Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides

Journal Pre-proof Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides

Hexiang Zhang, Jiangchao Zhao, Hongmei Shang, Yang Guo, Shilun Chen PII:

S0141-8130(19)40241-9

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.194

Reference:

BIOMAC 14498

To appear in:

International Journal of Biological Macromolecules

Received date:

12 December 2019

Revised date:

15 January 2020

Accepted date:

20 January 2020

Please cite this article as: H. Zhang, J. Zhao, H. Shang, et al., Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.01.194

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© 2018 Published by Elsevier.

Journal Pre-proof Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides1

Hexiang Zhanga, Jiangchao Zhaob, Hongmei Shanga,c,d,e,∗, Yang Guoa, Shilun Chena

a

College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118,

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c

Department of Animal Science, University of Arkansas, Fayetteville 72701, Arkansas, USA Key Laboratory of Animal Production, Product Quality and Security, Ministry of Education,

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b

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China

d

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Jilin Agricultural University, Changchun 130118, China

Jilin Provincial Key Lab of Animal Nutrition and Feed Science, Jilin Agricultural University,

Joint Laboratory of Modern Agricultural Technology International Cooperation, Ministry of

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e

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Changchun 130118, China

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Education, Jilin Agricultural University, Changchun 130118, China

* Corresponding author at: College of Animal Science and Technology, Jilin Agricultural University, No. 2888 Xin-Cheng Street, Changchun 130118, China. E-mail address: [email protected] (H. Shang).

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Journal Pre-proof ABSTRACT Hot water extraction was applied to extract red clover (Trifolium pratense L.) polysaccharides (RCP) and the extraction conditions were optimized using the response surface methodology (RSM). An RCP yield of 12.72 ± 0.14% was achieved under the optimum extraction conditions: extracting time of 95 min, extracting temperature of 93 °C, and solvent-material ratio of 21 mL/g. A component named RCP-1.1 with the molecular weight of 7528.81 kDa was purified

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from RCP. RCP-1.1 was composed of glucose, galacturonic acid, arabinose, and galactose, with

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molar percentages of 52.54, 1.04, 16.31, and 30.11%, respectively. At the determination

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concentration of 10 mg/mL, the α-glucosidase inhibition ability of RCP-1.1 reached 86.72% of that of acarbose. The scavenging rates of RCP-1.1 (3.0 mg/mL) for DPPH and ABTS radicals

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reached 91.82% and 98.95% of that of ascorbic acid (3.0 mg/mL), respectively. Based on these

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results, RCP-1.1 possesses the potential to be used as a natural hypoglycemic agent or an

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Keywords:

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antioxidant.

Polysaccharides Activities

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Red clover (Trifolium pratense L.)

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Journal Pre-proof 1. Introduction Red clover (Trifolium pratense L.) is a short-term perennial herb of the Trifolium genus, Leguminosae family. It is an important perennial forage herb in temperate climate regions of the world [1]. Because of the high crude protein content and low content of neutral detergent fibers, red clover is one of the important nutrient sources for grazing animals [2]. In addition, red clover is also a popular medicinal plant because it is enriched in bioactive substances such as

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isoflavones [3]. Red clover is a natural substitute for antioxidants, treats menopausal symptoms,

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and is an anti-aging agent [4]. It helps treat high cholesterol, premenstrual syndrome, breast pain,

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and menopausal symptoms [5].

Polysaccharides are composed of monosaccharides connected by glycosylic bonds.

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Polysaccharides have been reported to possess a variety of biological activities, such as

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regulating lipid metabolism [6], having therapeutic potential against colitis [7], reducing the

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oxidative damage of HaCaT cells [8], regulating the intestinal microbiome [9], having anti-tumor activity [10], having antihyperglycemic activity [11], having anti-aging properties [12], and

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regulating the immune system [13]. Currently, polysaccharides are used to design functional

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products for the cosmetics, agriculture, medical, and health food industries [14]. Few reports are available describing red clover polysaccharides (RCP). Buchala and Meier isolated a galactoglucomannan from red clover and studied the structural characteristics of the polysaccharide [15]. Relevant studies of RCP will broaden the applications of these polysaccharides and improve the utilization value of red clover. Therefore, this study was designed to extract and purify RCP and estimate its activity. First, the optimum extraction parameters for RCP were obtained using the Box-Behnken design (BBD) of response surface methodology (RSM). Second, the optimized crude RCP was sequentially purified with a DEAE-

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Journal Pre-proof Cellulose 52 column and a Sepharose CL-6B column. Third, the physicochemical properties, hypoglycemic activity, and antioxidant activity of the purified RCP were evaluated. 2. Materials and methods 2.1. Red clover and reagents Red clover was harvested in the bloom stage at the Grassland Science Professional Experiment Practice Base of Jilin Agricultural University (Changchun, China). The fresh

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harvested red clover was dried at 50 °C and ground into a powder (1 mm) in preparation for

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extracting RCP. 2,2-Azino-bis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), DEAE-52

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cellulose, and acarbose were purchased from Beijing Biotopped Science & Technology Co., Ltd. (Beijing, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was obtained from Tokyo Chemical

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Industry (Tokyo, Japan). 1-Phenyl-3-methyl-5-pyrazolone (PMP) was purchased from Shanghai

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McLean Biochemical Technology Co., Ltd. (Shanghai, China). Sepharose CL-6B, dextran, α-

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amylase, monosaccharide standards, ascorbic acid, and α-glucosidase were obtained from Hefei Bomei Biotechnology Co., Ltd. (Hefei, China).

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2.2. Optimization of the extraction conditions for RCP

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2.2.1. Single-factor design

Red clover powder (20 g) was extracted with deionized water in a beaker. Each extraction procedure was performed in triplicate. The effects of three extraction variables on the RCP yield were estimated. The three extraction variables were the solvent-material ratio (X1) (10, 15, 20, 25, and 30 mL/g), extracting time (X2) (30, 60, 90, 120, and 150 min), and extracting temperature (X3) (60, 70, 80, 90, and 100 °C). The first single-factor experiment was conducted with the solvent-material ratio ranging from 10 mL/g to 30 mL/g. The other two variables, the extracting time and extracting temperature, were 120 min and 90 °C, respectively. The single-factor

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Journal Pre-proof experiment of another extraction variable was performed based on the optimal result from the previous experiment. After the extraction procedure, centrifugation, alcohol precipitation, deproteinization and other steps were carried out to isolate RCP. Initially, the extract was centrifuged (3000 g) for 15 min and the supernatant of the extract was concentrated using the reduced pressure evaporation technique until the final volume was one-quarter of the original volume. Subsequently, the

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concentrated supernatant was precipitated with ethanol (12 h, 4 °C). A centrifugation procedure

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(3000 g, 15 min) was performed to obtain the precipitate. Afterwards, the protein in the

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precipitate was removed using Sevag reagent. The Sevag reagent was composed of n-butanol and chloroform at a ratio of 1:4 (v/v). Finally, the extract was subjected to dialysis using a

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semipermeable membrane with a 1400 Da molecular weight cut off (Union Carbide). The crude

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RCP was obtained after freeze drying. The RCP yield (%) was calculated using the following

RCP weight (g) 100 red clover powder weight (g)

(1)

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RCP yield (%) 

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formula (1):

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2.2.2. BBD design

After performing the single-factor experiment, a three-variable BBD design was generated using Design Expert 8.0.6 Software (Stat-Ease Inc., USA). Each variable contained three levels. The RCP yield (%) was the response. Seventeen trial runs, including 5 central points, constituted the whole design (Table 1). The quadratic polynomial equation (2) used in the BBD experiment is presented below. 3

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i =1

i =1

2

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Y  0 + i X i + ii X i2 +  ij X i X j

(2)

i 1 j=i 1

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Journal Pre-proof where Xi and Xj are the variables; β0 is the intercept; βij, βii, and βi are the regression coefficients for the interaction, quadratic, and linear terms, respectively; and Y is the RCP yield. 2.3. Purification of RCP According to the optimum extraction parameters obtained in the BBD design mentioned above, RCP was extracted from red clover powder. Subsequently, the procedures including centrifugation, concentration, alcohol precipitation, protein removal, and freeze drying were

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performed as described in section 2.2.1, and then the crude RCP was obtained.

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Afterwards, the crude RCP was first purified using a DEAE-52 cellulose column (3.5 × 20

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cm). An aqueous RCP solution (50 mg/mL, 1 mL) was loaded onto the DEAE-52 column. Components were successively eluted (2 mL/min) from the column with distilled water and a

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series of NaCl solutions (0.1, 0.3, 0.5, and 1.0 mol/L). The eluent was collected in 8 mL per tube,

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and the concentrations of polysaccharides were monitored with the phenol-sulfuric acid method

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[16]. Briefly, 60 μL of eluent was removed from each tube and then reacted with 30 μL of phenol solution (6%, w/v) and concentrated sulfuric acid (150 μL). The absorbance (490 nm) of the

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mixture was recorded with a microplate reader (Model ST-360, Shanghai Kehua Experimental

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System Co., Ltd., Shanghai, China) after an incubation for 30 min. After drawing the elution curve for RCP from the DEAE-52 cellulose column, the main fraction of polysaccharides was purified using a Sepharose CL-6B column (2.6 × 100 cm). Briefly, the collected RCP fraction (30 mg/mL, 1 mL) was loaded onto the column and elution (0.9 mL/min) was carried out with distilled water. One tube of eluent (4.05 mL) was collected every 4.5 min and the polysaccharide content was detected [16]. The main fraction was collected and subjected to freeze drying for further analyses of the physicochemical properties and biological activities.

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Journal Pre-proof 2.4. Chemical composition of the purified RCP The polysaccharide content of the purified RCP was analyzed using the phenol-sulfuric acid method [16]. Briefly, after preparing the D-glucose standard solution (100 μg/mL), different volumes (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL) of the D-glucose standard solution were accurately transferred to different stoppered test tubes, and distilled water was added to each tube to achieve a total volume of 1.0 mL. The first tube served as the blank control. Afterwards, a phenol

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solution (0.5 mL; 6%, w/v) and concentrated sulfuric acid (2.5 mL) were added into each tube

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and quickly mixed. After cooling for 30 min, the absorbance (490 nm) of the reaction mixture

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was recorded. The standard curve was drawn with the D-glucose content (μg) as the independent variable (x) and absorbance as the dependent variable (y), and the linear regression equation y =

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0.0171 x + 0.0045 (R2 = 0.9968) was obtained. Following the addition of 1.0 mL of the RCP

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solution, the absorbance of RCP was determined using the procedure described above by

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constructing a standard curve. Subsequently, the absorbance was substituted into the regression equation to calculate the polysaccharide content in the purified RCP.

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The protein content of the purified RCP was analyzed using the Bradford method [17]. Ten

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milligrams of bovine serum albumin was weighed and dissolved in distilled water to obtain a standard protein solution with a concentration of 100 μg/mL. After transferring different volumes (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL) of the standard protein solution into different stoppered tubes, distilled water was added to each tube to achieve a total volume of 1.0 mL. The first tube served as the blank. Afterwards, a Coomassie blue G250 solution (5.0 mL) was added to each tube. The absorbance of the mixture was recorded at 595 nm after incubation for 5 min. The standard curve was established with the protein content (μg) on the x axis and the absorbance value on the y axis. The linear regression equation was obtained as y = 0.0045 x + 0.0157 (R2 = 0.9928). The

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Journal Pre-proof RCP solution (1 mL) was added and the absorbance of RCP was determined according to the procedure described above for the standard curve. Then, the absorbance value of the RCP sample was substituted into the regression equation to calculate the soluble protein content in the purified RCP. The uronic acid content of the purified RCP was measured using the sulfuric acid-mhydroxybiphenyl method described in a previous report, with some modifications [18]. The m-

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hydroxybiphenyl solution (0.15%, w/v) was prepared by dissolving 15 mg of m-

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hydroxybiphenyl in 10 mL of a sodium hydroxide solution (0.5%, w/v). Sodium tetraborate

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(Na2B4O7·10 H2O, 0.476 g) was dissolved in 100 mL of concentrated sulfuric acid to prepare the sodium tetraborate-concentrated sulfuric acid solution (0.0125 mol/L). D-Glucuronic acid (60

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μg/mL) was used as the standard solution. Different volumes (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL) of

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the standard solution were transferred to different test tubes. Distilled water was mixed with

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standard solutions to obtain a total volume of 0.5 mL in each tube. The first tube served as the blank. Subsequently, the sodium tetraborate-concentrated sulfuric acid solution (0.0125 mol/L, 3

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mL) was added to each tube. After vortexing, the mixture was incubated in a boiling water bath

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for 5 min and then cooled to room temperature in an ice water bath. The m-hydroxybiphenyl solution (0.05 mL; 0.15%, w/v) was added to the mixture. The absorbance was measured at 520 nm, and the standard curve was established by setting the D-glucuronic acid content (μg) as the horizontal coordinate (x) and the absorbance value as the vertical coordinate (y). The linear regression equation was obtained as y = 0.0252 x － 0.029 (R2 = 0.9954). Afterwards, an appropriate concentration of RCP solution (approximately 0.1－0.8 mg/mL) was prepared. The RCP solution (0.5 mL) was subjected to this procedure and the absorbance value of RCP was determined as described above. The absorbance value of the RCP sample was substituted into the

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Journal Pre-proof regression equation to calculate the uronic acid content in the purified RCP. 2.5. Monosaccharide composition of the purified RCP HPLC (2010AHT, Kyoto, Japan) was used to determine the monosaccharide composition of the purified RCP. Briefly, the RCP sample (2 mg) was hydrolyzed (80 °C, 16 h) in the presence of a hydrochloric acid solution (1 mol/L, 2.5 mL) prepared in anhydrous methanol. After drying the hydrolysate under a nitrogen stream, 0.5 mL of a trifluoroacetic acid (TFA) solution (2

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mol/L) was added to the hydrolysis tube and the hydrolysis process continued for 1 h at 120 °C.

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The hydrolysate was co-evaporated at 45 °C with ethanol to remove the excess TFA. After

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hydrolysis, the hydrolysates of the purified RCP and the monosaccharide standards were derivatized with PMP using the procedure described in our previous study [19]. Subsequently,

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the PMP-labeled products were detected with the HPLC system equipped with a Sepax Amethyst

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C18 column (5 μm, 4.6 mm × 250 mm, Delaware, USA) and a UV detector (245 nm). The

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injection volume was 10 μL. The temperature of the C18 column was set to 25 °C. The mobile

at a 20:80 (v/v) ratio.

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phase (1 mL/min) was a mixture of acetonitrile and phosphate-buffered saline (pH 7, 0.1 mol /L)

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2.6. Molecular weight of the purified RCP The molecular weight of the purified RCP was analyzed using gel filtration chromatography technology with a Sepharose CL-6B column (2.6 × 100 cm). The eluent was distilled water applied at a flow rate of 0.9 mL/min. The eluates (4.05 mL in one tube) were collected. The polysaccharide contents in the collected eluates were determined using the method described in section 2.3. Dextran, including T-10, T-40, T-70, and T-500, was used as the standard. 2.7. Ultraviolet (UV) spectrum of the purified RCP Distilled water was used as the blank. UV spectrum of the purified RCP (0.5 mg/mL) were

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Journal Pre-proof measured with a UV-visible spectrophotometer (Model T6 New Century, Beijing Puxi General Instrument Co., Ltd., Beijing, China). The wavelengths ranged from 200-400 nm. 2.8. Congo red test of the purified RCP The purified RCP solution (0.5 mg/mL, 2 mL) was added to 2 mL of Congo red solution (80 μmol/L). Subsequently, a NaOH solution (4.0 mol/L) was added to the mixture, and solutions with a series of concentrations of NaOH (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol/L) were obtained. The

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RCP solution was replaced with deionized water (2 mL) in the blank. After a 10 min incubation,

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the maximum absorption wavelength (λmax) of the reaction solution was recorded.

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2.9. Dissolution time and pH determination of the purified RCP

The dissolution time of the purified RCP was measured. The purified RCP (0.1 g) was placed

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in 50 mL of distilled water in a beaker. Afterwards, water baths with temperatures of 20, 40, 60,

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80, and 100 °C were used to heat the beaker. The purified RCP solution in the beaker was stirred

recorded.

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at a speed of 150 rpm/min. When the purified RCP was dissolved, the dissolution time was

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The pH of the purified RCP was determined. An RCP solution (2 mg/mL) was prepared with

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distilled water and its pH was determined with a pH meter (PHS-3C-02, Aipu, Quzhou, China). 2.10. Hypoglycemic and antioxidant activities of the purified RCP The hypoglycemic property of the purified RCP was estimated by determining its ability to inhibit the activities of two digestive enzymes (α-amylase and α-glucosidase) using previously described methods [20]. Acarbose served as the positive control. Serial dilutions of RCP (0.5, 1, 2, 4, 6, 8, and 10 mg/mL) were used in the experiment. ABTS radical and DPPH radical scavenging activities were measured to evaluate the antioxidant activities of the purified RCP. Ascorbic acid served as the positive control. The

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Journal Pre-proof determination methods were performed as described in a previous report [21]. RCP was prepared with distilled water at different concentrations (0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, and 3.0 mg/mL) to determine its antioxidant activities. 2.11. Statistical analysis All experiments were performed in triplicate. The values were compared using one-way analysis of variance (ANOVA) with SPSS 19.0 software. The values are presented as means ±

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standard deviations. Comparisons of means between multiple groups was performed using

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Duncan’s multiple range test. Curve estimation was carried out to evaluate the effects of the

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polysaccharide levels on the antioxidant and hypoglycemic activities. P<0.05 was recognized as statistically significant.

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3. Results and discussion

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3.1. Single-factor design for RCP extraction

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3.1.1. Solvent-material ratio

As presented in Fig. 1A, the highest RCP yield (9.84%) was obtained at the solvent-material

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ratio of 20 mL/g and then significantly decreased (P<0.05). The potential explanation for this

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difference is that when the solvent-material ratio was relatively low, saturation would readily be achieved in the extraction system, resulting in the incomplete extraction of RCP [22]. As the solvent-material ratio increased, the viscosity of the extraction solution decreased and the number of polysaccharide molecules dissolved in water increased, leading to an increase in the RCP yield. However, when the volume of extraction solvent was too large, the distance of RCP diffusion from plant tissues increased, which might inhibit the dissolution of RCP. At the same time, an excessive solvent-material ratio would also cause the solvent to be wasted, increasing

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Journal Pre-proof the burden of subsequent concentration processes. Thus, the optimum solvent-material ratio was 20 mL/g. 3.1.2. Extracting time As displayed in Fig. 1B, the RCP yield increased remarkably as the extracting time was extended from 30 to 90 min (P<0.05). The maximum RCP yield of 10.52% was obtained at the extracting time of 90 min. The RCP yield decreased significantly at the extracting time of 120

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and 150 min compared with 90 min (P<0.05), which might potentially due to the degradation of

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RCP under the long extracting time [23]. Therefore, the optimum extracting time was 90 min.

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3.1.3. Extracting temperature

As presented in Fig. 1C, the RCP yield first increased significantly and then decreased

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slightly thereafter as the extracting temperature increased; the maximum yield of 10.64% was

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obtained at the extracting temperature of 90 °C. A potential explanation for this finding is that as

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the extracting temperature increased, the molecular motion accelerated and the yield of RCP increased. However, when the extracting temperature was excessively high, part of the

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polysaccharides degraded, leading to a reduction in the RCP yield [24]. As a result, the optimum

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extracting temperature for RCP was 90 °C. 3.2. BBD for RCP extraction

3.2.1. Prediction model and ANOVA The three variables for RCP extraction were optimized by 17 runs using the BBD. The runs and the corresponding results are provided in Table 1. The relationship between the response value Y and the three extraction variables (X1, X2, and X3) was a second-order polynomial equation (3): Y  64.09+2.20X 1 +0.16 X 2 +1.00 X 3  4.11E-004X 1 X 2  4.63E-003X 1 X 3  5.42E-004X 2 X 3 0.04 X 12  5.37E-004 X 2 2  4.56E-003X 32

(3)

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Journal Pre-proof where X1, X2, and X3 are the solvent-material ratio, extracting time, and extracting temperature, respectively; Y is the RCP yield. The results of the ANOVA of the second-order polynomial model is shown in Table 2. The validity of the polynomial model was judged by the lack of fit. The P-value (0.6173) of the lack of fit was greater than 0.05, suggesting that the difference between the polynomial model and the experiment results was not significant relative to the experimental error, implying a good

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reliability of the polynomial model. The F-value (41.57) and P-value (<0.0001) of the regression

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model indicated its significance. The determination coefficient (R2 = 0.9816) suggested that the

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difference between the regression model and the empirical data was only 1.84%. In addition, the R2 value (0.9816) and the adjusted determination coefficient (Adj R2, 0.9580) were near unity,

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suggesting a good fit between the experimental data and the predicted data [25]. This good fit

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was also observed in the linear fitting diagram (Fig. 2A). Moreover, the low value for the

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coefficient of variation (C.V., 1.41%) suggested a high reliability and precision of the polynomial model. Adeq Precision was used to estimate the signal-to-noise ratio. The Adeq Precision of

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19.815 suggested that the signal of the polynomial model was adequate.

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Furthermore, the adequacy of the polynomial model was estimated from the normal probability graph of residuals (Fig. 2B), which showed that residuals were distributed along a straight line, indicating that residuals were normally distributed. As shown in Fig. 2C, the comparison between the internally studentized residuals and the actual runs revealed that all data points were within the acceptable range (±3.00). In Fig. 2D, the residual distribution of the residual and predicted response graph was random, indicating that the variance of the original observed values was constant for all values. All diagrams (Fig. 2A, B, C, and D) revealed the applicability and accuracy of BBD for the design of the optimized RCP extraction method.

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Journal Pre-proof The significances of the coefficients obtained were estimated from their P-values (Table 2). Smaller P-values of the variables indicated a more significant contribution of the variables to the polynomial model. The P-values of all linear coefficients (X1, X2, and X3), cross-product coefficients (X1X3), and all quadratic coefficients (X12, X22, and X32) were less than 0.05, suggesting that these terms exerted significant effects on RCP yield. By comparing the F-values

material ratio > extracting temperature > extracting time.

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of the three extraction parameters, the order of their effects on the RCP yield was solvent-

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3.2.2. Response surface plots and contour plots of the prediction model

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The response surface plot and the contour plot are graphs displaying the regression equation. In each contour plot, two variables were in the test ranges, while other variables were fixed at the

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0 level. As displayed in Fig. 3, for the solvent-material ratio and extracting time (Fig. 3B), the

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circular shape of the contour plot suggested that the mutual interaction between these two

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parameters was not significant. For the solvent-material ratio and extracting temperature (Fig. 3D), the shape of the contour plot was elliptical, suggesting that the mutual interaction between

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these two parameters was significant. For the extracting temperature and extracting time (Fig.

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3F), the shape of the contour plot was circular, indicating that the mutual interaction between these two parameters was not significant. 3.2.3. Regression model for the optimization of RCP extraction conditions The calculated extraction parameters for the RCP extraction were obtained from the predictive regression equation (3): a solvent-material ratio of 21.04 mL/g, an extracting time of 95.18 min, and an extracting temperature of 92.88 °C. The predicted RCP yield was 13.01%. Consequently, the three extraction parameters were set to 21 mL/g, 95 min, and 93 °C. Under these conditions, a reliability compliance test was performed and an RCP yield of 12.72 ± 0.14%

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Journal Pre-proof (n = 3) was obtained, which was consistent with the predicted RCP yield of 13.01%. Therefore, the regression model obtained in this study was adequate for predicting the extraction yield of RCP. 3.3. Purification of RCP Using the optimal parameters described in section 3.2.3 and the procedure described in section 2.2.1, crude RCP was extracted and prepared. Crude RCP was purified using a DEAE-52

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cellulose column. As presented in Fig. 4A, three elution peaks were obtained and named RCP-1,

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RCP-2, and RCP-3, which were eluted from distilled water, a 0.1 mol/L NaCl solution, and a 0.5

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mol/L NaCl solution, respectively. As the main elution fraction, RCP-1 was collected and lyophilized. Subsequently, RCP-1 was further purified using a Sepharose CL-6B column, and

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two elution peaks named RCP-1.1 and RCP-1.1 were obtained (Fig. 4B). As the main

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homogeneous fraction, RCP-1.1 was pooled, lyophilized, and generated as the purified RCP.

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3.4. Physicochemical properties of RCP-1.1

3.4.1. Preliminary characterization of RCP-1.1

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The polysaccharide content of RCP-1.1 was measured as 77.94±2.93%. The uronic acid

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content of RCP-1.1 was 5.16±0.24%. Protein was not detected in RCP-1.1. The pH of RCP-1.1 was detected as 6.80±0.04. As presented in Fig. 4C, RCP-1.1 showed one elution peak, indicating that RCP-1.1 was a homogenous polysaccharide. The molecular weight of RCP-1.1 was calculated to be 7528.81 kDa. The monosaccharide composition of RCP-1.1 is presented in Fig. 5 and includes glucose, galacturonic acid, arabinose, and galactose with the molar percentages of 52.54, 1.04, 16.31, and 30.11%, respectively. 3.4.2. UV spectrum of RCP-1.1 The UV spectrum of RCP-1.1 is presented in Fig. 6A. No absorbance was detected at the

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Journal Pre-proof wavelengths of 260 nm or 280 nm, suggesting that RCP-1.1 did not contain nucleic acids or proteins [26]. This result was consistent with the analysis of the chemical composition of the purified RCP described in section 3.4.1, in which proteins were not detected in RCP-1.1. 3.4.3. Congo red test of RCP-1.1 As a useful and simple method, the Congo red test has been widely applied to judge the helical conformation of polysaccharides [27]. Polysaccharides with a triple-helix conformation

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form special complexes with Congo red in an alkaline solution. Due to the transformation of the

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polysaccharide conformation in alkaline solution, the λmax of the complexes show a

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bathochromic shift compared with pure Congo red. The mixed solution of Congo red and RCP1.1 presented longer λmax values than the pure Congo red solution when the concentrations of the

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NaOH solution ranged from 0.1 to 0.4 mol/L (Fig. 6B). However, when the NaOH

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concentrations were greater than 0.4 mol/L, the λmax values of the mixed solution decreased,

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indicating that the well-organized triple-helix conformation of RCP-1.1 shifted to a random coil [28]. The results of the Congo red test revealed a triple-helix conformation of RCP-1.1.

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3.4.4. Dissolution time of RCP-1.1

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As shown in Fig. 6C, the dissolution time of RCP-1.1 significantly and gradually decreased as the test temperature increased in the range of 20–100 °C (P<0.05). The dissolution time (y) displayed a linear dependence on the temperature (x), and the equation was y = – 3.2x + 21.8 (R2 = 0.9846). Water solubility is one of the indexes used to evaluate the polysaccharides applied in the pharmaceutical and food industries. Polysaccharides used in drug sustained-release carriers, dressings, and food additives must have short dissolution time and good water solubility [29]. RCP-1.1 exhibited a short dissolution time, indicating that it might have potential in certain applications in food, medicine and other fields.

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Journal Pre-proof 3.5. Hypoglycemic activity of RCP-1.1 The percent inhibition of α-amylase and α-glucosidase activity were determined to estimate the hypoglycemic effects of the test samples. Inhibitors of α-amylase and α-glucosidase may delay or reduce the postprandial blood glucose level. The inhibitory effects of RCP-1.1 on the two test enzyme activities improved as the analyzed concentration increased (Fig. 7). In addition, the inhibitory effects of acarbose and RCP-1.1 on α-amylase activity (y) both showed a quadratic

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relationship to the test concentration (x). The formulas were y = –0.3886x2 + 8.0284x + 8.0274

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(R2 = 0.9308) and y = –0.1941x2 + 4.8265x + 4.9823 (R2 = 0.9474), respectively (Fig. 7A). As

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displayed in Fig. 7B, the abilities of acarbose and RCP-1.1 to inhibit α-glucosidase activity (y) exhibited the same trends with the increase in the tested concentration (x). The quadratic

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formulas were y = –0.6055x2 + 9.4564 x + 6.3231 (R2 = 0.9350) and y = –0.6091x2 + 9.4633x +

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2.0478 (R2 = 0.9788), respectively. At each test concentration in the range from 0.5 to 10 mg/mL,

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RCP-1.1 exerted significantly lower inhibitory effects on α-amylase and α-glucosidase activities than acarbose (P<0.05). The greatest inhibitory effects of RCP-1.1 (10 mg/mL) on α-amylase

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activity and α-glucosidase activity observed were 67.91% and 86.72%, respectively, of the

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activities of acarbose. Based on these results, RCP-1.1 exerted an effect on hypoglycemia in vitro. The carboxyl and hydroxyl groups on the branched chain of polysaccharides have been reported to interact with the amino acid residues of the digestive enzymes, thus inhibiting the activities of digestive enzymes [30]. Acarbose is used in the clinic to treat type 2 diabetes. However, acarbose also has some side effects in clinical applications, such as abdominal distension and diarrhea. The excessive suppression of α-amylase activity by acarbose might cause undigested carbohydrates to be fermented by the bacteria in the colon [31]. Therefore, the weaker α-amylase inhibitory activity

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Journal Pre-proof would help reduce the side effects of acarbose during the treatment of type 2 diabetes. In the present study, the α-amylase inhibitory activity of RCP-1.1 (10 mg/mL) reached 67.91% of the value of acarbose, indicating that RCP-1.1 might be a natural and safe α-amylase inhibitor. Therefore, RCP-1.1 potentially represents a candidate for treating type 2 diabetes in the future. 3.6. Antioxidant activities of RCP-1.1 3.6.1. DPPH radical scavenging activity

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This assay is a common strategy used to estimate the antioxidant activity of a compound. In

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the presence of antioxidants, the color of the reaction system changes to yellow from purple. As

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presented in Fig. 8A, the DPPH radical scavenging activity of RCP-1.1 (y) exhibited a quadratic relationship to the test concentration (x, 0.0625–3.0 mg/mL), and this relationship was

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represented by the formula y = –5.83x2 + 25.235x + 30.596 (R2 = 0.8448). In the test

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concentration range (0.0625–3.0 mg/mL) analyzed in this study, RCP-1.1 showed lower

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scavenging activity than ascorbic acid. However, the scavenging rate of RCP-1.1 (3.0 mg/mL) was 55.79%, which reached 91.82% of the value of ascorbic acid. The ability of the

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polysaccharides to scavenge the DPPH radical was related to the molecular weight, chemical

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composition, and monosaccharide composition of polysaccharides, because this scavenging activity was ultimately rooted in the electron supply capacity of polysaccharides [30]. Thus, RCP-1.1 possessed a relatively significant scavenging activity toward the DPPH radical, particularly at the test concentration of 3.0 mg/mL. 3.6.2. ABTS radical scavenging activity This assay has the advantages of simplicity and speed, and it is a well-known method used to estimate the antioxidant ability of test samples. As presented in Fig. 8B, the ability of RCP-1.1 to scavenge the ABTS radical (y) showed a quadratic relationship to the test concentration (x,

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Journal Pre-proof 0.0625–3.0 mg/mL) with the formula of y = –24.104x2 + 96.504x + 19.63 (R2 = 0.8666). At the test concentrations of 1.0, 2.0, and 3.0 mg/mL, the ABTS radical scavenging activities of RCP1.1 were 97.10%, 98.35%, and 98.94%, respectively. The scavenging rate of RCP-1.1 (3.0 mg/mL) toward the ABTS radical reached 98.95% of ascorbic acid. Moreover, noticeable differences in the ABTS radical scavenging activities were not observed between RCP-1.1 and ascorbic acid at these three test concentrations (1.0, 2.0, and 3.0 mg/mL) (P>0.05), indicating

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that polysaccharides with a notable ability to scavenge the ABTS radical were extracted from red

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clover.

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Reactive oxygen species and free radicals react with biomolecules such as proteins, carbohydrates, and lipids, causing damage to the body and health problems [32]. Therefore, the

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identification of exogenous natural antioxidants that will improve the antioxidant capacity of the

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body is an important goal. Plant extracts are considered a valuable source of natural antioxidants.

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Polysaccharides with radical scavenging activities have attracted increasing attention and are very important in the production of natural drugs [33]. Based on the results obtained in this

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study, RCP-1.1 possessed relatively significant scavenging activities toward ABTS and DPPH

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radicals, indicating the RCP-1.1 might be a candidate free radical scavenger. In fact, several factors affect the antioxidant activity of polysaccharides, such as their chemical composition and monosaccharide composition. Uronic acid is postulated to scavenge free radicals [34]. The uronic acid content of RCP-1.1 was 5.16±0.24%, which might have contributed to the antioxidant activity of RCP-1.1. 4. Conclusions In this study, the RSM design was used to optimize the conditions for RCP extraction. A maximum RCP yield of 12.72% was obtained under the optimum extraction conditions of an

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Journal Pre-proof extracting time of 95 min, an extracting temperature of 93 °C, and a solvent-material ratio of 21 mL/g. After purification, the purified polysaccharide component RCP-1.1 with a molecular weight of 7528.81 kDa was obtained. The monosaccharide composition of RCP-1.1 was detected as glucose, galacturonic acid, arabinose, and galactose with molar percentages of 52.54, 1.04, 16.31, and 30.11%, respectively. RCP-1.1 showed good hypoglycemic activity and antioxidant properties. Based on these results, RCP-1.1 might serve as potential natural antioxidant and

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hypoglycemic agent. RCP-1.1 deserves further analysis of its potential applications in the fields

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of food and medicine.

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Declaration of interest

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The authors declare that there is no competing interest.

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Acknowledgements

This research was financially supported by the Science and Technology Department of Jilin

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Province of China (20190201158JC); the Education Department of Jilin Province of China

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(JJKH20190912KJ); the China Postdoctoral Science Foundation (2019T120244; 2017M621224); the National Natural Science Foundation of China (31601972); the China Scholarship Council (201805965018).

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extraction of polysaccharides from Hovenia dulcis: extraction, structure, antioxidant activity and hypoglycemic, Int. J. Biol. Macromol. 137 (2019) 676–687. [31] H. Zhang, J. Zhong, Q. Zhang, D. Qing, C. Yan, Structural elucidation and bioactivities of a novel arabinogalactan from Coreopsis tinctoria, Carbohyd. Polym. 219 (2019) 219–228. [32] M. Jridi, R. Nasri, Z. Marzougui, O. Abdelhedi, M. Hamdi, M. Nasri, Characterization and assessment of antioxidant and antibacterial activities of sulfated polysaccharides extracted from cuttlefish skin and muscle, Int. J. Biol. Macromol. 123 (2019) 1221–1228. [33] P. Mutaillifu, K. Bobakulov, A. Abuduwaili, H. Huojiaaihemaiti, R. Nuerxiati, H.A. Aisa, A.

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Journal Pre-proof Table 1 Box-Behnken design and response values for RCP yields.

90 90 90 90 80 80 100 100 80 80 100 100 90 90 90 90 90

RCP yields (%) Actual values 10.60 11.66 11.21 12.03 10.21 11.75 11.51 12.13 11.32 12.06 12.15 12.25 12.89 12.85 12.85 12.66 13.17

Predicted values 10.58 11.71 11.16 12.04 10.33 11.80 11.47 12.01 11.22 12.00 12.22 12.35 12.88 12.88 12.88 12.88 12.88

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1 15 60 2 25 60 3 15 120 4 25 120 5 15 90 6 25 90 7 15 90 8 25 90 9 20 60 10 20 120 11 20 60 12 20 120 13 20 90 14 20 90 15 20 90 16 20 90 17 20 90 RCP, red clover polysaccharides.

Extracting temperature (X3) (°C)

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Extracting time (X2) (min)

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Solvent-material ratio (X1) (mL/g)

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Runs

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Journal Pre-proof Table 2 ANOVA for the second-order polynomial model of RCP yield. F value 41.57 71.76 14.56 32.41 0.54 7.56 3.73 156.92 34.72 30.92

P value < 0.0001* < 0.0001* 0.0066* 0.0007* 0.4876 0.0285* 0.0946 < 0.0001* 0.0006* 0.0009*

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0.6173

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Source Sum of squares DF Mean square Model 10.61 9 1.18 X1 2.03 1 2.03 X2 0.41 1 0.41 X3 0.92 1 0.92 X1X2 0.015 1 0.015 X1X3 2.10E-01 1 2.10E-01 X2X3 0.11 1 0.11 X12 4.45 1 4.45 X22 0.98 1 0.98 X32 0.88 1 0.88 Residual 0.2 7 0.028 Lack of fit 0.066 3 0.022 Pure error 0.13 4 0.033 Cor total 10.81 16 R2 0.9816 Adj R2 0.9580 C.V.% 1.41 Adeq Precision 19.815 * P values < 0.05 were considered statistically significant. X1, solvent-material ratio; X2, extracting time; X3, extracting temperature. RCP, red clover polysaccharides.

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Journal Pre-proof Fig. 1. Single-factor design for extracting RCP: (A) solvent-material ratio; (B) extracting time; (C) extracting temperature. RCP, red clover polysaccharides. Letters (a-d) different in the same graph differ statistically (P<0.05). Fig. 2. Diagnostic graphics of the prediction model: (A) predicted vs. actual RCP yields; (B) normal probability of the internally studentized residuals; (C) internally studentized residuals vs. actual runs; (D) internally studentized residuals vs. predicted RCP yield. RCP, red clover

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polysaccharides.

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Fig. 3. Response surface (A, C, and E) and contour (B, D, and F) plots for RCP yield. RCP, red

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clover polysaccharides.

Fig. 4. Elution curve of RCP on the DEAE-52 cellulose (A), elution curve of RCP-1 on the

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Sepharose CL-6B column (B), and molecular weight distribution of RCP-1.1 (C). RCP, red

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clover polysaccharides.

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Fig. 5. HPLC chromatogram showed the monosaccharide composition of the purified RCP: (A) monosaccharide standards; (B) the purified RCP. 1, glucuronic acid; 2, galacturonic acid; 3,

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glucose; 4, galactose; 5, arabinose; 6, fucose. RCP, red clover polysaccharides.

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Fig. 6. Ultraviolet spectrum (A), Congo red test (B), and dissolution time (C) of the purified RCP. Letters (a-e) different in Fig.6C differ statistically (P<0.05). RCP, red clover polysaccharides.

Fig. 7. Hypoglycemic activity of the purified RCP: (A) inhibition ability on α-amylase activity; (B) inhibition ability on α-glucosidase activity. RCP, red clover polysaccharides. Fig. 8. Antioxidant activity of the purified RCP: (A) DPPH radical scavenging activity; (B) ABTS radical scavenging activity. RCP, red clover polysaccharides.

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Journal Pre-proof Author statement

Hexiang Zhang: Investigation. Jiangchao Zhao: Writing-Reviewing and Editing. Hongmei Shang: Conceptualization, Methodology, Data curation, Writing-Original draft preparation,

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Project administration. Yang Guo: Investigation. Shilun Chen: Investigation.

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Figure 1

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