Method for fermenting gastrodia elata through Armillaria Galaululifera, fermentation product of gastrodia elata and application of fermentation product

Abstract

The invention discloses a method for fermenting gastrodia elata by Armillaria Galaululifera and a fermentation product and application thereof, and the method comprises the following steps: preparing gastrodia elata powder; preparing a PDB culture medium; the preparation method comprises the following steps: firstly, inoculating a PDA plate culture medium with a Garubus armillaria strain for activation culture, inoculating the strain into a PDB culture medium after hyphae grow to bacterial colonies with the diameter of 1cm, and performing shaking culture on a shaking table to obtain a strain suspension; mixing gastrodia elata powder, a PDB solution and water to obtain a gastrodia elata mixed culture medium; and inoculating the strain suspension into the gastrodia elata mixed culture medium, and carrying out shake culture to obtain the gastrodia elata fermentation product. The gastrodia elata leavening obtained by the invention is obtained by fermenting Armillaria Galauracil, has the characteristic of increasing the content of gastrodin, and shows remarkable effects of resisting depression and improving sleep under the condition that the dosage is 200-800mg / kg.

Description

Technical Field

[0001] The present invention relates to a Gastrodia elata fermentation method, in particular to a method for fermenting Gastrodia elata with Armillaria gallica, and its fermentation products and applications. Background Art

[0003] With the development of modern science and technology, the processing of traditional Chinese medicine by microbial fermentation has gradually changed from traditional natural fermentation to pure strain fermentation. The effects of microbial fermentation of traditional Chinese medicine are mainly reflected in the change of the active ingredients and efficacy of traditional Chinese medicine by microorganisms ("double-effect" modification) and the influence of traditional Chinese medicine components on the secondary metabolism of microorganisms. Based on its advantages such as short growth cycle, mild conditions, few by-products, and strong stereoselectivity, the research on modifying traditional Chinese medicine by microbial fermentation has attracted increasing attention. In recent years, in actual industrial production, the production of some novel, highly active, and health-beneficial foods or drugs through the fermentation of microorganisms has been widely applied.

[0004] Literature review shows that there are relevant reports on fermenting Gastrodia elata with microorganisms, but there are still deficiencies. For example, Cai Ni et al. (The influence of yeast and lactic acid bacteria as fermentation agents on the total phenol content and reducing ability of Gastrodia elata, Modern Food. 2020(20): 193-197+203.) fermented Gastrodia elata with Saccharomyces cerevisiae, Lactobacillus bulgaricus, and Streptococcus thermophilus as fermentation agents. The results showed that compared with the control group, the total phenol content decreased in the fermented Gastrodia elata group, and Saccharomyces cerevisiae had a greater impact on the decrease in total phenol content. Another example is Gao Ming et al. (An antidepressant-active enzymatically modified Gastrodia elata extract and its preparation and application, CN113666804A), who used a mixture of yeast: lactic acid bacteria: acetic acid bacteria = 1:1:1 as a fermentation agent for Gastrodia elata fermentation. Although its antidepressant activity increased compared with the Gastrodia elata powder group, there are disadvantages such as poor stability and difficult-to-control fermentation conditions. Therefore, continuously exploring new microbial species is one of the essential key links to promote the development of the Gastrodia elata industry.

[0005] Armillaria belongs to Basidiomycetes, Agaricales, and Fungi. It is a fungus that can be used as both medicine and food and is a symbiotic fungus of Gastrodia elata. Gastrodia elata is a non-autotrophic organism that has established a symbiotic relationship with Armillaria during long-term evolution. Armillaria is the bridge for Gastrodia elata to obtain nutrients, and Armillaria is required to accompany the cultivation of Gastrodia elata throughout its asexual reproduction stage. Modern research shows that the fruiting body of Armillaria and its fermentation products exhibit similar effects to Gastrodia elata. However, the active substances contained in the fermentation products formed by different Armillaria species fermenting Gastrodia elata are different, and there is no relevant research on fermenting Gastrodia elata with Armillaria gallica as a medium in the prior art. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a method for fermenting Gastrodia elata by Armillaria gallica, its fermentation product and application. The Gastrodia elata fermented product obtained in the present invention is obtained by fermentation with Armillaria gallica, has the characteristics of increasing the content of gastrodin and ensuring that the content of other active substances changes little, and shows significant antidepressant and sleep improvement effects under the condition of a dosage of 200-800 mg / kg.

[0007] The technical solution of the present invention: A method for fermenting Gastrodia elata by Armillaria gallica, comprising the following steps:

[0008] S1. Preparation of Gastrodia elata powder;

[0009] S2. Preparation of PDB solution;

[0010] S3. Cultivation of strains and preparation of strain suspension:

[0011] The Armillaria gallica strain is first inoculated into a PDA plate medium for activation culture. After the mycelium grows to a colony with a diameter of 1 cm, it is inoculated into a PDB medium and cultured with shaking on a shaker to obtain a strain suspension;

[0012] S4. Fermentation of Gastrodia elata:

[0013] S41. Take Gastrodia elata powder, PDB solution and water and mix them to obtain a Gastrodia elata mixed medium;

[0014] S42. Inoculate the strain suspension into the Gastrodia elata mixed medium and culture with shaking to obtain a Gastrodia elata fermented product.

[0015] In the aforementioned method for fermenting Gastrodia elata by Armillaria gallica, the preparation of the PDB medium in step S2 is specifically as follows: Cut the potatoes into pieces, boil them with water, then filter the potato pieces, and add granulated sugar to the filtrate and dissolve it evenly to obtain the PDB medium.

[0016] In the aforementioned method for fermenting Gastrodia elata by Armillaria gallica, in step S3, the temperature for activation culture is 20-30 °C, the culture time is 10-15 days; the temperature for shaking on the shaker is 20-26 °C, the shaking culture time on the shaker is 10-15 days; the rotation speed is 100-150 r / min.

[0017] In the aforementioned method for fermenting Gastrodia elata by Armillaria gallica, in step S41, the ratio of Gastrodia elata powder: PDB solution: water is 1 g: (5-15) mL: (10-20) mL.

[0018] In the aforementioned method for fermenting Gastrodia elata by Armillaria gallica, step S42 is specifically as follows: Inoculate the strain suspension into the Gastrodia elata mixed medium and culture with shaking at 20-32 °C for 8-14 days to obtain a Gastrodia elata fermented product.

[0019] The present invention also provides a Gastrodia elata ferment, which is obtained by the above fermentation method.

[0020] In the aforementioned Gastrodia elata ferment, the contents of gastrodin, p-hydroxybenzyl alcohol, parishin E, parishin B, parishin C, and parishin A are respectively above 2500, 750, 2800, 2000, 1250, and 100 μg / g.

[0021] The present invention also provides the application of the Gastrodia elata ferment in the preparation of antidepressant drugs or foods for improving neurotransmitter transmission, increasing the release of neurotrophic factors, inhibiting inflammatory factors, enhancing nerve remodeling and neurogenesis, or regulating intestinal microbiota disorders.

[0022] The present invention also provides the application of the Gastrodia elata ferment in the preparation of sleep-improving drugs or foods for shortening the sleep latency, prolonging the sleep time, reducing the contents of TNF-α, IL-6, and IL-1β, increasing the contents of GSH-Px and SOD, increasing the neurotransmitter content, and reducing the structural damage of hippocampal neurons.

[0023] In the aforementioned application, the dosage of the Gastrodia elata ferment is 200 - 800 mg / kg.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention ferments the strain of Armillaria gallica and Gastrodia elata under certain conditions to obtain a Gastrodia elata ferment. The contents of active ingredients in unfermented Gastrodia elata and different Gastrodia elata ferment samples are determined by HPLC method. It is known that the total content of 6 Gastrodia markers in the Gastrodia elata ferment after fermentation by Armillaria gallica can increase by about 28.85% compared with the unfermented Gastrodia elata extract. In particular, the content of GAS can increase by up to 3.8 times compared with that before fermentation, and the contents of the remaining Gastrodia markers are also relatively high, and the biological activity is significantly improved compared with that before fermentation.

[0026] In addition, the present invention also constructs a mouse depression model and an insomnia model by chronic unpredictable stress (CUMS) induction and PCPA induction respectively, and explores the improvement effect of the Gastrodia elata ferment on depressive and insomnia symptoms by combining aspects such as brain neurochemical substances, nerve generation pathways, and intestinal microbiota.

[0027] In a mouse depression model, through the analysis of active ingredients, the health status of hippocampal nerve cells, the regulation of protein pathways, and the comprehensive multi-omics analysis of maintaining intestinal homeostasis, the present invention demonstrated the complex mechanism by which Gastrodia elata fermentum effectively alleviates depression. Among them, the Gastrodia elata fermentum fermented by Armillaria gallica significantly increased the content of GAS, enhanced the motor ability and exploration desire of CUMS mice, and alleviated the depressive-like behaviors of CUMS mice, etc.; by improving the transmission of neurotransmitters in the mouse brain, increasing the release of neurotrophic factors, and regulating indicators such as reducing the inflammatory factors in the brain and serum, it effectively alleviated the depressive symptoms of mice; the Armillaria gallica Gastrodia elata fermentum could also regulate the BDNF / NMDA / CREB protein pathway and PSD-95 / SYN1 protein, enhancing nerve remodeling and neurogenesis; regulating the disorder and correlation of the intestinal microbiota in depression, increasing the content of beneficial bacteria in the intestinal microbiota, reducing the content of harmful bacteria, and promoting the ecological balance of the intestinal microbiota. All in all, the Armillaria gallica Gastrodia elata fermentum improved the biological activity compared with the unfermented Gastrodia elata extract, verified the protein pathways regulated by the Armillaria gallica Gastrodia elata fermentum in the treatment of depression, and comprehensively provided the comprehensive multi-omics information of the Armillaria gallica Gastrodia elata fermentum effectively improving the depressive symptoms of CUMS mice.

[0028] In a mouse insomnia model, by observing the relevant biochemical indexes and protein expression in the serum and brain tissue of mice, the present invention demonstrated that the Armillaria gallica Gastrodia elata fermentum improved the biological activity compared with the unfermented Gastrodia elata extract, could significantly prolong the sleep time of mice, shorten the sleep latency, and down-regulate the contents of TNF-α, IL-6 and IL-1β, up-regulate the contents of GSH-Px, SOD, 5-HT, DA, GABA, NE, NGF and GDNF, and down-regulate the expression of proteins such as AMPK, p-JNK, JNK, p-p38, p38, etc., thereby improving the insomnia situation of mice induced by PCPA. Brief Description of the Drawings

[0029] Figure 1 are the HPLC chromatograms of Gastrodia elata extract and Gastrodia elata fermentum; among them, (A) is the chromatogram of the blank group (unfermented Gastrodia elata extract), (B) is the chromatogram of Gastrodia elata fermented by MHJ, (C) is the chromatogram of Gastrodia elata fermented by MHJ2-1, (D) is the chromatogram of Gastrodia elata fermented by MHJ3-1, (E) is the chromatogram of Gastrodia elata fermented by MHJ-4, (F) is the chromatogram of Gastrodia elata fermented by MHJ6-1, (G) is the chromatogram of the mixed standard product, and (H) is the content diagram of Gastrodia elata markers.

[0030] Figure 2Antidepressant-like effects of different samples on SPT, OFT, TST, and FST in CUMS model mice; data are expressed as mean ± SEM; compared with the control group, ## indicates p < 0.01, # indicates p < 0.05; compared with the model group, ** indicates p < 0.01, * indicates p < 0.05.

[0031] Figure 3 Protective effects of different samples on CUMS-induced hippocampal neuron damage; Figure A is a cell staining image; the arrows in Figure A indicate abnormal neurons in H&E staining and damaged DNA labeled by TUNEL staining; Figures B, C, and D represent neuron cell count graphs; data are expressed as mean ± SEM, compared with the control group, p < 0.001, ## p < 0.01, # p < 0.05; compared with the model group, *** p < 0.001, ** p < 0.01, * p < 0.05.

[0032] Figure 4 Statistical graphs of biological indicators of brain tissues of different samples improving depressive mice; data are expressed as mean ± SEM; compared with the control group, indicates p < 0.001, ## indicates p < 0.01, # indicates p < 0.05; compared with the model group, *** indicates p < 0.001, ** p < 0.01, * indicates p < 0.05.

[0033] Figure 5 Effects of different samples on hippocampal synaptogenesis and synaptic plasticity proteins in CUMS-induced mice; Figure A is a Western blot analysis of protein expression bands; Figure B is a graph of the expression of different proteins.

[0034] Figure 6 Effects of different samples on the structure and abundance of the gut microbiota in CUMS-induced depressive mice.

[0035] Figure 7 Effects of different samples on the gut microbiota of depressive mice; data are expressed as mean ± SEM; compared with the control group, indicates p < 0.001, ## indicates p < 0.01, # indicates p < 0.05, compared with the model group, *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05.

[0036] Figure 8 Statistical correlation graph between serum biochemical indicators and gut microbiota.

[0037] Figure 9 Statistical graph of the effects of different samples on the sleep latency of subthreshold dose of sodium pentobarbital; among them, compared with the blank group, ####Indicates p < 0.0001; compared with the model group, * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0038] Figure 10 It is a statistical chart of the effects of different samples on the sleeping time of sodium pentobarbital. Among them, compared with the blank group, #### Indicates p < 0.0001; compared with the model group, * indicates p < 0.05, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0039] Figure 11 It is a diagram of the effects of different samples on the contents of TNF-α, IL-6, IL-1β, GSH-Px, and SOD in the serum of mice.

[0040] Figure 12 It is a diagram of the effects of different samples on the contents of 5-HT, DA, GABA, NE, NGF, and GDNF in the brain tissue of mice.

[0041] Figure 13 It is a diagram of the effects of different samples on the protein expression in the brain tissue of mice; among them, A is a Western blot analysis diagram of the protein expression band; B-D are diagrams of the expression of different proteins. Specific implementation mode

[0042] The present invention will be further described below in conjunction with embodiments, but it shall not be used as a basis for limiting the present invention.

[0043] Embodiment 1:

[0044] Material preparation: Red Gastrodia elata was purchased from Dafang Jiulong Gastrodia elata Company. The Armillaria mellea strain was isolated from the cultivation fungus bag for Gastrodia elata growth in the planting base of Dafang Jiulong Company and was identified as Armillaria gallica by 16S rRNA technology.

[0045] A method for fermenting Gastrodia elata with Armillaria gallica includes the following steps:

[0046] S1. Preparation of Gastrodia elata samples:

[0047] Select fresh red Gastrodia elata, wash it, steam it in a water bath for 30 minutes until it is thoroughly cooked, cut it into slices, place it in an oven at 50 °C, dry it by blowing for 28 hours, use a high-speed traditional Chinese medicine grinder to crush the dried Gastrodia elata, and sieve it through a 60-mesh sieve to obtain Gastrodia elata powder for standby.

[0048] S = 2. Obtaining of strains:

[0049] The Armillaria gallica strain MHJ6-1, CFCC 5187 (Accession No. of Armillaria gallica Marxm. & Romagn. GenBank is KY474051) was isolated from the cultivation fungus bag.

[0050] S3. Preparation of PDB solution (Potato Dextrose Broth):

[0051] Cut 200 g of potatoes into pieces, add 1 L of pure water and boil for 30 min. Then remove the potato pieces by filtration. Add 20 g of granulated sugar to the filtrate and dissolve evenly, and make up the volume to 1000 mL to obtain the PDB solution.

[0052] S4. Cultivation of the strain and preparation of the strain suspension:

[0053] First inoculate the strain MHJ6-1 onto a PDA plate medium and carry out activation culture at 26°C for 12 days. After the mycelium grows into a colony with a diameter of 1 cm, inoculate it into 50 mL of sterilized PDB medium and culture it on a shaker for 12 days. The culture temperature is 26°C and the rotation speed is 130 r / min. Observe in time for any contamination. The obtained Armillaria mellea mycelium with uniform overall size and clear seed solution is used as the strain suspension with the number of mycelial pellets being 15 - 20 per 1 mL.

[0054] S5. Fermentation of Gastrodia elata:

[0055] S51. Take 2.0 g of Gastrodia elata powder, add 20 mL of PDB solution and 30 mL of pure water, mix and sterilize to obtain the Gastrodia elata mixed medium;

[0056] S52. Inoculate 4 mL of the strain suspension into the Gastrodia elata mixed medium and culture it by shaking at 26°C for 5 days to obtain the Gastrodia elata fermentation product.

[0057] Example 2:

[0058] This example is basically the same as Example 1, except that the strain used is Armillaria sinapina MHJ3-1 (Accession No. in Genbank is OL411631.1), which is isolated from a cultivated spawn bag.

[0059] Example 3:

[0060] This example is basically the same as Example 1, except that the strain used is Armillaria sp. MHJ-4 (Accession No. in Genbank is PP467623.1), which is isolated from a cultivated spawn bag.

[0061] Example 4:

[0062] This example is basically the same as Example 1, except that: the strain used is Armillaria gallica MHJ2-1 (Accession No. in Genbank of Armillaria sp. is PP467625.1), which is isolated from the surface mycelial cords of wild Gastrodia elata Blume.

[0063] Comparative Example 1:

[0064] This comparative example is basically the same as Example 1, except that: the strain used is Armillaria mellea MHJ, CFCC 5898 (Armillaria mellea (Vahl) P. Kumm), which is purchased from Ningbo Minghai Biotechnology Co., Ltd.

[0065] Respectively take 2 g of the Gastrodia elata Blume fermentation products of Examples 1-4 and Comparative Example 1 as the experimental groups. Crush 2 g of the original Gastrodia elata Blume (unfermented) using a blender, freeze-dry it under reduced pressure at -80 °C, then add methanol with a volume percentage of 60% to make up to 50 mL, and perform ultrasonic extraction for 1 h to obtain the unfermented Gastrodia elata Blume extract, which is used as the blank group.

[0066] Use HPLC to detect the contents of Gastrodia elata Blume markers in the experimental groups and the blank group respectively.

[0067] The Gastrodia elata Blume markers are: gastrodin (GAS), p-hydroxybenzyl alcohol (HBA), parishin E (PE), parishin B (PB), parishin C (PC), and parishin A (PA) components.

[0068] The chromatographic conditions of HPLC are: select ultraviolet light with a wavelength of 220 nm as the determined detection condition according to the method of the Chinese Pharmacopoeia. Mobile phase A is 0.1% formic acid water, and mobile phase B is acetonitrile. The system is gradient eluted. The phenomenex C18 chromatographic column (250×4.6 mm, 5 μm) is used, and the elution gradient is (0-10 min, 97%-90% A, 3%-10% B; 10-15 min, 90%-88% A, 10%-12% B; 15-25 min, 88%-82% A, 12%-18% B; 25-40 min, 82% A, 18% B; 40-45 min, 82%-5% A, 18%-95% B); the column temperature is 30 °C, the injection volume is 10 μL, and the flow rate is 1.0 mL / min.

[0069] The detection results of the active ingredient contents in different Gastrodia elata Blume fermentation products and Gastrodia elata Blume are shown in Table 1, Figure 1 as shown.

[0070] Table 1. Contents of Gastrodia elata Blume markers obtained by fermenting different strains under the same conditions

[0071]

[0072] According to the results, after Gastrodia elata was fermented by the transformed strain MHJ6-1 in Example 1, the total content of 6 markers in Gastrodia elata increased by about 28.85%. The content of gastrodin increased the most, and the increase rate of gastrodin content compared with the blank group was 3.8 times. Moreover, the content changes of other Gastrodia elata markers in the fermented product of Armillaria gallica and Gastrodia elata were not significant compared with unfermented Gastrodia elata (except for PA), while the content of each Gastrodia elata marker in the fermented product of Armillaria mellea (MHJ) and Gastrodia elata decreased significantly.

[0073] 1. CUMS depression animal experiment:

[0074] 1.1 Define the Gastrodia elata fermented product in Example 1 as FG, define the positive drug escitalopram as ESC, and define the Gastrodia elata extract as GE. The Gastrodia elata extract was obtained by crushing the original Gastrodia elata (unfermented) using a blender, freeze-drying under reduced pressure at -80°C, then adding methanol with a volume percentage of 60% to make up to 50 mL, and ultrasonic extraction for 1 h; provide standard feed and water to the animals without restriction, and maintain the mice in the following environment: the temperature is set at 23 ± 2°C, and follow the principles of laboratory animal care (NIH publication No. 80-23, revised in 1996), and adapt to feeding for 7 days. Divide 70 male C57 / BL mice (20 - 22 g) into 7 groups, namely: (1) control group (Control); (2) model group (CUMS); (3) CUMS + ESC (10 mg / kg); (4, 5, 6) CUMS + FG (FGL: 200 mg / kg, FGM: 400 mg / kg, FGH: 800 mg / kg); (7) GE group (administered dose 400 mg / mL). Except for the control group, mice in all groups were subjected to mild unpredictable stress. Different stressors: water deprivation, food deprivation, day-night exchange, night-time strobe light, cage tilt (45°), restricted activity space, wet bedding, no bedding, tail clamping, forced swimming, foot shock, and noise. Receive variable mild unpredictable stress for 8 weeks, randomly use 2 stimuli every day, and detect whether depressive-like behavior phenotypes occur in mice during this process by behavioral methods on the 30th day. After determining that the model was successfully established, start FG administration treatment, and use behavioral methods such as the sucrose preference test (SPT), open field test (OFT), tail suspension test (TST), and forced swimming test (FST) to detect their depressive-like behavior from 10:00 for 4 h. Then anesthetize and sacrifice the mice with sodium pentobarbital, and take their whole brains, hippocampal tissues, sera, and cecal feces. The experimental results of SPT, OFT, TST, and FST are shown in Figure 2 as follows.

[0075] According to Figure 2It can be seen that, compared with the control group, the sugar water consumption of SPT in the CUMS group was significantly reduced, the movement distance in the OFT was decreased, and the immobility time in the TST and FST was prolonged, indicating that the CUMS model was successfully established.

[0076] Among them, the SPT results showed that FG at all concentrations could stimulate depressed mice to consume more sugar water, reaching a consumption equivalent to that of the ESC treatment group, enhancing the hedonic state of the mice (see Figure 2 A).

[0077] The OFT results showed that there were significant differences between the model group and the CUMS group. The total movement distance of the CUMS group was significantly decreased, and the exploratory desire was reduced. After the treatment with ESC, FGM, and FGH, the exploratory desire and the total movement distance increased, recovering to be equivalent to that of the control group (see Figure 2 B).

[0078] In the TST results, compared with the control group, the CUMS group had a longer immobility time and a shorter struggling time. After the ESC treatment, the immobility time was shortened and recovered compared with the CUMS group. The FGH results showed a treatment effect equivalent to that of ESC, with no significant difference from the control group (see Figure 2 C).

[0079] The results of the FST were similar to those of the TST. CUMS mice had a shorter struggling time and swimming time in the FST forced swimming test, and the suspended immobility time was prolonged. FG could improve and reverse this situation. That is, the depressed mice treated with FG showed a much shorter immobility time in both the TST and FST. And at the same drug dosage of 400 mg / mL, the unfermented Gastrodia elata extract (GE) had a weaker effect on alleviating depressive-like behaviors (see Figure 2 D).

[0080] Therefore, the administration of FG and ESC reversed the hedonic state of CUMS-induced depressed mice, enhanced the motor ability and exploratory desire, and indicated that high-dose FG could effectively alleviate depressive-like behaviors in mice.

[0081] 1.2 HE whole brain tissue section staining test: Put the whole brain tissue into paraformaldehyde fixative, dewax and hydrate, section, dewax and dehydrate, rinse with pure water and then use for HE and Nissl staining, then rinse with gradient ethanol, and finally perform microscopic examination, image acquisition and analysis. The TUNEL method was used to detect the apoptosis of nerve cells and neurons in the hippocampal region, and a TUNEL apoptosis assay kit was used for determination. The cell nucleus was counterstained with DAPI to promote the accurate visualization of apoptotic cells. The sections were washed 3 times with PBS (pH = 7.4) for 5 minutes each time. After removing the PBS, the DAPI dye solution was added to the circle and incubated at room temperature in the dark for 10 minutes. The ImageJ software was used to calculate the number of Nissl-stained cells to determine the survival rate of neurons. The detection results are shown inFigure 3 shown.

[0082] like Figure 3 As shown in Figure A, H&E staining was used to observe damaged or necrotic cells in the mouse hippocampus. In the control group, neurons in the hippocampal DG and CA3 regions had round, intact nuclei and were well-arranged. In the CUMS group, neurons in the hippocampal DG and CA3 regions were loosely arranged with obvious gaps between them. The number of cells was reduced, and pyknosis, nuclear condensation, partial nuclear fragmentation, and cell damage were stained dark, indicating that the neurons were severely damaged. In the FG and ESC groups, the morphology of damaged neurons in the hippocampal DG and CA3 regions was restored to a complete state.

[0083] like Figure 3 As shown in A, TUNEL staining can dye apoptotic cells into red fluorescence. There are a lot of enhanced fluorescence signals in the brain of CUMS depressed mice, indicating that a large number of neurons in the mouse brain have apoptosis. In contrast, few fluorescent spots were observed in the DG region of the hippocampus in the FG and ESC treatment groups. The apoptotic cells in the DG region were counted and analyzed for significant differences (see Figure 3 B), The number of apoptotic cells in CUMS was significantly increased.

[0084] like Figure 3 As shown in A, Nissl staining was used to observe the number and degree of Nissl bodies in neuronal cells. Compared with the control group, the staining in the CUMS group became lighter, and hippocampal pyramidal cells showed nuclear shrinkage, neuronal loss, and neuronal degeneration. However, FG and ESC significantly alleviated CUMS-induced neuronal damage. By quantitatively analyzing the number of Nissl bodies in healthy neurons in the DG and CA3 regions of the hippocampus ( Figure 3 C, D), Compared with the control, the number of surviving neurons in CUMS was significantly reduced, and this condition was significantly reversed after treatment with FGM and FGH, and the cell status was no different from that in the control group.

[0085] From the above, we can see that FG can regulate the recovery of nerve cells in the DG and CA3 areas of the hippocampus.

[0086] 1.3 Hippocampal tissue enzyme-linked immunosorbent assay: Rinse the hippocampal tissue with pre-cooled PBS (0.01M, pH = 7.4) to remove residual blood. After weighing, mince the hippocampal tissue and add 9 times the amount of PBS (proteinase inhibitors are added to PBS) of the corresponding tissue sample. Grind the homogenate at -4°C. Finally, centrifuge the homogenate for 5-10 minutes, take the supernatant, and use Elisa kits to determine the levels of neurotransmitters (5-HT, NE, DA), neurotrophic factors (NGF, BDNF, GDNF) and inflammatory factors (TNF-α, IL-6, IL-1β). The test results are shown in Figure 4 shown.

[0087] Figure 4 The results showed that the levels of neurotransmitters measured and analyzed in the cerebral cortex and hippocampus of CUMS mice were significantly lower, and ESC treatment could significantly restore the decline of the above indicators. After FG treatment, the contents of 5-HT, DA, and NE increased, especially at medium and high doses, significantly increasing the levels of neurotransmitters 5-HT, DA, and NE and comparable to the blank group (see Figure 4 A-C).

[0088] Similar induction to neurotransmitters was also found in the expression of neurotrophic factors: the neurotrophic factors in CUMS model mice decreased, and high-dose FG treatment was very obvious in increasing the expression of NGF, BDNF, and GDNF in the hippocampal region (see Figure 4 D-F).

[0089] From the expression maps of key inflammatory factors (IL-1β, IL-6, and TNF-α) in the hippocampus of mice, it can be seen that compared with the control group, the inflammatory factors in the hippocampus and serum of CUMS-treated mice increased significantly; compared with the CUMS-treated group, FG and ESC significantly inhibited the excessive release of inflammatory factors in hippocampal nerve cells mediated by CUMS (see Figure 4 G-I).

[0090] As can be seen from the above, FG can regulate the levels of neurotransmitters and neurotrophic factors to return to normal levels, and at the same time inhibit the release of inflammatory factors.

[0091] 1.4 Immunoblot assay (Western blot): Take the hippocampal tissue of the mouse brain and homogenize it by ultrasound. Total protein was extracted from these brain regions using RIPA buffer and low-temperature centrifugation. The protein concentration of each group was quantified by the BCA method. 10 micrograms of protein in each sample was separated on a 10% or 12% SDS-PAGE gel and then transferred to a PVDF membrane. Then, the membrane was blocked with a 5% low-fat milk solution in 0.01% Tween-Tris buffered saline (TBST) for 1 hour at room temperature. The samples were incubated with goat anti-rabbit secondary antibody for 1 hour at room temperature. The protein expression level was detected using an enhanced chemiluminescence kit. The protein expression level was quantified using ImageJ, and the shadow intensity of each protein band was measured and compared. Each protein band was normalized to the loading control protein (β-Actin).

[0092] The expressions of NMDAR1, N-methyl-D-aspartic acid receptor 2A (NMDAR2A), N-methyl-D-aspartic acid receptor 2B (NNDAR2B), calcium / calmodulin-dependent protein kinase IIα (CaMKIIα), phosphorylated cAMP response element-binding protein (p-CREB), phosphorylated extracellular signal-regulated kinase (p-ERK), brain-derived neurotrophic factor (BDNF), PSD-95, and Syn1 proteins in the hippocampus of mice were detected by Western blotting.

[0093] The detection results are as Figure 5 shown. The expressions of NMDAR1 and NMDAR2A in the CUMS group of mice were upregulated. After treatment with FG and ESC, NMDAR1 and NMDAR2A were downregulated, showing significant improvement, and FG showed an obvious dose dependence. Compared with the control group, the expression of NMDAR2B protein decreased in the CUMS group, and the expression level increased after drug administration. The calcium channel protein in the CUMS group was significantly upregulated, and after treatment with FG and ESC, the expression of this protein was significantly inhibited. It was also found that the levels of p-CREB and p-ERK in the CUMS group were significantly decreased, while treatment with FG and ESC could significantly reverse the upregulation of the above protein expressions. These results indicate that FG attenuated the negative changes in NMDA receptors and CaMKIIα proteins induced by CUMS. Moreover, it was also confirmed that high-dose FG could significantly increase the reduction in the expressions of BDNF, PSD-95, and Syn1 proteins induced by CUMS, and it was comparable to the control group. Therefore, the results of both immunofluorescence and Western blotting tests showed that CUMS severely reduced the expression distribution of NMDAR1, PSD-95, and Syn1 proteins in the hippocampal region. FG could regulate the activation of CREB and ERK proteins, inhibit the phosphorylation reaction, reverse the reduction in spinal synaptic density and the expression levels of synaptic-related proteins induced by CUMS, significantly enhance hippocampal synaptic plasticity and neurogenesis, and restore the expression of these key proteins, thus playing a role in relieving depression.

[0094] 1.5 16S rRNA sequencing analysis of bacteria in mouse feces: Genomic DNA of gut microbiota in feces was extracted using a DNA extraction kit. Specific primers with 12bp barcodes were used to amplify 16S rRNA containing the V3-V4 region to label each PCR product. By ™ DNA sample preparation kit (Illumina, San Diego, CA) was used to prepare sequencing libraries. High-throughput sequencing was performed on an Illumina Miseq (PE 300) platform according to the standard protocol of Shanghai Meiji Biotechnology Co., Ltd. FLASH (version 1.2.11) was used to trim, fill, and merge the raw reads from 16S sequencing. Operational taxonomic units (OTUs) were generated by clustering the sequences at 97% sequence similarity using Uprase (version 7.0.1090). Community analysis, principal coordinate analysis (PCoA), and related heat maps were calculated using R software (version 3.4.1).

[0095] like Figure 6 As shown in A, based on the analysis of species composition based on OUTs composition, CUMS interference led to a decrease in the diversity and richness of intestinal flora compared with the control group mice. Figure 6 As shown in B, the species composition of each group in the principal coordinate analysis (PCoA) of β diversity was significantly similar, and CUMS was clearly separated from each group. Figure 6 As shown in CF, α diversity analysis of ACE, Chao, Shannon and Simpson, all indexes showed a significant decrease in the diversity and richness of the microbial community in CUMS mice, which indicated that the occurrence of CUMS destroyed the host intestinal flora, and the microbial community structure had significant differences, with reduced diversity and richness, which improved after FG treatment.

[0096] The degree of bacterial taxonomic similarity at the phylum, family, and genus levels was assessed, and statistical and one-way ANOVA analyses were performed on genera with abundance >1 at the genus level to discuss the differences in microbial community richness in order to gain a more comprehensive understanding of the overall gut microbiome composition. Figure 7 .like Figure 7 As shown in A, DF, at the phylum level, the distribution of Firmicutes and Bacteroidota in the CUMS group mice was as high as 70-80% compared with the control group, and the F / B ratio decreased, proving that CUMS caused intestinal disorders. After FG treatment, the abundance of Bacteroides was significantly reduced. Figure 7 As shown in B and GH, compared with the control group, the CUMS group mice showed an increased abundance of Muribaculaceae and a decreased abundance of Lachnospiraceae. After FG intake, the abundance of Lachnospiraceae showed an upward trend, and the abundance of Muribaculaceae returned to normal. Figure 7As shown by C and I-M, the abundances of norank_f__Muribaculaceae, Lactobacillus, and Desulfovibrio increased in the CUMS group of mice, while that of Lachnospiraceae_NK4A136_group decreased, and Akkermansia was almost completely disrupted. Similarly, ESC and FG treatments could reverse the changes in the relative abundances of these five bacteria to some extent, restoring them to a level comparable to that of the control group. As described above, CUMS-induced depression led to disorders in the intestinal microbiota of mice, and FG regulated the homeostasis of the intestinal microbiota in CUMS-treated mice.

[0097] 1.6 The indicators of neurotransmitters (5-HT, NE, DA), neurotrophic factors (NGF, BDNF, GDNF), and inflammatory cytokines (TNF-α, IL-6, IL-1β) were screened, and Spearman correlation analysis was performed with the top 20 abundant intestinal microorganisms with significantly changed abundances at the genus level. The analysis results are shown in Figure 8 as follows. From Figure 8 it can be seen that neurotransmitters and neurotrophic factors were positively correlated with the relative abundances of Akkermansia, Allobaculum, and norank_o__Clostridia_UCG014, and negatively correlated with the relative abundances of norank_f__Ruminococcaceae, Ruminococcus, norank_f__Lachnospiraceae, and norank_f__Muribaculaceae. Inflammatory cytokines were positively correlated with the relative abundances of norank_f__Ruminococcaceae, Ruminococcus, norank_f__Lachnospiraceae, norank_f__Muribaculaceae, and Lactobacillus, and negatively correlated with the relative abundances of Akkermansia and Allobaculum. Therefore, the structure of the intestinal microbiota affects the levels of neurotransmitters, neurotrophic factors, and inflammatory factors, indicating that FG treatment can improve depressive-like effects by maintaining the structure of the body's intestinal flora.

[0098] Through comprehensive multi-omics analysis of the active ingredients, the health status of hippocampal neurons, the regulation of protein pathways, and the maintenance of intestinal homeostasis, the present invention proves the complex mechanism by which Gastrodia elata fermentum effectively alleviates depression. Among them, Gastrodia elata fermented by Armillaria gallica significantly increased the content of GAS, enhanced the motor ability and exploratory desire of CUMS mice, and alleviated the depressive-like behavior of CUMS mice, etc.; by improving the transmission of neurotransmitters in the mouse brain, increasing the release of neurotrophic factors, and regulating indicators such as reducing inflammatory factors in the brain and serum, it effectively alleviated the depressive symptoms of mice; Armillaria gallica Gastrodia elata fermentum can also regulate the BDNF / NMDA / CREB protein pathway and PSD-95 / SYN1 protein, enhancing neural remodeling and neurogenesis; regulating the disorder and correlation of the gut microbiota in depression, increasing the content of beneficial bacteria in the gut microbiota, reducing the content of harmful bacteria, and promoting the ecological balance of the gut microbiota. All in all, Armillaria gallica Gastrodia elata fermentum has increased the component content and biological activity compared with the sample of unfermented Gastrodia elata extract, and verified the protein pathway regulated by Armillaria gallica Gastrodia elata fermentum in the treatment of depression, comprehensively providing comprehensive omics information on the effective improvement of CUMS depressive symptom mice by Armillaria gallica Gastrodia elata fermentum.

[0099] 2. PCPA insomnia animal experiment:

[0100] 2.1 Modeling and administration: Divide 70 ICR male mice into 7 groups, with 10 mice in each group, namely: blank group (Control), model group (Model), clonazepam group (Clonazepam, 0.65 mg / kg), low, medium, and high dose groups of Gastrodia elata fermentum in Example 1 (FGL: 200 mg / kg; FGM: 400 mg / kg; FGH: 800 mg / kg), and GE group (Gastrodia elata extract obtained by ultrasonic extraction with 60% methanol after crushing and freeze-drying unfermented Gastrodia elata, administration dose 400 mg / mL). Each group of mice was housed in a barrier-level animal house at a temperature of 20 - 25 °C and a relative humidity of 50% - 60%, and adaptively fed for one week under the condition of 12 h light-dark cycle alternation, with free access to food and water during this period. One week later, except for the blank control group, mice in other groups were intraperitoneally injected with p-chlorophenylalanine (PCPA) suspension (450 mg / kg, dissolved in physiological saline, pH = 7 - 8), once a day for 5 consecutive days. During the modeling period, the behavior of mice was observed. Compared with the blank group, mice in other groups showed significant behavioral changes such as circadian rhythm disorder, frequent daytime activities, messy hair, increased aggression, significant weight loss, and grayish-white stools, indicating successful preparation of the insomnia model. After successful modeling, intragastric administration was carried out. The administration volume was 0.1 mL / 10 g. The normal control group and the model group were intragastrically administered the same volume of physiological saline in proportion. Each group of mice was intragastrically administered once a day at 8:00 - 9:00 in the morning for 7 consecutive days.

[0101] 2.2 Pentobarbital Sodium Sleep Time and Sleep Latency Test: Seven days after administration, animals in each group were intraperitoneally injected with pentobarbital sodium at a dose of 50 mg / kg (this dose was measured as the minimum threshold dose that caused 100.00% of mice to sleep in the pre-experiment). Based on the disappearance of the righting reflex, the sleep latency and sleep duration of mice in each group were observed and recorded. The test results are shown in Figure 9 and 10 as shown.

[0102] From Figure 9 it can be seen that compared with the blank group, the sleep latency of the model group was significantly prolonged (p < 0.05), further indicating that the PCPA insomnia model was successfully established. Compared with the model group, the sleep latency of the low, medium, and high-dose FG groups was significantly shortened (p < 0.05), and was shorter than that of the GE group. From Figure 10 it can be seen that in terms of sleep time, the sleep time of the low, medium, and high-dose FG groups was significantly prolonged (p < 0.05), and was longer than that of the GE group. It shows that FG can significantly shorten the sleep latency of PCPA insomnia model mice, prolong the sleep time, play a sedative and hypnotic role, and its effect is better than that of gastrodia extract, and its activity is stronger than that of gastrodia extract.

[0103] 2.3 Detection of Neurotransmitter, Inflammatory and Oxidative Stress Indexes: Seven days after administration, mice in each group were fasted for more than 12 h, whole blood was taken from the vein, and serum was separated at 3500 r / min for standby, for the detection of the contents of TNF-α, IL-6, IL-1β, GSH-Px and SOD. The test results are shown in Figure 11 .

[0104] After blood collection, the mice were sacrificed by cervical dislocation, and the brain tissues and hippocampi of mice in each group were dissected. The brain and hippocampal tissues were rinsed with physiological saline in the raphe nucleus, weighed and recorded. Part of the hippocampus was fixed with 10% neutral formaldehyde, and part of the hippocampus was added with 10% physiological saline according to the weight, homogenized, centrifuged at 4 °C and 8000 r / min for 10 min, and the supernatant was taken for the detection of the contents of 5-HT, DA, GABA, NE, NGF and GDNF. The measurement method was carried out according to the operation steps of the Elisa kit. The test results are shown in Figure 12 as shown.

[0105] From Figure 11It can be seen that the contents of TNF-α, IL-6 and IL-1β in the serum of the mice in the model group were significantly higher than those in the blank group (p<0.05). Compared with the model group, the contents of TNF-α, IL-6 and IL-1β in the serum of the mice in the clonazepam group and each dose group of the fermented product of Armillaria mellea and Gastrodia elata were significantly decreased (p<0.05). Compared with the blank group, the contents of GSH-Px and SOD in the serum of the mice in the model group were significantly decreased (p<0.05). Compared with the model group, the contents of GSH-Px and SOD in the serum of the mice in the positive control group and each dose group of the fermented product of Armillaria mellea and Gastrodia elata were significantly increased (p<0.05), and there was a dose-dependent relationship.

[0106] From Figure 12 It can be seen that the contents of 5-HT, DA, GABA, NE, NGF and GDNF in the brain tissue of the mice in the model group were significantly lower than those in the blank group (p<0.05). Compared with the model group, the contents of neurotransmitters in the brain tissue of the mice in the positive control group and each dose group of the fermented product of Armillaria mellea and Gastrodia elata were significantly increased (p<0.05).

[0107] The above results indicate that the fermented product of Armillaria mellea and Gastrodia elata can positively regulate the imbalanced oxidative stress response and neurotransmitter content in the body of insomnia mice, thereby playing a role in improving sleep.

[0108] 2.4 Investigation of the expression of related proteins in the brain tissue of mice: The expression of AMPK, p-JNK and p-p38 proteins in the brain tissue of mice in each group was detected by western blot method. Hippocampal tissue was taken and homogenized by ultrasonic wave, and total protein was extracted from these brain regions by RIPA buffer and low-temperature centrifugation method. The protein concentration of each group was quantified by BCA method. 20 micrograms of protein in each sample was separated on a 10% or 12% SDS-PAGE gel, and then transferred to a PVDF membrane. Then, the membrane was blocked with 5% low-fat milk solution in 0.01% Tween-Tris buffered saline (TBST) at room temperature for 1.5 hours. Immunoblotting was performed using a set of antibodies. The samples were incubated with the secondary antibody (goat anti-rabbit) at room temperature for 1 hour. The protein expression level was detected using an enhanced chemiluminescence kit. The protein expression level was quantified using ImageJ, and the intensity of each protein band was measured and compared. Each protein band was normalized to the loading control protein (β-Actin) for calculation.

[0109] As Figure 13 shown, compared with the blank group, the expression of proteins such as AMPK, p-JNK and p-p38 in the model group increased. Compared with the model group, after intragastric administration of the fermented product of Armillaria mellea and Gastrodia elata, the expression of proteins such as AMPK, p-JNK, JNK, p-p38 and p38 in the brain tissue decreased.

[0110] The present invention proves that the fermented product of Armillaria mellea and Gastrodia elata can significantly prolong the sleeping time of mice, shorten the sleep latency, has stronger activity than the extract of Gastrodia elata, and can also down-regulate the contents of TNF-α, IL-6 and IL-1β, up-regulate the contents of GSH-Px, SOD, 5-HT, DA, GABA, NE, NGF and GDNF, and down-regulate the expressions of proteins such as AMPK, p-JNK, JNK, p-p38 and p38 by observing the relevant biochemical indexes and protein expression in the serum and brain tissue of mice, so as to improve the insomnia situation of mice induced by PCPA.

[0111] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. For those skilled in the art, the technical solutions recorded in the above embodiments can be modified, or some of the technical features can be equivalently replaced; and all such modifications and replacements should fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for fermenting Gastrodia elata by Armillaria gallica, characterized in that: It includes the following steps: S1. Preparation of Gastrodia elata powder; S2. Preparation of PDB solution; S3. Cultivation of strains and preparation of strain suspension: The Armillaria mellea strain of Gallic is first inoculated into a PDA plate medium for activation culture. After the mycelia grow into colonies, they are inoculated into a PDB medium and cultured with shaking on a shaker to obtain a strain suspension; S4. Fermentation of Gastrodia elata: S41. Mix Gastrodia elata powder, PDB solution and water to obtain a Gastrodia elata mixed medium; S42. Inoculate the strain suspension into the Gastrodia elata mixed medium and culture with shaking to obtain a Gastrodia elata fermentation product.

2. The method for fermenting Gastrodia elata with Armillaria gallica according to claim 1, wherein: The preparation of the PDB medium in step S2 is specifically as follows: Cut potatoes into pieces, boil them with water, then filter the potato pieces, and add granulated sugar to the filtrate and dissolve evenly to obtain the PDB medium.

3. The method for fermenting Gastrodia elata by Armillaria gallica according to claim 1, characterized in that: In step S3, the temperature for activation culture is 20 - 30 °C, and the culture time is 10 - 15 days; the temperature for shaking on the shaker is 20 - 26 °C, and the culture time for shaking on the shaker is 10 - 15 days; the rotation speed is 100 - 150 r / min.

4. A method for fermenting Gastrodia elata with Armillaria gallica according to claim 1, characterized in that: In step S41, the ratio of Gastrodia elata powder : PDB solution : water is 1 g : (5 - 15) mL : (10 - 20) mL.

5. A method for fermenting Gastrodia elata by Armillaria gallica according to claim 1, characterized in that: Step S42 is specifically that the strain suspension is inoculated into the Gastrodia elata mixed medium and cultured with shaking at 20 - 32 °C for 8 - 14 days to obtain a Gastrodia elata fermentation product.

6. A Gastrodia elata fermentation product, obtained by fermenting with the Armillaria mellea of Gallic according to any one of claims 1 - 5.

7. The gastrodia elata ferment according to claim 6, characterized in that: The contents of gastrodin, p-hydroxybenzyl alcohol, parishin E, parishin B, parishin C and parishin A in the Gastrodia elata fermentation product are respectively above 2500, 750, 2800, 2000, 1250, 100 μg / g.

8. Use of the Gastrodia elata fermentation product according to claim 6 in the preparation of an anti-anxiety and antidepressant drug or food for improving neurotransmitter transmission, increasing the release of neurotrophic factors, inhibiting the secretion of inflammatory factors, enhancing nerve remodeling and neurogenesis or regulating intestinal microbiota disorders.

9. Use of the Gastrodia elata fermentation product according to claim 6 in the preparation of a sleep-improving drug or food for shortening sleep latency, prolonging sleep time, reducing the contents of TNF-α, IL-6 and IL-1β, increasing the contents of GSH-Px and SOD, increasing the content of neurotransmitters, and reducing the damage of hippocampal neuron structure.

10. Use of the Gastrodia elata ferment according to claim 8 or 9, characterized in that: The dosage of the Gastrodia elata fermentation product is 200 - 800 mg / kg.

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