Identification of Anticancer Active Compound from GC-MS Test Results of Zodia Leaves ( Evodia suaveolens ) Ethanol Extract

ABSTRACT


Introduction
Cancer is a disease that characterized by excessive cell growth can attack adjacent body parts or spread to other organs. Based on the WHO (World Health Organization) cancer is estimated to be the second leading cause globally in 2018 (Organization, 2019). The prevalence of cancer in Indonesia in 2018 reached 775,120 people with mortality reaching 207,210 of 348,809 cases. Some approaches used to treat cancer ar surgery, chemotherapy, radiotherapy, immunoterapy, targeted therapy, and hormone therapy (Health, 2019). Therapy can be given separately or in combination as adjuvant or neoadjuvant depending on the stage of the cancer (Nurhayati & Lusiyanti, 2006;Sari, Wahid, & Suchitra, 2019). Surgery and radiotherapy treatment are effective for early cancer, but not for metastatic cancer. Chemotherapy, targeted therapy, and hormone therapy are more appropriate for metastatic cancer (Chabner & Roberts, 2005). But these treatments cause side effects, chemotherapy can affect normal cells and some drugs cannot be combined because it can strengthen the work of other drugs that can increase their side effects to cause death (Lander et al., 2001;Sari et al., 2019). Radiotherapy can affect normal cells causing xerostomia, central nerve complications, and RIHD (Radiation Induced Heart Desease) (Fitriatuzzakiyyah, Sinuraya, & Puspitasari, 2017). To reduce these side effects it is necessary to find anticancer agents from natural products to prevent and cure cancer.
Herbal medicine is an alternative therapy because it is considered to have lower side effects and can even reduce the side effects of chemotherapy and radiotherapy (Radji, Aldiarti, Harahap, & Irawan, 2010). Based on the Minister of Health Decree in 2007, Indonesia has 30,000 plant species which 300 species have been used as traditional medicinal materials from 9,600 species as medicinal plants. Some plants that are used as anticancer include crown god (Phaleria macrocarpa), turmeric (Curcuma longa), white turmeric (Curcuma zedoaria), kopasanda (Chromolaena odorata L.) and red fruit (Pandanus conoideus) (Fitrah, Winarno, & Simanjuntak, 2017;Lisdawati, 2009;Mutiah, 2015;Radji et al., 2010). Research related to curcumin activity as an anticancer has been widely carried out both in vivo, in vitro, until clinical studies. Based on research that has been done, the administration of curcumin monotherapy can reduce the volume of bladder cancer using human bladder cancer on orthotopic mouse models. In silico and in vivo research shows that curcumin plays a role in inhibiting cell apoptosis and regulating transcription factors (Mutiah, 2015).
Indonesian endemic plants that have the potential to be anticancer namely zodia (Evodia suaveolens) which is commonly found as ornamental plants in Java (Kusnoto, 2007). Zodia is often used as an anti-mosquito by rubbing it directly on the body, formed as soap, and spray (Muhamat, Wahyuni, Rusmiati, & Jumar, 2016;Simaremare, Sinaga, & Agustini, 2017). Studies that have been done stated that the content of evodiamine in Zodia (Evodia sauveolens) has been shown to have anticancer activities by inhibiting proliferation, invasion, and metastasis, and inducing apoptosis in various types of tumor cells (Jiang & Hu, 2009).
Testing of plant compounds as potential drugs can be done in vivo and in vitro, but this method requires a lot of cost and time, so it is necessary to first examine the interaction of compounds with receptors while eliminating compounds that have low activity through the in silico test (Ekins, Mestres, & Testa, 2007;Ruswanto, 2015). Untuk mengurangi kekurangan tersebut dapat diatasi dengan menggunakan uji secara komputasi atau in silico. The computational testing or in silico can reduce these deficiencies and could be done by many methods, one of which is molecular docking. Molecular docking is a computational procedure that describes interactions between drug molecules as ligands and protein receptors (Suhud, 2015). Before conducting the in silico test, it is necessary to identify the active compound contained in a sample to be tested.
There are many methods that can be used to identify active compounds contained in a sample. One of method used is Gas Chromatography -Mass Spectrum (GC-MS), a chromatographic technique to detect volatile compounds and identify molecular weights and molecular formulas (Darmapatni, Basori, & Suaniti, 2016). This study aims to identify and determine the active compound from ethanol extract of zodia leaves from GC-MS analysis which has the potential as an anticancer.

Material and Methode
This research was conducted in April 2019 at the Physiology Laboratory of the Biology Department of the Faculty of Mathematics and Natural Sciences, University State of Surabaya. The tools used in this study include blenders, rotary evaporators, and GC-MS tools. The ingredients used are zodia leaves, 96% ethanol, and filter paper. The steps taken are:

Sampling
The zodia (Evodia suaveolens) leaves collection was obtained from several locations of gardens and yards in the Surabaya that cultivated the plants to obtain sufficient quantities for extraction.

Extraction Zodia Leaves (Evodia suaveolens)
Zodia leaves are washed and then dried for three days and avoided from the sun. The leaves are dried using an oven for two to three days at 60 o C, then mashed with a blender until it becomes powder and sifted using a 40 mesh sieve. The zodia leaves powder macerated using 96% polar solvent for 3 days with the concentration of the solvent compared to the first day was 1 : 3 (weight : volume) while the concentration on the second and third days was 1: 1 (weight : volume). The results of maceration were evaporated using a rotary evaporator to form a paste. The extraction results are 100% extract of zodia leaf.

Identification Active Compound
The identification of active compounds contained in zodia leaf extract will be carried out using GC-MS analysis. Analysis using the AGILENT 6980N Network GC System with an autosampler. The sample is heated at a temperature of 50-280 °C with an increase in temperature of 100 °C / minute and leave it for 10 minutes. Helium gas is used as a career with a flow of 1.3 ml / minute. Heat the injector at 280 °C, the size of the injection of 1µl neat, with a split ratio of 1:10. The interface and source of the MS ion are maintained at 230 °C and 150 °C carefully. The mass spectrum is obtained at 70 eV and in the reading range 200 -700 amu. The data obtained was analyzed in the GC-MS solution software. This stage was carried out in the Testing Service Unit (ULP), Faculty of Pharmacy, Airlangga University Surabaya.

Results and Discussions
This research was a descriptive experimental study by identifying potentially anticancer compounds from the results of GC-MS test ethanol extract of zodia leaves (figure 1).
Evodone has the potential as an anticancer ovary through its interaction with the influential 3HHM protein on the PI3KCA pathway. This pathway is one of the signal transduction pathways that plays a role in cell proliferation and differentiation. In cancer cells, this pathway experiences hyperactivation resulting in excessive activation of protein kinase B (PKB) (Brown & Toker, 2015). Excessive activity of PKB causes inhibition of proapoptotic protein activation, activation of anti-apoptotic proteins, and increased metastasis (McDonald, 2008). Compounds that have the potential to be anticancer lungs, namely evodone; 3,5,7-Octatrien-2-Ol, 2,6-Dimethyl; 2,4-Dimethyl-2,4-Heptadienal; Phytol; and Squalene with protein targets namely EGFR, CCNE1, and CYP450. EGFR (Epidermal Growth Factor Receptor) is a 170-kDa glycoprotein protein tyrosine kinase which is present in cell membranes. In normal cells EGFR expression includes epidermal, mesenchymal, and neurogenic cells. EGFR expression when binding to EGF activates cell proliferation and differentiation. DNA mutations in EGFR as detected by polymerase chain reaction (PCR) can occur in the appropriate area with parts of extracellular or intracellular proteins. In non-small cell lung cancer, overexpression of EGFR or mutations in intracellular EGFR (Bethune, Bethune, Ridgway, & Xu, 2010;Inamura, Ninomiya, Ishikawa, & Matsubara, 2010).

Figure 1. GC-MS Test Results Ethanol Extract of Zodia Leaves
Based on searching through a Swiss Target webserver, it can be seen that the compounds of Beta-Tocopherol and Stigmasta-5,23-dien-3.beta.-ol have potential to be lung anticancer and colorectal by targeting epidermal growth factor erbB1 (EGFR) which is on protein kinase. The EGFR gene starts when the ligand is bound to the outermost part of the EGFR receptor protein so that another homodimer or heterodimer structure will be formed. The results of the formation of this structure will activate the receptor kinase and autophosphorylate specific tyrosine residues that have the ends of the -COOH group. The autophosphorylation process will activate the signal transduction pathways that are regulated by EGFR such as the AKT and ERK pathways that control several cellular processes (Ladanyi & Pao, 2008).