to 14 days for the toxic symptoms and mortality.

Two sub-maximal doses, 1/5^th and 1/10^th cut-off doses for extracts 200 and 400 mg/kg, p.o. were found to be safe (1/10^thof LD₅₀) and used for further pharmacological investigations.

Evaluation of Hepatoprotective activity⁵:

Hepatotoxicity was induced by single intra peritoneal injection of carbon tetrachloride: olive oil in 1:1 ratio. Animals were divided into five groups consisting of six animals in each group. First group of animals were received only vehicle (1% CMC, p.o.) once daily for 7 days and served as normal group. Second group of animals received vehicle (1% CMC, p.o.) for 1-7 days and CCl₄(1.25ml/kg) on day 6 and served as disease control group. Third group of animals received silymarin (10mg/kg) p.o once daily for 7 days and CCl₄(1.25ml/kg, i.p.) on day 6 and served as standard group, fourth and fifth groups received MEAM (200 & 400 mg/kg p.o) once daily for 7 days and CCl₄(1.25 ml/kg, i.p.) on day-6.

Estimation of biochemical parameters:

After 48 h, following CCl4 administration, blood samples were collected from the retro orbital plexus and serum was separated for analyzing SGOT, SGPT, ALP, serum total bilirubin and serum triglycerides^6-10.

Histopathological studies:

At the end of the study, animals were decapitated and cut open to excise the liver. The liver was immediately removed and a small piece was placed in 10% formalin for histopathological assessment.

Estimation of post mitochondrial supernatant (PMS):

The livers were perfused with ice cold saline (0.9% sodium chloride) and homogenized in chilled potassium chloride (1.17%) using a homogenizer. The homogenates were centrifuged at 800 rpm for 5 minutes at 4^oC to separate the nuclear debris. The supernatant so obtained was centrifuged at 10,500 rpm for 20 minutes at 4^oC to get the PMS.

Estimation of lipid peroxidation (LPO) from PMS¹¹:

0.5 ml of PMS was taken and to it 0.5 ml of Tris hydrogen chloride buffer was added and incubated at 37⁰ C for 2 hours and then 1 ml of ice cold trichloroacetic acid was added, centrifuged at 1000 rpm for 10 min. from the above, 1 ml of supernatant was taken and added to 1 ml of thiobarbituric acid and the tubes were kept in boiling water bath for 10 min. the tubes were removed and brought to room temperature and 1 ml of distilled water was added. Absorbance was measured at 532 nm by using spectrophotometer. Blank was prepared without tissue homogenate.

Estimation of reduced glutathione from PMS¹²:

1.0 ml of PMS was precipitated with 1.0 ml of sulphosalicylic acid (4%). The samples were kept at 4⁰ C for at least 1 hour and then subjected to centrifugation at 1200 rpm for 15 minutes at 4⁰C. The assay mixture contained 0.1 ml filtered aliquot and 2.7 ml phosphate buffer (0.1 M, Ph 7.4) in a total volume of 3.0 ml. The yellow colour developed was read immediately at 412 nm using a spectrophotometer. Blank was prepared without tissue homogenate.

Estimation of catalase (CAT) from PMS¹³:

The assay mixture consisted of 1.95 ml phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M), and 0.05 ml of PMS (10%) in a final volume of 3 ml. Changes in absorbance were recorded at 240nm. The blank was prepared without tissue homogenate. Catalase activity was calculated in terms of k minutes.

Statistical analysis:

All the data were expressed as Mean ± Standard Error (SEM). Data obtained from various groups was subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. Significant values were set accordingly.

RESULTS:

The phytochemical analysis conducted on methanolic extract of A. madraspatana revealed the presence of alkaloids, flavonoids, tannins, glycosides and carbohydrates. In case of acute toxicity study, and during the seven days of study period, there was no death record, nor signs of any changes in the physiopathological activities or in the neurological behavior in the treated groups.

Administration of single dose of CCl₄(1.25 ml/kg, p.o.) raised the serum levels of all biochemical parameters significantly (p<0.05) compared to normal group animals. The elevated levels of serum SGOT, SGPT, ALP, total bilirubin and triglycerides were significantly (p<0.05) reduced in the rats treated with A. madraspatana extract as depicted in Table 1.

Results presented in table 2 clearly revealed the significantly increased level of MDA in Group-II (disease control) compared to Group-I (normal control).Treatment with A. madraspatana extract significantly prevented this rise in level. Levels of antioxidant enzymes, GSH and CAT were significantly increased in A. madraspatana extracts. Animals treated with high dose of A. madraspatana extract (400mg/kg) demonstrated maximum hepatoprotection.

The liver section of the normal animals showed normal hepatocyte architecture and hepatic lobules without any fatty changes (figure 1A). The liver section of the animals after acute treatment with CCl₄ showed complete loss of hepatic architecture with intense peripheral and central vein necrosis, apoptosis, hepatocellular vacuolization, fatty changes and congestion of hepatocyte sinusoids (figure 1B). Liver sections of animals treated with low dose of A. madraspatana extract displayed mild Swelling of hepatocyte, hepatocyte atrophy, mild fatty change with central vein damage, mild fatty change with central vein damage given in figure. 1D. Where liver sections of animals Pre-treated with silymarin (10 mg/kg) and high dose of A. madraspatana extract (400 mg/kg) exhibited normal architecture without any fatty changes as shown in figure 1C, 1E.

Table-1: Effect of methanolic leaf extract of A. madraspatana on biochemical parameters

S.no. Group SGOT SGPT ALP Total bilirubin Triglycerides

1. Normal control 56.74±4.47 59.81±5.74 42.9±3.85 0.59±0.12 68.41±3.92

2. CCl₄168.8±7.62^a 163.59±5.63^a 130.2±7.82^a 3.27±0.11 134.23±6.51

3 Silymarin 82.8±6.76^b 72.78±5.04^b 58.4±3.67^b 0.72±0.18 0.65±0.11

4. MEAM 89.21±6.14^b 91.60±3.85^b 73.8±3.28^b 0.65±0.11 97.43±3.22

(200 mg/kg)

5. MEAM 83.54±4.75^b 84.8±4.79^b 63.7±2.85^b0.54±0.12 81.08±3.12

(400 mg/kg)

Values are expressed as mean ±SEM for six animals; MEAM=Methanolic Extract of A. madraspatana

a= p<0.05 as compared to normal control; b= p<0.05 as compared to CCl₄ treated group

Table-2: Effect of methanolic leaf extract of A. madraspatana on Catalase, Reduced glutathione and Lipid peroxidation

S.no. Group Catalase Reduced glutathione Lipid peroxidation

(μM/gm tissue) (μM/gm tissue) (μM/gm tissue)

1. Normal control 1.23±0.09 0.168±0.009 0.073±0.002

2. CCl₄ 0.098±0.091^a 0.061±0.002^a 0.33±0.014^a

3. Silymarin 1.618±0.083^b 0.241±0.009^b 0.063±0.009^b

4. MEAM 1.303±0.119^b 0.171±0.009^b 0.113±0.012^b

(200 mg/kg)

5. MEAM 1.419±0.47^b 0.196±0.013^b 0.101±0.01^b

(400 mg/kg)

Values are expressed as mean ±SEM for six animals; MEAM=Methanolic Extract of A. madraspatana

a= p<0.05 as compared to normal control; b= p<0.05 as compared to CCl₄treated group

Histopathological studies on rat liver

Figure 1A.Normal control

No fatty change, normal hepatocyte

Figure 1B. CCl₄ treated

Loss of hepatic architecture with intense

Peripheral central vein necrosis, apoptosis

Hepatocellular vascuolization, fatty changes

Figure 1C. Silymarin treated

Apparently normal hepatocyte

Figure 1D. MEAM-200

Swelling of hepatocyte, hepatocyte atrophy, mild fatty change with central vein damage

Figure 1D. MEAM-400

Normal hepatic architecture was seen with only

Moderate accumulation of fatty lobule

DISCUSSION:

Carbon tetrachloride is a highly toxic compound. A single exposure of CCl₄ to hepatocytes leads to centrizonal necrosis and steatosis¹⁴. CCl₄is metabolized in the liver to form highly reactive trichloromethyl radical that leads to autooxidation of fatty acids present in the cytoplasmic membrane phospholipids and causes morphological and functional changes in the cell membrane. It also causes influx of extracellular calcium into the cell leading to cell death. It also plays a significant role in inducing triacylglyceral accumulation, depression of protein synthesis, depletion of GSH and loss of enzyme activities in liver.

In the present study, silymarin was used as the standard hepatoprotective drug. On treatment with silymarin, all the biochemical parameters elevated by CCl₄ were reversed back to normal. Several mechanisms have been proposed for the hepatoprotective activity of silymarin. They include autooxidation¹⁵, inhibition of lipid peroxidation¹⁶, enhanced liver detoxification, protection of glutathione depletion ¹⁷, anti-inflammatory^18,19 by increasing hepatocyte synthesis and promoting hepatic tissue regeneration. The antioxidant properties of silymarin have been attributed to their flavonoids²⁰, which can prevent lipid peroxidation, changes in composition of the membrane phospholipids, hepatic glutathione depletion and improve the functional markers of liver damage.

The present study revealed that serum SGOT, SGPT, ALP, total bilirubin and triglycerides levels were significantly elevated in rats intoxicated with CCl₄ in comparison with normal group. Zimmerman et al., showed that elevation of serum SGOT and SGPT indicate hepatic injury by CCl₄ and its metabolites, which resulted from cell membrane damage and mitochondrial damage in liver cells respectively and the release of more than 80% of total hepatic enzymes from the mitochondria²¹. The activity of serum alkaline phosphatase (ALP) was also elevated during CCl₄ administration. ALP is excreted normally via bile by the liver. In liver injury due to CCl₄, there is a defective excretion of bile by the liver which is reflected by the increased level of ALP in serum²².

Oral administration of A. madraspatana to rats caused a dose dependent decrease in the activity of the liver enzymes, which may be a consequence of the stabilization of plasma membrane as well as repair of hepatic tissue damage caused by CCl₄. This is supported by the view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and regeneration of hepatocytes²³.

Acute administration of CCl₄ elevated the levels of bilirubin by interfering in its synthesis and discharge of bile acids and bilirubin whereas increased levels of triglycerides was attributed to decreased lipase activity that leads to reduced triglyceride hydrolysis²⁴. Depletion of elevated bilirubin level and triglyceride parameters were observed with the preadministration of A. madraspatana extract suggesting the possibility of the extract being able to stabilize biliary dysfunction and improving the lipase activity. The improvement in the levels of biochemical parameters was further supported by histopathological changes noticed in the liver sections of different groups.

During hepatic injury, superoxide radicals generate at the site of damage and modulate MDA, TBARS and CAT, resulting in the loss of activity and accumulation of superoxide radical, which damages liver. The high significant elevation of MDA level in liver homogenate of rats treated with CCl₄in the present study indicated excessive formation of free radicals and activation of lipid peroxidation of the hydrophobic core and cell damage²⁵. CCl₄ also induced highly significant reduction in the level of reduced glutathione and catalase levels in liver homogenates of the treated animals.

In contrast, groups treated with low and high dose of methanolic extract of A.madraspatana showed a significant decrease in MDA levels compared to the CCl₄ control group. It may thus be possible that supplementation of the extract were potentially effective in blunting lipid peroxidation, suggesting that the extract possibly had antioxidant property to reduce toxicant induced membrane lipid peroxidation and thereby preserving the membrane structure. On the other hand, significant amplification in the concentration of CAT and GSH for the extracts as compared to CCl₄ control group may be due maintenance of enzymatic activity that could be related to their scavenging capacity for O2•− and mainly, •OH, decreasing the oxidative damage of hepatic proteins (e.g. enzymes) and thereby enhancing the enzymatic activity which may possibly detoxify the reactive oxygen species and avert CCl₄ induced liver toxicity²⁶.

From the results it is evident that the preadministration of A. madraspatana extract resulted in suppression of ccl₄ –induced adverse effects on liver antioxidant status and there by suggesting that this plant has antioxidant activity based on free radical scavenging or modulation of antioxidant status and tissue regeneration. The ability of natural compounds to attenuate carcinogen induced hepatotoxicity is believed to be related to their intrinsic antioxidant properties²⁷. Phytochemical screening of this study revealed the presence of flavonoids, which has been reported as potent antihepatoxic compound in the previous studies²⁸. The protective mechanism of flavonoids are related to their ability to inhibit peroxidative damage caused by potent toxicants. In conclusion the flavonoids present in leaves of A. madraspatana may have, perhaps played a major role in the hepatoprotective action. However further work is obviously required to identify the compounds responsible for the hepatoprotective activity and to elucidate their exact mechanism of action.

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Received on 17.09.2014 Modified on 01.10.2014

Res. J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 176-180