Cytotoxic Activity of Ethanol and Ethyl Acetate Extract of Kenikir (Cosmos caudatus) against Cervical Cancer Cell Line (HELA)

Dwira S.¹*, Fadhillah M. R.¹, Fadilah F.¹, Azizah N. N¹, Putrianingsih R.¹, Kusmardi K.²

¹Department of Medical Chemistry, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia

²Department of Anatomical Pathology, Faculty of Medicine, Universitas Indonesia, Jakarta-10430, Indonesia

ABSTRACT:

Cervical cancer is one of the most frequent cancer that occur among reproductive women in the world. The main modality of treatment is chemoradiotherapy and surgery. But, its wide-ranged success therapy and various side effects are still remaind the issues. To solve this problem, ethanol extract of kenikir leaves (EEKL) and kenikir leaves ethyl acetate extract of kenikir leaves (EAKL) have been thought to contain various substrate that could promote anticancer activities. This study comprised qualitative and quantitative test. Qualitative test consists thin layer chromatography and phytochemistry analysis while the quantitative test, MTT assay was used. MTT assay has done by using eight variety of concentrations of EEKL and EAKL on HeLa cells. Qualitative test showed that EAKL and EEKL has at least 5 compounds, which are flavonoid, tanin, steroid, alkaloid, dan glycoside. MTT assay revealed that EEKL and EAKL has strong cytotoxicity activity with IC50 17,46 ppm dan 6,31 ppm, respectively. In addition, each extract exhibited significant difference in some variants of concentration.

INTRODUCTION:

Cancer is the number one cause of death globally. According to data from the WHO, cancer causes the death of at least 8.8 million people in 2015 and 1 of 6 people in the world die of cancer. [1] Cervical cancer is listed as the fourth most common cancer in women in the world with a total estimation cases of 530,000 in year 2012 and deaths of 266,000 people. [1,2]

Human papilloma virus (HPV) is the primary cause of cervical cancer, especially HPV with oncogenic subtypes, subtypes 16 and 18. HPV infects squamous epithelial cells in areas that are damaged or in immature squamous cell at Squamocolumnar junction area. [3][4]

In this era, cancer therapy with plant extracts as a complementary medicine is developing. One of the plant extracts that are being studied for that purpose is kenikir leaf extract. Kenikir (Cosmos caudatus kunth.) is a source of antioxidants and potent anticancer. [5][6][7]

In methanolic extract, some compounds that play a role in this high antioxidant activity include quercetin and its glycoside derivatives, phenolic acid (CGA, cryptochlorogenic acid, neochlorogenic acid, caffeic acid, and ferulic acid), (+)(-) catechins. and proanthocyanidin. [5] Quercetin is a natural flavonol derived from plant pigments that can be found in fruits and vegetables, such as apples, onions, manga and tomatoes. Quercetin is a dominant flavonoid in kenikir leaves with a percentage of approximately 51%.8 In methanol extract of kenikir, quercetin can be isolated in the form of glycosylated or Quercetin Glycoside. [5][8] The differ forms of glycosylation caused difference in the bioavailability of quercetin, depend on the bound sugar group. This form of glycosylation and non-glycosylation can be found in blood plasma and will go to certain body organs which needed it. [5]

Other flavonoid components contained in kenikir are myricetin, kaempferol, epicathecin, luteolin, and naringenin. Proantocyanidine are tannin groups with varying concentrations depending on plant age and isolation method. [5]

Phenolic acid in kenikir included in the subclass of hydroxycinamic acid. Some groups of other substrates contained in kenikir include saponins, tannins, phytosterols, alkaloids, carotenoids, fatty acids, organic acids, hydroxyugenol, sesquiterpene lactone, terpenes, sugar acids, quaternary ammonium salts, and phenylpropanoids which can only be isolated from roots. [5]

Fig.1: chemical structure of Quercetin

MATERIALS AND METHODS:

Kenikir leaves (Cosmos caudatus) was from Bogor, West Java, Indonesia. The kenikir leaves along with their taxonomy. [10] HeLa cervical cancer cells that used in this study were available at the Laboratory of the Department of Anatomical Pathology, Faculty of Medicine, Universitas of Indonesia. Cisplatin was used as a positive control. Ethanol extract of kenikir leaves (EEKL) and ethyl acetate extract of kenikir leaves (EAKL)

Extraction of Kenikir leaves powder:

Kenikir leaf powder was immersed in both of 96% ethanol and ethyl acetate for 48 hours. Then filtrat with whatman paper and the solvent was evaporated by heating the extract at about 57˚C-60˚C using a rotary evaporator. 

Thin Layer Chromatography (TLC):

The purpose of the TLC was to determine the number of compounds in the extract. This test was able to separate the components in the extract based on the polarity of the content. The extract was spotted on the plate (approximately 1 cm x 20 cm x 0.2 cm ). Then, the plate was kept into a container containing methanol and chloroform with a ratio of 5: 1. The spot moved towards the upper line. After separation, the plate was observed under a UV lamp and Rf was calculated.

Phytochemical screening test:

Phytochemical test was a qualitative test. It was carried out to find out what kind of compound exist in leaves kenikir are. The procedures below are taken from Harborn. [11]

Flavonoid screening, 1 ml extract of solution was evaporated. After the solution was dry, it was moisten with acetone. Before heating carefully, a small amount of oxalic acid powder and boric acid was added to this mixture. The remaining are added with 10 mL ether. The results are observed in UV light 366 nm. If there was intensive yellow light, the solution contains flavonoids.

Saponin screening, The extract solution was shaken vigorously for 10 seconds. After the foam was formed with a height of 1-10 cm which is stable within 10 minutes, give 1 drop of 2N HCl into the solution. If the foam still stays, it was suggested the positive saponin in the solution. Tanin screening, 1 mL of extract solution was reacted with 10% iron (III) chloride solution. If dark blue or greenish black was observed, a solution contains tannins.

Triterpenoids and steroids screening, 2 mL of extract solution was evaporated. After the volume was half from the beginning, it was added by 0.5 mL of anhydrous acetic acid and 0.5 mL of CHCl3. Then, a few drops of H2SO4 was poured through the wall of the test tube. If there is a brown or violet ring, a positive solution contains triterpenoid. If there is a greenish blue ring, a positive solution contains steroids.

Glycosides screening, 2 mL of extract solution was evaporated in a water bath. After the volume of the solution becomes half of the initial, 5 mL of anhydrous acetic acid was addded. Then, 10 mL of concentrated H2SO4 was given. If the solution contains a blue or green ring indicated that the solution contains glycosides.

Alkaloids screening, 2 mL of the solution is evaporated so that the initial volume is reduced to half. After cooling, 5 ml of 2N HCL solution was added, the result of this solution was divided into three tubes. The first tube was used as a blank, the second tube was given three drops of Mayer solution, and the third tube was given three drops of Dragendorff's solution. The presence of yellow deposit in the second tube or orange deposit indicated the presence of alkaloid.

Cytotoxicity test using MTT assay:

MTT [3-4, 5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide method was used in the Cytotoxicity tests11. The procedure was begun by taking the cell from the CO2 incubator, then the cell's condition was observed. In order to to be harvested, cell cultures were confluent in 80% and counted in number. Then the cell dilution was performed with complete culture media (ccm). The cell then was transferred into a 96-well plate with a number of 5x103 cells/wells continued by incubating cells for 24 hours. The extracts with various concentrations (A series of concentration extract of kenikir leaves was made;6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, 100 ppm, 200 ppm, 400 ppm, and 800 ppm and cisplatin as a positive control ; 2 ppm, 4 ppm, 8 ppm, 16 ppm ,and 32 ppm) then were added to the well and were incubated at 37oC with a flow 5% of CO2. After incubation for 24 hours, 100 μl of MTT reagent concentration (0.5 mg / ml) in DMEM was added into each well. Incubation was continued at 37 °C for 3 hours until formazan is formed. Sodium dodecyl sulphate 10% in 0.1 N HCl as a stopper solution was then added. Optical Density (OD) was read by ELISA reader at λ 595 nm. Optical Density or absorbance value was converted to percent of inhibition by the formula :

            OD of negative control – OD of extract concentration

% Inhibition =-------------------------------------------------- X 100

                              OD of negative control

RESULT AND DISCUSSION:

In this study The extract’s compounds of EAKL and EEKL were identified by TLC with methanol: chloroform solvent (5:1). The spots on the TLC plate then were measured to find out Rf. The results are shown in Table 1 that EAKL had more compounds than EEKL.

Table 1: TLC test and Rf of Cosmos caudatus

There were two spots on TLC that had adjacent values, namely 0,89 and 0,80. These two spots might be the same components.

Phytochemical Test of EEDK and EADK:

In accordance with the results of phytochemical analysis carried out on EEKL and EAKL, both of them contained flavonoids, tannins, steroids, glycosides and alkaloids as it is shown in Table 2. The absence of saponins in the phytochemical test results was different from the study that conducted by Liliwirianis et al. In Liliwirianis’s research, foam that formed on saponin test could stay for 3 hours. [12] This difference in results was likely due to the small quantity of sample tested.

Table 2: Phytochemical Test of kenikir

Ethanol is a polar solvent and ethyl acetate is a semipolar solvent that’s why alkaloid, flavonoids, tannins appeared as spots. [13]

Cytotoxicity Assay:

The OD value of control (cell without addition of extract) were 2,372, 2,358 and 2,346. The average of OD control was 2,359. Concentration, OD value, and percentage of inhibition of EEKL are shown in the Table 3.

Table 3: Result for EEKL with Concentration, OD value, and percentage of inhibition

In the equation y = 30,99x + 11,50 , if y is replaced by the number 50, then the x value will be obtained 1,24. So, IC50 of EEKL is 17,46.

Table 4: Result for EAKL with Concentration, OD value, and percentage of inhibition

Table 5: Result for cisplatin with Concentration, OD value, and percentage of inhibition

The concentration of Cosmos caudatus extract (in x axis) were plotted against the percentage of inhibition (in y-axis). The line equation obtained from Microsoft excel is y = 30,99x + 11,50 . Concentration, OD value, and percentage of inhibition of EAKL are shown in the table 4. From the equation above, y = 23.361x + 31.312, IC50 of EAKL can be determined, that was 6.31

The same way was used to calculate IC50 of cisplatin. Concentration, OD value, and percentage of inhibition of cisplatin are shown in the table 5, from the linear equation of cisplatin, y = 25,83x+47,33, the IC50 is 1,49.

The data resulted from the cytotoxic test in this study were the percentage of the number of surviving HeLa cancer cells (or percentage of cell viability) after addition a series concentration of kenikir extract leaves. The lower the value of the cell viability means the higher percentage of inhibition, indicating that the more potent kenikir extract in inducing HeLa cancer cell death.

In general, both extracts (EEKL and EAKL) showed that the greater the concentration of extract, the greater the percentage of inhibition, the more cancer cells die. EEKL has the highest percentage of inhibition at a concentration of 800 ppm, that is 88.606%, while EAKL has the highest percentage of inhibition at a concentration of 200 ppm, that is 88.3365%.

IC50 of EEKL and EAKL is 17.46 ppm and 6.31 ppm respectively. The value of IC50 EAKL was 6.31, it was smaller than the value of IC50 of EEKL which was 17.46ppm. It might be caused more anticancer component from kenikir leaves could be extracted by ethyl acetate. Both extracts are categorized as strong cytotoxic based on classification National Cancer Institute. [14]

As it is seen in table 2, EEKL and EAKL contained flavonoids, tannins, quercetin glycosides and alkaloids. Flavonoids are the most common secondary metabolites in plant tissues.15 Those compounds had cytotoxic activity 16,17. Lotulung et al. mentioned that kenikir leaves contained compounds that had quite high antioxidant power18. Antioxidant activity is commonly associated with its benefits in cancer therapy 19. As an antioxidant, flavonoids play a role in donating hydrogen atoms or in chelating metals, in the form of glucosides (containing glucose side chains) or in the free forms which is called aglikon. [20] From the research conducted by Lee and Vairappan, it was known that ethanol extract of kenikir had the potential antioxidant with the value of Ascorbic Acid Equivalent Antioxidant Capacity (AEAC) was 3200.37 ± 54.11 mg AA / 100 g extract. Kenikir also had strong potential in cytotoxic testing of murine leukemia P388 cancer cells with the value of IC50 was 25 µg / mL. [21]

Quercetin glycosides are included in the class of flavonoids that can regulate the cancer cell cycle by binding to several targets. Quercetin is also able to induce apoptosis through the mitochondrial pathway. [22] Tarapdhar reported that quercetin could induce Caco-2 and HT-29 colon cancer apoptosis and HL-60 leukemia cancer cells by stimulating the release of cytochrome c from mitochondria. [23]

Research related to kenikir toxicity test on HeLa cells is still very rare. Several previous studies have discussed the cytotoxic activity of kenikir extract using different solvents and test cells. The previous test showed that the IC50 value of cytotoxic activity of methanol extract of kenikir leaves on breast cancer cells TD47 was 344.91 ppm [24] and of cytotoxic activity of ethanol extract of kenikir leaves on P388 mouse leukemia cells was 25 ppm. [21]

Data analysis was normally with distributed if p > 0.05. On EELK, groups of data that were not normally distributed were at concentrations of 25 ppm, 200 ppm and 400 ppm, whereas in EAKL extract, groups that were not normally distributed were in the group of 100 ppm concentration. After data transformation, it turned out that the data was still abnormally distributed or p < 0.05. Post hoc tests performed to determine which groups had significant differences. On EEKL, there were found the pair of concentration group of 200 ppm with 400 ppm that did not differ significantly with p = 0.822. Whereas on EAKL, there were three pairs of concentrations that were not significantly different ie 200 ppm and 400 ppm (p=0.827), 200 ppm and 800 ppm (p = 0.513), and 400 ppm and 800 ppm (p = 0,658).

This study concludes the EEKL and EAKL of Cosmos caudatus were having significant anti-cervical cancer activity when compared to cisplatin as standard. As this study is a preliminary analysis of pdytochemical test and it needs to be purified further to identify of active compounds. This study warrants further investigation including in vivo test and the mechanism of action.

ACKNOWLEDGMENT:

Authors are very grateful to Faculty of medicine Universitas Indonesia, for PITTA grand and for providing facilities to full fill the work.

CONFLICT OF INTREST:

The Authors declare no conflict of interest.

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Received on 22.11.2018         Modified on 12.12.2018

Accepted on 26.12.2018      © RJPT All right reserved