Antioxidant Activity Evaluation from Tomatoes’ N- Hexane, Ethyl Asetate, and Water Fraction with DPPH

Antioxidants are compounds that can stabilize free radicals in the body. Free radicals are highly reactive molecules as they have unpaired electrons to interact with body cell molecules. Tomatoes contain flavonoids, saponins, solanine tannins, folic acid, malic acid, citric acid, protein, fat, vitamins, minerals, and histamine, which can be used as antioxidants. This study aims to evaluate the antioxidant activity of n-hexane, ethyl acetate, water fraction, and ethanol extracts of Tomatoes and to determine the greatest antioxidant activity between n-hexane, ethyl acetate, water and vitamin C. Tomatoes (Lycopersicum esculentum Mill.) was extracted using the maceration method with ethanol followed by fractionation using nhexane and ethyl acetate solvents. The test of antioxidant activity to DPPH radical was conducted on n-hexane, ethyl acetate, water, and vitamin C. The antioxidant activity results, expressed by IC50 value to the n-hexane, ethyl acetate, water fraction of Tomatoes fruit, were 4.4603 ppm; 4.0868 ppm; and 4.0527 ppm, respectively. Thus, the greatest antioxidant activity was the water fraction.


INTRODUCTION
Tomato is one type of fruit with polyphenolic compounds, carotenoids, and vitamin C, which can act as antioxidants. 1 Tomatoes contain a carotenoid compound called lycopene. 2 Atherosclerosis is strongly related to Low-Density Lipoprotein (LDL)-cholesterol. Unsaturated lipid acid components in the LDL-Cholesterol are easy to bind with free radicals and transform into oxidative LDL. It triggers sponge cells, releases reactive oxygen species (ROS), and induces oxidative stress. ROS can form * Corresponding author, e-mail: sariratna339@gmail.com atherosclerosis, plague, lipid oxidation, and endothelial disorder. Simultaneously oxidative stress will decrease the immune response to keep the body healthy. It can be avoided through high antioxidant food consumption.
The antioxidant is easily obtained from various foods containing vitamin E, vitamin C, polyphenol, and carotenoid, especially lycopene and β-carotene. Tomato is one of the abundant and affordable fruit or vegetables. Tomato (Lycopersicum esculentum Mill.) is a species from the Solanaceae family. It contains a lot of natural antioxidants, such as vitamin E, vitamin C, and carotenoid. Based on the chemical composition, carotenoid is divided into two chemical compound groups, namely, carotene and xanthophyll. The most abundant carotene in tomatoes is lycopene and β-carotene. 3 Carotene is an antioxidant that can reduce malignant cells of cancer. Of all these carotenoid compounds, it turns out that lycopene is relatively more efficient as a singlet oxygen scavenger than other carotenoids (higher than beta-carotene and alphatocopherol). 4 Singlet oxygen is a nonradical electrophilic ROS. 5 Lycopene is a special carotenoid that is potential to prevent prostate cancer and degenerative cardiovascular disease and anti-aging. 6 Lycopene is the main carotenoid in tomatoes which is a powerful antioxidant and has received much attention as it is associated with a lycopene-rich diet and reduces the risk of heart disease, cancer and disease in old age. 7 Aging is a biological process that occurs naturally and affects all living things, including all body organs such as the heart, lungs, brain, kidneys, and skin. 8 The community prepares various techniques of tomato processing. Some people eat fresh tomatoes directly without any treatment. Meanwhile, most Indonesian people always cook tomatoes in various processing, such as steaming, boiling and frying. The cooking process techniques are predicted to impact antioxidant amounts inside tomatoes. Understanding the antioxidant capacity found in tomato fruits requires studying the antioxidant activity using the DPPH method to identify the antioxidant compounds in the tomato sample.
The research on samples of fruit tomato leaves (Lycopersicon esculentum Mill, var. Pyriforme Alef.) and vegetable tomato leaves (Lycopersicon esculentum Mill, var. Commune Bailey.) showed that it has antioxidant activity with IC50 values of 279.482 µg each. / mL and 280.190 µg / mL. It indicated the ethanol extract of fruit tomato leaves (Lycopersicon esculentum Mill, var. Pyriforme Alef.) and vegetable tomato leaves (Lycopersicon esculentum Mill, var. Commune Bailey.) have very weak activity as antioxidants. 9 Lycopene can hinder endometrial cancer growth, breast cancer, and lung cancer on cell culture with a higher activity than α and beta-carotene. 10 Generally, tomatoes have high antioxidants. However, high temperatures can reduce the antioxidant amount. 11 The vitamin C was measured using ascorbic acid colorimetric assay kit, and the number of α-tocopherol analyses was performed using an ELISA kit for αtocopherol. The antioxidant activities were measured by using 1-1-diphenyl-2pic-1-1-diphenyl-2-pic-rylhydrazyl (DPPH) and stoichiometry method. 12 DPPH provided maximum absorption on 516 nm wavelength and resulted in purple color. 13 This method was utilized due to its simple, easy, rapid, sensitive ability and only involved a minimum sample. 14

METHOD
This research used 14 kg tomatoes (Lycopersicum esculentum Mill.) obtained from Desa Sepanjang, Tawangmangu, Karanganyar. Centra Java, Indonesia and were collected through purposive random sampling. The tomatoes' criteria included ellipse form, red color, and approximately weighted 1,5-2 ons, fresh and peel. Tomatoes processed in 6 different ways were then extracted by the same technique. This experimental study was conducted at the Laboratory of Biochemistry, Department of Biology, Universitas Ahmad Dahlan. This research used a complete random design with a post-test Only Randomized Controlled Group Design.

RESULTS AND DISCUSSION
The results of data analysis showed that all data were normally distributed and homogeneous. The data were then tested with IC50 (Inhibition Concentration 50). Phytochemicals The below compounds were identified to determine the chemical compounds in tomatoes. Phytochemical screening was carried out to determine the secondary metabolites in the extract and powder on tomatoes. Secondary metabolites tested qualitatively included alkaloids, flavonoids, saponins, tannins, steroids/triterpenoids.

Fractionation
The results of the extraction of tomato fruit samples (Lycopersicum esculentum Mill.) were then fractionated. Fractionation was carried out to separate a class of compounds from other groups based on the difference in polarity of the solvent. N-hexane, ethyl acetate and water were the solvents used in fractionation in this study.

Antioxidants activity
Several methods can determine antioxidant activity. In this study, the method used was antioxidant testing with DPPH free radical scavenger. Antioxidant activity was characterized by trapping electrons by free radicals, which caused electrons in free radicals to become paired electrons, resulting in a color change from purple to yellow.

Inhibition Concentration 50
The results of the measuring absorbance were used to obtain the percentage of inhibition value. The antioxidant activity of the n-hexane fraction, ethyl acetate fraction, and the water fraction of the ethanol extract of tomato fruit can be expressed by IC50 (Inhibition Concentration 50). IC50 is the sample concentration needed to capture 50% of DPPH free radicals during operating time. The data from n-hexane, ethyl acetate, and water fractions were then calculated for the IC50 value using a linear regression equation based on the formula Y = a + bx. Furthermore, the water fraction had an IC50 value of 4.0527 ppm, indicating that the antioxidant activity of the water fraction was greater than the antioxidant activity of the n-hexane fraction due to its triterpenoid compounds, tannins, saponins, and flavonoids that might capture radicals.

CONCLUSION
Based on the results of this research, it can be concluded that the n-hexane fraction had an antioxidant activity with an IC50 value of 4.4603 ppm; the ethyl acetate fraction had an antioxidant activity with an IC50 value of 4.0868, and the water fraction from the tomato extract had an antioxidant activity with an IC50 of 4.0527 ppm. Therefore, the water fraction of the tomato fruit extract had the best antioxidant activity with an IC50 value of 4.0527 ppm.