Antioxidant activity of ethanolic extract of Tinospora cordifolia on N-nitrosodiethylamine (diethylnitrosamine) induced liver cancer in male Wister albino rats.

BACKGROUND: Cancer is a disease that evokes wide spread fear among people and is one of the leading causes of deaths in the world. Diethylnitrosamine (DEN) is a known carcinogen in rodent liver. DENs reported to undergo metabolic activation by cytochrome P450 enzymes to form reactive electrophiles that cause oxidative stress leading to cytotoxicity, mutagenicity and carcinogenicity.
OBJECTIVE: The present study was carried out to evaluate the antioxidant activity of ethanolic extract of Tinospora cordifolia (EETC) in N-nitrosodiethylamine (DEN) induced liver cancer in male Wister albino rats.
MATERIALS AND METHODS: The antioxidant activity was assessed by the levels of lipid peroxidation (LPO), enzymic and nonenzymic antioxidants. RESULT: A significant levels of LPO was increased as the enzymic and nonenzymic antioxidants values were decreased in liver cancer bearing animals.
CONCLUSIONS: The administration of EETC to cancer bearing animals reverted the LPO levels, enzymic and nonenzymic antioxidants to near normal.

Cancer is a disease that evokes wide spread fear among people and is one of the leading causes of deaths in the world, resulting in more than 10 million patients and 6 million deaths/year.[12] It is characterized by uncontrolled, irreversible, independent, autonomous and abnormal growth of tissue.[3] Hepatocellular carcinoma (HCC) is one of the most malignant diseases and has become increasingly important all over the world. HCC is currently the fifth most common solid tumor world-wide and the fourth leading cause of cancer related death.[4] Risk Factors of HCC is unique in that it largely occurs with an established back ground of chronic liver disease and cirrhosis (70–90% of all detected HCC cases) Major causes of cirrhosis in patients with HCC include hepatitis B, hepatitis C, alcoholic liver disease, and possibly nonalcoholic steatohepatitis. Less common causes include hereditary hemochromatosis, α-1 antitrypsin deficiency, autoimmune hepatitis, and some porphyries.[5] The other factors are aflatoxin exposure, heavy alcohol intake, oral contraceptives, tobacco and obesity. Epidemiological and experimental evidence indicate that the exposure to chemicals also contributes to the development of HCC). Such cancer producing chemicals are called as carcinogens. These include hydrocarbons such as dibenz anthracene, benzylpyrenol, nitroso compounds, aromatic amines and azodyes.[6] Diethylnitrosamine (DEN), a representative chemical of a family of N-nitroso compounds has been found distributed in processed meats, tobacco smoke, alcoholic beverages, and ground water having high level of nitrates.[7] Metabolism of certain therapeutic drugs is also reported to produce DEN.[8]

Diethylnitrosamines is a known carcinogen in rodent liver. DENs reported to undergo metabolic activation by cytochrome P450 enzymes to form reactive electrophiles that cause oxidative stress leading to cytotoxicity, mutagenicity and carcinogenicity.[9] Excessive generation of oxygen free radicals can cause oxidative damage to biomolecules resulting in lipid peroxidation (LPO), mutagenesis and carcinogenesis. Natural antioxidants show strong defense against the damages of the cellular organelles caused by free radical induced oxidative stress.[10] Since free radicals are very unstable, they are immediately neutralized by antioxidants. A major defense mechanism involves antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), which neutralize reactive oxygen species (ROS) in cells.[11] Tinospora cordifolia Miers, belonging to the family Menispermaceae, is commonly known as Guduchi or Giloe is a large, extensively spreading, glabrous, perennial deciduous shrub widely distributed throughout the plains of India.[12] In Indian Ayurvedic system of medicine, the powder and aqueous extract of the plant is used in debility, hepatitis, dyspepsia, jaundice and other liver afflictions. Its antidiabetic, antiinflammatory[13] and diuretic activities[14] have also been reported. A variety of constituents has been isolated from T. cordifolia belonging to different classes such as alkaloids, diterpenoid lactones, glycosides, steroids and sesquiterpenoids.[15] Though a number of studies have been documented the biological activities of T. cordfolia against various diseases, there is a paucity of information, especially against DEN induced liver cancer with reference to LPO and its antioxidant status. Hence, the present investigation was under taken to evaluate the therapeutic role on the status of LPO, enzymic and nonenzymic antioxidants levels during DEN induced liver cancer in Wister albino rats.[16]

Materials and Methods

Plant source

The plant T. cordifolia was collected during July 2006 in Chennai, Tamil Nadu, India. The plant was authenticated by botanist, Captain Srinivasa Murti Drug Research Institute for Ayurveda and Siddha, Chennai, Tamil Nadu, India. A voucher specimen (No. 00625) has been deposited in the herbarium of the same department.

Preparation of ethanolic extract of Tinospora cordifolia

The whole plant T. cordifolia was shade dried (1 kg), coarsely powdered and soaked in 95% ethanol and kept for 10 days at room temperature for maceration. It was filtered, and the process was repeated for 3 times. The menstruum was concentrated at 55°C to obtain the semi-solid residue and dried in a vacuum evaporator. The weight of the residue was noted. The yield of total ethanolic extract was 13% w/w and refrigerated until use.

Chemicals

N-nitrosodiethylamine (DEN) and phenobarbital (PB) were purchased from Sigma Chemical Company, St. Louis, MO, USA. All other chemicals including solvents used were of high purity and analytical grade marketed by Glaxo Laboratories, Mumbai, and Sisco Research Laboratories Pvt. Ltd., Mumbai, India.

Animals

Male adult albino rats of Wister strain weighing between 100 ± 20 g were procured from “Tamil Nadu Veterinary and Animal Sciences University” Chennai, India. The animal house was well ventilated, and the animals had 12 ± 1 h day and night rhythm throughout the experimental period. The animals were housed in large spacious polypropylene cages, and they were given food and water ad libitum. During the course of the experiments, the temperature remained between 27°C and 37°C. Guidelines for breeding and Experiments on Animals, 1998 defined by the Ministry of Social Justice and Empowerment of India was followed (IAEC/CSMDRIA/04/2007).

Experimental design

The rats were divided into four groups with six animals in each group and were given do + se regimen as given below.

Group I: Control animals were given normal Saline (0.9%).

Group II: Animals received a single intraperitoneal injection of N-nitrosodiethylamine at a dose of 200 mg/kg body weight in saline to induce liver cancer. Two weeks after administration of DEN, PB at a concentration of 0.05% was incorporated into rat chow for up to 14 successive weeks to promote the cancer (liver cancer bearing animals)

Group III: Liver cancer bearing animals were treated with ethanolic extract of Tinospora cordifolia (EETC) orally at a concentration of 300 mg/kg body weight for 30 days.

Group IV: Animals received EETC orally at a concentration of 300 mg/kg body weight for 30 days.

Collection of blood and tissues

After the experimental period, the animals were sacrificed. Blood was collected, and serum was separated for the assays. The liver and kidney tissues were collected and homogenized by a Teflon homogenizer in phosphate buffered saline, pH 7.4 and samples were stored at −80°C for further assays.

Estimation of macromolecular damages

Assay of lipid peroxidation

Macromolecular damage such as LPO was estimated using the method of.[17]

Assay of enzymic antioxidants

Superoxide dismutase levels in the serum and tissue were determined using the method of.[18] The enzyme activity was expressed as U/mg protein for tissues and as mg/dL for serum. CAT was determined according to the method of[19] and the enzyme activity was expressed as μmol of decomposed H2O2/min/mg protein. The activity of GPX was assayed by the method of.[20] Using glutathione (GSH) as substrate and the activity was expressed as nmol of oxidized GSH/min/mg protein.

Assay of nonenzymic antioxidants

GSH was measured according to the method of.[21] Using DTNB and the enzyme activity was expressed as μmol/g of wet tissue. Ascorbic acid (Vitamin C) and alpha tocopherol (Vitamin E) were estimated in tissue homogenate according to the methods of[2223] respectively. Their levels were expressed as milligram per gram for wet tissue.

Statistical analysis

Values are expressed as mean ± standard deviation. The results were statistically evaluated using one-way analysis of variance by SPSS 10.0 (Minitab: Turkey's multiple comparison method, Brandon Court unit, E1-E2, Coventry, United kingdom) student version followed by Turkey's multiple comparison method to compare means of different groups. The mean difference is significant at the 0.05 levels.

Results

Lipid peroxidation enzymes

Lipid peroxidation of polyunsaturated fatty acids which is an important consequence of oxidative stress and has been investigated extensively.[24] ROS and organic free radical intermediates formed as a result of biotransformation are suggested to initiate various macromolecular changes like LPO, DNA damage, necrosis and apoptosis. These macromolecular changes are a reflection of xenobiotics interfering with normal cellular metabolism and the enzyme regulation.

Table 1 shows the effect of T. cordifolia on LPO in the liver of control and experimental animals. The levels of LPO were found to be significantly increased in group II cancer bearing animals (P < 0.001) when compared with control animals under basal conditions and also in the presence of inducers. Conversely, the administration of T. cordifolia significantly reduces the peroxidation reaction in group III T. cordifolia treated animals (P < 0.01). However, no significant changes were observed in group IV T. cordifolia control animals when compared to Group I control animals.

Table 1

The levels of lipid peroxidation in liver of control and experimental animals

Antioxidant enzymes

The toxicity of xenobiotic chemical can be effected by the parent compound by metabolism to free radicals are electrophilic metabolites and stimulation of ROS production.[252627]

In general, the biological effects of ROS are quite similar in terms of their toxicity and the denomination ROS is useful when there is no need for identification of a biochemical or biological mechanism. However, it is important to note that three of these reactive species, superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (HO+) are the result of the partial reduction of oxygen and consequences of the transferase of one, two or three electrons to molecular O2 respectively.

Therefore, the antioxidant status has been suggested as a useful tool in assessing the risk of oxidative damage due to the dynamic imbalance between the antioxidant defense and pro-oxidation conditions.[28]

Table 2 shows the activities of SOD, CAT, GPx, GSH, Vitamin C and Vitamin E in the serum of control and experimental animals. Serum of the DEN-induced cancer bearing animals show a significant decrease of enzymic antioxidants (SOD, CAT, GPx is (P < 0.001)) and nonenzymic antioxidants (GSH, Vitamin C and Vitamin-E (P < 0.001) the levels of these antioxidants were increased significantly in T. cordifolia treated group III animals (SOD, CAT, GPx and GSH, Vitamin C and Vitamin E (P < 0.001). No remarkable changes were observed in group IV T. cordifolia control animals.

Table 2

Effect of T. cordifolia extract on enzymic and nonenzymic antioxidants in serum of control and experimental animals

Table 3 shows the activities of SOD, CAT, GPx, GSH, Vitamin C and Vitamin E in the liver of control and experimental animals. Liver of DEN-induced cancer bearing animals show a significant decrease of enzymic antioxidants (P < 0.001) and nonenzymic antioxidants (P < 0.001). The levels of these antioxidants were increased significantly in T. cordifolia treated group III animals (SOD, CAT, GPx, GSH, Vitamin C and Vit-E are (P < 0.001). No remarkable changes were observed in group IV, T. cordifolia control animals.

Table 3

Effect of T. cordifolia extract on enzymic and nonenzymic antioxidants in liver of control and experimental animals

Discussion

Liver cancer is a major health problem, being the serious form of cancer in Liver after lung cancer. Great efforts have been made in the treatment of liver cancer. However, current treatments used for Liver cancer like radiation, antihormonal therapy, resection of liver and chemotherapy produce various side effects. Considering these facts, it is of interest to find and alternate with fewer side effects. Hence, its of interest to screen anticancer agents from Plant products that fulfills the criteria.

Number of research work has been carried out to evaluate the therapeutic effect of medicinal plants from natural sources for the cure of various human diseases, including cancer.[29] The use of plant drugs has been reported in the traditional systems of India medicine for the treatment of cancer, without any side effects. Cancer and its medical complications are receiving greater attention day-by-day.[30] It is generally accepted that numerous medicinal plants and their formulations are used to cure cancer in the Indian systems of medicine.[31] Aerobic organs, such as liver generate ROS that will induce oxidative tissue damage. These radicals, react with a cell membrane and induce LPO or cause inflammation, have been implicated as important pathological mediators in many clinical disorders.[32] LPO has long been known and has been suggested to be responsible for numerous deleterious effects observed in biological systems, especially because after initiation it concurrently proceeds by a free radicals reaction mechanism. LPO is regarded as one of the basic mechanism of cellular damage caused by free radical.[33] It is initiated by the obstruction of a hydrogen atom from the side chain of polyunsaturated fatty acids (PUFAs) in membrane lipids. Increased LPO alters membrane fluidity and membrane potential and thereby leads to loss of cellular function and cell death.[34] LPO is the presumptive markers for free radicals generation and the development of oxidative damage. Free radicals are short-lived reactive chemical species involved in a variety of functions like oxidation of polyunsaturated fatty acids in cell membranes. The generation of free radicals can be measured by the rate of LPO.[35] LPO was measured by indirectly measuring the thiobarbituric reactive material, malonedialdehyde (MDA) and it is one of the products of LPO. DEN induced oxidative damage is generally attributed to the formation of highly reactive hydroxyl radical (OH−), a stimulator of LPO and the source of destruction and damage to the cell membrane.[36] Have reported that animals treated with DEN resulted in increased lipid peroxide formation and cell membrane damage and decreased levels of antiperoxidative enzymes. In the present investigation, the LPO levels were found to be significantly increased in DEN induced rats. Stimulation of LPO is a consequence of tissue injury, a relationship between LPO and hepatotoxicity has been explained by[37] reported that alkaloids of T. cordifolia inhibits LPO and increases the antioxidant status in animals intoxicated with xenobiotic compounds. In the present investigation, increased levels of LPO were observed in liver of cancer bearing animals. This may be due to free radicals induced by DEN. However, the administration of EETC decreased the LPO levels in cancer bearing animals.

Antioxidant enzymes are agents that catalytically scavenge the toxic free radicals and producing compounds into extractable nontoxic substance.[38] Antioxidants have been proposed as a remedy against the deleterious effects of ROS. ROS is thought to be involved in the etiology of several diseases cancer, hepatic injury, diabetes, arthritis and ageing.[39]

However, they are reported of being responsible for liver damage and carcinogenesis.[4041] Several epidemiological studies have shown the beneficial effect of vegetables and fruits[42] part of which has been attributed to the antioxidant effects. Therefore, the development and utilization of more effective antioxidant of natural origin are desired.

Thus, antioxidants play an important role to protect the human body against damage by ROS. The antioxidant system classified as the first line, second line, third-line defenses. The first-line defense comprises of preventive antioxidants that act by quenching of O2-by decomposition of H2O2 and sequestration with metal ions. The antioxidant belonging to this category is enzymes like SOD, CAT and GPx and GSH reductase and nonenzymic molecules like minerals and some proteins.[43]

Antioxidant in intracellular and extracellular fluids is involved in the scavenging of free radicals. Presently in enzymic antioxidants provide a major intracellular antioxidant protection by removing superoxide radicals and hydrogen peroxide (H2O2), superoxide radical are reduced by the enzyme SOD to form H2O2 and CAT converts H2O2 to neutral products of O2 and H2O. GPx catalyzes destruction of H2O2 and other lipid hydrogen peroxide in the presence of GSH as an electron donor.

Superoxide dismutase is a group of metalloenzyme that catalyze the conversion of reactive superoxide radical anions to yield hydrogen peroxides. It is widely distributed in cells with high oxidative metabolism and has been proposed to protect cells against the deleterious effects of superoxide anions.[44] In the present investigation, the decreased activity of SOD was observed in liver and Serum of cancer bearing animals. CAT is an enzyme that is, presents in most cells and catalyzes the decomposition of hydrogen peroxide to water and oxygen. Since CAT is localized in the peroxisomes of most cells and is involved in fatty acid metabolism. GPx enzyme is a well-known first-line defense against oxidative stress, which in turn requires GSH as a cofactor. GPx catalyzes the oxidation of GSH to Glutathione disulfide (GSSG) at the expense of H2O2. Furthermore, the hepatic activities of antioxidant enzymes SOD, CAT and GPx are known to decrease in rats treated with DEN.[37] The production of hydroxyl radical stimulates the LPO and destruction of cells. The levels of major antioxidant enzymes SOD, CAT and GPx are significantly decreased in DEN induced rats. But rats pretreated with EETC showed level of antioxidant enzymes to be near normal.

The nonenzymic antioxidant systems are second line of defense against free radical damage. They are GSH, Vitamin C (Ascorbic acid), Vitamin E (α-tocopherol). GSH (γ-glutamylcystinyl glycine) is the most abundant nonprotein thiol, synthesized in the liver and acts as a substrate for GPx enzyme. GSH plays a critical role in important cellular functions, which includes the maintenance of thiol status of protein, the destruction of H2O2, lipid peroxides and translocation of aminoacids across cell membrane.[45] In oxidative stress condition, GSH is converted to GSSG and depleted leading to LpO. Therefore, the role of GSH as a responsible marker for evaluation of oxidative stress is important as it act as an antioxidant both extra cellular and intracellular and produced in the liver.[46] The decreased level of GSH in DEN induced rats may be due to its utilization by excessive amount of free radicals. Increased LPO also correlates with depletion of GSH and this effect is prior to alteration of PUFA[20] In the present study, it was observed that the decreased level of GSH in cancer bearing animals are reverted to near normal in EETC treated animals.

The presence of alkaloids may be contributed to this beneficial effects, because they are potent inhibitors of enhanced spontaneous production of MDA and are able inhibit LPO[47] GSH is also the most important biomolecule protecting against chemically induced cytotoxicity, by participating in the elimination of reactive intermediates by conjugation and hydroperoxide reduction or free radical by direct quenching.[48] Similarly, Vitamin E and C are important scavenging vitamins, which cannot be synthesized by most mammals including human being, and therefore are required from the diet. Vitamin C and dehydroascorbic acid interact directly with radicals like O2− and OH− in plasma, thus preventing damage to RBC. It probably assists α-tocopherol in inhibition of LPO by recycling the tocopherol radical.

Vitamin E is one of the most important and least toxic of all lipid soluble antioxidant vitamins. It scavenges peroxyl radical intermediates in LPO and is responsible for protecting polyunsaturated fatty acid present in the cell membrane against LPO. In the present study, decreased level of Vitamin E and C was observed in cancer bearing animals. The recoupment of Vitamin E and C to near normal level in EETC treated rat was found near to normal levels. Therefore in this study EETC might render protection to macromolecules to avoid damage from xenobiotic such as DEN by maintaining the redox balance thereby exhibit anticancer activity during DEN induced liver cancer.