IN-VIVO ANTICANCER ACTIVITY OF RED ALGAE (GELIDIELA ACEROSA AND ACANTHOPHORA SPICIFERA)

Received on 13 February, 2014; received in revised form, 03 April, 2014; accepted, 13 June, 2014; published 01 August, 2014

IN-VIVO ANTICANCER ACTIVITY OF RED ALGAE (GELIDIELA ACEROSA AND ACANTHOPHORA SPICIFERA)

K. Duraikannu*¹, K. Shameem rani ¹, R. Anithajothi², G. Umagowsalya¹ and C. M. Ramakritinan¹

Department of Marine and Coastal Studies ¹, Madurai Kamaraj University, Madurai-21, Tamil Nadu, India

Department of Zoology, MSS Wakf Board College ², Madurai, Tamilnadu, India

INTRODUCTION: Cancer is one of the most dangerous threats to human being in the world. Chemotherapy is the only standard remedy for cancer treatment. Most of the anticancer drugs currently used in chemotherapy are cytotoxic to normal cells and cause immunotoxicity; this affects not only the tumor development, but also aggravates patient’s recovery. The discovery and identification of new anticancer drug with low side effects on immune system have become an essential purpose in much research in immunopharmacology ¹.

Hence, so many researches were interested to finding new drugs from terrestrial plants, marine organisms/microorganisms including marine macroalgae etc. Now a day, drug discovery has been developed greatly in finding a pure organic compounds or crude extracts to provide new lead. Marine algae have been historically an extremely rich source of pharmacologically active metabolites with antineoplastic, antimicrobial and antiviral effects ^{2, 3}. And various biological effects are found in marine algae ⁴.

The Marine macroalgae are ecologically and commercially important to many regions in the world, especially in India. They are a valuable food resource which contains low calories and rich vitamin, minerals, proteins, polysaccharides, steroids and dietary fibers ^{5, 6}. They were also considered as important as traditional remedies ⁷. Macroalgae have been one of the richest and most prospective sources of bioactive metabolites ²⁸ and their discovery has significantly prolonged ^{7, 8}. The algae are synthesizing a variety of compounds such as Carotenoids, Terpenoids, xanthophylls, chlorophyll, vitamins and amino acids etc ⁹.

Macroalgae act as allelopathic, antimicrobial, antifouling and herbivore deterrents, or as ultraviolet screening agents ¹⁰. They are also used by the pharmaceutical industry in drug development to treat diseases like cancer, Acquired Immune-Deficiency Syndrome (AIDS), inflammation, pain, arthritis, infection for virus, bacteria and fungus ¹¹. Currently, algae represent about 9% of biomedical compounds obtained from the sea ¹².

The low side effects of bioactive compounds were isolated from natural resources; scientists are   interested in working on them to finding new medications. Finding anticancer agents from plant sources started in the earliest 1950s with the discovery and development of vinca alkaloids, vinblastine and vincristine and the isolation of the cytotoxic podophyllotoxins ¹³. Marine algae are one of the natural resources in the marine ecosystem. They contain various thousands of compounds have been isolated from macroalgae populations ¹⁴. Recently the marine algae contained antiviral ¹⁵, antibacterial, antifungal ¹⁶ and antitumor⁴ potentials, among numerous others. And then the polysaccharides and peptides compounds were isolated from macroalgae have become a substance of great interest for cancer therapy. The mechanisms of their anticancer activity are related to their ability to suppress the growth of cancer cells ¹⁷.

Previous studies have proved that macroalgae possess broad range of biological activities such as antibiotics ¹⁸. Since, the demand for screening of natural bioactive compounds has widened in interest of research. To date, research on anticancer activity substance from red algae species is rather and limited of Gulf of Mannar, southeast coast of India. Therefore the present study was aimed to evaluate in anticancer activity of marine red algae against Dalton’s Ascitic Lymphoma (DAL) cells in mice. This preliminary research studies reported here in could serve as a basic to isolated and identify the anticancer compounds from red algae extracts as a source of natural anticancer agents for pharmaceutical leads.

MATERIALS AND METHODS:

Preparation of algae extract: TheG. acerosa and A. spicifera belongs used in this study to red algae. The macroalgae were collected freshly from Gulf of Mannar, Southeast coast of India (Long N 9°16.313 Lat E 79°00.073). The collected samples were immediately rinsed with water to remove all kind of epiphytes. The macroalgae was shade dried at room temperature for 3-4 days. The shade dried macroalgae was powdered individually and used for experiment. The powdered samples (15.0 g) were soaked in 100 mL methanol for 2 days at room temperature twice, then filtered and evaporated under reduced pressure below 40°C. The crude samples were subjected to anticancer assay with DAL cells bearing in mice.

Acclimation of animal: The healthy adult male Swiss Albino mice (20-25g) sizes were used throughout the experiments. the experiment room was maintained at micro nylon boxes at suitable environmental condition i.e., temperature at 25 ±2 °C and 12 hours light / dark cycles with standard laboratory diet and water ad libitum ¹⁹. This study was conducted after obtaining institutional ethical clearance (Proposal Number: K. Durai/ MKU/IAEC/KMCP/60/2012) as per standard practice. After sufficient period of acclimatization, they were used to anticancer activity experiment.

 Grouping of animals: Swiss albino mice were divided into five groups of each six mice. Four groups (G₂-G₅) of animals were injected with DAL cells (1×10⁶ cells/mouse) intraperitonealy and remaining one group (G₁) was treated as a normal control group ²⁰.

• Group 1 served as Normal Control (G₁)
• Group 2served as  Cancer Control (G₂)
• Group 3 served as  Positive Control, was treated with injection flurouracil at 20mg/kg body weight, oral route (G₃) ²¹
• Group 4served as Treatment Control, which was treated with methanol extract of G. acerosa at 200mg/kg body weight, in i.p, (as per LD₅₀ value) (G₄).
• Group 5 served as Treatment Control (G₅), which was treated with (methanol extract of A. spicifera) at 200mg/kg body weight, in i.p, (as per LD₅₀ value).

In the present investigation, treatment was given after 24 hrs inoculation, once daily for 14 days. At the end of 15^th days sacrificed in all five groups was observed. The blood was withdrawn in all mice separately by Retro-Orbital plexus methods and the collected blood was stored in refrigerator at 4°C ²⁷.

Cancer cell count: The fluid (100 µl) from the peritoneal cavity of each animal was withdrawn by sterile syringe and diluted with 800µl of ice cold normal saline or sterile phosphate buffer solution (PBS) and 100µl of tryphan blue and total numbers of the living cells were counted using haemocytometer ²².

Total number of cells per µl = average no of cell × dilution factor 2×10⁴   

Animal weight: All mice were weighted from the beginning to 15^th day of the experiment; average increase in body weight on the 15^th day was calculated.

Life span (%): The effect of methanolic extract of G. acerosa and A. spicifera on cancer cells growth and the life span (%) were calculated as follow.

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

Mean survival time (MST) = [1^st Death + Last Death] / 2

Haematological parameters: Such as WBC, RBC, and Platelet count and haemoglobin count of all five groups were analysed (pentra-120 Automated Hematology Analyzer). Similarly; Total cholesterol (TC), Triglycerides (TG), Aspartate amino transferase (AST), Alanine amino transferase (ALT) and Alkaline phosphatise (ALP) were analyzed the blood serum.

• All biochemical investigation was done by using COBAS MIRA PLUS-S Auto analyzer from Roche Switzerland.
• Haematological test was carried out in COBAS MICROS OT 18 from Roche
• Biochemical investigation of blood sample were analysed in added Hi-Tech instruments MAX-MAT used for auto analyzer.

Statistical analysis: The total experimental results were expressed as the mean ± S.E.M. The haematological and biochemical parameters were subjected to statistical analysis by one way analysis of variance to determine the significant difference between the groups. ANOVA was done with graph pad prism software.  The data were accepted as statistically significant different was obtained at p< 0.05.

RESULTS: The intraperitoneal inoculation of DAL cells in the mice produces increased proliferation of cells. The methanol extract of G. acerosa and A. spicifera samples was reduced the cancer cell count to 1.88±0.30×10⁶, 1.80±0.25×10⁶ cells in the treated mice. The methanol extract of G. acerosa and A. spicifera treated mice survived upto 35 days where as cancer control mice survived upto 20 days only. And packed cell volume in cancer control mice was found 31.40±3.25% to be high.

The oral dose administration of Methanol extract of G. acerosa and A. spicifera extract had reduced the Packed Cell Volume 22.40±1.70% and 23.26±1.85% respectively. Whereas the methanol extracts of G. acerosa and A. spicifera at the dose of 200mg/kg body weight increased by 82% and 84% respectively.

Therefore, the extract treatment was reduces the tumor weight and hence increased the life span of cancer induced mice (Table 1).

TABLE 1: THE EFFECT OF G. ACEROSA AND A. SPICIFERA EXTRACT ON THE LIFE SPAN, BODY WEIGHT AND CANCER CELL COUNT OF CANCER INDUCED MICE.

G₁-Normal Control, G₂-Cancer Control_, G₃-Standard_, G₄-Methanolic extract of G. acerosa_, G₅-Methanolic extract of A. spicifera. All data vale are mean ± S.E.M (n=6); *Values are significantly different from normal control (G1) at P < 0.01; **Values are significantly different from cancer control (G2) at P < 0.01

On the subject of haematological parameters, cancer control mice showed reduced RBC count but also increased WBC count than normal group. The treatment of methanol extract G. acerosa and A. spicifera also raised the RBC count significantly to 3.35±0.68 mill/cumm and 3.15±0.60 mill/cumm respectively. Similarly both are restored, the WBC value to 12.22±1.90 cells/mL × 10³, 12.10±1.65 cells/mL × 10³ respectively. Haemoglobin content in cancer control mice reduced significantly when compared with normal group. But, the methanol extracts of G. acerosa and A. spicifera doses were increased Hb contents in 10.40±1.32 gm/dL, 10.25±1.05 gm/dL. The methanol extract G. acerosa and A. spicifera restored the normal platelet count in treated mice (Table 2).

TABLE 2: THE EFFECTS OF G. ACEROSA AND A. SPICIFERA EXTRACT ON HAEMATOLOGICAL PARAMETERS

G₁-Normal Control, G₂-Cancer Control_, G₃-Standard_, G₄-Methanolic extract of G. acerosa_, G₅-Methanolic extract of A. spicifera. All values are expressed as mean ± S.E.M (n=6); *Values are significantly different from normal control (G₁) at P < 0.01; **Values are significantly different from cancer control (G₂) at P < 0.01.

The inoculation of DAL cells caused significantly increase in the level of total cholesterol, TGL, AST, ALT and ALP in the cancer control animals (G2), when compared to the normal group (G1). The treatment with G. acerosa and A. spicifera at the dose of 200mg/kg body weight reversed these changes towards the normal level. All the values were found to be significant. However the treatment with standard 5- FU at the dose 20 mg/kg body weight also produced better result in all parameters (Table 3).

TABLE 3: THE EFFECTS OF G. ACEROSA AND A. SPICIFERA EXTRACT ON BIOCHEMICAL PARAMETERS

G₁-Normal Control, G₂- Cancer Control, G₃- Standard_, G₄-Methanolic extract of G. acerosa, G₅-Methanolic extract of A. spicifera. All values are expressed as mean ± S.E.M (n=6); *Values are significantly different from normal control (G₁) at P < 0.01; **Values are significantly different from cancer control (G₂) at P < 0.01

DISCUSSION: The present study shows, the methanol extract of G. acerosa and A. spicifera significantly inhibited the cancer volume, packed cell volume, cancer cell (visible) count and haematological parameters in normal levels. Intraperitoneal inoculation of DAL cells in the mice produces huge cells of cancer count, which indicate development of cancer in the mice. The consistent criterion for judging the anticancer effect of methanol extract samples was reduction in viable cell count towards normal. It may be due to stimulate the immune cells activity ²³.

The reliable form for judging the value of any anticancer drug is the continuation of lifespan of the animal and decreased WBC count level. The reduce RBC or haemoglobin percentage in tumor bearing mice may be due to iron deficiency (anaemia) or due to haemolytic or myelopathic conditions ²⁴. Usually, myelo suppression and anaemia are the major problems encountered in cancer chemotherapy ²⁵. The treatment of G. acerosa and A. spicifera brought back the haemoglobin content, RBC and WBC count level is significantly. The haematological parameter results are clearly indicated the G. acerosa and A. spicifera possess protective action on the hemopoietic system. The previous report shows that the presence of cancer in the human body or in the experimental animals is known to affect may function of the liver.

The significantly elevated level of total cholesterol, TG, AST, ALT, ALP in serum of cancer inoculated animal indicated liver damage and defeat of functional reliability of cell membrane ²⁶. Haematological and biochemical observed in cancer bearing mice by Chondrococcus hornemanni and Spyridia fusiformis extract was effective in inhibiting the tumor growth in ascetic tumor model ²².

The biochemical assessment of DAL inoculated animals showed obvious changes indicating the toxic effect of the cancer. The normalization of these effects observed in the serum treated with methanol extracts red algaeG. acerosa and A. spicifera at a dose of 200mg/kg body weight supported the potent anticancer effects.

CONCLUSION: The present studies were showed a decrease in cancer cell count as a confirmatory evidence for protection against DAL bearing mice. Consequently increased life span was observed with methanol extracts treated mice. The haematological and biochemical variation observed in cancer bearing animals in this study may be due to the reduction level of cancer proliferation. Similarly, administration of red algae G. acerosa and A. spicifera extracts significantly alters this level in cancer-bearing animals. Thus, from the above observations on other parameters it was concluded that the red algae methanol extract of G. acerosa and A. spicifera possesses anticancer activity against DAL bearing mice.

ACKNOWLEDGEMENTS: The work was supported financially by grants of UGC-BSR Fellowship funded in Madurai Kamaraj University, Madurai. I thanks to Dr. N. Chidambaram, K.M. Pharmacy College in Madurai support for in-vivo study.

REFERENCES:

1. Xu H, Yao L, Sung H and Wu L: Chemical composition and antitumor activity of different polysaccharides from the roots Actinidia eriantha. Carbohydrate Polymer 2009; 78: 316-322.
2. Faulkner DJ: Marine natural products. Natural Product Reports2000; 17: 7–55.
3. Tziveleka  LA, Vagias C and Roussis V: Natural products with anti-HIV activity from marine organisms. Current Topics in Medicinal Chemistry2003; 3(13): 1512–1535.
4. Harada H, Noro T and Kamei Y: Selective antitumor activity in vitro from marine algae from Japan coast. Biological & Pharmaceutical Bulletin 1997; 20(5): 541–546.
5. Ito K and Hori K: Seaweed: Chemical composition and potential uses. Food Reviews International 1989; 5: 1101-1144.
6. Lahaye M: Marine algae as a source of dietary fibers: Determination of soluble and insoluble dietary fiber contents in some ‘sea vegetable’. Journal of the Science of Food and Agriculture1993; 54: 523-535.
7. Smit AJ: Medical and pharmaceutical uses of seaweeds natural products: A review. Journal of Applied Phycology2004; 16: 245-262.
8. O’Sullivan L, Murphy B, McLoughlin P, Duggan P, Lawlor PG, Hughes, H and Gardiner GE: Prebiotics from marine macroalgae for human and animal health application. Marine Drugs2010; 8: 2038-2064.
9. Paniagua-Michel J, Capa-Robles W, Olmos-Soto J and Gutierrez-Millan LE: The carotenogenesis pathway via the isoprenoid beta-carotene interference approach in a new strain of Dunaliella salina isolated from Baja California Mexico. Marine Drugs 2009; 7: 45-56.
10. Ianora A, Boersma M, Casotti R, Fontana A, Harder J, Hoffmann F, Pavia H, Potin P, Poulet SA and Toth G: New trends in marine chemical ecology. Estuaries Coasts 2006; 29: 531-551.
11. Deig EF, Ehresmann DW, Hatch MT and Riedlinger DJ: Inhibition of herpesvirus replication by marine algae extracts. Antimicrobial Agents and Chemotherapy 1974; 6: 524-525.
12. Jha RK and Zi-rong X: Biomedical compounds from marine organisms. Marine Drugs 2004; 2: 123-146.
13. Cragg GM and Newman DJ:  Plants as source of anticancer agents. Journal of Ethnopharmacology 2005; 100: 72-79.
14. Manilal A, Sujith S, Kiran GS, Selvin J and Shikar C: Cytotoxic potentials of red alga, laurencia brandenii collected from the Indian Coast. Global Journal of Pharmacology 2009; 3: 90-4.
15. Matsuhiro B, Conte AF, Damonte EB, Kolender AA, Matulewicz MC, Mejias EG, Pujol CA and  Zuniga EA:  Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales Rhodophyta). Carbohydrate Research 2005; 340: 2392-2402.
16. Li XC, Jacob MR, Ding Y, Agarwal AK, Smillie TJ, Khan SI, Nagle DJ, Ferreira D and Clark AM: Capisterones A and B, which enhance fluconazole activity in Saccharomyces cerevisiae, from the marine green alga, Penicillus capitatus. Journal of Natural Products 2006; 69: 542-546.
17. Wang YY, Khoo KH, Chen ST, Lin CC, Wong CH and Lin CH: Studies on the immunomodulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: Functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorganic & Medicinal Chemistry 2002; 10: 1057–62.
18. Bhosale SH, Nagle VL and Jagtap TG: Antifouling Potential of Some marine Organisms from India against Species of Bacillus and Pseudomonas. Marine Biotechnology 2002; 4: 111–118.
19. Unnikrishnan MC and Kuttan R: Tumor reducing and anticarcinogenic activity of selected spices. Cancer letters 1990; 51: 85-89.
20. Nagineni S, Santhanalakshi  R, Venketela V, Meenakshisundaram M and  Brindha P: Antitumorpotential of passiflora incarnatall against ehrlich ascites carcinoma. International journal of pharmacy and pharmaceutical sciences 2012; 4: 17- 20.
21. Sathiyanarayanan L, Shinnathambi A and Chindhambaranathan N: Anti carcinogenic activity of Leptadenia reticulate against Dalton’s ascetic lymphoma. Iranian journal of pharmacology and toxicology 2006; 6: 133-136.
22. Subbiah M and Sundaresan B: Antitumor activity of Chondrococcus hornemanni and Spyridia fusiformis on Dalton’s lymphoma ascites in mice.  Bangladesh Journal of Pharmacology 2012; l 7, 173-177.
23. Bhist M, Bist SS and Dhasmana DC: Biological response modifiers: current use and future propects in cancer therapy. Indian Journal of Cancer 2010; 47 (4): 443-451.
24. Steensma DP: Management of anemia in patients with cancer. Current Oncology Reports 2004; 6: 297-304.
25. Preston-Martin S, Pike MC, Ross RK and Jones PA: Increased cell division as cause of human cancer. Cancer Research 1990; 50: 7415-7422.
26. Zahan R, Alam, MB, Islam MS, Sarker GC, Chowdhury NS, Hosain SB, Mosaddik MA, Jesmin M and Haque ME: Anticancer activity of Alangium salvifolium flower in Ehrlich Ascites carcinoma bearing mice. International Journal of Cancer Research2011; 7: 254-262.
27. Janet Hoff, LVT and RLATG: Methods of Blood Collection in the Mouse. Lab Animal 2000; 29 (10): 47-53.
28. Faulkner DJ: Marine natural products. Natural Product Reports 2002; 19: 1-48.
    

    ---

    How to cite this article:

    Duraikannu K*, Shameem rani K, Anithajothi R , Umagowsalya G  and  Ramakritinan CM: In - vivo anticancer activity of red algae (Gelidiela acerosa and Acanthophora spicifera). Int J Pharm Sci Res2014; 5(8): 3347-52.doi: 10.13040/IJPSR.0975-8232.5(8).3347-52

    ---

    All © 2014 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.