INTRODUCTION:

Cancer is one of the ailments which cannot be completely subdued by chemotherapy. The chemotherapeutic agents though effective against various types of tumor are not totally free from side effects. This fostered our attempts to evaluate some plant products against cancer, as they are less likely to cause serious side effects¹. Many Indian plants like black pepper, asafetida and garlic are quoted to be useful in different types of cancer^2,3. Antioxidant principles derived from plants are reported to have antitumor activity⁴. Hence plants containing flavonoids are constantly being screened for antitumor activity⁵. Some of the active principles present in this plant are reported to be flavonoids⁶. It is also used by the tribals of Kolli Hills, Tamil Nadu, India for various types of tumors and by practitioners of traditional systems of medicine against acute tumors.

The plant Pseudarthria viscida is also called as Hedysarum viscidum family (Fabaceae). The plant is perennial, diffuse, prostrate, stems 60-120 cm long, slender, more or less clothed with soft whitish hairs. It is distributed throughout South India, up to 900m in the hills and also in Gujarat. Literature review reveals that root and leaves contain proteins, tannins and flavonoids and also showed significant inhibitory activity against some fungal pathogens causing major diseases in crop plants and stored food grains⁷. Traditionally plant is used in cases of biliousness, rheumatism, excessive heat, intestinal poison, fever, diarrhea, asthma, heart diseases, worms, piles and tumors^8,9. Realizing these facts, this work was carried out to evaluate the antitumor and antioxidant activity of the ethanol extract of Pseudarthria viscida roots (EEPV) against Ehrlich ascites carcinoma in Swiss albino mice.

MATERIALS AND METHODS:

Preparation of extract:

The roots of Pseudarthria viscida Linn were collected from Kolli Hills, Nammakal Dist, Tamilnadu, India and authenticated by the Botanical Survey of India, Southern Circle, TNAU campus, Coimbatore. Further the roots were dried under shade and then powdered with mechanical grinder, defatted with petroleum ether in a Soxhlet apparatus and subsequently extracted with ethanol (95%). After exhaustive extraction, the ethanol extract was made solvent free by distillation under reduced pressure and the resulting semisolid mass was vacuum dried to yield a solid ethanol extract .Yield was 18.2% w/w.

Animals:

Studies were carried out using male adult Swiss albino mice weighing 18-25 gm. They were maintained under standard laboratory conditions (Temperature 25 ± 2°C) with dark/light cycles (14/10 h). They were allowed free access to standard dry pellet diet and water ad libitum. The mice were acclimatized to laboratory conditions for 10 days before commencement of the experiment¹⁰. All procedures described were reviewed and approved by the Institutional Animal Ethical committee of J.K.K. Nataraja College of Pharmacy, Komarapalayam.

Tumor cells:

EAC cells were obtained under the courtesy of Amala Cancer Research Center, Thrissur, Kerala, India. They were maintained by weekly intraperitoneal inoculation of 2 X 10⁶ cells / mouse¹¹.

Antitumor activity:

Male adult Swiss albino mice were then divided into 5 groups (n=8). All the groups were injected with EAC cells (0.2 ml of 2x10⁶cells/mouse) intraperitoneally except the normal group. This was taken as day zero. On the first day, 5 ml/ kg of normal saline (0.9% NaCl w/v) was administered in Group 1 (Normal). Propylene glycol, 5 ml/kg/day was administered in Group 2 (EAC control). EEPV at different doses (100 mg/kg, 200 mg/kg per day) and the standard drug Vincrystine (0.8 mg/kg) were administered in groups 3, 4 and 5 respectively for 14 days intraperitoneally. After the last dose and 18 h fasting, 4 mice from each group were sacrificed for the study of antitumor activity, hematological and liver biochemical parameters. The rest of the animal groups were kept to check the survival time of EAC tumor bearing hosts^11-13.

Tumor growth response:

The Antitumor effect of EEPV was assessed by change in the body weight, Ascites tumor volume, packed cell volume, viable and non viable tumor cell count, mean survival time (MST) and percentage increased life span (% ILS). MST of each group containing four mice was monitored by recording the mortality daily for 6 weeks and % ILS was calculated using following equation¹⁴.

MST = (Day of first death and Day of last death) / 2

% ILS = [(MST of treated group / MST of control group) – 1] x 100

Hematological studies:

Hemoglobin, red blood cells (RBC) and white blood cells (WBC)¹⁵ were measured from freely flowing tail vein blood^{16, 17}. Differential leukocyte count was measured from Leishman stained blood smears of normal, EAC control and EEPV treated groups, respectively¹⁸.

Biochemical assays:

After the collection of blood samples, the mice were sacrificed. Then their liver was excised, rinsed in ice-cold normal saline followed by cold 0.15 M Tris-HCL (pH 7.4), blotted dry and weighed. A 10 % w/v homogenate was prepared in 0.15M Tris-HCL buffer; a portion was utilized for the estimation of lipid peroxidation^19,20 and a second portion after precipitating proteins with TCA, was used for the estimation of Glutathione²¹. The rest of the homogenate was centrifuged at 1500 rpm at 4 C for 15 min²². The supernatant thus obtained was used for the estimation of superoxide dismutase, catalase and protein^23-27_.

RESULTS AND DISCUSSION:

The present investigation indicates that the ethanol extract of Pseudarthria viscida (EEPV) showed significant antitumor and antioxidant activity (in vitro and in vivo) in EAC bearing mice. The effect of EEPV at the dose of 100 mg/kg and 200 mg/kg on various biochemical and biological parameters are discussed below.

Effect of EEPV on mean survival time and tumor growth:

In the EAC control group the mean survival time was 22.10 ± 0.05 days, while it was increased to 29.50 ± 0.55 (100 mg/kg) and 34 ± 0.2 (200 mg/kg) days, respectively in EEPV treated mice. The mice treated with the standard drug Vincrystine (0.8 mg/kg) showed 37.70 ± 0.10 days for the same. Treatment with EEPV at the dose of 100 mg/kg and 200 mg/kg reduced the body weight, tumor volume, packed cell volume and viable tumor cell count in dose dependant manner as compared to that of EAC control mice. Further, non-viable tumor cell counts at different doses of EEPV were increased when compared with EAC control.

The increase in the life span of tumor bearing mice treated with EEPV and Vincrystine (P < 0.01) as compared to the control group²⁸_.

Effect on hematological parameters:

Hemoglobin content and RBC count in the EAC control mice were decreased when compared to normal group. Treatment with EEPV at the dose of 100 and 200 mg/kg in EAC treated mice were increased the hemoglobin content (11.42 ± 0.47 and 12.6 ± 0.49 g/dl respectively). Moderate change in the RBC count was observed in the extract treated mice. The total WBC counts were found to be increased in EAC control mice when compared with normal mice. Administration of EEPV at the dose of 100 mg/kg and 200 mg/kg in EAC bearing mice reduced WBC count when compared with EAC control. In differential count, the percentage of lymphocyte decreased and percentage of granulocyte increased in EAC control, while EEPV treated mice lymphocyte was increased and granulocyte was decreased as compared with EAC control mice²⁹_.

TABLE – 1: Effect of the ethanol extract of Pseudarthria viscida (EEPV) on body weight, mean survival time, % ILS, tumor volume, packed cell volume and viable and non-viable tumor cell count of EAC-bearing mice

Parameters	EAC Control (2× 10⁶cells/ml/mice)	EEPV (100mg/kg) + EAC	EEPV (200mg/kg) + EAC	Standard Vincrystine (0.8 mg/kg) + EAC
Body weight (g)	27.5 ± 0.14	23.3 ± 0.17	22.1 ± 0.12	20.7 ± 0.19
Mean survival time (days)	22.1 ± 0.05	29.5 ± 0.55	34 ± 0.2	37.7 ± 0.10
Increase life span (%)	-	33.48 %	53.84 %	70.58 %
Tumor volume (ml)	3.1 ± 0.20	2.2 ± 0.19	1.6 ± 0.17	0.8 ± 0.13
Packed cell volume (ml)	1.9 ± 0.11	1.0 ± 0.14	0.6 ± 0.11	0.3 ± 0.15
Viable tumor cell count( x 10⁷ cells/ml)	8.9 ± 0.27	4.1 ± 0.24	2.3 ± 0.22	0.9 ± 0.17
Nonviable tumor cell count (x 10⁷cells/ml)	0.3 ± 0.01	0.4 ± 0.71	0.4 ± 0.31	0.3 ± 0.19

Values are mean ± SEM of 8 animals each. P < 0.01, statistically significant when compared with EAC control group

TABLE – 2: Effect of the ethanol extract of Pseudarthria viscida (EEPV) on hematological parameters of EAC-bearing mice

Parameters	Normal (0.9 % NaCl W/V)	EAC control (2x10⁶cells/mouse)	EAC + EEPV (100 mg/kg)	EAC + EEPV (200 mg/kg)	EAC + Vincrystine (0.8 mg/kg)
Hemoglobin (g/dl)	13.15 ± 0.35	9.95 ± 0.12^a	11.42 ± 0.47^b	12.60 ± 0.49^b	13.12 ± 0.36^b
RBC (cells/ml X 10⁶)	5.37 ± 0.13	3.87 ± 0.80^a	4.12 ± 0.35^b	4.69 ± 0.66^b	4.92 ± 0.42^b
WBC (cells/ml X 10⁶)	7.87 ± 0.45	15.32 ± 0.23^a	12.12 ± 0.35^b	9.71 ± 0.07^b	8.50 ± 0.04^b
Lymphocyte (%)	68 ± 1.31	35 ± 1.56^a	45 ± 1.10^b	54 ± 1.61^b	63 ± 1.29^b
Monocyte (%)	2.2 ± 1.50	1.6 ± 0.50^a	1.6 ± 1.20	1.7 ± 1.50^b	1.7 ± 0.80^b
Neutrophil (%)	27 ± 1.10	65 ± 1.40^a	52 ± 1.60^b	35 ± 1.10^b	30 ± 0.1^b

Values are mean ± SEM of 8 animals each.

^a P < 0.01, Statistically significant when compared with normal group.

^b P < 0.05, Statistically significant when compared with EAC control group.

TABLE – 3: Effect of the ethanol extract of Pseudarthria viscida (EEPV) on biochemical parameters of EAC bearing mice

Parameters	Normal saline (0.5 ml/kg)	EAC control (2 X 10⁶ cells / mouse)	EAC + EEPV (100 mg/kg)	EAC + EEPV (200 mg / kg)	EAC + Vincrystine (0.8 mg/kg)
Lipid peroxidation (n moles of MDA/g of tissue)	0.85 ± 0.11	1.71 ± 0.01	1.53 ± 0.09	1.33 ± 0.01	-
Catalase (U/mg tissues)	2.49 ± 0.61	1.77 ± 0.18	1.45 ± 0.13	2.28 ± 0.22	-
Protein content (g/100 ml)	13.07 ± 0.41	17.16 ± 0.63	16.45 ± 0.15	15.97 ± 0.50	14.85 ± 0.51
GSH/mg.g-¹(wet tissue)	2.35 ± 0.09	1.63 ± 0.07	1.90 ± 0.09	2.09 ± 0.25	-
SOD/U.mg-¹(protein)	4.49 ± 0.35	2.89 ± 0.26	3.58 ± 0.29	3.97 ± 0.32	-

Values are mean ± SEM of 8 animals each. P < 0.05, statistically significant when compared with EAC control group.

Effect on biochemical parameters:

Table 3 showed that the level of lipid peroxidation in liver tissue was increased to EAC control mice 1.71±0.01 n moles as compared to the normal mice 0.85 ± 0.11 n moles. Treatment with EEPV (100 mg/kg and 200 mg/kg) were significantly decreased the lipid peroxidation level (1.53 ± 0.09 and 1.33 ± 0.01 n moles) in a dose dependant manner. The catalase level was decreased in EAC control mice (1.77 ± 0.18 unit/mg of protein tissue) when compared with normal mice (2.49 ± 061 unit/mg of protein in tissue) treatment with EEPV at the doses of 100 mg/kg and 200 mg/kg brought back to normal level (1.45 ± 0.13 and 2.28 ± 0.22 unit/mg of protein in tissue)^30,31_.

In EAC tumor bearing mice, a regular rapid increase in ascitic tumor volume was observed. Ascitic fluid is the direct nutritional source for tumor cells and a rapid increase in ascetic fluid with tumor growth would be a means to meet the nutritional requirement of tumor cells. Treatment with EEPV inhibited the tumor volume, viable tumor cell count and increased the life span of the tumor bearing mice. The reliable criteria for judging the value of any anticancer drug are the prolongation of life span of animals. It may be concluded that EEPV by decreasing the nutritional fluid volume and arresting the tumor growth increases the life span of EAC-bearing mice³²_.

In cancer chemotherapy the major problems are of myelosuppression and anemia. The anemia encountered in tumor bearing mice is mainly due to reduction in RBC or hemoglobin percentage and this may occur either due to iron deficiency or due to hemolytic or myelopathic condition. Treatment with EEPV brought back the hemoglobin content, RBC and WBC cell count near to normal values. This indicates that EEPV posses protective action on the heamoto-pioetic system³³_.

Excessive production of free radicals resulted in oxidative stress, which leads to damage of macromolecules such as lipids can induce lipid peroxidation in-vivo. Increased lipid peroxidation would cause degeneration of tissues. Lipid peroxide formed in the primary site would be transferred through the circulation and provoked damaged by propagating the process of lipid peroxidation. MDA, the end product of lipid peroxidation was reported to be higher in carcinomatous tissue than in non-diseased organs. EEPV reduced the elevated level of lipid peroxidation³⁴_.

Hydrogen peroxide itself not very reactive, but it can sometimes be toxic to cell because of it may give rise to hydroxyl radical in the cells³⁵. Thus removing of H₂O₂ is very important for antioxidant defence in cell or food systems.

Nitric oxide is produced by several different types of cells, including endothelial cells and macrophages. The early release of nitric oxide through the activity of constitutive nitric-oxide synthase is important in maintaining the dilation of blood vessels the much higher concentrations of nitric oxide produced by inducible nitric-oxide synthase in macrophages can result in oxidative damage. Nitric oxide reacts with free radicals, thereby producing the highly damaging peroxynitrite. Nitric oxide injury takes place for the most part through the peroxynitrite route because peroxynitrite can directly oxidize LDLs, resulting in irreversible damage to the cell membrane.

Lipid peroxidation has gained more importance nowadays because of its involvement in the pathogenesis of many diseases like atherosclerosis, cancer, diabetes mellitus, myocardial infarction, immunological incompetence, neurodegenerative disorders and also in aging³⁶.

CONCLUSION:

We propose that the additive and synergistic antioxidant activity of phytochemicals such as flavonoids, proteins and tannins, etc, present in EEPV are responsible for its potent antitumor activity which can be inferred from the increased life span of EAC tumor bearing mice. Further investigations are in progress in our laboratory to identify the active principles involved in this antitumor and antioxidant activity and investigate their mechanism.

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Received on 09.08.2010 Modified on 22.08.2010

Asian J. Research Chem. 4(1): January 2011; Page 110-113

Antitumor Activity and Antioxidant Role of Ethanolic Extract of Pseudarthria viscida Linn against Ehrlich Ascites Carcinoma