Anticancer Effect of Cymodocea Serrulata Sea grass Crude Extract against Breast Cancer Cell Line

Nandini Palanivel, P Sivaperumal^*, D Ezhilarasan, Elumalai and Lakshmi T

^*Correspondence: P Sivaperumal, Department of Pharmacology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Chenna, India, Email:

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Introduction

Sea grass is a plant that lives in tidal areas, possesses unique secondary metabolites, which have roles as anticancer bioactive compounds. The method used in the experimental method which continues the extraction towards sea grass leaves using n-hexane (non-polar), ethyl acetate (semi polar) and ethanol (polar) [1]. Extract obtained was analyzed for its anticancer activity by using some cells. Breast cell lethality by semi polar extract, polar extract and doxorubicin are 48.11%, 15.32 % and 48.75%, respectively. Ethyl acetic crude extract of fresh sea grass contains phytochemical compounds of alkaloid, terpenoids, polyphenol and flavonoid. Cancer disease has been a major health problem in the world and also in Indonesia. Based on WHO data on 2013, cancer incidence increased from 12.7 million cases (2008) to 14.1 million cases (2012). It is predicted that in 2030, cancer incidence could reach up to 26 million people, in which 17 million people could die because of cancer, particularly in underdeveloped countries and developing countries [2,3].

Several treatments to prevent and cure cancer have been done, such as radiotherapy, chemotherapy, or using synthetic medicine. In general, anticancer therapy has been felt to be sufficient to give good results, but it has side effects and the costs are quite. Radiotherapy and chemotherapy have limitations [4]. The effectiveness of rays used for radiotherapy will decrease along with the increase of tumor size, meanwhile the increase of dosage given to the level exceeds its toxicity level will give effect to normal human tissues and organs. The use of chemical drugs such as chemotherapy kill not only the tumor cells but also damage blood cells, which causes the decrease in immunity or even death, resulting from complications due to side effects of drugs. Several cervical cancer cells have become resistant towards treatment using radiation and chemotherapy [4,5]. Therefore, many people have turned to traditional medicines that are considered more safe and economical, because they use natural ingredients. Natural ingredients contain several active compounds which give pharmacological effects. In general, those active compounds are secondary metabolites. Secondary metabolites have been known as sources of medical therapy, for example as antibacterial and anticancer medicines, etc. Majority of secondary metabolites are synthesized by organisms to adapt to their environment, thus, the search of secondary metabolites that are able to act as anticancer bioactive mainly focused on organisms that live in extreme environmental condition. One of the extreme environments is in a tidal area. Organisms which live in the tidal area and have been reported to contain anticancer bioactive compounds are sponges and their entophytic microbes, Ascidia (sea squirt) [6]. Microbes that associated with sea biota, such as marine derived fungi, mangroves and their entophytic microbes, and sea grass and its endophytic microbes [7]. Symbiotic or epiphytic bacteria on sea grass, a term for bacterial colony that lives, grows and associates with sea grass, has been reported to contain similar bioactive compounds to its symbiotic. Sea grass is a hydrophyte that lives in tropical or subtropical tidal coastal areas. Around the world, there are 52 types of sea grass, 15 of which are present in Indonesia. Several researches showed that sea grass has potential as antioxidant, anticancer and antibacterial agents. Compounds in sea grass that are suspected to be able to inhibit cancer cell proliferation and inactivate pathogenic bacteria are flavonoid, saponin, steroid, terpenes, tannin and alkaloid [8]. It is stated that sea grass Enhalus acoroides contains stigmasterol, sitosterol and alkaloid. Sea grass Thalassia testudinium contains glycosides and phenol compounds which are potential to act as antifungal and anticancer, while seagrass Halodule uninervis contains steroid compounds which acts as antibacterial and anticancer [2,3,9,10]. It have stated that crude extract from seagrass Thalassia hemprichii,Cymodocea serrulata acoroides had high phenolic content. Moreover, reported the cytotoxicity of crude extract from sea grass [11]. Enhalus acoroides Cymodocea serrulata toward Artemia salina concentration of 404.88 ppm and 136.398 ppm, respectively. The highest phenolic content is on the leaves part. One of seagrass that can be found in tidal coastal areas in Indonesia is Cymodocea serrulata [11,12]. Our team has extensive knowledge and research experience that has translated into high quality publications [13-25]. The types of sea grass were reported to contain anticancer bioactive compounds, another research to determine the potential of sea grass and Cymodocea serrulata as a source of anticancer bioactive compounds should also be conducted. Therefore, the aim of this research was to determine the potential of sea grass Cymodocea serrulata fresh leaves extract as an anticancer agent.

Materials and Methods

Preparation of Extract

A 10g of dried powdered sea grass samples were mixed with 100ml of methanol/Ethanol (V/V) and allowed to place for 24 hours at ambient temperature. Then the mixture was passing through what man filter paper (No. 4) then the filtrate was centrifuged at 3000rpm for 10min and further filtered by 0.45μm syringe micro filter. At last, the solvents are evaporated via a vacuum rotary evaporator until samples obtain in powder form. Then the sample was stored in a shadowy aluminium container at 4ºC for further analysis.

MTT Assay

The proliferation of MCF-7 cells was assessed by MTT assay. MCF-7 cells were plated in 48 well plates at a concentration of 2x10 4 cells/well 24 hours after plating, cells were washed twice with 500μl of serum-free medium and starved by incubating the cells in serum-free medium for 3 hours at 37ºC. After starvation, cells were treated with sea grass extract in different concentrations for 24 hours. At the end of treatment, the medium from control and Sea grass extract treated cells were discarded and 200μl of MTT containing DMEM (0.5mg/ml) was added to each well. The cells were then incubated for 4h at 37ºC in the CO2 incubator.

The MTT containing medium was then discarded and the cells were washed with 1x PBS. The crystals were then dissolved by adding 200μl of solubilisation solution and this was mixed properly by pipetting up and down. Then the formed crystals were dissolved in dimethylsulfoxide (200μl) and incubated in dark for an hour. Then the intensity of the colour developed was assayed using a Micro ELISA plate reader at 570 nm. The number of viable cells was expressed as the percentage of control cells cultured in serum-free medium. Cell viability in the control medium without any treatment was represented as 100%. The cell viability is calculated using the formula: % cell viability=[A570 nm of treated cells/A570 nm of control cells]×100.

Results and Discussion

The crude extract obtained from Cymodocea serrulata showed potential anticancer activities against Breast cancer cell lines. The cancer forms in either the lobules or the ducts of the breast. Cancer can also occur in the fatty tissue or the fibrous connective tissue within your breast. Cymodocea serrulata (Family Cymodoceaceae).

Figure 1: The picture represent the sea grass Cymodocea serrulata present under the sea.

Figure 2:The picture represents the single plant of the Cymodocea serrulata.

Figure 3:The picture represents the dried sea grass samples.

Figure 4:The picture represents the crude extracts.

Figure 5AB:5AB: The picture represent about the control of the breast cancer cell line and treated with sea grass Cymodocea Serrulata crude extracts.

Figure 6:The % of cell viability against different crude extract concentration.

The Results showed that non-polar extract has no anticancer activity, while semi polar and polar extract showed their potential as an anticancer agent. Breast cell lethality level by semi polar extract was higher than polar extract, but not significantly different with cancer medicine doxorubicin [2]. The extract of C. serrulata reveals the presence of phytocompounds that are biologically active. According to the chromatogram obtained by GCMS ethanol extract of C. serrulata consists of palmitic acid, myristic acid, and pentadecanoic acid as a major component. They may be produced by the plant defenses itself from stress as secondary metabolites. These cytoprotectants proved to possess pharmacological activity in a similar way as synthetic drugs. The palmitic acid reported possessing anticancer activity, antimicrobial, and nematicide activity. The palmitic acid increases the number of probiotic bacteria in the gut; thus, they are involved in the development of the intestine. It is required in the biosynthesis of lung lecithin, which is related to fetal maturation as well as it has been reported that presence of palmitic acid in the Nigerian meal can partly be related to the low incidence of respiratory disease. Palmitic acid reported inhibiting human hepatoma cell growth in a dose dependent and time-dependent manner. Thus, they possess anticancer. One of seagrass that can be found in tidal coastal areas in Indonesia is Cymodocea serrulata. Since other types of seagrass were reported to contain anticancer bioactive compounds, another research to determine the potential of seagrass Cymodocea serrulata as a source of anticancer bioactive compounds should also be conducted [26].

Cymodocea serrulata had high phenolic content. Moreover, reported the cytotoxicity of crude extract from seagrass Enhalus acoroides Cymodocea serrulata toward Artemia salina concentration of 404.88 ppm and 136.398 ppm, respectively.it is stated that the highest phenolic content is on the leaves part. Sea grasses were known to possess remarkable bioactivities. Cymodocea serrulata exhibited more potent antioxidant activity than ascorbic acid, Gallic acid. Another finding of seaweed Gracilaria tenuistipitata was antioxidant inactive. The water extracts of seaweed Gracilaria tenuistipitata were proved to contain bioactive compounds such as phenolic, flavonoid, and ascorbic acid, however their DPPH free radical scavenging activity was relatively weak, with 63.37% DPPH free radicals scavenged by 4 mg/ml-1 extract [27]. From the test results of cytotoxic, antimicrobial and antioxidant activities of 57 seaweed and sea grass extracts in this study, it could be concluded that samples extracted from closely taxonomic species were not completely homogeneous in biological activities. The reason may result from the divergence in geographic distribution, ages of seaweed and sea grass samples. We can further study using more volume of samples and this can be done as an antimicrobial and anti-cytotoxicity study against breast cancer cell line in future. WOS cite [28-44]

Conclusion

The anticancer effect of Cymodocea serrulata crude extract against breast cancer cell line is found. Therefore, many people have turned to traditional medicines that are considered more safe and economical, because they use natural ingredients. Natural ingredients contain several active compounds which give pharmacological effects. In general, those active compounds are secondary metabolites. Secondary metabolites have been known as sources of medical therapy, for example as antibacterial and anticancer medicines, etc.

Acknowledgement

We would like to thank Saveetha Dental College for providing the opportunity to conduct this study.

Conflict of Interest

There is no conflict of interest.

Source of Funding

Saveetha Dental College and Hospital, Chennai and Palanivel group of companies, Dindigul, Tamil Nadu.

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