CYTOTOXIC ACTIVITY AGAINST L1210 LEUKEMIA CELLS FROM THE ETHYL ACETATE FRACTION OF KENIKIR LEAVES (Cosmos. Caudatus) PRESERVED BY GAMMA IRRADIATION

Kenikir leaves (Cosmos caudatus) Has been used as traditional medicine, especially as an anti-cancer, this plant has been in Indonesia both in herbs and capsules. Microbial contamination of herbal medicinal raw materials occurs when the storage process is done. One of the preservation techniques used in the industry is using gamma irradiation techniques to reduce microbial and fungal contamination. The purpose of this research was to study the effect of gamma irradiation for the preservation of kenikir leaves (C. caudatus) as an anti-cancer based on cytotoxic activity using L1210 leukemia cells. The simplicia was gamma-irradiated by Co-60 source with variation doses of 0 (control); 5; 7.5; 10; and 15 kGy. Then the irradiated and control samples were macerated successively using n-hexane, ethyl acetate, and ethanol. The active extract (ethyl acetate) was further fractionated using column chromatography, obtained seven fractions (F1 F7). The seven fractions' cytotoxic activity against L1210 leukemia cells showed that fraction 3 (F3) was the most active fraction with an IC50 of 1.26 μg/mL. Each dose's F3 cytotoxic activity showed that the IC50 7.5 kGy irradiation sample did not change significantly with control (0 kGy) based on ANOVA analysis using SPSS 24 with a 95% confidence level. In comparison, irradiation samples of 10 and 15 kGy showed a change in the IC50 value is significant with the control (0 kGy). These results indicate that gamma irradiation can be used as an alternative for preserving C. caudatus with a maximum dose of 7.5 kGy, so that its anti-cancer properties do not change with those without irradiation.


INTRODUCTION
Indonesians from generation to generation to treat various diseases [2].

One of the medicinal plants used by
Indonesian people is kenikir (Cosmos caudatus). C. caudatus has properties healing high blood pressure, anti-diabetes, anti-inflammatory, anti-bacterial and anticancer [3]. The secondary metabolite of C. caudatus leaf extract consists of flavonoids, especially quercitrin glycosides. The results revealed that secondary metabolites of flavonoid compounds act as anti-cancer, which induce apoptosis [4]. Methanol extract from kenikir leaves has potential as chemotherapy derived from natural ingredients. The methanol extract of kenikir leaves can against T47D breast cancer cells through apoptotic mechanisms to develop as an anti-cancer drug [5]. The ethanol extract of kenikir leaves has a high antioxidant content 181.64 µg chatecin/mg extract, so it can prevent free radicals and can inhibit the development of hella cancer cells with IC 50 89.90 µg/mL [6] C. caudatus, as traditional medicine has been widely used in Indonesia either in herbs or capsules. Microbial contamination in herbal medicinal products is common and usually comes from raw materials, processing to the equipment used [6]. Although the number of bacteria and fungi are found still under the recommendation and regulation of BPOM No. 12 of 2014 that the number of bacteria should not be more than 10 7 colonies/gram, and the number of fungi should not be more than 10 4 colonies/gram. Can be dangerous if the bacteria and fungi are aflatoxins [7]. The unirradiated temu putih (Curcuma zedoaria) capsule product has the number of bacteria and fungi 28.5x10 2 colonies/gram and 19x10 2 colonies/gram. After irradiated gamma of 5 kGy the number of bacteria and fungi decreased to 18.6x10 2 colonies/gram and 0.57x10 2 colonies/gram [8].
One of the preservation techniques used in the industry is the gamma irradiation technique. According to the BPOM regulation, no 18 of 2019 concerning irradiate food, preservation using gamma irradiation techniques can reduce the number of bacteria, fungi, and storage time longer than before [9]. Preservation techniques using gamma irradiation can reduce the number of bacteria and fungi and extend these products' shelf life. Preservation techniques are currently widely used because it has many advantages such as more efficient, easy to control, does not cause residues, and is very environmentally friendly. Therefore the cost of production is cheaper [10]. Irradiation of peach fruit using a dose 5 kGy increased to 17 days longer shelf life than unirradiated samples [11].

Irradiation of samples
The samples were irradiated using gamma irradiator IRKA (source: Co-60) at PAIR-BATAN with the irradiation doses of 0 (control), 5, 7.5, 10 15 kGy with each irradiation dose were two replications.

Extraction
After irradiation, each carried out maceration of the sample successively using

RESULTS AND DISCUSSION
The moisture content of C. caudatus after drying for one week at room temperature (22°C) is 6.58%. This value follows the NAFDAC (Badan POM) requirements that the water content in herbal plants must be below 10% [7].
Drying is carried out to reduce the moisture content in the simplicia and prevent decay caused by microorganisms to affect the quality and efficacy of the simplicia finally.
Maceration was carried out in stages starting from non-polar (n-hexane) to polar solvents (ethanol) to separate the secondary metabolites in the C. caudatus simplicia. The yield of each extract can be seen in Table 1. compared to n-hexane and ethanol extract [12]. Therefore fractionation was carried out on ethyl acetate extract to determine the most active fraction to inhibit L1210 leukemia cells.
The fractionation of ethyl acetate extract using column chromatography obtained seven fractions, and the weight of the fractions obtained, as shown in Table 2. values <20 µg/mL.