Traditional Chinese medicine ointment for improving insomnia and preparation method thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine ointment, in particular to traditional Chinese medicine ointment for improving insomnia and a preparation method thereof. The traditional Chinese medicine ointment for improving insomnia is prepared from the following components in percentage by weight: 15-20% of agilawood extract, 20-25% of thuja sutchuenensis extract, 2-3% of radix aucklandiae, 5-10% of herba elsholtziae, 8-10% of spina date seed, 1-2% of nacre mother of pearl, 2-3% of polygala tenuifolia, 10-15% of cortex albiziae, 3-5% of caulis spatholobi, 3-5% of rhizoma corydalis, 3-5% of realgar, 1-2% of cinnabar, 3-5% of borneol and 0.5-1% of brain-derived neurotrophic factors. Compared with chemical drugs, the natural plant components adopted by the invention have small side effects, can directly act on the body surface by being smeared on the skin as an ointment, and can be continuously and slowly released in the body for a long time by absorbing the drug through the skin, so that a relatively stable nerve soothing effect can be maintained; the medicine is prevented from entering the gastrointestinal system, the gastrointestinal tract burden is reduced, meanwhile, blood circulation can be improved, internal secretion is adjusted, body tension is relieved, the sleep quality is improved, and sleep is deeper.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine plasters, and particularly relates to a traditional Chinese medicine plaster for improving insomnia and a preparation method thereof. Background Art

[0002] With the rapid development of society, the increase in work pressure and the acceleration of the pace of life, insomnia has become a common health problem. Modern people are facing various pressures, such as work pressure, family problems, irregular life, etc. These factors have made the sleep problem increasingly serious; with the popularization of Western medicine, many drug-based treatment programs have entered the field of insomnia treatment. For example, benzodiazepine drugs and non-benzodiazepine drugs have become common choices for treating insomnia. These drugs act on the neurotransmitters in the brain to help patients fall asleep quickly. However, these drugs often bring some side effects, such as dependence, drug resistance, memory loss, etc. Long-term use will affect health.

[0003] In traditional Chinese medicine, insomnia is regarded as a manifestation of restlessness of the mind or deficiency of qi and blood. The idea of traditional Chinese medicine in treating insomnia is to restore the balance of the body by regulating the yin, yang, qi and blood, and zang-fu organs of the body to improve sleep; Chinese Patent No. CN107007666A discloses a composition for treating and improving insomnia, its preparation method and uses. It uses semen ziziphi spinosae, cloves, nutmeg, black pepper and cinnamon as raw materials, and obtains a composition for improving insomnia through extraction. However, through testing, it is found that its improvement effect on insomnia is limited; Chinese Patent No. CN118787701A discloses a traditional Chinese medicine composition, a traditional Chinese medicine preparation for improving insomnia and a preparation method thereof. Its synergistic compatibility effect of monarch drug, ministerial drug, adjuvant drug and guiding drug can significantly improve insomnia symptoms, but as an oral preparation, it needs to be specially prepared and is inconvenient to use.

[0004] In view of the above problems, the present invention provides a solution. Summary of the Invention

[0005] The purpose of the present invention is to provide a traditional Chinese medicine plaster for improving insomnia and a preparation method thereof, which can significantly improve the sleep condition of insomnia patients.

[0006] To achieve the above purpose, the present invention adopts the following technical scheme: A traditional Chinese medicine plaster for improving insomnia is composed of the following percentage components: 15-20% of agarwood extract, 20-25% of thuja occidentalis extract, 2-3% of costus root, 5-10% of elsholtzia ciliata, 8-10% of semen ziziphi spinosae, 1-2% of nacre, 2-3% of polygala tenuifolia, 10-15% of cortex albiziae, 3-5% of polygala tenuifolia willd, 3-5% of rhizoma corydalis, 3-5% of realgar, 1-2% of cinnabar, 3-5% of borneol and 0.5-1% of brain-derived neurotrophic factor;

[0007] The preparation method of brain-derived neurotrophic factor includes the following steps:

[0008] A1: Disinfect the operating table with ultraviolet light for 30 minutes. Then, prepare the culture medium: 44.5 ml of DMEM + 5 ml of FBS + 0.5 ml of double antibody. Quickly take out the CHO cells from liquid nitrogen with forceps and immediately place them in a water bath to thaw and melt.

[0009] A2: Aspirate and transfer the CHO cell suspension into a 15-ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, evenly spread the resuspended cells in a 60-mm culture dish, shake the dish up, down, left, and right evenly, and then place the culture dish in a 5% CO 2 cell incubator for culture;

[0010] A3: After the CHO cells are cultured to a density of 85%, rinse them with PBS, digest with trypsin for 2 minutes, and stop the trypsin digestion with the culture medium containing FBS. Aspirate and transfer the CHO cell suspension into a 15-ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, take 100 μl of the cell suspension, add 900 μl of PBS to resuspend the cells, then take 10 μl of the cell suspension for cell counting, take 2000 cells, add them to a 6-well plate containing 2 ml of culture medium, and place it in a 5% CO 2 cell incubator for 5 days;

[0011] A4: Construct the recombinant plasmid pcDNA3.1-TOPO-BDNF of human brain-derived neurotrophic factor using an empty vector. Subsequently, use the three-plasmid system of pcDNA3.1-TOPO-BDNF, pVSV-G, and pCMVΔR8.91 for lentivirus packaging. The tool cell is CHO cell. Infect the CHO cells with the virus solution and screen out the CHO cells stably expressing brain-derived neurotrophic factor. After the CHO cells stably expressing brain-derived neurotrophic factor are cultured to a density of 85%, rinse them with PBS, digest with trypsin for 2 minutes, and stop the trypsin digestion with a medium containing FBS. Subsequently, aspirate and transfer the suspension of CHO cells stably expressing brain-derived neurotrophic factor into a 15 ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for a total of 3 minutes;

[0012] A5: After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish up, down, left, and right evenly, and then place the culture dish in a cell culture incubator with 5% CO 2 culture. After the CHO cells stably expressing brain-derived neurotrophic factor are cultured to a density of 85%, rinse them with PBS, and then add serum-free medium and culture for 5 days. After the culture is completed, collect the serum-free medium;

[0013] A6: Add 5% mannitol to the collected serum-free medium, then dispense it into a 15 cm culture dish, discard the top cover of the culture dish, cover the culture dish with tin foil, punch 15 small holes in the tin foil with a 20 μl pipette tip, and place the culture dish in a freeze dryer and freeze-dry for 8 h to obtain the freeze-dried powder of brain-derived neurotrophic factor.

[0014] Furthermore, the mass ratio of the serum-free medium to 5% mannitol in step A6 is 20:1;

[0015] Furthermore, the realgar is a yellowish-brown powder of diarsenic disulfide with a specification of 500 mesh; the agarwood extract is a brown agarwood powder with a specification of 500 mesh; the thuja extract is a fat-soluble reddish-brown thuja essential oil distilled from thuja wood chips.

[0016] Furthermore, a preparation method of a traditional Chinese medicine ointment for improving insomnia comprises the following steps:

[0017] B1: Add costus root, elsholtzia, wild jujube seed, mother-of-pearl, polygala root, cortex albiziae, caulis polygoni multiflori, rhizoma corydalis, realgar, cinnabar, and borneol into a pulverizer, mix and pulverize. Set the particle size of the pulverizer to 500 mesh, pulverize for 30 min, then pass through a 500-mesh sieve. The material that fails to pass through the sieve is re-added to the pulverizer, pulverized for 15 min again, and passed through the 500-mesh sieve repeatedly to collect the sieved product. Then add agarwood extract and thuja occidentalis extract to obtain the raw medicine;

[0018] B2: Add ethanol to the raw medicine and mix well with a magnetic stirrer. Set the rotation speed of the stirrer to 600 - 800 rpm, mix and stir for 30 min. After stirring, add propylene glycol, heat up to 45 - 50 °C, set the rotation speed of the magnetic stirrer to 400 - 600 rpm, stir and extract for 30 min. After extraction, add deionized water and boil for 5 - 6 h to obtain the concentrated solution of the raw medicine;

[0019] B3: Add the concentrated solution of the raw medicine to the ointment base, heat up to 50 - 55 °C, set the rotation speed of the stirrer to 1000 - 1200 rpm, stir for 10 - 15 min, and transfer to a homogenizer after stirring for 30 min for homogenization;

[0020] B4: After homogenization, put the ointment into an autoclave and sterilize at 121 °C for 20 min. After sterilization, add freeze-dried brain-derived neurotrophic factor to obtain a traditional Chinese medicine ointment for improving insomnia.

[0021] Further, the ointment base is a mixed base of silicone oil base, vegetable oil base, and cream base, and the mass ratio of the silicone oil base, vegetable oil base, and cream base is 4:2:4; in step B2, the mass ratio of the raw medicine, ethanol, propylene glycol, and deionized water is 1:2:2:5; in step B3, the mass ratio of the concentrated solution of the raw medicine and the ointment base is 1:3.

[0022] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: the present invention uses natural plant components, which can significantly improve the sleep condition of insomnia patients, has less side effects compared with chemical drugs, and as an ointment, it can directly act on the body surface by being applied on the skin. The drug can be continuously and slowly released through skin absorption to maintain a certain concentration in the body for a long time, which helps to maintain a relatively stable calming effect, avoids the drug entering the gastrointestinal system and reduces the gastrointestinal burden. At the same time, it can improve blood circulation, regulate endocrine, relieve physical tension, thereby improving sleep quality and making sleep deeper. Specific Embodiments

[0023] In order to make the technical means, creative features, achieved purposes, and functions of the present invention easy to understand, the present invention will be further described below with reference to specific embodiments.

[0024] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0025] Example 1

[0026] 1: Ultraviolet disinfect the operating table for 30 minutes. Subsequently, prepare the culture medium: 44.5 ml of DMEM + 5 ml of FBS + 0.5 ml of double antibiotics. Quickly take out CHO cells from liquid nitrogen with forceps and immediately place them in a water bath to thaw and melt.

[0027] 2: Pipette the CHO cell suspension into a 15 ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish evenly up, down, left and right, and then place the culture dish in a cell culture incubator with 5% CO 2 for culture.

[0028] 3: When the density of CHO cells reaches 85%, rinse with PBS, digest with trypsin for 2 minutes, and stop the trypsin digestion with the culture medium containing FBS. Pipette the CHO cell suspension into a 15 ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, take 100 ul of the cell suspension, add 900 ul of PBS to resuspend the cells, then take 10 ul of the cell suspension for cell counting, take 2000 cells, add them to a 6-well plate containing 2 ml of culture medium and place it in a cell culture incubator with 5% CO 2 for 5 days of culture.

[0029] 4: Construct a recombinant plasmid of human brain-derived neurotrophic factor pcDNA3.1-TOPO-BDNF using an empty vector. Subsequently, use the three-plasmid system of pcDNA3.1-TOPO-BDNF, pVSV-G and pCMVΔR8.91 for lentivirus packaging. The tool cells are CHO cells. Infect CHO cells with the virus solution and screen out CHO cells stably expressing brain-derived neurotrophic factor. When the density of CHO cells stably expressing brain-derived neurotrophic factor reaches 85%, rinse with PBS, digest with trypsin for 2 minutes, and stop the trypsin digestion with the culture medium containing FBS. Then pipette the cell suspension of CHO cells stably expressing brain-derived neurotrophic factor into a 15 ml centrifuge tube, add 6 ml of DMEM(+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes;

[0030] 5: After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, evenly spread the resuspended cells in a 60-mm culture dish, shake the dish evenly up, down, left, and right, and then place the culture dish in a cell culture incubator with 5% CO 2 cultivate until the density of the stably expressing brain-derived neurotrophic factor CHO cells reaches 85%, then rinse with PBS, then add serum-free medium, cultivate for 5 days, and collect the serum-free medium after cultivation is completed;

[0031] 6: Add 5% mannitol to the collected serum-free medium, then dispense it into a 15-cm culture dish, discard the top cover of the culture dish, cover the culture dish with tin foil, make 15 small holes in the tin foil with a 20-μl pipette tip, and place the culture dish in a freeze dryer and freeze-dry for 8 h to obtain the freeze-dried powder of brain-derived neurotrophic factor prepared in Example 1.

[0032] Table 1, Reagent Parameter Table for Example 1

[0033] Drug Name Source Model Parameter DMEM ThermoFisher 11320033 500ml FBS BBI E510002 500ml Double Antibody BBI A430977 500mg CHO Cells Jinshaoyuan Biology JSY-CC4287 <![CDATA[1×10 6 cell]]> Serum-Free Medium Cytiva SH30941.02 1L Mannitol BBI A430632 100mg Trypsin Diamond A100458 50g

[0034] Example 2

[0035] 1: Disinfect the operating table with ultraviolet light for 30 minutes, then prepare 44.5 ml of DMEM + 5 ml of FBS + 0.5 ml of double antibody, quickly take out the CHO cells from liquid nitrogen with forceps and immediately place them in a water bath to thaw and melt;

[0036] 2: Pipette the CHO cell suspension into a 15-ml centrifuge tube, add 6 ml of DMEM (+ / +), balance and centrifuge at 1000 rpm / min for a total of 3 minutes. After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, evenly spread the resuspended cells in a 60-mm culture dish, shake the dish evenly up, down, left, and right, and then place the culture dish in a cell culture incubator with 5% CO 2 cultivate;

[0037] 3: When the density of the CHO cells reaches 85%, rinse with PBS, digest with trypsin for 2 minutes, stop the trypsin digestion with medium containing FBS, pipette the CHO cell suspension into a 15-ml centrifuge tube, add 6 ml of DMEM (+ / +), balance and centrifuge at 1000 rpm / min for a total of 3 minutes. After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, take 100 μl of the cell suspension, add 900 μl of PBS to resuspend the cells, then take 10 μl of the cell suspension for cell counting, take 2000 cells, add them to a 6-well plate containing 2 ml of medium, and place it in a cell culture incubator with 5% CO 2 cultivate for 5 days;

[0038] 4: Construct the recombinant plasmid pcDNA3.1-TOPO-BDNF of human brain-derived neurotrophic factor using an empty vector. Subsequently, use the three-plasmid system of pcDNA3.1-TOPO-BDNF, pVSV-G, and pCMVΔR8.91 for lentivirus packaging. The tool cell is CHO cell. Infect CHO cells with the virus solution and screen out the CHO cells stably expressing brain-derived neurotrophic factor. After the CHO cells stably expressing brain-derived neurotrophic factor are cultured to a density of 85%, rinse them with PBS, digest with trypsin for 2 minutes, and stop trypsin digestion with a medium containing FBS. Subsequently, aspirate and transfer the suspension of CHO cells stably expressing brain-derived neurotrophic factor into a 15 ml centrifuge tube, add 6 ml DMEM(+ / +), balance and centrifuge at 1000 rpm / min for a total of 3 minutes;

[0039] 5: After centrifugation, discard the supernatant, add 1 ml of medium to resuspend the cells, evenly spread the resuspended cells in a 60 mm culture dish, shake the dish up, down, left, and right evenly, and then place the culture dish in a cell culture incubator with 5% CO 2 cultivate. After the CHO cells stably expressing brain-derived neurotrophic factor are cultured to a density of 85%, rinse them with PBS, then add serum-free medium, and culture for 5 days. After the culture is completed, collect the serum-free medium;

[0040] 6: Add 5% mannitol to the collected serum-free medium, then dispense it into a 15 cm culture dish, discard the top cover of the culture dish, cover the culture dish with tin foil, pierce 15 small holes in the tin foil with a 20 ul pipette tip, and place the culture dish in a freeze dryer and freeze-dry for 8 h to obtain the freeze-dried powder of brain-derived neurotrophic factor prepared in Example 2.

[0041] Table 2, Reagent Parameter Table for Example 2

[0042] Drug Name Source Model Parameter DMEM ThermoFisher 11320033 500ml FBS BBI E510002 500ml Double Antibody BBI A430977 500mg CHO Cells Jinshaoyuan Biology JSY-CC4287 <![CDATA[1×10 6 cell]]> Serum-Free Medium Cytiva SH30941.02 1L Mannitol BBI A430632 100mg Trypsin Diamond A100458 50g

[0043] Example 3

[0044] 1: Add 30 g of Aucklandia lappa, 100 g of Elsholtzia ciliata, 100 g of Ziziphus jujuba var. spinosa, 20 g of Concha Margaritifera Usta, 30 g of Polygala tenuifolia, 100 g of Cortex Albiziae, 50 g of Caulis Sinomenii, 50 g of Corydalis yanhusuo, 50 g of Realgar, 20 g of Cinnabaris, and 50 g of Borneol into a pulverizer, mix and pulverize. Set the particle size of the pulverizer to 500 mesh, pulverize for 30 min, then pass through a 500-mesh sieve. The material that does not pass through the sieve is re-added to the pulverizer, repeat pulverizing for 15 min, repeat passing through the 500-mesh sieve, collect the sieved product, and then add 200 g of Aquilaria sinensis extract and 250 g of Thuja sutchuenensis extract to obtain 1000 g of mixed medicinal powder;

[0045] 2: Add 2000 g of ethanol to the mixed medicinal powder and stir evenly using a magnetic stirrer. Set the stirrer speed to 800 rpm and mix and stir for 30 min. After stirring is completed, add 2000 g of propylene glycol, heat up to 50 °C, set the magnetic stirrer speed to 600 rpm, and stir and extract for 30 min. After extraction is completed, add 5000 g of deionized water and boil for 6 h to obtain 900 g of concentrated crude drug solution;

[0046] 3: Add the concentrated crude drug solution to the ointment matrix obtained by mixing 1080 g of silicone oil matrix, 540 g of vegetable oil matrix and 1080 g of cream matrix, heat up to 55 °C, set the stirrer speed to 1200 rpm, stir for 15 min, and transfer to a homogenizer after stirring is completed and homogenize for 30 min;

[0047] 4: After homogenization is completed, put the ointment into an autoclave and sterilize at 121 °C for 20 min. After sterilization is completed, add 10 g of the freeze-dried brain-derived neurotrophic factor prepared in Example 1 to obtain a traditional Chinese medicine ointment for improving insomnia prepared in Example 3.

[0048] Table 3, Reagent parameter table for Example 3

[0049] Drug Name Source Model Parameter Costus Root Bozhou Henggu Pharmaceutical Co., Ltd. 002 500g Mosla Chinensis Juancheng Lujian Medicinal Herbs Warehouse 24 1kg Semen Ziziphi Spinosae Bozhou Heentang Pharmaceutical Co., Ltd. None 500g Concha Margaritifera Usta Bozhou Yuanshengtang Pharmaceutical Co., Ltd. ZZM-01 500g Radix Polygalae Anhui Kangweifu Pharmaceutical Co., Ltd. YZ-500 500g Cortex Albiziae Bozhou Wufangjiuru Pharmaceutical Co., Ltd. WFJRYY 500g Caulis Polygoni Multiflori Bozhou Quanle Pharmaceutical Co., Ltd. YJT-02 500g Rhizoma Corydalis Bozhou Baichuan Pharmaceutical Co., Ltd. BZBC 500g Realgar Bozhou Yurunfu Agricultural Products Business Department YRF 500g Cinnabar Herbal Shop ECN000219 500g Borneol Herbal Shop ECN000215 500g Agarwood Extract Guangzhou Artemisia argyi Biotechnology Co., Ltd. AHC-02 500g Thuja sutchuenensis Franch. Extract Shenzhen Guoxin Biotechnology Co., Ltd. GX 500g

[0050] Example 4

[0051] 1: Add 20 g of Aucklandia lappa, 50 g of Elsholtzia ciliata, 80 g of Ziziphus jujuba var. spinosa, 10 g of Margaritifera, 20 g of Polygala tenuifolia, 100 g of Albizia julibrissin, 20 g of Sinomenium acutum, 30 g of Corydalis yanhusuo, 30 g of Realgar, 10 g of Cinnabaris and 30 g of Borneol into a pulverizer, mix and pulverize. Set the particle size of the pulverizer to 500 mesh and pulverize for 30 min. Then pass through a 500-mesh sieve. The material that does not pass through the sieve is added back to the pulverizer and pulverized again for 15 min. Repeat passing through the 500-mesh sieve, collect the sieved product, and then add 150 g of Aquilaria sinensis extract and 200 g of Thuja sutchuenensis extract to obtain 750 g of mixed medicinal powder;

[0052] 2: Add 1500 g of ethanol to the mixed medicinal powder and stir evenly using a magnetic stirrer. Set the stirrer speed to 600 rpm and mix and stir for 30 min. After stirring is completed, add 1500 g of propylene glycol, heat up to 45 - 50 °C, set the magnetic stirrer speed to 600 rpm, and stir and extract for 30 min. After extraction is completed, add 3750 g of deionized water and boil for 6 h to obtain 720 g of concentrated crude drug solution;

[0053] 3: Add the concentrated solution to the ointment matrix obtained by mixing 864 g of silicone oil matrix, 432 g of vegetable oil matrix and 864 g of cream matrix, heat up to 50 °C, set the stirrer speed to 1200 rpm, stir for 15 min, and transfer to a homogenizer after stirring is completed and homogenize for 30 min;

[0054] 4. After homogenization, put the paste into an autoclave and sterilize it at 121 °C for 20 min. After sterilization, add 5 g of the freeze-dried brain-derived neurotrophic factor prepared in Example 2 to obtain a traditional Chinese medicine paste for improving insomnia prepared in Example 4.

[0055] Table 4, Reagent Parameter Table for Example 4

[0056]

[0057]

[0058] Comparative Example 1

[0059] Select Example 1 of the Chinese patent with the publication number CN107007666A as Comparative Example 1.

[0060] Comparative Example 2

[0061] Select Example 1 of the Chinese patent with the publication number CN118787701A as Comparative Example 2.

[0062] Cytotoxicity Test

[0063] Take one pair of the insomnia-improving medicaments prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2 respectively, prepare them into a 100 mg / L solution with sterile water, and then dilute them to 10% and 1% with sterile water respectively to make concentration gradients for cytotoxicity detection;

[0064] Use 293T cells and the MTT kit with the product number M1020 produced by Solarbio for the cytotoxicity experiment; Resuscitate and culture 293T cells, collect logarithmic-phase 293T cells after 1 day, and distribute them in 96-well plates, 180 μl per well, 3000 - 10000 cells / well; Place the 96-well plates in a 37 °C, 5% CO 2 temperature constant incubator for 6 hours; Add 10 μl of the test sample to each well, set 3 groups for each gradient, aspirate the supernatant and then add 90 μl of fresh medium, add 10 μl of MTT solution, continue to culture for 4 h, then aspirate the supernatant and add 110 μl of Formazan solution to each well, place it on a shaker and shake at a low speed for 10 min to fully dissolve the crystals. After shaking, transfer the 96-well plates to a microplate reader to measure the absorbance value of each well at 490 nm, and set blanks (culture medium, MTT solution, Formazan solution), control wells (293T cells, sterile water, culture medium, MTT solution, Formazan solution), and repeat 3 for each well;

[0065] Table 5, Cytotoxicity Test Results

[0066] Grouping Cytotoxicity Cell Activity 1% Example 3 Non-Toxic / 1% Example 4 Non-Toxic / 1% Comparative Example 1 Non-Toxic / 1% Comparative Example 2 Non-Toxic / 10% Example 3 1.47％ 98.53％ 10% Example 4 1.45％ 98.55％ 10% Comparative Example 1 2.77％ 97.23％ 10% Comparative Example 2 2.38％ 96.62％ 100% Example 3 5.33％ 94.67％ 100% Example 4 5.29％ 94.71％ 100% Comparative Example 1 7.45％ 92.55％ 100% Comparative Example 2 6.14％ 93.86％

[0067] It can be seen from the analysis of Table 5 that the medicaments prepared in Example 3 and Example 4 are non-toxic to cells at a concentration of 1%, slightly toxic to cells at 5%, and moderately toxic to cells at 100%. The reason is that the cream contained in the ointment matrix will slightly damage the phospholipid bilayer on the cell membrane surface when the concentration is too high. However, the medicaments prepared in Example 3 and Example 4 are ointments, and when applied to the skin surface, they will be blocked by the skin barrier. Compared with Comparative Examples 1 and 2 taken orally, their cytotoxicity can be ignored.

[0068] Rat insomnia improvement experiment

[0069] Dissolve p-chlorophenylalanine in physiological saline to prepare a suspension, and intraperitoneally inject rats at a dose of 250 mg / (kg / rat) once a day for 3 consecutive days. It is observed that the circadian rhythm of the rats disappears, they are active day and night without sleep until it disappears, their food and water intake increases, their body hair is messy and dull, and they are listless. When stimulated again, their resistance is significantly weakened. Overall observation shows obvious differences from normal animals, and an insomnia rat model is obtained.

[0070] Select 20 insomnia rats, use a 6% sodium sulfide solution to remove the hair on one side of the insomnia rats as the drug application site. Divide the hairless insomnia rats into 2 groups, with 10 rats in each group. Apply the traditional Chinese medicine ointments prepared in Example 3 and Example 4 respectively, 4 g each time, once a day, and observe the improvement of the insomnia situation of the rats for 7 days. The results are averaged.

[0071] Use physiological saline to prepare the medicaments obtained in Comparative Examples 1 and 2 into medicament samples with a concentration of 100 mg / kg. Select 30 insomnia rats, divide them into 3 groups, with 10 rats in each group. Intraperitoneally inject the prepared medicament samples, with a dosage of 4 ml each time, once a day. The third group is the control group, which is intraperitoneally injected with physiological saline, 4 ml each time, once a day. Observe the improvement of the insomnia situation of the rats for 7 days. The results are averaged.

[0072] Table 6, Results of rat insomnia improvement experiment

[0073] Grouping Number of Fallen Asleep Falling Asleep Rate Sleep Latency Sleep Duration Example 3 9 90％ 4.92min 118.27min Example 4 10 100％ 4.77min 117.65min Comparative Example 1 8 80％ 6.28min 98.43min Comparative Example 2 10 100％ 4.74min 120.63min Control Group 0 0％ 18.26min 52.44min

[0074] It can be seen from the analysis of Table 6 that the traditional Chinese medicine ointments prepared in Example 3 and Example 4 can significantly improve the insomnia situation of rats, and at the same time, the sleep quality is good and the sleep onset rate is high.

[0075] Insomnia improvement evaluation

[0076] Forty insomnia patients were recruited, including 8 patients with difficulty in falling asleep, 12 patients with difficulty in maintaining sleep, 4 patients with early awakening insomnia, 8 patients with chronic insomnia, and 8 patients with physiological insomnia; they were evenly divided into 4 groups and were respectively administered the medicaments prepared in Example 3, Example 4, Comparative Example 1, and Comparative Example 2. Among them, the patients using the ointments prepared in Example 3 and Example 4 applied 5 g at the temple position 30 minutes before going to bed each time, once a day, for 14 days; the patients using the medicaments prepared in Comparative Example 1 and Comparative Example 2 took one dose of the medicament orally each time before going to bed, for 14 days. The improvement of insomnia in the volunteers was recorded, and the results were averaged;

[0077] Table 7, Improvement of Insomnia in Volunteers

[0078] Grouping Number of Fallen Asleep Falling Asleep Rate Sleep Latency Sleep Duration Example 3 8 80％ 12.42min 375.85min Example 4 9 90％ 14.83min 391.79min Comparative Example 1 5 50％ 18.65min 268.16min Comparative Example 2 9 90％ 14.25min 422.45min

[0079] During the 14-day experience, the improvement of the patients with difficulty in maintaining sleep was the best. All the patients with difficulty in maintaining sleep who used the ointment prepared in Example 4 had more than 5 hours of sleep, and the insomnia conditions of other insomnia types of patients were also significantly improved; at the same time, it can be seen from the analysis of Table 7 that the ointments prepared in Example 3 and Example 4 can significantly reduce the sleep latency of insomnia patients and increase the sleep duration of insomnia patients.

[0080] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A Chinese medicine ointment for improving insomnia, characterized in that: The invention is composed of the following percentage ingredients: 15-20% agarwood extract, 20-25% thuja extract, 2-3% costusroot, 5-10% elsholtzia, 8-10% jujube seed, 1-2% mother of pearl, 2-3% polygala, 10-15% julibrissin bark, 3-5% strychnos nux vomica, 3-5% yanhusuo, 3-5% realgar, 1-2% cinnabar, 3-5% borneol and 0.5-1% brain-derived neurotrophic factor; The preparation method of brain-derived neurotrophic factor comprises the following steps: A1: Disinfect the operating table with ultraviolet light for 30 minutes, then prepare the culture medium 44.5ml DMEM+5ml FBS+0.5ml double antibody, quickly take out the CHO cells from the liquid nitrogen with tweezers and immediately place them in a water bath to thaw; A2: Pipette the CHO cell suspension into a 15 ml centrifuge tube, add 6 ml DMEM (+ / +), balance and centrifuge at 1000 rpm / min for 3 minutes. After centrifugation, discard the supernatant, add 1 ml culture medium to resuspend the cells, and evenly spread the resuspended cells in a 60 mm culture dish. Shake the dish evenly up and down and left and right, and then place the culture dish in a cell culture incubator with 5% CO2 for culture; A3: After the CHO cells are cultured to a density of 85%, they are rinsed with PBS and trypsinized for 2 minutes. The trypsinization is terminated with a medium containing FBS, and the CHO cell suspension is pipetted into a 15 ml centrifuge tube. 6 ml of DMEM (+ / +) is added, and the tube is balanced and centrifuged at 1000 rpm / min for 3 minutes. After the centrifugation is completed, the supernatant is discarded, 1 ml of culture medium is added to resuspend the cells, 100 ul of the cell suspension is taken, 900 ul of PBS is added to resuspend the cells, and then 10 ul of the cell suspension is taken for cell counting. 2000 cells are taken, added to a 6-well plate containing 2 ml of culture medium, and placed in a cell culture incubator with 5% CO2 for 5 days; A4: Use an empty vector to construct the human brain-derived neurotrophic factor recombinant plasmid pcDNA3.1-TOPO-BDNF, then use the three-plasmid system of pcDNA3.1-TOPO-BDNF, pVSV-G and pCMVΔR8.91 for lentiviral packaging, select CHO cells as tool cells, use the virus solution to infect CHO cells and screen out CHO cells that stably express brain-derived neurotrophic factor, and after the stably expressed brain-derived neurotrophic factor CHO cells are cultured to a density of 85%, they are rinsed with PBS and trypsinized for 2 minutes. The trypsinization is terminated with a medium containing FBS, and then the stably expressed brain-derived neurotrophic factor CHO cell suspension is pipetted into a 15 ml centrifuge tube, 6 ml of DMEM (+ / +) is added, balanced and centrifuged at 1000 rpm / min for a total of 3 minutes; A5: After centrifugation, discard the supernatant, add 1 ml of culture medium to resuspend the cells, and evenly spread the resuspended cells in a 60 mm culture dish. Shake the dish evenly up and down and left and right. Then place the culture dish in a cell culture incubator with 5% CO2 for culture. After the stably expressed brain-derived neurotrophic factor CHO cells are cultured to a density of 85%, rinse with PBS, then add serum-free culture medium, and culture for 5 days. After the culture is completed, collect the serum-free culture medium; A6: Add 5% mannitol to the collected serum-free culture medium, then divide it into 15 cm culture dishes, discard the top cover of the culture dishes, cover the culture dishes with tin foil, use a 20ul pipette tip to poke 15 small holes in the tin foil, put the culture dishes into a freeze dryer, and freeze-dry for 8 hours to obtain brain-derived neurotrophic factor freeze-dried powder.

2. A Chinese medicinal ointment for improving insomnia according to claim 1, characterized in that: The mass ratio of serum-free medium and 5% mannitol in step A6 is 20:

1.

3. A Chinese medicinal ointment for improving insomnia according to claim 1, characterized in that: The realgar is yellow-brown arsenic disulfide powder with a specification of 500 meshes.

4. A Chinese medicinal ointment for improving insomnia according to claim 1, characterized in that: The agarwood extract is brown agarwood powder with a specification of 500 meshes.

5. A Chinese medicinal ointment for improving insomnia according to claim 1, characterized in that: The thuja extract is fat-soluble brown-red thuja essential oil extracted by distilling thuja sawdust.

6. The method for preparing a Chinese medicinal ointment for improving insomnia according to claim 1, characterized in that: The following steps are involved: B1: Add costus root, elsholtzia ciliata, spiny jujube seed, mother-of-pearl, polygala root, julibrissin bark, ramie vine, corydalis yanhusuo, realgar, cinnabar and borneol into a grinder, mix and grind, set the grinder particle size to 500 mesh, grind for 30 minutes, then filter through a 500-mesh filter screen, add the product that does not pass through the filter screen back into the grinder, repeat the grinding for 15 minutes, repeatedly filter through a 500-mesh filter screen to collect the sieved product, then add agarwood extract and thuja extract to obtain the raw material drug; B2: Add ethanol to the raw material drug and stir and mix with a magnetic stirrer, set the stirrer speed to 600-800 rpm, mix and stir for 30 minutes, add propylene glycol after stirring, heat to 45-50°C, set the magnetic stirrer speed to 400-600 rpm, stir and extract for 30 minutes, add deionized water after extraction, and boil for 5-6 hours to obtain the raw material drug concentrate; B3: Add the API concentrate to the ointment matrix, raise the temperature to 50-55°C, set the agitator speed to 1000-1200 rpm, stir for 10-15 minutes, and transfer to a homogenizer for 30 minutes after stirring. B4: After homogenization, the ointment is placed in a high pressure sterilizer and sterilized at 121° C. for 20 min. After sterilization, freeze-dried brain-derived neurotrophic factor powder is added to obtain a traditional Chinese medicine ointment for improving insomnia.

7. The method for preparing a Chinese medicinal ointment for improving insomnia according to claim 6, characterized in that: The ointment matrix is ​​a mixed matrix of a silicone oil matrix, a vegetable oil matrix and a cream matrix, wherein the mass ratio of the silicone oil matrix, the vegetable oil matrix and the cream matrix is ​​4:2:

4.

8. The method for preparing a Chinese medicinal ointment for improving insomnia according to claim 6, characterized in that: The mass ratio of the raw material drug, ethanol, propylene glycol and deionized water in step B2 is 1:2:2:

5.

9. The method for preparing a Chinese medicinal ointment for improving insomnia according to claim 6, characterized in that: The mass ratio of the raw material drug concentrate to the ointment base in step B3 is 1:3.

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