Clerodendranthus spicatus fructo-oligosaccharide and preparation method and application thereof

Abstract

The invention discloses a codonopsis lanceolata fructooligosaccharide compound, a derivative, a composition and a preparation method thereof, and application of the codonopsis lanceolata fructooligosaccharide compound in the aspect of regulating intestinal flora, and belongs to the field of biological medicine and food. The Clerodendranthus spicatus fructo-oligosaccharide compound provided by the invention has a structure as shown in a formula (I). The Clerodendranthus spicatus fructo-oligosaccharide compound, the derivative or the composition thereof can remodel intestinal flora and regulate metabolite balance, and has the activity of improving and treating diseases related to the intestinal flora. Therefore, the clerodendranthus spicatus fructo-oligosaccharide compound, the derivative or the composition thereof can be used for preparing products such as medicines and foods for preventing and / or treating intestinal flora related diseases.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and food, specifically relating to fructooligosaccharides and their derivatives, compositions containing them, their preparation methods, and their application in the preparation of drugs, foods, and other products for the treatment and / or prevention of intestinal flora-related diseases. Background Technology

[0002] The gut microbiota is a community of microorganisms that colonize the human gut and coexist with the body for a long time. Because its cell count is 10 times that of human cells and it encodes more than one million genes, it is often referred to as the body's "second genome." It not only affects food digestion and absorption to provide energy for the body, but also plays a role in regulating immune function and participating in metabolism. It can also influence endocrine function (regulating energy, blood sugar, and inflammatory responses). However, today, with increasing social pressure, dietary changes (including high-sugar, high-fat, high-salt, and high-spicy diets), and reduced physical activity, people are experiencing various sub-health conditions (such as anxiety, chronic imbalances in metabolic and immune processes), and in severe cases, even depression, diabetes, hyperlipidemia, obesity, proctitis, and cancer. Research shows that gut microbiota dysbiosis plays an important role in the development and progression of these diseases, and that restoring the balance of the gut microbiota can improve and even treat many diseases.

[0003] In recent years, more and more people have begun to pay attention to personal health issues, and the idea of ​​improving health through diet therapy has gradually gained acceptance, especially the use of dietary fiber to improve sub-health conditions. Studies have shown that dietary fiber can act as a prebiotic, improving health by regulating the balance of gut microbiota. *Codonopsis bulleynana*, the root of a plant in the genus *Codonopsis* of the family Campanulaceae, is widely cultivated in Yunnan Province and is a local specialty edible resource. *Codonopsis bulleynana* emits a distinctive odor, clearly different from medicinal *Codonopsis*, hence its alternative name, "stinky medicine." It has a long history of use as a folk tonic in Yunnan, commonly seen in stews made with chicken or pork ribs. Consuming it is said to promote flatulence, expel accumulated gas, and relieve constipation, while also having the effects of replenishing qi and blood, strengthening the spleen and lungs. Research reports that its water extract has good effects on lowering blood lipids, combating fatigue, and promoting gastrointestinal motility. Therefore, *Codonopsis bulleynana* offers numerous health benefits and possesses high edible, medicinal, and health-promoting value. However, *Stachys affinis* has not yet been developed into a high-value-added product, and the industry has remained largely based on extensive farming and retail in farmers' markets. Polysaccharides, as water-soluble dietary fiber, are an important class of bioactive substances in *Stachys affinis*, possessing immense potential for developing high-value-added products.

[0004] To date, there are no reports in the existing technology regarding fructooligosaccharides, their preparation methods, or their application in the preparation of drugs, food products, or other products for the prevention and treatment of intestinal flora-related diseases. Summary of the Invention

[0005] To address the aforementioned shortcomings in existing technologies, this invention has discovered that fructooligosaccharides derived from *Gynostemma pentaphyllum*, with a molecular weight distribution between 3.0 and 7.0 kDa, significantly regulate intestinal flora, promote the growth of various beneficial bacteria, and reduce the abundance of harmful bacteria. They also regulate intestinal metabolites. Therefore, the purpose of this invention is to provide fructooligosaccharides derived from *Gynostemma pentaphyllum*, or their derivatives, or combinations thereof, their preparation method, and their application in the preparation of pharmaceuticals, food products, and other products for the prevention and / or treatment of intestinal flora-related diseases.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] This invention first provides an oligofructose derived from ginseng, which has a structure as shown in chemical formula (I):

[0008]

[0009] In chemical formula (I),

[0010] A is α-D-glucose (α-D-Glc); B is β-D-fructose (β-D-Fru); C is β-D-fructose (β-D-Fru); D is β-D-fructose (β-D-Fru).

[0011] n is an integer between 0 and 100.

[0012] The weight-average molecular weight (Mw) of the fructooligosaccharide compound described in this invention is in the range of 3.0. ~7.0 kDa, and its dispersion index (PDI) value is less than or equal to 2.0.

[0013] The stag ginseng oligofructose of the present invention has a monosaccharide composition including glucose and fructose, wherein the glucose is α-D-glucose and the fructose is β-D-fructose.

[0014] The stag ginseng oligofructose of the present invention has a monosaccharide linkage in which α-D-glucose is linked to β-D-fructose by a (1→2) glycosidic bond, and β-D-fructose is linked to β-D-fructose by a (2→1) glycosidic bond.

[0015] Furthermore, the fructooligosaccharide of the present invention has a structure of [→1)-D-Fru-β-(2→] repeating linkage, and a D-Glc-α-(1→) at the end is connected to the 2 position of β-D-Fru.

[0016] The fructooligosaccharide of the present invention is derived from the root of Codonopsis bulleynana, a plant belonging to the genus Codonopsis in the family Campanulaceae. It is obtained by extraction and purification from the washed fresh or dried roots of Codonopsis bulleynana.

[0017] The method for preparing the fructooligosaccharide from *Gynostemma pentaphyllum* according to the present invention includes the following steps:

[0018] Step (1): The crude sugar of Codonopsis bulleynana is obtained by defatting, water extraction and alcohol precipitation from the roots of Codonopsis bulleynana, including but not limited to fresh or dried roots;

[0019] Alternatively, in step (two): the crude ginseng sugar described in step (one) can be purified by ion exchange chromatography, molecular sieve gel chromatography, and then dialysis and freeze-drying to obtain ginseng oligofructose with different molecular weight distributions.

[0020] Currently, only *Codonopsis bulleynana* has been found to contain the *Corydalis oryzae* oligofructose defined in this invention. However, studies have shown that different species of *Corydalis oryzae* are cultivated and consumed in Yunnan Province, including *Codonopsis subglobosa*, *Codonopsis micrantha*, *Codonopsis cordifolioidea*, and *Codonopsis pilosula*. Those skilled in the art will understand that although few *Corydalis oryzae* varieties have undergone isolation and purification of *Corydalis oryzae* oligofructose, the *Corydalis oryzae* varieties containing *Corydalis oryzae* oligofructose as defined in this invention are not limited to *Codonopsis bulleynana*.

[0021] The fructooligosaccharide composition of the present invention is fructooligosaccharide or its derivative.

[0022] A ginseng oligofructose composition containing the aforementioned ginseng oligofructose or its derivatives.

[0023] A ginseng oligofructose pharmaceutical composition containing an effective amount of the ginseng oligofructose or its derivatives.

[0024] The use of the aforementioned ginseng oligofructose or its derivatives, or the aforementioned ginseng oligofructose composition or pharmaceutical composition, in the preparation of medicaments for the treatment and / or prevention of intestinal flora-related diseases.

[0025] The application of the aforementioned ginseng oligofructose or its derivatives, or the aforementioned ginseng oligofructose composition, in the preparation of food.

[0026] The gut microbiota-related diseases mentioned above refer to gut microbiota-related diseases occurring in the gastrointestinal tract, liver, kidneys, cardiovascular system, brain, and nervous system.

[0027] The preparation method of the ginseng oligofructose composition involves first obtaining ginseng oligofructose or its derivatives according to the preparation method of ginseng oligofructose or its derivatives, and then adding a pharmaceutically acceptable carrier or conventional food excipients.

[0028] The preparation method of the ginseng oligofructose pharmaceutical composition involves first obtaining ginseng oligofructose or its derivatives according to the preparation method of ginseng oligofructose or its derivatives, and then adding a pharmaceutically acceptable carrier.

[0029] Gut microbiota dysbiosis plays a crucial role in the development and progression of various metabolic and immune-related chronic diseases, and reshaping the gut microbiota balance can improve, and even treat, many diseases. In recent years, the idea of ​​improving sub-health conditions through dietary fiber supplementation has gained increasing acceptance.

[0030] The fructooligosaccharide (FOS) described in this invention can be used as dietary fiber to regulate the abundance of specific gut microbiota and their metabolites, with significant effects. At the species level, compared with the control group, the FOS treatment group significantly reduced the abundance of Akkermansia sp., Akkermansia muciniphila, Ileibacterium valens, and Hungatella sp. in the mouse intestine, while significantly increasing the abundance of Dorea sp. and various probiotics. It also regulates the expression of metabolites in multiple metabolic pathways, including Arachidonic acid metabolism, Linoleic acid metabolism, Phenylalanine metabolism, and Primary bileacid biosynthesis. Furthermore, FOS also significantly affects the microorganisms in human fecal in vitro fermentation products. After treatment, the abundance of Bifidobacterium, Segatella, and Catenibacterium species in the fecal fermentation broth significantly increased. Notably, the effects of FOS on gut microbiota and metabolites are significantly different from those of the positive control Inulin treatment group. This indicates that the effects of fructooligosaccharides from *Syngonium sibiricum* on gut microbiota and metabolites are unique.

[0031] Therefore, the *Syngonium sibiricum* oligofructose or its derivatives, or combinations thereof, described in this invention have the effect of regulating the abundance of intestinal flora and its metabolites, and have application value in the development and application of drugs, food products, etc., for the prevention and / or treatment of intestinal flora-related diseases. Therefore, this invention provides a drug or food ingredient for the prevention and treatment of intestinal flora-related diseases, containing an effective dose of the *Syngonium sibiricum* oligofructose or its derivatives, or combinations thereof. Attached Figure Description

[0032] Figure 1 The chemical structural formula of fructooligosaccharides from *Gynostemma pentaphyllum*.

[0033] Figure 2 The OHpak SB-804 HQ size exclusion chromatograms of five different molecular weights of ginseng oligofructose.

[0034] Figure 3 The molecular weight distribution of five different molecular weights of fructooligosaccharides from *Syngonium sibiricum* is shown in the diagram.

[0035] Figure 4 Infrared spectrum of fructooligosaccharides from *Syngonium sibiricum*.

[0036] Figure 5 The diagram shows the monosaccharide composition analysis of five different molecular weights of fructooligosaccharides from *Syngonium sibiricum* (A in the diagram represents PMP derivatization analysis; B in the diagram represents direct analysis via acid hydrolysis).

[0037] Figure 6 Gas chromatography-mass spectra of monosaccharide sugar alcohol acetate derivatives of five different molecular weights of fructooligosaccharides from *Syngonium sibiricum*.

[0038] Figure 7 For ginseng oligofructose 1 H(A) and 13 C (B) NMR spectrum;

[0039] Figure 8 The two-dimensional NMR spectrum of fructooligosaccharides from *Stachys pubescens* (A in the figure is...). 1 H- 13 C HSQC; B in the diagram is 1 H- 13 C HSQC-TOCSY, where C in the diagram represents... 1 H- 1 H COSY is, and D in the diagram is... 1 H- 1 H TOCSY, where E in the diagram is... 1 H- 13 C HMBC, where F in the diagram is... 1 H- 1 HROESY);

[0040] Figure 9The results of the regulation of gut microbiota and its metabolites by fructooligosaccharides from *Syngonium stenoptera* are shown in the figure (A in the figure shows the effect on the abundance of genera in the mouse gut microbiota, B in the figure shows the effect on high abundance species in the mouse gut microbiota, C in the figure shows the effect on probiotics in the mouse gut, D in the figure shows the effect on the metabolic pathways in the mouse gut, and E and F in the figure show the effect on in vitro fermentation microorganisms in human feces). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Example 1

[0043] Extraction, separation and purification of fructooligosaccharides from *Gynostemma pentaphyllum*.

[0044] 1.1 Experimental Materials

[0045] Ingredients: Fresh Codonopsis bulleynana, purchased commercially.

[0046] Reagent: YKTS (Cl) strong anion exchange resin - ), Tianjin Yunkai Resin Technology Co., Ltd.; 1000 Da dialysis bags, Spectrum Laboratories, USA; Dextran standards D1 (2500 Da), D2 (4600 Da), D3 (7100 Da), D4 (10000 Da), D5 (21400 Da), D6 (41100 Da), D7 (84400 Da), D8 (133800 Da) and D2000 (2000000 Da), China National Institute for Food and Drug Control; NaCl and ethanol and other commonly used reagents are commercially available analytical grade reagents.

[0047] 1.2 Extraction, Separation and Purification

[0048] (1) 95% ethanol defatting treatment: Take 2.0 kg of fresh rhizome of Codonopsis bulleynana, grind it with a soymilk maker, add 95% ethanol at a material-to-liquid ratio of 2:5 and place it at room temperature for overnight extraction twice. After each extraction, the extract and residue are separated by centrifugation and filtration. The extracts are combined and concentrated to extract to remove fat-soluble substances from Codonopsis bulleynana. The residue is air-dried to remove ethanol.

[0049] (2) Extraction of crude polysaccharides from ginseng: Take the air-dried residue, add deionized water at a material-to-liquid ratio of 1:8 and place it in a water bath at 50°C for 4 hours. Extract twice. After each extraction, separate the extract and residue by centrifugation and filtration. Combine the extracts to obtain the total crude polysaccharide extract from ginseng.

[0050] (3) Fractional alcohol precipitation of crude polysaccharides from *Gynostemma pentaphyllum*: 95% ethanol was added to the extract to a final concentration of 40%, allowed to stand and precipitate, and centrifuged (4000 rpm × 15 min). The precipitate was rinsed twice with 95% ethanol to obtain 40 precipitated crude polysaccharides (CS40). 95% ethanol was added to the supernatant to a final concentration of 60%, allowed to stand and precipitate, and centrifuged (4000 rpm × 15 min). The precipitate was rinsed twice with 95% ethanol to obtain 60 precipitated crude polysaccharides (CS60). 95% ethanol was added to the supernatant to a final concentration of 80%, allowed to stand and precipitate, and centrifuged (4000 rpm × 15 min). The precipitate was rinsed twice with 95% ethanol to obtain 80 precipitated crude polysaccharides (CS80). CS40, CS60, and CS80 were reconstituted with deionized water and then freeze-dried to obtain the crude polysaccharides CS40, CS60, and CS80 solids.

[0051] (4) Purification of ginseng polysaccharides: Take CS40 sample, add 20 times deionized water, vortex to dissolve for 10 min, and centrifuge at 10000 rpm to separate soluble polysaccharides (supernatant) and insoluble polysaccharides (precipitate). Add 10 times deionized water to the precipitate and repeat the above operation to completely remove soluble substances, obtaining soluble polysaccharides (supernatant) and insoluble polysaccharides (precipitate). Take the soluble polysaccharide solution, filter, add to YKTS anion exchange chromatography column, elute with 3 column volumes of membrane-passed deionized water, collect the flow-through, concentrate under reduced pressure at 55°C, dialyze overnight using a dialysis bag (MWCO 1000 Da), and freeze-dry to obtain pure polysaccharide CS40-1 (CCP1). Freeze-dry the insoluble polysaccharide directly to obtain pure polysaccharide CS40-2 (CCP2).

[0052] The same method was used to process crude CS60 polysaccharide to obtain soluble pure polysaccharide CS60-1 (CCP3) and insoluble pure polysaccharide CS60-2 (CCP4). The same method was used to process crude CS80 polysaccharide to obtain only soluble pure polysaccharide CS80-1 (CCP5), and no insoluble polysaccharide was obtained.

[0053] 1.3 Analysis of Impurities in Panax notoginseng Polysaccharides

[0054] Common impurities in polysaccharides are nucleic acid and protein macromolecules. In this experiment, ultraviolet-visible light spectroscopy and ninhydrin colorimetric method were used to analyze and detect the protein and nucleic acid components in *Gynostemma pentaphyllum* polysaccharide.

[0055] UV-Vis spectroscopy analysis: Weigh out polysaccharide samples CCP1~CCP5 and add an appropriate amount of deionized water to prepare 0.2 mg / mL. Use a UV spectrophotometer to scan the UV absorption in the full wavelength range of 190-600 nm.

[0056] Ninhydrin colorimetric analysis: The ninhydrin colorimetric method was used to qualitatively detect whether there are protein components in the polysaccharide of *Gynostemma pentaphyllum*.

[0057] 1.4 FT-IR analysis

[0058] 1 mg of each of the polysaccharide samples CCP1-CCP5 were weighed and prepared using the KBr pellet method. The samples were then measured using a Fourier transform infrared spectrophotometer at 400-4000 cm⁻¹. -1 Absorption intensity within the range.

[0059] 1.5 Analysis of the homogeneity and molecular weight distribution of *Gynostemma pentaphyllum* polysaccharides

[0060] The homogeneity and molecular weight distribution of polysaccharides were analyzed and determined by high-performance gel permeation chromatography (HPGPC).

[0061] (1) Preparation of standard solution and ginseng sample solution

[0062] Preparation of standard solutions: Weigh out dextran standards of known molecular weight (2700, 5250, 9750, 13050, 36800, 646500, 135350 and 300600 Da), add an appropriate amount of deionized water to prepare a 5 mg / mL standard solution, place it in a 1.5 mL centrifuge tube, centrifuge (13000 rpm, 10 min, 20 °C), filter through a 0.22 μm microporous membrane, and analyze by high performance liquid chromatography.

[0063] Preparation of ginseng sample solution: Weigh polysaccharide samples CCP1~CCP5 separately, add an appropriate amount of deionized water to prepare a sample solution of 5 mg / mL, place in a 1.5 mL centrifuge tube, centrifuge (13000 rpm, 10 min, 20 °C), then filter through a 0.22 μm microporous membrane and analyze by high performance liquid chromatography.

[0064] (2) Chromatographic conditions

[0065] Instruments: Agilent Technologies 1260 series high performance liquid chromatograph; Column: Shodex OHpak SB-804 HQ (8.0 × 300 mm); Injection volume: 30 μL; Temperature: column temperature 35°C, flush valve 40°C; Mobile phase: 0.1 M NaCl solution; Flow rate: 0.5 mL / min; Detectors: refractive index detector (RID), diode array detector (DAD).

[0066] (3) Data Analysis

[0067] Data were processed using GPC software to plot a standard curve, which was then used to determine the molecular weight of the ginseng polysaccharide.

[0068] 1.6 Experimental Results

[0069] Following the extraction, separation, and purification steps described in this embodiment, five polysaccharides, CCP1, CCP2, CCP3, CCP4, and CCP5, were obtained from fresh ginseng, with masses (yield relative to fresh samples) of 4.49 g (0.22%), 12.43 g (0.62%), 12.67 g (0.63%), 3.79 g (0.19%), and 42.1 g (2.11%), respectively.

[0070] Ultraviolet spectrophotometry analysis showed that none of the five polysaccharide samples exhibited significant absorption at 260 nm and 280 nm, indicating the absence of nucleic acids and proteins in the polysaccharides. Ninhydrin colorimetric analysis further confirmed that the *Gynostemma pentaphyllum* polysaccharides did not contain proteins or amino acids.

[0071] Furthermore, analysis of the lyophilized samples by high-performance gel permeation chromatography (HPGPC) showed that all five polysaccharide samples exhibited single peaks on the gel size exclusion column, with good peak symmetry and narrow peak widths, indicating a narrow molecular weight distribution of the polysaccharides and good sample homogeneity. Figure 2 Furthermore, the chromatographic peak retention times were relatively long, with elution times of 19.6, 18.5, 19.5, 18.5, and 19.2 min, indicating a low molecular weight of the *Gynostemma pentaphyllum* polysaccharide. The standard curve fitted to the polysaccharide molecular weight using HPGPC results from a series of dextran standards with known molecular weights was LogMw = 10.96 - 0.501 Rt + 0.006049 Rt. 2 -0.00004485Rt 3 (R) 2 = 0.9999). Based on this standard curve, the weight-average molecular weights (Mw) of the *Gynostemma pentaphyllum* polysaccharides were calculated to be 3.19, 6.44, 4.29, 6.01, and 4.36 × 10⁻⁶, respectively. 3 Da ( Figure 3 ).

[0072] The main functional groups and configuration of *Gynostemma pentaphyllum* polysaccharide were analyzed using Fourier transform infrared spectroscopy (FT-IR). The results showed that ( Figure 4 All five ginseng polysaccharides exhibited similar infrared absorption peaks, with the peak at 3368 cm⁻¹ being the most prominent. −1 The broad and strong absorption peak at 2931 cm⁻¹ is attributed to the OH stretching vibration; −1 The absorption peak at 1638 cm⁻¹ originates from the stretching vibration of CH in the sugar ring. −1 The weak characteristic absorption may be caused by bound water; 1417 cm −1 The peaks at 1130 and 1031 cm⁻¹ correspond to the bending vibrations of CH; −1 The significant absorption peaks at 936 and 811 cm⁻¹ indicate that the polysaccharide has a pyran ring structure; −1 The signal at this location indicates the presence of a β-glycosidic bond. It is noteworthy that at 1700 cm⁻¹... −1 The absence of characteristic absorption peaks nearby confirms that the polysaccharides from *Gynostemma pentaphyllum* do not contain unsaturated groups such as benzene rings, double bonds, and carbonyl groups.

[0073] Example 2

[0074] Monosaccharide composition analysis of ginseng polysaccharide.

[0075] PMP derivatization is a commonly used analytical method for determining monosaccharide composition. However, PMP derivatization is only applicable to monosaccharides with a hemiacetal at the reducing end, and cannot be used for ketoses. Therefore, this experiment used PMP derivatization combined with acid hydrolysis direct analysis to analyze the monosaccharide composition of polysaccharides.

[0076] 2.1 Experimental Materials

[0077] Raw materials: Polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 derived from *Gynostemma pentaphyllum* prepared according to the method in Example 1.

[0078] Reagents: D-glucose (D-Glc), D-galactose (D-Gal), D-galacturonic acid (D-GalA), D-fructose (D-Fru), Sigma-Aldrich, USA; D-mannose (D-Man); L-rhamnose (L-Rha), D-arabinose (D-Ara), Alfa Aesar, Inc.; D-fucose (D-Fuc), JD Chemical Industry Co., Ltd.; anhydrous ethanol, Shanghai Titan Technology Co., Ltd.; 1-phenyl-3-methyl-5-pyrazolone (PMP), trifluoroacetic acid (TFA), Shanghai Aladdin Biochemical Technology Co., Ltd.; acetonitrile, Beijing Merida Technology Co., Ltd. (chromatographic grade); dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0079] 2.2 PMP Derivatization Analysis Method

[0080] (1) Preparation of standard solution and sample solution

[0081] Preparation of standard solutions: Weigh out the monosaccharide standards of D-Man, L-Rha, D-GalA, D-Glc, D-Gal, D-Ara and L-Fuc respectively, and add an appropriate amount of deionized water to prepare a standard solution of 10 mg / mL.

[0082] Preparation of sample solutions: Weigh out polysaccharide samples CCP1~CCP5 respectively, add an appropriate amount of deionized water to prepare a sample solution of 1 mg / mL.

[0083] Preparation of monosaccharide mixed standard solution: Take 100 μL of each of the 10 mg / mL standard solutions (D-Man, L-Rha, D-GlcA, D-GalA, D-Glc, D-Gal, L-Xyl, D-Ara and L-Fuc) into the same COD tube, add deionized water to 1 mL, and prepare monosaccharide mixed standard solution.

[0084] (2) Acid hydrolysis

[0085] Take 1 mL of sample solution into a COD tube, add 1 mL of 4 M TFA solution, hydrolyze at 110 °C for 4 h, remove, cool to room temperature, evaporate to dryness, add 2 mL of methanol and evaporate to dryness again (repeat 3 times) until there is no sour smell of TFA. The hydrolysis steps for each monosaccharide standard solution, the monosaccharide mixed standard solution and the blank solution are as above.

[0086] (3) PMP Derivatization

[0087] Add 200 μL of deionized water to the residue of the sample solution, standard solution, and monosaccharide mixed standard solution. Then add 200 μL of 0.6 M NaOH solution and 400 μL of 0.5 M PMP methanol solution, mix well, and incubate at 70 °C for 1 h. Remove from heat, cool to room temperature, add 200 μL of 0.6 M HCl solution, add 2 mL of chloroform, shake vigorously, let stand, and discard the lower layer (repeat 3 times). Transfer the upper layer to a 1.5 mL centrifuge tube, centrifuge (13000 rpm, 10 min, 20 °C), filter through a 0.22 μm microporous membrane, and analyze in a high-performance liquid chromatograph.

[0088] (4) Chromatographic conditions

[0089] Instrument: Agilent Technologies 1260 series high performance liquid chromatograph; Column: XSelect HSS T3 (4.6 × 250 mm, 5 µm); Injection volume: 5 μL; Temperature: Column temperature 25 °C; Mobile phase: 0.1 M phosphate buffer (pH 6.8): acetonitrile = 82:18; Flow rate: 1.0 mL / min; Detector: Diode array detector (DAD); Detection wavelength: 250 nm.

[0090] 2.3 Direct Analysis Method Based on Acid Hydrolysis

[0091] (1) Acid hydrolysis

[0092] Weigh 10 mg each of polysaccharide samples CCP1-CCP5 into COD tubes, add 1 mL of 0.5 M TFA to dissolve them, incubate at 80 °C for 40 min, remove, cool to room temperature, evaporate to dryness, add 2 mL of methanol and evaporate to dryness again (repeat the operation 3 times) until there is no sour smell of TFA. Add 1 mL of deionized water to dissolve the residue, place in a 1.5 mL centrifuge tube, centrifuge (13000 rpm, 10 min, 20 °C), filter through a 0.22 μm microporous membrane, and analyze by high performance liquid chromatography.

[0093] Preparation of monosaccharide mixed standard solutions: Take 1 mL each of 10 mg / mL standard solutions (D-Man, L-Rha, D-Glc, D-Ara and D-Fru) into the same COD tube, add 1 mL of 0.5 M TFA, and proceed in the same manner as the sample solutions.

[0094] (2) Chromatographic conditions

[0095] Instruments: Agilent Technologies 1260 series high performance liquid chromatograph; Column: Cosmosil Sugar-D (4.6 × 250 mm, 5 µm); Injection volume: 5 μL; Temperature: Column temperature 30 °C; Purge valve 35 °C; Mobile phase: Water:acetonitrile = 15:85; Flow rate: 0.2 mL / min; Detector: Refractive index detector (RID).

[0096] 2.4 Experimental Results

[0097] The monosaccharide composition of *Gynostemma pentaphyllum* polysaccharides was analyzed using pre-column derivatization (PMP). Liquid chromatography analysis showed that all five *Gynostemma pentaphyllum* polysaccharides contained only one monosaccharide signal, which, compared to the standard, was glucose (Glc). Figure 5 (A). The glucose content in each polysaccharide was calculated to be 5.60%, 2.68%, 4.45%, 2.75%, and 3.82% using a standard curve of glucose. This indicates that the glucose content in each polysaccharide is very low, and other types of monosaccharides may also be present in high concentrations. Since PMP derivatization primarily targets aldoses with a hemiacetal at the reducing end 1-position, and cannot be performed on ketoses, this invention employs fructose analysis, i.e., direct analysis of acid hydrolysis products. The results showed that all *Gynostemma pentaphyllum* polysaccharides contained a large amount of fructose (…). Figure 5 (B). Therefore, it can be preliminarily determined that the polysaccharides in *Gynostemma pentaphyllum* are likely all fructooligosaccharides.

[0098] Example 3

[0099] Methylation analysis of ginseng polysaccharides determined the linkage mechanism between monosaccharides.

[0100] 3.1 Experimental Materials

[0101] Raw materials: Polysaccharide samples CCP1~CCP5 from *Gynostemma pentaphyllum* prepared according to the method in Example 1.

[0102] Reagents: Dimethyl sulfoxide (DMSO), Shanghai Aladdin Biochemical Technology Co., Ltd.; Sodium hydroxide, Tianjin Damao Chemical Reagent Factory; Iodomethane, Chengdu Aikeda Chemical Reagent Co., Ltd.; Chloroform, Tianjin Damao Chemical Reagent Factory; Trifluoroacetic acid, Shanghai Aladdin Biochemical Technology Co., Ltd.; Sodium borodeuteride (AR), Tianjin Damao Chemical Reagent Factory; Glacial acetic acid (AR), Tianjin Fuyu Fine Chemical Co., Ltd.; Pyridine, Shanghai Aladdin Biochemical Technology Co., Ltd.; Acetic anhydride, Tianjin Lianlong Bohua Pharmaceutical Chemical Co., Ltd.

[0103] 3.3 Methylation and GC-MS Analysis

[0104] (1) Methylation reaction

[0105] Weigh 2 mg each of polysaccharide samples CCP1-CCP5 into COD tubes, add 1 mL of DMSO to dissolve them, then add 1 mL of 50 mg / mL NaOH / DMSO suspension (prepared in advance), stir well, and add 1 mL of iodomethane dropwise under ice bath conditions. Stir for 2 h in the dark, sealed environment, and then add 1 mL of deionized water to stop the reaction. Then add 2 mL of chloroform for extraction (repeat 3 times), take the chloroform layer, add 2 mL of deionized water for extraction again (repeat 3 times), and evaporate the chloroform to dryness to obtain the methylated polysaccharide products.

[0106] (2) Acid hydrolysis

[0107] Add 2 mL of 0.5 M TFA to the methylated polysaccharide and hydrolyze at 70 °C for 3 h. Remove the contents, cool to room temperature, evaporate to dryness, add 2 mL of methanol and evaporate to dryness again (repeat 3 times) until there is no sour taste of TFA.

[0108] (3) Restoration

[0109] Add 1 mL of deionized water to the residue after acid hydrolysis, add 1 M NaOH solution to bring the pH to 10, then add 15 mg of NaBD4 solid, stir at 50 °C for 2 h, then add 500 μL of glacial acetic acid and evaporate to dryness.

[0110] (4) Acetylation of sugar alcohols

[0111] Add 1 mL of pyridine and 1 mL of acetic anhydride to the reduced residue and react at 100 °C for 1 h. Stop the reaction by adding 1 mL of deionized water. Extract with 2 mL of dichloromethane (repeat 3 times), and extract again with 2 mL of deionized water (repeat 3 times). Dry the dichloromethane layer by rotary evaporation. Add 1 mL of dichloromethane, place in a 1.5 mL centrifuge tube, centrifuge (13000 rpm, 10 min, 20 °C), filter through a 0.2 μm microporous membrane, and analyze by GC-MS.

[0112] (5) GC conditions

[0113] Instrument: Agilent Technologies 7890A gas chromatograph; Column: DB-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm); Injection volume: 1 μL; Injector temperature: 250 °C; Carrier gas: high-purity helium; Column flow rate: 1.5 mL / min;

[0114] Temperature program: Start at 80 °C, hold for 1 min, then increase to 250 °C at a rate of 5 °C / min and hold for 40 min.

[0115] (6)MS conditions

[0116] Ionization method: EI; Electron energy: 70 eV; Transfer line temperature: 290 °C; Ion source temperature: 230 °C; Quadrupole temperature: 150 °C; Mass range: 50~600 Da.

[0117] (7) Data Analysis

[0118] The mass spectra were compared with the Complex Carbohydrate Research Center, University of Georgia (CCRC) database (https: / / www.ccrc.uga.edu) to confirm the type of sugar alcohol derivative.

[0119] 3.4 Experimental Results

[0120] Methylation analysis showed that the total ion chromatograms of the five polysaccharides all showed the same signal, with retention times of 20.24, 22.02, and 22.12 min, respectively. Figure 6 (A). Mass spectrometry analysis and database comparison analysis showed that the signal eluting at 20.24 min was 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucanol, attributed to terminal glucose; the peak signals at 22.02 and 22.12 min were 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-D-mannitol and 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-D-glucanol, respectively. Figure 6 (B and C). Since the 2-position of fructose can produce two isomers, R and S, namely mannose and glucose derivatives, during derivatization, the two subsequent peaks are attributed to 2,1-linked fructose. Therefore, this indicates that *Gynostemma pentaphyllum* polysaccharides contain only terminally linked glucose and 2,1-linked fructose, and no monosaccharide derivatives with complex branched linkages.

[0121] Example 4

[0122] NMR analysis of ginseng polysaccharide.

[0123] 4.1 Experimental Materials

[0124] Samples: Polysaccharide samples CCP1~CCP5 derived from *Gynostemma pentaphyllum* prepared according to the method in Example 1.

[0125] Reagent: Deuterated water.

[0126] 4.2 NMR Spectrum Acquisition

[0127] Approximately 10 mg of each of the polysaccharide samples CCP1~CCP5 were weighed, and after three exchanges with deuterium water, D2O was added to prepare a sample solution of 20 mg / mL. Using 3-(trimethylsilane)phosphate (TMSP) as an internal standard, 1D NMR was detected using an 800 MHz NMR spectrometer. 1 H and 13 C) Spectrum and 2D NMR ( 1 H- 1 H COSY, 1 H- 1 H TOCSY, 1 H- 13 C HSQC, HMBC, HSQC-TOCSY, ROESY) spectra.

[0128] 4.3 Experimental Results

[0129] Comparison of five types of ginseng polysaccharides 1 H and 13 The C-ray spectral signals revealed that all five polysaccharides exhibited the same NMR signal peaks, indicating that the five *Scutellaria baicalensis* polysaccharides have identical chemical structures. Their difference lies in the degree of fructose polymerization (i.e., the molecular weight of the polysaccharides differs, as shown in Example 1). The following NMR analysis uses CCP5 as an example only.

[0130] exist 1 The H NMR spectrum shows a set of 7 main signals ( Figure 7 (A), respectively δ H 4.26 (d, J = 8.5 Hz, 1H), δ H 4.10 (d, J = 8.5 Hz, 1H), δ H 3.93 (d, J = 10.2 Hz, 1H), δ H 3.86 (d, J = 7.3 Hz, 1H), δ H 3.84 (d, J = 11.0 Hz, 1H), δ H 3.77 (dd, J = 11.0,7.3 Hz, 1H), δ H 3.71 (d, J = 10.2 Hz, 1H), and a set of resolvable micro-signals δ H 5.44 (d, J = 3.9 Hz, 1H), δ H 3.55 (dd, J = 1.0, 3.9 Hz, 1H), δ H3.48 (t, J = 9.6 Hz, 1H). Since fructose is a ketose, its structure lacks terminal hydrogen signals from the reducing end, and its chemical shifts are all at a high field, typically below 4.5 ppm. Furthermore, the coupling constant between hydrogens on the same carbon atom in the sugar ring is usually close to or greater than 10 Hz, while the chemical shifts between hydrogens on adjacent carbons are usually less than 10 Hz, and the coupling constants of the two coupled hydrogens are the same. Therefore, it can be inferred that the high-content signal is from fructose, and the chemical shifts of 3.93 and 3.71, and 3.77 and 3.84, are the hydrogen signals from the 1 and 6 carbons of fructose, respectively. For glucose, the terminal hydrogen signals from the reducing end are usually at a relatively low field, with the chemical shifts of the terminal hydrogen signals of α-configured sugars generally greater than 4.8 ppm. Therefore, the high-field δ... H 5.44 may be a terminal hydrogen signal of α-glucose.

[0131] exist 13 In the C NMR spectrum, the polysaccharide of *Gynostemma pentaphyllum* also exhibits a set of major signals and some trace signals. Figure 7 (B) In the polysaccharide structure, because the 2-position of fructose and the 1-position of glucose are acetals, with carbon atoms bonded to two oxygen atoms, their chemical shifts are at a lower field, at 96.3 (α configuration) and 106.1 (β configuration) ppm, respectively. Meanwhile, the 1 and 6 positions of fructose and the 6-position of glucose are secondary alcohols, while the alcohols at other positions are tertiary alcohols. Therefore, the carbon signals at the 1 and 6 positions of fructose and the 6-position of glucose are at a higher field than the signals of other carbons, at δ... C 63.7, 64.9 and 62.9 ppm.

[0132] To further elucidate the structure of the ginseng polysaccharide and determine its chemical shift, this invention conducted a detailed 2DNMR signal correlation analysis (…). Figure 8 HSQC can detect the carbon-hydrogen interactions. 1 J-related signals, through HSQC spectra, can be clearly assigned to hydrogen atoms directly bonded to carbon atoms, and some signals with specific NMR characteristics can be identified, such as the G1 (δ) terminal group signal of α-glucose. H 5.44; δ C 95.3) and the 1-bit signal F1 (δ) of fructose H 3.93, 3.71; δ C 63.7) and 6-bit signal F1 (δ) H 3.84, 3.77; δ C 64.9). 1 H- 1 H COSY correlation spectrum can detect 1 H- 1Between H 2-3 By analyzing the correlation signal, two hydrogen signals on adjacent or the same carbon atom can be identified. Starting from a specific signal, the hydrogen signal can be attributed to another hydrogen atom through the correlation between consecutively connected hydrogen atoms. For example, from H... G1 H can be found at the beginning G1 / H G2 / H G3 Continuous correlation, can be attributed to H G2 and H G3 The signals are δ H 3.55 and δ H 3.77. From H F3 H can be found at the beginning F3 / H F4 / H F5 Continuous correlation, can be attributed to H F4 and H G5 The signals are δ H 4.10 and δ H 3.86. TOCSY spectra can detect... 1 H- 1 Remote correlation signals between H, such as H G1 and H G2 / H G3 / H G4 The correlation between them can be used to determine H. G4 The signal is δ H 3.85. In addition, H F3 and H F4 / H F5 / H F6 Further evidence confirms that these signals are fructose signals. The HSQC-TOCSY spectrum combines the characteristics of both HSQC and TOCSY spectra, enabling simultaneous detection of long-range signals. 2 J and multi-order HC cross-correlation signals can be used to demonstrate the carbon-hydrogen signals on the same sugar ring. For example, H... F3 With C F6 / C F4 / C F5 The correlation indicates that these signals are all fructose signals. However, H F1 It is unrelated to other carbons because it is associated with C in fructose. F1 The connected carbons are quaternary carbons at position 2, which may terminate the crossover signal in the HSQC-TOCSY spectrum. The NMR signals of glucose and fructose in *Gynostemma pentaphyllum* polysaccharide can be attributed to these four 2D NMR correlation spectra, as shown in Table 1.

[0133] In addition, HMBC correlation spectra are commonly used for detection 2-3The HC correlation within J is unaffected by intermediate heteroatoms and quaternary carbons; therefore, the HMBC correlation spectrum can be used to assign carbon and hydrogen signals on sugar rings and to determine the glycosidic bond linkage sites between sugar rings. Figure 8 In E, H G1 / C F2 and H F1 / C F2 The correlations between glucose and fructose indicate that the glycosidic bonds between glucose and fructose are linked via a 1→2 connection, as are those between fructose and fructose. Other correlation signals further confirm the chemical shift assignments of the monosaccharides. ROESY is a technique used to detect spatial distance correlation signals between hydrogen atoms. When two hydrogen atoms are spatially close, cross-correlation signals appear, unaffected by the chemical bonds between hydrogen atoms. Therefore, ROESY spectra can often be used to determine the connection sites between glycosidic bonds. However, because the 2-position of fructose is a quaternary carbon and lacks a hydrogen atom, no glycosidic bond correlation signals appeared in the ROESY spectrum; only hydrogen atom correlation signals within the sugar ring were observed.

[0134] Based on comprehensive analysis of monosaccharide composition, methylation, and NMR, the structure of *Gynostemma pentaphyllum* polysaccharide was ultimately determined to be a β2→1 linked oligofructose, in which the terminal glucose and fructose are linked via α1→2, and its structure is shown in the chemical structural characteristics of formula (I):

[0135]

[0136] In chemical formula (I),

[0137] A is α-D-glucose (α-D-Glc); B is β-D-fructose (β-D-Fru); C is β-D-fructose (β-D-Fru); D is β-D-fructose (β-D-Fru). n is an integer from 0 to 40.

[0138] Example 5

[0139] Stinking ginseng polysaccharide remodels the gut microbiota of mice.

[0140] 5.1 Materials

[0141] Samples of *Gynostemma pentaphyllum*: Polysaccharide samples CCP2 and CCP5 derived from *Gynostemma pentaphyllum* prepared according to the method in Example 1.

[0142] Positive control: Inulin (CAS:308066-66-2), Beijing Mairuida Technology Co., Ltd.

[0143] Laboratory mice: 24 male SPF-grade C57BL / 6 mice, approximately 8 weeks old and weighing approximately 20 g. The housing was kept under light and dark cycles every 12 hours, with free access to water and food. Animal experiments were conducted strictly in accordance with the U.S. National Research Council's "Guidelines for the Care and Use of Laboratory Animals" and were approved by the Research Ethics Committee of the Kunming Institute of Botany, Chinese Academy of Sciences (Approval No.: SYXK-K2018-0004).

[0144] 5.2 Experimental Methods

[0145] Animal experiments: After one week of acclimatization, mice were randomly divided into four groups (n=6 per group): a positive control (Inulin) group, a blank (saline) group, a CCP2 group, and a CCP5 group. During the experiment, mice were administered specific sample treatments orally via gavage every other day at a dose of 6 mg / 20 g body weight (approximately 0.2 ml per mouse) for four weeks. Mouse weight was recorded every other day. At the end of the treatment period, all mice were euthanized using cervical dislocation. Cecal contents were collected under aseptic conditions, immediately flash-frozen in liquid nitrogen, and stored at -80°C for subsequent metagenomic analysis.

[0146] Metagenomic analysis: Total genomic DNA was extracted from fecal samples using the FastPure fecal DNA extraction kit (magnetic bead method; Shanghai Maijie Medical Technology Co., Ltd., China). Sequencing libraries were constructed using the NEXTFLEX Rapid DNA-Seq kit (PerkinElmer, USA), with an average insert size of 350 bp. Paired-end sequencing (2 × 150 bp) was performed on the Illumina NovaSeq XPlus platform (Illumina, San Diego, USA). Fastp (v0.23.0) was used for quality control of the raw sequencing data, removing adapter sequences, low-quality bases (quality value <20), and reads shorter than 50 bp. High-quality reads were de novo assembled into contigs using MEGAHIT (v1.2.9) with default parameters. Open reading frames (ORFs) were predicted using MetaGeneMark (v3.38), and a non-redundant gene catalog was constructed using CD-HIT (v4.8.1) under conditions of 95% sequence consistency and 90% coverage. Non-redundant gene sequences were compared with the NCBI NR database using DIAMOND (v2.0.9) (e-value threshold set to 1×10⁻). 5Species annotation was completed. Functional annotations were performed using the DIAMOND-KEGG, CAZy, and Probiotics databases, based on the same e-value threshold. All bioinformatics analyses were performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com).

[0147] 6.3 Experimental Results

[0148] Metagenomic sequencing analysis was performed on the contents of the mouse cecum (24 samples in 4 groups) using erythrogenomic sequencing. Alpha diversity analysis revealed a significant increase in the Chao index in the CCP2 group compared to the control group (p = 0.0222), while no significant change was observed in the Inulin and CCP5 groups. This indicates that CCP2 can significantly increase the richness of the gut microbiota. Similarly, the Shannon index in the CCP2 group also showed a significant increase compared to the control group (p = 0.009), although the Shannon index in the Inulin and CCP5 groups also showed an increasing trend, but the difference was not statistically significant. This indicates that CCP2 can significantly increase the diversity of the gut microbiota. In Beta diversity analysis, hierarchical cluster analysis revealed that the drug-treated groups and the control group were differentiated at a high level, and at the next higher level, the Inulin group was also differentiated from the CCP2 and CCP5 groups. At the next lower level, the CCP2 and CCP5 groups were also differentiated, indicating significant differences in the microbial communities among the groups, with the most significant difference between the control group and the drug-treated group. PCoA analysis revealed a significant difference in the microbial community structure between the blank control group and the drug-treated groups. Among the three drug-treated groups, the Inulin group and the CCP2 group were more closely connected, with some overlap between the two groups. This indicates a significant difference in microbial community structure among the different treatment groups (p = 0.001), with the most significant difference between the blank control group and the drug-treated groups. Beta diversity analysis further confirmed significant differences in microbial community composition among the groups, allowing for inter-group microbial species difference analysis.

[0149] NR species annotation was performed on genes detected in all samples, annotating gut microbiota of 203 phyla, 2961 genera, and 11629 species. Venn diagrams showed 2315 genera shared across the four groups, with unique genera for each group being 37 in the control group, 41 in the Inulin group, 52 in the CCP2 group, and 26 in the CCP5 group. At the phylum level, species composition analysis showed that Bacteroidota, Bacillota, Verrucomicrobiota, Actinomycetota, and Thermodesulfobacteriota were the five most abundant phyla in all samples, with significant differences in community abundance at the phylum level among groups, particularly between Bacteroidota, Bacillota, and Verrucomicrobiota. Compared to the control group, fructooligosaccharide treatment significantly decreased the abundance of Bacteroidota and Verrucomicrobiota, while increasing the abundance of Bacillota. Figure 9 (A). Among them, the abundance of Verrucomicrobiota was most significantly reduced after CCP2 and CCP5 treatment, decreasing by 99.5% and 81.2% respectively, while the abundance in the Inulin treatment group decreased by only 10.1%. Figure 9 Figure A shows the 10 genera with the highest bacterial abundance across all samples. Several genera showed significant abundance differences between different treatment groups, with the most significant differences observed in the abundance of *Akkermansia* and *Ileibacterium*. For *Akkermansia*, after treatment with fructooligosaccharides, the abundance in the Inulin group decreased by only 9.7% compared to the control group, while the CCP2 and CCP5 groups showed decreases of 99.4% and 81.2%, respectively. For *Ileibacterium*, the abundance in the Inulin group increased by 406.6% compared to the control group, while the CCP2 and CCP5 groups showed decreases of 99.0% and 88.3%, respectively. At the species level ( Figure 9 In group B, compared with the control group, the oligofructose-treated groups showed significant changes in Akkermansia sp., Ileibacterium valens, Akkermansia muciniphila, Hungatella sp., and Dorea sp., with significant decreases in Akkermansia sp. and Akkermansia muciniphila in both CCP2 and CCP5 groups. Ileibacterium valens was significantly increased in the Inulin group. Furthermore, various probiotics showed significant increases in both CCP2 and CCP5 groups. Figure 9 (C)

[0150] Example 6

[0151] Stinking ginseng polysaccharide regulates intestinal metabolites in mice.

[0152] 6.1 Materials

[0153] Same as Example 5.

[0154] 6.2 Experimental Methods

[0155] Animal experiments: Same as in Example 5.

[0156] Untargeted metabolomics analysis: Approximately 20 mg of sample was accurately weighed and extracted using a low-temperature grinding technique with 400 μL of methanol / water (4:1, v / v) mixture. The extract was analyzed using an AB SCIEX UHPLC-Triple TOF 6600 system equipped with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm inner diameter, 1.8 μm; Waters, USA). The mobile phase consisted of a water / acetonitrile solution containing 0.1% formic acid (95:5, v / v; solvent A) and an acetonitrile / isopropanol / water solution containing 0.1% formic acid (47.5:47.5:5, v / v / v; solvent B). The chromatographic flow rate was 0.40 mL / min, the column temperature was 45 °C, and the injection volume was 10 μL. Mass spectrometry analysis was performed using an electrospray ionization source in both positive and negative ion modes, with the following parameters: scan range m / z 50–1200, ion source temperature 500℃, ion spray voltage +5500 V (positive ion mode) and -4500 V (negative ion mode), and collision energy 40 ± 20 eV. All bioinformatics analyses were performed on the Majorbio CloudPlatform (https: / / cloud.majorbio.com).

[0157] 6.3 Experimental Results

[0158] Metabolomics analysis of mouse cecal contents revealed 2075 effective metabolites, including 848 in cationic mode and 1227 in anionic mode. The metabolites detected in the four sample groups were: blank group (2026), Inulin group (1967), CCP2 group (2062), and CCP2 group (1923). Significant differences in metabolites were observed between the fructooligosaccharide (FOS) treatment groups and the blank control group. In the Inulin and CCP5 groups, over 200 metabolites were downregulated, while only 63 and 47 differentially regulated metabolites were upregulated, respectively. In contrast, the CCP2 treatment group showed downregulation of 60 metabolites compared to the blank control group, and 88 metabolites were downregulated compared to the blank control group. This indicates significant changes in metabolites after FOS treatment compared to the blank group, and significant differences exist among the different FOS treatment groups. To elucidate the effects of fructooligosaccharide (FOS) treatment on bodily functions from a metabolomics perspective, 633 differentially expressed metabolites compared to the control group were merged and annotated using the KEGG compound classification system (Compounds with biological roles), identifying 21 differentially expressed metabolites. Variable importance (VIP) values ​​and expression heatmaps were performed on these differentially expressed metabolites, revealing that 10 metabolites had a VIP value greater than 1, indicating that these metabolites have a significant impact on FOS treatment. Furthermore, this invention also performed KEGG pathway enrichment analysis on the 633 differentially expressed metabolites, enriching a total of 145 pathways. Among these, linoleic acid metabolism, arachidonic acid metabolism, serotonergic synapse, phenylalanine metabolism, neuroactive ligand-receptor interaction, and primarybile acid biosynthesis pathways were the most frequently detected (n > or = 4) and showed the most significant differences (P < or = 0.005). Figure 9(D). Although the enrichment factors for the Asthma, Melanogenesis, and Human cytomegalovirus infection pathways were the highest (greater than 0.3), the enriched metabolites were relatively few (equal to 2), and therefore not representative. This invention performed expression heatmap analysis on the differentially enriched metabolites in the top six signaling pathways, finding that compared to the control group, Inulin treatment showed a decreasing trend in metabolites in the Arachidonic acid metabolism and Neuroactive ligand-receptor interaction pathways, while CCP5 treatment showed a decreasing trend in metabolites in the Arachidonic acid metabolism, Linoleic acid metabolism, Serotonergic synapse, Phenylalanine metabolism, and Primary bile acid biosynthesis pathways.

[0159] Example 7

[0160] The effect of ginseng polysaccharide on in vitro fermentation culture of human intestinal flora.

[0161] 7.1 Materials

[0162] Samples of *Gynostemma pentaphyllum*: Polysaccharide samples CCP1, CCP2, and CCP5 derived from *Gynostemma pentaphyllum* prepared according to the method in Example 1.

[0163] Positive control: Inulin (CAS:308066-66-2), Beijing Mairuida Technology Co., Ltd.

[0164] 7.2 Experimental Methods

[0165] In vitro fermentation experiment using human feces: Fecal samples were collected from six healthy adult volunteers (three men and three women), all of whom had not taken any antibiotics in the past three months. Samples were homogenized in PBS buffer (0.1 mol / L) to prepare a 10% (w / v) fecal suspension, followed by centrifugation at 270 × g for 5 minutes. The supernatant was collected for subsequent fermentation experiments. Specific CCP samples and inulin (positive control) were added to glucose-free MRS broth to achieve a final polysaccharide concentration of 1%. The culture medium was autoclaved at 121°C for 30 minutes. The fecal supernatant was inoculated at 10% (v / v) into the sterilized culture medium and placed in anaerobic culture bags for fermentation at 37°C under anaerobic conditions for 24 hours. After fermentation, samples were aliquoted and stored at -80°C for microbial composition analysis.

[0166] Microbial composition analysis: Changes in microbial composition were analyzed using 16S rRNA sequencing. Total genomic DNA of the microbial community was extracted using the TIANMicrobeMagnetic Environment DNA Kit 4 (Tiangen, China) according to the manufacturer's instructions. The V3–V4 variable region of the 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Liuet al., 2016). The purified PCR products were used to construct libraries using the NEXTFLEX RapidDNA-Seq Kit and sequenced on an Illumina Nextseq 2000 platform (San Diego, USA). The raw paired-end reads were quality controlled using fastp (v0.19.6) and assembled using FLASH (v1.2.11). The quality-filtered assembled sequences were de-noised using the DADA2 plugin in the QIIME2 workflow with default parameters. Finally, based on the Naive Bayes classifier built into QIIME2, species classification annotation of amplicon sequence variants (ASVs) was performed using the SILVA 16S rRNA gene database (v138.2). All bioinformatics analyses were performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com).

[0167] 7.3 Experimental Results

[0168] The in vitro fermentation products of human feces treated with ginseng polysaccharide were detected using 16S ribosomal RNA. Alpha diversity analysis revealed that, compared with the blank control group, the oligofructose treatment groups, including Inulin, CCP1, CCP2, and CCP5, significantly reduced the chao index (p = 0.0023) and Shannon index (p = 0.00023) of the microbial community, indicating the abundance and diversity of oligofructose. PCoAf analysis at the ASV level showed that the differences between groups were greater than the differences within groups (R = 0.76, P = 0.001), with significant differences between the blank control group and the oligofructose treatment groups. Although there was partial overlap between the Inulin group and the CCP1 group, and between the CCP2 and CCP5 groups, the overlapping groups were further separated. The Venn diagram at the genus level shows 111 genera shared by the five groups, while the genera unique to each group are: 69 in the control group, 13 in the Inulin group, 15 in the CCP1 group, 14 in the CCP2 group, and 10 in the CCP5 group. Microbial community composition is easily altered by environmental factors. Based on the different sensitivities of gut microbiota to fructooligosaccharides (FOS), the detected gut bacteria were classified into Transient, Intermittent, and Persistent types. Core microbiota analysis revealed that FOS treatment reduced the abundance and quantity of Transient and Intermittent types, while increasing the abundance and quantity of Persistent types, indicating that FOS treatment increased the abundance and quantity of core microbiota, resulting in a more stable microbiota. Phylum-level microbial structure analysis revealed that Bacillota, Bacteroidota, Pseudomonadota, and Actinomycetota were the four most abundant phyla, with their combined abundance exceeding 95%. Among them, Bacillota had the highest abundance in the control group, followed by Bacteroidota. However, after treatment with fructooligosaccharides, the abundance of Bacillota decreased significantly, while that of Bacteroidota increased significantly. The changes were most pronounced in the CCP5 treatment group, with Bacteroidota even exceeding the abundance of Bacillota. For Pseudomonadota, compared to the control group, the abundance of Pseudomonadota in the fructooligosaccharide treatment groups showed a decreasing trend, with the most significant decreases in the CCP2 and CCP5 groups. Conversely, Actinomycetota showed an increasing trend after fructooligosaccharide treatment, with the most significant increase in the CCP1 group. At the genus level ( Figure 9The five most abundant species (E and F) were Segatella, Megasphaera, Sutterella, Catenibacterium, and Bifidobacterium. Among them, the abundance of Segatella, Catenibacterium, and Bifidobacterium species increased after treatment with fructooligosaccharides (FOS), with the most significant increase in Segatella abundance in CCP2 and CCP5 groups. Catenibacterium had the highest abundance in CCP2, while Bifidobacterium had the highest abundance in CCP1. Megasphaera and Sutterella species showed a decreasing trend after FOS treatment, with the most significant decreases in abundance after treatments in CCP2 and CCP5.

[0169] The above results all reveal that *Syngonium odoratum* polysaccharide has the function of reshaping the abundance of intestinal flora and regulating intestinal metabolites. Moreover, this effect is significantly different from that of the positive control (inulin) sold on the market, indicating that *Syngonium odoratum* polysaccharide can play a unique role in regulating human and animal health and has application value.

[0170] Example 8

[0171] Polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 droplets derived from *Gynostemma pentaphyllum*.

[0172] 8.1 Materials

[0173] Polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 derived from *Gynostemma pentaphyllum* were prepared according to the method in Example 1.

[0174] 8.2 Prescription:

[0175] Raw material names Dosage The polysaccharide samples derived from *Gynostemma pentaphyllum* include any one or any combination of CCP1, CCP2, CCP3, CCP4, and CCP5. 100 g polyethylene glycol 6000 g Made into 1000 pills

[0176] 8.3 Preparation process

[0177] Weigh out any one or any combination of the polysaccharide samples CCP1, CCP2, CCP3, CCP4, and CCP5 from the above-mentioned prescription. Add an appropriate amount of anhydrous ethanol, dissolve by gentle heating, and then add to the prescribed amount of polyethylene glycol melt. Stir and mix thoroughly until the ethanol evaporates completely. Let stand in a 60°C water bath for 30 minutes to remove air bubbles. Transfer the bubble-free melt to a storage container and, under the condition of 80-85°C, control the dripping rate and add it drop by drop into the condensate. After complete condensation, pour off the condensate and collect the pellets. The temperature and density of the condensate should be appropriate to ensure that the pellets can fully solidify and do not stick together. Drain and remove the condensate from the pellets with filter paper, and dry under reduced pressure. Remove the completely condensed pellets from the condensate and wash them with an appropriate solvent to remove the surface condensate. Dry the washed pellets in a low-temperature oven. Select pellets that are uniform in size and round from the prepared pellets, and remove unqualified pellets. The pellets are coated to increase their stability.

[0178] Example 9

[0179] Capsules containing any one or any combination of the polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 derived from *Gynostemma pentaphyllum*.

[0180] 9.1 Materials

[0181] The polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 obtained by the same method as in Example 1, or any combination thereof, are food or pharmaceutical grade starch.

[0182] 9.2 Prescription:

[0183] Raw material names Dosage The polysaccharide samples derived from *Gynostemma pentaphyllum* include any one or any combination of CCP1, CCP2, CCP3, CCP4, and CCP5. 500 g starch 300 g Made into 10,000 pills

[0184] 9.3 Preparation process

[0185] Weigh out the prescribed amount of any one or any combination of the polysaccharide samples CCP1, CCP2, CCP3, CCP4, and CCP5 from *Periplaneta americana*, and starch, and stir until completely mixed. Add an appropriate amount of talc, granulate using a wet ethanol method, sieve and dry, and then fill into No. 2 capsule shells. Each capsule is filled with 50 mg of *Periplaneta americana* dextran (PaN) to obtain capsules.

[0186] Example 10

[0187] Oral solutions of polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 derived from *Gynostemma pentaphyllum*.

[0188] 10.1 Materials

[0189] The polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 obtained from *Gynostemma pentaphyllum* using the same method as in Example 1, with food or pharmaceutical grade flavoring agent and caramel flavoring.

[0190] 10.2 Prescription

[0191] Raw material names Dosage The polysaccharide samples derived from *Gynostemma pentaphyllum* include any one or any combination of CCP1, CCP2, CCP3, CCP4, and CCP5. 100 g sucrose 0.6 g Caramel flavoring 0.1 g Purified water 2000 mL Made into 500 pieces

[0192] 10.3 Preparation process

[0193] Weigh out the prescribed amount of any one or any combination of the polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 from ginseng, sucrose and caramel flavoring, add purified water to completely dissolve, filter through a 0.2μm microporous membrane, and fill each bottle with 4mL of the filtrate using an oral liquid filling machine. After sealing, sterilize to obtain the final product.

[0194] Example 11

[0195] Lyophilized powder injections of polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 from Panax notoginseng.

[0196] 11.1 Materials

[0197] The polysaccharide samples CCP1, CCP2, CCP3, CCP4 and CCP5 derived from *Gynostemma pentaphyllum* obtained by the same method as in Example 1, were used with water for injection.

[0198] 11.2 Prescription:

[0199] Raw material names Dosage The polysaccharide samples derived from *Gynostemma pentaphyllum* include any one or any combination of CCP1, CCP2, CCP3, CCP4, and CCP5. 20 g Water for Injection 1000 mL Made into 1000

[0200] 11.3 Preparation process

[0201] Weigh out any one or any combination of CCP1, CCP2, CCP3, CCP4, and CCP5 from the polysaccharide sample derived from *Gynostemma pentaphyllum* in the prescribed amount, add water for injection to the total volume, stir until completely dissolved, and sterilize by intermittent autoclaving. After the content is qualified, filter through a 0.22 μm microporous membrane; dispense into controlled vials, 0.5 mL per vial, half-stop, place in a freeze-drying oven, freeze-dry according to the set freeze-drying curve, stopper, remove from the oven, cap, visually inspect for compliance, and package to obtain the finished product.

[0202] Freeze-drying process: Place the sample in the chamber, lower the partition temperature to -50℃, and hold for 5 hours; lower the cold trap to -50℃ and begin evacuation to 200 μbar. Sublimation begins: Heat to -15℃ at a constant rate for 2 hours and hold for 3 hours; heat to -5℃ at a constant rate for 3 hours and hold for 6 hours, maintaining a vacuum of 100~250 μbar; Drying continues: heat to 5℃ for 2 hours and hold for 2 hours, maintaining a vacuum of 150~200 μbar; heat to 15℃ for 1 hour and hold for 2 hours, maintaining a vacuum of 80~100 μbar; heat to 40℃ for 0.5 hours and hold for 4 hours, then evacuate to the lowest possible vacuum.

Claims

1. The fructooligosaccharide or its derivatives shown in structural formula (I) below, in, A is one or more of the esterification, etherification or amination products of α-D-glucose or its hydroxyl groups, or the oxidation products of its non-reducing terminal hydroxyl groups or one or more derivatives of its oxidation products, wherein the oxidation products include aldehydes or carboxyl groups; B is one or more of the esterification, etherification or amination products of β-D-fructose or its hydroxyl groups, or the oxidation products of its non-reducing terminal hydroxyl groups or one or more derivatives of its oxidation products, wherein the oxidation products include aldehydes or carboxyl groups; C is one or more of the esterification, etherification or amination products of β-D-fructose or its hydroxyl groups, or the oxidation products of its non-reducing terminal hydroxyl groups or one or more derivatives of its oxidation products, wherein the oxidation products include aldehydes or carboxyl groups; D is one or more of the esterification, etherification or amination products of β-D-fructose or its hydroxyl groups, or the oxidation products of its non-reducing terminal hydroxyl groups or one or more derivatives of its oxidation products, wherein the oxidation products include aldehydes or carboxyl groups; n is an integer between 0 and 100.

2. The fructooligosaccharide or its derivative according to claim 1, characterized in that, The monosaccharide composition includes glucose and fructose, wherein the glucose is one or more of α-D-glucose or its derivatives, and the fructose is one or more of β-D-fructose or its derivatives.

3. The fructooligosaccharide or its derivative according to claim 1 or 2, characterized in that, Its monosaccharide linkage is that α-D-glucose or its derivatives are linked to β-D-fructose or its derivatives by (1→2) glycosidic bonds, and β-D-fructose or its derivatives are linked to β-D-fructose or its derivatives by (2→1) glycosidic bonds.

4. The fructooligosaccharide or its derivative according to claim 1, 2 or 3, characterized in that, Its structure is [→1)-D-Fru-β-(2→] repeated connection, and at the end there is a D-Glc-α-(1→ connected to the 2-position of β-D-Fru.

5. The method for preparing the fructooligosaccharide or its derivatives according to any one of claims 1 to 4, characterized in that, The fructooligosaccharides were obtained from the roots of Codonopsis pilosula, including but not limited to fresh or dried roots, through water extraction and alcohol precipitation, followed by centrifugation, ion exchange chromatography purification, dialysis, and freeze drying.

6. A ginseng oligofructose composition comprising ginseng oligofructose or its derivatives as described in any one of claims 1 to 4.

7. A ginseng oligofructose pharmaceutical composition comprising an effective amount of ginseng oligofructose or a derivative thereof as described in any one of claims 1 to 4.

8. The use of the fructooligosaccharide or its derivatives according to any one of claims 1 to 4, or the fructooligosaccharide composition according to claim 6, or the fructooligosaccharide pharmaceutical composition according to claim 7 in the preparation of medicaments for the treatment and / or prevention of intestinal flora-related diseases.

9. The use of the ginseng oligofructose or its derivatives according to any one of claims 1 to 4, or the ginseng oligofructose composition according to claim 6, in the preparation of food.

10. The application according to claim 8 or 9, characterized in that, The gut microbiota-related diseases mentioned above refer to gut microbiota-related diseases occurring in the gastrointestinal tract, liver, kidneys, cardiovascular system, brain, and nervous system.

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