Invitro Antioxidant and Cytotoxic Potential of Ficus carica L. and Olea europeae L. Against Cervical Cancer

INTRODUCTION:Cervical cancer is a malignant infectious disease caused by Human Papilloma Virus (HPV) in the cervix. Fig and olive oil containing flavonoid has been shown to have antioxidant and anticancer activity.OBJECTIVE:This study aimed to evaluate antioxidant and cytotoxic activity of combined Ficus carica L. and Olea europeae L. against HeLa cervical cancer cells.METHODS: This is an experimental study with a post-test only control group design. The HeLa cells were divided into 5 groups: fig extract, olive oil, combined fig extract and olive oil (at the ratio of 1:3, 1:1, 3:1), positive control (doxorubicin). The cytotoxic and antioxidant activity were evaluated by using MTT Assay and DPPH, respectively. The cytotoxic results were analyzed using probit and antioxidant activity was analyzed by using linear regression to obtain IC50 values.RESULTS:The IC50 cytotoxic of fig extract, olive oil, combined fig extract and olive oil (at the ratio of 1:3, 1:1, 3:1) with positive control (doxorubicin) were 13063,915 �g/mL, 679,593 �g/mL, 1562,356 �g/mL, 746,923�g/mL, 563,626 �g/mL and 13,707 �g/mL respectively. The IC50 antioxidant of fig extract, olive oil, and combination of fig extract and olive oil (3:1) was 105.9272 ppm, 23.1276 ppm, and 21,0689 ppm respectively.CONCLUSION:The combination of fig extract and olive oil (3:1) was shown to have the highest antioxidant and cytotoxic activity against HeLa cervical cancer cells.


INTRODUCTION
Figs (Ficus carica L.) containing active compounds such as phenols, terpenoids, flavonoids and alkaloids in vitro have shown to have effect in inhibiting the proliferation of various cancer cells and antioxidant properties (Joseph and Justin Raj, 2011). Research was conducted by Ghanbari et al., (2012) show that olive oil has the main class content of phenol compounds namely phenolic acids, flavonoids, lignans and biophenol which have potential as antioxidants and anticancer.
Cancer is one of the life-threatening diseases in the world. In Indonesia, the prevalence of cancer is the number seven cause of death (Wahyuni et al., 2015). The prevalence of cervical cancer in Indonesia is in the first rank, followed by breast cancer (Dasar, 2013). Cervical cancer is a malignant disease caused by infection from Human Papilloma Virus (HPV) that occurs around the cervical tissue (Fitriana and Ambarini, 2012). The prevalence of HPV infection as many as 75% occurs in women who have had free sex before marriage (Cancer Foundation of Indonesia, 2017).
Patients infected with HPV are treated with chemotherapy, radiation therapy, or even to undergo surgery (Lui and Grandis, 2012). Chemotherapy can produce very strong toxic effects to cancer cells and can attack normal cells so that they cause adverse side effects (Baqutayan, 2012). Therefore, there is a need for alternative treatments for cervical cancer, one of which uses medicinal plants mentioned in the Qur'an, in the first and second verse of At-Tin which means "For the sake of figs and olives", these figs and olive plants are mentioned together (Ministry of Religion of the Republic of Indonesia, 2014).
In the results of a study by Kichaoui et al., (2016) it was shown that fig fruit (Ficus carica L.) extract contains the compound of Quercetin-3-O-glucoside which has been proven to have an intrinsic mechanism that is capable of reducing HeLa cell proliferation. Likewise, in the study of Yao et al., (2014), it was stated that olive oil extract (Olea europaea L.) contains Oleuropein compounds and intrinsically has a cytotoxic effect on HeLa cells. Because both can potentially inhibit the proliferation of HeLa cervical cancer cells, a research related to the combination of figs and olive oil as anticancer agents is needed, so that they can be used as effective and maximum treatment and expected to have a synergistic and mutual reinforcing mechanism to inhibit proliferation of HeLa cervix cancer cell (Yao et al., 2014). Threfore, aim of the study to evaluate antioxidant and cytotoxic activity of combined Ficus carica L. and Olea europeae L. against HeLa cervical cancer cells

Figs Extraction
Extraction was carried out through maceration method using 70% ethanol with a ratio of 1:4 for 5 days, stirred every day. Extracts were separated from the solution by evaporation with temperatures below 50 o C until thick extracts were formed and stored at 4 o C (Jasmine et al., 2015).

Flavonoid Identification
As many as 1 ml of ethanol extract samples of figs and olive oil were put into the test tube, then added sulfuric acid (H2SO4) 2N as many as 2 drops and strongly shaken. Samples positively contain flavonoids when the solution undergoes a very striking color change to yellow, red or brown (Lisi, Runtuwene and Wewengkang, 2017).

Total Flavonoid Level Test a. Preparation of Quercetin Raw Curves
As many as 10.0 mg quercetin raw was dissolved using aquadest solvent until the volume limit of the pumpkin volume was 10.0 mL (quercetin level to 1000 µg / mL or 1 mg / mL). Then, standardized curves from mother liquor 1000 µg / mL with 5 different concentrations (20,40,60,80 and 100 μg / mL) were made, added 0.3 ml of 5% NaNO2, added 0.3 ml of AlCl3 10% which was left to stand for 5 minutes, plus 2 ml of 1M NaOH and aquadest until the pumpkin volume limit was 10.0 ml. The raw series is left to the appropriate operating time and measured at maximum wavelength (John et al., 2013).

Cytotoxic Test
Cells were planted on 96-well plates with 5x103 cells/wells incubated for 24 hours. Cells were treated through samples with a ratio of 1:0; 1:3; 1:1; 3:1; 0:1 for 24 hours. The media were removed and washed with 100μl PBS (Sigma), adding MTT reagent 5 mg / mL to each well and incubated at 370C for 4 hours. Alive cells can convert MTT reagent to purple formazan after incubation by adding SDS 10% HCL 0.01 N reagent stopper and incubating it for 24 hours. To read absorbance with an ELISA reader is at λ 595 nm (Cancer Chemoprevention Research Center, 2015).

Test of antioxidant activity using the DPPH method
To make a DPPH ( Then it is calculated as the percentage of DPPH color reduction by using the equation: Antidote to free radical activity (%) = x100% (Munte, Runtuwene and Citraningtyas, 2015) Data analysis of cytotoxic test was using probit analysis to obtain IC50 values of samples that showed a percentage of culture death cell as much as 50%. Then, Data analysis of antioxidant test was using linear regression.

RESULTS
The The phytochemical test results are presented in the Table 1. Test results for total flavonoids levels are presented in Table 2.  According to (Tiwary et al., 2015), the potential of an extract is said to have cytotoxic activity if the