Chuanminshen violaceum crude polysaccharide as well as extraction method and application thereof

Abstract

The invention discloses a Chuanminshen violaceum crude polysaccharide and an extraction method and application thereof.The extraction method comprises the steps that a deep-eutectic solvent composed of choline chloride and citric acid in the molar ratio of 1: 1 is adopted, the water content of the deep-eutectic solvent is 30%, Chuanminshen violaceum powder and the deep-eutectic solvent are extracted under the heating condition according to the liquid-material ratio of (19-20) mL: 1 g, the extraction temperature is 70-80 DEG C, the extraction time is 1 h, and the extraction time is 30 h; the extraction time is 110 to 130 minutes; after the extraction is finished, centrifugally collecting supernate, and precipitating by adopting absolute ethyl alcohol; taking the precipitate, redissolving the precipitate with water, carrying out deproteinization treatment, concentrating the treatment liquid, transferring the concentrated treatment liquid into a dialysis bag with the molecular weight cut-off of 3500 Da for dialysis, and freeze-drying the dialyzed polysaccharide liquid to obtain a crude Chuanminshen violaceum polysaccharide sample named as CVP. According to the method, the extraction rate of Chuanminshen violaceum crude polysaccharide can be remarkably increased, the obtained polysaccharide is uniform in structure and excellent in thermal stability, and the molecular weight is remarkably reduced compared with that of a traditional water extraction method.

Description

Technical Field

[0001] The present invention relates to a method for extracting polysaccharides, and particularly to a crude polysaccharide from Chuanminshen, its extraction method and application. Background Art

[0002] Chuanminshen ( Chuanminshen violaceum M. L. Sheh&R. H. Shan ), also known as Adenophora stricta, Ming Adenophora, and Tumen Adenophora, is a perennial herb of the Umbelliferae family. Its roots are used for moistening the lungs and resolving phlegm, strengthening the spleen and nourishing the stomach, etc. As the main active substance in Chuanminshen, polysaccharides have rich biological activities and health benefits. Polysaccharides not only contain important food nutritional value, but also are regarded as ideal objects for the development of functional foods due to their significant health care functions. Chuanminshen polysaccharides are the main bioactive components in Chuanminshen, with significant physiological activities such as antioxidant, hypoglycemic, immunomodulatory, and intestinal flora regulating effects. Therefore, Chuanminshen polysaccharides ( Chuanminshen violaceum polysaccharides, CVP) have high market value and application prospects.

[0003] Currently, the main methods for extracting polysaccharides are: water extraction method, alkali extraction method, acid extraction method, enzyme leaching method, etc. Among them, the water extraction method has low efficiency, high energy consumption, and long time consumption. The acid extraction method has a short time consumption and is easy to operate, but the volume of the extraction solution is large, and the subsequent concentration and purification processes are more difficult, and it is easy to cause environmental pollution. The alkali extraction method has a high yield, but causes greater damage to the structure of polysaccharides. The enzyme extraction method has high efficiency, but the cost is high and the reaction conditions are more stringent. For the extraction of Chuanminshen polysaccharides, the ultrasonic-microwave synergistic method is used to extract Chuanminshen polysaccharides, and the extraction rate of crude polysaccharides is 26.12%; the ethanol reflux combined with the enzyme method is used to extract Chuanminshen polysaccharides, and the extraction rate of crude polysaccharides is 5.41%. Generally speaking, traditional methods have problems such as low extraction rate and poor process adaptability.

[0004] Deep eutectic solvent (DES) is a new type of green and environmentally friendly extraction solvent, which is composed of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD). Compared with traditional solvents and ionic liquids, it has the advantages of non-toxicity, low price, simple synthesis, high efficiency, etc. At present, DES has attracted much attention in the extraction of bioactive components. As an economical green solvent, it is widely used in many fields such as food, biomedicine, and chemistry. The extraction rate of polysaccharides by DES is high, the purity is high, and there are fewer impurities. Compared with the traditional hot water extraction method, DES has stronger ability to remove proteins and calcium carbonate. At present, there are few studies on the application of DES in the extraction of Chuanminshen polysaccharides.

[0005] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure, and thus may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention

[0006] The object of the present invention is to provide a Chuanminshen crude polysaccharide, its extraction method and application, which solves the problem of low extraction rate of the existing Chuanminshen crude polysaccharide, can significantly improve the extraction rate of Chuanminshen crude polysaccharide, and the obtained polysaccharide has a uniform structure, excellent thermal stability, and its molecular weight is significantly lower than that of the traditional water extraction method.

[0007] To achieve the above object, the present invention provides an extraction method for Chuanminshen crude polysaccharide, which includes: using a deep eutectic solvent composed of choline chloride and citric acid with a molar ratio of 1:1, the water content of the deep eutectic solvent is 30%, and extracting the Chuanminshen powder with the deep eutectic solvent at a liquid-solid ratio of (19 - 20) mL: 1 g under heating conditions, the extraction temperature is 70 - 80 °C, and the extraction time is 110 - 130 min; after the extraction is completed, centrifuge to collect the supernatant and precipitate with absolute ethanol; take the precipitate, redissolve it with water, perform deproteinization treatment, concentrate the treatment solution, transfer it to a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis, and freeze-dry the dialyzed polysaccharide solution to obtain a Chuanminshen crude polysaccharide sample, named CVP.

[0008] Preferably, the molecular weight of the CVP is 3.82 kDa.

[0009] Preferably, the extraction method further includes: performing gradient elution on the Chuanminshen crude polysaccharide sample using a DEAE-52 cellulose ion exchange chromatography column, and using ultrapure water and 0.10 mol / L NaCl solution for gradient elution. The ultrapure water elution fraction is named CVP-1, and the 0.1 mol / L NaCl solution elution fraction is named CVP-2. By eluting the Chuanminshen crude polysaccharide sample with a DEAE-52 cellulose ion exchange chromatography column, acidic polysaccharides are removed to obtain neutral polysaccharides.

[0010] More preferably, the molecular weight of the CVP-1 is 4.33 kDa, and the molecular weight of the CVP-2 is 18.65 kDa.

[0011] More preferably, the extraction method further includes: separately eluting CVP-1 and CVP-2 using a Sephadex G-100 gel column, with the eluent being ultrapure water, and the collected fractions being named CVP-1-1 and CVP-2-1 respectively, where the molecular weight of CVP-1-1 is 9.47 kDa and the molecular weight of CVP-2-1 is 18.15 kD. By eluting with a Sephadex G-100 gel column, polysaccharides with different molecular weights are obtained.

[0012] Preferably, the liquid-to-material ratio is 20 mL: 1 g, the extraction temperature is 80 °C, and the extraction time is 120 min.

[0013] Preferably, the Chuanminshen powder is screened through an 80-mesh sieve; and / or, for the centrifugation, it is centrifuged at 4000 r / min.

[0014] Preferably, for the precipitation with absolute ethanol, 4 times the volume of absolute ethanol is added to the supernatant for precipitation, and the precipitation is allowed to stand at low temperature at 4 °C.

[0015] Preferably, for the protein removal treatment, the Sevage method is used, with the volume ratio of chloroform to n-butanol being 4:1 and the volume ratio of polysaccharide to Sevage reagent being 3:1.

[0016] Preferably, for the freeze-drying, the polysaccharide solution is first frozen in an environment of -80 °C for more than 4 h and finally freeze-dried in a freeze-dryer.

[0017] The second object of the present invention is to provide the crude polysaccharide of Chuanminshen obtained by the extraction method described above.

[0018] Preferably, the crude polysaccharide of Chuanminshen is any one or more of CVP, CVP-1, CVP-2, and CVP-1-1; among them, CVP, CVP-1, CVP-2, and CVP-1-1 are composed of glucose and galactose, and CVP-2-1 is composed of glucose, galactose, and rhamnose.

[0019] The third object of the present invention is to provide the application of the crude polysaccharide of Chuanminshen in improving the intestinal microecology.

[0020] Preferably, the crude polysaccharide of Chuanminshen can increase the short-chain fatty acids produced by the intestinal flora, and / or improve the richness of the intestinal flora.

[0021] More preferably, the crude polysaccharide of Chuanminshen can up-regulate the ratio of Bacteroidetes / Firmicutes.

[0022] More preferably, the crude polysaccharide of Chuanminshen can increase the relative abundance of beneficial bacteria and reduce the relative abundance of pathogenic bacteria; among them, the beneficial bacteria include: BacteroidesBacteroides ), Roseburia Phascolarctobacterium ), and Lactococcus Lactococcus ), etc.; the pathogenic bacteria include: Enterobacteriaceae Enterobacteriaceae ), Shigella Escherichia-Shigella ), etc.

[0023] The Chuanminshen crude polysaccharide, its extraction method and application of the present invention solve the problem of low extraction rate of the existing Chuanminshen crude polysaccharide, and have the following advantages: (1) The present invention adopts the DES method, optimizes the extraction process of Chuanminshen polysaccharide through single factor experiment combined with response surface experiment, and obtains purified polysaccharide through ion exchange column and gel column chromatography, which can significantly improve the extraction rate of Chuanminshen crude polysaccharide, and the obtained polysaccharide has a uniform structure and excellent thermal stability, and its molecular weight is significantly lower than that of the traditional water extraction method, indicating that the Chuanminshen polysaccharide extracted by DES may have higher antioxidant activity potential; (2) The Chuanminshen crude polysaccharide obtained by the method of the present invention, CVP, CVP-1, CVP-2, CVP-1-1 are composed of glucose and galactose, and CVP-2-1 is composed of glucose, galactose and rhamnose; (3) The Chuanminshen crude polysaccharide obtained by the method of the present invention, the molecular weights of CVP, CVP-1, CVP-2, CVP-1-1 and CVP-2-1 are 3.82 kDa, 4.33 kDa, 18.65 kDa, 9.47 kDa, 18.15 kD respectively, CVP-1-1 and CVP-2-1 are homogeneous polysaccharides, and the molecular weight of the Chuanminshen polysaccharide extracted by DES is much smaller than that of the polysaccharide extracted by the water extraction method; (4) The Chuanminshen crude polysaccharide obtained by the method of the present invention reveals the interaction law between CVP and intestinal flora through an in vitro fermentation model, analyzes its action mechanism, and predicts potential functional pathways, which can improve the intestinal microecology, provide a reference for the further development and utilization of Chuanminshen resources, and lay a theoretical foundation for its application in the fields of health food and medicine. Description of the Drawings

[0024] Figure 1 Shows the effects of different types of DES on the extraction rate of CVP.

[0025] Figure 2 Shows the effects of different single factors on the extraction rate of CVP; (a) solid-liquid ratio; (b) extraction temperature; (c) extraction time.

[0026] Figure 3 Shows the response surface (A, C, E) and contour plot (B, D, F) of the interaction of each factor.

[0027] Figure 4It is a figure of the elution curve of cellulose DE-52 (a) and the elution curves of Sephadex G-100 chromatography columns for dextran (b, c).

[0028] Figure 5 It is the determination result of the monosaccharide composition of 5 polysaccharides.

[0029] Figure 6 It is the determination result of the infrared spectra of 5 polysaccharide components.

[0030] Figure 7 It is the scanning electron microscope results of 5 polysaccharide components; (a) CVP; (b) CVP-1; (c) CVP-2; (d) CVP-1-1; (e) CVP-2-1.

[0031] Figure 8 It is the thermal stability results of 5 polysaccharide components; (a) CVP; (b) CVP-1; (c) CVP-2; (d) CVP-1-1; (e) CVP-2-1.

[0032] Figure 9 It is the change in pH value (a) and the change in gas production (b) during in vitro fermentation of the blank group and the polysaccharide group.

[0033] Figure 10 It is the Chao1 (a), Ace index (b), Venn diagram (c) and PcoA analysis diagram (d) of the blank group and the polysaccharide group during in vitro fermentation.

[0034] Figure 11 Bacterial abundance distribution maps and heat maps (c) at the phylum level (a) and genus level (b) of different treatment groups after 48 h of fermentation.

[0035] Figure 12 It is the KEGG function prediction map of the polysaccharide; (a) Results of three-level KEGG enrichment analysis; (b) GO enrichment analysis is classified into biological process BP at the three-level classification; (c) Cellular component CC; (d) Molecular function MF.

[0036] Figure 13 Pearson correlation analysis of polysaccharides and fecal flora at the genus level (a); Pearson correlation analysis of short-chain fatty acids (SCFAs) and fecal flora at the genus level (b). Specific implementation manners

[0037] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0038] It should be noted that for those not specifying specific conditions in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the instruments without specifying the manufacturer, they are all conventional products that can be obtained through commercial purchase. For the raw materials and reagents without specifying the manufacturer, they are all commercially available products or can be prepared by known methods.

[0039] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of simplicity and convenience. Accordingly, the description of the numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range (including integers and fractions).

[0040] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features. All possible combinations should be considered as the scope described in this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0041] Experimental Example 1 Preparation and Screening of DES 1. Preparation of DES To investigate the effect of DES with different hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) on the extraction ability of Chuanminshen polysaccharide, the specific steps are as follows: Seven kinds of DES were prepared with choline chloride (ChCl) as the hydrogen bond acceptor (HBA) and urea (U), propylene glycol (PG), 1,4-butanediol (BDO), ethylene glycol (EG), lactic acid (LA), citric acid (CA), and malic acid (MA) as the hydrogen bond donors (HBD), respectively, and the water content was selected to be 30%. Among them, the molar ratios of ChCl-U, ChCl-PG, and ChCl-EG are 1:2; the molar ratios of ChCl-LA, ChCl-CA, and ChCl-MA are 1:1; the molar ratio of ChCl-BDO is 1:4.

[0042] The HBD and HBA were mixed in the corresponding molar ratio and then water was added. The mixture was stirred in a water bath at 80 °C until completely dissolved to obtain a homogeneous and transparent liquid for standby.

[0043] 2. Screening of DES Fresh Chuanminshen was dried at a constant temperature of 60 °C until constant weight, pulverized, and passed through an 80-mesh sieve to obtain Chuanminshen powder. An appropriate amount of Chuanminshen powder was accurately weighed, and DES with the corresponding molar ratio and water content was added according to the liquid-to-solid ratio of 20:1 (mL / g). The extraction temperature was set at 80 °C and the extraction time was 30 min.

[0044] After extraction, centrifuge at 4000 r / min for 10 min and collect the supernatant. Add 4 times the volume of absolute ethanol to the supernatant for precipitation, and let it stand at 4 °C overnight for low-temperature precipitation.

[0045] Take the precipitate, dissolve it in deionized water, and use the Sevage method to remove proteins from the aqueous solution. The volume ratio of chloroform to n-butanol is 4:1, and the volume ratio of polysaccharide to Sevage reagent is 3:1. Concentrate the treated solution with a rotary evaporator and transfer it to a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis. Freeze the dialyzed polysaccharide solution at -80 °C for more than 4 h, and finally freeze-dry it in a freeze dryer to obtain the CVP sample.

[0046] As Figure 1 shown, the extraction rate results of different DESs on Chuanminshen polysaccharide in Experimental Example 1 of the present invention are presented. It can be seen that the extraction rates of the seven DES combinations for Chuanminshen polysaccharide are arranged in descending order as ChCl-CA > ChCl-MA > ChCl-LA > ChCl-EG > ChCl-PG > ChCl-U > ChCl-BDO. Among them, the extraction rate of the DES with the ChCl-CA combination is (37.47 ± 0.47)%, which is significantly higher than other combinations. Therefore, ChCl-CA is selected as the extraction solvent for Chuanminshen polysaccharide, and subsequent process optimization is carried out.

[0047] Experimental Example 2 Optimization of the extraction process of Chuanminshen polysaccharide 1. Single-factor experimental design The molar ratio of ChCl-CA is 1:1, and the water content is 30%. Based on a liquid-to-solid ratio of 20:1 (mL / g), an extraction temperature of 80 °C, and an extraction time of 90 min, the effects of three factors, namely the liquid-to-solid ratio, extraction temperature, and extraction time of DES, on the extraction rate of Chuanminshen polysaccharide were investigated. The liquid-to-solid ratio (mL / g) was set at 5:1, 10:1, 15:1, 20:1, and 25:1 respectively; the extraction time was set at 60 min, 90 min, 120 min, 150 min, and 180 min; the extraction temperature was 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C.

[0048] As Figure 2 shown, the single-factor experimental results of the optimization of the extraction process of Chuanminshen polysaccharide in Experimental Example 2 of the present invention are presented. The results show that in the investigation of the liquid-to-solid ratio, the extraction rate of Chuanminshen polysaccharide is the best at a liquid-to-solid ratio of 20:1 (mL / g); in the investigation of the extraction temperature, the extraction rate of Chuanminshen polysaccharide is the best at an extraction temperature of 70 °C; in the investigation of the extraction time, the extraction rate of Chuanminshen polysaccharide is the best at an extraction time of 120 min.

[0049] 2. Response surface experimental design The Box-Behnken design was carried out using Design-Expert 12.0 software to optimize the conditions for extracting CVP with a deep eutectic solvent. A response surface experiment design with three factors and three levels was adopted, namely the liquid-to-solid ratio A (mL / g), extraction temperature B (°C), and extraction time C (min). The response value was the extraction rate of Chuanminshen polysaccharide (see Table 1). The results of variance analysis and significance test are shown in Table 2. The quadratic regression equation fitted by statistical analysis was Y = 45.15 - 1.82*A + 5.31B - 0.145*C + 1.30*AB + 0.9275*AC - 2.11*BC - 8.64*A² - 11.86*B² - 4.17*C².

[0050] Table 1 Factors and levels of the response surface experiment

[0051] Table 2 Variance analysis and significance test

[0052] As Figure 3 shown, it is the response surface and contour results of the extraction process optimization of Chuanminshen polysaccharide in Experimental Example 2 of the present invention. After the optimization of the response surface experiment, the verified optimal conditions were: liquid-to-solid ratio 20:1 (mL / g), extraction temperature 80 °C, extraction time 120 min, and the CVP extraction rate reached (44.17 ± 0.19)%, which was close to the predicted value of 45.84%. This was significantly higher than the CVP extraction rates obtained by the traditional water extraction method (30.45%) used by Dong Hongmin (Research on the Extraction, Analysis and Antioxidant Activity of Alcohol Extracts and Polysaccharides from Chuanminshen [D]. Sichuan: Sichuan Agricultural University, 2017), the microwave-assisted extraction method (34.59%), and the ultrasonic microwave-synergistic extraction method (26.12%) used by Wei Ruilin (Research on the Effect of Chuanminshen Polysaccharide on Regulating Lipid Metabolism and Intestinal Flora in Mice Fed a High-Fat Diet [D]. Sichuan: Xihua University, 2024). Moreover, the extraction temperature was significantly lower than that of the conventional high-temperature reflux method (80 - 100 °C), effectively avoiding polysaccharide degradation.

[0053] Experimental Example 3 Separation, purification and component identification 1. Separation and purification The CVP was gradient eluted using a DEAE-52 cellulose ion exchange chromatography column, and gradient elution was carried out using ultrapure water and 0.10 mol / L NaCl solution. The elution curve is shown in Figure 4 (a). The component eluted with ultrapure water was named CVP-1, and the component eluted with 0.1 mol / L NaCl solution was named CVP-2.

[0054] Then, Sephadex G-100 gel column was used to elute CVP-1 and CVP-2 respectively, with ultrapure water as the eluent. The collected components were named CVP-1-1 and CVP-2-1 respectively, and the elution curves are shown in Figure 4 the (b) and (c) of

[0055] 2. Determination of chemical composition (1)Determination of total sugar content After establishing the standard curve for polysaccharide content by the phenol-sulfuric acid method (Y = 7.6581X + 0.1077, R 2 = 0.9994), for the Chuanminshen polysaccharide sample obtained above (1 mg / mL, dissolved in water to prepare a polysaccharide aqueous solution), 0.5 mL was taken, added with water to 2 mL, then 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid were added, shaken well and allowed to stand for 30 min, and then the absorbance was measured at 490 nm. Thus, the total sugar contents of the purified polysaccharides CVP-1-1 and CVP-2-1 reached (94.87 ± 0.80)% and (92.01 ± 1.77)%, respectively.

[0056] (2)Determination of uronic acid content After establishing the uronic acid standard curve by the m-hydroxybiphenyl method (Y = 1.3436X + 0.0555, R 2 = 0.9977), the Chuanminshen polysaccharide sample obtained above was prepared into a 0.5 mg / mL solution. 100 μL was taken, added with 1 mL of sodium tetraborate-sulfuric acid solution and mixed well. After reacting at 95 °C for 15 min, it was taken out and cooled to room temperature, then 20 μL of 3-phenylphenol was added, and the reaction was carried out in the dark for 10 min. Finally, the absorbance was measured at 520 nm. The results showed that only trace amounts of uronic acid were contained in the 5 polysaccharide components.

[0057] (3)Determination of protein content For the detection of protein content in polysaccharides, a standard curve was established by the Coomassie brilliant blue method (Y = 0.2555X + 0.0371, R 2 = 0.9966). 200 μL of 1 mg / mL Chuanminshen polysaccharide sample was mixed with 1 mL of Coomassie brilliant blue solution, allowed to stand for 10 min, and the absorbance was measured at 595 nm. Finally, the protein contents of CVP-1-1 and CVP-2-1 were (0.89 ± 0.97)% and (1.54 ± 1.77)%, respectively.

[0058] The specific results of the above determination experiments are shown in Table 3.

[0059] Table 3 Total sugar content and chemical composition of CVP, CVP-1, CVP-2, CVP-1-1, and CVP-2-1

[0060] 3. Structural Characterization (1)Determination of Monosaccharide Composition Accurately weigh 5 mg of the Chuanminshen polysaccharide sample, add 2 mL of 3 M TFA solution (trifluoroacetic acid), and hydrolyze at 121 °C for 3 hours. Blow dry with nitrogen, then centrifuge at 12,000 r / min for 5 min, and transfer to a chromatographic vial for measurement. Analyze its monosaccharide composition by an ion chromatography system.

[0061] As Figure 5 shown, the results show that CVP, CVP-1, CVP-2, and CVP-1-1 are composed of glucose and galactose, and CVP-2-1 is composed of glucose, galactose, and rhamnose.

[0062] (2)Molecular Weight Determination The high performance gel permeation chromatography (HPGPC) method was used, and detection was carried out using an HPGPC tandem column.

[0063] As shown in Table 4, the molecular weights of CVP, CVP-1, CVP-2, CVP-1-1, and CVP-2-1 are 3.82 kDa, 4.33 kDa, 18.65 kDa, 9.47 kDa, and 18.15 kD, respectively. CVP-1-1 and CVP-2-1 are homogeneous polysaccharides. The molecular weight of the Chuanminshen polysaccharide extracted by DES is much smaller than that of the polysaccharide extracted by the water extraction method. Generally, low molecular weight polysaccharides have a large number of reducing terminal hydroxyl groups, which can better scavenge free radicals and exert antioxidant activity. This indicates that the Chuanminshen polysaccharide extracted by DES may have greater potential in antioxidant performance.

[0064] Table 4 Molecular Weights of CVP, CVP-1, CVP-2, CVP-1-1, and CVP-2-1

[0065] (3)Infrared Spectroscopy Determination Accurately weigh 1 - 2 mg of the five dried Chuanminshen polysaccharide components and thoroughly grind them with dried potassium bromide powder (100 - 200 mg). After grinding until evenly mixed, press them into a transparent or semi-transparent thin film. Using potassium bromide as the blank background, scan and analyze in a Fourier transform infrared spectrometer within the wavenumber range of 4000 - 400 cm -1 -1.

[0066] The results are as Figure 6As shown, the absorption peaks of CVP, CVP-1, CVP-2, CVP-1-1, and CVP-2-1 at 3397 cm -1 are caused by the stretching vibration of -OH in the polysaccharide molecule, indicating that the sample contains free hydroxyl groups. The absorption peak at 2932 cm -1 is mainly caused by the stretching vibrations of CH, CH2, and CH3. The absorption peak at 1028 - 1052 cm -1 is a typical characteristic absorption peak of pyranose, usually considered to be the stretching vibrations of the C−O−C and C−O−H glycosidic bonds in the pyranose ring, indicating that the samples are all pyranose. The characteristic absorption at 862 cm -1 indicates that the sugar molecules in the polysaccharide contain α-glycosidic bonds. Here, the peak intensity of the crude polysaccharide is lower than that of the purified polysaccharide. At the same time, the functional groups detected by infrared spectroscopy are the same as those contained in glucose and galactose, which also proves that Chuanminshen polysaccharide is composed of glucose and galactose. The results show that the Chuanminshen polysaccharide extracted by DES is a pyranose polysaccharide containing α-glycosidic bonds.

[0067] (4)Scanning electron microscopy analysis Take the Chuanminshen polysaccharide sample and place it on the black glue of the sample stage. After gold spraying treatment, observe it through a Scanning Electron Microscope (SEM) with magnification of 200 times and 500 times and a voltage of 10 kV.

[0068] The results are as Figure 7 shown. The surface of CVP (a) is relatively rough, showing irregular small spherical shapes, tending to a dispersed structure. This may be because the low-molecular-weight polysaccharide chains are shorter, and the intermolecular forces are weakened, so it shows a dispersed state; at 500 times magnification, CVP-1 (b) is shaped like coral clusters, with irregular shapes and a rough and porous surface, and the intertwined structures are stacked layer by layer; CVP-2 (c) shows relatively smooth fragmented surfaces with cracks; CVP-1-1 (d) shows an irregular filamentous structure with many voids inside, and these voids may be formed by the spatial arrangement between polysaccharide chains; CVP-2-1 (e) aggregates into clusters, showing irregular shapes and a loose and porous surface.

[0069] (5)Thermal stability determination Weigh appropriate amounts of CVP, CVP-1, CVP-2, CVP-1-1, and CVP-2-1 respectively, and analyze them using a thermogravimetric analyzer under nitrogen conditions. Set the flow rate at 40 mL / min, the heating rate at 10 ℃ / min, and the measured temperature range at 40 - 600℃ to perform thermogravimetric analysis (TG / DTG) on the samples.

[0070] The results are as Figure 8As shown, when the measurement temperature rises to 100 °C, the moisture in the polysaccharide is evaporated, and all five polysaccharide components have a slight mass loss. The most obvious weight loss stage occurs in the range of 200 - 400 °C. The appearance of this peak indicates that these five components start to degrade at this temperature. Due to heating, the polysaccharide molecules decompose, resulting in a rapid loss of their mass. Between 400 - 600 °C, the decomposition rate of the polysaccharide gradually stabilizes, and the mass loss rate is the smallest. The percentage of weight loss of the five components of Chuanminshen polysaccharide is as follows: CVP (74.12%), CVP-1 (72.4%), CVP-2 (70.53%), CVP-1-1 (71.85%), CVP-2-1 (69.15%). The weight loss rate of CVP-2-1 is significantly lower than that of the crude polysaccharide and other components, and it has better thermal stability. The weight loss of CVP-2-1 is the most obvious in the range of 200 - 400 °C. This stage corresponds to the thermal decomposition of the main chain of the polysaccharide. The decrease in the weight loss rate may be related to the higher molecular weight of CVP-2-1.

[0071] (6)NMR Nuclear Magnetic Resonance Measurement Using nuclear magnetic resonance analysis to further obtain the structural characteristic information of the polysaccharide, and conducting one-dimensional nuclear magnetic 1 H-NMR, 13 C-NMR measurements. Weigh approximately 30 mg of CVP-1-1 and CVP-2-1 samples, dissolve them in 0.55 mL of D2O, prepare a concentration of approximately 55 g / mL, and place them in a 5 mm nuclear magnetic resonance tube. Use a Bruker nuclear magnetic resonance instrument to collect the 1 H NMR and 13 chemical shifts of C NMR of the samples and record them. The chemical shifts are expressed in ppm. Probe type: 5 mm dual-core probe; sample treatment temperature: 298 K (approx. 24.85 °C); 1 Resonance frequency of H-NMR: 400 MHz; 13 Resonance frequency of C-NMR: 100 MHz; Analysis software: MestReNova.

[0072] Generally, in the hydrogen spectrum, the anomeric protons with δ > 5.0 ppm are in the α configuration, and the anomeric protons with δ < 5.0 ppm are in the β configuration. In this experiment, the strong signal at 1H δ 4.70 ppm is attributed to the solvent peak D2O. The proton signals of the sugar ring are in the range of δ 3.50 - 4.00 ppm, which is a typical characteristic of polysaccharides. The chemical shifts of the more obvious resonance peaks of CVP-1-1 and CVP-2-1 are δ 5.35 ppm. Therefore, it can be known that CVP-1-1 and CVP-2-1 mainly have α-configured glycosidic bonds, which is consistent with the results of infrared spectroscopy analysis.

[0073] Generally, the chemical shift signals of α-anomeric carbons are in the region of δ 98 - 103 ppm, and those of β-anomeric carbons are in the region of δ 101 - 105 ppm. In the 13 13C NMR of CVP-1-1 and CVP-2-1, the anomeric carbon signals are at δ 99.64 ppm and δ 99.65 ppm, indicating that the anomeric configurations of CVP-1-1 and CVP-2-1 are α configurations. The strong signal at about chemical shift δ 70 ppm is for the glucose anomeric carbon residue. The 13 13C NMR spectra of CVP-1-1 and CVP-2-1 both contain glucose. There is no signal peak at about δ 170 ppm, indicating that this polysaccharide does not contain uronic acid, which is consistent with the monosaccharide composition analysis.

[0074] Experimental Example 4 In vitro fermentation The basic nutrient medium for in vitro fermentation of polysaccharides was sterilized at 121 °C for 20 min. The nutrient components of the basic nutrient medium (1.0 L, pH 7.0) are shown in Table 5.

[0075] Fresh fecal samples were collected from 4 healthy volunteers (aged 22 - 30 years, with no history of gastrointestinal diseases, not using antibiotics within 3 months, 2 males and 2 females). The feces were diluted with 0.9% sterile normal saline (w / v) to prepare a fecal suspension (10%, w / v). The fecal suspension was centrifuged at 1000 r / min for 5 min, and the supernatant was collected to prepare a fecal homogenate (referred to as fecal slurry).

[0076] The fermentation group consisted of 9.0 mL of basic nutrient medium, 1.0 mL of fecal slurry, and 100 mg of Chuanminshen polysaccharide, including the CVP group, CVP-1 group, CVP-2 group, CVP-1-1 group, and CVP-2-2 group. The basic nutrient medium without the sample was used as the negative control (BLANK group). The 6 treatment groups were incubated in a carbon dioxide anaerobic incubator at 37 °C for 48 h. The fermentation products were collected at 0, 24, and 48 h and immediately frozen for further analysis. After centrifuging all the collected samples, the supernatant was taken to measure its pH value, gas production, and short-chain fatty acids. In addition, using the fermentation broth after 48 h of fermentation, the effects on the intestinal flora composition and metabolic pathways were measured.

[0077] Table 5 Nutrient components of the basic medium

[0078] The change in pH value is one of the important indicators reflecting the fermentation degree of polysaccharides during in vitro fermentation. As Figure 9As shown in (a) of, during the fermentation process, the pH values of the CVP group, CVP-1 group, CVP-2 group, CVP-1-1 group, and CVP-2-1 all decreased significantly. At 0 h of fermentation, the initial pH value of the fermentation broth was greater than 7. As fermentation progressed, the pH values of the fermentation broth in the CVP, CVP-1, CVP-2, CVP-1-1, and CVP-2-1 groups showed a stable downward trend. After 48 h of fermentation, the pH value of the BLANK group slightly decreased from 7.47 to 7.21, and all five polysaccharide components promoted the pH of the fermentation broth to drop to 4.68 - 5.11, among which the CVP group had the largest decrease in pH value; all five polysaccharide components produced gas, and the CVP-1-1 group had the highest gas production during the fermentation process, probably because more gas-producing bacteria were generated in the CVP-1-1 group ( Figure 9 of b).

[0079] Through the determination of short-chain fatty acids (SCFAs), it was found that the total acid content of the polysaccharide groups was higher than that of the blank group. Among them, the production of acetic acid was the highest, followed by propionic acid and butyric acid. The total production of SCFAs in the CVP-2-1 group was the highest, significantly promoting the production of acetic acid and propionic acid (Table 6); the Venn diagram, Alpha diversity analysis, and PcoA analysis of the intestinal flora during in vitro fermentation were as Figure 10 shown, and these results all indicated that Chuanminshen polysaccharide had an impact on the richness of the intestinal flora.

[0080] Table 6 Changes in short-chain fatty acid content during fermentation

[0081] As Figure 11 shown, in the microbial composition, the polysaccharide groups upregulated the Bacteroidetes / Firmicutes ratio, which helped reduce weight gain, obesity, and insulin resistance caused by a high-fat diet. The relative abundances of beneficial bacteria such as Bacteroides ( Bacteroides ), Phascolarctobacterium ( Phascolarctobacterium ), and Lactococcus ( Lactococcus ), etc. were increased, while the relative abundances of pathogenic bacteria such as Enterobacteriaceae ( Enterobacteriaceae ), Shigella ( Escherichia-Shigella ), etc. were decreased. They could affect the AMPK signaling pathway, IL-17 signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, MAPK signaling pathway, and PI3K-Akt signaling pathway, as well as the pathways related to inflammation and immune regulation, thereby maintaining the ecological health of the intestinal microbiota ( Figure 12 ). Glucose and galactose in the polysaccharide groups were positively correlated with beneficial bacteria (such as Phascolarctobacterium and Lactococcus), while negatively correlated with pathogenic bacteria (such as Escherichia coli-Shigella). In addition, a significant correlation was observed between the production of SCFAs and the microbial community ( Figure 13). The above results indicate that Chuanminshen polysaccharide can effectively regulate the composition of intestinal microbiota, increase the production of beneficial metabolites, and promote intestinal health.

[0082] In the present invention, DES is applied to the extraction of Chuanminshen polysaccharide, and its process is optimized through single-factor experiments combined with response surface experiments. Its extraction rate is significantly improved compared with traditional methods. Then, the crude polysaccharide is separated and purified by anion exchange column chromatography and gel column chromatography to obtain CVP-1, CVP-2, and the homogeneous polysaccharides CVP-1-1 and CVP-2-1. The structures of the crude polysaccharide and the purified polysaccharides are analyzed. The structures of the polysaccharides are solved by means such as monosaccharide composition determination, molecular weight determination, thermal stability determination, scanning electron microscopy determination, and one-dimensional nuclear magnetic resonance analysis. The obtained polysaccharides have uniform structures and excellent thermal stability, and their molecular weights are significantly lower than those obtained by the traditional water extraction method, indicating that the Chuanminshen polysaccharide extracted by DES may have higher potential antioxidant activity. Then, the polysaccharide is applied to in vitro fermentation experiments. The experimental results confirm that Chuanminshen polysaccharide can promote the production of SCFAs by regulating the metabolism of intestinal flora and can improve the intestinal microenvironment by affecting different metabolic pathways. The research results provide a new theoretical reference for the green and efficient extraction of Chuanminshen polysaccharide and the improvement of intestinal microecology.

[0083] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A method for extracting Chuanminshen crude polysaccharide, characterized in that, The extraction method includes: Using a deep eutectic solvent composed of choline chloride and citric acid with a molar ratio of 1:1, the water content of the deep eutectic solvent is 30%. Chuanminshen powder and the deep eutectic solvent are extracted at a liquid-to-solid ratio of (19-20) mL:1 g under heating conditions, the extraction temperature is 70-80 °C, and the extraction time is 110-130 min; After the extraction is completed, the supernatant is collected by centrifugation and precipitated with absolute ethanol; The precipitate is redissolved in water and then subjected to protein removal treatment. After the treatment solution is concentrated, it is transferred to a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis. The dialyzed polysaccharide solution is freeze-dried to obtain a crude polysaccharide sample of Chuanminshen, named CVP.

2. The extraction method according to claim 1, characterized in that, The extraction method also includes: Gradient elution of the crude polysaccharide sample of Chuanminshen is carried out using a DEAE-52 cellulose ion exchange chromatography column, and gradient elution is carried out using ultrapure water and 0.10 mol / L NaCl solution. The elution fraction with ultrapure water is named CVP-1, and the elution fraction with 0.1 mol / L NaCl solution is named CVP-2.

3. The extraction method according to claim 2, wherein The extraction method also includes: Using a Sephadex G-100 gel column to elute CVP-1 and CVP-2 respectively, the eluent is ultrapure water, and the collected fractions are named CVP-1-1 and CVP-2-1 respectively. The molecular weight of CVP-1-1 is 9.47 kDa, and the molecular weight of CVP-2-1 is 18.15 kD.

4. The extraction method according to claim 1, wherein The Chuanminshen powder is screened through an 80-mesh sieve; Or / and, for the centrifugation, it is centrifuged at 4000 r / min.

5. The extraction method according to claim 1, wherein For the precipitation with absolute ethanol, 4 times the volume of absolute ethanol is added to the supernatant for precipitation, and the precipitation is allowed to stand at a low temperature of 4 °C.

6. The extraction method according to claim 1, wherein For the protein removal treatment, the Sevage method is used, the volume ratio of chloroform to n-butanol is 4:1, and the volume ratio of polysaccharide to Sevage reagent is 3:

1.

7. The extraction method according to any one of claims 1 to 6, characterized in that, For the freeze-drying, the polysaccharide solution is first frozen in an environment of -80 °C for more than 4 h, and finally freeze-dried in a freeze-dryer.

8. The crude polysaccharide of Chuanminshen obtained by the extraction method according to any one of claims 1 to 7.

9. The application of the crude polysaccharide of Chuanminshen according to claim 8 in improving the intestinal microecology.

10. The application according to claim 9, characterized in that, The crude polysaccharide of Chuanminshen can increase the short-chain fatty acids produced by intestinal flora, or / and improve the richness of intestinal flora.

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