Ultrasonic extraction, structural characterization, and antioxidant activity of oligosaccharides from red yeast rice

Abstract Red yeast rice is consumed as a medicinal food to lower blood lipids. Besides, it is used to color food, make wine, etc. In this study, water‐soluble oligosaccharides in red yeast rice were extracted by ultrasonic‐assisted extraction method. The parameters to extract oligosaccharides from red yeast rice were optimized by the Box‐Behnken design under the following optimal extraction conditions: extraction temperature, 60°C; extraction time, 97 min; and liquid/material ratio, 25 ml/g. The structure and the antioxidant activity of the new oligosaccharide were preliminarily investigated. Total carbohydrates extracted from red yeast rice with 80% ethanol–water solution (v/v) were first removed from pigments using D101 macroporous adsorption resin. The total sugar contents were then purified by DE52 resins and Sephadex G‐25 resins to obtain red yeast rice oligosaccharides, coded as RYRO1. Structural characterization experiments indicated that RYRO1 is an oligosaccharide with a weight average molecular weight of 874 Da and a theoretical degree of polymerization of 4.86. RYRO1 is composed of mannose, glucosamine, glucose, and galactose with a molar ratio of 0.248:0.019:1:0.026. The ABTS, DPPH, and hydroxyl free radical scavenging assays showed antioxidant nature of RYRO1.

are proved to possess a wide variety of bioactivities. Bai et al. ex-tracted an oligosaccharide with the average relative molecular weight of 318 Da from Codonopsis pilosula and found it could increase the secretion of cytokines (TNFα, NO, etc.) by triggering the MAPK signaling pathway (Bai et al., 2020). Lan et al. demonstrated that chi-tosan oligosaccharides are capable of maintaining intestinal integrity under oxidative stress by modulating the intestinal oxidative status and the release of inflammatory cytokines (Lan et al., 2021). Yu et al. extracted three novel oligosaccharides from Kunlun chrysanthemum flower tea and proved their inhibitory effects on α-amylase and αglucosidase (Yu et al., 2021). What's more, oligosaccharides derived from fungi have attracted the interest of researchers. For example, some oligosaccharides composed of mannose were extracted from Tremella fuciformis Berk. They were characterized as (1 → 3)-mannan oligosaccharides with a straight chain. Furthermore, an in vitro study demonstrated some of them possessed cytokine-stimulating activity (Gao et al., 2020). Ding et al. (2015) isolated one major oligosaccha-  (Loncar et al., 2021).
RYR is a fermented product; in our previous in vivo study indicated that, the 3.49-kDa nonstarch polysaccharides were isolated from RYR and that it possessed the gastrointestinal-protective effect (Luo et al., 2020). Most of the modern research into RYR focused on small molecule substances such as statins and pigments; yet, oligosaccharides extracted from medicinal foods have been proved to have many beneficial effects, which provides more evidence for further development of these substances. However, the studies on the structural characterization and bioactivity of oligosaccharides in RYR are limited. Hence, studies on the content, structural information, and bioactivity of oligosaccharides in RYR are essential.
Here, RYR oligosaccharides were isolated and purified from RYR, and the extraction flow chart is shown in Figure 1. Subsequently, precolumn derivatization HPLC, UPLC-TOF-MS, HPGPC, FT-IR, NMR, and SEM were employed to elucidate the structure of RYRO1, and finally, its antioxidative activity was investigated by DPPH, ABTS, and hydroxyl free radical assays.

| Ultrasonic-assisted extraction of oligosaccharides from RYR
Oligosaccharides were extracted from RYR by ultrasonic-assisted extraction. The dried red yeast rice was ground into a fine powder and was prepared for use. An appropriate amount of RYR fine powder was placed in a conical flask, and 95% ethanol solution was added at a liquid/material ratio of 20 ml/g and then sealed. The sample was then shaken at 150 rpm for 30 min at 40°C to initially remove the pigments and monosaccharides. Finally, the sample was filtered under reduced pressure and the filter residue was collected. The above procedure was repeated three times. The sample (3.0 g) was extracted with 80% ethanol solution using a 40-kHz ultrasonic cleaner (KQ5200DE; Kunshan Ultrasonic Co., Kunshan, China) equipped F I G U R E 1 Flowchart showing isolation and purification of RYRO1 from red yeast rice with a digital timer and temperature controller, and a single-factor experimental design was performed under the following conditions: liquid/material ratio (10-30 ml/g), extraction time , and extraction temperature (25-60°C). When optimizing factors in each experiment, one factor was changed while the other factors were kept constant. After extraction, the supernatant was obtained by centrifugation (L-550; Xiangyi Centrifuge Instrument Co., Changsha, China) and filtration under reduced pressure. Each sample was extracted three times and the filtrates were combined. The content of oligosaccharides obtained from red yeast rice was determined by the phenol sulfuric acid method (Zhu et al., 2020). The yield of oligosaccharides from red yeast rice was calculated by [oligosaccharide mass]/[sample mass] ×100%.

| Optimization of the extraction conditions by the Box-Behnken design
Based on the preliminary single-factor experimental results, a threelevel, three-factor Box-Behnken design method was used to optimize the study by Design-Expert software. Liquid/material ratio (X 1 ; 20, 25, and 30 ml/g), extraction time (X 2 ; 60, 90, and 120 min), and extraction temperature (X 3 ; 50, 60, and 70°C) were the independent variables to be optimized for the extraction of the RYR oligosaccharides (RYRO). The yield of oligosaccharide (%) (Y) was employed as the response of the design experiment. Based on the Box-Behnken design experimental data, a second-order polynomial equation was utilized to describe the relationship between the predicted response and the variables as follows: where Y is the predicted response, x i and x j are the levels of the independent variables, and β 0 , β i , β ii , and β ij are the intercept, linear coefficient, quadratic coefficient, and interaction coefficient of the model, respectively. For subsequent studies, RYRO was purified using an ionexchange chromatography column and size exclusion column.

| Purification of RYRO
After elution, the total sugar contents were determined by the phenol sulfuric acid method and the content of uronic acid was determined by the sulfamate/meta-hydroxydiphenyl assay using glucuronic acid as the standard (Blumenkrantz & Asboe-Hansen, 1973). The RYRO1 aqueous solution was scanned with a UV-VIS spectrophotometer in the range 200-400 nm to determine whether it contained protein and nucleic acid. RYRO was dissolved in 8 ml deionized water and then purified with a DEAE (Biobying, Beijing, China) column (2.8 × 40 cm, i.d.) that had been equilibrated using deionized water. After loading, elution was performed with deionized water, 0.1 M, and 0.3 M NaCl solution at a flow rate of 0.8 ml/min, and fractions were collected with an automatic fraction collector (8 ml/tube). The elution profile is shown in Figure 2a.
The first aqueous fraction was named RYRO1 and collected, lyophilized, and dissolved in 3 ml deionized water, and loaded onto a   (Zhao et al., 2020). Briefly, saccharide samples (40 mg) were mixed with 8 ml TFA (2 mol/L) in a stoppered colorimetric tube, sealed and placed in an oven at 110°C for 5 hr hydrolysis. The mixture was then cooled to room temperature, and TFA was evaporated under reduced pressure. After adding methanol and evaporating under reduced pressure six times to remove residual TFA, the obtained monosaccharides were dissolved with 5 ml deionized water.

2) Precolumn derivatization of mixed monosaccharide standards and samples
A volume of 0.2 ml of a mixed standard solution (2 mM) containing glucose, glucosamine, mannose, and galactose, and 0.2 ml of the RYRO1 hydrolysate were mixed in an EP tube, and an isometric PMP-methanol solution (0.5 mol/L) and NaOH solution (0.3 mol/L) were sequentially added. The mixture was incubated at 70°C in a water bath for 1 hr, cooled to room temperature, and neutralized with HCl solution (0.3 mol/L). One milliliter of chloroform was added and shaken for 1 min, and the chloroform layer was discarded. The extraction process was repeated three times. Finally, the aqueous layer was supplemented to 5 ml with deionized water, and 1 ml was filtered through a 0.45μm microporous membrane prior to further HPLC analysis.

3) HPLC analysis
PMP derivatives of monosaccharides were analyzed by an

| Determination of molecular weight of RYRO1
The molecular weight distribution of RYRO1 was determined by highperformance gel permeation chromatography (HPGPC) employing an Agilent Infinity 1260 system, equipped with a refractive index detector (Agilent, California, USA). The tested sample (5 mg/ml) was filtered through a 0.45μm polyether sulfone filter before injecting into the system. The columns in series were two PL aquagel-OH MIXED-H

| DPPH radical scavenging assay
The DPPH scavenging ability of RYRO1 was measured by a previously reported method with minor modifications (Zhao et al., 2020).

| ABTS radical scavenging assay
The ABTS radical scavenging ability of RYRO1 was evaluated by a previously reported method with minor modifications (Zhao et al., 2020). The ABTS radical cation solution (ABTS•+) was pre-

| Single-factor experiment analysis
The effect of single factors (liquid/material ratio, extraction time, and extraction temperature) on the extraction yield of oligosaccharides is shown in Figure 3.
According to Figure 3a, when the extraction temperature was 40°C and the liquid/material ratio was 20 ml/g, the yield of oligosaccharides increased remarkably from 30 to 90 min. However, the yield stopped rising when the extraction time extended due to the extraction equilibrium (Liu et al., 2019). Hence, 90 min was selected as the center point for the response surface methodology based on the Box-Behnken design. As can be seen from Figure 3b, when the extraction time was 90 min and the liquid/material ratio was 20 ml/g, the extraction yield increased significantly at the temperature between 25 and 40°C. However, when the temperature was between 40 and 60°C, the increase in yield was not significant. However, when the temperature reached 70°C, the yield dropped sharply. This may be due to the degradation of oligosaccharides caused by high temperatures (Gu et al., 2021). Therefore, an extraction temperature of 60°C was selected as the center point for the following optimization. Based on Figure 3c, when the extraction temperature of 60°C and extraction time was 90 min, the extraction rate of oligosaccharides increased significantly when the liquid/material ratio increased from 10 to 25 ml/g but decreased significantly when the liquid/material ratio increased from 25 to 30 ml/g. This may be due to the tendency of the red yeast rice powder to clump together at low ratios, which makes it difficult for water to enter, while higher liquid/material ratios result in lower concentrations and viscosity of the extractant, leading to the dissolution of oligosaccharides in water (Nuerxiati et al., 2019).
Therefore, the liquid/material ratio of 25 ml/g was selected for the following optimization. where Y is the extraction yield of RYRO and X 1 , X 2 , and X 3 represented liquid/material ratio, extraction time, and extraction temperature, respectively.

| Optimization of the extraction conditions of RYRO
The ANOVA was used to assess the significance and fitness of the regression models, and the results are shown in Table 2.
According to Table 2, the high F-value (67.42) and low p-value (p < .0001) indicate that the model is highly significant. The F and p values of lack of fit were 4.62 and 0.0868, respectively. The results showed that the model was able to predict the yield of RYRO.
The values of R 2 , adj-R 2 , and coefficient of variation were 0.9972, 0.9935, and 1.47, respectively, indicating that the model predicted the yield of RYRO accurately and reliably. The p-value was utilized as a tool to test the significance of each coefficient. A smaller pvalue indicated that the corresponding coefficient was more significant. As shown in Table 2, the extraction yield of RYRO was significantly (p < .05) affected by the linear coefficients (X 1 , X 2, , and X 3 ) and the quadratic coefficients (X 1 2 , X 2 2 , X 3 2 ). However, the two interaction coefficients (X 1 X 2 and X 2 X 3 ) had less effect on the extraction yield of RYRO due to the higher p-value (p >.05).
Three-dimensional response surface plots and contour plots The optimal conditions were slightly modified: that is, the liquid-tomaterial ratio was 25 ml/g, the extraction time was 97 min, and the extraction temperature was 60°C. A validation experiment was performed under these conditions. The actual extraction rate of 1.52% was in good agreement with the predicted value, indicating that the process can be directly used to guide the extraction of RYRO.

| General properties of RYRO1
RYRO was obtained from RYR through ultrasonic-assisted 80% ethanol extraction. Then, it was purified to obtain RYRO1. The sugar content was 93.7% and no uronic acid was detected. The UV-Vis spectrum showed no absorption in the range 200-400 nm, indicating that RYRO1 contains no nucleic acids or proteins . The results of UPLC-TOF-MS indicate that RYRO1 was mainly composed of monosaccharides, disaccharides, trisaccharides, and tetrasaccharides, and trace amounts of oligosaccharides with DP from 5 to 7 ( Figure 5).
TA B L E 2 ANOVA for the response surface quadratic model F I G U R E 4 3D response surface and 2D contour plots showing the effects of liquid/material ratio (X 1 ), extraction temperature (X 2 ), and extraction temperature (X 3 ) on the yield of RYRO

| Determination of molecular weight of RYRO1
The molecular weight of RYRO1 was determined by HPGPC

| FT-IR spectrum
FT-IR spectroscopy is a tool commonly used to characterize the functional groups of carbohydrates Zhao et al., 2020). As shown in Figure 8, RYRO1 has eight absorption bands, and the broadstretching intense peak near 3253 cm -1 is assigned to the stretching vibration of the hydroxyl group (He et al., 2016). A weak-stretching band at 2929 cm -1 is ascribed to the C-H-stretching vibration (You et al., 2021). The peak at 1024 cm -1 is the characteristic absorption peak of asymmetric stretching vibrations of the ether bond on the sugar ring, indicating that there are pyranose units in RYRO1 You et al., 2021). The signal at 1623 cm -1 was the characteristic absorption of bonded water, indicating the N-H of primary amine (Xiang et al., 2019). The signal at 1398 cm

| NMR
In 1 H NMR spectra of oligosaccharides, chemical shifts usually appear in the 1.0-6.0 ppm range. From 4.3 ppm to 5.9 ppm was considered to be associated with anomeric protons. The region of 3.0-4.2 ppm is related to the most protons present on C2-C6 with overlap problems (Agrawal, 1992). While in 13 C NMR spectra of oligosaccharides, the anomeric carbon signals distributed in the range 90-112 ppm (Agrawal, 1992). The structural characterization of RYRO1 was further elucidated by 1 H-NMR and 13 C-NMR. Chemical shifts ( 1 H) between 4.9 and 5.5 ppm are α-anomeric protons, while signals between 4.4 and 4.9 ppm are assigned to β-anomeric protons.
As seen from 1 H-and 13 C NMR (Figure 9a

| SEM analysis
SEM has been used to effectively illustrate the surface microstructure of polysaccharides and oligosaccharides (Chaari et al., 2016;. In the present study, the microstructure of RYRO1 was examined by SEM ( Figure 10). The SEM image of RYRO1 at 200 magnification shows that it has a folded paper-like surface.
The image at 2000 magnification shows that its surface is relatively intact and smooth, but with some folds and holes. This may be due to the low degree of branching and short chain structures of RYRO1 .

| Antioxidant activity
3.5.1 | DPPH radical scavenging activity DPPH is a colored, remarkably stable free radical at room temperature and is often used to assess the scavenging capacity of antioxidants (Foti, 2015). The scavenging ability of RYRO1 and Vc for DPPH was measured, and the results are shown in Figure 11a.
Overall, the ability of RYRO1 to scavenge DPPH was relatively weak. The scavenging effect of RYRO1 on DPPH was concentration dependent and reached a maximum inhibitory capacity of 34.29% at a concentration of 1 mg/ml, after which there was no further increase. As a positive control, ascorbic acid reached a strong DPPH scavenging capacity at a very low concentration (0.1 mg/ml) (Liu et al., 2019). Figure 11b shows the ability of RYRO1 and ascorbic acid to scavenge ABTS radicals. The total antioxidant capacity of a compound is usually estimated in terms of its ability to scavenge ABTS•+.

| ABTS radical scavenging activity
In the range 0.025-1.5 mg/ml, the ability of RYRO1 to scavenge ABTS•+ was concentration dependent. The scavenging capacity of both compounds for ABTS continued to increase overall at the concentrations tested, but ascorbic acid reached maximum scavenging capacity before RYRO1. The ABTS radical scavenging capacity of RYRO1 was observed to be greater than 100% at a concentra-

| Hydroxyl free radical scavenging activity
Hydroxyl radical is a kind of reactive oxygen species that can cause serious damage to the body (Lipinski, 2011). The scavenging ability of RYRO1 and the positive control (Vc) for hydroxyl radicals are shown in Figure 11c. The scavenging ability of both was concentration dependent. The scavenging capacity of ascorbic acid rose sharply with increasing concentration over the concentration interval tested, while the scavenging capacity of RYRO1 rose slowly. When the concentration reached 0.5 mg/ml, the scavenging capacity of RYRO1 and the positive control reached 26.96 U/ml and 69.16 U/ml, respectively.
The test results showed that although the hydroxyl radical scavenging ability of RYRO1 tended to increase in the tested concentration interval, it was generally not as strong as that of ascorbic acid. Free radicals such as reactive oxygen species (ROS) are of paramount relevance in intracellular signaling pathways. It is believed that dysregulated ROS signaling contributed to a plethora of human diseases, such as neurological disorders, cardiovascular system diseases, and diabetes (Finkel, 2011;Sies & Jones, 2020). Antioxidants can counter the negative effects of ROS and thereby avoid or eliminate related diseases, and they are abundant in natural resources.

| CON CLUS ION
The antioxidant effect of polysaccharides is determined by a combination of many factors, such as molecular weight, protein, phenolic substances, and uronic acid content . This conclusion may also apply to oligosaccharides, as they are all glycans. Previous studies have shown that the antioxidant capacity of polysaccharide conjugates is directly related to their molecular weight and the content of uronic acid (Chen et al., 2004(Chen et al., , 2008.
RYRO1 does not contain uronic acid, but it has a small heavy average molecular weight. This may be one of the reasons that explain the results of antioxidant activity in this study. Moreover, the relationship between the antioxidant activity of RYRO1 and its structure remains to be further investigated. The results of this study provide a reference for the development of RYR oligosaccharides as natural antioxidants in food and medicine and the extended application of RYR.

ACK N OWLED G M ENTS
This study was supported by the National Natural Science Foundation of China (81703706).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C S A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.