Abstract Elephantopus mollis Kunth is a medicinal plant known for its diverse phytochemical constituents and potential pharmacological properties. This study aimed to evaluate the tyrosinase inhibitory activity, antioxidant capacity, and total phenolic and flavonoid contents of various extracts from E. mollis Kunth, and to isolate and characterize the bioactive compounds responsible for these effects. The leaves, bark, and roots were extracted using n-hexane, ethyl acetate, and methanol through Soxhlet extraction, while whole-plant extracts were prepared separately via maceration with the same solvents. All extracts were analyzed for total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity using the DPPH assay, and tyrosinase inhibitory activity through in vitro enzymatic assays. Bioactive compounds were isolated using chromatographic techniques and structurally characterized by UV-Vis, FTIR, MS, and 1D/2D NMR spectroscopy. Among the whole-plant extracts, the ethyl acetate fraction showed the highest TPC (21.39 ± 0.70 mg GAE/g) and TFC (4.06 ± 0.60 mg QE/g), as well as the strongest antioxidant (IC₅₀: 54.17 ± 2.48 µg/mL) and tyrosinase inhibitory activities (IC₅₀: 91.98 ± 3.90 µg/mL), followed by n-hexane and methanol extracts, although all were less potent than kojic acid (IC₅₀ = 29.58 ± 0.53 µg/mL). Six compounds were isolated: epifriedelanol, stigmasterol, deoxyelephantopin, molephantinin, tricin, and 3,4-di-O-caffeoylquinic acid. Among these, stigmasterol and molephantinin exhibited notable tyrosinase inhibitory activity, with IC₅₀ values of 57.96 and 62.00 µg/mL, respectively. These findings highlight E. mollis Kunth as a promising natural source of antioxidant and tyrosinase inhibitors for potential application in cosmeceutical and pharmaceutical products targeting hyperpigmentation.

Keywords: Elephantopus mollis Kunth, Antioxidant, Tyrosinase inhibition, Bioactive compounds, Natural skin-whitening

Funding: The authors gratefully acknowledge the financial support from the Directorate of Research and Community Service, Ministry of Research (DP2M), Technology, and Higher Education of the Republic of Indonesia (Kemenristek-Dikti RI), for the PUPT project under Grant No. 030/SP2H/PL/Dit.Litabmas/II/2015.

Citation:  Efendi, M.R., Bakhtiar, A., and Putra, D.P. 2026. Tyrosinase inhibitory activity of extracts and bioactive compounds from Elephantopus mollis Kunth. Natural and Life Sciences Communications. 25(3): e2026047.

Graphical Abstract:

INTRODUCTION

Indonesia is renowned for its exceptional biodiversity, particularly its richness in medicinal plants, which have long been utilized in traditional healthcare systems (Ngezahayo et al., 2025). These plants play a crucial role in managing various ailments, including degenerative diseases (Antika et al., 2024). Despite their widespread empirical use, only a small proportion has been scientifically investigated, especially in the context of cosmetic and dermatological applications (Ansari et al., 2025). Growing global interest in plant-derived bioactive compounds has further encouraged systematic exploration of their antioxidant, antimicrobial, and enzyme-inhibitory properties, as demonstrated by recent studies across diverse botanical species (Zengin et al., 2021; Babacan et al., 2023).

Elephantopus mollis Kunth, a member of the Asteraceae family, is one such underexplored species. While its close relative, Elephantopus scaber, has been widely studied for its pharmacological potential, research on E. mollis Kunth remains comparatively limited. Ethnobotanical reports describe its traditional use for inflammatory conditions, bacterial infections, wound healing, and metabolic disorders (Rao et al., 2024). A review by Rusdi and Efendi (2021) identified at least 35 secondary metabolites within this species, including sesquiterpene lactones, steroids, and phenolic compounds, with molephantin reported as one of the major constituents. Previous studies have also documented several pharmacological activities of E. mollis Kunth, such as antioxidant, antibacterial, anti-inflammatory, antifungal, antitumor, antiprotozoal, antidiabetic, hepatoprotective, and bone-regenerative effects (Wu et al., 2017; Rao et al., 2024). Although these studies highlight the therapeutic relevance of E. mollis Kunth, none have specifically investigated its potential role in melanogenesis regulation or tyrosinase inhibition. This represents an important knowledge gap, particularly considering that many Asteraceae plants have demonstrated strong tyrosinase-inhibitory and depigmenting activities, an area that remains unexplored for E. mollis Kunth.

Hyperpigmentation disorders, such as melasma, freckles, and age spots, arise from excessive melanin production and continue to pose significant cosmetic and clinical challenges (Li et al., 2024). Melanin biosynthesis is regulated primarily by tyrosinase, the rate-limiting enzyme responsible for the oxidation of L-tyrosine to L-DOPA and subsequently to dopaquinone (Wang et al., 2024). Conventional depigmenting agents, including hydroquinone, kojic acid, arbutin, and several metallic compounds, are widely used but often limited by safety concerns, including cytotoxicity, irritancy, and potential carcinogenicity (Munchinamane et al., 2024; Shivaram et al., 2024). As a result, there is an increasing demand for natural tyrosinase inhibitors with improved safety profiles.

Given the limited scientific attention to E. mollis Kunth and the absence of studies addressing its potential effects on melanogenesis pathways, this work aims to isolate and characterize its secondary metabolites and evaluate their tyrosinase-inhibitory activity. By addressing this gap, the study provides new insight into the biochemical properties of E. mollis Kunth and its potential application as a natural depigmenting agent. This research aligns with current efforts to develop safer and more effective alternatives for cosmetic and pharmaceutical use.

MATERIALS AND METHODS

General procedure

¹H and ¹³C NMR spectra were recorded using a JEOL JNM-ECZ500R spectrometer (500 and 125 MHz, respectively; JEOL Ltd., Tokyo, Japan). FTIR spectra were obtained on a Spectrum Two FTIR spectrophotometer (PerkinElmer Inc., Waltham, MA, USA). Mass spectra were recorded on a Xevo G2-XS QTof UPLC-MS system (Waters Corp., Milford, MA, USA). UV spectra were measured using a UV-1800 Pharmaspec spectrophotometer (Shimadzu Corp., Kyoto, Japan), and melting points were determined with a Fisher–Johns apparatus (Fisher Scientific, Pittsburgh, PA, USA). Quantification of total phenolic and flavonoid contents, as well as antioxidant and tyrosinase inhibitory assays, was performed using an xMark™ microplate absorbance reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). All reagents, including Folin–Ciocalteu reagent, aluminum chloride, sodium acetate, sodium hydroxide, DPPH, L-DOPA, kojic acid, and dimethyl sulfoxide (DMSO)—were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Column chromatography was carried out using silica gel 60 (70–230 mesh) and Diaion HP-20 resin (Mitsubishi Chemical Corp., Tokyo, Japan), with monitoring by TLC on silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany). TLC spots were visualized under UV light at 254 and 366 nm.

Plant material

The plant material used in this study was the whole plant of E. mollis Kunth. Specimens were collected from the vicinity of Andalas University, located in Limau Manis, Pauh District, Padang City, West Sumatra, Indonesia. Botanical identification was performed by Dr. Nurainas, M. Si., a taxonomist at the Herbarium of Andalas University (ANDA). The identification was verified and officially documented under the reference number 327/K-ID/ANDA/XII/2016.

Preparation extract for screening tyrosinase inhibitor

The tyrosinase inhibitory activity was evaluated for the n-hexane, ethyl acetate, and methanol extracts obtained from the root, stem, and leaf parts of E. mollis Kunth. These extracts were previously obtained through sequential Soxhlet extraction, as reported by (Efendi et al., 2024).

Extraction, purification and isolation

A total of 7.5 kg of E. mollis Kunth powder was extracted sequentially using 25 L each of n-hexane, ethyl acetate, and methanol by maceration. The extraction was carried out over three days with two repetitions for each solvent. The resulting extracts were concentrated under reduced pressure using vacuum distillation at controlled temperatures to obtain crude extracts. This process yielded 110 g of n-hexane extract, 105 g of ethyl acetate extract, and 435 g of methanol extract. Each extract was then standardized by determining its total phenolic, flavonoid content, antioxidant, and inhibitor tyrosinase activity.

From the evaporation of the n-hexane extract, a white solid precipitate (5.3 g) was obtained. This precipitate was pre-adsorbed with 5 g of stationary phase and subjected to column chromatography using silica gel 60 (50 g) as the stationary phase. A step-gradient polarity (SGP) elution system was employed, utilizing a solvent mixture of n-hexane: ethyl acetate: methanol with increasing polarity. The separation was monitored using thin-layer chromatography (TLC) on silica gel 60 F254 plates. Visualization of the chromatograms was performed using Liebermann–Burchard (LB) reagent and UV light at 254 and 365 nm. Fractions showing similar TLC profiles were combined, resulting in five pooled fractions. The fourth fraction displayed two distinct dominant spots on TLC and was subsequently recrystallized using an ethyl acetate: n-hexane solvent system. Ethyl acetate was added in small volumes, followed by the gradual addition of n-hexane until turbidity was observed. The resulting precipitate was collected and analyzed by TLC, which confirmed the presence of two pure crystalline compounds, designated as compound 1 (875 mg) and compound 2 (247 mg).

A portion of the ethyl acetate extract (53 g) was pre-adsorbed with 50 g of stationary phase and subjected to normal-phase column chromatography using silica gel 60. Separation was performed using the SGP elution system (n-hexane: ethyl acetate: methanol) with increasing polarity, yielding 18 fractions. Compound 3 (25.8 mg) was isolated from the fifth fraction as needle-shaped crystals following recrystallization in 90% acetone: water. Compound 4 (74 mg) was obtained from the seventh fraction, eluted with n-hexane: ethyl acetate (40%, 500 mL), and recrystallized using 90% acetone: water. Fractions 12–14 were partitioned using ethyl acetate: n-hexane, resulting in a precipitate that was subjected to TLC analysis. TLC revealed four yellow-colored spots under 365 nm UV light after spraying with citroborate reagent. The precipitate (230 mg) was further purified by column chromatography on Sephadex LH-20 (150 g) using 70% methanol: water as the mobile phase, resulting in compound 5 (17.4 mg), which was obtained as yellow crystals.

The methanol extract (93.41 g) was suspended in distilled water and loaded onto a Diaion HP-20 resin column until saturation. Elution was carried out using an SGP system with a water: methanol gradient (from 100% water to 10% water) followed by 100% methanol. Fractions 3–7 (26.99 g) were combined and further purified by Sephadex LH-20 column chromatography (200 g) using 70% methanol: water as the eluent, yielding compound 6 (230 mg) as a yellow amorphous solid.

The isolated compounds were characterized by organoleptic properties and specific chemical reagents. Further structural elucidation involved melting point (MP) determination, UV spectroscopy for chromophore analysis, IR spectroscopy for functional group identification, and molecular weight determination via LC-MS. The chemical structures were elucidated using ¹H-NMR and ¹³C-NMR spectroscopy, with spectral data compared against reference literature.

Determination of total phenolic content (TPC)

The TPC of E. mollis Kunth extract was determined using the Folin–Ciocalteu colorimetric method adapted to a 96-well microplate format (Marliani et al., 2022). A gallic acid stock solution (400 µg/mL) was prepared by dissolving 10 mg of gallic acid (≥98% purity) in 25 mL of absolute ethanol. Serial dilutions of the stock solution were made to obtain final concentrations of 25, 30, 45, 50, and 60 µg/mL. In each well of a 96-well microplate, 50 µL of either the standard or extract solution, 50 µL of distilled water, and 50 µL of 7.5% Folin–Ciocalteu reagent were added sequentially. After incubation at room temperature for 8 minutes, 50 µL of 1% sodium hydroxide (NaOH) solution was added. The plate was subsequently incubated in the dark at room temperature for 1 hour. For sample preparation, 0.2 g of dry extract was dissolved in 100 mL of ethanol, and the same assay procedure was followed. Absorbance was recorded at 750 nm using a microplate reader. All measurements were conducted in triplicate. The TPC was quantified based on the gallic acid calibration curve (R² > 0.99) and expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g).

Determination of total flavonoid content (TFC)

The TFC of E. mollis Kunth extract was determined following the method described in the Indonesian Herbal Pharmacopoeia, adapted to a 96-well microplate format (Ministry of Health Indonesia 2017; Razali et al., 2024). A quercetin stock solution (400 µg/mL) was prepared and serially diluted twofold to obtain a series of standard concentrations. For each well, 50 µL of either the standard or sample solution was mixed with 30 µL of 10% aluminum chloride (AlCl₃) solution and 30 µL of 1 M sodium acetate (NaOAc). Finally, 90 µL of distilled water was added, and the mixture was incubated at room temperature for 30 minutes. A blank was prepared similarly, but without the addition of aluminum chloride. Absorbance was measured at 438 nm using a microplate reader. Sample solutions were prepared by dissolving 0.2 g of dry extract in 100 mL of ethanol and processed as described above. All measurements were performed in triplicate. The total flavonoid content was calculated from a quercetin calibration curve (R² > 0.99) and expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g).

Antioxidant activity evaluation

The antioxidant activity of E. mollis Kunth extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, following a modified method described by (Doungsaard et al., 2023). A 100 µg/mL DPPH working solution was prepared by dissolving 5 mg of DPPH in 50 mL of analytical-grade methanol. The extract (10 mg) was dissolved in 5 mL of methanol to yield a 2,000 µg/mL stock solution, followed by serial twofold dilutions in a 96-well microplate to obtain final concentrations of 1,000, 500, 250, 125, and 62.5 µg/mL. Gallic acid and vitamin C were used as positive control standards. For the assay, 50 µL of each sample dilution was mixed with 150 µL of the DPPH working solution. The reaction mixtures were incubated in the dark at room temperature for 30 minutes. Absorbance was then measured at 515 nm using a microplate reader. A blank solution was prepared by replacing the sample with 50 µL of methanol under identical conditions. All measurements were conducted in triplicate. The percentage of radical scavenging activity (RSA%) was calculated using the following equation:

RSA (%)= [(Acontrol  - Asample)/Acontrol] × 100

The IC₅₀ value (the concentration of sample required to inhibit 50% of DPPH radicals) was determined by plotting RSA (%) against sample concentration and applying linear regression analysis (y = a + bx). The IC₅₀ was calculated using the equation:

_IC50= (50 - a) / b

Colorimetric tyrosinase inhibition assay

The melanin inhibition activity of E. mollis Kunth extract and its isolated compounds was assessed based on the method described by (Efendi et al., 2023), with kojic acid used as a positive control. In a 96-well microplate, 50 μL of each sample solution at the desired concentration was added, followed by 20 μL of mushroom tyrosinase solution (250 U/mL in 50 mM phosphate buffer, pH 6.8). An additional 30 μL of phosphate buffer (pH 6.8) was added to bring the total volume to 100 μL. The mixture was incubated at room temperature for 5 minutes. Subsequently, 100 μL of freshly prepared 5.07 mM L-DOPA solution was added to initiate the enzymatic reaction. Final concentrations tested for the extract were 15.62, 31.25, 62.5, 125, and 250 μg/mL, while those for the isolated compounds and kojic acid were 1.5, 3.1, 6.2, and 12.5 μg/mL. The microplate was incubated at room temperature for 30 minutes, and absorbance was recorded at 492 nm using a Bio-Rad xMark Microplate Reader. The tyrosinase inhibition percentage was calculated using the following formula:

The Inhibition Percentage (%) = [(B - S)/B] × 100

where B is the absorbance of the blank (without inhibitor) and S is the absorbance in the presence of the sample. The IC₅₀ value, representing the concentration required to inhibit 50% of enzyme activity, was determined by plotting the percentage of inhibition against the logarithm of the concentration. A linear regression model was applied to the dose–response curve to calculate the IC₅₀. All measurements were conducted in triplicate.

Statistical analysis

All data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test for post hoc comparisons. Differences were considered statistically significant at P < 0.05.

RESULTS

Screening of tyrosinase inhibitory activity from different plant parts of Elephantopus mollis Kunth

Table 1 summarizes the tyrosinase inhibitory activity of various extracts from the leaves, bark, and roots of E. mollis Kunth. Among the leaf extracts, the crude methanol extract exhibited the highest activity with the lowest IC₅₀ value (116.70 ± 1.22 µg/mL), followed by the ethyl acetate (351.70 ± 6.43 µg/mL), n-hexane (531.10 ± 1.33 µg/mL), and methanol extracts (4,208.54 ± 65.95 µg/mL). In the bark, the n-hexane extract showed the strongest inhibition (IC₅₀ = 94.03 ± 0.30 µg/mL), significantly outperforming the crude methanol (2,608.87 ± 15.89 µg/mL), methanol (4,037.45 ± 73.86 µg/mL), and ethyl acetate extracts (4,903.82 ± 109.76 µg/mL). Similarly, in the roots, the n-hexane extract exhibited the highest potency (IC₅₀ = 485.63 ± 7.93 µg/mL), followed by the crude methanol (1,322.03 ± 7.09 µg/mL), ethyl acetate (2,364.04 ± 30.73 µg/mL), and methanol extracts (2,328.97 ± 29.02 µg/mL). Statistical analysis (P < 0.05) confirmed significant differences among extracts within each plant part, as indicated by different superscript letters. These findings suggest that E. mollis Kunth contains bioactive constituents with varying levels of tyrosinase inhibitory activity, influenced by both plant part and extraction solvent. Notably, the crude methanol leaf extract and the n-hexane bark extract exhibited the most potent inhibition.

Table 1. Screening of tyrosinase inhibitory activity of parts of E. mollis Kunth plant.

Note: Values are presented as mean ± SD. Different letters in the column (a,b,c,d) indicate a significant difference among extracts (P < 0.05).

Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, and tyrosinase inhibitory activity of whole plant extracts of E. mollis Kunth

This study investigated the phytochemical profile and bioactivity of E. mollis Kunth whole-plant extracts, focusing on total phenolic content (TPC), total flavonoid content (TFC), antioxidant capacity, and tyrosinase inhibitory activity (Figure 2). Extracts were obtained through successive maceration using n-hexane, ethyl acetate, and methanol. Among these, the ethyl acetate extract consistently demonstrated superior performance across all parameters. It exhibited the highest TPC (21.38 ± 0.69 mg GAE/g) and TFC (4.06 ± 0.59 mg QE/g), markedly higher than those of the n-hexane (1.80 ± 0.04 mg GAE/g; 1.72 ± 0.42 mg QE/g) and methanol extracts (10.85 ± 0.59 mg GAE/g; 0.39 ± 0.07 mg QE/g). In the DPPH assay, the ethyl acetate extract showed strong antioxidant activity (IC₅₀ = 54.17 ± 2.48 µg/mL), outperforming the methanol (88.04 ± 5.10 µg/mL) and n-hexane extracts (945.09 ± 3.13 µg/mL). Similarly, for tyrosinase inhibition, the ethyl acetate extract exhibited the greatest potency (IC₅₀ = 91.98 ± 3.90 µg/mL), significantly more active than the methanol (780.62 ± 6.07 µg/mL) and n-hexane (530.72 ± 2.21 µg/mL) extracts, although less potent than the positive control, kojic acid (IC₅₀ = 29.58 ± 0.53 µg/mL). Statistical analysis (P < 0.05) confirmed significant differences among the extracts. These findings indicate that ethyl acetate is the most effective solvent for extracting phenolic and flavonoid compounds from E. mollis Kunth, yielding bioactive-rich extracts with notable antioxidant and tyrosinase inhibitory activities, thus highlighting its potential as a promising natural source of therapeutic agents.

Figure 1. TPC (A), TFC (B), antioxidant activity (C), and tyrosinase inhibitory activity (D) of E. mollis Khunt. whole-plant extracts. Different letters in the row (a, b, c) indicate a significant difference among groups (P < 0.05), based on Duncan’s Multiple Range Test.

Characterization of isolated compounds

In this study, six secondary metabolites were successfully isolated from E. mollis Kunth, including two compounds from the n-hexane extract, three from the ethyl acetate extract, and one from the methanol extract. These compounds belong to diverse chemical classes, namely triterpenoids, steroids, sesquiterpenes, flavonoids, and phenolics. Their structures were comprehensively elucidated using UV–Vis, IR, mass spectrometry, and NMR spectroscopy, providing detailed insights into their molecular frameworks.

Compound 1: colorless crystals, m.p: 280–282 °C. UV (MeOH): λmax 206 nm. IR (KBr) υmax: 3468.93, 3006.29, 2932.51, 2832.51, 1462.47, 1384.93, 1173.69 cm⁻¹. ¹H-NMR (CDCl₃, 500 MHz): δ 3.64 (1H, bd, H-3, J = 2.6 Hz), δ 1.81 (1H, bd, H-2a),  1.66 (1H, dt, H-6a, J = 3.2, 12.35 Hz), 1.49–1.17 (18H, m, H-18, H-2b, H-8, H-4, H-15, H-1a, H-1b, H-7a, H-7b, H-12a, H-12b, H-11a, H-11b, H-19a, H-21, H-16a, H-16b, H-22a), 1.09 (3H, s, H-27), 0.93 (3H, s, H-24), 0.912 (3H, s, H-29), 0.91 (3H, s, H-23), 0.87 (3H, s, H-26), 0.85 (3H, s, H-25), 0.84 (3H, s, H-30), 0.77 (3H, s, H-28). ¹³C-NMR (CDCl₃, 125 MHz): δ 72.6 (C-3), 49.5 (C-9), 42.0 (C-17), 41.3 (C-29), 40.4 (C-20), 38.7 (C-5), 37.3 (C-18), 36.7 (C-13), 35.5 (C-4), 35.1 (C-30), 34.3 (C-10), 32.3 (C-19), 31.4 (C-6), 29.6 (C-14), 29.5 (C-22), 27.8 (C-21), 27.5 (C-12), 27.2 (C-8), 26.7 (C-15), 24.4 (C-26), 22.9 (C-7), 22.3 (C-16), 21.9 (C-11), 21.3 (C-28), 20.7 (C-25), 20.1 (C-2), 18.3 (C-27), 16.2 (C-24), 15.8 (C-1), 11.5 (C-23).

Compound 2: colorless needle-like crystals, m.p. 169–170 °C. UV (MeOH) λmax: 202.40 nm. IR (KBr) υ_max: 3419.96, 2934.99, 1461.68, 1050.16 cm⁻¹. LC-MS (ESI⁺) m/z: 413 [M+H]⁺ (C₂₉H₄₈O). ¹H-NMR (CDCl₃, 500 MHz): δ 5.35 (1H, d, J = 5.2 Hz, H-6), 5.17 (1H, dd, J = 15.5 Hz, H-22), 5.03 (1H, dd, J = 14.9 Hz, H-23), 3.53 (1H, m, H-3), 1.02 (3H, s, H-19), 0.99 (3H, d, J = 8.45 Hz, Me-21), 0.84 (3H, d, J = 5.85 Hz, Me-26), 0.81 (3H, d, J = 6.5 Hz, Me-29), 0.70 (3H, s, H-18). ¹³C-NMR (CDCl₃, 125 MHz): δ 140.91 (C-5), 138.52 (C-22), 129.43 (C-23), 121.90 (C-6), 71.98 (C-3), 57.03 (C-14), 56.10 (C-17), 51.41 (C-24), 50.32 (C-9), 42.46 (C-25), 42.37 (C-13), 40.70 (C-20), 39.84 (C-12), 37.41 (C-1), 36.68 (C-10), 32.07 (C-8), 31.83 (C-7), 29.11 (C-16), 25.60 (C-28), 24.54 (C-15), 21.39 (C-21), 21.29 (C-11), 21.24 (C-27), 19.58 (C-26), 19.15 (C-19), 12.45 (C-29), 12.23 (C-18).

Compound 3: colorless needle crystals, m.p. 198–200 °C. UV (MeOH) λmax: 202.80 nm. IR (KBr) νmax: 3095.79, 1737.18, 1394.79 cm⁻¹. LC-MS (ESI⁺) m/z: 367.13876 [M+Na]⁺(C₁₉H₂₀O₆Na). ¹H-NMR (CDCl₃, 500 MHz): δ 7.10 (1H, d, H-1), 6.23 (1H, dd, H-13b), 6.16 (1H, dd, H-19b), 5.68 (1H, dd, H-19a), 5.66 (1H, dd, H-13a), 5.48 (1H, d, H-2), 5.16 (1H, d, H-6), 4.79 (1H, d, H-5), 4.67 (1H, d, H-8), 3.04 (1H, dd, H-9b), 2.96 (1H, m, H-7), 2.87 (1H, dd, H-3b), 2.80 (1H, dd, H-9a), 2.71 (1H, dd, H-3a), 1.95 (1H, s, H-18), 1.86 (1H, s, H-14). ¹³C-NMR (CDCl₃, 125 MHz): δ 172.6 (C-15), 169.5 (C-12), 166.5 (C-16), 153.4 (C-1), 136.1 (C-17), 135.8 (C-4), 134.2 (C-11), 133.9 (C-5), 128.6 (C-10), 126.7 (C-19), 123.9 (C-13), 81.5 (C-2), 78.1 (C-6), 71.7 (C-8), 52.6 (C-7), 41.5 (C-3), 33.7 (C-9), 20.3 (C-14), 18.4 (C-18).

Compound 4: white needle crystals, m.p. 223–225 °C. UV (MeOH) λmax: 206.00 nm; IR (KBr) νmax: 3426.29, 2941.92, 2160.74, 1754.24⁻¹. LC-MS (ESI⁺) m/z: 383.168 [M+Na]⁺(C₂₀H₂₂O₆Na). ¹H-NMR (CDCl₃, 500 MHz): δ 6.92 (1H, br m, H-19), 6.35 (1H, d, J = 2.5 Hz, H-13a), 6.21 (1H, s, H-1),6.00 (1H, s, H-3), 5.46 (1H, br s, H-5), 5.28 (1H, d, J = 2.0 Hz, H-13b), 5.24 (1H, dt, J = 11.0, 3.8 Hz, H-8), 4.23 (1H, d, J = 3.25 Hz, H-6), 3.38 (1H, m, H-7), 2.76 (1H, dd, J = 12.3, 3.85 Hz, H-9d), 2.50 (1H, d, J = 11.0 Hz, H-9c), 2.01 (3H, s, H-14), 1.84 (3H, s, H-20), 1.84 (3H, s, H-18). ¹³C-NMR (CDCl₃, 125 MHz): δ 174.3 (C-4), 170.8 (C-16), 165.7 (C-15), 153.2 (C-2), 145.5 (C-12), 135.2 (C-17), 134.6 (C-10), 131.4 (C-11), 126.5 (C-19), 124.3 (C-13), 84.2 (C-1), 81.0 (C-3), 79.8 (C-5), 72.4 (C-8), 52.3 (C-6), 38.9 (C-7), 33.1 (C-9), 20.8 (C-14), 18.1 (C-20), 17.5 (C-18).

Compound 5: yellow needle-like crystals. m.p.: 289–291 °C. UV (MeOH) λmax: 350 and 269.4 nm. IR υmax: 3275–3628.75, 2947.81, 2615, 1604.57, 1451.72, 1324.60, 1249.49, 1200, 1179.18, 1029.98 cm⁻¹. LC–MS (ESI⁺) m/z 331.108 [M+H]⁺(calculated for C₁₇H₁₄O₇). ¹H NMR (acetone-d₆, 500 MHz): δ 13.02 (1H, s, 5-OH), 7.38 (2H, s, H-2′, H-6′), 6.74 (1H, s, H-3), 6.55 (1H, d, J = 2 Hz, H-8), 6.25 (1H, d, J = 2 Hz, H-6), 3.96 (6H, s, OCH₃). ¹³C NMR (acetone-d₆, 125 MHz): δ 183.1 (C-4), 165.1 (C-2), 164.9 (C-7), 163.4 (C-5), 158.8 (C-9), 149.1 (C-3′, C-5′), 141.0 (C-4′), 122.4 (C-1′), 105.3 (C-2′, C-6′), 105.2 (C-10), 104.7 (C-3), 99.7 (C-6), 94.9 (C-8), 56.9 (OCH₃).

Compound 6: pale yellow crystals. m.p.: 234–238 °C. UV (MeOH) λmax: 218, 245.4, 301.2, and 327.4 nm. IR υmax: 3223.13, 1684, 1527, 1251, 1168 cm⁻¹. ¹H NMR (DMSO-d₆, 500 MHz): δ 7.60 (1H, d, J = 16 Hz), 7.50 (1H, d, J = 16 Hz), 7.02 (1H, d, J = 2 Hz), 6.99 (1H, d, J = 2 Hz), 6.90 (1H, dd, J = 8.0, 2.0 Hz), 6.88 (1H, dd, J = 8.0, 2.0 Hz), 6.74 (1H, d, J = 8.0 Hz), 6.73 (1H, d, J = 8.0 Hz), 6.29 (1H, d, J = 16 Hz), 6.20 (1H, d, J = 16 Hz), 5.68 (1H, dt, J = 10.0, 5.0 Hz), 5.12 (1H, dd, J = 10.0, 3.0 Hz), 4.34 (1H, dt, J = 3.0, 2.5 Hz), 2.29 (1H, dd, J = 14.3 Hz), 2.20 (2H, m), 2.04 (1H, dd, J = 14.6 Hz); ¹³C NMR (DMSO-d₆, 125 MHz) δ: 168.72 (C-1″), 168.51 (C-1′), 149.73 (C-7′, C-7″), 147.74 (C-3″), 147.59 (C-3′), 146.83 (C-6′, C-6″), 123.25 (C-9′, C-9″), 116.55 (C-8′, C-8″), 115.18 (C-5′, C-5″), 114.84 (C-2′, C-2″), 112.66 (C-1), 109.75 (C-2), 70.13 (C-5), 69.38 (C-3), 38.73 (C-6).

Tyrosinase inhibitor activity of isolated compound of E. mollis Kunth

Table 2 summarizes the tyrosinase inhibitory activities of compounds isolated from E. mollis Kunth. Stigmasterol exhibited the strongest activity (IC₅₀ = 57.96 µg/mL), followed closely by molephantinin (IC₅₀ = 62.00 µg/mL). Both compounds demonstrated notable inhibition relative to other isolates. Deoxyelephantopin and epifriedelanol showed moderate effects (IC₅₀ = 104.21 and 177.68 µg/mL, respectively), while tricin displayed the weakest activity (IC₅₀ = 241.94 µg/mL). Kojic acid, used as a positive control, had the highest potency (IC₅₀ = 29.58 µg/mL), confirming the assay's reliability. These findings highlight stigmasterol and molephantinin as potential leads for natural tyrosinase inhibitor development.

Table 2. Tyrosinase inhibitor activity of isolated compound and purified methanol extract of E. mollis Kunth.

DISCUSSION

This study evaluated the antioxidant and tyrosinase inhibitory activities of extracts and isolated compounds from E. mollis Kunth using in vitro assays. Tyrosinase is a copper-containing oxidase that plays a central role in melanin biosynthesis by catalyzing the hydroxylation of L-tyrosine to L-DOPA and the subsequent oxidation of L-DOPA to dopaquinone (Doungsaard et al., 2023; Li et al., 2024). As the rate-limiting enzyme in melanogenesis, elevated tyrosinase activity leads to excessive melanin accumulation, contributing to hyperpigmentation disorders such as melasma and age spots. Therefore, inhibition of tyrosinase is a widely accepted strategy for controlling melanin synthesis and developing skin-lightening agents (Li et al., 2024).

In this study, antioxidant and tyrosinase inhibitory activities were assessed in the leaf, stem, and root extracts of E. mollis Kunth, prepared using n-hexane, ethyl acetate, methanol, and crude methanol via Soxhlet extraction (Efendi et al., 2024). All extracts showed measurable degrees of tyrosinase inhibition, with activity influenced by both plant part and extraction solvent. The most potent activity was observed in the bark n-hexane extract (IC₅₀ = 94.03 ± 0.30 µg/mL), followed by the crude methanol leaf extract (IC₅₀ = 116.70 ± 1.22 µg/mL), and the root n-hexane extract (IC₅₀ = 485.63 ± 7.93 µg/mL) as shown in Table 1. These findings indicate that non-polar to medium-polar constituents concentrated in the bark and leaves of E. mollis Kunth substantially contribute to tyrosinase inhibition. Consistent with this, Hasegawa et al. (2010) reported that E. mollis Kunth extract reduced melanin production in B16 murine melanoma cells by downregulating tyrosinase, supporting the relevance of this species as a source of depigmenting agents.

Differences in tyrosinase inhibition are likely attributed to the phytochemical diversity among plant parts. Bioactive constituents such as flavonoids, sesquiterpene lactones, and phenolic acids are known to inhibit tyrosinase either by direct interaction with the copper center at the active site (e.g., via metal chelation) or by modulating gene and protein expression of melanogenic enzymes (Seo et al., 2003). Solvent polarity plays a crucial role in extracting these constituents, thereby affecting the biological activity of the resulting extracts (Dai and Mumper, 2010; Do et al., 2014).

The study also determined total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity. According to Chang (2009), polyphenols, particularly flavonoids, are often potent tyrosinase inhibitors because their catechol-like or hydroxylated aromatic structures can chelate copper ions and stabilize radical intermediates. In the present work, the ethyl acetate extract exhibited the highest TPC (21.38 ± 0.69 mg GAE/g) and TFC (4.06 ± 0.59 mg QE/g), followed by the methanol and n-hexane extracts, as shown in Figure 1.

In terms of antioxidant capacity (DPPH assay), the ethyl acetate extract displayed the strongest activity (IC₅₀ = 54.17 ± 2.48 µg/mL), followed by methanol (88.04 ± 5.10 µg/mL), and n-hexane (945.09 ± 3.13 µg/mL). Likewise, the ethyl acetate extract displayed the most potent tyrosinase inhibition (IC₅₀ = 91.98 ± 3.90 µg/mL), although it was less active than kojic acid (IC₅₀ = 29.58 ± 0.53 µg/mL). These results support a positive contribution of phenolic and flavonoid constituents to both antioxidant and tyrosinase inhibitory properties, in agreement with previous studies that reported strong correlations between polyphenol content, free-radical scavenging capacity, and antityrosinase activity (Zuo et al., 2018; Lang et al., 2021; Gębalski et al., 2023)

A total of six compounds were successfully isolated from the aerial parts of E. mollis Kunth and their structures were elucidated using a combination of spectroscopic techniques, including one-dimensional (¹H and ¹³C NMR) and two-dimensional NMR (COSY, HSQC, and HMBC), along with UV, IR, and mass spectrometry (MS) data. Structural assignments were confirmed by detailed interpretation of spectroscopic data and comparison with previously reported literature values. The isolated compounds were identified as: Epifriedelanol (Compound 1), Stigmasterol (Compound 2), Deoxyelephantopin (Compound 3), Molephantinin (Compound 4), Tricin (Compound 5), and 3,4-di-O-caffeoylquinic acid (Compound 6). The chemical structures of these compounds are shown in Figure 2.

Figure 2. Compounds isolated from the aerial parts of E. mollis Kunth.

From the n-hexane extract of E. mollis Kunth, two colorless crystalline compounds were obtained, both giving a positive Liebermann–Burchard reaction, confirming a triterpenoid/steroid framework. UV spectra indicated the absence of aromatic chromophores. Compound 1 exhibited a high melting point (280–282 °C) and IR bands characteristic of hydroxyl (3,468.93 cm⁻¹) and aliphatic moieties, consistent with a pentacyclic triterpenoid. NMR data revealed a hydroxylated methine at C-3 (δ H 3.64; δ C 72.6), multiple methyl singlets, and COSY/HMBC correlations supporting the epifriedelanol skeleton (But et al., 1996; Kundu et al., 2000; Salazar et al., 2000). Compound 2 had a lower melting point but similar IR characteristics, with ¹H NMR showing six methyl singlets, an H-3 proton (δ H 3.53, m), and three olefinic protons; ¹³C NMR indicated C-3 (δ C 72.0) and four sp² carbons. LC–MS (ESI⁺) yielded m/z 413 [M+H]⁺(C₂₉H₄₈O), confirming the structure as stigmasterol (Jain and Bari, 2010; Srinivasan et al., 2011).

The ethyl acetate extract of E. mollis Kunth yielded three compounds (3–5). Compounds 3 and 4, obtained as needle-like colorless crystals, gave a positive Liebermann–Burchard reaction and showed no aromatic chromophores in their UV spectra. Both exhibited strong IR absorptions at 1,737 and 1,754 cm⁻¹, characteristic of γ-lactone groups, and ¹H/¹³C NMR data consistent with a sesquiterpene lactone bearing α-methylene-γ-lactone functionality. Each displayed three carbonyl carbons (δ C ~165–175 ppm) and multiple olefinic protons (δ H ~5.0–7.1 ppm). Compound 4 differed from compound 3 by the presence of an additional methyl group and an oxygenated carbon (δ C 84.2), indicating higher substitution on the side chain. LC–MS (ESI⁺) analysis identified compound 3 (m/z 367.13876 [M+Na]⁺, C₁₉H₂₀O₆Na) as deoxyelephantopin (Koe et al., 2013) and compound 4 (m/z 383.168 [M+Na]⁺, C₂₀H₂₂O₆Na) as molephantinin (Lee et al., 1980).

Compound 5, isolated as yellow crystals, displayed yellow fluorescence on TLC under UV 365 nm after citroborate spraying and heating. UV absorptions at 350 and 269.4 nm, positive citroborate test, and IR data indicated a flavonoid skeleton. ¹H NMR showed a chelated 5-OH proton (δ H 13.02, s), meta-coupled protons at H-6 (δ H 6.25, d, J = 2 Hz) and H-8 (δ H 6.55, d, J = 2 Hz), and symmetric B-ring protons at δ H 7.38 (s, 2H), along with two methoxy groups at δ H 3.96 (s, 6H) at C-3’ and C-5’. ¹³C NMR confirmed a carbonyl at δ C 183.1, three C–OH, and two C–OCH₃ signals. LC–MS (ESI⁺) at m/z 331.108 [M+H]⁺(C₁₇H₁₄O₇) identified compound 5 as tricin (Li et al., 2016).

From the methanol extract, compound 6 was obtained as pale-yellow crystals (m.p. 234–238 °C) and gave a positive FeCl₃ test for phenolics. IR spectra showed broad O–H stretching (3,223 cm⁻¹) and an ester/lactone C=O band at 1,684 cm⁻¹. UV maxima at 218, 245.4, 301.2, and 327.4 nm were characteristic of caffeoyl chromophores. The ¹H-NMR spectrum displayed trans-olefinic protons and aromatic signals from two caffeoyl units, together with resonances typical of quinic acid. The ¹³C-NMR data confirmed two ester carbonyls, aromatic carbons, and quinic acid carbons. These features, supported by literature, established the structure as 3,4-di-O-caffeoylquinic acid (Geng et al., 2011; Ooi et al., 2011).

The isolated compounds were evaluated for their tyrosinase inhibitory activity. As shown in Table 2, all compounds exhibited moderate to weak inhibition compared to kojic acid (IC₅₀ = 29.58 µg/mL). Among them, stigmasterol (Compound 2) and molephantinin (Compound 4) showed the strongest effects, with IC₅₀ values of 57.96 µg/mL and 62.00 µg/mL, respectively. Deoxyelephantopin (Compound 3; IC₅₀ = 104.21 µg/mL) and epifriedelanol (Compound 1; IC₅₀ = 177.68 µg/mL) displayed weaker activity, whereas tricin (Compound 5) showed the lowest potency (IC₅₀ = 241.94 µg/mL). Interestingly, 3,4-di-O-caffeoylquinic acid (Compound 6) tested at a single concentration (93.75 µg/mL) enhanced tyrosinase activity, indicating a potential pro-melanogenic effect rather than inhibition.

The differences in activity can be rationalized by considering the chemical structures and functional groups of the isolates. Stigmasterol is a phytosterol with a tetracyclic steroid nucleus, a 3β-hydroxyl group, and two double bonds at Δ⁵ and Δ²². The rigid hydrophobic core likely promotes interactions within the non-polar regions of the tyrosinase active site, while the 3β-OH group can participate in hydrogen bonding with amino acid residues or coordinated water molecules near the copper center. Previous reports have shown that phytosterols, including mixtures of β-sitosterol and stigmasterol, exhibit detectable antityrosinase activity, supporting the contribution of this scaffold to enzyme inhibition (Muhammad et al., 2023).Thus, the moderate potency of stigmasterol observed in this study is consistent with its amphiphilic sterol structure, which allows sufficient binding to interfere with substrate access without reaching the potency of classical chelating inhibitors such as kojic acid.

Molephantinin, a sesquiterpene lactone bearing an α-methylene-γ-lactone moiety and multiple conjugated carbonyls, also demonstrated relatively strong tyrosinase inhibition. The α-methylene-γ-lactone motif is a well-known Michael acceptor that can interact with nucleophilic residues (e.g., cysteine and histidine) in proteins, leading to covalent or quasi-covalent modifications and conformational changes. In the context of tyrosinase, such reactive centers may perturb the geometry of the active site or interfere with access to the dinuclear copper center, thereby reducing enzymatic activity. The comparable IC₅₀ values of molephantinin and stigmasterol suggest that both hydrophobic interactions (in the case of stigmasterol) and electrophilic Michael acceptor reactivity (in the case of molephantinin) provide effective but moderate inhibition of tyrosinase.

When compared with related species, the tyrosinase inhibitory profile of E. mollis Kunth appears broadly consistent with, yet distinct from, other members of the genus. (Xu et al., 2022) reported several constituents from E. scaber with significant antityrosinase activity, in some cases surpassing arbutin in potency. More recently, E. scaber leaf and root extracts have been shown to possess antioxidant and antityrosinase properties, further supporting the dermatological potential of the genus (Anuar et al., 2025). The IC₅₀ values obtained for stigmasterol and molephantinin from E. mollis Kunth fall within the same order of magnitude as those reported for active constituents from E. scaber, suggesting that sesquiterpene lactones and sterols are recurring pharmacophores for tyrosinase inhibition within Elephantopus. At the same time, the present work extends existing literature by providing a systematic comparison of multiple isolated compounds from E. mollis Kunth, alongside comprehensive extract-level data and detailed structural assignments.

Skin-lightening agents inhibit tyrosinase primarily through competitive mechanisms, in which compounds occupy the active site and prevent substrate binding, or through non-competitive and mixed-type mechanisms, in which binding at peripheral or allosteric sites induces conformational changes that lower catalytic efficiency (Zolghadri et al., 2019). Flavonoids, terpenoids, and steroids, including those isolated in the present study, have been previously reported as non-competitive or mixed-type inhibitors, often interacting via hydrophobic contacts, hydrogen bonding, and, in some cases, weak coordination to the copper center (Dolorosa et al., 2019; Masum et al., 2019; El-Nashar et al., 2021). Their appeal lies in low toxicity, multiple biological targets, and potential for skin-lightening applications (Kumari B M, 2019; Zhao et al., 2022). In addition, stigmasterol has recently been shown to reduce melanin synthesis and modulate reactive oxygen species and apoptosis pathways in melanoma cells, supporting its dual potential as both a skin-whitening and anti-melanoma agent (Han et al., 2024).

The present findings indicate that E. mollis Kunth-derived stigmasterol and molephantinin exhibit moderate tyrosinase inhibition and could be further optimized via structural modification or nanoformulation to enhance solubility, skin penetration, and stability. The combination of antioxidant and tyrosinase inhibitory activities offers dual benefits for dermocosmetic applications, particularly in preventing oxidative stress-induced hyperpigmentation. Nonetheless, this study is limited by its in vitro design, absence of kinetic characterization, and lack of synergy assessment among active compounds. Future work should address these gaps through enzyme kinetics, in vivo or ex vivo skin models, and formulation studies to improve delivery and efficacy.

CONCLUSION

E. mollis Kunth contains a variety of bioactive compounds, including terpenoids, flavonoids, and phenolic acids, with potential as natural skin-whitening agents. The ethyl acetate extract showed high total phenolic and flavonoid contents, correlating with significant antioxidant activity, which may help mitigate oxidative stress-related hyperpigmentation. Among the isolated compounds, stigmasterol and molephantinin exhibited moderate tyrosinase inhibitory activity. Although less potent than kojic acid, the dual antioxidant and anti-tyrosinase activities offer synergistic benefits for dermo-cosmetic use. These findings underscore the potential of E. mollis Kunth as a source of multifunctional, plant-based skin-lightening agents.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to the Universitas Andalas and Biota Sumatra laboratory for facilitate this research.

AUTHOR CONTRIBUTIONS

Deddi Prima Putra: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead),Validation (Lead), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Investigation (Equal), Supervision (Lead), Project administration (Lead); M. Rifqi Efendi: Methodology (Supporting), Data Curation (Equal), Formal Analysis (Equal), Validation (Supporting), Writing – Original Draft (Equal), Writing – Review & Editing (Equal), Investigation (Lead); Amri Bakhtiar: Conceptualization (Equal), Methodology (Lead), Formal Analysis (Equal), Data Curation (Equal) Validation (Lead), Investigation (Equal), Resource (Equal), Writing – Original Draft (Equal), Writing – Review & Editing (Equal).

CONFLICT OF INTEREST

The authors declared that no conflict of interest.

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