In Silico Anticancer Activity and In Vitro Antioxidant of Flavonoids in Plectranthus amboinicus

Exposure to carcinogenic substances will damage Deoxyribose Nucleic Acid (DNA). If the Deoxyribose Nucleic Acid (DNA) repair fails, it will lead to cancer.1 Based on research results from the International Agency for Research on Cancer, the most common cancers in Indonesia are breast, cervical, lung, ovarian, rectum, thyroid, colon, liver and nasopharyngeal cancer.2 The main strategy of choice in the treatment of colon cancer is chemotherapy with 5-fluorouracil (5-FU) but it is very toxic to other normal tissues.3-4


INTRODUCTION
Exposure to carcinogenic substances will damage Deoxyribose Nucleic Acid (DNA). If the Deoxyribose Nucleic Acid (DNA) repair fails, it will lead to cancer. 1 Based on research results from the International Agency for Research on Cancer, the most common cancers in Indonesia are breast, cervical, lung, ovarian, rectum, thyroid, colon, liver and nasopharyngeal cancer. 2 The main strategy of choice in the treatment of colon cancer is chemotherapy with 5-fluorouracil (5-FU) but it is very toxic to other normal tissues. [3][4] The diversity of plants in Indonesia is one of the important opportunities in developing Indonesia potential in the era of globalization. 5 Natural sources of antioxidants that come from food ingredients are found in spices, leaves, seeds, vegetables. 6 Most sources of natural antioxidants are plants, which are generally phenolic compounds, including the flavonoid group. Flavonoids that are active as antioxidant and anticancer compounds. The leaves of Plectranthus amboinicus (Lour.) Spreng contain many flavonoids Chrysoeriol, Cirsimaritin, Eriodictyol, Luteolin, Rutin, Salvigenin, Thymoquinone, Quercetin, Apigenin, and 5-O-Methyl-Luteolin. 7 The ability of flavonoids as antioxidants has been widely researched recently, because flavonoids have the ability to reduce free radicals. This study aims to determine the antioxidant activity of the ethanol extract of Plectranthus amboinicus (Lour.) Spreng leaves and 10 pure flavonoid compounds contained in the leaves of Plectranthus amboinicus (Lour.) Spreng by in vitro. The research was also continued by testing the anticancer activity of 5-fluorouracil and 10 pure flavonoid compounds contained in the leaves of Plectranthus amboinicus (Lour.) Spreng against several cancer receptor by in silico.

Tools Preparation of Extract
Leaves of Plectranthus amboinicus (Lour.) Spreng were obtained from Dolok Marlawan Village, Jorlang Hataran District, Simalungun Regency, North Sumatra Province, Republic of Indonesia, Postal Code 21172. Determination of plant materials was carried out at Herbarium Medanense, Faculty of Mathematics and Natural Sciences, University of North Sumatera. The samples used were collected, washed, drained, sliced, dried, blended, and sieved. A total of 500 grams of powder were added 3.75 L of 96% ethanol, left for 5 days while stirring frequently, filtered the extract, readded 1.25 L of 96% ethanol, left for 5 days while stirring frequently, filtered the extract, concentrated with a rotary evaporator, evaporated in a water bath until a crude extract was obtained. 8

In Silico Anticancer Test
The molecule obtained from the Protein Data Bank is a combination of the native ligand and the binding pocket molecule. Native ligand molecule ATP and binding pocket molecule P-Glycoprotein-1 with protein code 1MV5; native ligand molecule IMN and binding pocket molecule Cyclooxygenase-2 with protein code 4COX; native ligand molecule 1YG and binding pocket molecule Cyclin Dependent Kinase-2 with protein code 4LYN; native ligand molecule 1UK and binding pocket molecule Phosphoinositide-3-Kinase with protein code 4KZC; native ligand molecule GCP and binding pocket molecule Phosphoenolpyruvate Carboxykinase with protein code 1KHB. The three dimensional conformation of native ligand molecules were redocking into the each binding pocket molecule then calculated as the Root Mean Square Deviation value is valid if it is less than 2 Å. The three dimensional conformation of the test molecule and the reference molecule were docking to binding pocket molecule, and the binding energy value of each conformation of the molecule of the test molecule and the reference molecule were obtained in the various binding pocket molecule. 9 In Vitro Antioxidant Test Free radical solution 1,1-diphenyl-2-picryhydrazil with a concentration of 200 µg/mL was fresh prepared, pipetted as much as 5 mL, inserted in a 25 mL volumetric flask, added a certain volume of the extract solution in methanol or flavonoid compound solution in methanol with a concentration of 100 µg/mL (obtained a concentration of 2.5 µg/mL to 20 µg/mL), diluted with methanol, allowed for 60 minutes, measured at a wavelength of 516 nm, calculated the Inhibitory Concentration 50% with Statistical Package for Social Sciences version 26 year 2019. 10

In Silico Anticancer Test
The native ligand molecule was extracted from the protein obtained from the protein data bank and redocking native ligand molecule against the binding pocket molecule to determine the validity of the binding pocket molecule. The results of validation of the binding pocket molecule validation can be seen in Table 1.
The in silico molecular docking process of all native ligand compounds in each binding pocket molecule was valid, because the value of Root Mean Square Deviation obtained was less than 2 Å. 11 So that the entire binding pocket molecule can be used to docking the test compound molecule by in silico. The binding energy of the test molecule and reference molecule as a result of the molecular docking test can be seen in Table 2.
The binding energy value by in silico docking result of the whole test molecule were lower than the reference molecule against the Cyclooxygenase-2 and Phosphoenolpyruvate Carboxykinase. This means that all the test molecules have a stronger interaction than the reference molecules against the Cyclooxygenase-2 and Phosphoenolpyruvate Carboxykinase. On the P-Glycoprotein-1, Cyclin Dependent Kinase-2, and Phosphoinositide-3-Kinase, the test molecule gave results that were not significantly different from the reference. This means that all the test molecules have a similar interaction to the reference molecules against the P-Glycoprotein-1, Cyclin Dependent Kinase-2, and Phosphoinositide-3-Kinase. The binding energy resulted by in silico molecular docking is reflection of the affinity between the test molecule and the binding pocket molecule. 12 The lower the binding energy value, the stronger the interaction between the test molecule and the binding pocket molecule. 13 The in silico molecular docking results of flavonoid compounds in the glycoside form gives a lower bond energy yield than flavonoid compounds in the non glycoside form. This occurs because in silico molecular docking testing of glycoside compounds has bigger molecule (steric barrier effect), making it difficult to enter the binding pockets and difficult to bind with receptors. However, in reality in the body, most of the ether and ester after penetrate in the cell will undergo hydrolysis. So that in this study the 5-O-Methyl-Luteolin molecule is undergoes hydrolysis to become a Luteolin molecule which has better bonding energy (lower) and better bond affinity (higher). 14

In Vitro Antioxidant Test
The test for the antioxidant activity of the ethanol extract of Plectranthus amboinicus (Lour.) Spreng leaves and pure flavonoid compounds contained in the ethanol extract of Plectranthus amboinicus (Lour.) Spreng leaves was carried out by the free radical scavenging method. The use of the compound 1,1-diphenyl-2picryhydrazil is a free radical that has an unpaired nitrogen atom. The reaction between 1,1-diphenyl-2-picryhydrazil with hydrogen atoms in the antioxidant will cause a color change from purple to yellow. The results of antioxidant activity of the extract and pure flavonoid compounds can be seen in Table 3.
The results showed that the Inhibitory Concentration 50% value of the extract was higher than the pure compound, but this difference was not significantly different from the Inhibitory Concentration 50% value compared to pure compounds. These results indicate that the antioxidant activity of the extracts and pure compounds is similar. So that the use of extracts is very effective and efficient in terms of acquisition and price, because pure compounds have a higher price because the isolation process is longer. 15 Inhibitory Concentration 50% is a number that can indicate a concentration that can inhibit 50% of free radical activity. 15 Inhibitory Concentration 50% is used to compare the antioxidant activity so that the Inhibitory Concentration 50% value is inversely proportional to the ability of antioxidants to reduce free radicals. The lower the Inhibitory Concentration 50% value, the higher the antioxidant activity.