Liver is a vital organ among the vertebrate bodies. Even the heart and kidneys are now-a-days being transplanted, but the transplantation of liver is still at large. The advanced civilization and luxury in life are bringing a lot of testing and trials to our body in the form of pollution. Even the innocent human beings without taking toxic substances in the food or water, their blood contents are enriched with pollutants like lead, arsenic, cadmium and mercury through the inhalation of polluted atmosphere, triggered by the industrial exhaust, cosmetics, automobiles, etc. (Bray and Bettger, 1990; Williams, 1984; Muriel et al., 2001).
After the publication of the presence of excess heavy metals in the Ayurvedic drugs in the JAMA article (Saper et al., 2004), the importance given to Ayurvedic herbal drugs as alternative medicine has been brought to a question mark. The ban order on Ayurvedic products either in the form of pharmaceuticals or neutraceuticals has surrogated the popularity of Ayurvedic drugs. Under such circumstances, it is very essential to protect the traditional system particularly herbal medicines. The results of such testing have tarnished the validity of herbal drugs.
Keeping this in mind, the herbal plants here, are exposed for such studies. Such efforts have already been taken by many researchers on plants like Spirulina maxima, Picrorhiza kurroa, Eclipta alba, Boehemeia nieva, Cichurium intybus, etc. (Anandan et al., 1999). In spite of confirmatory results in the hepatoprotective effects for the above mentioned plants, there are still many more herbal plants which have to be studied for the hepatoprotective effects. Once the question on the veracity of herbal medicine is brought forward, it is felt that without any further delay such work has to be expedited. With that aim, the plant Terminalia arjuna has been taken up here for the research work.
Terminalia arjuna is an ornamental tree as well as a tree of shade.
It is also known as an adaptogen. The bark of this tree has been commonly used
in Ayurvedic preparations to bring hepatoprotective effect in a drug. It is
considered to be a common plant in the lower Himalayas. However, in the southern
tip of Tamil Nadu, it is found in the coastal ridges. While scanning the literature,
a scientific approach using this plant for understanding the hepatoprotective
effect could not be seen but for a few papers which have adopted either as antioxidant
(Gupta et al., 2001) or powerful cardiotonic (Karthikeyan et al.,
2003) or the salutary effect (Bharani et al., 1995). The scientific studies
using Terminalia arjuna for the natural treatment has been effected by
either aqueous extract or 50% ethyl alcohol has been studied. In spite of the
efforts made in understanding the hepatoprotective effects from Terminalia
arjuna, hydroalcoholic extract has not been applied so far. Under such status,
it is felt that a study using hydroalcoholic extract against the hepatotoxin
CCl4 may throw better light on the hepatoprotective effect from the
plant Terminalia arjuna.
While screening the literature, it is observed that a number of pharmacological and chemical agents act as hepatotoxin and produce variety of liver ailments (Liv et al., 1994). Among them, carbon tetrachloride intoxication in mice is an experimental model widely used for necrotic and steatotic changes in hepatic tissue (Parola et al., 1992). The study on hepatoprotective effect using CCl4, for any medicinal plants extraction will be of great interest and significant in standardizing the formulation and declare globally, the validity of the herbal products (Mehmet Kanter et al., 2005; Nagi et al., 1999; Al-Gharably et al., 1997).
MATERIALS AND METHODS
Plant materials: Bark of Terminalia arjuna (TA) was collected from Tirunelveli, Tamil Nadu, India. The plant was previously identified and authenticated by experts in the Rabinat Herbarium, St.Joseph College, Trichy, St. Xaviers College, Palayamkottai and Botanical Survey, CCRAS Unit, Govt. Medical College, Palayamkottai. The collected materials were washed thoroughly in water, chopped and kept for air drying. Then, the plants were shade dried for nearly 15 days. Once dried, they were crushed to get coarse powder.
The pulverized plant materials were taken up for extraction using Hydro Alcoholic Extract (HAE) in the proportion of 30:70. The extraction was carried out by cold percolation method. The extracts were then dried in vaccuo and they were stored in desiccators and kept in refrigerator for further use.
Experimental animals: Twenty-four male and female Albino Mice of Wister
strain weighing 25-35 g were taken from SASTRA Animal House, Thanjavur. The
animals were housed in polypropylene cages and maintained in controlled temperature
(22±2°C) and 12 h light and dark cycle. They were fed with the normal
rodent feed supplied by Venkateswara Animal House, Bangalore. Water was supplied
ad libitum. They were given a weeks time to get acclimatized with
the laboratory conditions. Initial body weight of each animal was recorded.
Ethical clearance for the use of animals was obtained from the committee constituted
for the purpose in SASTRA.
Experimental induction of hepatic damage: Liver damage was induced in mice by administering CCl4 in a form of suspension of Liquid Paraffin (LP) in the ratio of 3% V/V at the dose of 1 mL CCl4/kg body weight of each animal by adopting the route of intra-peritoneal (ip) in the lower abdomen (Rajesh and Latha, 2001; Liv et al., 1994). CCl4 was administered twice a week on every first and fourth day of all the 22 days.
Experimental design: Mice were divided into 6 groups of 6 animals each as revealed in OECD guidelines (OECD, 2000).
Group I animals served as control and given ip administration of LP only at the dose of 3 mL kg-1 b.w. Group II animals constituted hepatotoxic mice and provided ip administration of LP+CCl4 twice a week for 22 days. Group III animals were treated with standard drug of silymarin (25 mg kg-1 bw) + Ccl4. Group IV, V and VI animals were the herb-treated animals and given oral administration of Terminalia arjuna. After treating animals with drug at different doses of 50, 100, 200 mg kg-1 for 21 days CCl4 was administered.
A known quantity of fresh food daily at 9.30 am was provided. Thereby, measuring the difference in the food intake in a day, the daily food consumption of the previous day was carried out. Once the measurement of food intake is over, the LP, LP+CCl4, daily food consumption Std+CCl4, LP+CCl4+TA were administered at the same time between 10-11 am. Body weights of Mice were recorded weekly to assess percentage of weight gained in each group. General well being and behaviour of the animals were observed daily throughout the period of study. The lither in the cage was renewed twice a week to ensure maximum comfort for the animals.
Animals were kept starved overnight on the 21st day. On the next day, after recording the weight in each case, they were sacrificed by decapitation and making an incision on jugular vein to collect blood. The liver, heart, kidney, spleen and testes were dissected out, blotted off blood, washed in saline and weighed instantaneously. This was kept in frozen containers and proceeded to biochemical estimations.
Biochemical parameters: Plasma was prepared from the collected blood
and subjected to biochemical estimation of different hepatic markers such as
GOT (Rellman and Frankel, 1957), GPT (Rellman and Frankel, 1957) ALP (King and
King, 1954), Protein (Lowry et al., 1951) and Thio barbituric acid reactive
substances (TBARS) (Yagi et al., 1979), Reduced glutathione (Beutler
et al., 1963).
observation of macroscopic characters
10% liver homogenate in Tris HCL buffer of 0.1 M (pH 7.4) was also used for
the estimation of enzymes.
Histopathology: Gross pathology of animal organs has been macroscopically examined and the observed characters are qualitatively tabulated (Table 1). A portion of liver tissue in each group was fixed in 10% formalsal (formalin diluted to 10% with normal saline.) and proceeded to histopathology. After paraffin embedding and block making, several sections were made, stained with Haematoxylin and Eosin and examined under microscope. A few photomicrographs of representative types were also taken.
Statistical analysis: Statistical analysis was carried out using student t-test and the values of mean and ±SD.
Food consumption and weight gain: We observed that food consumption and weight gained significantly increased in group IV, V and VI as compared to other groups. In group II mice, there was a lesser weight gain as compared to group I animals (Table 2).
Animal organ weight: Organs like liver and kidney were found to be increased in disease control groups. But, on herb extract treated animals decrease in weight of organs like liver and kidney was observed (Table 3).
Haematological parameters: Hematological parameters like Haemoglobin,
Erythrocytes and Leucocytes were observed in both control and treated groups.
of change in body weight (values are mean±SEM)
Haemoglobin and erythrocytes level were found to decrease on CCl4
induced mice, whereas in the herb extract treated animals, the Haemoglobin was
found to increase in a dose dependent manner. At the same time, the leucocytes
were seen to increase in CCl4 induced mice. But, in herb extract
treated animals a decrease in a dose dependent manner was noticed (Table
Hepatic marker enzymes: Hepatic marker enzymes like ALP, GOT and GPT were found to go high to a greater extent in plasma to those animals treated with CCl4. But, in the case of TA herb extract treated animals dose dependent increase in activity of those enzymes could be accounted significantly (Athar et al., 1997). The level of these enzymes in hepatic tissue was found to decrease in disease control groups, whereas, in those herb extract treated animals, the level of these enzymes were noticed to increase and p<0.05 significant difference was noted at the highest dose of Terminalia arjuna extract treatment. Though the effect is very low when compared to standard drug treatment group, the results indicate that the extract is able to exhibit hepatoprotective activity to some extent (Table 5).
Antioxidant study: The level of reduced Glutathione in CCl4 treated mice were observed to be high while at the same time, the TA herb extract treated animals also indicated a significant raise in glutathione level (Anandan et al., 1999). Instead, the thio barbuturic acid (TBARS) level in hepatic cell of those animals treated with CCl4 though displayed an increase in values, unlike the above case, the values of TBARS in the case TA herb extract treated animals, were decreased (Table 6). This study reveals that Terminalia arjuna is also antioxidant in nature.
The levels of protein in plasma and hepatic tissue were much decreased in CCl4 treated animals. It was the other way in the case of TA herb extract treated animals. However, the same swang to normal level at higher dose of extract.
Gross pathology/necropsy report
Group I (1, 2, 3): Livers of all the three mice showed no gross pathological
of changes in organ weight (values are mean±SEM)
of standard drug and Terminalia arjuna on the activity of plasma
and hepatic enzymes (values are mean±SD)
of standard drug and Terminalia arjuna on the activity of plasma
and hepatic protein, hepatic reduced glutathione and TBARS level (values
Group II (4, 5): Liver of mouse no: 4 reflected patchy areas of necrosis throughout the external surface. The mouse no: 5 indicated a pale diffuse of discolouration of liver.
Group III (6, 7, 8, 9): While the Livers of mice 6, 8 and 9 were apparently normal, the mouse no: 7 registered a very pale discolouration.
Group IV (10, 11): Only mouse no: 11 indicated relatively pale colour, while the other one was normal.
Group V (12, 13 and 14): No gross pathological changes were observed except for a pale discolouration of liver in mouse no: 13.
Group VI (15, 16 and 17): Pale focal areas of discolouration were present on the dorsal surface of the liver of mice no: 16. No gross changes were detected in all other mice.
Group I (1, 2, 3): The liver sections of all the three mice revealed normal hepatocytes and mild to moderate congestion of blood vessels which correlated with the gross findings (Fig. 1).
Group II (4, 5): Severe colliquative necrosis of the hepatocytes especially
in the peripheral areas of the most of the portal triads i.e., the periportal
hepatocytes was consistently observed in the sections of both the mice. There
were large vacuolated spaces in the areas of necrosis where the necrotic debris
had been completely phagocytosed. Massive mononuclear cell infiltration was
consistently observed. Around 50-60% of the liver parenchyma was found to be
necrosed. The histopathological findings correlated with the necrotic patches
observed during necropsy (Fig. 2, 5 and
Group III (6, 7, 8, 9): The liver sections revealed diffuse colliquative
necrosis along with severe mononuclear cell infiltration.
liver section showing apparently normal hepatocytes (Group I-Control)
induced hepatotoxicity. Note areas of diffuse necrosis (arrow) around
the portal triads (Group II) 10X
of CCl4 treated mice with periportal necrosis of hepatocytes
and haemorrhages (arrow) (Group III) 10X
The necrosed hepatocytes were having intensely eosinophilic cytoplasm with
nuclear remnants, seen as karyorhectic fragments. The area involved was relatively
less than that of the group II (Fig. 3 and 4).
Group IV (10, 11): Moderate hydropic degeneration along with very few areas of spotty necrosis was observed consistently in both the sections. There were no areas of diffuse necrosis as observed in the groups II and III (Fig. 7).
treated liver section revealing apparently normal hepatocytes with engorged
blood vessels. (Group III) 10X
hepatocytes around the blood vessels with intensely eosinophilic cytoplasm
and karyolytic/pyknotic nuclei (Group II) 50X
normal hepatocytes of TA2 treated mice. Note the presence of
mild hepatocytes swelling and thin sinusoidal spaces (arrow) (Group II)
Group V (12, 13 and 14): The hepatocytes were near normal with a few areas of spotty necrosis. No diffuse necrosis was observed (Fig. 8).
Group VI (15, 16 and 17): Focal areas of hydropic changes were observed throughout the sections along
necrosis (arrow) along with haemorrhages in CCl4 treated mice
liver section (Group IV) 50X
treated mice liver with normal hepatocytes and sinusoids (Group V) 50X
with few areas of mononuclear infiltration and occasional spotty necrosis. No diffuse necrotic changes were observed in all the three sections.
Recknagel (1967) has established the CCl4 metabolization in the liver by the highly reactive trichloromethyl radical and auto-oxidation of the fatty acids present in the cytoplasmic membrane phospholipids by the free radical made available by CCl4 metabolisation and functional and morphological changes taking place in the cell membrane. The same has been attested due to the elevation of marker enzymes like GOT, GPT and ALP in serum. Above all, they have also proved the hepatoprotectivity against CCl4 induced liver damages by using a polyherbal formulation, known as Himoliv which is a combination of 25 different herbal plants (Bhattacharyya et al., 2003a, b). However, in those 25 plants they have not included the Terminalia arjuna which is taken up for the present study. Here, it is noticed that Terminalia arjuna is producing a better hepatoprotective activity against CCl4 induced liver damages.
On induction of disease with CCl4 GOT, GPT and ALP level in plasma were found to be increased significantly (p<0.01) and it was found to be 3.87, 3.48, 7.5 times, respectively when compared to the normal animals. But on Terminalia arjuna treated animals at the doses of 50, 100, 200 mg kg-1 bw, a decrease in the enzyme level according to the dosage levels has been observed. When compared to the control group, in the case of GOT, the level in plasma is found to be 3.5, 2.5 and 1.9 times, respectively while GPT level remains to be of 2.7, 2.4 and 1.8 times and ALP level of 6.3, 5.6 and 4.9 times at 50, 100 and 200 mg kg-1 bw, respectively. It has been noticed that the different levels of enzyme changes are in very close range in activities to the standard modern drug silymarin, which is also bringing a decrease in the level of enzyme in plasma like GOT by 1.4 times, GPT by 1.7 times and ALP by 4.6 times.
Instead of the serum plasma when the homogenated livers of both CCl4 inducted and extract treated are examined GOT, GPT and ALP levels in liver is found to be decreased by 0.6, 0.4 and 0.4 times, respectively. But, for Termianlia arjuna extract treated animals keep the variation in the enzyme level according to the dose rate such as 50, 100 and 200 mg kg-1 b. w. As revealed from the percentage values of GOT from 14 to 71%, GPT from 33.3 to 91.7% and ALP 20 to 40%, it is suggested that the extract provides due protection from liver damage. In other words, it enables the prevention of liver damage. The observed values of GOT, GPT and ALP in Terminalia arjuna extract treated cases are comparable to the standard drug silymarin values in such a way silymarin provides a preventive level to the tune of GOT by 61.6% while GPT by 52.4% and ALP by 38.4%.
Likewise, in the protein levels of plasma and liver, are found to be decreased significantly in the CCl4 inducted animals (p<0.01) whereas the Terminalia arjuna extract treated, it is noticed to have increase to the tune of the normal level at 200 mg kg-1 bw. Hypo-albuminaemia is the most frequently observed one in advanced chronic liver diseases. Hence, decline in total protein content can be deemed as a useful index of the severity of cellular dysfunction in chronic liver diseases (Venukumar et al., 2002). The lowered level of total proteins recorded here in the plasma as well as liver of CCl4 inducted mice reveals the severity of hepatopathy. The attainment of near normalcy in total protein contents of both plasma and liver of herb extract-treated mice further, attests the hepatoprotective effect of Terminalia arjuna.
It is well known that the hepatotoxic effect of carbon tetrachloride is due to the oxidative damage by free radical generation and antioxidant property is claimed to be one of the mechanisms of hepatoprotective drugs (Pandit et al., 2004).
Malondialdehyde level in liver is found to increase very rapidly to a significant level of p<0.05. On Terminalia arjuna treated animals the level of malondialdehyde is observed to decrease. Though significant difference is not noted in any dose when compared to the disease control animals, the extract treated is found to reflect a decrease to some extent. At 200 mg kg-1 bw of extract treated case, malondialdehyde level is found to increase by 1.6 times but in disease condition, it is seen to have an increase by 2.9 times when compared to the normal animals. But, Silymarin drug is able to exhibit significant difference p<0.05 when compared to the disease control animals.
Liver is the major site for the synthesis of GSH (Parola et al., 1993). The detoxification of different drugs and xenobiotics in the liver interferes with GSH (Seven et al., 2004). It was found to be decreased significantly by p<0.05. No significant difference was found on treating animals with different doses like 50, 100, 200 mg kg-1 b. w. But the extract was found to be able to exhibit some percent of protection in liver by increasing the level of GSH by 1.95 times higher than the disease control animals. But on silymarin treated animals reduced glutathione level is recorded to have a significant increase in comparison to the disease control animal. As suggested by Prasenjit Manna et al. (2006) in their studies on aqueous extract of Terminalia arjuna, here too, decrease in Reduced Glutathione level in liver of CCL4 toxicity is attributed to the decreased availability of GSH, resulted during the enhanced lipid peroxidation.
Lu (1999) has put forth that the decreased hepatic GSH in CCl4 intoxicated mice could be as a result of Hexose Monophosphate (HMP) which shunts the impairment due to CCL4 intoxication. Thereby, NADPH availability is reduced and the ability to recycle GSSG in GSH is decreased. Likewise, it is surmised that Termianlia arjuna extract may exhibit its activity by blocking oxidative damage through lipid peroxidation and protein oxidation which must have enabled the prevention of the loss of membrane permeability and dysfunction of cellular proteins and in turn, a decrease in the endogenous level of hydroxyl radical.
Noguchi et al. (1982) have observed the metabolisation of CCl4 in the liver by the cytochrome P450-dependent monooxygenase systems followed by its conversion to more chemically active form, trichloromethyl radical. Rajesh and Latha (2004) explained that the enzymes involved in this process are located in the endoplasmic reticulum of the liver and their activities are dependent on many environmental factors. Prasenjit Manna et al. (2006) have attributed the cause of metabolisation CCl4 to cytochrome P450 and other environmental factors to the results obtained for the aqueous extract of Terminalia arjuna. In the present case too, it is presumed that hepatoprotective and antioxidant activity of the hydroalcoholic extract of Terminalia arjuna must have been the result of what Prasenjit Manna et al. (2006) has proposed. However, the nature of this extract being different to the above one, the enzymes of endoplasmic reticulum involved in trichloromethyl radical formation has to be evaluated for confirmation.
The authors record their thanks to Prof. R. Sethuraman, Vice-Chancellor and his able administrative colleagues for allowing us to complete this work. We also place our thanks to the Drugs and Pharmaceuticals Division of Department of Science and Technology, Govt. of India, New Delhi for their financial support during the course of this work. We also thank our colleagues in CARISM, SASTRA for their extraordinary support and encouragement at every stage of this study.