INTRODUCTION:

Many studies suggest that free radicals are essential in developing some human skin disorders. For example, oxidative stress has been widely reported and has been proposed to be involved in skin aging, as well as the formation of lines and wrinkles, dryness and dullness, skin with a rough texture, uneven skin tone, and the appearance of age spots^1,2. Recent epidemiological studies have found that high antioxidant consumption is correlated with a lower risk of skin aging disorders^3,4. Some synthetic antioxidants, including BHA and BHT, have been associated with both carcinogenicity and toxicity^5,6. As a result, there is a rising interest in exploring natural antioxidants. Because the complex structure of natural substances makes it challenging to screen for active antioxidants rapidly. Several assay methods were developed and used to search for potential antioxidant sources, such as the relatively high throughput DPPH radical scavenging capacity (RDSC) test⁷, the hydroxyl radical scavenging capacity (HOSC) test⁸, and the thin-layer chromatography (TLC) bioautography test⁹ Compared to other procedures, the TLC bioautography method can rapidly detect and separate component active compounds in complicated experimental specimen extracts. It also has additional benefits such as simplicity, ease of operation, and no requirement for specialized equipment. This article describes the guided detection of bioactive substances. The TLC bioautography method evaluated Various marine sponge species for their natural antioxidant activity.

Secondary metabolites from sponges have a lot of ecological roles, such as inhibiting predation, infection, and microbial proliferation in the sponge body^10,11. Sponge secondary metabolites possess diverse medicinal properties and are used in a few applications in life. Several studies show that active compounds from the terpenoid, steroid, and alkaloid groups of sponges Stylotella sp., Agelas dispar, Neopetrosia sp., Aaptos sp., Haliclona sp., Stylissa flabelliformis, and Hyrtios reticulatus have antioxidant capacity, is tyrosinase inhibitors, suppress melanin synthesis, and many important biological activities^11–13. This study investigated the potential of marine sponge secondary metabolites for antihyperpigmentation therapy by inhibiting the tyrosinase enzyme and monitoring antioxidant activity against Vero and B16F10 melanoma cells to identify active components.

MATERIAL AND METHODS:

Materials:

Marine Sponge Materials:

Marine sponges were collected, identified, and prepared by recognized techniques¹⁴. Table 1 contains information on the marine sponge that was collected.

Preparation of Marine Sponge Extracts:

Marine sponge crude extracts were produced using a maceration process with chloroform and ethanol (96%) at a 1:10 ratio of marine sponge material to solvent, as previously reported¹⁴.

Antioxidant Activity Through the Utilization of DPPH Radical Scavenging Activity:

The activity of CHCl₃ and EtOH extract (31.25, 62.5, 125, 250, and 500μg/mL) in scavenging DPPH radicals was measured through the addition of 3.0mL of seed extract with 1.0mL of 0.1mM DPPH (2,2-diphenyl-l-picrylhydrazyl) in methanol. The absorbed wavelength at 517nm was measured using a UV/Vis spectrophotometer (123) after it was incubated for 30 minutes at 37^oC^15,16.

Antioxidant Profiling by Bioautography Assay:

Direct deposit (as spots) of each marine sponge extract with a 100mg/mL concentration of 5µL onto the TLC plate. In a saturated solvent chamber, TLC plates were developed using a developing reagent mixture of n-hexane, toluene, ethyl acetate, and formic acid (2:5:2.5:0.5) until the solvent front was one centimeter below the top of the plates. The generated TLC plates were then taken out of the chamber and given 30 minutes to air dry before being sprayed with a 0.2 mg/mL DPPH methanol solution to derivatize. As for a purple background, white-yellow bands exhibiting DPPH scavenging activity were visible. Every TLC plate was observed at 254 and 366nm under UV illumination.

Inhibition of Tyrosinase Assay:

L-tyrosine was employed as the substrate, and tyrosinase as the enzyme in the tyrosinase-blocking analysis. In 96-well plates, 10µL of the extract and 170µL of 50mM phosphate buffer containing NaH2PO4-NaHPO4 (pH 6.5) were added. After treatment with 20µL of tyrosinase, the well was incubated for 30 minutes at 37 °C. At 475nm, the absorbance was measured with a microplate reader^17,18.

Culture of The Cell:

Vero cells and B16F10 rodent skin melanoma were obtained from the UGM FKKMK parasitology laboratory collection. Vero cells were cultivated at 37^oC in RPMI-1640 (Roswell Park Memorial Institute) with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 units/mL penicillin, and 100µg/mL streptomycin. B16F10 cells were cultured in DMEM (Dulbecco's modified Eagle). The cells were incubated in a 5% CO₂ environment at 37°C^19,20.

Antitoxicity Assay against B16F10 and Vero Cells:

In living cell mitochondria, yellow MTT is converted to purple formazan. The absorbance of this colored solution at a particular wavelength (500-600 nm) can be measured using a spectrophotometer²¹. As a result, the application may analyze the extract's antitoxicity based on cell survival or viability. The efficacy of an agent in inducing cell death can be assessed by comparing the quantity of purple formazan generated by cells treated with an agent to that produced by untreated control cells. In 96-well microplates, 100µl of B16F10 and/or Vero cell suspension was added at a density of 1.5 x 10⁴ cells. Cell cultures were treated with extract concentrations that ranged from 15.63 to 1000µg/ml and doxorubicin concentrations that ranged from 1.5 to 50µg/ml, respectively. Three evaluations were conducted for each treatment group. After being treated, the cell cultures were incubated at 37°C and 5% CO₂ for 24 hours. Post-incubation, the culture media was eliminated. After adding 100 µl of MTT solution to each well, the wells were incubated for four hours at 37^oC with 5% CO₂. After incubation, 100 µl of 10% SDS reagent was added as a stopper and left overnight. The ELISA reader was employed for measuring the cell absorbance at 595 nm ²². A curve was established between the concentration and the percentage of live cells to ascertain the IC₅₀value of the test drug against the B16F10 and Vero cell lines. The following equation was employed to determine the significant percentage test of cell viability:

TC - CM

% Cell viability = -------------- x 100

CC - CM

The symbols TC, CC, and CM represent treatment cell absorbance (TC), control cell absorbance (CC), and control media absorbance (CM).

Statistical Analysis:

All data values' means and standard deviations (SD) were presented. The Student's t-test was implemented to detect statistically significant differences, with a significance threshold of p<0.05.

RESULT:

Effect of Extracts on Antioxidant Activity:

Table 1. The IC₅₀ values of marine sponge and kojic acid extracts were obtained using DPPH.

Sample			^IC₅₀(µg/mL)	^{Antioxidant Activity Category}
Extracts	Chloroform	Stylotella sp.	132.94±0.26	Moderate
		Agelas dispar	114.99±0.21	Moderate
		Neopetrosia sp.	60.80±0.14	Strong
		Aaptos sp.	87.36±0.24	Strong
		Haliclona sp.	91.05±0.19	Strong
	Ethanol	Stylotella sp.	207.80±0.64	Moderate
		Agelas dispar	94.47±0.16	Strong
		Neopetrosia sp.	69.90±0.19	Strong
		Aaptos sp.	68.60±0.17	Strong
		Haliclona sp.	102.95±0.15	Moderate
Kojic acid^a			12.10±0.35	Very strong

Note: ^a Antioxidant standard; Antioxidant activity IC₅₀ (µg/mL): very strong <20 µg/mL, strong <100 µg/mL, moderate 100-500 µg/mL, weak >500 µg/mL ²³. IC₅₀ was reported as mean values ± SD of three independent assays.

Antioxidant Substances are Present From Marine Sponge Extracts:

Figure 1. TLC plates stained with a 0.2 mg/mL DPPH solution in methanol were observed under (A.1; B.1) visible light, (A.2; B.2) UV 254 nm, and (A.3; B.3) UV 366 nm. (1) Stylotella sp., (2) Agelas dispar, (3) Neopetrosia sp., (4) Aaptos sp., (5) Haliclona sp. 100 mg/mL methanol extracts, (6) Stylotella sp., (7) Agelas dispar, (8) Neopetrosia sp., (9) Aaptos sp., (10) Haliclona sp. 100 mg/mL chloroform extracts were placed in five microliters as dots on a TLC layer. Yellow spot regions show the compounds with DPPH-scavenging capabilities. The sponge Neopetrosia sp., for instance, has the markings a, b, c, d, and e. A.1, A.2, and A.3 were eluted with the solvent system n-hexane/AcOEt (87.5/12.5), and then B.1, B.2, and B.3 were eluted with the solvent system CHCl₃/acetic acid (9/1).

Effect of Extracts on Tyrosinase Activity:

Table 2. The IC₅₀ values of chloroform extracts of marine sponge and kojic acid against tyrosinase inhibition.

Sample		^Inhibition(%)
Extracts	Stylotella sp.	43.35±0.02
	Agelas dispar	47.73±0.21
	Neopetrosia sp.	67.18±0.23
	Aaptos sp.	55.27±0.14
	Haliclona sp.	35.09±0.24
Kojic acid^a		55.13±0.10

Note: ^a Tyrosinase inhibitor standard; chloroform extracts of marine sponge (200 µg/mL) and kojic acid (21.32 µg/mL) at the fourth testing minute. IC₅₀ was reported as mean values ± SD of three independent assays.

Effect of Extracts on Cytotoxic Activity:

Table 3. The IC₅₀ values of marine sponge extracts and doxorubicin against B16F10 and Vero cells.

Sample			^IC₅₀(µg/mL)		^{Cytotoxicity Category}
Sample			B16F10	Vero	B16F10	Vero
Extracts	Chloroform	Stylotella sp.	391.01±0.43	750.80±0.26	Moderate	Moderate
		Agelas dispar	253.73±0.31	555.85±0.45	Moderate	Moderate
		Neopetrosia sp.	284.04±0.40	626.74±0.30	Moderate	Moderate
		Aaptos sp.	258.44±0.27	446.92±0.38	Moderate	Moderate
		Haliclona sp.	329.56±0.62	918.66±0.44	Moderate	Moderate
	Ethanol	Stylotella sp.	936.25±0.23	835.05±0.82	Moderate	Moderate
		Agelas dispar	272.60±0.46	619.69±0.62	Moderate	Moderate
		Neopetrosia sp.	251.41±0.39	1022.41±0.36	Moderate	Non-toxic
		Aaptos sp.	236.14±0.54	258.87±0.44	Moderate	Moderate
		Haliclona sp.	251.87±0.31	1475.06±0.36	Moderate	Non-toxic
Doxorubicin^a			1.21±0.36	1.33±0.74	Potentially toxic	Potentially toxic

Note: ^a Cytotoxic standard; cytotoxic activity IC₅₀ (µg/mL): potential <100 μg/mL, moderate 100-1000 μg/mL, non-toxic >1000 μg/Ml ²⁴. IC₅₀ was reported as mean values ± SD of three independent assays.

DISCUSSION:

Stylotella sp., Agelas dispar, Neopetrosia sp., Aaptos sp., Haliclona sp., which resemble marine sponges, have been shown to possess antioxidant and anti-cancer effects²⁵. Studies on the benefits of skincare have been conducted in a limited amount. Phenols, lactones, steroids, terpenoids, peptides, macrolides, alkaloids, fatty acids, polysaccharides, glycoproteins, and other compounds are responsible for biological activity from sponges^26,27. Compounds that have been isolated from various types of sponges include bromopyrrole alkaloids such as oroidin and quinolone ageloline A from Agelas aoroides with antioxidant, cytotoxic, and antimicrobial activities^28,29, β-carboline derivative alkaloids such as Manzamine from Haliclona sp. with antimicrobial, antiviral, cytotoxic, and antimalarial activities^30–32, peptides such as Discodermin A from Discodermia kiiensis as antimicrobials³³, peptides such as Kapakahines from Cribrochalina olemda as antileukemia and antimalarial^34–36, Triterpenes such as stelletin a from Stelleta tenuis³⁷, sesterpenoids such as manoalide from Luffariella variabilis³⁸, sesquiterpenes such as avarol and avarone derivatives from Dysidea avara with antiviral activity³⁹, and Hamigeran B from Hamigera tarangaensis with antiviral activity⁴⁰. In any case, a study showed that phenolic, alkaloid, and flavonoid compounds suppressed transcriptional expression of tyrosinase (TYR), tyrosinase-related proteins (TYRPs), and microphthalmia-associated transcription factor (MITF), several of which play a role in melanin formation, through B16F10 mouse skin melanoma cells^41–43. Furthermore, the ethanol extract of the marine sponge was found to have antioxidant and antityrosinase activity⁴⁴. Another study showed that adding phenolic, alkaloid, and flavonoid compounds to potato supernatant decreased enzymatic browning and increased antioxidant capacity over time⁴⁵.

The capacity test and conventional DPPH spectrophotometry are employed to identify possible antioxidant extracts from marine sponges. The marine sponge extract absorbed the DPPH dose-dependent manner, with IC₅₀ values ranging from 60.80±0.14 to 207.80±0.64µg/mL (Table 1). Neopetrosia sp. was shown to have remarkably higher levels of free radical scavenging activity than other sponge species. This is probably because Neopetrosia sp. has high concentrations of secondary metabolite components like quinones, terpenoids, sterols, and alkaloids⁴⁶. The high concentration of these substances may support Neopetrosia sp. antioxidant activity and other health advantages⁴⁷. The TLC bioautography method is then used to monitor this activity as a guide separation since it offers quick access for identifying and localizing active chemicals⁴⁸. The technique showed yellow-white dots on a purple backdrop as the DPPH scavenging activity. Under visible light, Figure 1 (A.1) displays the profile of antioxidant components found in marine sponges' ethanol and chloroform extracts. It was found that the chromatogram had at least five points (such as points a–e) with DPPH scavenging activity. The biological activity generated by the various types of sponges that were tested indicates that there is activity that is not specific to any particular family or species of sponges. The results of the antioxidant activity test indicated that each extract possesses antioxidant activity; however, the secondary metabolites involved are inconsistent across different sponge varieties. Concurrently, the tyrosinase inhibition test showed Neopetrosia sp. and Agelas dispar possess a higher level of tyrosinase inhibitory activity than other sponge species. The results of the antioxidant activity test shown are consistent with this.

We utilized the B16F10 melanoma cell model for evaluating the cytotoxic effect of marine sponge extract when applied as an antihyperpigmentation agent in inhibiting melanogenesis involving tyrosinase enzyme activity and ensuring its safety against Vero cells. Furthermore, marine sponge extracts showed vigorous antioxidant activity using the DPPH assay. These findings correlated with previous studies. Phenolic compounds from marine sponge extracts will scavenge hydroxyl radicals (OH) in oxidative stress in mesenchymal stem cells. The mechanism to protect DNA and mesenchymal stem cells from oxidative damage will likely involve metal chelation and direct radical scavenging⁴⁹. Another study showed that phenolic, alkaloid, and flavonoid from plant extracts on fresh-cut fruits and vegetables enhanced antioxidant capacity^16,45,50,51. Antioxidants can inhibit the initial stage of enzymatic browning through their interaction with oxygen. By disrupting chain processes and inhibiting melanin synthesis, they can interact with intermediate products^52,53. The antioxidant activity is correlated with the enzyme inhibitor if it is associated with the tyrosinase enzyme inhibitory activity. Compared to extracts from other marine sponges, Neopetrosia sp. exhibits the highest inhibitory activity at a concentration of 5 mg/mL with a percentage of inhibition of 67.18±0.23 % (Table 2).

Safety is crucial in the pharmaceutical and cosmetic industries when employing particular compounds as therapeutic or cosmetic agents. The safety of using sea sponges as a medicine or cosmetic has not been investigated despite reports that they are safe for human consumption and rich in nutrients and secondary metabolites. The findings of this investigation show that, in B16F10 and Vero cells, the marine sponge extract does not result in cellular toxicity at concentrations up to 250µg/mL but that, at concentrations above that, it marginally impairs cell viability. Depending on the intended use, more investigation should be done to examine both the immediate and lasting adverse inhibitory effects of Neopetrosia sp. and Aaptos sp. in several animal models. Furthermore, observation is required to assess the anti-melanogenic activity of tyrosinase inhibitors on B16F10 cells.

Due to their limited reproductive rate, sponges are eligible for protection as marine biota. Nevertheless, sponges possess a significant potential for producing bioactive compound metabolites with various novel characteristics. Therefore, the subsequent phase involves utilizing biotechnological methods to augment the production of secondary metabolites from sponges with the assistance of endophytic microorganisms.

CONCLUSION:

The CHCl₃ and EtOH extracts from Agelas dispar, Neopetrosia sp., Aaptos sp., Haliclona sp., and Stylotella sp. exhibited potent antioxidant and tyrosinase inhibitory properties, as shown by our research findings. In addition, anti-toxic efficacy against B16F10 and Vero cells was demonstrated by marine sponge extract at a concentration of less than 250µg/mL. These results show that marine sponge extract can inhibit the tyrosinase enzyme and absorb free radicals without causing damage to test cells. This suggests that marine sponge extract could be used as an antihyperpigmentation agent, but more investigation is required to determine whether it also has anti-melanogenic properties. We presented the antioxidant and anti-tyrosinase properties of CHCl₃ and EtOH extracts and found that these substances were isolated from these extracts.

CONFLICT OF INTEREST:

The authors confirm that they have no conflicts of interest.

ACKNOWLEDGMENTS:

Indonesia Education Scholarship (BPI); Center for Higher Education Funding and Assessment Ministry of Higher Education, Science, and Technology of Republic Indonesia (PPPAPT); and Indonesia Endowment Fund for Education (LPDP) have supported this work.

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Received on 02.08.2024 Revised on 17.02.2025

Accepted on 14.06.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3579-3585.

DOI: 10.52711/0974-360X.2025.00515

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