INTRODUCTION:

Fucoxanthin, a xanthophyll carotenoid wholly found in brown seaweeds viz., Fucus vesiculous, Laminaria digitata, L. japonica, L. religiosa, Padina tetrastomatica, Petalonia binghamie, Sargassum horneri, S. weightii, Saccharina latissima, Scytosiphon lomentaria, Turbinaria ornata and Undaria pinnatifida¹. Fucoxanthin is known for its vast bioactive properties like anti-obesity, anti-inflammatory, anti-tumor and anti-cancer^2-4.

Fucoxanthin has found various food, pharmaceutical and cosmetic applications as nutraceuticals. However, its stability is poor owing to its highly unsaturated molecular structure, making it liable to degradation by heat, light, and oxidation. When exposed to heat, light, oxygen, enzymes, unsaturated lipids, and other pro-oxidant compounds during processing, storage, and ingestion, fucoxanthin was found easily degrade which can limit its efficacy in therapeutic and nutritional applications⁵. Studies have demonstrated degradation of fucoxanthin under light, air, acids and illumination^6-7. Koo, (2016) studied the effect of heat (2, 10 and 26°C) on the stability of fucoxanthin and found 30% degradation after 4 weeks of storage⁸.

Fucoxanthin being a lipophilic in nature makes it poorly aqua soluble and less bioavailable from digestive tract^9,10. In the systemic circulation and tissues, fucoxanthin undergoes metabolic/oxidative conversion, into highly active fucoxanthinol and amarouciaxanthin A^11,12. Fucoxanthin was found to degrade by 75% in the process of digestion¹³. These studies shows that fucoxanthin is rapidly degraded and cleared from the circulation and degrades and hence needs higher or repeated dosing to maintain effective levels to achieve maximum functionality⁷.

Therefore, encapsulation is a novel approach to overcome degradation by encapsulating fucoxanthin in a suitable protective layer, such as liposomes or micelles. Encapsulation is reported to improve fucoxanthin stability by protecting it from heat, light, pH, gastric acids and enzymes¹⁴. Thus, to maximise the intestinal absorption and bioavailability of fucoxanthin, it is important to protect its structure for higher functionality. To achieve this, micro and nano-encapsulation have been used to improve the stability and bioavailability of fucoxanthin^1,7. In this regard, choice of protective excipient for encapsulation is essential to influence the slow and sustainable release of fucoxanthin for higher bioavailability¹⁵. Encapsulating fucoxanthin with wall material also protect the fucoxanthin against adverse reactions. Proteins (whey proteins, gelatin), oligosaccharides (maltodextrins, cyclodextrin), and polysaccharides (gum acacia, alginates) are commonly used as encapsulation materials¹⁶. Pectin, gelatin, corn starch, maltodextrin, inulin, and gum acacia are considered effective encapsulants due to their unique biodegradable property and economically viable that facilitate their effective application to protect bio-actives for controlled release^5,17-20. Pectin and gelatin form gels that create stable, protective layer around encapsulated substances, making them ideal for safeguarding light and heat sensitive compounds¹⁷. Corn starch and maltodextrin serve as effective encapsulating agents by forming matrices that provide good protection and controlled release, with maltodextrin additionally contributing to improved texture and stability¹⁸. Inulin, a dietary fibre, forms a gel-like structure that can shield and release encapsulated ingredients slowly¹⁹. Gum acacia is valued for its emulsifying properties, which help create stable encapsulation systems and enhance the release of encapsulated compound²⁰. Together, these materials offer a range of functional benefits that make them versatile for various encapsulation applications in the food, pharmaceutical, and cosmetic industries. Although there are studies on the application of these polysaccharides for encapsulation of bio-actives, a comparative study of these protective excipients on the stability of fucoxanthin at different condition of time and pH is limited²¹. Hence, the current study is designed to screen suitable encapsulating materials to improve stability and bio-availability of fucoxanthin in in vitro in gastric condition.

MATERIALS AND METHODS:

Extraction and purification of fucoxanthin:

Fucoxanthin was extracted from Padina tetrastomatica (P.tetrastomatica) (Dictytaceae) procured from AquAgri laboratory, Manamadurai, Tamil Nadu (9°48’0.504” N, 78°28’31.6956” E). Fucoxanthin standard was purchased from Sigma-Merck. Solvents like acetone, hexane, diethyl ether were of analytical grade and dichloromethane, methanol and acetonitrile of HPLC grade were purchased from Himedia and Sigma-Merck (Bengaluru, India). Protective excipients pectin, gelatin, corn starch, maltodextrin, inulin and gum acacia of analytical grade were purchased from Himedia and Sigma-Merck (Bengaluru, India). Other chemicals used were of analytical grade.

The cleaned and dried (40℃ for 24 hours) seaweed (leaves and stem) was used to extract fucoxanthin as per the procedure explained by Ravi et al., (2015)⁷. Briefly, the dried seaweed was macerated in acetone and methanol in the ratio of 9:1 (ice cold extraction). The crude extract was concentrated using flash evaporator, and the chlorophyll was removed by phase separation using hexane and diethyl ether. The diethyl ether phase was dried and re-dissolved in hexane and subjected to open column chromatography (Chromatography column 25x450mm – Biohall Lifesciences) with silica gel (mesh size 60-120). β-carotene and chlorophyll were eluted using hexane and hexane acetone mixture (9:1) respectively. Fucoxanthin was eluted in the third fraction with hexane acetone (7:3). The dried fucoxanthin fraction was re-dissolved in mobile phase and quantified through HPLC⁷.

FT-IR analysis:

Purity of extracted fucoxanthin was further conformed against standard fucoxanthin by FT-IR analysis using FT-IR spectrometer (Bruker Optik GmBH, Germany). Briefly, a pinch of the sample was placed on the prism of the FT-IR spectrometer and the spectrum was recorded. The spectra were analysed for different functional groups in the region between 4000 and 400 cm⁻¹ wavenumbers.

Stability study:

Preparation of micelles:

Mixed micelles were prepared containing glycerol (2.5µM), oleic acid (75µM), sodium taurocholate (12µM), cholesterol (0.5µM) and fucoxanthin (200nM) in methanol. The mixture was vortexed and sonicated to obtain a clear orange coloured solution (micelles). This mixture was used as control.

Experimental design:

Stability of fucoxanthin was studied at 37˚C at different pH (2, 4, 6 and 8) and time intervals (15, 30, 60, 90, 120 and 150minutes). Fucoxanthin micellised without protective excipients was considered as control. Pectin, gelatine, maltodextrin, inulin, corn starch and gum acacia micellised with fucoxanthin with considered as test groups. The excipients were dissolved in distilled water before experimentation. All the test group samples were prepared as per the procedure mentioned above, where, 100µL of the protective excipients were added to the buffer along with micellised fucoxanthin. Different buffers were used to simulate the pH range for the study, namely HCl-KCl buffer for pH 2, citrate buffer for pH 4-6, and phosphate buffer for pH 8. 6µL (200nM) of the fucoxanthin mixed micelles prepared was taken separately in 200µL of the respective buffer. The samples were incubated in a water bath at 37˚C for 15, 30, 60, 90, 120 and 150 minutes. Fucoxanthin was extracted using a mixture of methanol-hexane (1:1) and centrifuged at 2000rpm for 3 minutes. The upper hexane layer was discarded and the extraction was repeated using diethyl ether and water (2:1). The upper diethyl ether phase collected was dried in a vacuum dryer (Speed vacuum concentrator, Savant 1SS110, Thermoscientific), re-dissolved in mobile phase containing acetonitrile, methanol and dichloromethane and analysed using HPLC explained elsewhere²².

Gastrointestinal digestion of micellar fucoxanthin (in-vitro):

In vitro simulated digestion was performed as per the method by Nidhi and Bhaskaran (2011). Fucoxanthin (200nM) was solubilised in oleic acid (50µL) with selected protective excipient dissolved in distilled water. To mimic the mouth phase of digestion, α-amylase (2000 units) was added to the micelles and incubated at 37˚C for 5minutes with shaking. This mixture was then acidified with 1M HCl (pH 2) to stimulate the acidic environment of the stomach and 2mL of porcine pepsin (40mg/mL in 0.1M HCl). The homogenate was incubated at 37˚C in a shaking water bath at 120rpm for 1h in dark. Following the stomach phase of digestion, the pH of this mixture was raised to 5.3 by addition of 1M sodium bicarbonate and a mixture of bile salts and pancreatin enzyme solution (2mg/mL pancreatin and 12mg/mL bile extract in 100mMol/L sodium bicarbonate solution) to stimulate the intestinal phase. The pH was raised to 7.5 by adding 1N NaOH and the mixture was kept at 37˚C in a shaking water bath at 120rpm for 2h in dark to complete the digestion process. At the end of the experiment, fucoxanthin was extracted and analysed by HPLC as explained elsewhere²³.

Fucoxanthin permeation by franz cell diffusion (in-vitro accessibility):

In vitro permeation of fucoxanthin was performed using franz cell diffusion apparatus. Goat’s small intestine collected from the local market was used as a permeable membrane. This membrane was set between the donor and acceptor compartment of the apparatus. The permeability and absorption of fucoxanthin through the small intestine from the donor cell to the acceptor cell was estimated after an interval of 2h²³.

In brief, 7mL of the digesta (obtained at the end simulated intestinal phase) was poured into the donor compartment and 20mL receptor fluid (70% 1X PBS and 30% ethanol of pH 7.4) was placed in the receptor compartment. The goat intestine was placed in between the donor and receptor compartments to stimulate permeation. Fucoxanthin was allowed to permeate through freshly excised goat intestine for a period of 2h at 37°C using a water circulator. 300µL of the receptor fluid was withdrawn after 2h from the receptor cell and fucoxanthin was extracted and analysed by HPLC as explained elsewhere.

Extraction of fucoxanthin from digesta:

Fucoxanthin was extracted using methanol-hexane (1:1) and centrifuging at 3000rpm for 3minutes. The upper hexane layer was discarded, and the extraction was repeated using diethyl ether and water (2:1). The extraction was repeated three to four times and the upper diethyl ether was collected and dried in a vacuum dryer. The dried sample is re-dissolved in mobile phase and analysed using HPLC⁷.

Analysis of fucoxanthin by HPLC:

The extracts were dissolved in mobile phase and were analysed using HPLC (Waters RP-HPLC system Model-1525) equipped with photodiode array detector 2998, USA and C18 column (4.6 × 150mm, 5μM). Fucoxanthin was eluted with a mobile phase containing acetonitrile: methanol: dichloromethane-1 (6:2:2) with 0.1% ammonium acetate at a flow rate of 1.0mL/min. The samples were detected at 445nm²².

Statistical analysis:

All tests were done in triplicates (n=3) and values represented as mean±SD. T-test for two groups and ANOVA for more than two groups was performed using GraphPad Prism software.

RESULTS:

Extraction and purification of fucoxanthin:

Fucoxanthin was extracted from P. tetrastomatica using ice-cold acetone method and was quantified using HPLC at 445nm. The purity of fucoxanthin was found by 97% at a retention time of 3.153min (Figure 1). The fucoxanthin content in P. tetrastomatica was 6.64mg/100g (dry weight).

Figure 1. HPLC chromatographs of extracted fucoxanthin (a) and standard fucoxanthin (b) at 445nm

FT-IR analysis of fucoxanthin:

FT-IR analysis showed various bonds and functional groups in the fucoxanthin isolated from P. Tetrastomatica and in the commercially available standard. The FT-IR pictograph (Figure 2) shows the presence of various carbon bonds and functional groups. Prominently the unique allenic bond present in fucoxanthin was observed in the standard and extracted fucoxanthin.

Figure 2. FTIR spectra of the standard fucoxanthin and fucoxanthin extracted from Padina tetrastomatica

Stability of fucoxanthin:

Fucoxanthin reacts unfavourably to heat, light and pH, and therefore determining suitable protective excipients is of necessity. Bright-field microscopy was used to detect the micellization of fucoxanthin at a 20x magnification (BX53 Fluorescent Microscope, Olympus) (Figure 3). The micelle was surrounded by the aqueous medium that contained the protective excipient^24-26. The compatibility of protective excipients with fucoxanthin was studied under in vitro gastric conditions at different pH (pH 2, 4, 6 and 8) and time intervals (15, 30, 60, 90, 120 and 150 minutes) at 37˚C (Figure 4). At pH 2 (Figure 4A), micellized free fucoxanthin (control), exhibited the lowest stability, with 97% disintegration by the end of 150 minutes. After 15 minutes, gelatin retained approximately 1.5-fold higher fucoxanthin compared to the control (0.565nM). However, as the time progressed, reduction in the stability of both control and micellized fucoxanthin with different protective excipients were noted. At the end of 150 minutes, inulin, maltodextrin, and corn starch demonstrated higher (>1-fold) retention of fucoxanthin compared to gelatin. At pH 4, gum acacia exhibited a 2-fold higher stability up to 30 minutes relative to the control (1.025nM). Corn starch, gelatin, and inulin also showed fucoxanthin retention ~ of 1.5-fold for 90 minutes. However, pectin displayed significant degradation of fucoxanthin by the end of the study. Improved fucoxanthin retention were observed at pH 6 and 8 compared to 2 and 4, likely due to their proximity to neutral to alkaline pH (Figure 4). Gum acacia, maltodextrin, inulin, and corn starch showed 2–2.5-fold increase in fucoxanthin retention compared to the control (1.105nM), while gelatin and pectin exhibited a gradual decline, with a 1-fold decrease in retention after 15 minutes. Notably, pH 8 demonstrated the highest stability, with gum acacia, maltodextrin, inulin, and corn starch showing better fucoxanthin stability up to 150 minutes. The study demonstrates, free fucoxanthin (control) exhibiting minimal stability across various pH levels and undergoes consistent degradation over time at 37 °C.

Figure 3. Bright-field microscopy images of micellised fucoxanthin with schematic representation (a) of micelles

Note: Control: - Free fucoxanthin, PcFx:- Pectin-Fucoxanthin, GlFx:- Gelatin-Fucoxanthin, CSFx:- Corn starch-Fucoxanthin, GAFx:- gum acacia-Fucoxanthin, MdFx :- Maltodextrin-Fucoxanthin, InFx:- Inulin-Fucoxanthin

Gastrointestinal digestion of micellar fucoxanthin (in-vitro):

Influence of the protective excipients on the digestion of fucoxanthin is depicted in the Figure 5. Fucoxanthin micellized with inulin retained the highest fucoxanthin (2.5-fold) at the end of the digestion, followed by gum acacia (0.5-fold) and maltodextrin (1-fold). This study demonstrates that protective excipients fucoxanthin retention during in vitro gastric digestion.

Figure 5. Retention of fucoxanthin after In vitro simulated digestion

Note: Control- Free fucoxanthin. All tests were done in triplicates and results are represented as mean ± SD. Bars with asterisks represent significant (p≤0.05) difference between different wall materials compared with control (free fucoxanthin) as calculated using one-way ANOVA and Dunnett’s multiple comparison test

Fucoxanthin permeation by franz cell diffusion (in-vitro accessibility):

Fucoxanthin permeation (absorption) from the in vitro digested sample (digesta) was carried out using franz diffusion method and the results are given in Figure 6. Freshly excised goat intestine was used for fucoxanthin absorption from the digested samples (digesta). Fucoxanthin was extracted from the digesta after a 2 h incubation. 500 µL of digesta was collected from the acceptor cell, permeated fucoxanthin was then extracted and quantified using HPLC. It is evident from the results that gum acacia influenced 3-fold permeability of fucoxanthin compared to control. Inulin showed higher permeation of 2.5-fold followed by maltodextrin compared to control.

Figure 6. Permeation of micellised fucoxanthin using goat intestine

DISCUSSION:

Ravi et al (2018) reported a fucoxanthin concentration of 18mg/100g (dry weight), whereas, another study reported the fucoxanthin content to be 17mg/100g (dry weight) from P. tetrastomatica^27,28. Studies reported varied fucoxanthin content in Sargassum weightii from 0.12 – 175mg/g across different parts of the world²⁹. Other brown seaweed containing fucoxanthin like Fucus vesiculus and Laminaria digitata ranged from 0.65 – 5.05 and 0.183 – 5.13mg/g respectively ^30,31. Differences in the concentration of fucoxanthin from the same brown algae could be due to vast environmental conditions. Oceanic temperature, sea nutrients, harvest conditions are a few detrimental factors of varied fucoxanthin conditions.

The absorption frequencies of purified fucoxanthin at 3460 cm^-1 and 3461 cm^-1 showed the presence of -OH bonds and 174 cm^-1and 1711 cm^-1 confirming the presence of ketones with -C=0 bonds. Absorption at 1316-1436 cm^-1, and 1381-1435 cm^-1 showed the presence of scissoring and bending alkane’s with-C-H bonds. The presence of esters with -C-O bonds were presented at 1033 cm^-1 and 1038 cm^-1. Presence of an allenic bond at 1951 and 1937 cm^-1 is considered as a unique functional group of fucoxanthin³². The peak at C conjugate system and C=C stretching vibration showed at 1511cm-1 and 1556 cm-1, which are characteristically found in fucoxanthin. The presence of these functional groups in the spectrum validates the HPLC results confirming fucoxanthin.

The micelles were found to be enclosed in layers, with the hydrophilic layer on the outside comprising the carboxylate group of oleic acid, the hydroxyl head of cholesterol, and the hydrophilic nucleus of sodium taurocholate. The aqueous layer contains glycerol, which aids in the micelles' polarity and viscosity even though it does not form micelles. The hydrophobic layer at the innermost was made up of sodium taurocholate's hydrophobic end, the steroid backbone of cholesterol, and the hydrocarbon tail of oleic acid. The lipid containing fucoxanthin is located within this hydrophobic layer.

Studies on stability of fucoxanthin showed greater stability under basic conditions of pH 9 and 50% retention when stored with ascorbic acid³³. Kawai-ee et al. reported better stability (1.4mg/L) of free fucoxanthin ranging between pH 6 to 10 and poor stability (0.4mg/L) from pH 2 to 4³⁴. Another study, reported fucoxanthin in an oil in water emulsion was stable (retaining 50% of initial value till 60 days) at pH 7.5, due to reduced protonation and increased reduction potential at neutral pH whereas complete degradation was reported at pH 1.2 after 22 hours⁶. The above results demonstrate the efficacy of the protective excipients to protect fucoxanthin from unfavourable extrinsic conditions.

Protective excipients that are enzymatically digestible can enhance the release and absorption of carotenoids⁷. Although there are studies on fucoxanthin’s unstable nature to exo- and endogenous conditions, only few have demonstrated the effect of digestive factors on protective excipients encapsulated with fucoxanthin for the suitable for fucoxanthin. In-vitro digestion of control along with fucoxanthin micellized with protective excipients was performed simulating the human digestion system. Inulin, a naturally occurring polysaccharide which is not digested in the stomach may aid in passing of fucoxanthin through the stomach without being affected by gastric fluids and enzymes¹⁹. A study reported that fucoxanthin was greatly protected in acidic environments and had better thermal stability (60.72, 56.03 and 54.57%) at 90 ℃ when encapsulated with biopolymers like whey protein isolate, gum arabica, and maltodextrin than in free form (2.49%). In addition to the encapsulation increased the rate of fucoxanthin release in the intestinal tract, indicating improved bioaccessibility³⁵.Sorasitthiyankarn, (2024) reported chitosan and alginate coated nanoparticles with fucoxanthin improved in vitro oral bioaccessibility and stability of fucoxanthin³⁶. The present results also establish that fucoxanthin is unstable at acidic pH highlighting the need for a stable or compatible protective excipient to aid fucoxanthin stability and bioavailability.

The study demonstrates influence of the type of protective excipients on fucoxanthin permeating property. They can be critical in protecting the compound from degradation, enhancing its solubility, and improving its release and absorption in the gastrointestinal tract³⁷. Results demonstrate that gum acacia aids in higher permeation of fucoxanthin compared to the other two wall materials which may be due to providing a strong barrier property and protect the fucoxanthin from degradation at gastrointestinal environment apart from improving the dispersion in aqueous medium. However, corn starch provided minimal (0.6238µg/mL) permeation of fucoxanthin compared to control (0.38µg/mL). Corn starch being a hydrophilic compound lacks the specific molecular interactions necessary to hold hydrophobic compounds like fucoxanthin³⁸. Free fucoxanthin maybe degraded in the intestinal digestion due to the presence of gut microbiota causing rapid degradation hindering fucoxanthin permeation where only 2% was available in the intestinal phase of digestion after 2h³⁹. This, along with intestinal pH and competing nutrient absorption could be one of the few reasons contributing to poor fucoxanthin permeation in the intestine.

CONCLUSION:

Selecting an appropriate protective excipient for is essential to protect fucoxanthin from varying temperatures, gastric fluids and enzymes to improve its bio-accessibility and therapeutic efficacy significantly. The objective of this study was to screen the protective excipients (pectin, gelatin, corn starch, gum acacia, maltodextrin and inulin) for improved stability and permeation of fucoxanthin. Inulin, gum acacia, and maltodextrin demonstrated better stability, retention, and permeability, with gum acacia exhibiting the highest permeation. These materials demonstrated effective protection against degradation and facilitated enhanced release and permeation in a simulated gastric environment, compared to the control. This study demonstrates the compatibility of these excipients as protective agents in enhancing the stability and absorption of fucoxanthin. The selected excipients, maltodextrin, gum arabica and inulin can be further tested as suitable wall materials during micro/ nanoencapsulation of fucoxanthin.

ABBREVIATIONS:

HCl-Hydrochloric acid; PBS-Phosphate buffered saline

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

The authors express their sincere gratitude to Nitte (DU) for the facility provided to carry out the research work. The authors acknowledge the financial support provided by ICMR and UGC to carry out the research work.

This study was funded by ICMR (Project grant no: F.N. 5/9/1412/2022Nut) and UGC-SJSGC fellowship (UGCES-22-OB-KAR-F-SJSGC-4076).

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Received on 12.12.2025 Revised on 16.04.2026

Accepted on 20.06.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3089-3096.

DOI: 10.52711/0974-360X.2026.00439

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