Antitumor And Antioxidant Activity Of Artemisia Nilagirica (Clarke) Against Ehrlich’s Ascites Carcinoma In Swiss Albino Mice.
P P, R V, S V, G S, S R, J S, P Pal, S P
Keywords
anticancer, antioxidants, artemisia nilagirica, lipid peroxidation
Citation
P P, R V, S V, G S, S R, J S, P Pal, S P. Antitumor And Antioxidant Activity Of Artemisia Nilagirica (Clarke) Against Ehrlich’s Ascites Carcinoma In Swiss Albino Mice.. The Internet Journal of Pharmacology. 2009 Volume 9 Number 1.
Abstract
The present study was carried out to assess the pharmacological potential towards Antitumor and antioxidant activity of
Introduction
Herbal medicine derived from plant extracts are being increasingly utilized to treat a wide variety of clinical diseases, though relatively little knowledge about their mode of action is available. There is a growing interest in the pharmacological evaluation of various plants used in Indian traditional system of medicine.
Plant derived natural products such as flavonoids, terpenoids and steroids etc have received considerable attention in recent years due to their diverse pharmacological properties including antioxidant and anticancer activity. There has been growing interest in the analysis of certain flavonoids, triterpenoids and steroids due to their potential benefits to human health. One of their main properties in this regard is their antioxidant activity, which enables them to attenuate the development of tumor and inflammatory disease. Antioxidant plays an important role in inhibiting and scavenging radicals, thus providing protection to human against infection and degenerative diseases.1
Plants have been prime source of highly effective conventional drugs for the treatment of many forms of cancer, and while the actual compounds isolated from the plant frequently may not serve as the drugs, they provides lead for the development of potential novel agents.2
Realizing the fact, this research was carried out to evaluate the antioxidant and anticancer activity of Ethanolic Extract of
Material and Methods
Cancer Cell Lines
EAC cells were obtained from the courtesy of Amla cancer research centre, Trissur, kerala. They were maintained weakly by intraperitoneal inoculation of 106cells/mouse.
Chemical and Reagents
Chemicals used in this study were of the highest purity. Vincristine was purchased from Sigma-Aldirch. Tricholoro acetic acid (TCA), Thiobarbituric acid (TBA), 5,5 dithio bis 2-nitro benzoic acid (DTNB), Phenazine metho sulphate, n-butanol, Nitro blue tetrazolium chloride (NBT) were purchased from Sigma Aldrich Pvt.ltd, Bangalore, India. All other chemicals and biochemicals used in our study were of high analytical grade.
Animal
Swiss albino mice weighing between 20-25gm were used for the present study. They were maintained under standard environmental conditions and were fed with standard pellet diet and water
Tumor Transplantation
EAC cells were maintained by serial transplantation from tumor bearing swiss albino mice. Ascitic fluid was drawn out from tumor bearing mice at log phase (day 7-8 of tumor bearing) of tumor cells. The freshly drawn fluid was diluted with ice-cold normal saline (0.9 %) and tumor cell number was adjusted to 2x106 cells/ml for inoculation.3
Treatment Schedule
Swiss albino mice were used for Antitumor studies. All groups were treated with EAC ascetic carcinoma cell lines (0.2 ml of 2x106 cells/mice) intraperitoneally except normal group. This was taken as day ‘zero’. Prior to administration of EAC cell to mice, the animal were divided into 5 groups (n=8) and given water and food
Tumor Growth Response
Antitumour effect of ethanolic extract of Artemisia nilagirica (Clarke) was ascertained by observing the changes in body weight, ascetic tumor volume, packed cell volume, viable and non-viable tumor cell count, Mean survival time (MST) and percentage increase in life span (% ILS). The tumor cell volume was measured by taking it in a graduated centrifuge tube and packed cell volume determined by centrifuging tumor volume at 1000 rpm for 5min. viable and non-viable count were stained by the trypan blue (0.4 % in normal saline) and count was determined in a Neubauer counting chamber. The antitumor efficacy of drug was compared with that of standard drug vincristine. Mean Survival Time (MST) was noted with reference to control. Mean survival time of treated groups (T) were compared with those of control groups (C) using following equation.4
Increase of life span = T-C/C x 100.
Percentage increase in lifespan is calculated using the formula
ILS % = (MST of treated group/MST of control group -1) x 100
Hematological Study
Blood was obtained from cardiac puncture, 24 hr after last dose. For the total count, blood was drawn into RBC and WBC pipette, diluted and counted in Neubauer counting chamber. Sahli’s hemoglobinometer is used determine the hemoglobin concentration. Differential count of leucocyte was done on a freshly drawn blood using leishman’s stain. Hemoglobin content, RBC and WBC count and differential leucocyte were determined for normal, EAC control and treated animal groups.7
Biochemical Assay
Estimation of lipid peroxidation:
Livers was excised and immediately frozen in dry-ice and stored at 200C degree centigrade. Frozen tissue from each rat was homogenized in ice cold 0.1M Tris-HCL buffer (pH 7.4) and assayed for degree of lipid peroxidation by measuring thiobarbituric acid reactive substances ( TBARS ) according to Ohkawa et al9. To 0.5ml tissue homogenate, 0.5ml saline and 1.0ml 10% TCA were added, mixed well and centrifuged at 3000 rpm for 20 minutes. To 1ml of the protein free supernatant, 0.25ml of thiobarbituric acid ( TBA ) reagent was added; the contents were mixed and heated for one hour at 950C. The tubes were cooled to room temperature under running water and absorbance were measured at 532nm. The levels of lipid peroxides were expressed as nmoles of thiobarbituric acid reactive substances (TBARS)/mg protein.
Estimation of reduced Glutathione:
To measure reduced glutathione (GSH), 0.2ml of tissue homogenate was mixed with 1.8ml of EDTA solution. To this 3.0ml precipitating reagent (1.67g of met phosphoric acid, 0.2g of EDTA disodium salt, 30g sodium chloride in 1L of distilled water) was added, mixed thoroughly and kept for 5min before centrifugation. To 2.0ml of the supernatant, 4.0ml of 0.3-M disodium hydrogen phosphate solution and 1.0ml of DTNB (5, 5-dithio bis 2-nitro benzoic acid) reagent were added and was read at 412nm. Absorbance values were compared with a standard curve generated from known GSH10.
Estimation of catalase:
Catalase was assayed according to the method of Aebi et al11. The estimation was done spectrophotometrically following the decrease in absorbance at 230nm. The tissue was homogenized in M/150 phosphate buffer (pH 7.0 ) at 1-40C and centrifuged at 5000rpm. The reaction mixture contained 0.01M phosphate buffer (pH 7.0), 2mM H2O2 and the enzyme extract. The specific activity of catalase was expressed in terms of units/mg protein. Absorbance values were compared with a standard curve generated from known CAT.
Estimation of super oxide dismutase:
The activity of SOD in tissue was assayed by the method of Karkkar et al12. The assay mixture contained 1.2ml sodium pyrophosphate buffer (pH 8.3, 0.025 mol/L), 0.1ml phenazine methosulphate (186 mM ), 0.3ml NBT ( 300 mM ), 0.2ml NADH (780mM) and approximately diluted enzyme preparation and water in a total volume of 3ml. After incubation at 30 degree centigrade for 90 sec, the reaction was terminated by the addition of 1.0ml glacial acetic acid. The reaction mixture was stirred vigoursly and shaken with 4.0ml n-butanol. The color intensity of the chromogen in the butanol layer was measured at 560nm against n-butanol and concentration of SOD was expressed as units/mg protein. Absorbance values were compared with a standard curve generated from known SOD.
Figure 1
Survival time and Percentage increase in life span in EAC bearing mice
Values are mean± SEM (n=5) in each group
**P<0.01, experimental groups were compared with EAC control
*P<0.5, experimental groups were compared with EAC control
Figure 2
Values are mean ±SEM (n=3)
**P<0.01, experimental groups were compared with EAC control
*P<0.5, experimental groups were compared with EAC control
Figure 3
Values are mean
**P<0.01, experimental groups were compared with EAC control
*P<0.5, experimental groups were compared with EAC control
Figure 4
Values are mean
**P<0.01, experimental groups were compared with EAC control.
*P<0.5, experimental groups were compared with EAC control
Figure 5
Values are mean
No. of mice in each group (n = 3)
**P<0.01, experimental groups were compared with EAC control.
*P<0.05, experimental groups were compared with control groups
Stastistical Analysis
All data were analysed by using one way analysis of variance (ANOVA) and results are expressed as mean ± SEM.
Result and Discussion
The present investigation indicates that ethanolic extract of Artemisia nilagirica (Clarke) showed significant anti-tumor and antioxidant activity in EAC bearing mice. The effect of ethanol extract of Artemisia nilagirica (Clarke) on survival time, body weight, tumor volume, viable and non-viable cell count and survival time are shown in table 1 and 2. Administration of ethanolic extract of Artemisia nilagirica (Clarke) reduced the tumor volume, packed cell volume and viable cell count, when compared to EAC control mice.
In EAC control mice, the mean survival time is 21±0.38, whereas in the case of group treated with 400mg/kg ethanol extract of
The percentage increase in life span of ethanolic extract of extract treated goup bearing EAC cancer cell lines were 28.57, whereas group treated with standard drug showed 59.52% increase in life span when compared to EAC control group.
The percentage decrease in tumor volume, packed cell volume and viable cell count in animals treated with ethanolic extract of Artemisia nilagirica (Clarke) was found to be 32.3%, 41.36% and 39.54% when compared to EAC control group animals. whereas the group treated with standard drug showed a decrease of 57.47% of tumor volume, 56.77% packed cell volume and 43.48% decrease in viable cell count compared to EAC control group.
As shown in table 3, the hemoglobin content in the experimental groups were compared with EAC control group. Carcinoma bearing control animals showed a fall in hemoglobin content when compared to normal group. A significant increase in hemoglobin content were noted in group treated with 400mg/kg extract. Treatment group showed 22% rise in hemoglobin compared to EAC control group. Number of red blood cells in EAC control group was found to be low when compared to normal animals. In extract treated animal showed a significant rise in RBC compared to EAC control group. The total WBC count were significantly high in EAC control mice when compared to normal mice, whereas the extract treatment reduced the number of WBC compared to EAC control animals.
As shown in table 4, EAC control group showed increased in neutrophil percentage when compared to control group animals. The extract treatment decreased the percentage of neutrophil to about 30% when compared to EAC control animals.
A fall in lymphocyte percentage was noted in EAC control group when compared to control group animals. The extract treated group raised the percentage to about 79.2% when compared to EAC control group animals.
The levels of lipid peroxidation, catalase, protein content, SOD and GSH were summarized in Table 5. The lipid peroxidation in EAC control group were significantly increased when compared to control group. A 74% increase was noted when compared to control group animals. The extract treated group decreased lipid peroxidation when compared to EAC control. A 20% decrease was noted in extract treated group.
The catalase was found to be significantly reduced in EAC control group when compared to control group. A 51% decrease was noted when compared to control group. The ethanolic extract of
The protein content was significantly reduced in EAC control group when compared to control animals. A 16% reduction in protein content was noted in EAC control animals. The extract treated group significantly raised the protein content to about 12% when compared to EAC control animals.
SOD levels were reduced in EAC group animals when compared to control animals. A 50% decrease in SOD levels were noted in EAC control group animals. The extract treated group significantly raised the levels of SOD compared to EAC control group. A percentage increase of about 81% was noted in treated group animals.
Glutathione levels were significantly reduced in EAC control group animals when compared to control group animals. A 31% decrease was noted in EAC group animals. The extract treated group showed significant raise in Glutathione levels to about 33% compared to EAC control group.
The reliable criteria for judging the value of any anticancer drug are prolongation of life span and decrease of WBC count. The results demonstrate the indirect inhibitory effect of the extract in EAC bearing group, which is probably mediated by the enhancement and deactivation of either macrophages or cytokine production. The result of present study has shown Antitumour effect of ethanolic extract of
Myelosupression is a frequent and major complication of cancer chemotherapy. Compared to the EAC control animals, ethanolic extract of
Lipid peroxidation mediated by free radicals is considered as a primary mechanism of cell membrane destruction and cell damage. Increase in level of TBARS indicates enhanced lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanism to prevent the formation of excess free radicals. From the results it is clear that EAC induced oxidative stress caused enhanced lipid peroxidation. The group treated with ethanolic extract of Artemisia nilagirica (Clarke) showed significant degree of protection against oxidative damage caused by EAC by decreasing lipid peroxidation.
Glutathione peroxidase, superoxide dismutase and catalase constitute a mutually supportive team of defence against ROS. Superoxide dismutase, one of the important intracellular antioxidant enzymes present in all aerobic cells has an antitoxic effect against superoxide anion. Catalase is a hemoprotein and it protects cells from the accumulation of H2O2 by dismutating it to form H2O and O2. Glutathione peroxidase is a selenoenzyme, it functions are concerned with removal of free radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase and GST.
In the present study, decline in the level of antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase are observed in EAC group animals. Therefore reduction in the activity of these enzymes may result in a number of deleterious effects due to accumulation of superoxide radicals and H2O2. This decline in activity of the enzymes was significantly increased by administration of the ethanolic extract of Artemisia nilagirica (Clarke).
Conclusion
It may be possible that the natural antioxidants strengthen the endogenous antioxidant defence from ROS ravage and restore the optimal balance by neutralizing the reactive species. They are gaining immense importance by virtue of their critical role in disease prevention. To conclude, the results of the present study demonstrated that the ethanolic extract of Artemisia nilagirica (Clarke) exhibited antitumour and antioxidant activity against Ehrlich’s ascites carcinoma in swiss albino mice. Thus the plausible mechanism of antitumour effect may be due to its antioxidant effect. Further study is needed to identify and isolate the active principle of Artemisia nilagirica (Clarke).
Acknowledgement
The authors are thankful to the management of JKK Nataraja College of Pharmacy, komarapalayam, Tamilnadu, INDIA for providing necessary facilities to carry out the research work.