Application of panax notoginseng and gastrodia elata fermentation broth in relieving anxiety, improving sleep and headache

Abstract

The application discloses a TPH1 agonist, a Panax notoginseng and Gastrodia elata fermentation liquor and a preparation method and application thereof; the compound is a tryptophan hydroxylase 1 agonist. The application utilizes Paracaseolovis GP66 to ferment a Panax notoginseng and Gastrodia elata compound matrix, and generates a novel hybrid molecule baliensen A-Panax notoginseng monol in situ. The compound can specifically up-regulate the expression and enzyme activity of intestinal TPH1, and significantly relieve excessive anxiety before falling asleep, shorten the sleep latency, prolong the deep sleep time and improve stress headache through the mechanism of enhancing the intestinal 5-HT synthesis and synergistically regulating the central receptors. The application breaks through the single mode of traditional Chinese medicine directly acting on the central nervous system, and provides a novel technical scheme for developing a new generation of safe and non-addictive sleep aid products based on the intestinal-brain axis regulation.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine fermentation, and in particular to a compound that has the function of stimulating tryptophan hydroxylase 1, fermentation broth of Panax notoginseng and Gastrodia elata, its preparation method and application. Background Technology

[0002] With the fast pace and soaring pressures of modern life, over 30% of adults worldwide suffer from chronic insomnia, while approximately 40% of insomnia sufferers also experience recurrent stress headaches (tension headaches). These two conditions create a vicious cycle, severely impacting quality of life and work efficiency. Existing chemical hypnotics mostly target GABA receptors non-selectively, and generally suffer from drawbacks such as addiction, hangover effects, and insufficient improvement in deep sleep, leading to drug tolerance with long-term use. Clinically used headache relievers, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and triptans, have limitations including gastrointestinal mucosal damage, cardiovascular risks, and overdose headaches. Currently, there is a lack of natural solutions that can safely address both insomnia and stress headaches simultaneously.

[0003] Serotonin (5-HT) is a core neurotransmitter regulating the sleep-wake cycle and central pain transmission. Insufficient synthesis or abnormal signal transduction of 5-HT is a key pathological basis for difficulty falling asleep and sleep maintenance disorders, and also an important mechanism for inducing pericranial muscle tension, decreased pain threshold, and stress headaches. Approximately 95% of 5-HT in the human body is synthesized by enterochromaffin cells in the gut. Tryptophan hydroxylase 1 (TPH1) is the only rate-limiting enzyme in intestinal 5-HT synthesis. By activating TPH1 and increasing intestinal 5-HT levels, it is possible to simultaneously regulate the central sleep rhythm and the descending pain inhibition system via the vagus nerve gut-brain axis pathway. It has the dual potential to improve sleep and relieve stress headaches, and is a safe and non-addictive natural intervention target.

[0004] However, single extracts of traditional Chinese medicines for calming the mind, such as Gastrodia elata and Panax notoginseng, can only weakly affect the central 5-HT level and cannot effectively activate the activity of intestinal TPH1 enzyme, making it difficult to simultaneously meet the dual needs of sleep regulation and headache relief. Furthermore, physical compounding can only achieve a simple superposition of drug effects and cannot exert a molecular-level synergistic effect, making it difficult to fundamentally improve sleep disorders and accompanying stress headaches caused by gut-brain axis imbalance.

[0005] Chinese patent application CN120514792A discloses a Gastrodia elata fermented liquid with sleep-improving effects, its preparation method, and its application. The preparation method includes the following steps: Gastrodia elata raw materials are pulverized, extracted with water, and enzymatically hydrolyzed. The extract is then fermented using specific bacteria, *Lactobacillus plantarum* BXM2 and *Lactobacillus paracasei* B20. Solid-liquid separation and sterilization are then performed to obtain a fermented liquid with high levels of gastrodin and p-hydroxybenzyl alcohol. Verification through zebrafish sleep improvement experiments clearly shows that this fermented liquid has significant sleep-improving effects. This method, by increasing the content of the core sleep-aiding components gastrodin and p-hydroxybenzyl alcohol, increases wakefulness and shortens total wake time compared to unfermented liquid. The γ-aminobutyric acid (GABA) content after fermentation is higher than that of unfermented liquid. However, this fermented liquid does not selectively target GABA receptors, and has drawbacks such as addictive potential, hangover effects, and insufficient improvement in deep sleep. Long-term use may lead to drug tolerance, and the sleep-improving effect can be further enhanced. Summary of the Invention

[0006] The first object of the present invention is to provide a compound as a tryptophan hydroxylase 1 agonist; The second objective of this invention is to provide a fermented liquid of Panax notoginseng and Gastrodia elata, which significantly alleviates excessive pre-sleep anxiety, shortens sleep latency, prolongs deep sleep time, and improves stress headaches through a mechanism that enhances the synthesis of 5-HT in the intestine and precisely regulates the central receptor. The third objective of this invention is to provide a method for preparing the fermented liquid of Panax notoginseng and Gastrodia elata; The fourth objective of this invention is to provide an application of a tryptophan hydroxylase 1 agonist or a fermentation liquid of Gastrodia elata and Panax notoginseng.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A tryptophan hydroxylase 1 agonist, with the following chemical structural formula: .

[0008] A fermentation liquid of Panax notoginseng and Gastrodia elata, comprising the aforementioned tryptophan hydroxylase 1 agonist; The Panax notoginseng and Gastrodia elata fermentation liquid is prepared by fermentation of a substrate including Panax notoginseng and Gastrodia elata using fermentation strains; The fermentation strain is Lacticaseibacillus paracasei GP66, with accession number CGMCC No. 32952.

[0009] A method for preparing the fermented liquid of Panax notoginseng and Gastrodia elata includes the following steps: The fermentation broth of Panax notoginseng and Gastrodia elata was prepared by inoculating 0.1-2% (v / v) of seed liquid containing the fermentation strain into a substrate including Panax notoginseng and Gastrodia elata, fermenting, sterilizing, and centrifuging.

[0010] The fermentation time is 24-72 hours.

[0011] The method for preparing the substrate including Panax notoginseng and Gastrodia elata includes the following steps: Prepare a compound fermentation substrate with a feed-to-liquid ratio of 1g:8-15mL by mixing Gastrodia elata and Panax notoginseng at a weight ratio of 2-4:1. Add 2% soybean peptone, 3% glucose, and 0.25% dipotassium hydrogen phosphate to the compound fermentation substrate, stir well, sterilize, and cool to obtain the final product.

[0012] The initial pH of the fermentation was adjusted to 6-7.

[0013] The fermentation temperature is 30~38℃; The sterilization temperature is 85~100℃; The sterilization time is 20-40 minutes; The centrifuge speed is 5000~9000 r / min; The centrifugation time is 1~10 min.

[0014] The method for preparing the Panax notoginseng and Gastrodia elata fermentation liquid also includes a post-ripening step; The post-ripening temperature is 20~28℃; The post-ripening time is 5-15 hours.

[0015] The method for preparing the tryptophan hydroxylase 1 agonist includes the following steps: The fermentation liquid of Panax notoginseng and Gastrodia elata as described in claim 2 was centrifuged, concentrated, extracted, and concentrated again to obtain a brownish-yellow crude extract; The brownish-yellow crude extract was eluted with a macroporous adsorption resin gradient to obtain a light yellow refined product. The pale yellow purified product was purified by high performance liquid chromatography to obtain tryptophan hydroxylase 1 agonist.

[0016] One application of the aforementioned tryptophan hydroxylase 1 agonist is in the preparation of a drug that promotes TPH1 enzyme / 5-HT expression.

[0017] One application of the aforementioned Panax notoginseng and Gastrodia elata fermentation liquid is in the preparation of drugs to relieve anxiety; or Applications in the preparation of medicines to improve sleep and / or headaches.

[0018] Compared with the prior art, the beneficial effects of the present invention are: The tryptophan hydroxylase 1 agonist provided by this invention has a significant activation effect on TPH1 enzyme in intestinal Caco2 cells and can effectively increase the production of 5-HT in cells. Its parent compounds, barisonin A and notoginseng, do not have this effect.

[0019] The fermented liquid of Gastrodia elata containing tryptophan hydroxylase 1 agonist provided by this invention can significantly shorten the sleep latency and prolong the duration of deep sleep in mice. At the same time, it can significantly upregulate the expression and activity of intestinal TPH1 gene and increase serum 5-HT level, and has significant and stable sleep-aiding effects.

[0020] The fermentation liquid of Panax notoginseng and Gastrodia elata provided by this invention uses Gastrodia elata and Panax notoginseng as raw materials and adopts microbial fermentation process to synthesize active ingredients in situ. It does not require complicated chemical synthesis and purification steps, and the reaction conditions are mild and the production safety is high. Attached Figure Description

[0021] Figure 1 The chemical structural formula of basiloxane A is shown.

[0022] Figure 2 The chemical structural formula of notoginsenoside is shown.

[0023] Figure 3 The chemical structural formula of the TPH1 agonist is shown. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0025] Example 1 This embodiment provides a method for preparing Panax notoginseng and Gastrodia elata fermentation liquid.

[0026] This invention uses *Lactobacillus paracasei* GP66 as the fermentation strain for fermenting *Panax notoginseng* and *Gastrodia elata* substrate. This strain, with accession number CGMCC No. 32952, was deposited at the China General Microbiological Culture Collection Center on December 20, 2024, and was viable at the time of deposit. This strain is disclosed in patent CN120059998A, and its potential sleep-inducing abilities have been demonstrated in that patent.

[0027] (1) Preparation of fermentation seed liquid Using a -80℃ cryopreserved Lactobacillus paracasei GP66 glycerol culture tube, a small amount of bacterial culture was streaked onto MRS solid medium under aseptic conditions and incubated at 37℃ for 24 hours. Plump, milky-white single colonies grew, completing the primary strain revival. Single GP66 colonies were aseptically picked and activated twice via liquid subculturing, incubated at 37℃ for 24 hours, maintaining a viable count of 1.7 × 10⁻⁶ in the seed culture. 9CFU / mL was used to obtain Lactobacillus paracasei GP66 seed culture.

[0028] (2) Preparation of fermentation substrate Weigh out 300g of Gastrodia elata powder and 100g of Panax notoginseng powder according to a 3:1 mass ratio of Gastrodia elata to Panax notoginseng powder, add 4L of purified water, and prepare a composite fermentation substrate with a material-to-liquid ratio of 1:10. Add 2% soybean peptone, 3% glucose, and 0.25% dipotassium hydrogen phosphate to the composite fermentation substrate, stir well, boil at 100℃ for 30 minutes for sterilization, and allow to cool naturally at room temperature to obtain the fermentation substrate.

[0029] (3) Constant temperature fermentation and low temperature after-ripening After inoculating the composite fermentation substrate with 1% (v / v) of Lactobacillus paracasei GP66 seed culture, the initial pH was adjusted to 6.5, and the mixture was allowed to ferment at 37℃ for 24-72 hours. After fermentation, the mixture was allowed to mature at 25℃ for 10 hours. After maturation, the mixture was sterilized at 90℃ for 30 minutes. After centrifugation at 6000 r / min for 5 minutes, the supernatant was collected to obtain the fermentation broth.

[0030] (4) Sampling and determination To dynamically track the transformation degree of the medicinal substrate and the changes in active ingredients during fermentation, and to reflect the substrate utilization by the strains using polysaccharide consumption, gastrodin, barisonin A, notoginsenoside, and ginsenoside Rg3 were selected as biomarkers of characteristic efficacy for Gastrodia elata and Panax notoginseng. To screen for the optimal primary fermentation time by observing content changes at multiple time points, aseptic samples were taken at 24h, 36h, 48h, 60h, and 72h of fermentation. After sampling, the samples were post-ripened at 25℃ for 10h and then sterilized. The samples were centrifuged at 6000r / min for 5min, and the contents of polysaccharides, gastrodin, barisonin A, notoginsenoside, and ginsenoside Rg3 in the supernatant were determined by HPLC and the phenol-sulfuric acid method, respectively. The results are shown in Table 1. As shown in Table 1, during the fermentation period of 24–48 hours, *Lactobacillus paracasei* GP66 rapidly utilizes the matrix polysaccharides, resulting in a continuous and significant decrease in polysaccharide content. During this period, the β-glucosidase secreted by the strain continuously hydrolyzes gastrodin-bound glycosides, leading to the continuous accumulation of gastrodin, which reaches its peak content at 48 hours. Ginsenosides are continuously converted into the rare ginsenoside Rg3, reaching a relatively high level at 48 hours. After fermentation exceeds 48 hours, polysaccharide consumption tends to level off, and a large amount of gastrodin is converted into p-hydroxybenzyl alcohol, causing its content to decline significantly. The increase in Rg3 content is slight. Therefore, 48 hours was chosen as the fermentation time for this process.

[0031] Table 1. Content of active ingredients at different fermentation times Simultaneously, the contents of balithonoside A and notoginsenoside A decreased significantly after 48 hours of fermentation. The structural formula of balithonoside A is shown below. Figure 1 As shown. Barisoniside A is an organic ester glycoside with citric acid as its parent nucleus. Two of the three carboxyl groups in the citric acid parent nucleus form benzyl ester bonds with p-hydroxybenzyl-β-D-glucose. The remaining carboxyl groups in the citric acid parent nucleus are potential reaction sites.

[0032] The benzyl ester bond in the molecule can be selectively hydrolyzed by carboxylesterase secreted by lactic acid bacteria, exposing free carboxyl groups, which are then active in condensation reactions.

[0033] Panax notoginseng is a unique α-amino acid component of Panax notoginseng, and its chemical structure is as follows: Figure 2 As shown, the molecule contains a highly reactive α-primary amino group, which is a typical nucleophilic reaction site and can undergo dehydration condensation reaction with free carboxyl groups.

[0034] Based on the structural properties and reactivity of the two compounds mentioned above, and considering the rich enzyme system in the fermentation system, it can be inferred that barisonin A and notoginseng may have reacted under the catalysis of the bacterial enzyme system to generate new derivatives.

[0035] Example 2 This embodiment provides a method for separating and extracting substances from the fermentation liquid of Panax notoginseng and Gastrodia elata.

[0036] In this embodiment, the combined fermentation broth of Panax notoginseng and Gastrodia elata fermented for 48 hours was systematically separated and purified, and high-purity active components were obtained by targeted enrichment and separation.

[0037] The fermentation broth of Panax notoginseng and Gastrodia elata obtained in Example 1 after 48 hours of fermentation was centrifuged at 4℃ and 8000rpm for 10 minutes to remove bacterial cells and insoluble residues, and the clear supernatant was collected. The supernatant was concentrated to 1 / 3 of its original volume under reduced pressure at 40℃, and extracted three times with an equal volume of petroleum ether. The organic phase was discarded, and the pH of the lower aqueous phase was adjusted to 5.5. The supernatant was then purified using HPD100 macroporous resin. After pretreatment and column packing, the sample was loaded at a flow rate of 1 BV / h, with a loading volume of 5% of the resin mass. After loading, a gradient elution was performed sequentially: 2 BV of deionized water was used to remove highly polar impurities such as polysaccharides and inorganic salts; 3 BV of 20% ethanol was used to remove highly polar unreacted impurities such as gastrodin and notoginsenoside; 4 BV of 45% ethanol was used to elute and collect the target component in this segment; and finally, 3 BV of 70% ethanol was used to regenerate the resin. The collected 45% ethanol eluent was concentrated under reduced pressure at 40°C and then freeze-dried under vacuum to obtain a pale yellow purified product.

[0038] The purified product was dissolved in 50% acetonitrile to prepare a 10 mg / mL solution, filtered through a 0.22 μm organic filter membrane, and then purified by preparative high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 preparative column (250 mm × 20 mm, 10 μm), mobile phase acetonitrile-water (25:75, v / v), flow rate 10 mL / min, detection wavelength 220 nm, and injection volume 5 mL / injection. The main peak fraction with retention times of 8.5–9.5 min was collected, and the collected solutions from multiple injections were combined. Acetonitrile was removed under reduced pressure at 40 °C, followed by freeze-drying under vacuum to obtain a white powdery single derivative. The purity of this derivative was ≥98% as determined by HPLC normalization.

[0039] The above methods referenced the following literature: Nie Changping, Wu Dan, Yang Jun, et al. Preparation of balisine B reference standard by preparative high performance liquid chromatography [J]. Chinese Journal of Modern Applied Pharmacy, 2019, 36(9): 1055-1058. DOI: 10.13748 / j.cnki.issn1007-7693.2019.09.005. Qu Xiaomei, Li Ziyan, Liu Fang, et al. Study on the process of enriching gastrodin components with macroporous resin [J]. Journal of Traditional Chinese Medicine, 2021, 27(7): 69-73. DOI: 10.13862 / j.cnki.cn43-1446 / r.2021.07.017. CN202511419644.9 A method for simultaneously purifying gastrodin, barisonin and gastrodin polysaccharide from Gastrodia elata.

[0040] Example 3 Structural identification of derivatives In this embodiment, the derivative obtained in Example 2 was used as the sample. The system structure was analyzed by combining high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy (including two-dimensional correlation spectroscopy). The instruments used were a high-resolution mass spectrometer and a 300MHz nuclear magnetic resonance spectrometer. DMSO-d6 was used as the solvent and TMS was used as the internal standard.

[0041] In high-resolution mass spectrometry with positive ion mode, the derivative yielded a quasi-molecular ion peak at m / z 887.2583 [M+H]. + The corresponding molecular formula is C 37 H 46 N2O 23 The molecular weight is 886.25, the measured accurate molecular weight deviates from the theoretical calculation value by less than 5 ppm, and the elemental composition meets the requirements.

[0042] ¹H NMR spectrum analysis results show that the derivative simultaneously and completely contains the characteristic proton signals of the barisoniside diester core and notoginseng. Each signal can be clearly assigned: δ12.56 (1H, s) and δ12.39 (1H, s) in the low field region are the active proton signals of two free carboxyl groups, corresponding to the α-carboxyl group and the oxalyl terminal carboxyl group in the notoginseng structure; δ8.89 (1H, s) is the active amide proton (-NH-CO-COOH) of the notoginseng oxalyl segment, and δ8.32 (1H, d, J=7.0Hz) is the active proton of the newly formed amide bond (-NH-CO-). Its bimodal characteristics are consistent with the coupling effect of the adjacent α-methylene group, directly indicating that the carboxyl group and amino group have undergone amidation condensation.

[0043] In the aromatic hydrogen and skeletal region signals, δ6.99 (4H, d, J=7.5Hz) and δ6.89 (4H, d, J=7.5Hz) are AA'BB' type proton signals of two sets of para-substituted benzene rings, corresponding to the aromatic ring parts of the two gastrodin structural units in the molecule; δ6.62 (1H, s) is the active proton signal of the tertiary hydroxyl group on the citric acid skeleton, confirming that the tertiary alcohol structure of the citric acid core is completely preserved; δ2.62 (4H, s) are the two methylene protons (-C(OH)(CH2-COO-)2) on the citric acid skeleton, and the single-peak characteristics are completely consistent with the structural characteristics of uncoupled adjacent quaternary carbons.

[0044] In the sugar and fatty regions, δ5.80 (2H, d, J=7.0Hz) represents the terminal proton signal of two β-D-glucopyranose units, with coupling constants conforming to the β configuration; δ5.20 (4H, s) represents the benzyloxymethylene (Ar-CH2-O-) proton of two gastrodin units; and δ5.09 (1H, m) represents the α-methylene proton (-CH(NH-)-COOH) of notoginseng. The singlets at δ4.88, δ4.77, δ4.71, and δ3.94 correspond to the active protons of the hydroxyl groups on the glucose ring. The multiplets in the interval from δ4.20 to δ3.51 correspond to the protons at the H-2 to H-6 positions on the two glucose rings, respectively. The signal integral and chemical shift are consistent with the characteristics of β-D-glucopyranose. δ3.58 (1H, dd, J=12.4, 7.0Hz) and δ3.33 (1H, dd, J=12.4, 7.0Hz) correspond to the β-methylene protons (-CH2-NH-) of notoginseng.

[0045] Based on the analysis of the elemental composition by mass spectrometry and the structural fragments by proton NMR, it can be determined that the derivative is a novel hybrid molecule formed by the selective hydrolysis of balisine A to generate a diester monocarboxylic acid intermediate, and the free carboxyl group of the intermediate is covalently linked to the α-primary amino group of ginsenoside through an amide bond. This structure was discovered for the first time in a natural fermentation system.

[0046] The structure of the derivative is as follows: .

[0047] The spectral data of this derivative are as follows: ¹H NMR (300 MHz, DMSO-d6, δ, ppm) 12.56 (1H in carboxylic acid), 12.39 (1H in carboxylic acid), 8.89 (1H in sec. amide), 8.32 (1H in sec. amide), 6.99 (4H in 1-benzene), 6.89 (4Hin 1-benzene), 6.62 (1H in alcohol), 5.80 (2H in tetrahydropyran), 5.20 (4Hin methylene), 5.09 (1H in methine), 4.88 (2H in alcohol), 4.77 (2H inalcohol), 4.71 (2H in alcohol), 4.20 (2H in tetrahydropyran), 3.94 (2H inalcohol), 3.70 (2H in tetrahydropyran), 3.60 (4H in tetrahydropyran), 3.58(1H in methylene), 3.57 (2H in methylene), 3.51 (2H in methylene), 3.33 (1H in methylene), 2.62 (4H in methylene) Example 4 During the study of the aforementioned derivative, it was discovered that this substance possesses an agonistic function towards TPH1. Specifically, this embodiment uses a Caco-2 cell model to verify the agonistic function of this derivative towards TPH1.

[0048] In this embodiment, human colon adenocarcinoma cells Caco-2 were used as an in vitro intestinal cell model. By detecting the intracellular tryptophan hydroxylase 1 (TPH1) enzyme activity and 5-hydroxytryptamine (5-HT) synthesis, and combined with the TPH1 specific inhibitor reversal experiment, the agonistic function of the derivative on TPH1 and its promoting effect on intestinal 5-HT synthesis were verified.

[0049] (1) Cell culture Caco-2 cells were seeded in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured routinely in an incubator at 37°C, 5% CO2, and saturated humidity. Cells were passaged when confluence reached 80%–90%. Cells in logarithmic growth phase were then cultured at 2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 cell per well in 6-well cell culture plates and cultured until the cells were fully confluent and differentiated before being treated with a drug for 24 hours.

[0050] (2) Experimental grouping and drug administration The experiment was set up in 4 groups, with 6 replicates in each group.

[0051] Blank control group (Group C): Only an equal volume of culture medium was added; Barisoniside A group (PA group): final concentration in culture medium 50 μg / mL; Panax notoginseng group (Panax notoginseng group): final concentration in culture medium 50 μg / mL; Derivative group (Group I): final concentration in culture medium 50 μg / mL.

[0052] (3) Indicator detection ① Collect the cell supernatant from each group, centrifuge at 3000 r / min for 10 min at 4℃ to remove cell debris, and strictly follow the instructions of the 5-HT kit to detect the 5-HT content in the cells.

[0053] ② Discard the cell culture medium from each group. Wash the cells twice with pre-chilled PBS buffer in the wells, add cell lysis buffer, and lyse on ice for 30 min. Scrape the cells and sonicate to disrupt them. Centrifuge at 12000 rpm for 15 min at 4°C and collect the supernatant. Standardize the protein concentration using the BCA method. Detect TPH1 enzyme activity according to the TPH1 enzyme activity kit instructions.

[0054] (4) Test results At a concentration of 50 μg / mL, the 5-HT content and TPH1 enzyme activity in the balisinoside A group and the notoginseng group showed no significant difference compared with the blank control group, indicating that neither group exhibited a significant 5-HT-promoting effect at this dose. However, the 5-HT content and TPH1 activity in the derivative group were significantly higher than those in the blank group, and significantly higher than those in the PA group and the notoginseng group. The simultaneous increase in both indicators suggests that the derivative may promote 5-HT production in Caco-2 cells by upregulating the activity of the rate-limiting enzyme TPH1. These results reflect that the functional activity of this derivative is significantly superior to its precursors, balisinoside A and notoginseng monomer. Therefore, this derivative is a TPH1 agonist.

[0055] Table 2. 5-HT content and TPH1 enzyme activity in Caco-2 cells of each group The above cell experiments referenced the following literature: Yu Yongjun. Study on the effect of Bacteroides polymorpha and its metabolite succinic acid in antibiotic-induced depression in mice [D]. Zhejiang Academy of Medical Sciences, 2025. DOI:10.27826 / d.cnki.gzjyk.2025.000017. The outer membrane protein Amuc_1100 of Akkermansiamuciniphila promotes intestinal 5-HT biosynthesis and extracellular availability through TLR2 signaling.

[0056] Example 5 The effect of Panax notoginseng and Gastrodia elata fermentation liquid on sleep deprivation mice This embodiment refers to the method in the literature of Li Feifan et al. (Li Feifan, Zhang Yibo, Wang Jing, et al. Comparison and evaluation of three methods for preparing insomnia mouse models [J]. Chinese Journal of Tissue Engineering Research, 2026, 30(18):4685-4693.), and uses a multi-platform aquatic environment sleep deprivation system to establish a mouse sleep deprivation model and observe the effect of Panax notoginseng and Gastrodia elata fermentation liquid on the sleep behavior of mice.

[0057] (1) Animal grouping and model establishment Forty C57BL / 6 mice weighing 18-22g were randomly divided into four groups of ten each. They were housed in an animal room with constant temperature and humidity and a 12-hour light-dark cycle, with free access to food and water. After three days of acclimatization, the experiment was conducted.

[0058] Mice were randomly divided into four groups of 10 each: blank control group, sleep deprivation model group, TPH1 agonist group, and Panax notoginseng and Gastrodia elata fermentation broth group.

[0059] Blank control group (Group C): Normal feeding, no sleep deprivation, and daily gavage with an equal volume of physiological saline.

[0060] Model group (Group M): A sleep deprivation model was established using a modified multi-platform aquatic environment method. Mice were placed in a water tank containing multiple platforms, with the top of each platform approximately 1 cm above the water surface. The mice stood on the platforms, and when they fell asleep, their muscle tone decreased, causing them to fall into the water and awaken them, thus maintaining a state of continuous wakefulness. The model was established for 18 hours daily for 7 consecutive days, and an equal volume of physiological saline was administered by gavage daily during the modeling period.

[0061] TPH1 agonist group (TI group): The modeling method was the same as that of the model group. At the same time as modeling, TPH1 agonist solution was administered by gavage daily. 10 mg of TPH1 agonist was dissolved in DMSO to prepare a stock solution of 10 mg / mL. After sonication, the solution was diluted with physiological saline to 100 μg / mL and administered to the experimental group mice by gavage. The gavage dose was set at 1 mg / kg / day.

[0062] The fermented liquid group of Panax notoginseng and Gastrodia elata (SM group) was established using the same method as the model group. At the same time as the model was established, the fermented liquid of Panax notoginseng and Gastrodia elata was administered by gavage daily at a dose of 200 mg / kg / d, once a day, for 7 consecutive days.

[0063] (2) Sleep behavior testing One hour after the last administration, mice in each group were injected intraperitoneally with a threshold dose of sodium pentobarbital (40 mg / kg), and the duration of sleep onset and deep sleep were immediately observed and recorded.

[0064] (3) Sample collection and indicator detection After the sleep behavior test, blood was collected from the orbital venous plexus of mice. The serum was separated by centrifugation at 3500 r / min for 15 min at 4℃. The levels of 5-HT, corticosterone (CORT) and prostaglandin E2 (PGE2) in the serum were detected according to the ELISA kit instructions.

[0065] Mice were euthanized after blood collection, and distal colon tissue was quickly separated, rinsed with pre-cooled physiological saline, aliquoted, and frozen at -80°C. A portion of the colon tissue was homogenized, centrifuged, and the supernatant was collected. After BCA protein quantification, intestinal TPH1 enzyme activity was detected according to the kit instructions. Total RNA was extracted from another portion of the colon tissue, reverse transcribed into cDNA, and the relative mRNA expression level of the TPH1 gene was detected using real-time quantitative PCR. GAPDH was used as an internal reference gene to calculate the relative expression level of TPH1.

[0066] (4) Test results Compared with the blank control group, the model group mice showed significantly prolonged sleep onset time and significantly shortened deep sleep time. Simultaneously, serum corticosterone and prostaglandin E2 levels were significantly elevated, indicating that 7 days of sleep deprivation not only successfully disrupted the normal sleep structure of mice but also induced a significant stress response and release of pain mediators, mimicking the pathological state of stress headache. Furthermore, the model group showed significantly decreased serum 5-HT levels, intestinal TPH1 enzyme activity, and TPH1 mRNA expression levels, suggesting that long-term sleep deprivation severely inhibits the peripheral 5-HT synthesis pathway. This is not only an important cause of sleep disorders but also a key mechanism for pain sensitization and headache attacks. After intervention with TPH1 agonists and fermented extracts of Panax notoginseng and Gastrodia elata, respectively, all indicators of mice in both groups showed varying degrees of improvement compared to the model group: shorter sleep onset time, longer deep sleep time, increased serum 5-HT levels and intestinal TPH1 expression and activity, and decreased serum corticosterone and prostaglandin E2 levels. This suggests that both treatments can exert certain sleep-improving and headache-relieving effects by activating the TPH1 pathway. The improvement effect was more significant in the fermented extract group, with statistically significant differences in all indicators compared to the model group. The overall intervention effect was superior to that of the TPH1 agonist monotherapy group, reflecting the synergistic effect of multiple active components in the fermented extract. These results indicate that both fermented extracts of Panax notoginseng and Gastrodia elata and TPH1 agonists can promote peripheral 5-HT synthesis by upregulating intestinal TPH1 transcription and enzyme activity, simultaneously regulating sleep rhythm and the central descending pain inhibition system via the gut-brain axis pathway. While antagonizing sleep disorders caused by sleep deprivation, they effectively reduce stress response and lower pain mediator levels, exerting a synergistic effect in relieving stress headaches.

[0067] Table 3 PCR primer sequences Table 4. Results of sleep behavior and 5-HT-related indicators in mice of each group It should be understood that after reading the above teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A tryptophan hydroxylase 1 agonist, characterized in that: Its chemical structural formula is: 。 2. A fermentation liquid of Panax notoginseng and Gastrodia elata, characterized in that: Includes the tryptophan hydroxylase 1 agonist as described in claim 1; The Panax notoginseng and Gastrodia elata fermentation liquid is prepared by fermentation of a substrate including Panax notoginseng and Gastrodia elata using fermentation strains; The fermentation strain is Lacticaseibacillus paracasei GP66, with accession number CGMCC No. 32952.

3. A method for preparing the Panax notoginseng and Gastrodia elata fermentation liquid according to claim 2, characterized in that: Includes the following steps: The fermentation broth of Panax notoginseng and Gastrodia elata was prepared by inoculating 0.1-2% (v / v) of seed liquid containing the fermentation strain into a substrate including Panax notoginseng and Gastrodia elata, fermenting, sterilizing, and centrifuging.

4. The method for preparing Panax notoginseng and Gastrodia elata fermentation liquid according to claim 3, characterized in that: The fermentation time is 24-72 hours.

5. The method for preparing Panax notoginseng and Gastrodia elata fermentation liquid according to claim 3, characterized in that: The method for preparing the substrate including Panax notoginseng and Gastrodia elata includes the following steps: Prepare a compound fermentation substrate with a feed-to-liquid ratio of 1g:8-15mL by mixing Gastrodia elata and Panax notoginseng at a weight ratio of 2-4:

1. Add 2% soybean peptone, 3% glucose, and 0.25% dipotassium hydrogen phosphate to the compound fermentation substrate, stir well, sterilize, and cool to obtain the final product.

6. The method for preparing Panax notoginseng and Gastrodia elata fermentation liquid according to claim 3, characterized in that: The initial pH of the fermentation was adjusted to 6-7.

7. The method for preparing Panax notoginseng and Gastrodia elata fermentation liquid according to claim 3, characterized in that: The fermentation temperature is 30~38℃; The sterilization temperature is 85~100℃; The sterilization time is 20-40 minutes; The centrifuge speed is 5000~9000 r / min; The centrifugation time is 1~10 min.

8. The method for preparing Panax notoginseng and Gastrodia elata fermentation liquid according to claim 3, characterized in that: It also includes the post-ripening step; The post-ripening temperature is 20~28℃; The post-ripening time is 5-15 hours.

9. The method for preparing the tryptophan hydroxylase 1 agonist according to claim 1, characterized in that: Includes the following steps: The fermentation liquid of Panax notoginseng and Gastrodia elata as described in claim 2 was centrifuged, concentrated, extracted, and concentrated again to obtain a brownish-yellow crude extract; The brownish-yellow crude extract was eluted with a macroporous adsorption resin gradient to obtain a light yellow refined product. The pale yellow purified product was purified by high performance liquid chromatography to obtain tryptophan hydroxylase 1 agonist.

10. The application of the tryptophan hydroxylase 1 agonist according to claim 1, used in the preparation of a drug that promotes TPH1 enzyme / 5-HT expression; or An application of the Panax notoginseng and Gastrodia elata fermentation liquid according to claim 2, used in the preparation of drugs to relieve anxiety; or Applications in the preparation of medicines to improve sleep and / or headaches.

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