INTRODUCTION:

The liver has an enormous task of maintaining the body‘s metabolic homeostasis. This includes, the processing of dietary amino acids, carbohydrates, lipids, and vitamins; synthesis of serum proteins; and detoxification and excretion into bile of endogenous waste products and pollutant xenobiotics. In addition, human beings consume a lot of synthetic drugs during diseased conditions which are alien to body organs. All these compounds produce a variety of toxic manifestations (Athar et al., 1997). The use of natural remedies for the treatment of liver diseases has a long history, starting with the Ayurvedic treatment, and extending to the Chinese, European and other systems of traditional medicines. The 21^st century has seen a paradigm shift towards therapeutic evaluation of herbal products in liver disease. A large number of plants and formulations have been claimed to have hepatoprotective activity. Nearly 160 phytoconstituents from 101 plants have been claimed to possess liver protecting activity.

In India, more than 87 plants are used in33 patented and proprietary multi ingredient plant formulations. (Handa et al., 1986). India is well known for a plethora of medicinal plants. The medicinal use of many plants (as hepatoprotectants) like Flacourtia indica (Marina et al., 2009), Annona squamosa (Saleem et al., 2008), Silybum marianum and Cichorium intybus (Madani et al., 2008), Aegle marmelos (Vinodhini et al., 2007). Sida veronicaefolia (Sharma et al., 2012) has already been reported in literature. Nyctanthes arbor-tristis ‘a night flowering sad tree’ of family Oleaceae (nyctaginaceae) is well known in India and its neighbouring countries. It is a perennial plant having life span of 5-20 years. It is a large shrub growing up to 10 m tall, with quadrangular branches and flasky grey rough bark, leaves are decussately opposite, simple, 6-12 cm long, 2-6.5 cm broad. flowers are fragrant, sessile and 5-8 lobed corolla with an orange red center. Nyctanthes arbor-tristis is found on rocky ground in dry hill sides as undergrowth in dry deciduous forests. In India , it grows in the outer Himalayas, East Assam, Bengal through central region upto Godavari in the south. It also grows well in average garden situations (Subhash et al., 2012). Flowers and seeds of Nyctanthes arbor-tristis L. have CNS depressant activity, little antispasmodic activity and also have antipyretic and anti inflammatory activities. (Das et al., 2002) This plant has also been found to possess anti allergic (Gupta et al., 1993) Antimalarial (Badam et al., 1988; Misra et al., 1991) amoebicidal (Chitravanshi et al., 1992) and anthelminthic activities (Lal et al., 1976). The present study is to investigate the hepatoprotective activity of hydro-ethanol extracts of stem bark of Nyctanthes arbor-tristis.

MATERIAL AND METHOD:

Collection and authentication of the leaves:

The stem bark of Nyctanthes arbor-tristis was collected from the garden of National Botanical Research Institute, Lucknow, India in month of July 2012. The leaves were authenticated by Dr. Sayeeda Khatoon, Chemotaxonomist and the voucher specimens were deposited in the departmental herbarium for future reference.

Preparation of extracts of Nyctanthes arbor-tristis linn:

The collected, cleaned and powdered stem bark (500 gm) were sequentially extracted using various solvents from non polar to polar such as petroleum ether, chloroform and acetone in Soxhlet apparatus. The hydro-ethanol extraction was carried out by cold maceration process.

The extracts were concentrated by vacuum distillation. (Kokate et al., 2008).

Preliminary phytochemical screening (Gothoskar et al., 1971; Kokate et al.,2008; Khandelwal, 2004).

Extracts of Nyctanthes arbor-tristis was subjected to qualitative tests for the identification of various active constituents viz. carbohydrate, glycoside, alkaloid, amino acids, flavanoids, fixed oil, tannins, gum and mucilage, phytosterols etc. The phytoconstituents were identified by chemical tests, which showed the presence of various constituents in the different extracts.

Pharmacological studies:

Animals:

Female Wistar albino rats (150-200 g) of approximately the same age, were procured from central drug research institute, Lucknow, and were used for acute toxicity studies. They were housed in polypropylene cages and fed with standard rodent pellet diet (Hindustan Lever Limited, Bangalore) and water ad libitum. The rats were exposed to alternate cycle of 12 hrs of darkness and light each. Before the test, the rats were fasted for at least 12 hrs; the experimental protocols were exercised according to CPCSEA guidelines (CPCSEA, 2003) for care of laboratory animals and the ethical guideline for investigations of experimental pain in conscious animals. The standard orogastric cannula was used for oral drug administration in rats.

Chemicals and reagents:

Paracetamol as inducing agent, silymarin as standard drug and 50% hydro-ethanol extract of stem bark N. arbor-tristis as test compound.

Acute toxicity studies:

Organization for economic co operation and development (OECD) regulates guideline for oral acute toxicity study. Acute toxic category method (Guideline 423) is a method for assessing acute oral toxicity that involves the identification of a dose level that causes mortality. This test involves the administration of a simple bolus dose of test substances to fasten healthy young adult rodents by oral gavage, observation for upto 15 days. Observations were done daily for changes in skin and fur, eyes, mucus membrane (nasal), respiratory rate, circulatory signs (heart rate), autonomic effect (salivation, lacrimation, perspiration, urinary incontinence and defecation) and central nervous system (drowsiness, gait, tremors and convulsion) changes of all the animals. In this method pre specified fixed doses of the test substances were used i.e., 5 mg/kg, 50 mg/kg, 300 mg/kg, 2000 mg/kg and 5000 mg/kg. the mortality due to these doses were observed. Generally female animals were used for this study and each dose group should consist of 3 animals.

Experimental design:

Rats of either sex were divided into 4 groups of six animals in each group. (n= 6) (Haque et al., 2011; Balakrishnan et al., 2011) Group I: Received water (5 ml/kg, p.o.) for 9 days once daily, and served as normal control. Group II: Received water (5 ml/kg, p.o.) for 9 days once daily and paracetamol (1g/kg, p.o.) on the 7th day. Group III: Received standard drug silymarin (25 mg/kg, p.o.) for 9 days once daily and paracetamol (1 g/kg, p.o.) on the 7th day.Groups IV: Received 50% hydro-ethanol extract (500 mg/kg) for 9 days once daily and paracetamol (1 g/kg, p.o.) on the 7th day. On the last day, functional parameters i.e. onset of sleep and duration of sleep, morphological parameters i.e. liver weight and liver volume, serum marker enzyme parameters i.e. Serum glutamic Pyruvate transaminase (SGPT), (Reitman and Frankel, 1957) Serum Glutamic Oxaloacetic Transaminase (SGOT) (Reitman and Frankel, 1957) and Alkaline phosphatase (ALP), (Kind and King, 1954) biochemical parameters i.e. Total bilirubin (Amour et al.,1965) and Total protein (Lowry et al., 1951) were analyzed according to the reported methods.

RESULTS AND DISCUSSION:

Preliminary phytochemical studies:

Phytoconstituents like flavonoids (Paya et al., 1993), glycoside (Bhaskar and Balakrishnan, 2009), alkaloids (Vijayan et al., 2003), saponins ( Boots et al., 2008) are known to possess hepatoprotective activities in animals.

Acute toxicity study:

Based on the phytochemical results, 50% hydro-ethanol extract of N. arbor-tristis was selected and screened for acute toxicity study by the acute toxicity studies were done by as per OECD Guideline 423. The results showed that there was no mortality amongst the graded dose groups of animals and they did not show any toxicity or behavioral changes at a dose level of 5000 mg/kg. This finding suggests that all the extracts were safe in or non toxic to rats and belonging to category 5 (>5000), hence doses of 500 mg/kg, p.o. were selected for the study.

Effect of selected plant extracts on Functional Parameters:

Thiopentone sodium (40 mg/kg, i.p) induced sleep in experimental animals in all groups. It was found that rats treated with paracetamol showed a marked decrease in onset of sleep (sec) and increase the duration of sleeping time (min) when compared against normal control group. Onset of sleep had significantly increased in the rats pretreated with 50%hydro-ethanol extract (500 mg/kg, po) and silymarin treated rats while the duration of sleeping time had significantly decreased for the same, when compared to paracetamol treated group.

S.No.	Treatment/ Dose	Onset of sleep (Sec)	Duration of sleep (Min)
1	Normal	170.0 ± 2.06	110.2 ± 2.80
2	Induced(paracetamol)	98.4 ± 6.28*	255.8 ± 5.90*
3	Standard(silymarin)	176.6 ± 4.48***	140.2 ± 4.49***
4	Heena(500 mg/kg)	149.8 ± 5.81***	192.3 ± 4.55***

Effect of selected plant extracts on physical parameters:

Liver weight

In paracetamol treated rats, enlargement of liver was observed, which was evident of increase in the liver weight. The group treated with Heena (500 mg/kg,p.o) and silymarin showed significant restoration of liver weight nearer to normal.

Liver volume

In paracetamol treated animals, the volume of the liver was significantly increased, but it was normalized in Heena (500 mg/kg, po) and silymarin treated groups of animals. A significant reduction in liver volume supports hepatoprotective effects of selected extracts.

S.No.	Treatment/ Dose	Liver weight (wt/100gm bw)	Liver Volume
1	Normal	6.84 ±0.06	6.97 ± 0.05
2	Induced (paracetamol)	8.84 ± 0.48*	9.02 ± 0.49*
3	Standard (silymarin)	7.02 ± 0.46***	7.36 ± 0.49***
4	HEENA(500 mg/kg)	7.38 ± 0.80***	7.59 ± 0.83***

Effect of selected plant extracts on serum marker enzymes

There was a significant elevation in the levels of serum marker enzymes like SGOT (serum glutamate oxalacetate transminase), SGPT (serum glutamate pyruvate Transamainse) and ALP (alkaline phosphatase) in hepatotoxicated animals. Pretreatment with Heena (500mg/kg, po) and silymarin (25 mg/kg, po) exhibited an ability to counteract the hepatotoxicity by decreasing the elevated level of serum marker enzymes.

S.No.	Treatment/ Dose	SGPT U/L	SGOT U/L	ALP U/L
1	Normal	62.0 ± 3.71	168.04 ± 2.80	190.0 ± 8.01
2	Induced(paracetamol)	154.8 ± 8.64*	248.4 ± 9.24*	360.20 ± 8.82*
3	Standard(silymarin)	86.86 ± 8.63***	176.16 ± 8.17***	166.35 ± 4.27***
4	HEENA(500 mg/kg)	92.24 ± 8.24***	186.48 ± 8.52***	193.0 ± 6.14***

Effect of selected plant extracts on biochemical parameters

In paracetamol treated groups, there was a significant increase in total bilirubin and significant reduction in total protein. Whereas in Heena (500mg/kg, po) and silymarin treated groups, significant reduction in total bilirubin and significant increase in total protein was observed.

S.No.	Treatment/ Dose	Total Bilirubin mg/dl	Total Protein gm/dl
1	Normal	0.38 ± 0.06	9.57 ± 0.24
2	Induced(paracetamol)	5.42 ± 0.11*	5.42 ± 1.46*
3	Standard(silymarin)	0.45 ± 0.82***	9.21 ± 1.26***
4	HEENA(500 mg/kg)	0.52 ± 0.48***	8.28 ± 0.84***

Values are mean ± SEM, n = 6. (One way ANOVA Followed by Dunnette multiple comparisons test). Statistically significance of ** P<0.01, *** P<0.001, when compared with paracetamol induced group and * P<0.05, when compared with normal group.

Histopathology

Histopathological studies provided a supportive evidence to biochemical analysis that the selected extractes posses the hepatoprotective activity. Histological changes occurs due to hepatocytes were restored to normal by selected extracts treatment. (Yue et al., 2006).

Effect of selected plant extracts on histopathological diagram of liver tissue in hepatotoxic rats.

CONCLUSION:

Every year 18,000 people are reported to die due to liver cirrhosis caused by hepatitis. Traditional systems of medicine, especially Ayurveda contains number of preparations for treating liver and GIT disorders. Modern medicine provides only symptomatic relief with side effects in the treatment of liver disease. By considering such aspects, the present proposal of study is designed for the development and evaluation for effective management of liver diseases using some of the indigenous plants. on the basis of literature review and discussion with the traditional medical practitioners of the remote areas of Ujjain, near Mandideep (Bhopal) Madhya Pradesh and Jashpur, Chhattisgarh, India. Stem bark of Nyctanthes arbor-tristis Linn was selected for evaluation of the hepatoprotective activity using animal model. Hence, it is concluded that the hydro-ethanol extract of the selected plant possesses hepatoprotective activity and thus supports the traditional application of the same under the light of modern science. The plant selected for the present study has demands for further phytochemical as well as pharmacological research such as isolation of principle active phytoconstituents, evaluation of various pharmacological activities.

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Received on 13.07.2015 Modified on 25.07.2015

Res. J. Pharmacology & P’dynamics. 7(3): July-Sept., 2015; Page 124-128

DOI: 10.5958/2321-5836.2015.00023.3