Evaluation of the Hepatoprotective Effect of Fumaria parviflora and Momordica balsamina from Saudi Folk Medicine Against Experimentally Induced Liver Injury in Rats

INTRODUCTION

Natural products still represents a vital source for biologically active drugs with unique mechanisms of actions. Many traditional plants are used for the treatment of liver problems. Study of these led to the discovery of active compounds yet developed to successful drugs. Silymarin (Saller et al., 2007), schisandrin B (Zhu et al., 1999; Cyong et al., 2000), phyllanthin, hypophyllanthin, picroside I and kutkoside (Ram, 2001) are examples of natural antihepatotoxic compounds derived from traditional herbs. In Saudi Arabia, many plants are claimed to treat liver problems (Mossa et al., 1987), however, most of these claims lack the scientific prove. Here was an interest in screening of such plants for their hepatoprotective effect and in advanced steps identify their active constituents. The aerial parts of Fumaria parviflora are used in Saudi folk medicine for bile secretion, spleen disorders and jaundice. Other used includes diaphoretic, diuretic, anthelmintic and laxative. It is also claimed to have antimicrobial activity so used for treatment of bacterial and skin diseases (Mossa et al., 1987; Ghazanfar, 1994; El-Shanawany, 1996). Extract of the aerial parts of Fumaria parviflora showed significant anticholinesterase activity (Orhan et al., 2004). It also proved to be active against larvae of Haemonchus contortus (Hordegen et al., 2006). Pharmacological studies indicated that Fumaria parvi posses anti-inflammatory, anti-nociceptive and antidiabetic activities (Akhtar et al., 1984; Heidari et al., 2004). Different parts of Momordica balsamina are used as galactagogue, anthelmintic, laxative and tonic. It is also used for disorders of the liver (Mossa et al., 1987; El-Shanawany, 1996). Recent study indicated that the fruit pulp extract of Momordica balsamina has promising anti-HIV, antimicrobial and antiplasmodial effects (Bot et al., 2007; Benoit-Vical et al., 2006; Jigam et al., 2004).

MATERIALS AND METHODS

Plant Materials
The aerial parts of Fumaria parviflora Lam. (Asteraceae) and the leaves of Momordica balsamina Linn. (Cucurbitaceae) were purchased from the local market in Riyadh, in April 2003. The plants were identified by Dr. Mohammad Atiqur Rahman, taxonomist of the Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

Extraction
The dried ground aerial parts of Fumaria parviflora and leaves of Momordica balsamina (50 g from each plant) were extracted to exhaustion by percolation at room temperature with 90% ethanol and the extract was evaporated in vacuo to leave 7.4 and 6.3 g of residue.

Animals
Wistar albino rats (150-200 g) of either sex roughly the same age (8-10 weeks), obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh were used. The animals were housed under constant temperature (22±2 °C), humidity (55%) and light/dark conditions (12/12 h). They were provided with Purina chow and free access to drinking water ad libitum (Abdel-Kader and Alqasoumi, 2008). The experiments and procedures used in this study were approved by the Ethical Committee of the College of Pharmacy, King Saud University.

Chemicals
Silymarin (Sigma Chemical Company, USA).

Hepatoprotective Activity
Male Wistar rats were divided into five groups six animals each. Group 1 was kept as a control group. Groups 2, 3, 4 and 5 received 0.125 mL of CCl₄ in liquid paraffin (1:1) per 100 g body weight intraperitoneally. Group 2 received only CCl₄ treatment. Group 3 was administered silymarin at a dose of 10 mg kg^-1 p.o. Groups 4 and 5 were treated with 250 and 500 mg kg^-1 of extracts respectively. Drug treatment was started 5 days prior to CCl₄ administration and continued till the end of the experiment. After 48 h, following CC1₄ administration the animals were sacrificed using ether anesthesia. Blood samples were collected by heart puncture and the serum was separated for evaluating the biochemical parameters. The liver was immediately removed and a small piece was fixed in 10% formalin for histopathological assessment.

Determination of the Enzyme Levels
The biochemical parameters such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin were estimated by reported methods (Boon et al., 2006). The enzyme activities were measured using diagnostic strips (Reflotron^®, ROCHE) and were read on a Reflotron^® Plus instrument (ROCHE).

Statistical Analysis
For each set of experiments where two or more than two groups were compared, an analysis of variance (ANOVA) test was used to determine the significance of the differences. Differences between the control and CCl₄-treated group were compared for significance using student`s t-test for non paired samples (Woolson and Clarke, 2002). All the values shown are the mean±SE mean.

Histopathology
The livers of treated animals were immediately removed and a small piece was fixed in 10% formalin for histopathological assessment. All specimens were placed in cassettes and loaded into tissue baskets. The specimens were subjected to dehydration, clearing and inflatration by immersion in different conc of ethanol (70-100%), xylene (3 times, 1 h each) and finally paraffin wax (4 times, 1 h each). The tissues were then transferred into moulds filled with paraffin wax. After orienting the tissues by hot forceps the moulds were chilled on cold plates and excess wax were trimmed off using a knife. The rotary microtome (Leitz 1512) was used for making thin sections (3°m). The sections were placed onto clean slides that were drained vertically for several minutes before placing them onto a warming table at 37-40°C (Prophet et al., 1994). The slides were then deparaffinized, hydrated and to stained in Mayer`s hematoxylin solution for 15 min. The slides were then washed in lukewarm running tap water for 15 min, placed in distilled water, 80% ethyl alcohol for 1 to 2 min then counterstained in eosin-phloxine solution for 2 min. The slides were then dehydrated and cleared through 2 changes each of 95% ethyl alcohol, absolute ethyl alcohol and xylene, 2 min each and finally mounting with resinous medium.

RESULTS AND DISCUSSION

Male Wistar rats were used to estimate the serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin serum enzymes as indicator for liver protection against carbon tetrachloride hepatotoxicity. Treatment of animals with the hepatotoxic agent carbon tetrarchloride resulted in significant increase of transaminases (SGOT and SGPT) and alkaline phosphate levels (ALP) due to hepatocytes damage (Zafar and Ali, 1998). Sever jaundice was reflected by increase level of serum bilirubin (Lin et al., 1997) (Table 1).

Treatment of animals with the known hepatoprotective agents silymarin as a reference standard resulted in significant decrease in the elevated levels of SGOT, SGPT, ALP and bilirubin (p<0.001) (Table 1). Silymarin act as an antioxidant by scavenging prooxidant free radicals and by increasing the intracellular concentration of GSH. It also exhibits a regulatory action of cellular membrane permeability and increase in its stability against xenobiotics injury; increasing the synthesis of ribosomal RNA by stimulating DNA polymerase-I, exerting a steroid like regulatory action on DNA transcription and stimulation of protein synthesis and regeneration of liver cells (Saller et al., 2007; Dehmlow et al., 1996a, b; Gakova et al., 1992). Silymarin efficacy is not limited to the treatment of toxic and metabolic liver damage; it is also effective in acute, chronic hepatitis and in inhibiting fibrotic activity (Hikino and Kiso, 1988; Mayer et al., 2005).

Treatment of the animals with doses of 500 mg kg^-1 body weight of Fumaria parviflora aerial parts ethanol extract prior to CCl₄ administration resulted in decrease in the levels of SGOT (24.1%), SGPT (21.6%), ALP (20.5%) and bilirubin (11.3%) levels. The lower dose of the extract (250 mg kg^-1, body weight) show only significant decrease of the levels of APL (15.2%) (Table 1).

Table 1:	Effects of ethanolic extracts of Fumaria parviflora and Momordica balsamina on serum biochemical parameters

p<0.05; p<0.01; **p<0.001, ^aas compared with the normal saline (control) group; ^bas compared with the CCl₄ only group

Momordica balsamina (Leaves) crude extract when given to rats in the two used doses (250 and 500 mg kg^-1, body weight) before CCl₄treatment resulted in highly significant (p<0.001) decrease in the levels of SGOT, SGPT, ALP and bilirubin (Table 1). The obtained results indicated a high degree of protection against the hepatotoxic effect of CCl₄.

The histological appearance of the hepatocyte reflects their conditions (Fig. 1A). Exposure of hepatocytes to toxic agents such as CCl₄leads to histopathological changes from the normal cell appearance. The hepatocytes of rat livers treated with a single dose of 1.25 mL CCl₄ kg^-1, showed centrilobular hepatocyte necrosis, microvesicular fatty change and extensive fatty change were observed on the midzonal or entire lobe at 24 h (Fig. 1B). Liver tissue of rats treated with CCl₄ and silymarin showed good recovery with absence of necrosis, fatty depositions and recovery to normal histological appearance of hepatocytes (Fig. 1C). The central vein has minimal portal inflammation. Histological appearance of rat liver treated with 500 mg kg^-1 Fumaria parviflora aerial parts total extract showed minimal portal inflammation and more or less normal appearance (Fig. 1D). Sections of the rat liver treated with CCl₄ and Momordica balsamina (500 mg kg^-1) showed significant recovery with disappearance of fatty deposition and necrosis. The portal tracts appeared almost clear indicating a potent hepatoprotective activity (Fig. 1E).

The current study aimed to evaluate the effect of Fumaria parviflora and Momordica balsamina as protective for hepatocytes. The higher dose of Fumaria parviflora showed significant decrease in the level of biochemical markers as well as notable recovery of the histological characters of the hepatocytes. Momordica balsamina showed superior protective effect in both biochemical markers assessment and histopathological appearance of hepatocytes.

Fig. 1:

Histopathological appearance of liver cells, (A) normal cells, (B) liver cells of rats treated with CCl₄, (C) liver cells of rats treated with CCl₄ and silymarin, (D) liver cells of rats treated with CCl₄ and Fumaria parviflora, (E) liver cells of rats treated with CCl₄ and Momordica balsamina

ACKNOWLEDGMENTS

The authors are grateful to Mr. Ali D. Al-Sawadi and Mr. Malik Sawood for technical assistance. The research was supported by grants from King Abdul Aziz City of Science and Technology (KACST), Riyadh, Saudi Arabia to Dr. Alqasoumi.