INTRODUCTION:

Liver is involved in several vital functions such as metabolism, secretion and storage. Furthermore, the detoxification of variety of drugs and xenobiotics occurs in liver. Liver disease is mainly caused by toxic chemicals, excess of consumption of alcohol, and infections, and sometimes autoimmune. Plant drugs are known to play a vital role in the management of liver disease ^[1]. Hence, many folk medicines are evaluated for its anti-oxidant and Hepatoprotective effect against different chemical –induced liver damages in the experimental animals.

Ixora javanica D. C. (Rubiaceae) is an evergreen shrub found throughout India, depending on the medical condition, the flowers, leaves, roots and stem are used to treat various ailments in both Indian traditional and Ayurvedic system of medicine^[2]. Flowers are small golden orange to red, corolla is mostly 4-5 lobed, occasionally with 3 or upto 10 lobes, gynoecium consists of single compound pistil of 2 or seldom more carpels, epigynous nectroy disk is usually present^[3], used as astringent, skin disease, carminative, bronchitis, chronic ulcers^[4]. Flowers extract of Ixora javanica inhibited growth and delayed onset of Papilloma formation in mice with DMBA and promoted using croton oil ^[5].

The methanolic extract of Ixora coccinea is reported to posses potent antioxidant activity ^[6]. Since many hepatic injuries are free radical medicated, it is possible that the different Ixora species may show potent Hepatoprotective activity. Hepatoprotective activity of this particular species i.e. Ixora javanica plant has not been reported so far. In this study, hepatoprotective property of ethyl acetate and ethanloic extracts of Ixora javanica flowers in CCl₄ induced toxicity is being reported.

MATERIALS AND METHODS:

Plant material

Flowers of Ixora javanica were collected from the local areas of Hyderabad. The plant was authenticated by Dr. B. Bhadraiah (HOD, Dept. of Botany, Osmania University, Hyderabad). Voucher specimen (IJ/2012/23) was kept at Malla Reddy College of Pharmacy, Dhulapally, Hyderabad, Andhra Pradesh, India.

Chemicals

All the chemicals and solvents were of analytical grade and were procured from Ranbaxy Fine Chemicals Ltd., Mumbai, Maharashtra, India. The standard drug silymarin was obtained as a gift sample from Micro Lbs, India. Standard kits for SGOT, SGPT, ALP and bilirubin were obtained from Span Diagnostics Ltd., India.

Preparation of extracts

Flowers of Ixora javanica were (3 Kg) dried under shade and subjected for continuous successive extractions with ethyl acetate and ethanol using a soxhlet apparatus for about 48 h. Both solvents were removed from the extracts under reduced pressure by using a rotary vacuum evaporator. The yield of ethyl acetate and ethanolic extracts was found to be 7.45 % and 8.15 % w/w, respectively.

Phytochemical Analysis

Both extracts were subjected to identify the presence of various phytoconstituents namely, triterpenoids and sterols (Leiberman-Burchard test), flavoniods (shinoda test), carbohydrates (Benedict’s test), alkaloids (Dragendroff’s test), proteins (Biuret test), glycosides (legal test), etc., by usual methods prescribed in standard texts ^[7].

Behavioural effect and toxicity study:

The preliminary pharmacological studies were conducted to assess the acute pharmacological effects and LD₅₀ of ethyl acetate and ethanolic extracts. Both the extracts were subjected to acute oral toxicity studies in adult female albino rats according to OECD guidelines 423.

Selection of dose

For assessment of Hepatoprotective activity, two dose level were chosen, i.e, lower dose was one-tenth of LD₅₀ cut-off value observed, and a high dose was twice that of one-tenth dose (200 and 400 mg/Kg respectively).

Carbon tetrachloride-induced hepatotoxicity

Rats of either sex were divided into seven groups of six animals each (n=6) ^[1].

Group I: Received water (5ml/Kg, p.o.) for 9 days once daily, and served as normal control.

Group II: Received water (5ml/Kg, p.o.) for 9 day once daily and carbon tetra chloride (1ml/Kg in 50 % v/v olive oil, s.c.) on the 7^th day.

Group III: Received standard drug silymarin (100 mg/Kg, p.o.) for 9 days once daily and carbon tetrachloride (1ml/Kg in 50 % v/v olive oil, s.c.) on the 7^th day.

Group IV, V, VI and VII: Received ethyl acetate and ethanolic extracts (200 and 400 mg/Kg) for 9 days once daily and carbon tetrachloride (1ml/Kg in 50% v/v)

Biochemical assay

After 48 h of carbon tetrachloride administration, the blood was obtained from animals from animals by puncturing the retroobital plexus. The blood samples were allowed to clot for 45 min at room temperature. The serum was separated by centrifugation at 2500 rpm at 30 °C for 15 min and utilized for the estimation of various biochemical parameters including SGPT, SGOT ^[8], ALP ^[9], serum bilirubin ^[10] and serum cholesterol ^[11]. After collection of blood samples, the animals were sacrificed under deep ether anesthesia and their livers were excised immediately and washed with ice cold saline, and 10% homogenate was prepared in a phosphate buffer (pH7.0). The homogenate was centrifuged at 3000 rpm for 15 min at 4°C.

Histopathological study

The liver was examined grossly, stored in formalin 10 % and were processed for paraffin embedding using the standard micro technique. A section of liver (5 µm) stained with alumhemotoxylin and eosin was microscopically for histopathological studies and was analyzed by a pathologist.

Statistical analysis

The results are expressed as Mean ±SEM (n=6) and were analyzed statistically using analysis of variance (ANOVA) followed by Dunnett’s test. Values of p<0.05 were considered significant.

RESULTS:

Phytochemical screening

The preliminary phytochemical investigation of the ethyl acetate and ethanolic extracts of Ixora javanica flower showed various phytochemicals including sterols, flavonoids, triterpenoids, glycosides, tannins and some phenolic compounds.

Acute toxicity study

Both extracts (ethyl acetate and ethanolic) did not show any sign of toxicity or mortality. Hence, at a dose level 2000 mg/Kg the LD₅₀ cut off value of both extracts was found to be infinite.

Carbon tetrachloride-induced hepatotoxicity

The results of ethyl acetate and ethanolic extracts of Ixora javanica flowers at the two dose levels (200 and 400 mg/Kg, p.o.) on serum marker enzymes and serum biliribin and cholesterol in CCl₄ –induced liver damage are shown in Table1. Liver damage induced by CCl₄ caused significant rise in marker enzymes SGPT, SGOT, ALP and serum bilirubin and cholesterol, when compared to normal group.

Table no:1 Effects of ethyl acetate and ethanolic extracts of Ixora javanica serum biochemical marker in CCl₄ induced liver damage in rats.

Treatment /group	Dose (mg/kg)	SGPT (U/l)	SGOT (U/l)	ALP (U/l)	Bilirubin (mg/dl)	Cholestrol (mg/dl)
Normal	----	58.60±2.10	132.3±2.49	83.3±0.8	0.28±0.25	7.22±1.52
Positive control (CCl₄ control)	1.0(ml/Kg)	257.27±3.89*	289.47±8.48*	135.2±0.9*	7.14±2.16*	21.17±4.26*
Silymarin	100	72.69±1.92***	153.64±4.62***	94.2±0.9***	0.76±2.12***	12.12±6.28***
Ethyl acetate extract +CCl₄	200	178.45±2.21**	196.63±6.88**	119.4±0.8***	0.72±2.16***	16.51±8.21**
Ethyl acetate extract +CCl₄	400	94.34±0.99***	165.33±4.21***	108.6±0.5***	0.65±4.26***	13.42±8.28***
Ethanolic extract +CCl₄	200	188.23±2.01**	199.76±6.22**	126.4±0.1***	0.52±0.18***	18.68±2.68***
Ethanolic extract +CCl₄	400	99.12±1.19***	176.83±4.28***	111.3±0.8***	0.49±4.28***	14.22±8.20***

Values are Mean ± SEM, n=6 (One-way ANVOA followed by Dunnett’s test);

*P <0.05, **P<0.01, ***P<0.001 denotes statistical significance

Histopathological study of carbon tetrachloride-induced hepatotoxicity

The central vein, sinusoids and portal triads appeared normal. The hepatocytes showed moderate cytoplasm and round-to-oval nuclei. There was no periportal inflammation. A section of the liver tissue of control rats showing normal histology [Figure 1]. The liver carbon tetra chloride-intoxification rats showed massive fatty changes, gross necrosis and broad infiltration of lymphocytes and Kupffer cells around the central vein, and loss of cellular boundaries [Figure 2]. Silymarin-treated rats showed the central vein dilated and congested. The hepatocytes showed feathery degeneration. The portal triads showed mild portal inflammation composed of lymphocytes [Figure 3]. The rats treated with ethyl acetate and ethanolic extracts (200 and 400 mg/Kg, p. o.) showed mild dilatation and congestion in central veins. The hepatocytes were normal. The portal triads appeared normal [Figure 4, 5, 6 and 7].

Figure 1: Liver cells of normal rats (Negetive control).

Figure2: Liver cells of rats intoxicated with CCl₄ (positive control).

Figure 3: Liver cells of rats treated with Silymarin (100 mg/Kg) and intoxicated with CCl₄

Figure 4 and 5: Liver cells of rats treated with ethyl acetate extract 200 and 400 mg/Kg respectively.

Figure 6 and 7: Liver cells of rats treated with ethanolic extract 200 and 400 mg/Kg respectively.

DISCUSSION:

The purpose of this study was to explore the Hepatoprotective effect of the ethyl acetate and ethanolic extracts from the flowers of Ixora javanica against carbon tetrachloride –induced hepatotoxicity in rats.

It is well established that CCl₄ induces hepatoprototoxicity by metabolic activation, therefore, it selectively causes toxicity in liver cells maintaining semi-normal metabolic function. CCl₄ is bio-transformed by the cytochrome P₄₅₀ system in the endoplasmic reticulum to produce trichloromethyl free radical (^.CCl₃). Trichloromethyl free radical then combined with cellular lipids and proteins in the presence of oxygen to form a trichloromethylperoxyl radical, which may attack lipid on the membrane of endoplasmic reticulum faster than trichloromethyl free radical. Thus, trichloromethylperoxyl free radical leads to elicit lipid peroxidation, the destruction of Ca²⁺ homeostasis and finally, results in cell death ^[12]. This in turn lead to changes in structure of endoplasmic reticulum and other membrane, loss of enzymes metabolic enzymes activation, reduction of protein synthesis and loss of Glucose-6-Phosphatase activation, leading in liver damage ^[13].

From the results of ethyl acetate and ethanolic extracts of Ixora javanica flowers (200 and 400 mg/Kg) showed significant hepatoprotective activity, as evidenced by the biochemical and histopathological parameters. The protective effect of both extracts against carbon tetrachloride may be due to the presence of flavonoids, sterols, triterpenoids, and phenolic compounds. Flavonoids, triterpenoids and phenolic compounds are known antioxidants, free radicals scavengers and antilipoperoxidants leading to hepatoprotection ^[14]. We now intend to isolate the active constituent and clarify in more detail the pathway concerning the protective mechanism of ethyl acetate and ethanolic extracts against carbon tertracholoride-induced liver injuries.

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Received on 17.09.2012 Modified on 18.10.2012

Research J. Pharm. and Tech. 5(11): Nov. 2012; Page 1438-1441