V. Lavakumar et al. / Journal of Pharmacy Research 2012,5(3),1363-1367 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Anti cancer and Antioxidant Effect of spicifera against EAC induced carcinoma in mice

V. Lavakumar 1, 2, KFH Nazeer Ahamed1 and V.Ravichandiran* 1,2 1 Vels College of Pharmacy, Pallavaram, Chennai 117, Tamil Nadu, India 2 The Tamilnadu Dr. MGR Medical University, Guindy, Chennai- 600 032, Tamil Nadu, India Received on:10-12-2011; Revised on: 15-01-2011; Accepted on:12-02-2012

ABSTRACT Acanthophora spicifera is important seaweed in folklore remedies as well as food supplement in Gulf of Mannar, coastal region of Tamil Nadu. In the present study, the anticancer and anti-oxidant activities of the alcoholic extract of A. spicifera have been evaluated. The anti-tumour effect of the extract at the dose of 100 and 200 mg/kg oral route was carried out in mice treated with EAC cancerous cell lines. 5 Flurouracil (5FU) at the dose of 20 mg/kg given intraperitoneally to EAC treated mice serving as standard drug control. After 21 days of test and standard drug administration, anti-cancer effect was assessed by measuring tumor volume, tumor weight, mean survival day (MSD) and various haematological parameters. In addition, anti-oxidant status of the liver tissue was assessed. The alcohol extract of A. spicifera exhibits significant anti-cancer effect by reducing tumor volume and weight with increasing mean survival day (MSD) in EAC cell lines treated cancerous mice. The extract also showed anti-oxidant effect by significantly increasing endogenous anti-oxidant and decreasing oxidative stress marker in liver homogenates. The results of these study indicated that alcohol extract of A. spicifera possess anti-tumour and anti-oxidant activity and the beneficial effect may be due to the presence of bioactive components like flavonoids, terpenoids and tannins.

Key words: , A.spicifera, EAC, Antioxidant, Anticancer INTRODUCTION Free radical and reactive oxygen species (ROS) are by products produced in terpenes, enzymes, polysaccharides, alkaloids, flavonoids (Mohamed et al., body due to various physiological and biochemical processes. Generally, 2012). More recent reports revealed that marine algae possess rich sources of most of these free radicals generated from cellular metabolism are scavenged antioxidant compounds with potential free radical scavenging activity as in by endogenous defence system such as superoxide dismutase, Catalase and Halimeda tuna (Ananthi et al.,2008) and Acanthophora spicifera (Vasanthi et Peroxidase–glutathione system (Govindarajan et al., 1992). However, in many al.,2006). In this study Acanthophora spicifera (Vahl) Borgrsen cases, such as in unhealthy physical condition, ageing or under stress (:Rhodophyta) are commonly known as spiny seaweed, is widely environments, the endogenous antioxidants are either exhausted or insufficient distributed throughout the tropic and subtropics throughout the Gulf of to scavenge these free radicals. Imbalance of these free radicals can cause Mannar, Rameswaram. (Umamaheswara Rao, 1970). To the date, research oxidative damage to biomolecules like lipids, proteins and DNA (Halliwell, on biologically active substances from this species is rather limited or not 1994; Niki, 1997). These reactive oxygen species (ROS) has been implicated been clearly established (Ahira et al, 1968; Prakash et al, 1989; Wahidulla et and having major role on human biological system which leads to diseases, al,1986, Wang et al,1998). Therefore the aim of the present study was to like ageing, cancer, and neurodegenerative disorders etc (Harman, 1994; evaluate anticancer and antioxidant activity of Acanthophora spicifera, a red Simonian and Coyle, 1996). Among this, Cancer is a complex disease algae in suitable experimental conditions. characterized by proliferation (uncontrolled cell division), cell transformation, and cape of apoptosis, invasion, angiogenesis and metastasis. Cancerous MATERIALS AND METHODS cells are to produce reactive oxygen species (ROS) and their inflammatory mediators. ROS may cause DNA mutation, which may be followed by Marine algae collection oncogenes activation and down regulation of tumour suppressor genes (Ames, The red algae Acanthophora spicifera (Family: , Ceramiales) 1983; Loft and Poulsen, 1996). By the activity of ROS, scavenging system was collected from Mandapam, during the month of March 2008 from gets altered in tumour cells (Trachootham et al., 2009). Over the past decades, Ramaeshwaram coast, Tamil Nadu, India. It is identified and authenticated seaweeds or their extracts have been shown to produce a variety of compounds by Dr. Krishnamurthy, Institute of algology, Anna nagar, Chennai. The and some of them have been reported to possess biological activity of potential voucher specimen (VCP/09-345) was deposited in the Department of Phar- medicinal values (Heo et al., 2005). The identification and exploitation of macognosy, Vels College of Pharmacy, Chennai 117. potent anticancer molecules from the marine environment such as marine algae has generated great interest in recent years. Extensive screening of Extraction of Acanthophora spicifera marine microalgae has led to the isolation and chemical determination of over Dried, pulverized A. spicifera (1 kg) was extracted with 5 liters of ethanol 15,000 compounds, including fatty acids, sterols, phenolic compounds, using soxhlet apparatus for 24 hrs. The extract was filtered, and the filtrate was evaporated by rotary vacuum evaporator and sample was freeze dried for further use. The percentage yield of ethanol extract was found to be *Corresponding author. 20.22% w/w. The ethanolic extract was subjected to qualitative chemical test V. Lavakumar and thin layer chromatography studies (Kokate, 1994; Harborne, 1998). Vels college of Pharmacy Old Pallavaram, Chennai 117 Chemicals and drugs Tamilnadu, India. The chemicals used were 5-Fluorouracil (Ranbaxy Laboratories, Ltd., India), Coboxy methyl cellulose (CMC) (Ranbaxy Laboratories, Ltd., India), Adrena-

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1363-1367 V. Lavakumar et al. / Journal of Pharmacy Research 2012,5(3),1363-1367 line bi-tartrate (Sisco chemicals, Mumbai), Thiobarbituric acid (Sisco chemi- Measurement of Antioxidant / Oxidative stress markers cals, Mumbai), Elman’s reagent (SRL Chemicals, Mumbai) and all other Animals (n=3) were sacrificed from each group. Blood samples were col- chemicals and reagents used were purely of analytical grade. lected transcordially for the following haematological and biochemical esti- mations. Animals Swiss male albino mice weighing around 20-22g were used for present inves- Biochemical parameters tigation. Animals were obtained from the central animal house, Vels College After the collection of blood samples, the mice were sacrificed. Liver was of Pharmacy, Chennai. Mice were grouped and housed in poly acrylic cages excised, rinsed in ice cold normal saline followed by ice-cold 10% KCl solu- (n=6) and maintained in standard laboratory conditions under the tempera- tion, blotted, dried and weighed. A 10% w/v liver homogenate was prepared ture 25 ± 20 C dark/light cycle. They were allowed free access to standard in ice-cold KCl solution and centrifuged at 1500rpm for 15 min at 4oC. The dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The supernatant thus obtained were used for the estimation of thio-barbituric mice were acclimatized into a laboratory conditions for 7 days before the acid reactive substances (TBARS) (Okhawa et al., 1979) glutathione (GSH) experiment. All procedures described were reviewed and approved by the (Jollow et al, 2009), superoxide dismutase (SOD) (Magwere et al., 1997) Institutional Animal Ethical Committee. catalase (CAT)( Clairborne,1989), glutathione peroxidase (GPx) and glu- tathione-S-transferase (GST) (Rajkumar et al.,2010 ) and total protein (TP) Acute Toxicity Study (Lowry et al., 1951). Toxicity study - up and down procedure was carried out as per the guidelines set by Organization for Economic Co-operation and Development (OECD). Haematological parameters Oral toxicity study was done according to OECD guidelines 423. In this At the end of the experimental period, next day after an overnight fasting, experiment two groups of wistar rats (n=3) were used. Animals were fasted blood was collected from freely flowing tail vein and used for the estimation over night with water ad libitum and foods were withheld for 3-4 hrs after of haemoglobin (Hb) content, red blood cell (RBC) count, white blood cell oral administration of the extracts. One group of animals were treated with (WBC) count, Protein content, Packed cell volume (PCV) and differential starting dose of the Acanthophora spicifera extract of 2000mg/kg b.wt orally. count of WBC was measured using standard procedures. Another group of rats were treated with normal saline. Observation includes mortality and clinical signs, which includes changes in skin, fur, eyes and Statistical analysis mucous membranes. The gross behaviors like body positions, locomotion, All the data were expressed as Mean ± SEM. All the groups were compared rearing, tremors, gait was observed. The effect of Acanthophora spicifera on by one way (ANNOVA) followed by Dunnett’s post hoc test. Probability of passivity, grip strength, pain response, stereotypy, vocalization, writhing P< 0.05 was considered as significant. reflex, body weight and water intake was observed (Lipnic et al., 1995). RESULTS AND DISCUSSION Transplantation of tumour The preliminary phytochemical analysis of Acanthophora spicifera showed Ehrlich ascites carcinoma (EAC) cells were obtained through the courtesy of that the presence of flavonoids, tannins, terpenoids and glycosides. Amala Cancer Research Centre, Thrissur, Kerala. The EAC cells were main- tained in vivo in Swiss albino mice by intraperitoneal inoculation of 2 × 106 Effect of Acanthophora spicifera on Tumour weight, Tumour volume cells per mouse. From the peritoneal cavity of the mice, the EAC cells were and Mean survival day aspirated, washed with saline and were given intraperitoneally to develop Fig:1 represents the effect of the acanthophora spicifera on tumour weight in ascitic tumor (Rajkapoor et al, 2004). EAC in mice. Inoculation of EAC cell lines into mice, significantly (P <0.001) increased the body weight of the mice as measured the indices of the tumour Animal grouping and Drug treatment mice. Oral administration of ethanolic extract of Acanthophora spicifera (100 Animals were divided into five groups (n=6) and 200 mg/kg bd wt) significantly (P<0.001) decreased the tumour weight in Group-I was served as normal control treated with saline control (5 ml/kg EAC treated carcinoma mice than the saline treated control tumour mice. In i.p.) addition 5-FU also significantly (P<0.001) decrease the tumour weight in Group-II was served as EAC treated control group EAC inoculated mice as compared with saline treated EAC bearing tumour Group-III was served as EAC tumour control administered with 5 FU (20 mice. mg/kg, i.p.) Group-IV was served as EAC treated tumour control treated with alcoholic 20 extract of Acanthophora spicifera (100 mg/kg/p.o) Group-V was served as EAC treated tumour control treated with alcoholic 15 extract of Acanthophora spicifera (200 mg/kg/p.o) ** *** The standard 5-FU and the test drug Acanthophora spicifera extract were 10 *** administered after 72 hrs of tumour inoculation. All the drugs were adminis- tered for 21 days continuously and the following parameters were evaluated 5 periodically. Tum our wt i n g

Tumor volume, tumour weight and Mean survival Day 0 The ascetic fluid was collected from the peritoneal cavity. The volume was measured by taking it in a graduated centrifuge tube. The tumor weight was 5 FU measured by taking the weight of the mice before and after the collection of the ascetic fluid (Bala et al., 2010).

The Mean survival day were calculated by animals survived from the date of Tumor Control ASE 100 mg/kg ASE 200 mg/kg tumor inoculation to the date at which the animal death occurs. Mean survival day is calculated by using the formula given below. *** represents the P<0.001, 5FU Vs tumour control No. of days animals survived in test group ** represents P<0.01, ASE (100 & 200 mg/kg) Vs Tumour control Mean survival day = ————————————————————— × 100 Fig: 1 Effect of Acanthophora spicifera on tumour weight in EAC treated No. of days animals survived in control group mice Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1363-1367 V. Lavakumar et al. / Journal of Pharmacy Research 2012,5(3),1363-1367 Fig: 2 represent the effect of the ethanolic extract of Acanthophora spicifera Fig: 4 represents the effect of Acanthophora spicifera extract on various on tumour volume in EAC treated cancerous mice. Increase in tumour vol- blood haematological parameters treated with mice bearing with EAC cell ume was noted in saline treated EAC mice during the 3 weeks of study lines. period. The tumour volume decreases significantly (P<0.01) in EAC mice treated with Acanthophora spicifera (100 and 200 mg/kg bd wt). The effect 30 was found to be dose dependent. In addition there was a significant (P< *** Control 0.001) decrease in tumour volume in 5-FU treated tumour mice than saline Tumour control treated tumour mice. *** *** ASE 100 mg/kg 10 20 *** ASE 200 mg/kg *** 8 ** *** *** 10 *** ** 6 ** ** *** 4 0 V o l ume in ml 2

Hb gm % PCV mm 0 Protien mg %

5FU RBC mill cells/cuWBCmm mill cells/cumm *** P<0.001 represents Tumour control Vs normal control ** P<0.01 represents ASE 200 mg/kg Vs Tumour control Tumor Control ASE 100 mg/kg ASE 200 mg/kg Fig: 4. The effect of Acanthophora spicifera on various blood *** P<0.001represents Tumour control Vs normal control haematological parameters treated with mice bearing with EAC cell ** P<0.01 represents ASE 200 mg/kg Vs tumour control lines Fig: 2. The effect of the Acanthophora spicifera on tumour volume in EAC treated cancerous mice Effect of Acanthophora spicifera on haemoglobin Mice treated with EAC had showed significant decrease in (P<0.001) hae- Fig: 3 represents the mean survival day of the tumor bearing mice treated moglobin (Hb) content as compared with saline treated control normal mice. with 5 FU and ethanolic extract of Acanthophora spicifera 100 & 200 mg/kg/ Intrestingly, the tumor bearing mice administered with high dose of ethanolic orally. It was observed from the fig 3 that significant increase (P<0.001) in extract of Acanthophora spicifera (200mg/kg) significantly (p<0.01) increases mean survival day in 5 FU treated tumor mice group as compared with saline the haemoglobin content as compared to that of tumor control group animals. treated tumor control. Oral administration of Acanthophora spicifera had significantly (P< 0.01) increased the MSD of the tumor bearing mice as Effect of extracts on WBC: compared to that of tumor control group. The increase in mean survival day Induction of tumor in mice had significantly (P<0.001) increase the WBC of the tumor bearing mice treated with Acanthophora spicifera extract was count as compared to that of vehicle treated control normal animal. Oral dose dependent administration of Acanthophora spicifera extracts to EAC bearing mice showed significant reduction (P<0.001) in WBC levels as compared to that 40 of vehicle treated control tumor mice. The effect was dose dependant and the *** ** high dose of Acanthophora spicifera (200 mg/kg/bd wt) almost reverses the WBC count towards normal. 30 ** Effect of Acanthophora spicifera extract on packed cell volume: 20 There was a significant increase in PCV cells in EAC treated cancer mice as compared to that of saline treated control group. It was interesting to observe that administration of Acanthophora spicifera ethanolic extract at the dose of MSD (Days) 10 100 and 200mg /kg bd wt orally significantly (P<0.001) decreases PCV levels towards the normal in treated tumor treated mice.

0 Fig: 5 represents the blood haematological parameters (lymphocytes, neu- trophils and monocytes) of control and EAC treated tumor mice treated with 5FU Acanthophora spicifera extract. Significant (P<0.001) increase in lympho- cytes and decreases in neutrophils was observed in EAC challenged tumor bearing animal group compared to that of saline treated control group. Ad- Tumour control ASE 100 mg/kg ASE 200 mg/kg ministration of Acanthophora spicifera significantly (P<0.001) increases the lymphocytes percentage as compared to that of EAC treated tumor control *** P<0.001 represents 5FU Vs Tumour control mice whereas the high dose level of 200mg/kg orally of Acanthophora spicifera ** P< 0.01 represents ASE 100 & 200 mg/kg Vs Tumour control extract exhibited significant decrease (P<0.01) in the neutrophil percentage Fig: 3. Mean survival day of the tumour bearing mice treated with 5 FU of tumor bearing mice group. However no effect was observed in monocytes and ASE (100 & 200 mg/kg) percentage of vehicle treated and EAC treated cancer group.

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1363-1367 V. Lavakumar et al. / Journal of Pharmacy Research 2012,5(3),1363-1367

80 *** Control *** Tumour control 60 *** ASE 100 mg/kg ASE 200 mg/kg 40 *** Pe r cen tage 20 **

0

Neutrophils % Monocytes % Lymphocytes %

*** P<0.001 represents Tumour control Vs normal control ** P<0.01 represents ASE 200 mg/kg Vs Tumour control Fig: 5. The effect of Acanthophora spicifera on blood haematological parameters like Lymphocytes, Nuetrophils and Monocytes 50 Control

40 *** Tumour control *** ASE 100 mg/kg *** *** 30 *** *** * ASE 200 mg/kg *** * 20 *** *** ***

10

0 SOD CAT LPO GPX GST

*** P<0.001 represents Tumour control Vs normal control ** P<0.01 represents ASE 200 mg/kg Vs Tumour control *p<0.05 represents ASE 100 mg/kg Vs Tumour control

Fig: 6. The effect of Acanthophora spicifera on biochemical parameters Fig: 6 represents the effect of 5FU and A. spicifera extract on antioxidant and Acanthophora spicifera extract in EAC treated mouse model. In this present TBARS level in the liver homogenate of EAC treated cancer group. Admin- investigation, the oral administration of Acanthophora spicifera extract given istration of EAC significantly decreases (P<0.001) SOD and CAT level as for 21 days has significantly decreased the tumor volume and tumor weight compared to that of vehicle treated normal control group. The high dose of following the EAC inoculation in mice. The assessment of tumour volume Acanthophora spicifera extract administered orally to the EAC treated mice and tumor weight is the direct parameter for assessment of tumour growth as significantly (P<0.01) increases the SOD and CAT levels of the EAC treated well as anti tumour efficacy of the test drugs. The results from Mean survival mice as compared to that vehicle treated tumor control group. Whereas EAC day suggested that EAC treated mice shows decrease in MSD than the 5FU inoculation to normal mice significantly (P<0.001) increases the levels of and Acanthophora spicifera treated EAC mice. The decreased tumour vol- LPO, GPx & GST as compared to that of vehicle treated control mice group ume and tumour weight, with increased MSD in EAC bearing mice treated and this effect was significantly decreases (P<0.01) significantly elevated with Acanthophora spicifera 100 and 200 mg /kg body weight suggested the LPO, GPx & GST levels as compared to that of saline treated tumor control anti tumour properties of the extracts. It has been shown earlier that the group. decrease in life span of the tumour control was directly proportional to the propagation to the tumor growth. It has been reported by many investigators DISCUSSION that the life span expectancy or mean survival day get increased with the The present study high lights the in vivo antitumor effects of crude extract of decrease in tumour size (Sreelatha et al., 2011). These results clearly sug-

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1363-1367 V. Lavakumar et al. / Journal of Pharmacy Research 2012,5(3),1363-1367 gested that direct antitumor efficacy of Acanthophora spicifera in in-vivo 10. Ananthi S, SaravanaBabu C, Chandronitha C, Vasanthi Hannah R, model. It was further observed that beneficial impact of EAC inoculation on In-vitro antioxidant activity of the Green algae Halimeda tuna, Sea Weed Res.Utilin, 2008, 30,1-7. haematological parameters of the EAC treated mice in which there was de- 11. Vasanthi, Hannah R, Charles Dorni AI, Vidhyalakshmi KS, crease in RBC, increase in WBC with decrease in haemoglobin levels in tumor Rajamanickam GV, Free radical scavenging and antioxidant activity bearing mice. The reversal and restoration of haematological parameters like of red algae Acanthophora spicifera- relation to its chemical RBC, WBC and haemoglobin (Hb) levels in EAC treated mice with composition, Seaweed Res. Utilin, 2006, 28, 1–7. 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