INTRODUCTION:

Infectious diseases are a type of disease that many Indonesians. One of the infectious diseases that often occurs is gastrointestinal infections, such as diarrhea. Diarrhea is a bowel movement with a more liquid stool consistency with a frequency of >3 times a day.

Diarrhea disease is an endemic disease that has the potential to cause Extraordinary Events and is still a contributor to mortality in Indonesia, especially in toddlers¹. Escherichia coli causes diarrhea because it produces toxins that irritate intestinal membranes (Rahayu et al., 2018). Escherichia coli makes enterotoxin, which causes excretion of electrolyte fluids in the body, resulting in diarrhea and dehydration. According to Zaunit et al. (2019), Staphylococcus enterotoxin is the main cause of food poisoning accompanied by diarrhea. Diarrhea occurs due to the interaction between enterotoxin and the enteric nervous system, which is the nerve contained in the digestive tract wall that stimulates inflammation in the intestine and diarrhea². Infectious diseases are the leading cause of death worldwide, and the use of antibiotics has revolutionized the treatment of various bacterial infections³.

The people of Senduro village, Lumajang, East Java, use the unripe fruit peel of the kayu banana (Musa paradisiaca L. Var. Kayu) as an empirical medicine to treat diarrhea. In Senduro Lumajang, unripe fruit peels from kayu bananas (Musa paradisiaca L. Var. Kayu) were used by boiling, steaming, and burning as an antidiarrheal. The ethanol extract from the unripe fruit of the kayu banana gives Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration (MIC) on bacterial growth at a concentration of 50%, according to research data from Ningsih et al. (2021). Ningsih et al. (2022) report that at a dosage of 200 mg/kg body weight, an ethanol extract from the unripe fruit of the kayu banana (Musa paradisiaca L. Var. Kayu) showed Oleum Ricini-induced antidiarrheal activity. Therefore, it is important to prevent diarrheal infectious diseases by exploiting new drugs with optimal therapeutic activity^4,5,6,7.

Phenolic compounds in some plants are known to have antibacterial properties. According to Borrás-Linares et al. (2015), it disrupts the function of bacterial cell membranes, thereby inhibiting the growth or proliferation of bacteria. Other mechanisms are put forward by Zainol et al. (2013) that at high concentrations, phenolic compounds can easily diffuse into cells and inhibit bacterial growth using hydroxyl groups in the structure of organic components and nutrient transport, which eventually causes toxic effects on bacteria^8,9.

Fractionation is the process of separating the types of compounds found in plants. Fractionation is carried out to separate the content of primary compounds from other classes of compounds. Separation of fractions is carried out by considering the properties of the desired compound. Fractionation is generally done using solvents with different polarities so that polar compounds enter the polar solvent while nonpolar compounds enter the nonpolar solvent (selfiana, 2019). n-hexane fraction, ethyl acetate fraction, and water fraction of antibacterial research so far have never been conducted¹⁰.

As one of the efforts to use the unripe fruit of kayu bananas as part of infection treatment, it is necessary to analyze and determine the effect of phenolic levels on antibacterial activity. Based on the above background will be determined phenolic content and antibacterial activity of n-hexane fraction, ethyl acetate fraction, and water fraction of unripe fruit peel of kayu banana (Musa paradisiaca L.Var. kayu). Hopefully, this study can provide an overview of the relationship between phenolic compounds and the greatest antibacterial activity of each fraction.

RESEARCH METHODS:

Material:

Folin Ciocalteu reagents, gallic acid, nutrient agar, nutrient broth, and muller hinton agar were purchased from Merck. Mayer reagent, 96% ethanol, Dreagendroff reagent, Wagner reagent, Mg powder, FeCl₃ solution, gelatin salt, concentrated HCl, sterile equates, 70% alcohol, chloramphenicol, BaCl₂ solution, H₂SO₄ solution, 0.9% NaCl. The solvent used in the study was of the analytical type and was purchased at Merck.

Plant:

The unripe fruit of kayu bananas was taken from Lumajang Regency, East Java Province, and selected as unripe, with the characteristics of green banana skin, and the fruit was still hard and approximately 3 months old after flowering. The determination of kayu banana plants was carried out at LIPI Purwodadi and the Lumajang City Agriculture Office.

Method:

Extraction method:

A 96% ethanol solvent was used to macerate 500 grams of dry powder unripe fruit peel of kayu banana. The solvent was repeated eight times until it became clear and colorless again, and then filtering was carried out to separate the residue and filtrate. The resulting fiber was then evaporated in a rotary evaporator until it became a viscous extract¹¹. The extraction method carried out in this study was repeated remaceration was a method of extracting powder using a solvent with replacement or repetition of the application of solvent to the same powder after the maceration process 3x24 hours is complete, for the remaceration process lasts for 1x24 hours by adding solvent repeatedly and until clear with a solvent ratio of 1: 4 with the required amount of solvent as much as 1600 ml. The duration of maceration was 24 hours with 2 stirrings after 24 hours filtered and soaked for another 24 hours. The viscous extract was calculated as the yield value¹².

Fractionation method:

After using the liquid-liquid method for fractionation, three solvents with different degrees of polarity—N-hexane, ethyl acetate, and water—were used to fractionate into a viscous extract. The fractionation process was carried out by dissolving 10 grams of unripe fruit peel extract from kayu bananas, then dissolving it in 100 ml of warm aquadest and putting it into a separate funnel. It added 100 ml of n-hexane and then was beaten until 2 phases were formed, separating the n-hexane phase and water. Fractionation was carried out in 4 repetitions with a total solvent of 400 ml. The n-hexane phase was separated from the aqueous phase. The water phase was 100 ml of ethyl acetate, and then the steps were repeated as above until clear results were obtained. The filtrate was thickened with a rotary evaporator until viscous¹³.

Phytochemical screening:

1. Alkaloid screening:

The fraction on the KLT plate was then eluted with ethyl acetate: methanol: water (6: 4: 2) and was observed in UV light 254 nm and 366 nm, then was given dragendoff reagent if brown or orange color arose then it was shown the presence of alkaloid compounds if without chemical reagents, under 366 nm UV light, alkaloids will fluoresce blue, blue-green or purple⁴.

2. Saponin screening:

Spotted the fraction on the KLT plate then was eluted with n-hexane: ethyl acetate (4: 1), then was given sulfuric acid anisaldehyde reagent if a red, purple, or purple color is formed, the fraction contains saponin compounds⁴.

3. Flavonoid screening:

Spotted the fraction on the KLT plate and was eluted using butanol: acetic acid: water (3: 1: 1), then was observed at 254 nm and 366 nm UV lamps and then was given ammonia reagent on the KLT plate. If it showed a yellow color, then the fraction contained flavonoid compounds⁴.

4. Tanin and polifenol screening:

Spotted the fraction on the KLT plate and expanded it with chloroform: ethyl acetate: acetic acid (0.5: 9: 0.5), then sprayed the 10% FeCl₃ reagent on the KLT plate, if black appears on the KLT plate, the positive fraction contained tannin compounds⁴.

6. Triterpenoid and Steroid screening:

Spotted the fraction on the KLT plate then was eluted with n-hexane: ethyl acetate (4: 1), then was given sulfuric acid anisaldehyde reagent if a red, purple, or purple color was formed, the fraction contained terpenoid compounds or steroids⁴.

7. Antrakuinon screening:

Spotted the fraction on the KLT plate then was eluted with toluene: ethyl acetate: acetic acid (75:24:1), then was given a 10% KOH action if yellow, yellow-brown, red-purple, or purple-green stains were formed, the fraction showed the presence of anthraquinone compounds⁴.

8. Glikosida screening:

The added fraction of 0.5 mL HCl and Mg metal was then observed, and the color change occurred. If the color changes from green to blue, it contains compounds⁴.

Determination of Total Phenol Content:

After mixing and having the mixture sit for 4–8 minutes, The goal was for the process of the reaction of compounds with reagents to be optimal. One milliliter of unripe kayu banana fruit fraction solution and 0.4 milliliters of Folin Ciocalteau reagent was added. The mixture was beaten homogenous. After the aquadestillata was added to 10 mL, let it remain at room temperature for two hours. Measure the absorption at a maximum absorption wavelength of 745 nm. Three replications were necessary to get the phenol levels in mg to match the gallic acid/g fraction. Gallic acid was the standard. A solution of gallic acid was made with a 1000 ppm concentration. For analysis, 10 milligrams of gallic acid were weighed and then diluted in 10 milliliters of methanol. After considering the 10 mg fraction, transfer it to a 10 ml measuring flask, allowing enough capacity to contain the limit of the methanol p.a⁴.

Determination of the inhibitory zone:

Each petri dish received up to 15 milliliters of MHA medium, which was then infected with 1 milliliter of bacterial suspension and left to stand until hardened. After 100 μL of the sample was analyzed, the paper disc had been put on an agar plate and was incubated for 24 hours at 37°C¹⁵. Six-millimetre disc paper was utilized. Dimethyl sulfoxide, or DMSO, was used as the negative control, and chloramphenicol was used as the positive control¹⁶. Using a caliper instrument, a horizontal line was selected on the clear zone around the paper disc to measure the inhibitory zone—next, each sample's inhibitory zone diameter (mm). Category very strong between 2-=30 mm, strong between 10-20 mm, medium between 5-10 mm and weak < 5 mm¹⁷.

Determination of Minimum Inhibition Concentration (MIC) at a Concentration of 1mg/ml:

Determination of MIC was carried out using 1 ml of NB (Nutrient Broth) media inserted into a test tube, then 1 ml of bacterial suspension and 1 ml of test solution, with positive control and negative control. After that, all samples were incubated at 37⁰C for 24 hours until bacterial growth occurred¹⁸. The positive control was 0.0015 grams of chloramphenicol and dissolved in 10 ml of sterile aquades. The negative control preparation was 5 ml of concentrated DMSO dissolved in 100 ml sterile aquades. An extract test solution of 1 gram was dissolved in 1 ml of solvent. The test method used was the OD dilution with λ max 625 nm for Escherichia coli and Staphylococcus aureus of 292 nm. The turbidity of the culture can be used to calculate the number of bacterial cells. Next, the solution's turbidity was noted, and both before and after Incubation, the absorbance value was determined^3,20.

Determination of Minimum Bactericidal Concentration (MBC) at a Concentration of 1mg/ml:

MBC was determined with EMB (Eosin Methylene Blue) media for Escherichia coli bacteria and MHA (Muller Hinton Agar) for Staphylococcus aureus bacteria. A total of 15 ml of media was put into a petri dish, and then 1 ml of incubated test solution was inoculated on the press and homogenized. Then, the media was allowed to solidify and set at 37⁰C for 24 hours, then observed. The indicator was the amount of or lack of bacterial growth on agar media, which was shown by the presence or absence of spots or white patches on the medium¹⁹.

Data Analysis:

The data obtained were seen whether they included normal and homogeneous distributions or not using normality tests and homogeneity tests, if the data was normally distributed, then continued with the ANOVA test, and if not normally distributed, continued with the Kruskal Wallis test. The Correlation Test was determined using the Pearson correlation coefficient (r) to see the relationship between Total Phenol Content and it was antimicrobial activity. Analysis of this data was calculated using SPSS version 21.0 using the 95% confidence level.

RESULTS AND DISCUSSION:

In the manufacture of fractionation using the liquid fractionation method - liquid uses 3 different solvents, namely n-hexane solvent, ethyl acetate, and water, with varying levels of polarity.

Table 1 Fractionation results

Solven	Weight extract (gr)	Amount of solvent used (ml)	Fraction Weight (gr)	% Fraction Yield
Aquadest	10 gr	870 ml	0,53 gram	5,3%
Etil Asetat		2.637 ml	3,7 gram	37%
N-Heksana		1.570 ml	0,50 gram	5%

Table 4. Inhibitory Zone Diameter Measurement Results

No	Sample	Replication	Inhibitory Diameter(mm)	Average ± SD	Category (CLSI,2013)
Escherichia coli
1	etil asetat Fraction 6 mg/60 µl	1	20	19±1^a	Strong
		2	19
		3	20
2	n-hexane Fraction 6 mg/60 µl	1	16	16±0^b	Strong
		2	15,7
		3	15,17
3	Water fraction 6 mg/60 µl	1	19	18±1^a	Strong
		2	18,5
		3	16,33
4	K+ 15 µg/100 µl	1	32.5	33 ± 1^c	Strong
		2	34
		3	33.5
Staphylococcus aureus
5	etil asetat Fraction 6 mg/60 µl	1	11	9 ± 1,6^a	Strong
		2	7
		3	9
6	n-hexane Fraction 6 mg/60 µl	1	0	0 ± 0^b	Inactivity
		2	0
		3	0
7	Water fraction 6 mg/60 µl	1	0	0 ± 0^b	Inactivity
		2	0
		3	0
8	K+ 15 µg/100 µl	1	20	20 ± 0,5^c	Strong
		2	20,5
		3	19,3

The result is expressed as the mean of the inhibitory zone ± SD of 3 replications. The average SPSS test result of the Kruskal Wallis inhibitory zone is significantly different at a p-value of < 0.05. Follow-up test between sample groups using the Mann-Whitney test. The same superscript letters show no noticeable difference. Other superscript letters indicate there is a noticeable difference. The confidence level used is 95%. Category very strong between 2-=30 mm, strong between 10-20 mm, medium between 5-10 mm and weak < 5 mm.

Table 5. Measurement Results Optical Density (OD) at Minimum Inhibition Concentration (MIC) .

No.	Sample	Average OD MIC score			Category
No.	Sample	Before Incubation	after Incubation	∆OD ± SD	Category
Escherichia coli
1	etil asetat Fraction 0.1 mg/ml	0.84525	0.478	-0.295±0.11^b	down
2	n-hexane Fraction 0.1 mg/ml	0.6775	0.476	-0.2015±0.04^c	down
3	Water fraction 0.1 mg/ml	0.166	0.10525	-0.0607±0.002^b	down
4	K+0.1 mg/ml	2.2850	1.0678	-1.2173 ± 0.112^a	down
5	K-0.1 mg/ml	0.0383	0.2970	0.2588±0.172^d	up
Staphylococcus aureus
1	etil asetat Fraction 0.1 mg/ml	3.996	3.041	-0.931 ± 0.436^a	down
2	n-hexane Fraction 0.1 mg/ml	2.859	2.465	-0.330 ± 0.05^c	down
3	Water fraction 0.1 mg/ml	3.655	3.145	-0.511 ± 0.32^b	down
4	K+0.1 mg/ml	2.119	1.902	-0.216 ± 0.03^c	down
5	K-0.1 mg/ml	2.161	2.496	+0.335 ± 0.07^d	up

Note: The indicator "Up" shows that there has been bacterial growth if the absorbance value after Incubation is greater than the absorbance value before Incubation; the indicator "Down" shows that bacterial growth has been inhibited. Optical density (OD). The average SPSS Anova test result of MIC is significantly different at a p-value of < 0.05 with further tests using the Tukey test. The same superscript letters show no noticeable difference. Different superscript letters indicate there is a noticeable difference. The confidence level used is 95%.

Table 6. Bacterial Growth Observation Results

No	Sample	Replication 1	Replication 2	Replication 3
Escherichia coli
1	Etil asetat Fraction 0.1 mg/ml	-	-	-
2	n-hexane Fraction 0.1 mg/ml	-	-	-
3	Water fraction 0.1 mg/ml	-	-	-
4	K+0.1 mg/ml	-	-	-
5	K-0.1 mg/ml	+	+	+
Staphylococcus aureus
1	Etil asetat Fraction 0.1 mg/ml	-	-	-
2	n-hexane Fraction 0.1 mg/ml	-	-	-
3	Water fraction 0.1 mg/ml	-	-	-
4	K+0.1 mg/ml	-	-	-
5	K-0.1 mg/ml	+	+	+

Information:

+: growing colonies of Escherichia coli and Staphylococcus aureus

-: does not grow colonies of Escherichia coli and Staphylococcus aureus

Etil Asetat Fraction Heksan Fraction

Water Fraction

Figure 2. Minimum Bactericidal Concentration 0.1 mg/ml

DISCUSSION:

The fractionation process uses 3 solvents with different polarity properties. Namely, n-hexane is a nonpolar solvent, ethyl acetate is a semipolar solvent, and aquadest is a polar solvent. The highest % yield is the ethyl acetate fraction of 3.7 grams. The higher the fraction yield in polar solvents, the more polar compounds are contained²⁰. The content of semipolar compounds is more than that of nonpolar and polar compounds. Phenolic compounds are polar compounds, so they can dissolve in polar solvents in accordance with the principle of like dissolve like. Yield is a comparison of the weight of the extract obtained with the weight of dry simplisia used to make the extract. The greater the yield, the more substances are attracted from a simplisia. The yield of the ethyl acetate fraction is greater than that of the aquadest and n-hexane fractions. The solvents ethyl acetate and aquadest have a higher dielectric constant than n-hexane. The higher the dielectric constant, the more polar the solvent. The ethyl acetate solvent and aquades are more polar than the n-hexane solvent and can attract more phenolic compounds according to the like dissolve like principle. The yield of the ethyl acetate fraction is greater than that of the water fraction, and the difference in levels can occur due to several factors, including the level of solubility of secondary metabolites. Ethyl acetate solvent has semipolar properties so that it can attract polyhydroxy polyhydroxy aglycone phenolic compounds, while phenolic glycosides are soluble in water. The ethyl acetate fraction shows the greatest phenolic content, which shows that the ethyl acetate solvent can attract phenolic compounds better than the water fraction. The yield of the ethyl acetate fraction is greater than that of the water fraction, and the difference in levels can occur due to several factors, including the level of solubility of secondary metabolites. Ethyl acetate solvent has semipolar properties so that it can attract polyhydroxy polyhydroxy aglycone phenolic compounds, while phenolic glycosides are soluble in water. The ethyl acetate fraction shows the greatest phenolic content, which shows that the ethyl acetate solvent can attract phenolic compounds better than the water fraction²¹.

Different types of solvents have a significant influence on the total phenolic content produced. Ethyl acetate solvents can significantly produce higher total phenolic levels than aquades and n-hexane solvents. This result is directly proportional to the yield of the resulting fraction. This shows that the higher the yield produced, the higher the levels of compounds contained in the extract. Similar research results were shown in the study of Miranti et al.,(2023), the test results determined total flavonoid extract levels of 0.233±0.0025%, water fraction of 0.324±0.0116%, and ethyl acetate fraction of 1.079±0.028%. This shows that the ethyl acetate fraction of limpasu fruit has the highest total flavonoid levels²².

In the alkaloid test, silica gel plates sprayed with Dragendorff produced an orange color, so they were positive for alkaloids. Dragendorff reagents are generally used to detect the presence of nitrogen compounds²³. In the test of flavonoid compounds sprayed with ammoniac, a yellow color appears that quickly fades on the KLT plate, so it is positive that it contains flavonoid compounds. Tanin identification using FeCl₃ as a spot appearance and producing blue-black stains shows a reaction between FeCl₃and hydroxyl groups in tannin compounds. The identification of saponins by KLT is detected by the appearance of anisaldehyde-sulfuric acid spots, producing a blue color. The anthraquinone compound test is sprayed with 10% KOH, and brownish-yellow, purplish-red, or purplish-green stains appear on the KLT plate, so it is positive to contain anthraquinone compounds. A glycoside compound test sprayed H2SO4, and a turquoise stain appeared on the KLT plate. It is positive that it contains glycoside compounds²⁴.

Active compounds in the form of tannin, alkaloids, saponins, flavonoids, polyphenols, and positive anthraquinone are contained in fractions, including polar compounds, so they are easily soluble in ethyl acetate and water solvents. Triterpenoid compounds are composed of long chains of C30 hydrocarbons, so they are nonpolar and easily soluble in n-hexane solvents. Polar compounds that dissolve in nonpolar solvents and polar solvents are likely in the form of glycosides. Negative results are shown in steroid tests, and steroids are composed of isoprenes from long chains of hydrocarbons, so they are very nonpolar.

Data analysis was carried out using Kruskal-Wallis non-parametric statistical tests. The test results obtained a value of p = 0.002 (p < 0.05), meaning that there is a real difference in the average total phenol content between groups of ethyl acetate fraction, n-hexane fraction, and water fraction. Based on Table 3, the ethyl acetate fraction group has the largest total phenol content of 270.88 mg, equivalent to gallic acid/gram fraction, then continued by the water fraction, has a total phenol content of 126.67 mg equivalent to gallic acid/gram fraction and the n-hexan fraction group has the lowest total phenol content of 214.72 mg equivalent to gallic acid/gram fraction.

Analysis of total phenol content using UV-Vis spectrophotometer at maximum wavelength (λ max) 745 nm. Phenolic ions are formed by proton dissociation of phenolic compounds, but this reaction runs slowly. Therefore an alkaline atmosphere carrier solution is needed for the reaction to take place quickly, so sodium carbonate is used as an alkaline atmosphere carrier. The type of solvent influences Total Phenol content. Phenol is a polar compound, so that its solubility is highest in polar solvents. Polar solvents can dissolve phenol better so that its levels in fraction become high²⁵.

Colourimetric oxidation and reduction reactions to measure all phenolic compounds in a test sample. Measurement of phenolic compounds is added with the folin reagent Ciocalteu. These reagents oxidize phenolic (alkali salts) or phenolic hydroxyl groups, reducing heteropoly acid (phosphomolybdate-phosphotungstate) present in the Folin-Ciocalteu reagent to molybdenum tungsten complexes. Phenolic compounds that react with the Folin-Ciocalteu reagent in an alkaline atmosphere to separate protons in phenolic compounds into phenolic ions, and to create alkaline conditions, 7% Na₂CO₃ is used. The hydroxyl group in the phenolic compound reacts with the Folin-Ciocalteu reagent to form a blue molybdenum-tungsten complex that can be detected with a spectrophotometer. The greater the concentration of phenolic compounds, the more phenolic ions will reduce hetero polyacid (phosphomolybdate-phosphotungstate) into molybdenum-tungsten complexes so that the blue color produced is more concentrated. The total phenol content is affected by the type of solvent. Since phenol is a polar molecule, polar fluids are where its solubility is greatest. Polar solvents can more effectively dissolve phenol, increasing its concentration in the extract^26,29.

Data analysis was carried out using Kruskal-Wallis non-parametric statistical tests. In Escherichia coli, the test results were obtained p = 0.009 (p < 0.05) and p = 0.008 values in Staphylococcus aureus, meaning that there was a real difference in the average inhibitory zone between groups of ethyl acetate fraction, n-hexane fraction, water fraction, and positive control. Based on Table 8, the ethyl acetate fraction group has the largest inhibitory zone, which is 19 mm in Escherichia coli and 9 mm inhibitory zone in Staphylococcus aureus, in the n-hexane fraction group, and the inhibition zone water fraction formed close to the ethyl acetate fraction group. In the bacteria Staphylococcus aureus, the n-hexane fraction of water fraction has no obstacles. The antibacterial activity test aims to measure the antibacterial power activity of unripe fruit extract, unripe fruit flesh extract, and unripe kayu banana peel extract, which is thought to have the ability to inhibit growth.

Data analysis was carried out using ANOVA parametric statistical tests. In Escherichia coli and Staphylococcus aureus, the test results obtained p = 0.001 (p < 0.05), meaning that there was a real difference in the average decrease in Optical Density (OD) between groups of ethyl acetate fraction, n-hexane fraction, water fraction, and positive control. Based on Table 8, the ethyl acetate fraction group has the largest average decrease in Optical Density (OD), namely -0.295 in Escherichia coli and -0.931 in Staphylococcus aureus. In the results of Tukey's follow-up test on Escherichia coli, there are 5 different superscript letters, which means there are 5 groups that are not the same. Group “a” in the positive control had the highest average decrease in Optical Density (OD), then group “b” in the ethyl acetate fraction had the average decrease in Optical Density (OD) second after the positive control, Group “c” was the n-hexane fraction group, Group “d” was the water fraction group and Group “e” was the negative control fraction group. In the results of Tukey's follow-up test on Staphylococcus aureus, there are 3 different superscript letters, which means there are 3 groups that are not the same. Group “a” in the ethyl acetate fraction and the water fraction has the highest average decrease in Optical Density (OD), then group “b” is the n-hexane fraction, and the positive dick has an average decrease in Optical Density (OD) second only to group 1, “c” is the group of negative control fraction group.

The test method used is the dilution method of OD λ max 625 nm in Escherichia coli and OD λ max 292 nm in Staphylococcus aureus with an incubation time of 24 hours. Bacteria used in Escherichia coli. This antibacterial test aims to determine the ability to inhibit bacterial growth within a certain incubation time. The cloudier a culture, the greater the number of cells. The light emitted by the spectrophotometer will hit the cell so that some light will be absorbed and some passed on. The amount of light in the spectrophotometer absorbed by the cell in the cuvette is calculated as the absorbance value. Minimal inhibitory concentration (MIC) at 1mg/ml is the lowest antibacterial substance concentration in inhibiting bacterial growth within a certain incubation time. The MIC at a concentration of 1mg/ml value is obtained by looking at bacterial growth in a test tube using a UV-Vis spectrophotometer. Bacteria that grow and die can be observed by looking at the difference in absorbance from before to after, called the change in Optical Density (ΔOD).

Based on Table 5, it can be seen that ethyl acetate fraction, n-hexane fraction, and unripe fruit peel water fraction show negative ΔOD values. This suggests that bacterial growth is inhibited, characterized by a decrease in ΔOD after the incubation period. The same was established in the positive control of negative ΔOD. This indicates inhibited bacterial growth. The unripe fruit peel fraction of kayu bananas is thought to have penetrated the bacterial cell wall and can suppress its growth characterized by negative ΔOD. The ethyl acetate fraction showed a large ΔOD of -0.295 in Escherichia coli and -0.931 in Staphylococcus aureus compared to other fractions. This shows the ability of the ethyl acetate fraction of unripe fruit peel to inhibit bacterial growth more optimally than the water fraction and the n-hexane fraction.

Minimum Inhibitor Concentration (MIC) at a concentration of 1mg/ml is the concentration of an antibacterial substance inhibiting bacterial growth within a certain incubation time. The MIC at a concentration of 1mg/ml value is obtained by looking at bacterial growth in a test tube using a UV-Vis spectrophotometer. Bacteria that grow and die can be observed by looking at the difference in absorbance from before to after, called the change in Optical Density (ΔOD). The number of bacterial cells can be measured by knowing the turbidity of the culture. The cloudier a culture is the greater the number of cells. The light emitted by the spectrophotometer will hit the cell so that some light will be absorbed and some passed on. The amount of light absorbed by the cell in the cuvette in the spectrophotometer is calculated as the absorbance value²⁷.

Based on Table 5, it can be seen that ethyl acetate fraction, n-hexane fraction, and water fraction show negative ΔOD values. This shows the ability of the unripe fruit skin fraction to inhibit the growth of Escherichia coli and Staphylococcus aureus. The fraction has antibacterial activity, but its mechanisms and molecular pathways have not been studied.

Based on Table 6, ethyl acetate fraction, n-hexane fraction, and water fraction can kill bacteria because there is no bacterial colony growth in bacterial growth media. The ability of the fraction to kill bacteria is the same as the ability of positive control (chloramphenicol) because the positive control media also has no bacterial growth. The ability of fractions to kill bacteria differs from negative control because negative control media has bacterial growth. This is related to the results of the study by Pusmarani et al. (2022), who stated that ethyl acetate and banana peel water fraction showed the highest antioxidant activity compared to chloroform and n-hexane fractions. According to Paju et al. (2013), an antibacterial substance can inhibit or kill bacteria that cause infection. Among the bacteria that can cause infection are Staphylococcus aureus and Escherichia coli. Tanin has antibacterial power by precipitating because it is suspected that tannins have the same effect as phenolic compounds. The antibacterial mechanism of tannins is through reactions with inhibition of cell membrane biosynthesis, enzyme inactivation, and destruction or inactivation of genetic material function^28,29.

Antibacterial tests of unripe fruit peel extract of kayu banana (Musa paradisiaca L var. Kayu) against Staphylococcus aureus and Escherichia coli showed that all treatments tested were able to kill Staphylococcus aureus and Escherichia coli, a decrease in the number of colonies at each treatment concentration showed this. Based on the results of the study, the fraction and positive control were able to inhibit bacterial growth. The mechanism of positive control is blocking the activity of the peptidyl transferase enzyme, which is an enzyme that works in the process of protein synthesis in the microbial body. As a result, the process of protein synthesis will stop instantly and cause inhibition of microbial growth. Chloramphenicol contains antibacterial properties that can inhibit bacterial growth. According to Syafna et al., (2021), banana unripe fruit skin contains phenolics and active ingredients such as tannin and flavanoid, plants that contain tannins and flavonoids are antiseptic and can be used as antibacterial^30,31.

The results of the antibacterial activity test of the n-hexane fraction of the unripe fruit peel of kayu banana have the weakest antibacterial activity compared to the fraction of water and ethyl acetate. This is because the levels of compounds in the extract are more dissolved in ethyl acetate, so the compounds extracted or absorbed are greater semipolar and polar compounds compared to nonpolar compounds. Compounds contained in the unripe fruit peel of kayu bananas tend to have semipolar and polar properties, so the compounds dissolved in the n-hexane fraction are less because n-hexane is nonpolar. Nonpolar compounds have potential as antibacterial compounds, but the results are less effective than water fractions and ethyl acetate³².

According to research by Ajeng et al., (2023), antibacterial extract of unripe fruit peel of banana kepok (Musa paradisiaca x balbisiana) can inhibit the growth of Staphylococcus aureus. The concentration of unripe fruit peel extract of banana kepok (Musa paradisiaca x balbisiana), which is good in inhibiting the growth of Staphylococcus aureus bacteria, is at a concentration of 100%. This shows that banana peels contain antibacterial compounds to be used as antibiotics against Staphylococcus aureus infections³³.

The antibacterial activity test aims to measure antibacterial power activity, which is thought to have the ability to inhibit growth and even kill bacteria. In the diffusion method, the growth of test organisms can be inhibited so as not to spread along antibacterial diffusion (a clear zone forms around the disc) so that it can be said that the bacteria are bacteria that are sensitive to antibacterial compounds. The inhibitory zone is one of the parameters for determining the antibacterial sensitivity of fractions in test bacteria³⁴.

The presence of antibacterial inhibitory activity is due to compounds in unripe fruit peel extracts that contribute to antibacterial effects. According to research Nair (2010), The mechanism of the active compound responsible for the antimicrobial activity is unknown³⁵. Still, preliminary phytochemical results have shown that in the presence of various compounds, the inhibitory potential of the extract may be due to its secondary metabolite content³⁶. The antibacterial activity of the extract is due to the presence of secondary metabolites of phenolic groups such as tannin, flavonoid, phenolic, and polyphenol in the extract. The hydroxyl group is thought to be related to its toxicity activity to microorganisms, whereas increased hydroxylation results in increased toxicity. The extract mechanism in bacterial inhibition may be related to inhibitions in electron transport, protein translocation, phosphorylation, and other reactions involving enzymes, leading to increased plasma membrane permeability and, eventually, ion imbalance in the cell wall. In addition, the mechanism may also be related to the surface permeability of the bacterial cell wall by fraction. According to research, The phenolic compound artocarpanone provides antibacterial activity against Escherichia coli by interfering with membrane permeability. The antibacterial mechanism is still unclear, and it is suspected that the bacterial cell wall membrane is one of the sites of action that interacts on the lipophilic side of the cell wall membrane, causing damage to the cell wall membrane. Research Rita et al..(2020) state that Broken Banana Thousand has flavonoid and phenolic content of 2033.53 mg QE/100g and 250.25 mg GAE/100g respectively, which can inhibit the growth of Staphylococcus aureus by 12.27 mm at a concentration of 20% and 13.60 mm at a concentration of 50%, while Escherichia coli by 11.87 mm at a concentration of 20% and 12.73 mm at a concentration of 50% [36]. The difference in sensitivity between ethyl acetate fraction, water fraction, and n-hexan fraction of unripe banana peel is due to the difference in the content of secondary metabolites. The absorption of phenolic compounds by bacterial cell membranes and the interaction of phenolic compounds with active enzymes in bacterial cell walls greatly affect their antibacterial acti. In the banana peel fraction, unripe kayu has optimal activity with the largest total phenol content¹⁹.

According to research, The phenolic compound artocarpanone provides antibacterial activity against Escherichia coli by interfering with membrane permeability. The antibacterial mechanism is still unclear, and it is suspected that the bacterial cell wall membrane is one of the sites of action that interacts on the lipophilic side of the cell wall membrane, causing damage to the cell wall membrane. The absorption of phenolic compounds by bacterial cell membranes and the interaction of phenolic compounds with active enzymes in bacterial cell walls greatly affect their antibacterial activity. Unripe banana peel extract has optimal activity and the largest total phenol content.

A correlation test was carried out to determine the relationship between the effect of Total Phenol Content on the minimum inhibitory concentration (MIC) at a concentration of 1mg/ml and the inhibitory zone. The results of the correlation test on Escherichia coli showed that the Pearson correlation value was -0.750 in the relationship of Total Phenol Content with minimum inhibitory concentration (MIC) at a concentration of 1mg/ml and 0.521 in the relationship of Total Phenol Content with the inhibition zone. The results of the correlation test on Staphylococcus aureus showed that the Pearson correlation value was -0.462 in the relationship of Total Phenol Content with minimum inhibitory concentration (MIC) at concentration of 1mg/ml and 0.834 in the relationship of Total Phenol Content with the inhibition zone. The value shows a value that has a significant relationship meaning because of the value of recognition (p-value) <0.05³⁸.

At the minimum inhibitory concentration (MIC) at a concentration of 1mg/ml, there is a significant relationship (meaningful correlation) between the variable Total Phenol Content with the minimum inhibitory concentration (MIC) and a negative correlation, which means that between the 2 variables tested in the opposite direction, namely the greater the total phenol content, the more the optical density value in the minimum inhibitory concentration parameter (MIC) at a concentration of 1mg/ml. In other words, the greater the total content of phenols, the higher their antibacterial activity. The correlation value is -0.750 in Escherichia coli, which means that the relationship between total phenol content to the minimum inhibitory concentration (MIC) at a concentration of 1mg/ml has a strong correlation and the correlation value of -0.462 in Staphylococcus aureus a which means that the relationship between total phenol content to the minimum inhibitory concentration (MIC) at a concentration of 1mg/ml has a moderate correlation.

In the inhibitory zone, there is a significant relationship (meaningful correlation) between the variable Total Phenol Content with the value of the diameter of the inhibitory zone and has a positive correlation, which means that between the 2 variables tested in the same direction, namely the greater the total phenol content, the greater the value of the diameter of the inhibitory zone. In other words, the greater the total content of phenols, the higher their antibacterial activity. The correlation value is 0.521 in Escherichia coli, which means the relationship between the total phenol content and the decrease in Optical Density (OD) has a moderate correlation. The correlation value is 0.834 in Staphylococcus aureus, which means the relationship between the total phenol content and the decrease in Optical Density (OD) has a very strong correlation.

The results of research on the antibacterial activity of unripe fruit peel extract of kayu banana (Musa paradisiaca L var kayu) against Staphylococcus aureus and Escherichia coli can conclude that ethyl acetate fraction, N-hexane fraction, and water fraction of unripe fruit peel of kayu banana (Musa paradisiaca L var. kayu) have different antibacterial activity. Of the three samples, the unripe fruit peel fraction of kayu banana (Musa paradisiaca L var. kayu) that has active antibacterial activity in inhibiting bacteria is the ethyl acetate fraction with the most effective minimum inhibitory concentration results, namely ethyl acetate fraction of -0.931 in Staphylococcus aureus and -0.295 in Escherichia coli. The ethyl acetate fraction's most optimal minimum kill concentration is evidenced by the absence of growth in the medium. The results of this research are the same as the research by Hyun (2018), that the ethyl acetate fraction of Crowberry raw fruit with phenolic content (579±22 mg GAE/g extract) shows that solvent polarity is important for the extraction of phenolic compounds³⁹. The increase in antibacterial activity also depends on the phenolic content. In the ethyl acetate fraction with the highest phenolic content, it has the greatest antibacterial activity. In addition, fractions using non-polar solvents showed weak antibacterial activity, suggesting that solvents with different polarities significantly affected antibacterial activity. In the MIC results, the ethyl acetate fraction showed greater activity against Gram-positive bacteria than Gram-negative bacteria (Table 5) because the decrease in absorbance was greater. Structural differences in the cell walls of Gram-positive and Gram-negative bacterial bacteria may cause this activity. Gram-negative bacteria have a cell wall located between the inner and outer membranes of the cell, whereas Gram-positive bacteria have a cell wall, which is the outer layer of the cell. Therefore, Gram-negative bacteria are more resistant to the peel fraction of the unripe fruit of kayu bananas³⁹.

This research is linear with research by Maria Anastasiadi et al (2009), who found that phenol compounds in the grape extract have synergistic activity as natural antibacterial through the determination of their minimum inhibitory concentration. Phenol compounds in grape extract can inhibit bacterial growth⁴⁰. According to Anyasi et al. (2018), Phenol derivatives use hydrogen bonds during adsorption to interact with bacterial cells. Phenol protein complexes with weak bonds are created at low concentrations and quickly break down. This is followed by phenol entering the cell and precipitating and denaturing proteins. Phenols cause lysis of cell membranes and coagulation of proteins at high concentrations^41,42,43. Antibacterial activity is carried out by denatureizing bacterial proteins. Others are more specific, altering the structure of membranes or inactivating key compounds or important cell functions. Therefore, these biomolecules are bactericidal or bacteriostatic, depending on their properties and concentration^44,45.

CONCLUSION:

The unripe fruit peel fraction of kayu banana has antibacterial potential in Escherichia coli and Staphylococcus aureus. The ethyl acetate fraction has a total phenol content of 270.88 mg, equivalent to gallic acid/gram fraction. Phenolic compounds can inhibit bacterial growth. A diffusion test was conducted to prove that all fractions have antibacterial potential. The minimum inhibitory concentration test results demonstrate that all fractions can inhibit bacteria, and the ethyl acetate fraction has optimal potential compared to other fractions. The Minimum Bactericidal Concentration results prove that all fractions can kill all bacterial growth. The correlation of the value of total phenol content (TPC) and antibacterial activity is established to have a linear relationship between total phenol content and antibacterial potential. The greater the total phenol content, the greater the antibacterial potential.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

This research was carried out thanks to everyone's support. Thank you to Universitas Airlangga for its support. Thank you to Anwar Medika University, lecturers, and students for their support in completing this research.

REFERENCES:

1. Kemenkes Ri, Profil Kesehatan Indonesia 2021. 2022.

2. C. H. Lescano. Ivan PO. Tiago Z. Campomanesia Adamantium Peel Extract In Antidiarrheal Activity: The Ability Of Inhibition Of Heat-Stable Enterotoxin By Polyphenols. Plos One. 2016. 11(10) 1–15. Doi: 10.1371/Journal.Pone.0165208.

3. M. K. R. Konduri, K. B. Uppuluri, R. Chintha, S. Mulla, And R. Peruri. In-Vitro Antimicrobial Activity Of Four Indigenous Medicinal Plants Belonging To Bapatla, A.P. Res. J. Pharm. Technol. 2010; 3(2): 461–465.

4. A. W. Ningsih, M. Rochmanti, And A. Basori, Effectiveness Of Antidiarrheal Unripe Wooden Banana (Musa Paradisiaca L.) In Male Balb-C/Mice Induced With Escherichia Coli. Folia Medica Indones. 2021; 56(3): 208.Doi: 10.20473/Fmi.V56i3.24558.

5. A. W. Ningsih, E. Pratama, S. Komariyah, D. P. Astuti, I. C. S. Klau, And D. Rahmawati. Differences In Extraction Methods To Antidiarrheal Activity In Vitro And In Vivo In Unripe Kayu Banana Fruit ( Musa Paradisiaca L. Var. Kayu). Gac Méd Caracas. 2022; 130(Supl 5): 1133–1146. Doi: 10.47307/Gmc.2022.130.S5.38.

6. S. R. Patel, A. P. Suthar, A. M. Shah, H. V Hirpara, V. D. Joshi, And M. V Katheria. Antimicrobial Activity Of Methanolic Extracts Of Mucuna Pruriens, Semecarpus Anacardium, Anethum Graveolens By Agar Disc Diffusion Method. Res. J. Pharm. Technol. 2010; 3(1).

7. S. R. Kane, V. A. Apte, S. B. Patil, And C. S. Magdum. Antimicrobial Potential Of Euphorbia Thymifolia Linn. Res. J. Pharm. Technol. 2009; 2(1): 208–209.

8. I. Borrás-Linares. Fernandez A. D. Arraez.Characterization Of Phenolic Compounds, Anthocyanidin, Antioxidant And Antimicrobial Activity Of 25 Varieties Of Mexican Roselle (Hibiscus sabdariffa). Ind. Crops Prod. 2015; 69: 385-394. Doi: 10.1016/J.Indcrop.2015.02.053.

9. M. I. Zainol, K. Mohd Yusoff, And M. Y. Mohd Yusof. Antibacterial Activity Of Selected Malaysian Honey. Bmc Complement. Altern. Med. 2013; 13. Doi: 10.1186/1472-6882-13-129.

10. Selfiana, A. Identifikasi Senyawa Aktif Antrakuinon Fraksi Etil Asetat Kayu Songga (Strychnos ligustrida) Sebagai Anti Malaria Melalui Uji Aktivitas Penghambatan Polimerisasi Heme. Skripsi. 2019. Universitas Islam Indonesia

11. A. W. Ningsih, I. Hanifa, And A. Hisbiyah. Pengaruh Perbedaan Metode Ekstraksi Rimpang Kunyit (Curcuma domestica) Terhadap Rendemen Dan Skrining Fitokimia. J. Pharm. Care Anwar Med. 2020; 2(2): 96–104. DOI: http://dx.doi.org/10.36932/jpcam.v2i2.27

12. Rosida, Sukardiman, And J. Khotib. The Increasing Of Vegf Expression And Re-Epithelialization On Dermal Wound Healing Process After Treatment Of Banana Peel Extract (Musa Acuminata Colla). Int. J. Pharm. Pharm. Sci. 2014; 6(11): 427–430.

13. P. Jambwa, F. N. Makhubu, G. Matope, G. Fouche, And L. J. Mcgaw. Bioassay Guided Fractionation Of Senna Singueana And Its Potential For Development Of Poultry Phytogenic Feed Additives. Front. Vet. Sci. 2022; 8(1): 1–14. Doi: 10.3389/Fvets.2021.800272.

14. R. Karamian, M. Asadbegy, And S. Yari. Protective Potency Of Meristotropis xanthioides Against Nephrotoxicity In A Rat Model Along With Its Antioxidant And Antibacterial Activities. Asian Pac. J. Trop. Med. 2017; 10(10): 960–966 Doi: 10.1016/J.Apjtm.2017.09.007.

15. E. N. Siju. GR Rajalakshmi. D Vivek. Antimicrobial Activity Of Leaf Extracts Of Cleodendrum viscosum. Vent. Res. J. Pharm. Technol. 2009; 2(3): 599–600.

16. G. College And K. Naka. Antimicrobial And Antifungal Activity Of Centella asiatica (L.) Urban , Umbeliferae. Res. J. Pharm. Tech. 2009; 2(2): 328–330.

17. N. Benchikha. Imane C. Hannane D. Lobularia Libyca: Phytochemical Profiling, Antioxidant And Antimicrobial Activity Using In Vitro And In Silico Studies. Molecules. 2022; 27(12): 1–18. Doi: 10.3390/Molecules27123744. https://doi.org/10.3390/molecules27123744.

18. S. T. Sumathi R, Pavni S. Antimicrobial Evaluation Of Lipid Extract Of Pongamia Pinnata Leaves. Res. J. Pharm. Tech. 2009; 2(4): 714–718.

19. K. Ács, V. L. Balázs, B. Kocsis, T. Bencsik, A. Böszörményi, And G. Horváth. Antibacterial Activity Evaluation Of Selected Essential Oils In Liquid And Vapor Phase On Respiratory Tract Pathogens. Bmc Complement. Altern. Med. 2018; 18(1): 1–9. Doi: 10.1186/S12906-018-2291-9.

20. L. Chen. B Yu. Y Zhang. X Gao. Bioactivity-Guided Fractionation Of An Antidiarrheal Chinese Herb Rhodiola Kirilowii (Regel) Maxim Reveals (-)-Epicatechin-3-Gallate And (-)-Epigallocatechin-3-Gallate As Inhibitors Of Cystic Fibrosis Transmembrane Conductance Regulator. Plos One. 2015; 10(3): 1–12. Doi: 10.1371/Jurnal.Pon.0119122.

21. S. Susanti, R. S. Sundari, L. R. Rizkuloh, And R. Mardianingrum. Effect Of Solvents Extraction On Total Phenolic Content And Antioxidant Activity Of Gadung Extract (Dioscorea hispida Dennst.). Biopropal Ind. 2021; 12(1); 43 Doi: 10.36974/Jbi.V12i1.6482.

22. R. M. Miranti, S. Nashihah, F. Farmasi, And U. Muhammadiyah. Phytochemical Screening And Determination Of Total Flavonoid Content Of Limpasu Fruit Extracts And Fractions (Baccaurea Lanceolate (Miq.) Müll.Arg. J. Curr. Pharm. Sci. 2023; 7(1): 652–656.

23. D. Wigati And R. R. Rahardian. Penetapan Standarisasi Non Spesifik Ekstrak Etanol Hasil Perkolasi Umbi Bawang Dayak (Eleutherine Palmifolia (L.)Merr). Jiffk J. Ilmu Farm. Dan Farm. Klin. 2018; 15(2): 36. Doi: 10.31942/Jiffk.V15i2.2564.

24. D. Yanti And N. Nurhayati. Identifikasi Senyawa Anti Mikroba Ekstrak Etanol Batang Brotowali (Tinospra Crispa (L.) Terhadap Staphylococcus Aureus, Bacillus Substillis, Dan Candida Albicans Dengan Metode Klt Bioautografi. J. Ayurveda Medistra. 2022; 4(1): 26–33. Doi: 10.51690/Medistra-Jurnal123.V4i1.54.

25. A. Mulia, M. S. Masfria, A. D. S. Si, And M. Si. Pengujian Kandungan Total Fenol Ekstrak Etanol Tempuyung (Shoncus Arvensis L.) Talenta Conference Series Pengujian Kandungan Total Fenol Ekstrak Etanol Tempuyung (Shoncus Arvensis L.). In Talenta Conference Series: Tropical Medicine (Tm) Paper. 2018; 1(1); 284–290.DOI:10.32734/tm.v1i1.75

26. T. E. Wado, S. Suleman, And T. Mohammed. Antimicrobial Evaluation Of Sequentially Extracted Leaf Of Vernonia Auriculifera Hiern (Rejicho). Bmc Complement. Med. Ther. 2022; 22(1): 1–10.Doi: 10.1186/S12906-022-03690-2.

27. H. Soetjipto, A. I. Kristijanto, And R. S. Asmorowati. Toksisitas Ekstrak Kasar Bunga Dan Daun Ketepeng Cina (Senna Alata L. Roxb.) Terhadap Larva Udang Artemia Salina Leach. Biota J. Ilm. Ilmu-Ilmu Hayati. 2019; 11(2): 78–82. Doi: 10.24002/Biota.V12i2.2645.

28. Pusmarani, J., Ulfa, U., Dewi, C., and Nasir, N. H. Aktivitas antioksidan fraksi air, etil asetat dan N-Heksan kulit Pisang Raja (Musa paradisiaca var. Sapientum). Jurnal Mandala Pharmacon Indonesia. 2022; 8(2): 275-283.

29. Paju, N., Yamlean, P. V., and Kojong, N. Uji efektivitas salep ekstrak daun binahong (Anredera cordifolia (Ten.) Steenis) pada kelinci (Oryctolagus cuniculus) yang terinfeksi bakteri Staphylococcus aureus. Pharmacon. 2013; 2(1).

30. Syafna, E. Y.. Uji Aktivitas Antibakteri Tepung Kulit Pisang Kepok (Musa balbisiana Colla) pada Staphylococcus aureus. 2021. (Doctoral dissertation, Universitas Perintis Indonesia).

31. Sari, R., & Ferdinan, A.. Pengujian aktivitas antibakteri sabun cair dari ekstrak kulit daun lidah buaya. Pharmaceutical Sciences and Research. 2017; 4(3): 1.

32. P. Devi. Diuretic And Antimicrobial Activity Of Leaves Of Limnophila Rugosa. Res. J. Pharm. Tech. 2008; 2(1): 25–27.

33. Ajeng, R.. Pemanfaatan Ekstrak Kulit Pisang (Musa Paradisiaca L.) sebagai Pestisida Alami Hama Wereng Coklat (Nilaparvata Lugens) pada Tanaman Padi. 2023. (Doctoral dissertation, Uin Raden Intan Lampung)..

34. N. Raaman, S. Selvarajan, D. Balakrishnan, And G. Balamurugan. Preliminary Phytochemical Screening, Antimicrobial Activity And Nutritional Analysis Of Methanol Extract Of Asparagus Racemosus (Willd) Roots. Res. J. Pharm. Technol. 2009; 2(4): 777–779.

35. D. U. S. Nair M. P. Narra Kishore Pully N. R.. Phytochemical And Antimicrobial Evaluation Of Luffa Cylindrica Linn. Leaf And Flower Extracts - An In-Vitro Study,” Res. J. Pharm. Technol. 2010; 3(2): 438–441.

36. D. U.S. Nair M. P. Narra Kishore Pully N. R.. Phytochemical And Antimicrobial Evaluation Of Luffa Cylindrica Linn. Leaf And Flower Extracts An In-Vitro Study. Res. J. Pharm. Technol. 2010; 3(2); 438–441.

37. W. S. Rita, I. H. Resaputra, And I. M. Sukadana. Aktivitas Antibakteri Ekstrak Metanol Kulit Pisang Pecah Seribu (Musa X Paradisiaca L.) Terhadap Bakteri Staphylococcus aureus Dan Escherichia coli. Cakra Kim. [Indonesian E-Journal Appl. Chem. 2020; 8(2); 82–91.

38. T. A. Anyasi, A. I. O. Jideani, And G. R. A. Mchau. Phenolics And Essential Mineral Profile Of Organic Acid Pretreated Unripe Banana Flour. Food Res. Int. 2018; 104(5): 100–109. Doi: 10.1016/J.Foodres.2017.09.063.

39. T. K. Hyun, J. H. Ra, S. H. Han, And J. S. Kim. Antioxidant, Antimicrobial, And Antidiabetic Activities Of Crowberry Fruits. Indian J. Pharm. Sci. 2018; 80(3): 489–495. Doi: 10.4172/Pharmaceutical-Sciences.1000382.

40. S. A. H. Maria Anastasiadi, Nikos G. Chorianopoulos, George-John E. Nychas. Antilisterial Activities Of Polyphenol-Rich Extracts Of Grapes And Vinification Byproducts. 2009. J. Agric. Food Chem. 2009; 57: 457–463. https://doi.org/10.1021/jf8024979

41. T. A. Anyasi, A. I. O. Jideani, And G. R. A. Mchau. Phenolics And Essential Mineral Profile Of Organic Acid Pretreated Unripe Banana Flour. Food Res. Int. 2018; 104(5): 100–109. Doi: 10.1016/J.Foodres.2017.09.063.

42. N. Raaman, S. Selvarajan, D. Balakrishnan, And G. Balamurugan. Preliminary Phytochemical Screening, Antimicrobial Activity And Nutritional Analysis Of Methanol Extract Of Asparagus Racemosus (Willd) Roots. Res. J. Pharm. Technol. 2009; 2(4): 777–779.

43. D. U.S. Nair M. P. Narra Kishore Pully N. R.. Phytochemical And Antimicrobial Evaluation Of Luffa Cylindrica Linn. Leaf And Flower Extracts - An In-Vitro Study. Res. J. Pharm. Technol. 2010; 3(2): 438–441.

44. K. Kaboré, C. I. Dibala, H. Sama, M. Diao, M. K. Somda, And M. H. Dicko.Phenolic Content, Antioxidant Potential, And Antimicrobial Activity Of Uvaria Chamae (Annonaceae), A Food Plant From Burkina Faso. Biochem. Res. Int. 2024. Doi: 10.1155/2024/1289859.

45. N. ‘Aini H. Dan A. N. Hery Muhamad Ansory, Prietta Khania Kusuma Putri. Analisis Senyawa Minyak Atsiri Biji.Pala Secara Gc-Ms Dan Uji Aktivitas Antibakteri Terhadap Escherichia coli dan Staphylococcus aureus. Pros. Snst. 2018; 9: 19–25.

Received on 22.02.2024 Revised on 17.06.2024

Accepted on 28.09.2024 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2137-2148.

DOI: 10.52711/0974-360X.2025.00307

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

No.	Phytochemical screening	Reagent	Result	EAF	HF	WF
1	Alkaloid	Dragendorf	Brownish-orange	+	+	+
2	Flavonoid	Wilstater	orange	+	+	+
3	Glikosida	Molisch	Brown color	+	+	+
4	Saponin	Busa	Forming foam	+	+	+
5	Tanin	FeCl3	Blackish green	+	+	+
6	Triterpenoid	Lieberman-Burchard	red or purple color	+	+	+
7	Steroid	Lieberman-Burchard	green color	+	+	-
8	Polifenol		green to black, blue color	+	+	+
9	Antrakuinon	KOH	Red	+	+	+

No.	Sample	Average absorbance	Total Phenolic Content (mgGAE/g fraction) ± SD
1	ethyl acetate Fraction	0,787	270,88 ± 0,36^a
2	n-hexane Fraction	0,328	126,67 ± 0,50^b
3	Water fraction	0,608	214,72 ± 0,50^c

Table 2. Phytochemical screening results of unripe fruit peel extract of kayu banana (Musa paradisiaca L.var.Kayu)