Antioxidant and anticancer activity of Eleutherine bulbosa (Mill.) Urb on leukemia cells L1210

Antioxidant and anticancer activity test on Leukemia cells L1210 were carried out on four fractions (ethanol extract, n-hexane, chloroform, and water fraction) of Eleutherine bulbosa [Mill.] Urb. The purpose of this research is to obtain IC50 antioxidant and anticancer activity of the active fraction of Eleutherine. bulbosa [Mill.] Urb on Leukemia cells L1210. The research method includes antioxidant activity test with DPPH (2,2-diphenyl-1-picrylhydrazyl) method, anticancer activity test of active fraction on Leukemia cell L1210, and GC-MS analysis to determine the active compound. Chloroform fraction has the best antioxidant activity with IC50 value 19.694 ppm (very strong category). The IC50 value of the chloroform fraction on L1210 Leukemia cells was 9.56 ppm (very strong category). Based on GC-MS analysis of the chloroform fraction, some compounds such as hexadecanoic acid, 9,12-octadecadienoic acid, linolenic acid, octadecanoic acid, androstan-17-one, and 1-(2,3,5,6-tetramethylphenyl)- ethanone were detected.


Introduction
Leukemia is a proliferation that occurs irregularly or a buildup of white blood cells in the bone marrow so that the elements in the normal bone marrow are replaced [1]. Old cells will die and be replaced by new cells, but this regular process does not work properly, due to the growth of abnormal white blood cells [1]. Eleutherine is a plant that is known to contain naphthoquinone and has been used as a drug and poison since prehistoric times. Naphthoquinone compounds are known to be very toxic that can be used as antimicrobial and antioxidant [2]. "Bawang Tiwai" (Eleutherine bulbosa) is widely used by the Dayak and Kutai tribes as traditional medicines that can treat various diseases including colon cancer, breast cancer, diabetes mellitus, hypertension, cholesterol-lowering, stroke, boils, anti-bleeding and abdominal pain. [3]. Secondary metabolite of Eleutherine bulbosa has the potential as a medicinal plant with various properties. Currently, the use of Eleutherine bulbosa as an additive in cooking is increasing, but studies of Eleutherine bulbosa about its activity as an anti-cancer blood or leukemia have not been widely studied. Several studies have shown that Eleutherine bulbosa has moderate potential antibacterial activity against Escherichia coli, with a strong category and has potential as an antidiabetic agent that is useful in the prevention and protection of diabetes mellitus [2,4,5]. Eleutherine bulbosa is widely used in Kalimantan to cure breast cancer, heart problems, increase endurance, anti-inflammatory, anti-tumor and can stop bleeding [5]. This article discusses the antioxidant and anticancer activity test of Eleutherine bulbosa on Leukemia cells.

Sample preparation
The simplisia of mashed Eleutherine bulbosa bulbs were put in a bottle. Ethanol was added to the bottle and the sample was soaked for 24 hours, filtered, and the filtrate was concentrated. The maceration process in the same sample was repeated twice. The remaceration results were mixed together. All concentrated filtrates are called ethanol extracts. The ethanol extract was partitioned with n-hexane:methanol:water (5:9:1) using a separating funnel until 2 layers were formed, then each part was concentrated with a rotary evaporator to obtain n-hexane fraction and methanol-water fraction. The extract from methanol-water fraction was partitioned with chloroform:water (1:1) using a separating funnel until 2 layers were formed, then each part was concentrated with a rotary evaporator to obtain chloroform fraction and water fraction. Identification of the compounds in the active fraction was conducted using a Gas Chromatography-Mass spectrophotometer (GC-MS) Shimadzu QP 2010.

Phytochemical Screening
The ethanol extract and all fractions were subjected to phytochemical screening tests, including identification of alkaloids, steroid and triterpenoid groups, flavonoids, saponins and tannin groups with a method describe in Harborne [7] and Meyer et al. [8].

Antioxidant activity test with DPPH (2,2-diphenyl-1-picrylhydrazyl)
The antioxidant activity test with DPPH was conducted with a method described in Sundu et al. [9] Antioxidant activity test was carried out on ethanol extract, n-hexane fraction, chloroform fraction and water fraction of Eleutherine bulbosa. Vitamin C was used as a positive control. Determination of absorbance of the samples was carried out using Spectrophotometer UV Vis Shimadzu 1800. The values of free radical inhibition activity were calculated.

Cytotoxicity test for L1210 leukemia cells
The cytotoxicity test for L1210 leukemia cells was carried out with a method described in Hayshi et al. [10].

Preparation of media.
RPMI-1640 (10.4 g) containing L-glutamine was dissolved in 1 L of sterile water (solution A). NaHCO3 (1.3 g) was dissolved in 50 mL of sterile water (solution B). Twenty-five mL of solution B was added to 475 mL of solution A to obtain 500 mL of media (solution C). In the testing process, 15 mL of serum calf bovine was added to 85 mL of solution C. All work was carried out in a sterile room.

5. Cell planting and testing of cytotoxicity activity.
Leukemia cells L1210 were added to the media containing bovine calf serum with 2x10 5 cells/mL. Leukemia cancer cells used were from The Institute of Chemical Research Japan. Testing of cytotoxic activity was carried out on chloroform extract from tiwai onions. The dosage variations of the extract were 5, 10, 20, 40, 80 μg/mL. The media containing L1210 leukemia cell suspension (2x10 5 cells/mL) and the test substance were inserted into a multi well plate tissue's culture so that the total volume was 1 mL in each well. As a control, 10 μL methanol was added with 990 μL cell suspension. The experiment was conducted twice, then the cell suspension filled with the test substance was incubated for 48 hours at 37°C in an incubator (5% CO2).

Calculation of cells.
Cell calculations were carried out using the Haemocytometer Neubauer Improved. To differentiate between living cells and dead cells, before counting, 90 μL of the suspension was inserted into the sero cluster plate (96 wells) and was added with 10 μL of 1% tryphan blue solution and homogenized. A total of 10 μL of the solution was flowed into the Haemocytometer Neubauer Improved. The number of surviving cells was calculated under a microscope. Living cells were seen as clear spheres with blue spots of the cell nucleus in the middle of the circle, while dead cells appeared as dark blue patches that were irregular in shape.

Calculation of percentage of inhibition.
Percentage of inhibition of test substances on the growth of L1210 leukemia cells was calculated as follows: Where: A: the number of living cells in a medium containing test substances. B: number of living cells in a medium that does not contain a test substance (control).

Phytochemical screening
Phytochemical screening aims to determine the secondary metabolites contained in the extract and fractions of Eleutherine bulbosa. The results of the phytochemical screening are presented in Table 1. activity including antioxidants and anticancer. Flavonoids are known to be good antioxidants because they have at least two hydroxyl groups in ortho positions and para which can capture free radicals by freeing hydrogen atoms from their hydroxyl groups. Flavonoids as antioxidants have a higher potential as anticancer drugs than vitamins and minerals. Flavonoid compounds can prevent the reaction of carcinogen molecules joining cell DNA so as to prevent cell DNA damage; the bioactive components of flavonoids can prevent the initial process of cancer cell formation. Flavonoids can stimulate the regenaration process of mutated DNA cell so that the cells become normal again. In addition, quinone compounds have also been reported to have antioxidant activity. It is likely that quinone originates from the oxidation of the corresponding phenols namely catechol to form ortho-quinones and quinol producing para-quinones.

Antioxidant activity test
The antioxidant activity test was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl) method. This method is a simple and common method to measure the antioxidant activity of extract and each fraction. The principle of this method is the measurement of scavenging free radical in solvents such as ethanol/methanol at room temperature by a compound that has antioxidant activity [11]. The IC50 value of chloroform fraction was 19.649 which categorized as very strong antioxidant activity. Chloroform fraction was detected to have very strong antioxidant activity because it contains a lot of compounds such as flavonoids, phenolic and alkaloid. Those compounds are known to exhibit antioxidant activity. Flavonoids can provide one electron to the free radical compounds and form complex compounds with metal.

Anticancer activity on L1210 cells
Based on the results of the antioxidant test, chloroform fraction of Eleutherine bulbosa has IC50 value which is categorized as very strong antioxidant. Chloroform fraction was chosen because of the potential for anticancer activity testing on L1210 cells. This test used L1210 leukemia cancer cells which is one of the leukemia cell lines derived from inoculation of rat spleen. Anticancer activity test of chloroform fraction of Eleutherine bulbosa was carried out at the Health Materials Laboratory, the Center for Radioisotope and Radiation (PAIR) of the National Atomic Energy Agency (BATAN) Jakarta. Anticancer activity test results from the chloroform fraction of Eleutherine bulbosa using doxorubicin as a positive control on L1210 leukemia cancer cell growth can be seen in table 3. Table 3. Anticancer activity on L1210 cells.
Anticancer activity test of chloroform fraction of Eleutherine bulbosa on L1210 leukemia cells in this study showed an average IC50 value of 9.56 ppm (very strong activity). The value of IC50 on anticancer activity test result was carried out in duplicate and obtained values of 9.22 ppm and 9.90 ppm with an average value of 9.56 ppm. This value indicated that the chloroform fraction had high anticancer potential against L1210 leukemia cells. GC-MS analysis of chloroform fraction of Eleutherine bulbosa was performed to determine its chemical profile (Figure 1). In this study, there were several compounds identified such as hexadecanoic acid, 9,12-octadecadienoic acid, linolenic acid, octadecanoic acid, androstan-17-one, and 1-(2,3,5,6-tetramethylphenyl)-ethanone.