INTRODUCTION:

Indonesia has a wider ocean area than the mainland. The Indonesian sea has around 8,500 species of fish, 555 species of seaweed and 950 species of coral reef biota¹. One of the potential marine biotas in Indonesian waters is macroalgae or in a trade known as seaweed which is taxonomically grouped into the Thallophyta division. The four classes in the division Thallophyta are Chlorophyceae (green algae), Phaeophyceae (brown algae), Rhodophyceae (red algae) and Cyanophyceae (blue-green algae)². East Java is one of the potential locations for the development of seaweed cultivation, namely Pacitan, Banyuwangi and Sumenep³.

Poteran Island is one of the small islands of 126 islands in Sumenep Regency which is included in the administrative area of Talango district. Sumenep Regency is famous for its seaweed harvest⁴. The water temperature in several areas in Sumenep Regency ranges from 26-30°C. The temperature of the seas that sustain the existence of many species of seaweed, according to Nugrahadi and Yanagi (2003), varies from 24-31°C. The temperature in the Sumenep regency is suitable for seaweed development. Hence, the ideal salinity conditions for seaweed growth in Sumenep regency are between 25 and 30 degrees Celsius. Sumenep regency has water currents that range from 26 to 32cm/s and pH levels of 6.9 to 7.6, which are ideal for seaweed development. The speed of water currents in Sumenep regency is between 26-32cm/s and pH conditions of 6.9-7.6, which are optimal conditions for seaweed growth⁵.

Algae are a source of bioactive compounds such as carotenoids, dietary fibre, protein, vitamins, essential fatty acids and minerals. Brown seaweed is one of the marine natural resources whose existence is very abundant and grows naturally in the coastal waters of Indonesia, especially in the waters of Madura, but this potential has not been utilized optimally. In general, Agar, alginate, and carrageenan are the three kinds of hydrocolloids found in brown seaweed⁶. Algin or alginic acid from Padina australis is used in the culinary and pharmaceutical industries to make ice cream, tablets, ointments, teeth cleaners, lotions and creams, and it may also be utilized for its high nitrogen and potassium content, as well as its low phosphorus level^7,8.

Approximately 500 products derived from marine algae have been identified with the largest percentage in the form of secondary metabolites. In general, the chemical content in Padina australis that has been known is fucoxanthin content of 0.6368mg/g wet weight and carotenoid pigments includingcarotene, diadinoxanthin, diatoxanthin, fucoxanthin, chlorophyll-A and chlorophyll-C⁹. Phaeophyta macroalgae are potential sources of bioactive compounds that are beneficial for the development of the pharmaceutical industry as antibacterial, anti-tumour, anti-cancer and agrochemical industries, especially for fungicides and herbicides¹⁰. The results of phytochemical screening with acetone extract showed that Padina australis contains secondary metabolites in the form of steroids, terpenoids, polyphenols and saponins that have the potential as¹¹.

According to (Salem et al., 2011), several types of macroalgae from the Phaeophyta division have antimicrobial properties, including Sargassum sp and Turbinaria sp. In addition to these two types, Padina australis also contains potential bioactive compounds as antibacterial^12,13. Algae Padina australis Hauck is a macroalga belonging to the brown algae (Phaeophyta) group, living attached to hard substrates¹⁴. Macroalgae extracts of Padina australis and Laurencia nidifica species have potential as antibacterials, namely Escherichia coli and Staphylococcus aureus. Escherichia coli bacteria have a more sensitive response than Staphylococcus aureus¹⁵.

Various studies have shown that Padina australis species have antibacterial activity against various types of bacteria. Research on the activity of Padina australis shows that Padina australis has antibacterial activity against Escherichia coli. Another study on Padina australis from Totok Bay waters using the resazurin method showed that there was antibacterial activity both for gram-positive bacteria Staphylococcus aureus and gram-negative bacteria Escherichia coli^15,16. Research by El-fatimy and Said (2011) showed that the methanol and chloroform extracts (2:1) Padina sp had a significant effect when tested against Escherichia coli and Staphylococcus aureus. Another study that tested antibacterial ethanol and water extracts of Padina pavonica were positive against Staphylococcus aureus^17-21. Based on this background, this study was conducted to prove the antibacterial potential of the 96% ethanol extract of Padina australis originating from the Sumenep Islands against Escherichia coli ATCC 25922.

MATERIALS AND METHODS:

Materials:

The tools used in this study are analytical balance, a set of glassware, glass jars, grinding tools, rotary evaporators, tweezers, vortex, Petri dishes, incubators, gloves, masks, nurse cap, Laminar Air Flow (LAF), micropipettes and sterile holes. Materials used in this study include samples of Padina australis, ethanol 96% (for maceration), pure culture suspension of Staphylococcus aureus ATCC 25923 obtained from BBLK (Center of Health Laboratory) Surabaya, NA media (Nutrient Agar), chloramphenicol 0, 1%, and dimethylsulfoxide (DMSO) 0.1%. HCl 2N, NaCl (Bratachem), Mayer and Wagner reagents, NH₄OH 28% (Merck), methanol (Merck), ethyl acetate (Merck), Dragendorf reagents, anhydrous acetic acid, H₂SO₄ (Merck), n-hexane (Merck), water, H₂SO₄ (Merck), sulfuric acid anisaldehyde, HCl (p), chunks of magnesium, butanol, glacial acetic acid (Merck), 10% NaCl, gelatine, chloroform (Merck), FeCl₃ (Merck), toluene (Merck), HNO₃ (Merck).

Brown Seaweed Extraction (Padina australis):

Padina australis simplex powder was extracted using the maceration method. Comparison of samples with solvents is 1: 4, 1: 3, and 1: 3 (w/v) for 3 days, then filtered to get the filtrate. Then the extract was concentrated using a rotary evaporator at 40^oC until a thick extract was obtained²².

Preparation of Escherichia coli ATCC 25922 Suspension:

The test bacteria, namely Escherichia coli ATCC 25922 which had been inoculated, were taken ±1 ose and then suspended into a tube containing 5 mL of 0.9% NaCl solution so that the turbidity was the same as the turbidity standard of Mac Farland's solution 0.5²³.

Preparation of Padina australis Extract Concentration:

Ethanol extract 96% was made with a concentration of 15% and 5%(w/v). Preparation of a parent standard solution of 20% with 2grams of extract dissolved in 10 mL 0.1% DMSO. Then prepare work standards with concentrations of 15% and 5% (w /v).

Antibacterial Activity Test with Wells Diffusion Method:

In this method, a clear area is produced around the well. Antibacterial activity test is done by pouring the base layer media and seed layer into a sterile petri dish. The seed layer was added with a 40µL suspension of Escherichia coli ATCC 25922 which was adjusted to the standard turbidity of 0.5 Mc Farland and according to the transmittance of ±25%. Then the well was made into the media and filled with 96% ethanol extract Padina australis as much as 40µL with a concentration of 15% and 5%. The same thing was done in the positive control treatment using 0.1% chloramphenicol solution and negative control using 0.1% DMSO solution. Then incubated at 37°C for 24 hours, a clear zone was observed and measured using callipers^23,24.

RESULT:

Phytochemical screening of the 96% ethanol extract Padina australis:

Phytochemical screening of the ethanol extract Padina australis contained many compounds like tannin, alkaloids, terpenoids, flavonoids. For further information as seen in table 1.

Table 1: Phytochemical Screening Ethanol Extract 96% Padina australis

No.	Test	Reagents	Result	Information
1.	Alkaloid	Mayer Wagner	+ +	White sediment formed Brown sediment formed
2.	Flavonoid	Wilstater	+	Red formed
3.	Terpenoid	Liebermann-burchard	+	Formed a red ring
4.	Steroid	Liebermann-burchard	+	Formed a green ring
5.	Saponin	Forth	+	Formed stable foam ± 10 minutes
6.	Polyphenols	FeCl₃ 1%	+	Blackish green formed

The Inhibition zone of 96% ethanol extract Padina australis:

The diameter of inhibition zone of the ethanol extract Padina australis showed a strong activity against gram negative bacteria (Eschericia coli). The higher concentration of the extract, the wider diameter zone resulted from the study. For more details as presented in table 2.

DISCUSSION:

The goal of this exploratory investigation is to show that the 96% ethanol extract of Padina australis has antibacterial activity against Escherichia coli ATCC 25922. The research was preceded by a determination made by Airlangga University's Faculty of Fisheries in Surabaya, which declared that the sample utilized was Padina australis. The next stage is phytochemical screening, which determines the composition of the sample's components²⁵. The screening results may be seen in table 1. Padina australis includes steroids, terpenoids, polyphenols, saponins, alkaloids, and flavonoids, according to other research^26-29.

The results of the bacterial activity test showed that the chloramphenicol inhibition zone was large, the test concentrations of 5%, 10%, and 15% were 11.64±0.13 mm, 14.27±0.54mm, and 15.19±0.46mm which was indicated strong ability as antimicrobial, based on the Clinical and Laboratory Standard Institute (CLSI, 2016) regarding the standard of antimicrobial sensitivity testing, inhibiting the development of Escherichia coli, it falls under the sensitive group³⁰. This is in line with the findings of Amudha and Rani (2016) and Silvia et al (2020), who found that evaluating antibacterial activity against Escherichia coli with a methanol extract of brown algae was difficult. The demonstrates that the presence of active chemicals in the brown algae extract Padina australis causes the inhibitory zone generated by the extract, exhibits antibacterial action against the bacterium Escherichia coli³¹. The inhibitory zone was also determined by specific solvent and extraction method which was chosen for extracting secondary metabolites^30-32.

Padina australis contains antibacterial chemicals, particularly flavonoids, which work as antibacterial by building complex molecules against extracellular proteins that impair bacterial cell membrane integrity³². Flavonoids can cause cell death by denaturing cell proteins and destroying cell membranes. According to Anyanwu et al (2018), terpenoid chemicals can inhibit microorganisms. Cell membrane damage can occur when antibacterial active compounds react with the active site of the membrane or by dissolving lipid constituents and increasing their permeability^32,33.

The bacterial cell membrane is composed of phospholipids and protein molecules. With an increase in permeability, antibacterial compounds can enter the cell and can lyse the cell membrane or coagulate the cytoplasm of the bacterial cell. The mechanism of steroids as an antibacterial is related to lipid membranes and sensitivity to steroid components that cause leakage in liposomes^33-35. Steroids can interact with cell phospholipid membranes that are permeable to lipophilic compounds, causing decreased membrane integrity and cell membrane morphology to change which causes cells to become brittle and lyse³⁶.

Saponin compounds have a mechanism of action, namely by interfering with cell permeability which causes intracellular compounds such as cytoplasm to come out and cause cell death. The mechanism of action of saponins as an antibacterial is to decrease surface tension, resulting in greater permeability of cell leaks and the release of internal chemicals^36,37. The statement (Silva et al., 2013) is reinforced by (Mendes et al., 2013), that this compound diffuses through the outer membrane and vulnerable cell walls, then binds to the cytoplasmic membrane, and disrupts and reduces its stability. This causes the cytoplasm to leak out of the cell resulting in cell death. Antimicrobial agents that disrupt the cytoplasmic membrane are bactericidal. The mechanism of alkaloids in cell death is thought to be by interfering with the constituent components of peptidoglycan in cells so that the cell wall layer is not fully formed and causes cell death and the mechanism of action of tannins as an antibacterial by shrinking the cell wall or cell membrane so that it interferes with the permeability of the bacterial cell wall itself. Alkaloids have toxicity that makes them effective against cells originated from other species. Alkaloids attach to cell DNA, interfering with cell activity and ultimately leading to cell death. Tannin is one of the chemicals that can cause the protein to precipitate. Tannins can inactivate enzymes, react with cell membranes, and inactivate the function of genetic material in bacterial cells, making them antibacterials^37-39.

From the data on the average diameter of the inhibition zone of the 96% ethanol extract of Padina australis obtained as listed in Table 2, the results show that the higher the concentration of the 96% ethanol extract of Padina australis, the higher the content of antibacterial active compounds so that the ability to inhibit the growth of Escherichia coli is higher and continue raising⁴⁰. This shows that the concentration of 96% ethanol extract of Padina australis 5% (w/v) and 15% is in a strong category⁴¹.

CONCLUSION:

Ethanol extract 96% Padina australis can inhibit the growth of Eschericia coli bacteria, characterized by increasing concentrations, the greater the diameter of the inhibitory zone produced.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The subjects are acknowledged for participating in this study. The authors also thank you to Lunardhi Susanto, Ana Khusnul Faizah and Maya Indrawati for supporting and constructing this article.

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Received on 04.08.2022 Modified on 18.09.2023

Research J. Pharm. and Tech 2024; 17(7):2999-3003.

DOI: 10.52711/0974-360X.2024.00468

Ethanol Extract Concentration 96% *Padina* *australis* (% b/v)	Inhibited zone (mm)			Rate ± SD (mm)	Interpretation
	Replication
	1	2	3
5%	11.66	11.75	11.50	11.64 ± 0.13	Strong
10%	14.66	14.50	13.66	14.27 ± 0.54	Strong
15%	15.41	14.66	15.50	15.19 ± 0.46	Strong
Chloramphenicol 0.1% (positive control)	34.4	34.4	34.4	34.30 ± 0.17	Very strong
DMSO 0.1% (negative control)	0	0	0	0 ± 0	Weak