Preparation method and application of a silver radix bupleuri alkaloid A

Abstract

This invention discloses a method for preparing siliceous alkaloid A and its application. It belongs to the field of organic synthesis technology. This invention provides a method for synthesizing the compound siliceous alkaloid A, which employs a Pictet-Spengular reaction, causing L-tryptophan methyl ester hydrochloride to undergo a cyclization condensation reaction with methylglyoxal under acid catalysis, followed by ammonolysis to obtain the target compound. This method has the following advantages: simple reaction steps, eliminating the need for complex separation and purification, improving operational convenience; high reaction selectivity, yielding a high-purity target compound, improving synthesis efficiency; and low-cost reagents, reducing synthesis costs. This invention is the first to study the anti-anxiety activity of siliceous alkaloid A and elucidate its mechanism of action.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and more specifically to a method for preparing silagene A and its application. Background Technology

[0002] Radix Stellariae, a traditional Chinese medicine, is slightly cold in nature and sweet in taste. It clears deficiency heat and eliminates fever caused by infantile malnutrition. It is the dried root of *Stellaria dichotoma* L. var. *lanceolata* Bge., a perennial herb belonging to the Caryophyllaceae family. It is a commonly used medicinal material for treating febrile diseases (such as fever due to prolonged illness and fever caused by infantile malnutrition). Modern pharmacological studies have shown that Radix Stellariae possesses antipyretic, anti-inflammatory, anti-allergic, anti-cancer, neuroprotective, and vasodilatory activities. Current basic research on Radix Stellariae mainly focuses on the extraction and separation of its chemical components. Alkaloids, sterols, and cyclic peptides are the main components and have shown good biological activity, providing a sufficient material basis for screening bioactive lead compounds from Radix Stellariae.

[0003] Research on the chemical constituents of *Stellaria media* is still in its initial stages. To date, only one total synthesis report has been published involving stellaria alkaloid A: In 2021, Tao Sheng et al. synthesized a series of β-carboline alkaloids starting from L-tryptophan and conducted antifungal activity studies, including the synthesis of stellaria alkaloid A through a three-step reaction. However, the high cost of its raw materials and the limited availability of active compounds restrict the study of the mechanism of action, efficacy, and application and development of *Stellaria media*.

[0004] In summary, how to provide a method for synthesizing the compound siliceosaine A and explore its activity is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing silagenicine A and its application.

[0006] On the one hand, this invention provides a method for synthesizing the compound silagene A. This method employs a Pictet-Spengular reaction, in which L-tryptophan methyl ester hydrochloride undergoes a cyclization condensation reaction with methylglyoxal under acid catalysis, followed by ammonolysis to obtain the target compound. This method has the following advantages: the reaction steps are simple, eliminating the need for complex separation and purification, thus improving operational convenience; the reaction selectivity is high, yielding the target compound with high purity, thereby improving synthesis efficiency; and the reagents are inexpensive, reducing synthesis costs. On the other hand, this invention is the first to study the anti-anxiety activity of silagene A and elucidate its mechanism of action.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing silagenicine A includes the following steps:

[0009] (1) L-tryptophan methyl ester hydrochloride was reacted with methylglyoxal under the catalytic condition of a catalyst to obtain methyl 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid;

[0010] (2) Dissolve methyl 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid in methanol and react with ammonia to generate silver bupleurum alkaloid A.

[0011] The synthesis route is as follows:

[0012]

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

[0014] Furthermore, in 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 for step (1) is 10 to 48 hours.

[0016] Furthermore, in step (1), after adding concentrated sulfuric acid, the mixture is stirred until L-tryptophan methyl ester hydrochloride is completely dissolved before adding methylglyoxal to ensure complete utilization of the raw materials. Oxygen is then introduced to accelerate the reaction and increase the yield.

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

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

[0019] Furthermore, in step (2), dissolving ammonia in methanol and then taking the supernatant for reaction can improve the purity of the product.

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

[0021] Furthermore, it was used to reverse changes in the levels of 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] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0025] (1) In this invention, L-tryptophan methyl ester hydrochloride and methyl glyoxal are used as raw materials. After Pictec-Spengular reaction, methyl 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid is obtained. Then, after ammonolysis, silver alkaloid A is obtained, which can realize the chemical synthesis of silver alkaloid A.

[0026] (2) The synthetic route of the present invention has the advantages of being simple, efficient, easy to operate and low in cost. It is suitable for the large-scale synthesis of silagenicine A and provides an important material basis for the evaluation of the bioactivity of silagenicine A.

[0027] (3) This invention is the first to propose the anti-anxiety activity of silagene A, which provides strong data support and reference for the development of highly effective and low-toxic natural anti-anxiety drugs. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 For example, the silver-based alkaloid A in Example 1 of this invention 1 HNMR spectrum;

[0030] Figure 2 For example, the silver-based alkaloid A in Example 1 of this invention 13 C NMR spectrum;

[0031] Figure 3 This diagram illustrates the docking of Stellarine A and diazepam with ERK, CREB, and BDNF in Embodiment 2 of the present invention. A represents Stellarine A docking with ERK; B represents diazepam docking with ERK; C represents Stellarine A docking with CREB; D represents diazepam docking with CREB; E represents Stellarine A docking with BDNF; and F represents diazepam docking with BDNF.

[0032] Figure 4 This is the effect of Stellarine A on the behavior of acute restraint stress-induced anxiety rats in EPM in Example 2 of the present invention;

[0033] Figure 5 This illustrates the effect of Stellarine A on the behavior of rats with uncertainty-induced empty bottle stress in EPM, as described in Example 2 of this invention.

[0034] Figure 6 This is the effect of Stellarine A on the behavior of acute restraint stress-induced anxiety rats in OFT in Example 2 of the present invention;

[0035] Figure 7 This illustrates the effect of Stellarine A on the behavior of uncertain empty bottle stress-induced anxiety rats in OFT in Example 2 of this invention.

[0036] Figure 8 This illustrates the effect of Stellarine A on the behavior of acute restraint stress-induced anxiety rats in LDT in Example 2 of this invention.

[0037] Figure 9 This illustrates the effect of Stellarine A on the behavior of rats with uncertainty-induced empty bottle stress in the LDT in Example 2 of this invention.

[0038] Figure 10 This illustrates the effect of Stellarine A on serum ACTH and CORT levels in rats with acute restraint stress-induced anxiety, as described in Example 2 of this invention.

[0039] Figure 11 This illustrates the effect of Stellarine A on serum ACTH and CORT levels in rats with uncertain empty bottle stress, as described in Example 2 of this invention.

[0040] Figure 12 The effect of Stellarine A on the levels of DA and 5-HT in the brain tissue of rats with acute restraint stress-induced anxiety, as described in Example 2 of this invention;

[0041] Figure 13 This refers to the effect of Stellarine A on the levels of DA and 5-HT in the brain tissue of rats with uncertain empty bottle stress in Example 2 of the present invention;

[0042] Figure 14 This invention illustrates the effect of Stellarine A on the morphology of hippocampal neurons in rats with acute restraint stress-induced anxiety, as described in Example 2 of this invention. A represents the blank control group; B represents the model group; C represents the diazepam group; and D represents the high-dose Stellarine A group. The total number of doses is 40 × 10⁻⁶, and n = 3.

[0043] Figure 15This invention illustrates the effect of Stellarine A on the morphology of hippocampal neurons in rats with uncertain empty bottle stress-induced anxiety, as described in Example 2 of this invention. A represents the blank control group; B represents the model group; C represents the diazepam group; and D represents the high-dose Stellarine A group. The total number of doses is 40 × 10⁻⁶, n = 3.

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

[0045] Figure 17 The image shows the expression of CB1 in the CA1 region of rats with acute restraint stress-induced anxiety in Example 2 of this invention. A represents the blank control group; B represents the model group; C represents the diazepam group; and D represents the high-dose Stellarine A group. The values ​​are: 400 μm, 20 × 10⁻⁶, n = 3.

[0046] Figure 18 The image shows the expression of CB2 in the CA1 region of acute restraint stress-induced anxiety rats in Example 2 of this invention.

[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; 400μm, 20×, n=3;

[0048] Figure 19 The expression of CB1 in the CA1 region of rats with uncertain empty bottle stress-induced anxiety in Example 2 of the present invention is shown. In this example, A represents the blank control group; B represents the model group; C represents the diazepam group; and D represents the high-dose Stellarine A group. The values ​​are 400 μm, 40 × 10⁻⁶, and n = 3.

[0049] Figure 20 The expression of CB2 in the CA1 region of rats with uncertain empty bottle stress-induced anxiety is shown in Example 2 of this invention. In this example, A represents the blank control group; B represents the model group; C represents the diazepam group; and D represents the high-dose Stellarine A group. The values ​​are 400 μm, 40 × 10⁻⁶, and n = 3.

[0050] Figure 21 The relative expression levels of BDNF, CREB, and ERK mRNA in the hippocampus of rats with uncertain empty bottle stress-induced anxiety in Example 2 of this invention;

[0051] Figure 22 This invention illustrates the effect of Stellarine A on the expression of BDNF, p-CREB, and p-ERK proteins in the frontal cortex of rats experiencing anxiety due to uncertain empty bottle stress, as described in Example 2 of this invention.

[0052] Figure 23This invention relates to the effect of Stellarine A on the expression of BDNF, p-CREB, and p-ERK proteins in the hippocampus of rats with anxiety induced by uncertain empty bottle stress, as described in Example 2 of this invention. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] The reagents required for this invention are conventional experimental reagents, purchased from commercially available 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 (Annegi Company) was dissolved in water, 1 mL of concentrated sulfuric acid and 3.6 mL of 40% methylglyoxal (Annegi Company) aqueous solution were added, oxygen was introduced, and the mixture was stirred overnight at room temperature, precipitating a yellow solid of 5.54 g. The filter cake was washed with water after filtration and dried. 1.01 g of the yellow solid was dissolved in 1 L of methanol (Annegi Company), and the mixture was stirred continuously. The supernatant was collected, and 1.5 L of 25% ammonia water was added. The mixture was stirred overnight at room temperature for 2 days, and then filtered and dried to obtain 0.38 g of solid A.

[0058] Solid A is a yellow powder, readily soluble in DMSO, with the molecular formula 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 (101MHz, 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). The above... 1 HNMR ( Figure 1 )and 13 C NMR ( Figure 2 The spectral data are basically consistent with the data in existing reports (Cui ZH, Li GY, Qiao L, et al. Two New Alkaloids From Stellaria dichotoma var. Lanceolata. Natural Product Letters, 2006, 7: 59-64.), therefore the compound was identified as Stellarine A.

[0059] The purity was determined to be 92.54% by liquid chromatography-mass spectrometry (LC-MS) analysis, and the calculated yield was 34.75%.

[0060] The yield calculation method for the target product, silagenoside 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] Research on the anti-anxiety effects and mechanisms of Stellarine A based on the ERK / CREB / BDNF signaling pathway

[0064] The following experiments used GraphPad Prism 9 software (GraphPad-software Inc., CA, USA) for plotting and SPSS Statistics 26 for data analysis. One-way ANOVA was used for comparisons between groups, the LSD test was used for homogeneity of variance, and the 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 a highly significant difference.

[0065] (I) Molecular docking

[0066] The SDF files and SMILES information of cinnamomea alkaloid A and diazepam were retrieved using PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) and SwissADME (http: / / www.swissadme.ch) websites. Diazepam was used as a positive docking control. The above small molecules were then input and optimized using Discovery Studio 2021 software, and defined as ligands.

[0067] The target proteins ERK (1ZC0), CREB (5CGP), and BDNF (6KZD) were queried and saved from the PDB protein database (https: / / www.rcsb.org / ). The obtained PDB files were imported into Discovery Studio 2021 software for appropriate protein modification, removing water and ligand molecules, and adding hydrogen. Then, the Clean Protein function was used to remove redundant protein structures, and the defined proteins were used as receptors to generate docking sites. Molecular docking was performed using LibDock with the selected target small molecules, and the affinity of the molecular conformation was evaluated using the LibDock score. The LibDock scores are shown in the table below. The docking status of Stellarine A and diazepam with ERK, CREB, and BDNF is shown in the table below. Figure 3 As shown.

[0068] Table 1 LibDock Ratings

[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 docked with ERK(1ZC0), it formed five hydrogen bonds with protein residues ARG284, GLU254, SER256, GLY264, and SER168, exhibiting strong hydrogen bonding. The anion of GLU258 formed a conjugation effect with the benzene ring of the ligand. ILE266 and PRO263 exhibited π-alkyl interactions with alkyl groups, and VAL143 was linked via Pi-Sigma interaction. The LibDock score was 105.92. When Stellarine A docked with CREB(5CGP), it connected with protein residues MET1133 and ASN1168 via hydrogen bonds, and formed a weaker C-H bond with PHE1111. The benzene ring formed a conjugation effect with the anions of VAL1115, ALA1164, PRO1110, LEU1120, and VAL1174. The LibDock score was 83.34. When Stellarine A docks with BDNF (6KZD), it is linked via hydrogen bonds at GLU618, and PHE617 forms a stable π-π hydrophobic stacking interaction with the aromatic ring of Stellarine A. VAL601 exhibits π-alkyl interactions with the alkyl group. The LibDock score is 97.81. These results indicate that Stellarine A shows high affinity for all three proteins, and its LibDock scores are higher than those of the positive control diazepam. Therefore, it can be predicted that Stellarine A exerts its anti-anxiety activity through its target proteins (ERK, CREB, and BDNF). Based on the LibDock score, the following experiments will investigate the anti-anxiety activity and mechanism of Stellarine A through the ERK / CREB / BDNF signaling pathway.

[0071] (II) Establishment of the anxiety model and medication regimen

[0072] SPF-grade male SD rats were randomly divided into six groups of six each: a control group, a model group, low-dose (SAD), medium-dose (SAZ), and high-dose (SAG) Stellarine A groups, and a positive control group (DZP). The control group was housed in a group, while the other groups were housed individually. (An acute restraint stress-induced anxiety model and an uncertain empty bottle stress-induced anxiety model were established, using a total of 72 rats).

[0073] The dosage was determined, and the positive control group was administered DZP (1.0 mg / kg). The Stellarine A group was administered 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 administered the corresponding volume of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution via gavage. Administration was continued for 7 days. Starting on day 5, except for the blank control group, all other groups were placed in restraint devices after gavage administration to establish an acute restraint model. During this period, food and water were withheld, and only the rats' activity range was restricted; respiration was not suppressed. Stimulation was performed for 30 minutes daily for 3 consecutive days. The model was established on day 7, marking the end of the acute restraint stress-induced anxiety model in rats.

[0074] Rats were trained to drink water at set times for 7 days. Except for the control group, all other groups were given water for 10 minutes at fixed times in the morning and evening, and the water bottles were removed at other times. After the water training period, an uncertain empty bottle stress model was established, with rats receiving uncertain empty water bottle stimulation at fixed time intervals for two consecutive weeks. Following the stress, the positive control drug DZP (1.0 mg / kg) was administered. The Stellarine A treatment groups were administered SAD (2.5 mg / kg), SAZ (5.0 mg / kg), and SZG (10.0 mg / kg), respectively. The control group and the model group were administered the corresponding volume of 0.5% CMC-Na solution via gavage. Administering the drugs via gavage for 7 consecutive days established an uncertain empty bottle stress-induced anxiety model in rats.

[0075] (III) Behavioral Experiments

[0076] Behavioral tests were conducted on the acute restraint stress group after modeling on day 7. Behavioral tests were conducted on the uncertain empty bottle stress group on day 7, 1 hour after the last administration of the blank control group, model group, and drug administration group, and 0.5 hours after the positive drug group was administered the drug via gavage, in order to evaluate the anxiety-like behavior of rats.

[0077] (1) Elevated Cross Maze Experiment

[0078] The elevated cross maze experiment utilizes animals' exploratory nature towards novel environments and their fear of suspended open arms to create ambivalent behavior. The elevated cross maze device is shaped like a cross and consists of two opposing open arms, two opposing closed arms, and a central area, placed at a height of 50 cm above the ground. Before the experiment, rats are placed in the testing chamber for 30 minutes to acclimatize, and the testing environment is kept relatively quiet. Each rat is placed individually in the central area, facing one open arm at the start of the test, and is allowed 5 minutes of free exploration. The movement trajectories of all rats are tracked by a target tracking system (SMART V3.0) consisting of cameras fixed above the maze. Anxiety levels are assessed using the time spent in the open arms (percentage of total exploration time with both arms open, OT%) and the number of times rats enter the open arms (percentage of entries into the open arms out of all arm entries, OE%). After each test, feces are cleaned and odor is eliminated by spraying with 75% alcohol.

[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 significantly reduced the percentage of rats entering the open arm and the percentage of time spent in the open arm (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the positive control groups DZP, SAZ, and SAG significantly increased the percentage of rats entering the open arm (P<0.01) and the percentage of time spent in the open arm (P<0.05, P<0.01). This indicates that acute restraint stress can induce anxiety in rats during the elevated cross maze experiment, and Stellarine A can exert significant anti-anxiety effects at medium (5 mg / kg) and high (10 mg / kg) doses.

[0080] Table 2. Effects of Stellarine A on the behavior of rats under acute restraint stress-induced anxiety 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 significantly reduced the percentage of rats entering the open arm and the percentage of time spent in the open arm (P<0.05, P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the positive control drug DZP group significantly increased the percentage of rats in the open arm (P<0.01), and the SAZ (P<0.05, P<0.01) and SAG groups also significantly increased the percentage of rats entering the open arm and the percentage of time spent in the open arm (P<0.01). These results indicate that uncertain empty bottle stress can induce anxiety in rats during the elevated cross maze test, and Stellarine A exerts significant anti-anxiety effects at medium (5 mg / kg) and high (10 mg / kg) doses.

[0084] Table 3. Behavioral effects of Stellarine A on uncertain empty bottle stress-induced anxiety in rats during EPM.

[0085]

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

[0087] Pharmacodynamic and behavioral results showed that, compared with the blank control group, the number of times rats entered the open arms of the elevated cross maze and the percentage of time spent in the acute restraint stress and uncertain empty bottle stress-induced anxiety model group were significantly reduced; compared with the acute restraint stress and uncertain empty bottle stress-induced anxiety model group, the number of times rats entered the open arms of the elevated cross maze and the percentage of time spent in the elevated cross maze were significantly increased in the Stellarine A administration group.

[0088] (2) Open field experiment

[0089] The open field test is primarily used to evaluate the neurobiological basis of anxiety and to detect anxiety-like behaviors in animals. The open field test is conducted the day after the EPM test. Rats are allowed at least 30 minutes to acclimatize to the test environment before the test, ensuring a relatively quiet environment. The open field box is a square area (80cm, 80cm, 40cm) with plastic walls and a floor. The bottom is divided into a 50% central area and a 50% peripheral area using software settings and placed in a dimly lit isolation chamber. Rats are placed in the central area of ​​the box and allowed free movement for 5 minutes. A camera fixed above the floor records the distance and time the rats walk, and the data is analyzed using a video tracking system (SMART V3.0) to count the number of times the rats entered the central area and the time spent there. After each test, feces are removed, and the test box is wiped with 75% alcohol to eliminate odor and avoid interference.

[0090] From Table 4, Figure 6The results showed that, compared with the blank control group, the number of times rats entered the central area and the time spent in the central area were significantly reduced in the acute restraint stress-induced anxiety model group (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the time spent in the central area was significantly increased in the diazepam positive control group (P<0.01), the number of times rats entered the central area was significantly increased in the SAZ group (P<0.05), and the number of times rats entered the central area was significantly increased in the SAG group (P<0.01) and the time spent in the central area was significantly increased (P<0.05). These results indicate that acute restraint stress can induce anxiety in rats in the open field experiment, and high doses of Stellarine A (10.0 mg / kg) can exert a significant anti-anxiety effect.

[0091] Table 4. Effects of Stellarine A on behavioral behavior in acute restraint stress-induced anxiety rats during 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 entered the central area and the time spent in the central area were significantly reduced in the uncertain empty bottle stress-induced anxiety model group (P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the number of times rats entered the central area and the time spent in the central area were significantly increased in the positive drug diazepam group and the SAG group (P<0.01), and the time spent in the central area was significantly increased in the SAZ group (P<0.01). The results indicate that uncertain empty bottle stress can induce anxiety in rats in the open field experiment, and can exert a significant anti-anxiety effect at high doses of Stellarine A (10.0 mg / kg).

[0096] Table 5. Effects of Stellarine A on behavioral behavior in uncertain empty bottle stress-induced anxiety rats during OFT.

[0097]

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

[0099] (3) Dark and Bright Box Experiment

[0100] The light-dark box test is a widely used experimental method for evaluating anxiety levels in rodents. The light-dark box test is conducted the day after the OFT test. The test is performed in a box consisting of a dark chamber and a light chamber (45×30×15cm), with a small hole between the two chambers allowing the rat to pass freely. At the start of the test, the rat is placed in the center of the light chamber for 5 minutes. The number of times and the duration of the rat's movement in the light chamber within 5 minutes are recorded using a video tracking system (SMART V3.0). After each test, the rat's feces are cleaned, and the light-dark box is wiped with a drop of 75% alcohol 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 significantly reduced the number of rats shuttling and the time spent in the bright box (P<0.01). Compared with the acute restraint stress-induced anxiety model group, the positive control drug DZP and SAG groups significantly increased the number of rats shuttling and the time spent in the bright box (P<0.05, P<0.01), while the SAZ group significantly increased the number of rats shuttling (P<0.01). These results indicate that acute restraint stress can induce anxiety in rats during the light-dark box experiment, and high doses of Stellarine A (10.0 mg / kg) can exert a significant anti-anxiety effect.

[0102] Table 6. Effects of Stellarine A on the behavior of rats under acute restraint stress-induced anxiety in the 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, Figure 9 The results showed that, compared with the blank control group, the uncertain empty bottle stress-induced anxiety model group significantly reduced the number of rat shuttles and the time spent in the bright box (P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the positive control group DZP and SAD significantly increased the number of rat shuttles (P<0.05), while the SAZ and SAG groups significantly increased the number of rat shuttles and the time spent in the bright box (P<0.05, P<0.01). These results indicate that uncertain empty bottle stress can induce anxiety in rats during the light-dark box experiment. Stellarine A at medium (5.0 mg / kg) and high (10.0 mg / kg) doses exhibits significant anti-anxiety effects, with the effect increasing with increasing dose.

[0106] Table 7. Effects of Stellarine A on behavioral behavior in uncertainty bottle stress-induced anxiety rats during the 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 show that Stellarine A can significantly improve anxiety-like behaviors induced by acute restraint stress and uncertain empty bottle stress in rats.

[0110] (iv) Specimen Collection

[0111] After anesthetizing rats, three rats from each group were randomly selected for whole-brain extraction via cardiac perfusion. The brains were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for HE staining and immunohistochemical studies. The remaining rats were fixed to a binding board for blood collection from the abdominal aorta. After standing for 20 minutes, the blood was centrifuged, and the supernatant was collected and aliquoted. After blood collection, the rats were decapitated, and the hippocampus and frontal cortex were dissected on ice. The brain tissue was then flash-frozen in liquid nitrogen and stored at -80°C for later use.

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

[0113] Rat frontal cortex was thawed on ice, and the levels of dopamine (DA) and serotonin (5-HT) in the rat frontal cortex were measured according to the ELISA kit instructions (Shanghai Enzyme-Link Biotechnology Co., Ltd.). Rat serum was thawed on ice, and the levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in the rat serum were measured according to the ELISA kit instructions.

[0114] As shown in Table 8, Figure 10 The results showed that, compared with the blank control group, the levels of ACTH and CORT in 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 dose groups of Stellarine A were all 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, Figure 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, the medium- and high-dose Stellarine A groups could significantly reduce the levels of ACTH and CORT in rats with uncertain empty bottle stress-induced anxiety (P<0.05, P<0.01).

[0119] Table 9. Effects of Stellarine A on serum ACTH and CORT levels in rats with uncertainty-empty-bottle stress-induced anxiety.

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

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

[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, Figure 13The results showed that, compared with the blank control group, the level of dopamine (DA) was significantly increased and the level of 5-HT was significantly decreased in the uncertain empty bottle stress-induced anxiety model group (P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the DZP group, the medium- and high-dose Stellarine A groups all significantly reduced the DA level in rats with uncertain empty bottle stress-induced anxiety (P<0.01), while the DZP group and the high-dose Stellarine A group significantly increased the level of 5-HT (P<0.05, P<0.01).

[0128] Table 11 Effects of Stellarine A on DA and 5-HT levels in brain tissue of rats induced by uncertainty 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 significantly reversed changes in serum levels of glucocorticoids ACTH and CORT, as well as monoamine neurotransmitters DA and 5-HT in rat brain tissue, suggesting that its anti-anxiety effect may be related to the regulation of monoamine neurotransmitter levels and the HPA axis in the brain.

[0133] (vi) Observation of organizational morphology

[0134] (1) HE staining

[0135] The sections were baked in a 65°C oven for 2 hours. The dewaxing and hydration process was as follows: First, the sections were soaked in xylene I and II for 20 minutes, followed by soaking in anhydrous ethanol I and II and 75% alcohol for 5 minutes, and finally washed with distilled water. Hematoxylin and eosin staining: Hematoxylin differentiation solution was used for differentiation, followed by rinsing with tap water, hematoxylin blueing solution, and rinsing with tap water. The sections were dehydrated in a gradient of 85% and 95% alcohol for 5 minutes, and then soaked in eosin staining solution for 5 minutes. Dehydration and mounting: The sections were soaked in anhydrous ethanol I, II, and III, and xylene I and II for 5 minutes, and then cleared and mounted. Microscopic images were analyzed.

[0136] like Figure 14The results showed that, compared with the blank control group, the neurons in the acute restraint stress-induced anxiety model group were loosely and irregularly arranged, the cell bodies were shrunken and deformed, the boundary between the cell nucleus and cytoplasm was unclear, and the neurons were atrophied and stained more deeply. Compared with the acute restraint stress-induced anxiety model group, the high-dose Stellarine A group showed improvements in the arrangement of cells, cell morphology and nucleolus morphology in the hippocampus.

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

[0138] (2) Immunohistochemical studies

[0139] The tissue sections were immersed in hematoxylin for 5 minutes, then rinsed with tap water. Next, they were immersed in environmentally friendly dewaxing solutions I, II, and III for 10 minutes, followed by immersion in anhydrous ethanol solutions I, II, and III for 5 minutes each, and finally washed with distilled water. For antigen retrieval, the sections were immersed in antigen retrieval buffer and subjected to microwave antigen retrieval treatment to prevent drying. After cooling, the sections were washed three times with PBS (pH 7.4) for 5 minutes each time. 3% hydrogen peroxide was added, and the sections were incubated at room temperature in the dark for 25 minutes, followed by three more washes with PBS (pH 7.4) for 5 minutes each time. 3% bovine serum albumin (BSA) was evenly applied to the tissue, and a blocking treatment was performed for 30 minutes. After removing the blocking solution, diluted primary antibody was added to the tissue, and the tissue was incubated overnight at 4°C. The primary antibody was removed, and the sections were washed three times with PBS (pH 7.4) for 5 minutes each time, followed by incubation at room temperature for 50 minutes with secondary antibody. The slide was then washed three times, 5 minutes each time. After a short time to allow it to air dry slightly, freshly prepared DAB staining solution was added to the circle. A brownish-yellow color indicated a positive result. The slide was then rinsed with tap water and the staining process was stopped. Hematoxylin counterstaining and hematoxylin differentiation solution were then applied. The slide was rinsed and hematoxylin blue-return solution was used to gradually restore the blue color. Finally, the slide was rinsed with running water. The slide was then dehydrated by soaking in 75% ethanol, 85% ethanol, anhydrous ethanol I and II, n-butanol, and xylene I for 5 minutes, and then dried. Finally, the slide was mounted. Images were acquired, and the positive expression of CB1 and CB2 in the CA1 region was observed. The average optical density values ​​for each group were calculated.

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

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

[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 systems CB1 and CB2 in rats experiencing uncertainty-empty bottle stress-induced anxiety.

[0145]

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

[0147] These results indicate that Stellarine A can exert its anxiolytic effect through the expression of CB1 and CB2 in the cannabinoid system.

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

[0149] (vii) RT-qPCR technology analysis

[0150] First, the hippocampus was removed from the -80℃ freezer, thawed on ice, and homogenized in a homogenizer using lysis buffer. Total RNA was extracted from the hippocampus of anxiety-inducing rats using a total RNA extraction kit, and the purity and concentration of total RNA were determined using a micro spectrophotometer. The RNA was reverse transcribed into cDNA using a reverse transcription kit. Then, real-time quantitative PCR was performed using a cDNA amplification kit. Primer design is shown in the table below:

[0151] Table 14 Primer Sequences

[0152]

[0153]

[0154] As shown in Table 15, Figure 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 rats with uncertain empty bottle stress-induced anxiety.

[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 extraction solution according to the instructions of the whole protein extraction kit and keep it on ice. Add 0.5-1 mL of protein extraction solution to each brain tissue sample and homogenize it at low temperature using a homogenizer. After homogenization, centrifuge at 12000 rpm for 5 min at 4°C; collect the supernatant, which is the whole protein extract. Use the BCA protein quantification kit to plot a standard curve, measure the absorbance of the samples, and calculate the protein concentration. Mix the protein extract with loading buffer (reduced, 5×) at a ratio of 1:4, heat in a metal bath at 100°C for 10 min to fully denature the protein, cool to room temperature, centrifuge briefly, and store at -80°C for later use. Separate the denatured protein samples by 10% SDS-polyacrylamide gel electrophoresis and transfer the protein bands to polyvinylidene fluoride (PVDF). After the transfer is complete, wash the bands three times with TBST for 5 min each time to remove the transfer buffer. Place the membrane in rapid blocking buffer and block for 15-20 min. Wash the strips three times with TBST to remove blocking buffer, 10 min each time. Dilute the primary antibody to the appropriate concentration and incubate overnight at 4°C. Recover the primary antibody, wash the membrane three times with TBST, 10 min each time, add the diluted secondary antibody, and incubate at room temperature for 1 h. Discard the secondary antibody, wash the strips three times with TBST, 10 min each time. Prepare developer A and developer B at a 1:1 ratio, mix well, and expose the strips in a developing apparatus.

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

[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, Figure 23 The results showed that, compared with the blank control group, the relative expression levels of BDNF protein, p-CREB, and p-ERK proteins in the hippocampus of rats in the uncertain empty bottle stress-induced anxiety model group were significantly decreased (P<0.05) (P<0.01). Compared with the uncertain empty bottle stress-induced anxiety model group, the relative expression levels of BDNF protein in the hippocampus of rats in the DZP, SAD, SAZ, and SAG groups were significantly increased (P<0.05), and the relative expression levels of p-CREB and p-ERK proteins in the hippocampus of rats in the DZP, SAZ, and SAG groups 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 rats experiencing anxiety due to uncertainty about empty bottles.

[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 significantly upregulated the expression levels of BDNF, p-CREB / CREB, and p-ERK1 / 2 / ERK1 / 2 proteins. Therefore, Stellarine A may exert its anti-anxiety effect by activating the ERK / CREB / BDNF pathway.

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

[0172] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of silagenitine A in the preparation of anti-anxiety drugs, characterized in that, The preparation method of silagene A includes the following steps: (1) L-tryptophan methyl ester hydrochloride was reacted with methylglyoxal under the catalytic condition of a catalyst to obtain methyl 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid; (2) Dissolve methyl 1-acetyl-9H-pyrido[3,4-b]indole-3-carboxylic acid in methanol and react with ammonia to generate silver bupleurum alkaloid A.

2. The application as described in claim 1, characterized in that, The catalyst mentioned in step (1) is concentrated sulfuric acid.

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

2.

4. The application as described in claim 1, characterized in that, The reaction time for step (1) is 10 to 48 hours.

5. The application as described in claim 1, characterized in that, In step (2), the volume ratio of methanol to ammonia is 1:2 to 1:

3.

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

7. The application as described in claim 1, characterized in that, It was used to reverse changes in serum glucocorticoids ACTH and CORT, as well as monoamine neurotransmitters DA and 5-HT in rat brain tissue.

8. The application as described in claim 1, characterized in that, It is used to regulate the expression of CB1 and CB2 proteins in the CA1 region of the hippocampus.

9. The application as described in claim 1, characterized in that, Used to regulate the expression of BDNF, CREB, and ERK.

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