INTRODUCTION:

Seaweed is one of the biological resources that is often found in coastal and marine areas. Sargassum sp. is a species of brown algae with approximately 400 species in the world and 12 species known in Indonesia.¹ Sargassum polycystum is a brown algae from the species Sargassum sp. which is known in Indonesia.² S. polycystum has many benefits so its economic value is quite high.^1,3 The largest carotenoid found in brown macroalgae is fucoxanthin.⁴

Fucoxanthin from a group of compounds called carotenoid^5,6, and it is an orange pigment found in S. polycystum.^2,7 The active fucoxanthin component showed properties as an antibacterial agent at 500 and 1000 μg/disk concentrations against Staphylococcus aureus.⁸ In this study, the antibacterial activity of fucoxanthin against Staphylococcus aureus was evaluated, and its formulation into a nanogel, with the goal of developing an anti-acne product.

Staphylococcus aureus is a gram-positive bacteria responsible for keratin material accumulation, leading to blockages and inflammation in the pilosebaceous layer, resulting in a skin condition commonly referred to as acne.⁹ The antibacterial activity of the pure fucoxanthin isolate and fucoxanthin in nanogel formulation was examined using the well diffusion method. The experts formulated the nanogel with fucoxanthin to address the algae physical form and fishy odor, which makes it less appealing for use as a cosmetic ingredient. Therefore, the purpose of this study was to develop an effective cosmetic product that is beneficial as well as acceptable to the public.

MATERIALS AND METHODS:

Materials:

Sargassum polycystum samples were collected from the rivers of North Lombok, Indonesia, in August 2021. The samples were then placed in plastic containers filled with sterile seawater and stored in a coolbox to maintain the freshness.^10,11,12 The Sargassum polycystum samples used are shown in Figure 1.

Figure 1. Sargassum polycystum

METHODS:

Extraction and Isolation of Fucoxanthin:

The obtained Sargassum polycystum was thoroughly washed with water, and CaCO₃ was added to help rinse. The maceration method was used to carry out the extraction process, where the samples were soaked in an acetone: methanol (7:3) solvent at a sample-to-solvent ratio of 1:10 for 15 - 20 minutes, and then filtered.^13,14 The rotary evaporator was then used to concentrate the filtrate at a temperature less than 35°C. In this case, n-hexane was used to fractionate the concentrated filtrate, and a saturated saline solution and tap water were added to separate the mixture into two layers.¹⁵ The fractions were then concentrated under N₂ gas and stored in a dark place. Also, column chromatography was utilized to carry out the isolation process with the eluent being n-hexane: ether: acetone (6:3:2). The fractions collected were sorted based on color and analyzed by Thin Layer Chromatography (TLC) using the same eluent. In addition, the fractions were concentrated again under N₂ gas and stored in a dark place at 4–10°C and tested for purity using the TLC method on three different mobile phases, including n-hexane: acetone (6:4), n-hexane: ethylacetate (4:6), and chloroform: ethanol (7:3).¹⁶

Fucoxanthin Identification by UV-Vis Spectrophotometer:

A UV-Vis spectrophotometer was utilized to measure the absorption spectrum of the carotenoid compounds.^17,18 The samples were dissolved in acetone and measured at wavelengths between 400 and 500 nm.¹⁹ In this context, the samples were considered to contain pure fucoxanthin when the spectrum of absorption showed three distinct peaks in the 400–500 nm range, with minimal absorption in other regions, or two peaks around 450 nm with two additional smaller peaks flanking the main peak.¹³

Fucoxanthin Identification by UV-Vis Spectrophotometer:

In order to carry out further identification of fucoxanthin, Fourier-Transform Infrared (FTIR) was used to scan 0.5–1.5 mg of the samples. Moreover, the functional groups were identified based on specific wavenumbers and compared to a fucoxanthin standard.²⁰

Formulation and Preparation of Fucoxanthin Nanogel:

The nanoemulsion was prepared through the homogenization of Virgin Coconut Oil (VCO), Tween 20, and PEG 400 using a vortex mixer for 5 minutes. Furthermore, the fucoxanthin compound was dissolved into the nanoemulsion base, homogenized for 5 minutes, and finally sonicated for 15 minutes. The mixture was diluted with distilled water to a total volume of 5 ml, and followed by a transmittance test. The gel base was prepared by swelling carbopol in hot distilled water, at 20 times its weight.²¹ Subsequently, the swollen carbopol was mixed with glycerin and stirred until homogeneous, then propylene glycol was added and stirred again. The nanoemulsion was then mixed with the gel base, and triethanolamine was slowly added drop by drop to achieve final consistency and volume.^21,22 The nanoemulsion and nanogel formulation are shown in Table 1 and Table 2 respectively.

Table 1. Nanoemulsion formula for preparing the nanogel with fucoxanthin from Sargassum polycystum

Material	Concentration (%)			Function
Material	0.1%	0.2%	0.3%	Function
Fucoxanthin	0.1	0.2	0.3	Antibacterial against Staphylococcus aureus
VCO (Virgin Coconut Oil)	0.24	0.24	0.24	Oil phase
Tween 20	0.72	0.72	0.72	Surfactant
Polyethylene-glycol 400	0.24	0.24	0.24	Co-surfactant
Aquadest	Ad 5 ml	Ad 5 ml	Ad 5 ml	Solvent

Table 2. Nanogel Formula with the active substance fucoxanthin from Sargassum polycystum

Material	Concentration (%)			Function
Fucoxanthin Nanoemulsion	16.67	16.67	16.67
Carbopol	1	1	1	Gelling agent
Glycerin	8	8	8	Humectant
Triethanolamine	0.5	0.5	0.5	Alkalizing agent
Propylene glycol	15	15	15	Co-solvent
Aquadest	Ad 30 g	Ad 30 g	Ad 30 g	Solvent

The final preparation was evaluated for its physical characteristics, including organoleptic properties, homogeneity, spreadability, adhesion, pH, and viscosity.

Evaluation of Physical Characteristics of Nanoparticles:

After ultracentrifugation, the nanoparticles were resuspended in distilled water, and the particle size, zeta potential, and polydispersity index (PDI) were measured using a Particle Size Analyzer (PSA) (Beckman Coulter).²³

Antibacterial Activity:

Activity tests were conducted on fucoxanthin isolates and fucoxanthin-loaded nanogel using the diffusion method. A Petri dish was filled with 10 mL of Mueller-Stokes Agar (MSA) medium and allowed to solidify. A cylinder cup was then placed on the solidified layer, and an additional 20 mL of MSA medium, inoculated with test bacteria, was poured into the dish as the second layer, with the dish placed in a laminar airflow to allow the medium to harden. Once it had hardened, the cylinder cup was carefully removed, creating a well-like hole. Subsequently, 20 µL of each sample and control were introduced into the wells. The plates were incubated at 37°C for 24 hours, after which the clear zone around each well was measured using a caliper. The observed diameter of the clear zone was adjusted for the diameter of the cylinder cup. ^24,25

RESULT:

The yield of the extract was 85.98%, while the yield of the n-hexane fraction was 2.62%, which was analyzed using Thin-Layer Chromatography (TLC) to identify fucoxanthin compounds, and the results are shown in Figure 2.

Figure 2. TLC of n-Hexane Fraction with Silica Gel GF 254 Stationary Phase, n-Hexane: Ether: Acetone (6:3:2) Mobile Phase

TLC analysis showed that the fucoxanthin compound had an Rf value of 0.54, showing clear separation. This result showed that TLC was suitable for use in column chromatography during the isolation process. The concentrated orange pigment collected was compared to a fucoxanthin standard using TLC.

Figure 3. Fucoxanthin isolate (A) and fucoxanthin standard (B)

The TLC analysis showed that the isolated compound was fucoxanthin, as it showed the same Rf value of 0.54 as the standard. Purity tests were then conducted using TLC with three different mobile phase systems, as shown in Figure 4.

(a) (b) (c) (d)

Figure 4. Fucoxanthin Purity Test with TLC

Image caption: Mobile Phase (a) n-Hexane: Ether: Acetone (6:3:2), (b) n-Hexane: Acetone (6:4), (c) n-Hexane: Ethylacetate (4:6) , (d) Chloroform: Ethanol (7:3)

Furthermore, fucoxanthin isolates were identified by spectrophotometry (Figure 5.)

Figure 5. Spectrum Pattern of Fucoxanthin Compound

Image caption: A: Standard fucoxanthin; B: Isolated fucoxanthin

Further identification was performed using FTIR spectrophotometry to detect the functional groups present in the fucoxanthin compounds. The FTIR analysis results are shown in Figure 6 and Table 3.

Figure 6. FTIR Spectrum Pattern of Red Fucoxanthin Isolate and Blue Standard Fucoxanthin

Table 3. Results of FTIR Analysis of Fucoxanthin from Sargassum polycystum

No.	Functional Group	V_max cm^-1 (Haugan et al., 1992)	V_max cm^-1 (Sample)	V_max cm^-1 (Standard)
1.	OH	3483
2.	CH	3030-2856	2954, 2922, 2853	2967, 2924
3.	Aliene	1930
4.	C=O, ester	1732	1733	1733
5.	C=C conjugated	1654	1653	1650
6.	CH₂	1607,1576, 1530, 1471,1456	1576, 1457	1606, 1577, 1472, 1456
7.	C-O, acetat	1335, 1261, 1245	1260	1243
8.	C-O-C	1032	1032	1032
9.	C=C, trans substituted	1201,1175, 1171, 1051, 1053, 958	1070, 1153, 958	1200,1155, 1051, 958

The purified and concentrated fucoxanthin compounds were tested for antibacterial activity against Staphylococcus aureus. The results of this antibacterial activity test are presented in Figure 7 and Table 4.

Figure 7. Antibacterial Activity Test of Fucoxanthin Compound Against Staphylococcus aureus

The results of the antibacterial activity test against Staphylococcus aureus are presented in Table 4. The data showed the concentrations of fucoxanthin compounds influenced the size of the inhibition zone, where a higher concentration led to larger inhibition zones.

Table 4. Results of Antibacterial Activity Test for Fucoxanthin Compound

Concentration	Diameter of inhibition (mm)
K+ (Clindamycin 0.05%)	15.40 ± 0.002
K- (DMSO 10%)	0 ± 0
Fucoxanthin 0.1%	13.60 ± 0.001
Fucoxanthin 0.2%	14.50 ± 0.002
Fucoxanthin 0.3%	15.30 ± 0.002

Fucoxanthin compounds at 0.1%, 0.2%, and 0.3% concentrations produced inhibition zones, which led to formulation into nanogel preparations. The results of the nanogel formulation are presented in Figure 8.

The tests that were carried out on the nanogel preparations included the assessment of organoleptic properties, homogeneity, spreadability, adhesion, pH, and viscosity (Table 5).

After conducting these characteristic tests, a PSA was performed to confirm the nanogel nature of the preparation. The PSA results are shown in Table 6.

Table 6. Evaluation Results of Fucoxanthin Nanogel

Formula	Nano size (nm)	Zeta Potensial (mV)	Polydispersity Index
Fucoxanthin 0.1 %	117±0.015	-0.21 ±0.56	0.33 ± 0.01
Fucoxanthin 0.2 %	392±0.02	-0.34 ± 0.17	0.75 ± 0.03
Fucoxanthin 0.3 %	505±0.02	-0.83 ± 0.43	0.82 ± 002

Fucoxanthin nanogel preparation that met the required specifications was tested for antibacterial activity against Staphylococcus aureus. The results showed fucoxanthin nanogel concentrations affected the size of the inhibition zone, and higher concentrations led to larger inhibition zones, as shown in Figure 8 and Table 7.

Figure 9. Antibacterial Activity of Fucoxanthin Nanogel 0.1%, 0.2%, 0.3%, and positif control

Table 7. Antibacterial Activity of Fucoxanthin Nanogel

Concentration	inhibition zone (mm)
K+ (Clindamycin gel 1%)	8.82 ± 0.002
K- (Nanogel basis)	0 ± 0
Nanogel fucoxanthin 0.1%	5.42 ± 0.002
Nanogel fucoxanthin 0.2%	6.62 ± 0.001
Nanogel fucoxanthin 0.3%	8.15 ± 0.002

DISCUSSION:

The spectrum pattern of the isolated compound matched that of the standard (Figure 5,), with both exhibiting similar maximum wavelengths, 445.60 nm for the isolated fucoxanthin and 446.20 nm for the standard fucoxanthin. These findings were consistent with literature, where the maximum wavelength of fucoxanthin to be between 445 and 447 nm.²⁶

The FTIR results showed the spectrum pattern of the fucoxanthin isolate was consistent with the standard. Additionally, the functional groups identified in the fucoxanthin isolate, such as CH, C=O, conjugated C=C, CH2, C-O acetate, C-O-C, and substituted C=C.²⁷

The concentrations of fucoxanthin compounds in the nanoemulsion preparations affected the particle size, as shown by the relationship between concentrations and percent transmittance. Specifically, higher concentration of fucoxanthin correlated with lower percent transmittance values. The observed percent transmittance values were 95% for 0.1%, 93% for 0.2%, and 91% for 0.3% fucoxanthin. Following their transmittance assessment, the nanoemulsion preparations were incorporated into a gel base and analyzed for physical characteristics.

Organoleptic tests on the concentration of fucoxanthin nanogel preparations showed the following characteristics, a semi-solid shape, a distinctive odor from the base, and an orange color that intensified with higher concentration of fucoxanthin. The homogeneity tests showed that all concentration of fucoxanthin nanogel preparations were homogeneous. The homogeneity test showed that the tested nanogel preparations were homogeneous and free from coarse grains.²⁸ A preparation is considered homogeneous when it exhibits uniform color distribution and lacks visible particulate matter.^29,30

The pH test assessed the acidity of the nanogel preparations, and it was found that higher concentrations of fucoxanthin in the nanogel resulted in increased pH values. As shown in Figure 13, the pH of the preparation increased with higher fucoxanthin concentration. Since fucoxanthin has a basic pH of 7.61, increased concentration led to a more alkaline pH in the nanogel preparation.

Viscosity testing was carried out using a Brookfield viscometer to determine how the viscosity of the nanogel affected its spreadability.^31,32 The results in Table 5 showed the viscosity of the fucoxanthin nanogel preparation was within the acceptable range of 2000-4000 cps.^31,33 In this study, the goal of the spreadability test was to determine how well the nanogel preparations spread when applied to the skin.^{28, 30, 34} Greater spreadability allows for contact with a wider area of skin.³⁴ According to the results in Table 7, the fucoxanthin nanogel preparation showed adequate spreadability, falling within the acceptable range of 5-7 cm.²⁸

The adhesion test assessed the ability of the nanogel to adhere to the skin. Prolonged adhesion ensured extended contact between the nanogel and the skin, maximizing drug delivery and prolonging the therapeutic effect.^31,35,36 Also, a higher viscosity value correlates with better adhesion.³⁶ The results in Table 5 showed that the fucoxanthin nanogel preparation met the requirement with an adhesion time of not less than 4 seconds.^31,36

The resulting nanoparticles were sized between 10-1000 nm,³⁷ confirming that the three nanogels were nano-sized. The zeta potential value is generally used to characterize the surface charge properties of nanoparticles, which significantly impact their stability. Particles with zeta potential values greater than +30mV or less than -30mV were generally sidered stable during storage, because they were less likely to aggregate.³⁸ Moreover, the zeta potential value obtained from the nanoparticles preparation was close to +30 mV, which showed the nanoparticles were relatively stable.

The particle size distribution of a nanoparticle system was measured by polydispersity index. In this context, a PDI value of less than 0.3 showed a very narrow size distribution, which can be seen in the small and homogeneous particle size. Meanwhile, a polydispersity index value greater than 0.3 (PI>0.3) showed broad size distribution and greater particles aggregation, which can lead to instability in the preparation.³⁹

Statistical analysis was carried out on the antibacterial activity test results of fucoxanthin nanogel against Staphylococcus aureus. The data were shown to be normally distributed and homogeneous, making them suitable for One-Way ANOVA testing. The results of this analysis showed there were significant differences among the three concentration, with a p-value of 0.001 (p < 0.05). Subsequently, a post hoc test was carried out to further analyze the differences. The post hoc test results showed significant differences in the inhibition zones across the three fucoxanthin nanogels concentration, with a p-value of 0.001 (p < 0.05). A correlation test between the concentrations and the inhibition zone produced an r-value of 0.9977, indicating a very strong positive relationship. This shows the concentrations of fucoxanthin nanogel increased, and the inhibition zone also increased.

CONCLUSION:

In conclusion, the extracted fucoxanthin from Sargassum polycystum produced pure compounds that have similar properties to the standard. The isolate had antibacterial activity against Staphylococcus aureus at 0.1%, 0.2%, and 0.3% concentrations. In addition, the fucoxanthin nanogel at these concentrations showed good characteristics, including nanoparticle-sized particles, and the antibacterial activity of the nanogel was directly influenced by the concentrations of fucoxanthin.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like the thank Yayasan Pharmasi Semarang for financial support to this research through HIBAH Yayasan Stifar 2023 research grant scheme and World Class University Program, Indonesia Endowment Fund for Education 2024.

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Received on 18.01.2025 Revised on 08.05.2025

Accepted on 19.07.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):153-159.

DOI: 10.52711/0974-360X.2026.00024

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

Physical Characteristics Test	Fucoxanthin Nanogel 0.1%	Fucoxanthin Nanogel 0.2%	Fucoxanthin Nanogel 0.3%
Organoleptic: Form Smell Color	Half solid Typical base Light Orange	Half solid Typical base Dark Orange	Half solid Typical base Brownish orange
Homogeneous	Homogeneous	Homogeneous	Homogeneous
pH	5.05±0.02	5.32±0.0153	7.14±0,03
Viscosity (cP)	2173±2.00	2732± 1.5275	2997.67±1.5275
Spread power (cm)	5.1±0.1	5.4± 0.1	6.7± 0.2517
Sticking power (second)	5.12±0.02	5.42±0.0153	6.96±0.0153