AIP Conference Proceedings 2092, 030017 (2019); https://doi.org/10.1063/1.5096721 2092, 030017
© 2019 Author(s).
Phytochemistry test and cytotoxic activity of macroalgae Eucheuma Sp. as a growth inhibitor of breast MCF-7 cancer cells
Cite as: AIP Conference Proceedings 2092, 030017 (2019); https://doi.org/10.1063/1.5096721 Published Online: 09 April 2019
Elvira Lesmana, Ade Arsianti, Trivani Putri, and Priscilla Aya Maheswari Subroto
ARTICLES YOU MAY BE INTERESTED IN
Phytochemical analysis and antioxidant activity of marine algae Eucheuma Sp.
AIP Conference Proceedings 2092, 030016 (2019); https://doi.org/10.1063/1.5096720 Phytochemical analysis and anticancer activity of seaweed Eucheuma Sp. against colon HCT-116 cells
AIP Conference Proceedings 2092, 030015 (2019); https://doi.org/10.1063/1.5096719
Liver and kidney function analysis on mice given ethyl acetate fraction of Garcinia mangostana as potential colorectal cancer therapeutic agent
AIP Conference Proceedings 2092, 030020 (2019); https://doi.org/10.1063/1.5096724
Phytochemistry Test and Cytotoxic Activity of Macroalgae Eucheuma sp . as a Growth Inhibitor of Breast MCF-7
Cancer Cells
Elvira Lesmana
1,a), Ade Arsianti
2,3,b), Trivani Putri
1,c)and Priscilla Aya Maheswari Subroto
1,d)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)elvira.lesmana@ui.ac.id, b)ade.arsianti@ui.ac.id, c)trivani.putri@ui.ac.id,
d)priscilla.aya@ui.ac.id
Abstract. In this research, we develop Indonesia marine macroalgae of Eucheuma sp. as an anti-breast cancer agent.
Macroalgae Eucheuma sp. which was collected from Labuhan Aji beach, Lombok, Nusa Tenggara Barat, Indonesia, were extracted into four different organic solvents, which are n-hexane, ethyl acetate, chloroform and ethanol. The extracts were subjected to Phytochemistry test and were analyzed by Thin Layer Chromatography (TLC). Subsequently, cytotoxic activity of n-hexane, chloroform, ethyl acetate, and ethanol extracts of Eucheuma sp. were evaluated as a growth inhibitor for breast MCF-7 cancer cells. Based on the phytochemical analysis, the extracts of Eucheuma sp. containing secondary metabolite of flavonoid, triterpenoid, alkaloid and tannin, while TLC analysis indicated that the extracts were composed of one to four chemical compounds. Extracts of Eucheuma sp. demonstrated a strong cytotoxic activity against breast MCF- 7 cancer cells. The results suggesting that macroalgae Eucheuma sp. should be developed as a promising anti-breast cancer agent.
Keywords: breast MCF-7 cells, cytotoxic activity, Eucheuma sp., macroalgae, MTT assay, phytochemistry
INTRODUCTION
Cancer signifies a rapid and uncontrollable growth and deployment of cells. Breast cancer, as the most common malignancy happens in women, turns out to be the leading cause of death by cancer in women globally, especially in Indonesia. Around 1.67 million new cases were diagnosed in 2015, accounts for around 25% of cancer prevalence worldwide. In 2015, 570.000 death or 15% of death in women worldwide were caused by breast cancer. Nevertheless, the death incidence of breast cancer stood in second place after lung cancer, 12.9% out of 8.2 million deaths caused by cancer worldwide in 2012. This prevalence rate is estimated to increase annually until reaching 23.6 million new cases in 2030 [1,2,4,5].
Thus, the development of research and technology regarding this issue has become a major concern of the world.
Scientists have found a lot of innovation in finding ways to decrease this incidence rate as well as to find the best way to cure this rate of prevalence effectively. Hence, we are still in need of innovations and further research to help us cope up with this problem. Depart from a variety of ways to resolve this problem or at least decrease its incidence, marine biota is one in the top listed substance that yields hope due to its benefits for nutritional support and health enhancer. However, the nutritional composition and details regarding different kind of algae need to be traced further.
With all the possibilities in finding innovations regarding marine algae, experiments, as well as collaborations in algae conservation, continue to increase [3].
In Asia, algae are more often consumed and based on research, Asian has a lower incidence rate for breast cancer due to its apoptosis activity detected in a cancer cell with algae exposure. In this study, phytochemistry analysis is conducted to specify its secondary metabolite content of Eucheuma cottoni and Eucheuma spinosum from Labuan Aji beach, Lombok, Nusa Tenggara Barat, Indonesia, followed by in vitro anticancer activity test to the two marine algae extract towards MCF-7 cancer cells by MTT cell proliferation assay [1].
(a) (b)
FIGURE 1. Eucheuma cottonii (a) and Eucheuma spinosum (b)
METHODS
Extraction and Fractionation Procedure of Macroalgae Samples
The process was started with collecting samples from Eucheuma spinosum and Eucheuma cottoni, proceed with drying, grinding and maceration in n-hexane (first solvent), ethyl acetate, chloroform and ethanol in a sealed container one by one, twice per solvent, respectively. The soaking period lasted for 24 hours per cycle with 500 mL solvent involved. Those extracts were then concentrated with a rotary evaporator, generating each n-hexane, ethyl acetate, chloroform and ethanol extracts before thereafter being analyzed with thin layer chromatography to discover its phytochemistry component in the extracts [6].
Thin Layer Chromatography Procedure
Thin Layer Chromatography (TLC) procedure is conducted to separate mixture compounds based on its polarity.
This procedure starts with a dripping of 1.5cm base plate which made of glass, plastic or aluminum foil overlaid by silica gel or cellulose with the sample. The thin layer of adsorbent overlaying the plate is known as a static phase.
Afterward, solvent or solvent mixture would be flowed up through the plate based on its capillarity (known as dynamic phase). Different analytes will flow up the TLC plate at different rates so that component separation occurs. Finally, the distance of the stain from the solvent to the sample will appear and is stated as Retention Factor (RF). The quotation for RF is [6]:
solvent by reached distance
sample by reached distance
RF
1)Phytochemistry
A phytochemistry test was carried out to identify the metabolite components of Eucheuma cottoni and Eucheuma spinosum extracts which were concentrated in n-hexane, chloroform, ethanol and ethyl acetate. Several tests done to
figure out seaweeds components were saponin test, flavonoid test, triterpenoid test, steroid test, alkaloid test, tannin test and glycoside test [6].
Saponin Test
Saponin test was done by shaking the sample tube for 10 minutes and left it vertically until 10 seconds. If a form of 1-10 cm foam was seen for less than or exactly 10 minutes and lasted with an addition of one drop HCl 2N, the result is positive [6].
Flavonoid Test
Flavonoid test was conducted by evaporating the test solution until it dried. The rest of the solution was then dampened by acetone and added with fine boric acid and oxalate. Then, careful heating and mixing with 10 mL ether were conducted, the result is positive if an intensive yellow fluorescent solution is found when observed with 366 nm UV rays [6,9].
Triterpenoid and Steroid Test
This test was carried out with Liebermann-Burchard reaction, started with 2 mL test solution evaporated in a porcelain container. Next, the residue was dissolved in 0.5 mL chloroform, added by 0.5 mL anhydrate acetic acid and also sulfuric acid through its wall. The test is considered positive if violet or brown ring formation on the limits of the solution. The steroid is interpreted as positive if the blue-green ring is seen [6,10,13].
Alkaloid Test
In Alkaloid test, approximately 2 mL of solution test was placed in the porcelain container and dried until the residue was made. Thereafter, 5 mL of HCl 2N was added and the solution was separated into three different containers in which, nothing, HCl 2N and three drops of Dragendorff reagent are added, respectively. A positive result will be shown if an orange precipitation is seen in the second tube and a yellow precipitation is seen in the third tube [6, 11].
Tannin Test
This Phytochemistry Tannin test was conducted by reacting a 1 mL solution test with 10% FeCl3. Positive is measured by the existence of dark blue and greenish black color [6, 12].
Glycoside Test
The Phytochemistry Glycoside test was done by evaporating 0.1 mL solution in a water bath, proceeded with dissolving the solution with anhydrate acetic acid and added by 10 drops of concentrated sulfuric acid. The result is defined glycoside positive if a blue or green product is found [6].
In vitro Cytotoxic Test of Macroalgae Extracts with MTT Assay
MTT Assay is aimed to find out the inhibitory effect from Eucheuma sp. extracts towards MCF-7 cancer cells.
Extracts of both samples were dissolved and added to target cell MCF-7 with final concentrations of 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, and 0.4 μg/ml. After that, cells were then incubated for 48 hours and added by 20 μg MTT 5 mg/mL at phosphate-buffered saline to samples then proceeded to incubation for another 4 hours. The result was centrifuged and separated from the medium. Then, the solution was added by 200 μl dimethyl sulfoxide to every container in order to dissolve the bluish purple precipitate. Subsequently, absorbance rate was read with 590 nm wave on a microplate reader. IC50 (50% inhibitory concentration) is enumerated with bliss assay [6].
Inhibition rate is counted with:
% group 100
control from
absorbance
group treatment from
absorbance 1
= rate(%)
Inhibition x
2)RESULTS AND DISCUSSION Phytochemistry Configuration
Through the phytochemical test analyzed, we figured out that both Eucheuma cottoni and Eucheuma spinosum contains four kinds of secondary metabolites, which are flavonoid, triterpenoid, alkaloid and tannin, in contrary showing negative outcome for saponin and glycoside as shown in Table 1 and Table 2.
TABLE 1: Phytochemistry configuration of Eucheuma cottonii extracts
No Secondary Metabolite n-hexane ethyl acetate chloroform ethanol
1 Saponin - - - -
2 Flavonoid + + + +
3 Triterpenoid and Steroid + + + +
4 Alkaloid + + - -
5 Tannin - - - +
6 Glycoside - - - -
TABLE 2: Phytochemistry configuration of Eucheuma spinosum extracts
No Secondary Metabolite n-hexane ethyl acetate chloroform ethanol
1 Saponin - - - -
2 Flavonoid + + + +
3 Triterpenoid /Steroid + + + - /+
4 Alkaloid + - - -
5 Tannin - + - -
6 Glycoside - - - -
By analyzing secondary metabolites from two different types of Eucheuma sp., it turns out that both Eucheuma cottonii and Eucheuma spinosum extracts all contain Flavonoid and Triterpenoid, except ethanol extract of Eucheuma spinosum (steroid positive in ethanol extract of Eucheuma spinosum). Nowadays, the flavonoid is known as an indispensable ingredient for a variety of pharmaceutical and medicinal applications. It roles as an antioxidant, anti- inflammatory, antimutagenic, anticarcinogenic component that also has a capacity to modulate the functions of a key cellular enzyme. Flavonoid anticarcinogenic effect might further be used for cancer prevention since it has chemo- preventive properties and able to induce apoptosis by holding back cell cycle, regulate the metabolism of carcinogen and its ontogenesis expression. Ubiquitously found in plants, on the other hand, Triterpenoid is also prompt an anticancer, anti-inflammatory, and anti-proliferative as well as pro-apoptotic effect in both in vitro and in vivo test. It regulates various kind of transcription and growth factors, cytokines pro-inflammatory and cell proliferation’s intracellular signaling pathways which are apoptosis and angiogenesis of tumor [9, 10, 13].
Besides flavonoid and triterpenoid, Alkaloid and Tannin are also found in both seaweeds. However, alkaloid can be found in two out of four solvents in Eucheuma cottoni and only in one out of four solvents in Eucheuma spinosum.
The alkaloid is also one promising secondary metabolite, but as an anticancer, however, some clinical trials found alkaloid to be ineffective. Other research using alkaloid is focusing more interest for its antineoplastic activity, but this study is still being conducted as it shows potentials. It works by targeting protein synthesis or DNA replication on tumor cells that leads to apoptosis of the neoplastic cells. Last finding, tannin, which configure both seaweeds in one solvent and has already shown a high potency as an anticancer for breast cancer, could be discovered in one out of four solvent both in Eucheuma cottonii and Eucheuma spinosum [11, 12].
Thin Layer Chromatography
Thin Layer Chromatography (TLC) results in analysis and Retention Factor (RF) from Eucheuma spinosum and Eucheuma cottonii are shown below in Table 3 and Table 4, respectively.
Table 3: TLC Analysis, RF from Eucheuma cottonii extracts
Extract Rf value
1 2 3 4
n-Hexane 0.593 0.843 0.937 -
ethyl acetate 0.906 - - -
chloroform 0.218 0.687 0.843 0.937
ethanol 0.188 0.968 - -
Table 4: TLC Analysis, RF from Eucheuma spinosum extracts
Extract Rf value
1 2 3 4
n-Hexane 0.188 0.281 0.625 0.906
ethyl acetate 0.281 0.675 0.906 -
chloroform 0.188 0.313 0.593 0.938
ethanol 0.219 0.281 0.656 -
The values from Table 3 and Table 4 shows a Retention Factor of the components in each extract. Furthermore, this test also able to classify composition based on its polarities with higher retention factor represent a less polar compound as well as determining the number of compounds towards each extract (one value represents one compound). Looking at Table 3, chloroform has the most number of bands (four bands), followed by n-hexane with three bands, ethanol with two bands and ethyl acetate with only one band. Both chloroform and n-hexane have more similar components since they possess two exactly same RF value. In Table 4, we could see that n-hexane and chloroform composed of four different compounds while ethyl acetate and ethanol only composed of three different compounds [14].
MTT Assay
Eucheuma cottonii Extracts and Its Inhibition towards Breast Cancer
FIGURE 2. Correlation between concentration of Eucheuma cottonii extracts and percent of inhibition to MCF-7 cancer cells Figure 2 exhibits the ability of Eucheuma cottonii extracts to hamper the growth of MCF-7 breast cancer cells.
The fluctuating patterns ranging from 26.3% until 64.1% shows us that the capacities of the extracts to act as inhibitors increase as the extracts get more concentrated until it reaches the concentration of 50 μl/mL (dose-dependent), in which the most percentage inhibition is seen in all four extracts and decreasing after further increase.
0 10 20 30 40 50 60 70
1.5 3.125 6.25 12.5 25 50 100 200
Percentage Inhibition (%)
Extracts Concentration (μg/ml) n-hexane ethyl acetate chloroform ethanol
Eucheuma spinosum Extracts and Its Inhibition towards Breast Cancer
FIGURE 3. Correlation between concentration of Eucheuma spinosum extracts and percent of inhibition to MCF-7 cancer cells.
As seen in Fig. 3, we could clearly interpret how Eucheuma spinosum extracts correlate with percentage inhibition of MCF-7 breast cancer cells. There is a significant increasing trend in all four extracts with its peak seen to inhibit the most in 50 μl/mL concentration. The percentage inhibition is ranging from -0.5% to 59.9%.
Table 5: IC50 values of Eucheuma sp. extracts towards MCF-7 breast cancer cells
No Solvent IC50 value (μg/mL)
E. cottonii
IC50 value (μg/mL) E. spinosum
1 n-hexane 14.589 33.841
2 ethyl acetate 15.987 37.328
3 chloroform 15.924 51.981
4 ethanol 18.449 41.523
Table 5 depicted the requirements of Eucheuma sp. extracts needed to do a 50% inhibition of MCF-7 breast cancer cells growth. Anticancer activity is composed of four group, < 20 μg/mL accounted as active compound, 20-100 μg/mL accounted as a moderately active compound, 100-1000 μg/mL accounted as weak compound and more than 1000 μg/mL accounted as an inactive compound in relation to its anticancer activity. The lower the IC50 value of a compound is, the more potent anticancer activity of one solvent. From Table 5 it is shown that Eucheuma cottonii in all extracts have a lower score than Eucheuma spinosum. Eucheuma cottoni which its IC50 value spread below 20 μg/mL is considered active compound and Eucheuma spinosum which its IC50 value spread from 38.422 μg/mL to 57.075 μg/mL are considered moderately active compound. It means, those two seaweeds only need a small number of extracts (especially n-hexane) to be able to inhibit 50% of MCF-7 breast cancer cells, especially for Eucheuma cottonii which has a greater effect in less amount. These incredible results suggest Eucheuma cottonii and Eucheuma spinosum in being used as a potential anticancer drug for breast cancer [15].
CONCLUSION
Macroalgae Eucheuma cottoni and Eucheuma spinosum, sequentially exhibit a promising anticancer activity against MCF-7 breast cancer cells to be evolved as potential anticancer for breast cancer based on its inhibition rate
-10 0 10 20 30 40 50 60 70
1.5 3.125 6.25 12.5 25 50 100 200
Percentage Inhibition (%)
Extracts Concentration (μg/ml) n-hexane ethyl acetate chloroform ethanol
and IC50 values ranging from 14.589 μg/mL to 51.981 μg/mL, especially Eucheuma cottoni (with all IC50 in all extracts scored below 20 μg/mL). Further drug development research with both ingredients are suggested to be conducted.
ACKNOWLEDGMENT
We are granting our gratitude towards Faculty of Medicine Universitas Indonesia and Directorate of Research and Public Service Universitas Indonesia for Publikasi Internasional Terindeks Scopus untuk Tugas Akhir Mahasiswa (PITTA) research grant fiscal year 2018, contract number 2040/UN2.R3.1/HKP.05.00/2018.
REFERENCES
1. M.L. Wells, P. Potin, J.S. Craigie, J.A. Raven, S.S. Merchant, K.E. Helliwell, A.G. Smith, M.E. Camire, S.H.
Brawley, J Appl Phycol 29(2), 949–82 (2017).
2. World Health Organization - Cancer Country Profiles, [Internet]. World Health Organization; 2014 [cited 15 January 2018]. Available from: http://www.who.int/cancer/country-profiles/idn_en.pdf
3. H. Funahashi, T. Imai, T. Mase, M. Sekiya, K. Yokoi, H. Hayashi, A. Shibata, T. Hayashi, M. Nishikawa, N.
Suda, Y. Hibi, Y. Mizuno, K. Tsukamura, A. Hayakawa, S. Tanuma, Jpn J Cancer Res 92(5), 483-487 (2001).
4. WHO | Breast cancer [Internet]. WHO. [cited 2018 Mar 15]. Available from:
http://www.who.int/cancer/prevention/diagnosis-screening/breast-cancer/en/
5. Kementerian Kesehatan Republik Indonesia. Oktober 2016 Bulan Peduli Kanker Payudara. Jakarta: Pusat Data dan Informasi; 2016 p. 2-3.
6. A. Arsianti, L.K. Wibisono, N.N. Azizah, R. Putrianingsih, T. Murniasih, A. Rasyid, F. Fadilah, K. Suid, F.
Yazid, R. Pangestuti, Asian J Pharm Clin Res 9(6), 115-119 (2016).
7. Dried-Eucheuma-spinosum-MC-38-below-Impurity.jpeg_350x350.jpeg (JPEG Image, 350 × 262 pixels) [Internet]. [cited 2018 May 10]. Available from: https://sc02.alicdn.com/kf/UT8kIKXXshaXXaFIFrXs/Dried- Eucheuma-spinosum-MC-38-below-Impurity.jpeg_350x350.jpeg
8. 15601.jpeg (JPEG Image, 259 × 194 pixels) [Internet]. [cited 2018 May 10]. Available from:
https://id.all.biz/img/id/catalog/15601.jpeg
9. A.N. Panche, A.D. Diwan, S.R. Chandra, J Nutri Sci 5, e47 (2016).
10. S. Sawai, K. Saito, Front Plant Sci 2, 25 (2011).
11. T. Isah, Pharmacogn Rev 10(20), 90–99 (2016).
12. C.J. Bridgeman, T.U. Nguyen, V. Kishore, J Biomater Sci Polym Ed 29(4), 412–27 (2018).
13. J.M. Patlolla, C.V. Rao, Curr Pharm Biotechnol 13(1), 147-155 (2012).
14. S. Kumar, K. Jyotirmayee, M. Sarangi, J Pharm Sci Rev Res 18(1), 126-132 (2013).
15. K. Atjanasuppat, W. Wongkham, P. Meepowpan, P. Kittakoop, P. Sobhon, A. Bartlett, P.J. Whitfield, J.Ethnopharmacol 123(3), 475–482 (2009).
