Abstract Sea cucumbers have long been valued for their nutritional and pharmacological properties, yet their processing generates substantial viscera residues that remain undervalued and underexploited. This study aimed to isolate, characterize, and evaluate the bioactivities of polysaccharides extracted from the viscera of Phyllophorella kohkutiensis, designated as PKV. Compositional characterization and FTIR analysis indicated that PKV is a crude sulfated heteropolysaccharides preparation composed mainly of galactose (Gal), galacturonic acid (GalA), glucuronic acid (GlcA), fucose (Fuc), and N-acetylgalactosamine (GalNAc). PKV demonstrated dose-dependent angiotensin I-converting enzyme (ACE) inhibitory activity, with an IC₅₀ value of 1.30 ± 0.23 mg/mL. In addition, PKV exhibited preliminary growth-inhibitory effects against Gram-positive bacteria, including S. aureus, B. subtilis, and K. rhizophila, and showed moderate antioxidant properties as indicated by DPPH and ABTS radical scavenging activities and ferric-reducing power. Thermogravimetric analysis showed a maximum decomposition temperature of 247°C, indicating relatively high thermal resistance under dynamic heating conditions. Collectively, these findings highlight P. kohkutiensis viscera, a common processing by-product, as a sustainable source of bioactive sulfated polysaccharides with potential applications in nutraceuticals and functional foods.

Keywords: Sulfated polysaccharides, ACE inhibition, Antioxidant, Antibacterial, Phyllophorella kohkutiensis, By-product valorization

Funding: This research was supported by funding from the Faculty of Science, Burapha University (Grant number: SC07/2563).

Citation: Aramsangtienchai, P., Tarakam, Y., Pombubpha, A., Sirirak, T., and Watanachote, J. 2026. Biofunctional potential of sulfated polysaccharides from the visceral by-products of Phyllophorella kohkutiensis. Natural and Life Sciences Communications. 25(4): e2026087.

Graphical Abstract:

INTRODUCTION

Sea cucumbers have long been valued in Asian cultures for both nutritional and medicinal purposes. Their processed and dried body wall is a high-value export commodity and a highly regarded seafood delicacy throughout Asia. In the South Pacific, the sea cucumber export fishery ranks second in profitability, following tuna (Muhsin et al., 2023). Dried sea cucumbers are highly nutritious, containing up to 83% protein by weight and rich in essential fatty acids, vitamins, and minerals such as calcium, magnesium, and zinc (Bordbar et al., 2011; Xia et al., 2022). Recent research has revealed diverse therapeutic properties, primarily attributed to bioactive compounds isolated from their body walls. These compounds, including sulfated polysaccharides, peptides, and triterpene glycosides, have demonstrated anticoagulant, antioxidant, antimicrobial, anti-inflammatory, anticancer, and antihypertensive activities (Hossain et al., 2020). Sulfated polysaccharides are polysaccharides containing sulfate ester groups attached to the hydroxyl groups of monosaccharide residues. The introduction of sulfate groups alters their physicochemical properties by increasing electrostatic repulsion, water solubility, and molecular flexibility, which may enhance interactions with biological targets (Huang et al., 2019). Consequently, sulfated polysaccharides generally exhibit stronger biological activities than their non-sulfated counterparts, particularly immunomodulatory, antiviral, and antioxidant activities (Xie et al., 2016).

Although the body wall of sea cucumbers has been extensively studied, the viscera, including the digestive, respiratory, and reproductive organs, remain comparatively underexplored. These internal organs can account for up to 50% of the sea cucumber’s total weight and are commonly treated as processing waste and discarded (Hossain et al., 2023). Recent studies, however, have reported that the viscera contain valuable compounds such as proteins, polysaccharides, and saponins (Hossain et al., 2023). The high levels of omega-3 PUFAs and glycine further highlight their potential for development into functional foods and dietary supplements (Liu et al., 2021). Beyond their nutritional value, viscera-derived polysaccharides have demonstrated notable bioactivities. A glycosaminoglycan isolated from the viscera of Apostichopus japonicus exhibited anti-inflammatory activity both in vitro and in vivo (Ye et al., 2024). Fucosylated glycosaminoglycans from sea cucumber viscera activated TLR4-mediated MAPK and NF-κB pathways in macrophages, suggesting immunomodulatory potential (Yang et al., 2020). Moreover, sulfated polysaccharides from sea cucumber intestines have shown inhibitory effects on α-glucosidase and α-amylase, with enhanced activity after fermentation with Enterococcus hirae GS22 (Tan et al., 2024).

The edible sea cucumber Phyllophorella kohkutiensis, from the family Phyllophoridae and order Dendrochirotida, is distributed across the Andaman Sea, the Gulf of Thailand, and the South China Sea down to Singapore (Heding and Panning, 1954; Ong et al., 2016). The fishery for this species has expanded, with both fresh and dried body walls consumed locally and exported. Recently, a sulfated polysaccharide, predominantly chondroitin sulfate, was isolated from the body wall of P. kohkutiensis and shown to possess anti-inflammatory effect by suppressing nitric oxide production in LPS-induced macrophages (Aramsangtienchai et al., 2025). Nevertheless, its viscera are typically discarded, and limited information is available regarding their extraction and bioactivities of compounds within them.

This study aimed to extract and characterize sulfated polysaccharides from the viscera of P. kohkutiensis, specifically by analyzing sulfate content and monosaccharide composition. Additionally, their thermal stability and biological activities were evaluated, focusing on angiotensin-converting enzyme (ACE) inhibition, antimicrobial and antioxidant. These findings highlight the potential of these polysaccharide extracts for future bioactive products and support the valorization of sea cucumber by-products for sustainable resource utilization.

MATERIALS AND METHODS

Materials

Cetylpyridinium chloride (CPC), DEAE–Sepharose fast flow resin, papain (from papaya latex), and 3-methyl-1-phenyl-2-pyrazolin-5-one were purchased from Sigma–Aldrich (USA). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), and monosaccharide standards were supplied by Tokyo Chemical Industry Co., Ltd. (Japan). All reagents and solvents were of analytical grade unless otherwise specified.

Viscera preparation and polysaccharide extraction

Viscera samples of P. kohkutiensis were collected from Bandon Bay, Surat Thani Province, Thailand, and kindly provided by Dr. Wannapa Kasiroek (Institute of Marine Science, Burapha University). The viscera were cleaned, thoroughly oven-dried at 65°C, and subjected to polysaccharide extraction following a modified method reported previously (Yang et al., 2020). Specifically, 40 g of dried viscera was ground into powder and incubated in a 5% (w/v) papain solution prepared in 50 mM sodium acetate buffer (pH 5.6) containing 5 mM EDTA and 5 mM cysteine at 60°C for 24 hours. After centrifugation at 2,000×g for 20 minutes, the supernatant was collected and precipitated with 5% (w/v) CPC at room temperature for 24 hours. The resulting pellet was dissolved in a 3 M NaCl:ethanol (10:1.5, v/v) solution and further precipitated with four volumes of cold 95% ethanol at 4°C for 24 hours. The precipitate was collected, washed twice with 80% ethanol, and dialyzed against distilled water three times (MW cut-off: 14 kDa, Sigma). Finally, the sample was lyophilized, and the percentage yield (w/w) was calculated.

Total carbohydrates, sulfate, uronic acid and proteins determination

Total carbohydrate content was determined using the phenol–sulfuric acid method (DuBois et al., 1956). Briefly, 0.5 mL of sample solution (1 mg/mL) was mixed with 0.5 mL of 5% (w/v) phenol, followed by the addition of 2.5 mL of concentrated sulfuric acid. After reaction, absorbance was measured at 490 nm using a UV–Vis spectrophotometer, with glucose as the calibration standard.

Sulfate content was quantified using the BaCl₂–gelatin assay as previously described (Dodgson and Price, 1962). Samples were dissolved in 1 M hydrochloric acid and hydrolyzed at 100°C for 6 hours. After cooling, 0.2 mL of each sample was mixed with 3.8 mL of 3% (w/v) TCA. Then, 1 mL of 0.5% (w/v) BaCl₂–gelatin reagent was added, mixed thoroughly, and incubated at room temperature for 20 minutes before measuring absorbance at 360 nm.

Uronic acid concentration was determined using the m-hydroxydiphenyl assay (Blumenkrantz and Asboe-Hansen, 1973). Briefly, 0.2 mL of each sample was mixed with 1.2 mL of 12.5 mM sodium tetraborate in sulfuric acid, placed on ice, briefly agitated, and heated at 100°C for 5 minutes. After cooling on ice for 5 minutes, 20 μL of 0.15% (w/v) hydroxydiphenyl reagent in 0.5% (w/v) sodium hydroxide was added. The solution was vortexed and allowed to stand for 2 minutes before measuring absorbance at 520 nm.

Total protein content was examined using the Bradford’s assay (Bradford, 1976). A 100 µL aliquot of each sample was mixed with 1,000 µL of Bradford reagent and incubated at room temperature for 5 min. Absorbance was measured at 595 nm, and protein amount was calculated from a BSA standard curve.

Monosaccharide composition

Monosaccharide composition was analyzed following a previously described method with slight modifications (J. Yang et al., 2015). A 5 mg sample was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110°C for 9 hours. Residual TFA was removed by co-evaporation with methanol. Monosaccharides were derivatized using PMP (1-phenyl-3-methyl-5-pyrazolone). Briefly, 100 μL of the sample was mixed with 100 μL of 0.1 M NaOH and 100 μL of 0.5 M PMP in methanol, incubated at 70°C for 30 minutes, and neutralized with 0.1 M HCl. Excess PMP was extracted three times with chloroform. Derivatized samples were analyzed using HPLC with a C18 column (Phenomenex Luna^® 5 µm C18(2), 250 × 4.6 mm), using 50 mM phosphate buffer (pH 6.7) and acetonitrile (80:20, v/v) as the mobile phase at 1 mL/min, with detection at 254 nm.

FT-IR spectroscopy

Polysaccharide functional groups were characterized using ATR-FTIR (PerkinElmer Frontier, USA) with 250 scans per sample over 4,000–400 cm⁻¹ at a resolution of 4 cm⁻¹.

Thermal properties determination

Thermal decomposition was evaluated by thermogravimetric analysis (TGA) using a PerkinElmer TGA4000 instrument. Samples were heated from 30°C to 450°C at 10°C/min under a constant nitrogen flow of 20 mL/min.

ACE inhibition assay

ACE inhibitory activity was determined using a commercial ACE activity assay kit (Sigma-Aldrich, USA), which quantifies the cleavage of a synthetic fluorogenic peptide substrate by ACE. The resulting fluorescence intensity is directly proportional to ACE activity. The assay was carried out following the manufacturer’s protocol with minor modifications. Briefly, 50 µL of polysaccharide solutions at different concentrations were prepared in water, diluted with assay buffer, and dispensed into a 96-well flat-bottom black microplate. Each sample was pre-incubated with 50 µL of ACE solution at 37°C for 5 min. A control reaction (without polysaccharide) was included in parallel. The reaction was initiated by adding 50 µL of the fluorogenic substrate, and fluorescence was recorded at Ex/Em = 320/405 nm every minute for 5 min using a microplate reader (Agilent Cary Eclipse, USA). All assays were performed in triplicate. ACE activity was calculated from the linear slope of fluorescence increase over time, using a standard curve to convert RFU/min into nmol/min. One unit of ACE activity was defined as the amount of enzyme that releases 1 nmol of fluorescent product per minute at 37°C. The percentage of ACE inhibition was calculated as:

%ACE Inhibition = (Ac−As)/Ac × 100

where Ac is the ACE activity of the control (without polysaccharide) and As is the ACE activity in the presence of the polysaccharide.

Antibacterial activity

The antibacterial activity of PKV was evaluated using a modified disc diffusion assay in accordance with the CLSI M02-A11 guidelines. Tested strains included E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and Kocuria rhizophila ATCC 9341. 

Samples were dissolved to yield 100 µg/disc (in 75% DMSO) and 75 µg/disc (in 50% DMSO). Sterile paper discs (6 mm) were loaded with 20 µL of each solution and placed on Mueller–Hinton agar previously inoculated with bacterial suspensions (0.5 McFarland standard). Gentamicin (10 µg/disc) and solvent-only discs served as positive and negative controls, respectively. Plates were incubated at 37°C for 18 h, and inhibition zones were measured using a vernier caliper. Assays were performed in triplicate, and data are expressed as mean ± SD.

Fractionation of PKV polysaccharides

A crude viscera polysaccharide extract (150 mg) was dissolved in distilled water (6 mL) and the resulting supernatant was subjected to DEAE-Sepharose column (2.7 × 20 cm). The column was eluted stepwise with 1.0, 1.5, and 2.0 M NaCl at a flow rate of 1 mL/min, collecting 4-mL fractions throughout the run. Polysaccharide presence was monitored using the phenol–sulfuric acid method (Aramsangtienchai et al., 2023), and conductivity confirmed NaCl concentration. Fractions corresponding to each major elution peak were combined, dialyzed three times against deionized water, and lyophilized for subsequent antioxidant analyses.

Antioxidant assays

DPPH assay

The DPPH radical scavenging assay was performed according to a previously published procedure (Schinella et al., 2010). Briefly, sample solutions (0-4 mg/mL) were mixed with 0.15 mM DPPH in a 96-well plate and incubated for 30 minutes in the dark at 25°C. Absorbance was measured at 517 nm. The percentage of DPPH inhibition was calculated using the formula:

% Inhibition = [(A-B)-(C-D)]/(A-B) × 100

where A, B, C, and D represent the absorbance of the DPPH control, methanol, sample with DPPH, and sample blank, respectively. All analyses were performed in triplicate. The results were also expressed as mg ascorbic acid equivalent antioxidant capacity (AEAC) per gram of sample, based on an ascorbic acid calibration curve.

ABTS assay

The ABTS radical scavenging capacity was evaluated as previously described with minor modifications (Cai et al., 2003). A working solution of ABTS˙⁺ was prepared by reacting 7 mM ABTS with 2.45 mM potassium persulfate and incubating the mixture overnight in the dark. This solution was then diluted with 0.1 M sodium acetate buffer (pH 4.5) to an absorbance of 0.70 ± 0.02 at 734 nm. Samples (0–4 mg/mL) were mixed with the diluted ABTS˙⁺ solution in a 96-well plate, incubated for 30 minutes at 25°C, and the absorbance was read at 734 nm. The scavenging percentage was calculated using the formula:

% Inhibition = [(A-B)-(C-D)]/(A-B) × 100

where A, B, C, and D represent the ABTS solution, buffer, sample with ABTS, and sample blank, respectively. All tests were performed in triplicate. The results were also expressed as AEAC per gram of the sample, based on an ascorbic acid calibration curve.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was performed to evaluate the electron-donating capacity of the samples, adapting a published method (Schinella et al., 2010). A fresh FRAP reagent was prepared from 10 mM TPTZ and 20 mM FeCl₃ in 0.28 M acetate buffer (pH 3.6). In a 96-well plate, 20 µL of each sample (0–4 mg/mL) was mixed with 180 µL of the FRAP reagent and incubated for 30 minutes in the dark. Absorbance was measured at 596 nm. The reducing activity was calculated as:

A₅₉₆ = (A-B)-(C-D)

where A, B, C, and D correspond to the sample with FRAP reagent, sample blank, FRAP reagent blank, and buffer blank, respectively. All measurements were performed in triplicate, and results were expressed as µmol Fe²⁺ equivalents (FE) per gram of the sample using a FeSO₄ calibration curve.

Statistical analysis

All data are expressed as the mean ± standard deviation (S.D.) based on a minimum of three independent experiments. Statistical significance was set at P < 0.05.

RESULTS

Isolation and compositional characterization of polysaccharides

Crude polysaccharides were extracted from the viscera of P. kohkutiensis using papain digestion followed by CPC precipitation, yielding a fraction designated as PKV. The extraction produced an average yield of 8.80 ± 1.84% (w/w). PKV contained 27.23 ± 0.43% sulfate, 6.14 ± 0.95% uronic acid, and 3.37 ± 0.52% total protein. Chemical composition analysis indicated that PKV is a crude sulfated polysaccharide preparation containing sulfate and uronic acid residues. A relatively low level of residual protein remained in the preparation. The monosaccharide composition of PKV was further analyzed following acid hydrolysis and PMP-derivatization (Figure 1). PKV consisted primarily of Gal (58.08%) and GalA (23.20%), with minor components of GlcA (7.80%), Fuc (5.81%), and GalNAc (5.10%).

Figure 1. HPLC chromatograms of (A) the monosaccharide composition of PKV and (B) mixed monosaccharide standards.

FT-IR spectroscopy

The FTIR spectrum of PKV exhibits characteristic features of a polysaccharide (Figure 2). A broad and intense band around 3,400 cm⁻¹ corresponds to O–H stretching vibrations, indicative of hydroxyl groups. The absorption signal at 2,920 cm⁻¹ is attributed to aliphatic C–H stretching, characteristic of sugar backbones. A distinct peak near 1,640 cm⁻¹ is assigned to C=O stretching vibrations of uronic acid moieties. Notably, the absorption at ~1,250–1,220 cm⁻¹ is consistent with asymmetric S=O stretching, thereby confirming the presence of sulfate ester groups. In addition, a band around 1,550 cm⁻¹, particularly in samples containing GalNAc, is indicative of N-acetyl functionalities (Cui et al., 2024). The prominent band at ~1,030 cm⁻¹ reflects combined C–O–C and C–O–H stretching within the polysaccharide backbone. Collectively, these spectral features, together with the compositional analysis, support that PKV is a crude preparation of sulfated polysaccharides.

Figure 2. FTIR of PKV polysaccharides.

Thermogravimetric analysis

Thermogravimetric analysis (TGA) of PKV revealed a three-stage thermal degradation profile, indicative of its compositional heterogeneity (Figure 3). The initial mass loss of ~6% up to 150°C was attributed to the evaporation of moisture and loosely bound volatiles. A major second stage commenced at ~244°C, with a Tₘₐₓ of 247°C and a mass loss (ΔY) of 29.53%, which can be ascribed to the decomposition of low-molecular-weight fractions and thermally labile components (Zhu et al., 2020). A minor third weight-loss event began near 337°C (ΔY = 28.76%, Tₘₐₓ = 353°C). This stage corresponds to pyrolytic degradation of the more resilient polysaccharide backbone and subsequent char formation (Zhuang et al., 2023).

Figure 3. TGA analysis curve of PKV polysaccharides.

Anti-ACE activity of PKV

ACE is a key enzyme of the renin–angiotensin system (RAS), which regulates blood pressure. In this system, renin first converts angiotensinogen to angiotensin I, which is then cleaved by ACE to produce the active peptide angiotensin II. This molecule raises blood pressure by constricting blood vessels and stimulating aldosterone release, which increases sodium and water retention in the kidneys (Riordan, 2003). Because of its pivotal role in RAS regulation, ACE is a major therapeutic target for hypertension, heart failure, and related conditions (Sjakoer et al., 2021).

To assess the inhibitory potential of PKV, ACE activity was measured using a commercial fluorogenic assay in the presence and absence of the polysaccharide. As shown in Figure 4, PKV suppressed ACE activity in a concentration-dependent manner. Modest inhibition was detected at low concentrations, whereas inhibition increased progressively with dose, reaching a maximum of 78.07% at 2 mg/mL. The IC₅₀ value was calculated as 1.30 ± 0.23 mg/mL. These findings highlight the potential of PKV as a natural ACE modulator.

Figure 4. ACE inhibitory activity of PKV at different concentrations. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences among concentrations according to Tukey's multiple comparison test (P < 0.05).

Anti-bacterial activity of PKV

PKV exhibited selective growth-inhibitory effects against Gram-positive bacteria in the disc diffusion assay. As shown in Table 1, clear zones of inhibition were observed for S. aureus, B. subtilis, and K. rhizophila. The activity was concentration-dependent, with the strongest effect observed against K. rhizophila at 100 µg/disc. Although the inhibition zones were modest compared to gentamicin, the activity underscores the potential of sea cucumber viscera—often discarded as waste—as a promising source of functional polysaccharides. In contrast, no inhibition was detected against the Gram-negative strains E. coli and P. aeruginosa.

Table 1.  Antibacterial activity of PKV determined by disc diffusion assay.

Note: – indicates no detectable inhibition.

Fractionation of PKV

The crude polysaccharide PKV was further fractionated using a DEAE–Sepharose column. Each fraction was analyzed for polysaccharide content using the phenol–sulfuric acid assay. As shown in Figure 5, three major peaks were separated: one unbound peak (PKV-P0) and two bound peaks (PKV-P1 and PKV-P2) eluted with 1.0 and 1.25 M NaCl, respectively. PKV-P1 and PKV-P2 indicated the presence of negatively charged polysaccharides. All the fractionated products were subsequently evaluated for their antioxidant activities.

Figure 5. Fractionation profile of the sulfated polysaccharides PKV using a DEAE–sepharose anion-exchange column with stepwise NaCl elution.

Antioxidant activities of PKV

The antioxidant potential of the crude polysaccharide preparation (PKV) isolated from sea cucumber viscera and its DEAE fractions (PKV-P0, PKV-P1, and PKV-P2) was evaluated using the DPPH, ABTS, and FRAP assays (Figure 6, S1 and Table 2). As shown in Figure 6(A) and Table 2, all DEAE fractions exhibited dose-dependent scavenging of DPPH radicals, increasing steadily with concentration (0–4 mg/mL). The DPPH scavenging capacities ranged from 2.23 ± 0.34 to 2.75 ± 0.71 mg AEAC/g extract, with no significant differences observed among PKV, PKV-P0, PKV-P1, and PKV-P2 (P > 0.05). 

In contrast, ABTS scavenging (Figure 6(B)) showed a distinct pattern. Specifically, PKV and PKV-P0 exhibited the highest activities (2.89 ± 0.23 mg AEAC/g and 3.28 ± 0.33 mg AEAC/g, respectively), whereas PKV-P1 and PKV-P2 were significantly lower.

The FRAP results are shown in Table 2. The crude PKV exhibited the strongest reducing activity (4.07 ± 0.40 µmol FE/g extract), which was significantly higher than that of PKV-P2 and PKV-P1, whereas PKV-P0 showed no detectable activity.

Figure 6. Antioxidant activities of PKV polysaccharides.  Scavenging of DPPH radicals (A) Scavenging of ABTS radicals (B).

Table 2. Antioxidant activities of PKV polysaccharides.

DISCUSSION

Isolation and characterization of polysaccharides

Crude polysaccharides (PKV) were extracted from the viscera of P. kohkutiensis, yielding 8.8%. This yield was slightly higher than that reported for viscera-derived polysaccharides from A. japonicus (4.9%) under comparable extraction conditions (Ye et al., 2024). The PKV yield, however, was lower than that obtained from the body wall polysaccharides of P. kohkutiensis (~12.4%) (Aramsangtienchai et al., 2025). This difference is likely attributable to the distinct tissue compositions of the two organs. The body wall contains abundant extracellular matrix components, including collagenous connective tissue, glycosaminoglycans, and fucosylated chondroitin sulfate, which contribute substantially to its polysaccharide content (H. Yang et al., 2015). In contrast, the viscera consist predominantly of softer tissues with a higher proportion of intracellular components and generally contain higher levels of proteins and lipids but a lower polysaccharide content (Muhsin et al., 2023). Compositional analysis showed that PKV consisted primarily of Gal, GalA, GlcA, Fuc, and GalNAc. This monosaccharide profile was partially consistent with that of the viscera-derived polysaccharide SCVP2 from A. japonicus, which contained Fuc, Glc, Gal, GalNAc, and GlcN in a molar ratio of 1.61:1.00:4.79:1.32:3.29 (Ye et al., 2024).

Thermogravimetric analysis

The TGA analysis showed a maximum decomposition temperature (T_max) of 247°C, indicating relatively high thermal resistance of PKV under the dynamic heating conditions used for thermogravimetric analysis. This temperature is higher than those commonly encountered during food processing, including pasteurization, sterilization, blanching, and frying (Al Faruq et al., 2022; Maurya et al., 2025). Since T_max represents the temperature of the maximum decomposition rate rather than the onset of thermal degradation, it does not directly predict the isothermal stability of PKV during food processing (Vyazovkin et al., 2011). Therefore, the present results characterize the thermal behavior of PKV under dynamic heating conditions but do not directly demonstrate its stability during isothermal processing.

Anti-ACE activity of PKV

ACE plays a central role in blood pressure regulation through the renin–angiotensin system, making it a key therapeutic target for hypertension. PKV inhibited ACE activity in a concentration-dependent manner, achieving 78.07% inhibition at 2 mg/mL with an IC₅₀ of 1.30 ± 0.23 mg/mL. These results indicate that PKV exhibits moderate in vitro ACE-inhibitory activity. ACE-inhibitory activity has also been reported for polysaccharides from both marine and terrestrial sources. For instance, sulfated polysaccharides isolated from squid (Loligo vulgaris) skin exhibited 86.3% ACE inhibition at 1 mg/mL (Abdelmalek et al., 2015), while sulfated glycosaminoglycans from Atlantic bluefin tuna (Thunnus thynnus) skin achieved 70.81% inhibition at 0.8 mg/mL with an IC₅₀ of 0.66 mg/mL (Krichen et al., 2018). Furthermore, sulfated polysaccharides from smooth-hound (Mustelus mustelus) fishes displayed IC₅₀ values of 1.04 and 0.75 mg/mL depending on the precipitation method used (Abdelhedi et al., 2016). In terrestrial sources, polysaccharides from Gastrodia elata Blume, a traditional Chinese medicinal herb, reached 74.40% inhibition at 1 mg/mL (IC₅₀ 0.66 mg/mL) (Zhu et al., 2019).

These differences in IC₅₀ values between PKV and previously reported polysaccharides may be attributed to differences in sample composition, structural characteristics as well as variations in assay conditions. Because PKV was evaluated as a crude sulfated polysaccharide preparation, the specific component responsible for the observed ACE inhibition remain unclear. Further structural characterization is therefore needed to identify the active constituents and clarify their contribution to ACE inhibition. Nevertheless, the concentration-dependent inhibition observed in this study suggests that P. kohkutiensis viscera represent a promising source of bioactive polysaccharide-rich preparations for further investigation as natural ACE inhibitors.

Anti-bacterial activity of PKV

PKV produced detectable inhibition zones against the Gram-positive bacteria S. aureus, B. subtilis, and K. rhizophila, with the largest zone observed against K. rhizophila at 100 μg/disc. In contrast, no inhibition zone was detected against the Gram-negative strains under the tested conditions. These findings suggest preliminary selective growth-inhibitory effects of PKV against the tested Gram-positive bacteria. This selectivity may be related to structural differences in the bacterial cell envelope. In particular, the outer membrane of Gram-negative bacteria restricts the penetration of high-molecular-weight and hydrophilic compounds, including polysaccharides and their complexes (Silhavy et al., 2010). In addition, the observed differences may also be influenced by compound diffusion through the agar matrix and the concentration of active constituents in the crude extract (Balouiri et al., 2016).

Several mechanisms have been proposed for the antimicrobial effects of polysaccharides, including disruption of the cell envelope, interference with adhesion or biofilm formation, and disturbance of cellular metabolic processes (Wang et al., 2021). These effects may depend on structural features such as molecular weight, charge density, monosaccharide composition, branching, and the degree and pattern of sulfation. Among these characteristics, sulfation has been recognized as an important determinant of antibacterial activity, with sulfated polysaccharides generally exhibiting stronger antibacterial effects than their non-sulfated counterparts (Gunasekaran et al., 2021). Sulfated polysaccharides such as fucoidans, sulfated galactans, and ulvans may interact with bacterial surface components through electrostatic interactions and hydrogen bonding, potentially affecting cell attachment and biofilm integrity (Abdalla et al., 2021). Because PKV was evaluated as a crude sulfated polysaccharide preparation and its antibacterial activity was assessed only by the disc diffusion assay, the specific active components and underlying mechanisms remain to be elucidated. Further evaluation using broth microdilution-based MIC and MBC assays is therefore warranted to more rigorously characterize the antibacterial potential of PKV.

Antioxidant activities of PKV

PKV exhibited approximately 24% DPPH radical scavenging activity at 4 mg/mL. Compared with polysaccharides from other natural sources, sulfated polysaccharides isolated from the edible mushroom Pleurotus eous showed more than 60% DPPH scavenging activity at the same concentration (Gunasekaran et al., 2021), whereas polysaccharides isolated from the sporocarp of Tricholoma lobayense exhibited less than 20% scavenging activity (Moorthy et al., 2024). These comparisons indicate that the DPPH scavenging activity of PKV falls within the range reported for naturally derived polysaccharides, which may reflect differences in structural characteristics and experimental conditions.

Antioxidant activities of PKV and its DEAE fractions showed assay-dependent changes rather than a uniform loss of activity after fractionation. DPPH scavenging activity did not differ significantly among PKV, PKV-P0, PKV-P1, and PKV-P2, indicating that the hydrogen-donating capacity measured by this assay was retained across the charge-separated fractions.

In the ABTS assay, PKV and PKV-P0 exhibited comparable scavenging capacities, whereas PKV-P1 and PKV-P2 showed significantly lower activities. In contrast, crude PKV exhibited the highest FRAP value, although PKV-P2 retained appreciable ferric-reducing activity. The different trends observed in the ABTS and FRAP assays indicate that the components contributing to radical scavenging and ferric reduction were not distributed identically among the DEAE fractions. DEAE–Sepharose chromatography separated the heterogeneous PKV preparation according to charge, and the resulting fractions may differ in degree and pattern of sulfation and monosaccharide composition (Wang et al., 2016). Previous studies have shown that higher proportions of Gal, Ara, and uronic acids are associated with stronger DPPH and ABTS radical-scavenging activities, suggesting that these monosaccharides contribute to the antioxidant capacity of polysaccharides (Zhang et al., 2026). Accordingly, the broader antioxidant profile observed for crude PKV may reflect additive or cooperative effects among multiple polysaccharide components that were separated during DEAE fractionation. Characterization of the carbohydrate composition and sulfate content of the individual fractions would provide further insight into the structural basis of these differences.

When comparing the antioxidant activities of PKV extracted from the viscera with those of polysaccharides previously isolated from the body wall P. kohkutiensis (Aramsangtienchai et al., 2025), similar radical scavenging activities were observed. However, the ferric-reducing power of all PKV samples was considerably lower than that reported for the body wall polysaccharides. This difference may be related, in part, to the intrinsically lower water solubility of PKV, which has been reported to influence the apparent ferric-reducing capacity of polysaccharides (Christodoulou et al., 2022). In the present study, all PKV samples were fully dissolved at the concentrations tested; therefore, the observed differences are more likely to reflect intrinsic physicochemical properties than incomplete dissolution. Nevertheless, other structural factors may also contribute to the lower ferric-reducing activity, including monosaccharide composition, degree of sulfation, molecular weight, chain conformation, and associated biomolecules. Previous studies have shown that lower molecular weight can enhance the electron-donating ability and ferric-reducing capacity of polysaccharides (Wang et al., 2016). Additional studies focusing on structural characterization will help identify the features primarily responsible for the lower FRAP activity of the viscera-derived polysaccharides.

CONCLUSION

The crude sulfated polysaccharides (PKV) were isolated from the viscera of P. kohkutiensis, an underutilized sea cucumber processing by-product. Compositional characterization showed that PKV contained Gal, GalA, GlcA, Fuc, and GalNAc as the major monosaccharides, together with sulfate and uronic acid residues. PKV exhibited antioxidant activity, preliminary growth-inhibitory effects against selected Gram-positive bacteria, and moderate ACE-inhibitory activity. Although PKV contained a relatively low level of residual protein (3.37 ± 0.52%), a partial contribution of proteinaceous components to the observed bioactivities cannot be completely excluded. Further studies involving detailed structural elucidation of the fractionated polysaccharides are warranted to better define the structure–activity relationships and therapeutic potential of PKV. Overall, these findings highlight P. kohkutiensis viscera as a promising and underutilized source of bioactive sulfated polysaccharide-rich preparations and support the valorization of sea cucumber processing by-products into value-added functional ingredients.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Sumaitt Putchakarn for assistance in species identification. This work was financially supported by the Faculty of Science, Burapha University and was partially supported by the Science Innovation Facility, Faculty of Science, Burapha University (SIF-IN-60300003); the Center of Excellence for Innovation in Chemistry (PERCH-CIC), the Commission on Higher Education, Ministry of Education, Thailand; and the Scientific Instruments Center, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang.

AUTHOR CONTRIBUTIONS

Pornpun Aramsangtienchai: Conceptualization (Lead), Methodology (Lead), Investigation (Equal), Formal Analysis (Lead), Writing – Original Draft (Lead), Review & Editing (Lead), Project Administration (Lead), Funding Acquisition (Lead), Supervision (Lead); Yanin Tarakam: Investigation (Equal); Akkhayanee Pombubpha: Investigation (Equal); Thanchanok Sirirak: Investigation (Supporting), Formal Analysis (Supporting); Janjarus Watanachote: Sources Acquisition (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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Supplementary

Figure S1 Antioxidant assays. DPPH inhibition of ascorbic acid (A), ABTS inhibition of ascorbic acid (B), and the absorbance of FeSO₄ standards at 596 nm in the FRAP assay (C).