INTRODUCTION:

The pathogen microbes found in fish can be the bacteria of Aeromonas hydrophila, Aeromonas salmonicida, Streptococcus agalactiae, Edwarsiella Ictaluri, and Flavobacterium Columnare.

Aeromonas infecting fish can cause Motile Aeromonas Septicaemia (MAS) marked with lesion, abscess, and local bleeding in the gills, kidneys, liver, and spleen. The MAS disease can also cause death in fish. Streptococcus agalactiae causes meningoencephalitis in fish.¹ The exposure to bacteria toward fish can decrease the quality of fish cultivated. To prevent this, the fish farmers use antibiotics with the purpose to control the pathogen bacterial infection in fish.²

The antibiotic risks and its resistance toward the aquaculture/fishery products become a problem for the customers who will enjoy the protein food from fish. It is necessary to have a solution to avoid the use of antibiotics, with the natural substances that are environmentally friendly and easily degradable. Besides antibiotics, it is also necessary to have a compound having the activity as antioxidant that can function to increase the fish immunity so that they will not be easily exposed to pathogen microbes. Islam et al. stated that the natural substances having the potential as antibacterial and antioxidant are Eleocharis dulcis herbs. The herbal plant E. dulcis can be utilised to cure diseases in humans, such as jaundice, stomach mass, conjunctive congestion, throat swelling and pain, bleeding diarrhoea, hypertension, chronic nephritis, and constipation.³

Previous research reported that the extract of E. dulcis at the right concentration, having the antibacterial activity at Aeromonas hydrophila of 6.25 mg/mL, Aeromonas salmonicida of 3.125 mg/mL, and Streptococcus agalactiae of 1.5625 mg/mL. The extract of ethyl acetate has the antibacterial and antioxidant activities compared with the extracts of N-Hexane and methanol that do not have the antibacterial and antioxidant activities at all.⁴

The result that becomes the foundation for a further test is in a form of fractionation of the ethyl acetate and N-Hexane solution. It is with the purpose to obtain a single compound having the antibacterial activity in fish diseases tested and giving an opportunity in the discovery of new compounds to deal with fish diseases.⁵

MATERIALS AND METHODS:

Materials:

This research uses the water plant E. dulcis coming from Marullah Swamp of Bireuen Regency of Aceh Province (5°11'26.1"N 96°47'08.7"E). The species determination was conducted by the National Innovation Research Agency (BRIN) in Cibinong of West Java, Indonesia, with the number of herbarium B-2503/11.6.2/D1.05.07/8/2022.

The tested bacteria used are the negative gram bacteria, Aeromonas hydrophila and Aeromonas salmonicida, and the positive gram bacteria, Streptococcus agalactiae. These bacteria are isolates from the Fish Health Laboratory, the Faculty of Fishery and Maritime, IPB University, Bogor.

The chemical substances used are hexane (Brataco, Indonesia), ethyl acetate (Brataco, Indonesia), methanol (Brataco, Indonesia), saline solution (NaCl 0.9%) (Otsuka, Indonesia), acetone (Merck, Germany), Whatman filter paper No. 1, paper disc (Oxoid, the UK); chloramphenicol disc (Oxoid, the UK); silicone gel TLC plate (Merck, Germany); silicone gel (Merck, Germany), FeCL₃ (Merck, Germany), DMSO (Merck, Germany), DPPH (Sigma-Aldrich, the US), sulphate acid (Merck, Germany), aluminium foil (Total, Indonesia), chloramphenicol, Trypton Soy Agar (TSA) Media (Oxoid, the UK), Trypton Soy Broth (MHB) Media (Merck, Germany), Brain-Heart Infusion Agar (BHIA) Media (Merck, Germany), Brain-Heart Infusion Broth (BHIB) Media (Merck, Germany), sterile cotton swab (One Med, Indonesia), chloroform (Merck, Germany), ascorbic acid (Sigma-Aldrich, the US).

The equipment used in this research includes the glass equipment commonly utilised in a laboratory of organic chemistry and equipment for testing the antibacterial activity. For the equipment, the instruments used are UV lamp (Camag), Ultrasonic-Assisted Extraction (Krisbow, Indonesia), spectrophotometer UV-VIS (Camag T80+ UV/Vis Spectrometer), 96-well Microplate Reader (Versa Max, the US), Spectrophotometer Massa (Thermo HPLC-DIONEX ULTIMATE -TSQ Quantum Access MAX Triple Quadrupole Mass Spectrometer), and the essence magnetic resonance spectrophotometer (JNM-ECZ500R/S1; 500/125 Mhz).

Fractionation of the extracts of N-Hexane and Ethyl Acetate:

The extraction of E. dulcis based on Amalia et al. (2023) used the levelling extraction with the solutions of N-Hexane, Ethyl Acetate, and Methanol. From the results of Amalia et al. (2023), the extraction of the solutions of N-Hexane and Ethyl Acetate has the antibacterial and antioxidant activities. Meanwhile, the extract of Methanol lacks the antibacterial and antioxidant activities. The derivatives of the extracts of N-Hexane and Ethyl Acetate were fractioned with column chromatography using the silicone gel silent phase.⁴

The separation principle with this chromatography is adsorption and partition by utilising the silent phase and the moving phase. In the silent phase, the substances or materials that can be used are silicon gel, sephadex, and alumina, while the moving phase uses the organic solutions.⁶ The elution process was conducted by mixing the solutions having different polarity levels, starting from mixing the solutions with the lowest polarity to the mixing with the highest polarity. The elution process started by using 100% solution of N-Hexane as much as 1000 ml, then elution was continued in levelling with the mixing of N-Hexane and Ethyl Acetate with the comparison of (95:5) (90:10) (85:15) (80:20) (75:25) (70:30) (65:35) ( 60:40) (55:45) (50:50) (45:55) (40:60) (35:65) (30:70) (25:75) (20:80) (15:85) (10:90) (5:95), and it was ended with 100% Ethyl Acetate without the solution of N-Hexane.

Afterwards, it was continued with the moving phase using ethyl acetate-methanol: starting from 100% of the ethyl acetate solution as much as 1000ml, then proceeded in levelling with the mixing of ethyl acetate and methanol with the comparison of (95:5) (90:10) (85:15) (80:20) (75:25) (70:30) (65:35) ( 60:40) (55:45) (50:50) (45:55) (40:60) (35:65) (30:70) (25:75) (20:80) (15:85) (10:90) (5:95), and ended with 100% of the methanol solution. The process was carried out until it obtained the eluents with different polarity gradients. The result of the elution process was put in a number of bottles with the volume of 100mL each. Furthermore, the fractions were evaporated and tested using Thin Layer Chromatography (TLC). The result of TLC from each fraction produced a chromatogram profile. The fractions having the similar TLC profile were united and dried.⁷

Isolation and purifying:

From the fractionation result, 6 subfractions of N-Hexane (NH 1, NH2, NH3, NH4, NH 5, NH 6) and 8 subfractions of Ethyl Acetate (EA 1, EA2, EA3, EA 4, EA 5, EA6, EA7, EA 8) were obtained. The best result in the subfraction of Ethyl Acetate 1 (EA1) has the weight of 1,180.1mg. Subfraction EA 1 was repurified by using the column chromatography using the silicon gel of the silent phase with the moving phase of the levelling gradient system, which is N-Hexane and Ethyl Acetate with the concentration comparison starting with 100% N-Hexane to 100% Ethyl Acetate (like the previous phase).

The fractions obtained were put in 163 glass bottles with the volume of ±50 mL each, and then the solutions were evaporated. Furthermore, the fractions were evaporated and their TLC chromatogram profile was observed; for the fractions having the same TLC profile, they were united and dried. Out of 163 fraction bottles, around 27 bottles are EA1 subfraction seen to have crystal like a white mixed with green needle. The purifying process was continued with the deposition using the N-Hexane solution. The separation result produced white powder with the weight of 11.56 mg. Around 0.5 mg isolate was dissolved in ethyl acetate and then analysed with TLC with the N-Hexane and Ethyl Acetate eluents with the comparison of 4500:500 μL. The TLC result had one spot under the UV light and sprayed with anisaldehyde and heated until there was a change of colour. The isolates obtained were then restored in glass vial that was tightly sealed. The structure of the compounds obtained was then identified, and their antimicrobial activity was tested toward the tested microbes.

The antioxidant activity test:

a) Testing the fraction antioxidant activity:

Each fraction sample solution as much as 500 μL was put into vial and added with as much as 1.0 mL DPPH, then added with 96% methanol so that the volume became 5mL and the concentration became 100ppm. Furthermore, it was incubated for 30minutes and applied measurement was conducted using spectrophotometer of UV-Vis at the wave length of 515nm. Afterwards, the calculation of the inhibition percentage was conducted and the value of IC50 is with the following formula.⁸

Abs Blanko-(Abs blanko sample)

%Inhibition = ------------------------------------- x 100%.

Abs Blank

b) Testing the isolate antioxidant activity:

Testing the isolate antioxidant activity was conducted with the microdilution method in the microplate of 96 well. 1 mg isolate was dissolved into 1mL 96% ethanol solution. Each sample solution was put into vial and added with as much as 60μL DPPH, and then added with 96% methanol until the volume became 200μL so that the final concentration of every sample was 100 ppm. Moreover, it was incubated for 30 minutes and applied measurement was conducted using a microplate reader with the wave length of 515nm.⁹

Compound structure determination:

The structure determination with NMR (Nuclear Magnetic Resonance) was with, among others, 1H-NMR, carbon 13C-NMR, and HMBC (Heteronuclear Multiple Bond Correlation).¹⁰

The antibacterial activity test:

a) Minimum inhibitory concentration:

The antibacterial activity test uses the subfractions NH1-NH6 and the subfractions EA1-EA8 with the concentration of 500ppm. Then the Minimum Inhibitory Concentration (MIC) test was conducted using the media of TSA (Tryptic Soy Agar), TSB (Tryptic Soy Broth), and BHIA (Brain Heart Infusion Agar) & BHIB (Brain Heart Infusion Broth) ⁴. The Minimum Inhibitory Concentration (MBC) testing was conducting in levelling.

Step1. As much as 50μL of TSB and BHIB media was put into a microplate added with 50μL of the tested subfractions NH1-NH6 and subfractions EA1-EA8. The subfraction concentration became 250ppm.

Step 2. As much as 50µL solution of every fraction of phase 1 was taken and put into a microplate that had been filled in with 50μL of TSB dan BHIB media. The fraction concentration became 125ppm.

Step 3. As much as 50µL solution of every fraction of phase 2 was taken and put into a microplate that had been filled in with 50μL of TSB dan BHIB media. The fraction concentration became 62.5ppm.

Step 4. As much as 50µL solution of every fraction of phase 3 was taken and put into a microplate that had been filled in with 50μL of TSB dan BHIB media. The fraction concentration became 31.25ppm.

Step 5. As much as 50µL solution of every fraction of phase 4 was taken and put into a microplate that had been filled in with 50μL of TSB dan BHIB media. The fraction concentration became 15.625ppm. As much as 50µL of aliquot of the step 5 fraction was thrown out so that the final volume in step 5 became 50µL with the concentration of 7.8125ppm.

b) The minimum bactericidal concentration test:

The tested bacteria of Minimum Bactericidal Concentration (MBC) were determined by growing the tested bacteria again from the aliquot of Minimum Inhibitory Concentration (MIC) in TSA and BHIA media. The bacteria were incubated for 24 hours for the bacteria of Aeromonas Hydrophyla and Aeromonas Salmonisida, and 48 hours for Streptococcus agalactiae. Observation and determination of MBC were based on whether there is bacterial growth or not in the growing media.

RESULT AND DISCUSSION:

Fractionation of the extracts of N-Hexane and Ethyl Acetate:

From the hexane extract fractionation, it resulted in 159 bottles of fractional derivatives with the volume of 100 mL each, and there are around 187 bottles of the ethyl acetate extract fractional derivatives with the same volume of 100mL each. Those fractions were analysed for their profile of Thin Layer Chromatography (TLC) and observed under the UV lamp of 366/254nm to see the profile of every fraction. The fractions having the same TLC profile were united in a new container so that the final result obtained as many as 6 fractions of N-Hexane and 8 fractions of Ethyl Acetate. The fractionation results from the extracts of hexane and ethyl acetate are shown in Table 1.

Isolation and single compound purifying:

From 6 fractions of N-Hexane and 8 fractions of ethyl acetate, there is one best fraction, which is the fraction of ethyl acetate no 1 (EA1) with the isolate weight of 7.16gram. The fraction of EA1 produced 26 subfractions. Out of 26 subfractions, the fraction no 8 was purified because it had white crystal mixed with a little greenish colour around the crystal. The green colour around the isolate dissolved faster than the white crystal that would dissolve if given a little shake or stirring. After the subfractions were believed to have been pure, marked with the loss of the green colour around the white colour in fractions. Then spot monitoring was conducted under the light of UV so that the TLC profile was seen with one spot, meaning the isolate was already pure. This isolate had the weight of 7.61gram in a form of white coloured powder. Isolate identification in the TLC of the silent phase of silicon gel F254 and of the moving phase of N-Hexane: Ethyl Acetate with the comparison of 9:1 shows the value of RF (Retention Factor) of 0.5. The observation results of isolate TLC are shown in Figure 1.

Figure 1. The observation results of the isolate TLC

The isolates obtained were sprayed with anisaldehide showing the red colour single spot (Figure 1). This shows that the isolates obtained are assumed to be the steroid/terponoid type of compound. The purifying test with the TLC 2 dimension shows 1 single spot under UV of 254 nm using the eluent I with methanol:chloroform (95:5). Moreover, the plate was rotated 900 and eluted with theeluents of N-Hexane: Ethyl Acetate (9:1) also showing a single spot. The results of the isolate 2D TLC under UV of 254 nm are shown in Figure 2.

Figure 2. Isolate 2D TLC Profile

The antioxidant activity test:

a) Antioxidant of fractions:

The antioxidant activity of a fraction is determined by measuring the inhibition percentage at the concentration of 100 ppm. The antioxidant strength with IC50 at 100 ppm is still categorised as weak antioxidant. The categorisation of antioxidant strength can be seen in Table 2.

Table 2. The categorisation of antioxidant strength ¹¹

IC₅₀	Antioxidant Strength
< 10	Very Strong
10-50	Strong
50-100	Medium
100-250	Weak
>250	Not Active

The antioxidant activity testing results of the fraction N-Hexane and the fraction Ethyl Acetate are seen in Table 3. The research results show that only fractions EA5 and EA6 have the best inhibition percentage, which is 81.55% for EA5 and 65.77% for EA6. Out of 16 fractions, there is only one isolate from EA1. This EA1 isolate does not have the antioxidant activity.

Table 3. The antioxidant activity testing results of the fraction N-Hexane and the fraction Ethyl Acetate

Fraction	%Inhibition 100 ppm	Fraction	%Inhibition 100 ppm
EA1	27.34	NH1	6.66
EA2	7.92	NH2	34.74
EA3	18.12	NH3	10.01
EA4	37.82	NH4	14.48
EA5	81.55	NH5	15.18
EA6	65.77	NH6	13.79
EA7	24.83
EA8	15.04

Notes: NH= N-Hexane Fraction; EA = Ethyl Acetate Fraction

Determining the structure of the isolate single compound:

Determining the structure of the single compound found in the isolate of EA1 was based on the testing results of LC-MS. The molecular ion peak at m/z=412 showing the isolate compound has the molecule weight of 412 g/mol so that it is assumed that the isolate obtained is the stigmasterol compound based on the database of LC-MS. The analysis of LC-MS shows that precursor ion as [M+H-H₂O] at m/z=395.31 provides information that the main ionisation path from the stigmasterol compound is through the loss of water molecules. The fragmentation of losing water molecules from the stigmasterol structure is shown in Figure 3. This fragmentation pattern is suitable with what is stated by Jiang et al. (2019) that the stigmasterol compound analysed with mass spectrometer has the main fragmentation pattern by losing water molecules ¹².

Table 4. The NMR Chemistry Shift Value at the Isolates and Stigmasterols of References

Position of Atom C	Stigmasterol Control		Stigmasterol - Isolate EA1				Note
	H-NMR	C-NMR	H-NMR		C-NMR
1	1.02 (m);1.07(m)	37.3		1.0 (m);1.07(m)		37.4		CH₂
2	1.82 (m)	31.7		1.83 (m)		31.8		CH₂
3	3.52 (m)	71.8		3.52 (m, J=4.5 Hz)		71.9		CH-OH
4	0.99(m);2.30 (m)	42.3		0.98(dd, J=5 Hz);2.30 (dd, J=5.5 Hz)		42.4		CH₂
5	-	140.8		-		140.9		C=C
6	5.35 (d, 5.5)	121.7		5.34 (d, J=5.2 Hz)		121.9		CH
7	1.50 (m)	33.0		1.50 (m)		34.8		CH₂
8	1.49 (m)	31.9		1.45 (m)		32.0		CH
9	0.92(m)	50.2		0.91 (m)		50.2		CH
10	-	36.5		-		36.6		C
11	1.46;1.49 (m)	25.4		1.45;1.48 (m)		25.6		CH₂
12	1.15;1.95 (m)	39.7		1.15;1.97 (m)		39.9		CH₂
13	-	42.3		-		42.3		C
14	1.03 (m)	56.9		1.02 (m)		57.0		CH
15	1.07;1.56 (m)	24.4		1.07;1.50 (m)		24.4		CH₂
16	1.26;1.67 (m)	28.9		1.27;1.65 (m)		28.4		CH₂
17	1.13 (m)	56.0		1.07 (m)		56.9		CH
18	0.67 (s)	12.1		0.68 (s)		12.1		CH₃
19	1.00 (s)	19.4		1.00 (s, )		19.2		CH₃
20	2.02 (m)	40.4		2.02 (m)		40.7		CH
21	0.92 (d, 6.5)	21.2		0.91 (d, J=6.5 Hz)		20.0		CH₃
22	4.98 (m)	138.3		4.98 (m)		138.5		C=C
23	5.02 (dd, 8.5;15.0)	129.4		5.01 (dd, J=8.5;15.0 Hz)		129.4		C=C
24	1.53 (m)	51.3		1.55 (m)		51.4		CH
25	1.45 (m)	31.9		1.45 (m)		31.3		CH
26	0.84 (d, 9)	18.9		0.83 (d, J= 9 Hz)		18.9		CH₃
27	0.82 (d, 4.5)	21.1		0.81 (d, J=4.5 Hz)		21.2		CH₃
28	1.15 (t)	26.0		1.15 (t)		26.2		CH₂
29	0.80 (t, 8.5)	12.2		0.79 (t, J=8.5 Hz)		12.4		CH₃

Table 5. The MIC testing results of the fractions N-Hexane and Ethyl Acetate of E. dulci

	Kons. (ppm)	AS	AHA	SA		Kons. (ppm)	AS	AHA	SA		Kons. (ppm)	AS	AHA	SA
PC	30	+	+	+	NH5	125	-	+	+	EA4	125	+	+	+
NH1	125	+	+	+		62,5	-	-	+		62,5	-	+	+
	62,5	-	+	+		31,25	-	-	+		31,25	-	-	+
	31,25	-	-	+		15,625	-	-	+		15,625	-	-	+
	15,625	-	-	+		7,8125	-	-	-		7,8125	-	-	-
	7,8125	-	-	+	NH6	125	-	+	-	EA5	125	-	+	-
NH2	125	+	+	+		62,5	-	-	-		62,5	-	+	-
	62,5	-	+	+		31,25	-	-	-		31,25	-	-	-
	31,25	-	-	+		15,625	-	-	-		15,625	-	-	-
	15,625	-	-	-		7,8125	-	-	-		7,8125	-	-	-
	7,8125	-	-	-	EA1	125	+	+	+	EA6	125	-	-	-
NH3	125	-	+	+		62,5	-	+	+		62,5	-	-	-
	62,5	-	+	+		31,25	-	-	+		31,25	-	-	-
	31,25	-	-	+		15,625	-	-	+		15,625	-	-	-
	15,625	-	-	+		7,8125	-	-	+		7,8125	-	-	-
	7,8125	-	-	+	EA2	125	+	+	+	EA7	125	-	-	-
NH4	125	-	+	+		62,5	-	+	+		62,5	-	-	-
	62,5	-	+	+		31,25	-	-	+		31,25	-	-	-
	31,25	-	-	+		15,625	-	-	+		15,625	-	-	-
	15,625	-	-	-		7,8125	-	-	+		7,8125	-	-	-
	7,8125	-	-	-	EA3	125	-	+	+	EA8	125	-	-	-
GC		-	-	-		62,5	-	-	+		62,5	-	-	-
MC		-	-	-		31,25	-	-	+		31,25	-	-	-
SolC		-	-	-		15,625	-	-	-		15,625	-	-	-
NC		-	-	-		7,8125	-	-	-		7,8125	-	-	-

Notes: + : No bacterial growth - : there is bacterial growth inhibition

PC: Positive Control; GC: Germ Control; MC:Media Control; SolC: Solution Control; NC: Negative Control

AHA: Aeromonas Aydrophila; AS: Aeromonas salmonicida; SA: Streptococcus agalactiae

Table 6. The MBC testing results of the fractions hexane and ehtyl acetate of E. dulcis

	Kons. (ppm)	AS	AHA	SA		Kons. (ppm)	AS	AHA	SA		Kons. (ppm)	AS	AHA	SA
PC	30	+	+	+	NH5	125	-	+	+	EA4	EA4	125	-	+
NH1	125	-	+	+		62,5	-	-	+			62,5	-	-
	62,5	-	+	+		31,25	-	-	+			31,25	-	-
	31,25	-	-	+		15,625	-	-	+			15,625	-	-
	15,625	-	-	+		7,8125	-	-	-			7,8125	-	-
	7,8125	-	-	+	NH6	125	-	+	-	EA5	EA5	125	-	+
NH2	125	-	+	+		62,5	-	-	-			62,5	-	-
	62,5	-	+	+		31,25	-	-	-			31,25	-	-
	31,25	-	-	+		15,625	-	-	-			15,625	-	-
	15,625	-	-	-		7,8125	-	-	-			7,8125	-	-
	7,8125	-	-	-	EA1	125	+	+	+	EA6	EA6	125	-	-
NH3	125	-	+	+		62,5	-	+	+			62,5	-	-
	62,5	-	-	+		31,25	-	-	+			31,25	-	-
	31,25	-	-	+		15,625	-	-	+			15,625	-	-
	15,625	-	-	+		7,8125	-	-	+			7,8125	-	-
	7,8125	-	-	+	EA2	125	-	+	+	EA7	EA7	125	-	-
NH4	125	-	+	+		62,5	-	-	+			62,5	-	-
	62,5	-	-	+		31,25	-	-	+			31,25	-	-
	31,25	-	-	+		15,625	-	-	+			15,625	-	-
	15,625	-	-	-		7,8125	-	-	+			7,8125	-	-
	7,8125	-	-	-	EA3	125	-	+	+	EA8	EA8	125	-	-
GC		-	-	-		62,5	-	-	+			62,5	-	-
MC		-	-	-		31,25	-	-	-			31,25	-	-
SolC		-	-	-		15,625	-	-	-			15,625	-	-
NC		-	-	-		7,8125	-	-	-			7,8125	-	-

Note:

+: No bacterial growth

-: There is bacterial growth

PC: Positive Control; GC: Germ Control; MC:Media Control; SolC: Solution Control; NC: Negative Control

AHA: Aeromonas Aydrophila; AS: Aeromonas salmonicida; SA: Streptococcus agalactiae

The tests for the isolates of MIC and MBC show the isolates having the inhibition and killing activities toward three tested bacteria. The bacterial growth inhibition of Aeromonas hydrophila happens at the concentration of 62.5 ppm. The bacterial growth inhibition of Aeromonas salmonicida is at the concentration of 125 ppm and for the bacterium Streptococcus agalactiae it is at the concentration of 31.25 ppm. The concentration for MBC is similar with that for MIC for all three bacteria. The testing results of MIC and MBC can be seen in Table 7.

Table 7. The isolate testing results of MIC and MBC

	Konsentration (ppm)	AS	AHA	SA
Chloramphenicol	30	+	+	+
MIC	125	+	+	+
	62,5	-	+	+
	31,25	-	-	+
	15,625	-	-	-
	7,8125	-	-	-
Chloramphenicol	30	+	+	+
MBC	125	+	+	+
	62,5	-	+	+
	31,25	-	-	+
	15,625	-	-	-
	7,8125	-	-	-

The antibacterial activity testing results show that there are differences of the antibacterial activity between the positive gram and the negative gram. This might occur because there are differences of cell formation components between positive gram bacteria and negative gram bacteria. Negative gram bacteria are surrounded by two membranes, cytoplasmic cell membrane and outer membrane. Positive gram bacteria do not have the additional outer membrane layer just like what negative gram bacteria have. Negative gram bacteria tend to be more resistant toward antimicrobial agents than positive gram bacteria because there is extra protection given by the outer membrane¹⁴.

The stigmasterol compound is called as one of the compounds having the antibacterial and antifungal activities with a wide spectrum so that it has the potential to be developed as a new antimicrobial agent ¹⁵. The research on the antibacterial activity in vitro from the stigmasterol compound isolated is from the root of Caylusea Abyssinica toward S. Aureus, E. Coli, Pseudomonas Aeruginosa, and Salmonella Typhimurium with the inhibition zone starting from 11mm to 18mm at the concentration of 50mg/mL¹⁶. Other research conducted on the stigmasterol compound provides a lower inhibition zone (9mm–10mm) toward S. Aureus and E. Coli at the concentration of 250µg/mL¹⁷. The stigmasterol compound mechanism as an antibacterial agent is assumed by disrupting the microbial cell membrane. Besides that, the steroid group compound is also reported to be able to prevent transpeptidase by inhibiting sortase protein at the bacterial cell membranes ¹⁵.

CONCLUSION:

The research results show that the fractions NH1 and EA1 have a better antibacterial activity than other fractions. Compound characterisation from the isolates obtained from the fraction EA1 shows the stigmasterol compound structure. The isolate MIC determination toward the bacterium Aeromonas hydrophila is at the concentration of 62.5 ppm, toward the bacterium Aeromonas salmonicida is at the concentration of 125 ppm, and toward the bacterium Streptococcus agalactiae is at the concentration of 31.25 ppm. MBC determination also shows a similar concentration with MIC concentration. This research shows that stigmasterol has the potential to be developed as an antibiotic for fish. The development of antibiotics for fish is still very limited, so the results of this research are expected to provide great benefits for developing drugs to treat fish diseases.

ACKNOWLEDGEMENTS:

The research from 2022-2023 was funding by Insinas Kemenristek Dikti and Grand Research Ministry of health Republic of Indonesia. The authors also wish to extend a sincere appreciation to Faculty of Marine and Fisheries IPB University for allowing the use of the laboratory facilities and for the assistance provided by the laboratory technicians. All authors in the study are the main contributors.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Austin, Brian DAA. Bacterial Fish Pathogens:Disease of Farmed and Wild Fish Fifth Edition. Published online 2012. doi:10.1007/978-94-0074884-2

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Received on 29.08.2023 Modified on 17.02.2024

Research J. Pharm. and Tech 2024; 17(10):4817-4825.

DOI: 10.52711/0974-360X.2024.00741

Ethyl Acetate Ekstract				N-Hexane Ekstract
Fraction	No Bottle	Weight (mg)	% Fraction Weight	Fraction	No Bottle	Weight (mg)	% Fraction Weight
1	1-20	1180,1	5.9005	1	1-31	1962,5	9.8125
2	21-34	1078,6	5.393	2	31-47	4733	23.665
3	35-43	1794.9	8.9745	3	48-52	2277,2	11.386
4	44-49	5313.5	26.5675	4	53-68	3340,1	16.7005
5	50-58	1540	7.7	5	69-85	6612	33.06
6	59-99	6403.9	32.0195	6	86-159	388,8	1.944
7	99-150	596.7	2.9835
8	151-187	879.5	4.3975