The Ability Free Radical Binding of Dengen’s Stem Bark Extract (Dillenia serrata) From Luwu District Indonesia

Non-communicable diseases or degenerative diseases are diseases that cause damage to body organs. Some degenerative diseases that are mostly suffered are diabetes mellitus, hypertension, cancer, coronary heart disease (CHD), cardiovascular disease and lung disease. Based on WHO data in 2008 there were 57 million deaths in the world, mostly caused by non-communicable diseases. Degenerative diseases not only kill old people, they can also kill young people.1


INTRODUCTION
Non-communicable diseases or degenerative diseases are diseases that cause damage to body organs. Some degenerative diseases that are mostly suffered are diabetes mellitus, hypertension, cancer, coronary heart disease (CHD), cardiovascular disease and lung disease. Based on WHO data in 2008 there were 57 million deaths in the world, mostly caused by non-communicable diseases. Degenerative diseases not only kill old people, they can also kill young people. 1 One of the causes of degenerative diseases is free radicals. Free radicals are active electrons in the body that do not have a partner so that they can bind normal cells in the body and develop an abnormal cell called a tumor and / or cancer. 2 The binding of free radicals with normal cells in the body can be prevented by the presence of antioxidant ingredients. This antioxidant material can reduce free radicals so as to prevent binding with normal cells. Apart from chemicals, these antioxidant ingredients can also be obtained from natural ingredients. The government program for Back to Nature can be encouraged to find Indonesian endemic plants that contain secondary metabolites as a preventative and curative basis for disease.
Dengen (Dillenia serrata) is one of Indonesia's endemic plants that can only be found on the island of Sulawesi and its surroundings, especially in the area of Luwu Regency. Utilization of this plant is still limited to the fruit, which has been made in addition to juice and sour taste in cooking, has also been developed into dodol. Other parts of dengen plants that potentially contain secondary metabolites of polyphenols are the bark. Utilization of bark in the community, usually cooked with water, and drunk for patients with vomiting blood. [3][4][5] This is supported by the large content of polyphenols in dengen stem bark ethanol extract that is equal to 444.8 mg GAE / g or 44.48%. 6 Seeing the use and content of dengen stem bark above, the researchers will examine the ability of dengue skin ethanol extract in counteracting free radicals. The formulation of the problem in this research is how much is the ability of dengue stem ethanol extract in counteracting free radicals.

MATERIALS AND METHODS
The study was conducted in July -December 2019 at the Laboratory of Chemistry, Department of Pharmacy, Poltekes Ministry of Health, Makassar. The tools used are maceration vessels, analytical scales, rotary evaporators, glassware, UV-Vis spectrophotometers. While the ingredients used are dengen bark, 70% ethanol, 96% ethanol, DPPH, Vitamin C. Selected good stem bark, then washed with running water. The part is made dry simplicia. Dillenia serrata stem bark that has been pollinated is weighed and put into a maceration container, then added 70% ethanol until completely submerged. The extraction process is carried out for 5-6 days accompanied by stirring so that the extraction process is perfect. Filtering is done, if the solvent has been colored then the solvent is always replaced. The filtering results are evaporated to obtain a thick extract. Extraction was carried out 3 times.

Making a Standard Curve
As much as 4.0 mL of a 40 ppm DPPH solution was added with 1.0 mL of 96% ethanol to the vial, then the absorbance was measured in the wavelength range of 400 nm-800 nm. Obtained maximum wavelength at 516 nm.

Manufacture and measurement of Vitamin C solutions
Weighed 500 ppm standard vitamin C as much as 50 mg and was dissolved with 96% ethanol to 100 mL. A dilution series of Vitamin C solution was made from stock solutions which were 5 ppm, 10 ppm, 15 ppm and 20 ppm. Each concentration series was carefully piped as much as 1.0 mL and each of them was added 4.0 mL of a 40 ppm DPPH solution to the vial. The solution was incubated for 30 minutes, then the absorbance was measured at a wavelength of 516 nm.

Preparation of test solutions
Each extract extracted from the extracted replication was weighed carefully as much as 200 mg of the extract was put into a 20 ml volumetric flask diluted with 96% ethanol and sufficient to the mark (10,000 ppm). Then piped stock solutions as much as 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml, put into a 10 ml volumetric flask and sufficient volume with 96% ethanol to the mark, so that the solution is obtained with a concentration of 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm.
Each concentration series was carefully piped as much as 1.0 mL and each of them was added 4.0 mL of a 40 ppm DPPH solution to the vial. The solution was incubated for 30 minutes, then the absorbance was measured at a wavelength of 516 nm.

Data analysis
IC50 value is obtained by linear regression equation which states the relationship between extract concentration with the x-axis and the percent radical capture as the y-axis. % sample binding / inhibition of free radicals (DPPH solution). The percentage of free radical binding is calculated by the formula:

% Inhibition
After obtaining the percentage of inhibition of each concentration, then the equation y = a + bx is determined by calculating a linear regression where x is the concentration (ppm) and y is the percentage of inhibition (%). Antioxidant activity is expressed by 50% inhibition concentration or IC50, which is a sample concentration that can reduce DPPH radicals by as much as 50%. IC50 values obtained from the value of x after replacing y = 50. From the equation y = a + bx IC50 values can be calculated using the formula:

RESULT
From this study, the following data were obtained (Tables 1 and 2).

DISCUSSION
In this study, the determination of the ability to bind free radicals from Dengen stem bark ethanol extract (Dillenia serrata) originating from the Malangke area of Luwu Regency Indonesia by using a UV-Vis spectrophotometer. This plant is an endemic plant (Figures 1-3), which only grows in the Sulawesi islands. This plant contains many      secondary metabolites. Based on the use of the bark by the community as a medicine to vomit blood, while the fruit is usually eaten directly. But because of its sour taste, people use it as an acidic ingredient in food, for example in fish cooking. Based on this utilization, the ability to bind free radicals from the bark is examined. 2 The use of dengen plants as a medicine to vomit blood was allegedly due to the content of polyphenol compounds. Polyphenol compounds are useful for human health because they have antioxidant properties, free antiradical, anticarogenogenic, and antimicrobial properties so that they can inhibit food pathogens, antiproliferation and antimutagenic, can inhibit the oxidation of low density cholesterol compounds (LDL) on endhothelial cells, can increase high-density cholesterol (antiproliferation and antimutagenic), can inhibit the oxidation of low density cholesterol compounds (LDL) HDL), and can reduce triglyceride content. These polyphenol compounds will be responsible for the antioxidant activity of these parts. The higher levels of polyphenol compounds contained, the higher the antioxidant activity of these parts. In a previous study, 3 the total polyphenol content of dengen bark extract ethanol extract was 444.8 mg GAE / g or 44.48%.
Determination of the ability to bind to free radicals is expressed as an antioxidant activity. Potential antioxidant activity in vitro is carried out using DPPH reagents. The ability to bind to free radicals is illustrated by the reduction in color intensity of DPPH. DPPH has a purple color. The less intensity of the purple color, the higher the ability to bind free radicals. This measurement is carried out at a maximum wavelength of 516 nm. 4 DPPH is a stable free radical. The principle of this antioxidant activity is the occurrence of a hydrogen or electron donation process that reduces DPPH to diphenyl picrilhydrazine which is no longer radical. This change is seen in the intensity of the DPPH color changing from purple to yellow (pikril group). The change in DPPH color intensity is proportional to the number of electron donations followed by a decrease in DPPH absorbance in the wavelength range of 515 nm -520 nm. Absorption reads on the spectrophotometer at Wavelength 516 illustrate the remaining DPPH molecules contained in the solution.