Evaluation of antitumor activity and in vivo antioxidant status of Anthocephalus cadamba on Ehrlich ascites carcinoma treated mice

Journal of Ethnopharmacology 142 (2012) 865–870

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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Ethnopharmacological communication

Evaluation of antitumor activity and in vivo antioxidant status of Anthocephalus cadamba on Ehrlich ascites carcinoma treated mice Narayan Dolai a, Indrajit Karmakar a, R.B. Suresh Kumar a, Biswakanth Kar a, Asis Bala a,b, Pallab Kanti Haldar a,b,n a b

Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, W.B., India Himalayan Pharmacy Institute, Majhitar, East Sikkim 737136, Sikkim, India

a r t i c l e i n f o

abstract

Article history: Received 5 November 2011 Received in revised form 12 May 2012 Accepted 25 May 2012 Available online 15 June 2012

Ethnopharmacological relevance: Anthocephalus cadamba (Roxb.) Miq. (Family: Rubiaceae) is commonly known as ‘‘Kadamba’’ in Sanskrit and Hindi in India. Various parts of this plant have been used as a folk medicine for the treatment of tumor, wound healing, inflammation and as a hypoglycemic agent. Aim of study: The purpose of this investigation was to evaluate the antitumor activity and antioxidant status of defatted methanol extract of A. cadamba (MEAC) on Ehrlich ascites carcinoma (EAC) treated mice. Materials and methods: In vitro cytotoxicity assay has been evaluated by using the trypan blue method. The determination of in vivo antitumor activity was performed by using different EAC cells (2  106 cells, i.p.) inoculated mice groups (n ¼ 12). The groups were treated for 9 consecutive days with MEAC at the doses of 200 and 400 mg/kg b.w. respectively. After 24 h of last dose and 18 h of fasting, half of the mice were sacrificed and the rest were kept alive for assessment of increase in life span. The antitumor potential of MEAC was assessed by evaluating tumor volume, viable and nonviable tumor cell count, tumor weight, hematological parameters and biochemical estimations. Furthermore, antioxidant parameters were assayed by estimating liver and kidney tissue enzymes. Results: MEAC showed direct cytotoxicity on EAC cell line in a dose dependant manner. MEAC exhibited significant (P o0.01) decrease in the tumor volume, viable cell count, tumor weight and elevated the life span of EAC tumor bearing mice. The hematological profile, biochemical estimations and tissue antioxidant assay were reverted to normal level in MEAC treated mice. Conclusion: Experimental results revealed that MEAC possesses potent antitumor and antioxidant properties. Further research is going on to find out the active principle(s) of MEAC for better understanding of mechanism of its antitumor and antioxidant activity. & 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Anthocephalus cadamba Ehrlich ascites carcinoma Antitumor Cytotoxicity Antioxidant

1. Plant The stem bark of Anthocephalus cadamba (Roxb.) Miq. (Rubiaceae) was collected from the middle hill region of Sikkim in the month of September 2010 and authenticated by Botanical Survey of India, Gangtok, India. A voucher specimen (SHRC-5/5/2010/ Tech.47A) was deposited at Phytotherapy and Pharmacology Research Laboratory, Department of Pharmaceutical Technology, Jadavpur University, Kolkata. First the collected stem bark was shade dried at room temperature for 7 days and then powdered in a mechanical grinder. Next, the powdered plant material (200 g) was successively extracted by petroleum ether (60–80 1C)

n

Corresponding author at: Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, W.B., India E-mail address: [email protected] (P.K. Haldar). 0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.05.050

followed by methanol using Soxhlet extraction apparatus. Then the solvent was completely evaporated under a reduced pressure and stored in a vacuum desiccator for future use. The yield of the petroleum ether and defatted methanol extract was about 8% and 20% w/w respectively. The preliminary phytochemical study of the defatted methanol extract indicated the presence of triterpenoid, glycosides, saponins, alkaloids and flavonoid (Evans, 1996). Finally, the defatted methanol extract was sealed in a glass beaker and stored at 20 1C for the assessment of the present experiment.

2. Uses in traditional medicine and reported activities A. cadamba (Roxb.) Miq. (Rubiaceae) is commonly known as ‘‘Kadamba’’ in Sanskrit and Hindi, which is frequently found in moist deciduous evergreen forests and widely distributed throughout the greater part of India (Umachigi et al., 2007).

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Various plant parts are used as folk medicine in the treatment of fever, uterine complaints, hematological diseases, skin diseases, leprosy, dysentery, tumor and it also improves the quality of semen (Umachigi et al., 2007; Dr. Duke’s Phytochemical and Ethnobotanical Database, 2007; Alam et al., 2008). In traditional system of remedies, bark is used as a tonic, febrifuge, and to reduce pain and inflammation. The traditional healers of Chhattisgarh prefer the decoction of leaves in place of bark for the same purpose (Ambujakshi et al., 2009). The bark and leaf extract has also been reported to be traditionally used as a hypoglycemic agent in Bangladesh and tribal areas of Orissa specially Ganjam district Acharyya et al., 2010. Until now, scientific bioactivity determination studies have revealed its antimalarial (Sianne and Fanie, 2002), antihepatotoxic (Kapil et al., 1995), analgesic (Ambujakshi et al., 2009), anti-inflammatory (Kodangula et al., 2010), antidiabetic Acharyya et al., 2010, antifungal (Patel Divyakant et al., 2011), anti-diarrheal (Alam et al., 2008), antioxidant, wound healing and antimicrobial activities (Umachigi et al., 2007). This plant is used for antitumor purpose in folklore medicine. Although like most traditional usage of ethno medicines, here in this case too, there is a dearth of scientific evidences. Due to the aforementioned reason, present study was performed to evaluate the antitumor activity and antioxidant status of defatted methanol extract of A. cadamba (MEAC) on Ehrlich ascites carcinoma (EAC) treated mice.

3. Previously isolated constituents The principal constituents of stem bark circumscribe a wide range of chemical class of molecules namely—triterpenes, triterpenoid glycosides, saponins, indole alkaloids; cadambine, 3adihydrocadambine, cadamine, isocadamine and isodihydrocadambine (Gurjar et al., 1998). It has been reported that heartwood, leaves, flower and seed contain typical alkaloid cadambine and cholorogenic acid (Kapil et al., 1995).

4. Materials and methods 4.1. Animals Swiss albino mice of about 8 weeks of age with an average body weight of 20–25 g were used for the experiment. The mice were grouped and housed in poly acrylic cages (38 cm  23 cm  10 cm) with not more than 6 animals per cage. The animals were maintained under standard laboratory conditions (temperature 25–30 1C and 55–60% relative humidity with dark/light cycle 14/10 h) and were allowed free access to standard dry pellet diet and water ad libitum. The mice were acclimatized to laboratory conditions for 7 days before the commencement of the experiment. All the described procedures were reviewed and approved by the Jadavpur University Animal Ethics Committee (367001/C/CPCSEA).

4.3. Transplantation of tumor cells The EAC cells were obtained from the Chittaranjan National Cancer Institute, Kolkata, India. The ascitic fluid was drawn out from EAC tumor bearing mouse at the log phase (days 7–8 of tumor bearing) of the tumor cells. The EAC cells were maintained in vivo in Swiss albino mice by intraperitoneal transplantation of 2  106 cells per mouse after every 10 days and it is used for both in vivo and in vitro study (Haldar et al., 2010a). 4.4. Assay for in vitro cytotoxicity study In vitro cytotoxicity assay of MEAC was performed by using EAC cell line. Briefly, 1  106 EAC cells were suspended in 0.1 ml of phosphate buffered saline (PBS, 0.2 M, pH 7.4) and mixed with 100 ml of various concentrations of MEAC (25, 50, 100, 150, 200 and 300 mg/ml). The final volume was adjusted to 1 ml with PBS and was incubated at 37 1C for 3 h. After the completion of incubation, the viability of the cells was determined using trypan blue (0.4% in normal saline) method and the percentage of cytotoxicity was determined by calculating percent inhibition and IC50 values (Manojkumar et al., 2009). 4.5. Treatment schedule for assessment of in vivo antitumor potential The Swiss albino mice (20–25 g) were divided into five groups (n ¼12). Except Group-I all the animals in each groups were being injected EAC cells (2  106 cells/mouse, i.p.). This was marked as day ‘‘0’’. Group-I was served as normal saline control (5 ml/kg, i.p.) and group-II was served as EAC control. After 24 h, EAC transplanted Group-III and -IV were being injected MEAC (200 and 400 mg/kg b.w, i.p.) once daily for 9 consecutive days. GroupV received standard drug 5-FU (20 mg/kg i.p) for 9 consecutive days (Bala et al., 2010). After administration of last dose, 6 mice from each group were kept fasting for 18 h and blood was collected by direct cardiac puncture for the estimation of hematological and serum biochemical parameters determination and then those animals were sacrificed for collection of liver and kidney tissues to check the different antioxidant parameters. Rest of the animals in each groups were kept alive with food and water ad libitum to check the percentage increase in life span of the tumor host to determine the mean survival time (MST). Antitumor activity and antioxidant status of MEAC was assessed by observation of changes with respect to the following parameters (Haldar et al., 2010a). 4.6. Tumor volume and weight The mice were dissected and the ascitic fluid was collected from the peritoneal cavity. Volume of the fluid was measured by taking it in a graduated centrifuge tube and expressed in milliliter (ml). Tumor weight was measured by taking the weight of the mice before and after the collection of the ascitic fluid from peritoneal cavity and expressed in gram (g). 4.7. Percentage increase life span (% ILS)

4.2. Acute toxicity and dose calculation The acute oral toxicity of MEAC in Swiss albino mice was performed as per OECD guideline 425 (OECD, 2008). The extract was safe up to the dose of 2 g/kg b.w. p.o. for mice. Generally 1/ 5th to 1/10th of the lethal dose was taken for effective dose calculation. So, 200 and 400 mg/kg b.w. doses were used in the present study (Bala et al., 2010).

The effect of MEAC on tumor growth was monitored by recording the mortality of the experimental mice. The percentage increase in life span (% ILS) was calculated by the following formula: Mean survival time (MST) in days ¼(day of the first death þday of the last death)/2 ILS (%)¼ [(MST of the treated group/MST of the control group)  1]  100

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Table 1 Effect of MEAC on tumor volume (ml), tumor weight (g), viable (cells  107 cell/ml) and nonviable cell count (cells  107 cell/ml), median survival time (MST), percentage increase life-span (% ILS) and hematological parameters like RBC (cells  106ml  1), WBC (cells  103ml  1) and Hb content (g/dL) in EAC bearing mice. Parameters

Normal control (5 ml/kg)

EAC control (2  106 cell/ml)

EAC þMEAC (200 mg/kg)

EACþ MEAC (400 mg/kg)

EACþ 5-FU (20 mg/kg)

Tumor volume Tumor weight Viable cell Nonviable cell MST (days) % ILS RBC WBC Hemoglobin

– – – – – –

2.92 70.20 2.75 70.25 8.35 70.31 0.33 70.05 20.5 70.51 00 3.16 70.40a,* 11.16 7 0.75a,* 7.25 70.61a,*

1.76 7 0.18b,* 1.30 7 0.14b,* 3.61 7 0.19b,* 1.23 7 0.12b,* 29.5 7 0.80 43.90 4.11 7 0.28b,* 8.33 7 0.51b,* 9.16 7 0.98b,*

1.18 70.17b,* 0.957 0.09b,* 1.36 70.18b,* 2.53 70.22b,* 367 0.51 75.61 5.16 70.22b,* 6.36 71.16b,* 10.917 0.80b,*

0.63 70.07b,* 0.61 70.10b,* 0.807 0.06b,* 3.03 70.15b,* 40.5 70.67 97.56 5.65 7 0.44b,* 5.81 7 0.32b,* 11.83 7 0.98 b,*

6.03 70.08 5.16 70.98 12.33 71.21

Values are represented as mean 7 SEM, where n¼ 6. a b n

EAC control group vs. normal group. All treated groups vs. EAC control group. P o 0.01.

4.8. Tumor cell (viable/nonviable) count The ascitic fluid was taken in a WBC pipette and diluted upto 20 times with PBS solution. Then a drop of the diluted cell suspension was placed on Neubauer’s counting chamber and the number of cells in the 64 small squares were counted. The viability and non-viability of the cell were determined by trypan blue assay. The cells were stained with trypan blue (0.4% in normal saline) dye. The cells that did not take up the dye were viable and those that took the dye were nonviable. These viable and nonviable cells were counted by using the under-scribbled formula: Cell count¼(number of cells  dilution factor)/(area  thickness of liquid film) 4.9. Hematological parameters The collected blood was used for the estimation of hemoglobin (Hb), red blood cell (RBC) and white blood cell (WBC) count by standard procedures (D’Armour et al., 1965; Wintrobe et al., 1961). 4.10. Biochemical parameters The blood samples were allowed to clot and the serum was separated by centrifugation at 5000 rpm for 10 min. Serum was utilized for the estimation of various biochemical parameters like total protein, serum glutamic oxaloacetate transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase (ALP). All the analysis was performed by using commercially available kits manufactured by the Span Diagnostics Ltd., Surat, India. 4.11. Tissue antioxidant assay parameters The tissue antioxidant assay was performed with liver and kidney tissues and the evaluation was carried out by measuring the level of total protein (Span Diagnostics kit), protein carbonylation (Abraham et al., 1999), lipid peroxidation (Ohkawa et al., 1979), the amounts of enzymatic Catalase (Aebi, 1974) and superoxide dismutase (Kakkar et al., 1984) and nonenzymatic antioxidant system such as reduced glutathione (Ellman, 1959). 4.12. Statistical analysis All the experimental data are expressed as the mean7SEM. The data was statistically analyzed by using one way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test by Instat

software (version 4). Po 0.01 was considered as statistically significant.

5. Results In the assay for in vitro cytotoxicity study, MEAC showed direct cytotoxic effect on the EAC cell line in a concentration dependent manner and the IC50 value was found to be 99.45 76.94 mg/ml. Antitumor activity of MEAC against EAC tumor bearing mice was assessed by the parameters such as tumor volume, tumor weight, cell count (viable and nonviable), mean survival time and percentage increase in life span. The tumor volume, tumor weight and viable cell count were found to be significantly (Po0.01) increased and nonviable cell count was significantly (Po0.01) declined in EAC control animals, when compared with normal control animals (Table 1). Administration of MEAC at the doses of 200 and 400 mg/kg significantly (Po0.01) decreased the tumor volume and viable cell count. Nonviable cell count was significantly (Po0.01) higher in MEAC treated animals when examined with respect to EAC control animals. Furthermore, the median survival time was increased to 29.570.80 (% ILS ¼43.90) and 3670.51 (% ILS¼ 75.61) on administration of MEAC in a dose dependant manner. There was significantly (P o0.01) elevated level of WBC and significantly (Po0.01) reduced level of RBC and hemoglobin (Hb) in EAC control group as compared to normal control group (Table 1). But, treatment with MEAC at the doses of 200 and 400 mg/kg in EAC bearing mice significantly (Po0.01) increased both the RBC count and Hb content while WBC count was deduced significantly (Po0.01) when compared with the EAC control group. The biochemical parameters like the amount of SGOT, SGPT and ALP in the EAC control group were significantly (Po0.01) increased as compared to the normal group. The total protein content was found to be significantly (Po0.01) declined in the EAC control group when compared with the normal group. Administration of MEAC significantly (Po0.01) increased the total protein content as compared with the EAC control mice (Fig. 1A).Treatment with MEAC in EAC bearing mice significantly (Po0.01) decreased the SGOT, SGPT and ALP levels in a dose dependant manner as compared to EAC control groups (Fig. 1B–D). Total protein and protein carbonylation were significantly (P o0.01) raised in both liver and kidney in EAC control mice when compared to normal control mice. Administration of MEAC significantly (P o0.01) attenuates the protein carbonylation of both liver and kidney tissue (Fig. 2A and B) in a dose dependant manner as compared to EAC control mice.

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20

MEAC 200 mg/kg

Normal control EAC control

150

MEAC 400 mg/kg 5-Fluorouracil

MEAC 200 mg/kg

EAC control

MEAC 400 mg/kg 5-Fluorouracil

b,

15

Normal control

a, #

b, b,

10

b,

IU/L

g/dl

100

a, #

b, b,

50

5

0 100

0 Normal control

MEAC 200 mg/kg

EAC control

MEAC 400 mg/kg

200

5-Fluorouracil

a, #

Normal control

MEAC 200 mg/kg

EAC control

MEAC 400 mg/kg 5-Fluorouracil

80

150

a, #

40

b, b,

20 0

KA Unit

IU/L

b,

60

b,

100

b,

b,

50

0

Fig. 1. Effect of methanol extract of Anthocephalus cadamba (MEAC) on serum biochemical parameters such as Total protein count (1A), SGOT (1B), SGPT (1C) and ALP (1D) in EAC bearing mice. Values are represented as mean 7 SEM, where n¼ 6. a: EAC control group vs. normal control group, #: P o 0.01; b: All treated groups vs. EAC control group, n: P o0.01.

Lipid peroxidation results in the formation of ROS species and subsequently elevates the level of malondialdehyde (MDA) which induces cancer in liver and kidney tissues. In the present study the MDA level was significantly (Po0.01) increased in EAC control animals when compared with normal control animals. Interestingly, treatment with MEAC significantly (P o 0.01) reduced the MDA levels as compared with EAC control group (Fig. 2C). The levels of Catalase, reduced GSH and SOD were significantly (Po0.01) decreased in EAC control group when compared with normal control group. Administration of MEAC in a dose dependent manner significantly (Po0.01) raised the Catalase, reduced glutathione and SOD levels as compared with EAC control animals (Fig. 2D–F).

6. Discussion Ethno medicines constitute a common substitute for cancer prevention and treatment in disparate countries around the globe. Due to the aforementioned concerns, such studies investigating medicinal herbs have been steadily held with interests. Currently, the number of plants reported to possess anticancer properties are around more than 3000 (Dai and Mumper, 2010). The present study was carried out to evaluate the antitumor effect and antioxidant activity of MEAC in EAC bearing mice. The Ehrlich ascites carcinoma (EAC) cells were initially described as a spontaneous murine mammary rapidly growing adenocarcinoma with a very aggressive behavior and can proliferate in almost all strains of mice (Haldar et al., 2010a). The Ehrlich ascitic tumor implantation induces per se a local inflammatory reaction, with increasing vascular permeability, which results in an intense edema formation, cellular migration, progressive ascitic fluid formation which is essential for tumor growth since it constitutes a direct nutritional source for tumor cells (Bala et al., 2010; Haldar et al.,

2010a). From the present experiment, it is limpid that treatment with MEAC at the doses 200 and 400 mg/kg significantly reduced the tumor volume, tumor weight, tumor cell count (viable and non-viable) when compared to the tumor control group. These results could connote either a direct cytotoxic effect of MEAC on tumor cells or an indirect local effect, which may involve macrophage activation and vascular permeability inhibition (Bala et al., 2010). The prolongation of the animal life span was being considered as a reliable criterion for the depiction of efficacy of an anticancer agent (Haldar et al., 2010b). The increase of life span of tumor bearing mice by reduction of nutritional fluid volume and seization of the tumor growth is a positive result and further corroborates the antitumor effect of MEAC. The major problems encountered in cancer chemotherapy are myelosuppression and anemia. The anemia exhibited in tumor bearing mice is mainly due to reduction of RBC or hemoglobin percentage and etiology is either iron deficiency or hemolytic/ myelopathic conditions (Haldar et al., 2010a). Pharmacotherapy with MEAC replenishes the hemoglobin (Hb) content, RBC and WBC count to the normal levels. It is evident from the result that MEAC possess protective action on hemopoietic system. From many years, serum enzymes have been studied as both early possible indicators of neoplasia and as an aid in following the progression and regression of the disease. In certain circumstances they can be carcinogenic and may engender hepatoxicity (Kathiriya et al., 2010). Furthermore, results of the experiments concluded that EAC control group exhibited raised levels of liver enzymes such as SGOT, SGPT, ALP and the levels of total protein were declined due to hepatocellular damages. Treatment with the MEAC restored the elevated biochemical parameters almost within the normal range, indicating the protection against tumor cell induced hepatotoxicity. Protein carbonylation (PC) is a process of irreversible nonenzymatic oxidation or carbonylation of protein. The carbonylation

N. Dolai et al. / Journal of Ethnopharmacology 142 (2012) 865–870

Normal control EAC control

a,#

b,*

4

b,*

b,* b,*

2

b,*

b,*

b,*

b,* b,*

4

Liver

b,*

100

b,*

a,#

b,* b,*

b,* b,*

50

Kidney

Normal control EAC control

25

MEAC 200 mg/kg MEAC 400 mg/kg 5-Fluorouracil

KU/min/mg protein

Normal control EAC control a,#

nM/mg-protein

6

0

150

b,* b,*

MEAC 200 mg/kg MEAC 400 mg/kg 5-Fluorouracil

b,*

20

b,*

b,*

15 10

b,*

b,*

a,# a,#

5 0

0 Kidney

30 b,*

15

MEAC 200 mg/kg MEAC 400 mg/kg 5-Fluorouracil

Normal control EAC control

b,*

b,*

20

b,* a,#

Kidney

Liver

b,*

b,*

a,#

mUnit/mg protein

Liver

µg GSH/mg protein

a,#

Kidney

Liver

10

MEAC 200 mg/kg MEAC 400 mg/kg 5-Fluorouracil

2

0

40

Normal control EAC control a,#

a,#

b,*

g/dl

10 8

b,*

6

MEAC 200 mg/kg MEAC 400 mg/kg 5-Fluorouracil

µM/cm

8

869

Normal control EAC control

MEAC 200 mg/kg MEAC 400 mg/kg 5-Fluorouracil

b,*

10

b,* b,*

b,*

b,*

b,*

5 a,#

a,#

0

0 Liver

Kidney

Liver

Kidney

Fig. 2. Effect of methanol extract of Anthocephalus cadamba (MEAC) on tissue antioxidant defense parameters like Total protein count (2A), protein oxidation (2B), lipid peroxidation (2C), catalase (2D), reduce glutathione (2E), and superoxide dismutase (2F) in EAC bearing mice. Values are represented as mean 7 SEM, where n¼6. a: EAC control group vs normal control group, #: Po 0.01; b: All treated groups vs. EAC control group, n: P o 0.01.

of protein often leads to a loss of protein function, which is considered as a widespread marker of severe oxidative stress, damage and disease-derived protein dysfunction (Dalle-Donne et al., 2006a). Moreover, PC has been found in various types of cancer including hematological malignancies and also in neurodegenerative diseases (Dalle-Donne et al., 2006b). The present results demonstrate that the PC levels were high in EAC control mice and it indicates the high free radical generation in the hematopoietic cells. These high levels of free radicals may cause the oxidative stress in hematopoietic cells if the antioxidant defense system is not effective. Administration of MEAC restored the increased PC levels more or less to normal range signifies the antioxidant and free radical scavenging property of MEAC. The oxidative stress may lead to damage of the macromolecules such as lipids and can induce lipid peroxidation in vivo (Haldar et al., 2010b). In EAC bearing mice, the level of lipid peroxide in liver was significantly raised, which was however reduced near to normal level in MEAC treated animal groups. This reflects the decline in free radical production and subsequent reduction in oxidative stress, which are the main risk factors of the ailment.

Glutathione (GSH), a potent inhibitor of neoplastic proliferation process, plays a crucial role as an endogenous antioxidant system. It was found particularly in high concentration in liver and is known to have a key function in the protective process (Haldar et al., 2010a). The level of GSH was depleted in EAC mice which is probably due to its utilization by the excessive amount of free radicals. Treatment with MEAC was found to raise the GSH content in the liver and kidney as compared to EAC control animals. The cells are also equipped with enzymatic antioxidant mechanisms that play an imperative role in the elimination of free radicals. It has been reported that a decrease in SOD activity in EAC-bearing mice may be due to the loss of Mn2 þ containing SOD activity in EAC cells and the loss of mitochondria which finally leads to a decrease in total SOD activity in liver (Rushmore and Picket, 1993). The inhibition of both SOD and CAT activities as a result of tumor growth was also reported (Haldar et al., 2010b). Similar findings were observed in the present investigation with EAC-bearing mice. The administration of MEAC not only significantly elevated the SOD and CAT levels in a dose dependent

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manner but also restores the lipid peroxide and GSH content near to normal level; which indicates the antioxidant and free radical scavenging property of MEAC.

7. Conclusion The present investigation is quite encouraging as it explores that MEAC have potent antitumor and antioxidant activity. The phytochemical study confirmed the presence of compounds namely triterpenoid, glycosides, saponins, alkaloids and flavonoid in MEAC which may possess these properties. Finally our findings suggest that the defatted methanol extract of A. cadamba exhibits potential antitumor and antioxidant activities which further shed light as a novel source of phytomedicines in the field of free radical and tumor biology. Moreover, investigations are working in progress at our laboratory to identify the active principle(s) involved for demonstrating antitumor and antioxidant activity.

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