Affinity chromatography screening method of medicine with sleep aiding effect

Abstract

The invention discloses an affinity chromatography screening method of a medicine with a sleep aiding effect, and belongs to the technical field of biological medicine. Comprising the following steps: respectively combining an MAO silica gel stationary phase and a TPH2 silica gel stationary phase with a high performance liquid chromatography system, and screening out components with MAO inhibitory activity or TPH2 activation activity from a medicinal and edible material extracting solution. According to the affinity chromatography screening technology of the sleep-aiding composition, efficient adsorption and separation of target components are achieved, HPLC is combined to screen eluted components, the sleep-aiding activity of the components is further confirmed, and scientificity and accuracy of analysis of the sleep-aiding components of traditional Chinese medicine are improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an affinity chromatography screening method for drugs with sleep-aiding effects. Background Art

[0002] Good sleep can make people healthy both physically and mentally, but with the progress of society and the accelerated pace of life, more and more people are suffering from insomnia. There are many factors that cause insomnia, including neurobiological factors, genetic factors and psychological factors. Among neurobiological factors, substances such as catecholamines, orexins and histamine promote wakefulness, while substances such as gamma-aminobutyric acid (GABA), adenosine, 5-hydroxytryptamine (5-HT), melatonin and prostaglandin D2 help sleep. When the balance of these substances is broken, the regulation of the sleep-wake cycle is affected, which may lead to insomnia.

[0003] 5-HT is widely involved in regulating emotions, cognition, memory, sleep and many other physiological processes in the human body. TPH (tryptophan hydroxylase) and MAO (monoamine oxidase) are key enzymes for the synthesis and degradation of 5-HT, and play an important role in sleep regulation in the human body. TPH is the rate-limiting enzyme for the synthesis of 5-HT. It catalyzes the conversion of tryptophan into 5-hydroxytryptophan, which in turn generates 5-hydroxytryptamine (5-HT). There are two subtypes of TPH, TPH1 is expressed in the neurons of the intestinal myenteric layer, and TPH2 is mainly expressed in the raphe neurons of the brainstem. Due to the restriction of the blood-brain barrier, TPH2 has a low content, low activity and high specificity, and plays an important role in the metabolism of 5-HT. MAO is an important enzyme in the human body, which is mainly responsible for catalyzing the oxidative deamination reaction of monoamine neurotransmitters (such as norepinephrine, dopamine and 5-hydroxytryptamine), thereby regulating the level and activity of these neurotransmitters in the body. Therefore, MAO and TPH2 are responsible for regulating the metabolism of monoamine neurotransmitters and the biosynthesis of serotonin in the human body, respectively, and together maintain the normal function and homeostasis of the nervous system.

[0004] Common sleep aids are mainly sedative and hypnotic chemical drugs, including benzodiazepines Drugs such as antidepressants and melatonin have serious side effects and are highly addictive, making them unsuitable for long-term use. Traditional Chinese medicine has a long history of helping people sleep, and it has significant therapeutic effects and few side effects, making it a safe and effective sleep aid. However, the composition of traditional Chinese medicine is complex, containing a variety of active and inactive ingredients. Although traditional Chinese medicine extraction and screening methods, such as solvent extraction and column chromatography, can extract effective ingredients from traditional Chinese medicine to a certain extent, they are inefficient and it is difficult to accurately locate ingredients with sleep-inducing activity, resulting in complex extracts and low levels of active ingredients, which affects the further development of the sleep-inducing effect of traditional Chinese medicine.

[0005] In recent years, with the progress of technology, people have begun to explore more advanced traditional Chinese medicine extraction and screening techniques in order to more accurately locate and extract the ingredients with sleep-promoting activity. As a result, the method of screening traditional Chinese medicine sleep-promoting active ingredients based on affinity chromatography combined with enzyme technology has emerged. Affinity chromatography is a method that uses the inherent specific interactions of biomolecules for highly selective separation of samples. Specific interactions include hormones and receptors, enzymes and substrates, antibodies and antigens, etc. Affinity chromatography uses immobilized enzymes or receptors as ligands and utilizes the specific binding ability of biomolecules to directly screen out the active ingredients that interact with sleep regulation targets from the extract, realizing the integration of "separation - screening". This method not only improves the screening efficiency but also ensures the purity and specificity of the active ingredients, laying a solid foundation for the modern research and application of traditional Chinese medicine sleep-promoting ingredients. Summary of the Invention

[0006] The purpose of the present invention is to provide an affinity chromatography screening method for drugs with sleep-promoting effects to solve the problems existing in the above-mentioned prior art. The present invention specifically screens the active ingredients in traditional Chinese medicine that bind to sleep regulation targets and provides a food-drug homologous composition that can improve the sleep quality of mice and has low toxicity and its applications.

[0007] To achieve the above purpose, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention, an affinity chromatography screening method for drugs with sleep-promoting effects, includes the following steps:

[0009] Connect the MAO silica gel stationary phase and the TPH2 silica gel stationary phase with a high-performance liquid chromatography system respectively, and screen out the ingredients with MAO inhibitory activity or TPH2 activating activity from the extract of food-drug homologous materials.

[0010] Another technical solution of the present invention, a food-drug homologous composition with sleep-promoting effects, includes the following raw materials in parts by weight: 10 - 15 parts of wild jujube seeds, 10 - 15 parts of Poria cocos, 10 - 15 parts of moringa seeds, 10 - 15 parts of Ganoderma lucidum, 5 - 10 parts of hawthorn, 5 - 10 parts of mulberry, 5 - 10 parts of wolfberry, 5 - 10 parts of walnut, 2 - 3 parts of jasmine, 2 - 3 parts of gardenia, 2 - 3 parts of Chinese date, 2 - 3 parts of longan, 2 - 3 parts of Hericium erinaceus, 2 - 3 parts of mangosteen, 2 - 3 parts of lotus seeds and 2 - 3 parts of tremella.

[0011] Another technical solution of the present invention, the preparation method of the food-drug homologous composition as claimed in claim 5 or 6, includes the following steps: crush the raw materials into powder, mix them with water, perform ultrasonic treatment and then heat extraction, collect the extract, filter and concentrate it; add excipients and prepare granules through the wet granulation process.

[0012] Based on the above technical solutions, the present invention has the following technical effects:

[0013] (1) The present invention establishes an affinity chromatography screening method for a sleep aid composition. By using an affinity chromatography column with MAO silica gel and TPH2 silica gel as the stationary phase in combination with HPLC, 16 sleep aid components that inhibit MAO activity or activate TPH2 activity are screened out from various traditional Chinese medicine raw materials of medicine and food homology. After reasonable compatibility of the extracted components of Ziziphus jujuba var. spinosa, Poria cocos, Moringa oleifera seeds, Ganoderma lucidum, Hawthorn, Mulberry, Lycium barbarum, Walnut, Jasminum sambac, Gardenia jasminoides, Chinese date, Longan, Hericium erinaceus, Mangosteen, Lotus seed, Tremella fuciformis, and supplemented with erythritol and maltodextrin to make granules, it can significantly improve the sleep quality of mice and regulate the protein expression of MAO and TPH2 in the brains of mice.

[0014] (2) The affinity chromatography screening technology of the sleep aid composition of the present invention realizes the efficient adsorption and separation of target components, combines HPLC to screen the eluted components, further confirms their sleep aid activities, and improves the scientificity and accuracy of the analysis of traditional Chinese medicine sleep aid components.

[0015] (3) According to the screened and formulated sleep aid traditional Chinese medicine composition, which uses traditional Chinese medicine of medicine and food homology as the main raw material, it has no damage to the livers and kidneys of mice, has high safety, no toxic and side effects, and can be taken for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 SEM images of SiO2 with different properties. Among them, A: SEM image of unactivated SiO2; B: SEM image of activated SiO2; C: SEM image of the SiO2 chromatographic stationary phase wrapped with MAO; D: SEM image of the SiO2 chromatographic stationary phase wrapped with TPH2.

[0018] Figure 2 Effect of the drug of the present invention on the sleep time of mice. Among them, A: Sleep latency; B: Sleep time (*: P < 0.01; **: P < 0.001).

[0019] Figure 3 Effect of the drug of the present invention on the autonomous activity distance and time of insomnia mice. Among them, A: Autonomous activity distance of mice; B: Autonomous activity time of mice (**: Compared with the blank group, P < 0.001; ##: Compared with the model group, P < 0.001).

[0020] Figure 4Effects of the drug of the present invention on sleep latency and sleep duration in insomnia mice. Among them, A: sleep latency; B: sleep duration (**: compared with the blank group, P < 0.001; ##: compared with the model group, P < 0.001).

[0021] Figure 5 It is a HE staining section diagram of mouse liver and kidney.

[0022] Figure 6 It is the effect of the granule of Example 3 on MAO and TPH2 proteins in the cerebral cortex of mice.

[0023] Figure 7 It is the content of MAO and TPH2 proteins in the cerebral cortex of mice. Among them, A is the relative content of MAO protein, and B is the relative content of TPH2 protein.

[0024] Figure 8 It is a schematic diagram of the principle and process of the construction and screening of the affinity chromatography column in the present invention. Detailed implementation manners

[0025] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation manners of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0029] As used herein, terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0030] The technical solutions of the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0031] The embodiments of the present invention provide an affinity chromatography screening method for a drug with sleep-aiding effects, including the following steps:

[0032] Couple the MAO silica stationary phase and the TPH2 silica stationary phase with a high-performance liquid chromatography system respectively, and screen out the components with MAO inhibitory activity or TPH2 activation activity from the extract of medicated and edible homologous materials.

[0033] In some specific embodiments, the preparation method of the MAO silica stationary phase is: mix the activated SiO2, PBS solution and MAO, stir magnetically and centrifuge under ice bath conditions to obtain the MAO silica stationary phase MAO@SiO2.

[0034] In some specific embodiments, the preparation method of the TPH2 silica stationary phase is: mix the activated SiO2, PBS solution and TPH2, stir magnetically and centrifuge under ice bath conditions to obtain the TPH2 silica stationary phase TPH2@SiO2.

[0035] In some specific embodiments, the chromatographic conditions of the affinity chromatography screening method are: column temperature 30°C, flow rate 0.2 mL / min, mobile phase is 10 mM ammonium acetate (pH 7.2 - 7.4), injection volume 20 μL, using an ELSD1260 detector: evaporation temperature 60°C, drift tube temperature 60°C, high-purity nitrogen gas flow rate 1.6 L / min.

[0036] The embodiments of the present invention also provide a medicated and edible homologous composition with sleep-aiding effects, including the following raw materials in parts by weight: 10 - 15 parts of wild jujube seeds, 10 - 15 parts of poria cocos, 10 - 15 parts of moringa seeds, 10 - 15 parts of ganoderma lucidum, 5 - 10 parts of hawthorn, 5 - 10 parts of mulberry, 5 - 10 parts of wolfberries, 5 - 10 parts of walnuts, 2 - 3 parts of jasmine flowers, 2 - 3 parts of gardenia jasminoides, 2 - 3 parts of Chinese dates, 2 - 3 parts of longans, 2 - 3 parts of hericium erinaceus, 2 - 3 parts of mangosteens, 2 - 3 parts of lotus seeds and 2 - 3 parts of tremella fuciformis.

[0037] In some specific embodiments, the raw materials include the following parts by weight: 15 parts of wild jujube seeds, 15 parts of Poria cocos, 15 parts of moringa seeds, 15 parts of Ganoderma lucidum, 10 parts of hawthorn, 10 parts of mulberries, 10 parts of wolfberries, 10 parts of walnuts, 3 parts of jasmine flowers, 3 parts of gardenia fruits, 3 parts of Chinese dates, 3 parts of longans, 3 parts of Hericium erinaceus, 3 parts of mangosteens, 3 parts of lotus seeds, and 3 parts of tremella.

[0038] The embodiment of the present invention also provides a preparation method of the medicine and food homologous composition, including the following steps: crushing the raw materials into powder, mixing with water, performing ultrasonic treatment and then heating for extraction, collecting the extract, filtering and concentrating; adding auxiliary materials, and preparing granules through a wet granulation process.

[0039] In some specific embodiments, the material-liquid ratio of the raw materials to water is 1:(6 - 12) (kg / L);

[0040] The frequency of the ultrasonic treatment is 100 Hz, and the time is 30 min;

[0041] The conditions for the heating extraction are: water bath heating to 80 - 90 °C for extraction for 3 h, repeating 2 - 3 times;

[0042] The conditions for the concentration are: concentrating to 40% - 60% of the original weight;

[0043] The auxiliary materials are: 10% - 30% of erythritol and 20% - 40% of maltodextrin.

[0044] The affinity chromatography stationary phase of the present invention is MAO silica gel and TPH2 silica gel. Connect them to HPLC respectively, and online screen 16 components with MAO inhibitory activity or TPH2 activating activity from 60 medicine and food homologous materials, which are wild jujube seeds, Poria cocos, moringa seeds, Ganoderma lucidum, hawthorn, mulberries, wolfberries, walnuts, jasmine flowers, gardenia fruits, Chinese dates, longans, Hericium erinaceus, mangosteens, lotus seeds, and tremella. After extracting and concentrating the above components, granules are made with the addition of erythritol and maltodextrin. The granules can significantly improve the sleep quality of mice, and can regulate the protein expression of MAO and TPH2 in the brains of mice, and have no damage to the livers and kidneys of mice. This technology can be widely applied to pharmaceutical research and development, functional food and natural product development, and provides an efficient and low-cost solution for screening active ingredients for sleep aid products.

[0045] An affinity chromatography screening of a sleep aid composition adopts the following steps:

[0046] (1) Activation of silica gel: Weigh about 20 g of silica gel into a three-necked flask, add 200 mL of 10% HCl and stir, heat to 105 °C, reflux for acidification treatment for 8 h, filter, wash with distilled water until neutral, place in an oven at 110 °C for drying for 24 h, and store in a desiccator for standby.

[0047] (2) Preparation of affinity chromatography stationary phase: Weigh 20 mg of activated SiO2 precisely and place it in a 100 mL round-bottom flask. After adding 20 mL of PBS solution, add 98 μL (5 U / 490 μL) of MAO and TPH2 solutions respectively. Stir magnetically for 3 h under ice bath conditions, then centrifuge. Wash the precipitate with PBS solution repeatedly five times, and the SiO2 chromatography stationary phase (MAO@SiO2, TPH2@SiO2) wrapped with MAO and TPH2 is obtained after centrifugation.

[0048] (3) Preparation of affinity chromatography column: Select a column core with a size of 50 mm × 4.6 mm for the chromatography column. After washing the column core and column jacket with ultrapure water, use low-pressure wet packing: Install the column core into the column jacket, pad gaskets and hoof plates at both ends, and slowly inject the PBS suspension of the stationary phase into the column core. Apply pressure with a high-pressure pump at a low flow rate to make the stationary phase settle rapidly until the pressure in front of the column no longer rises significantly. Open the upper hoof plate and add the PBS suspension of the stationary phase again. Repeat this several times until the chromatography column is filled.

[0049] (4) Screening of hypnotic activity of affinity chromatography: Connect the MAO@SiO2 affinity chromatography column and TPH2@SiO2 affinity chromatography column to HPLC respectively, and online screening of hypnotic activity can be carried out. The chromatographic conditions are as follows: column temperature 30 °C, flow rate 0.2 mL·min -1 , the mobile phase is 10 mM ammonium acetate (pH 7.2 - 7.4), the injection volume is 20 μL, and an ELSD1260 detector is used: evaporation temperature 60 °C, drift tube temperature 60 °C, and the high-purity nitrogen gas flow rate is 1.6 L / min.

[0050] Each sample is extracted and concentrated with purified water and then formulated into a sample with a sample concentration of 1 mg / mL.

[0051] A medicine-food homologous composition with hypnotic effect, adopting the following steps:

[0052] (1) Weigh the selected raw materials according to the ratio, clean and dry them, and pour them into a pulverizer for pulverization;

[0053] (2) Add the powder obtained in step (1) to pure water according to a solid-liquid ratio of 1:6 to 1:12 and mix evenly; After ultrasonic treatment for 30 min, heat in a water bath to 80 °C to 90 °C for extraction for 3 h, repeat 2 - 3 times, collect the extract, filter and concentrate;

[0054] (3) Add erythritol and maltodextrin to the concentrated solution and prepare granules by wet granulation process.

[0055] Example 1

[0056] Preparation of SiO2 chromatography stationary phase wrapped with MAO and TPH2:

[0057] Weigh about 20 g of silica gel into a three-necked flask, add 200 mL of 10% HCl, stir, heat to 105 °C, reflux and acidify for 8 h, filter, wash with distilled water until neutral, dry in an oven at 110 °C for 24 h, and store in a desiccator for later use.

[0058] Precisely weigh 20 mg of activated SiO2 and place it in a 100 mL round-bottom flask. After adding 20 mL of PBS solution, add 98 μL (5 U / 490 μL) of MAO and 98 μL (5 U / 490 μL) of TPH2 solution respectively. Stir magnetically for 3 h under ice bath conditions, centrifuge, wash the precipitate with PBS solution repeatedly five times, and obtain the SiO2 chromatographic stationary phase (MAO@SiO2, TPH2@SiO2) wrapped with MAO and TPH2 after centrifugation.

[0059] Observe the prepared silica gel powder with a scanning electron microscope. First, stick the conductive tape on the sample stage. Second, dip a small amount of silica gel powder with a toothpick and sprinkle it evenly on the conductive tape. Then use compressed air (or an ear bulb) to remove the unadhered powder to ensure a monolayer distribution for observation. For non-conductive or poorly conductive samples, they need to be coated before being placed in the scanning electron microscope for observation; while for conductive samples, they can be directly placed in the scanning electron microscope for observation.

[0060] As can be seen from Figure 1 A and B, there are no significant morphological differences in SiO2 before and after activation. Figure 1 As shown in C and D, the surfaces of SiO2 wrapped with MAO and TPH2 are rough, indicating that there are substances adsorbed on the surface.

[0061] Example 2

[0062] Use the MAO@SiO2 and TPH2@SiO2 affinity chromatography columns combined with HPLC to screen for sleep aid ingredients:

[0063] Select a column core with a size of 50 mm × 4.6 mm for the chromatographic column and use low-pressure wet packing: Install the column core in the column sleeve, pad gaskets and hoof plates at both ends, slowly inject the PBS suspension of the MAO@SiO2 and TPH2@SiO2 stationary phase into the column core, pressurize with a high-pressure pump at a low flow rate to make the stationary phase settle rapidly until the pressure in front of the column no longer rises significantly. Open the upper hoof plate and add the PBS suspension of the stationary phase again. Repeat several times to fill the chromatographic column.

[0064] Connect the MAO@SiO2 affinity chromatography column and the TPH2@SiO2 affinity chromatography column to HPLC respectively for online screening of sleep-aiding activity. The chromatographic conditions are as follows: column temperature 30°C, flow rate 0.2 mL / min, mobile phase 10 mM ammonium acetate (pH 7.2 - 7.4), injection volume 20 μL, using an ELSD1260 detector: evaporation temperature 60°C, drift tube temperature 60°C, high-purity nitrogen flow rate 1.6 L / min.

[0065] After each sample is extracted and concentrated with purified water, a sample with a sample concentration of 1 mg / mL is prepared. The peak elution times of 60 components on the two affinity chromatographies are shown in Tables 1 - 2.

[0066] Table 1 Peak elution time table of MAO@SiO2 affinity chromatography for different sleep-aiding components

[0067]

[0068] Table 2 Peak elution time table of TPH2@SiO2 affinity chromatography for different sleep-aiding components

[0069]

[0070] In addition, there are no obvious chromatographic peaks for Chinese yam, purslane, smoked plum, papaya, liquorice root, cassia seed, lily, cinnamon, gorgon fruit, malt, fructus aurantii immaturus, boat-fruited sterculia seed, peach kernel, mulberry leaf, tangerine peel, semen coicis, polygonatum sibiricum, kudzu root, sophora japonica flower, selfheal, angelica sinensis, kaempferia galanga, saffron, tsaoko fruit, turmeric, shiitake mushroom, mushroom, flammulina velutipes, chrysanthemum, green tea, mint, orange peel, dandelion, almond, matsutake mushroom, morel, flower mushroom, straw mushroom, agaric, dictyophora indusiata, boletus edulis, agrocybe cylindracea, oyster mushroom, blueberry, waxberry, yacon, etc.

[0071] Therefore, by connecting the MAO@SiO2 affinity chromatography column and the TPH2@SiO2 affinity chromatography column to HPLC, 16 components with inhibitory activity on MAO or activating activity on TPH2 are screened online, namely semen ziziphi spinosae, poria cocos, moringa seeds, ganoderma lucidum, hawthorn, mulberry, wolfberry fruit, walnut, jasmine flower, gardenia fruit, Chinese date, longan, hericium erinaceus, mangosteen, lotus seed, tremella.

[0072] Example 3

[0073] A sleep-aiding composition and its preparation method

[0074] Raw materials by weight parts: 15 parts of wild jujube seeds, 15 parts of Poria cocos, 15 parts of moringa seeds, 15 parts of Ganoderma lucidum, 10 parts of hawthorn, 10 parts of mulberry, 10 parts of wolfberry, 10 parts of walnut, 3 parts of jasmine, 3 parts of gardenia, 3 parts of Chinese date, 3 parts of longan, 3 parts of Hericium erinaceus, 3 parts of mangosteen, 3 parts of lotus seed, 3 parts of Tremella fuciformis are poured into a pulverizer and pulverized into powder. The obtained powder is mixed with pure water according to a solid-liquid ratio of 1:8 (m / v, kg / L); after ultrasonic treatment (100 Hz) for 30 min, it is heated in a water bath to 85 °C for extraction for 3 h, and the process is repeated 2 - 3 times. The extract is collected, filtered and concentrated. The concentrate accounts for 50% of the total mass, and the excipients are erythritol (20%) and maltodextrin (30%). The granules are prepared by a wet granulation process.

[0075] Example 4

[0076] Verify its sleep-promoting effect and mechanism at the mouse level

[0077] 1 Sleep situation of normal mice after administration.

[0078] Before the experiment, the mice were acclimatized to the environment in the animal house for 3 days. Fifty healthy male ICR mice (body weight 20 ± 2 g) were selected as experimental animals and randomly divided into 6 groups: blank group, positive control group (diazepam), high, medium and low dose groups of Example 3, with 10 mice in each group.

[0079] Mice in the high, medium and low dose groups of Example 3 and the diazepam group were intragastrically administered different doses of Example 3 (100 mg / kg, 50 mg / kg, 25 mg / kg) and diazepam suspended in 0.5% CMC-Na (2 mg / kg), and the blank group was intragastrically administered with the same volume of 0.5% CMC-Na solution. One hour after administration, the mice were placed in a multi-functional mouse voluntary activity recorder to record their activity, including the activity distance and activity time, and the results were recorded for statistical analysis. Subsequently, the sleep latency and sleep time of the mice were recorded. The sleep latency was the time from intraperitoneal injection to the disappearance of the righting reflex, and the sleep time was the time from the disappearance of the righting reflex to recovery.

[0080] As Figure 2 shown, compared with the mice in the blank group, the positive control group, the medium and high dose groups of Example 3 could significantly reduce the sleep latency of the mice, and there was no significant difference in the low dose group of Example 3. The positive control group, the high, medium and low dose groups of Example 3 could all prolong the sleep time of the mice, and there was a significant difference (P < 0.01). The effect of Example 3 on the sleep latency of the mice decreased with the increase of the administration dose, and the sleep time of the mice increased. It shows that Example 3 has a sleep-promoting effect.

[0081] 2 Establishment of the insomnia model in insomnia mice and the free activity and sleep situation of the mice after administration

[0082] Sixty healthy male ICR mice (body weight 20±2 g) were selected as experimental animals and randomly divided into 6 groups: blank group, model group, positive control group (diazepam), high, medium and low dose groups of Example 3, with 10 mice in each group. Except for the blank group, the other groups were intraperitoneally injected with p-chlorophenylpropionic acid solution (PCPA) to induce an insomnia mouse model. Each mouse was injected continuously with PCPA solution at a dose of 300 mg / kg for 2 days. The disappearance of the animal's circadian rhythm and significant activity were observed, indicating the success of the model.

[0083] After successfully establishing the PCPA insomnia model, the mice in the high, medium, low dose groups of Example 3 and the diazepam group were intragastrically administered different doses of Example 3 (100 mg / kg, 50 mg / kg, 25 mg / kg) and diazepam (1 mg / kg) once a day for 7 days. In addition, the mice in the control group and the model group were intragastrically administered the same volume and equal amount of normal saline. The fixed administration time was 10:00 am every day for 3 consecutive days. 1 h after the last administration, each group of mice was intraperitoneally injected with sodium pentobarbital (50 mg / kg). The mice were placed in a multi-functional mouse voluntary activity recorder to record the activity, including the activity distance and activity time, and the results were statistically processed, and the sleep latency and sleep time were recorded.

[0084] As Figure 3 shown, compared with the blank group, the moving distance of the mice in the model group was significantly increased. The positive control group, the high and medium dose groups of Example 3 could significantly reduce the voluntary activity distance and time of the mice (P<0.001), and there was no significant difference in the low dose group of Example 3. From the overall trend, the granules prepared in Example 3 could reduce the voluntary activity of insomnia mice, indicating that it has a sedative effect and can improve the tension and anxiety behaviors of insomnia mice.

[0085] As Figure 4 shown, the sleep latency of the mice in the model group was the longest and the sleep time was the shortest, indicating that the mouse insomnia model was successfully induced. The positive control group, the low, medium and high dose groups of Example 3 could shorten the sleep latency of the mice and prolong the sleep time of the mice, and there was a significant difference (P<0.001). The effect of the granules prepared in Example 3 on the sleep latency of insomnia mice decreased with the increase of the administration dose, and the sleep time of the mice increased, indicating that Example 3 had a promoting effect on the sleep of insomnia mice caused by PCPA. The mechanism of action of PCPA is to inhibit the activity of tryptophan hydroxylase, block the synthesis of 5-hydroxytryptamine (5-HT), and cause a significant decrease in the level of 5-HT in the brain, inducing continuous insomnia in mice.

[0086] Therefore, the use of this sleep aid product can significantly prolong the sleep time, voluntary activity time and voluntary activity distance of mice, and shorten the sleep latency, suggesting that this product has the effect of helping sleep.

[0087] Safety investigation on the liver and kidney

[0088] After the insomnia model mice regained consciousness and resumed normal movement, the mice were sacrificed, the brains were removed and frozen for later use. Liver and kidney tissues were taken, immersed in formalin for fixation, dehydrated, cleared, embedded in paraffin and then sectioned. After staining the sections with eosin-hematoxylin, observation was carried out.

[0089] From Figure 5 It can be seen that under microscopic observation, there were no differences in the livers and kidneys of the high-dose granules of Example 3 after HE-stained sections compared with the normal livers and kidneys of the blank group. The boundaries of liver and kidney cells were clear, and no swelling or necrosis was observed, indicating that the granules of Example 3 had high safety.

[0090] 4 Mechanism study: WB detection of the protein expressions of MAO and TPH2

[0091] Prepare a lysis buffer by mixing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM PMSF, protease inhibitor (1:100), and phosphatase inhibitor (1:50). After cutting the brain tissues of the insomnia model mice into small pieces, mix them with the lysis buffer at a ratio of (100 mg / mL), perform ultrasonic fragmentation on ice (5 s pulse × 3 times), transfer them to a refrigerator at 4 °C for lysis for 30 min, centrifuge at 12,000 rpm for 15 minutes at 4 °C, and take the tissue supernatant. The protein concentration is detected by the BCA method.

[0092] SDS-PAGE electrophoresis:

[0093] Prepare the gel: Separating gel (10%): 3.3 mL of 30% acrylamide + 2.5 mL of separating gel buffer + 4.0 mL of H2O + 50 μL of APS + 5 μL of TEMED. Stacking gel (5%): 0.5 mL of 30% acrylamide + 1.0 mL of stacking gel buffer + 2.3 mL of H2O + 30 μL of APS + 3 μL of TEMED.

[0094] Loading and electrophoresis: Mix the protein sample with Loading Buffer (volume ratio 4:1), boil at 100 °C for 5 minutes, cool and then load the sample (20 - 40 μg of protein per well). Constant voltage 80 V (stacking gel) → 120 V (separating gel), stop when the bromophenol blue reaches the bottom of the gel.

[0095] Transfer the membrane: Use a PVDF membrane (activated with methanol) and filter paper, transfer the membrane at a constant current of 250 mA for 2 hours (MAO is about 60 kDa, TPH2 is about 55 kDa) (verify the protein transfer effect by staining with Ponceau S after membrane transfer).

[0096] Blocking and antibody incubation blocking solution: 5% non-fat milk (for non-phosphorylated proteins) or 3% BSA (for phosphorylated proteins), at room temperature for 1 hour to reduce non-specific binding.

[0097] Primary antibody: Anti-MAO / TPH2 primary antibody, diluted according to the instruction manual (commonly 1:1000 - 1:5000), incubated at room temperature for 1 hour, and incubated overnight at 4°C.

[0098] Secondary antibody: HRP / fluorescently labeled secondary antibody (1:5000 - 1:10000), incubated at room temperature for 1 hour, and the membrane was washed 3 times with PBST (10 minutes each time).

[0099] Detection by chemiluminescence method: Develop with ECL reagent, exposure time 10s - 5 minutes, and analyze the gray value of the band with Image Lab software.

[0100] From Figure 6 and Figure 7 It can be concluded that the granule of Example 3 can regulate the expression of MAO and TPH2 proteins in the mouse brain, reduce the content of MAO protein, increase the content of TPH2 protein, and play a sleep-promoting role.

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A method for screening drugs with sleep-inducing effects by affinity chromatography, characterized in that: The following steps are involved: The MAO silica gel stationary phase and the TPH2 silica gel stationary phase were respectively connected to a high performance liquid chromatography system to screen out components with MAO inhibitory activity or TPH2 activating activity from the extract of the medicinal and edible materials.

2. The affinity chromatography screening method according to claim 1, characterized in that: The preparation method of the MAO silica gel stationary phase comprises: mixing activated SiO2, PBS solution and MAO, magnetically stirring and centrifuging under ice bath conditions to obtain the MAO silica gel stationary phase MAO@SiO2.

3. The affinity chromatography screening method according to claim 1, characterized in that: The preparation method of the TPH2 silica gel stationary phase is: mixing activated SiO2, PBS solution and TPH2, magnetically stirring and centrifuging under ice bath conditions to obtain the TPH2 silica gel stationary phase TPH2@SiO2.

4. The affinity chromatography screening method according to claim 1, characterized in that: The chromatographic conditions of the affinity chromatography screening method are: column temperature 30°C, flow rate 0.2 mL / min, mobile phase 10 mM ammonium acetate (pH 7.2-7.4), injection volume 20 μL, ELSD1260 detector: evaporation temperature 60°C, drift tube temperature 60°C, high-purity nitrogen flow rate 1.6 L / min.

5. A medicine-food composition having a sleep-inducing effect, characterized in that: The invention comprises the following raw materials in parts by weight: 10-15 parts of sour jujube kernels, 10-15 parts of poria cocos, 10-15 parts of moringa seeds, 10-15 parts of ganoderma lucidum, 5-10 parts of hawthorn, 5-10 parts of mulberry, 5-10 parts of wolfberry, 5-10 parts of walnut, 2-3 parts of jasmine, 2-3 parts of gardenia, 2-3 parts of jujube, 2-3 parts of longan, 2-3 parts of hericium erinaceus, 2-3 parts of mangosteen, 2-3 parts of lotus seeds and 2-3 parts of white fungus.

6. The medicine-food composition according to claim 5, characterized in that: The invention comprises the following raw materials in parts by weight: 15 parts of sour jujube kernels, 15 parts of poria cocos, 15 parts of moringa seeds, 15 parts of ganoderma lucidum, 10 parts of hawthorn, 10 parts of mulberry, 10 parts of wolfberry, 10 parts of walnut, 3 parts of jasmine, 3 parts of gardenia, 3 parts of jujube, 3 parts of longan, 3 parts of hericium erinaceus, 3 parts of mangosteen, 3 parts of lotus seeds and 3 parts of white fungus.

7. The method for preparing the edible-medicinal composition according to claim 5 or 6, characterized in that: The following steps are involved: The raw materials are crushed into powder, mixed with water, subjected to heating extraction after ultrasonication, and the extract is collected, filtered and concentrated; Add auxiliary materials and prepare granules through wet granulation process.

8. The preparation method according to claim 7, characterized in that: The material-liquid ratio of the raw material to water is 1:(6-12) (kg / L); The frequency of the ultrasound is 100 Hz and the time is 30 min; The conditions of the heating extraction are: heating in a water bath to 80-90°C for 3 hours, and repeating 2-3 times; The concentration conditions are: concentrating to 40%-60% of the original weight; The auxiliary materials are: 10%-30% erythritol and 20%-40% maltodextrin.

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