Preparation method and application of starwort alkali A

Abstract

The invention discloses a preparation method and application of starwort alkali A. Belongs to the technical field of organic synthesis. The invention provides a synthesis method of a compound starwort alkali A. The method comprises the following steps: carrying out a cyclization condensation reaction on L-tryptophan methyl ester hydrochloride and methylglyoxal under the catalysis of acid by adopting a Pictet-Spengula reaction, and carrying out an ammonolysis reaction to obtain a target compound. The method has the following advantages: the reaction steps are simple, complicated separation and purification are not needed, and the operation convenience is improved; the reaction selectivity is relatively high, a target compound with relatively high purity can be obtained, and the synthesis efficiency is improved; reaction reagents are low in price, so that the synthesis cost is reduced. The anti-anxiety activity of the stelline A is researched for the first time, and the action mechanism of the stelline A is clarified.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis, and more particularly to a preparation method of arginine A and application thereof. Background Art

[0002] The Chinese herbal medicine Radix Stellariae is slightly cold in nature, sweet in taste, and has the functions of clearing away heat and removing malnutrition fever. It is the dried root of Stellaria dichotoma L.var.lanceolata Bge., a perennial herb of the Caryophyllaceae family. It is a commonly used medicinal material for treating fever-related diseases (such as chronic fever and malnutrition fever in children). Modern pharmacological studies have shown that Radix Stellariae has antipyretic, anti-inflammatory, anti-allergic, anti-cancer, neuroprotective, and vascular relaxation activities. At present, the basic research on Radix Stellariae is mainly focused on the extraction process, separation and preparation of chemical components. Among them, alkaloids, sterols, and cyclic peptide compounds are the main components and show good biological activity, which provides a sufficient material basis for screening biologically active lead compounds from Radix Stellariae.

[0003] The study of the chemical composition of Bupleurum argyi is still in its initial stage. So far, only one total synthesis report involves Bupleurum argyi alkaloid A: In 2021, Tao Sheng et al. synthesized a series of β-Carboline alkaloids from L-tryptophan and conducted antifungal activity studies, in which Bupleurum argyi alkaloid A was synthesized through a three-step reaction. However, the high price of its raw materials and the limited access to active compounds have restricted the mechanism of action, efficacy research, and application and development of Bupleurum argyi.

[0004] In summary, how to provide a synthesis method of compound arginine A and explore its activity is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a preparation method of argyranthirine A and application thereof.

[0006] On the one hand, the present invention provides a method for synthesizing a compound, arginine A, which adopts the Pictet-Spengular reaction to cause L-tryptophan methyl ester hydrochloride and methylglyoxal to undergo a cyclization condensation reaction under acid catalysis, and utilizes an aminolysis reaction to obtain a target compound. The method has the following advantages: the reaction steps are simple, and no complicated separation and purification are required, which improves the convenience of operation; the reaction selectivity is high, and a target compound of higher purity can be obtained, which improves the efficiency of the synthesis; the reaction reagent is low in price, which reduces the cost of the synthesis. On the other hand, the present invention studies the antianxiety activity of arginine A for the first time and clarifies its mechanism of action.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing argyrocatecholamine A comprises the following steps:

[0009] (1) reacting L-tryptophan methyl ester hydrochloride with methylglyoxal under catalytic conditions to obtain 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0010] (2) 1-Acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester is dissolved in methanol and reacted with aqueous ammonia to generate argyrocatechol A.

[0011] The synthetic route is as follows:

[0012]

[0013] Furthermore, the catalyst in step (1) is concentrated sulfuric acid.

[0014] Furthermore, in the step (1), the molar ratio of L-tryptophan methyl ester hydrochloride to methylglyoxal is 1:1 to 1:1.2.

[0015] Furthermore, the reaction time of step (1) is 10 to 48 hours.

[0016] Furthermore, in the step (1), after the concentrated sulfuric acid is added dropwise, the mixture is stirred until the L-tryptophan methyl ester hydrochloride is completely dissolved, and then methylglyoxal is added to ensure that the raw materials are fully utilized, and then oxygen is introduced to accelerate the reaction speed and improve the yield.

[0017] Furthermore, in step (2), the volume ratio of methanol to aqueous ammonia is 1:2 to 1:3.

[0018] Furthermore, in the step (2), the mass volume ratio of 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester to methanol is 0.001 g / ml.

[0019] Furthermore, in the step (2), after dissolving aqueous ammonia in methanol, the supernatant is reacted, which can improve the purity of the product.

[0020] The application of silver bupleurum A prepared by the above preparation method in the preparation of anti-anxiety drugs.

[0021] Furthermore, it is used to adjust the changes in glucocorticoids ACTH and CORT in rat serum and monoamine neurotransmitters DA and 5-HT in brain tissue.

[0022] Furthermore, it is used to regulate the expression of CB1 and CB2 proteins in the CA1 region of the hippocampus.

[0023] Furthermore, it is used to regulate the expression of BDNF, CREB, and ERK.

[0024] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0025] (1) The present invention uses L-tryptophan methyl ester hydrochloride and methylglyoxal as synthetic raw materials, obtains 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester through a Pictec-Spengular reaction, and then obtains arginine A through an aminolysis reaction, thereby realizing the chemical synthesis of arginine A.

[0026] (2) The synthetic route of the present invention has the advantages of simplicity, high efficiency, simple operation and low cost, and is suitable for the large-scale synthesis of argyrocatecholamine A, providing an important material basis for the biological activity evaluation of argyrocatecholamine A.

[0027] (3) The present invention proposes for the first time the antianxiety activity of arginine A, providing strong data support and reference for the development of highly effective and low-toxic natural antianxiety drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 is the silver bupleurum alkaloid A in Example 1 of the present invention 1 HNMR spectrum;

[0030] Figure 2 is the silver bupleurum alkaloid A in Example 1 of the present invention 13 C NMR spectrum;

[0031] Figure 3 The docking conditions of Stellarine A and diazepam with ERK, CREB and BDNF in Example 2 of the present invention, wherein A is the docking of Stellarine A with ERK; B is the docking of diazepam with ERK; C is the docking of Stellarine A with CREB; D is the docking of diazepam with CREB; E is the docking of Stellarine A with BDNF; and F is the docking of diazepam with BDNF;

[0032] Figure 4 The effect of Stellarine A on the behavior of anxious rats induced by acute restraint stress in EPM in Example 2 of the present invention;

[0033] Figure 5 The effect of Stellarine A on the behavior of anxious rats induced by uncertain empty bottle stress in EPM in Example 2 of the present invention;

[0034] Figure 6 The effect of Stellarine A on the behavior of anxious rats induced by acute restraint stress in OFT in Example 2 of the present invention;

[0035] Figure 7 The effect of Stellarine A on the behavior of anxious rats induced by uncertain empty bottle stress in OFT in Example 2 of the present invention;

[0036] Figure 8 The effect of Stellarine A on the behavior of anxious rats induced by acute restraint stress in LDT in Example 2 of the present invention;

[0037] Fig. 9 The effect of Stellarine A on the behavior of anxious rats induced by uncertain empty bottle stress in LDT in Example 2 of the present invention;

[0038] Fig.10 The effect of Stellarine A on the levels of ACTH and CORT in the serum of rats with anxiety induced by acute restraint stress in Example 2 of the present invention;

[0039] Fig.11 The effect of Stellarine A on the levels of ACTH and CORT in the serum of anxious rats induced by uncertain empty bottle stress in Example 2 of the present invention;

[0040] Fig.12 The effect of Stellarine A in Example 2 of the present invention on the content of DA and 5-HT in the brain tissue of rats with anxiety induced by acute restraint stress;

[0041] Fig.13 The effect of Stellarine A in Example 2 of the present invention on the content of DA and 5-HT in the brain tissue of anxious rats induced by uncertain empty bottle stress;

[0042] Fig.14 The effect of Stellarine A on the neuronal morphology of hippocampal region of rats with anxiety induced by acute restraint stress in Example 2 of the present invention, wherein A is a blank control group; B is a model group; C is a diazepam group; D is a high-dose Stellarine A group; 40×, n=3;

[0043] Fig.15The effect of Stellarine A on the neuronal morphology of the hippocampus of anxious rats induced by uncertain empty bottle stress in Example 2 of the present invention, wherein A is a blank control group; B is a model group; C is a diazepam group; D is a high-dose Stellarine A group; 40×, n=3;

[0044] Fig.16 The effect of Stellarine A in Example 2 of the present invention on CB1 and CB2 proteins in anxious rats induced by acute restraint stress (A) and uncertain empty bottle stress (B);

[0045] Fig.17 The expression of CB1 in CA1 region of rats with anxiety induced by acute restraint stress in Example 2 of the present invention, wherein A is a blank control group; B is a model group; C is a diazepam group; D is a high-dose Stellarine A group; 400 μm, 20×, n=3;

[0046] Fig.18 The expression of CB2 in the CA1 region of rats with anxiety induced by acute restraint stress in Example 2 of the present invention, wherein:

[0047] A represents the blank control group; B represents the model group; C represents the diazepam group; D represents the high-dose Stellarine A group; 400 μm, 20×, n=3;

[0048] Fig.19 The expression of CB1 in the CA1 region of the anxious rats induced by the uncertain empty bottle stress in Example 2 of the present invention, wherein A represents the blank control group; B represents the model group; C represents the diazepam group; D represents the high-dose Stellarine A group; 400 μm, 40×, n=3;

[0049] Fig. 20 The expression of CB2 in the CA1 region of the anxious rats induced by the uncertain empty bottle stress in Example 2 of the present invention, wherein A represents the blank control group; B represents the model group; C represents the diazepam group; D represents the high-dose Stellarine A group; 400 μm, 40×, n=3;

[0050] Fig.21 is the relative expression level of BDNF, CREB and ERK mRNA in the hippocampus of the anxious rats induced by the uncertain empty bottle stress in Example 2 of the present invention;

[0051] Fig. 22 The effect of Stellarine A on the expression of BDNF, p-CREB and p-ERK proteins in the frontal cortex of anxious rats induced by uncertain empty bottle stress in Example 2 of the present invention;

[0052] Fig.23This is the effect of Stellarine A in Example 2 of the present invention on the expression of BDNF, p-CREB and p-ERK proteins in the hippocampus of anxious rats induced by uncertain empty bottle stress. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0055] Example 1

[0056] Synthesis of Stellarine A

[0057] 12.05 g of L-tryptophan methyl ester hydrochloride (Annage) was dissolved in water, 1 mL of concentrated sulfuric acid and 3.6 mL of 40% methylglyoxal (Annage) aqueous solution were added, oxygen was introduced, and the mixture was stirred overnight at room temperature. 5.54 g of a yellow solid was precipitated. After filtration, the filter cake was washed with water and dried. 1.01 g of the yellow solid was dissolved in 1 L of methanol (Annage), and the supernatant was taken after continuous stirring. 1.5 L of 25% ammonia water was added, and the mixture was stirred overnight at room temperature for 2 days. 0.38 g of solid A was obtained after filtration and drying.

[0058] Solid A, yellow powder, easily soluble in DMSO, molecular formula is C 14 H 11N3O2. 1H-NMR (400MHz, DMSO) δ12.15 (s, 1H, 9-NH), 9.07 (s, 1H, H-4), 8.41 (d, J = 7.8Hz, 1H, H-5), 8.23 ​​(s, 1H, 17-NH2), 7.8 2(d,J=8.2Hz,1H,H-8),7.64(s,1H,17-NH2),7.61(dd,J=8.2,1.1Hz,1H,H-7),7.33(dd,J=11.0,4.0Hz,1H,H-6),2.89(s, 3H, -CH3); 13C-NMR (101 MHz, DMSO) δ 201.48 (C-14), 166.73 (C-16), 142.50 (C-13), 139.01 (C-3), 135.07 (C-10), 134.20 (C-1), 132.10 (C-11), 129.58 (C-7), 122.39 (C-5), 121.11 (C-6), 120.48 (C-12), 118.23 (C-4), 113.49 (C-8), 26.23 (C-15). 1 HNMR Figure 1 )and 13 C NMR ( Figure 2 ) spectrum data are basically consistent with the data of the existing report (Cui ZH, Li GY, Qiao L, et al. Two New Alkaloids From Stellaria dichotoma var. Lanceolata. Natural Product Letters, 2006, 7: 59-64.), so the compound was identified as Stellarine A.

[0059] The purity of the product was 92.54% by liquid chromatography-mass spectrometry, and the calculated yield was 34.75%.

[0060] The yield calculation method of the target product silver bupleurum alkaloid A is as follows:

[0061] Yield (%) = [actual yield of target product × purity of target product) / theoretical yield of target product] × 100%.

[0062] Example 2

[0063] Study on the anxiolytic effect and mechanism of Stellarine A based on ERK / CREB / BDNF signaling pathway

[0064] In the following experiments, GraphPad Prism 9 software (GraphPad-software Inc., CA, USA) was used for drawing, and SPSS Statistics 26 statistical software was used for data statistics. One-way analysis of variance was used for comparison between groups, LSD test was used for variance homogeneity, and Dunnett T3 test was used for variance heterogeneity. Data are expressed as mean ± standard error (± SEM). #P<0.05 or *P<0.05 indicates a significant difference, and ##P<0.01 or **P<0.01 indicates an extremely significant difference.

[0065] (I) Molecular docking

[0066] PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) and SwissADME (http: / / www.swissad me.ch) websites were used to search and obtain the sdf files and SMILES information of arginine A and diazepam. Diazepam was used as a positive docking control. The above small molecules were then input and optimized through Discovery Studio 2021 software, and defined as ligands.

[0067] The PDB protein database (https: / / www.rcsb.org / ) is used to query and save the pdb files of the target proteins ERK (1ZC0), CREB (5CGP), and BDNF (6KZD). The obtained pdb files are imported into the Discovery Studio 2021 software for corresponding protein modification, removal of water and ligand small molecules, and addition of hydrogen. The Clean Protein function is then used to remove redundant protein structures, and then the defined protein is set as a receptor, and a docking site is generated. Molecular docking is performed using LibDock with the selected target small molecules, and the LibDock score is used to evaluate the affinity of the molecular conformation. The LibDock score is shown in the following table. The docking status of Stellarine A and diazepam with ERK, CREB, and BDNF is shown in the following table. Figure 3 shown.

[0068] Table 1 LibDock scores

[0069] Compound ERK CREB BDNF StellarineA 105.92 83.34 97.81 Positive control (diazepam) 94.52 82.47 94.53

[0070] The results showed that when Stellarine A was docked with ERK (1ZC0), it formed five hydrogen bonds with protein residues ARG284, GLU254, SER256, GLY264, and SER168, with strong hydrogen bonding. The anion of GLU258 formed a conjugated effect with the benzene ring of the ligand. ILE266 and PRO263 had a π-alkyl interaction with the alkyl group, and were connected to VAL143 through Pi-Sigma interaction. The LibDock score was 105.92. When Stellarine A was docked with CREB (5CGP), it was connected to protein residues MET1133 and ASN1168 through hydrogen bonds, formed a weak carbon-hydrogen bond with PHE1111, and the benzene ring formed a conjugated effect with the anions of VAL1115, ALA1164, PRO1110, LEU1120, and VAL1174. The LibDock score was 83.34. When A docked with BDNF (6KZD), GLU618 was connected by hydrogen bonds, PHE617 formed a stable π-π hydrophobic stacking interaction with the aromatic ring of Stellarine A, and VAL601 had a π-alkyl interaction with the alkyl group, with a LibDock score of 97.81. The results showed that Stellarine A showed a high affinity with the above three proteins, and the LibDock scores were all higher than the positive control diazepam. Therefore, it can be predicted that silver bupleurum A exerts anxiolytic activity through target proteins (ERK, CREB, BDNF). According to the Libdock score, the following experiment will study the anxiolytic activity and mechanism of Stellarine A based on the ERK / CREB / BDNF signaling pathway.

[0071] (II) Establishment of anxiety model and drug administration regimen

[0072] SPF male SD♂ rats were randomly divided into six groups, 6 rats in each group, including blank control group (Control), model group (Model), Stellarine A low-dose (SAD), medium-dose (SAZ), high-dose group (SAG), and positive drug diazepam group (DZP). The blank control group was group-housed, and the rats in the other groups were housed in single cages. (Acute restraint stress-induced anxiety model and uncertain empty bottle stress-induced anxiety model were established, with a total of 72 rats).

[0073] The dosage was determined, and the positive drug DZP (1.0 mg / kg) was given. The Stellarine A group was given drugs according to SAD (2.5 mg / kg), SAZ (5.0 mg / kg), and SAG (10.0 mg / kg). The blank control group and the model group were gavaged with the corresponding volume of 0.5% sodium carboxymethylcellulose (CMC-Na) solution. The drugs were gavaged for 7 consecutive days. Starting on the 5th day, except for the blank control group, the animals in the other groups were placed in a restraint fixture after gavage for acute restraint modeling. During this period, the rats were fasted and deprived of water. Only the range of activity of the rats was restricted, and breathing was not inhibited. Stimulation was performed for 30 minutes every day for 3 consecutive days. At the end of the modeling on the 7th day, an acute restraint stress-induced anxiety model in rats was established.

[0074] Rats were trained to drink water regularly for 7 days. Except for the blank control group, rats in other groups were given drinking water for 10 minutes at fixed time in the morning and evening, and the water bottles were removed at other times without water. After the training water feeding period, the uncertain empty bottle stress model was established. The uncertain empty water bottle stimulation was given at a fixed time period for two consecutive weeks. The positive drug DZP (1.0 mg / kg) was given after stress, and the Stellarine A administration group was given according to SAD (2.5 mg / kg), SAZ (5.0 mg / kg), and SZG (10.0 mg / kg). The blank control group and the model group were gavaged with the corresponding volume of 0.5% CMC-Na solution. The drug was administered by gavage for 7 consecutive days. Anxiety model of rats induced by uncertain empty bottle stress was established.

[0075] (III) Behavioral Experiment

[0076] The acute restraint stress group was subjected to behavioral testing after modeling on the 7th day. The uncertain empty bottle stress group was subjected to behavioral testing on the 7th day, the blank control group, the model group, and the drug group 1 hour after the last drug administration, and the positive drug group 0.5 hour after intragastric administration to evaluate the anxiety-like behavior of rats.

[0077] (1) Elevated plus maze test

[0078] The elevated plus maze experiment uses the animals' exploratory nature towards novel environments and their fear of high-hanging open arms to form contradictory and conflicting behaviors in animals. The elevated plus maze device is in the shape of a "cross" and consists of two opposing open arms, two opposing closed arms and a central area, placed at a height of 50 cm from the ground. Before the experiment, the rats were placed in the test room for 30 minutes to adapt, and the test environment was kept relatively quiet. Each rat was placed individually in the central area, facing an open arm at the beginning of the test, and allowed to explore freely for 5 minutes. The movement trajectory of all rats was tracked by a target tracking system (SMART V3.0) composed of cameras fixed above the maze. The degree of anxiety was assessed by the time spent in the open arms (the percentage of time in open arms to the total exploration time, OT%) and the number of times entering the open arms (the number of times the ...

[0079] As shown in Table 2, Figure 4 The results showed that compared with the blank control group, the acute restraint stress-induced anxiety model group could significantly reduce the percentage of rats entering the open arms and the percentage of time (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the positive drugs DZP, SAZ, and SAG groups could significantly increase the percentage of rats entering the open arms (P<0.01) and the percentage of time (P<0.05, P<0.01). This shows that in the elevated plus maze experiment, acute restraint stress can induce anxiety in rats, and StellarineA can exert significant anxiolytic effects at medium (5 mg / kg) and high doses (10 mg / kg).

[0080] Table 2 Effects of Stellarine A on the behavior of rats induced by acute restraint stress in EPM

[0081]

[0082] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0083] From Table 3, Figure 5The results showed that compared with the blank control group, the uncertain empty bottle stress-induced anxiety model group could significantly reduce the percentage of rats entering the open arm and the percentage of time (P<0.05, P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the positive drug DZP group could significantly increase the percentage of rats in the open arm (P<0.01), and the SAZ (P<0.05, P<0.01) and SAG groups could significantly increase the percentage of rats entering the open arm and the percentage of time (P<0.01). The results showed that in the elevated plus maze test, uncertain empty bottle stress can induce anxiety in rats, and Stellarine A can exert significant anxiolytic effects at medium (5mg / kg) and high doses (10mg / kg).

[0084] Table 3 Effects of Stellarine A on the behavior of anxious rats induced by uncertain empty bottle stress in EPM

[0085]

[0086] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0087] The behavioral results of the pharmacodynamic experiment showed that compared with the blank control group, the number of times the rats in the anxiety model groups induced by acute restraint stress and uncertain empty bottle stress entered the open arms of the elevated plus maze and the percentage of time they stayed there were significantly reduced; compared with the anxiety model groups induced by acute restraint stress and uncertain empty bottle stress, the number of times the rats in the Stellarine A-treated group entered the open arms of the elevated plus maze and the percentage of time they stayed there were significantly increased.

[0088] (2) Open field test

[0089] The open field test is mainly used to evaluate the neurobiological basis of anxiety and to detect anxiety-like behaviors in animals. After the EPM test, the open field test was conducted the next day. Rats were adapted to the test environment for at least 30 minutes before the test, and the test environment was kept relatively quiet. The open field box is a square area with plastic walls and floors (80cm, 80cm, 40cm). The bottom is divided into 50% central area and 50% peripheral area by software settings, and is placed in an isolated room with weak light. The rat is placed in the central area of ​​the box and allowed to move freely for 5 minutes. The distance and time the rat walks are recorded with a camera fixed above the floor, and analyzed with a video tracking system (SMART V3.0), and the number of times the rat enters the center and the time it stays in the center are counted. After each test, the feces are removed and the test box is wiped with 75% alcohol to eliminate the smell and avoid interference.

[0090] From Table 4, Figure 6The results showed that compared with the blank control group, the number of rats in the acute restraint stress-induced anxiety model group entering the central area and the time they stayed in the central area were significantly reduced (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the time the rats in the positive drug diazepam group stayed in the central area (P<0.01), the number of rats in the SAZ group entering the central area were significantly increased (P<0.05), and the number of rats in the SAG group entering the central area (P<0.01) and the time they stayed in the central area were significantly increased (P<0.05). The results showed that in the open field test, acute restraint stress can induce anxiety in rats, and Stellarine A can play a significant anti-anxiety effect at a high dose (10.0 mg / kg).

[0091] Table 4 Effects of Stellarine A on the behavior of rats induced by acute restraint stress in OFT

[0092]

[0093]

[0094] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0095] From Table 5, Figure 7 The results showed that compared with the blank control group, the number of times rats in the anxiety model group induced by uncertain empty bottle stress entered the central area and the time they stayed in the central area were significantly reduced (P<0.01). Compared with the anxiety model group induced by uncertain empty bottle stress, the number of times rats in the positive drug diazepam group and SAG group entered the central area and the time they stayed in the central area were significantly increased (P<0.01), and the time rats in the SAZ group stayed in the central area was significantly increased (P<0.01). The results showed that in the open field test, uncertain empty bottle stress can induce anxiety in rats, and Stellarine A can play a significant anti-anxiety effect at a high dose (10.0 mg / kg).

[0096] Table 5 Effects of Stellarine A on the behavior of anxious rats induced by uncertain empty bottle stress in OFT

[0097]

[0098] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0099] (3) Light and dark box experiment

[0100] The light-dark box test is an experimental method widely used to evaluate the anxiety state of rodents. After the OFT test, the light-dark box test was conducted the next day. The light-dark box test was conducted in a box consisting of a dark room and a light room (45×30×15cm), with a small hole between the dark room and the light room for rats to pass freely. At the beginning of the test, the rat was placed in the center of the light room for 5 minutes. A video tracking system (SMART V3.0) was used to record the number and time of rat activities in the light box within 5 minutes. After each test, the feces was cleaned up and the light-dark box was wiped with 75% alcohol drops for the next test.

[0101] From Table 6, Figure 8 The results showed that compared with the blank control group, the acute restraint stress-induced anxiety model group could significantly reduce the number of rats' shuttles and the time they stayed in the light box (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the positive drugs DZP and SAG groups could significantly increase the number of rats' shuttles and the time they stayed in the light box (P<0.05, P<0.01), and the SAZ group could significantly increase the number of rats' shuttles (P<0.01). The results showed that in the light-dark box experiment, acute restraint stress could induce anxiety in rats, and Stellarine A at a high dose (10.0 mg / kg) could exert a significant anxiolytic effect.

[0102] Table 6 Effects of Stellarine A on the behavior of rats induced by acute restraint stress in LDT

[0103]

[0104] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0105] From Table 7, Fig. 9 The results showed that compared with the blank control group, the anxiety model group induced by uncertain empty bottle stress could significantly reduce the number of rats' shuttles and the time they stayed in the light box (P<0.01). Compared with the anxiety model group induced by uncertain empty bottle stress, the positive drug DZP group and SAD group could significantly increase the number of rats' shuttles (P<0.05), and the SAZ and SAG groups could significantly increase the number of rats' shuttles and the time they stayed in the light box (P<0.05, P<0.01). The results showed that in the light-dark box experiment, uncertain empty bottle stress could cause anxiety in rats, and Stellarine A could play an obvious anti-anxiety effect at medium doses (5.0 mg / kg) and high doses (10.0 mg / kg), and the effect was better with increasing doses.

[0106] Table 7 Effects of Stellarine A on the behavior of anxious rats induced by uncertain empty bottle stress in LDT

[0107]

[0108] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0109] The results of this study showed that Stellarine A could significantly improve the anxiety-like behavior of rats induced by acute restraint stress and uncertain empty bottle stress.

[0110] (IV) Specimen collection

[0111] After the rats were anesthetized, three rats were randomly selected from each group for whole brain extraction by cardiac perfusion, fixed with 4% paraformaldehyde, and then embedded in paraffin for HE staining and immunohistochemistry research; the remaining rats were fixed on a mouse tie board for blood extraction from the abdominal aorta, centrifuged after standing for 20 minutes, and the supernatant was aspirated and packaged. After blood extraction, the heads were cut off, and the brains were removed on ice, the hippocampus and frontal cortex tissues were removed, and then quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for later use.

[0112] (V) ELISA method for determination of ACTH, CORT, DA, and 5-HT levels

[0113] The rat frontal cortex was thawed on ice, and the contents of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the rat frontal cortex were determined according to the instructions of the ELISA kit (Shanghai ELISA Biotechnology Co., Ltd.). The rat serum was thawed on ice, and the contents of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in the rat serum were determined according to the instructions of the ELISA kit.

[0114] As shown in Table 8, Fig.10 The results showed that compared with the blank control group, the levels of ACTH and CORT in the rats in the acute restraint stress-induced anxiety model group were significantly increased (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the DZP group and the low, medium and high doses of Stellarine A groups were able to significantly reduce the levels of ACTH and CORT in rats with acute restraint stress-induced anxiety (P<0.05, P<0.01).

[0115] Table 8 Effects of Stellarine A on serum ACTH and CORT levels in rats with acute restraint stress-induced anxiety

[0116]

[0117] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0118] As shown in Table 9, Fig.11 The results showed that compared with the blank control group, the levels of ACTH and CORT in the uncertain empty bottle stress-induced anxiety model group were significantly increased (P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the DZP group and the medium and high doses of Stellarine A groups were able to significantly reduce the levels of ACTH and CORT in the uncertain empty bottle stress-induced anxiety rats (P<0.05, P<0.01).

[0119] Table 9 Effects of Stellarine A on serum ACTH and CORT levels in anxious rats induced by uncertain empty bottle stress

[0120]

[0121]

[0122] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0123] As shown in Table 10, Fig.12 The results showed that compared with the blank control group, the DA content in the anxiety model group induced by acute restraint stress was significantly increased, while the 5-HT content was significantly decreased (P<0.01). Compared with the anxiety model group induced by acute restraint stress, the DZP group and the medium and high doses of Stellarine A could significantly reduce the DA content (P<0.01) and significantly increase the 5-HT content (P<0.01) in the anxiety rats induced by acute restraint stress.

[0124] Table 10 Effects of Stellarine A on DA and 5-HT levels in brain tissue of rats with acute restraint stress-induced anxiety

[0125]

[0126] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0127] As shown in Table 11, Fig.13The results showed that compared with the blank control group, the DA content in the anxiety model group induced by uncertain empty bottle stress was significantly increased, while the 5-HT content was significantly decreased (P<0.01). Compared with the anxiety model group induced by uncertain empty bottle stress, the DZP group, the medium-dose and high-dose Stellarine A groups were able to significantly reduce the DA content of the anxious rats induced by uncertain empty bottle stress (P<0.01), and the DZP group and the high-dose Stellarine A group were able to significantly increase the 5-HT content (P<0.05, P<0.01).

[0128] Table 11 Effects of Stellarine A on DA and 5-HT levels in brain tissue of anxious rats induced by uncertain empty bottle stress

[0129]

[0130]

[0131] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0132] Stellarine A can significantly reverse the changes in the levels of glucocorticoids ACTH and CORT in rat serum and monoamine neurotransmitters DA and 5-HT in brain tissue, suggesting that its anxiolytic effect may be related to regulating the content of monoamine neurotransmitters in the brain and the HPA axis.

[0133] (VI) Histomorphological observation

[0134] (1) HE staining

[0135] Bake the slices in a 65℃ oven for 2h. The steps for dewaxing and hydrating the tissue are as follows: first, soak in xylene I and II for 20min, then soak in anhydrous ethanol I, II, and 75% alcohol for 5min, and finally wash with distilled water. Hematoxylin and eosin staining: differentiate with hematoxylin differentiation solution, rinse with tap water, blue with hematoxylin blueing solution, and rinse with tap water. Dehydrate the slices in 85% and 95% graded alcohol for 5min, then soak in eosin staining solution for 5min. Dehydrated slides: Soak the slices in anhydrous ethanol I, II, III, xylene I, II for 5min, and transparent slides. Analyze the images under a microscope.

[0136] like Fig.14The results showed that compared with the blank control group, the neurons in the acute restraint stress-induced anxiety model group were arranged loosely and irregularly, the cell bodies were shrunk and deformed, the boundaries between the nucleus and cytoplasm were unclear, the neurons were atrophied and stained darker; compared with the acute restraint stress-induced anxiety model group, the arrangement of cells in the hippocampus, the morphology of cells and the morphology of nucleoli in the high-dose Stellarine A group were improved.

[0137] like Fig.15 The results showed that compared with the blank control group, more neurons in the uncertain empty bottle stress-induced anxiety model group were scattered, loose, and irregularly arranged; compared with the uncertain empty bottle stress-induced anxiety model group, the neurons in the hippocampus of the positive drug diazepam group and the high-dose Stellarine A group had regular morphology, were relatively neat and densely arranged, with clear nucleoli, abundant cytoplasm, large and round nuclei, and their nuclear chromatin was uniformly light blue or blue.

[0138] (2) Immunohistochemical study

[0139] The slices were soaked in hematoxylin for 5 minutes and then rinsed with tap water. Then soaked in environmentally friendly dewaxing solution I, II, and III for 10 minutes, then soaked in anhydrous ethanol I, II, and III for 5 minutes, and finally washed with distilled water. In order to repair the antigen, the slices must be soaked in antigen repair buffer and microwave antigen repair treatment to avoid drying of the slices. After cooling, PBS (PH = 7.4) solution was used to shake the wash three times, each wash for 5 minutes. Add 3% hydrogen peroxide, incubate at room temperature in the dark for 25 minutes, and then use PBS (PH = 7.4) solution to shake the wash three times, each wash for 5 minutes. Use 3% bovine serum albumin (BSA) to evenly cover the tissue and perform a blocking treatment for 30 minutes. After removing the blocking solution, add the diluted primary antibody to the tissue and incubate it overnight at 4°C. Remove the primary antibody, then use PBS (PH = 7.4) solution to shake the wash three times, each wash for 5 minutes, and then add the secondary antibody to incubate at room temperature for 50 minutes. Then wash it on the slide three times, each time for 5 minutes. Wait for a while to let it dry slightly, then add freshly prepared DAB color developing solution in the circle. When it shows brown-yellow color, it indicates that the corresponding circle is positive. Rinse with tap water and stop coloring. Then perform hematoxylin counterstaining and hematoxylin differentiation solution differentiation, rinse and use hematoxylin blueing solution to gradually return to blue, and finally rinse with running water. Soak in 75% alcohol, 85% alcohol, anhydrous ethanol I, II, n-butanol, and xylene I for 5 minutes for dehydration, and pay attention to drying, and finally mount the slide. Collect images, observe the positive expression of CB1 and CB2 in the CA1 area, and calculate the average optical density value of each group.

[0140] As shown in Table 12 and Table 13, Figures 16 to 20 The results showed that compared with the blank control group, the average optical density values ​​of CB1 and CB2 in the CA1 area of ​​rats in the acute restraint stress-induced anxiety and uncertain empty bottle stress-induced anxiety model groups were significantly reduced (P<0.05, P<0.01); compared with the model group, the average optical density values ​​of CB1 and CB2 in the CA1 area of ​​rats in the DZP group and the high-dose Stellarine A group were significantly increased (P<0.05, P<0.01).

[0141] Table 12 Effects of Stellarine A on cannabinoid systems CB1 and CB2 in rats with acute restraint stress-induced anxiety

[0142]

[0143] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0144] Table 13 Effects of Stellarine A on the cannabinoid system CB1 and CB2 in anxious rats induced by uncertain empty bottle stress

[0145]

[0146] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0147] The above results indicate that Stellarine A can exert anxiolytic effects through the expression of CB1 and CB2 of the cannabinoid system.

[0148] Stellarine A can improve the expression levels of CB1 and CB2 proteins in the CA1 region of the rat hippocampus and upregulate the expression of CB1 and CB2 proteins in the endocannabinoid system. Stellarine A treatment has a significant anxiolytic effect on it, which may be associated with enhancing the expression of CB1 and CB2 in the endocannabinoid system.

[0149] (VII) RT-qPCR technical analysis

[0150] First, the hippocampus was taken out of the -80℃ freezer, thawed on ice, and lysed in a grinder at low temperature. The total RNA was extracted from the hippocampus of the rats with anxiety caused by the uncertain empty bottle using a total RNA extraction kit, and the purity and concentration of the total RNA were determined by an ultra-micro spectrophotometer. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Then a cDNA amplification kit was used for real-time fluorescence quantitative PCR reaction. The primer design is as shown in the following table:

[0151] Table 14 Primer sequences

[0152]

[0153]

[0154] As shown in Table 15, Fig.21 The results showed that compared with the blank control group, the mRNA levels of ERK, CREB and BDNF in the hippocampus of rats in the uncertain empty bottle stress-induced anxiety model group were significantly decreased (P<0.01); compared with the uncertain empty bottle stress-induced anxiety model group, the mRNA levels of CREB and BDNF in the hippocampus of rats in the DZP group were significantly increased (P<0.01), and the high-dose Stellarine A group significantly upregulated the mRNA levels of ERK, CREB and BDNF in the hippocampus of rats (P<0.01).

[0155] Table 15 Relative expression levels of BDNF, CREB and ERK mRNA in the hippocampus of anxious rats induced by uncertain empty bottle stress

[0156]

[0157] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0158] (VIII) Western Blot Analysis

[0159] Prepare the protein extract according to the instructions in the whole protein extraction kit and place it on ice for use. Add 0.5-1 mL of protein extract to each brain tissue sample and homogenize it at low temperature using a grinder. After grinding, centrifuge at 4°C, 12000r / min for 5 minutes; aspirate the supernatant, which is the whole protein extract. Use the BCA protein quantification kit to draw a standard curve, measure the sample absorbance value, and calculate the protein concentration. Mix the measured protein extract with Loading Buffer (reduced, 5×) in a ratio of 1:4, place it in a metal bath and heat it at 100°C for 10 minutes to fully denature the protein, then cool it to room temperature, centrifuge it briefly, and store it in a -80°C refrigerator for use. Use 10% SDS polyacrylamide gel electrophoresis to separate the denatured protein sample, and transfer the protein band to polyvinylidene fluoride (PVDF). After the transfer is completed, wash the strips with TBST three times, 5 minutes each time, and wash off the transfer solution. Place the membrane in a rapid blocking solution and block it for 15-20 minutes. Wash the strips three times with TBST to remove the blocking solution, each time for 10 minutes. Dilute the primary antibody to an appropriate concentration and incubate at 4°C overnight. Recover the primary antibody, wash the membrane three times with TBST, each time for 10 minutes, add the diluted secondary antibody, and incubate at room temperature for 1 hour. Discard the secondary antibody and wash the strips three times with TBST, each time for 10 minutes. Prepare developer A and developer B in a 1:1 ratio and mix them well. Place the strips into the developer for exposure.

[0160] As shown in Table 16, Fig. 22 The results showed that compared with the blank control group, the relative expression of BDNF protein in the frontal cortex of the rats in the uncertain empty bottle stress-induced anxiety model group (P<0.05), as well as the relative expression levels of p-CREB and p-ERK proteins were significantly decreased (P<0.01); compared with the uncertain empty bottle stress-induced anxiety model group, the relative expression levels of BDNF, p-CREB and p-ERK proteins in the frontal cortex of the rats in the high-dose Stellarine A group were significantly increased (P<0.05, P<0.01), and the relative expression level of BDNF protein in the frontal cortex of the rats in the DZP group was increased, and the relative expression levels of p-CREB and p-ERK proteins were significantly increased (P<0.05, P<0.01).

[0161] Table 16 Effects of Stellarine A on the expression of BDNF, p-CREB and p-ERK proteins in the frontal cortex of anxious rats induced by uncertain empty bottle stress

[0162]

[0163]

[0164] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0165] As shown in Table 17, Fig.23 The results showed that compared with the blank control group, the relative expression of BDNF protein in the hippocampus of rats in the uncertain empty bottle stress-induced anxiety model group (P<0.05), as well as the relative expression levels of p-CREB and p-ERK proteins were significantly decreased (P<0.01); compared with the uncertain empty bottle stress-induced anxiety model group, the relative expression of BDNF protein in the hippocampus of rats in the DZP group, SAD group, SAZ group and SAG group were significantly increased (P<0.05), and the relative expression of p-CREB and p-ERK proteins in the hippocampus of rats in the DZP group, SAZ group and SAG group were significantly increased (P<0.05, P<0.01).

[0166] Table 17 Effects of Stellarine A on the expression of BDNF, p-CREB and p-ERK proteins in the hippocampus of anxious rats induced by uncertain empty bottle stress

[0167]

[0168]

[0169] Note: Compared with Control, #P<0.05, ##P<0.01; Compared with Model, *P<0.05, **P<0.01.

[0170] Stellarine A treatment can significantly upregulate the expression levels of BDNF, p-CREB / CREB and p-ERK1 / 2 / ERK1 / 2 proteins. Therefore, Stellarine A may exert its anxiolytic effect by activating the ERK / CREB / BDNF pathway.

[0171] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0172] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing argyrocatecholamine A, characterized in that: The steps include: (1) reacting L-tryptophan methyl ester hydrochloride with methylglyoxal under catalytic conditions to obtain 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester; (2) 1-Acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester is dissolved in methanol and reacted with aqueous ammonia to generate argyrocatechol A.

2. The preparation method according to claim 1, characterized in that The catalyst in step (1) is concentrated sulfuric acid.

3. The preparation method according to claim 1, characterized in that: In the step (1), the molar ratio of L-tryptophan methyl ester hydrochloride to methylglyoxal is 1:1 to 1:1.

2.

4. The preparation method according to claim 1, characterized in that: The reaction time of step (1) is 10 to 48 hours.

5. The preparation method according to claim 1, characterized in that: In the step (2), the volume ratio of methanol to aqueous ammonia is 1:2 to 1:

3.

6. The preparation method according to claim 1, characterized in that: In the step (2), the mass volume ratio of 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester to methanol is 0.001 g / ml.

7. Use of arginine A prepared by the preparation method according to any one of claims 1 to 6 in the preparation of antianxiety drugs.

8. The use according to claim 7, characterized in that Used to adjust the changes in glucocorticoids ACTH and CORT in rat serum and monoamine neurotransmitters DA and 5-HT in brain tissue.

9. The use according to claim 7, characterized in that Used to regulate the expression of CB1 and CB2 proteins in the CA1 region of the hippocampus.

10. The use according to claim 7, characterized in that Used to regulate the expression of BDNF, CREB, and ERK.

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