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AIP Conference Proceedings 2092, 030015 (2019); https://doi.org/10.1063/1.5096719 2092, 030015

Phytochemical analysis and anticancer activity of seaweed Eucheuma Sp. against colon HCT-116 cells

Cite as: AIP Conference Proceedings 2092, 030015 (2019); https://doi.org/10.1063/1.5096719 Published Online: 09 April 2019

Priscilla Aya Maheswari Subroto, Ade Arsianti, Trivani Putri, and Elvira Lesmana

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Phytochemical Analysis and Anticancer Activity of Seaweed Eucheuma Sp. Against Colon HCT-116 Cells

Priscilla Aya Maheswari Subroto

^1,a)

, Ade Arsianti

^2,3,b)

, Trivani Putri

¹

, and Elvira Lesmana

¹

1Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia

2Department of Medical Chemistry, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia

3Drug Development Research Cluster, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia

Corresponding author: ^a)[email protected], ^b)[email protected], [email protected]

Abstract. Seaweed macroalgae is one of marine natural product which shows a potent anticancer activity. In this work, we developed seaweed Eucheuma Sp. collected from east Lombok beach, Nusa Tenggara Barat, Indonesia, as potential anti- colorectal cancer agents. Seaweed macroalgae Eucheuma Sp. were extracted in four kind of organic solvents of hexane, ethylacetate, chloroform and ethanol, respectively. The extracts were tested for its phytochemical composition, and were then analyzed by Thin Layer Chromatography (TLC). Furthermore, anticancer activity of hexane, ethyl acetate, chloroform and ethanol extracts of Eucheuma Sp. were examined against colon HCT-116 cells by MTT cell proliferation assay.

Phytochemical test of the extracts of Eucheuma Sp. showed positive result for metabolite of flavonoid, triterpenoid, alkaloid and tannin, whereas the TLC analysis revealed that the extracts containing five chemical compounds. Ethanol, hexane, chloroform, and ethyl acetate extracts of Eucheuma Sp. exhibited a good anticancer activity against colon HCT-116 cells with IC50 value ranging from 16.82 μg/mLto 40.45 μg/mL. The results suggest that seaweed Eucheuma Sp. could be further developed as a candidate for the new anti-colorectal cancer agents.

Keywords: anticancer activity, Eucheuma Sp., HCT-116 cells, MTT assay, phytochemistry, seaweed

INTRODUCTION

Cancer is one of the most common causes of death in the world. In 2015, cancer caused 8.8 million deaths in the world [1]. To be specific, colorectal cancer is one of the highest prevalent cancer, which is the third highest for man and second highest for woman. According to the early screening and health promotion division of Dharmais Hospital in Jakarta, Indonesia, colorectal cancer is one of the 10 cancer with highest prevalence [2]. Globocan data in 2012 showed that the incidence of colorectal cancer in Indonesia is 12.8 per 100.000 people [3].Colorectal cancer is the abnormal growth of cell in the large intestine and rectum. This high incidence is risked by the lifestyle of Indonesian people whose diet contains high amount of fats but low in fiber. This high incidence contributes in decreasing life expectancy of Indonesian people and their quality of life. In accordance to that, there needs to be prevention and treatment to decrease mortality caused by colorectal cancer [4].

Algae as a sea product drew the attention of researchers around the globe because it has been said that algae have good effect for health and can help the development of treatment of some diseases including cancer. Algae are commonly used for food material, beverages and pharmacological agents due to its high concentration in polysaccharides. Algae have the potential to be biological agents that have anticoagulant, antioxidant, and antitumor effect [5]. One of the algae that has the potential to be developed as antitumor agent is Eucheuma cottonii. It has been reported that Eucheuma cottonii from Salema Island in South Sulawesi, Indonesia has anti proliferative effect against

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breast cancer MCF-7 cells and colorectal cancer HCT-116 cells [6]. The methanol extract of Eucheuma cottonii from Eastern Malaysia also shows anti-tumor effect against cervix cancer [7]. The suppression of growth of cells is done by the induction of apoptosis.

In this research, the activity of Eucheuma cottonii (Figure 1a) and Eucheuma spinosum (Figure 1b) from Labuhan Baji, East Lombok beach, Nusa Tenggara Barat Indonesia were evaluated as an anti-colorectal cancer agent. We did phytochemical analysis to determine the secondary metabolite content of the species. Then, in vitro anticancer activity evaluation of Eucheuma cottonii and Eucheuma spinosum from Labuhan Haji, Lombok were conducted against colorectal cancer HCT-116 cells using MTT cell proliferation assay.

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FIGURE 1. (a) Eucheuma cottonii, (b) Eucheuma spinosum

METHODS

Extraction and Fractionation of Seaweed Samples

Eucheuma sp. was dried, grinded to powder form, and macerated in organic solvents such as hexane chloroform, ethyl acetate and ethanol two times for each solvent. Maceration was done in stages in which seaweed that had been soaked using 500 mLof hexane was filtered and the filtrate solution was collected while the residue was soaked again in the next solvent. The filtrate solution from each solvent was then concentrated using rotary evaporator, producing extracts of seaweed in hexane, chloroform, ethyl acetate, and ethanol. These extracts were then analyzed using thin layer chromatography to determine number of metabolites in the fraction [6].

Thin Layer Chromatography

Thin layer chromatography is a technic carried out to separate the mixture of compounds. This technic is done using a sheet of glass, plastic, or aluminum foil coated by a thin layer of absorbent material called the stationary phase.

Materials that can be used are silica gel, aluminum oxide or cellulose. The sample is applied to the plate and solvent, which is eluent, was drawn up the plane as the mobile phase. In this research, the mixture of chloroform (CHCl3)- methanol(CH3OH) in ratio 3:1 was used as a mobile phase, and silica gel as a stationary phase. Different compounds in the sample moved at different rates due to the difference in polarity against stationary phase and solubility in eluent.

After separation, the spots of chemical components were visualized under UV lamp with wavelenght of 254 nm and 366 nm and the Rf (retention factor) value of each spot was recorded

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The Rf value can be calculated using this following formula [8]:

Rf (retention factor) = Distance traveled by sample / distance traveled by solvent

Phytochemical Analysis

For this phytochemical analysis, sample as a form of paste was dissolved in its each solvent and used as solution for the test [6].

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Alkaloid Test

Samples in the form of paste were dissolved in each solvent for phytochemical analysis. 2 ml of sample solution was put in a porcelain cup and evaporated to get the residue. 5 ml of 2N chloric acid was then added to the sample.

The solution that had been mixed was divided into three tubes. First tube is for blanko, added with 2N chloric acid.

Second tube was added with 3 drops of Dragendorff and the third tube was added with 3 drops of Mayer’s reagent.

Orange precipitate formed in the second tube and yellow precipitate in the third tube showed positive sign of alkaloid [6].

Steroid and Triterpenoid Test

The Liebermann-Burchard reaction is used in steroid test. 2 ml of sample solution was evaporated in a porcelain cup. The residue was then dissolved in 0.5 ml of chloroform and 0.5 ml of anhydrate acetate acid. 2 ml of concentrated sulfuric acid was then added through the tube wall. Formation of a green ring shows positive result of steroid and formation of brown or purple ring at the border of sample shows positive result of triterpenoid [6].

Saponin Test

Sample is dissolved in its extract and 10 ml of the solution as added to a test tube and shook vertically for 10 seconds. The solution was then left to rest for 10 seconds. Positive result of saponin in the sample is showed by foam formed 1-10 cm thick for 10 minutes or more. The foam that withstand when given one drop of 2N of chloric acid confirms the result [6].

Flavonoid Test

1 ml of sample solution was dried, and then acetone and soft powder of boric acid and oxalate acid were added. It was then warmed with hot water and the residue was mixed with 10 ml ether solution. Yellow ray that will be seen shows positive result of flavonoid in 366 nm ultraviolet rays [6].

Tannin Test

1 ml of sample was reacted with solution of 10% iron (III) chloride. Dark blue or greenish black color in the solution shows positive result of tannin [6].

Glycoside Test

0.1 ml of sample solution was dried and dissolved in 5 ml of acetate acid anhydrate. Then 10 drops of concentrated sulfuric acid were added. Blue or green precipitation shows positive result of glycoside [6].

MTT Assay for Anticancer Activity

MTT assay was conducted to observe in vitro anticancer activity of Eucheuma sp. against colorectal cancer HCT- 116 cells. Samples were dissolved with their solvents and made with concentration as the following, 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, and 0.4 μg/ml. These samples were then reacted with the target cell. Following that, the target cells were incubated for 48 hours. After incubation, 20 μl of MTT solution with 5 mg/ml concentration in phosphate- buffered saline was added to the samples and incubated for 4 more hours. The samples were then centrifuged. Samples were then added with 200 μl of dimethyl sulfide. Finally, absorbance was read using 590 nm wave in the microplate reader. The inhibition rate is counted with this following formula [6]:

Inhibition rate (%) = (1 – (absorbance of the sample with treatment / absorbance of control)) x 100%

IC50 or the concentration of sample that cause 50% inhibition of the target cell, is determined using the Bliss assay.

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RESULTS AND DISCUSSION Phytochemical Composition

Eucheuma cottonii

Phytochemical test of Euchema cottonii extracs shows positive result for flavonoid, triterpenoid, alkaloid and tannin.

Table 1. Phytochemical analysis of extracts of Eucheuma cottonii

Metabolites Solvent

Hexane Ethyl acetate Chloroform Ethanol

Saponin - - - -

Flavonoid + + + +

Triterpenoid + + + +

Steroid - - - -

Alkaloid + + - -

Tannin - - - +

Glycoside - - - -

All extracts contain flavonoid and triterpenoid while only two of them contain alkaloid and one of them contain tannin, as shown in Table 1. The result means that concentration of flavonoid and triterpenoid is high in this sample.

Flavonoids have anti-allergic, anti-inflammatory, antioxidant, anti-mutagenic, and anti-carcinogenic activity [9]. The mechanisms of anticancer include increasing apoptosis rate, inhibit proliferation of cells, and inhibit lipid peroxidation [9]. Flavonoids also affect the immunological process in the development and progression of cancer [10].

Triterpenoids are metabolites of isopentenyl pyrophosphate. They have been used as pharmacological agents such as anti-inflammatory, analgesic, and antipyretic. Research showed that triterpenoids also have antioxidant, antimicrobial, antiviral and anticancer activity [11]. These results show that Eucheuma cottonii that contains these metabolites is a potent pharmacological agent including anti-cancer medicine.

Our previous research showed that Eucheuma cottonii that came from Parangtritis Beach, Yogyakarta showed positive result of flavonoid, steroid, and glycosides [6]. Different metabolites contained in these seaweeds are steroid and glycosides while flavonoids are contained in both. Difference of metabolites contained can be affected by the habitat of the seaweed [6].

Eucheuma spinosum

TABLE 2. Phytochemical analysis of extracts of Eucheuma spinosum

Metabolites Solvent

Hexane Ethyl acetate Chloroform Ethanol

Saponin - - - -

Flavonoid + + + +

Triterpenoid + + + -

Steroid - - - +

Alkaloid + + - -

Tannin - - - +

Glycoside - - - -

Table 2 shows that all extracts of Eucheuma spinosum contain flavonoid while only extract of hexane, ethyl acetate, and chloroform contains triterpenoid. Extract of ethanol contains steroid instead of triterpenoid. Only extract of hexane and ethyl acetate contains alkaloid while only extract of ethanol contains tannin. Flavonoid and triterpenoid, as explained above, have promising medicinal activity. In addition to that, steroid, alkaloid, and tannin also have some pharmacological activity. According to Ikeda T et al, steroidal glycosides showed good cytotoxic activity against PC- 12 and HCT-116 cell lines [12]. Alkaloid isolated different parts of Solanum pseudocapsicum exhibited good cytotoxic activity against many cells such as HT-29 colon cancer, A-549 lung carcinoma, HEp-2 HeLa cells, and Vero cell lines with cytotoxic concentration ranges from 0.39 to 5.65 μg/mL. Cytotoxic concentration is the concentration of the extract needed to cause the death of 50% targeted cells [13]. Lastly, tannin from Pimenta dioica leaves showed

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cytotoxic activity against solid tumor cancer cells [14]. To sum up, metabolites such as saponin, flavonoid, triterpenoid, steroid, alkaloid, tannin, and glycoside have showed that their anticancer activity is significant against cell lines. This can conclude that Eucheuma spinosum that contains these metabolites can be a potent anticancer agent.

Thin Layer Chromatography

The results of TLC analysis of E. spinosum and E. cottonii are displayed in Table 3 and Table 4, as follows.

TABLE 3. TLC analysis and Retention factor (Rf) of E. spinosum extracts

FIGURE 2. TLC Figures of Eucheuma spinosum extract TABLE 4. TLC analysis and Retention factor (Rf) of E. cottonii extracts

*Rf= Retention factor

FIGURE 3. TLC Figures of Eucheuma cottonii extract

Extract Rf value

1 2 3 4

Hexane 0.187 0.281 0.625 0.906

Ethyl acetate 0.281 0.625 0.906 -

Chloroform 0.187 0.312 0.593 0.937

Ethanol 0.218 0.281 0.656 -

Extract Rf value

1 2 3 4

Hexane 0.539 0.843 0.937 -

Ethyl acetate 0.906 - - -

Chloroform 0.218 0.687 0.843 0.937

Ethanol 0.187 0.968 - -

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As shown in Table 3 and Figure 2, the TLC analysis of hexane and chloroform extracts show that they have 4 spots of chemical components, while ethyl acetate and ethanol extracts only have 3 spots of chemical components.

Chloroform and hexane extracts tend to contain non-polar chemical compounds, while ethyl acetate extract contains semi polar chemical compounds and ethanol extract contains polar chemical compounds.

Based on the TLC analysis of E. cottonii extracts in Table 4 and Figure 3, non-polar extracts of hexane and chloroform have 3 and 4 spots of chemical components, respectively. Semi-polar extract of ethyl acetate has only one spot, and polar extract of ethanol has 2 spots of chemical components. Table 3 shows three Rf values which are the same between hexane and ethyl acetate extract, which are 0.281, 0.625 and 0.906. These same Rf values show that hexane and ethyl acetate extracts contain similar metabolites.

MTT Assay

Extracts of Eucheuma spinosum Against Colorectal Cancer HCT-116 Cells

FIGURE 4. The relationship between concentration of Eucheuma spinosum extracts and percentage inhibition of colorectal cancer HCT-116 cells

Figure 4 shows the percentage inhibition of HCT-116 cells when reacted to the samples. The range of percentage inhibition starts from 26.1 to 59.7%. The higher the percentage inhibition, the stronger the anti-proliferation activity.

The overall graph shows that percentage inhibition increases when concentration of extracts is higher. However, some values of percentage inhibition shows fluctuative pattern.

Extracts of Eucheuma cottonii Against Colorectal Cancer HCT-116 Cells

FIGURE 5. The relationship between concentration of Eucheuma cottonii extracts and percentage inhibition of colorectal cancer HCT-116 cells

Figure 5 shows percentage inhibition of HCT-116 cells. The percentage inhibition ranges from 16.5 to 61.8%. In general, the graph shows increasing pattern of percentage inhibition and directly proportional relation to concentration of extract. As the concentration of extract increases, the percentage inhibition mostly increases. However, in some concentration, value of percentage inhibition show fluctuation, such as in concentration 3.125 μg/mL. Percentage inhibition showed by Eucheuma spinosum and Eucheuma cottonii is similar as the highest value inhibition is around 60%.

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TABLE 5. IC50 values of Eucheuma spinosum and Eucheuma cottonii extracts against colorectal HCT-116 cells

Extract IC50 value (μg/mL)

Eucheuma spinosum Eucheuma cottonii

Ethanol 37.59 28.45

Ethyl acetate 16.82 24.40

Hexane 22.84 24.83

Chloroform 26.87 40.45

Table 5 shows the IC50 values of HCT-116 cells. The smaller the value, the stronger the anti-cancer activity is.

These results revealed that Eucheuma spinosum and Eucheuma cottonii extracts are moderately active for anticancer with IC50 < 100 μg/mL. According to Atjanasuppat et al., anticancer activity level of the extracts were categorized into four groups: IC50 < 20 μg/mL is considered as active, IC50>20-100 μg/mL considered as moderately active, value IC50 >100-1000 μg/mL considered as weak, and IC50 > 1000 μg/mL considered as inactive compounds [15]. IC50 was calculated using graph ploted with concentration of extract in the x-axis and percentage inhibition in y-axis.

Previous study on Eucheuma cottonii from Parangtritis beach in Yogyakarta also shows active activity as anticancer against HCT-116 cells, with IC50 value as the following, 65.3 μg/mL for ethanol extract, 99.3 μg/mL for chloroform extract, 43.0 μg/mL for hexane extract, and 21.4 μg/mL for ethyl acetate extract [6]. These values show same order from the best antiproliferation activity to worst, which starts from ethyl acetate extract, followed by hexane, ethanol, and chloroform extract has the highest IC50 value.

Table 5 shows that choloroform extract of Eucheuma spinosum and Eucheuma cottonii have similar IC50 value, which are 26.87 and 26.46 μg/mL. IC50 value of the ethyl acetate extract is slightly lower in Eucheuma spinosum while IC50 value of hexane extracts is slightly higher in Eucheuma spinosum compared to Eucheuma cottonii. However, IC50 in ethanol extract of Eucheuma spinosum is significantly higher than in Eucheuma cottonii. Among all samples, the best inhibition activity to the targeted cells has shown by ethyl acetate extract of Eucheuma spinosum, as it has the lowest value of IC50, which is 16.82 μg/mL.

Extract of ethyl acetate and hexane has two lowest value of IC50 in both Eucheuma species. As explained in Table 1 and Table 2 above, hexane and ethyl acetate extracts have similar metabolites, which are flavonoid, triterpenoid and tannin. Chloroform and ethanol extract shows negative result of alkaloid. The difference in IC50 value can be caused by the presence of alkaloid. As explained above, alkaloid has shown good anticancer activity against many cancer cells [13]. Table 3 above shows TLC analysis of Eucheuma spinosum extract. This result shows similar Rf values of hexane and ethyl acetate, which suggesting that hexane and ethyl acetate extract contain similar metabolites, thus can have also similar antiproliferative activity.

Overall, phytochemical analysis, thin layer chromatography and the result of MTT assay shows that ethyl acetate extract of Eucheuma spinosum is the most potent anti-colorectal cancer agent on HCT-116 cells.

CONCLUSION

Seaweed Eucheuma spinosum and Eucheuma cottonii which demonstrated anticancer activity against HCT-116 cells with IC50 value ranging from 16.82 to 40.45 μg/mL, are potential to be developed as anti-colorectal cancer agents.

Ethyl acetate extract of Eucheuma spinosum showed the strongest anti-proliferative activity against colon HCT-116 cells compared to other extracts, so it can be further developed as one of the most potential anti-colorectal cancer drug.

ACKNOWLEDGMENTS

Our gratitude should greatly be expressed to Directorate of research and Public Service University of Indonesia for the research grant PITTA (Publikasi Internasional Terindeks Scopus Untuk Tugas Akhir Mahasiswa) research grant fiscal year 2018, contract number 2040/UN2.R3.1/HKP.05.00/2018.

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