阅读说明：本技术 查尔酮化合物在制备改善睡眠药物中的应用 (Application of chalcone compound in preparation of sleep improvement medicine ) 是由 吕翠 王岱杰 崔莉 张宇 张�浩 武玉波 谢惠 冯彦祥 于 2020-11-30 设计创作，主要内容包括：本发明提供查尔酮化合物在制备改善睡眠药物中的应用,属于生物医药技术领域。本发明提供一种从刺果毛茛中分离获得的查尔酮化合物4-Benzyloxylonchocarpin,经试验证明其具有较好的催眠效果,且安全性好,其可以增加中枢神经系统内最重要的抑制性神经递质GABA的水平,同时其与水合氯醛具有良好的协同催眠作用,因此本发明的查尔酮化合物4-Benzyloxylonchocarpin具有良好的实际应用之价值。(The invention provides an application of a chalcone compound in preparation of a sleep improvement medicine, and belongs to the technical field of biological medicines. The invention provides a chalcone compound 4-Benzyloxylonchocarpin separated from ranunculus spinosus, which has better hypnotic effect and good safety, can increase the level of the most important inhibitory neurotransmitter GABA in the central nervous system, and has good synergistic hypnotic effect with chloral hydrate, so the chalcone compound 4-Benzyloxylonchocarpin has good practical application value.)

1. The application of chalcone compound in preparing medicine for improving sleep;

wherein the structural formula of the chalcone compound is shown as the formula (I):

2. the use according to claim 1, wherein the improving sleep specifically comprises:

1) tranquilizing and hypnotizing;

2) the sleep latency period is shortened;

3) prolonging sleep duration;

4) increasing GABA content in brain tissue.

3. The use according to claim 1, wherein the dose of the chalcone compound in the sleep improvement medicament is controlled to be not less than 20mg/kg/d, preferably 20 to 80mg/kg/d, and more preferably 20 to 40 mg/kg/d.

4. A pharmaceutical composition for improving sleep, comprising a chalcone compound in combination with at least one other pharmaceutically active ingredient;

wherein the structural formula of the chalcone compound is shown as the formula (I):

5. the pharmaceutical composition of claim 4, wherein the additional pharmaceutically active ingredient is an ingredient that increases GABA activity; including flavonoids, alkaloids, coumarins and diterpene quinones; preferably, the peanut shell contains one or more of luteolin, eriodictyol and 5, 7-dihydroxy chromone which are flavonoid components in peanut shells, and proanthocyanidin components in peanut skin.

6. The pharmaceutical composition of claim 4, wherein the additional pharmaceutically active ingredient is chloral hydrate.

7. The pharmaceutical composition according to claim 4, wherein the mass ratio of the chalcone compound to the chloral hydrate is 1-4: 12 to 24.

8. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition has a use comprising:

1) tranquilizing and hypnotizing;

2) the sleep latency period is shortened;

3) prolonging sleep duration;

4) increasing GABA content in brain tissue.

9. A pharmaceutical formulation for improving sleep comprising a pharmaceutical composition according to any one of claims 4 to 8 and at least one other non-pharmaceutically active ingredient.

10. The pharmaceutical formulation of claim 9, wherein the other non-pharmaceutically active ingredient comprises a pharmaceutically acceptable carrier, excipient and/or diluent.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a chalcone compound in preparation of a medicine for improving sleep.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Sleep is the active physiological process of human body, and can restore spirit and relieve fatigue. 1/3, the sleep accounts for human life, and sufficient sleep is the basis for guaranteeing normal life; research shows that the sleep deficiency is inversely related to happiness; meanwhile, the risk of cardiovascular diseases, hypertension, coronary heart disease, dementia and other diseases is increased for people with poor sleep quality and short sleep time, and the life quality is reduced; good sleep quality can improve cognitive and emotional functions and help better participate in social activities. Nowadays, the rhythm of social life is gradually accelerated, the mental pressure of people is gradually increased, and the problem of sleep disorder caused by the gradual increase of mental pressure is increasingly highlighted. One Meta analysis result shows that 25.7% of 11 ten thousand college students in china have sleep disorder; another cross-sectional survey data related to sleep disorders of the elderly in China shows that the incidence rate of sleep disorders is 35%.

The sleep improving medicines include benzodiazepine medicines and non-benzodiazepine medicines, such as zolpidem and eszopiclone, which can prolong sleep time and/or improve sleep quality. The two drugs have similar structural functions and are targeted on a gamma-aminobutyric acid (GABA) signal path. These drugs have fast onset and definite action mechanism, but long-term administration is accompanied by the disadvantages of drug resistance and dependence. Therefore, there is still an urgent need to develop a drug with good efficacy, safety and low toxic and side effects for improving sleep, which is of great significance for improving national health level and quality of life.

The traditional Chinese medicine has the advantages of rich resources, high multi-target activity, small toxic and side effects and the like, and is an important source for medicine development. Chalcone compounds are organic compounds with a basic skeleton structure of 1, 3-diphenyl acrylketone, widely exist in various medicinal plants such as liquorice, safflower and the like, are precursors for synthesizing flavone in plants, are important organic medical intermediates, can be combined with different receptors due to large flexibility of molecules, show various biological activities, and particularly have remarkable activities in the aspects of insomnia resistance, aging resistance, tumor resistance, parasite resistance, virus resistance, bacteria resistance, inflammation resistance, platelet aggregation resistance and the like. Therefore, research and development of chalcone compounds become a research hotspot of medicinal chemistry.

Disclosure of Invention

Based on the defects of the prior art, the invention provides the application of the chalcone compound in preparing the sleep improvement medicine. The invention reports a chalcone compound 4-Benzyloxylonchocarpin separated from ranunculus spinosus, and experiments prove that the chalcone compound 4-Benzyloxylonchocarpin has a better hypnotic effect and good safety, can increase the level of the most important inhibitory neurotransmitter GABA in the central nervous system, and simultaneously has a good synergistic hypnotic effect with chloral hydrate, so the chalcone compound 4-Benzyloxylonchocarpin has a good practical application value.

In a first aspect of the invention, there is provided the use of a chalcone compound in the manufacture of a medicament for improving sleep. And thus, chalcone compounds are effective for treating sleep disorder-related diseases.

Wherein the structural formula of the chalcone compound is shown as the formula (I):

according to the present invention, the concept of "treatment" means any measure suitable for the treatment of a disease associated with sleep disorders, or the prophylactic treatment of such manifested disease or manifested symptoms, or the avoidance of recurrence of such disease, e.g. recurrence after the end of a treatment period or treatment of symptoms of an already established disease, or the prevention or suppression or reduction of the occurrence of such disease or symptoms with prior intervention.

More specifically, the sleep improvement specifically comprises:

1) tranquilizing and hypnotizing;

2) the sleep latency period is shortened;

3) prolonging sleep duration;

4) increasing GABA content in brain tissue.

In a second aspect of the present invention, there is provided a pharmaceutical composition for improving sleep, which comprises the above chalcone compound and at least one other pharmaceutically active ingredient.

Wherein the other pharmaceutical active ingredients are ingredients for improving GABA activity, including but not limited to flavonoids, alkaloids, coumarins and diterpene quinones.

According to experimental research, the chalcone compound and chloral hydrate have a synergistic sedative hypnotic effect, and the mass ratio of the chalcone compound to the chloral hydrate is (1-4): 12 to 24.

The pharmaceutical composition has the following uses, including:

1) tranquilizing and hypnotizing;

2) the sleep latency period is shortened;

3) prolonging sleep duration;

4) increasing GABA content in brain tissue.

In a third aspect of the present invention, there is provided a pharmaceutical formulation for improving sleep comprising the above pharmaceutical composition and at least one other pharmaceutically active ingredient.

The beneficial technical effects of one or more technical schemes are as follows:

the chalcone compound disclosed by the technical scheme has a good hypnotic effect and good safety, and can increase the level of the most important inhibitory neurotransmitter GABA in the central nervous system; meanwhile, the compound has good synergistic effect with chloral hydrate on hypnosis and sedation, so that the compound has better application prospect in preparing sleep-improving drugs.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a COSY and HMBC correlation spectrum of the novel chalcone compound 4-benzyloxynechoracaripin prepared in the example of the present invention;

FIG. 2 shows the preparation of 4-benzyloxychlorociapin, a novel chalcone compound prepared in the examples of the present invention¹H NMR Spectrum (400MHz, CDCl)₃)；

FIG. 3 shows the preparation of 4-benzyloxychlorociapin, a novel chalcone compound prepared in the examples of the present invention¹³C NMR Spectrum (400MHz, CDCl)₃)；

FIG. 4 is a COSY spectrum (400MHz, CDCl) of the novel chalcone compound 4-benzyloxychlorocarpin prepared in the example of the present invention₃)；

FIG. 5 shows the HSQC spectrum (400MHz, CDCl) of 4-benzyloxynecropin, a novel chalcone compound prepared in the examples of the present invention₃)；

FIG. 6 shows the HMBC spectrum (400MHz, CDCl) of 4-benzyloxychlorocarpin, a novel chalcone compound prepared in the examples of the present invention₃)；

FIG. 7 is a HRESIMS spectrum of 4-benzyloxynocharpin, a novel chalcone compound prepared in example of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As mentioned above, the sleep-improving drugs include those that can prolong sleep time and/or improve sleep quality, such as benzodiazepine drugs and non-benzodiazepine drugs, which have fast onset of action and definite action mechanism, but have disadvantages of drug resistance and dependence when taken for a long time.

In view of this, in the present invention, a novel chalcone compound 4-benzyloxychlorococcapin is first isolated from Ranunculus spinosus. By adopting chloral hydrate synergistic sleep experiments and taking the sleep-in rate, the sleep latency and the sleep duration of a mouse as observation indexes, the new chalcone compound is verified to be capable of obviously increasing the sleep-in rate of the mouse, shortening the sleep latency of the mouse, obviously prolonging the sleep duration and having the function of obviously improving the sleep. On the basis, by utilizing an enzyme-linked immunosorbent assay and measuring the GABA content of mouse brain tissues, the mechanism research of the sleep improvement effect of the novel chalcone compound is developed. Meanwhile, the safety evaluation of the novel chalcone compound is carried out by measuring the change of the organ coefficient of the mouse. The result shows that the compound related to the invention has no organ damage to mice, can obviously increase the GABA concentration of brain tissues of the mice, and is supposed to play a role in improving sleep through GABA signal pathways.

In particular, in an exemplary embodiment of the present invention, there is provided a use of a chalcone compound for the manufacture of a medicament for improving sleep. And thus, chalcone compounds are effective for treating sleep disorder-related diseases.

Wherein the structural formula of the chalcone compound is shown as the formula (I):

according to the present invention, the concept of "treatment" means any measure suitable for the treatment of a disease associated with sleep disorders, or the prophylactic treatment of such manifested disease or manifested symptoms, or the avoidance of recurrence of such disease, e.g. recurrence after the end of a treatment period or treatment of symptoms of an already established disease, or the prevention or suppression or reduction of the occurrence of such disease or symptoms with prior intervention.

In another embodiment of the present invention, the improving sleep specifically comprises:

1) tranquilizing and hypnotizing;

2) the sleep latency period is shortened;

3) prolonging sleep duration;

4) increasing GABA content in brain tissue.

In another embodiment of the present invention, in the sleep improvement drug, the dose of the chalcone compound is controlled to be not less than 20mg/kg/d, preferably 20 to 80mg/kg/d, and more preferably 20 to 40 mg/kg/d. Experimental research proves that the chalcone compound with the medium and low dose can obviously improve the GABA content in the brain tissue of a mouse, thereby playing a role in calming and hypnotizing; meanwhile, the medicine is safe and has no toxic or side effect.

According to the present invention, not only the use of the chalcone compounds for the preparation of a medicament for improving sleep, but also the enhancement of such effects when the chalcone compounds are administered in combination with at least one other pharmaceutically active ingredient is disclosed. The chalcone compounds may also be used in combination with other non-pharmaceutically active ingredients, instead of or in addition to other pharmaceutically active ingredients.

In view of the above, in another embodiment of the present invention, a pharmaceutical composition for improving sleep is provided, which comprises the chalcone compound described above and at least one other pharmaceutically active ingredient.

In yet another embodiment of the present invention, the other pharmaceutically active ingredient is an ingredient that enhances GABA activity, including but not limited to flavonoids, alkaloids, coumarins and diterpene quinones.

In another embodiment of the present invention, the other pharmaceutically active ingredients are one or more of luteolin, eriodictyol and 5, 7-dihydroxy chromone, which are flavonoid ingredients in peanut shell, and procyanidin ingredients in peanut skin.

In yet another embodiment of the present invention, the other pharmaceutically active ingredient is chloral hydrate.

According to experimental research, the chalcone compound and chloral hydrate have a synergistic sedative hypnotic effect, and the mass ratio of the chalcone compound to the chloral hydrate is (1-4): 12 to 24.

In yet another embodiment of the present invention, the pharmaceutical composition has the following uses, including:

1) tranquilizing and hypnotizing;

2) the sleep latency period is shortened;

3) prolonging sleep duration;

4) increasing GABA content in brain tissue.

In yet another embodiment of the present invention, there is provided a pharmaceutical formulation for improving sleep comprising the above pharmaceutical composition and at least one other non-pharmaceutically active ingredient.

Other non-pharmaceutically active ingredients include, but are not limited to, pharmaceutically acceptable carriers, excipients, and/or diluents.

Other non-pharmaceutically active ingredients include pharmaceutically acceptable carriers, excipients and/or diluents. Such as pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, pharmacologically innocuous salts such as sodium chloride, flavoring agents, vitamins such as vitamin a or vitamin E, tocopherols or provitamins, antioxidants such as ascorbic acid, and stabilizers and/or preservatives for extending the use and shelf life of the pharmaceutically active ingredient or formulation, and other common non-pharmaceutically active ingredients or adjuvants and additives known in the art, and mixtures thereof.

The pharmaceutical formulation may be administered in unit dosage form. The administration form may be a usual formulation form of the chalcone compound, or other feasible formulations, such as those skilled in the art can select a formulation suitable for the chalcone compound among conventional formulations, by adding carriers, excipients, binders, diluents, etc. compatible with the chalcone compound. Conventional dosage forms such as liquid dosage forms, solid dosage forms, external preparations, sprays, and the like described herein, such as the following: true solutions, colloids, microparticles, emulsion, suspension, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, and liniment.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

The method comprises the following steps:

1. isolation and identification of novel chalcone compounds

1.1 Experimental Equipment

Nicolet-510P type Infrared Spectroscopy (USA), v_maxIn cm^-1Is a unit; the NMR spectrometer was of the Bruker AMX-400 type, TMS was used as an internal standard, chemical shifts were expressed in delta and coupling constants were expressed in J.

1.2 extraction and separation methods

Crushing Ranunculus spinosus 3.4kg, extracting with 10L ethanol at room temperature, filtering the filtrate, and concentrating under reduced pressure to obtain 6.5g of total extract. Separating the total extract by silica gel column chromatography, and gradient eluting with n-hexane/ethyl acetate system sequentially with mixed solvent of n-hexane/ethyl acetate at volume ratio of 10:90, 7.5:92.5 and 5:95 to obtain 8 fractions (F1-8), wherein fraction F3(120mg) is subjected to silica gel chromatography repeatedly (1.8:8.2, v/v) with n-hexane/ethyl acetate system to obtain 4-benzyloxynocarpin (neochalcone compound, 7.9mg) and 4-methoxynocarpin (5.3 mg).

2. Establishment of cooperative chloral hydrate hypnosis model

2.1 Experimental animals

8 week old SPF grade male C57BL/6J mice, body weight (22. + -.2) g. Purchased from Beijing Weitonglihua laboratory animal technology Co., Ltd., certification number: SCXK (Jing) 2012 and 0001. The animals were housed in the analytical testing center of the academy of sciences of Shandong province. Animal feeding was in full compliance with the regulations of the laboratory animal care Committee of the analytical testing center of Shandong province. The humidity of the animal room is 50% -55%, the temperature is (23 +/-2) DEG C, light and shade are alternately daylighted for 12h, sterile common rat food is fed, and water is freely drunk. The animal experiments in this study were reviewed and supervised by the ethical committee of medical science of the analytical testing center of Shandong province.

2.2 drugs and reagents

Chloral hydrate was purchased from Shanghai Michelin Biochemical science and technology, Inc. (Cat # C804539, Shanghai Macklin Biochemical Co., Ltd.) and distilled water was formulated to the concentrations required for the experiments. Diazepam tablets, Beijing Yimin pharmaceutical industry Co., Ltd, lot number 20170802, and physiological saline were prepared to the concentrations required for the experiments.

2.3 Main Equipment

BSA124S model electronic balance (sartorius, germany), 1ml syringe, heating blanket, timer, and weight scale, etc.

2.4 Chloral hydrate suprathreshold and subthreshold sleep dosimetry

2.4.1 Experimental groups

100 mice were taken. Each group of mice was injected with chloral hydrate at different doses per dose, 10 mice per dose, and the injection amount was 0.1ml/10 g. Before injecting the medicine, weighing and recording the body mass of each mouse; after injection of the drug, the mice were placed on a heating blanket (adjusted to 37 ℃ C.) for the experiment.

2.4.2 judgment index

The disappearance of the righting reflex means that the four limbs of the mouse are not turned over within 1min after lying on the back, namely the disappearance stage of the righting reflex is entered. Righting reflex recovery means that the mice can turn freely and do not turn any more within 1 min. The sleep latency is the time taken from the start of chloral hydrate injection to the disappearance of the righting reflex. The sleep time is the time taken from disappearance of the righting reflex to restoration of the righting reflex. The standard for judging sleep onset is that the disappearance of righting reflex exceeds 1min, and the sleep onset percentage (the percentage (%) of sleeping onset) is equal to the number of animals falling asleep/the total number of animals multiplied by 100%.

2.4.3 Chloral hydrate subthreshold sleep dosimetry

The subthreshold sleep dose refers to the maximum dose at which 100% of mice positive reflex has not disappeared. 50 mice were selected and randomly divided into 5 groups and 10 mice/group. Chloral hydrate 0.24, 0.26, 0.28 and 0.30mg/g is injected into the abdominal cavity respectively, and the blank control group is injected with physiological saline with the same volume. And observing and recording the sleep condition of each group of mice within 30min after the chloral hydrate is injected, recording the sleep number of each group of mice, and calculating the sleep rate of each group of mice. This experiment was repeated 3 times with an interval of 24h between each experiment.

2.4.4 Chloral hydrate suprathreshold sleep dosimetry

Suprathreshold sleep dose refers to the minimum dose at which 100% of mice have a positive reflex abolition. 50 mice were taken and randomly divided into 5 groups and 10 mice per group. Respectively injecting 0.34, 0.36, 0.38, 0.40 and 0.42mg/g of chloral hydrate into the abdominal cavity. Observing and recording the sleeping condition of each group of mice within 30min after the chloral hydrate is injected, and recording the sleeping number, the sleeping incubation period and the sleeping time of each group of mice; the rate of falling asleep was calculated for each group of mice. This experiment was repeated 3 times with an interval of 24h between each experiment.

2.5 study of the hypnotic Effect of New chalcone compounds on mice Using the synergistic Chloral hydrate model 2.5.1 Experimental groups

50 male C57BL/6J mice were taken and, after one week of acclimation, randomized into 5 groups: blank control group, diazepam positive control group (2mg/kg), new chalcone compound low, medium and high dose groups (20mg/kg, 40mg/kg, 80mg/kg), 10 groups per group. The new chalcone compounds were formulated in the doses with normal saline, and each of the dose groups was administered with the corresponding dose of the drug per mouse by daily gavage at a volume of 0.2ml/10g, and the blank control group was administered with an equal volume of normal saline. 1/d, 7d of continuous administration. The diazepam positive control group is perfused with physiological saline with the same volume as the gastric lavage, 2mg/kg diazepam is continuously administered by intragastric administration at 7 days, and the administration volume is 0.2ml/10 g.

2.5.2 Effect of New chalcone Compounds on the Rate of sleep in subthreshold dose chloral hydrate mice

Each group of mice was gavaged continuously for 7d according to the corresponding drug. And after the last administration for 30min, the mice of each group are given a subthreshold dose of chloral hydrate by intraperitoneal injection, and the administration volume is 0.1ml/10 g. And observing and recording the sleep condition of each group of mice within 30min after administration, recording the number of the mice in each group, and calculating the sleep rate of each group of mice.

2.5.3 Effect of New chalcone Compounds on sleep latency and sleep time in suprathreshold dose chloral hydrate mice

Each group of mice was gavaged continuously for 7d according to the corresponding drug. And after the last administration for 30min, the mice of each group are given suprathreshold dose of chloral hydrate by intraperitoneal injection, and the administration volume is 0.1ml/10 g. The sleep onset latency and sleep duration of each group of mice were observed and recorded.

2.5.4 measurement of GABA content in mouse brain tissue

30min after the last drug, mice were killed by dislocation, brains were taken out, washed with ice-cold physiological saline, blotted with filter paper, weighed, homogenized by a homogenizer, centrifuged at 3000r/min for 10min, and the supernatant was taken and measured for GABA content by ELISA according to the instructions of the kit (GABA kit, Wuhan cloud technologies GmbH, cat # CEA900 Ge).

2.5.5 organ coefficients

Weighing mice in each group before death due to dislocation, dissecting to remove heart, liver, spleen, lung and kidney tissues, removing fat connective tissue around the organs, sucking blood on the surface of organs with filter paper, weighing, and calculating organ coefficient, i.e. organ wet weight (mg)/body weight (g)

2.6 statistical analysis

Statistical analysis of the data using SPSS 25.0 software, results are expressed as mean ± standard deviation. Independent sample two-sided T-test was performed for mean differences between independent experimental groups.

As a result:

1. structural analysis of the novel chalcone compound 4-Benzyloxyconchocarpin:

4-Benzyloxyconchocarpin (novel chalcone compound): yellow solid, IR (KBr) v_max：3310、1655、1610、1420、1000cm^-1，HRESIMS：m/z 413.1711[M+H]⁺：(calcd for C₂₇H₂₅O₄ ⁺，413.1747，M 8.8ppm)。¹H-NMR showed 1-proton bimodal signals with 16.0Hz coupling constants at delta 7.45 and 7.86, typical characteristics of H-alpha and H-beta for trans-chalcones, from delta 27.8 (C-beta), 144.0 (C-alpha) and 191.9 (conjugated ketones)¹³This is further confirmed by the C-MR signal. In addition, the hydrogen spectrum shows double peaks at δ 6.76(J ═ 10Hz, H-1 ") and 5.59(J ═ 10Hz, H-2"), and a 6 proton single peak at δ 1.47. The pyran ring is formed by cyclization of a typical prenyl group with an adjacent hydroxyl group.¹H(400MHz,CDCl₃) And¹³C NMR(100MHz,CDCl₃) The results are shown in Table 1.

The structural formulas of the novel chalcone compound 4-benzyloxylonchocarpin and the compound 4-methoxylonchocarpin are as follows:

wherein, 4-benzoxylonchocarpin, R ═ Bn,

4-methoxylonchocarpin，R＝Me。

TABLE 14 of benzyloxylonchocaripin¹H NMR (400MHz) and¹³c NMR (100MHz) results

2 establishment of cooperative chloral hydrate hypnosis model

2.1 subthreshold sleep dose of chloral hydrate

As shown in Table 2, after the chloral hydrate with the dose of 0.24mg/g is injected into the abdominal cavity, the mice have no obvious gait abnormality or activity reduction and other expressions, and have no obvious difference with the mice before administration, and the sleep rate is 0%; in the 0.26mg/g dosage group, some mice have unstable crawling gait after about 10min, the active activity is obviously reduced under the condition of no external stimulation, the righting reflex exists but is weakened (the self righting speed of the mice after being placed in a supine position is obviously slower than that before administration), the active activity begins to increase and gradually returns to normal after lasting for about 30min, and the sleeping rate is 0%. The phenomena of unstable crawling gait and reduced autonomic activity occur about 8min in the mice in the 0.28mg/g dose group, the falling asleep rate is (14.44 +/-3.14)%, the phenomena of unstable crawling gait and reduced autonomic activity occur about 6min in the mice in the 0.30mg/g dose group, and the falling asleep rate is (30.00 +/-2.72)%. Therefore, the subthreshold sleep dose of chloral hydrate was determined to be 0.26 mg/g.

2.2 Chloral hydrate suprathreshold sleep dose

As shown in Table 2, the mice injected with chloral hydrate at doses of 0.34mg/g, 0.36mg/g and 0.38mg/g all exhibited reduced autonomic activity, and the sleep onset rates were (36.67. + -. 5.44)%, (63.33. + -. 2.72)%, and (84.44. + -. 1.57)%, which were all less than 100%. The sleep rate of the mice is 100 percent after the mice of each age group are given 0.40mg/g and 0.42mg/g chloral hydrate. After the righting reflex reaction of all mice is recovered, the active activities gradually return to normal, and no obvious difference exists between the activity and the activity before administration. Therefore, the suprathreshold sleep dose of chloral hydrate was determined to be 0.40 mg/g.

TABLE 2 Effect of varying doses of chloral hydrate on sleep in mice: (n＝30)

3. Effect of novel chalcone Compounds on sleep in mice at subthreshold doses induced by chloral hydrate

As shown in table 3, the sleeping rates of the diazepam positive control group, the new chalcone compound low dose group and the middle dose group were significantly increased compared to the blank control group, and the differences were statistically significant (P < 0.05). While the sleep rate of the mice in the high dose group of the novel chalcone compound is not increased obviously. The low and medium dosage groups of the novel chalcone compound and the chloral hydrate subthreshold dosage group are prompted to have synergistic effect, and the sleep rate of mice can be improved.

4. Effect of novel chalcone Compounds on sleep in mice at suprathreshold doses with chloral hydrate

As shown in table 3, the diazepam positive control group, the new chalcone compound low dose group and the middle dose group all reduced the sleep latency and prolonged the sleep duration of mice significantly (P <0.05) compared to the blank control group. The high dose groups of the novel chalcone compounds reduced sleep latency and extended sleep duration in mice, but the differences were not statistically significant (P > 0.05). The low and medium dosage groups of the novel chalcone compound and the chloral hydrate suprathreshold dosage group are prompted to have synergistic effect, so that the sleep latency of the mice can be shortened, and the sleep duration of the mice can be prolonged.

TABLE 3 Effect of New chalcone Compounds on the Rate of onset of sleep and the sleep latency and sleep time of mice at subthreshold doses of chloral hydrate: (n＝10；*P<0.05)

5. Effect of novel chalcone compounds on GABA content in mouse brain

As shown in table 4, the high dose group of the novel chalcone compound increased GABA content in mouse brain compared to the blank control group, but the difference was not statistically significant (P > 0.05); however, the content of GABA in the brain of mice in a diazepam positive control group and a new chalcone compound is obviously increased, and the difference is obvious (P < 0.05). Suggesting that the low and medium dose groups of the novel chalcone compound may increase GABA content in the mouse brain.

6. Effect of novel chalcone Compounds on mouse organs

As shown in table 5, the organ coefficients of the new chalcone compounds in the dose groups were statistically insignificant (P >0.05) compared to the control blank group, indicating that the new chalcone compounds did not damage the organs of the mice in the high dose range. See table 5.

TABLE 4 comparison of GABA content in brain tissue of mice in each group: (n＝10；*P<0.05)

TABLE 5 organ coefficients of mice of each group: (n＝10；*P<0.05, coefficient units: mg/g)

And (4) conclusion:

the results of the embodiment of the invention show that compared with a blank control group, the combination of the low and medium dosage groups and the subthreshold dosage chloral hydrate of the novel chalcone compound can obviously increase the sleep rate of mice; the combination of the low and medium dosage groups of the novel chalcone compound and the suprathreshold dosage of chloral hydrate can obviously shorten the sleep latency of mice and prolong the sleep duration of the mice, and the novel chalcone compound has the synergistic effect of hypnosis of the chloral hydrate.

GABA is the most important inhibitory neurotransmitter in the central nervous system and can cause a large number of chloride ions (Cl) by binding to GABA receptors (GABAA)^－) The internal flow causes the hyperpolarization of the cell membrane to reduce the excitability of the neuron, thereby playing the role of sedation and hypnosis. The study found that the low and medium dose groups of the novel chalcone compound can significantly increase the GABA content in the brain tissue of mice compared to the blank control group.

The weight changes of the heart, liver, spleen, lung and kidney can reflect the function changes of some organs in the animal body. The results herein show that each dose group of the novel chalcone compounds had no damaging effect on each organ of the mice compared to the blank control group.

In conclusion, the novel chalcone compound 4-Benzyloxyconchocarpin has low hypnotic effect in the medium-dose group and good hypnotic effect, and has no damage to animal organs. And the new chalcone compound is preliminarily speculated to possibly play a sedative-hypnotic effect by improving GABA (gamma-aminobutyric acid) level in the brain of the mouse.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.