Leaf samples of R. mucronata and R. apiculata were collected from the BAPPL Serang mangrove conservation area in Indonesia. The samples consisted of fresh, intact, mature leaves attached to the branches. The leaves were transported by land in polybags to the laboratory. The collected mangrove leaves were then re-sorted to check for any damage, followed by thorough washing of each leaf under running water. The leaves were wiped dry, and the moisture content of the fresh leaves was measured. The samples were subsequently dried at room temperature (airdrying) (Arifin et al., 2023) for approximately 20 days or until a moisture content of <15% was achieved. The dried samples were ground using a grinder and extracted with methanol at concentrations of 70%, 80%, and 90% (v/v) to determine the optimal concentration. Extraction was performed by soaking the samples in the solvent at a ratio of 1:5 (w:v). Maceration was performed on an orbital shaker (SCILOGEX-SKo330-Pro) for 48 h at room temperature at a speed of 180 rpm. The macerate was filtered using Whatman No. 42 filter paper and evaporated with a rotary vacuum evaporator (BUCHI-Heating bath B-100) at under reduced pressure until most of the solvent was removed, obtaining a viscous crude extract rather than a completely dry extract. The crude extract was subsequently analyzed for antioxidant activity (DPPH), phytochemical composition, total phenolic content, and total flavonoid content (TFC). The extract yield was weighed and calculated using the following formula:

This analysis consisted of several components and followed the procedure described by Tiwari et al. (2011).

Alkaloid test: A total of 0.05 g of the extract was dissolved in 10 drops of 2N filtered, and divided into three test tubes. Each tube was then treated with Wagner’s reagent (brown precipitate), Dragendorf ’s reagent (orange–red precipitate), or Mayer’s reagent (yellowish-white precipitate) as indicators of a positive result. Flavonoid test: A total of 0.05 g of extract was mixed with 0.1 mL of Mg powder, 0.4 mL of amyl alcohol, and 4 mL of ethanol. A positive reaction was indicated by the formation of red, yellow, or orange color in the amyl alcohol layer.

Saponin test: A total of 0.05 g of extract was mixed with hot water, shaken for 1 min, and then 1 drop of 2N HCl was added. The formation of stable foam indicated a positive result.

Tannin test: A total of 0.05 g of extract was infused with hot water for 3 min, filtered, and then treated with 1% FeCl₃. A dark blue or greenish-black color indicated a positive reaction. Phenol test: A total of 0.05 g of extract was mixed with 2.5 mL of ethanol and two drops of 5% . The appearance of green, red, purple, or blue colorations indicated a positive result. Steroid and triterpenoid test: A total of 0.05 g of extract was mixed with 2 mL of chloroform, 10 drops of acetic anhydride, and 3 drops of Blue coloration indicates the presence of steroids, whereas reddish-brown or purple coloration indicates the presence of triterpenoids.

The antioxidant activity of the mangrove leaf extract was evaluated using the DPPH method (Boeing et al., 2014). The assay was conducted by mixing 300µL of the sample extract with 600 µL of 0.1 mM DPPH solution, followed by vortexing for 30 s. All mixtures were incubated for 30 min in the dark at room temperature, and the absorbance was measured at 517 nm using a spectrophotometer. The samples were tested at concentrations of 50, 100, 150, 200, 250, and 300 ppm, while ascorbic acid was used as a positive control at concentrations ranging from 1–6 ppm. The percentage inhibition and were calculated using the following formula:

This analysis was conducted to determine the total phenolic content in the mangrove leaf extract, which contributed to its antioxidant properties, following the method described by Alara et al. (2020), with modifications. In this assay, 50% Folin– Ciocalteu reagent was prepared by mixing 3 mL of Folin reagent with 3 mL of distilled water. A 5% solution was prepared by dissolving 0.5 g of in 10 mL distilled water. A gallic acid standard solution (1:1, w:v) was prepared at concentrations of 0, 20, 40, 60, 80, and 100 ppm.

A total of 0.125 mL of the standard solution was mixed with 0.125 mL of ethanol, 0.625 mL of distilled water, and 62.5 µL of 5% Folin reagent and incubated for 5 min. After incubation, 0.125 mL of the 5% solution was added, followed by a 60-minute incubation in the dark. After incubation, 180–200 µL of the mixture was transferred into a microtube and measured using a spectrophotometer at 725 nm wavelength.

This analysis began with the preparation of a quercetin standard solution using ethanol and quercetin (1:1) at a concentration of 1000 ppm, followed by the preparation of a 2% AlCl₃ solution. A series of quercetin standard concentrations (0, 20, 40, 60, 80, and 100 ppm) was prepared. A total of 500 µL of the sample was mixed with 500 µL of 2% AlCl₃, vortexed, and incubated for 10 min. The absorbance was measured at 415 nm using a spectrophotometer. The same procedure was performed for the blank and all quercetin standard concentrations Alara et al. (2020).

The data obtained in this study were analyzed using statistical testing tools. The analysis was performed using a factorial completely randomized design (CRD) ANOVA with two mangrove species and three solvent concentrations. Variables showing significant efects were further evaluated using Tukey multiple range test.

Mangrove plants have been reported to contain important bioactive compounds with potential applications in various industries. The distribution of phytochemicals varies among diferent plant parts, including leaves, fruits, flowers, stems, and roots.

Phytochemical analysis (Table 2) revealed that the leaf extracts of R. mucronata and R. apiculata contained steroids, tannins, phenols, and flavonoids. These findings are consistent with previous studies reporting that mangrove leaf extracts generally contain various bioactive compounds, including alkaloids, phenols, flavonoids, tannins, saponins, steroids, and terpenoids. (Priyanto & Rimba, 2023; Akasia et al., 2021; Dahibhate et al., 2018; Eswaraiah et al., 2020; Krisnafi et $a l . ,$ 2024; Rahmawati et $a l . ,$ 2025; Syahidah & Subekti, 2019; Thao et al., 2022). Sachithanandam et al. (2022) reported that the mangrove leaf extract of R. mucronata contains a diverse range of bioactive compounds, including phytochemicals or secondary metabolites such as alkaloids, flavonoids, steroids, saponins, tannins, and phenolic compounds. Steroids are derived from various plants, animals, and microorganisms and are characterized by 17 carbon atoms arranged in four fused rings. Plant-derived steroids possess a wide range of medically, pharmaceutically, and agrochemically relevant properties (B. Gunaherath & Gunatilaka, 2014). The leaf extract of R. mucronata was found to contain high levels of steroids, whereas the levels of alkaloids, saponins, tannins, phenols, and flavonoids were relatively low. Steroids are known to enhance stamina (aphrodisiac efects) and exhibit anti-inflammatory activities (Egra et al., 2023; Rahmawati et al., 2025). Steroids also play important roles as antioxidants, anticancer, anticholesterol, and antiviral agents (Lein et al., 2025).

Phenols are characterized by an aromatic ring structure attached to one or more hydroxyl groups. Phenolic compounds are generally found in various plant parts, such as leaves, fruits, and bark (Sachithanandam et al., 2022). Flavonoids consist of two benzene rings (A and B) connected through a heterocyclic pyran ring (C). Flavonoids are an important group of polyphenolic compounds that possess a benzo-γ-pyrone structure and are primarily found in plants. Flavonoids exhibit strong antioxidant and fungicidal activities and are used as therapeutic agents for menstrual and skin disorders, as well as for wounds, including leprosy and dermatitis (Liu et al., 2021; Parthiban et al., 2022). Alkaloids are a group of secondary metabolites and the most diverse nitrogen-based bioactive compounds, typically characterized by heterocyclic rings. Alkaloids are commonly found in flowering plants (Maldoni, 1991). Mangrove plants that contain alkaloids are known to be toxic or medicinal (Parthiban et al., 2022).

The results showed that the extract did not contain alkaloid compounds. This outcome is presumed to be influenced by several factors. The polarity of the solvent significantly afects the number of chemical components present, the eficiency of the extraction process, and the resulting biological activity (Ali et al., 2021; Truong et al., 2019). This finding is consistent with the study of Akasia et al. (2021), which reported that phytochemical tests of mangroves from Tuban (Bali) extracted using methanol revealed that R. mucronata was positive for phenols, flavonoids, tannins, saponins, and terpenoids, but negative for alkaloids and steroids. Similarly, Hardiningtyas et al. (2024) reported that alkaloid compounds were not detected in the leaf, flower, and fruit extracts of R. mucronata, as indicated by the absence of white precipitates following the addition of Mayer’s reagent.

The compounds detected in the phytochemical analysis were associated with the antioxidant capacity of the sample. Several previous studies have reported that high levels of phenolic compounds, alkaloids, and terpenoids are linked to strong antioxidant activity. In addition, phytochemical compounds such as alkaloids, tannins, saponins, and terpenoids are known to act as free radical scavengers and chain-breaking agents in oxidation reactions (Bulan et al., 2022; Miranti et al., 2018; Nguyen et al., 2021).

The solvent concentration range used in this study (70–90%) was not suficiently wide to alter the solubility threshold required for these metabolites to yield diferent qualitative outcomes. In other words, although the concentrations of phenolic, flavonoid, tannin, or other compounds may vary quantitatively, as reflected in the TPC, TFC, and values, their levels remain above the detection limit of the qualitative assays across all treatments, resulting in similar positive reactions. This phenomenon is consistent with previous reports stating that qualitative phytochemical tests are not sensitive enough to distinguish moderate variations in compound concentrations within polar solvent systems (Tiwari et al., 2011).

DPPH is a stable free radical compound widely used to evaluate the free radical-scavenging potential of antioxidants (Jha et al., 2022). The antioxidant activity of the extract was indicated by its value. The value of DPPH scavenging activity is inversely proportional to the percentage of DPPH scavenging activity, indicating that the highest antioxidant activity is represented by the lowest value (Priyanto & Rimba, 2023). Analysis of variance (ANOVA) showed thSat mangrove leaf type, solvent concentration, and their interaction had a highly significant efect on the value of the extract (Table 3).

This indicates that the efectiveness of the extract in scavenging free radicals is influenced by variations in leaf type and the solvent concentration used. Tukey’s posthoc test showed that the values difered significantly among the treatments. The 90% concentration yielded the lowest value and was categorized as having strong antioxidant activity, which was significantly diferent from the 80% and 70% concentrations.

The values showed significant variation among the treatments, which was attributed to the diferences in the levels of bioactive compounds present in the samples. In the R. mucronata extract, the value decreased with increasing solvent concentration, from 141.58 ppm (70%) to 89.00 ppm (80%) and 74.33 ppm (90%). A similar trend was observed for the R. apiculata extract, with the values decreasing from 100.79 ppm (70%) to 88.49 ppm (80%) and 72.55 ppm (90%). These findings are consistent with previous studies reporting that methanolic extracts of R. mucronata have an value of 117.498 µg/mL (Kasitowati et al , 2017), methanolic extracts of R. apiculata have an value of 92.75 µg/mL and R. mucronata 93.21 µg/mL (Vittaya et al., 2022), and methanolic extracts of R. apiculata have an value of 68.00 µg/mL (Chelliah et al , 2023). According to Ramli et al. (2025), increasing the methanol concentration enhanced the antioxidant activity of the extract, with the 90% methanol extract showing the lowest value (62.42±3.23 ppm), whereas the 80% and 70% methanol extracts showed lower antioxidant capacities. The study also demonstrated that the highest total phenolic content (TPC) was obtained from the 80% methanol extract, while the 90% methanol extract contained the highest total flavonoid content (TFC). The decrease in the values indicates that the use of solvents at higher concentrations enhances the antioxidant activity of the extract. Among the ten diferent methanolic extracts of mangrove plants, R. mucronata was shown to possess eficient phytochemicals that exhibit strong antimicrobial and antioxidant activities. The methanolic extract of R. mucronata has an value of 70.93 µg/mL, whereas R. apiculata has an value of 127.79 µg/mL (Sachithanandam et al., 2022).

The results show a relationship between the total phenolic content and the values of the mangrove leaf extracts (Table 3). This relationship indicates that a higher phenolic content results in a lower value, reflecting stronger antioxidant activity. The antioxidant activity in the samples is presumed to originate from compounds capable of donating protons to neutralize the free radicals. Phenolic and flavonoid compounds are known to possess these capabilities (Fidrianny et al., 2014, 2015).

The total phenolic content is closely related to the antioxidant activity of black mangrove leaves (Arifin et al., 2023). Phenolic compounds are known to donate hydrogen atoms to neutralize free radicals, thereby contributing to the reduction of values as their concentration increases in the extract. These findings are consistent with previous studies reporting strong negative correlations between total phenolic, flavonoid, and carotenoid contents and IC₅₀ values in DPPH radical scavenging assays. This indicates that the higher the levels of total phenols, flavonoids, and carotenoids, the lower the value, reflecting stronger DPPH radical-scavenging activity (Heim et al., 2002, Fitriansyah et al., 2018). The higher the total phenolic content, the stronger the antioxidant activity (Sobuj et al., 2021).

The phenolic content of the material was expressed in gallic acid equivalents (GAE). Analysis of variance (ANOVA) showed that mangrove leaf type significantly afected total phenolic content, whereas solvent concentration did not. The interaction between leaf type and solvent concentration also did not have a significant influence. Tukey’s HSD post-hoc test indicated that variations in solvent concentration (70%, 80%, and 90%) did not result in significant diferences in phenolic content, as all concentration groups fell within the same homogeneous subset. The analysis further showed that the total phenolic content in the leaf extracts of R. mucronata and R. apiculata increased with the use of higher methanol concentrations during the extraction process. Although minor fluctuations in the mean values were observed across concentrations, no consistent directional trend was evident. The absence of significance is likely attributable to the relatively large within-group variability, as indicated by substantial standard deviations, which reduced the ability to detect diferences among treatments. The overlapping variability suggests that the solvent concentration did not meaningfully influence the total phenolic extraction under the tested conditions. The total phenolic content of the R. mucronata extract at 90% solvent concentration was 101.24±46.18 mg GAE/g, and no significant diferences were observed among the tested solvent concentrations. This increase indicates that methanol 90% is more efective in dissolving phenolic compounds. Phenols are key bioactive compounds that contribute to antioxidant capacity; therefore, optimal extraction enhances the levels of these components (Banerjee et al., 2008; Mokhtar et al., 2022).

The study conducted by Hardiningtyas et al. (2024) showed that the leaves, flowers, and fruits of R. mucronata are rich in phenolic compounds. A positive result in the phenol test was indicated by a color change to blue. According to Haryati et al. (2015), this color change occurs due to a reaction between FeCl₃ and the –OH groups of phenolic compounds. Another study by Rahmawati et al. (2025) reported that the total phenolic content in the ethanol extract of R. mucronata leaves from Surabaya waters was 6.70%. In addition, Kaur et al. (2019) showed that the total phenolic content in the methanolic extract of R. mucronata leaves reached 21.25 mg/g. Black mangrove leaves contain phenolic compounds, including flavonoids. Nearly all mangrove species are capable of producing flavonoids because they inhabit relatively extreme environments. These compounds play an essential role in protecting mangroves from various environmental stresses (Arifin et al., 2023; Mierziak et al., 2014). The use of solvents with diferent polarities results in varying total phenolic contents (Hardiningtyas et al., 2024).

Analysis of variance (ANOVA) showed that neither mangrove leaf type nor solvent concentration had a significant efect on total flavonoid content. Their interaction also had no significant efect. The average flavonoid content was 5.00±0.85 mg QE/g. This indicates that within the solvent concentration range used (70–90%), the ability of methanol to extract flavonoids remains relatively stable, and variations in leaf type do not result in meaningful diferences in total flavonoid levels. Previous studies have reported that the amount of flavonoids extracted is strongly influenced by the extraction method applied and the conditions or characteristics of the material during the extraction process (Zhu et al., 2021). In fresh mangrove leaves, moisture content may afect the stability of flavonoid compounds because water contributes to the vulnerability of flavonoids, which are sensitive to temperature and humidity (Prayitno et al., 2025).

In plant tissues, flavonoids generally occur as glycosylated derivatives with diverse functions. Their distinctive colors, as seen in flavones, flavonols, and anthocyanidins, may serve as visual signals for pollinators. Compounds with astringent tastes, such as catechins and other flavanols, are presumed to function as defense mechanisms against herbivorous insects (Mazza & Miniati, 2018). In addition, flavonoids may act as catalysts in the light phase of photosynthesis or as regulators of ion channels involved in phosphorylation (Harborne, 2017), protecting plant cells from stress by scavenging reactive oxygen species generated during photosynthesis (Alara et al., 2020). Phenolic compounds, such as flavonoids, function as antioxidants because they contain hydroxyl groups attached to aromatic carbon rings, enabling them to scavenge free radicals (Phillipson, 2001).