Use of acetyldopamine compounds in the preparation of drugs for improving insomnia

Abstract

The application provides application of an acetyl dopamine compound in preparation of a medicine for improving insomnia, and belongs to the technical field of traditional Chinese medicine medicines, and the acetyl dopamine compound is acetyl dopamine dimer A extracted from cicada slough. Experiments prove that the acetyl dopamine dimer A can increase the content of brain-derived neurotrophic factor (BDNF) which plays an important role in the occurrence process of insomnia, reduce dopamine (DA) level, and has obvious effects on DA metabolites homovanillic acid (HVA) and dihydroxyphenyl acetic acid (DOPAC), thereby effectively improving insomnia. Through research on the effect of the acetyl dopamine dimer A on improving insomnia, the application obtains the basis of using the acetyl dopamine dimer A for improving insomnia, provides data support for research and development of the cicada slough as a medicine, and also provides a new medicinal source for improving insomnia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to application of an acetyldopamine compound in preparation of a medicine for improving insomnia. BACKGROUND

[0002] According to statistics of the World Health Organization, the global incidence of insomnia is about 35%, and insomnia has become the second most common mental illness. In China, the number of patients with insomnia is as high as 38%, and about 300 million middle-aged people suffer from sleep disorders and have a gradually increasing trend. Insomnia is often closely related to neurodegenerative diseases, cardiovascular diseases, type II diabetes, anxiety, depression, drug abuse, and suicide tendency. At present, benzodiazepine drugs, melatonin receptor agonists, and anticonvulsants, antidepressants, and antipsychotic drugs with hypnotic effects are mainly used in the clinical treatment of insomnia. However, the above drugs have many adverse reactions such as tolerance, addiction, withdrawal syndrome, daytime residual effect, and rebound after drug withdrawal. Therefore, there is an urgent need to develop new drugs with more effective and fewer side effects to treat insomnia.

[0003] Molting is the exuviae of the nymph of the insect Cryptotympana pustulata of the family Cicadidae, which can relieve convulsions and calm down. According to Records of Famous Doctors, molting is “mainly used for treating infantile convulsions and night crying”, and because of its remarkable effect on treating infantile convulsions and night crying, it is extended to treat insomnia, and its clinical application range is expanded. For example, molting can be used with honey, molting and ephedra soup (molting as the monarch drug), Shengjiang powder (molting as the minister drug), and molting and jujube soup to treat insomnia. However, the pharmacodynamic material basis of molting for improving insomnia is unknown, and the mechanism of action is less studied. At present, there is no specific acetyldopamine compound extracted from molting for treating insomnia, which limits the development of the medicinal value of molting. SUMMARY

[0004] Therefore, the application provides application of an acetyldopamine compound in preparation of a medicine for improving insomnia.

[0005] The technical solution adopted by the application to solve the technical problem is as follows:

[0006] The application of an acetyldopamine compound in preparation of a medicine for improving insomnia, wherein the acetyldopamine compound is acetyldopamine dimer A extracted from molting, and the structural formula is as follows:

[0007]

[0008] Preferably, the medicine takes acetyldopamine dimer A as an active ingredient, can increase the content of BDNF in brain tissue, reduce the content of DA, and effectively affect the content of DA metabolites HVA and DOPAC, so as to improve insomnia.

[0009] Preferably, the administration dosage of the acetyl dopamine dimer A is 20.154 mg / kg.

[0010] Preferably, the preparation method of the acetyl dopamine dimer A comprises the following steps:

[0011] (1) Taking dried meimena as raw material, adding 70% volume concentration of ethanol aqueous solution at a material liquid ratio of 1:9, soaking at room temperature for 3-4 hours, then refluxing and extracting at 110-130 DEG C for 2-3 times, each time for 1.5-2 hours, filtering the solution, and evaporating the filtrate to dryness to obtain meimena extract;

[0012] (2) Taking the meimena extract to HP-20 macroporous resin, gradient eluting with 30%, 50%, 80% and 100% methanol aqueous solution at room temperature to obtain different elution parts, and evaporating the 80% methanol aqueous solution elution part to dryness to obtain meimena 80% methanol macroporous resin part;

[0013] (3) Passing the meimena 80% methanol macroporous resin part through MCI column chromatography at room temperature, and eluting with 50%, 60%, 70%, 80%, 90% and 100% methanol aqueous solution in sequence to obtain different elution parts, evaporating the 50% methanol part solution to dryness to obtain a sample, and subjecting the sample to ODS reverse phase fast preparation chromatographic column, gradient eluting with 20%-100% methanol aqueous solution at a flow rate of 10 mL / min to obtain 26 tube sample solutions, combining the sample solutions to obtain 5 sections, taking the sample in the third section to pass through dextran gel chromatographic column, and eluting with pure methanol to obtain eluent, combining the sample to obtain 13 sections, and taking the sample in the ninth section to purify by semi-preparation liquid phase, and eluting with 25% methanol to obtain the acetyl dopamine dimer A.

[0014] It can be known from the above technical solution that the application provides an application of an acetyl dopamine compound in preparation of a medicine for improving insomnia, and the beneficial effects thereof are as follows: the application provides a new use of the acetyl dopamine compound, and the acetyl dopamine compound is acetyl dopamine dimer A extracted from meimena. It is proved by experiments that the acetyl dopamine dimer A can increase the content of brain-derived neurotrophic factor (BDNF) which plays an important role in the process of insomnia, reduce the dopamine (DA) level, and has obvious effects on DA metabolites homovanillic acid (HVA) and dihydroxyphenyl acetic acid (DOPAC), thereby effectively improving insomnia. The application obtains the basis of using the acetyl dopamine dimer A to improve insomnia by studying the effect of the acetyl dopamine dimer A on improving insomnia, provides data support for research and development of meimena as a medicine, and provides a new medicinal source for improving insomnia. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 This is the proton NMR spectrum of acetyldopamine dimer A.

[0016] Figure 2 This is the carbon NMR spectrum of acetyldopamine dimer A.

[0017] Figure 3 This is a bar chart showing the effect of acetyl-dopamine dimer A on the hypothalamus DA in insomniac rats.

[0018] Figure 4 This is a bar chart showing the effect of acetyl-dopamine dimer A on the hypothalamic HVA in insomniac rats.

[0019] Figure 5 This is a bar chart showing the effect of acetyl-dopamine dimer A on DOPAC in the hypothalamus of insomniac rats.

[0020] Figure 6 This is a bar chart showing the effect of acetyl-dopamine dimer A on BDNF in the hypothalamus of insomniac rats.

[0021] Figure 7 This is a pathological section of the hypothalamus tissue of an insomniac rat, showing the effects of acetyl-dopamine dimer A. Detailed Implementation

[0022] The technical solutions and effects of the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Dopamine (DA) plays a role in maintaining wakefulness and behavioral arousal. Normal levels reduce the number of awakenings and ensure the integrity of sleep, while excessively high levels can cause insomnia. Changes in DA levels affect its metabolites, thereby influencing the dopaminergic neuronal system. Homantanilic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) are DA metabolites. Changes in the levels of HVA and DOPAC in patients can indicate changes in DA metabolism and activity.

[0024] Brain-derived neurotrophic factor (BDNF) is a key factor regulating synaptic development and plasticity. It is widely distributed in the central nervous system, and its expression is directly and closely related to central nervous system function. BDNF can regulate synaptic plasticity, promote axonal growth, and facilitate neuronal repair. Decreased BDNF levels lead to nervous system damage, increased neuronal apoptosis, and can result in insomnia.

[0025] The insomnia model induced by p-chlorophenylalanine (PCPA) can hinder the synthesis of 5-hydroxytryptamine (5-HT), and 5-HT as a monoamine neurotransmitter plays an important role in the induction and maintenance of slow wave sleep (SWS). The insomnia animal model made by PCPA can further explore the mechanism of cicada slough in improving insomnia, and the experimental model is classical, simple and practical, and the modeling effect is accurate.

[0026] The content of DA, HVA, DOPAC and BDNF in the brain tissue of insomnia rats induced by PCPA was determined by using acetyldopamine dimer A prepared from cicada slough, and the pathological changes of hypothalamic sections of insomnia rats were observed, and the present application was further described in detail, which was an explanation of the present application but not a limitation.

[0027] 1. Establishment of insomnia rat model induced by PCPA

[0028] 1.1 Experimental animals

[0029] SPF grade SD male rats weighing 180-220 g were provided by Beijing Vito Lihua Experimental Animal Technology Co., Ltd. The animals were kept in a ventilated, dry, room temperature 22-25℃, humidity 50%-70% animal room, and the rats were fed with quantitative feed and free water.

[0030] 1.2 Reagents and drugs

[0031] Acetyldopamine dimer A was extracted from cicada slough (purchased from Zhengzhou medicinal material market); diazepam (Huazhong Pharmaceutical Co., Ltd., national drug code H42021528); PCPA (SIGMA-ALDRICH); Tween-80 (laboratory self-prepared); DA kit (Nanjing Jiancheng Biological Engineering Institute, batch number H170-1-2); HVA kit (enzyme-free, batch number MM-70643R1); DOPAC kit (enzyme-free, batch number MM-70438R1); BDNF kit (enzyme-free, batch number MM-0209R1).

[0032] 1.3 Instruments

[0033] Enzyme marker (infinite F50); centrifuge (SIGMA); analytical balance (Henan Deep Blue Instrument Equipment Co., Ltd.)

[0034] 1.4 Preparation of solvents

[0035] Preparation of Acetyldopamine Dimer A: (1) Take 1010 g dried T. chinensis as raw material, add 70% volume concentration of ethanol aqueous solution at a solid-liquid ratio of 1:9, soak at room temperature for 3 hours, then reflux extract twice at 130°C, each for 2 hours, filter the solution, and evaporate the filtrate to dryness to obtain T. chinensis extract; (2) take the T. chinensis extract and load it on HP-20 macroporous resin, and sequentially elute it with 30%, 50%, 80% and 100% methanol aqueous solution at room temperature to obtain different elution parts, about 300 mL of 30% part, about 500 mL of 50% part, and about 800 mL of 80% part, evaporate the 80% methanol aqueous elution part to dryness to obtain the 80% methanol macroporous resin part of T. chinensis; (3) sequentially elute the 80% methanol macroporous resin part of T. chinensis with 50%, 60%, 70%, 80%, 90% and 100% methanol aqueous solution at room temperature by MCI column chromatography (model: CHP 20P) to obtain different elution parts, evaporate the 50% methanol part to dryness to obtain a sample; perform gradient elution on the sample by ODS reverse-phase preparative chromatography column with 20%-100% methanol aqueous solution to obtain 26 sample solutions, combine the samples to obtain 5 sections, take the sample of the 3rd section and pass it through a Sephadex gel chromatography column (model: Sephadex LH-20) to elute it with pure methanol, combine the eluate to obtain 13 sections, take the sample of the 9th section and purify it by semi-preparative liquid chromatography (liquid phase: semi-preparative liquid chromatography of Sepsys; column: Cosmosil RP-C18; flow rate: 3 mL / min, peak time: 30 min) to obtain the acetyldopamine dimer A by elution with 25% methanol.

[0036] The compound obtained by the above separation was dissolved in deuterated methanol, and was loaded into a nuclear magnetic tube for determination of nuclear magnetic hydrogen spectrum and carbon spectrum. Please refer to Figure 1 As shown in the figure, the hydrogen spectrum shows 6 benzene ring proton signals at δ 6.68-6.81, two oxymethylene signals at δ 5.64 (d, J = 7.5 Hz, 1H, H-3), 4.66 (d, J = 7.5 Hz, 1H, H-2), two methylene signals at δ 3.27 (m, 2H, H-2”), 2.64 (t, J = 7.5 Hz, 2H, H-1”), and two methyl proton signals at δ 1.86 (s, 3H) and 1.83 (s, 3H). Please refer to Figure 2 As shown in the figure, the carbon spectrum data is 13CNMR (126 MHz, MeOD): δ 173.2 (C-7c), 173.2 (C-3b), 147.1 (C-4'), 146.4 (C-3'), 144.2 (C-8a), 142.1 (C-4a), 134.1 (C-7), 128.8 (C-1'), 123.2 (C-6), 120.6 (C-6'), 118.0 (C-5), 116.1 (C-5'), 115.6 (C-2'), 78.2 (C-2), 78.2 (C-3), 42.1 (C-2”), 35.7 (C-1”), 22.6 (CH3), 22.6 (CH3). By comparison with the literature, it is finally determined that this compound is acetyldopamine dimer A, and the structure is shown above.

[0037] Acetyldopamine dimer A quantitative analysis: First, the preparation of standard sample, take dopamine dimer A 0.01 g, dissolved in methanol to prepare a concentration of 1.00 mg / mL standard solution; with a micropipette 0.5, 1, 1.5, 2, 2.5, 3 mL of standard solution, placed in 6 10 mL flask, diluted to the mark with methanol, in turn to prepare 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mg / mL mass concentration of series standard solution. Then take 1.0 g of cydistin ethanol extract in a 250 mL round bottom flask, according to 1:8 liquid ratio, 70%(volume fraction) ethanol solution, in 150 ℃ water bath reflux extraction two times, each 2 h, the collection of extract evaporated to dryness, fine, about 0.1 g, accurately weighed, placed in 100 mL flask, add methanol, ultrasonic treatment(powers 250 W, frequency 40 kHz) 30 min, cool, add methanol to the mark, take the supernatant through 0.22 μm microporous filter membrane, namely the mass concentration of 1 mg / mL of test solution. Finally, with the standard solution to establish the standard curve, with the same chromatographic conditions into 10 μL of test solution, record the peak area, combined with the established standard curve calculation acetyldopamine dimer A content of 11.03%.

[0038] PCPA solution preparation(350mg / kg): take the appropriate amount of PCPA with a small amount of Tween-80 to help dissolve, then add distilled water to prepare 350mg / kg PCPA solution, this solution is prepared now.

[0039] Diazepam solution preparation(3mg / kg): the appropriate amount of diazepam is crushed, dissolved in distilled water to prepare 0.3mg / mL diazepam solution.

[0040] Acetyldopamine dimer A solution preparation(20.154mg / kg): take the appropriate amount of acetyldopamine dimer A dissolved in distilled water to prepare 20.154mg / kg acetyldopamine dimer A solution.

[0041] 1.5 Model grouping and establishment

[0042] SPF level SD male rats, after adaptive feeding for 8 days, were randomly divided into normal control group (CON) 9, model group (MOD) 9, diazepam positive group (DIA) 9, acetyl dopamine dimer A monomer group (NAND) 9, a total of 4 groups. On the 9th day, the normal group was injected with an appropriate amount of distilled water, and the other groups were injected with PCPA solution 350 mg / kg intraperitoneally once a day for 2 consecutive days. After the injection was completed, the success of the rat modeling was marked by the appearance of circadian rhythm loss, restlessness, and agitation. On the 11th day, the normal control group and the model group were given an appropriate amount of distilled water by gavage daily, the diazepam group was given diazepam solution 3 mg / kg by gavage daily, and the monomer group was given acetyl dopamine dimer A solution 20.154 mg / kg by gavage daily, once a day, for 7 consecutive days.

[0043] 2. Detection of relevant biochemical indicators in each group

[0044] 2.1 Determination of hypothalamic DA, HVA, DOPAC, and BDNF

[0045] On the seventh day of administration, each group began fasting at 8 pm, and the next day, intraperitoneal injection of chloral hydrate anesthesia was performed. The hypothalamus was taken and washed with physiological saline. A portion of 30 mg of the hypothalamus was crushed and added to the lysis solution on ice in a proportion of weight: volume (mg / μL) = 1:9. Homogenate was obtained by grinding and dissociating, and the supernatant was obtained by centrifugation at 4000 r / min for 10 min. The experiment was performed according to the DA, HVA, DOPAC, and BDNF kit instructions. Finally, the OD value of each well was measured on a microplate reader at 450 nm wavelength, and the standard curve was fitted. The OD value was substituted to obtain the corresponding concentration, and the content changes of DA, HVA, DOPAC, and BDNF in the hypothalamus of each group were obtained.

[0046] 2.2 Pathological detection of hypothalamic sections

[0047] Half of the hypothalamus of two random rats in each group was taken and fixed in 4% paraformaldehyde. HE staining was performed, and the pathological condition of the hypothalamus was observed under a light microscope.

[0048] 2.3 Statistical method

[0049] The experimental data was analyzed by one-way ANOVA test of statistical software SPSS Statistics 26.

[0050] 3. Effect of acetyl dopamine dimer A on insomnia rat model

[0051] 3.1 Effect of acetyl dopamine dimer A on the content of DA, HVA, DOPAC, and BDNF in the brain tissue of insomnia rats

[0052] CON is normal control group, MOD is model group, DIA is diazepam group, NADA is acetyldopamine dimer A monomer group (the same below). Compared with the normal control group, P<0.05, ##P<0.01, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0053] See Figure 3 As shown in the figure, the hypothalamic DA content determination results of each group, compared with the normal group, the level of DA in the hypothalamus of the model group increased (P<0.01), with statistical significance, indicating that the insomnia model induced by PCPA was successfully established. Compared with the model group, the DA level of diazepam group decreased, and the acetyldopamine dimer A monomer group could significantly reduce the DA level (P<0.01), which was better than the positive control group.

[0054] See Figure 4 As shown in the figure, the hypothalamic HVA content determination results of each group, compared with the normal group, the level of HVA in the hypothalamus of the model group increased significantly (P<0.01), with statistical significance, indicating that the insomnia model induced by PCPA was successfully established. Compared with the model group, the HVA content of diazepam group and acetyldopamine dimer A monomer group decreased significantly (P<0.05), and the acetyldopamine dimer A monomer group was better than the positive group.

[0055] See Figure 5 As shown in the figure, the hypothalamic DOPAC content determination results of each group, compared with the normal group, the level of DOPAC in the hypothalamus of the model group increased significantly (P<0.01), indicating that the insomnia model induced by PCPA was successfully established. Compared with the model group, the DOPAC of diazepam group and acetyldopamine dimer A monomer group decreased significantly (P<0.05), and the acetyldopamine dimer A monomer group was better than the positive control group.

[0056] See Figure 6 As shown in the figure, the hypothalamic BDNF content determination results of each group, compared with the normal group, the level of BDNF in the hypothalamus of the model group decreased significantly (P<0.05), indicating that the insomnia model induced by PCPA was successfully established. Compared with the model group, the BDNF level of diazepam group and acetyldopamine dimer A monomer group increased significantly (P<0.01), and the acetyldopamine dimer A monomer group was better than the positive control group.

[0057] 3.2 Pathological detection results of hypothalamic sections

[0058] See Figure 7As shown, as shown in A, normal group of rats hypothalamic neuron cell complete clear, neat arrangement, neuron cytoplasm is rich, light dye, nucleus in the middle, nucleolus clear. As shown in B, the model control group of rats hypothalamic cell deformation is serious, a large number of cells show vacuole phenomenon, loose arrangement, cell boundary performance fuzzy. As shown in C and D, the positive group and acetyl dopamine dimer A monomer group neuron cell vacuole phenomenon is lighter than the model group. Acetyl dopamine dimer A monomer group (D) cell vacuole phenomenon is not obvious, basically tend to normal. Positive group (C) also has more obvious vacuole phenomenon, the possible reason is that the drug dose is too high.

[0059] In summary, the hypothalamus DA, HVA, DOPAC, BDNF and insomnia closely related biochemical indicators, compared with the model group, acetyl dopamine dimer A can significantly increase the level of BDNF, reduce the content of DA, indicating that acetyl dopamine dimer A can improve insomnia.

[0060] The above determination of neurotransmitters related to insomnia biochemical indicators, found that the DA index, diazepam group (3mg / kg) significant difference is greater than 0.05, the possible reason is that the drug dose is too high, thereby producing toxic effect. Analysis of hypothalamic HVA and DOPAC determination results can be obtained, acetyl dopamine dimer A monomer group (20.154mg / kg) reduces the level of two indicators than the positive control group; analysis of hypothalamic BDNF determination results, acetyl dopamine dimer A monomer group can significantly increase the level of BDNF. Comprehensive analysis of the above indicators can be obtained from the extracted acetyl dopamine dimer A in cicada exuviae has the medical use of improving insomnia.

[0061] The above disclosed only for the preferred embodiments of the present application, of course, can not be limited by the scope of the present invention, those skilled in the art can understand the implementation of the above embodiment of all or part of the process, and according to the invention claims made by equivalent changes, still belong to the scope of the present application.

Claims

1. The use of an acetyldopamine compound in the preparation of a drug for improving insomnia, wherein the acetyldopamine compound is acetyldopamine dimer A extracted from cicada molts, and its structural formula is as follows:

2. Use according to claim 1, characterized in that: The medicine takes acetyl dopamine dimer A as the active ingredient, can increase the BDNF content in brain tissue, reduce the DA content and effectively affect the HVA and DOPAC contents of DA metabolites, thereby improving insomnia.

3. Use according to claim 1, characterized in that: The administration dose of the acetyl dopamine dimer A is 20.154 mg / kg.

4. The use according to claim 1, characterized in that: The preparation method of the acetyl dopamine dimer A comprises the following steps: (1) taking dried cicada slough as raw material, adding 70% ethanol aqueous solution with a volume concentration of 70% at a solid-liquid ratio of 1:9, soaking at room temperature for 3-4 hours, then refluxing and extracting at 110-130 DEG C for 2-3 times, each time for 1.5-2 hours, filtering the solution, and evaporating the filtrate to dryness, to obtain cicada slough extract; (2) taking the cicada slough extract and loading on HP-20 macroporous resin, gradient eluting with 30%, 50%, 80% and 100% methanol aqueous solution at room temperature to obtain different elution parts, evaporating the 80% methanol aqueous elution part to dryness, and obtaining the 80% methanol macroporous resin part of cicada slough; (3) passing the 80% methanol macroporous resin part of cicada slough through MCI column chromatography at room temperature, and eluting with 50%, 60%, 70%, 80%, 90% and 100% methanol aqueous solution to obtain different elution parts, evaporating the 50% methanol part solution to dryness to obtain a sample; passing the sample through ODS reversed-phase fast preparation chromatographic column, gradient eluting with 20%-100% methanol aqueous solution, and obtaining 26 tube sample solutions at a flow rate of 10 mL / min, combining the samples to obtain 5 parts, taking the 3rd part sample and passing it through dextran gel chromatographic column, eluting with pure methanol, combining the eluate to obtain 13 parts, and taking the 9th part sample and purifying it by semi-preparation liquid phase, eluting with 25% methanol to obtain the acetyl dopamine dimer A.

Images