Application of quinic acid derivative

Abstract

The invention relates to the field of biological medicine, in particular to application of a quinic acid derivative. The quinic acid derivative comprises one or two of 3-O-p-coumaroyl quinic acid and 4-O-p-coumaroyl quinic acid, and the quinic acid derivative comprises one or two of 3-O-p-coumaroyl quinic acid and 4-O- The applications include treatment and / or prevention of depression. The quinic acid derivatives 3-O-p-coumaroyl quinic acid and 4-O-p-coumaroyl quinic acid can be used for improving the behavioristics of depressive mice; the compound can significantly increase dopamine and noradrenaline in the brain of a depressive mouse, reduce the levels of inflammatory factors IL-6 and TNF-alpha in the brain of the depressive mouse, increase the intestinal microbial diversity of the depressive mouse and significantly increase the BDNF level in the brain of the depressive mouse, and can be used as a drug for treating and / or preventing depression.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a quinic acid derivative. Background Technology

[0002] Depression is a major disease of the 21st century, characterized by high incidence, high suicide rates, high disability rates, and high relapse rates, making it a serious global public health problem and a prominent social issue. The World Health Organization predicts that by 2030, depression will become the most economically burdensome disease worldwide. Currently, treatment for depression mainly includes medication, psychotherapy, and other treatment methods, with medication being the most commonly used and effective. Clinical drug treatment primarily relies on chemically synthesized drugs, such as first-generation antidepressants: tricyclic antidepressants (TCAs) imipramine and amitriptyline; second-generation antidepressants: serotonin reuptake inhibitors (SSRIs) fluoxetine, paroxetine, sertraline, and citalopram; and third-generation antidepressants: serotonin / norepinephrine dual uptake inhibitors (SNRIs) venlafaxine and duloxetine. These drugs generally suffer from drawbacks such as significant side effects, slow onset of action, and high cost, resulting in less than ideal treatment outcomes and an urgent need to develop new antidepressant drugs. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art and provide an application of quinic acid derivative in the prevention and treatment of depression.

[0004] The technical solution adopted by the present invention to solve its technical problem is as follows: the application of a quinic acid derivative, wherein the quinic acid derivative comprises 3- O -p-Coumarylquinic acid, 4- O - One or both of coumaroylquinic acids; the applications include the treatment and / or prevention of depression.

[0005] 3- O - Coumaroylquinic acid, abbreviated as compound 1, has the following structural formula: .

[0006] 4- O - p-Coumaranoylquinic acid, abbreviated as compound 2, has the following structural formula: .

[0007] Preferably, the pure form of the quinic acid derivative or a mixture containing the quinic acid derivative is used for the treatment and / or prevention of depression.

[0008] Preferably, the quinic acid derivative is obtained by one or more of the following methods: chemical synthesis, biological extraction, and biological fermentation.

[0009] Preferably, the quinic acid derivative is synthesized from dextrorotatory quinic acid.

[0010] Preferably, the mixture containing the quinic acid derivative comprises one or two of the following: biological extract, biological fermentation product, and quinic acid derivative dispersion.

[0011] Preferably, the mixture containing the quinic acid derivative includes daylily extract.

[0012] Preferably, the daylily extract is extracted using water as a solvent and by cold soaking extraction.

[0013] Preferably, the daylily extract is extracted using water as a solvent by cold soaking, and then further purified by adsorption onto macroporous resin: eluted with an ethanol-water solution, and the eluent is collected.

[0014] Preferably, the macroporous resin includes one or more of AB-8, D101, HZ-816, and HPD100.

[0015] Preferably, the volume percentage of ethanol in the ethanol-water solution is 30% to 40%.

[0016] This invention has the following beneficial effects: Research has found that quinic acid derivative 3- O -p-Coumarylquinic acid and 4- O - Coumaroylquinic acid can improve the behavior of depressed mice; the quinic acid derivative can significantly increase dopamine and norepinephrine in the brain of depressed mice, reduce the levels of inflammatory factors IL-6 and TNF-α in the brain of depressed mice, increase the diversity of gut microbiota in depressed mice, and significantly increase the level of BDNF in the brain of depressed mice, and can be used as a drug for the treatment and / or prevention of depression.

[0017] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 It is compound 1 1 H-NMR spectrum; Figure 2 It is compound 1 13 C-NMR spectrum; Figure 3 It is compound 2.1 H-NMR spectrum; Figure 4 It is compound 2. 13 C-NMR spectrum; Figure 5 The graph shows the behavioral results of mice in evaluating the antidepressant activity of compounds 1 and 2: Figure 5 (A) Sugar water preference index Figure 5 (B) The immobility time in the tail suspension experiment Figure 5 (C) The immobility time during the forced swimming experiment; Figure 6 The graph shows the effects of compounds 1 and 2 on monoamine neurotransmitters in the mouse brain: Figure 6 (A) DA content in the cerebral cortex Figure 6 (B) DA content in the hippocampus Figure 6 (C) Norepinephrine (NE) content in the cerebral cortex Figure 6 (D) Hippocampal NE content, Figure 6 (E) 5-HT content in the cerebral cortex Figure 6 (F) Hippocampal NE content; Figure 7 This is a graph showing the effects of compounds 1 and 2 on inflammatory factors in the mouse brain: Figure 7 (A) IL-6 content in the cerebral cortex Figure 7 (B) Hippocampal IL-6 content, Figure 7 (C) TNF-α content in the cerebral cortex Figure 7 (D) Hippocampal TNF-α content; Figure 8 The graph shows the effects of compounds 1 and 2 on BDNF levels in the mouse brain: Figure 8 (A) Cerebral cortex, Figure 8 (B) Hippocampus; Figure 9 The following is a graph showing the results of behavioral tests in mice during a study on the effects of inhibitor H-89 on the antidepressant activity of compounds 1 and 2: Figure 9 (A) Sugar water preference index Figure 9 (B) The immobility time in the tail suspension experiment Figure 9 (C) The immobility time during the forced swimming experiment; Figure 10 The graph shows the effect of inhibitor H-89 on compounds 1 and 2: Figure 10 (A) BDNF content in the mouse cerebral cortex Figure 10 (B) BDNF content in the mouse hippocampus; Figure 11 (A) and Figure 11 (B) are the HPLC-Q-TOF-MS total ion chromatogram and UV spectrum of 35% HCW daylily extract; Figure 12 The following is a graph showing the behavioral results of mice in the evaluation of the antidepressant activity of 35% HCW extract of daylily: Figure 12 (A) Sugar water preference index Figure 12 (B) The immobility time in the tail suspension experiment Figure 12 (C) Incubation period after feeding; Figure 13 Here is a graph showing the effect of 35% HCW extract of daylily on monoamine neurotransmitters in the mouse brain: Figure 13 (A) DA content in the cerebral cortex Figure 13 (B) DA content in the hippocampus Figure 13 (C) Norepinephrine (NE) content in the cerebral cortex Figure 13 (D) Hippocampal NE content, Figure 13 (E) 5-HT content in the cerebral cortex Figure 13 (F) Hippocampal 5-HT content; Figure 14 The graph shows the effect of 35% HCW extract of daylily on BDNF levels in the mouse brain: Figure 14 (A) Cerebral cortex, Figure 14 (B) Hippocampus; Figure 15 This is a graph showing the effect of 35% HCW extract of daylily on inflammatory factors in the mouse brain: Figure 15 (A) IL-6 content in the cerebral cortex Figure 15 (B) Hippocampal IL-6 content, Figure 15 (C) TNF-α content in the cerebral cortex Figure 15 (D) Hippocampal TNF-α content; Figure 16 The graph shows the effect of 35% HCW extract of daylily on oxidative stress factors in the mouse brain: Figure 16 (A) MDA content in the cerebral cortex, Figure 16 (B) MDA content in the hippocampus Figure 16 (C) SOD content in the cerebral cortex, Figure 16 (D) SOD content in the hippocampus. Detailed Implementation

[0019] To make the objectives, solutions, and beneficial technologies of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be noted that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0020] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0021] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, "multiple" in "one or more" means two or more, and "more than" in "one or more" means two or more.

[0022] This article contains some technical terms from the field. Below is an explanation of some of these terms: Dopa: Dopa; DA: Dopamine; NE: Norepinephrine; BDNF: Brain-derived neurotrophic factor; 5-HT: 5-hydroxytryptamine; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; 4-Hydroxycinnamic acid: 4-hydroxycinnamic acid; Tyrosine: Tyrosine; MDA: Malondialdehyde; SOD: Superoxide dismutase.

[0023] An embodiment of the present invention provides an application of a quinic acid derivative, the quinic acid derivative comprising 3- O -p-Coumarylquinic acid, 4- O - One or both of coumaroylquinic acids; the applications include the treatment and / or prevention of depression.

[0024] 3- O - Coumaroylquinic acid, abbreviated as compound 1, has the following structural formula: .

[0025] 4- O - p-Coumaranoylquinic acid, abbreviated as compound 2, has the following structural formula: .

[0026] In some embodiments of the present invention, a pure form of the quinic acid derivative or a mixture containing the quinic acid derivative is used for the treatment and / or prevention of depression.

[0027] In some embodiments of the present invention, the quinic acid derivative is obtained by one or more of the following methods: chemical synthesis, biological extraction, and biological fermentation.

[0028] In some embodiments of the present invention, the quinic acid derivative is derived from synthesis using dextrorotatory quinic acid as a raw material.

[0029] In some embodiments of the present invention, the mixture containing the quinic acid derivative includes one or two of the following: biological extracts, biological ferments, and quinic acid derivative dispersions.

[0030] In some embodiments of the invention, the mixture containing the quinic acid derivative includes daylily extract. The daylily extract was detected to contain 3- O -p-Coumarylquinic acid and 4- O - p-Coumarylquinic acid.

[0031] In some embodiments of the present invention, the daylily extract is extracted using water as a solvent and by cold soaking extraction.

[0032] In some embodiments of the present invention, the daylily extract is extracted using water as a solvent by cold soaking, and then further purified by adsorption onto macroporous resin: eluted with ethanol-water solution, and the eluent is collected.

[0033] In some embodiments of the present invention, the macroporous resin includes one or more of AB-8, D101, HZ-816, and HPD100.

[0034] In some embodiments of the present invention, the volume percentage of ethanol in the ethanol-water solution is 30% to 40%.

[0035] Example The following embodiments describe the disclosure of the present invention in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of the present invention.

[0036] In the elution process, the mobile phase ratio is based on volume ratio by default; otherwise, unless otherwise stated, all parts, percentages and ratios reported in the following examples are based on weight.

[0037] Unless otherwise stated, all reagents used in the examples are available commercially or synthesized using conventional methods and are ready for use without further processing. Unless otherwise stated, all instruments used in the examples are available commercially.

[0038] The following is some information about the experimental materials involved in the examples: AB-8, D101, HZ-816, HPD100 macroporous adsorption resins and ethanol were purchased from Tianjin Guangfu Fine Chemical Research Institute and Shanghai Guoyao Group for the crude separation of water extracts from flowering daylilies; fluoxetine hydrochloride dispersible tablets (Elilly Suzhou Ltd., USA) were used as positive control drugs; Mouse 5-HT kit, Mouse NE kit, Mouse BDNF kit, Mouse DA kit, Mouse TNF-α kit and Mouse IL-6 kit (MU30044 100 tubes / 96 samples) were purchased from Bioswamp (China) for the determination of monoamine neurotransmitters, BDNF and inflammatory factors in the brain; malondialdehyde kit (MDA) and superoxide dismutase kit (SOD) (MS1401 100 tubes / 96 samples, Suoqiao Biotechnology, China) were used for the determination of peroxides.

[0039] Statistical methods used in the examples: SPSS 16.0 was used for statistical analysis. The level of statistical significance was set at P ≤ 0.05. Quantitative data were expressed as mean ± standard deviation (x ± s). Leven's test was used to test for normality and homogeneity of variance. If normality and homogeneity of variance were met (P > 0.05), one-way ANOVA and LSD test were used for statistical analysis. If normality and homogeneity of variance were not met (P ≤ 0.05), the Kruskal-Wallis test was used. If the Kruskal-Wallis test was statistically significant (P ≤ 0.05), Dunnett's test (a non-parametric method) was used for comparative analysis. Statistical differences and biological significance were considered in the evaluation.

[0040] To date, the pathophysiological basis of depression remains unclear, with several theories vying for dominance. The main neurophysiological bases for depression include the monoamine hypothesis, the neurotrophic hypothesis, and the stress hypothesis. The monoamine hypothesis is currently the most well-established and classic, and the vast majority of antidepressants are developed based on it. However, with the continuous advancements in human health and gut microbiota research in recent years, studies have shown a close link between the occurrence of depression and changes in gut microbiota, giving rise to the gut microbiota hypothesis. Therefore, research into the mechanisms of antidepressants may involve the monoamine hypothesis, the neurotrophic hypothesis, the stress hypothesis, and the gut microbiota hypothesis.

[0041] Monoamine Hypothesis: First proposed in the 1960s, the monoamine hypothesis revealed that reserpine, an antihypertensive drug, caused depressive symptoms in 20% of patients. Subsequent studies showed that reserpine depletes 5-HT and catecholamines (including NE and DA) in the central nervous system, thus demonstrating a direct correlation between the occurrence of depression and the reduction of monoamine neurotransmitters. The classic monoamine hypothesis posits that the release of monoamine neurotransmitters 5-HT, NE, and DA, which play a crucial role in synaptic signal transmission in the brain's diffuse regulatory system, is directly related to the onset of depression. When the release of monoamine neurotransmitters 5-HT, NE, and DA decreases, symptoms of depression appear, such as depressed mood, sadness, loss of interest, and feelings of guilt. Clinically, antidepressants based on the monoamine hypothesis dominate the use of drugs, such as selective 5-HT reuptake inhibitors like fluoxetine hydrochloride and paroxetine, and dual 5-HT / NE reuptake inhibitors like venlafaxine and duloxetine. Currently, the mainstream approach to developing antidepressants based on the monoamine hypothesis is a multi-target strategy.

[0042] The neurotrophic hypothesis: Clinical observations have revealed that deficiency and metabolic disorders of brain-derived neurotrophic factor (BDNF), responsible for neurogenesis, nutrition, and remodeling in the hippocampus, are important mechanisms contributing to depression. Studies have shown that direct perfusion of BDNF into the hippocampus of animal receptors can produce a significant antidepressant effect, while depriving animals of BDNF or its receptors results in depressive symptoms and interferes with the effects of antidepressants. Therefore, low BDNF levels are closely related to the development of depression.

[0043] The stress hypothesis: Stress refers to a series of non-specific responses that occur in the body after being stimulated by stressors, before specific pathological damage occurs. This hypothesis mainly involves hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis. When the body suffers prolonged stress, the stress acts on the hypothalamus, prompting it to release corticotropin-releasing hormone (CRF). CRF then promotes the pituitary gland to release adrenocorticotropic hormone (ACTH), which in turn promotes the adrenal gland to release cortisol (CORT). When the concentration of CORT in plasma and urine is significantly higher than normal, it damages the hippocampus, leading to a decrease in neurotransmitters and brain-derived neurotrophic factor, thereby inducing depression. Simultaneously, elevated CORT concentrations produce inflammatory factors (such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α),) causing central nervous system inflammation, impairing the conduction and plasticity of hippocampal neurons, and inducing or exacerbating depression. Recent studies have shown that oxidative stress products caused by long-term stress induce damage to neurons in the central nervous system, which is also one of the important mechanisms leading to the pathogenesis of depression.

[0044] The Gut Microbiota Hypothesis: This hypothesis posits a close link between depression and an individual's gut microbiota. Both clinical evidence and animal studies demonstrate significant differences in the gut microbiota between individuals with depression and healthy individuals. Researchers transplanted fecal matter from depressed patients and healthy individuals into germ-free mice (lacking gut microbiota). Mice receiving fecal matter from depressed patients exhibited typical depressive behaviors, and high-throughput 16S rRNA sequencing revealed significant differences in the gut microbiota between these two groups. Furthermore, researchers disrupted the gut microbiota of mice using antibiotics and unhealthy diets, finding an increased risk of depression. Restoring gut microbiota balance through probiotics, prebiotics, a healthy diet, and fecal microbiota transplantation improved depression-related symptoms. All these studies indicate a close association between abnormal gut microbiota and depression. Gut microbiota primarily influence brain function through the gut-brain axis system. Currently, the specific relationship between abnormalities in the gut microbiota-gut-brain axis system and the occurrence of depression is unclear. The possible mechanism is that abnormalities in the gut microbiota lead to abnormalities in carbohydrate and amino acid metabolism, resulting in abnormal intestinal function. Abnormal intestinal function affects the release of monoamine neurotransmitters in the brain and neuronal plasticity through pathways such as the vagus nerve, inflammatory factors, and the hypothalamus-pituitary-adrenal axis, thus leading to the occurrence of depression.

[0045] The embodiments will focus on the mechanism research of various hypotheses on the formation of depression.

[0046] Example 1 (a) Synthesis of Compound 1 Starting with dextrorotatory quinic acid ((-)-quinic acid), esterification of the 1-carboxyl group and 5-hydroxyl group in (-)-quinic acid was carried out in the presence of toluenesulfonic acid-hydrate (p-TsOH) and N,N-dimethylformamide solution (DMF) to generate Cp1. Cp1 was dissolved in N,N-dimethylformamide, and imidazole and tert-butyldimethylchlorosilane were added. The mixture was refluxed to generate a mixture of Cp2 and Cp3. The mixture of Cp2 and Cp3, along with Cp4, was dissolved in dichloromethane, and DMAP and N,N'-dicyclohexylcarboimide (DCC) were added for catalytic reaction. After purification, 3- O - p-Coumaroylquinic acid (yield 40%).

[0047] The synthesis reaction formula for compound 1 can be written as: The specific synthetic method of compound 1 is as follows: (1) Synthesis of Cp1: Dextrorotatory quinic acid (-quinic acid, 5 g, 26 mmol) and p-toluenesulfonic acid hydrate (495 mg, 2.6 mmol) were dissolved in 50 mL of toluene, and then 12 mL of N,N-dimethylformamide solution (DMF) was added. The mixture was refluxed in an oil bath at 150 °C for 18 h. After cooling to room temperature, 100 mL of water was added to the reaction solution, and the layers were separated. The upper layer was extracted multiple times with n-butanol. The extracts and lower layers were combined and concentrated under reduced pressure. The solution was separated by column chromatography to obtain a white solid, which was the target product; the yield was approximately 82%. The mass spectrometry, carbon spectroscopy, and proton spectroscopy data are as follows: white solid, HRMS (ESI) － ):theory m / z : 173.0450, Molecular formula: C7H9O5 － [MH] － ;actual m / z : 173.0456. 1 H-NMR (400 MHz, DMSO-d6): δ 5.90 (s, 1H), 5.23 (d, J = 4 Hz, 1H), 4.84 (d, J = 8 Hz, 1H), 4.62 (t, J = 4 Hz, 1H), 3.82 (q, J = 4 Hz, 1H),3.54-3.47 (m, 1H), 2.29-2.25 (m, 1H), 2.14-2.09 (m, 1H), 1.88-1.83 (m, 1H),1.70 (t, J= 12 Hz, 1H). 13 C-NMR (100 MHz, DMSO-d6): δ 178.10, 76.33, 71.97, 65.99, 65.62, 37.13 (2×CH).

[0048] (2) Synthesis of a mixture of Cp2 and Cp3 Lactone Cp1 (6 g, 34.50 mmol) was dissolved in N,N-dimethylformamide (65 mL), and imidazole (128.52 mmol) and tert-butyldimethylchlorosilane (6 g, 40.00 mmol) were added at 0 °C. The reaction was allowed to proceed for 30 min, and then stirred at room temperature for another 1 h. The reaction mixture was poured into 100 mL of water and repeatedly extracted with ethyl acetate (3 times, 80 mL each time), washed with water (3 times, 200 mL each time), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified to obtain a white solid. Nuclear magnetic resonance (NMR) analysis determined that this was a mixture of the target products Cp2 and Cp3 (difficult to separate due to their similar polarities), with a yield of approximately 62%. The mass spectrometry, carbon spectroscopy, and proton NMR data are as follows: white solid, HRMS (ESI). + ):theory m / z : 289.1466, Molecular formula: C 13 H 25 O5Si + [M+H] + ;actual m / z : 289.1479. 1 H-NMR (400 MHz, DMSO-d6): δ 5.96 (s, 1H), 5.02 (d, J = 4 Hz, 1H), 4.64 (t, J = 4 Hz, 1H), 3.84 (q, J = 4 Hz, 1H), 3.71-3.65 (m, 1H), 2.32 (d, J = 12 Hz, 1H), 2.15-2.10 (m, 1H), 1.88-1.76 (m, 2H), 0.87 (s, 9H), 0.05 (d, J = 8 Hz, 6H). 13 C-NMR (100 MHz, DMSO-d6): δ177.93, 177.88, 76.29, 76.11, 71.98, 71.81, 68.11, 67.80, 65.09, 65.79, 39.90, 39.51, 37.23, 36.93, 26.22 (6×CH), 18.41, 18.37, -3.89, -4.34, -4.44, -4.68.

[0049] (3) Synthesis of Cp4 Under ice bath conditions (0°C), 4-dimethylaminopyridine (DMAP) was dissolved in pyridine solution (20 mL), and then 4-hydroxycinnamic acid (10 g, 60.92 mmol) was added to form a suspension; the reaction was carried out at 0°C for 30 min, followed by the addition of acetic anhydride (9.32 g, 91.38 mmol); the mixture was then placed at room temperature and stirred for 3 h. After the reaction was completed, the reaction solution (yellow-orange solution) was poured into ice water (100 mL), and then HCl was added to slowly precipitate a white precipitate; the precipitate was filtered using a vacuum filtration flask while being slowly washed with a large amount of distilled water, the waste liquid was discarded, the white precipitate was collected, and concentrated under reduced pressure to obtain the target product Cp4, with a yield of approximately 90%. Its mass spectrometry, carbon spectroscopy, and proton spectroscopy data are as follows: white solid, HRMS (ESI) + ):theory m / z : 207.0652, Molecular formula: C 11 H 11 O4 + [M+H] + ;actual m / z : 207.0655. 1 HNMR (400 MHz, DMSO-d6): δ 12.41 (s, 1H), 7.74 (d, J = 8 Hz, 2H), 7.63 (d, J =16 Hz, 1H), 7.18 (d, J = 8 Hz, 2H), 6.52 (d, J = 16 Hz, 1H), 2.28 (s, 3H). 13 C-NMR (100 MHz, DMSO-d6): δ 169.47, 167.99, 152.29, 143.41, 132.39, 129.87 (2×CH), 122.80 (2×CH), 119.77, 21.30.

[0050] (4) Synthesis of Compound 1 The reactants Cp4 (0.83 g, 4 mmol), a mixture of Cp2 and Cp3 (576 mg, 2 mmol), were dissolved sequentially in dichloromethane (80 mL) and stirred at 0 °C. Then, DMAP (244 mg, 2 mmol) and N,N'-dicyclohexylcarboimide (DCC) catalyst (1.24 g, 2 mmol) were added, and the mixture was stirred for 10 min. The mixture was then transferred to room temperature and reacted for 24 hours. After the reaction was completed, the solvent was removed, and the mixture was purified by column chromatography to obtain a white solid. This white solid was still a mixture, making it difficult to obtain a single substance. The white solid (300 mg) obtained in the previous step was purified and dissolved in tetrahydrofuran (3 mL) solution. 2M hydrochloric acid (9 mL) was added, and the mixture was stirred at 40 °C for 96 h. After the reaction was complete, excess saturated sodium chloride solution was added, and the organic layer was extracted with ethyl acetate (3 times, 30 mL each time). The mixture was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The white solid, the target product compound 1 (3-), was obtained by high-speed countercurrent chromatography. O - p-Coumaroylquinic acid), yield approximately 40%. Compound 1 1 See H-NMR image Figure 1 Compound 1 13 C-NMR spectrum can be seen Figure 2 .

[0051] (ii) Synthesis of Compound 2 First, Cp1 was synthesized using the same method as in the synthesis of compound 1. Cp1 was dissolved in DMF, and DMAP, a special catalyst tetrabutylammonium iodide, and triethylamine were added sequentially. Finally, tert-butyldimethylsilyl chloride was added to protect the 3-hydroxyl group in the Cp1 structure, thus generating Cp3. Cp3 and Cp4 were dissolved in dichloromethane, and DMAP and DCC catalysts were added. After the reaction was completed and purified, 4- O - p-Coumaroylquinic acid (yield 45%).

[0052] The synthesis reaction formula for compound 2 can be written as: The specific synthesis method of compound 2 is as follows: (1) The synthesis of Cp1 is the same as that of compound 1.

[0053] (2) Synthesis of Cp3 Maintaining an environment of -10°C, Cp1 (3.54 g, 20.3 mmol) was dissolved in DMF (40 mL), followed by the sequential addition of DMAP (350 mg, 2.86 mmol), a special catalyst tetrabutylammonium iodide (378 mg, 1.02 mmol), and triethylamine (3.4 mL, 24.40 mmol), and finally tert-butyldimethylsilyl chloride (3.8 g, 25.00 mmol). The solution was stirred at -10°C for 1 h, then transferred to room temperature and stirred for another 5 h. After the reaction was complete, the reaction solution was diluted with ethyl acetate (200 mL) to obtain a yellow solution (containing a large amount of white precipitate), which was filtered using a vacuum filtration flask. The filtrate was washed sequentially with 1M hydrochloric acid (150 mL) and saturated sodium chloride solution (3 times, 150 mL each time), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Column chromatography yielded a white solid, the target product Cp3, with a characteristic odor, and a yield of approximately 65%. Its mass spectrometry, carbon spectrum and proton spectrum data are as follows: white solid, HRMS (ESI) + ):theory m / z : 289.1466, Molecular formula: C 13 H 25 O5Si + [M+H] + ;actual m / z : 289.1479. 1 H-NMR (400MHz, DMSO-d6): δ 5.96 (s, 1H), 5.02 (d, J = 4 Hz, 1H), 4.64 (t, J = 8 Hz, 1H),3.85-3.82 (m, 1H), 3.71-3.65 (m, 1H), 2.32 (d, J = 8 Hz, 1H), 2.15-2.10 (m,1H), 1.88-1.76 (m, 2H), 0.87 (s, 9H), 0.05 (d, J = 4 Hz, 6H). 13 C-NMR (100 MHz, DMSO-d6): δ 177.93, 76.30, 71.81, 68.11, 65.90, 39.55 (3×CH), 36.93, 26.22 (3×CH), 18.41, -4.33, -4.43.

[0054] (3) The synthesis of Cp4 is the same as that of compound 1.

[0055] (4) Synthesis of compound 2 The reactants Cp4 (0.83 g, 4 mmol) and Cp3 (576 mg, 2 mmol) were dissolved sequentially in dichloromethane (80 mL). The mixture was stirred at 0 °C to dissolve the reactants. Then, DMAP (244 mg, 2 mmol) and DCC catalyst (1.24 g, 2 mmol) were added, and the mixture was stirred for 10 min. The reaction was then allowed to proceed at room temperature for 24 hours. After the reaction was complete, the solvent was removed, and the product was purified by column chromatography to obtain a white solid (crude product).

[0056] The obtained white solid (300 mg) was simply purified and dissolved in tetrahydrofuran (3 mL) solution. 2M hydrochloric acid (9 mL) was added, and the mixture was stirred at room temperature for 48 h. After the reaction was complete, excess saturated sodium chloride solution was added to the reaction solution, followed by extraction with ethyl acetate (3 times, 30 mL each time). The mixture was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The white solid, the target product compound 2 (4-), was obtained by high-speed countercurrent chromatography. O - p-Coumaroylquinic acid), yield approximately 45%. Compound 2 1 See H-NMR image Figure 3 Compound 2 13 C-NMR spectrum can be seen Figure 4 .

[0057] (III) Study on the antidepressant activity and mechanism of action of compounds 1 and 2 (1) Evaluation of the antidepressant activity of compound 1 and compound 2 One hundred and twenty male ICR mice, weighing 18.0 - 22.0 g, were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., with the experimental animal production license number: SCXK(Xiang) 2019 - 0004. Except for 10 mice in the normal control group, the remaining mice were subjected to chronic unpredictable stress (CUMS) modeling, including food and water deprivation (24 h), 4°C ice water bath (5 min), foot shock (1 min, 2 mA), noise (6 h), tail clamping (2 min), wet cage (adding 150 mL of water to the bedding, 24 h), circadian rhythm inversion and restraint (12 h) (placing the mice in a 50 mL centrifuge tube with 4 - 5 ventilation holes with a diameter of 0.5 mm on the tube wall), etc. The animals needed to be deprived of food and water during restraint. Forty-two days after modeling the mice, sucrose preference test was conducted, and according to the test results and the body weight of the mice, they were randomly grouped into: normal group (N), model group (M), fluoxetine hydrochloride group (Y, 5.2 mg / kg / day), compound 1 group and compound 2 group (30 mg / kg / day) (a total of 5 groups, with 10 mice in each group). The normal group and the model control group were given 0.4 mL of distilled water by gavage every day, and the remaining groups were given the corresponding medicinal solutions, with the administration volume of 0.4 mL. The administration was continued for 35 days, and chronic unpredictable stress was continued for the mice after each administration. Finally, after the last administration, behavioral tests such as sucrose preference test (SPT), tail suspension test (TST) and forced swimming test (FST) were conducted on the mice, and the results are shown in Figure 5 .

[0058] Results of sucrose preference test: As shown in Figure 5 (A), compared with the normal group (N), the sucrose preference index of the mice in the model group (M) decreased significantly (P < 0.05); compared with the model group (M), the sucrose preference index of the mice in the fluoxetine hydrochloride group (Y) increased significantly (P < 0.05); the preference indices of the compound 1 group (P < 0.01) and the compound 2 group (P < 0.001) increased significantly.

[0059] Results of tail suspension test: As shown in Figure 5 (B), compared with the normal group (N), the tail suspension immobility time of the mice in the model group (M) increased significantly (P < 0.01); compared with the model group (M), the tail suspension immobility time of the mice in the fluoxetine hydrochloride group (Y) and the compound 1 group decreased significantly (P < 0.05); the tail suspension immobility time of the mice in the compound 2 group decreased significantly (P < 0.01).

[0060] Results of forced swimming test: As shown in Figure 5 (C), compared with the normal group (N), the forced swimming immobility time of the mice in the model group (M) increased significantly (P < 0.05); compared with the model group (M), the forced swimming immobility time of the mice in the fluoxetine hydrochloride group (Y), the compound 1 group and the compound 2 group decreased significantly (P < 0.05).

[0061] (2) Study on the mechanism of action of compound 1 and compound 2 in antidepressant activity The research methodology is as follows: 1) In vivo metabolic studies based on the CA / Dopa / DA / NE pathways After the behavioral tests of mice were completed, the last administration was recorded as 0h. At 1.0h, the mice were enucleated and blood was collected. The blood was dropped into EP tubes containing anticoagulant. The blood samples were allowed to stand at room temperature for 1 hour and then centrifuged at 3000 rpm for 30 minutes at 4°C. The supernatant was used to detect DOPA in the mouse blood. After collecting blood from the orbital cavity, the mice were euthanized under ether anesthesia. The abdominal cavity was opened with surgical scissors, the intestines were cut open, and the intestinal contents were divided into two equal portions and placed in sterile EP tubes. One portion was used for LC-MS mass spectrometry analysis of intestinal metabolites (including 4-hydroxycinnamic acid, tyrosine, and DOPA), and the other portion was used for 16S DNA sequencing to detect changes in the types of microorganisms in the mouse intestinal contents. Simultaneously, mouse cerebral cortex and hippocampus were separately taken on ice and divided into two parts. One part was used to detect the levels of inflammatory factors (including IL-6 and TNF-α), monoamine neurotransmitters (including DA, NE and 5-HT) and BDNF in the cerebral cortex and hippocampus using an ELISA kit. The cerebral cortex in the other part was homogenized with 20 times PBS, and after centrifugation, the supernatant was collected. The levels of Dopa, DA and NE in the cerebral cortex were detected using mass spectrometry.

[0062] The mass spectrometry detection method for the CA / Dopa / DA / NE pathway is as follows: An Agilent 1290 HPLC tandem with a 6530Q-TOF-MS mass spectrometer was used, with an Agilent ZORBAX SB-C18 (4.6×250mm, 5µm) column. The elution system consisted of aqueous phase (A) 0.1% formic acid and organic phase (B) 0.1% formic acid and acetonitrile. Gradient elution program: 0–30 min, 5–45 vol% B; 30–40 min, 45–90 vol% B; 40–50 min, 90–95 vol% B; column temperature: 30℃; flow rate: 0.3 mL / min; injection volume: 5 μL. Negative ion full scan mode; full scan range: m / z 100~1000; Sheath gas temperature: 350℃; Sheath gas flow rate: 11.0L / min; Drying gas temperature: 300℃; Drying gas pressure: 45psi; Drying gas flow rate: 10L / min; Capillary voltage: 3500V; Fragmentation voltage: 175V.

[0063] Studies on mouse inflammatory factors and mouse gut microbiota: The determination of inflammatory factors (including IL-6 and TNF-α) in the mouse cerebral cortex and hippocampus was performed using ELISA kits, and the operation procedures were strictly followed according to the ELISA kit instructions. Gut microbiota 16S RNA sequencing was conducted in collaboration with Megagene Biotechnology Co., Ltd., who performed the 16S RNA sequencing and analysis of the mouse gut.

[0064] The following conclusions were reached after testing: Compounds 1 and 2 significantly increased dopamine and norepinephrine levels in the brains of depressed mice. Figure 6 The effects of compounds 1 and 2 on monoamine neurotransmitters in the mouse brain were statistically analyzed; for example... Figure 6 (A) Figure 6 As shown in (B), compared with the normal group (N), the DA levels in the cerebral cortex and hippocampus of mice in the model group (M) were significantly decreased (P<0.001); compared with the model group (M), the DA levels in the fluoxetine hydrochloride group (Y) were significantly increased in both the cerebral cortex and hippocampus (P<0.01, P<0.001), the DA levels in the compound 1 group were significantly increased in both the cerebral cortex and hippocampus (P<0.001), and the DA levels in the compound 2 group were also significantly increased in both the cerebral cortex and hippocampus (P<0.05, P<0.01); it can be found that compound 1 is more effective than the first-line clinical antidepressant fluoxetine hydrochloride in increasing DA in the cerebral cortex and hippocampus of depressed mice; like Figure 6 (C) Figure 6 As shown in (D), compared with the normal group (N), the NE levels in the cerebral cortex and hippocampus of the model group (M) mice were significantly decreased (P<0.0001); compared with the model group (M), the NE levels in the fluoxetine hydrochloride group (Y) mice were significantly increased in both the cerebral cortex and hippocampus (P<0.0001, P<0.001), compound 1 significantly increased the NE level in the hippocampus of depressed mice (P<0.05), and compound 2 significantly increased the NE level in the cerebral cortex of depressed mice (P<0.05); the positive control group (Y) of fluoxetine hydrochloride significantly increased the 5-HT levels in the cerebral cortex and hippocampus of depressed mice (P<0.001, P<0.005); Figure 6 (E) Figure 6 As shown in (F), compounds 1 and 2 also increased the level of 5-HT in the cerebral cortex and hippocampus of depressed mice, but the increase was not significant (P>0.005).

[0065] Compounds 1 and 2 can increase the levels of dopamine (DA) and norepinephrine (NE) in the cerebral cortex and hippocampus of depressed mice, but their specific mechanisms of action are unknown. To investigate how compounds 1 and 2 increase the levels of DA and NE in the brains of depressed mice, the metabolism of compound 1 (compound 1 and 2 are isomers) in mice was studied, using compound 1 as a representative. Mass spectrometry was used to discover that compound 1 ( m / z 337.0923) is metabolized in the intestine to 4-Hydroxycinnamicacid ( m / z 163.0395), and Tyrosine, a metabolite of 4-Hydroxycinnamic acid, was also detected in the mouse intestine. m / z 180.0661) and Dopa ( m / z The compound 1 (196.0610) metabolites were difficult to detect in the intestinal contents of mice in the normal group (N). Therefore, we hypothesized that compound 1 is metabolized in the intestine to 4-Hydroxycinnamic acid, which is then converted to Tyrosine by intestinal transaminases, and Tyrosine is converted to Dopa by hydroxylases. Dopa was also detected in the blood of mice, but the Dopa content in the compound 1 group was significantly higher than that in the normal group (N), indicating that Dopa produced in the intestine is absorbed into the blood. In the hippocampus of the brain, the levels of Dopa, dopamine, and norepinephrine in the compound 1 group were higher than those in the normal group (N), indicating that Dopa in the blood enters the hippocampus of the brain, where more dopamine and norepinephrine can be synthesized. Compound 1 increases dopamine and norepinephrine in the brains of depressed mice through the gut-brain axis. Compound 1 and Compound 2 are isomers, and Compound 2 should follow the same metabolic pathway in vivo. Therefore, we believe that Compound 1 and Compound 2 are metabolized into Dopa in the intestine. Dopa is absorbed into the bloodstream through the small intestine, circulates through the blood-brain barrier, and is synthesized into dopamine and norepinephrine in the hippocampus of the brain, thereby achieving an antidepressant effect. This is the specific reason why they increased the levels of dopamine and norepinephrine in depressed mice.

[0066] The effects of compounds 1 and 2 on the inflammatory factors IL-6 and TNF-α in the cerebral cortex and hippocampus were statistically analyzed. Figure 7 In; as Figure 7 (A) Figure 7 As shown in (B), compared with the normal group (N), the levels of IL-6 in the cerebral cortex and hippocampus of mice in the model group (M) were significantly increased (P<0.0001, P<0.05), indicating that an inflammatory response was induced in the mouse brain during the CUMS modeling process, resulting in the release of inflammatory factors;Figure 7 As shown in (A), compared with the model group (M), compounds 1 and 2 significantly reduced the level of the inflammatory cytokine IL-6 in the mouse cerebral cortex (P<0.0001); the reduction in the level of the inflammatory cytokine IL-6 in the mouse cerebral cortex by compounds 1 and 2 was consistent with that of the positive control drug fluoxetine hydrochloride, and the activity of compound 2 was stronger than that of compound 1; Figure 7 As shown in (B), compounds 1 and 2 were also able to reduce the level of the inflammatory factor IL-6 in the hippocampus of the mouse brain; like Figure 7 (C) and Figure 7 As shown in (D), compared with the normal group (N), the levels of TNF-α in the cerebral cortex and hippocampus of mice in the model group (M) were significantly increased (P<0.01); Compound 1 (P<0.05, P<0.01) and Compound 2 (P<0.01, P<0.01) significantly reduced the levels of the inflammatory factor TNF-α in the cerebral cortex and hippocampus of mice; the reduction of the levels of the inflammatory factor TNF-α in the cerebral cortex and hippocampus of mice by Compound 1 and Compound 2 was similar to that of the positive control drug fluoxetine hydrochloride, and the activity of Compound 2 was stronger than that of Compound 1.

[0067] Effects of Compound 1 and Compound 2 on gut microbiota: In mice in the normal control group, the number of gut microbial OTUs decreased by 24.6% after depression. However, treatment with compound 1 (P<0.05) and compound 2 (P<0.01) increased the number of gut microbial OTUs by 25.5% and 58.6%, respectively, indicating that administration of compounds 1 and 2 enriched the gut microbiota of depressed mice, with compound 2 showing a superior effect compared to compound 1. From the gut microbiota... α Diversity index data showed that, compared to the model group, compound 2 significantly increased the Sobs (P<0.001), Chao1 (P<0.001), ACE (P<0.001), and Shannon (P<0.01) indices of the gut microbiota in depressed mice, while compound 1 significantly increased the Shannon (P<0.05) index of the gut microbiota in depressed mice. These data indicate that both compound 1 and compound 2 can increase the diversity of gut microbiota. α The diversity of compounds is evident, and compound 2 is more effective than compound 1. Among all samples, the top 5 in terms of bacterial abundance are Firmicutes , Bacteroides , Actinobacteria、 Patescibacteria and Desulfobacterota Compared to the normal group (N), the model group (M) showed... Firmicutes Gate abundance significantly increased ( P <0.01), while after treatment with compound 1 (P<0.05) and compound 2 (P<0.01), itsFirmicutes Gate abundance was significantly reduced; compared with the normal group (N), the model group (M) showed a significant decrease. Bacteroides Gate abundance was significantly reduced ( P <0.05); while after treatment with compound 1 (P<0.05) and compound 2 (P<0.01), its Bacteroides Gate abundance increased significantly, while Bacteroides An increase in gut microbiota can significantly alleviate depressive symptoms.

[0068] Compounds 1 and 2 significantly increased BDNF levels in the cerebral cortex and hippocampus of depressed mice. Figure 8 The effects of compounds 1 and 2 on BDNF levels in the mouse brain were statistically analyzed. Clinical observations revealed that deficiency and metabolic disorders of brain-derived neurotrophic factor (BDNF), responsible for neurogenesis, nutrition, and remodeling in the cerebral cortex and hippocampus, are important causes of depression. Figure 8 As shown, the study found that after depression in normal mice, the levels of BDNF in the cerebral cortex and hippocampus were significantly reduced (P<0.001, P<0.01); while after treatment with compound 1 (P<0.05, P<0.01) and compound 2 (P<0.0001), the levels of BDNF in the cerebral cortex and hippocampus were significantly increased, and the increase in BDNF level was stronger than that of the positive control drug fluoxetine hydrochloride.

[0069] 2) Methods based on PKA / CREB / BDNF pathway research Evaluation of the antidepressant activity of compounds 1+H-89 and 2+H-89: Seventy male ICR mice (18.0~22.0g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (Experimental Animal Production License No.: SCXK (Xiang) 2019-0004). After one week of acclimatization feeding, 10 mice were randomly selected as the normal group (N), and the remaining mice were housed in isolation to establish a mouse model of depression induced by chronic unpredictable stress. Forty-two days after modeling in mice, based on sucrose preference data and body weight, the mice were randomly divided into seven groups: normal group (N), model group (M), fluoxetine hydrochloride group (5.2 mg / kg / d), compound 1 group (30 mg / kg / d), compound 1+H-89 group (30 mg / kg / d + 5 mg / kg / d), compound 2 group (30 mg / kg / d), and compound 2+H-89 group (30 mg / kg / d + 5 mg / kg / d), with 10 mice in each group. The normal group (N) and model group (M) were administered 0.4 mL of distilled water by gavage daily, while the other groups were administered the corresponding drug solutions in 0.4 mL volumes for 35 consecutive days. After each administration, the mice were subjected to chronic unpredictable stress. Finally, after the last administration, the mice were subjected to behavioral tests such as the sugar water preference test (SPT), tail suspension test (TST), and forced swimming test (FST). The levels of BDNF in the cerebral cortex and hippocampus of the mice were measured according to the instructions accompanying the corresponding ELISA kits.

[0070] PKA / CREB / BDNF pathway protein assay method: After the behavioral test of mice, the mice were euthanized by ether anesthesia, and the cerebral cortex and hippocampus of the mice were collected. Three cerebral cortex samples and three hippocampal samples were collected from each group (42 samples in total) and sent to Wuhan Youpin Biotechnology Co., Ltd. The protein extraction experimental procedure was carried out according to the product instructions. The company's protein immunoblotting experimental protocol includes SDS-PAGE electrophoresis, membrane transfer, immunoassay, chemiluminescence, and gel image analysis. Primary antibodies included CREB Rabbit mAb (1:1000 or 1:2000, UPharm Biotech, catalog number YP-Ab-17877), PKAα / β / γ cat (phospho Thr197) polyclonal antibody (1:1000 or 1:2000, UPharm Biotech, catalog number YP-Ab-14339), and CREB-1 (phospho Ser133) polyclonal antibody (1:1000 or 1:2000, UPharm Biotech, catalog number YP-Ab-01236). The expression levels of PKA and CREB in the mouse cerebral cortex and hippocampus were analyzed by Western blotting. The grayscale values ​​from ImageJ analysis were imported into an Excel spreadsheet, and bar charts were created for grayscale analysis.

[0071] The following conclusions were reached after testing: Compounds 1 and 2 increased BDNF levels via the PKA / CREB / BDNF pathway. Figure 9 These are the results of behavioral studies in mice investigating the effects of the inhibitor H-89 on the antidepressant activity of compounds 1 and 2. Figure 9 (A) It can be seen that compounds 1 and 2 significantly increased the saccharin preference index in depressed mice (P<0.0001), while when compounds 1+H-89 and 2+H-89 were administered to depressed mice, the saccharin preference index of the depressed mice was significantly decreased (P<0.0001, P<0.01). Figure 9 (B) It can be seen that compounds 1 and 2 significantly reduced the tail immobility time in depressed mice (P<0.0001), while when compounds 1+H-89 and 2+H-89 were administered to depressed mice, we found that the tail immobility time in depressed mice significantly increased (P<0.0001). Figure 9(C) It can be seen that compounds 1 and 2 significantly reduced the immobility time during forced swimming in depressed mice (P<0.001, P<0.0001), while the immobility time during forced swimming in depressed mice significantly increased when compounds 1+H-89 and compounds 2+H-89 were administered (P<0.0001). The above sucrose preference index, tail suspension, and forced swimming experiments once again confirmed that compounds 1 and 2 could improve the behavior of depressed mice. However, the addition of the inhibitor H-89 to compounds 1 and 2 did not significantly improve the behavior of depressed mice, indicating that compounds 1 and 2 no longer exhibited antidepressant activity after the addition of the inhibitor H-89.

[0072] Regarding BDNF, compared with the normal group (N), the BDNF content in the model group (M) mice was significantly decreased in both the cerebral cortex and hippocampus (P<0.0001). Figure 10 Compared with the model group (M), the levels of BDNF in the cerebral cortex and hippocampus of depressed mice were significantly increased in the compound 1 group (P<0.0001), while the BDNF levels in the cerebral cortex and hippocampus of depressed mice were significantly decreased after administration of H-89 (P<0.0001). The compound 2 group also showed similar activity, significantly increasing the levels of BDNF in the cerebral cortex and hippocampus of depressed mice (P<0.0001, P<0.001), while the levels of BDNF in the cerebral cortex and hippocampus of the compound 2+H-89 group were significantly decreased (P<0.05). These experimental results further demonstrate that compounds 1 and 2 can significantly increase the levels of BDNF in the cerebral cortex and hippocampus of depressed mice, but the levels of BDNF in the cerebral cortex and hippocampus of depressed mice were significantly decreased after administration of the inhibitor H-89, indicating that H-89 significantly inhibited the expression of BDNF in the cerebral cortex and hippocampus of mice. To investigate how the inhibitor H-89 affects BDNF production through the PKA / CREB / BDNF pathway, Western blotting analysis was performed on PKA, p-PKA, CREB, and p-CREB in the PKA / CREB / BDNF pathway. Results showed that compared to the normal group (N), the expression levels of p-PKA in the cerebral cortex and hippocampus of the model group (M) mice were decreased; compared to the model group (M), the expression levels of p-PKA in the cerebral cortex and hippocampus of mice treated with compounds 1 and 2 were increased; compared to the compound 1 group, the expression levels of p-PKA in the compound 1 + H-89 group were decreased in both the cerebral cortex and hippocampus; compared to the normal group (N), the expression levels of p-CREB in the cerebral cortex and hippocampus of mice treated with compounds 1 and 2 were increased. The expression level of EB was significantly reduced (P<0.0001); compared with the model group (M), the expression level of p-CREB in the cerebral cortex and hippocampus of mice in the fluoxetine hydrochloride group (Y) was significantly increased (P<0.001), and the expression level of p-CREB in the cerebral cortex and hippocampus of depressed mice in the compound 1 and compound 2 groups was significantly increased (P<0.01); compared with the compound 2 group, the expression level of p-PKA in the cerebral cortex and hippocampus of mice in the compound 2+H-89 group was decreased.

[0073] The above experimental results demonstrate that H-89 inhibits PKA activity, thereby significantly reducing the expression of the downstream protein CREB, ultimately leading to a significant decrease in BDNF levels in both compound 1 and compound 2 groups. This further illustrates that compounds 1 and 2 achieve their antidepressant effect by activating the PKA / CREB / BDNF pathway and increasing BDNF levels in the cerebral cortex and hippocampus of depressed mice.

[0074] Example 2 (I) Preparation and component analysis of daylily extract (1) Daylily ( Hemerocallis citrina Daylily (Hemerocallis fulva) belongs to the genus Hemerocallis in the family Asphodelaceae and is a traditional plant used for both food and medicine. Samples of daylily (variety Mengzihua) were collected from Huangtupu Town, Qidong County, Hunan Province (26°53′23.75″N, 111°52′22.44″E). 300 kg of fresh daylily was chopped and placed in five 300-liter containers. A total of 900 liters of water was added to completely submerge the chopped daylily samples, and cold extraction was performed for 24 hours. After filtration, the extract was concentrated to 35 liters using a large rotary evaporator and adsorbed onto AB-8 macroporous adsorption resin. Elution was performed using water or ethanol-water solutions to obtain extracts including the water-washed fraction (0% HCW), the 35 vol% ethanol-eluted fraction (35% HCW), and the 50 vol% ethanol-eluted fraction (50% HCW).

[0075] (2) The extracts of each daylily were analyzed by HPLC-Q-TOF-MS.

[0076] Chromatographic conditions: Huapu X-aqua C18 column (150×2.1 mm, 2.8 μm, Akram Technologies, China); elution system: aqueous phase (A) 0.1% formic acid water, organic phase (B) 0.1% formic acid acetonitrile; gradient elution program: 0~30 min, 5~45 vol% B, 30~40 min, 45~90 vol% B, 40~45 min, 90~90 vol% B; column temperature: 30℃; flow rate: 0.3 mL / min; injection volume: 5 μL; detection wavelength: 300 nm.

[0077] Mass spectrometry conditions: Ion source: Agilent Dual AJS ESI source; negative ion full scan mode; full scan range: m / z 100~1000; Sheath gas temperature: 350℃; Sheath gas flow rate: 11.0 L / min; Gas temperature: 300℃; Nebulizer pressure: 45 psi; Drying gas flow rate: 10 L / min; Capillary voltage: 3500 V; Fragmenter voltage: 175 V. Data acquisition software: Agilent MassHunter acquisition Workstation Software (B.05.00). High-resolution secondary mass spectrometry data (HRMS / MS) were acquired using target MS / MS mode, with collision energies selected from 10 to 80 eV. Reference solution was used ( m / z The time-of-flight mass spectrometer (112.9855 and 966.0007) was continuously calibrated to obtain high-precision mass measurements.

[0078] Analysis revealed that 35% HCW contained compounds 1 and 2. The total ion chromatogram of 35% HCW is shown below. Figure 11 (A). The UV detection pattern at 254nm is shown below. Figure 11 (B).

[0079] (3) Separate compound 1 and compound 2 in 35%HCW: Take 2.0g of 35%HCW, dissolve it in 50% methanol solution by ultrasonication, and filter it through a 0.45μm microporous membrane for later use. The separation of compounds 1 and 2 was mainly performed using an Agilent 1260 preparative liquid chromatography system. The specific separation conditions were as follows: Agilent ZORBA SB-C18 (4.6 × 250 mm, 5 μm), mobile phase: 0.1% formic acid in water (pump A), 0.1% formic acid in acetonitrile (pump B), gradient elution: 0.01–10.00 min, B: 15 vol%, 10.00–15.00 min, B: 25 vol%, 15.00–20.00 min, B: 30 vol%, 20.00–30.00 min, B: 90 vol%; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 60 μL; detection UV: 300 nm. Sample 1 (12.7 mg) and sample 2 (24.6 mg) were finally obtained. After high-resolution mass spectrometry and nuclear magnetic resonance identification, sample 1 and sample 2 were identified as compounds 1 and 2, respectively. The specific identification is as follows: Sample 1: White powder, HRMS: 337.0928 [MH] - (C 16 H 17 O8), UV(MeOH)λ max : 220 and 310 nm. The theoretical mass-to-charge ratio for the deprotonation of chlorogenic acid is . m / z 353.0873 [MH] - The difference between sample 1 and chlorogenic acid m / z The value is 15.9945, indicating that sample 1 has one less hydroxyl group than chlorogenic acid. According to the secondary mass spectrum, the parent nucleus ( m / z 337.0928) After the ester bond is broken, deprotonated fragment ions are formed ( m / z 163.0388), and this fragment ion will continue to neutrally lose one CO2 molecule to form m / z The fragment ion concentration of 119.0496 indicates that Sample 1 has only one hydroxyl group on its benzene ring. The proton NMR spectrum of the compound shows two pairs of overlapping hydrogen atoms (δ 7.51 and 6.83), and the carbon NMR spectrum also shows two pairs of overlapping carbon atoms (δ 133.8 and 116.8), indicating that the hydroxyl group on the benzene ring is located at position 14. In the proton NMR spectrum of Sample 1, the chemical shift of H-3 (δ 4.58) is greater than that of H-4 (δ 4.31) and H-5 (δ 4.22), indicating that the ester group is attached to C-3, causing the hydrogen at position 3 to shift to a lower field. The H-... 1 -H 1COSY, HMBC, and HMQC all indicate that the carboxyl group of 4-hydroxycinnamic acid undergoes esterification condensation with the hydroxyl group at C-3 of quinolinic acid. Therefore, sample 1 was identified as compound 1. The hydrogen and carbon signals of sample 1 were assigned as follows: 1 H-NMR (800MHz, MeOD) δ H 7.72(1H, d , J = 16 Hz, H-10), 7.51 (2H, d , J = 16 Hz, H-12-16), 6.83 (2H, d , J = 8.8 Hz, H-13-15), 6.45(1H, d , J = 15.2 Hz, H-9), 4.58 (1H, d ,H-3),4.31(1H, d ,H-4),4.22(1H, m , H-5), 2.01~2.50 (4H, m H-2, 6). 13 C-NMR (200MHz, MeOD) δ C 115.4(C-9), 116.8(C-13,15), 131.1(C-11), 133.8(C-12,16), 146.7(C-10), 161.2(C-14), 168.9(C-8).

[0080] Sample 2: White powder, HRMS: 337.0928 [MH] - (C 16 H 17 O8), UV(MeOH)λ max 220 and 310 nm. Sample 2 and Sample 1 are isomers, and their primary and secondary mass spectra are almost identical. From the 1H NMR spectrum of Sample 2, the chemical shift H-4 (δ 4.81) is higher than that of H-3 (δ 4.31) and H-5 (δ 4.22), indicating that the carboxyl group in 4-hydroxycinnamic acid undergoes deesterification condensation with the 4-hydroxyl group of quinoline acid to form Sample 2. From the H NMR spectrum... 1 -H 1 According to COSY, H-4 is only coupled to H-3 and H-5, but not to H-2 and H-6. HMBC data shows... δ H 4.81 (H-4) and δ C 168.9 (C-8) has remote coupling.δ H 4.31 (H-3) and δ H 4.22 (H-5) and δ C The presence of long-range coupling at 79.1 (C-4) further indicates that the ester group should be attached to the C-4 position of the quinolinic acid. Therefore, sample 2 was identified as compound 2. The hydrogen and carbon signals of sample 2 were assigned as follows: 1 H-NMR (800MHz, MeOD) δ H 7.72(1H, d , J = 16 Hz, H-10), 7.51 (2H, d , J = 16 Hz, H-12-16), 6.83 (2H, d , J = 8.8 Hz, H-13-15), 6.45(1H, d , J = 15.2 Hz, H-9), 4.81 (1H, m ,H-4),4.31(1H, m ,H-3),4.22(1H, m , H-5), 2.01~2.50 (2H, m H-2, 6). 13 C-NMR (200MHz, MeOD) δ C 38.7(C-2), 42.3(C-6), 65.8(C-5), 69.4(C-3), 76.7(C-1), 79.1(C-4), 115.4(C-9), 11 6.8(C-13,15), 127.3(C-11), 131.4(C-12,16), 146.7(C-10), 168.9(C-8), 178.2(C-7).

[0081] (4) The contents of compounds 1 and 2 in 35% HCW were determined using high performance liquid chromatography (HPLC): Chromatographic conditions: A ZORBAX SB-C18 (4.6×250 mm, 5 µm) column was used with acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B. Gradient elution was used: 0–10 min, 9 vol%–9.6 vol% A; 10–20 min, 9.6 vol%–9.8 vol% A; 20–26 min, 9.8 vol%–9.9 vol% A; 26–30 min, 9.9 vol%–12 vol% A; flow rate was 1 mL / min; injection volume was 10 μL; column temperature was 30 °C; UV detection wavelength was 300 nm. Sample solution preparation: Accurately weigh 0.1 g of the dried 35% HCW sample into a 14 mL centrifuge tube, precisely add 6 mL of Merck methanol, sonicate for 60 min until completely dissolved, take 1 mL of the sample solution, filter it through a 0.22 μm microporous membrane into a sample vial, and store the remaining sample at 4 °C for later use; accurately weigh 10 mg of each of compound 1 and compound 2, place them in 10 mL volumetric flasks, add Merck methanol, sonicate to dissolve and dilute to the mark, preparing a 1 mg / mL solution for each sample; then take 100 μL from each sample solution, place it in a 1 mL volumetric flask, add Merck methanol and dilute to the mark to prepare reference solutions of compound 1 and compound 2; store at 4 °C for later use. Linearity investigation: Accurately pipette 2 mL of the mixed reference solution of compound 1 and compound 2 into a graduated cylinder, add methanol for twofold dilution to obtain a mixed standard solution, measure its peak area and calculate its regression equation to prepare a standard curve; Methodological evaluation and content determination: The established method was evaluated, mainly including precision, stability, repeatability and recovery experiments, and the RSD or recovery rate of the corresponding experiments were calculated. The contents of compound 1 and compound 2 in 35% HCW were determined according to the established HPLC method, the peak area of ​​each component was recorded and the mass fraction of each component in the sample was calculated.

[0082] A method for detecting and determining the content of compounds 1 and 2, potential antidepressant components, in 35% HCW extract of daylily was established using HPLC. The results showed that within the range of 1.5625 μg / mL to 100.00 μg / mL, the regression equations for compounds 1 and 2 were Y = 26764x - 36415(R²). 2 =0.9999) and Y=26869x-37821(R 2=0.9999). The detection instrument had good precision, the test sample had good stability, and the detection method had good repeatability. The average recovery rate was 98.33%~100.43%, and the RSD was 0.55%~2.70%. Finally, the contents of compound 1 and compound 2 in 35% HCW were detected to be 7.71 mg / g and 7.61 mg / g, respectively.

[0083] (II) Analysis of the antidepressant activity of daylily extract (1) Antidepressant animal behavior experiment Male ICR mice (No. 43004700048460) weighing 18.0–22.0 g were purchased from Hunan Silek Jingda Experimental Animal Co., Ltd. [SCXK (Xiang) 2019-0004]. The ICR mice were housed in a barrier environment at the Hunan Provincial Drug Safety Evaluation and Research Center (Experimental Animal Use License No.: SYXK (Xiang) 2015-0016). Ten mice were randomly selected as the normal control group (routinely fed), while the remaining mice were housed individually for 28 days to establish a chronic unpredictable stress (CUMS) model. After modeling, based on the sucrose preference test results and mouse body weight, mice were randomly divided into six groups of ten mice each: a blank control group, a model group, a positive control group (fluoxetine hydrochloride 5.2 mg / kg / day), and three different doses of 35% HCW (denoted as 35%HCW-L, 35%HCW-M, and 35%HCW-H, administered at 7.5, 15, and 30 mg / kg / day, respectively). The normal control and model control groups were administered 0.4 mL of distilled water by gavage daily, while the other groups received the same volume of the corresponding drug solution by gavage for 35 consecutive days. During the administration period, mice continued to receive CUMS treatment after each gavage. After the last administration, behavioral tests were performed on the mice, including the sucrose preference test (SPT), the tail suspension test (TST), and the novelty inhibition feeding test. The test results are shown in [link to results]. Figure 12 .

[0084] The antidepressant activity of 35% HCW was evaluated using a CUMS mouse depression model. Figure 12 It can be seen that 35% HCW, regardless of low or high doses, significantly increased sucrose preference, significantly reduced feeding latency, and significantly increased tail suspension in depressed mice. Furthermore, the effect of 35% HCW from low to high doses was dose-dependent, consistent with the trend observed with compound 1 or compound 2. These experimental results indicate that 35% HCW possesses significant antidepressant activity.

[0085] (2) After the behavioral tests were completed, blood was collected from the posterior orbital venous plexus of the mice. After euthanasia by cervical dislocation, the brain tissue of the experimental mice was first removed under sterile conditions using a sterile scalpel. The cerebral cortex and hippocampus tissue were removed using a sterile scalpel and sterile forceps. After removal, the tissue was immediately stored on an ice pack in a sterile cryovial. Then, the intestinal segment was removed, and the intestinal contents were removed using a sterile scalpel. The sample size was 200-500 mg / vial. After removal, the tissue was immediately labeled with sterile centrifuge tubes on an ice pack, aliquoted, and stored in liquid nitrogen at -80°C for relevant testing. The determination of monoamine neurotransmitters (including DA, NE, and 5-HT), BDNF, inflammatory factors (including IL-6 and TNF-α), and oxidative stress factors (including SOD enzyme and MDA) in the cerebral cortex and hippocampus of mice was performed using an ELISA kit. The operation steps were strictly performed according to the ELISA kit's operating instructions. The 16S RNA sequencing of gut microbiota was conducted in collaboration with Novogene Biotechnology Co., Ltd., who completed the 16S RNA sequencing and analysis of the mouse gut.

[0086] The following conclusions were reached after testing: 1) Daylily extract (35% HCW) can significantly increase the level of monoamine neurotransmitters in the brain. Figure 13 The effects of 35% HCW extract of daylily on monoamine neurotransmitters in the mouse brain were statistically analyzed; from Figure 13 It can be seen that when CUMS induced depression in normal mice, the levels of dopamine and norepinephrine in the cerebral cortex and hippocampus were significantly reduced. However, after administration of fluoxetine hydrochloride, the levels of dopamine and norepinephrine in the cerebral cortex and hippocampus were significantly increased, indicating that the mouse depression model and the selection of the positive drug in this study were both successful. After administration of low, medium, and high doses of 35% HCW, the levels of dopamine and norepinephrine in the cerebral cortex and hippocampus of depressed mice were significantly increased. The high dose of 35% HCW significantly increased 5-HT in the cerebral cortex of depressed mice.

[0087] 2) Daylily extract (35% HCW) can significantly increase the level of BDNF in the brain. Figure 14 The effect of 35% HCW extract of daylily on BDNF levels in the mouse brain was statistically analyzed; from Figure 14 It can be seen that when CUMS induced depression in normal mice, the BDNF content in the cerebral cortex and hippocampus was significantly reduced. After administration of low, medium and high doses of 35% HCW, the BDNF content in the cerebral cortex and hippocampus of depressed mice was significantly increased, with the BDNF content in the cerebral cortex increasing by 32.4% and the BDNF content in the hippocampus increasing by 35.8%. The effect of 35% HCW on increasing the BDNF level in depressed mice was higher than that of the positive control drug fluoxetine hydrochloride.

[0088] 3) Daylily extract (35% HCW) can reduce brain inflammation and oxidative stress. Figure 15 The effects of 35% HCW extract of daylily on inflammatory factors in the mouse brain were statistically analyzed; from Figure 15 It can be seen that during the CUMS modeling process, the levels of inflammatory factors (IL-6 and TNF-α) in the cerebral cortex and hippocampus of mice increased. However, after administration of low, medium, and high doses of 35% HCW, the levels of IL-6 and TNF-α in the cerebral cortex and hippocampus of depressed mice decreased to some extent. The high dose of 35% HCW showed the best activity, significantly reducing the level of IL-6 in the hippocampus of depressed mice (P<0.05), bringing it close to the normal value.

[0089] Figure 16 The effects of 35% HCW extract of daylily on oxidative stress factors in the mouse brain were statistically analyzed; from Figure 16 It can be seen that during the CUMS modeling process, both the cerebral cortex and hippocampus of mice exhibited a certain degree of oxidative stress response, manifested as a significant increase in MDA levels and a significant decrease in SOD levels in both the cerebral cortex and hippocampus. After administration of low, medium, and high doses of 35% HCW, the MDA levels in the cerebral cortex and hippocampus of depressed mice were significantly reduced (P<0.05, P<0.01), showing a dose-response relationship. Medium and high doses of 35% HCW significantly increased the SOD level in the hippocampus, with effects superior to the positive control drug fluoxetine hydrochloride.

[0090] 4) Daylily extract 35% HCW can improve gut microbiota. In the model group, bacterial α-diversity indices (Sobs, Chao1, ACE, and Shannon) were all decreased, while different doses of 35% HCW all increased bacterial α-diversity indices and improved the gut microbiota of depressed mice; the high dose of 35% HCW was the most significant, increasing the abundance of gut microbiota by 21.8% compared to the model group. These results indicate that 35% HCW can significantly increase the abundance of gut microbiota. PcoA analysis showed that the model group and the positive group, as well as different doses of 35% HCW, were well separated, further demonstrating that 35% HCW can improve the bacterial community of depressed mice.

[0091] Bacteroidetes ( BacteroidetesFirmicutes are the dominant gut microbiota, responsible for fermenting carbohydrates, participating in the metabolism of bile acids, steroids, and polysaccharides, and maintaining normal gut physiology; they are considered beneficial gut bacteria. In mice after depression, the abundance of Bacteroides significantly decreased (P<0.05), while administration of a low dose of 35% HCW significantly increased the abundance of Bacteroides (P<0.05). Compared to the control group, the abundance of Firmicutes in the model group was significantly increased (P<0.05), and compared to the model group, the abundance of Firmicutes in the low-dose 35% HCW group (35%L) was significantly decreased (P<0.05). The gut microbiota changes in the 35% HCW group were consistent with those of compound 1 or compound 2. Community composition analysis at the genus level revealed significant differences among groups in eight bacterial genera: norank_f__Muribaculaceae, Lachnospiraceae_NK4A136_group, unclassified_f__Lachnospiraceae, Streptococcus, Enterorhabdus, Ruminiclostridium_9, unclassified_o__Lactobacillales, and Ruminiclostridium_5. In mice with depression, the abundance of norank_f__Muribaculaceae bacteria significantly decreased (P<0.05), while low-dose 35% HCW significantly increased the abundance of norank_f__Muribaculaceae bacteria (P<0.05). Increased abundance of norank_f__Muribaculaceae bacteria reduced intestinal inflammatory factors. Streptococcus is a common pyogenic coccus, widely found in the nose, throat, and feces of humans and animals. It can cause various purulent inflammations, such as scarlet fever, neonatal sepsis, meningitis, puerperal fever, and streptococcal allergic diseases. Compared with the control group, the abundance of Streptococcus bacteria was significantly increased in the model group (P<0.01). Compared with the model group, the abundance of Streptococcus bacteria was significantly decreased in different doses of 35% HCW (P<0.01), indicating that 35% HCW reduces intestinal inflammation by decreasing the abundance of Streptococcus bacteria, thereby achieving an antidepressant effect.

[0092] Experimental results showed that 35% HCW extract of daylily possessed significant antidepressant activity, significantly increasing sucrose preference in depressed mice, significantly reducing feeding latency and tail immobility time in depressed mice. The antidepressant activity of 35% HCW was closely related to increasing dopamine, norepinephrine, and BNDF levels in the cerebral cortex and hippocampus of depressed mice, reducing inflammatory factors and MDA, increasing SOD, and improving gut microbiota.

Claims

1. An application of a quinic acid derivative, characterized in that: The quinic acid derivative includes 3- O -p-Coumarylquinic acid, 4- O - One or both of coumaroylquinic acids; the applications include the preparation of medicines for the treatment and / or prevention of depression.

2. The application of the quinic acid derivative according to claim 1, characterized in that, The pure form of the quinic acid derivative or a mixture containing the quinic acid derivative is used to prepare a medicine for treating and / or preventing depression.

3. The application of the quinic acid derivative according to claim 1 or 2, characterized in that, The quinic acid derivative is obtained by one or more of the following methods: chemical synthesis, biological extraction, and biological fermentation.

4. The application of the quinic acid derivative according to claim 1 or 2, characterized in that, The sources of the quinic acid derivatives include synthesis using dextroquinic acid as a raw material.

5. The application of the quinic acid derivative according to claim 2, characterized in that, The mixture containing the quinic acid derivative includes one or two of the following: biological extracts, biological ferments, and quinic acid derivative dispersions.

6. The application of the quinic acid derivative according to claim 1 or 5, characterized in that, Mixtures containing the quinic acid derivative include daylily extract.

7. The application of the quinic acid derivative according to claim 6, characterized in that, The daylily extract was extracted using water as a solvent via cold soaking.

8. The application of the quinic acid derivative according to claim 6, characterized in that, The daylily extract was extracted using water as a solvent by cold soaking, and then further purified by adsorption onto macroporous resin: eluted with ethanol-water solution, and the eluent was collected.

9. The application of the quinic acid derivative according to claim 8, characterized in that, The macroporous resin includes one or more of AB-8, D101, HZ-816, and HPD100.

10. The application of the quinic acid derivative according to claim 8 or 9, characterized in that, The ethanol-water solution contains 30% to 40% ethanol by volume.

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