ANTIBACTERIAL ACTIVITY OF METHANOL EXTRACT FROM INGGILI FRUIT ( Xylocarpus granatum ) AGAINST Propionibacterium acnes AND Escherichia coli USING WELL DIFFUSION METHOD

Inggili is a type of mangrove plant that has been traditionally used by the community for medicine. It contains many active compounds that function as antibacterials. Based on empirical data, inggili fruit is used to remove acne on the skin and diarrheal diseases. This study aims to determine the antibacterial activity of methanol extract of inggili pulp in inhibiting Propionibacterium acnes and Escherichia coli bacteria using the sumuran diffusion method. The research conducted is experimental. The object under study was the antibacterial activity of inggili fruit extract against Propionibacterium acnes and Escherichia coli bacteria. Antibacterial activity was tested using the sumuran diffusion method with extract concentrations of 10%, 15%, and 20%. The negative control used was 1% DMSO and the positive control used was 0.1% chloramphenicol.


Introduction
Mangrove plants are one of the plants that can live in extreme environments, where the physical and chemical properties of their habitats always change as a result of tides, freshwater or rivers, silt deposition, decomposition of organic matter, and others so that this plant has excellent potential for research on secondary metabolite compounds it contains (Fahlifi et al., 2018).
Secondary metabolites are defined as compounds synthesized by organisms such as microbes, plants, insecticides, and so on (Maradona, 2013).Xylocarpus granatum mangroves have seeds, fruits, and tree bark that are useful as drugs for various types of diseases because they contain secondary metabolite compounds (Kumoro, 2015).The content of flavonoids, tannins, and saponins prevents the risk of skin hyperpigmentation due to exposure to ultraviolet radiation from sunlight.Phytochemical components in bark and stem extracts, both consist of alkaloids, flavonoids, and tannins (Hendrawan et al., 2015;Adelberg et al., 2008).
Propionibacterium acnes is a normal bacterial flora of the skin but can be the cause of opportunistic infections in the form of acne (Nakase et al., 2017).According to the Indonesian cosmetic dermatology study, there was an increase in the incidence of acne which showed 60% of acne sufferers in 2014, 80% in 2015, and 90% in 2016(Zahrah et al., 2019)).
Escherichia coli is a gram-negative bacterium, a normal flora germ found in the ORIGINAL ARTICLE human large intestine.Escherichia coli often causes infections of the urinary tract, bile ducts, and other places in the abdominal cavity.Escherichia coli is also a cause of diarrhea and urinary tract infections (Erlangga, 2016).
The use of good natural materials as topical therapy, one of which is mangrove plants, namely inggili plants.Based on the research of (Gabariel et al., 2019), the results of the inhibitory test of inggili plant extracts (leaves, fruits, stems) have antibacterial inhibitory against the growth of pathogenic bacteria (P.aeruginosa, E.coli, and V.alginolyticus).Although the resistance zone formed differs markedly for each extract concentration, the resulting inhibitory power is directly proportional to the increase in concentration.The higher the concentration, the higher the bacterial resistance zone formed.This is thought to be because with every increase in concentration, the amount of bioactive compounds increases so that the ability to inhibit bacterial growth increases.
Based on the above background, it is necessary to do a study testing the antibacterial activity of methanol extract of inggili fruit against Propionibacterium acnes and Escherichia coli bacteria using the sumuran diffusion method.

Materials and Methods
The peel of the fruit as much as 2 kg obtained is carried out wet sorting, aiming to separate the skin of the fruit from the seed.Washed using running water to separate impurities attached to the fruit, then cut to reduce the sample size, so that the drying process is faster than dried by aerating the aim is to reduce the moisture content so that simplisia is not easily damaged and can be used in the long term.Simplisia that has been dried is ground into coarse powder using a blender and sifted using a mesh 60 sieve and 400 mg of fine powder is obtained.This research stage starts with determination, collection of raw materials, making simplisia, and making extracts with methanol solvents, to determine antibacterial activity.The study was conducted in the laboratory of the Samarinda College of Health Sciences from April to June 2023.

Extraction
The skin of the inggili fruit is taken on a large fruit.The fruit skin used in this study was obtained from Tepian Village, Sembakung District, Nunukan Regency, North Kalimantan Province.Weighing fruit simplisia 300 g of simplisia powder was extracted by maceration using 2 L of methanol solvent.Stirring was carried out for the first 2 hours, then soaked for 24 hours and filtered.Remaceration is carried out with 1 L of methanol stirred and soaked for 24 hours.All the maserat obtained is evaporated until a viscous extract is obtained (Mukhriani, 2014).

Flavonoid
A total of 5 drops of filtrate were added 0.1 g Mg powder and concentrated hydrochloric acid 1 mL, amyl alcohol 2 mL, shaken and left to separate.28 Flavonoids are positive if red, yellow, and orange colors form in the amyl alcohol layer (Pardede et al., 2013).

Tanin
A total of 10 drops of filtrate, added aquades until the color fades, added 2-3 drops of FeCl reagent, if a blue or blackish-green color is formed, it shows the presence of tannin compounds in the extract (Dewatisari et al., 2018).

Saponin
A total of 10 drops of filtrate, and 5 drops of hot water are added and then cooled, then shaken vigorously for ± 10 seconds.If a lot of foam is formed for 10 minutes as high as 1 cm to 10 cm does not disappear with the addition of hydrochloric acid 2N 1 drop, then it indicates the presence of saponin compounds (Pardede et al., 2013).

Antibacterial activity
Antibacterial activity testing used several concentrations with two kind of bacteria, which include Propionibacterium acnes and Escherichia coli.Antibacterial activity method following the previous reported research (Mpila et al., 2012;Nugroho and Widayati, 2013).

Results and Discussion
Extraction has been carried out and produced methanol extract from the skin of the inggili fruit.Data from phytochemical screening showed that the extract contained flavonoid compounds and alkaloids, but specifically, alkaloids were only positive using dragendrof tests while alkaloid tests with other reagents gave negative test results.This indicates that the alkaloid content in the extract is not able to be detected properly because the intensity or quantity of alkaloids contained in the extract is only small.Other tests such as tannins and saponins showed negative results or the extract did not contain these compounds (table 1).This phytochemical screening data shows that the compounds responsible for antibacterial activity are flavonoids because alkaloids are difficult to detect due to their small intensity or quantity in the extract.
Flavonoids are polar compounds that dissolve in polar solvents such as methanol (Marjoni, 2016).Flavanoids have effective properties to inhibit bacterial growth by damaging the bacterial cell membrane in the phospholipid section to reduce permeability which causes bacteria to be damaged so that it affects the inhibitory zone formed from antibacterial activity tests.

Antibacterial Activity
Antibacterial activity was carried out with several concentrations, which aimed to determine the sensitivity of the methanol extract of Inggili fruit peel to the growth of Propionibacterium acnes and Escherichia coli 105 1 bacteria.This method was chosen because it has the advantage that the procedure is simple, easy, and practical to perform can also be used to see the sensitivity of various microbes to antimicrobials at certain concentrations and is often used in antibiotic sensitivity tests in quality control programs (Mpila et al., 2012;Melliawati, 2014;Mulia, 2018).
In this study, antibacterial testing used several series of concentrations of methanol extract of inggili fruit peel which aimed to determine the effect of each concentration on Propionibacterium acnes and Escherichia coli bacteria.Methanol extract from inggili fruit peel is made into 3 different concentrations namely 10%, 15%, and 20%.The positive control used in this test was chloramphenicol while the negative control used was DMSO 1%.This antibacterial activity test uses the sumuran diffusion method, the reason for choosing this method is because it has advantages that are easy to do and relatively cheap.The results of the antibacterial activity test of methanol extract of inggili fruit against Propionibacterium acnes and Escherichia coli bacteria with concentrations used namely 10%, 15% and 20% can be seen in Table 2.The parameter measured in the antibacterial activity test is the formation of an inhibitory zone around the well that has been inserted with a solution of methanol extract concentration of inggili fruit peel (figure 1).The antibacterial activity test showed that Propionibacterium acnes bacteria with an extract concentration of 10% had an inhibitory zone diameter of mm 14.3 and at an extract concentration of 15% an inhibitory zone diameter of 15 mm was formed and at an extract concentration of 20% the diameter of the inhibitory zone obtained was 15 mm.The results of antibacterial activity tests show that the skin of the inggili fruit also has activity as an inhibitor of Escherichia coli bacteria, at a concentration of 10% the extract of the inggili fruit has an inhibitory zone diameter of 14.3 mm, and at a concentration of 15% has an inhibitory zone diameter of 14.6 mm, and at a concentration of 20% the diameter of the inhibitory zone is 15.3 mm.Antibacterial activity increased with increased concentrations from 10%, 15%, to 20% but still below the antibacterial activity of positive controls.

Compliance with ethical standards
Meyer reagent of 5 drops of filtrate, put into a test tube added 2-3 drops of HCl 2N and 2 drops of Meyer reagent.When a white or yellow precipitate is formed, it indicates the presence of alkaloid compounds.Bounchardat reagent of 5 drops of filtration, put into the test tube added 2-3 drops of HCl 2N and 2 drops of Bounchardat reagent.If a brown-to-black precipitate is formed, it indicates the presence of alkaloid compounds.Dragendrof reagent as much as 5 drops of filtrate, put into a test tube added 2-3 drops of HCl 2N and 2 drops of dragendrof reaction.If an orange-to-red-brown precipitate is formed, it indicates the presence of alkaloids (Pardede et al., 2013).

Figure 1 .
Figure 1.Inhibition Zone of Methanol extract of Inggili Fruit Skin against a.Propionibacterium acnes and b.Escherichia coli

Table 1 .
Phytochemical Screening Results of Methanol Extract of Inggili Fruit

Table 2 .
Results of bacterial inhibition zones of Propionibacterium acnes and Escherichia coli