Antibacterial test of Rhizophora apiculata extract originated from Mangrove Wonorejo Surabaya against Staphylococcus aureus and Escherichia coli

Rhizopora apiculata is a type of mangrove that has the potential as a source of antibacterial. This plant is used as medicine by coastal communities in Indonesia because it contains useful active ingredients. This research aims to study Rhizophora apiculata antibacterial potential against pathogenic bacteria Staphylococcus aureus and Escherichia coli . The samples used were Rhizophora apiculata leaves from Mangrove Wonorejo Surabaya. The research stages include extracting mangrove leaves, media preparation, and testing the antibacterial inhibition zone. The results of the research showed that the antibacterial test with Escherichia coli bacteria had a weak inhibition zone of less than 10 mm with details of a 0.1 ml sample around 5.35 mm and a 0.2 ml sample around 6.78 mm at 24 hour incubation, a 0.1 ml sample measuring around 7.12 mm and the 0.2 ml sample measuring around 6.98 mm at 48 hours incubation, and for the Staphylococcus aureus bacteria there was no zone of inhibition at 24 and 48 hours incubation for both the 0.1 ml and 0.2 ml samples.


INTRODUCTION
Mangroves are located in tropical and subtropical areas which are commonly called tidal forests.Mangroves are found in areas protected by sand dunes, islands, coral reefs and seagrass beds.Mangrove ecosystems thrive in protected areas such as beaches, bays and lagoons [1].Mangroves have been the subject of conservation studies because of the many natural products with beneficial activities and commercial values resulting from them.Mangrove habitat is a productive source of endophytic microorganisms.Mangroves' roots and leaves serve as a habitat for many bacteria, fungi, and algae [2].The benefits of mangroves for human life are to cure muscle pain, back pain, rheumatic malaria, wounds, tuberculosis and spermicides and benefits for the environment [3].
The biological function of mangrove forests will work perfectly if their existence is maintained and sustainable.Several types of mangrove leaves that have been identified as bioactive as producing antiviral, antioxidant, antibacterial, bactericidal and fungicide sources and their effectiveness have been tested against bacterial diseases in a laboratory.One of the mangrove plants that needs to be preserved is Kopasanda and Eupatorium inulifolium.Kopasanda leaf extract can function as a bactericidal against vibrio bacteria [4].The indirect benefits of mangroves for fisheries as a food supply for marine communities through the detritus food chain which starts from all mangrove leaf litter.The decomposition of organic matter, also called the mineralization process, is a microbial process (decomposer) in obtaining energy for its propagation [5].
Mangrove leaves contain bioactive compounds such as antioxidants, anti-bacterial and anti-aging [6].In addition, compounds that can protect free radicals by inhibiting the initiation or spread of oxidation chain reactions [7].Mangrove is a plant that is rich in bioactive compounds, these compounds do not always come from the mangrove plant itself, but from other organisms that synthesize bioactive compounds in the mangrove section.The presence of bacteria in the plant body is important because it affects the physical, chemical and biological properties of the soil.The diversity of mangrove endophytic bacteria indicates that mangrove plants are a rich source of isolated endophytic bacteria, namely Enterobacter sp.UB-R [8].Bacteria that are beneficial to these plants are types of bacteria that provide many benefits to host plants and help withstand various biological and abiotic stresses that can affect growth.Bacteria that live outside the host plant are epiphytes that live on the surface of plant leaves or root balls that live on plant roots in the soil.Bacteria that live and reproduce in the host plant are called endogenous bacteria, but all of these classes of bacteria share many essential properties that promote the growth of the host plant [9].
Most parts of the Rhizophora apiculata, or often called oil mangrove, are used as medicine by coastal communities in Indonesia because they contain useful active ingredients.This plant is rich in alkaloids, flavonoids, triterpenoids, steroids, saponins and tannins.There are many sources that we can use, starting from the stems, roots and bark of Rhizophora apiculata, all of which contain natural antioxidants [10].Mangrove Rhizophora apiculata has several bioactive compounds and is widely used as various preparations.Therefore it is necessary to know the antibacterial contained in the mangrove leaves of Rhizophora apiculata.This research 6 goals is to study the antibacterial activity of Rhizophora apiculata extract from the Mangrove Wonorejo Surabaya against Escherichia coli and Staphylococcus aureus bacteria.

METHODS Location and Time of Research
This research is an experimental observational research conducted in December 2022 at the Laboratory of the Faculty of Science and Technology, Universitas Islam Negeri Sunan Ampel, Surabaya.Sampling of Rhizophora apiculata mangrove leaves was carried out in the Wonorejo Surabaya mangrove ecosystem.

Tool Sterilization
The tools used in this study were sterilized beforehand.Glassware and media (NA, NB, and MHA) were sterilized in an autoclave at 121°C for 1 hour in solid mode (wet sterilization).

Media Preparation
For the antibiotic test, solid media are needed, one of which is NB media for the cultivation of the test bacteria.The powdered NB medium was weighed as much as 1.95 grams and put into an Erlenmeyer then added 150 ml of distilled water.Mix and cover with cotton, aluminum foil, and plastic wrap.Then, heat on a hot plate until homogeneous.After that, it is sterilized by wet sterilization.If it has been sterilized, the media solution is poured into a test tube and covered with aluminum foil and plastic wrap.Meanwhile, NA and MHA media are poured into petri dishes for cultivation or planting.

Mangrove Leaves Extraction
Mangrove leaves extraction are shown in Figure-1.Mangrove leaves that have been prepared are first washed thoroughly, then dried with the help of an oven at 100°C for 24 hours.The dried mangrove leaves were crushed using a mortar pestle, then sifted and weighed as much as 40 grams.The extraction process is carried out on the refined leaves.Mangrove leaf extraction was carried out by maceration method.Maceration is done by soaking mangrove leaf powder using ethanol solvent until submerged for 1×24 hours, 1×17 hours, and 1×7 hours.Mangrove leaves were weighed as much as 40 grams and then put into the maceration container, then soaked with ethanol until submerged.The next stage is the vacuum process and then evaporated using a rotary evaporator at 70°C to produce a thick mangrove leaf extract or paste.The evaporation process is carried out until a viscous extract (paste) is obtained which is characterized by no more solvent dripping into the holding flask.Vacuum and evaporation processes were also carried out at 1×17 hours and 1×7 hours of maceration.After it becomes a paste, the mangrove leaf extract is placed in a vial and combined with the evaporation results from 1×17 hours and 1×7 hours of maceration, then placed in the freezer before further treatment.

Inhibition Ability Test and Inhibition Zone Diameter Measurement
The extraction results were planted in combined media with 2 extracted samples (0.1 ml and 0.2 ml) each in 1 place on 2 petri dishes with symmetrical spacing.The round-shaped filter paper is dipped into the mangrove leaf extraction solution (previously dissolved with 20% DMSO solvent), then placed on the right and left of the petri dish that has been given Staphylococcus aureus bacteria and the petri dish which already has media and previously divided into 2 parts.This was also done for Escherichia coli bacteria for the next petri dish.After that incubated at room temperature (25°C) (Figure -2).The inhibition zone of each sample of mangrove leaf extraction was observed after 1×24 hours and observed again 1×24 hours later.Measurement of the inhibition zone was carried out using a digital caliper by inverting the petri dish and measuring the diameter of the clear area.

RESULTS AND DISCUSSIONS
Based on the experiment that has been carried out, the results of marine natural products from Rhizopora apiculata extraction and antibacterial tests can be seen from the measurements of the bacterial inhibition zone that has been made on the media in a petri dish.
The first step is to prepare tools and materials and then sterilize them.Then weigh 1.95 grams of NB media and put it in an Erlenmeyer and pour 150 ml of distilled water.Next, cover the Erlenmeyer with cotton, aluminum foil and plastic wrap and then label it on the Erlenmeyer.Then heated on a hot plate until homogeneous.

Figure-2. Inhibition ability test process
Then several tools that have not been used and the media solution are put into the autoclave for 1 hour at 121°C in solid mode.After that, the tool in the autoclave was removed and the petri dish was opened, then the MHA and NA media solution was poured into the petri dish, then waited until it was slightly frozen and covered with plastic wrap.After that, pour the NB media solution into the test tube and wait for it to freeze slightly then cover it with aluminum foil and plastic wrap.
At the extraction stage, the mangrove leaves Rhizophora apiculata were used.First, the selected Rhizophora apiculata mangrove leaves are dried and placed on a baking sheet in an oven for 24 hours at 100°C.Furthermore, the dried mangrove leaves were mashed with a mortal pestle and then weighed as much as 40 grams and put into a 500 ml Erlenmeyer.Then added ethanol until submerged then covered with aluminum foil and plastic wrap after that it was labeled on the Erlenmeyer and waited for 1×24 hours.Furthermore, the solution was poured to be vacuumed and then rotated until a paste extract was formed.The extracted paste that was formed was taken and put into a vial and put in the freezer.Then it was macerated again with ethanol for 17 hours and then vacuumed again and rotated and then the extract of the paste that was formed was taken.After that, maceration was carried out again with ethanol and waited for 7 hours and then in a vacuum and in a rotary, the paste extract was taken and put into a vial.
Then there is the antibacterial test stage, the first thing to do is to calculate the weight of the sample to be used and then weigh the 0.1 and 0.2 ml samples.Then 10 ml of 20% DMSO solution was added and then dissolved with a vortex mixer.Then divide the two media in the petri dish.Open the isolated media and then take 20μ of Staphylococcus aureus bacteria and place it on the agar media and then flatten it with a spreader.Rinse the spreader with alcohol after each treatment.Then a 0.1 ml sample was taken with disc paper and then planted on the media.Furthermore, the same treatment was carried out on a sample 0.2 ml.After that it is labeled on the media and wrapped in plastic wrap.Incubated for 2×24 hours and observed every 1×24 hours.
In the results of observations made on a sample of 0.1 ml on the 24 hours incubation, the diameter of the inhibition zone was 5.35 mm in Escherichia coli bacteria and there was a change on the 48 hours incubation, namely 7.12 mm.Whereas in Staphylococcus aureus bacteria there was no inhibition zone at all in sample 0.1 of mangrove leaf extract Rhizophora apiculata (Table -1).
Then, on the observation of 0.2 ml sample on Escherichia coli bacteria, the diameter of the inhibition zone was 6.78 mm during 24-hour incubation.Then there was a change in the diameter of the inhibition zone on the 48-hour incubation in Escherichia coli bacteria of 6.98 mm.Meanwhile, in the 0.2 ml sample of Staphylococcus aureus bacteria, no inhibition zone was found at all (Table-2).
The criteria for the strength of antibacterial activity are as follows, an inhibition zone diameter of <5 mm is categorized as weak, an inhibition zone diameter of 5-10 mm is categorized as moderate, an inhibition zone diameter of 10-20 mm is categorized as strong and an inhibition zone > 20 mm is categorized as very strong [11].The average antibacterial activity found in Escherichia coli bacteria is included in the moderate category on the criteria for the strength of antibacterial activity.Meanwhile, in samples of Staphylococcus aureus bacteria, no inhibition zones were found in samples 0.1 ml and 0.2 ml.
There are several factors that affect the antibacterial activity in the content of secondary metabolites.Several factors that affect the inhibition of bacterial growth are the selection of media, the solvent used, the thickness of the media, the solvent used and the concentration of the methanol extract used [12].From this statement, it can be concluded that there were several factors that caused no inhibition zones to be found in the Staphylococcus aureus sample.
From all observations, it was found that there was antibacterial activity of Rhizophora apiculata mangrove leaf extract on E.coli bacteria samples with moderate strength so that the leaf extract could be used in making a product that provides economic value with potential ingredients in the pharmaceutical field and can be developed in other fields.Meanwhile, no antibacterial activity was found in the Staphylococcus aureus bacteria samples, which may be caused by several factors.
The results of another research showed that all Rhizophora apiculata leaf extracts were unable to inhibit the growth of the MDR (Multi-Drug Resistant) pathogenic bacteria used [13].These results contradict several previous studies which showed that Rhizophora apiculata extract has antibacterial activity against several human pathogenic bacteria.Rhizophora apiculata mangrove had the highest antibacterial activity of 23.9 ± 21.3 mm for Escherichia coli bacteria while Staphylococcus aureus bacteria had an antibacterial activity of 14 ± 10.8 mm [14].
Rhizophora apiculata leaf extract with methanol and dichloromethane solvents was able to inhibit the growth of the Gram-negative bacteria Pseudomonas aeruginosa [15].Rhizophora apiculata leaf extract with nhexane, ethyl acetate and methanol solvents was able to inhibit the growth of Gram-positive bacteria: Bacillus cereus and Staphylococcus aureus as well as Gramnegative bacteria: Escherichia coli and Salmonella typhi.This difference can be influenced by the type of pathogen strain used [16].

CONCLUSIONS
The mangrove leaves of Rhizopora apiculata contain many antibacterial bioactive compounds, namely tannins, saponins, terpenoids, alkaloids, and flavonoids, which are often used as traditional medicines for antivirus, anti-cancer, anti-fungal and anti-tumor.So that many mangrove antibacterial compounds are contained in the leaves.
It can be seen from the observation of the inhibition zone in the antibacterial test with Escherichia coli bacteria which has a weak inhibition zone of around less than 10 mm with details of the 0.1 ml sample around 5.35 mm and the 0.2 ml sample around 6.78 mm on the 24 hours incubation, on the 48 hours incubation the 0.2 ml sample was around 7.12 mm and the 0.2 ml sample was around 6.98 mm and while for Staphylococcus aureus bacteria no inhibition zone was found on the 24 and 48 hours incubation.Further experiments are needed to determine the antibacterial potential of Rhizophora apiculata mangrove leaf extract against Staphylococcus aureus bacteria.

Table - 1
. The diameter of the inhibition zone (mm) for Rhizopora apiculata leaf extract in the 0.1 ml sample

Table - 2
. The diameter of the inhibition zone (mm) for Rhizopora apiculata leaf extract in the 0.2 ml sample