1. Introduction
Acanthopanax senticosus (AS) is a shrub plant belonging to
Acanthaceae, mainly distributed in Russia, Japan, Heilongjiang, and Jilin of China. AS contains multiple nutrients and components as a medicinal and food homologous plant. AS has a variety of pharmacological activities, such as immunomodulatory [
1], antioxidant [
2], anti−inflammatory [
3], and anti−tumor [
4]. Based on the numerous pharmacological functions of polysaccharides, it has become a hot topic to study the extraction methods of polysaccharides to enhance their content without destroying the active ingredients.
Acanthopanax polysaccharides are extracted mainly through hot water extraction, acid−base extraction, ultrasonic−assisted extraction, and enzyme−assisted extraction. However, each method has its shortcomings. For instance, hot water extraction uses the principle of “similar compatibility” to extract polysaccharides while other water−soluble components are also extracted, and high extraction temperature results in low purity of polysaccharides, long time consumption, and low extraction rate. The acid−base extraction method has the disadvantage of easily destroying the structure of polysaccharides and reducing the pharmacological activity of polysaccharides [
5]. Although the ultrasonic−assisted extraction method can destroy the cell wall of plants and improve the extraction rate, it has the problems of slow and time−consuming temperature rise and high energy consumption [
6]. Enzyme−assisted extraction technology uses the property of enzymes to break down plant tissues and accelerate the release of polysaccharides. It is an effective extraction method that is gentle, specific, quick, high rate, environmentally friendly, and maintains the biological activity of polysaccharides [
7]. Various enzymes are widely used to extract bioactive components, such as cellulase, pectinase, hemicellulase, and amylase. Cellulases are enzymes that break down cellulose into glucose, cellobiose, and higher glucose polymers; Pectinases can break down pectin compounds [
8]; Hemicellulases can break down hemicellulose in plant cell walls and accelerate polysaccharide solubilization [
9]. Several studies have demonstrated the advantages of enzymes in extracting plant polysaccharides. Gao et al. [
10] extracted
E. sibiricumbulb polysaccharides with amylase, and the yield reached 59.71 ± 2.72% after optimization of extraction conditions, which was much higher than that of hot water extraction (37.25 ± 0.17%). Shi et al. [
11] extracted
Dendrobium polysaccharides with the aid of a combination of cellulase and pectinase, resulting in a much higher extraction rate. Wang et al. [
12] extracted
pumpkin seed polysaccharides with cellulase and compared the yield with hot water extraction and ultrasonic extraction and concluded that the enzyme extraction (3.22% ± 0.04%) gave a higher yield than hot water extraction (2.18%) and ultrasonic extraction (2.29%). The molecular weights of the polysaccharides obtained by the three extraction methods were different, indicating that the extraction methods affect the molecular weight of polysaccharides.
The bioactivity of polysaccharides is influenced by molecular weight [
13]. Screening the molecular weight range for better activity is conducive to improving the bioavailability of polysaccharides and developing polysaccharides with better action. Ethanol graded precipitation method is often used to separate polysaccharides of different molecular weight sizes. The precipitation using different ethanol concentrations can yield polysaccharide fractions of various molecular weights, which have other properties and can be used in different pathways [
14]. Therefore, ethanol−graded precipitation is widely used. In studying
dandelion polysaccharides, one researcher used ethanol graded precipitation method to obtain two polysaccharide fractions of DRP−2b and DRP−3a with sizes of 31.8 kDa and 6.72 kDa, respectively, and the antioxidant capacity of DRP−3a was higher than that of DRP−2b at a concentration of 1 mg/mL, and the clearance of DPPH by DRP−2b and DRP−3a was 28.82 ± 2.45% and 71.58 ± 3.11%, respectively [
15]. Long investigated the binding ability of different fractions of hot water−extracted
Caulerpa lentillifera polysaccharides to bile acids after graded precipitation by ethanol [
16]. The findings demonstrated that the smaller molecular weights of WCLP−55 and WCLP−75 had a much better bile acid binding ability than the other fractions [
17]. In their study of the hypoglycemic effect of
senna polysaccharides precipitated from different ethanol concentrations (20%, 40%, 60%, and 80%), they found that HP80 with the minimum molecular weight of molecular weight showed the most potent inhibitory activity against α−amylase and α−glucosidase when they investigated the hypoglycemic effect of senna polysaccharides precipitated by different ethanol concentrations. Gao et al. [
18] isolated
lychee polysaccharides using four different methods and discovered that those with the highest molecular weight had the most antioxidant action. In recent years, more and more researchers have obtained polysaccharides of different molecular weight sizes by graded precipitation of ethanol, intending to develop a highly bioavailable polysaccharide.
At present, most of the studies on Acanthopanax polysaccharides have focused on their pharmacological effects. Few studies have been reported on the extraction process and physicochemical properties of Acanthopanax polysaccharides, which are the key factors for their practical application. Therefore, it is worthwhile to conduct research into the Acanthopanax polysaccharide extraction procedure and its physicochemical characteristics. In this paper, three enzymes, cellulase, hemicellulase, and pectinase, were selected for the extraction of Acanthopanax polysaccharides by the complex enzyme−assisted hot water method. The response surface was used to optimize the extraction procedure. Ethanol−graded precipitation was used to separate polysaccharides with different molecular weights. Physicochemical properties of different molecular weights of polysaccharides were compared by HPGPC, ion chromatography, FT−IR, UV, SEM, TGA, XRD, Congo red, and I2−KI. It provides some technical support for the clinical application of Acanthopanax polysaccharide.
3. Materials and Methods
3.1. Materials and Chemicals
A. senticosus was obtained from Limin Pharmacy (Liaocheng, China), ground into a powder using a grinder (H8422, Hebei Huicai Technology Co., Ltd., Hebei, China), and sieved through a 60−mesh sieve. Cellulase (1 × 104 U/g), Pectinase (3 × 104 U/g), and Hemicellulase (2 × 105 U/g) were purchased by Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). All other reagents were at least of analytical grade.
3.2. Preparation of A. senticosus Polysaccharide
The powder (1.0 g) was precisely weighed and placed in a clay−shaped bottle. The enzyme quantity, solid–liquid ratio, enzymatic digestion time, enzymatic digestion temperature, and pH were analyzed in a constant−temperature water bath boiler. After enzymatic digestion, the solution was heated to 95 °C for 5 min to activate the enzyme. Filtration was performed with filter paper, and the filtrate was collected to obtain the polysaccharide extracts. The extraction solution was added 95% ethanol to reach an 80% ethanol final concentration and then left at 4 °C overnight. After the alcoholic precipitation, the polysaccharide extract from
A. senticosus was obtained by centrifugation and drying. The polysaccharide yields were determined using the following equation:
3.3. Experimental Design
3.3.1. Single Factor Experiment
In order to determine the optimal dosage of each enzyme, tests were carried out according to the optimal enzymatic conditions for the three enzymes (pectinase temperature 50 °C, pH 3.4, cellulase temperature 50 °C, pH 5, hemicellulase temperature 30 °C, pH 5). A total of 1.0 g of Acanthopanax powder was precisely weighed to screen the amount of cellulase addition (25 U/g, 50 U/g, 100 U/g, 200 U/g, 400 U/g), pectinase addition (12.5 U/g, 25 U/g, 50 U/g, 100 U/g), hemicellulase addition (25 U/g, 50 U/g, 100 U/g, 200 U/g, 400 U /g), respectively.
3.3.2. Compound Enzyme Ratios Optimized by Orthogonal Experiment
Based on the above single−factor test results, we used the orthogonal test L
9(3
3) to design the optimal concentration ratios of cellulase, pectinase, and hemicellulase. The fixed extraction conditions were as follows: the solid–liquid ratio was 1:40 (g/mL), the extraction temperature was 50 °C, the extraction time was 60 min, and the pH was 5.0. The experimental design is shown in
Table 5. The experiment was conducted in nine groups of parallel tests, and the extracts were collected and treated according to the method in 3.2.
3.3.3. Single−Factor Experiment
The 1.0 g of Acanthopanax powder was weighed precisely. The five factors were investigated according to the optimal compound enzyme ratio determined by the orthogonal test, which included the amount of compound enzyme (600 U/g, 800 U/g, 1000 U/g, 1200 U/g, 1400 U/g), enzymatic pH (3, 4, 5, 6, 7), enzymatic temperature (30 °C, 40 °C, 50 °C, 60 °C, 70 °C), enzymatic time (20, 40, 60, 80, 100 min), solid–liquid ratio (1:15, 1:20, 1:30, 1:40, 1:50). The polysaccharide yield was determined according to the steps in 3.2, and the extraction was conducted in triplicate.
3.3.4. Response Surface Test
Based on single−factor tests, the original range of independent variables was chosen. A four−factor, three−level response surface experiment was designed utilizing the response surface methodology (RSM) and Box–Behnken design (BBD). The enzyme treatment pH, temperature, time, and solid–liquid ratio were used as independent variables, and the extraction rate of polysaccharides was used as the dependent variable. These are shown in
Table 6.
3.3.5. Validation Tests
Three repetitions of enzyme−assisted aqueous extraction of Acanthopanax polysaccharides using the optimal conditions derived from the response surface.
3.4. Ethanol−Graded Precipitation of Polysaccharides
The optimum technique for extracting Acanthopanax polysaccharides was utilized for the extraction procedure based on the experiments mentioned above. The extraction liquid was mixed with 95% ethanol until it had a final concentration of 80% ethanol in water, and then put in a refrigerator at 4 °C for 12 h. Remove the supernatant to obtain crude polysaccharide. By adding the right amount of distilled water, the polysaccharide was dissolved. It was then deproteinized with the Sevage method, depigmented with H2O2, and then precipitated with 80% ethanol to get pure polysaccharide. Add distilled water to dissolve the purified polysaccharide, add 95% ethanol to the ethanol final concentration of 40%, rest for 12 h at 4 °C in the refrigerator, centrifuge at 3000 r/min for 5 min, and the supernatant was separated, the precipitate was placed at 105 °C and dry for 1.5 h to obtain ASPS−40. The supernatant was added with 95% ethanol to 60% ethanol concentration and left to stand for 12 h at 4 °C in the refrigerator. The subsequent operation was the same as above to obtain ASPS−60 and ASPS−80.
3.5. Component Analysis
The phenol−sulfuric acid method was employed to measure the total amount of sugar, with glucose serving as a reference [
48]. The Coomassie Brilliant Blue G−250 staining method was used to determine the protein content with bovine serum albumin as standard [
49]. The glucuronide content was determined by the m−hydroxyphenyl colorimetric method using D−glucuronide as a standard [
50].
3.6. Molecular Weight Determination
HPGPC determined the molecular weights of ASPS−40, ASPS−60, and ASPS−80. The samples and standards were carefully weighed, the samples were made into a 5 mg/mL solution, spun at 10,142× g for 10 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane. The sample was then put into an injection tube with 1.8 mL. The chromatographic column used in this study was a BRT105−104−102 tandem gel column with dimensions of 8 × 300 mm. The mobile phase employed was a 0.05 M NaCl solution, and the flow rate was set at 0.6 mL/min. The column temperature was maintained at 40 °C throughout the experiment. A volume of 25 μL was injected into the column, and the detector used was the Differential detector RI−10A.
3.7. Monosaccharide Composition Analysis
The 5 mg of sample was placed in an ampoule, 3 M trifluoroacetic acid (TFA) 2 mL added, and hydrolyzed at 80 °C for 3 h. The acid hydrolysis solution was accurately aspirated, transferred to a tube with nitrogen blowing, and dried. 5 mL of water, vortexed to mix and centrifuged at 10,142× g for 5 min. The supernatant was taken into the ion chromatograph for analysis.
The chromatographic column used in this study was the Dionex Carbopac TM PA20, with dimensions of 3 × 150 mm. The mobile phase consisted of three components: A, which was composed of H2O; B, which contained 15 mM NaOH; and C, which consisted of a mixture of 15 mM NaOH and 100 mM NaOAc. The flow rate of the mobile phase was set at 0.3 mL/min. A volume of 25 µL was injected into the column. The column temperature was maintained at 30 °C throughout the experiment. An electrochemical detector was employed to detect and analyze the compounds.
3.8. Fourier Transform Infrared (FT−IR) Analysis
The dried sample (1.0 mg) was mixed with 150 mg of KBr powder, uniformly ground, and pressed, and then FT−IR spectra were recorded from 400 to 4000 cm−1 using a Nicolet iS10 FT−IR spectrometer (Thermo Fisher Shanghai, China).
3.9. UV−Vis Spectroscopy
The UV−vis spectra of the polysaccharide solutions were recorded in the range of 200–500 nm using a UV−vis spectrophotometer (Nano Ready F−1100 Shanghai Metash Instruments Co., Ltd., Shanghai, China).
3.10. Thermogravimetric Analysis
Thermogravimetric analysis was performed in the temperature range of 30 °C–400 °C with a scanning speed of 10 °C/min using a NETZSCH 209 F1 instrument German, N2 environment.
3.11. SEM Analysis
This study compared the morphological characteristics of three different molecular weights polysaccharide by uniformly attaching the dried powder to the sample stage and spraying gold. Imaging was performed using a scanning electron microscope (Tokyo, Japan, Electron Optics Laboratory jsm−it100) at an accelerating voltage of 15.0 kV and a magnification of 5000×.
3.12. X-ray Diffraction (XRD) Analysis of ASPSs
The X-ray diffractograms were taken with a D8 Advance X-ray diffractometer utilizing copper lamp radiation (Cu−Ka) at 15 mA and 40 kV, 2°/min, 5–70 °C.
3.13. Congo Red and I2−KI Test
Congo red (80 μmol/L, 2.0 mL) was mixed with polysaccharide solution (2.0 mg/mL, 2.0 mL), and a specific volume of NaOH solution (1 mol/L) was added to make the final concentration of NaOH between 0.0 mol/L and 0.5 mol/L. After the reaction at room temperature for 5 min, the maximum absorption wavelength (λmax) was measured using a UV−Vis spectrophotometer (Nano Ready F−1100 Shanghai Metash Instruments Co., Ltd., Shanghai, China).
The I2−KI test involved mixing I2−KI (0.2%, 8.0 mL) with polysaccharide (2.0 mg/mL, 2.0 mL) and recording the absorbance at 300–700 nm with a UV−Vis spectrophotometer (Nano Ready F−1100 Shanghai Metash Instruments Co., Ltd., Shanghai, China).
3.14. Analysis of Antioxidant Activity
3.14.1. ABTS Radical Scavenging Activity Assay
A described approach was slightly modified to test the abilities of polysaccharides to scavenge ABTS radicals [
51]. The samples (4 mL) at different concentrations (5, 10, 20, 30, 40 mg/mL) were mixed with 5.8 mL of ABTS working solution. Then, the mixtures were shaken and kept at room temperature for 30 min in a dark room. The absorbance of the mixture was measured at 734 nm with a UV spectrophotometer. The same concentration of Ascorbic acid (Vc) was used as the positive control. Anhydrous ethanol was used as a blank control. The ABTS scavenging activity was calculated using the following formula:
where A
1, A
2, and A
3 were the absorbances of the ABTS blank, sample, and control, respectively.
3.14.2. DPPH Radical Scavenging Activity Assay
Polysaccharides were tested for their DPPH radical−scavenging activities using a previously reported slightly modified [
52]. In brief, 2.0 mL of samples at different concentrations (0.5, 1, 2, 3, 4, 5 mg/mL) was added to 2.0 mL of a methanolic solution (0.2 mM) of DPPH. The mixture was shaken and mixed and then reacted in the dark for 30 min. The absorbance of the mixture was measured at 517 nm. The same concentration of Ascorbic acid (Vc) was used as the positive control. Anhydrous ethanol was used as a blank control. The experiment was conducted in triplicate. This scavenging activity was calculated by following the equation.
where B
1, B
2, and B
3 were the absorbances of the DPPH control, sample, and blank, respectively.
3.14.3. Fe2+ Chelating Activity
The Fe
2+ chelating capabilities of ASPSs were mildly modified [
53]. For each sample (400 μL), 50 μL of FeCl
2 solution (2 mM), 100 μL of ferrozine solution (5 mM), and 2 mL of methanol were combined. The mixture was agitated well, reacted for 10 min at room temperature, and measured at 562 nm with ethylenediaminetetraacetic acid disodium salt as a positive control. The following formula calculated Fe
2+ chelating activity.
where C
1, C
2, and C
3 were the absorbances of the control, sample, and blank, respectively.
3.14.4. H2O2 Radical Scavenging Activity Assay
The ability to remove H
2O
2 was determined according to the reported method with minor modifications [
54]. A solution was created with 1 mL of freshly made H
2O
2 (0.1 mmol L
−1), 1 mL of diluted polysaccharide, 0.1 mL of ammonium molybdate (3%
w/
v), 10 mL of H
2SO
4 (2 mol L
−1), and 7 mL of KI (1.8 mol L
−1). The mixture was titrated with Na
2S
2O
3 (5 mmol L
−1) until the yellow color faded. For comparison, the removal capacity of L−ascorbic acid at the same sample concentration was determined. The removal activity was calculated according to the following equation:
where, V
0 was the volume of the Na
2S
2O
3 solution used to titrate the control mixture and V
1 was the titration volume of the sample−containing mixtures.
3.15. Statistical Analysis
The Box–Behnken experimental design was designed with Design−Expert 13, the graph was plotted with Origin 2021, and the data were analyzed with SPSS statistical software version 24.0. The results were shown as the mean ± standard deviation, and the data were analyzed using a one−way ANOVA.
4. Conclusions
The polysaccharides from A. senticosus had been extracted from the Acanthopanax root by enzymolysis−assisted hot water. The study showed that the content and extraction rate of Acanthopanax polysaccharides could be significantly improved. After optimization, the polysaccharide extraction rate from A. senticosus was 10.53 ± 0.682%, which was about 2.5 times higher than that of hot water extraction, and the polysaccharide content was directly affected by enzymolysis. Three polysaccharide fractions, namely, ASPS−40, ASPS−60, and ASPS−80, were obtained by ethanol−graded precipitation, and the three polysaccharide fractions differed significantly in total sugar, protein, and glyoxylate contents. ASPS−60 had the highest polysaccharide yield, ASPS−40 contained glyoxylate, and glucose and xylose were the main monosaccharides constituting the three polysaccharides. The FT−IR and TGA results showed that the three polysaccharides did not differ in structure and thermal stability, but SEM showed that the three polysaccharides had distinctly different microstructures. The XRD suggests that the internal structure of ASPSs is amorphous. The Congo red test proved that all three polysaccharides have a triple helix structure, and the I2−KI test confirmed that all three polysaccharides have longer branched chains and more side chains. The results of antioxidant tests conducted by four methods showed that the antioxidant activity of polysaccharides is not only related to the molecular weight size but also can be related to its composition and structure. This study can improve the yield of Acanthopanax polysaccharides and provide a research direction and reference for further development and utilization of A. senticosus.