Antioxidant and antibacterial activity test on a crude extract of sand (Holothuria atra) in Lemukutan water, Bengkayang District

Sea cucumbers are soft-bodied marine invertebrates from the Holothuroidea class whose habitat is in sandy areas. This study aims to determine the content of secondary metabolites in crude extracts of sea cucumber which have the potential as antioxidants and antibacterials. The type of sea cucumber used in this research is the sand sea cucumber (Holothuria atra). The research was carried out from October 2020-February 2021. Samples were taken using the exploratory method, the sea cucumber samples were processed with 5 treatments, namely cleaning and crushing sea cucumber samples, extraction using the maceration method, evaporation with a vacuum rotary evaporator, and drying using freeze-drying. The bioactive components found in sea cucumbers are alkaloids, flavonoids, saponins, and phenols. The antioxidant activity found in sea cucumbers has an IC50 1194 ppm which is classified as a weak antioxidant. The antibacterial activity found intraday sand sea cucumbers E. coli gave the highest inhibition zone at a concentration of 20% with an inhibition zone diameter of 20.08 mm while S. aureus gave the highest inhibition zone at a concentration of 30% with an inhibition zone diameter of 22.17 mm.


Introduction
Lemukutan Island is one of the islands in Bengkayang Regency, West Kalimantan [1]. The waters on this island have characteristics suitable for the survival of sea cucumbers. The most common sea cucumber found on the island is the sand sea cucumber (Holothuria atra). Sea cucumbers have a lot of potential, one of which is that their secondary metabolites can be used as natural antioxidants and antibacterials. Identification of secondary metabolite content is an important first step in the process of identifying new bioactive compounds from natural ingredients that can be precursors for the synthesis of new drugs or prototypes of drugs with certain activities [2,3]. The method that is generally used to determine secondary metabolites in sea cucumbers is to perform phytochemical screening, then proceed with the antioxidant activity test using the DPPH method and determine its antibacterial activity using the method paper disk. The increasing use of synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) in the long term will cause IOP Publishing doi: 10.1088/1755-1315/1221/1/012064 2 bad side effects for humans [4]. The addition of BHT in foodstuffs is thought to cause cancer and gene mutations in humans [2]. Therefore, there is a need for breakthroughs that can be used to produce antioxidants and antibacterials derived from natural ingredients.
The formulation of the problem in this study is that the use of synthetic antioxidants in foodstuffs is suspected to cause cancer and gene mutations in humans and synthetic antibiotics in overcoming various diseases caused by bacteria have begun to cause new problems because most the antibacterial materials used are hazardous chemicals and are characterized by their nature [5]. Unsafe for health.
The purpose of this study was to determine the content of secondary metabolites in the crude extract of sea cucumbers in the Lemukutan waters of Bengkayang Regency, to determine the antioxidant potential of the crude extract of sea cucumbers in the Lemukutan waters of Bengkayang Regency, and to determine the antibacterial potential of the crude extract of sea cucumbers in the Lemukutan waters of Bengkayang Regency using the test bacterium S.aureus and E.coli.

Time and place
This research was conducted from October 2020 to February 2021 which included sampling in Lemukutan Waters, Bengkayang Regency, and data collection and data analysis carried out at the Marine Science Laboratory, SKIPM Laboratory, UPT-PMHP laboratory, and Chemistry Laboratory, Faculty of Mathematics and Natural Sciences, University of Tanjugpura, Pontianak, West Kalimantan.

Sea cucumber extraction.
Frozen sea cucumber samples were melted at room temperature, then weighed 500 grams and cleaned with running water, and drained. The drained sample was then crushed using a blender. The maceration used methanol as a solvent and was macerated for 3 times 24 hours at room temperature (the ratio of the sample and the solvent was 1:1). The result of maceration in the form of a solution is then filtered with filter paper to obtain the filtrate. The filtrate obtained was then evaporated using a rotary vacuum evaporator at a temperature of 40 o C and a speed of 90 rpm until it thickened into a crude extract. After thickening and becoming a crude extract, it was dried using a freeze dryer at a temperature of -40 o C

Identification of secondary metabolites
 Flavonoid test: a total of 1 mL of the sample was added with 0.05 mg of Mg powder, then 3 drops of HCl were added. The test result is positive if the solution is red light, orange, red, or purple.  Phenol test: a total of 1 mL of the sample was put into a test tube and the FEC added 2 drops of 1%. Green/blue colors that change to red, green, purple, blue, and black strongly indicate the presence of phenolic compounds in the test material:  Alkaloid test: a total of 1 mL of the sample was put into a test tube and then added 3 drops of Meyer reagent by seeing a white or yellow precipitate, 3 drops of Wagner by looking at a brown precipitate and 3 drops of Dragendorff with a brownish-orange or brick red precipitate, if there is such a precipitate the sample is declared positive.  Tannins test: 1 mL of crude extract was put into a test tube, then 0.5 mL offer was added 1%.
The results of the tannin test are positive when a blue or black color is formed.  Saponin test: extract 1 mL coupled with 10 mL of H2O heat. The solution was shaken vigorously vertically for 10-15 minutes. Saponins are indicated by the formation of stable foam as high as 1-10 cm for 10 minutes and do not disappear when 1 drop of 2 N HCl is added.  Triterpenoids/steroids test: as much as 1 mL of the sample was added with 3 drops of Liebermann-Burchard reagent, if there was a green to blue color, it was declared positive.

Antioxidant activity iest
 DPPH solution preparation: preparation of 0.2 mM DPPH solution was carried out by weighing ± 0.000789 g of DPPH then dissolved with 10 mL of analyzed methanol then homogenized and placed in a dark bottle.
 Preparation of blank solution: the blank solution was made by pipetting 1 mL of 0.2 mM DPPH solution then put into a test tube and then made into 5 mL using pro-analytical methanol solvent, then homogenized.
 Preparation of test solution: preparation of the test solution was carried out by weighing 10 mg of crude sea cucumber extract using an analytical balance, then dissolved with proanalytical methanol to 10 mL (1000 ppm). This solution is the mother liquor. Then pipette 250 l, 500 l, 1000 l, and 2000 l of the mother liquor into a 5 mL test tube to obtain sample concentrations of 50 ppm, 100 ppm, 200 ppm, and 400 ppm.
 Preparation of vitamin C solution (positive control): Weighing 10 mg of vitamin C, then dissolved in methanol pro-analysis to 10 mL (1000 ppm) this solution is the mother liquor. Then 20 l, 30 l, 40 l, and 50 l of mother liquor were pipetted into a 5 mL measuring flask to obtain concentrations up to 4 ppm, 6 ppm, 8 ppm, and 10 ppm.
 Determination λmak DPPH: making a solution to get the maximum wavelength is pipetting 1 mL of 0.2 mM DPPH solution then put into a test tube and then made into 5 mL using preanalytical methanol solvent, homogenized and left for 30 minutes, then measured absorption 400-800 nm using a UV-Vis spectrophotometer.
 Antioxidant activity test with DPPH method: test solution and the positive control incorporated into each -each test tube and then added 1 mL of DPPH solution was then added methanol pro analysis of up to 5 mL, then homogenized with the reference solution, The result of the % resistance is made into a linear regression equation by entering the concentration value of the test solution as the x-axis and % resistance as the y-axis after which IC is calculated50 by entering the % resistance of 50% in the linear regression equation.

Antibacterial activity test
 Making nutrient agar : enter 20 grams of powder nutrient agar (NA) into an Erlenmeyer flask then dissolve it with 1000 mL of distilled water, then heat and stir until dissolved on a hotplate. After that, they were wrapped in plastic puppets and then sterilized in an autoclave at 121°C for 15 minutes.  Test Bacterial rejuvenation culture: the isolates of E. coli and bacteria S. aureus were inoculated as much as 1 ose into the nutrient broth (NB), then incubated using a rotary shaker for ± 12 hours, speed of 80 rpm at 28 o C. The antibacterial activity required fresh bacterial colonies.  Antibacterial test with paper disc diffusion method: crude extracts of sea cucumbers were made with various concentrations of 40%, 30%, 20%, and 10%. Antibacterial activity testing was carried out by soaking the paper discs in the crude extract of sea cucumbers in each concentration variant, then drying them in the air. Furthermore, the paper disc was placed on the surface of the medium Nutrient agar (NA) which already contained the test bacteria, in this case, E. coli and bacteria S. aureus, and then incubated at 37˚C for 24 hours. The clear area formed is then measured in diameter using a caliper.

Extraction components of coarse extract of sand sea cucumber (H.atra)
The crude sea cucumber (H.atra) extraction results in extract yields. Extract yield is a comparison of the total weight of the resulting extract with the initial weight of the extracted sample, this is to determine the value of the bioactive components contained in the material [6].   (2017), Alkaloids can be used as antibacterial [6].
The test results in this study were positive for flavonoids in the crude extract of sea cucumbers (H.atra). The positive test for flavonoids was indicated by the formation of orange color in the tested extract. According to Rustamaji (2017), the flavonoid group has antioxidant activity [6].
The results of this study indicate that the triterpenoid/steroid content is negative. This is supported by research by Akerin and Sangaji (2019) Analysis of Phytochemicals and Toxicity and Antioxidant Activities and Several Types of Sea Cucumbers in Kakara Village, North Halmahera where the triterpenoid/steroid test results were negative [8]. However, positive results were found in the research of Fitriana and Jayuska (2014), extracts on Holothuria scabra gave positive results for triterpenoids/steroids [9]. The difference in the results of the triterpenoid/steroid test was due to the different types of sea cucumbers. In this study, the sea cucumber used was Holothuria scabra.
The results of the crude sea cucumber extract (H.atra) produced foam which indicated that the saponin content was positive. According to Yanuartono et al (2017) Saponins have broad activities such as antibacterial and antifungal [10].
The test results in this study were a change in color to red in the crude extract of sea cucumbers (H.atra). According to Zuraida et al (2017) phenol has antioxidant activity [11].
The results of this study showed that in the crude extract of the sea cucumber (H.atrathere was no change), indicating that the tannin content was negative. The results of this test were also found in the research by Akerin and Sangaji (2019) [8]. Phytochemical analysis and the toxicity and antioxidant activity of several types of sea cucumbers in Kakara Village, North Halmahera had negative results on the tannin test. However, positive results were found in the study of Ceesay et al (2019) extract on the body wall of the sea cucumber H. leucospilota [12]. The difference in the results of the tannin test was due to the different types of sea cucumbers used during the extraction process. concentration of 400 ppm is equal to 15.23%, while the lowest value of inhibition contained at a concentration of 50 ppm is equal to -0.514%.  [13]. This is due to differences in sampling locations.
In this study, the positive control used was vitamin C. The IC50 for vitamin C was categorized as very strong. In research Oktaviani et al (2015) about antioxidants on offal H.atra with a value of 2.33 ppm [2]. The test results of the positive control antioxidant activity can be seen in Table 3. The results of inhibition of vitamin C increased with increasing concentration. The highest inhibition result was at a concentration of 10 ppm with a value of 38.61%, while the lowest inhibition value was at a concentration of 4 ppm with 17.51%. The IC50 for vitamin C is 12.96 ppm. This value belongs to the very strong category.

Antibacterial activity test
This study tested the antibacterial activity in inhibiting the bacteria E.coli, and S.aureus using the diffusion method, paper disc namely paper discs with a diameter of 5 mm. The negative control used was 10% DMSO solution, because 10% DMSO did not provide antibacterial activity against bacteria. DMSO 10% is not toxic so it does not interfere with observations [14]. The positive control used was 0.7% tetracycline. Tetracycline is an antibiotic that can interfere with the protein synthesis process and is the antibiotic of choice that can inhibit both gram-positive and gram-negative bacteria. The results showed that the crude extract sand sea cucumber (H.Atra) has the antibacterial activity of E. coli and S.aureus. So it can be proved that the sea cucumber extract coarse sand (H.Atra) has the ability to be an antibacterial. Sea cucumber extract coarse sand (H.Atra) is active as an antibacterial due to the chemical components contained in the extract. According to Rahmadani (2015), phytochemical screening of extracts containing flavonoid glycosides, saponins, tannins, and phenols could potentially have antibacterial activity [15]. Harbone (1987) also stated that chemical compounds that have the potential as antibacterial are flavonoids, saponins, steroids, glycosides, tannins, and phenols [3].   (2012) that the inhibition zone >20 mm is included in the very strong inhibitory response, the 11-20 mm inhibition zone is included in the strong inhibitory response, the 5-10 mm inhibition zone is included in the moderate inhibitory response and the inhibition zone >5 mm is considered a weak inhibitory response [16].
Several factors affect the diameter of the zone of inhibition of bacterial growth. According to Zeniusa et al (2019), the turbidity of the bacterial suspension [17]. If the suspension is less turbid, the diameter of the inhibition zone will be larger and vice versa if the suspension is more turbid, the diameter of the inhibition zone will be smaller. Incubation temperature can also be a factor that affects the diameter of the zone of inhibition of bacterial growth. The thickness of the media is also a factor affecting the diameter of the zone of inhibition of bacterial growth. The thickness of the effective agar is about 4 mm. Different types of bacteria are also a factor that affects the diameter of the inhibition zone. In this study the bacteria used were E. coli and S. aureus, E. coli are gramnegative bacteria while S. aureus is gram-positive.

Conclusion
Based on the results obtained in this study, it can be concluded several things as follows, Samples of coarse sea cucumber extract (H.atra) contain 4 types of secondary metabolite components, namely alkaloids with Mayer reagent, Dragendorff's reagent and Wagner's reagent, flavonoids, saponins, and phenols. The antioxidant activity of samples of sea cucumber extract coarse sand (H.atra) with IC50 1194 ppm was classified as a weak antioxidant. Antibacterial activity in the crude extract of sea cucumber (H.atra) against E. coli gave the highest inhibition zone at a concentration of 20% with an inhibition zone diameter of 20.08 mm (strong category) while S. aureus gave the highest inhibition zone at a concentration of 30% with an inhibition zone diameter of 22.17 mm (very strong category).