Hepatoprotective potential of ethanolic extract of Pandanus odoratissimus root against paracetamol-induced hepatotoxicity in rats.

BACKGROUND: Pandanus odoratissimus (Pandanaceae) is popular in the indigenous system of medicines like Ayurveda, Siddha, Unani and Homoeopathy. In the traditional system of medicine various plant parts such as leaves, root, flowers, and oils are used as anthelmintic, tonic, stomachic, digestive and in the treatment of jaundice and various liver disorders.
OBJECTIVE: The aim was to investigate the hepatoprotective activity of ethanolic extract of the root of P. odoratissimus against paracetamol (PCM) induced hepatotoxicity in rats.
MATERIALS AND METHODS: Hepatotoxicity was induced in male Wistar rat by PCM (2 g/kg b.w. p.o. for 7 days). The ethanolic extract of P. odoratissimus root was administered at the dose level of 200 mg/kg and 400 mg/kg b.w. orally for 7 days and silymarin (100 mg/kg b.w. p.o.) as standard drug was administered once daily for a week. The hepatoprotective effect of ethanolic extract was evaluated by assessment of biochemical parameters such as serum glutamic oxaloacetic transaminase, serum glutamic-pyruvic transaminase, serum alkaline phosphatase, total and direct bilirubin and triglycerides. Histopathological study of rat liver was also done.
RESULTS: Experimental findings revealed that the extract at dose level of 200 mg/kg and 400 mg/kg of b.w. showed dose dependant hepatoprotective effect against PCM induced hepatotoxicity by significantly restoring the levels of serum enzymes to normal that was comparable to that of silymarin, but the extract at dose level of 400 mg/kg was found to be more potent when compared to that of 200 mg/kg. Besides, the results obtained from histopathological study also support the study.
CONCLUSION: From the results, it can be concluded that ethanolic extract of the root of P. odoratissimus afforded significant protection against PCM induced hepatotoxicity in rats.

Liver is the largest internal organ in the body, weighing around 1.2-1.5 kg. It plays a pivotal role in protein synthesis, storage and metabolism of fats and carbohydrates, detoxification of drugs and other toxins, excretion of bilirubin and metabolism of hormones.[1] Toxins, infectious agents, medications, and serum inflammatory mediators result in a diverse range of disease processes, leading to loss of normal histological architecture, reduced cell mass and loss of blood flow. Consequently, functional liver capacity may be lost.[2]

Herbal medicines have recently attracted much attention as alternative medicines useful for treating or preventing lifestyle related disorders and relatively very little knowledge is available about their mode of action. There has been a growing interest in the analysis of plant products that has stimulated intense research on their potential health benefits.[3]

Pandanus odoratissimus Linn. f. (Pandanaceae), commonly known as Umbrella tree or Screw pine tree in English, Ketaki in Sanskrit, Kewda, Ketki, Gagandhul in Hindi, is a palm like small tree or shrub with fragrant flowers found along the coast of India and Andaman Islands. Tribal/folklore practitioners use this drug to treat many ailments. The root and flowers of the drug Ketaki acts as an abortifacient, and it is indicated for the treatment of skin diseases, leprosy, scabies and syphilis.[4] A mixture of the dried root powder along with one spoonful turmeric juice and supernatant from a clean lime water extract, taken early in the morning orally for 1-week will cure urinary disorders.[5] The young tender shoots of Pandanus spp. along with boiled rice water soaked overnight is given to patients in the early morning to treat their severe jaundice.[6] Therefore, the present study is planned to investigate the effect of ethanolic extract of P. odoratissimus (EEPO) roots in paracetamol (PCM) induced liver damage in Wistar rats.

Materials and Methods

Plant material

The roots of P. odoratissimus (Pandanaceae) were gathered from the coastal region of Odisha. The plant specimen was authenticated by Birbal Sahni Institute of Palaeobotany, Lucknow, India. A voucher specimen no. BSIP 09 was submitted in Birbal Sahni Institute of Palaeobotany, Lucknow and also in Department of Pharmacognosy, Teerthanker Mahaveer College of Pharmacy, Moradabad.

Preparation of extracts

The plant material was dried under shade and mechanically reduced to moderate coarse powder. The coarse powder was defatted with petroleum ether and then extracted with ethanol using a soxhlet apparatus. The extract was then concentrated by using a rotary evaporator at 40°C under reduced pressure. The concentrated extract was then transferred to a china dish and evaporated on a thermostat-controlled water bath until complete drying.

Phytochemical screening

The freshly prepared ethanolic extract was subjected to various qualitative chemical tests to know the presence or absence of phytoconstituents such as alkaloids, glycosides, flavonoids, etc., These were identified by characteristic color changes by using the standard procedure.[78]

Acute toxicity study

The acute toxicity study was carried out as per the guidelines set by Organization for Economic Co-operation and Development-423, received from CPCSEA, Ministry of Social Justice and Empowerment, Government of India.[9] The ethanolic extract was orally administered to adult Wistar albino rats. The groups were continuously observed for mortality and behavioral changes during first 24 h and then daily for a fortnight. The oral LD50 was found to be more than 3000 mg/kg.

Preparation of the drug solution

The EEPO was suspended in normal saline to prepare the dose of 200 and 400 mg/kg body weight for administration to the rats.

Experimental animals

Wistar rats (180-200 g) were housed in a group of four in separate cages under controlled conditions of temperature (22°C ± 2°C). All animals were given standard diet (Golden feed, New Delhi) and water ad libitum. Animal experiment was approved by Institutional Animal Ethical Committee of Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad (Reg. No. 1205/c/09/CPCSEA).

Assessment of hepatoprotective activity

Wistar rats were divided into five groups having six animals in each (normal control, toxic control, standard, test low dose and test high dose). Normal control group received saline 1 mL/kg for 1-week, toxic control group received saline 1 mL/kg for 1-week, standard group received silymarin (100 mg/kg, p.o.) once a day for 1-week and test groups received 200 mg/kg and 400 mg/kg oral dose of ethanolic extract once a day for 1-week. On the 5th day, after the administration of respective treatments, all the animals of groups II, III, IV and V were administered PCM at a dose of 2 g/kg (p.o.). On the 7th day that is, after 48 h of pharmacological treatments, 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.[10]

Biochemical analysis

Blood samples were centrifuged for 10 min at 7000 rpm using micro-centrifuge to separate the serum. The levels of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic-pyruvic transaminase (SGPT)[11] serum alkaline phosphatase (SALP), bilirubin (total and direct) and triglycerides[121314] were estimated.

Histopathological studies

The liver specimens obtained from the control and treated groups of animals were fixed in 10% neutral formalin for 24 h. The sections were taken (5 μm thickness) using microtome, processed in alcohol-xylene series and stained with alum-hematoxylin and eosin.[15] The sections were examined microscopically for the evaluation of histopathological architecture.

Statistical analysis

The results were expressed as mean ± standard error mean. The statistical significance was assessed using one-way analysis of variance, followed by Bonferroni's Multiple Comparison test.

Results

Preliminary phytochemical screening of ethanolic extract revealed the presence of carbohydrate, alkaloids, glycosides, flavonoids and phenolic compounds. Based on these phytoconstituents, the ethanolic extract was selected for pharmacological evaluation.

Acute toxicity

The extract was found to be safe in the dose used and no mortality up to a dose of 3000 mg/kg, b.w. for ethanolic extract was observed. Hence, 200 and 400 mg/kg b.w. p.o. were selected for the activity.

Hepatoprotective activity

The administration of PCM resulted in a marked increase in serum SGOT, SGPT, ALP, total and direct bilirubin and triglycerides. The protective actions of ethanolic extract at dose level of 200 and 400 mg/kg b.w on hepatotoxicity induced by PCM are summarized in Table 1. Pretreatment of the rats with ethanolic extract before PCM administration caused a significant reduction in the values of SGOT, SGPT, ALP, total and direct bilirubin, and triglycerides.

Table 1

Effect of ethanolic extract of root of Pandanus odoratissimus on paracetamol induced hepatotoxicity in rats

Histopathology

The histological observations of control rats did not show any histological alterations in the hepatocytes. The liver sections showed normal architecture with no damage in the central vein and no change in sinusoids and hepatocytes architecture [Figure 1]. In toxic control group, the liver sections showed hepatic cell necrosis along with severe damage associated with central vein due to PCM [Figure 2]. In group treated with silymarin, the liver sections showed mild to moderate diffuse granular degeneration and necrosis in hepatocytes [Figure 3]. In group treated with ethanolic extract (200 mg/kg and 400 mg/kg), the liver sections showed reduced severity of damage and regeneration of hepatocytes, bile duct, branch of hepatic portal vein and minimal necrosis caused by PCM intoxication [Figures 4 and 5].

Figure 1

Photomicrograph of liver of control animals showed normal architecture

Figure 2

Photomicrograph of liver of animals treated with paracetamol only showed severe damage in central vein

Figure 3

Silymarin treated group showed mild to moderate diffuse granular degeneration and very mild necrosis in hepatocytes

Figure 4

Ethanolic extract of Pandanus odoratissimus treated groups at 200 mg/kg reduced damage due paracetamol in hepatocytes, regeneration of hepatocytes and reduced necrosis

Figure 5

Ethanolic extract of Pandanus odoratissimus treated groups at 400 mg/kg reduced damage due paracetamol in hepatocytes, regeneration of hepatocytes and reduced necrosis

Discussion

Paracetamol is a common antipyretic agent that is safe in therapeutic doses, but can produce fatal hepatic necrosis in man, rats and mice with toxic doses.[1617] PCM toxicity is due to the formation of toxic metabolites when a part of it is metabolized by cytochrome P-450.[18] Introduction of cytochrome or depletion of hepatic glutathione is a prerequisite for PCM-induced hepatotoxicity. Due to liver damage, cellular leakage and loss of functional integrity results elevated serum enzymes levels.[19]

In the present study, PCM was caused significant elevation in the levels of SGOT, SGPT, SALP, total and direct bilirubin and triglycerides. Pretreatment with ethanolic extract was found to be significantly reversing PCM induced changes. Hence, a reduction in the levels of these enzymes demonstrates membrane stabilizing activity of the extract. Reduction in the levels of SGOT and SGPT towards the normal value is an indication of the regeneration process. The histological examination of the liver sections reveals that the normal cellular architecture was retained when compared to silymarin, thereby confirming the protective effect of the extract. Various phytoconstituents such as alkaloids, flavonoids and other phenolic constituents present in ethanolic extract of roots of P. odoratissimus could be responsible for the membrane stabilizing activity.

Conclusion

Ethanolic extract of roots of P. odoratissimus has dose dependent hepatoprotective action upon PCM induced hepatotoxicity in rats, which may be attributed because of the presence of the phytoconstituents such as alkaloids, flavonoids, glycosides, etc., However, there is a need to identify the exact mechanism (s) and active phytoconstituent (s) involved in this effect for future studies.