Preparation method of astragalus mongholicus fermented polysaccharide and application of astragalus mongholicus fermented polysaccharide in medicine and food for intervening premenstrual syndrome

Abstract

The invention discloses a preparation method of astragalus mongholicus fermented polysaccharide and application of the astragalus mongholicus fermented polysaccharide in medicines and foods for intervening premenstrual syndrome, relates to a preparation method and application of an astragalus mongholicus fermented product, and belongs to the technical field of crossing of traditional Chinese medicines and microorganisms. The preparation method comprises the following steps: inoculating the lactobacillus casei paracasei into an astragalus membranaceus culture medium according to the inoculum size of 5% under the culture condition of 37 DEG C; the fermented astragalus polysaccharide is used as an active ingredient in the premenstrual syndrome intervention medicines and foods for intervening the premenstrual syndrome. The astragalus membranaceus fermented polysaccharide is used for gavage of mice, has better biological activity of relieving premenstrual syndrome related emotional disorder, and is mainly used for relieving neuronal injury in brain tissues by increasing the weight of the premenstrual syndrome mice and the content of gamma-aminobutyric acid, tetrahydroprogesterone, 5-hydroxytryptophan and brain-derived neurotrophic factors, so that the premenstrual syndrome related emotional disorder can be relieved. The aim of treating the premenstrual syndrome is fulfilled.

Description

Technical Field

[0001] This invention relates to a method for preparing Astragalus membranaceus ferment and its application, belonging to the field of interdisciplinary technology of traditional Chinese medicine and microorganisms. Background Technology

[0002] Premenstrual syndrome (PMS) is a common disorder that occurs during the luteal phase of the menstrual cycle, characterized by a range of emotional and physical symptoms. PMS is most common in women aged 25-45, with 95% of women of reproductive age experiencing these symptoms. Symptoms include changes in appetite, weight gain, abdominal pain, anxiety, irritability, anger, fatigue, restlessness, and mood swings, which typically lessen or disappear with the onset of menstruation. Current treatments for PMS include medication such as gonadotropin-releasing hormone (GnRH) analogs, antidepressants, and oral contraceptives. Psychotherapy and lifestyle modifications are also important. However, while medication is highly effective, it also has many side effects. Side effects related to low estrogen levels may occur, such as hot flashes, sweating, mood swings, decreased libido, and osteoporosis; in severe cases, it can negatively impact sexual function. Currently, there is a lack of safe and effective treatments. Summary of the Invention

[0003] The purpose of this invention is to address the limitations of existing treatments for premenstrual syndrome by providing a method for preparing Astragalus fermented polysaccharide and its application in drugs and foods for intervening in premenstrual syndrome.

[0004] The preparation method of Astragalus fermentation polysaccharide is as follows:

[0005] After pulverizing Astragalus membranaceus, prepare a suspension at a ratio of 20-30 mg to 70 mL. Autoclave at 121°C for 15 min, cool to room temperature, and then sterilize under UV light for 30 min in a clean bench. Incubate at 37°C, and then inoculate with 5% of activated Lactobacillus casei (Lactobacillus rhamnosus) solution to achieve a Lactobacillus casei concentration of 10. 8 -10 10 The concentration of *Lactobacillus casei* in the activated bacterial culture solution was 5 × 10⁻¹⁰ CFU / mL, yielding the *Lactobacillus casei* fermentation broth. 8CFU / mL, the fermentation broth of *Lactobacillus casei* was cultured in an incubator at 37℃ for 2-5 days. After fermentation, the fermentation broth was dried at 60℃, ground, and 0.5 g of the ground powder was weighed. 25 mL of 100% methanol was added, and then the mixture was sonicated at 300 W and 40 kHz for 30 min. After being cooled to room temperature, the lost methanol was replenished with 100% methanol, filtered, and the filtrate was collected for later use to obtain astragalus polysaccharide fermented by *Lactobacillus casei*.

[0006] 500 g of Astragalus polysaccharide fermented with Lactobacillus casei was dissolved in 80% ethanol at a ratio of 1 g: 4 mL and refluxed for 2 h. This process was repeated twice to remove lipids, pigments, and small molecules. After air drying, the polysaccharide was dissolved in 80% ethanol at a ratio of 1 g: 10 mL and refluxed for 2 h, 2 h, 2 h, and 1 h, respectively. The four refluxes were combined, concentrated, and then anhydrous ethanol was added to bring the ethanol concentration to 80%. The mixture was left overnight, centrifuged at 3500 × g for 8 min, and the precipitate was collected. The precipitate was redissolved in pure water, concentrated, dialyzed, and freeze-dried to obtain Astragalus fermented polysaccharide.

[0007] The Lactobacillus casei described was obtained from the National Culture Collection Center (CICC) with accession number 20990.

[0008] The application of the prepared Astragalus fermented polysaccharide in drugs for the intervention of premenstrual syndrome, wherein the Astragalus fermented polysaccharide is the active ingredient in the drugs for the intervention of premenstrual syndrome.

[0009] The medication used to intervene in premenstrual syndrome is a liquid, solid, or semi-solid formulation.

[0010] The medication for intervening in premenstrual syndrome is an oral liquid, capsule, tablet, or granule.

[0011] Each 5g granule of the medication for treating premenstrual syndrome contains 200mg of Astragalus fermented polysaccharide.

[0012] The concentration of Astragalus fermented polysaccharide in the oral solution for treating premenstrual syndrome is 20 mg / mL.

[0013] The application of the prepared Astragalus fermented polysaccharide in foods for the intervention of premenstrual syndrome, wherein the Astragalus fermented polysaccharide is the active ingredient in foods for the intervention of premenstrual syndrome.

[0014] The foods mentioned for intervening in premenstrual syndrome include functional foods and health foods.

[0015] The functional food is a fermented beverage or a compressed candy; the fermented beverage contains 10 mg / mL of Astragalus fermented polysaccharide; the compressed candy contains 300 mg of Astragalus fermented polysaccharide per 0.5 g.

[0016] The Astragalus fermented polysaccharide is used to increase the levels of body weight, γ-aminobutyric acid (GABA), tetrahydroprogesterone (ALLO), 5-hydroxytryptophan (5-HT), and brain-derived neurotrophic factor (BDNF) in patients with premenstrual syndrome, and to alleviate neuronal damage in brain tissue.

[0017] Astragalus polysaccharides have been found to have immunomodulatory, hypoglycemic, depressive-like behavior-improving, and gut microbiota-regulating effects. Astragalus polysaccharides can influence the structure of the gut microbiota and related metabolites, and regulate neurotransmitters, thus affecting brain function and achieving an antidepressant effect. Astragalus polysaccharides can affect neurotransmitter levels, especially serotonin (5-HT) and dopamine. By enhancing the activity of these neurotransmitters, they can alleviate anxiety and depressive symptoms, and have potential therapeutic effects on various mental disorders, including anxiety and depression.

[0018] Fermentation significantly enhances the bioactivity of plant polysaccharides due to changes in polysaccharide structure and the generation of extracellular polysaccharides. These changes effectively improve the gut microbiota structure, enhance antioxidant capacity, reduce inflammatory responses, and promote the production of short-chain fatty acids. These factors work together to improve the bioavailability and therapeutic effects of plant polysaccharides.

[0019] This invention utilizes bio-fermentation technology to obtain astragalus polysaccharides through the fermentation of *Lactobacillus casei* (Lactobacillus rhamnosus). This is the first time such polysaccharides have been applied to the treatment of premenstrual syndrome (PMS). Animal experiments have demonstrated that fermented astragalus polysaccharides can better regulate neurotransmitter function and alleviate neuronal damage, thereby affecting brain function and improving mood disorders, achieving the goal of treating PMS-related mood disorders. By studying the mechanism of action of fermented astragalus polysaccharides in PMS, this invention aims to elucidate the mechanism of action of fermented astragalus polysaccharides in PMS, and to expand the application of astragalus polysaccharides in the development of food-medicine homologous products and drugs for the protection of women's health.

[0020] This invention uses the Astragalus fermented polysaccharide obtained above to intervene in mice with premenstrual syndrome. This invention provides the first application of Astragalus fermented polysaccharide from *Lactobacillus casei* in the intervention of premenstrual syndrome-related mood disorders, opening up new uses for Astragalus polysaccharides. Through in vitro fermentation and in vivo experiments, this invention demonstrates that Astragalus fermented polysaccharide can better regulate neurotransmitter function and alleviate neuronal damage, thereby affecting brain function and improving mood disorders, achieving the goal of treating premenstrual syndrome-related mood disorders. It has good application prospects in the preparation of drugs and health foods for the intervention of premenstrual syndrome. Attached Figure Description

[0021] Figure 1 The image shows the high-performance liquid gel chromatogram of the molecular weight of Astragalus fermentation polysaccharide in Experiment 1.

[0022] Figure 2The effect of Astragalus fermented polysaccharide on anxiety and depression behavior in PMS model mice in Experiment 1 (mouse in open arm time during elevated cross maze experiment).

[0023] Figure 3 The effect of Astragalus fermented polysaccharide on anxiety and depression behavior in PMS model mice in Experiment 1 (mouse time in the closed arm during the elevated cross maze experiment).

[0024] Figure 4 The graph shows the effect of Astragalus fermented polysaccharide on anxiety and depression behavior in PMS model mice in Experiment 1 (time to surface of mice in the swimming experiment).

[0025] Figure 5 The graph shows the effect of Astragalus fermentation polysaccharide on body weight in Experiment 1.

[0026] Figure 6 This is a comparison chart of serum GABA levels in mice during Experiment 1. The horizontal axis represents the group, and the vertical axis represents the content.

[0027] Figure 7 This is a comparison chart of serum 5-HT levels in mice during Experiment 1. The horizontal axis represents the group, and the vertical axis represents the content.

[0028] Figure 8 This is a comparison chart of serum BDNF levels in mice during Experiment 1. The horizontal axis represents the group, and the vertical axis represents the content.

[0029] Figure 9 This is a comparison chart of serum ALLO levels in mice during Experiment 1. The horizontal axis represents the group, and the vertical axis represents the content.

[0030] Figure 10 The graph shows the effect of Astragalus fermentation polysaccharide on Nissl body staining in mice in Experiment 1. The horizontal axis represents the group and the vertical axis represents the number of deformed neurons.

[0031] Figure 11 The figure shows the effect of Astragalus fermentation polysaccharide on HE staining results of mouse hippocampus in Experiment 1.

[0032] Figure 12 The graph shows the effect of Astragalus fermentation polysaccharide on the expression of GABAAR4α mRNA in the amygdala of mice in Experiment 1. The horizontal axis represents the group and the vertical axis represents the content.

[0033] Figure 13 The graph shows the effect of Astragalus fermentation polysaccharide on the expression of GABAARβ2 mRNA in the amygdala of mice in Experiment 1. The horizontal axis represents the group and the vertical axis represents the content.

[0034] Figure 14 The figure shows the effect of Astragalus fermentation polysaccharide on the acetic acid content in mouse feces in Experiment 1.

[0035] Figure 15The figure shows the effect of Astragalus fermentation polysaccharide on butyric acid content in mouse feces in Experiment 1.

[0036] Figure 16 The figure shows the effect of Astragalus fermentation polysaccharide on the isovaleric acid content in mouse feces in Experiment 1. Detailed Implementation

[0037] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0038] Specific Implementation Method 1: The preparation method of Astragalus fermentation polysaccharide in this implementation method is as follows:

[0039] After pulverizing Astragalus membranaceus, prepare a suspension at a ratio of 20-30 mg to 70 mL. Autoclave at 121°C for 15 min, cool to room temperature, and then sterilize under UV light for 30 min in a clean bench. Incubate at 37°C, and then inoculate with 5% of activated Lactobacillus casei (Lactobacillus rhamnosus) solution to achieve a Lactobacillus casei concentration of 10. 8 -10 10 The concentration of *Lactobacillus casei* in the activated bacterial culture solution was 5 × 10⁻¹⁰ CFU / mL, yielding the *Lactobacillus casei* fermentation broth. 8 CFU / mL, the fermentation broth of *Lactobacillus casei* was cultured in an incubator at 37℃ for 2-5 days. After fermentation, the fermentation broth was dried at 60℃, ground, and 0.5 g of the ground powder was weighed. 25 mL of 100% methanol was added, and then the mixture was sonicated at 300 W and 40 kHz for 30 min. After being cooled to room temperature, the lost methanol was replenished with 100% methanol, filtered, and the filtrate was collected for later use to obtain astragalus polysaccharide fermented by *Lactobacillus casei*.

[0040] 500 g of Astragalus polysaccharide fermented with Lactobacillus casei was dissolved in 80% ethanol at a ratio of 1 g: 4 mL and refluxed for 2 h. This process was repeated twice to remove lipids, pigments, and small molecules. After air drying, the solution was dissolved in 80% ethanol at a ratio of 1 g: 10 mL and refluxed for 2 h, 2 h, 2 h, and 1 h, respectively. The four reflux solutions were combined, concentrated, and then anhydrous ethanol was added to bring the ethanol concentration to 80%. The solution was left overnight and centrifuged at 3500 × g for 8 min. The precipitate was collected, redissolved in pure water, concentrated, dialyzed, and freeze-dried to obtain Astragalus fermented polysaccharide (FAPS).

[0041] High performance liquid chromatography-gel chromatography of FAPS molecular weight Figure 1 Calculations show that the relative molecular mass Mw of the peak is 6.4 × 10⁻⁶. 4Da, accounting for 46.804%; contains 4.31% arabinose (Ara), 1.83% galacturonic acid (GalA), 1.84% galactose (Gal), 11.67% glucuronic acid (GlcA), 4.17% glucose (Glu), 1.69% rhamnose (Rha), and 0.64% mannose (Man).

[0042] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the *Lactobacillus casei* described is sourced from the National Culture Collection Center (CICC), with accession number 20990. Everything else is the same as in Specific Implementation Method One.

[0043] Specific Implementation Method 3: The application of Astragalus fermented polysaccharide prepared in Specific Implementation Method 1 in drugs for intervening in premenstrual syndrome, characterized in that the Astragalus fermented polysaccharide is the active ingredient in drugs for intervening in premenstrual syndrome.

[0044] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method Three in that the drug used to intervene in premenstrual syndrome is a liquid, solid, or semi-solid formulation. Everything else is the same as in Specific Implementation Method Three.

[0045] Specific Implementation Method Five: This implementation method differs from Specific Implementation Method Three in that the medication used to intervene in premenstrual syndrome is in the form of an oral liquid, capsule, tablet, or granule. Everything else is the same as in Specific Implementation Method Three.

[0046] Preparation method of Astragalus fermented polysaccharide granules:

[0047] According to Specific Implementation Method 1, Astragalus fermented polysaccharide was obtained, and after freeze-drying, Astragalus fermented polysaccharide freeze-dried powder was obtained. 50g of Astragalus fermented polysaccharide freeze-dried powder was mixed evenly with excipients 10g sodium chloride, 30g soluble starch, 250g microcrystalline cellulose, 200g lactose, 150g mannitol, 20g β-cyclodextrin, and 80g dextrin. 80% ethanol was used as a wetting agent to prepare a soft mass, which was granulated through a 10-mesh sieve, dried in an oven at 60℃ for 1 hour, granulated through a 14-mesh sieve, and then dried in an oven at 60℃ for another 3 hours to produce Astragalus fermented polysaccharide granules.

[0048] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Three in that each 5g granule dose of the drug for intervening in premenstrual syndrome contains 200mg of Astragalus fermented polysaccharide. Everything else is the same as in Specific Implementation Method Three.

[0049] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Three in that the concentration of Astragalus fermented polysaccharide in the oral liquid for intervening in premenstrual syndrome is 20 mg / mL. Everything else is the same as in Specific Implementation Method Three.

[0050] Specific Implementation Method 8: Application of Astragalus fermented polysaccharide prepared in Specific Implementation Method 1 in food for intervening in premenstrual syndrome, wherein the Astragalus fermented polysaccharide is the active ingredient in food for intervening in premenstrual syndrome.

[0051] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Eight in that the foods mentioned for intervening in premenstrual syndrome include functional foods and health foods. Everything else is the same as in Specific Implementation Method Eight.

[0052] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Eight in that the functional food is a fermented beverage or compressed candy; the concentration of Astragalus fermented polysaccharide in the fermented beverage is 10 mg / mL; and each 0.5 g of the compressed candy contains 300 mg of Astragalus fermented polysaccharide. Everything else is the same as in Specific Implementation Method Eight.

[0053] Preparation method of Astragalus fermented polysaccharide compressed candy:

[0054] Astragalus fermented polysaccharide was obtained according to specific implementation method one, and then freeze-dried to obtain astragalus fermented polysaccharide freeze-dried powder. The powder was then mixed with 80% astragalus fermented polysaccharide, 15% lactose, 1.6% microcrystalline cellulose, 1% silica, 1% magnesium stearate, and 1.4% hydroxypropyl methylcellulose. The total mixture was pressed into thin sheets, granulated using a granulator, and passed through a 16-mesh sieve. Magnesium stearate and silica were added to the granules, and the mixture was stirred for 15 minutes to obtain the final mixture. The final mixture was then compressed into tablets to produce astragalus fermented polysaccharide compressed candy.

[0055] The following experiments were used to verify the effectiveness of the invention:

[0056] Experiment 1:

[0057] Preparation method of Astragalus polysaccharide:

[0058] Sample pretreatment: Take dried Astragalus membranaceus slices and dry them in a 40 ℃ forced-air drying oven to obtain Astragalus membranaceus slices, and store them in a desiccator.

[0059] Degreasing: Take 1 kg of dried Astragalus powder, add 80% ethanol at a ratio of 1 g to 5 L to defatt the powder, stir at room temperature for 24 h, and then heat under reflux for 2 h.

[0060] Hot water extraction: After drying the filter residue, add deionized water at a material-to-liquid ratio of 1 g:10 L, and extract 4 times at 80℃, 2 h each time. Combine the extracts and concentrate them to 1 / 5 of the original volume using a rotary evaporator at 60℃, then refrigerate for later use.

[0061] Alcohol precipitation: Anhydrous ethanol was slowly added to the concentrate until the ethanol concentration reached 70%, with the stirring direction constantly changing to ensure thorough mixing of the ethanol and the drug solution. When the ethanol concentration exceeded 80% as measured by an alcohol meter, the mixture was sealed after stirring and allowed to stand in a refrigerator at 4°C for 12 hours. After centrifugation at 3500 rpm for 8 minutes, the precipitate was collected. The precipitate was reconstituted with deionized water, and residual ethanol was removed by rotary evaporation under reduced pressure. Finally, it was freeze-dried to obtain crude Astragalus polysaccharide (ASP).

[0062] I. Materials and Methods

[0063] 1. Experimental Materials

[0064] One hundred and five female BALB / c mice, approximately five weeks old and weighing 15±3 g, were selected. The temperature in the mouse house was 22±2°C and the humidity was 55±5%. The mice were kept in a 12-hour day-night cycle and had free access to food, water, and feed.

[0065] 2. Animal grouping

[0066] A progesterone withdrawal (PWD) mouse model was established by simulating luteal phase hormonal changes through long-term use of exogenous progesterone followed by abrupt cessation of treatment. PWD mice exhibited anxiety- and depression-like behaviors, as well as irritability and aggression, very similar to those seen in women with progesterone-induced dysregulation (PMS). Sixty BALB / c female mice, approximately 5 weeks old and weighing 15-18g, were used as experimental animals. After purchase, the mice were fed a basal diet for one week to acclimate to their environment, with free access to water. The mice were then divided into 6 groups (n=10 / group): control group, model group, Astragalus polysaccharide group (APS), Astragalus fermented polysaccharide group (FAPS), fluoxetine group (FXT), and Xiangshao granule group (XSKL). The control group mice were injected daily with sesame oil (0.1 ml / mouse / day), while the other groups were injected intraperitoneally daily with progesterone (0.125 mg progesterone / mouse / day, suspended in 0.1 ml sesame oil) for 21 consecutive days. Starting on day 15 of model establishment, mice were administered medications via gavage: Astragalus polysaccharide (200 mg / kg / day), Astragalus fermented polysaccharide (200 mg / kg / day), Fluoxetine dispersible tablets (0.645 mg / kg / day), and Xiangshao granules (0.356 mg / kg / day). The control group received an equal volume of physiological saline (0.2 ml / mouse). All injections were terminated 48 hours before the behavioral test. Mouse weight was monitored weekly.

[0067] 3. Sample preparation

[0068] Forty-eight hours after the last progesterone injection, mice underwent the elevated cruciate maze test and the grueling swimming test. Whole blood was collected by enucleation and left at room temperature for 2–4 hours. Serum was then collected by centrifugation and aliquoted into 200 μL centrifuge tubes, stored at -20°C for later use. Mice were anesthetized with ether, euthanized by cervical dislocation, and then dissected after surface disinfection. The hippocampus and amygdala were rapidly removed, frozen in liquid nitrogen, and then transferred to a -80°C freezer for long-term storage.

[0069] 4. Serum 5-HT, GABA, ALLO, and BDNF assays

[0070] The levels of 5-HT, GABA, ALLO, and BDNF in mouse serum were detected using the Shanghai 5-HT, GABA, ALLO, and BDNF enzyme-linked immunosorbent assay kit.

[0071] 5. Procedure for Paraffin Embedding and Sectioning of Tissue

[0072] (1) Sampling: Fresh tissue was fixed in 4% paraformaldehyde for more than 24 hours. The tissue was removed from the fixative and trimmed in the target area with a scalpel in a fume hood. The trimmed tissue and corresponding labels were placed in a dehydration box.

[0073] (2) Dehydration: Place the dehydration box into the basket and perform gradient dehydration in the dehydrator in sequence. Dehydration process: 75% alcohol (volume concentration) 4h, 85% alcohol (volume concentration) 2h, 90% alcohol (volume concentration) 2h, 95% alcohol (volume concentration) 1h, anhydrous ethanol (first time) 30min, anhydrous ethanol (second time) 30min, benzene (alcohol concentration) 5-10min, xylene (first time) 5-10min, xylene (second time) 5-10min, wax (first time) 1h, wax (second time) 1h, wax (third time) 1h.

[0074] (3) Embedding: The tissue impregnated with wax is embedded in an embedding machine. First, the melted wax is placed into the embedding frame. Before the wax solidifies, the tissue is taken out from the dehydration box and placed into the embedding frame according to the requirements of the embedding surface and labeled accordingly. It is cooled on a -20°C freezing stage. After the wax solidifies, the wax block is removed from the embedding frame and the wax block is trimmed.

[0075] (4) Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm. Float the sections on 40℃ warm water in a slide spreader to flatten the tissue. Use a glass slide to lift the tissue and place it in a 60℃ oven to bake. After the water has dried and the wax has melted, remove the sections and store them at room temperature for later use.

[0076] 6. HE staining experimental procedure

[0077] (1) Dewaxing paraffin sections to water: The sections were placed in xylene (first time) I for 20 min, xylene (second time) for 20 min, anhydrous ethanol (first time) for 10 min, anhydrous ethanol (second time) for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water.

[0078] (2) Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0079] (3) Eosin staining of cytoplasm: Slices are stained in eosin staining solution for 1-3 min.

[0080] (4) Dehydration and mounting: The sections were placed in 95% alcohol (first time) for 5 min, 95% alcohol (second time) for 5 min, anhydrous ethanol (first time) for 5 min, anhydrous ethanol (second time) for 5 min, xylene (first time) for 5 min, and xylene (second time) for 5 min to dehydrate until transparent. The sections were removed from the xylene and dried slightly, and then mounted with neutral resin.

[0081] (6) Microscopic examination, image acquisition and analysis.

[0082] 7. Expression of related genes in brain tissue

[0083] (1) Extraction of total RNA from brain tissue

[0084] The hippocampus and amygdala, removed from liquid nitrogen, were placed in a pre-chilled mortar and pestle. 1 mL of Trizol reagent was added, and the mixture was rapidly ground into powder using liquid nitrogen. The homogenate was transferred to an RNase-free centrifuge tube and incubated at room temperature for 5 minutes (to allow for complete separation of nucleic acids and proteins). 0.2 mL of chloroform was added to the tube, the cap was tightened, and the tube was vigorously shaken for 15 seconds. The mixture was then incubated at room temperature for 2-3 minutes and centrifuged at 12000×g for 15 minutes at 4°C. The upper RNA phase was aspirated using an RNase-free pipette tip (avoiding the middle protein phase, as this would affect purity), and transferred to a new RNase-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was gently inverted to mix. The mixture was incubated at room temperature for 10 minutes and centrifuged at 12000×g for 10 minutes at 4°C. The supernatant was discarded, and a white RNA precipitate was visible at the bottom of the tube. Add 1 mL of 75% ethanol solution (prepared with RNase-free water) to a centrifuge tube, gently invert the tube, and wash the precipitate (avoid vigorous shaking to prevent RNA breakage). Centrifuge at 7500×g for 5 minutes at 4°C, and discard the supernatant. Repeat the washing once, centrifuge again, and then aspirate any residual ethanol with a pipette tip. Allow the precipitate to air dry at room temperature for 5-10 minutes. Add 20-50 μL of RNase-free ultrapure water to the centrifuge tube, incubate at 55-60°C for 10 minutes, and gently mix to ensure complete RNA dissolution. Ensure the entire process is performed in the dark. Wipe the laminar flow hood with chloroform, and use DEPC-treated equipment. Collect the extracted total RNA in RNA-free centrifuge tubes and store at -80°C.

[0085] (2) Detection of RNA purity and integrity

[0086] Two μL of RNA sample was subjected to agarose gel electrophoresis to determine the integrity of total RNA. A 1% agarose TAE buffer was prepared, and Gel Red was used as the fluorescent dye. Electrophoresis was performed at 150 V for 10 min. The bands at 28S, 18S, and 5S were observed under UV light using a gel imaging system and photographed. One μL of RNA sample was then subjected to OD260 and OD280 measurements to determine the purity and concentration of the RNA sample. RNA with OD260 and OD280 values ​​within the range of 1.8–2.1 was considered to be of good quality and suitable for subsequent experiments.

[0087] (3) Reverse transcription system

[0088] Following the PrimeScript™ RT kit instructions, the sample RNA concentration was adjusted appropriately, and a reverse transcription reaction was performed to obtain cDNA. The reaction system involved in the reverse transcription reaction is shown in Table 1. The specific reaction conditions were: reverse transcription at 37°C for 15 min; reverse transcriptase inactivation reaction at 85°C for 5 s. The reaction products were frozen at -20°C for subsequent experiments.

[0089] Table 1. Preparation of the reaction system

[0090]

[0091] (4) Real-time RT-PCR detection of brain-related gene mRNA expression levels

[0092] Real-time RT-PCR was performed on the cDNA obtained by reverse transcription according to the TB Green® Premix Ex Taq™ II kit instructions to detect the expression of related genes in mouse brain tissue by Astragalus fermentation polysaccharide. Specific primers for GABA (γ-aminobutyric acid) related factors (α4, β2) were designed using Primer 5.0 software (Table 2), and primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0093] Table 2 Primer Information

[0094]

[0095] 8. Data processing and statistical analysis

[0096] All data were analyzed using one-way ANOVA with SPSS software, and plotting analysis was performed using GraphPad Prism 8.02. Results are expressed as mean ± standard deviation (Mean ± SD), and P < 0.05 was considered statistically significant.

[0097] II. Experimental Results

[0098] 1. Chemical results and characterization of Astragalus membranaceus fermented polysaccharides

[0099] High performance liquid chromatography-gel chromatography of FAPS molecular weight Figure 1 Calculations show that the relative molecular mass Mw of the peak is 6.4 × 10⁻⁶. 4 Da, accounting for 46.804%; consists of 4.31% arabinose (Ara), 1.83% galacturonic acid (GalA), 1.84% galactose (Gal), 11.67% glucuronic acid (GlcA), 4.17% glucose (Glu), 1.69% rhamnose (Rha), and 0.64% mannose (Man).

[0100] 2. Effects of Astragalus fermented polysaccharides on elevated cruciate maze and strenuous swimming in mice

[0101] In the elevated cross maze experiment, such as Figure 2-4 As shown in the figure, compared with the control group mice, the model group mice spent more time in the closed arm and less time in the open arm. The shorter time spent in the open arm indicates anxiety, indicating successful model establishment. The intervention group mice, compared with the model group mice, significantly increased the time spent in the open arm, indicating that Astragalus polysaccharide administration effectively alleviated anxiety in mice. In the forced swimming experiment, as shown in the figure, the model group mice significantly increased the stillness time during forced swimming compared to the control group, indicating successful model establishment. The intervention group mice significantly shortened the stillness time during forced swimming compared to the model group, indicating that Astragalus polysaccharide administration effectively alleviated depression in mice.

[0102] 3. The effect of Astragalus on body weight

[0103] After 14 days of modeling, such as Figure 5 The model group showed a significant decrease in weight compared to the control group, indicating that the modeling effect was effective. After 7 days of intervention with Astragalus fermented polysaccharide, fluoxetine, and Xiangshao granules, the weight of the intervention groups increased compared to the model group, indicating that the Astragalus polysaccharide intervention had a certain effect.

[0104] 4. Effects of Astragalus fermented polysaccharide on serum GABA, ALLO, 5-HT, and BDNF levels

[0105] Compared with the control group, the GABA content in the model group decreased, and the difference was statistically significant (P < 0.001); compared with the model group, the serum GABA content in the APS group increased significantly, and the difference was statistically significant (P < 0.001). (See attached table.) Figure 6 Compared with the control group, the 5-HT level in the model group decreased significantly (P < 0.001); compared with the model group, the serum 5-HT level in the FAPS group increased significantly (P < 0.001). (See attached table.) Figure 7Compared with the control group, the BDNF content in the model group decreased significantly (P < 0.001); compared with the model group, the serum BDNF content in the FAPS group increased significantly (P < 0.001), as shown in Figure 8. Compared with the control group, the ALLO content in the model group decreased significantly (P < 0.001); compared with the model group, the serum ALLO content in the APS group increased significantly (P < 0.001), as shown in Figure 9. Among them, the FAPS group (200 mg / kg) showed the closest recovery effect to the normal level for each indicator (P < 0.01). These results indicate that Astragalus fermented polysaccharides fermented with Lactobacillus casei may improve the neuroendocrine hormone disorder of PMS by regulating neurotransmitters, providing experimental evidence for its clinical treatment of PMS.

[0106] 5. Effects of Astragalus fermented polysaccharides on neurons in brain tissue

[0107] Nissl body staining results in the hippocampus of mouse brain tissue from each group: Nissl bodies can synthesize neurotransmitters, including enzymes and peptides required for neurotransmitter synthesis. When neurons are damaged, Nissl bodies shrink, disintegrate, or even disappear. Figure 10 As shown, the control group mice had abundant neurons in the CA1 and CA3 regions of the hippocampus, with intact, smooth, round morphologies, and a neat, dense, and evenly distributed arrangement. In the model group mice, the neurons in the CA1 and CA3 regions of the hippocampus were significantly sparsely arranged, with numerous instances of Nissl body loss, deeply stained and condensed nuclei, and significantly widened intercellular spaces. After administration of Astragalus fermented polysaccharide, compared to the model group, the APS group showed mild abnormalities in brain tissue structure, while the FAPS group showed essentially normal brain tissue structure. A small number of neuronal degenerations were observed in the CA3 region of the hippocampus, with darker staining and no clear cell bodies, as indicated by the red arrows. The tissue structure was compact, with no edema or inflammatory cell infiltration. Statistical results of deformed neurons showed that the number of deformed neurons in each group significantly decreased after Astragalus fermented polysaccharide intervention (P < 0.0001).

[0108] 6. Effects of Astragalus fermented polysaccharides on brain tissue-related genes

[0109] ALLO is a potent orthoallosteric modulator of the γ-aminobutyric acid type A receptor (GABAAR), and ALLO-mediated changes in GABAAR sensitivity primarily depend on the receptor subunits. The GABAARα4 and GABAARβ2 subunits are thought to be associated with the regulation of anxiety behavior.

[0110] like Figure 11As shown, compared with the control group, the expression level of GABAARα4 mRNA in the amygdala of mice in the model group was significantly increased (P < 0.05); compared with the model group, the expression level of GABAARα4 mRNA in the amygdala of mice in the drug-treated group was significantly decreased (P < 0.01), with the most significant decrease in the expression level of GABAARα4 mRNA in the FAPS group.

[0111] like Figure 12 As shown, compared with the control group, the expression level of GABAARβ2 mRNA in the amygdala of mice in the model group was significantly decreased, and the difference was statistically significant (P < 0.05); compared with the model group, the expression level of GABAARβ2 mRNA in the amygdala of mice in the fluoxetine group was significantly increased, and the difference was statistically significant (P < 0.01).

Claims

1. A method for preparing Astragalus fermented polysaccharide, characterized in that The preparation method of the Astragalus membranaceus fermented polysaccharide is as follows: After the Astragalus membranaceus is crushed, it is configured into a suspension according to a ratio of 20-30 mg:70 mL, high-pressure sterilization at 121 ℃ for 15 min, cooling to room temperature, ultraviolet sterilization in an ultraclean bench for 30 min, and then inoculating into activated bacteria liquid of the Brevibacterium casei according to a 5% inoculation amount to make the Brevibacterium casei concentration 10 8 -10 10 cfu / mL, obtaining Brevibacterium casei fermentation liquid, the Brevibacterium casei concentration in the activated bacteria liquid of the Brevibacterium casei is 5×10 8 CFU / mL, the Brevibacterium casei fermentation liquid is cultured in a 37 ℃ incubator for 2-5 days, after the fermentation is completed, the fermentation liquid is dried at 60 ℃, crushed, 0.5 g of the crushed powder is weighed, 25 mL of 100% methanol is added, then ultrasonic treatment is carried out under the condition of a power of 300 W and a frequency of 40 kHz for 30 min, after being placed at room temperature, the lost methanol is made up with 100% methanol, filtration is carried out, and Astragalus membranaceus polysaccharide fermented by the Brevibacterium casei is obtained; After the Astragalus membranaceus polysaccharide fermented by the Subtileye cheese Kocuria is 500 g, it is dissolved in 80% ethanol at a ratio of 1 g:4 mL, heated to reflux for 2 h, repeated 2 times, dried in the shade, and then dissolved in 80% ethanol at a ratio of 1 g:10 mL, respectively heated to reflux for 2 h, 2 h, 2 h and 1 h, and then the four refluxed liquids are combined and concentrated, anhydrous ethanol is added to make the ethanol volume concentration 80%, and centrifuged at 3500xg for 8 min, the precipitate is collected, redissolved in pure water, concentrated, dialyzed, and freeze-dried to obtain the Astragalus membranaceus fermented polysaccharide.

2. The method of claim 1, wherein the preparation of the fermented polysaccharide of Astragalus membranaceus is characterized by, The Kocuria subtilis is derived from the National Culture Collection Center CICC, and the preservation number is 20990.

3. The application of the Astragalus fermented polysaccharide prepared by the method of claim 1 in the medicine for intervening premenstrual syndrome, characterized in that, The Astragalus membranaceus fermented polysaccharide is used as an active ingredient in a premenstrual syndrome intervention drug.

4. Use according to claim 3, characterized in that, The premenstrual syndrome intervention drug is a liquid preparation, a solid preparation or a semi-solid preparation.

5. Use according to claim 3, characterized in that, The premenstrual syndrome intervention drug is an oral liquid, a capsule, a tablet or a granule.

6. Use according to claim 5, characterized in that, The premenstrual syndrome intervention drug contains 200 mg of Astragalus membranaceus fermented polysaccharide per 5 g of heavy granule.

7. Use according to claim 5, characterized in that, The concentration of Astragalus membranaceus fermented polysaccharide in the premenstrual syndrome intervention drug oral liquid is 20 mg / mL.

8. The use of the Astragalus fermented polysaccharide prepared according to claim 1 in a premenstrual syndrome intervention food, characterized in that, The Astragalus membranaceus fermented polysaccharide is used as an active ingredient in a premenstrual syndrome intervention food.

9. Use according to claim 8, characterized in that, The premenstrual syndrome intervention food includes functional food and health food.

10. Use according to claim 8, characterized in that, The functional food is a fermented beverage or a tablet candy; the concentration of Astragalus membranaceus fermented polysaccharide in the fermented beverage is 10 mg / mL; and the tablet candy contains 300 mg of Astragalus membranaceus fermented polysaccharide per 0.5 g.

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