INTRODUCTION:

Indonesia is one of the countries with the longest coastlines in the world. The coastlines in Indonesia are abundantly covered with thriving mangrove plants. Mangrove forests form because mangrove plants grow vigorously and abundantly. Mangrove forests are excellent for the reproduction of various living organisms such as fish, crabs, clams, and various species of birds. Mangrove plants are also crucial in preserving nature, such as preventing erosion caused by sea waves.^1,2

Mangrove R. apiculate Blume belongs to the Rhizophoraceae family and is widespread in Palaes Village, North Sulawesi, Indonesia. Previous studies have demonstrated that the distribution of R. apiculata Blume includes Indonesia, Australia, Micronesia, Guam, Japan, Malaysia, Thailand, the Philippines, and Sri Lanka.³ Mangrove plants possess numerous secondary metabolites to survive and reproduce. The content of secondary metabolites in mangrove leaves is highly beneficial as a raw material for medicine. The utilization of natural substances as medicinal raw materials is evolving and extensively researched to determine their bioactivity.^4,5 Previous research results showed that R. apiculata Blume extract can be developed as an anti-hyperlipidemic preparation.⁶ Previous research results have been conducted to find sources of secondary metabolites in plants. Exploration to find sources of flavonoids, phenolics, and tannins has been conducted on Lantana camara Linn. which lives as a wild plant.^7,8

The characterization process of plant extracts is crucial to provide valid and scientific identification. Characterization and identification of extracts enhance public confidence in utilizing natural substances for therapeutic purposes. This refers to the results of previous studies on laboratory bioanalytical quantification.^9,10,11 Several processes of extract characterization and identification include determining plant identity as raw material, macroscopic organoleptic testing of the extract, testing the content of soluble compounds in the extract in water and ethanol solvents. Screening of chemical content, both qualitatively and through thin-layer chromatography is conducted to identify the secondary metabolite compounds present in the extract. Non-specific testing of the extract is also necessary to provide information on the extract's quality. Non-specific parameter data from the extract can also be used to determine the safety of the extract if used as raw material for medicines. The purpose of this research is to determine the identity, non-specific parameters, and various chemical compound contents of the R. apiculate Blume leaf extract, specifically collected from Palaes Village, North Sulawesi, Indonesia.

MATERIALS AND METHODS:

Biological materials:

Collection of R. apiculata Blume leaves from Palaes village, West Likupang, North Minahasa, North Sulawesi, Indonesia. Identification of plant samples was carried out at the Laboratorium Dasar Bidang Teknologi Farmasi, Universitas Sam Ratulangi, Manado, Indonesia.

Extraction:

The process of making simplicia and the extraction process were carried out at the Pharmaceutical Technology Laboratory, Pharmacy Study Program, Universitas Sam Ratulangi, Manado, Indonesia. R. apiculata Blume leaves were washed using running water, then dried using an oven. The dried leaves were made into powder, then maceration was carried out using 96% ethanol solvent.

The liquid extract from the maceration process was filtered, then concentrated to become a thick extract. The time of collection of R. apiculata Blume leaves from Palaes village, the extraction process and the entire series of research were carried out in July 2023 - June 2024.

Organoleptic test:

Organoleptic test of R. apiculata Blume mangrove leaf extract was carried out using the five senses of the body, namely the nose, tongue, and eyes. The color and shape test of the extract was carried out using the eyes to observe. The odor test of the extract was carried out using the nose to observe. The taste test of the extract was carried out by tasting it using the tongue as the sense of taste.^8,12,13

Dissolved compound content test:

A total of 5g of extract was dissolved in water to a limit of 100mL, then shaken using a shaker for 6hours. Pipette 25mL of sample solution into a porcelain cup. Heat the solution in the porcelain cup at a temperature of 105°C for 3hours. Weigh the weight of the sample, until a constant weight is obtained and record it as the content of compounds in the extract that are soluble in water.

The content of the extract that is soluble in ethanol is measured by dissolving 5g of extract in 96% ethanol to a limit of 100mL. The sample solution was shaken using a shaker for 6 hours at a constant speed. Pipette 25mL of sample solution and put it into a porcelain cup. The sample solution in the porcelain cup was heated at a temperature of 105°C for 3hours or until a constant weight residue was obtained. The constant weight of the resulting residue was recorded as the content of compounds in the extract of R. apiculata Blume leaves that are soluble in ethanol.^12,13

Non-specific parameters test:

Drying shrinkage test is carried out by inserting 2g of extract into a closed weighing bottle, which has been calibrated. The extract in the bottle is then heated at a temperature of 105°C for 30 minutes. After that, it is cooled in a desiccator until it reaches room temperature. Furthermore, the bottle cap is opened and dried at a temperature of 105°C to obtain a constant weight of the extract. The drying shrinkage value is calculated based on the weight of the extract before drying minus the weight of the extract after drying.^14,15

The moisture content test was carried out by weighing 2 g of extract to be put into a porcelain cup. Furthermore, the extract was heated using an oven at a temperature of 105°C until a constant weight was obtained. The water content value in the extract was calculated using the formula: weight of the extract before heating minus the weight of the extract after heating.¹⁴

The total ash content test was carried out by weighing 2 g of extract to be put into the silicate exchanger. Furthermore, the sample was heated at a temperature of 600°C, so that a combustion residue was obtained, which was white or light gray in color. The combustion residue was transferred into a desiccator and allowed to cool at room temperature. The total ash content was obtained by weighing the combustion residue. The total ash content was then compared with the initial weight of the extract.¹⁴

The acid-insoluble ash content test was carried out by adding 50mL of dilute hydrochloric acid (HCl 10%) to the residue from the total ash content test. Heat the residue solution in HCl until boiling for 10minutes. Filter the residue using ash-free filter paper then dry it in an oven. Put the filtered residue into the silicate exchanger then heat it in a furnace at a temperature of 600°C until it burns completely. After cooling, weigh the acid-insoluble residue and the percentage of acid-insoluble ash content compared to the initial weight of the extract.¹⁴

Mold and Yeast Count testing was carried out by weighing 1g of extract sample in a sterile Erlenmeyer flask, then adding 9mL of Letheen board media, and both were homogenized. Pipette 1mL of sample solution into a test tube, then adding 9mL of agar spot assay (ASA) media, then both were homogenized. Pipette 0.5 mL of sample solution, and the solution in ASA media, then inoculated evenly into potato dextrose agar media. Incubate at 28°C for 5 days, then observe the growth of mold and yeast colonies.¹⁴

Total Plate Count testing is carried out by weighing 1g of extract sample in a sterile Erlenmeyer flask, then adding 9mL of sterile water, then both are homogenized. Pipette 0.5mL of sample solution, then inoculated evenly on the plate count agar media, incubated at 37°C for 24hours. Observations are made on the growth colonies of microorganisms.¹⁴

Heavy Metal Content testing was carried out by weighing 0.5g of extract in a destruction flask, then adding 5mL of nitric acid (HNO₃) and 0.5mL of percolic acid (HClO₄). The solution was then homogenized until completely dissolved, then left at room temperature for 24hours. Furthermore, the solution was heated at a temperature of 150°C for 1hour, then continued at a temperature of 170°C for 1hour, and at a temperature of 200°C for 1hour. Furthermore, the solution was cooled to room temperature. The next stage was to dilute the solution with distilled water in a 50mL measuring flask. Heavy metal content testing used Atomic Absorption Spectroscopy (AAS) at a wavelength of 283.3nm for Lead (Pb) analysis, a wavelength of 253.7nm for mercury (Hg) analysis, and a wavelength of 324.7nm for copper (Cu) analysis.^12,16

Qualitative phytochemical test:

Alkaloids identification was done by inserting 40mg of extract sample into a test tube, then adding 3mL of methanol and 5mL of ammonia. The mixture of substances in the test tube was heated for 2 minutes, and shaken until the extract was completely dissolved, then cooled. After the solution cooled, it was filtered to obtain a filtrate in the test tube. Furthermore, 2 drops of Dragendroff reagent were added to the filtrate in the test

tube, and the results were observed. If after the reaction, an orange precipitate forms, the extract positively contains alkaloids.^17,18

Flavonoid identification was carried out by adding 40 mg of extract sample into a test tube containing 10mL of distilled water, then heated for 5 minutes, then cooled, and filtered. The resulting filtrate was added with 0.1g of magnesium powder, and 10 drops of concentrated HCl. The solution was shaken for a while until completely mixed and then left to stand. Observations were made on orange to red sediment as an indicator of the presence of flavonoids in the extract.^18,19

The phenolic content in the extract was tested by adding 40mg of sample into a test tube containing 10mL of aqueous solution. The extract solution was filtered to obtain a filtrate, then 5 drops of sodium hydroxide were added, and then shaken until homogeneous. The extract was declared to contain phenolics if the solution turned orange.^18,19

Identification of saponins in the extract was done by dissolving 40 mg of sample into 10mL of hot aqueous solution. Furthermore, the solution was cooled while being shaken vigorously for 5minutes. The extract was declared to contain saponins if a stable foam was formed on the top layer of the solution.^18,20

Identification of tannin content in the extract is done by adding 40mg of sample into 10mL of distilled water, then boiled for 5minutes. Next, the solution is cooled, then filtered to obtain a filtrate. The filtrate is dripped with 3 drops of iron (III) chloride reagent (Fe Cl₃ 1%). The extract solution is stated to contain tannin compounds if it shows a color change to dark blue or blackish green.^17,18

Identification of triterpenoids was done by soaking 40 mg of sample in 10mL of n-hexane for 2hours. Furthermore, the solution was homogenized and then filtered, so that the filtrate was obtained. The next step was, the filtrate was dripped with 3 drops of Liebermann-Burchard reagent. The presence of triterpenoid content in the extract sample was indicated by a change in the color of the solution which turned orange or brick red.^18,21

Secondary metabolite test:

GF₂₅₄ silica plates that have been activated by heating at 110°C for 30minutes on using thin layer chromatography (TLC) are used to detect secondary metabolites in extract samples. The vessel for the elution process is saturated using the mobile phase used for each test. Spotting of the extract test sample and standard or comparison marker is done using a white tip tool. The silica plate that has been spotted with the extract and comparison is inserted into a vessel containing the mobile phase and has been saturated. The process of extract movement propagation to the specified upper limit. After the extract movement propagation is complete, the TLC plate is removed from the vessel, to be dried by airing. Observation of spots and calculation of Retention Factor (Rf) values are carried out under UV light with a wavelength of 254nm.^22,23,24 The data obtained will include the color of the spots or compound stains and the retention factor (Rf) values, which will be compared with the marker compounds used.

In the tannin test, the mobile phase used ethyl acetate, formic acid, acetic acid, and water in a ratio of 100:5:5:13, and the marker was gallic acid. In the alkaloid test, the mobile phase was methanol, and ammonia in a ratio of 100:1.5, and the marker was caffeine. In the flavonoid test, the mobile phase was n-hexane, ethyl acetate, and formic acid in a ratio of 6:4:0.1, and the marker was quercetin. In the saponin test, the mobile phase was chloroform and methanol in a ratio of 95:5, and the marker was dihydroquercetin. In the phenolic test, the mobile phase was methanol and 10% formic acid in a ratio of 95:5, and the marker was gallic acid.^23,24

Measurement of total phenolic, flavonoid, and tannin level:

Measurement of phenolic content in R. apiculata Blume mangrove leaf extract was carried out using the modified Folin-Ciocalteu method as previously conducted by researchers.^7,8,25,26,27 The colorimetric method with aluminum chloride reagent (Al Cl₃) was used to measure flavonoid content in R. apiculata Blume mangrove leaf extract referring to previous researchers.^7,8,28 Measurement of tannin content in R. apiculata Blume mangrove leaf extract was carried out using the modified Folin-Ciocalteu method as previously conducted by researchers.^7,8,29,30

Measurement of antioxidant activity:

Antioxidants in the extract samples were measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent method. Measurement of antioxidant activity in extracted samples using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent method. Measurement of antioxidant activity in this study followed the steps taken by previous researchers.^31,32

RESULTS:

Collection and identification of plant materials:

The R. apiculata Blume plant was collected from Palaes Village, North Minahasa, North Sulawesi Province, Indonesia (1° 40' 6.71" N, 124° 58' 1.92" E).

The R. apiculata Blume plant is shown in Figure 1.

Figure 1. Rhizophora apiculata Blume. Photgrapher by Hosea J Edy (June 20, 2023).

The systematics of R. apiculata Blume are as follows:

Kingdom : Plantae

Division : Magnoliophyta

Order : Myrtales

Family : Rhizophoraceae

Genus : Rhizophora

Species : R. apiculata Blume

In Indonesia, R. apiculata Blume is known as “bakau minyak” (oil mangrove) or “bakau akik” (akik mangrove), while in Palaes Village, North Sulawesi, it is known by the local name of “bakau lolaro putih” (white lolaro mangrove). In this study, the part of the plant used was young mangrove leaves that are light green in color.

Organoleptic test:

The liquid extract of R. apiculata Blume leaves are dark green. After the 96% ethanol solvent in the liquid extract is evaporated, a thick extract (thick porridge) of R. apiculata Blume mangrove leaf extract is obtained. The thick extract is dark green to blackish dark green, with a distinctive mangrove aroma, and a bitter astringent taste.

Testing the compound content of extracts soluble in both water and ethanol:

R. apiculata Blume mangrove leaf extract has a solubility value in water of 15.65%, while the solubility in ethanol is 25.70%.

Non-specific parameters:

Standardization results of non-specific parameters of R. apiculata Blume mangrove leaf extract presented in table 1.

Table 1. Standardization results of non-specific parameters of R. apiculata Blume mangrove leaf extract

Non-Specific Parameter	Content
Drying Shrinkage	0.187 %
Moisture Content	7.254 %
Total Ash Content	3.940 %
Acid-Insoluble Ash Content	0.410 %
Mold and Yeast Count	< 1 x 10¹ cfu/gr
Total Plate Count (TPC)	1.9 x 10²cfu/gr
Heavy Metal Content - Lead (Pb)	0.30 mg/kg
Heavy Metal Content - Mercury (Hg)	166.48 µg/kg
Heavy Metal Content - Copper (Cu)	17.56 mg/kg

Abbreviation: %=percent, cfu/gr=colony-forming unit per gram, mg/kg=milligrams per kilogram, µg/kg=micrograms per kilogram.

R. apiculata Blume mangrove leaf extract contains alkaloids, flavonoids, phenolics, saponins, and tannins. The Rf value for alkaloid compounds in the extract is 0.76 with an orange-brown spot. The Rf value of the flavonoid compound in the extract is 0.64, characterized by the presence of yellow spots. The Rf value of phenolic compounds in the extract is 0.74 with the characteristics of blackish gray spots. The Rf value of saponin content in the extract is 0.47, with characteristic a bluish-purple spot. The Rf value for tannin in the extract is 0.37, which forms a grayish blue spot.

Secondary metabolit levels:

Regression equation to measure secondary metabolit levels of R. apiculata Blume mangrove leaf extract is presented in figure 2.

Total phenolic content in R. apiculata Blume mangrove leaf extract is presented in Table 3.

Table 3. Total phenolic content in R. apiculata Blume mangrove leaf extract

Sample	Replicants	Absorbance	Phenolic level (mg GAE/g)	Average of phenolic level (mg GAE/g)
R. apiculata Blume mangrove leaf extract	1	0.574	3.0346	3.0469
	2	0.578	3.0593
	3	0.576	3.0469
	4	0.575	3.0408
	5	0.577	3.0531
	6	0.576	3.0469

Abbreviation: mg GAE/g = milli gram of gallic acid equivalent per gram.

The total phenolic content of R. apiculata Blume mangrove leaf extract was calculated based on the absorbance value of the extract in the linear regression formula obtained from the gallic acid calibration curve, namely y = 0.1619x + 0.0827 with an R² value of 0.9884. The total phenolic content value of R. apiculata Blume mangrove leaf extract was 3.0469 mg gallic acid equivalent/g.

The flavonoid content in R. apiculata Blume mangrove leaf extract is presented in Table 4.

Tabel 4. Flavonoid level in R. apiculata Blume mangrove leaf extract

Sample	Replicants	Absorbance	Flavonoid level (mg QE/g)	Average of flavonoid level (mg QE/g)
R. apiculata Blume mangrove leaf extract	1	0.958	5.7456	5.7980
	2	0.972	5.8337
	3	0.969	5.8149
	4	0.962	5.7708
	5	0.969	5.8149
	6	0.968	5.8086

Abbreviation: mg QE/g = milli gram of quercetin equivalent per gram.

The flavonoid content in R. apiculata Blume mangrove leaf extract was calculated based on the equation of y = 0.1619x + 0.0827 with an R² value of 0.9884. The total flavonoid content in R. apiculata Blume mangrove leaf extract was 5.7980 mg quercetin equivalent/g.

The tannin content in R. apiculata Blume mangrove leaf extract is presented in Table 5.

Tabel 5. Tannic acid level in R. apiculata Blume mangrove leaf extract

Sample	Replicants	Absorbance	Tannin level (mg TAE/g)	Average of tannin level (mg TAE/g)
R. apiculata Blume mangrove leaf extract	1	0.473	5.5109	5.4736
	2	0.468	5.4758
	3	0.462	5.4349
	4	0.471	5.4968
	5	0.469	5.4828
	6	0.463	5.4407

Abbreviation: mg TAE/g = milli gram of tannic acid equivalent per gram.

The tannin content in R. apiculata Blume mangrove leaf extract was calculated based on the equation of y = 0.01425x + 0.3123 with an R² value of 0.9226. The total tannin content in R. apiculata Blume mangrove leaf extract was 5.4736 mg tannic acid equivalent/g.

Antioxidant Activity:

IC₅₀calibration curve of R. apiculata Blume mangrove leaf extract presented in Figure 3.

Figure 3. IC₅₀calibration curve of R. apiculata Blume mangrove leaf extract. DPPH = 2,2-diphenyl-1-picrylhydrazyl, ppm = part per million.

IC₅₀value of R. apiculata Blume mangrove leaf extract is presented in Table 6.

Tabel 6. IC₅₀value of R. apiculata Blume mangrove leaf extract

Number	Concentration	Absorbance	% Inhibition	Linear equation	IC₅₀(ppm)
Number	(ppm)	Absorbance	% Inhibition	Linear equation	IC₅₀(ppm)
1	10	0.670 ± 0.0104	19.02	y = 15.099x - 11.772	4.0911 ppm
2	20	0.578 ± 0.0125	30.11
3	40	0.411 ± 0.0122	50.34
4	60	0.263 ± 0.0096	68.16
5	80	0.203 ± 0.0101	75.49

Abbreviation: IC₅₀= half-maximal inhibitory concentration, ppm= part per million.

Linear regression equation à y = 15.099x – 11.772. The x value (is the IC₅₀ value) = 4.091ppm. IC₅₀ of R. apiculata Blume mangrove leaf extract 4.0911ppm.

DISCUSSION:

The thick extract is dark green to blackish dark green, with a distinctive mangrove aroma, and a bitter astringent taste. The distinctive odor, color, and taste of the extract, and the similarity to the R. apiculata Blume leaves as the source of the extract, are indicators that the extraction process was successful.^7,8

The standard quality for the solubility of the extract in water is 12%, while the solubility of the extract in ethanol is 6%. The solubility of R. apiculata Blume mangrove leaf extract in water shows that the active compounds in the extract have polar properties. These properties tend to form hydrogen bonds with water because they contain hydroxyl groups. Extract compounds that are soluble in water facilitate the formulation process when the extract is used as an active ingredient in liquid herbal preparations.^33,34 Ethanol is a solvent with a hydroxyl group (-OH) that can extract compounds such as flavonoids, saponins, tannins, and alkaloids in greater quantities compared to water as a solvent. The levels of extract compounds that can be dissolved in ethanol facilitate the process of formulating the extract into a solid pharmaceutical dosage form. Based on the data above, it can be concluded that the compounds contained in the ethanol extract of R. apiculata Blume leaves are more semipolar than polar compounds.³⁵

Non-specific testing of extract parameters aims to determine the quality and characteristics of the extract in general. The results of this test will provide information on the quality of the extract in general, and can be used as a reference in making similar extracts in the future. Non-specific testing is carried out without measuring or determining the levels and types of active substances contained specifically.³⁶ The drying shrinkage value of R. apiculata Blume mangrove leaf extract shows that the evaporation of substances was minimal due to heating. Although the drying process to evaporate solvents such as water, ethanol, oil, and other compounds contained in the extract used a temperature of 105^oC, the drying shrinkage value of R. apiculata Blume extract was low. These results are in accordance with previous research results that the drying process is an important factor in the preparation process.¹⁵ The water content value of R. apiculata Blume mangrove leaf extract meets the quality parameters of concentrated extract water content, which is 5% - 30%. The low water content of the extract produces a stable extract that is not easily decomposed by compounds. In addition, the low water content helps inhibit the growth of microorganisms.³⁷ The success of the drying or concentration process to obtain R. apiculata Blume mangrove leaf extract meets the established standard. The results of this study are in line with the results of previous studies which stated that the drying process needs optimization to obtain the desired results.³⁸

The total ash content value of R. apiculata Blume mangrove leaf extract provides insights into the natural mineral content. The value indicates the presence of minerals, including organic and inorganic salts. Mineral compounds that may be present in the extract include oxalate, pectate, carbonate, oxalate, and phosphate.³⁶ Total ash content values in the extract provide insight into the natural mineral content. In line with recent research on ash content demonstrated that J. adhatoda leaves contain 11.5% total ash.³⁹ The value of acid-insoluble ash content in the R. apiculata Blume mangrove leaf extract indicates the presence of impurities such as dust, silicate soil, sand, and heavy metals.¹⁴ A lower acid-insoluble ash value indicates a purer extract with fewer impurities.⁴⁰

The test of mold and yeast content in R. apiculata Blume mangrove leaf extract showed that the value was <1 x 10¹ cfu/g. This value indicates the presence of mold and yeast in the extract. However, the mold and yeast content values in R. apiculata Blume mangrove leaf extract in this study were still below the limit set by the Indonesian government, which is <1 x 10³ cfu/g. However, the reduction in the amount of mold and yeast content needs to be reduced, so that the quality of the preparation does not decrease.⁴¹ The Total Plate Count (TPC) value for R. apiculate Blume mangrove leaf extract is 1.9 x 10² cfu/g, so signifying the presence of microbial contamination in the extract. The results of previous studies have demonstrated that several types of microbes that may contaminate plant extracts include Escherichia coli, Bacillus subtilis, Aspergillus flavus, and Staphylococcus aureus. The presence of microbes that contaminate plant extracts can reduce the quality of the extract. In this study, the TPC value for fungi and molds in the R. apiculata Blume extract was still below the permitted limit, which is <1 x 10³ cfu/g, so it is safe to use as a raw material for natural medicines.^12,16 Some state that the TPC value guidelines that are safe to use as raw materials for herbal medicine are <1 x 10⁴cfu/g.¹⁴

Heavy metal content analysis is performed to ensure that the extract does not contain compounds that are toxic and harmful to human health. The determination of heavy metal content in the mangrove leaf extract of R. apiculata Blume contains lead (Pb) at 0.30mg/kg, which is below the maximum limit allowed by the Indonesian government (≤ 10mg/kg). Lead in the extract can cause kidney, heart, and lung disorders. The mercury (Hg) content in the extract is 166.48µg/kg with a maximum permissible limit of ≤ 0.5mg/kg. Mercury contamination in the extract can lead to damage to the kidneys, cardiovascular system, immune system, and reproductive system in humans. The copper (Cu) content in the mangrove extract is 17.56mg/kg, below the allowable maximum limit of 150mg/kg. Copper contamination can cause toxicity symptoms such as diarrhea, nausea, dizziness, and skin rash. Pb, Hg, and Cu are potentially toxic to various organs. Therefore, the content of these metals in preparations or plant extracts as raw materials of natural medicine needs to be tested.^42,43

The R. apiculata Blume extract contains alkaloid compounds, as evidenced by the formation of orange precipitates when the extract solution is reacted with Dragendorff's reagent. The alkaloid compounds in the extract are basic and react with H₂SO₄ solution (which is acidic) to form potassium-alkaloid bonds and cause precipitation. The R. apiculata Blume extract positively contains flavonoids, because the extract solution turns brick red with the appearance of precipitates. The color change to brick red with the appearance of the precipitate occurs because the flavonoid compounds in the extract react with H Cl and Mg powder which turn red, and a precipitate form.¹⁸ The extract from R. apiculata Blume leaves positively contains phenolics, as evidenced by the color change to orange yellow.⁴⁴ The phenolic compounds in the extract react with Fe Cl₃ solution, forming a brick red or orange yellow solution. Saponin compounds were also positive in the R. apiculata Blume extract, because stable foam appeared after the extract solution was heated and stirred vigorously. The R. apiculata Blume extract was positive for tannins, because the color of the extract solution changed to dark green after the addition of 1% Fe Cl₃. Triterpenoid compounds were also positive in the R. apiculata Blume extract, because the color of the solution changed to orange-brown after reacting with concentrated sulfuric acid. The results of qualitative tests on the content of alkaloids, flavonoids, phenolics, saponins, and tannins in the extract of R. apiculate Blume obtained the same results as previous studies.^18,44

Qualitative screening of the phytochemical content of R. apiculate Blume mangrove leaf extract was performed using color reactions.¹⁸ Qualitative chemical composition tests provide an overview of the types of chemicals contained in the extract. In addition, it also provides an overview of the groups of chemical compounds it contains. This test was carried out to provide certainty that the chemicals we expected were contained in the extract. The chemicals detected in the extract facilitate the next research steps to determine the levels of substances quantitatively.^18,21 Qualitative screening of the phytochemical content of R. apiculata Blume extract was carried out using a color reaction. The data of compound composition in R. apiculata Blume extract is useful in understanding the function, use, or indication of the compound in therapeutic applications.¹⁸

These characteristics are in accordance with the results of previous research which demonstrated that R. apiculata Blume extract contains alkaloids, flavonoids, and phenolic.^24,25 Saponin compounds are also positively present in the mangrove leaf extract of R. apiculata Blume, that observed with anisaldehyde sulfuric acid reagent. The mangrove leaf extract of R. apiculata Blume also positively contains phenolic compounds. The mangrove leaf extract of R. apiculata Blume also positively contains tannin compounds. The Rf value for tannin in the extract is 0.37, which forms a grayish blue spot according to the tannin marker.^22,23,24,25 The saponin and tannin content in the mangrove leaf extract of R. apiculata Blume is the same as the results obtained from the mangrove leaf extract of R. apiculata Blume collected from Pitchavaram, Chidambaram, Tamil Nadu, India.⁴⁶

The phenolic, flavonoid, and tannin content as secondary metabolites can be found not only in R. apiculata Blume but also in other plants. The phenolic and flavonoid content show variations due to differences in the types of plants extracted.^25,47,48 Based on the type of plant, R. apiculata Blumme mangrove leaf extract contains lower phenolic, flavonoid and tannin content compared to L. camara Linn. leaf extract.⁷ The type of solvent in the extraction process, and the part of the plant that is extracted also cause differences in phenolic and flavonoid content. This statement is based on data from this study that we compared with previous research results.^49,50,51

Based on the regression equation y = 15.099x - 11.772 with R² of 0.9803, the IC₅₀ value of R. apiculata Blume mangrove leaf extract is 4.0911ppm. Based on the classification of antioxidant activity which shows IC₅₀ <50ppm, the antioxidant activity of R. apiculata Blume mangrove leaf extract is very strong. These results are in accordance with the statement that a lower IC₅₀ value is an indication of stronger antioxidant activity.^31,52 The very strong antioxidant activity of R. apiculata Blume mangrove leaf extract is due to the content of secondary metabolites such as flavonoids, phenolics and tannins. Flavonoid compounds contained in R. apiculata Blume mangrove leaf extract have antioxidant properties and can minimize the toxic effects of free radicals.⁵³ The mechanism of action of flavonoid compounds as antioxidants is by donating hydrogen atoms from phenolic hydroxyl groups. The hydrogen atoms of flavonoid compounds will bind metals (free radicals) in the form of glucosides or aglycones.^54,55 Phenolic compounds have the same antioxidant activity as flavonoid compounds, namely capturing free radicals by donating electrons or protons. Free radical compounds captured by phenolic compounds will be stable and oxidative.^56,57 The tannin compounds contained in the extract could inhibit and stop the formation of free radical compounds. Tannin compounds will chelate iron ions in stable complex bonds and inhibit the oxidation process carried out by free radicals.⁵⁸ The combination of antioxidant activity capabilities of flavonoid, phenolic and tannin compounds contained causes the ethanol extract of R. apiculata Blume leaves to have good and strong antioxidant properties.⁵⁷

LIMITATIONS:

Although we have studied the phenolic, flavonoid, tannin content and antioxidant activity in R. apiculata Blume mangrove leaf extract, we have not tested the extract as an active ingredient in antibacterial preparations. Another limitation is that no measurements were made of mineral or metal content data⁵⁹ that could affect secondary metabolite levels or IC₅₀.

CONCLUSION:

R. apiculata Blume mangrove leaf extract has a solubility value in water of 15.65%, and in ethanol of 25.70%. R. apiculate Blume mangrove leaf extract contains alkaloids, phenolics, flavonoids, saponins, tannins, and triterpenoids. The total phenolic content in term of gallic acid was 3.0469mg/g, while the total flavonoid content in term of quercetin was 5.7981mg/g. The total tannin content in term of tannic acid is 5.4749 mg/g. The IC₅₀ value of R. apiculata Blume mangrove leaf extract is 4.0911ppm, so it has very strong antioxidant properties.

In the future, it is necessary to test the relationship between phytochemical content and antioxidant activity to antibacterial activity in preclinical tests. Furthermore, preparations containing active ingredients of R. apiculata Blume mangrove leaf extract need to be continued with clinical trials.

ACKNOWLEDGEMENT:

The authors are thankful to Universitas Sam Ratulangi, Manado, Indonesia for facilities and support.

FUNDING:

This study was funded by Universitas Sam Ratulangi through scheme grant DIPA UNSRAT RDUU_K1 2023 contract number of 441/UN12.13/LT/2023.

AUTHORS CONTRIBUTIONS:

All authors have contributed equally.

CONFLICT OF INTERESTS:

The authors declare that they have no conflict of interest.

CONTRIBUTIONS:

Conceptualization: HJE, EP. Data acquisition: HJE. Data analysis or interpretation: HJE, EP. Drafting of the manuscript: HJE. Critical revision of the manuscript: EP. Approval of the final version of the manuscript: EP, HJE.

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Received on 07.12.2024 Revised on 17.04.2025

Accepted on 24.06.2025 Published on 16.03.2026

Available online from March 18, 2026

Research J. Pharmacy and Technology. 2026;19(3):1015-1025.

DOI: 10.52711/0974-360X.2026.00144

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

Chemical Compound	Result	Appearance	Photo	Mobile Phase	Rf	Chromatogram on UV 254 nm
Alkaloid	+	spots and orange precipitates		methanol : ammonia (100:1.5)	+ (0.67)
Flavonoid	+	brick-red precipitate		n-hexane:ethyl acetate : formic acid (6:4:0.1)	+ (0.64)
Phenolic	+	orange		methanol : formic acid 10% (95:5)	+ (0.74)
Saponin	+	foam		chloroform : methanol (95:5)	0.47
Tannin	+	dark green		ethyl acetate : formic acid : acetic acid : water (100:5:5:13)	+ (0.37)