Anti-tumor and anti-oxidant activities of Pseudarthria viscida against Dalton's ascites lymphoma bearing Swiss albino mice.

 

M.Vijayabaskaran1, P. Sivakumar 1, R. Sambathkumar 1, P.Perumal1, T.Sivakumar2 and B.Jayakar3

 

1J.K.K.Nataraja College of Pharmacy, Komarapalayam, Tamilnadu, India.

2Nandha College of Pharmacy, Natural Products Research Laboratory, Erode, Tamilnadu, India-638052.

3Vinayaka Mission's College of Pharmacy, Sankari Main Road, Ariyanoor, (NH - 47), Salem - 636 308.

* Corresponding Author E-mail: sivaecp@hotmail.com

 

ABSTRACT

The study was aimed at evaluating the anti-oxidant and anti-tumor activities of methanol extract of Pseudarthria viscida (Family: Fabaceae). The methanol extract of Pseudarthria viscida (MEPV) at the doses of 100 and 200 mg/kg in mice for 14 days after 24 h of tumor inoculation. The effect of MEPV on the growth of transplantable murine tumor, life span of DAL bearing mice, hematological profile and liver biochemical parameters were estimated. Treatment with MEPV decreased the tumor volume and viable cell count thereby increasing the life span of DAL bearing mice and brought back the hematological parameter more or less normal level. The effect of MEPV decreases the level of lipid peroxidation and protein content and increased the level of Catalase (CAT) and Glutathione (GSH). The present work indicates that the methanol extract of Pseudarthria viscida roots exhibited significant anti-tumor and anti-oxidant activity in vivo.

 

KEY WORDS    Pseudarthria viscida, Anti-tumor activity, Anti-oxidant activity

 

INTRODUCTION:

Plant derived natural products such as flavonoids, terpenoids, and steroids have received considerable attention in recent years due to their diverse pharmacological properties including antioxidant and anti-tumor activity1,2  Antioxidants play an  important role in inhibiting and scavenging radicals, thus providing protection to humans against infection and degenerative. Antioxidants are compounds that help to inhibit   many   oxidation   reactions   caused   by   free radicals such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxy nitrate there by preventing or delaying damage to the cells and tissues3.

 

Plant   material   have   been   used   in   treatment   of malignant diseases for centuries; a comprehensive survey of literature both old and new describing plants used  against cancer.  The  plant Pseudarthria viscida (Family:Fabaceae) is semierect diffuse undershrub, distributed throughout all districts of south India, also reported from Srilanka and Timor.

 

Traditionally,  the  plant  used  in  treatment  of  intermittent fever, urinary diseases, tumors, oedema, burning sensation, difficult breathing and toxic conditions4. Herbal medicine is frequently a  part  of  a  larger  therapeutic  system  such  as traditional and folk medicine. It is necessary to evaluate, in a scientific base, the potential use of folk medicine for the treatment of many diseases a literature survey indicated only antifungal work5 and antihypertensive work6 Phytochemical screening of Pseudarthria viscida indicated the presence of flavonoids,  tannins  and  proteins5.  Epidemiologic observations show lower cancer rates in people whose diets are rich in fruits and vegetables. Although some classes of antineoplastic agents generate high levels of oxidative stress, others, including the  taxanes, vinca alkaloids, antifolates, and nucleoside and nucleotide analogues, generate only low levels. Nevertheless, all drugs generate some free radicals as they induce apoptosis in cancer cells. The scientific community has begun to unveil some of the mysteries surrounding this topic, and the media has begun whetting our  thirst  for  knowledge. The  drugs  of  many  classes  of antineoplastic agents are known to generate a high level of oxidative stress in biological systems7. If generation of ROS by a cancer chemotherapeutic agent or a free radical intermediate of  the  drug  plays  a  role  in  its  cytotoxicity, antioxidants may interfere with the drug’s anti-neoplastic activity. However, if  the  reactive species are  responsible only for the drug’s adverse effects, antioxidants may actually reduce  the  severity of  such  effects  without interfering  with  the  drug’s  anti-neoplastic  activity. Thus, it is important to distinguish between a drug’s ability to induce oxidative stress in biological systems and the role, if any that ROS or free radical intermediates play in the mechanism of action of the drug. This has lead to the theory that these diets contain substances, possibly antioxidants, which protect against the development of cancer. There is currently intense scientific investigation into this topic. Therefore, such plants should be investigated to better understand their properties, safety and efficiency.      Hence, studies involving the use of plants as therapeutic agents should be emphasized, especially the anti-tumor activity and anti-oxidant status of methanol extract of Pseudarthria viscida (MEPV) against DAL bearing mice.

 

MATERIALS AND METHODS:

Plant material and extraction

The roots of Pseudarthria viscida (Family: Fabaceae) were collected in the month of August 2005 from the Kolli Hills, Tamilnadu, India. The plant material was taxonomically identified  by  the  Botanical  survey of India, Coimbatore and the voucher specimen was retained  in  our  laboratory for  future  reference.  The dried  powered  roots  were  defattted  with  petroleum ether (60-80 °C) and further extracted with methanol in a soxhlet apparatus. The solvent was removed under reduced pressure and a semisolid mass obtained was vacuum dried to yield a solid residue (5.2 % w/w). The extract showed positive test for flavonoids, tannins and proteins.

 

Animals

Studies were carried out by using swiss albino mice weighing between 20 ± 2 g. They were obtained from the  Perundurai  Medical  College,  Perundurai, Tamilnadu, India. The mice were grouped and housed in polyacrylic cages (38 x 23 x 10 cm) with not more than  ten  animals  per  cage  and  maintained  under standard laboratory conditions (temperature 25 ± 2° C) with dark / light cycle (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 experiment. All procedure described were approved by the Institutional Animal Ethical Committee.

 

Chemicals and reagents

Trypan blue, Bovine serum albumin (Sigma chemicals, USA), Thiobarbituric acid (Loba chemie, Bombay, India) and other solvents and /  or  reagents were of analytical  grade.  Dalton's  ascites  lymphoma  (DAL) cells used were obtained from Amala cancer Research Center, Thrissur, Kerala, India. The DAL cells were maintained by intraperitoneal inoculation of 2 x 106 cells/mouse.

 

Toxicity Study

An acute toxicity study relating to the determination of LD50 was performed found that methanol extract of Pseudarthria viscida (MEPV) did not show any sign of mortality up to the dose of 2000 mg/kg. The extract showed mild muscle relaxant property and diarrhoea. There was no change in general behavior, indicated that extract had no any effect on CNS activity9.

 

Treatment protocols

Tumor was induced by injecting 0.2 ml of 2 x 106 cells/ml of DAL cells into peritoneal cavity of mice. Prior to administration of DAL cells to mice, the animals were divided into five groups (n = 10). All the groups were injected DAL cell except normal group and this was taken as day O. On the first day normal saline (0.9 % w/v, NaCl, 5 ml/kg / mouse / day) administered into normal group 1. DAL control mice were received only vehicle (propylene glycol 5ml /kg /day /mouse) as group 2. The different doses of MEPV (100 and 200 mg / kg/ mouse/ day) and standard drug vincristine (0.8 ml / kg) were subsequently administered in groups 3, 4 and 5 respectively for 14 days intraperitoneally. On 15th  day, after the last dose and 18h fasting, five mice from each group were sacrificed for the study of anti-tumor activity, hematological and antioxidant enzyme estimation and rest of animals of each group were kept to check the mean survival time (MST) and increase in the lifespan of tumor bearing mice10-12.

 

Tumor growth response

Anti-tumor effect of MEPV was assessed by observation of changes with respect to body weight, ascites tumor volume, packed cell volume, viable and non-viable tumor cell count, MST and percentage increase in life span (% ILS). Transplantable murine tumor was carefully collected with the help of sterile 3  ml syringe and measured the tumor volume and the ascitic fluid was with drawn in a graduated centrifuge and packed cell volume was determined by centrifuging at 1000 g for 5 min. Viable and nonviable cell count of ascitic cell were stained by trypan blue (0.4 % in normal saline) dye exclusion test and count was determined in a Neubauer counting chamber. The effect of MEPV on tumor growth was monitored daily by recording mortality and percentage increase in life span ( % ILS) was calculated using following formula12-

ILS (%) = [(Mean survival of treated group / mean survival of control group) -1] x 100.

 

Hematological studies

Blood was obtained from the tail vein, 24 h after last dose. For the total count blood was drawn into RBC or WBC pipettes,   diluted   and   counted   in   a   Neubaur   counting chamber. Sahli's Hemoglobinometer determined the hemoglobin concentration. Hemoglobin count13, RBC and WBC count14  was estimated from the peripheral blood of normal, DAL control and treated animal groups.

 

Statistical Analysis

Result expressed as mean ± SEM were analyzed by student's t-test.

 

Table :-1Effect of methanol extract of Pseudarthria viscida roots (MEPV) on mean survival time, % ILS, tumor volume, packed cell volume, viable and non viable tumor cell count of DAL bearing mice.

Parameters	DAL    control    (2x106 cell/ml) Cells / mouse / ml	DAL    +    MEPV 100mg/kg	DAL  +  MEPV 200mg/kg	DAL       +       Standard vincristine (0.8mg/kg)
Body weight	27.2± 0.14	24.2±0.11	22.6±0.09	21.4±0.13
Mean survival time (days)	21.13±0.37	25.13±03.23	30.63±0.23	34.75±0.43
Increase in life span (%)	--	18.93 %	44.98 %	64.53 %
Tumor volume (ml)	4.48±0.07	3.26±0.06	1.92±0.06
Packed cell Volume (ml)	1.78±0.05	1.36±0.05	0.76±0.05	--
Viable tumor cell count (x 107 cells / ml)	11.19±0.18	8.52±0.18	3.76±0.22	--
Non-viable tumor cell count (x107 cells / ml)	0.31±0.04	0.75±0.07	1.32±0.08	--

Values are mean ± SEM. Number of mice in each group (n=5). p<0.01, Experimental groups were compared with DAL control.

 

Table: 2 Effect of methanol extract of Pseudarthria viscida roots  (MEPV) on hematological parameters of DAL bearing mice.

Parameters	Normal Saline (0.5 ml/kg) (Groups 1)	DAL (2x10 6 cells) Control+(Vehicles) (Groups 2)	DAL (2x10 6 cells)+ 100 mg/kg (Groups3)	DAL (2x10 6 cells) + 200 mg/kg (Groups 4)
Hemoglobin (g %)	13.8 ±1.10	11.3 ± 0.39 a	11.8 ±1.03	12.6 ±1.62
Total RBC (cells/ml x109  )	6.4 ±0.54	4.5 ± 0.45	5.6 ± 0.53 b	6.2 ± 0.68
Total WBC (cells/ml x106)	6.7 ±0.58	18.9 ± 1.62 b	11.6 ± 0.75	7.1 ± 0.72
Cells/ femur 1 x106/ml	18.9 ±1.68	14.9 ±1.49 b	16.4 ±1.46 a	17.4± 1.60
Cells/ spleen 2x106/ml	16.7 ±1.86	28.4 ±1.43 b	20.2± 1.70 b	14.4± 1.42

Values are mean ± SEM (n = 5). A group 2 was compared with normal group (p<0.05). Experimental groups were compared with control. a

p < 0.01,. b p < 0.01.

 

Table : - 3 Effect of Methanol extract of Pseudarthria viscida roots (MEPV) on different biochemical parameters in liver of DAL

bearing mice.

Parameters	Normal (saline 5mg/kg)	DAL           control (2x106 cell/ml)	DAL+MEPV          (100 mg/kg)	DAL+   MEPV   (200 mg/kg)
Lipid Peroxidation (n moles MDA/g of tissue)	0.94±0.06	1.39±0.11	1.22±0.12	1.09±0.12
Glutathione (mg/g of tissue)	2.35±0.09	1.63±0.07	1.90±0.09	2.09±0.25
Catalase (units/mg tissues)	2.64±0.17	1.68±0.09	1.82±0.05	2.10±0.14
Protein content (gm/100 ml)	14.87±0.09	18.14±0.14	17.03±0.08	16.07±0.16

Values are mean ± SEM. Number of mice in each group (n=5). p<0.01, Experimental groups were compared with DAL control.

 

Biochemical assays

The liver was excised, rinsed in ice-cold normal saline followed by cold phosphate buffer (pH 7.4) blotted and weight. The homogenate was processed for estimation of lipid peroxidation, catalase activity and protein content. Assay for microsomal lipid peroxidation was carried out by the measurement of thiobarbituric acid reactive substance (TBARS) in  tissue reported by15. The pink chromogen produced by the reaction of malondialdehyde, which is a secondary product of lipid peroxidation reaction with thiobarbituric acid was estimated at 532 nm. The activity of CAT was assayed the method of16  .Proteins were estimated by the method17 using bovine serum albumin as the standard.

 

RESULTS:

The present investigation indicates that MEPV showed significant anti-tumor and anti-oxidant activity in DAL bearing mice. The effects of MEPV (100 and 200 mg/kg) at different doses on tumor volume, viable and non-viable cell count, survival time and ILS, were shown in table 1. Administration of MEPV reduces the tumor volume, packed cell volume and viable tumor cell count in a dose dependant manner when compared to DAL control mice. As shown in Table 2, the hemoglobin content   in   the   DAL   control   mice   (9.96   g   %)   was significantly decreased when compared with normal mice (12.14g %). MEPV at the dose of 100 and 200 mg/kg the hemoglobin content in DAL treated mice were increased to 10.46 and 11.24 g % respectively. Moderate changes in the RBC counts were also observed in the extract treated mice significantly higher in the DAL treated mice when compared with normal mice whereas, MEPV treated mice significantly reduced the WBC count as compared to that of control mice.

 

The levels of lipid peroxidation, catalase activity, GSH and protein content were summarized in Table 3. The levels of lipid peroxidation in liver tissue were significantly increased in DAL control mice (1.39 n moles MDA/g of tissue) as compared  to  the  normal  mice  (0.94  n  mole  MDA/g  of tissue). Treatment with MEPV (100 and 200 mg/ kg) was significantly decrease the lipid peroxidation levels (1.22 and 1.09 n moles MDA /g of tissue) in a dose dependant manner. The CAT level were decreased in DAL control mice (1.68 unit/mg tissue) when compared with normal mice (2.64 unit /mg tissue) while treatment with MEPV at dose of 100 and 200 mg/kg brought nearer to normal level (1.82 and 2.10 unit / mg tissue). The GSH content in liver tissues of normal mice   was   found   to   be   2.35   mg/g   wet   tissue. Inoculation of DAL drastically decreased the GSH content to 1.63 mg/g wet tissue.   Whereas, treatment with different doses of MEPV the GSH levels were reverts to normal level (1.90 and 2.09 mg/g wet tissue). The  protein content were  increased in  DLA control mice (18.14 g/100 ml) as compared to normal mice (14.87 g/100ml) Administration of the MEPV significantly decreased the protein content (17.03 and 16.07 g/ 100 ml) at the dose of 100 and 200 mg / kg respectively.

 

DISCUSSION:

The present study was carried out to evaluate the effect of  MEPV  on  DAL bearing  mice.  The  MEPV  were showed significant anti-tumor and antioxidant activity against the transplantable murine tumor. The reliable criteria for judging the value of any anticancer drug is the prolongation of life span of animals18.  The Ascitic fluid is the direct nutritional source to tumor cells and the rapid increase in ascites fluid with tumor growth could possible by a means to meet more nutritional requirement of tumor cells [19]. A reduction in the number of ascitic tumor cells may indicate either an effect of MEPV on peritoneal macrophages or other components   of   the   immune   system20,   therefore increasing their capacity of killing the tumor cells, or a direct effect on tumor cell  growth. MEPV inhibited significantly the tumor volume, viable cell count and enhancement in survival time of DAL bearing mice and thereby act as anti-neoplastic agent. Myelosupression is a frequent and major complication of cancer chemotherapy. Compared to the DAL control animals,   MEPV   treatment   and   subsequent   tumor inhibition  resulted  in  appreciable  improvement  in hemoglobin content; RBC and WBC count      (Table -2).  These  observations assume  great  significance as anemia is a common complication in cancer and the situation aggravates further during chemotherapy since a majority of antineoplastic agents exert suppressive effects on erythropoiesis21,22  and thereby limiting the use of these drugs.

 

Lipid peroxidation (LPO) is a free radical-related process that in biologic systems may occur under enzymatic control, e.g., for the generation of lipid- derived inflammatory mediators, or nonenzymatically. This latter form is associated mostly with cellular damage as a result of oxidative stress, which also involves cellular antioxidants in this process. The oxidation of unsaturated fatty acids in biological membranes leads to a reduction in membrane fluidity and disruption of membrane structure and function23. MDA, the end product of lipid peroxidation was also reported to be higher in carcinomatous tissue than in non-diseased organs24. Increase in the level of TBARS indicates  enhanced  lipid  peroxidation  foremost  to tissue injury and failure of the antioxidant defence mechanisms to prevent the formation of excess free radicals. Glutathione is the most abundant non-protein thiol in mammalian cells. It is present mainly in the reduced form (GSH).  The oxidised form (GSSG) is less than 10 % of the reduced one.  The glutathione is involved in many important cellular functions, ranging from the control of physico chemical properties of cellular proteins and peptides to the detoxification of free radicals25.

 

Glutathione carries out an important number of metabolic functions and one of the most important is protection of cells against  oxidants  and  other  xenobiotics.     Glutathione,  a potent inhibitor of neoplastic process plays an important role as an endogenous antioxidant system that is found particularly in high concentration in liver and is known to have key function in the protective process26. MEPV significantly  reduced  the  elevated  levels  of  lipid preoxidation and increased glutathione content in DAL bearing mice thereby it may act as an anti-tumor agent.

 

The free radical scavenging system of catalase, it is present in all major organs in the body of animals and human beings respectively. It is especially concentrated in liver and erythrocytes. Catalase is capable of scavenging the hydrogen peroxide  radical,  which  is  formed  during  various biochemical and metabolic reactions26. Catalase laid an important role in the elimination of ROS derived from the redox process of xenobiotic in liver tissues27,28. It was suggested that catalase are easily inactivates by lipid peroxides or ROS30. Inhibition of catalase activity in tumor cell  line  was  also  reported30. In  this  study,  catalase  was appreciable elevated by administration of MEPV suggesting that it can restore the levels of catalase enzymes.

 

The present study demonstrated the MEPV increased the life span of DAL tumor bearing mice and decreased the lipid peroxidation and thereby augmented the endogenous antioxidant enzymes in the liver. The above parameters are responsible for the anti-tumor and antioxidant activities of Pseudarthria viscida. Based on the above findings, it would be interesting and worthy to further investigate the potential effectiveness of this type of molecules in prevention and probably in treatment of diseases caused by the overproduction of radicals. Further investigations are in progress in our laboratory to identify the active principles involved in this anti-tumor and anti-oxidant activity and investigate their mechanism.

 

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Accepted on 28.07.2008   © RJPT All right reserved