INTRODUCTION:

Red Pidada or Sea Rambai (Sonneratia caseolaris L.) is a mangrove plant. Many people use sea rambai as traditional medicine, especially in South Kalimantan. The fruit and leaves of sea rambai are often used as medicine and ingredients for cold powder and scar removal¹. Besides, the fruit, bark, and leaves of the Sonneratia species have been used in traditional medicine to treat asthma, febrifuge, ulcers, hepatitis, sprains, hemorrhoids, and bleeding²

The Red Pidada plant (Sonneratia caseolaris L.) is one of the plants used in traditional ways, especially by

Borneo's people where the red Pidada leaves are used as a cool powder composition and applied to the face as a protection from sun rays³. A pidada plant is traditionally used as a cold powder concoction⁴. The isolation of Pidada Merah leaves contain fatty acid compounds, hydrocarbon sterols, and two flavonoids, namely luteolin and luteolin 7-O-β glucoside, which have high antioxidant power^1,5.

A study that has been conducted by Herwinda and Amir⁶ found that this plant contains alkaloid compounds, flavonoids, glycosides, saponins, and phenols⁷. Red Pidada extract has a very strong antioxidant activity of 21.62ppm because it has secondary metabolites, which are one of the medicinal ingredients and have a molecular structure, and have various biological activities. Research conducted by Jubaidah et al⁸show that the polar fraction of the leaves has a total phenolic content of 213.49 1.2227 mg GAE/g, which means that in every gram, the polar fraction is equivalent to 213.49 mg of gallic acid, while in the nonpolar fraction of sea rambai leaves it is obtained that 55.79 1.0809mg GAE/g, which means that in every gram of nonpolar fraction is equivalent to 55.79mg/g. The total phenolic content produced was more significant proportion in the polar fraction. The phenolic compounds found in plants serve to protect plant tissues against damage due to solar radiation. Phenolic compounds in flavonoids have a strong potential for antioxidants because of chromophore groups that can capture free radicals. The aim of this study was to determine the activity of Pidada red leaf extract and fraction as antioxidants.

METERIAL AND METHODS:

A. Tools and Materials:

The tools needed in this research are a set of maceration tools, glassware, UV-Visspectrophotometer, micropipette, analytical balance, and a brown bottle.

The ingredients are red pidada leaf Simplicia, ABTS powder, K2S2O8 powder, ascorbic acid (vitamin C), distilled water, 70% ethanol, n-hexane, ethylacetate, aluminum foil, filter paper, and tissue.

B. Procedure:

1. Plant Selection:

The correct species of the plant was selected carefully to ensure the correctness of the plants under study and avoid errors in the collection of research materials and determine the names and species of plants specifically. Plant determination was carried out at the Samboja Center for Natural Resources Conservation, East Kalimantan, Indonesia.

2. Making Simplicia:

The collected leaves of Red Pidada are separated from the dirt and washed with clean and running water. Then it is chopped and dried by keeping them in a shady place. The drying process is carried out for about one week. The dry Simplicia is weighed, then crushed into a powder, and sieved using a 60-mesh sieve. Sieving with 60 mesh aims to reduce the size of the powder and expand it.

3. Leaf Extraction and Fractionation:

The extraction method used is the maceration method. This method is a simple extraction method. The maceration method was chosen because it minimizes the possibility of damage to the sample compounds due to the heating process. The solvent used in the extraction process is 70% ethanol. To produce the optimal amount of active ingredient, 70% semipolar ethanol is used.

The ethanol extract was fractionated in stages using the liquid-liquid fractionation method with n-hexane and ethyl acetate solvents. The ethanol extract was dissolved in distilled water before being mixed with n-hexane solvent and shaken/separated using a separating funnel procedure. After that, it was set aside for a while until two layers were formed, namely the n-hexane layer (top layer) and the water layer (bottom layer). Furthermore, the n-hexane fraction is separated and accommodated to evaporate the solvent. In contrast, the water fraction is put back into the funnels to be continued in the following fractionation process to get an ethyl acetate fraction and an ethanol fraction (residual).

4. Phytochemical Screening:

Phytochemical screening was carried out on the ethanol extract of Red Pidada leaves, which included examining chemical compounds of alkaloids, flavonoids, saponins, tannins, and steroids.

C. Antioxidant Activity Test:

1. Red Leaves Preparation of 100 ppm Ethanol Extract:

The 100ppm main solution was prepared by weighing 25mg of the extract and dissolving it with 70% ethanol until the 25mL volumetric flask mark obtained a 1000 ppm solution. Then it was diluted, by adding 1mL of the 1000ppm solution, put it in a 10mL volumetric flask, and 70% ethanol was added to mark the 10mL volumetric flask limit.

2. preparation of a main solution containing 10 ppm ascorbic acid:

The 10ppm mother liquor was prepared by weighing 10 mg of ascorbic acid dissolved in 70% ethanol until the 10mL volumetric flask limit mark obtained a 1000ppm solution. Then diluted, 100L of the solution was added with 70% ethanol to mark the limit of the 10mL volumetric flask.

3. Making ABTS Stock Solutions:

a. The 0.0384g of ABTS powder was weighed and then dissolved in distilled water until the 10 mL volumetric flask was shaken until homogeneous mixture was obtained.

b. About 0.0066g of K2S2O8 powder was weighed and then dissolved in distilled water until the 10mL volumetric flask was shaken until homogeneous.

c. In a brown bottle, solutions a and b was mixed and incubated in a dark room for 12-16 hours to produce a dark blue or turquoise solution.

d. Diluted1 mL of ABTS stock solution was taken and 70% ethanol was added to the 10mL volumetric flask, which is shaken until homogeneous. This solution is called the aqueous ABTS solution.

4. Qualitative Antioxidant Activity Test:

Three test tubes were prepared.

a. In Tube 1, 1mL of diluted ABTS was taken and 9 mL of 70% ethanol was added.

b. Tube 2, 500 L of the solution was taken and 1mL of dilute ABTS and 8mL of 70% ethanol was added.

c. 500 L of ascorbic acid solution was added with 1mL of diluted ABTS and 8 mL of 70% ethanol to Tube 3.

Antioxidants are present when there is a color change from turquoise to colourless or transparent.

5. Quantitative Antioxidant Activity Test:

a. Determination of the maximum wavelength

The absorption of diluted ABTS in 2 mL pipettes was measured using a UV-V spectrophotometer with a wavelength of 650-800 nm.

b. Measurement of ABTS blank solution absorption

Dilute ABTS in a 1 mL pipette was added with 70% ethanol to the 10 mL volume tricflask mark. The absorption was measured using a UV-V is spectrophotometer at the maximum wave length.

c. Measurement of ABTS freer adical binding activity with samples

The main solution of 100 ppm red Pidada leaf ethanol extract is piped at 100, 200, 300, 400, and 500 L. Each 1 mL of dilute ABTS is added to a 10 mL volumetricflask, and then the volume is sufficient to mark the limit with 70% ethanol, then homogenized, so that the concentrations of 1, 2, 3, 4, and 5 ppm are obtained. Furthermore, the absorption was measured using a UV-Vis spectrophotometer at the maximum wavelength. Increased ABTS-related activity is associated with ascorbate-rich ascorbate.

The main solution of 10 ppm ascorbic acid was piped into 100, 200, 300, 400, and 500 L, and then 1 mL of dilute ABTS was added to 70% ethanol until the 10 mL was reached in the volumetric flask and then it was shaken until homogeneous. Several concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 ppm was obtained. Furthermore, the absorption was measured using a UV-Visspectrophotometer at the maximum wavelength.

RESULTS:

Based on table 1 phytochemical screening results, the ethanol extract of Pidada red leaves was positive for secondary metabolites of alkaloids, flavonoids, saponins, and tannins.

Table 1. Phytochemical Screening Results

S. No	Group	Result
1	Alkaloid	+
	Meyer	+
	Bouchardat	+
	Dragendorff	-
2	Flavonoid	+
3	Tannin	+
4	Saponin	+
5	Steroid	-

Table 2 Explains the IC50 values of Red Pidada leaf extract and ethanol fraction.

Table 2. IC₅₀valuesof Pidada red leaf extract and ethanol fraction

Sample	Testing	Concentration (ppm)	% Inhibition	IC₅₀(ppm)
Ethanolic Extract	Rep 1	0.6	19.70	1.3
		0.8	29.57
		1	38.32
		1.2	43.75
		1.4	55.12
	Rep 2	0.6	22.55	1.33
		0.8	27.48
		1	40.12
		1.2	44.64
		1.4	52.30
	Rep 3	0.6	19.54	1.32
		0.8	29.78
		1	39.80
		1.2	44.09
		1.4	52.54
N-Hexane fraction	Mean IC₅₀ (ppm)			1.32
	Rep 1	5	11.28	21.10
		7	16.42
		9	20.50
		11	25.83
		13	30.60
	Rep 2	5	17.26	18.82
		7	21.10
		9	25.79
		11	30.76
		13	36.57
	Rep 3	5	14.60	19.75
		7	18.35
		9	22.20
		11	28.04
		13	34.38
Ethylacetate fraction	Mean IC50 (ppm)			19.89
	Rep 1	0.1	5.88	0.66
		0.2	16.68
		0.3	20.97
		0.4	29.63
		0.5	38.20
	Rep 2	0.1	11.88	0.41
		0.2	24.30
		0.3	33.59
		0.4	47.68
		0.5	62.22
	Rep 3	0.1	9.32	0.42
		0.2	17.46
		0.3	32.54
		0.4	42.69
		0.5	56.76
Ethanolic Fraction	Mean IC50 (ppm)			0.50
	Rep 1	0.6	17.29	1.70
		0.8	22.49
		1	31.57
		1.2	36.21
		1.4	39.72
	Rep 2	0.6	19.05	1.51
		0.8	26.42
		1	36.19
		1.2	40.04
		1.4	44.82
	Rep 3	0.6	17.24	1.68
		0.8	22.29
		1	31.15
		1.2	36.16
		1.4	40.47
Ascorbic Acid	Mean IC50 (ppm)			1.63
	Rep 1	0.2	13.28	0.75
		0.4	23.32
		0.6	41.47
		0.8	54.30
		1	66.41
	Rep 2	0.2	11.77	0.76
		0.4	23.71
		0.6	41.56
		0.8	53.15
		1	64.83
	Rep 3	0.2	10.47	0.79
		0.4	22.00
		0.6	38.69
		0.8	51.71
		1	62.71
	Mean IC₅₀ (ppm)			0.77

The exact values are taken from experimental measurement.

DISCUSSION:

The ABTS method was chosen because the extract can dissolve in both organic and water solvents to detect both lipophilic and dhydrophilic compounds. The advantages of ABTS methods over other DPPH methods are that it has a specific absorbance at a visible wavelength and it has a fast reaction⁹. The ABTS principle is the loss of color of ABTS cations to measure the capacity of antioxidants to react directly with ABTS cationradicals¹⁰.

The qualitative antioxidant activity test aims to determine whether or not the antioxidant activity of Red Pidada leaf extract and its fraction is present. The qualitative antioxidant test results were that there was a change in colour from turquoise to faded color until it was nolonger colored. According to Setiawan et al.¹⁰, it is said to be positive for antioxidants if a turquoise color changes to faded or colorless due to ABTS reduction by antioxidants. The test results support the statement made by Setiawan et al¹⁰, implying that ethanol extract of Pidada leaf contains positive antioxidants.

The wavelength obtained in this study is 744nm. The IC50 value indicates the value of the antioxidant activity. IC50 is the concentration of the sample solution needed to inhibit ABTS free radicals by 50%. The smaller the IC50 value, the stronger the antioxidant activity¹¹. The antioxidant activity test of the red pidada leaf was carried out three times to minimize errors during measurement and obtain the maximum IC50 average value. Ascorbicacid is used as a comparison because ascorbic acidis is a strong flavonoid compound in the flavonol group widely found in plants and canact as an antioxidant.

The average IC₅₀value of ascorbic acid was 0.77ppm (very strong), and the ethanol fraction had an IC50 value of 1.32ppm (very strong), n-hexanefraction 19.89ppm (very strong), ethylacetate fraction 0.50ppm (very strong), and an ethanol fraction of 1.63ppm (very strong). According to traditional Red Perepat plants, it is known to contain bioactive compounds such as flavonoids, steroids, phenol hydroquinone, tannins, and two flavonoids, namely luteolin and luteolin 7-O-β glucoside¹. According to Yushar¹², tannins, steroids, phenols, and saponins are found in Sonneratia alba species.

According to Wulansari¹³, if the IC₅₀value is less than 50ppm, it can be said that the compound has very strong antioxidant activity. Based on the IC₅₀value, the ethanol extract of Pidada red leaves and ascorbic acid is included in the very strong antioxidant category. It has an IC₅₀value of less than 50ppm.

CONCLUSION:

Based on the results of the research that has been done, it can be concluded that the ethanol extract has an IC value of 1.32ppm (verystrong), n-hexanefraction 19.89 ppm (verystrong), ethylacetate fraction 0.50ppm (very strong), and ethanol fraction 1.63ppm (very strong). The present study demonstrates that the extract of Red Pidada Leaves possesses substantial antioxidant activities. The antioxidant potential of these leaves extract may be attributed to its phenolic content as well as the presence of the flavonoids, rutin. Thus, the free radical scavenging ability of Red Pidada Leaves could provide health benefits to humans by protection against oxidative stress.

ACKNOWLEDGEMENT:

The Research Team would like to thank the Directorate of Research and Community Dedication, Ministry of Research and Technology, the National Research and Innovation Agency, Indonesia for funding this research on the Applied Research Scheme with SPPK contract number: 191/SP2H/AMD/LT/DRPM/2019 November 12, 2019.

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Received on 21.09.2021 Modified on 11.12.2021

Research J. Pharm. and Tech 2022; 15(9):3957-3961.

DOI: 10.52711/0974-360X.2022.00663