Evaluation of Hepatoprotective Activity of Rhizomes of

Curculigo orchioides Gaertn

 

G. Babu*, N. K. Shalima, T. A. Divya, T. Divya

Department of Pharmaceutical Chemistry, Devaki Amma Memorial College of Pharmacy, Chelembra 673634, Malappuram District, Kerala

*Corresponding Author E-mail: gbjournals@gmail.com

 

The present study was conducted to evaluate the hepatoprotective activity of ethyl acetate extract of rhizomes of Curculigo orchioides. The extract at the dose of 200 mg/kg and 400 mg/kg b. wt. was tested for its hepatoprotection, by inducing hepatotoxicity with CCl₄ in Wistar albino rats and using silymarin (100 mg/kg) as the reference standard. Biochemical parameters like, SGOT, SGPT, SALP and serum bilirubin were determined to assess the hepatoprotective effect. The extract has shown significant hepatoprotection in albino rats in reducing SGOT, SGPT, SALP and serum bilirubin levels.

 

 

 

INTRODUCTION:

Plants and their secondary metabolites have a long history of use in modern ‘western’ medicine and in certain systems of traditional medicine. Plants are used in traditional system of medicine for the management of liver disorders. However many of them have not investigated for their described effects. Curculigo orchioides Gaertn, belonging to the family Amaryllidacae, is one such medicinal plant used in the treatment of liver disorders in folk medicine. The active compounds that have been reported are flavones, glycosides, steroids, saponins, triterpenoids and other secondary metabolites¹. It also has anticancer, antibacterial, antihistaminic, antidiabetic activitie², etc. This has triggered the authors to evaluate the hepatoprotective activity of ethyl acetate extract of Curculigo orchioides against liver intoxication by CCl₄ in wistar albino rats. Biochemical parameters like, SGOT, SGPT, SALP and serum bilirubin were determined to assess the hepatoprotective effect. The study revealed that ethyl acetate extract significantly reduced SGOT, SGPT, SALP and serum bilirubin levels. The preliminary findings suggest that the plant Curculigo orchioides possess potential hepatoprotective activity. The present study scientifically validated the traditional use of Curculigo orchioides for liver disorders.

 

MATERIALS AND METHODS:

Plant material:

The rhizomes of Curculigo orchioides was collected in the month of December and was authenticated by Dr. M Sabu, Professor and Head, Department of Botany, Calicut University, Malappuram, Kerala, and a voucher specimen was deposited in the Department (Specimen No: 107892).

 

The powdered plant material (1 Kg) was extracted with chloroform, ethyl acetate and distilled water separately, in a Soxhlet apparatus, by continuous hot extraction method. Each extracts were then concentrated under reduced pressure on a rotary evaporator to dryness to give the crude residue. The crude residues were employed for further investigation³. The extracts were subjected to qualitative chemical tests for the detection of various plant constituents like carbohydrates, glycosides, proteins and amino acids, fixed oils and fats, gums and mucilage, alkaloids, phytosterols, flavanoids, tannins and phenolic compounds, saponins, triterpenoids⁴, etc.

 

Animals:

Albino mice of Swiss strain and albino rats of Wistar strain were used for toxicological studies and pharmacological studies respectively. Female mice selected were nulliparus and non-pregnant. Female mice weighing 25 to 30 g and rats of either sex weighing 125 to 150 g were used for the study. Each animal, at the commencement of its dosing, was between 8 and 12 weeks old and their weight variation was within ± 20% of the mean weight of any previously dosed animals.  The temperature in the experimental animal room was 22ºC (± 3ºC) and the relative humidity was between 50-60%. These animals were fed with pellet diet manufactured by Amrut laboratory, Animal Feed Company, Sangli, Maharashtra and drinking water ad libitum. They were kept in 12 h/12 h light/dark cycle and maintained for at least 5 d prior to dosing to allow for acclimatization to the laboratory conditions. The animal experimental protocol has been approved by OACE of U win life science, Malappuram, where the animal studies were carried out (OACE No: ULSB/DAEC/KER/60/363/13).

 

Acute toxicity study:

Acute toxicity study was carried out as per OECD 423 (Organization of Economic Cooperation and Development) guidelines. Swiss albino mice weighing 20-25g, were selected and fastened overnight .The selected animals were grouped as three in one group. The animals were and the test sample SCO1 was given orally at a starting dose of 5mg/Kg body weight and observed for a period of 2 h and occasionally for 4 h to detect any toxic signs and mortality. Since no mortality was observed, same dose was repeated with another group of animals. The procedure was repeated for doses of 50, 300 and 2000 mg/kg in separate group of animals. The experiment was repeated for seven more days and also for fourteen days, no change was observed from the experiment⁵. So the maximum dose of 2000 mg/Kg can be used as safe dose. From this result 1/10^th and 1/5^th values of 2000 mg/Kg were taken for further studies⁶.

 

Hepatoprotective activity:

Albino rats of Wistar strain weighing 125-150 g of either sex were used for the study. They were housed in polypropylene cages with not more than six animals per cage and maintained under standard conditions. Thirty rats were divided into five groups of six animals each. Group I was given a single daily dose of Carboxy Methyl Cellulose (1 ml of 1% w/v, p.o. b. wt.)⁷. Group II received carbon tetrachloride (1 ml/kg b. wt., sc.1:1 v/v mixture of CCl₄ and liquid paraffin) was given for every 72 h for 14 days⁸. Group III received Silymarin, a known hepatoprotective compound, at a dose of 100 mg/Kg .p.o., along with carbon tetrachloride for every 72 h for 14 d⁹. While group IV and V received orally, 200 and 400 mg/kg b. wt. of SCO1 and SCO2 in 1 %w/v, CMC respectively¹⁰, along with carbon tetrachloride as in group II.

 

Replenishing a known quantity of fresh food daily at 8.00 a.m. and thereby measuring the food intake of the previous day and carried out measurement of daily food consumption. Body weight of rats was recorded weekly to assess percentage of weight gain of each animal. Animals were kept starved overnight on the last day. On the next day, after recording the weight of each animal, they were euthanized by decapitation under ether anesthesia, by making an incision on jugular vein; blood was collected in sterile centrifuge tubes and allowed to clot. The liver was dissected out immediately, rinsed with ice cold phosphate buffer and homogenized with 5% formalin solution immediately after removal from the animal to avoid decomposition. Embedding in paraffin wax was carried out by removal of water using alcohol from 30-100% and then stained with hemotoxylin, which has an aqueous base. The sections were dehydrated using increasing concentration of alcohol and then stained with eosin. They were treated with diphenylxylene (DPX) and examined under the microscope¹¹.

 

Serum was separated from the collected blood by centrifugation and subjected to various biochemical estimations like serum glutamate oxaloacetic transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase (SALP), and serum bilirubin.

 

Statistical analysis:

Statistical analysis was performed by one way (ANOVA) followed by using results were expressed as mean ± the standard error of the mean (SEM) for 6 rats in each group. P< 0.05 was considered significant.

 

RESULTS AND DISCUSSION:

Carbohydrates, tannins, flavonoids, saponins, and coumarins were found to be present in the ethyl acetate extract of Curculigo orchioides rhizhomes. CCl₄ is one of the most commonly used hepatotoxins in the experimental study of liver diseases and proves highly useful as an experimental model for the study of acute hepatic injury. In this study, CCl₄ administration to rats lead to marked elevation in the levels of serum enzymes like, SGOT, SGPT, SALP and serum bilirubin level. The Table 1 shows that CCl₄ causes significant increase in SGOT value from control 188.99 ± 9.36 to 640.25 ± 11.24, SGPT value from 74.25 ± 4.32 to 388 ± 12.25, SALP value from 402.54 ± 3.65 to 645.25 ± 11.25, and serum bilirubin value from 0.28 ± 0.01 to 1.45 ± 0.03. This might be due to release of these enzymes from the cytoplasm, into the blood stream rapidly after rupture of the plasma membrane and cellular damage¹². Treatments with ethyl acetate extract of rhizomes of Curculigo orchioides (200 mg/kg and 400 mg/kg) significantly reduced the levels of these marker enzymes in CCl₄ treated rats, when compared with standard drug Silymarin.

 

Table 1 Biochemical changes in albino rats treated with ethyl acetate extract of rhizomes of Curculigo orchioides against CCl₄ induced hepatic injury.

Values are the mean ± S.E.M of six rats/treatment, *P<0.05, **P<0.01 and ***P<0.001 significant when compared to CCl₄

 

 

This implies that the extract tends to prevent liver damage, suppresses the leakage of enzymes through cellular membranes, preserves the integrity of the plasma membranes and hence restores these enzymes levels. This is in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes¹³.

 

Effective control of SALP, and serum bilirubin levels point toward an early improvement in the secretary mechanism of the hepatic cells. Decrease in serum bilirubin after treatment with the extract in liver damage indicated the effectiveness of the extract in normal functional status of the liver¹⁴. So, the result of present investigation indicates that the ethyl acetate extract of Curculigo orchioides possess good hepatoprotective activity. Serum total bilirubin and total protein levels on other hand are related to the function of hepatic cell.

 

(a)

 

(b)

 

(c)

 

(d)

 

(e)

Fig 1 Histopathological changes in liver of albino rats. Hematoxylin and Eosin (x100)

 

(a) Rats treated with normal saline 1 ml/kg b. wt.: Normal structure of hepatic lobes is seen; white spot indicates the presence of vacuoles, a prominent blood vein with normal appearance.

(b) Rats intoxicated with CCl₄ 1 ml/kg b. wt.:

Massive fatty changes are seen; necrosis, ballooning degeneration and broad infiltration of the lymphocytes are seen.

(c) Rats treated with CCl₄+silymarin 100 mg/kg b. wt.: Drastic recovery of hepatic parenchyma, mild congestion and micro vesicular changes are seen.

(d) Rats intoxicated with CCl₄ + 200 mg/kg b. wt.: Marked recovery of hepatic cells, mild congestion and micro vesicular changes are seen.

(e) Rats intoxicated with CCl₄ + 400 mg/kg b. wt.:

Drastic recovery of hepatic parenchyma, mild congestion and micro vesicular changes are seen.

 

The above fact is supported by histopathological studies. The Fig 1 indicates that the normal architecture of liver was completely lost in rats treated with CCl₄ with the appearance of vacuolated hepatocytes and degenerated nuclei. Section of liver in Silymarin treated group shows liver parenchyma with intact architecture. Section of liver in test drug treated groups (200 and 400 mg/Kg) shows intact architecture, few regenerative hepatocytes, and scattered mononuclear inflammatory cells which is similar to Silymarin treated group. The livers of rats treated with SCO1 and SCO2 showed a significant attenuation from CCl₄ induced liver damage as evident from normal hepatocytes with well defined nuclei. This study also supported the hepatoprotective activity of liver.

 

 

CONCLUSION:

From the above results, it may be concluded that the ethyl acetate extract of rhizomes of Curculigo orchioides is non-toxic and is safe. As the preliminary studies indicated, the extract possesses significant hepatoprotection, and after carrying out a thorough study of clinical trials, the plant may be considered as a low cost, potent, herbal liver tonic. 

 

ACKNOWLEGEMENT:

The authors are sincerely thankful to the Manager, Devaki Amma Memorial College of Pharmacy, Chelembra, for providing the facilities for carrying out the work.

 

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Accepted on 07.08.2013         © RJPT All right reserved