A compound chewable tablet containing purified grifola frondosa polysaccharide, a preparation method and application thereof

Abstract

The present application belongs to the technical field of health food processing, and provides a compound chewable tablet containing purified Grifola frondosa polysaccharide and a preparation method and application thereof. The purified Grifola frondosa polysaccharide is obtained by multi-step purification of Grifola frondosa fruiting bodies through ultrasonic-assisted hot water extraction, alcohol precipitation and ultrafiltration, has a high neutral sugar content, and is mainly composed of glucose. The compound chewable tablet is prepared by mixing, granulating, drying, whole-granulating and tabletting with purified Grifola frondosa polysaccharide, fucoidan, Hericium erinaceus polysaccharide and milk powder as main functional raw materials, and with maltitol, maltodextrin and other various auxiliary materials. The compound chewable tablet has a suitable taste, good formability, is convenient to carry and take, and can improve the problems of abnormal increase of intragastric pH, gastric mucosal inflammatory infiltration, gland damage and intragastric flora imbalance caused by Helicobacter pylori infection. The present application provides an application scheme for natural auxiliary intervention of Helicobacter pylori infection related stomach discomfort.

Description

Technical Field

[0001] This invention belongs to the field of health food processing technology, and relates to a compound chewable tablet of polysaccharides from medicinal and edible fungi, specifically a compound chewable tablet containing purified Grifola frondosa polysaccharide, its preparation method, and its application. Background Technology

[0002] Helicobacter pylori (Hp) infection has become a global health problem, seriously affecting human gastric health. Hp infection is often accompanied by gastric flora imbalance, abnormal gastric acid secretion, and gastric mucosal damage. Long-term infection can further develop into chronic gastritis, peptic ulcers, and other diseases. Currently, traditional antibiotic treatments for eradicating Hp infection still have limitations, such as increased drug resistance and significant adverse drug reactions. Therefore, developing natural sources with flora-regulating and mucosal-protective effects is of great significance.

[0003] Grifola frondosa, also known as maitake mushroom or chestnut mushroom, is a medicinal and edible fungus. Its polysaccharide components possess potential effects such as immunomodulation, antioxidation, and regulation of the gut microbiota. However, existing research largely focuses on evaluating the activity of Grifola frondosa crude polysaccharide in other disease models, with limited research on its effects on gastric flora regulation, improvement of gastric mucosal damage, and product development in the context of Helicobacter pylori infection. Furthermore, existing natural polysaccharide preparations are mostly available in powder or capsule form, which suffers from poor palatability, a generally unpleasant user experience, and insufficient portability, making it difficult to meet the needs of Hp-infected patients for long-term, convenient, and gentle intervention.

[0004] Therefore, it is necessary to provide an application and formulation of Grifola frondosa polysaccharide to meet the needs of gastric health management related to Helicobacter pylori infection. Summary of the Invention

[0005] The purpose of this invention is to provide a compound chewable tablet containing purified Grifola frondosa polysaccharide, its preparation method and application, as well as the preparation method of the purified Grifola frondosa polysaccharide, thereby improving the problems of existing natural adjuvant intervention products having single dosage form, inconvenient administration and insufficient functional utilization.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a compound chewable tablet containing purified Grifola frondosa polysaccharide, wherein the compound chewable tablet comprises the following raw materials by mass percentage: 40-80% functional raw materials, 4-8% sweetener, 25-45% filler, 6-10% binder, 2-6% acidulant, and 0.5-2.5% lubricant; the functional raw materials are a mixture of functional excipients and milk powder, the functional excipients are a mixture of purified Grifola frondosa polysaccharide, fucoidan, and Hericium erinaceus polysaccharide, the purified Grifola frondosa polysaccharide being a polysaccharide component obtained by hot water extraction of Grifola frondosa fruiting bodies, alcohol precipitation, deproteinization, dialysis, gradient ethanol precipitation, and ultrafiltration purification; the mass ratio of the functional excipients to the milk powder is (0-3):(0-3); the mass ratio of the purified Grifola frondosa polysaccharide, fucoidan, and Hericium erinaceus polysaccharide is 2:1:1.

[0007] Preferably, the compound chewable tablet comprises the following ingredients by weight percentage: 50% functional ingredients, 6% sweetener, 30% filler, 9% binder, 3% acidulant, and 1% lubricant, wherein the mass ratio of the functional ingredients to the milk powder is 2:1.

[0008] Preferably, the sweetener is at least one of aspartame, glucose, sucrose, and maltitol; the filler is at least one of lactose, maltodextrin, corn starch, and mannitol; the binder is at least one of sodium carboxymethyl cellulose, ethyl cellulose, and crosporovinylpyrrolidone; the acidulant is citric acid; and the lubricant is magnesium stearate.

[0009] Preferably, the sweetener is maltitol, the filler is maltodextrin, and the binder is sodium carboxymethyl cellulose.

[0010] This invention further provides a method for preparing the above-mentioned compound chewable tablets.

[0011] Preferably, the following steps are included: (1) mixing purified Grifola frondosa polysaccharide, fucoidan, Hericium erinaceus polysaccharide, maltitol, maltodextrin, sodium carboxymethyl cellulose and citric acid; (2) adding a wetting agent to make a soft mass; (3) wet granulation; (4) drying at 40 ℃-70 ℃; (5) granulation; (6) adding magnesium stearate and then compressing to obtain purified Grifola frondosa polysaccharide complex chewable tablets.

[0012] Preferably, the method for preparing the purified Grifola frondosa polysaccharide is as follows: (a) drying and pulverizing the fruiting bodies of Grifola frondosa; (b) hot extraction with water, filtering and combining the extracts; (c) centrifuging, concentrating and precipitating the extract with alcohol to obtain crude polysaccharide; (d) deproteinizing and dialysis the crude polysaccharide; (e) precipitating the dialyzed polysaccharide with 40% ethanol; (f) purifying the obtained fraction by 30 kDa ultrafiltration and lyophilizing.

[0013] The present invention further provides the application of the above-mentioned compound chewable tablets in improving the gastric flora associated with Helicobacter pylori infection.

[0014] Preferably, the chewable tablet has at least one of the following effects:

[0015] (1) Reduce the abnormal increase in gastric pH caused by Helicobacter pylori infection;

[0016] (2) Reduce gastric mucosal inflammatory infiltration and / or atrophy of acid-secreting glands;

[0017] (3) Inhibit the excessive proliferation of opportunistic pathogens of the phylum Proteobacteria in the stomach;

[0018] (4) Promotes the colonization of beneficial bacteria of the genus Lactobacillus and / or Ekkermansia;

[0019] (5) Restore the gastric flora diversity caused by Helicobacter pylori infection.

[0020] The beneficial effects of this invention are:

[0021] 1. This invention provides a method for preparing purified Grifola frondosa polysaccharide and its application in improving gastric flora associated with Helicobacter pylori infection, providing a material basis and experimental evidence for the development of related functional products.

[0022] 2. Compared with the control group and the drug group, the purified Grifola frondosa polysaccharide of this invention showed better effects in regulating gastric flora, restoring gastric pH and improving gastric mucosal damage.

[0023] 3. The compound chewable tablets developed in this invention have a pleasant taste, good formability, and are easy to carry and take, which helps to improve the convenience of long-term use. Attached Figure Description

[0024] Figure 1 This is the high-performance gel permeation chromatogram (HPGPC) of the purified Grifola frondosa polysaccharide GFP-40% in this invention, showing a single symmetrical elution peak;

[0025] Figure 2 This is a comparison of ion chromatograms of purified Grifola frondosa polysaccharide GFP-40% and standard monosaccharides in this invention, showing that the monosaccharide composition is mainly glucose;

[0026] Figure 3 This is the Fourier Transform Infrared (FT-IR) spectrum of the purified Grifola frondosa polysaccharide GFP-40% from this invention, showing an 856 cm⁻¹ value. -1 Characteristic peaks of α-glycosidic bonds;

[0027] Figure 4 This is a schematic diagram of the repeating structural unit and structural model of the purified Grifola frondosa polysaccharide GFP-40% in this invention;

[0028] Figure 5 This invention relates to the effects of Helicobacter pylori infection, purified Grifola frondosa polysaccharide, and drug intervention on the pH in the stomach of mice.

[0029] Figure 6 This invention relates to the effects of Hp infection, purified Grifola frondosa polysaccharide, and drug intervention on histopathology.

[0030] Figure 7 This is a bar chart showing the relative abundance of gastric flora at the phylum (A) and genus (B) levels in each group of mice in this invention;

[0031] Figure 8 This is an α-diversity diagram of the gastric flora of each group of mice in this invention.

[0032] Figure 9 This is a β-diversity diagram of the gastric flora of each group of mice in this invention.

[0033] Figure 10 This is a single-factor plot of excipient screening in this invention.

[0034] Figure 11 This is a single-factor experimental diagram in this invention.

[0035] Figure 12 This is a response surface diagram showing the interaction between the amount of raw materials added and the amount of maltitol added on the sensory score in this invention.

[0036] Figure 13 This is a picture of the purified Grifola frondosa polysaccharide GFP-40% complex chewable tablets from this invention. Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example 1: Preparation of purified Grifola frondosa polysaccharide GFP-40%

[0041] The dried fruiting bodies of *Grifola frondosa* were pulverized in a high-speed grinder and passed through a 60-mesh sieve. They were then soaked in 95% ethanol at a ratio of 1:5 (g / mL) for 48 hours to defatt them, centrifuged at 5000 r / min for 10 minutes, and the precipitate was dried at 60 ℃. Deionized water was added at a material-to-liquid ratio of 1:40 (g / mL), and the mixture was sonicated for 30 minutes (600 W). Extraction was then carried out at 80 ℃ for 4 hours. The supernatant was collected by centrifugation, and the residue was extracted once more. The extracts were combined. After concentration by rotary evaporation, 95% ethanol was added to a final concentration of 80%, and the mixture was allowed to stand overnight at 4 ℃ to precipitate. The precipitate was collected by centrifugation, redissolved in water, and deproteinized using the Sevag method (chloroform:n-butanol = 4:1), repeated three times. The deproteinized polysaccharide solution was transferred to a 3500 Da dialysis bag and dialyzed with distilled water for 48 hours. The dialysate was concentrated to a 1 mg / mL solution, and anhydrous ethanol was slowly added with stirring to a final concentration of 40%, and the solution was allowed to stand overnight at 4 ℃. Centrifuge at 10000 r / min for 10 minutes, collect the precipitate, and wash three times with anhydrous ethanol. Redissolve the precipitate in deionized water, transfer it to a 30 kDa ultrafiltration tube, centrifuge, collect the retentate, and freeze-dry to obtain purified Grifola frondosa polysaccharide GFP-40%.

[0042] The yield of crude polysaccharide from Grifola frondosa was calculated to be 10.2% (based on dried fruiting bodies), and the yield of GFP-40% was 20.5% (based on crude polysaccharide).

[0043] Example 2 Structural characterization of purified Grifola frondosa polysaccharide GFP-40%

[0044] 1. Chemical composition

[0045] Neutral sugar content was determined using the phenol-sulfuric acid method, with glucose as a reference, at a wavelength of 490 nm. The results showed that the neutral sugar content of GFP-40% was 85.9 ± 0.7%.

[0046] Protein content was determined using the BCA protein quantification kit: the results showed that the protein content of GFP-40% was 2.3 ± 0.4%.

[0047] The content of uronic acid was determined using a modified sulfuric acid-carbazole method: glucuronic acid was used as a standard, and detection was performed at a wavelength of 520 nm. The results showed that uronic acid was not detected in GFP-40%.

[0048] Ash content determination: After the sample was fully carbonized, it was transferred to a muffle furnace and ignited at 500-600 ℃ until constant weight. The ash content was measured to be 3.2±0.5%.

[0049] Moisture content determination: The constant weight method was used. The sample was dried at 105 °C until the mass was constant, and the moisture content was measured to be 8.7 ± 0.2%.

[0050] 2. Monosaccharide composition analysis

[0051] Take 5 mg of GFP-40% sample, add 0.5 mL of 12 mol / L H2SO4 solution, and hydrolyze in an ice-water bath for 0.5 hours. Then, slowly add 2.5 mL of deionized water and hydrolyze in an oil bath at 100 °C for 2 hours. Dilute the hydrolysate to 20 μg / mL, filter through a 0.22 μm membrane, and detect using an ion chromatography system equipped with a pulsed amperometric detector. The chromatographic column was a CarboPac PA20 (3 mm × 150 mm), and the mobile phase was 250 mmol / L NaOH solution, ultrapure water, and 1 mol / L NaOAc solution, with gradient elution.

[0052] The ion chromatogram of GFP-40% hydrolysis product was compared with that of mixed monosaccharide standards (containing Fuc, Rha, Ara, Gal, Glc, Man, Xyl, Rib, GalA, and GlcA). The results showed ( Figure 2 In the chromatogram of GFP-40%, the Glc (glucose) peak is absolutely dominant. According to the standard curve calculation, the glucose molar ratio is 99.75%, and it also contains trace amounts of galactose (molar ratio 0.25%), indicating that GFP-40% is a neutral polysaccharide mainly composed of glucose.

[0053] 3. Homogeneity and molecular weight determination

[0054] High-performance gel permeation chromatography (HPGPC) was used for detection. GFP-40% was prepared as a 2 mg / mL solution, filtered through a 0.22 μm filter membrane, and then fed into the chromatography column. A Waters Ultrahydrogel™ Linear column (7.8 mm × 300 mm) was used. The detection system consisted of a differential refractive index detector and a UV detector connected in series. The mobile phase was 0.02% NaN3 solution, the flow rate was 0.6 mL / min, and the column temperature was 35 ℃.

[0055] The results show that ( Figure 1 The elution peak of GFP-40% was a single symmetrical peak, indicating its high homogeneity. A standard curve was established using dextran standards, and the relative molecular mass of GFP-40% was calculated to be 1623 kDa.

[0056] 4. Infrared spectroscopy analysis

[0057] Take 1-2 mg of GFP-40% sample, grind it together with KBr crystals until homogeneous, and press it into thin slices. Use a Nicolet 5700 Fourier transform infrared spectrometer at 4000-400 cm⁻¹. -1 Scan within the range.

[0058] The results show that ( Figure 3), GFP-40% at 3381 cm⁻¹ -1 (OH stretching vibration), 2930 cm -1 (CH stretching vibration), 1650 cm -1 and 1415 cm -1 (C=O asymmetric and symmetric stretching vibrations), 1024 cm -1 (COC stretching vibration), 856 cm -1 There is a significant absorption at the (α-glycosidic bond characteristic peak).

[0059] 5. Methylation-GC-MS analysis

[0060] Take 2-3 mg of GFP-40% sample, dissolve it in 2.0 mL of DMSO, add NaOH powder, stir for 3 hours, then slowly add iodomethane solution dropwise under ice bath conditions in the dark, and react for 2.5 hours. After the reaction is complete, add water to stop the reaction, extract with dichloromethane, wash, and dry. Add 0.5 mL of 4 mol / L TFA and hydrolyze at 100 °C for 6 hours. After drying, add sodium borohydride for reduction, remove boric acid with acetic acid-methanol solution, and acetylate with acetic anhydride to obtain partially methylated sugar alcohol acetate derivatives (PMAAs), which are then analyzed by GC-MS.

[0061] The results showed that GFP-40% mainly contained three types of glycosidic bond linkages: 1,4-D-Glcp (73.99%), TD-Glcp (12.98%), and 1,4,6-D-Glcp (9.06%).

[0062] 6. Nuclear magnetic resonance analysis

[0063] Take 30 mg of GFP-40% sample, dissolve it in 99.9% heavy water for proton exchange, repeat three times, then add 0.6-1 mL of heavy water to dissolve it again, and transfer it to a 5 mm NMR sample tube. Use a Bruker Ascend™ 400 MHz NMR spectrometer to acquire one-dimensional and two-dimensional NMR spectra at 298 K.

[0064] 1 The anomeric hydrogen signals in the 1H NMR spectrum mainly appeared at δ 5.29, 5.27, 5.12 and 4.54 ppm; 13 The anomeric carbon signals in the 13C NMR spectrum appeared at δ 99.81, 95.43, and 91.67 ppm, with no signal in the δ 170-180 ppm range, confirming the absence of uronic acid or acetyl groups. Comprehensive analysis of HSQC, HMBC, and NOESY spectra confirmed that the main chain is composed of linearly linked 1,4-α-D-Glcp residues, with branch points located at the O-6 positions of the 1,4,6-α-D-Glcp residues, and the branches being terminal glucose residues.

[0065] The above results indicate that the purified Grifola frondosa polysaccharide GFP-40% of this invention is an α-glucan, and its repeating structural unit can be described as follows: the main chain is composed of linearly linked →4)-α-D-Glcp-(1→ residues, the branch point is located at the O-6 positions of →4,6)-α-D-Glcp-(1→ residues, and the branch is the terminal α-D-Glcp residue. Based on the proportions of 1,4-D-Glcp (73.99%) and 1,4,6-D-Glcp (9.06%) in the methylation data, the branching degree is calculated to be approximately 11%, meaning that on average, one out of every 10 main chain glucose residues in the repeating unit has a branch (…). Figure 4 ).

[0066] Example 3: Effects of purified Grifola frondosa polysaccharide on improving gastric flora and inflammation in Hp-infected mice.

[0067] 1. Experimental Materials

[0068] Experimental animals: 32 male C57BL / 6 mice, 8 weeks old, purchased from Nanchang Keruis Biotechnology Co., Ltd. Strains: Helicobacter pylori standard strain ATCC 43504 (Hp strain), purchased from Shanghai Beidaguan Biotechnology Co., Ltd. Drug controls: Omeprazole, Amoxicillin, Clarithromycin. Test sample: Purified Grifola frondosa polysaccharide GFP-40% prepared in Example 1.

[0069] 2. Hp culture and preparation of bacterial suspension

[0070] Frozen *Helicobacter pylori* (Hp) cultures were inoculated onto Büchner agar solid medium containing 5% sheep blood and incubated for 2 days in a tri-gas incubator at 37 °C, 85% N2, 10% CO2, and 5% O2. Colonies were subcultured using a sterile inoculation loop and cultured for another 2 days. Colonies were then collected using Büchner broth medium, and the OD value was measured at 660 nm (1 OD = 10⁻⁶). 6 (CFU / mL), adjust the bacterial concentration to 1×10⁻⁶. 9 CFU / mL available for use.

[0071] 3. Establishment of a mouse model of *H. pylori* infection

[0072] Thirty-two C57BL / 6 mice were randomly divided into four groups of eight mice each.

[0073] Blank control group (con group): 0.2 mL of sterile double-distilled water was administered by gavage daily for 5 weeks;

[0074] Model infection group (Hp group): first received Hp strain by gavage (1×10⁻⁶). 9CFU / mL, 0.2 mL / time, administered by gavage every other day for a total of 5 times (modeling time 1 week), followed by daily gavage administration of 0.2 mL of sterile double-distilled water for 4 weeks;

[0075] Grifola frondosa polysaccharide treatment group (Hp-GFP group): The model was first established according to the method of the Hp group, and then 0.2 mL of GFP-40% solution (20 mg / mL) was administered by gavage daily for 4 weeks;

[0076] Drug treatment group (Hp-M group): The model was first established according to the method of Hp group, and then 0.2 mL of triple drug mixture (omeprazole + amoxicillin + clarithromycin) was administered by gavage daily for 4 weeks.

[0077] 4. Gastric pH measurement

[0078] Mice were euthanized after fasting for 15 hours. After dissection, 1.5 mL of distilled water was injected into the stomach, and the gastric juice was collected and measured using a pH meter.

[0079] The results show that ( Figure 5 The gastric pH in the blank control group was 3.21±0.15; the gastric pH in the model infection group was significantly increased to 4.68±0.22 (P<0.01); the gastric pH in the Grifola frondosa polysaccharide treatment group was significantly decreased to 3.42±0.18 (P<0.01 vs Hp group, P<0.01 vs Hp-M group); and the gastric pH in the drug treatment group was 3.89±0.20 (P<0.05 vs Hp group). These results indicate that the purified Grifola frondosa polysaccharide of this invention is more effective than triple therapy in alleviating the abnormal increase in gastric pH caused by Hp infection.

[0080] 5. Histopathological evaluation of gastric tissue

[0081] Mouse gastric tissue was fixed with 10% formalin, dehydrated, embedded, sectioned (4 μm), and then stained with hematoxylin and eosin (HE). Gastric mucosal inflammation, atrophy of acid-secreting glands, metaplasia, and dysplasia were scored according to established criteria.

[0082] HE staining showed ( Figure 6 In the figure, A, B, C, and D represent the con group (double-distilled water), Hp group (Helicobacter pylori infection), Hp-GFP group (Grifola frondosa polysaccharide), and Hp-M group (triple therapy), respectively. 100* and 400* represent magnifications of 100x and 400x. In the blank control group, the gastric mucosa structure was intact and the glands were neatly arranged. In the model infection group, the glands were disordered, the epithelial cells were atrophied, and there was obvious inflammatory cell infiltration, with bleeding and ulceration visible. In the Grifola frondosa polysaccharide treatment group and the drug treatment group, the gastric mucosa structure was relatively intact and the inflammation was significantly improved.

[0083] Pathological scoring results showed that the scores for inflammation, atrophy of secretory glands, metaplasia, and dysplasia in the model infection group were significantly higher than those in the blank control group (P<0.01); the scores for the above four items in the Grifola frondosa polysaccharide treatment group were significantly lower than those in the model infection group (P<0.01 or P<0.05), and there were no significant differences between the two groups except for the inflammation score.

[0084] 6. Immunohistochemical staining (CD45)

[0085] Immunohistochemistry was used to detect the expression of the inflammatory marker CD45 in mouse gastric mucosa. After dewaxing, hydration, antigen retrieval, endogenous peroxidase blockade, and serum blocking, CD45 primary antibody was added (incubated overnight at 4 ℃), and secondary antibody was added the next day (incubated at room temperature for 50 minutes). DAB staining was performed, hematoxylin was counterstained, and the slides were mounted for observation.

[0086] The results showed that CD45 expression was extremely low in the blank control group; CD45 expression in the gastric mucosa of the model infection group was significantly increased (staining dark brown); CD45 expression in the Grifola frondosa polysaccharide treatment group and the drug treatment group was significantly lower than that in the model infection group, with the Grifola frondosa polysaccharide treatment group being closer to the blank control group level.

[0087] 7. Analysis of gastric flora

[0088] 7.1 DNA Extraction and Sequencing

[0089] Total DNA was extracted from mouse gastric tissue and analyzed using the FastDNA® Spin Kit for soil. The 16S rRNA V3-V4 region was amplified using primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT). The PCR products were purified, quantified, and homogenized before being used to construct a MiSeq library, which was then sequenced using an Illumina MiSeq PE300 platform.

[0090] 7.2 Analysis of Microbial Community Composition

[0091] Gate Horizontal Analysis ( Figure 7In the blank control group, Bacteroides (62.25%) and Firmicutes (20.43%) were the dominant phyla. In the model infection group, Proteobacteria increased sharply to 57.47%, Bacteroides decreased to 24.83%, and Firmicutes decreased to 5.69%. In the Grifola frondosa polysaccharide treatment group, Proteobacteria decreased significantly to 6.88%, while Firmicutes increased back to 34.20%, with the flora structure similar to that of the blank control group. In the drug treatment group, Proteobacteria remained at a high level (52.50%). Excessive proliferation of Proteobacteria is a typical marker of gastric flora imbalance, and Grifola frondosa polysaccharide can effectively inhibit this abnormal change.

[0092] genus level analysis ( Figure 7 In the blank control group, *Muribaculum* spp. (60.05%) was the predominant species; in the model infection group, *Escherichia-Shigella* spp. was significantly enriched (8.79%); in the *Grifola frondosa* polysaccharide treatment group, *Lactobacillus* spp. increased to 30.65% and *Akkermansia* spp. increased to 22.10%, both significantly higher than in the model infection group (0.48% and 5.28%, respectively) and the drug treatment group (13.09% and 1.20%, respectively).

[0093] 7.2 Alpha diversity and beta diversity

[0094] Figure 8 Alpha diversity analysis showed that the blank control group had the highest Shannon index (4.781±0.462) and Chao1 index (1213); the model infection group had the lowest Shannon index (2.127±1.559), with large fluctuations within the group; the Grifola frondosa polysaccharide treatment group had higher Shannon index (2.639±1.460) and Chao1 index (842) than the model infection group; the drug treatment group had a further increase in Shannon index (3.017±2.171) and Chao1 index (951).

[0095] Figure 9 β-diversity analysis (PCoA) showed that the blank control group samples were tightly clustered and significantly spatially separated from the model infection group and the drug treatment group; the overall position of the Grifola frondosa polysaccharide treatment group was between the blank control group and the model infection group, suggesting that Grifola frondosa polysaccharide can guide the microbial community structure back to a normal state.

[0096] Example 4: Preparation and optimization of purified Grifola frondosa polysaccharide complex chewable tablets

[0097] The preparation method is as follows:

[0098] (1) The purified Grifola frondosa polysaccharide, fucoidan, and Hericium erinaceus polysaccharide were mixed evenly in a ratio of 2:1:1 and used as functional raw materials;

[0099] (2) Pass the functional ingredients (functional excipients of three mixed polysaccharides + milk powder), maltitol, maltodextrin, sodium carboxymethyl cellulose and citric acid through an 80-mesh sieve respectively;

[0100] (3) Place the sieved raw and auxiliary materials in a mixer and mix thoroughly until they are uniformly dispersed;

[0101] (4) Add a wetting agent (purified water or ethanol aqueous solution) and stir to make a soft material. The soft material should be able to be formed into a ball when squeezed by hand and dispersed when lightly pressed.

[0102] (5) Granulate the soft material through a 20-mesh sieve, and dry the resulting wet granules in an oven at 50-60℃ until the moisture content is ≤5%;

[0103] (6) The dried granules are sieved through a 20-mesh sieve for granulation;

[0104] (7) Add magnesium stearate and mix well;

[0105] (8) Put the tablets into a tablet press and press them into tablets to obtain purified Grifola frondosa polysaccharide complex chewable tablets.

[0106] 1. Selection of excipients

[0107] Sweetener screening: Aspartame, glucose, sucrose, and maltitol were used as sweeteners to prepare compound polysaccharide chewable tablets according to the same formula. Granule structure, soft mass condition, and tablet forming effect were used as evaluation indicators. Figure 10 The results from the Chinese A study showed that when maltitol was used as the sweetener, the tablet hardness was 61.32±0.10N, the particles were uniform, the viscosity was moderate, and the tablet shape was intact, resulting in the best overall performance.

[0108] Filler selection: lactose, maltodextrin, corn starch, and mannitol were selected as fillers. Figure 10 The results from the B-test showed that when maltodextrin was used as a filler, the tablet hardness was 61.24±0.23N, and the forming effect was optimal.

[0109] Adhesive selection: Sodium carboxymethyl cellulose, ethyl cellulose, and cross-linked polyvinylpyrrolidone were selected as adhesives. Figure 10 The results from the C-test showed that when sodium carboxymethyl cellulose was used as a binder, the tablet hardness was 58.51 ± 0.44 N, which was the best overall performance.

[0110] 2. Single-factor experiment

[0111] The effects of raw material ratios (three complex polysaccharide functional excipients: milk powder = 3:0, 2:1, 1:1, 1:2, 0:3 g / g), raw material addition (three complex polysaccharide functional excipients + milk powder) of 40-80%, maltitol addition (4-8%), maltodextrin addition (25-45%), sodium carboxymethyl cellulose addition (6-10%), citric acid addition (2-6%), and magnesium stearate addition (0.5-2.5%) on the sensory quality of chewable tablets were investigated. The optimal range for each component was determined to be: raw material ratio 2:1 (three complex polysaccharide functional excipients: milk powder, g / g), raw material addition 50%, maltitol 6%, maltodextrin 30%, sodium carboxymethyl cellulose 9%, citric acid 3%, and magnesium stearate 1%. Figure 11 China A- Figure 11 (G).

[0112] 3. Response surface optimization

[0113] Using the amount of raw material added (A), the amount of maltitol added (B), and the amount of citric acid added (C) as independent variables, and the sensory score as the response value (Y), a three-factor, three-level response surface experiment was conducted using a Box-Behnken design.

[0114] The regression equation is: Y = 89.7 - 2.0A + 2.5B - 0.2C + 0.2AB + 1.1AC + 0.6BC - 8.7A 2 -2.8B 2 -2.1C 2 .

[0115] Analysis of variance showed that the model p < 0.0001, reaching a highly significant level, and R < 0.0001. 2 =0.9883, R 2 =0.9731, signal-to-noise ratio 23.37>4, indicating that the model fits well and can be used for formula optimization. The order of influence of each factor is: maltitol addition (B) > raw material addition (A) > citric acid addition (C).

[0116] The results show that ( Figure 12 The response surface graph exhibits a steep slope, intuitively reflecting the interaction between the amount of raw materials and maltitol added on the sensory score. The optimal formulation was determined through optimization: 50% raw material, 6.5% maltitol, and 3% citric acid. The model predicted a sensory score of 90.3.

[0117] Three batches of validation experiments were conducted using the optimal formula. The average sensory score of the resulting chewable tablets was 90.4 points, with a relative error of 0.09% compared to the predicted value, indicating that the model is accurate and reliable.

[0118] 4. Quality Inspection

[0119] Tablet weight variation test: 20 tablets of compound polysaccharide chewable tablets were weighed, and the tablet weight variation was calculated to be 0.25%, which meets the Chinese Pharmacopoeia standard (±5%).

[0120] Granulation rate determination: The granulation rate is over 91%.

[0121] Sensory inspection ( Figure 13 The compound polysaccharide chewable tablets are uniformly light brown in appearance, with intact tablets of uniform size, without cracks or powdering; the texture is firm and compact, not loose; the taste is moderately sweet and sour, without any gritty feeling.

[0122] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A compound chewable tablet containing purified Grifola frondosa polysaccharide, characterized in that, The compound chewable tablets comprise the following ingredients by weight percentage: 40-80% functional ingredients, 4-8% sweetener, 25-45% filler, 6-10% binder, 2-6% acidulant, and 0.5-2.5% lubricant. The functional ingredients are a mixture of functional excipients and milk powder. The functional excipients are a mixture of purified Grifola frondosa polysaccharide, fucoidan, and Hericium erinaceus polysaccharide. The purified Grifola frondosa polysaccharide is a polysaccharide component obtained through hot water extraction of Grifola frondosa fruiting bodies, alcohol precipitation, deproteinization, dialysis, gradient ethanol precipitation, and ultrafiltration purification. The mass ratio of the functional excipients to the milk powder is (0-3):(0-3). The mass ratio of the purified Grifola frondosa polysaccharide, fucoidan, and Hericium erinaceus polysaccharide is 2:1:

1.

2. The compound chewable tablet according to claim 1, characterized in that, The compound chewable tablets comprise the following ingredients by weight percentage: 50% functional ingredients, 6% sweetener, 30% filler, 9% binder, 3% acidulant, and 1% lubricant. The functional ingredients and milk powder are mixed in a weight ratio of 2:

1.

3. The compound chewable tablet according to claim 1, characterized in that, The sweetener is at least one of aspartame, glucose, sucrose, and maltitol; the filler is at least one of lactose, maltodextrin, corn starch, and mannitol; the binder is at least one of sodium carboxymethyl cellulose, ethyl cellulose, and crosporovinylpyrrolidone; the acidulant is citric acid; and the lubricant is magnesium stearate.

4. The compound chewable tablet according to claim 3, characterized in that, The sweetener is maltitol, the filler is maltodextrin, and the binder is sodium carboxymethyl cellulose.

5. The method for preparing the compound chewable tablet containing purified Grifola frondosa polysaccharide according to any one of claims 1-4, characterized in that, The process includes the following steps: (1) mixing purified Grifola frondosa polysaccharide, fucoidan, Hericium erinaceus polysaccharide, maltitol, maltodextrin, sodium carboxymethyl cellulose and citric acid; (2) adding a wetting agent to make a soft mass; (3) wet granulation; (4) drying at 40 ℃-70 ℃; (5) granulation; (6) adding magnesium stearate and then compressing to obtain purified Grifola frondosa polysaccharide complex chewable tablets.

6. The method according to claim 5, characterized in that, The method for preparing purified Grifola frondosa polysaccharide is as follows: (a) drying and pulverizing Grifola frondosa fruiting bodies; (b) hot extraction with water, filtering and combining the extracts; (c) centrifuging, concentrating and precipitating the extract with alcohol to obtain crude polysaccharide; (d) deproteinizing and dialysis the crude polysaccharide; (e) precipitating the dialyzed polysaccharide with 40% ethanol; (f) purifying the obtained fraction by 30 kDa ultrafiltration and lyophilizing.

7. The use of the compound chewable tablets containing purified Grifola frondosa polysaccharide according to any one of claims 1-4 in improving the gastric flora associated with Helicobacter pylori infection.

8. The application according to claim 7, characterized in that, The chewable tablet has at least one of the following effects: (1) Reduce the abnormal increase in gastric pH caused by Helicobacter pylori infection; (2) Reduce gastric mucosal inflammatory infiltration and / or atrophy of acid-secreting glands; (3) Inhibit the excessive proliferation of opportunistic pathogens of the phylum Proteobacteria in the stomach; (4) Promotes the colonization of beneficial bacteria of the genus Lactobacillus and / or Ekkermansia; (5) Restore the gastric flora diversity caused by Helicobacter pylori infection.

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