Fermented gastrodia elata polysaccharide, and preparation method and application thereof

Abstract

The application discloses a preparation method and application of fermented Gastrodia elata polysaccharide, and belongs to the field of traditional Chinese medicine microbial transformation. The fermentation is carried out under the conditions that a water extract of Gastrodia elata is used as a substrate, Gucailin is inoculated, the addition amount is 5% to 10%, the inoculation amount is 5% to 10%, the pH is 4 to 6, and the temperature is 25 DEG C to 28 DEG C, and the fermentation time is 5 to 7 days, and then the fermented Gastrodia elata polysaccharide is obtained through alcohol precipitation, deproteinization, pigment removal, dialysis and freeze drying. The total polysaccharide content of the product is significantly improved compared with that of unfermented product, the sleep latency of an anxiety-like insomnia mouse can be shortened, and the sleep time can be prolonged, the product can play a sedative and hypnotic role by regulating the expression of 5-HT, GABA and HTR-1A and GABAA mRNA, and can be used for preparing related medicines or health products.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine microbial transformation technology, specifically relating to a method for preparing fermented Gastrodia elata polysaccharide, and the application of the fermented Gastrodia elata polysaccharide prepared by this method in the preparation of sedative-hypnotic drugs or health products. Background Technology

[0002] Gastrodia elata Bl., the dried tuber of an orchid, is a traditional and precious Chinese medicinal herb, rich in various active ingredients such as gastrodin, gastrodia polysaccharides, amino acids, and trace elements. Modern pharmacological studies have shown that Gastrodia elata possesses various pharmacological activities, including sedation and hypnosis, antioxidant effects, immune enhancement, and memory improvement. Among these, gastrodia polysaccharides, as one of the main active ingredients, have been proven to have biological activities such as improving sleep and delaying aging. However, natural gastrodia polysaccharides mostly exist in large molecular form, resulting in low bioavailability and difficulty in fully realizing their activity.

[0003] Microbial transformation technology for traditional Chinese medicine (TCM) utilizes extracellular enzymes (such as hydrolases and oxidoreductases) secreted by medicinal fungi or bacteria to perform targeted hydrolysis, structural modification, and activity enhancement of macromolecular components in TCM under mild and controlled fermentation conditions. This technology can degrade poorly absorbed and low-activity macromolecules in TCM into easily absorbed and highly active small molecules, while optimizing monosaccharide composition and altering glycosidic bond types, thereby significantly improving bioavailability and pharmacological activity. Currently, microbial fermentation technology has been widely applied in the deep processing of TCM materials such as Ganoderma lucidum and ginseng.

[0004] In the field of sleep aids and natural medicines, existing products generally suffer from drawbacks such as easy inactivation of active ingredients and low bioavailability of functional substances. When developing sleep aids using Gastrodia elata as a raw material, existing technologies mostly focus on directly extracting gastrodin or crude polysaccharides, or preparing Gastrodia elata oral liquid through fermentation with probiotics (such as lactic acid bacteria). These technologies emphasize utilizing the probiotic function of probiotics and the synergistic effect of gastrodin, but have not conducted systematic research on the changes in the content and structural modification of Gastrodia elata polysaccharides during the fermentation process of medicinal fungi, as well as the mechanism by which their sedative-hypnotic activity is enhanced.

[0005] For example, CN113308403A discloses a strain of *Lactobacillus plantarum* and a method for preparing its fermented *Gastrodia elata* oral liquid. This method involves enzymatic hydrolysis of *Gastrodia elata* with a compound enzyme followed by inoculation with *Lactobacillus plantarum* for fermentation. The resulting oral liquid is said to improve sleep and regulate intestinal flora. However, this technology uses *Lactobacillus plantarum* as the fermentation strain, primarily utilizing the probiotic function of lactic acid bacteria and the synergistic effect of gastrodin. The product is an oral liquid containing live bacteria, but it does not involve the liquid fermentation of *Gastrodia elata* water extract by medicinal fungi, nor does it investigate the mechanism by which the polysaccharide content of *Gastrodia elata* increases and its sedative-hypnotic activity is enhanced during fermentation.

[0006] Therefore, there are currently no reports on using medicinal fungi to ferment the aqueous extract of Gastrodia elata, thereby increasing the polysaccharide content through biotransformation and systematically verifying its sedative-hypnotic activity and mechanism of action. How to screen suitable medicinal fungal strains, optimize the fermentation process to increase the polysaccharide content of Gastrodia elata, and elucidate the molecular mechanism by which fermented Gastrodia elata polysaccharides exert their sedative-hypnotic activity has become a pressing technical problem to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing fermented Gastrodia elata polysaccharides, as well as the fermented Gastrodia elata polysaccharides prepared by this method. This method involves screening a specific medicinal fungal strain—Ganoderma lucidum CGMCC No. 4873—to perform directed liquid fermentation of Gastrodia elata water extract, significantly increasing the content of Gastrodia elata polysaccharides and enhancing its sedative-hypnotic activity, thus providing a novel technological option for the development of natural sedative-hypnotic drugs or health products.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing fermented Gastrodia elata polysaccharide includes the following steps: S1. Mix the aqueous extract of Gastrodia elata with liquid culture medium to obtain the fermentation substrate; S2. Inoculate the fermentation substrate with Ganoderma lucidum and carry out liquid fermentation for 5 to 7 days at a fermentation temperature of 25℃~28℃ and an initial pH of 4~6 to obtain fermentation broth. S3. The fermentation broth is subjected to termination of fermentation, centrifugation to obtain supernatant, alcohol precipitation, protein removal, pigment removal, dialysis and freeze-drying to obtain fermented Gastrodia elata polysaccharide.

[0009] According to a preferred embodiment of the present invention, in step S2, the amount of Gastrodia elata water extract added to the fermentation substrate is 5% to 10%, and the amount of Ganoderma lucidum inoculated is 5% to 10%.

[0010] According to a further preferred embodiment of the present invention, the amount of Gastrodia elata water extract added is 7.5%, the amount of Ganoderma lucidum inoculated is 7.5%, the initial pH is 5.0, the fermentation temperature is 26°C, and the fermentation time is 6 days.

[0011] According to a preferred embodiment of the present invention, in step S1, the liquid culture medium comprises: 200 g / L potato, 20 g / L glucose, 1.5 g / L magnesium sulfate, 3 g / L potassium dihydrogen phosphate and 0.2 g / L vitamin B1.

[0012] According to a preferred embodiment of the present invention, in step S1, the gastrodia elata water extract is prepared by the following method: gastrodia elata powder is added to water at a material-to-liquid ratio of 1:20, extracted at 90°C for 1 hour, the supernatant is collected by centrifugation, the extraction is repeated once, the supernatants are combined and concentrated.

[0013] According to a preferred embodiment of the present invention, in step S3, the fermentation is terminated by boiling at 100°C for 10-15 minutes; the deproteinization is carried out by enzymatic hydrolysis with 1% papain at 55°C for 4 hours; the pigment removal is carried out by macroporous adsorption resin AB-8; and the alcohol precipitation is carried out by adding 4 times the volume of anhydrous ethanol and standing overnight at 4°C.

[0014] The present invention also provides a fermented Gastrodia elata polysaccharide prepared by the above preparation method.

[0015] According to a preferred embodiment of the present invention, the total polysaccharide content of the fermented Gastrodia elata polysaccharide is 11.0 mg / mL to 12.5 mg / mL.

[0016] According to a preferred embodiment of the present invention, the fermented Gastrodia elata polysaccharide has sedative-hypnotic activity.

[0017] The present invention also provides the use of the above-mentioned fermented Gastrodia elata polysaccharide in the preparation of drugs or health products that exert sedative and hypnotic effects by regulating the levels of 5-hydroxytryptamine, γ-aminobutyric acid and the expression of HTR-1A and GABAA mRNA.

[0018] The present invention has the following advantages over the prior art: 1. For the first time, Ganoderma lucidum extract from Gastrodia elata was subjected to liquid fermentation, which significantly increased the polysaccharide content of Gastrodia elata. Under optimized conditions (7.5% addition, 7.5% inoculum, initial pH 5.0, fermentation at 26℃ for 6 days), the total polysaccharide content in the fermentation broth reached 11.89±0.60 mg / mL, which was significantly higher than that of the unfermented Gastrodia elata extract (8.11±0.12 mg / mL), with a polysaccharide increase rate of 45.80%.

[0019] 2. The fermented Gastrodia elata polysaccharide (FGEP) prepared in this invention exhibits significantly superior sedative-hypnotic activity compared to unfermented Gastrodia elata polysaccharide (GEP). In an anxiety-like insomnia mouse model, the high-dose FGEP (104 mg / kg) group had a sleep latency of 240.7 ± 19.4 s and a sleep duration of 1586.0 ± 294.0 s, which was superior to the GEP group (latency 287.9 ​​± 35.6 s, sleep duration 1355.0 ± 255.8 s) and closer to the levels of the positive control drug estazolam group and the normal control group.

[0020] 3. This invention elucidates for the first time the molecular mechanism by which fermented Gastrodia elata polysaccharides exert sedative-hypnotic activity. ELISA results showed that the serum 5-HT content in the high-dose FGEP group was 55.1±2.6 pg / mL and the GABA content was 60.8±4.4 pg / mL, significantly higher than the model group (5-HT: 13.6±4.5 pg / mL; GABA: 30.6±3.6 pg / mL), and close to the normal control group (5-HT: 57.6±4.9 pg / mL; GABA: 61.2±5.2 pg / mL). RT-qPCR results showed that the high-dose FGEP group significantly downregulated HTR-1A mRNA expression (1.16±0.05) and upregulated GABAAmRNA expression (1.60±0.48) in the brain tissue of anxiety-like insomnia mice, thereby exerting a sedative-hypnotic effect by regulating the 5-HT and GABA neurotransmitter systems.

[0021] 4. This invention employs liquid fermentation technology using medicinal fungi, with mild and controllable process conditions, suitable for industrial production, providing a technical basis for the development of novel natural sedative-hypnotic drugs or health products. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 The graph shows the screening results of polysaccharide enhancement rate after fermentation of Gastrodia elata water extract by different bacterial strains.

[0024] Figure 2 The graph shows the effect of the amount of Gastrodia elata water extract added on the polysaccharide enhancement rate.

[0025] Figure 3 The effect of Ganoderma lucidum inoculation amount on the polysaccharide enhancement rate.

[0026] Figure 4 The graph shows the effect of fermentation time on the polysaccharide enhancement rate.

[0027] Figure 5 This is a graph showing the effect of initial pH on the polysaccharide enhancement rate.

[0028] Figure 6 This is a comparison chart of the total polysaccharide content of Gastrodia elata water extract before and after fermentation with Rongbao Ganoderma lucidum.

[0029] Figure 7The graph shows the results of the sodium pentobarbital-induced sleep experiment in each group of mice, where A represents sleep latency and B represents sleep duration.

[0030] Figure 8 The graph shows the results of detecting 5-HT and GABA levels in the serum of mice in each group.

[0031] Figure 9 The figure shows the results of detecting the relative expression levels of HTR-1A and GABAA mRNA in the brain tissue of mice in each group. Detailed Implementation

[0032] The following description is based on specific embodiments.

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0034] In this invention, unless otherwise specified, all raw materials, reagents and instruments are commercially available or commonly used in the industry. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0035] In the following examples, Rongbao Ganoderma lucidum No. 1 was selected. All experimental data were statistically analyzed using SPSS 26.0 software. One-way ANOVA was used for comparisons among multiple groups, and LSD-t test or Dunnett's T3 test was used for pairwise comparisons between groups. Experimental data are expressed as mean ± standard deviation (SD). () indicates that P < 0.05 is considered statistically significant.

[0036] Example 1

[0037] This embodiment describes the screening of strains for fermenting Gastrodia elata polysaccharides.

[0038] (1) Pretreatment of Gastrodia elata Gastrodia elata is sourced from Guizhou Dafang Biotechnology Co., Ltd. After being washed, steamed, dried at 60℃, ultra-finely pulverized, and passed through a 100-mesh sieve, it is ready for use.

[0039] (2) Preparation of Gastrodia elata water extract Take 40 g of Gastrodia elata powder, add 800 mL of water (solid-to-liquid ratio 1:20), and extract at 90℃ for 1 h. Then centrifuge at 8000 r / min for 15 min and collect the supernatant. Repeat the above operation on the filter residue, combine the two supernatants, and concentrate by rotary evaporation to 200 mL to obtain the water extract of Gastrodia elata.

[0040] (3) Preparation of culture medium (a) Slant culture medium: potato 200 g / L, glucose 20 g / L, magnesium sulfate 1.5 g / L, potassium dihydrogen phosphate 3 g / L, agar 15 g / L, vitamin B1 0.2 g / L, pH natural. Sterilize in an autoclave (121℃, 30 min).

[0041] (b) Liquid seed culture medium: potato 200 g / L, glucose 20 g / L, magnesium sulfate 1.5 g / L, potassium dihydrogen phosphate 3 g / L, vitamin B1 0.2 g / L, pH natural. Sterilize in an autoclave (121℃, 30 min).

[0042] (c) Fermented Gastrodia elata liquid culture medium: potato 200 g / L, glucose 20 g / L, magnesium sulfate 1.5 g / L, potassium dihydrogen phosphate 3 g / L, vitamin B1 0.2 g / L, Gastrodia elata water extract 5%, pH natural. Sterilize in an autoclave (121℃, 30 min).

[0043] (4) Cultivation methods (a) Slant culture: Take about the size of a soybean, take the mycelium of Huizhou Ganoderma lucidum, Rongbao Ganoderma lucidum (CGMCC No.4873), purple Ganoderma lucidum, Pengzhou Ganoderma lucidum and snow Ganoderma lucidum mother culture, inoculate it in the middle of the slant, and incubate at 27℃ for 6 days.

[0044] (b) Liquid seed culture: Take about the size of a broad bean from each Ganoderma lucidum mycelium cultured on an slant and inoculate it into liquid seed culture medium. Incubate at 27°C and 150 r / min on a shaker for 6 days. A total of 3 generations were inoculated.

[0045] (c) Fermented Gastrodia elata liquid culture: Take each activated Ganoderma lucidum strain and add it to the fermented Gastrodia elata liquid culture medium at 5% of the inoculum amount. Culture on a shaker at 27℃ and 150 r / min for 6 days.

[0046] (5) Determination of total polysaccharide content—phenol-sulfuric acid method (a) Preparation of standard curve: A glucose standard solution with a concentration of 0.1 mg / mL was accurately prepared. 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution were accurately transferred into 25 mL graduated test tubes, and the volume was brought up to 2 mL with distilled water. Then, 1 mL of 5% phenol solution was added to each tube and mixed thoroughly. 5 mL of concentrated sulfuric acid was then quickly added, mixed thoroughly, and heated in a boiling water bath for 2 min. After cooling to room temperature, the absorbance of each tube was measured at 490 nm, and the standard curve was plotted.

[0047] (b) Sample preparation: Take 100 mL of fermentation broth and boil it in a water bath for 10 min to terminate fermentation. After cooling to room temperature, centrifuge at 6000 r / min for 30 min. Take 10 mL of the supernatant and add 4 times the volume of anhydrous ethanol. Place in a refrigerator at 4℃ and let stand overnight. Centrifuge again, take the precipitate, dissolve it in hot water, and make up to 100 mL. The procedure is the same as in (a). Repeat the experiment three times.

[0048] Preliminary experiments showed that the extracellular polysaccharide content of Ganoderma lucidum was very low, only 0.15 mg / mL, which is significantly lower than that of Gastrodia elata polysaccharide and can be ignored. Therefore, the formula for calculating the polysaccharide enhancement rate is as follows: Polysaccharide increase rate (%) = Polysaccharide content of unfermented Gastrodia elata - Polysaccharide content of fermented Gastrodia elata × 100% The results of calculating the polysaccharide enhancement rate of each strain of fermented Gastrodia elata water extract are shown below. Figure 1 See Table 1.

[0049] Table 1. Polysaccharide enhancement rate of Gastrodia elata water extract after fermentation by different strains

[0050] Depend on Figure 1 As shown in Table 1, the total polysaccharide content of Gastrodia elata water extract decreased after fermentation with Ganoderma lucidum from Pengzhou, Ganoderma sinense, and Ganoderma lucidum; however, the total polysaccharide content increased after fermentation with Ganoderma lucidum from Huizhou and Ganoderma lucidum from Rongbao, by 13.85% and 39.47%, respectively. Therefore, Ganoderma lucidum from Rongbao was selected for further optimization of the fermentation process.

[0051] Example 2

[0052] Gastrodia elata aqueous extract and culture medium were prepared according to the method in Example 1. Activated Ganoderma lucidum strain was taken, and at inoculation rates of 5.0% and initial pH of 5.0, 2.5%, 5%, 7.5%, 10%, and 12.5% ​​of the Gastrodia elata aqueous extract were added to 200 mL of Gastrodia elata fermentation liquid culture medium, respectively. Fermentation was carried out at 26°C for 6 days. The polysaccharide enhancement rate was determined according to the phenol-sulfuric acid method described in Example 1. The experimental results are shown in [Figure 1]. Figure 2 And Table 2.

[0053] Table 2. Polysaccharide enhancement rate with different amounts of Gastrodia elata water extract.

[0054] Depend on Figure 2 As shown in Table 2, the polysaccharide increase rate was the highest when the amount of Tianma water extract added was 7.5%, which was 30.00±2.32%.

[0055] Example 3

[0056] Aqueous extracts of Gastrodia elata and culture medium were prepared according to the method in Example 1. The amount of aqueous extract added was 7.5%, and the initial pH was 5.0. Ganoderma lucidum was inoculated at concentrations of 2.5%, 5.0%, 7.5%, 10.0%, and 12.5% ​​(v / v) into 200 mL of Gastrodia elata fermentation liquid culture medium, and fermented at 26°C for 6 days. The polysaccharide enhancement rate was determined according to the phenol-sulfuric acid method described in Example 1. The experimental results are shown in [Figure 1]. Figure 3 And Table 3.

[0057] Table 3. Effects of different inoculum amounts on polysaccharide enhancement rate

[0058] Depend on Figure 3 As shown in Table 3, the polysaccharide increase rate was the highest when the inoculum content was 7.5%, which was 47.51 ± 5.33%.

[0059] Example 4

[0060] Aqueous extracts of Gastrodia elata and culture medium were prepared according to the method in Example 1. The amount of Gastrodia elata aqueous extract added was 7.5%, the initial pH was 5.0, and the inoculum size was 7.5%. Fermentation was carried out at 26℃ for 2, 4, 6, 8, and 10 days, respectively. The polysaccharide enhancement rate was determined according to the phenol-sulfuric acid method described in Example 1, and the experimental results are shown in [Figure 1]. Figure 4 And Table 4.

[0061] Table 4. Effects of different fermentation times on polysaccharide enhancement rate

[0062] Depend on Figure 4 As shown in Table 4, the polysaccharide increase rate was greatest on day 6 of fermentation, which was 45.80 ± 8.44%.

[0063] Example 5

[0064] Aqueous extracts and culture media of Gastrodia elata were prepared according to the method in Example 1. The amount of aqueous extract added was 7.5%, and the inoculum size was 5.0%. The initial pH values ​​were adjusted to 1, 3, 5, 7, and 9, respectively. The culture media were inoculated into 200 mL of liquid culture medium for Gastrodia elata fermentation and fermented at 26°C for 6 days. The polysaccharide enhancement rate was determined according to the phenol-sulfuric acid method described in Example 1. The experimental results are shown in [Figure 1]. Figure 5 See Table 5.

[0065] Table 5. Polysaccharide enhancement rate at different initial pH levels

[0066] Depend on Figure 5 As shown in Table 5, the polysaccharide increase rate was the highest when the initial pH was 5, which was 18.20 ± 4.81%.

[0067] Example 6

[0068] This embodiment describes the determination of total polysaccharide content before and after fermentation and the preparation of fermented Gastrodia elata polysaccharide.

[0069] Based on the optimization results of Examples 2-5, the optimal fermentation conditions were determined to be: 7.5% addition of Gastrodia elata water extract, 7.5% inoculum, initial pH value of 5.0, and fermentation at 26℃ for 6 days.

[0070] The total polysaccharide content of the fermentation broth and the aqueous extract of Gastrodia elata was determined according to the phenol-sulfuric acid method described in Example 1. The results are shown below. Figure 6 See Table 6.

[0071] Table 6 Total polysaccharide content of Gastrodia elata aqueous extract before and after fermentation

[0072] Depend on Figure 6 As shown in Table 6, the total polysaccharide content in the fermentation broth is much higher than that in the water extract of Gastrodia elata.

[0073] Preparation of fermented Gastrodia elata polysaccharide (FGEP) and Gastrodia elata polysaccharide (GEP): After fermenting the aqueous extract of Gastrodia elata under the above-mentioned optimal fermentation conditions, all fermentation broth was collected. Fermentation was terminated by boiling at 100℃ for 15 min, cooled to room temperature, and centrifuged at 8000 r / min for 20 min. The supernatant was collected. The fermentation supernatant and the aqueous extract of Gastrodia elata were concentrated to a certain volume by rotary evaporation, and then 1% papain was added. The mixture was enzymatically hydrolyzed at 55℃ for 4 h to remove protein. Then, the samples were purified by removing pigments using macroporous adsorption resin AB-8. After concentration to a certain volume, 4 volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4℃. Both groups of samples were centrifuged at 6000 r / min for 15 min, the precipitate was collected, reconstituted with water, dialyzed, and lyophilized to prepare fermented Gastrodia elata polysaccharide (FGEP) and unfermented Gastrodia elata polysaccharide (GEP), respectively.

[0074] Example 7

[0075] This embodiment describes an in vivo anti-anxiety-like insomnia experiment using fermented Gastrodia elata polysaccharide in mice.

[0076] Laboratory animals: Male ICR mice (SPF grade, body weight 16±2 g, 3-4 weeks old) were provided by Chengdu Dashuo Experimental Animal Co., Ltd., and the experimental animal production license number was SCXK (Sichuan) 2025-0030. The experimental process was strictly carried out in accordance with the "Regulations on the Administration of Experimental Animals of the People's Republic of China" and the "3R" principle, and the ethics number was: SIIA20250302. All experimental mice were raised under the conditions of a temperature of 22°C and a humidity of 40% - 70%, with 12 hours of light per day, and free access to feed and water.

[0077] Establishment of an anxiety-like insomnia model: The chronic restraint experiment was combined with intraperitoneal injection of 450 mg / kg p-chlorophenylalanine (PCPA) to induce anxiety-like insomnia in mice. PCPA induces insomnia in mice by inhibiting the synthesis of serotonin (5-HT), and the injection dose was 450 mg / (kg·d). 5% Tween-80 was used as the solvent. The normal group was injected with an equal amount of sterile solvent. Mice were subjected to chronic restraint stress in a 50 mL centrifuge tube, and ventilation holes with a diameter of about 5 mm were punched in the tip and the lid. After the restraint ended, the mice were gently taken out, their hair was dried, and then they were put back into the mouse cage.

[0078] Animal grouping and dose design: Seventy male ICR mice were selected and evenly divided into 7 groups with 10 mice in each group according to the principle of random allocation. The groups were set as follows: normal control group (Control), anxiety-like insomnia model group (Model), gastrodia elata polysaccharide group (GEP), estazolam positive drug group (Estazolam), low-dose fermented gastrodia elata polysaccharide group (FGEP-L), medium-dose fermented gastrodia elata polysaccharide group (FGEP-M), and high-dose fermented gastrodia elata polysaccharide group (FGEP-H).

[0079] In view of the fact that the extracellular crude polysaccharide of Rongbao Ganoderma lucidum was only 0.15 mg / mL as determined in the previous stage, which was quite different from the contents of gastrodia elata polysaccharide and fermented gastrodia elata polysaccharide, following the "3R" principle, a control group of Rongbao Ganoderma lucidum was not set up in this experiment.

[0080] According to the "Chinese Pharmacopoeia" (2025 edition), the recommended dosage of gastrodia elata药材 is 3 - 10 g. In this experiment, 3 g of gastrodia elata was used as the administration dose, and 400 mg of gastrodia elata polysaccharide could be prepared. Then, according to the "Methodology of Traditional Chinese Medicine Pharmacological Research", with an adult human body weight of 70 kg and a conversion coefficient between humans and mice of 9.1, the administration doses of mice in the FGEP-M and GEP groups were finally determined to be 52 mg / kg, FGEP-L was 26 mg / kg, and FGEP-H was 104 mg / kg. The administration dose of mice in the estazolam group was 2 mg / kg, and the normal group and the model group were given the same volume of normal saline.

[0081] Animal experiment design: The experiment began after 7 days of acclimatization. From day 1 to day 3, p-chlorophenylalanine solution was injected into the model group and each drug-treated group for the first time to induce a decrease in 5-HT levels. From day 4 to day 9, the model group and each drug-treated group were subjected to 4 hours of chronic restraint stress daily. From day 20 to day 22, p-chlorophenylalanine solution was injected again for 3 consecutive days. Mice were administered the drug by gavage for 23 days (days 4 to 26). In addition, body weight was recorded every other day. On day 26, a sodium pentobarbital-induced sleep experiment was conducted. On day 27, serum, brain, liver, spleen, kidney, and colon samples were collected and stored at -80°C for later use.

[0082] Example 8

[0083] This embodiment describes a sodium pentobarbital-induced sleep experiment.

[0084] Two hours after the last gavage, the experiment was conducted. Each group of experimental mice was intraperitoneally injected with sodium pentobarbital solution at a dose of 50 mg / kg, and two key sleep-related indicators were recorded: the sleep latency from the end of administration to the disappearance of the righting reflex, and the total sleep duration from the disappearance of the righting reflex to its recovery.

[0085] The experimental results are shown in Figure 7 See Table 7.

[0086] Table 7. Results of sodium pentobarbital synergistic with sleep

[0087] Depend on Figure 7 As shown in Table 7, compared with the normal group, the model group had a prolonged sleep latency and a shortened total sleep time (P<0.001, P<0.0001), indicating successful model establishment. Compared with the model group, the sleep latency of each treatment group was shortened to varying degrees, and the total sleep time was prolonged to varying degrees. Among them, the high-dose fermented Gastrodia elata group (FGEP-H) had the most significant therapeutic effect (P<0.0001), with both sleep latency and sleep time being superior to the GEP group.

[0088] Example 9: ELISA detection of serum 5-HT and GABA levels Serum levels of 5-HT and γ-aminobutyric acid (GABA) were measured according to the method described in the kit instructions from Shanghai Yuanju Biotechnology Center. Both GABA and 5-HT are inhibitory neurotransmitters that regulate sleep.

[0089] The experimental results are shown in Figure 8 See Table 8.

[0090] Table 8. 5-HT and GABA content in mice of each group

[0091] Depend on Figure 8 As shown in Table 8, compared with the normal group, the GABA and 5-HT levels in the model group were significantly decreased (P<0.0001); compared with the model group, the GABA and 5-HT levels in the other treatment groups increased to varying degrees. Among them, the FGEP-H group was closer to the normal group, indicating that high doses of fermented Gastrodia elata polysaccharide can significantly restore the 5-HT and GABA levels in anxiety-like insomnia mice.

[0092] Example 10

[0093] This example describes the detection of HTR-1A and GABAA mRNA expression in brain tissue by RT-qPCR.

[0094] Total RNA was extracted from mouse brain tissue using a total RNA kit. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit and stored at -20°C. The relative expression of HTR-1A and GABAA mRNA in mouse brain tissue was detected by real-time quantitative PCR (qRT-PCR) using GAPDH as a reference gene. Primer information is shown in Table 9.

[0095] Table 9 Primer Sequences

[0096] qRT-PCR was performed according to the instructions of the Stormstar Sybr Green qPCR Master Mix kit, using a 20 μL system. The prepared system was added to 96-well plates, with 3 wells per sample. After addition, the plates were sealed, centrifuged at 2500 rpm for 2 min, and then loaded onto the qRT-PCR machine. The qRT-PCR program was as follows: Incubation: 95℃ for 2 min; Cycling: 95℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s, 40 cycles; Melting: 95℃ for 1 min, 55℃ for 1 min, 98℃ for 10 s.

[0097] 5-HT binds to HTR-1A receptors in the brain and transmits inhibitory neural signals, but decreased transmission levels may lead to mental illnesses such as anxiety and depression; while GABA binds to GABAA receptors, producing neuromodulation effects, promoting relaxation, relieving anxiety, and promoting sleep. Experimental results are shown below. Figure 9 See Table 10.

[0098] Table 10. HTR-1A and GABAA mRNA expression levels in each group of mice

[0099] Depend on Figure 9As shown in Table 10, compared with the normal group, the expression level of HTR-1A mRNA in the model group was significantly increased (P<0.05), and the expression level of GABAA mRNA was significantly decreased (P<0.001). Compared with the model group, the other treatment groups showed varying degrees of downregulation of HTR-1A mRNA (P<0.01, P<0.001) and upregulation of GABAA mRNA expression (P<0.05, P<0.01). Among them, the FGEP-H group showed the most significant effect, indicating that fermented Gastrodia elata polysaccharide can restore the balance of the 5-HT and GABA neurotransmitter systems by regulating the expression of HTR-1A and GABAA mRNA, thereby exerting a sedative-hypnotic effect.

[0100] In summary, this invention, through screening specific strains of Ganoderma lucidum and optimizing fermentation conditions, significantly improved the total polysaccharide content of fermented Gastrodia elata polysaccharide (FGEP). Furthermore, it exhibited superior sedative-hypnotic activity compared to unfermented Gastrodia elata polysaccharide (GEP) in an anxiety-like insomnia mouse model. This mechanism is closely related to the regulation of 5-HT and GABA neurotransmitter levels and the expression of HTR-1A and GABAAmRNA. This invention provides experimental evidence and a technical foundation for the development of novel natural sedative-hypnotic drugs or health products.

[0101] In the description of this invention, it should be understood that "-" and "~" represent a range between two values, and this range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0102] In the description of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist simultaneously, and B exists alone.

[0103] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0104] In the description of this invention, the terms “about” or “approximately” are used to express approximate values ​​or ranges, allowing for a certain degree of error to ensure the flexibility and practicality of the description, while remaining within an acceptable range of error, with the maximum error not exceeding 10% of the corresponding value or range.

[0105] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing fermented Gastrodia elata polysaccharide, characterized in that, Includes the following steps: S1. Mix the aqueous extract of Gastrodia elata with liquid culture medium to obtain the fermentation substrate; S2. Inoculate the fermentation substrate with Ganoderma lucidum and carry out liquid fermentation for 5 to 7 days at a fermentation temperature of 25℃~28℃ and an initial pH of 4~6 to obtain fermentation broth. S3. The fermentation broth is subjected to termination of fermentation, centrifugation to obtain supernatant, alcohol precipitation, protein removal, pigment removal, dialysis and freeze-drying to obtain fermented Gastrodia elata polysaccharide.

2. The preparation method according to claim 1, characterized in that, In step S2, the amount of Gastrodia elata water extract added to the fermentation substrate is 5%~10%, and the amount of Ganoderma lucidum inoculated is 5%~10%.

3. The preparation method according to claim 2, characterized in that, The amount of Gastrodia elata water extract added was 7.5%, the amount of Ganoderma lucidum inoculated was 7.5%, the initial pH was 5.0, the fermentation temperature was 26℃, and the fermentation time was 6 days.

4. The preparation method according to claim 1, characterized in that, In step S1, the liquid culture medium comprises: 200 g / L potato, 20 g / L glucose, 1.5 g / L magnesium sulfate, 3 g / L potassium dihydrogen phosphate and 0.2 g / L vitamin B1.

5. The preparation method according to claim 1, characterized in that, In step S1, the gastrodia elata water extract is prepared by the following method: gastrodia elata powder is added to water at a material-to-liquid ratio of 1:20, extracted at 90°C for 1 hour, the supernatant is collected by centrifugation, the extraction is repeated once, the supernatants are combined and concentrated.

6. The preparation method according to claim 1, characterized in that, In step S3, the fermentation is terminated by boiling at 100°C for 10-15 minutes; the deproteinization is carried out by enzymatic hydrolysis with 1% papain at 55°C for 4 hours; the pigment removal is carried out by macroporous adsorption resin AB-8; and the alcohol precipitation is carried out by adding 4 times the volume of anhydrous ethanol and standing overnight at 4°C.

7. A fermented Gastrodia elata polysaccharide, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. The fermented Gastrodia elata polysaccharide according to claim 7, characterized in that, The total polysaccharide content of the fermented Gastrodia elata polysaccharide is 11.0 mg / mL to 12.5 mg / mL.

9. The fermented Gastrodia elata polysaccharide according to claim 7, characterized in that, The fermented Gastrodia elata polysaccharide has sedative and hypnotic activity.

10. Use of the fermented Gastrodia elata polysaccharide according to any one of claims 7 to 9 in the preparation of a medicament or health product that exerts a sedative-hypnotic effect by regulating the levels of 5-hydroxytryptamine, γ-aminobutyric acid and the expression of HTR-1A and GABAA mRNA.

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