CN114507205A

CN114507205A - Dihydromyricetin pyrrolidine compound and preparation method and application thereof

Info

Publication number: CN114507205A
Application number: CN202210408113.XA
Authority: CN
Inventors: 梁京
Original assignee: Beijing Jiafurui Biological Technology Co ltd
Current assignee: Beijing Jiafurui Biological Technology Co ltd
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2022-05-17
Also published as: WO2023202477A1

Abstract

The invention discloses a dihydromyricetin pyrrolidine compound and a preparation method and application thereof, wherein the dihydromyricetin pyrrolidine compound has a molecular formula shown in formula (I); the preparation method of the dihydromyricetin pyrrolidine compound comprises the following steps: s1: adding anhydrous methanol into dihydromyricetin to obtain dihydromyricetin anhydrous methanol solution; s2: under the protection of inert gas, adding anhydrous methanol into the pyrrolidine to obtain anhydrous methanol solution of the pyrrolidine; s3: reacting the dihydromyricetin absolute methanol solution obtained in the step S1 with the pyrrolidine absolute methanol solution obtained in the step S2 at room temperature; s4: and (4) filtering the reaction residue obtained in the step (S3), washing a filter cake by using anhydrous methanol, and performing vacuum pumping to obtain the dihydromyricetin pyrrolidine compound. Application of dihydromyricetin pyrrolidine complex in preparation of medicine for treating nervous system dysfunction is provided.

Description

Dihydromyricetin pyrrolidine compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a dihydromyricetin pyrrolidine compound, and a preparation method and application thereof.

Background

Dihydromyricetin (DHM) is a natural flavonoid compound extracted from hovenia and ampelopsis grossedentata, and has been widely used in the tonic market due to its various health benefits.

Since DHM is a natural flavonoid compound which has some disadvantages such as easy degradation and oxidation in oxygen, light and high temperature exposure environments and low solubility, there are many limitations in developing it as a therapeutic agent for alzheimer's disease (hereinafter abbreviated as AD) and other neurodegenerative diseases, and many difficulties are faced in the development of formulations, which affects the production of formulations. Therefore, maintaining the original efficacy of DHM, reducing the above-mentioned drawbacks to obtain more excellent efficacy is the key point in developing new dosage forms of DHM.

In the journal of modern food science and technology of China, No. 10 in 2014, Cao Minhui et al published the preparation of ampelopsis grossedentata dihydromyricetin theanine compound and the research on antioxidant activity, and discloses that the dihydromyricetin is up to 25.6 percent in natural ampelopsis grossedentata, has various physiological functions and is widely concerned. However, the dihydromyricetin has poor water solubility at normal temperature and low bioavailability, the application range of the dihydromyricetin is limited, in order to improve the water solubility of the dihydromyricetin, the dihydromyricetin-theanine compound is prepared by performing molecular modification on the dihydromyricetin, the compound is subjected to structural characterization by using an ultraviolet spectrum, an infrared spectrum, a mass spectrum and a nuclear magnetic resonance hydrogen spectrum, the physicochemical properties of the compound are measured, and the antioxidant activity of the compound is investigated. Through structural analysis, the compound is formed by combining C3-OH, C5-OH, C7-OH, C3 '-OH and C5' -OH in a dihydromyricetin molecule with active hydrogen of theanine through hydrogen bonds; the capability of the compound for removing OH free radicals is higher than that of dihydromyricetin and Vc, and the capability of removing O2 < - >. free radicals and DPPH free radicals is equivalent to that of the dihydromyricetin and is higher than that of the Vc. The combination of dihydromyricetin and theanine not only improves the water solubility of the dihydromyricetin, but also plays a role in strengthening the antioxidant activity.

In the journal of Chinese patent drug, vol 43 and 12 th of 2021, 12 months, Wei Yongge, etc., the preparation of dihydromyricetin phospholipid complex and its dripping pills and the comparison of in vivo pharmacokinetics are published, and the preparation of dihydromyricetin phospholipid complex and its dripping pills are disclosed, and the comparison of in vivo pharmacokinetics is made. The dripping pill can improve the accumulative dissolution rate and oral bioavailability of dihydromyricetin phospholipid complex.

Although the dihydromyricetin-theanine compound and the dihydromyricetin phospholipid compound have certain effects on water solubility and antioxidant activity, the application of the actual biological efficacy to medicines is not ideal, and especially the industrialized production and synthesis can be realized.

Disclosure of Invention

The invention aims to solve the technical problem of providing a dihydromyricetin pyrrolidine compound which has improved solubility and stability and improved biological efficiency and can be applied to the technical field of medicines.

In order to solve the technical problems, the technical scheme adopted by the invention is that the molecular formula of the dihydromyricetin pyrrolidine compound is shown as the formula (I):

（I）。

through modification and change of the dihydromyricetin DHM, the solubility and stability are improved, and the biological efficiency is improved, so that industrial production and synthesis and future usability for patients are realized, and the dihydromyricetin DHM can be applied to the technical field of medicines; water solubility (solubility) and stability are the essential physicochemical properties of small organic molecule drugs and are also very important issues in drug discovery. Improved water solubility generally results in better drug efficacy and more satisfactory pharmacokinetic profiles. It is emphasized that there are many methods for salifying dihydromyricetin DHM, but it is crucial that the pharmacological properties of DHM that have been found are not altered.

Another problem to be solved by the present invention is to provide a method for preparing a dihydromyricetin pyrrolidine complex, comprising the following steps:

s1: adding anhydrous methanol into dihydromyricetin to obtain dihydromyricetin anhydrous methanol solution;

s2: under the protection of inert gas, adding anhydrous methanol into the pyrrolidine to obtain anhydrous methanol solution of the pyrrolidine;

s3: reacting the dihydromyricetin absolute methanol solution obtained in the step S1 with the pyrrolidine absolute methanol solution obtained in the step S2 at room temperature to obtain a reactant;

s4: and (4) filtering the reactant obtained in the step (S3), washing a filter cake by using anhydrous methanol, and performing decompression and suction drying to obtain the dihydromyricetin pyrrolidine compound.

Preferably, the inert gas in step S2 is argon.

Preferably, the concentration of the dihydromyricetin anhydrous methanol solution in the step S1 is 150-350 mmol/L; the concentration of the pyrrolidine anhydrous methanol solution in the step S2 is 1-10 mol/L.

Preferably, the molar ratio of dihydromyricetin to tetrahydropyrrole in step S3 is 1: 0.7-1.3.

Preferably, the molar ratio of dihydromyricetin to tetrahydropyrrole in step S3 is 1: 1.

preferably, in step S1, the anhydrous methanol is added to the dihydromyricetin, and then the mixture is transferred to an ice-water bath to be stirred for 30 minutes; in step S3, after 2 drops/sec of anhydrous methanol solution of pyrrolidine was added to the anhydrous methanol solution of dihydromyricetin in an ice water bath, the reaction was carried out at room temperature.

Preferably, the preparation method of dihydromyricetin in step S1 includes:

s1-1: adding anhydrous dichloromethane and N, N-diisopropyl ethylamine into 2,4, 6-trihydroxyacetophenone, adding chloromethyl methyl ether at the speed of 1 drop/second, and obtaining 2-hydroxy-4, 6-dimethoxy methoxyidene acetophenone after the reaction is finished;

s1-2: under the protection of inert gas, mixing the 2-hydroxy-4, 6-dimethoxymethoxylideneacetophenone obtained in the step S1-1 with anhydrous tetrahydrofuran; then sodium hydride and chloromethyl methyl ether are added in sequence; after the reaction is finished, obtaining 2,4, 6-trimethoxy methoxylene acetophenone;

s1-3: under the protection of inert gas, mixing the 2,4, 6-trimethoxymethoxylideneacetophenone obtained in the step S1-2 with THF and water; then adding sodium hydroxide and 3,4, 5-trihydroxy benzaldehyde successively; after the reaction is finished, (E) -3-trihydroxybenzene-1- (2, 4, 6-trimethoxymethyleneoxyphenyl) -vinyl ketone is obtained;

s1-4: mixing the (E) -3-trihydroxybenzene-1- (2, 4, 6-trimethoxymethyleneoxyphenyl) -vinyl ketone obtained in the step S1-3 with 30% of hydrogen peroxide; after the reaction is finished, adding saturated sodium sulfite solution to quench the reaction, extracting by ethyl acetate, decompressing and removing the solvent, dissolving the residue after removing the solvent in methanol, adding dilute hydrochloric acid, decompressing and removing the solvent after the reaction is finished, and obtaining the dihydromyricetin tetrahydropyrrole compound.

The invention also provides the application of the dihydromyricetin pyrrolidine complex in the preparation of medicines for treating nervous system dysfunction diseases.

Preferably, the neurological disorder disease is sleep disorder, anxiety, depression, post-traumatic stress disorder, alzheimer's disease, dementia, parkinson's disease, stroke, epilepsy, autism, alcohol use disorder.

The dihydromyricetin pyrrolidine complex (DHMP) provided by the invention maintains the biological capability of Dihydromyricetin (DHM), improves the drug effect, improves the water solubility and enhances the GABA activity_AThe effect of Rs (as low as 0.1 nM), 1000-fold that of native DHM, replicates the efficacy of DHM and restores the expression of gephyrin level in experimental animals. Can improve recognition/memory impairment, improve behavioral impairment such as anxiety, depression, sleep disorder, and reduce cognitive impairmentThe attack and reduction of brain damage caused by stroke can be used as a treatment means for improving brain function related to motor function after stroke. DHMP shows activation of GABA_AR is similar potency and is more than 1000 times higher than DHM potency. Indications for DHM use include sleep disorders, anxiety, depression, early and intermediate stages of alzheimer's disease, dementia, parkinson's disease, other neurodegenerative diseases, stroke, epilepsy, autism, alcohol use disorders, and addiction to Benzodiazepines (Benzodiazepines) and other drugs.

Dihydromyricetin tetrahydropyrrole complex (DHMP) has biological safety. The experimental rats did not show any negative changes in the two-week functional observation combination (FOB) study at daily oral doses of 1, 10, and 100 mg/kg. There were no toxic side effects and no metabolic rate changes during the long-term evaluation of six months of 1, 10, and 500mg/kg dosing.

Drawings

The following further detailed description of embodiments of the invention is made with reference to the accompanying drawings:

FIG. 1 is a flow chart of the preparation of dihydromyricetin pyrrolidine complex (DHMP) of the present invention;

FIGS. 2a and 2b are nuclear magnetic resonance spectra of dihydromyricetin pyrrolidine complex (DHMP) prepared by the present invention, and FIGS. 2c and 2d are nuclear magnetic resonance spectra of Dihydromyricetin (DHM);

FIGS. 3a-3d are graphs of the dihydromyricetin tetrahydropyrrole complex (DHMP) versus GABA of the present invention_AA whole-cell patch clamp current comparison graph of positively regulated mouse brain sections of R;

FIGS. 4a and 4b are graphs comparing the effect of dihydromyricetin pyrrolidine complex (DHMP) of the present invention on mouse targeted proppant protein expression;

FIGS. 5, 6a and 6b are graphs comparing the improvement of dihydromyricetin pyrrolidine complex (DHMP) of the present invention on cognitive/memory disorders in mice;

FIGS. 7a-7f are graphs comparing the effect of dihydromyricetin pyrrolidine complex (DHMP) of the present invention on improving locomotor activity, reducing anxiety and seizure in mice;

FIGS. 8a-8c are graphs comparing the effect of dihydromyricetin pyrrolidine complex (DHMP) of the present invention on the amelioration of stroke-induced brain damage in mice;

FIGS. 9a-9f are graphs showing the effect of dihydromyricetin pyrrolidine complex (DHMP) of the present invention in reducing brain damage caused by stroke by inhibiting excessive release of glutamate;

FIG. 10 shows the dihydromyricetin tetrahydropyrrole complex (DHMP) versus GABA of the present invention_AA graph of the enhancement effect of R;

FIGS. 11a to 11j are graphs showing the results of non-toxicity safety tests of dihydromyricetin pyrrolidine complex (DHMP) according to the present invention.

Detailed Description

The molecular formula of the dihydromyricetin pyrrolidine complex is shown as the formula (I):

（I）。

the preparation method of the dihydromyricetin pyrrolidine compound comprises the following steps:

s4: and (4) filtering the reactant obtained in the step S3, washing a filter cake by using anhydrous methanol, and performing decompression and pumping to obtain the dihydromyricetin pyrrolidine compound.

The inert gas in step S2 is argon.

The concentration of the dihydromyricetin anhydrous methanol solution in the step S1 is 150-350mmol/L, preferably 250 mmol/L; the concentration of the anhydrous methanol solution of pyrrolidine in step S2 is 1-10mol/L, preferably 1 mol/L.

In step S3, the molar ratio of dihydromyricetin to tetrahydropyrrole is 1:0.7-1.3, and the preferred molar ratio of dihydromyricetin to tetrahydropyrrole is 1: 1.

in the step S1, adding absolute methanol to dihydromyricetin, and then transferring to an ice-water bath to stir for 30 minutes; in step S3, after 2 drops/sec of anhydrous methanol solution of pyrrolidine was added to the anhydrous methanol solution of dihydromyricetin in an ice water bath, the reaction was carried out at room temperature.

The preparation method of dihydromyricetin in the step S1 comprises the following steps:

The specific preparation examples are as follows:

as shown in fig. 1, the preparation process of dihydromyricetin pyrrolidine complex (DHMP) of this embodiment is shown, wherein compound 1 is 2,4, 6-trihydroxyacetophenone, compound 2 is 2-hydroxy-4, 6-dimethoxymethoxyacetophenone, compound 3 is 2,4, 6-trimethoxymethoxyacetophenone, compound 4 is 3,4, 5-trihydroxybenzaldehyde, compound 5 is (E) -3-trihydroxybenzene-1- (2, 4, 6-trimethoxymethyleneoxyphenyl) -vinyl ketone, and compound 6 is dihydromyricetin;

preparation of compound 2: compound 1 (10.0 mmol) was added to a 100 mL round bottom flask containing a stirrer under argon atmosphere, after purging, 20 mL of anhydrous Dichloromethane (dichromethane, DCM) and N, N-Diisopropylethylamine (N, N-diamopropylethylamine, DIPEA, 30 mmol) were added to the flask at room temperature. Chloromethyl Methyl Ether (Chloromethyl Methyl Ether, MOMCl, 30 mmol) was then added slowly at 0 ℃. After the reaction is finished, decompressing and removing the solvent, and performing column chromatography on the residue after the solvent is removed to obtain a compound 2;

preparation of compound 3: compound 2 (8.0 mmol) was added to a 100 mL round bottom flask containing a stir bar under argon atmosphere, after purging, 20 mL of anhydrous Tetrahydrofuran (THF) was added to the round bottom flask at room temperature. Then, Sodium hydride (NaH, Sodium hydride, 9.6 mmol) and MOMCl (9.6 mmol) were added successively at 0 ℃. After the reaction is finished, the solvent is removed by decompression and spinning, and the residue after the solvent is removed is subjected to column chromatography to obtain a compound 3;

preparation of compound 5: compound 3 (6.0 mmol) was added to a 100 mL round bottom flask containing a stirrer under argon blanket, after purging, 15mL THF and 10mL water were added to the round bottom flask at room temperature. Then, Sodium hydroxide (NaOH, Sodium hydroxide, 12.0 mmol) and compound 4 (12.0 mmol) were added sequentially at room temperature. After the reaction is finished, extracting for 3 times by using ethyl acetate, combining organic phases, decompressing and removing the solvent, and performing column chromatography on the residue after the solvent is removed to obtain a compound 5;

preparation of compound 6: compound 5 (6.0 mmol) and NaOH solution (5M, 60 mL) were added to a 100 mL round bottom flask containing a stirrer and 50 mL methanol. Adding into the above solution at room temperatureAdding 30% hydrogen peroxide (H)₂O₂1.8 mL, 18.0 mmol). After the reaction was completed, a saturated sodium sulfite solution was added to quench the reaction, followed by extraction three times with ethyl acetate, the organic phases were combined, the solvent was removed by evaporation under reduced pressure, the residue from which the solvent was removed was dissolved in 30 mL of methanol, and 2N dilute hydrochloric acid (HCl, 5 mL) was added to heat the reaction solution to 55 ℃ until the reaction was completed. And after the reaction is finished, decompressing, removing the solvent by spinning, and performing column chromatography to obtain the compound 6.

Preparation of dihydromyricetin pyrrolidine complex: adding the prepared compound 6 dihydromyricetin (DHM, dihydromyricetin, 1.6 g, 5.0 mmol) into a 100 mL round-bottom flask containing a stirrer under the protection of argon, and after purging, adding 20 mL of anhydrous methanol into the round-bottom flask at room temperature; stirring for 30 minutes at room temperature, transferring the round-bottom flask into an ice-water bath, continuing stirring for 30 minutes, and preparing 10mL of anhydrous methanol into a pyrrolidine anhydrous methanol solution (the purity of the compound is more than or equal to 98%) of pyrrolidine under the protection of argon; then, the prepared anhydrous methanol solution of pyrrolidine was added at a rate of 2 drops/sec to a flask containing DHM in an ice water bath, and allowed to naturally warm to room temperature to effect a reaction. After the reaction is finished, the solvent is removed by decompression and spinning, the residue of the reactant after the solvent is removed is filtered to obtain a filter cake, and the filter cake is washed by methanol for three times and then decompressed and dried to obtain the target product, namely the light brown dihydromyricetin pyrrolidine compound.

The nuclear magnetic resonance spectrum of the obtained dihydromyricetin pyrrolidine complex is shown in fig. 2a and 2b, and specifically comprises the following steps: 1H NMR (400 MHz, D)₂O): δ 1.94-1.97 (m, 4H, 2CH₂), 3.22-3.26 (m, 4H, 2CH₂), 4.52-4.56 (m, 1H, CH), 4.84 (d, 1H, J = 11.6, CH), 5.64 (s, 1H, ArH), 5.71 (m, 1H, ArH), 6.62 (s, 1H, ArH); 13C NMR (100 MHz, CD₃OD): δ 23.6, 45.4, 71.2, 82.9, 97.7, 98.6, 107.71, 107.75, 127.8, 133.9, 145.6, 162.3, 162.9, 178.4, 193.7；

The nuclear magnetic resonance spectrum of dihydromyricetin of the compound 6 is shown in fig. 2c and 2d, and specifically comprises the following steps: 1H NMR (400 MHz, CD)₃OD): δ 4.46 (d, 1H, J = 11.2 Hz, CH), 4.84 (d, 1H, J = 11.2 Hz, CH), 5.88 (d, 1H, J = 1.6 Hz, ArH), 5.92 (d, 1H, J = 1.6 HZ, ArH), 6.53 (s, 1H, ArH); ¹³C NMR (100 MHz, CD₃OD): δ 73.7, 85.3, 96.2, 97.3, 108.0, 129.1, 134.9, 146.9, 164.5, 165.3, 168.7, 198.3；

Comparing fig. 2c and 2d with fig. 2a and 2b, it can be seen from the nuclear magnetic resonance spectrum that the dihydromyricetin and tetrahydropyrrole form a compound, i.e., the dihydromyricetin tetrahydropyrrole compound of the present invention has a molecular formula shown in formula (I).

The dihydromyricetin pyrrolidine complex is applied to the medicines for treating the nervous system dysfunction diseases; the nervous system dysfunction diseases are sleep disorder, anxiety, depression, post-traumatic stress disorder, Alzheimer's disease, dementia, Parkinson disease, apoplexy, epilepsy, autism, and alcohol use disorder.

Aiming at the application of the dihydromyricetin pyrrolidine complex, the invention carries out the following application examples, in particular:

application example 1: evaluation of efficacy of dihydromyricetin-tetrahydropyrrole complex (DHMP) the efficacy of dihydromyricetin-tetrahydropyrrole complex and dihydromyricetin prepared in the preparation examples was examined in this example for comparison, and the specific experimental procedures were as follows:

1. experimental Material

Wild mice (Wild type, Wt mice) Wild type (Wt., C57BL/6, Charles river Lab, USA)) and transgenic mice (transgenic (TG) -SwDI, TG mice, Transgenic (TG) -SwDI (Jacson Lab, USA)) were male, 20 months old.

2. Experimental methods

Whole cell patch clamp recording of mouse brain sections:

we have demonstrated that (1) TG mouse models of alzheimer's disease AD have behavioral deficits such as lack of exploration/motor activity, increased anxiety, and susceptibility to epilepsy; (2) TG mice lost cognitive memory. These abnormal behavioral changes are consistent with human studies and are commonly found in patients with alzheimer's disease. So we used the same gene mouse in this example.

Lateral sections of dorsal hippocampus (400 μm thick brain sections) were obtained from Wt mice and TG-SwDI mice (Jacson Lab, USA, male, 20 months old) using a vibrating microtome (VT 100; international technical product), and the activity of all at least three layers of neurons in the brain sections was ensured. (Note: equivalent to a live brain tissue) sections were serially perfused with artificial cerebrospinal fluid (ACSF) consisting of the following (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂、2 MgCl₂、26 NaHCO₃And 10D-glucose. ACSF continuous input 95% O₂/5% CO₂To ensure that the sections are fully oxygenated, the pH is 7.4, and the sections are perfused while being maintained at 34 +/-0.5 ℃ to ensure the activity of the neurons in the brain slices (note: the cranial nerves can maintain physiological functions during the whole experiment). Tetrodotoxin (TTX) calcium channel blocker 0.5 mu M, D (-) -2-amino-5-phosphonopentanoate (D (-) -2-amino-5-phosphonopentanoate, APV) (40 mu M), 6-cyano-7-nitroquinoline-2, 3-dione (6-cyanoo-7-nitroquinoline-2, 3-dione, CNQX) 10 mu M and CGP54626 (1 mu M, GAB)_ABR antagonists) to ACSF for pharmacological isolation of GABA_AR-mediated mini inhibitory postsynaptic current (mlsc). The patch electrode was filled with an internal solution containing (in mM): 137 CsCl, 2 MgCl₂、1 CaCl₂11 EGTA, 10 HEPES and 3ATP, pH adjusted to 7.30 with CsOH. Recordings live cranial neuroamperometric recordings were made of dentate gyrus granular cells (DGC) from hippocampal slices.

Voltage-clamp whole-cell recordings were performed using patch-clamp amplifiers, and anxiety levels were tested in all animals on an elevated plus maze test (EPM). Animals were placed in the central area of the maze, tested for 5 minutes and video recorded. The following measurements were scored: the number of accesses to open arms, closed arms, or a central platform, and the time spent in these areas. The data is reported as a percentage of the number of weapon entries, a percentage of time spent in different entries, and the total number of entries.

3. Results of the experiment

To evaluate whether DHMP still enhances GABA_AR, whole cells of DGC in hippocampal gyrus in brain sections were tested using voltage clamp recording technique to test the extrasynaptic tension current (I) of DHMP and DHM on GABAergic (neuro-inhibitory)_tonic) Comparing the effect of postsynaptic mini-inhibitory postsynaptic current (mlsc), we increased the dose of DHMP and DHM in μ M as shown in fig. 3: 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0 and 30 μ M, respectively. Enhancement of GABAergic I with DHM_tonicIn comparison to mlscs, as shown in table 1. The results show that DHMP can maintain the biological activity of DHM, increase the efficacy of DHM and improve water solubility.

Table 1 is a summary of fig. 3a-3 d. DHM begins at 0.1. mu.M, a concentration-dependent enhancement, and DHMP begins at 0.001. mu.M to produce a significant and concentration-dependent enhancement, GABAergic Itonic. DHMP showed 100-fold greater efficacy compared to DHM. DHMP started at 0.001 μ M for the effect of mini-inhibitory postsynaptic current (mlsc), resulting in a significant enhancement of the concentration dependence on mini-inhibitory postsynaptic current (mlsc). Whereas DHM starts to produce a deliberate potentiation at 0.3 μ M. The results show that DHMP can maintain the biological activity of DHM, increase the efficacy of DHM and improve water solubility.

TABLE 1 comparison of the Effect of DHMP and DHM

3a-3d are combined with Table 1, and Table 1 compares the values in FIG. 3b and FIG. 3d, and clearly shows that the concentration of the change in I current caused by the DHMP group is 0.001, and that there is a significant change in the initial current caused by the same reason that 0.001 is the dominant current, while DHM starts to change at 0.1 (the effect of DHMP due to the fast absorption is more significant).

Application example 2: effect of Dihydromyricetin Tetrahydropyrrole Complex (DHMP) on mouse Targeted proppant level (gephyrin level) expression

In the application example, the influence of the dihydromyricetin pyrrolidine complex prepared in the preparation example on the Gephyrin level expression of the mouse is considered, and the specific experimental process is as follows:

1. experimental materials: animals used as in application example 1 were Wt mice (C57BL/6, charles river Lab, USA) and TG-SwDI (Jacson Lab, USA) mice, respectively, which were male and 20 months old.

2. The experimental method comprises the following steps: dividing two mice into three groups, namely a control group, an AD group and an AD + DHMP group; measuring Gephyrin amounts of a control group, an AD group and an AD + DHMP group respectively; taking out brains of all mice and analyzing, namely performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blot analysis respectively; the method specifically comprises the following steps: proteins were isolated on SDS-polyacrylamide gel (Sigma) using the Bio-Rad Mini-Protean 3 cell system; proteins were transferred to polyvinylidene fluoride (PDVF) membrane (Sigma) and blocked with 4% skim milk powder; the blot was incubated with rabbit anti-gephyrin (Santa Cruz, 1:200) and mouse anti-beta-actin (Sigma, 1:1000) overnight at 4 ℃ followed by two HRP-conjugated secondary antibodies (1:5000) for several hours (3-5 hours) at room temperature, and the strips were detected using ECL detection kit (Sigma) and exposed to X-ray film; the bands were analyzed by densitometry using ImageQuant5.2 (Molecular Dynamics), normalized to the corresponding β -actin signal and compared.

3. Results of the experiment

There has been a related report that DHM restores impaired GABAergic transmission in TG mouse models by restoring Gephyrin, a postsynaptic GABA_AR ankyrin, which can modulate GABAergic synapse formation and plasticity in TG mice; here, the previous method (i.e., testing Gephyrin protein levels in the hippocampus (left) and cortex (right) of mice) was repeated to test the effect of DHMP.

Gephyrin is a key scaffold protein of tissue inhibitory postsynaptic receptor density and relates to GABA_AAggregation of R at the postsynaptic site. Gephyrin not only has structural function at the site of the GABAergic synapse, but also plays a crucial role in synaptic dynamics. Therefore, we compared TG mice with Wt mice and scoredThe level of Gephyrin protein in hippocampus (left) and cortex (right) of TG mice and DHMP-treated TG mice were tested separately and from western blot analysis it was shown that, as shown in figure 4a, the level of Gephyrin was reduced in TG mice compared to Wt mice, i.e. the application of DHMP reversibly reduced the level of Gephyrin in AD animals with alzheimer disease.

In the experiment, the dosage of DHMP for the mouse is 0.5mg/kg per day for three months; the amount of DHMP administered orally is here only 1/4 doses of DHM. The lower dose was not chosen because it was necessary to confirm that: 1) whether DHMP and DHM have the same effect; 2) whether DHMP is more effective than native DHM.

After 1 month of mouse DHMP (0.5 mg/kg, 1/4 dose of DHM, oral daily) treatment, Gephyrin levels are partially restored, and after 3 months of mouse DHMP treatment, Gephyrin levels are restored to a normal level range, and oral administration of a small dose (1/4 dose of DHM) DHMP can reverse bridge protein levels in a short time, as shown in figure 4b, which indicates that DHMP can rapidly reach the brain through the blood brain barrier to play a role in reversing bridge protein, and further indicates that modified DHM (i.e. DHMP) can be rapidly absorbed into the blood circulation after being orally taken into the body, and can rapidly enter the brain to play a drug effect. The decrease in Gephyrin levels in TG mice may lead to GABA_AThe synaptic aggregation of R is reduced, which may play a role in impairment of gabaergic neurotransmission. Therefore, TG mouse models show loss of cognition and memory, lack of exploratory/motor activity, increased anxiety and susceptibility to epilepsy. The results indicate that DHMP treatment can restore Gephyrin levels, which is a potential mechanism for behavioral modification and improvement in learning and cognitive function in TG mouse animal models of the DHMP-treated AD group. DHMP has the same effect as DHM and is more effective than native DHM (DHMP is 1/4 dose of DHM, i.e. the same effect can be achieved with a smaller dose).

Application example 3: effect of dihydromyricetin tetrahydropyrrole complex (DHMP) on improving cognitive/memory disorder of mice

In this example, the improvement effect of dihydromyricetin pyrrolidine complex prepared in the preparation example on mouse cognitive/memory disorder was examined, and the specific experimental process is as follows:

1. experimental materials: animals used as in application example 1, Wt mice (C57BL/6, Charles river Lab, USA) and TG-SwDI (Jacson Lab, USA) mice, were male, 20 months old.

2. Grouping of laboratory animals

Mice were divided into four groups: (1) male mice, C57BL/6 Wt, were orally administered 2% sucrose; (2) male C57Bl/6 Wt mice were orally administered 0.5mg/kg DHMP (1/4 doses of DHM) in 2% sucrose; (3) TG-SwDI mice were orally administered 2% sucrose; (4) TG-SwDI mice were orally administered 0.5mg/kg DHMP (1/4 doses of DHM) in 2% sucrose.

3. Experimental methods

Target recognition test (NOR):

days 1-3: habituation (once a day).

Open top containers (open air) are used with cameras to record animal performance. The animals were placed in a container without the object for 5 minutes. The background was identical for each animal.

Day 4: familiarizing.

Two identical objects (toys (defined as FO): familiar objects) are placed in a specific location of the container. The animal is then placed in the container and the object is explored for 5 minutes. Retention test 1.5 hours: each animal was placed in its home cage for 1.5 hours after familiarity. One of the toys is replaced by a new one (a novel object, very different from FO). We then placed one animal into a container with the object for 3 minutes and recorded it for off-line scoring. The score is based on the time the animal explores each toy. The pair of subjects in one group has been previously tested to avoid natural preference of the mice for shape or light reflection; we used a total of 4 groups of subjects in this test.

Day 5: 24 hour retention test.

We have placed in the container another new toy (NO) and an original object (FO). We then placed the animals in containers and tested for 3 minutes. We scored the animal's behavior offline according to how long the animal explored each object. The Object Recognition Index (ORI) is calculated such that ORI = (tn-tf)/(tn + tf), where tf and tn represent exploring familiar and novel temporal objects.

New context recognition (NCR, fig. 5): days 1-3 are habitual (once per day), with the two contexts (containers) a and B with similar field areas being very different in shape. Context A is a rectangle and context B is a circle. The top of the container is open, with padding to minimize pressure, and a camera is mounted on the top to record the behavior of the animal. Each animal was left in environment a without the toy for 5 minutes and then returned to home for 30 minutes. The animals were then placed in environment B without the toy for 5 minutes. Day 4 was for familiarity. Two different sets of toys were used as familiar objects, referred to as FO1 and FO2, respectively. Each set consists of two identical toys, whereas the shape of FO1 and FO2 is very different. The two toys of FO1 are each placed in a particular location in scenario a. Each animal was placed in scenario a and allowed to explore FO 15 minutes; the animals were then returned to home cages for 30 minutes. The two toys of FO2 are each placed in a particular position in background B. Each animal was placed in background B and allowed to explore FO 25 minutes. Day 5 (24 hours after familiarity): memory trials were performed to determine the memory retention of each animal to a familiar subject. One toy of FO1 in context a is exchanged for one toy of FO 2. Each animal was then allowed to explore the subjects in context a for 3 minutes. The test is videotaped and analyzed off-line. The time taken to explore the familiar object (FO1) and to exchange the object (FO2) is calculated, where exploration is equivalent to touching the object with a nose or paw, or sniffing within 1.5 centimeters of the object.

Calculating a Recognition Index (RI) by using a formula; index = (tn-tf/(tn + tf), where tf represents the time to explore a previously encountered familiar object in the same context and tn represents the time to explore the object in a different context.

4. Results of the experiment

Studies have demonstrated that TG mice show signs of decreased cognitive memory. These abnormal behavioral changes are consistent with human studies and are common in AD patients. In this assessment study, we assessed the cognitive memory of mice using the NOR test (fig. 5 and fig. 6 a), and 3 months after treatment Wt mice spent more time exploring new objects (ORI = 68.8 ± 9.8%). In TG mice, ORI decreased to 49.8 ± 4.7%. Wt mice treated with DHMP (i.e., Wt mice of the DHMP group) showed similar recognition as the Wt mouse control group; the results showed that DHMP significantly improved NOR in TG mice (ORI = 68.6 ± 5.8%).

The evaluation of NCR was then performed (fig. 5 and 6 b), and RI was calculated. TG mice showed reduced RI (49.9 ± 2.6%) compared to Wt mouse control; DHMP significantly improved RI in TG mice (RI = 66.6 ± 5.8%). DHMP treatment reversed RI in TG mice and showed significant improvement in contextual memory. The results show that the daily oral administration of DHMP (1/4 dosage of DHM) can not only duplicate the DHM effect, but also the DHMP can improve the cognitive memory of mice with Alzheimer's disease TG in a short time, which indicates that the DHMP can rapidly reach the brain through the blood brain barrier to reverse the bridge protein, and further indicates that the modified DHM (namely DHMP) can be rapidly absorbed into the blood circulation after being orally taken into the body, and can rapidly enter the brain to exert the drug effect. (Note: Zongzhu)

Application example 4: improvement effect of dihydromyricetin pyrrolidine complex (DHMP) on behavior disorder and epileptic seizure of mice

In this example, the improvement effect of dihydromyricetin pyrrolidine complex prepared in the preparation example on the behavior disorder and the epileptic seizure of mice is considered, and the specific experimental process is as follows:

1. experimental materials: animals used as in application example 1, Wt mice (C57BL/6, Charles river Lab, USA) and TG-SwDI (Jacson Lab, USA) mice, were male, 20 months old TG2576 mice.

2. Experiment grouping

Mice were divided into three groups: (1) control group: c57BL/6 Wt male mice (2% sucrose, oral); (2) group AD: TG mice (2% sucrose, oral); (3) AD + DHMP group: TG mice were given DHMP (0.5 mg/kg (1/4 doses of DHM) in 2% sucrose).

3. Experimental methods

(1) Open field exercise test

To measure locomotor activity, total number of entries per animal was measured; determining statistical differences using analysis of variance; the effect of DHMP on anxiety levels was determined by these experiments.

(2) Elevated cross maze test (EPM)

In a quiet, dark room, with only low-powered red lights; rats were placed in the central area of the maze and video of their behavior was recorded for 5 minutes. The number of entries into the open arm, closed arm or central platform and the time spent in each of these areas were scored during the offline analysis. The data is reported as a percentage of the number of items in each group, the percentage of time spent in the different groups, and the total number of items.

4. Results of the experiment

The effect of DHMP on Alzheimer's Disease (AD) symptomatology behavior in aged TG2576 mice was determined using the open field assay. Running distance is one of the parameters quantifying the athletic activity (fig. 7 a). The weight control mice ran 922 ± 75 cm within 10 minutes. TG mice ran much shorter distances (235. + -.16 cm), while the AD + DHMP group (TG mice treated with DHMP) increased distances to 682. + -.109 cm. Control Wt mice showed frequent feeding (23.3 + -3.4 times), exploration of the empty field center 2.0 + -0.6 times, and 0.14 + -0.1 minute dwell at the center. The AD group TG-mice showed 12.1 + -2.1 fold rearing, exploration of the center of the open field (0.2 + -0.1 fold), and stay in the center for only 0.02 + -0.02 min. DHMP treatment of TG mice in the AD + DHMP group increased feeding (19.5 ± 2.0 fold), time to explore the center (1.4 ± 1.4 fold) and time to stay in the center (0.13 ± 0.05 min) (see figures 7b-7d for details). The results show that TG mice in AD group decreased exploration/locomotor activity, and daily oral DHMP (1/4 dose of DHM) treatment in AD + DHMP group mice improved locomotor activity and increased exploration activity, an instinct of the animals. Anxiety was measured by EPM. 48.5 + -, which spent total time in open arms in comparison to control group Wt mice7.5%, 33.5 ± 3.2% spent in the closed arms. TG-mice with AD spent significantly less time in the open arms and more time in the closed arms compared to the control Wt mouse control (statistical significance compared to Wt mouse control; as shown in FIG. 7 e), while TG-mice in the AD + DHMP group spent similar time on each arm. Pentylenetetrazole (PTZ) is GAB_ANon-competitive antagonists of the a receptor complex. PTZ (42 mg/kg) was used to induce seizures in mice. Seizure duration was 1.4 ± 0.6 min in control with Wt mice, with a significant increase in seizure duration (4.9 ± 0.8 min) in TG-mice with AD, and a significant decrease (1.8 ± 0.3 min) in AD + DHMP-treated mice (as shown in figure 7 f). These results indicate that TG mice with AD exhibit apathy-like behavioral deficits, anxiety and seizure sensitivity, and that daily DHMP treatment can ameliorate and prevent these symptoms. These results and behaviour DHMP behaviour are related to GABA_AThe notion that the Rs and GABAergic system contribute to AD behavioral changes is consistent.

Application example 5: improving effect of dihydromyricetin pyrrolidine complex (DHMP) on brain injury caused by mouse stroke

In this example, the effect of dihydromyricetin pyrrolidine complex prepared in the preparation example on improving the brain injury caused by stroke of mice is considered, and the specific experimental process is as follows:

1. experimental materials: SPD grade rats (Sprague Dawley, SD) Sprague Dawley (SD) rats (male, 200 + -20 g adult mouse, Charles river Lab, USA).

2. Experimental methods

(1) Apoplexy mouse model

On day 1, we injected silica gel through the left carotid artery and left cerebral stroke was surgically induced. Left stroke rats were divided into two groups, one experimental group (stroke + DHMP treatment), i.e. from day 2 we started gavage DHMP 0.5mg/kg (1/4 dose of DHM) once a day; the other group is a control group, and the rats after the operation receive 2 mL/kg of normal saline for the treatment of gastric lavage as drug control; grid walking and open-air walking were performed on treatment days (D) 10, 20 and 30.

(2) Mouse stroke model induced by unilateral carotid artery occlusion

DHMP effects were tested using a unilateral carotid clamp-induced stroke animal model. We clamped the left carotid artery and surgically stroke the left side. After 10 minutes, the brain is quickly taken out and a hippocampal slice is prepared for electrophysiological recording; hippocampal neuron whole-cell voltages were clamped at-70 mV holding potential using an Axopatch 200B amplifier (Molecular Devices). Before the electrical compensation, the resistance (< 25M) is switched in. The intracellular signal was low-pass filtered at 3kHz and digitized at a sampling frequency of 20 kHz. Pharmacologically isolated mini excitatory postsynaptic currents (mEPSCs) were recorded.

3. Results of the experiment

Stroke is the leading cause of limb dysfunction, but there is currently no pharmacotherapy available to promote limb functional rehabilitation. Recent studies have shown that the brain has limited ability to repair after stroke. Neural repair following stroke involves remapping cognitive functions in tissues adjacent to or associated with the stroke. Therefore, it is important to limit the neurotoxic damage caused by the release of additional glutamate by excitatory neurons for the first time. Fig. 8a-8c show the results of the day 10 measurements. In the 5-minute grid walking test (fig. 8a), the stroke rats were only able to perform for 0.8 ± 0.85 minutes and frequently dropped from the grid. Rats in the experimental group (stroke + DHMP treatment) can be treated for approximately 3.6 ± 0.5 minutes compared to 3.8 ± 0.3 minutes in the control group. In the open-air test (FIG. 8b), the control group of stroke rats could only run for a distance of 270 cm. And the stroke + DHMP treated mice in the experimental group can run for 700 cm. The stroke rats were unable to enter the center of the open field (fig. 8 c).

To understand the underlying mechanisms of stroke-induced brain injury, we also tested DHMP effects using a unilateral carotid clamp-induced stroke animal model. FIG. 9a is the brain slice recordings of the control group; FIG. 9b is a comparison of the peak postsynaptic mini-excitatory postsynaptic current (mEPSC) obtained for brain slices from control groups when different doses of DHMP were administered, and FIG. 9c is a dose-dependent significant reduction in stroke rat brain slice recordings mEPSC; figure 9d is a comparison of the peak values of mepscs obtained in brain slices of stroke mice when DHMP was administered at different doses. DHMP can reduce mepscs frequency to similar values as the control group (fig. 9 e); in addition, DHMP reduced the total charge transfer of mepscs to be similar to the control level of the ischemic group (fig. 9 f). The results indicate that DHM can inhibit excessive glutamate release, thereby reducing glutamate-induced neurotoxicity. Meanwhile, the results show that oral administration of a small dose of DHMP (1/4 dose of DHM) can show that DHMP can rapidly reach the brain through a blood brain barrier, and excessive release of glutamic acid is rapidly inhibited to reduce brain injury caused by stroke, so that the brain injury caused by stroke is limited, the stroke course is shortened, and the brain function is recovered to regulate the motor activity after stroke; the modified DHM (i.e. DHMP) is proved to be rapidly absorbed into the blood circulation after being orally taken into the body, and rapidly enters the brain to exert the drug effect.

Application example 6: dihydromyricetin tetrahydropyrrole complex (DHMP) on GABA_AEffect of enhancing R

In this example, the dihydromyricetin tetrahydropyrrole complex prepared in the preparation example was examined for GABA_AThe experimental exploration of the R enhancing effect comprises the following specific experimental processes:

1. experimental materials: human oocytes (professor aussler professor).

2. The experimental method comprises the following steps: GABAA5 was expressed on oocytes and DHMP was then recorded to enhance GABA in comparison to DHM.

3. The experimental results are as follows: DHM enhanced GABAA5 at doses from 0.3 to 300 μ M, whereas DHMP showed a 1000-fold efficacy in GABAA5 enhancement (at doses from 0.003 μ M), as shown in figure 10.

Application example 7: safety experiment exploration of dihydromyricetin pyrrolidine complex (DHMP)

In this example, the safety test of dihydromyricetin pyrrolidine complex (DHMP) was examined, and the specific experimental procedures are as follows:

1. experimental materials: sprague Dawley (SD) rats (male, body weight 200 + -20 g; female, body weight 190 + -20 g.n = 5/group/sex, Charles river Lab, USA).

Sprague Dawley adult rats, male, with body weights starting from 175 + -5 g were studied in a GLP-compliant facility.

2. Grouping experiments:

animal groups: the control group received 5% sucrose (SUC. 2 ml/100g body weight), DHMP 1mg/kg group, DHM P10 mg/kg group, and DHMP 100mg/kg group.

3. The experimental method comprises the following steps:

(1) the animals were grouped as described above for two weeks of functional observation in rats, the FOB parameters (body temperature, heart rate, respiration rate) were recorded, and DHMP was orally administered daily at doses of 1mg/kg, 10mg/kg and 100mg/kg, respectively.

(2) The animals were grouped for a long-term assessment study for 6 months and DHMP was administered orally daily at 1mg/kg, 10mg/kg and 500mg/kg, respectively, and FOB parameters (body temperature, heart rate, respiration rate) were recorded.

4. The experimental results are as follows:

after a single dose of SUC or DHMP, the animals were observed continuously for 4 hours after treatment, mainly concerning whether there were any negative changes in the animal's psychological behavior, voluntary activities, hair, glandular secretion, feces, death, etc. We found no significant change in these evaluations; in a two-week old rat Functional Observation (FOB) study, DHMP was administered orally daily at doses of 1mg/kg, 10mg/kg and 100mg/kg without any significant negative changes.

Within two weeks, we examined the animals for coat, response to treatment, etc.; all mice showed no loss of fur or negative reaction to treatment. No death or injury was observed during the experiment and we also ensured that animals were observed for 4 hours continuously after each treatment. There is a major concern about any negative changes in the mental behaviour, voluntary activity, hair, faeces, glandular secretion, death etc. of the animal. The FOB parameters, including body temperature, heart rate and respiration rate of the rats, are shown in table 2. We found no significant change in these evaluations and the results indicate that DHMP is a safe compound.

TABLE 2 FOB parameters for each group

T in the above Table 2 represents temperature in units of; p represents pulses in units of times/minute; b denotes breath and the unit is times/min.

To determine whether DHMP showed signs of toxicity, the effect of chronic DHMP administration on rats was tested to assess changes in metabolic rate. Body weight (fig. 11a, 11b), food intake (fig. 11c, 11d), water intake (fig. 11e, 11f), fecal excretion (fig. 11g, 11h) and urine excretion (fig. 11i, 11j) were measured as metabolic rate parameters; and the metabolic rates of all groups were compared.

In a long-term evaluation study over a 6 month period, DHMP was administered orally daily at doses of 1mg/kg, 10mg/kg, and 500mg/kg, and the results indicated that DHMP did not alter metabolic rate, i.e., the results indicated that DHMP was safe for daily oral use (shown in FIGS. 11 a-11 j).

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A dihydromyricetin pyrrolidine compound is characterized in that the molecular formula of the dihydromyricetin pyrrolidine compound is shown as the formula (I):

（I）。

2. a method for preparing dihydromyricetin pyrrolidine complex according to claim 1, comprising the steps of:

3. The method of claim 2, wherein the inert gas used in step S2 is argon gas.

4. The method for preparing dihydromyricetin pyrrolidine complex according to claim 2, wherein the concentration of the dihydromyricetin anhydrous methanol solution in step S1 is 150-350 mmol/L; the concentration of the pyrrolidine anhydrous methanol solution in the step S2 is 1-10 mol/L.

5. The method for preparing dihydromyricetin-pyrrolidine complex according to claim 2, wherein the molar ratio of dihydromyricetin to pyrrolidine in step S3 is 1: 0.7-1.3.

6. The method for preparing dihydromyricetin-pyrrolidine complex according to claim 5, wherein the molar ratio of dihydromyricetin to pyrrolidine in step S3 is 1: 1.

7. the method for preparing dihydromyricetin-pyrrolidine complex according to claim 2, wherein in step S1, anhydrous methanol is added to dihydromyricetin, and then the mixture is transferred to an ice water bath to be stirred for 30 minutes; in step S3, after 2 drops/sec of anhydrous methanol solution of pyrrolidine was added to the anhydrous methanol solution of dihydromyricetin in an ice water bath, the reaction was carried out at room temperature.

8. The method for preparing dihydromyricetin-pyrrolidine complex according to claim 2, wherein the method for preparing dihydromyricetin in step S1 comprises:

9. Use of dihydromyricetin pyrrolidine complex of claim 1 in a medicament for the treatment of neurological dysfunction.

10. Use according to claim 9, wherein the neurological disorder is sleep disorder, anxiety, depression, post traumatic stress disorder, alzheimer's disease, dementia, parkinson's disease, stroke, epilepsy, autism, alcohol use disorder.