Skip to Content
MDPIMDPI

Advanced

PolymersPolymers
PDF
  • Article
  • Open Access

11 February 2026

16 Pages

Ultrasound-Assisted Extraction of Adenophora triphylla Polysaccharides: Optimization and Characterization of Physicochemical and Functional Properties

,
,
,
,
,
and
1
School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, Republic of Korea
2
Research Institute of Tailored Food Technology, Kyungpook National University, Daegu 41566, Republic of Korea
3
Department of Biomedical Technology, School of Convergence, Kyungpook National University, Daegu 41566, Republic of Korea
4
Division of High-Tech Agricultural Industry, Kyongbuk Science University, Chilgok 39913, Republic of Korea
Polymers2026, 18(4), 457;https://doi.org/10.3390/polym18040457
This article belongs to the Special Issue Polysaccharides: Synthesis, Properties and Applications—2nd Edition
Version Notes
Order Reprints

Abstract

In this study, ultrasound-assisted extraction (UAE) of Adenophora triphylla root polysaccharides (ATRPs) was optimized using response surface methodology (RSM), and the physicochemical and functional properties of the resulting polysaccharides were investigated. A Box–Behnken Design (BBD) was applied to optimize the UAE conditions for ATRPs. The optimal UAE conditions for ATRPs with the maximum extraction yield were an extraction temperature of 34 °C, an extraction time of 41 min, and a solvent-to-solid ratio of 34 (mL/g). Under these conditions, the maximum extraction yield of UAE-ATRPs (12.46%) was significantly higher than that obtained by water extraction without sonication (WE-ATRPs, 9.76%). The results of monosaccharide composition showed that WE-ATRPs and UAE-ATRPs were heteropolysaccharides, mainly composed of glucose. In addition, FT-IR and 1H-NMR analyses indicated that both ATRPs had α-pyranose-type glycosidic structures. The optimal UAE process reduced the glucose content from 57.70% to 53.87% relative to WE-ATRPs. Moreover, UAE-ATRPs exhibited lower solution viscosity and improved the emulsifying properties relative to WE-ATRPs. Both ATRPs also exhibited anti-inflammatory activity by inhibiting nitric oxide synthesis. In summary, our findings suggest that UAE is an effective approach for improving the extraction yield and functional properties of ATRPs, highlighting their potential applications in the food industry.

1. Introduction

Polysaccharides have attracted considerable attention due to their diverse functional properties in food and biopolymer applications, including thickeners, food stabilizers, and emulsifiers, and their biological activities [1]. Among them, plant polysaccharides can acquire improved functionalities through processing steps such as extraction and purification, enzymatic treatment, and chemical modification, making them highly useful for industrial applications [2]. In this way, through appropriate processing methods, plant polysaccharides can be engineered into customized materials that extend beyond their inherent functions and are suitable for various industrial applications.
Adenophora triphylla is listed as Sasam in the Korean Pharmacopoeia for Herbal (Crude) Medicines (KHP). Its dried roots are referred to as Adenophora triphylla var. japonica Hara, which will hereafter be denoted as A. triphylla [3]. The origins have been recognized for their health benefits and used to alleviate symptoms such as febrile respiratory conditions, dry cough, dyspepsia, and heartburn [4]. Many significant chemical compounds are found in its roots, primarily including polysaccharides, triterpenoids, alkaloids, and essential oils [5]. Numerous studies have been conducted on the phytochemical properties of triterpenoids and phenolic glycosides in A. triphylla [6]. Notably, among these components, A. triphylla root polysaccharides (ATRPs) have gained attention for use in food fortification and pharmaceuticals due to their significant biological activities, including anti-aging, anti-obesity, anti-radiation, and hypocholesterolemic effects [5]. ATRPs are known for heteropolysaccharides, consisting of glucose, arabinose, rhamnose, mannose, and galacturonic acid [7]. Furthermore, a recent study by We et al. [5] reported that water-soluble ATRPs exhibit antidiabetic effects.
Conventional organic solvents, such as methanol, ethanol, ethyl acetate, butanol, and acetone, are commonly used to extract polysaccharides from plant materials; however, these solvent-based methods generate substantial chemical waste and raise environmental concerns [8]. To overcome these limitations, water extraction (WE) has been used to extract polysaccharides due to its simplicity and safety [9,10]. Currently, the extraction methods for polysaccharides mainly include traditional hot water extraction, ultrasound-assisted extraction, acid and alkali extraction, microwave-assisted extraction, enzyme-assisted extraction, supercritical fluid extraction, and other emerging techniques [11]. Among these methods, ultrasound-assisted extraction (UAE) offers several advantages, including a shorter processing time, lower costs, rapid extraction, low solvent consumption, high extraction efficiency, simple operation, and minimal damage to natural products [12]. In addition, UAE can enhance the functional properties of polysaccharides, such as emulsifying capacity and rheological behavior, making it a promising technique for polysaccharide extraction [13]. Despite the growing interest in ATRPs, studies on the optimization of UAE for ARTP remain limited. Moreover, comprehensive evaluations of the physicochemical and functional properties of the ARTP obtained via the UAE method have not been fully investigated.
Therefore, the objective of this study was to optimize UAE of ATRPs to maximize yield and to investigate how the optimized UAE process affects ATRP functionality. Several extraction conditions (temperature, solvent-to-solid ratio, and extraction time) were optimized using response surface methodology (RSM). Furthermore, the physicochemical properties (monosaccharide compositions, molecular weight, and color), structural characteristics (FTIR and 1H-NMR), and functional properties (viscosity, emulsifying ability, and anti-inflammatory) of the obtained ARTPs under the optimal UAE conditions were comparatively investigated with those of ARTPs obtained without sonication.

2. Materials and Methods

2.1. Material

Freeze-dried A. triphylla root was obtained from a local farm (Yecheon city, Gyeongsangbuk-do Province, Republic of Korea). The sample was pulverized using a grinder (RT-04, Mill powder, Tainan, Taiwan) for 25 s and then passed through 850 µm sieves. The resulting powder was stored in airtight bags at −18 °C before use. All other chemicals employed throughout the analysis were of analytical grade and standard were purchased from Duksan Chemicals (Ansan, Republic of Korea).

2.2. Optimization of Extraction of A. triphylla Root Polysaccharides (ATRPs)

2.2.1. Ultrasound-Assisted Extraction (UAE)

UAE process conditions, such as power and amplitude reported by Olawuyi et al. [14], were selected for ATRP extraction. Briefly, a sample solution (X3, mL/g) was prepared in distilled water and transferred to an ultrasonic bath (Kyung il Ultrasonic, KHC-1SUMP, Ansan, Republic of Korea) and sonicated at 100 kHz amplitude and 600 W at a temperature (X1, °C) for varied duration (X2, min). After extraction, the extract was cooled at 25 °C and centrifuged to recover the supernatant (Laborgene 1248R, Labogene Co., Ltd., Daejeon, Republic of Korea) at 6000 rpm for 10 min at 4 °C. Subsequently, ethanol was added to the supernatant at a final concentration of 80% for precipitation overnight at 4 °C. The precipitate was washed with ethanol three times and then redissolved in distilled water. Then, the extract was frozen and lyophilized using a freeze dryer (FDS8518, Ilsin 115 BioBase Co. Ltd., Dongducheon-si, Republic of Korea). The extraction yield of the ARTPs was calculated using Equation (1).
Extraction   yield   ( % ) = W 1 W 0 × 100
where W0 is the weight of the sample powder before extraction (g), and W1 is the weight of the freeze-dried ATRPs (g).

2.2.2. Response Surface Methodology (RSM)

RSM was employed to optimize UAE conditions and maximize yield using Design-Expert software version 11 (Stat-Ease, Inc., Minneapolis, MN, USA). A Box–Behnken design (BBD) was applied to assess the influence of three independent parameters: extraction temperature (X1), extraction time (X2), and solvent-to-solid ratio (X3). The ranges of extraction temperature (30, 40, and 50 °C), extraction time (15, 30, and 45 min), and solvent-to-solid ratio (30:1, 40:1, and 50:1 mL/g) were selected according to previous studies demonstrating their substantial influence on the extraction yield [15,16]. Each factor was coded at three levels: −1, 0, and +1. The experimental BBD matrix consisted of four center points and thirteen factorial points, resulting in a total of 17 experimental runs. A second-order polynomial model was developed based on the experimental data, and its adequacy for predicting the response was assessed using analysis of variance (ANOVA), lack-of-fit, and the coefficient of determination (R2).

2.3. Water Extraction (WE)

WE of ATRPs was conducted under the optimized extraction conditions determined by RSM, except for the use of sonication. Following extraction, the extract solution was centrifuged, precipitated, and lyophilized, and the extraction yield was calculated as previously described in Section 2.2.1.

2.4. Physicochemical Characteristics of ATRPs

2.4.1. Chemical Properties

The content of total polysaccharides was analyzed using the phenol-sulfuric acid method with a glucose standard [17]. The protein content was determined via Lowry’s method using bovine serum albumin (BSA) as a standard [18]. The total phenol (TPC) content was measured as described by Deng et al. [15].

2.4.2. Molecular Weight

The molecular weight (Mw) and distribution of ATRPs were investigated using the method reported by Du et al. [19]. Briefly, a sample at 5 mg/mL was prepared in distilled water and filtered through a 0.45 μm membrane. 50 μL of sample was inserted into Gel permeation chromatography (Thermo Dionex, HPLC Ultimate3000 RI System, JASCO International Co., Ltd., Tokyo, Japan) equipped with Waters Ultrahydrogel columns (120, 500, and 1000). Pulluan was used as a quantitative standard, and the results were analyzed using Chromeleon 6.8 Extension Peak software.

2.4.3. Color Analysis

The color parameters of the ATRPs were evaluated using a Chroma Meter colorimeter (CR-300, Minolta Co., Osaka, Japan). The L*, a*, and b* values were measured according to the CIE system of color measurement [20].

2.4.4. Monosaccharide Analysis

High-performance liquid chromatography (HPLC) was employed to determine the monosaccharide composition of the ATRPs, following hydrolysis with Trifluoroacetic acid (TFA) and 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization, described by Olawuyi and Lee [21] with minor modifications. Briefly, 20 mg of each polysaccharide sample was dissolved in 2 mL of 2 M TFA in a glass vial, sealed, and hydrolyzed in an autoclave at 121 °C for 60 min. After cooling, TFA was evaporated with the aid of methanol using a rotary evaporator, and the concentrate was re-dissolved in 1 mL of HPLC-grade water. An aliquot (0.5 mL) of the hydrolysate was mixed with 0.5 mL of 0.3 M NaOH and 0.5 mL of 0.5 M PMP in methanol, and the mixture was allowed to react at 70 °C for 90 min. The reaction solution was neutralized with 0.5 mL of 0.3 M HCl, followed by extraction with chloroform three times.
The resulting aqueous phase was filtered through a 0.45 µm membrane filter and injected into an HPLC apparatus (Jasco International Co., Ltd., Tokyo, Japan) equipped with an Athena C18 reverse-phase column (250 mm × 4.6 mm, 5 μm). The gradient elution, reported by Park et al. [22] consisted of two mobile phases: A: 8% acetonitrile in 0.1 M ammonium acetate buffer (pH 5.5), and B: 30% of acetonitrile in 0.1 M ammonium acetate buffer (pH 5.5). The condition was as follows: 0 min B 45%, 0–10 min B 50%, 10–40 min B 85%, and 40–42 min B 50%. The column oven temperature was set at 25 °C, a flow rate of 0.8 mL/min was used, and the wavelength was set to 245 nm. The monosaccharide composition was determined using monosaccharide standards (Sigma Aldrich, St Louis, MO, USA), including mannose (Man), rhamnose (Rha), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara).

2.5. Structural Analysis

2.5.1. FT-IR

The FTIR spectra of ATRPs were recorded within the range of 4000–400 cm−1 with a resolution of 4cm−1 using an FT-IR spectrophotometer (Frontier, Billerica, MA, USA).

2.5.2. NMR

The nuclear magnetic resonance (NMR) of ATRP measurement was assessed using a Bruker spectrometer (500 MHZ Bruker AscendTM, Billerica, MA, USA) equipped with a 5 mm probe at 298 K. The sample was dissolved in D2O (99.9% D, Merck, Darmstadt, Germany) at a concentration of 5% (w/v). The solution was freeze-dried and then reconstituted in D2O. This step was repeated two times, and the 1H-NMR spectra were acquired at 25 °C with 64 scans.

2.6. Functional Properties of Polysaccharides

2.6.1. Apparent Viscosity

Apparent viscosity was determined using a viscometer (DV-II+PRO, Brookfield, Middleboro, MA, USA) equipped with spindle No. 42. For measurement, 5 mL of a 0.5% (w/v) ATRP suspension was loaded into the machine. The viscosity (cP) curve of the sample was obtained at a range of shear rates at a constant temperature of 25 °C [19].

2.6.2. Emulsion Properties

The emulsifying capacity (EC) and emulsion stability (ES) of ATRPs were measured as described in our previous study [14]. Shortly, 200 mg of ATRPs was dissolved in 10 mL of distilled water in a 50 mL conical tube, followed by the addition of 10 mL of soybean oil. Subsequently, the mixture was homogenized at 12,000 rpm for 1 min using a PT-1200C homogenizer (KINEMATICA AG, Malters, Switzerland) to prepare an emulsion. For EC assessment, the emulsion was centrifuged at 4000 rpm for 10 min. The ES was determined by incubating the prepared emulsion in a water bath at 80 °C for 30 min, then centrifuging it at 4000 rpm for 10 min. The EC and ES were calculated using Equations (2) and (3), respectively:
EC   ( % )   =   E v T v × 100
where Ev is the emulsion volume, and Tv is the total volume of the emulsion phase.
ES   ( % ) = F e v I e v × 100
where Fev and Iev represent the final and initial emulsion volumes, respectively.
Microscopic observation of the produced emulsion droplets without dilution was performed using a digital optical microscope (Leica MC120 HD, Wetzlar, Germany) equipped with an HD microscope camera. Images were captured at a magnification of ×200.

2.6.3. Cell Viability and Nitric Oxide (NO) Production Assay

Murine origin macrophage cell line RAW264.7 cells were purchased from the Korean cell line bank (Seoul, Republic of Korea), and cultured in Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin under a humidified atmosphere at 5% CO2 and 37 °C.
The cell viability of RAW264.7 cells was analyzed using the cell counting kit-8 (CCK-8) assay. RAW264.7 cells (1 × 105 cells/mL) were seeded in 96-well plates and treated with various concentrations (50–200 μg/mL) of ATRPs for 24 h. Subsequently, CCK-8 solution was added to each well, then the absorbance at 450 nm was measured after 2 h using a SPECTROstar Nano microplate reader (BMG LABTECH, Ortenberg, Germany).
Nitric oxide production was quantified using the Griess reagent system kit (Promega, Madison, WI, USA). RAW264.7 cells (1 × 105 cells/mL) were seeded in 96-well plates and pretreated with various concentrations (50–200 μg/mL) of ATRPs for 1 h, then cotreated with lipopolysaccharide (LPS, 1 ug/mL) for 24 h. The supernatant from each well was transferred to new 96-well plates, and sulfanilamide solution and N-(1-naphtyl) ethylenediamine solution were sequentially added over a 10 min interval. The absorbance at 570 nm was measured using a SPECTROstar Nano microplate reader.

2.7. Statistical Analysis

All data are presented as the mean ± standard deviation of three independent experiments. The normality of the data distribution for continuous variables was assessed using the Shapiro–Wilk test at a significance level of 0.05. Statistical differences between samples were analyzed using a t-test at a significance level of p < 0.05. All statistical analyses were performed using SPSS software (version 20.0; IBM Corp., Chicago, IL, USA).

3. Results and Discussion

3.1. Optimization of Ultrasound-Assisted Extraction

Response surface methodology (RSM) was employed to investigate the interactions among the independent variables and to optimize UAE of ATRPs. In this study, the experimental design comprised three independent variables: extraction temperature (X1), ranging from 30 to 50 °C; extraction time (X2), ranging from 15 to 45 min; and solvent-to-solid ratio (X3), ranging from 30 to 50 mL/g. Seventeen experimental runs were conducted using a Box–Behnken design (BBD), and the design matrix along with the corresponding experimental conditions is presented in Table 1. The predicted extraction yield of ATRPs was determined according to the obtained second-order polynomial equation.
Y E x t r a c t i o n   y i e l d = 11.6460 − 0.3625 X 1 + 0.4125 X 2 − 0.4275 X 3 − 0.4700 X 1 X 2 − 0.4700 X 1 X 3 − 0.2350 X 2 X 3 − 0.3230 X 1 2 − 0.5180 X 2 2 − 0.3930 X 3 2
Table 1. The Box–Behnken experimental design and corresponding response values.
The regression coefficients and ANOVA results for the designed UAE optimization model are presented in Table 2. The ANOVA results indicated a highly significant model (p < 0.01), demonstrating that the model effectively described the relationship between the independent variables and the response. The polynomial regression model exhibited high goodness of fit, with an R2 of 0.9589 and an adjusted R2 of 0.9060, indicating that the observed variation in the data could be explained by the model with minimal experimental error. In addition, the lack of fit was not significant (p > 0.05), indicating that the obtained model is reliable for analyzing the data.
Table 2. Regression coefficients and ANOVA results for optimization of polysaccharide extraction.
The three-dimensional response surfaces and corresponding two-dimensional contour plots illustrating the interactions among variables on the ATRP extraction yield are shown in Figure 1. Figure 1A,B reveal that the extraction yield markedly increased with increasing extraction time at a low temperature of 35 °C. However, the response decreased when the temperature exceeded 35 °C. Similarly, as shown in Figure 1C,D, the extraction yield exhibited a decreasing trend with increasing extraction temperature. In addition, the response plateaued and then decreased as the solvent-to-solid ratio increased. In contrast, as presented in Figure 1E,F, the extraction yield significantly increased with increasing extraction time at a solvent-to-solid ratio of 35 mL/g, whereas further increases in the solvent-to-solid ratio reduced the response.
Figure 1. Response surface plots and corresponding contour plots of extraction yield. (A,B) Interaction of extraction temperature and extraction time. (C,D) Interaction of extraction temperature and solvent-to-solid ratio. (E,F) Interaction of extraction time and solvent-to-solid ratio.
These results imply that desirable extraction conditions require a balance among all three factors to achieve maximum extraction efficiency. Mechanistically, UAE relies on acoustic cavitation, in which collapsing bubbles generate microjets and shear forces that disrupt cell walls and enhance mass transfer of polysaccharides into the solvent [23]. At moderate temperatures, with appropriate extraction time and solvent-to-solid ratio, cavitation effectively promotes the release and diffusion of polysaccharides, thereby increasing extraction yield. However, excessively high levels of these variables can weaken cavitation efficiency or induce the degradation of ATRPs, ultimately reducing extraction yield.
According to the RSM analysis, the optimal conditions for the maximum extraction yield of ATRPs were predicted to yield 12.16%, with an extraction temperature of 34 °C, an extraction time of 41 min, and a solvent-to-solid ratio of 34 mL/g. A validation experiment under these optimized conditions resulted in an extraction yield of 12.47%, demonstrating that the experimental data aligned well with the predicted optimum, with a relative error of 2.49%. The close agreement between the predicted and experimental values confirmed the validity and reliability of the RSM model for optimizing UAE of ATRPs.
After the optimization process, the ATRPs obtained under optimal UAE conditions were designated as UAE-ATRPs. Meanwhile, the ATRPs obtained through water extraction under the same optimal UAE conditions but without ultrasonic treatment were designated as WE-ATRPs.

3.2. Chemical, Molecular, and Color Characterizations

The yield, composition, and color of WE-ATRPs and UAE-ATRPs are presented in Table 3. In terms of extraction yield, the UAE-ATRPs (12.47%) exhibited a significantly higher yield than WE-ATRPs (9.76%) (p < 0.05). The enhanced extraction yield under optimized UAE conditions could be attributed to acoustic cavitation during sonication, which disrupts cell walls and facilitates the release and diffusion of polysaccharides into the solvent [14]. In addition, the total polysaccharide content of UAE-ATRPs (78.57%) was higher than that of WE-ATRPs (69.79%). Both extraction methods yielded ATRPs with relatively low protein contents (4.77–5.63%), which may be due to the presence of proteinaceous components commonly co-extracted with plant polysaccharides [23]. Furthermore, total phenol was not detected in both samples, which may be due to the applied ethanol precipitation and washing steps [21].
Table 3. Extraction yield, chemical composition, molecular weight, and color of ATRPs.
No apparent shift in retention time between WE-ATRPs and UAE-ATRPs was observed (Figure S1). Accordingly, the molecular weight (Mw) and polydispersity (Mw/Mn) values of both ATRPs were similar, with Mw ranging between 7.37 and 7.40 × 103 g/mol and Mw/Mn valued at 1.02. The non-significant differences between these indicators indicate that the applied optimal UAE conditions did not induce notable depolymerization or chain scission of ATRPs. These trends are consistent with previous studies that employed the UAE method to extract polysaccharides from plant material [14,19].
Both ATRPs exhibited relatively higher L* values of 90.31–90.55, indicating bright, nearly white powders, but lower values of a* (0.12–0.13) and b* (−1.16–2.67). These characteristics imply that UAE does not induce noticeable pigment formation or thermal discoloration, which is advantageous for food-grade applications.
Overall, these results indicate that the optimized UAE conditions improved extraction yield without a significant reduction in molecular weight or undesirable coloration, suggesting the feasibility of UAE as an efficient and mild extraction technology for polysaccharides.

3.3. Monosaccharide Composition

The obtained component monosaccharides of WE-ATRPs and UAE-ATRPs are shown in Table 4 and Figure S2. The relative molar percentage of Man, Rha, GalA, Glu, Gal, and Ara in WE-ATRPs were 6.98%, 7.37%, 0.47%, 57.66% 13.44%, and 14.08%, respectively. The relative percentages of Man, Rha, GalA, Glu, Gal, and Ara in UAE-ATRPs were 6.87%, 7.87%, 1.17%, 53.87%, 13.10%, and 17.09%, respectively. These results indicate that ATRPs are heteropolysaccharides composed predominantly of glucose. Notably, the relative percentage of glucose was lower in UAE-ATRPs than in WE-ATRPs. This change may be associated with ultrasound-induced compositional modification rather than substantial depolymerization, as also reported in previous studies [14]. Thus, although the optimized UAE condition did not alter the monosaccharide types, it influenced the relative proportions of individual monosaccharides [24].
Table 4. Monosaccharide composition and molar ratios of ATRPs.

3.4. Structural Properties

3.4.1. Fourier Transform Infrared Spectroscopy (FT-IR)

FT-IR analysis was conducted to investigate the structural differences in ATRPs. The FT-IR spectra of WE-ATRPs and UAE-ATRPs within the range of 400 to 4000 cm−1 are shown in Figure 2. FT-IR spectra showed that both samples had similar structural configurations and peak patterns, indicating that the extraction method did not significantly alter the main structure of ATRPs. The observed broad peak at 3321–3323 cm−1 was assigned to O-H stretching vibrations, while weaker absorptions at 2925–2927 and 1330–1331 cm−1 corresponded to asymmetric and symmetric bending vibrations of C-H bonds [25,26]. The characteristic peak of methyl-ester (COO-R) near 1700 cm−1 was not detected in both spectra, indicating that ATRPs are neutral polysaccharides [24]. The peak at 1634–1644 cm−1 was associated with the C=O stretching, whereas the peak at 1413–1415 cm−1 reflects –COOH [21,27]. The strong band around 1000–1200 cm−1 was attributed to the C-O bond and the stretching vibration of C-O-C of glycosidic bonds, suggesting the potential presence of pyranose [28]. In addition, the absorption peak at 874 cm−1 may arise from an α-type linkage [29].
Figure 2. FT-IR spectra of WE-ATRPs and UAE-ATRPs. WE-ATRPs = ATRPs extracted by water; UAE-ATRPs = ATRPs extracted by ultrasound-assisted extraction.

3.4.2. 1H-NMR

The 1H-NMR spectra of WE-ATRPs and UAE-ATRPs are presented in Figure 3. The spectra of both samples showed structural similarities, consistent with the FT-IR results. The strong signal at 4.70 ppm in the spectrum was assigned to D2O, which served as a reference for other peaks. The signal at 5.35 ppm in the 1H-NMR spectrum was associated with the α-anomeric proton, and 3.4–4.2 ppm was attributed to ring protons [30]. These findings, together with the FTIR results, indicate that both ATRPs probably possess an α-pyranose-type glycosidic structure [31]. In addition, the low-intensity peaks at 1.0–1.12 ppm may be caused by the methyl proton group of rhamnose [21]. This phenomenon further supports the HPLC results. Overall, these structural analyses suggest that the UAE method did not markedly alter the structural features of ATRPs compared to the WE method.
Figure 3. 1H-NMR spectra of WE-ATRPs and UAE-ATRPs in D2O. WE-ATRPs = ATRPs extracted by water; UAE-ATRPs = ATRPs extracted by ultrasound-assisted extraction.

3.5. Functional Properties

3.5.1. Viscosity

The apparent viscosity of WE-ATRPs and UAE-ATRPs is presented in Figure 4A and Table S1. The results showed that UAE-ATRPs exhibited significantly lower viscosity than WE-ATRPs at shear rates ranging from 25 to 200 s−1 (p < 0.05), indicating shear-thinning behavior. In contrast, no significant differences were observed between the two samples at higher shear rates (200–750 s−1). The reduced viscosity of the UAE-ATRPs may be attributed to cavitation during UAE, which generates bubbles under strong shear forces, further disentangling intermolecular entanglements and increasing the structure’s flexibility [32]. Increased chain flexibility enhances interactions with water molecules and reduces flow resistance, thereby lowering viscosity [21]. Taken together with the GPC results, these findings suggest that UAE did not induce depolymerization but altered the structural conformation of ATRPs.
Figure 4. Functional properties of ATRPs. Viscosity curve of solutions of ATRPs (A). Emulsifying capacity (EC) and stability (ES) of ATRS solutions in oil-in-water emulsion system (B). Optical micrographs of emulsions of ATRPs at 200× magnification (C). WE-ATRPs = ATRPs extracted by water; UAE-ATRPs = ATRPs extracted by ultrasound-assisted extraction. Different letters indicate significant difference within same parameter (p < 0.05).

3.5.2. Emulsifying Properties

The emulsifying properties of WE-ATRPs and UAE-ATRPs are presented in Figure 4B. UAE-ATRPs exhibited higher emulsion capacity and stability (46.53% and 34.52%) than WE-ATRPs (37.37% and 21.03%) (p < 0.05). The superior emulsion properties of UAE-ATRPs may be associated with increased polymer chain flexibility and a looser conformation, which provides a more accessible surface for interaction with the oil phase and contributes to emulsion stabilization [33]. In addition, optical microscopy (Figure 4C) further revealed that emulsions stabilized with UAE-ATRPs contained smaller oil droplets with a more uniform size distribution. In contrast, WE-ATRPs emulsions displayed larger and non-uniform droplets, consistent with their lower ES value. Taken together, the functional properties suggest that UAE-ATRPs are more applicable in food systems due to ultrasound-induced conformational modifications.

3.5.3. Anti-Inflammatory Activities

Nitric oxide (NO), synthesized by macrophages, is reported to mediate various beneficial immunological responses in the body, including antiviral activity, the regulation of vascular tone, and wound healing [34]. However, the overproduction of NO can trigger inflammatory symptoms, such as pain, swelling, redness, and tissue damage, which may lead to chronic inflammatory diseases like rheumatoid arthritis [35]. Consequently, numerous studies have highlighted agents that can downregulate NO production as potential anti-inflammatory therapeutics [36,37].
In this study, LPS, the outer membrane constituting Gram-negative bacteria, is used to stimulate macrophage activation, thereby inducing overexpression of macrophage-derived NO [38]. Figure 5A shows the effects of WE-ATRPs and UAE-ATRPs on the LPS-induced NO production in macrophages. LPS treatment significantly increased the NO production in RAW264.7 cells (10.92 ± 0.52 μM), suggesting the induction of an inflammatory state. Notably, WE-ATRPs and UAE-ATRPs significantly ameliorated LPS-induced NO upregulation in a dose-dependent manner (9.24–1.48 μM and 8.99–1.21 μM, respectively). In addition, as shown in Figure 5B, the cell viability of both ATRPs showed no cytotoxicity (102.61–95.20% and 110.26–102.85%) compared to the non-treatment group (100 ± 3.03%). These findings confirm the NO inhibitory effects of ATRPs, suggesting their potential as an anti-inflammatory agent.
Figure 5. Anti-inflammatory activity of WE-ATRPs and UAE-ATRPs in LPS-induced activated RAW264.7 cells. RAW264.7 cells were pretreated with ATPRs for 1 h, then cotreated with or without LPS (1 μg/mL) for 24 h. Nitric oxide synthesis was measured by the Griess reagent (A). Cell viability was measured using the CCK-8 assay (B). WE-ATRPs = ATRPs extracted by water; UAE-ATRPs = ATRPs extracted by ultrasound-assisted extraction. The data are presented as mean ± SD. If the measurement value was too low or not observed, it is marked as N.D. (Not detected). # p < 0.01 comparing LPS with the non-treatment group (NC). ** p < 0.01 comparing the experimental group with the LPS-only treatment group (LPS). ‘ns’ refers to ‘not significant’ comparing non-treatment with the experimental group.
Although the optimized UAE method influenced the relative ratio of monosaccharide composition and structural conformation, no significant differences in anti-inflammatory activity were observed between WE-ATRPs and UAE-ATRPs. Hou et al. [38] suggested that the biological activities of polysaccharides are dependent on their glycosidic bond and monosaccharide composition. While the overall monosaccharide compositions of ATPRs obtained from the different methods showed differences, both samples were mainly composed of glucose and exhibited α-type pyranose glycosidic units. LPS is known to interact with toll-like receptor 4 (TLR4) on the surface of macrophages, which could trigger macrophage activation [39]. Liu et al. [40] reported that α-glucans exhibited anti-inflammatory activity by competitively interacting with TLR4 and inhibiting LPS binding at the cell membrane. Although ATRPs were not identified as α-glucans in the present study, both WE-ATRPs and UAE-ATRPs contained abundant α-pyranose glucose units, which may provide structural features relevant to such interactions. Therefore, it is plausible that ATRPs may also interfere with LPS–TLR4 interactions, thereby ameliorating macrophage activation.

4. Conclusions

In this study, the extraction of A. triphylla polysaccharides (ATRPs) was optimized using ultrasound-assisted extraction (UAE), and their physicochemical, structural, and functional properties were compared with those of ATRPs obtained without sonication. The RSM approach optimized the UAE conditions. The maximum extraction yield was determined under optimal conditions: an extraction temperature of 34 °C, an extraction time of 41 min, and a solvent-to-solid ratio of 34 (mL/g). Under these optimal conditions, the UAE-ATRPs showed a higher extraction yield than WE-ATRPs. Non-significant differences in the results of the molecular weight or structural features were observed between the two samples. The optimized UAE conditions altered the relative percentage of monosaccharide composition. In addition, UAE-ATRPs exhibited a more flexible polymer structure, resulting in lower viscosity and improved emulsifying properties compared to WE-ATRPs. Both ATRPs showed anti-inflammatory activity by inhibiting nitric oxide synthesis in LPS-induced RAW264.7 cells. These findings highlight that the UAE is an effective method for increasing yields and improving functional attributes, thereby making ATRPs value-added functional ingredients in food systems. Future studies should focus on in-depth structural characterization of ATRPs obtained via the UAE method to elucidate structure–activity relationships.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/polym18040457/s1, Table S1: Apparent viscosity of ATRPs solution at different shear rates; Figure S1: Gel permeation chromatograph (GPC) of WE-ATRPs and UAE-ATRPs; Figure S2: HPLC chromatogram of WE-ATRPs (top), UAE-ATRPs, and standard (bottom).

Author Contributions

Conceptualization, H.-I.L., H.-S.C., J.-H.P., J.-O.N., H.-M.J., M.-H.L. and W.-Y.L.; methodology and formal analysis, H.-I.L., H.-S.C., J.-H.P., J.-O.N., H.-M.J., M.-H.L. and W.-Y.L.; investigation, H.-I.L., H.-S.C. and J.-H.P.; data curation, H.-I.L., H.-S.C. and J.-H.P.; writing—original draft preparation, H.-I.L., H.-S.C. and J.-H.P.; writing—review and editing, H.-I.L., H.-S.C. and W.-Y.L.; visualization, H.-I.L. and H.-S.C.; resources and supervision, W.-Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Regional Innovation System & Education (RISE) program through the Gyeongbuk RISE CENTER, funded by the Ministry of Education (MOE) and the Gyeongsangbuk-do, Republic of Korea (2025-RISE-15-202).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ATRPsAdenophora triphylla root polysaccharides
UAEUltrasound-assisted extraction
WEWater extraction
RSMResponse surface methodology
BBDBox–Behnken design
MwHigh-performance liquid chromatography
ManMannose
RhaRhamnose
GalAGalacturonic acid
GluGlucose
GalGalactose
AraArabinose
ECEmulsifying capacity
ESEmulsifying stability
LPSLiposaccharide
NONitric oxide
TLR4Toll-like receptor 4

References

  1. Anal, A.K.; Boonlao, N.; Ruktanonchai, U.R. Emulsion systems stabilized with biopolymers to enhance oral bioaccessibility and bioavailability of lipophilic bioactive compounds. Curr. Opin. Food Sci. 2023, 50, 101001. [Google Scholar] [CrossRef] [Scilit]
  2. Niyigaba, T.; Liu, D.; Habimana, J.D.D. The extraction, functionalities and applications of plant polysaccharides in fermented foods: A review. Foods 2021, 10, 3004. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  3. Rho TaeWoong, R.T.; KeeDong, Y.K.Y. Phytochemical study of Adenophora stricta roots. Korean J. Pharmacogn. 2019, 50, 73–80. [Google Scholar]
  4. Park, W.S.; Kim, H.-J.; Choe, S.J.; Khalil, A.A.K.; Akter, K.-M.; Shin, M.C.; Chung, H.-J.; Park, J.H.; Ahn, M.-J. Anatomical characterization and LC-MS profiling of Adenophora roots from Korea. Rev. Bras. Farmacogn. 2019, 29, 695–701. [Google Scholar] [CrossRef] [Scilit]
  5. Wu, J.; Zhou, X.; Sun, H.; Yu, D. Optimization of Extraction Conditions for Water-Soluble Polysaccharides from the Roots of Adenophora tetraphylla (Thunb.) Fisch. and Its Effects on Glucose Consumption on HepG2 Cells. Molecules 2024, 29, 3049. [Google Scholar] [CrossRef] [Scilit]
  6. Han, Z.-Y.; Wu, J.-T.; Lin, Y.-X.; Bi, Y.; Naseem, A.; Hao, Z.-C.; Pan, J.; Guan, W.; Kuang, H.-X.; Chen, Q.-S.; et al. Seven new triterpenoids from the roots of Adenophora tetraphylla (Thub.) Fisch. Fitoterapia 2024, 175, 105902. [Google Scholar] [CrossRef] [Scilit]
  7. Nai, J.; Zhang, C.; Shao, H.; Li, B.; Li, H.; Gao, L.; Dai, M.; Zhu, L.; Sheng, H. Extraction, structure, pharmacological activities and drug carrier applications of Angelica sinensis polysaccharide. Int. J. Biol. Macromol. 2021, 183, 2337–2353. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  8. Olawuyi, I.F.; Heo, E.; Jeong, M.; Kim, J.H.; Park, J.-J.; Chae, J.; Gwon, S.; Lee, S.D.; Kim, H.; Ojulari, O.V.; et al. Acidic polysaccharide from the edible insect Protaetia brevitarsis seulensis activates antiviral immunity to suppress norovirus infection. Carbohydr. Polym. 2025, 347, 122587. [Google Scholar] [CrossRef] [Scilit]
  9. Wu, H.; Shu, L.; Liang, T.; Li, Y.; Liu, Y.; Zhong, X.; Xing, L.; Zeng, W.; Zhao, R.; Wang, X. Extraction optimization, physicochemical property, antioxidant activity, and α-glucosidase inhibitory effect of polysaccharides from lotus seedpods. J. Sci. Food Agric. 2022, 102, 4065–4078. [Google Scholar] [CrossRef] [Scilit]
  10. Huang, X.; Ai, C.; Yao, H.; Zhao, C.; Xiang, C.; Hong, T.; Xiao, J. Guideline for the extraction, isolation, purification, and structural characterization of polysaccharides from natural resources. eFood 2022, 3, e37. [Google Scholar] [CrossRef] [Scilit]
  11. Zhang, W.; Duan, W.; Huang, G.; Huang, H. Ultrasonic-assisted extraction, analysis and properties of mung bean peel polysaccharide. Ultrason. Sonochem. 2023, 98, 106487. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  12. Zhou, S.; Huang, G.; Ren, F. Ultrasonic-assisted extraction and the antioxidative mechanism of eggplant root polysaccharide. J. Funct. Foods 2024, 123, 106590. [Google Scholar] [CrossRef] [Scilit]
  13. Cho, H.-S.; Park, J.-H.; Olawuyi, I.F.; Nam, J.-O.; Lee, W.-Y. Optimization of ultrasound-assisted enzymatic hydrolysis Zophobas morio protein and its protective effects against H2O2-induced oxidative stress in RAW264. 7 cells. Int. J. Biol. Macromol. 2025, 298, 140111. [Google Scholar] [CrossRef] [Scilit]
  14. Olawuyi, I.F.; Kim, S.R.; Hahn, D.; Lee, W.Y. Influences of combined enzyme-ultrasonic extraction on the physicochemical characteristics and properties of okra polysaccharides. Food Hydrocoll. 2020, 100, 105396. [Google Scholar] [CrossRef] [Scilit]
  15. Deng, L.; Huang, G. Ultrasound-assisted extraction, optimization, characteristics and antioxidant activity of Piper nigrum L. polysaccharides. Ultrason. Sonochem. 2025, 116, 107309. [Google Scholar] [CrossRef] [Scilit]
  16. Yu, W.; Li, J.; Xiong, Y.; Wang, J.; Liu, J.; Baranenko, D.; Zhang, Y.; Lu, W. Optimization of ultrasound-assisted extraction of Imperata cylindrica polysaccharides and evaluation of its anti-oxidant and amelioration of uric acid stimulated cell apoptosis. Ultrason. Sonochem. 2024, 104, 106844. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  17. DuBois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350–356. [Google Scholar] [CrossRef] [Scilit]
  18. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265–275. [Google Scholar] [CrossRef] [Scilit]
  19. Du, H.; Olawuyi, I.F.; Said, N.S.; Lee, W.-Y. Comparative analysis of physicochemical and functional properties of pectin from extracted dragon fruit waste by different techniques. Polymers 2024, 16, 1097. [Google Scholar] [CrossRef] [Scilit]
  20. Khamsucharit, P.; Laohaphatanalert, K.; Gavinlertvatana, P.; Sriroth, K.; Sangseethong, K. Characterization of pectin extracted from banana peels of different varieties. Food Sci. Biotechnol. 2018, 27, 623–629. [Google Scholar] [CrossRef] [Scilit]
  21. Olawuyi, I.F.; Lee, W.Y. Structural characterization, functional properties and antioxidant activities of polysaccharide extract obtained from okra leaves (Abelmoschus esculentus). Food Chem. 2021, 354, 129437. [Google Scholar] [CrossRef] [Scilit]
  22. Park, J.-H.; Park, J.-J.; Chae, J.; Lee, S.D.; Gwon, S.; Kim, H.; Min, J.H.; Jung, Y.H.; Lee, J.H.; Lee, W.; et al. Structural characterization and immune-enhancing properties of an acidic heteroglycan from Protaetia brevitarsis seulensis larvae. Future Foods 2024, 10, 100439. [Google Scholar] [CrossRef] [Scilit]
  23. Kontogiorgos, V. Polysaccharides at fluid interfaces of food systems. Adv. Colloid Interface Sci. 2019, 270, 28–37. [Google Scholar] [CrossRef] [Scilit]
  24. Duan, Y.; Liu, Y.; Li, M.; Liu, Y.; Gu, J.; Zhou, W.; Sun, J.; Hu, Z.; Jin, M.; Li, G. Comparison on structural characterization, chain conformation, and neuroprotective activity of polysaccharides extracted from Albizia julibrissin Durazz. bark with hot water and ultrasound. Ultrason. Sonochem. 2025, 122, 107608. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  25. Hong, T.; Yin, J.-Y.; Nie, S.-P.; Xie, M.-Y. Applications of infrared spectroscopy in polysaccharide structural analysis: Progress, challenge and perspective. Food Chem. X 2021, 12, 100168. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  26. Bian, Z.; Ding, J.; Zhang, X.; Zhang, J.; Zhang, C.; Shang, G.; Zhu, L.; Zhang, Y.; Liu, Q.; Liu, Y. Multiple fingerprinting and chromatography-immunoreactivity relationship of polysaccharides from Dioscorea opposite Thunb. Food Chem. 2025, 464, 141891. [Google Scholar] [CrossRef] [Scilit]
  27. Chen, L.; Huang, G. Extraction, characterization and antioxidant activities of pumpkin polysaccharide. Int. J. Biol. Macromol. 2018, 118, 770–774. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  28. Li, H.; Gong, X.; Wang, Z.; Pan, C.; Zhao, Y.; Gao, X.; Liu, W. Multiple fingerprint profiles and chemometrics analysis of polysaccharides from Sarcandra glabra. Int. J. Biol. Macromol. 2019, 123, 957–967. [Google Scholar] [CrossRef] [Scilit]
  29. Zhou, L.; Duan, X.; Li, K.; Hill, D.R.A.; Martin, G.J.O.; Suleria, H.A.R. Extraction and characterization of bioactive compounds from diverse marine microalgae and their potential antioxidant activities. Chem. Biodivers. 2023, 20, e202300602. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  30. Kang, J.; Sha, X.-X.; Geng, C.-J.; Li, L.-X.; Chen, J.; Ren, F.-C.; Tian, M.-L. Ultrasound-assisted extraction and characterization of Penthorum chinense polysaccharide with anti-inflammatory effects. Ultrason. Sonochem. 2023, 99, 106593. [Google Scholar] [CrossRef] [Scilit]
  31. Xu, H.; Wang, W.; Li, X.; Sun, Y.; Li, Y.; Deng, C.; Song, X.; Zhang, D. A review of extraction and purification, biological properties, structure–activity relationships and future prospects of schisandrin c: A major active constituent of Schisandra chinensis. Chem. Biodivers. 2023, 20, e202301298. [Google Scholar] [CrossRef] [Scilit]
  32. Zhou, C.; He, S.; Gao, S.; Huang, Z.; Wang, W.; Hong, P.; Jia, R.-B. Effects of ultrasound-assisted treatment on physicochemical properties and biological activities of polysaccharides from Sargassum. Foods 2024, 13, 3941. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  33. Hammi, K.M.; Hammami, M.; Rihouey, C.; Le Cerf, D.; Ksouri, R.; Majdoub, H. Ultrasonication of polysaccharides from Tunisian Zizyphus lotus fruit: Emulsifying capacities, rheological properties and antioxidant activities. Chem. Afr. 2020, 3, 667–678. [Google Scholar] [CrossRef] [Scilit]
  34. Nam, H.-S.; Jung, J.-W.; Kim, D.-W.; Ha, H.-C. Antioxidant and anti-inflammatory activities of hot water extracts of Liguularia fischeri. Food Sci. Preserv. 2017, 24, 834–841. [Google Scholar]
  35. Park, J.H.; Lee, S.D.; Park, J.-J.; Olawuji, I.F.; Chae, J.; Lee, J.H.; Hwang, J.S.; Lee, W.; Hahn, D.; Nam, J.-O.; et al. Comparative Analysis of Antioxidant, Macrophage Proliferation, and Nitric Oxide Scavenging Properties of Molecular Weight–Fractionated Polysaccharides Derived from Protaetia brevitarsis seulensis Larvae. Entomol. Res. 2025, 55, e70063. [Google Scholar] [CrossRef] [Scilit]
  36. Cho, H.-S.; Park, J.-H.; Olawuyi, I.F.; Nam, J.-O.; Lee, W.-Y. Physicochemical characteristics and anti-inflammatory properties of Zophobas morio (super mealworm) protein extracted by different methods. Food Chem. 2025, 468, 142519. [Google Scholar] [CrossRef] [Scilit]
  37. Han, B.H.; Lee, Y.J.; Yoon, J.J.; Choi, E.S.; Namgung, S.; Jin, X.J.; Jeong, D.H.; Kang, D.G.; Lee, H.S. Hwangryunhaedoktang exerts anti-inflammation on LPS-induced NO production by suppressing MAPK and NF-κB activation in RAW264. 7 macrophages. J. Integr. Med. 2017, 15, 326–336. [Google Scholar] [CrossRef] [Scilit] [PubMed]
  38. Hou, C.; Chen, L.; Yang, L.; Ji, X. An insight into anti-inflammatory effects of natural polysaccharides. Int. J. Biol. Macromol. 2020, 153, 248–255. [Google Scholar] [CrossRef] [Scilit]
  39. Park, J.H.; Lee, E.; Nam, J.O. Bioconverted extract of Sophorae fructus modulates the innate immune response in RAW264. 7 macrophages and mouse splenocytes. J. Funct. Foods 2024, 116, 106202. [Google Scholar] [CrossRef] [Scilit]
  40. Liu, F.; Zhang, X.; Li, Y.; Chen, Q.; Liu, F.; Zhu, X.; Mei, L.; Song, X.; Liu, X.; Song, Z.; et al. Anti-inflammatory effects of a Mytilus coruscus α-d-Glucan (MP-A) in activated macrophage cells via TLR4/NF-κB/MAPK pathway inhibition. Mar. Drugs 2017, 15, 294. [Google Scholar] [CrossRef] [Scilit] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
The Effect of Non-Uniform Material Distribution on the Bending Strength and Energy Absorption of TPMS Structures
Previous
Flocculation Performance and Interfacial Adsorption Mechanism of Aluminum Hydroxide–Polyacrylamide in Coal Slime Water Treatment
Next