Radix pseudostellariae non-starch homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention provides heterophylly falsestarwort root non-starch homogeneous polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of natural substance extraction. The radix pseudostellariae non-starch homogeneous polysaccharide is divided into five types, the molecular weights of the five types are 2124Da, 85900Da, 72970Da, 35820Da and 20300Da respectively, and each type of the five types of radix pseudostellariae non-starch homogeneous polysaccharide is of a triple-helical structure. The monosaccharide of the non-starch homogeneous polysaccharide of the radix pseudostellariae with the molecular weight of 2124Da comprises glucose, galactose and mannose in a molar ratio of 95.47: 2.15: 2.37, and a repeatable primary structural unit of the non-starch homogeneous polysaccharide of the radix pseudostellariae comprises the following sugar residues: T-Glcp, 1, 3-Glcp, 1, 2-Galp, 1, 3, 6-Manp and 1, 4, 6-Glcp. The heterophylly falsestarwort root non-starch homogeneous polysaccharide has excellent prebiotic activity, the preparation method is simple, and the obtained heterophylly falsestarwort root non-starch homogeneous polysaccharide is stable in yield, high in purity, stable in structure and suitable for industrial scale production.

Description

Technical Field

[0001] The invention provides a homogeneous non-starch polysaccharide of Radix Pseudostellariae Radix and a preparation method and application thereof, belonging to the technical field of natural substance extraction. Background Art

[0002] Pseudostellariae Radix is ​​the dried root of the plant Pseudostellariae Radix. Pseudostellariae Radix has the effects of "replenishing qi and strengthening spleen, promoting body fluid and moistening lungs". It is often used clinically to treat symptoms such as spleen deficiency, fatigue, loss of appetite, weakness after illness, insufficient qi and yin, spontaneous sweating and thirst, dry cough due to lung dryness, etc. It is a tonic suitable for both young and old.

[0003] However, there are also the following problems: there is a large amount of starch in the tubers of Pseudostellaria chinensis, and the polysaccharides obtained by previous preparation methods have low solubility, which interferes with the structural characterization results; there is a lack of suitable extraction and purification processes to prepare Pseudostellaria chinensis non-starch uniform polysaccharides with clear chemical structure and uniform composition.

[0004] Chinese patent CN111647091B discloses a kind of active hexaaldehyde oligosaccharide of Radix Pseudostellariae and its preparation method and application, through alcohol extraction, water extraction, alcohol precipitation, enzyme-Sevage method protein removal, DEAE-52 anion exchange column separation and purification method, collect the product with molecular weight in the range of 1000Da to 2000Da, obtain the active hexaaldehyde oligosaccharide of Radix Pseudostellariae. Wherein the molecular weight of the active hexaaldehyde oligosaccharide of Radix Pseudostellariae is 1476.6185Da, and its monosaccharide composition is: mannose, glucose and galactose (molar ratio is 0.076:0.814:2.600), and this active hexaaldehyde oligosaccharide of Radix Pseudostellariae has anti-inflammatory and antioxidant activity, and has strong reducing ability. However, it only studies the effect of active hexaaldehyde oligosaccharide of Radix Pseudostellariae on intestinal diseases, and does not study the specific intestinal flora.

[0005] Xie Meilin et al. disclosed the purification, composition and protective effect of Radix Pseudostellariae polysaccharide on LPS-damaged Raw264·7 macrophages in Food Industry Science and Technology. The Radix Pseudostellariae polysaccharide extracted by the method has a purity of 80.52%, including galactose, D-mannose, rhamnose, arabinose, D-anhydrous glucose, D-glucuronic acid and D-lactobionic acid, which has a protective effect on Raw264.7 macrophages. The Radix Pseudostellariae polysaccharide does not remove starch, and there is a large amount of starch in the Radix Pseudostellariae tubers, so the polysaccharide obtained by the preparation method has low solubility, which interferes with the structural characterization results. Therefore, there is no specific structure and composition in the article, and the molecular weight is a range value. It is still a crude polysaccharide and not a uniform polysaccharide. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides a homogeneous non-starch polysaccharide of Radix Pseudostellariae and a preparation method and application thereof. The preparation method is simple and efficient in preparing the homogeneous non-starch polysaccharide of Radix Pseudostellariae and removing the starch therein, thereby improving the purity and reducing the interference of structural characterization. The homogeneous non-starch polysaccharide of Radix Pseudostellariaee obtained has a stable yield, high purity and stable structure, is suitable for industrial-scale production and has good intestinal beneficial properties.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a homogeneous non-starch polysaccharide of Radix Pseudostellariae, wherein the monosaccharide composition comprises glucose, galactose and mannose in the following molar ratio: 95.47:2.15:2.37; the molecular weight of the homogeneous non-starch polysaccharide of Radix Pseudostellariae is 2124Da; the homogeneous non-starch polysaccharide of Radix Pseudostellariae has a triple helix structure; the repeatable primary structural unit of the homogeneous non-starch polysaccharide of Radix Pseudostellariae comprises the following sugar residues: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, 1,4,6-Glcp, and the connection formula thereof is:

[0009]

[0010] In a second aspect, the present invention provides a homogeneous non-starch polysaccharide of Radix Pseudostellariae, wherein the monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 1.79:11.32:1.45:2.60:2.45:31.99:48.40; the molecular weight of the homogeneous non-starch polysaccharide of Radix Pseudostellariae is 85900Da; the homogeneous non-starch polysaccharide of Radix Pseudostellariae has a triple helical structure; the homogeneous non-starch polysaccharide of Radix Pseudostellariae has a repeatable primary The structural units include the following sugar residues: T-Araf, T-Rhap, 1,2-Araf, T-Glcp, T-GlcpA, T-Galp, 1,2-Galp, 1,2,3,5-Araf, 1,3,6-Manp, 1,3,6-Glcp, 1,4,6-Galp, 1,2,3,4-Galp, 1,2,3,4-GalpA, 1,2,3,6-Galp, 1,2,3,6-GalpA, and their connection formula is as follows Figure 1 shown.

[0011] In a third aspect, the present invention provides a homogeneous non-starch polysaccharide of Pseudostellaria chinensis, whose monosaccharide composition includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 13.04:15.18:0.82:19.83:51.14; the molecular weight of the homogeneous non-starch polysaccharide of Pseudostellaria chinensis is 72970Da; the homogeneous non-starch polysaccharide of Pseudostellaria chinensis has a triple helix structure; the repeatable primary structural unit of the homogeneous non-starch polysaccharide of Pseudostellaria chinensis includes the following sugar residues: T-Rhap, 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,2-Galp, 1,2,3-Araf, 1,3,5-Araf, 1,2,3-Galp, 1,3,4-Rhap, 1,2,3,4-Rhap, 1,2,3,5-Araf.

[0012] In a fourth aspect, the present invention provides a homogeneous non-starch polysaccharide of Pseudostellaria chinensis, whose monosaccharide composition includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 22.58:24.15:3.00:16.30:33.97; the molecular weight of the homogeneous non-starch polysaccharide of Pseudostellaria chinensis is 35820Da; the homogeneous non-starch polysaccharide of Pseudostellaria chinensis has a triple helix structure; the repeatable primary structural units of the homogeneous non-starch polysaccharide of Pseudostellaria chinensis include the following sugar residues: 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,2-Galp, 1,2,3-Araf, 1,3,5-Araf, 1,3,4-Rhap, 1,2,4-Rhap, 1,2,3,4-Glcp, 1,2,3,4-Rhap, 1,2,3,5-Araf.

[0013] In a fifth aspect, the present invention provides a homogeneous non-starch polysaccharide of Radix Pseudostellariae, wherein the monosaccharide composition comprises rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 24.75:45.64:2.19:11.37:16.04; the molecular weight of the homogeneous non-starch polysaccharide of Radix Pseudostellariae is 20300Da; the homogeneous non-starch polysaccharide of Radix Pseudostellariae has a triple helical structure; the homogeneous non-starch polysaccharide of Radix Pseudostellariae can be repeated once The primary structural units include the following sugar residues: 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,4-Rhap, 1,2,3-Araf, 1,3,5-Araf, 1,2,3-GalpA, 1,3,4-Rhap, 1,2,4-Rhap, 1,2,3,4-Glcp, 1,2,3,4-Rhap, 1,2,3,5-Araf.

[0014] In a sixth aspect, the present invention provides a method for preparing the above-mentioned Radix Pseudostellariae non-starch uniform polysaccharide, comprising the following steps:

[0015] (1) extracting Pseudostellariae Radix in ethanol, filtering to obtain a precipitate, and drying the precipitate to obtain Pseudostellariae Radix Reticulatae Radix dregs;

[0016] (2) taking the Radix Pseudostellariae Radix residue obtained in step (1), adding it into water for extraction, and obtaining Radix Pseudostellariae Radix aqueous extract;

[0017] (3) removing starch from the water extract of Radix Pseudostellariae obtained in step (2), centrifuging and concentrating, taking the supernatant and adding ethanol for alcohol precipitation, centrifuging again, collecting the precipitate, and obtaining crude Pseudostellariae polysaccharide;

[0018] (4) Separating and purifying the crude polysaccharide of Pseudostellariae Radix obtained in step (3), eluting, collecting the eluates corresponding to different gradient elution peaks, and concentrating to obtain the uniform non-starch polysaccharide of Pseudostellariae Radix.

[0019] Specifically, the extraction in step (1) is reflux extraction.

[0020] Specifically, the volume percentage of ethanol in the step (1) of producing the Pseudostellaria heterophylla non-starch uniform polysaccharide is 70-85%.

[0021] Specifically, the solid-liquid ratio of Pseudostellaria ginseng to ethanol in the step (1) of the Pseudostellaria ginseng non-starch uniform polysaccharide is 1g:10mL-1g:30mL.

[0022] Specifically, the extraction temperature of the Pseudostellaria heterophylla non-starch uniform polysaccharide step (1) is 85-90° C., the extraction time is 1-4 h / time, and the extraction is performed 1-3 times in total.

[0023] Specifically, the extraction of the Pseudostellaria heterophylla non-starch uniform polysaccharide in step (2) is reflux extraction.

[0024] Specifically, the solid-liquid ratio of the Pseudostellaria heterophylla residue to water in step (2) is 1g:10mL-1g:30mL.

[0025] Specifically, the extraction temperature in step (2) is 80-90° C., the extraction time is 1-4 h / h / time, and the extraction is performed 1-3 times in total.

[0026] Specifically, the starch removal in step (3) is as follows: adding amylase until the enzyme activity is 1-2 U / mL, the enzymatic hydrolysis temperature is 80-90° C., and the enzymatic hydrolysis time is 0.5-2 h.

[0027] Preferably, the amylase is an alpha-amylase.

[0028] Specifically, the ethanol concentration of the alcohol precipitation in step (3) is 70-90% by volume, the temperature is 4-8° C., and the time is 12 hours.

[0029] Preferably, the purification in step (4) uses an anion exchange chromatography column.

[0030] Specifically, the elution in step (4) is: elution with deionized water solutions of NaCl of different concentrations.

[0031] Specifically, the different concentrations are:

[0032] A, eluting with deionized water; obtaining the heterophyllotoxin non-starch uniform polysaccharide described in the first aspect;

[0033] B, eluting with 0.1M NaCl deionized water solution to obtain the heterophyllotoxin non-starch uniform polysaccharide described in the second aspect;

[0034] C. Elution with 0.2M NaCl deionized water solution yields a non-starch uniform polysaccharide mixture of Panax notoginseng.

[0035] Preferably, step (4) further comprises separating and purifying the non-starch uniform polysaccharide of Pseudostellaria heterophylla obtained by eluting with 0.1M NaCl deionized water solution, wherein the purification uses a gel column.

[0036] Specifically, step (4) further comprises separating and purifying the Pseudostellariae non-starch uniform polysaccharide mixture to obtain the Pseudostellariae non-starch uniform polysaccharides described in the third to fifth aspects respectively, and the purification uses a gel column.

[0037] Preferably, the concentration in step (4) also includes desalination.

[0038] In a seventh aspect, the present invention provides an application of a non-starch uniform polysaccharide of Pseudostellaria heterophylla in the preparation of intestinal probiotic foods and health products.

[0039] Preferably, the intestinal probiotics include BO bacteria, BT bacteria, BL bacteria and LGG bacteria.

[0040] Definition and Description

[0041] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered ambiguous or unclear without special definition, but should be understood according to the ordinary meaning.

[0042] The BO bacteria described in the present invention are Bacteroides ovatu bacteria.

[0043] The BT bacteria described in the present invention are Bacteroides thetaiotaomicro bacteria.

[0044] The BL bacteria described in the present invention are Bifidobacterium longum bacteria.

[0045] The LGG bacteria described in the present invention are Lactobacillus rhamnosus bacteria.

[0046] The technical effects achieved by the present invention are:

[0047] (1) The preparation process of the present invention has mild conditions, simple operation, and is environmentally friendly. The obtained Pseudostellaria heterophylla non-starch uniform polysaccharide has high purity, and the total content of neutral sugars and uronic acid can reach more than 85%.

[0048] (2) The non-starch uniform polysaccharide of Radix Pseudostellariae prepared by the present invention by removing starch with amylase has a clear structure, high yield and high solubility.

[0049] (3) The non-starch uniform polysaccharide of Radix Pseudostellariae of the present invention has good intestinal probiotic properties and has application potential in the preparation of intestinal probiotic foods and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the connection type of PHP-2a.

[0051] Figure 2 This is the UV spectrum of non-starch uniform polysaccharide from Pseudostellaria heterophylla.

[0052] Figure 3 This is the HPGPC diagram of non-starch uniform polysaccharides from Pseudostellaria heterophylla, where A is non-starch uniform crude polysaccharide from Pseudostellaria heterophylla; B is PHP-1; C is PHP-2a; D is PHP-3a; E is PHP-3b; and F is PHP-3c.

[0053] Figure 4 UPLC / Q-TOF-MS spectra of monosaccharide standard mixed standards and PHP-1, PHP-2a, PHP-3a, PHP-3b and PHP-3c, where the order of the mixed standards 1-8 is: mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and fucose.

[0054] Figure 5 Infrared spectra of PHP-1, PHP-2a, PHP-3a, PHP-3b and PHP-3c.

[0055] Figure 6 This is the Congo red test of non-starch uniform polysaccharides from Pseudostellaria heterophylla, where A is PHP-1, B is PHP-2a, C is PHP-3a, D is PHP-3b, and E is PHP-3C.

[0056] Figure 7The effects of PHP-1, PHP-2a, PHP-3a, PHP-3b and PHP-3c on the growth of BO, BT, BL and LGG bacteria (* compared with the Blank group, P < 0.05, ** compared with the Blank group, P < 0.01, *** compared with the Blank group, P < 0.001).

[0057] Figure 8 This is a graph of the in vitro experiment and fermentation characteristics test of non-starch uniform polysaccharides of Pseudostellaria heterophylla, where A is the pH value after fermentation, B is the reducing sugar content after fermentation, C is the neutral sugar content after fermentation, D is the carbohydrate consumption rate after fermentation, E is the molecular weight change after fermentation, and F is the change in monosaccharide composition at different time points during fecal fermentation (a, b and c indicate that there are significant differences between different treatment groups at the same time, P<0.05). DETAILED DESCRIPTION

[0058] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0059] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0060] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0061] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are common commercial products in the technical field.

[0062] Example 1: Preparation of non-starch uniform polysaccharide from Radix Pseudostellariae

[0063] (1) Take 150 g of dried Radix Pseudostellariae powder, add it to a 70% by volume ethanol solution at a solid-liquid ratio of 1:20, extract it at 90° C. for 2 h, and extract it three times in total. Collect the Radix Pseudostellariae powder, evaporate and remove the ethanol, and obtain Radix Pseudostellariae medicinal residue.

[0064] (2) Take 100 g of Radix Pseudostellariae Radix residue, add it into water at a solid-liquid ratio of 1:10, and heat reflux extraction at 90°C for 3 times, each time for 2 h. Combine the water extracts and concentrate them to a crude drug concentration of 0.5 g / mL to obtain Radix Pseudostellariae Radix water extract concentrate.

[0065] (3) Add α-amylase to the concentrated water extract of Radix Pseudostellariae to maintain the final enzyme activity at 1 U / mL, perform enzymolysis at 90°C for 0.5 h to obtain an enzymolysis solution; detect the enzymolysis solution with I2 / KI reagent until the starch is completely removed, centrifuge, collect the supernatant, and obtain the water extract of Radix Pseudostellariae after starch removal; perform alcohol precipitation, add anhydrous ethanol to the water extract of Radix Pseudostellariae after starch removal to make its final concentration be 90% by volume, perform alcohol precipitation at 4°C for 12 h, centrifuge, collect the precipitate, and obtain the non-starch uniform crude polysaccharide of Radix Pseudostellariae.

[0066] (4) Purification: The crude uniform non-starch polysaccharide of Pseudostellaria chinensis was dissolved in deionized water, and then separated and purified using a DEAE-52 anion exchange column. The eluate eluted with deionized water was collected, and the uniform non-starch polysaccharide of Pseudostellaria chinensis obtained was hereinafter referred to as PHP-1; the uniform non-starch polysaccharide of Pseudostellaria chinensis obtained by eluting with a 0.1M NaCl deionized water solution was hereinafter referred to as PHP-2; the uniform non-starch polysaccharide of Pseudostellaria chinensis obtained by eluting with a 0.2M NaCl deionized water solution was hereinafter referred to as PHP-3.

[0067] (4) PHP-2 was dissolved by ultrasonication, and the supernatant was centrifuged and loaded onto a Focurose CL-6B (2.5×100 cm) gel column and eluted with deionized water at a flow rate of 4 mL / 15 min. The purity of the eluate in each tube was tested by HPGFC-ELSD, and the high-purity eluates were combined and freeze-dried to obtain a uniform non-starch polysaccharide of Pseudostellaria heterophylla, hereinafter referred to as PHP-2a.

[0068] (5) PHP-3 was dissolved by ultrasonication, and the supernatant was centrifuged and loaded onto a Focurose CL-6B (2.5×100 cm) gel column and eluted with deionized water at a flow rate of 4 mL / 15 min. The purity and type of the eluate in each tube were detected by HPGFC-ELSD. The high-purity eluates of the same type were combined and freeze-dried to obtain three types of Pseudomonas heterophylla non-starch uniform polysaccharides, hereinafter referred to as PHP-3a, PHP-3b, and PHP-3c.

[0069] 1. Physicochemical properties of non-starch uniform polysaccharides from Radix Pseudostellariae

[0070] (1) Determination of carbohydrate content in non-starch homogeneous polysaccharides of Radix Pseudostellariae

[0071] The carbohydrate content of the non-starch uniform polysaccharide of Radix Pseudostellariae prepared in Example 1 was determined by the phenol-sulfuric acid method, and the absorbance was detected at 495 nm by an ultraviolet spectrophotometer. The carbohydrate content in PHP-1 was 89.15±2.75%. The total amount of carbohydrates was determined by the phenol-sulfuric acid method and the m-hydroxybiphenyl method, and the carbohydrate contents of PHP-2a, PHP-3a, PHP-3b and PHP-3c were 93.54±2.12%, 91.9±4.35%, 95.01±3.55% and 85.82±1.89%, respectively.

[0072] (2) UV spectroscopy analysis of non-starch uniform polysaccharides from Radix Pseudostellariae

[0073] The UV spectra of five non-starch homogeneous polysaccharides of Radix Pseudostellariae were scanned in the wavelength range of 200-400 nm, and no obvious absorption peaks appeared at 260 nm and 280 nm. Figure 2 , indicating that the non-starch uniform polysaccharides of the five types of Pseudostellariae do not contain nucleic acids and proteins.

[0074] (3) Determination of the uniformity of non-starch homogeneous polysaccharides from Radix Pseudostellariae

[0075] The homogeneity of five non-starch homogeneous polysaccharides from Pseudostellaria heterophylla was determined by high performance liquid gel permeation chromatography coupled with evaporative light detection (HPGFC-ELSD).

[0076] Chromatographic conditions: Polysep-GFC-P4000 column (300×7.8 mm, 35×7.8 mm); mobile phase: ultrapure water; flow rate: 0.4 mL / min; injection volume: 10 μL; column temperature: 30°C; ELSD detector parameters: N2 pressure 30 psi, drift tube temperature 100°C, nebulizer heating power 60%, gain value 10.

[0077] HPGFC-ELSD showed that compared with the crude non-starch uniform polysaccharide of Pseudostellaria heterophylla, the five non-starch uniform polysaccharides of Pseudostellaria heterophylla showed a single symmetrical peak, such as Figure 3 , indicating that it is a uniform polysaccharide.

[0078] 2. Chemical structure identification

[0079] (1) Molecular weight determination of non-starch homogeneous polysaccharides from Radix Pseudostellariae

[0080] The dried homogeneous polysaccharide sample of Pseudostellaria heterophylla was dissolved in a 0.9% NaCl aqueous solution to a sample concentration of 2 mg / mL, and filtered through a 0.45 μm filter membrane to obtain a test solution. 0.9% NaCl water was used to prepare dextran of different molecular weights (Mw=6300, 9800, 22000, 49400, 107000Da) at a concentration of 2 mg / mL, and the molecular weight of the homogeneous polysaccharide of Pseudostellaria heterophylla was detected by high pressure gel chromatography-differential refractive index detector (HPSEC-RID) technology. The mobile phase was a 0.9% NaCl solution at a flow rate of 0.5 mL / min. HPSEC-RID detected the retention time of the above samples, and the logarithm of the molecular weight of the dextran was used as the ordinate and the retention time as the abscissa to draw the equation of the dextran exclusion curve. The molecular weight of the homogeneous polysaccharide of Pseudostellaria heterophylla was calculated according to the exclusion curve equation. The results are shown in Table 1.

[0081] Table 1 Molecular weight of non-starch homogeneous polysaccharides from Radix Pseudostellariae

[0082] PHP-1 PHP-2a PHP-3a PHP-3b PHP-3c Molecular weight 2124Da 85900Da 72970Da 35820Da 20300Da

[0083] (2) Monosaccharide composition of non-starch homogeneous polysaccharides of Radix Pseudostellariae

[0084] The monosaccharide composition of the non-starch uniform polysaccharide of Radix Pseudostellariae was determined by pre-column PMP derivatization combined with UPLC / Q-TOF-MS. 1.0 mg of non-starch uniform polysaccharide of Radix Pseudostellariae was accurately weighed and placed in a clean and dry stoppered test tube. A 4M TFA solution was added and the mixture was placed in an oil bath at 120°C for 2 hours and then blown to dryness with nitrogen to obtain a completely acid-hydrolyzed sample. 0.5 mL of ammonia water (mass concentration 25%) and 0.5 mL of PMP-methanol solution (0.5 M) were added to the completely acid-hydrolyzed polysaccharide sample, mixed evenly, reacted at 70°C for 30 minutes, and then blown to dryness with nitrogen. Redissolved with 1 mL of methanol (LC-MS grade), centrifuged at 12000 rpm for 10 minutes, and the supernatant was taken for LC-MS detection.

[0085] Derivatization of monosaccharide standards: 1.0 mg of monosaccharide standards (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, fucose) was accurately weighed in a stoppered test tube, 0.5 mL of ammonia water and 0.5 mL of PMP-methanol solution (0.5 M) were added, mixed evenly, reacted at 70 ° C for 30 min, and blown to dryness with nitrogen. Redissolved with 1 mL of liquid grade methanol, centrifuged at 12000 rpm for 10 min, and the supernatant was taken for LC-MS detection.

[0086] UPLC / Q-TOF-MS analysis: chromatographic column Kinetex C18 100A (50×2.1 mm, 1.7 μm), column temperature 25°C, 0.05% acetic acid-20 mM ammonium acetate (phase A)-acetonitrile (phase B) as mobile phase for gradient elution, 0-10 min, 15% B, 10-16 min, 15%-17B, 16-19 min, 17%-70% B. The injection volume was 2 μL, the flow rate was 0.4 mL / min. Acquisition was performed in positive ion mode, and the mass acquisition range was 420-600 Da.

[0087] Compared with the mixed monosaccharide standard derivatives, PHP-1 mainly contains glucose (95.47%), and a small amount of mannose (2.37%) and galactose (2.15%). Figure 4 .

[0088] Compared with the mixed monosaccharide standard derivatives, PHP-2a mainly contains mannose (1.79%), rhamnose (11.32%), glucuronic acid (1.45%), galacturonic acid (2.60%), glucose (2.45%), galactose (31.99%) and arabinose (48.40%). Figure 4 .

[0089] Compared with the mixed monosaccharide standard derivatives, PHP-3a mainly contains rhamnose (13.04%), galacturonic acid (15.18%), glucose (0.82%), galactose (19.83%) and arabinose (51.14%); PHP-3b mainly contains rhamnose (22.58%), galacturonic acid (24.15%), glucose (3.00%), galactose (16.30%) and arabinose (33.97%); PHP-3c mainly contains rhamnose (24.75%), galacturonic acid (45.64%), glucose (2.19%), galactose (11.37%) and arabinose (16.04%). Figure 4 .

[0090] (3) Characteristic group analysis of non-starch homogeneous polysaccharides of Radix Pseudostellariae

[0091] Fourier transform infrared spectrometer was used to scan and analyze the characteristic groups of non-starch uniform polysaccharides of Radix Pseudostellariae. 2 mg of dried non-starch uniform polysaccharides of Radix Pseudostellariae were accurately weighed, and an appropriate amount of KBr (spectral grade) dry powder was added. The powder was ground in a dry agate mortar and vacuum pressed into tablets. The characteristic groups of non-starch uniform polysaccharides of Radix Pseudostellariae were determined at 4000-400 cm -1 Characteristic peaks in the range.

[0092] FT-IR spectrum of PHP-1 ( Figure 5) shows typical polysaccharide characteristic absorption peaks. 3394.38 and 2920.84 cm -1 The broad peak at 1610 cm is attributed to the stretching vibration of -OH and -CH3. -1 The left and right peaks are attributed to the shear vibration of bound water. -1 The absorption between them is attributed to the stretching vibration of the COC glycosidic bond on the pyranose ring. 1146.09cm -1 The band at 853.16 cm -1 The absorption peak at indicates the presence of α-type glycosidic bonds in PHP-1.

[0093] FT-IR spectrum of PHP-2a ( Figure 5 ) shows a typical polysaccharide characteristic absorption peak. 3419.70cm -1 The broad peak at 2924.76cm is attributed to -OH. -1 The absorption peak at 1324.21 cm is attributed to the stretching vibration of CH, and the bending vibration peak appears at 1324.21 cm -1 Nearby. 1000-1200cm -1 The absorption peak is attributed to the symmetrical stretching vibration of COC and COH. -1 The strong absorption peak at 894.14cm is attributed to the stretching vibration of C=O, indicating the presence of uronic acid. -1 The absorption peak at 856.03cm indicates the presence of a β-glycosidic bond. -1 The absorption peak at indicates the presence of α-glycosidic bond.

[0094] FT-IR spectra of PHP-3a, PHP-3b and PHP-3c ( Figure 5 ) shows similar polysaccharide characteristic absorption peaks. 3426.12cm -1 The broad peak at 2932.84cm is attributed to -OH stretching vibration. -1 The absorption peak at 1334.33 cm is attributed to the stretching vibration of CH, and the bending vibration peak appears at 1334.33 cm -1 Nearby. 1000-1200cm -1 The three absorption peaks indicate the presence of pyranose ring. 1743.19cm -1 The peak at 1615.60 cm indicates the stretching vibration of C=O and CO of the ester group (-COO-R) in the polysaccharide. -1 The strong absorption peak at 892.54cm is attributed to the stretching vibration of C=O. -1 The absorption peak at 823.13 cm indicates the presence of a β-glycosidic bond. -1 The absorption peak at indicates the presence of α-glycosidic bond.

[0095] (4) Analysis of glycosidic bond types of non-starch homogeneous polysaccharides of Radix Pseudostellariae

[0096] Gas chromatography-mass spectrometry (GC-MS) was used to analyze the glycosidic bond types of non-starch homogeneous polysaccharides of Pseudostellaria heterophylla. The polysaccharide samples were completely methylated, and the acidic polysaccharides (PHP-2a, PHP-3a, PHP-3b, and PHP-3c) were also subjected to carboxyl reduction reaction. The completely methylated polysaccharide samples were subjected to acid hydrolysis, reduction, and acetylation to obtain partially methylated PMAAs. GC-MS sampling was performed according to the working parameters in Table 2.

[0097] Table 2 GC-MS condition parameters

[0098] condition Working Parameters instrument Trace 1300-ISQ QD Chromatographic columns Agilent DB-5MS (3m×0.25mm, 0.25μm) Carrier gas Helium Flow rate 1mL / min Injection volume 2μL Temperature Program Keep at 60℃ for 1min, increase the temperature at 5℃ / min to 280℃ and keep it for 1min Quality scanning range 40-400Da

[0099] According to the retention time and mass fragments, the chromatographic peaks were assigned by comparing the CCRC database with the literature. The molar percentage of each derivative was calculated by dividing the peak area in the total ion chromatogram by the molecular weight of PMAAs. The molar ratio of mannose, glucose and galactose obtained by PHP-1 methylation analysis was 2.31:95.92:1.77, which was basically consistent with the molar ratio of mannose, glucose and galactose obtained by monosaccharide composition analysis (2.37:95.47:2.15). The results showed that PHP-1 mainly contained five types of sugar residue connection forms, as shown in Table 3: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, 1,4,6-Glcp. PHP-1 has a high degree of branching (DB=47%), indicating that PHP-1 is a heteropolysaccharide with a highly branched structure.

[0100] Table 3 Methylation analysis of the main glycosidic bonds of PHP-1

[0101]

[0102]

[0103] Note: NT, NB and NL are the numbers of terminal residues, branch residues and linear residues, respectively.

[0104] The results of methylation analysis showed that PHP-2a contained 15 types of glycosidic bonds as shown in Table 4. The contents of various monosaccharides in methylation analysis were similar to the monosaccharide composition content detected by UPLC-MS (Table 5), indicating that the methylation reaction of the polysaccharide was basically complete. The branching degree (DB) of PHP-2a was calculated to be 58.66%, which is a high-branching polysaccharide.

[0105] Table 4 Methylation analysis of the main glycosidic bonds of PHP-2a

[0106]

[0107]

[0108] Table 5 Comparison of PHP-2a methylation analysis and monosaccharide composition analysis results

[0109] Monosaccharide name Methylation Monosaccharide composition Man 2.73 1.79 Rha 10.88 11.32 GlcAc 1.17 1.45 GalAc 2.85 2.60 Glc 3.26 2.45 Gal 30.70 31.99 Ara 48.41 48.40

[0110] The contents of various monosaccharides in methylation analysis were similar to those in monosaccharide composition detected by UPLC-MS (Table 6), indicating that the methylation reaction of these three polysaccharides was basically complete. The results of methylation analysis showed that the glycosidic bond types of PHP-3a, PHP-3b and PHP-3c were relatively complex, with 12 different types of glycosidic bonds (Table 7). All three had a high degree of branching, with PHP-3a having the highest degree of branching (66.85%), which was consistent with the above result that PHP-3a had the highest degree of branching as determined by the sugar ratio.

[0111] Table 6 Comparison of methylation analysis and monosaccharide composition analysis results of PHP-3a, PHP-3b and PHP-3c

[0112]

[0113] Table 7 Methylation analysis of the main glycosidic bonds of PHP-3a, PHP-3b and PHP-3c

[0114]

[0115] (5) Attribution of NMR signals of non-starch homogeneous polysaccharides of Radix Pseudostellariae

[0116] The homogeneous non-starch polysaccharide of Radix Pseudostellariae (60 mg) was accurately weighed, and 600 μL D2O was added to fully dissolve it. The active hydrogen was replaced by freeze drying. The above process was repeated three times to completely replace the active hydrogen. 600 μL D2O was added to the dried polysaccharide sample to fully dissolve it, and the supernatant was taken by centrifugation. The homogeneous non-starch polysaccharide of Radix Pseudostellariae was detected on a Bruker Advance III 500MHz NMR spectrometer at a temperature of 298K. 1 H NMR, 13 C NMR and two-dimensional spectra (including 1 H- 1 H COSY, HSQC, HMBC and NOESY spectra). The NMR spectra were analyzed using MestReNova 12.0 software.

[0117] Combining the monosaccharide composition, glycosidic bond type, 2D NMR spectrum and data provided in relevant literature, the C / H chemical shifts of all monosaccharide residues in PHP-1 were obtained (Table 8). 1H and 13 The α- or β-configuration of isomeric hydrogen (H1) and isomeric carbon (C1) in sugar residues can be identified in the CNMR spectrum. In most cases, the isomeric region of the α-configuration appears at δ5.1-5.8ppm and δ98-103ppm (isomeric carbon), while the β-configuration corresponds to δ4.3-4.8ppm and δ103-106ppm (isomeric carbon) in the isomeric region. PHP-1 has multiple obvious anomeric hydrogen (H1) signals in the anomeric hydrogen region of δ4.5-5.5ppm. A large number of hydrogen signals (H2-H6) are concentrated in the δ3.0ppm-4.5ppm region, and the signals overlap severely, making it difficult to identify their attribution. 1 Compared with the H NMR spectrum of PHP-1 13 The C NMR spectrum has fewer spectral lines. The anomeric carbon region of δ90-110 ppm has five obvious signal peaks at δ92.46 ppm, δ96.78 ppm, δ98.70 ppm, δ99.75 ppm and δ99.87 ppm, corresponding to C1 of residues A, B, C, D and E, respectively.

[0118] COSY and HSQC spectra analyzed other signals in 1 H and 13 Assignment in C NMR. Take the sugar residue A→2)-α-Galp-(1→) as an example. COSY spectrum determined the cross peaks at δ5.22 / 3.49ppm, δ3.49 / 3.73ppm, δ3.73 / 3.19ppm, δ3.19 / 3.42ppm and δ3.42 / 3.70ppm. δ5.22ppm corresponds to H-1 of residue A, and δ3.49ppm, δ3.73ppm, δ3.19ppm, δ3.42ppm and δ3.70ppm correspond to H-2, H-3, H-4, H-5 and H-6 of residue A, respectively. HSQC spectrum determined the cross peaks

[0119] δ5.22 / 92.46ppm, δ3.49 / 73.38ppm, δ3.73 / 70.45ppm, δ3.19 / 71.83ppm, δ3.42 / 70.41ppm and δ3.70 / 60.52ppm, corresponding to H-1 / C-1, H-2 / C-2, H-3 / C-3, H-4 / C-4, H-5 / C-5 and H-6 / C-6 of A, respectively.

[0120] HMBC spectra and NOESY spectra were used to determine the glycosyl residues, main chain and substitution positions of polysaccharides. HMBC spectra can be used to calculate the bonds between adjacent sugar residues, reflecting the long-range coupling of about two or three bond distances between protons and carbon. NOESY spectra simultaneously reflect cross peaks within and between sugar residues. NOESY spectra reflect the correlation between sugar residues in PHP-1 (Table 9). The cross peak 5.19 / 3.57ppm reflects the long-range correlation between H-1 of sugar residue E and H-3 of sugar residue D, 5.19 / 5.37ppm reflects the long-range correlation between H-1 of sugar residue E and H-1 of sugar residue D, and 5.19 / 3.36ppm reflects the long-range correlation between H-1 of sugar residue E and H-3 of sugar residue C. The sequence between residues C, D and E was established: →3,6)-α-Manp-(1→3)-α-Glcp -(1→3)-α-Glcp-(1→α-Glcp. 5.37 / 3.57ppm reflects the long-range correlation between H-1 of sugar residue D and H-3 of sugar residue D, 5.37 / 3.83ppm reflects the long-range correlation between H-1 of sugar residue D and H-6 of sugar residue C, 5.37 / 3.49ppm reflects the long-range correlation between H-1 of sugar residue D and H-2 of sugar residue A, 3.57 / 3.64ppm reflects the long-range correlation between H-1 of sugar residue D and H-3 of sugar residue D, There is a long-range correlation between H-1 and H-4 of sugar residue B, 4.94 / 3.95 ppm reflects a long-range correlation between H-1 of sugar residue C and H-6 of sugar residue B, 4.63 / 3.73 ppm reflects a long-range correlation between H-1 of sugar residue B and H-4 of sugar residue B. Therefore, 5.22 / 3.57 ppm reflects a long-range correlation between H-1 of sugar residue A and H-3 of sugar residue D. In the HMBC spectrum, δ5.37 / 75.82 pp The cross peaks at m and δ4.94 / 73.53ppm represent the correlation between H-1 of residue D and C-4 of residue B, and H-1 of residue C and C-6 of residue B, respectively. The cross peak at δ4.63 / 75.82ppm indicates that there is a correlation between H-1 of residue B and C-4 of residue B. The HMBC spectrum reflects the correlation between the sugar residues of PHP-1 (Table 10). On this basis, the structure of PHP-1 is obtained by connecting residues A, B, C, D, and E:

[0121]

[0122] The main chain of PHP-1 is composed of T-α-Glcp-(1→, →3)-α-Glcp-(1→, →3,6)-α-Manp-(1→,

[0123] →4,6)-β-Glcp-(1→、→2)-α-Galp-(1→) are composed of five glycosidic bonds, and the branch chains are respectively

[0124] →3,6)-α-Manp-(1→'s C-3 and →4,6)-β-Glcp-(1→'s C-3 and C-6 extend Glc.

[0125] Table 8 PHP-1 1 H and 13 Summary of C NMR chemical shifts (unit: ppm)

[0126]

[0127]

[0128] Table 9 Correlation between sugar residues in PHP-1

[0129]

[0130] Table 10 Correlation between sugar residues in PHP-1

[0131] Glycosyl residues Proton Proton correlation A →2)-α-Galp-(1→ H-1(5.22) 77.06(D;C-3) B →4,6)-β-Glcp-(1→ H-1(4.63) 75.82 (B; C-4) C →3,6)-α-Manp-(1→ H-1(4.94) 73.53 (B; C-6) D →3)-α-Glcp-(1→ H-1(5.37) 75.82 (B; C-4), 74.49 (A; C-2) E T-α-Glcp-(1→ H-1(5.19) 77.06(D;C-3),99.75(D;C-1),76.74(C;C-3)

[0132] The precise structural information of PHP-2a was further analyzed by nuclear magnetic resonance spectroscopy. Combining the monosaccharide composition, glycosidic bond type, COSY spectrum and HSQC spectrum, the C / H chemical shifts of all monosaccharide residues were assigned (Table 11). Take the residue AT-Galp-(1→ as an example. The COSY spectrum determined the cross peaks at δ5.01 / 3.78ppm, δ3.78 / 3.99ppm, δ3.99 / 3.41ppm, δ3.41 / 3.64ppm and 3.64 / 3.73ppm. δ5.01ppm corresponds to H-1 of residue A, δ3.78ppm, δ3.99ppm, δ3.41ppm, δ3.64ppm and δ3.73ppm correspond to at H-2, H-3, H-4, H-5 and H-6 of residue A. The cross peaks determined by HSQC spectrum were δ5.01 / 99.66ppm, δ3.78 / 66.32ppm, δ3.99 / 69.15ppm, δ3.41 / 71.75ppm and δ3.73 / 61.17ppm, corresponding to H-1 / C-1, H-2 / C-2, H-3 / C-3, H-4 / C-4, H-5 / C-5 and H-6 / C-6 of A, respectively.

[0133] HMBC spectra and NOESY spectra were used to determine the glycosyl residues, main chain and substitution positions of the polysaccharide. Table 12 summarizes the correlations between the sugar residues of PHP-2a in the NOESY spectrum. The cross peak 5.18 / 4.08ppm reflects the long-range correlation between H-1 of sugar residue L and H-2 of sugar residue K, 5.01 / 3.87ppm reflects the long-range correlation between H-1 of sugar residue A and H-5 of sugar residue K, and the sequence between residues A, K and L was established: T-α-Galp(1→5)-α-1,2-Araf-(3→1)-Araf-(2→. 5.01 / 3.80ppm reflects the long-range correlation between H-1 of sugar residue A and H-2 of sugar residue N, 4.42 / 3.87ppm reflects the long-range correlation between H-1 of sugar residue D and H-5 of sugar residue K, 5.12 / 4.10ppm reflects the long-range correlation between H-1 of sugar residue I and H- 2, indicating that there are repeated sugar residue I units in PHP-2a, 5.09 / 4.29ppm reflects the remote correlation between H-1 of sugar residue H and H-3 of sugar residue F, 4.70 / 3.75ppm reflects the remote correlation between H-1 of sugar residue B and H-2 of sugar residue B, indicating that there are repeated sugar residue B units in PHP-2a, 3.75 / 5.12ppm reflects the remote correlation between H-2 of sugar residue B and H-1 of sugar residue I, 4.70 / 3.75ppm reflects the remote correlation between H-1 of sugar residue F and H-2 of sugar residue I, and 4.48 / 4.01ppm reflects the remote correlation between H-1 of sugar residue C and H-3 of sugar residue B.

[0134] The correlation between the sugar residues of PHP-2a in the HMBC spectrum is shown in Table 13. The cross peaks at δ5.01 / 69.26ppm and δ5.01 / 84.47ppm represent the correlation between H-1 of residue A and C-5 of residue K, and H-1 of residue A and C-2 of residue N, respectively. The cross peak at δ4.63 / 75.82ppm indicates that there is a correlation between H-1 of residue B and C-4 of residue B. On this basis, the structure of PHP-2a is obtained by the connection between sugar residues. Figure 1 .

[0135] The main chain of PHP-2a is composed of T-α-Galp, 1,2,3,5-α-Araf, 1,2-α-Araf and 1,2-β-Galp glycosidic bonds, and the side chains are mostly composed of T-α-Galp, T-β-Rhap and T-α-Araf.

[0136] Table 11 PHP-2a 1 H and 13 Summary of C NMR chemical shifts (unit: ppm)

[0137]

[0138]

[0139] Table 12 Correlation between sugar residues in PHP-2a

[0140]

[0141]

[0142] Table 13 Correlation between sugar residues in PHP-2a

[0143]

[0144] (6) Congo red test of non-starch homogeneous polysaccharides of Radix Pseudostellariae

[0145] Congo red solution (50 μM) was first mixed with an equal amount of Pseudo-Pseudo-starch homogeneous polysaccharide solution (0.5 mg / mL). Then NaOH solution (1 M) was added to produce a series of mixed solutions with a final NaOH concentration of 0.1 to 0.5 M. The UV absorption in the range of 400-600 nm was measured and C NaOH and the maximum absorption wavelength λ max In dilute alkaline solution, the λ max There is an obvious red shift from 495nm to 508nm; as C NaOH As the concentration increases, max The λ of the non-starch homogeneous polysaccharide solution of Pseudostellaria heterophylla max As the alkali concentration increases, the λ max The consistent trend of change indicates that the non-starch homogeneous polysaccharide of Radix Pseudostellariae has a triple helical structure ( Figure 6 ).

[0146] 3. In vitro experiments on non-starch uniform polysaccharides of Radix Pseudostellariae

[0147] (1) Preparation of culture medium

[0148] Preparation of GAM medium: Weigh 4.9 g of GAM medium and dissolve it in 100 mL of deionized water after boiling and deoxygenation, sterilize it by high pressure (121°C, 15 min). After cooling, add sterile hemin (1 mL, 5 mg / mL) and vitamin K1 (1 mL, 0.1%). Preparation of MM culture medium: accurately weigh KH2PO4 1.360g, NaCl 87.75g, MgCl2 925.11ng, (NH4)2SO4 112.319mg, L-cysteine ​​48.464g, L-histidine 3.103g, and FeSO4.7H2O3 8.927ng in a 150mL conical flask, add 100mL ultrapure water to dissolve, adjust pH to 7.4, add 100μL of resazurin indicator, sterilize by high pressure (121℃, 15min), cool and then add sterile hemin (100μL, 5mg / mL), vitamin K3 (100μL, 1mg / mL), and vitamin B12 (5μL, 0.1mg / mL), mix well and set aside.

[0149] (2) Effects of different concentrations of Pseudomonas heterophylla non-starch homogeneous polysaccharides on the in vitro activity of BO, BT, BL and LGG bacteria

[0150] BO, BT, BL and LGG bacteria were cultured in vitro anaerobically with GAM medium. After culturing at 37°C for 24h, obvious bacterial precipitation was observed. The supernatant was discarded by centrifugation (3000rpm, 5min). The bacterial precipitate was washed with MM medium and the bacterial solution was diluted to an OD600 value of 0.1-0.2. PHP-1, PHP-2a, PHP-3a, PHP-3b and PHP-3c were prepared at concentrations of 2.5mg / mL, 5mg / mL and 10mg / mL, respectively, as the only carbon source of the culture medium. Sterile water (Blank) was used as a blank control. 100μL of diluted bacterial solution of BO and BT bacteria and 100μL of different concentrations of Pseudostellaria heterophylla non-starch uniform polysaccharide solution or Blank solution were added to a 96-well plate. Five replicates were added for each sample. The culture was anaerobically cultured at 37°C for 72h. The bacterial solution concentration was observed with an ELISA reader at different time points, and the growth curve was drawn.

[0151] (3) Experimental results

[0152] The growth curves of Pseudostellaria heterophylla non-starch uniform polysaccharides PHP-1, PHP-2a, PHP-3a, PHP-3b and PHP-3c as the sole carbon source for BO, BT, BL and LGG strains showed that different concentrations of Pseudostellaria heterophylla non-starch uniform polysaccharides had a growth-promoting effect on the strains. When the concentration was 10 mg / mL, the above polysaccharides had the best growth-promoting effect on BO, BT, BL and LGG strains ( Figure 7); among them, PHP-1 has a better growth-promoting effect on BL and LGG bacteria than other polysaccharides; PHP-3b has a better growth-promoting effect on BO bacteria than other polysaccharides.

[0153] 4. Test on the glycolysis characteristics of non-starch uniform polysaccharides of Radix Pseudostellariae

[0154] (1) Preparation of human feces

[0155] Fresh feces were collected from three healthy volunteers (aged 20-25 years old, who had not received antibiotic or probiotic treatment for at least three months, and whose body mass index was normal). The volunteers wore masks and gloves, and used a sterile sampler to take a certain amount of fresh feces into a sterile feces collector, which was immediately sealed and placed in an anaerobic culture bag. Equal amounts of feces from each volunteer were mixed on a clean bench. The empty tube should be weighed in advance, and the feces should be put into the tube and weighed again, and then diluted to 32% (w / v). For example: 16g of mixed feces was placed in a sterile 50mL centrifuge tube, and immediately diluted with 50mL sterile phosphate buffer (0.1M, pH=7.0-7.2), and vortexed for 1min to obtain a fecal suspension (32%, w / v). The fecal suspension was centrifuged (265×g, room temperature, 5min) to remove food residues, and the supernatant was used as fecal slurry for further experiments. A sterile low-nutrient fermentation medium was prepared for use. There were three groups in the in vitro fermentation experiment. The culture medium without any other carbon source was the blank control (BLANK) group, the culture medium containing inulin (100.0 mg) was the positive control (INL) group, and the culture medium containing non-starch uniform polysaccharide of Pseudostellaria heterophylla (100.0 mg) was the experimental group. Three parallel experiments were performed in each group.

[0156] The non-starch uniform polysaccharide of Radix Pseudostellariae used in the fermentation characteristics test is the non-starch uniform crude polysaccharide of Radix Pseudostellariaee (cPHP) in step (3) of Example 1. Prepare appropriate sterile tubes, add 2.5mL of fecal slurry (32%, w / v) to 10.0mL of low-nutrient fermentation medium of BLANK group, INL group and experimental group respectively, spread a 1cm thick layer of liquid paraffin on the top of the liquid, plug the mouth of the test tube with silicone plug, and then put all of them into anaerobic gas production bags, put in anaerobic gas production bags and anaerobic indicators, and start in vitro simulated large intestine fermentation in a 37°C constant temperature incubator. The entire fermentation process was incubated at 37°C, and 2.5mL was sampled at 0, 6, 12, and 24h of fermentation. The fermentation product was quickly frozen and stored at -80°C, and the fermentation was stopped at 24h.

[0157] (2) Detection of physical and chemical properties of fermentation products

[0158] The pH value of the fermentation products at different culture time points was measured by pH meter. The reducing sugar content of the fermentation products at different culture time points was measured by DNS method. The molecular weight change of the fermentation products at different culture time points was measured by HPLC-ELSD method. The monosaccharide composition of the fermentation products at different culture time points was measured by PMP derivatization.

[0159] (3) Using pre-column derivatization UPLC / Q-TOF-MS to determine the short-chain fatty acids and lactic acid content of the supernatant of the fermentation product at different culture time points:

[0160] Preparation and derivatization of mixed reference solution: 11.42μL acetic acid, 14.82μL propionic acid, 17.62μL n-butyric acid, 22.00μL valeric acid, 22.00μL isovaleric acid, 11.68μL lactic acid were diluted to 20mL with 50% acetonitrile water to prepare a 10mM reference solution as the mother solution, and the mother solution was diluted in half to form a series of gradients. Take 100μL of mixed standard of each concentration, add 50μL 200mM 3-NPH-50% acetonitrile water and 50μL 120mM EDC-50% acetonitrile water (containing 6% pyridine), vortex to mix, derivatize at 37℃ for 30min, and quickly cool in an ice water bath for 1min. Add 10% acetonitrile to dilute to 1mL, vortex to mix, centrifuge at 12000rpm for 10min, and take the supernatant and store it at -20℃. Before sample injection and analysis, 400 μL of the internal standard derivative was mixed with 600 μL of the mixed standard derivative to obtain a mixed reference test solution.

[0161] Derivatization of fermentation products: Take 100 μL of the supernatant of the fermentation products at different culture time points after centrifugation at 4°C, add 100 μL of acetonitrile and mix well to obtain the sample solution. Take 100 μL of sample solution, add 50 μL of 200 mM 3-NPH-50% acetonitrile / water and 50 μL of 120 mM EDC-50% acetonitrile / water (containing 6% pyridine), derivatize at 37°C for 30 minutes, and quickly cool in an ice water bath for 1 minute. Add 10% acetonitrile to dilute to 1 mL, vortex to mix, centrifuge at 12000 rpm for 10 minutes, and take the supernatant and store it at -20°C. Before sampling and analysis, take 400 μL of the internal standard derivative and mix with 600 μL of the fermentation product derivative to obtain the test solution.

[0162] UPLC / Q-TOF-MS analysis: The chromatographic column was a BEH C18 column (50×2.1 mm, 1.7 μm), the column temperature was 40°C, the autosampler temperature was 4°C, the injection volume was 2 μL, and the flow rate was 0.35 mL / min. The mobile phase was 0.1% formic acid water (A)-acetonitrile containing 0.1% formic acid (B) for gradient elution, 0-2 min, 10%-15% B, 2-5 min, 15%-30% B, 5-6 min, 30%-45% B, 6-7 min, 45%-55% B, 7-8 min, 55%-100% B, 8-9 min, 100% B. ESI ion source, acquisition in negative ion mode, mass acquisition range m / z was 100-600 Da.

[0163] (4) Experimental results

[0164] The pH value changes during the 24h fermentation of Pseudostellaria heterophylla non-starch uniform polysaccharide showed that the pH value of the Pseudostellaria heterophylla non-starch uniform polysaccharide group decreased significantly from 6.86±0.15 to 4.85±0.24 ( Figure 8 A). At the beginning of fermentation, the neutral sugar content of the non-starch uniform polysaccharide group of Pseudostellaria heterophylla was 3.24±0.24 mg / mL, and at the end of fermentation, it was 0.35±0.03 mg / mL ( Figure 8 C). In the first 12 hours of fermentation, the sugar consumption rate reached 85.47%, and the sugar content slowly decreased in the later stage of fermentation, and the final consumption rate was 90.72% ( Figure 8 D). The non-starch uniform polysaccharide of ginseng seed of the present invention can be efficiently utilized by intestinal flora.

[0165] The molecular weight change results show ( Figure 8 E), the peak of Pseudostellaria heterophylla non-starch uniform polysaccharide kept moving towards the peak of the fermentation medium. At the 12th and 24th hours of fermentation, the peaks of Pseudostellaria heterophylla non-starch uniform polysaccharide group were basically the same, indicating that within the first 12 hours of fermentation, Pseudostellaria heterophylla non-starch uniform polysaccharide could be mostly degraded by intestinal microorganisms. This result was consistent with the result of carbohydrate consumption rate.

[0166] Short-chain fatty acids (SCFAs) are the main products of intestinal microbial fermentation of indigestible carbohydrates. As the fermentation progresses, the types and contents of SCFAs in the non-starch uniform polysaccharide group of Pseudostellaria heterophylla increase, and the short-chain fatty acids obtained are mainly acetic acid, propionic acid and butyric acid.

[0167] At the 12th hour of fecal fermentation, the total SCFAs content of the Pseudostellaria heterophylla non-starch uniform polysaccharide group reached the highest level (38.08±0.52mM), which was significantly higher than that of the blank control group (24.06±0.39mM), and was stronger than that of the INL group (36.23±0.32mM); among them, the butyric acid content produced by the fermentation of Pseudostellaria heterophylla non-starch uniform polysaccharide (7.38±1.21mM) was significantly higher than that of the INL group (5.53±0.55mM). At the 24th hour of the fermentation stage, the total SCFAs content of the Pseudostellaria heterophylla non-starch uniform polysaccharide group decreased (34.36±0.32mM), which corresponded to the increase in pH value in the late fermentation period.

[0168] The concentration of SCFAs is considered to be an important reflection of microbial activity, indicating that non-starch uniform polysaccharides from Pseudostellariae Radix have a stimulating effect on the production of SCFAs by intestinal flora. SCFAs can lower the pH value of the large intestine, thereby inhibiting the growth of adverse pathogens, affecting the activity of microbial enzymes, and preventing colorectal cancer. Butyrate in SCFAs is the main nutrient for intestinal epithelial cells. It is mainly produced and absorbed in the colon, which keeps colon cells stable and regulates intestinal flora imbalance. It can be used for the prevention and treatment of diseases such as irritable bowel syndrome, colitis, acute and chronic diarrhea, and intestinal cancer. Therefore, non-starch uniform polysaccharides from Pseudostellariae Radix have good beneficial properties and application value for regulating intestinal microorganisms and maintaining the body's intestinal health.

[0169] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A non-starch uniform polysaccharide from Radix Pseudostellariae Radix, characterized in that: Its monosaccharide composition includes glucose, galactose and mannose in the following molar ratios: 95.47:2.15:2.37; The molecular weight of the non-starch homogeneous polysaccharide of Radix Pseudostellariae is 2124Da; The Pseudostellaria heterophylla non-starch uniform polysaccharide has a triple helical structure; The repeatable primary structural units of the Pseudostellaria heterophylla non-starch uniform polysaccharide include the following sugar residues: T-Glcp, 1,3-Glcp, 1,2-Galp, 1,3,6-Manp, and 1,4,6-Glcp.

2. A non-starch uniform polysaccharide from Radix Pseudostellariae Radix, characterized in that: Its monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 1.79:11.32:1.45:2.60:2.45:31.99:48.40； The molecular weight of the non-starch uniform polysaccharide of Radix Pseudostellariae is 85900Da; The Pseudostellaria heterophylla non-starch uniform polysaccharide has a triple helical structure; The repeatable primary structural units of the non-starch uniform polysaccharide of Pseudostellaria heterophylla include the following sugar residues: T-Araf, T-Rhap, 1,2-Araf, T-Glcp, T-GlcpA, T-Galp, 1,2-Galp, 1,2,3,5-Araf, 1,3,6-Manp, 1,3,6-Glcp, 1,4,6-Galp, 1,2,3,4-Galp, 1,2,3,4-GalpA, 1,2,3,6-Galp, 1,2,3,6-GalpA.

3. A non-starch uniform polysaccharide from Radix Pseudostellariae Radix, characterized in that: Its monosaccharide composition includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 13.04:15.18:0.82:19.83:51.14； The molecular weight of the non-starch uniform polysaccharide of Radix Pseudostellariae is 72970Da; The Pseudostellaria heterophylla non-starch uniform polysaccharide has a triple helical structure; The repeatable primary structural units of the Pseudostellaria heterophylla non-starch uniform polysaccharide include the following sugar residues: T-Rhap, 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,2-Galp, 1,2,3-Araf, 1,3,5-Araf, 1,2,3-Galp, 1,3,4-Rhap, 1,2,3,4-Rhap, 1,2,3,5-Araf.

4. A non-starch uniform polysaccharide from Radix Pseudostellariae Radix, characterized in that: Its monosaccharide composition includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 22.58:24.15:3.00:16.30:33.97； The molecular weight of the non-starch uniform polysaccharide of Radix Pseudostellariae is 35820Da; The Pseudostellaria heterophylla non-starch uniform polysaccharide has a triple helical structure; The repeatable primary structural units of the Pseudostellaria heterophylla non-starch uniform polysaccharide include the following sugar residues: 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,2-Galp, 1,2,3-Araf, 1,3,5-Araf, 1,3,4-Rhap, 1,2,4-Rhap, 1,2,3,4-Glcp, 1,2,3,4-Rhap, 1,2,3,5-Araf.

5. A non-starch uniform polysaccharide from Radix Pseudostellariae Radix, characterized in that: Its monosaccharide composition includes rhamnose, galacturonic acid, glucose, galactose and arabinose in the following molar ratios: 24.75:45.64:2.19:11.37:16.04； The molecular weight of the non-starch uniform polysaccharide of Radix Pseudostellariae is 20300Da; The Pseudostellaria heterophylla non-starch uniform polysaccharide has a triple helical structure; The repeatable primary structural units of the Pseudostellaria heterophylla non-starch uniform polysaccharide include the following sugar residues: 1,2-Araf, T-Galp, 1,4-Galp, 1,4-GalpA, 1,4-Rhap, 1,2,3-Araf, 1,3,5-Araf, 1,2,3-GalpA, 1,3,4-Rhap, 1,2,4-Rhap, 1,2,3,4-Glcp, 1,2,3,4-Rhap, 1,2,3,5-Araf.

6. A method for preparing the non-starch uniform polysaccharide of Radix Pseudostellariae as claimed in any one of claims 1 to 5, characterized in that: The steps include: (1) extracting Pseudostellariae Radix in ethanol, filtering to obtain a precipitate, and drying the precipitate to obtain Pseudostellariae Radix Reticulatae Radix dregs; (2) taking the Radix Pseudostellariae Radix residue obtained in step (1), adding it into water for extraction, and obtaining Radix Pseudostellariae Radix aqueous extract; (3) removing starch from the water extract of Radix Pseudostellariae obtained in step (2), centrifuging and concentrating, taking the supernatant and adding ethanol for alcohol precipitation, centrifuging and collecting the precipitate to obtain crude Pseudostellariae polysaccharide; (4) Separating and purifying the crude polysaccharide of Pseudostellariae Radix obtained in step (3), eluting, collecting the eluates corresponding to different gradient elution peaks, and concentrating to obtain the uniform non-starch polysaccharide of Pseudostellariae Radix.

7. The preparation method according to claim 6, characterized in that: The starch removal in step (3) is as follows: adding amylase until the enzyme activity is 1-2 U / mL, the enzymatic hydrolysis temperature is 80-90° C., and the enzymatic hydrolysis time is 0.5-2 h.

8. The preparation method according to claim 6, characterized in that: The elution in step (4) is: eluting with deionized water solutions of NaCl of different concentrations; The different concentrations are: A, eluting with deionized water to obtain the Pseudostellaria heterophylla non-starch uniform polysaccharide according to claim 1; B, eluting with 0.1M NaCl deionized water solution to obtain the heterophyllotoxin non-starch uniform polysaccharide according to claim 2; C. Elution with 0.2M NaCl deionized water solution yields a non-starch uniform polysaccharide mixture of Panax notoginseng.

9. The preparation method according to claim 8, characterized in that: Step (4) further comprises separating and purifying the Pseudostellaria heterophylla non-starch uniform polysaccharide mixture to obtain the Pseudostellaria heterophylla non-starch uniform polysaccharides described in claims 3-5.

10. Use of the Pseudostellariae Radix non-starch uniform polysaccharide according to any one of claims 1 to 5 or the Pseudostellariae Radix non-starch uniform polysaccharide prepared by the preparation method according to any one of claims 6 to 9 in the preparation of intestinal probiotic foods and health products; The intestinal probiotics include BO bacteria, BT bacteria, BL bacteria and LGG bacteria.

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