Composite sleep-aiding composition based on agilawood and preparation method of composite sleep-aiding composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a composite sleep-aiding composition based on agilawood and a preparation method thereof. The preparation method comprises the following steps: carrying out ultrasonic-assisted enzymolysis on agilawood by adopting amylase and cellulase, carrying out mixed fermentation on the agilawood subjected to enzymolysis by inoculating lactobacillus plantarum, lactobacillus johnsonii and saccharomyces cerevisiae, and carrying out sterilization, filtration, concentration and drying treatment on the fermented agilawood to obtain the agilawood fermentation product. According to the method, the release rate of active substances in the agilawood is increased, the composite sleep-aiding composition containing the agilawood fermentation product has a remarkable sleep-aiding effect, the preparation method is simple, the problem that an existing agilawood product is poor in sleep treatment effect is solved, and the agilawood fermentation product is safe, free of irritation and high in application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a compound sleep-aid composition based on agarwood and a preparation method thereof. Background Art

[0002] The occurrence of sleep is an active process mainly involving the central nervous system, related special neurotransmitters, cytokines and other substances, and its physiological process is a complex network of interleaving. Sleep can restore human functions, consolidate memory and maintain the normal operation of brain functions, and is an important physiological function of the human body. With the acceleration of the modern life rhythm and the increase of work pressure, more and more people have sleep disorders, and the main symptoms are: difficulty in falling asleep, low sleep quality, and short sleep time. Long-term insomnia can cause symptoms such as physiological function disorders, fatigue, memory loss, etc. In severe cases, it may also cause other adverse symptoms and induce related diseases such as anxiety disorder, depression, and hypertension.

[0003] Currently, the recommended drugs for treating insomnia mainly include: melatonin receptor agonists; non-benzodiazepine receptor agonists; antidepressant drugs with sedative effects. Although drug treatment has a good effect on improving sleep, long-term use of chemical drugs by insomnia patients will cause damage to the liver and kidney functions, respiratory system, etc. of the human body. Traditional Chinese medicine has accumulated rich theoretical bases and treatment experiences in treating insomnia and has its inherent advantages. Moreover, compared with Western medicine, traditional Chinese medicine has the advantages of environmental protection, few adverse reactions, no drug dependence, and no addiction.

[0004] Agarwood is a medicinal spice formed by plants of the genus Aquilaria in the family Thymelaeaceae. It has a unique aromatic smell and is a precious material that can be used both as medicine and fragrance. It can act on the central nervous system, digestive system, respiratory system, circulatory system, etc. Agarwood has the functions of promoting qi and relieving pain, warming the middle-jiao and stopping vomiting, and receiving qi and relieving asthma, and also has the effect of helping sleep. Appropriate use of agarwood can help improve sleep problems and relieve insomnia symptoms. However, the health products containing agarwood on the market are not ideal for improving sleep. Therefore, providing an agarwood compound sleep-aid product with good curative effect and safety and no irritation is an urgent problem to be solved at present. Summary of the Invention

[0005] In view of the above defects or improvement requirements of the prior art, the present invention provides a compound sleep-aid composition based on agarwood and a preparation method thereof, so as to solve the problem of poor curative effect of existing agarwood products in treating insomnia.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a compound sleep aid composition based on agarwood. The compound sleep aid composition comprises the following raw materials in parts by weight: 3-5 parts of agarwood fermentation product, 10-15 parts of wild jujube seeds, 6-10 parts of semen platycladi, 6-9 parts of rose flowers, 3-6 parts of polygala tenuifolia, 10-15 parts of poria cocos, and 3-6 parts of roasted licorice root; The preparation method of the agarwood fermentation product comprises the following steps:

[0008] S1. Weigh agarwood powder, add sterile deionized water, mix evenly, then add a composite enzyme for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment, and cool to room temperature to obtain a mixture;

[0009] S2. Inoculate a composite bacterium into the mixture to obtain a mixed material, perform mixed bacterium fermentation on the mixed material, and after the fermentation is completed, sterilize and filter to obtain a filtrate;

[0010] S3. After performing vacuum concentration and freeze-drying on the filtrate, obtain the agarwood fermentation product, and store it sealed under low-temperature and light-proof conditions;

[0011] Preferably, the composite enzyme is composed of amylase and cellulase;

[0012] Preferably, the composite bacterium is composed of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae with a viable bacteria number ratio of 1-2:0.5-1:1. The preservation number of Lactobacillus plantarum is CGMCC NO.1.1856, the preservation number of Lactobacillus johnsonii is CGMCC NO.1.3221, and the preservation number of Saccharomyces cerevisiae is CGMCC NO.2.3854.

[0013] More preferably, the viable bacteria number ratio of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae is 1.5:0.7:1.

[0014] Preferably, the total viable bacteria number of the composite bacterium in the mixed material is 7×10 7 -4×10 8 CFU / mL.

[0015] Preferably, the compound sleep aid composition comprises the following raw materials in parts by weight: 4 parts of agarwood fermentation product, 12 parts of wild jujube seeds, 8 parts of semen platycladi, 8 parts of rose flowers, 4 parts of polygala tenuifolia, 12 parts of poria cocos, and 5 parts of roasted licorice root.

[0016] Preferably, in step S1, the dosage ratio of agarwood powder, sterile deionized water, amylase, and cellulase is 100 g:1-1.5 L:1-2 g:0.5-1.5 g. The enzyme activity of the cellulase is 40-60 U / mg, and the enzyme activity of the amylase is 30-40 U / mg.

[0017] Preferably, the ultrasonic frequency in step S1 is 20 - 40 kHz, the ultrasonic power is 100 - 120 W, the enzymatic hydrolysis temperature is 40 - 50 °C, the enzymatic hydrolysis pH is 5 - 6, and the enzymatic hydrolysis time is 60 - 90 min.

[0018] Preferably, the enzyme inactivation temperature in step S1 is 80 - 90 °C, and the enzyme inactivation time is 15 - 25 min.

[0019] Preferably, the fermentation temperature in step S2 is 30 - 34 °C, and the fermentation time is 48 - 72 h.

[0020] Preferably, the sterilization temperature in step S2 is 121 °C, and the sterilization time is 30 min.

[0021] Preferably, high-speed centrifugal filtration is used for filtration in step S2, the centrifugal rotation speed is 5000 r / min, and the centrifugal time is 10 min.

[0022] Preferably, vacuum concentration in step S3 is carried out under a pressure of 0.06 MPa until the volume is reduced to 30% of the original volume.

[0023] Preferably, spray freeze-drying equipment is used for freeze-drying in step S3, the cold trap temperature is -65 °C, the spray freezing temperature is -20 °C, the spray pressure is 0.3 MPa, and the spray freeze-drying is carried out until the water content in the agarwood fermentation product is 5 - 8 wt%.

[0024] In a second aspect, the present invention provides a method for preparing a compound sleep aid composition, comprising the following steps: crushing each raw material and passing through a 50-mesh sieve, weighing each raw material according to the formula amount and mixing them to obtain the compound sleep aid composition.

[0025] Advantages of the present invention:

[0026] In the present invention, amylase and cellulase are first used for ultrasonic-assisted enzymatic hydrolysis of agarwood, and then Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae are inoculated for mixed bacteria fermentation of the enzymatically hydrolyzed agarwood. Then, the fermented agarwood is sterilized, filtered, concentrated and dried to obtain the agarwood fermentation product. This method improves the release rate of active substances in agarwood. Among them, ultrasonic-assisted enzymatic hydrolysis can shorten the enzymatic hydrolysis time, effectively improve the enzymatic hydrolysis efficiency, minimize the damage of active ingredients in agarwood during enzymatic hydrolysis to the greatest extent, and increase its release rate; the metabolites produced during the fermentation of Lactobacillus plantarum and Lactobacillus johnsonii can inhibit the accumulation of harmful substances and the growth of harmful bacteria, and can also promote the absorption of active ingredients in agarwood; the enzymes produced during the fermentation of yeast can degrade macromolecular substances such as lignin in agarwood through enzymatic hydrolysis reactions, thereby promoting the release of active ingredients. At the same time, probiotic compound fermentation is conducive to the exertion of synergistic effects among various strains, rapid bacteria multiplication and nutritional complementarity, and accelerates the fermentation process. The composite sleep-aiding composition prepared from the agarwood fermentation product of the present invention has a significant sleep-aiding effect, and the preparation method is simple, solving the problem of poor sleep treatment efficacy of existing agarwood products, and is safe and non-irritating, with good clinical application value. Detailed Embodiments

[0027] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following examples are provided to illustrate in detail the specific embodiments, structures, features and their effects of the present invention.

[0028] For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0029] The sources of some raw materials are as follows:

[0030] Agarwood powder: purchased from Gansu Yishengxiang Biotechnology Co., Ltd.;

[0031] Semen Ziziphi Spinosae: purchased from Bozhou Ru'antang Pharmaceutical Co., Ltd.;

[0032] Semen Platycladi: purchased from Bozhou Ru'antang Pharmaceutical Co., Ltd.;

[0033] Flos Rosae Rugosae: purchased from Bozhou Ru'antang Pharmaceutical Co., Ltd.;

[0034] Radix Polygalae: purchased from Bozhou Ru'antang Pharmaceutical Co., Ltd.;

[0035] Poria: purchased from Anguo Desheng Pharmaceutical Co., Ltd.;

[0036] Honey-fried Licorice Root: purchased from Bozhou Wufangjiuru Pharmaceutical Co., Ltd.;

[0037] Lactobacillus plantarum: All were purchased from the China General Microbiological Culture Collection Center, with the preservation numbers being CGMCC NO.1.1856 and CGMCC NO.1.12974 respectively;

[0038] Lactobacillus johnsonii: Purchased from the China General Microbiological Culture Collection Center, with the preservation number being CGMCC NO.1.3221;

[0039] Saccharomyces cerevisiae: All were purchased from the China General Microbiological Culture Collection Center, with the preservation numbers being CGMCC NO.2.3854 and CGMCC NO.2.1527 respectively;

[0040] Cellulase: Purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 50 U / mg;

[0041] Amylase: Purchased from Shanghai Macklin Biochemical Co., Ltd., with an enzyme activity of 35 U / mg.

[0042] Example 1

[0043] A compound sleep-aiding composition based on Aquilaria sinensis, the compound sleep-aiding composition comprising the following raw materials in parts by weight: 3 parts of Aquilaria sinensis fermentation product, 15 parts of Ziziphus jujuba var. spinosa, 10 parts of Platycladus orientalis seed, 6 parts of Rosa rugosa, 3 parts of Polygala tenuifolia, 15 parts of Poria cocos, 3 parts of roasted Glycyrrhiza uralensis; the preparation method of the Aquilaria sinensis fermentation product comprises the following steps:

[0044] S1. Weigh Aquilaria sinensis powder, add sterile deionized water, mix evenly and then add a compound enzyme for ultrasonic-assisted enzymatic hydrolysis. After enzymatic hydrolysis is completed, perform enzyme inactivation treatment, and cool to room temperature to obtain a mixture;

[0045] S2. Inoculate a compound bacterium into the mixture to obtain a mixed material, perform mixed-bacterium fermentation on the mixed material, and after fermentation is completed, sterilize and filter to obtain a filtrate;

[0046] S3. After performing vacuum concentration and freeze-drying on the filtrate, obtain the Aquilaria sinensis fermentation product, and store it sealed under low-temperature and light-proof conditions;

[0047] The compound enzyme is composed of amylase and cellulase;

[0048] The compound bacterium is composed of Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae with a viable bacteria number ratio of 1:1:1. The preservation number of Lactobacillus plantarum is CGMCC NO.1.1856, the preservation number of Lactobacillus johnsonii is CGMCC NO.1.3221, and the preservation number of Saccharomyces cerevisiae is CGMCC NO.2.3854;

[0049] The total viable bacteria number of the compound bacterium in the mixed material is 7×10 7 CFU / mL;

[0050] In the step S1, the dosage ratio of agarwood powder, sterile deionized water, amylase and cellulase is 100 g: 1.5 L: 1 g: 1.5 g;

[0051] In the step S1, the ultrasonic frequency is 20 kHz, the ultrasonic power is 120 W, the enzymatic hydrolysis temperature is 40 °C, the enzymatic hydrolysis pH is 5, and the enzymatic hydrolysis time is 90 min;

[0052] In the step S1, the enzyme inactivation temperature is 80 °C and the enzyme inactivation time is 25 min;

[0053] In the step S2, the fermentation temperature is 30 °C and the fermentation time is 72 h;

[0054] In the step S2, the sterilization temperature is 121 °C and the sterilization time is 30 min;

[0055] In the step S2, filtration is carried out by high-speed centrifugal filtration, the centrifugal rotation speed is 5000 r / min, and the centrifugal time is 10 min;

[0056] In the step S3, vacuum concentration is carried out to concentrate to 30% of the original volume under a pressure of 0.06 MPa;

[0057] In the step S3, freeze-drying is carried out by a spray freeze-drying device, the cold trap temperature is -65 °C, the spray freezing temperature is -20 °C, the spray pressure is 0.3 MPa, and spray freeze-drying is carried out until the water content in the agarwood fermentation product is 5 wt%;

[0058] The preparation method of the composite sleep aid composition includes the following steps: crushing each raw material, passing through a 50-mesh sieve, weighing each raw material according to the formula amount, and mixing them evenly to obtain the composite sleep aid composition.

[0059] Example 2

[0060] A composite sleep aid composition based on agarwood, the composite sleep aid composition includes the following raw materials in parts by weight: 4 parts of agarwood fermentation product, 12 parts of wild jujube seed, 8 parts of semen platycladi, 8 parts of rose flower, 4 parts of polygala tenuifolia, 12 parts of poria cocos, 5 parts of roasted licorice; the preparation method of the agarwood fermentation product includes the following steps:

[0061] S1. Weigh agarwood powder, add sterile deionized water, mix evenly, add a composite enzyme for ultrasonic-assisted enzymatic hydrolysis, and after enzymatic hydrolysis is completed, carry out enzyme inactivation treatment, and cool to room temperature to obtain a mixture;

[0062] S2. Inoculate a composite bacterium into the mixture to obtain a mixed material, carry out mixed bacteria fermentation on the mixed material, sterilize and filter after fermentation is completed to obtain a filtrate;

[0063] S3. After vacuum concentration and freeze-drying of the filtrate, obtain the agarwood fermentation product, and store it sealed under low temperature and light-proof conditions;

[0064] The composite enzyme is composed of amylase and cellulase;

[0065] The composite bacteria are composed of Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae with a viable bacteria number ratio of 1.5:0.7:1. The preservation number of Lactobacillus plantarum is CGMCC NO.1.1856, the preservation number of Lactobacillus johnsonii is CGMCC NO.1.3221, and the preservation number of Saccharomyces cerevisiae is CGMCC NO.2.3854;

[0066] The total viable bacteria number of the composite bacteria in the mixed material is 1×10 8 CFU / mL;

[0067] In the step S1, the dosage ratio of agarwood powder, sterile deionized water, amylase and cellulase is 100g:1.2L:1.5g:1g;

[0068] In the step S1, the ultrasonic frequency is 30kHz, the ultrasonic power is 110W, the enzymolysis temperature is 45°C, the enzymolysis pH is 5.5, and the enzymolysis time is 75min;

[0069] In the step S1, the enzyme inactivation temperature is 85°C and the enzyme inactivation time is 20min;

[0070] In the step S2, the fermentation temperature is 32°C and the fermentation time is 60h;

[0071] In the step S2, the sterilization temperature is 121°C and the sterilization time is 30min;

[0072] In the step S2, high-speed centrifugal filtration is adopted for filtration, the centrifugal rotation speed is 5000r / min, and the centrifugal time is 10min;

[0073] In the step S3, vacuum concentration is carried out to concentrate to 30% of the original volume under a pressure of 0.06MPa;

[0074] In the step S3, spray freeze-drying is adopted for freeze-drying. The cold trap temperature is -65°C, the spray freezing temperature is -20°C, the spray pressure is 0.3MPa, and spray freeze-drying is carried out until the water content in the agarwood fermentation product is 6wt%;

[0075] The preparation method of the composite sleep aid composition includes the following steps: crushing each raw material, passing through a 50-mesh sieve, weighing each raw material according to the formula amount and mixing them to obtain the composite sleep aid composition.

[0076] Example 3

[0077] A compound sleep-aiding composition based on agarwood, the compound sleep-aiding composition comprising the following raw materials in parts by weight: 5 parts of agarwood fermentation product, 10 parts of wild jujube seeds, 6 parts of semen platycladi, 9 parts of rose flowers, 6 parts of polygala tenuifolia, 10 parts of poria cocos, 6 parts of roasted licorice root; the preparation method of the agarwood fermentation product comprises the following steps:

[0078] S1. Weigh agarwood powder, add sterile deionized water, mix evenly, then add a compound enzyme for ultrasonic-assisted enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment, and cool to room temperature to obtain a mixture;

[0079] S2. Inoculate a compound bacterium into the mixture to obtain a mixed material, perform mixed-bacterium fermentation on the mixed material, and after the fermentation is completed, sterilize and filter to obtain a filtrate;

[0080] S3. After performing vacuum concentration and freeze-drying on the filtrate, obtain the agarwood fermentation product, and store it sealed under low-temperature and light-proof conditions;

[0081] The compound enzyme is composed of amylase and cellulase;

[0082] The compound bacterium is composed of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae with a viable bacteria number ratio of 2:0.5:1. The preservation number of Lactobacillus plantarum is CGMCC NO.1.568, the preservation number of Lactobacillus johnsonii is CGMCCNO.1.10778, and the preservation number of Saccharomyces cerevisiae is CGMCC NO.2.3854;

[0083] The total viable bacteria number of the compound bacterium in the mixed material is 4×10 8 CFU / mL;

[0084] In step S1, the dosage ratio of agarwood powder, sterile deionized water, amylase, and cellulase is 100g:1L:2g:0.5g;

[0085] In step S1, the ultrasonic frequency is 40kHz, the ultrasonic power is 100W, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis pH is 6, and the enzymatic hydrolysis time is 60min;

[0086] In step S1, the enzyme inactivation temperature is 90°C, and the enzyme inactivation time is 15min;

[0087] In step S2, the fermentation temperature is 34°C, and the fermentation time is 48h;

[0088] In step S2, the sterilization temperature is 121°C, and the sterilization time is 30min;

[0089] In step S2, the filtration is performed by high-speed centrifugal filtration, the centrifugal rotation speed is 5000r / min, and the centrifugal time is 10min;

[0090] The decompression concentration in step S3 is carried out under a pressure of 0.06 MPa until the volume is reduced to 30% of the original volume;

[0091] The freeze-drying in step S3 uses a spray freeze-drying device. The cold trap temperature is -65 °C, the spray freezing temperature is -20 °C, the spray pressure is 0.3 MPa, and the spray freeze-drying is carried out until the water content in the agarwood fermentation product is 8 wt%;

[0092] The preparation method of the composite sleep aid composition includes the following steps: pulverize each raw material, pass through a 50-mesh sieve, weigh each raw material according to the formula amount, and mix them evenly to obtain the composite sleep aid composition.

[0093] Comparative Example 1

[0094] Compared with Example 2, the only difference is that ultrasonic treatment is not carried out in step S1 of Comparative Example 2. The specific steps of step S1 in Comparative Example 2 are: weigh agarwood powder, add sterile deionized water, mix evenly, add a composite enzyme for enzymatic hydrolysis, carry out enzyme inactivation treatment after enzymatic hydrolysis is completed, and cool to room temperature to obtain a mixture. Other conditions are the same as those in Example 2.

[0095] Comparative Example 2

[0096] Compared with Example 2, the only difference is that Lactobacillus plantarum is not added in Comparative Example 2. The composite bacteria are composed of Lactobacillus johnsonii and Saccharomyces cerevisiae with a viable bacteria number ratio of 0.7:1. The total viable bacteria number of the composite bacteria in the mixture remains unchanged. Other conditions are the same as those in Example 2.

[0097] Comparative Example 3

[0098] Compared with Example 2, the only difference is that Lactobacillus johnsonii is not added in Comparative Example 3. The composite bacteria are composed of Lactobacillus plantarum and Saccharomyces cerevisiae with a viable bacteria number ratio of 1.5:1. The total viable bacteria number of the composite bacteria in the mixture remains unchanged. Other conditions are the same as those in Example 2.

[0099] Comparative Example 4

[0100] Compared with Example 2, the only difference is that Saccharomyces cerevisiae is not added in Comparative Example 4. The composite bacteria are composed of Lactobacillus plantarum and Lactobacillus johnsonii with a viable bacteria number ratio of 1.5:0.7. The total viable bacteria number of the composite bacteria in the mixture remains unchanged. Other conditions are the same as those in Example 2.

[0101] Comparative Example 5

[0102] Compared with Example 2, the only difference is that the viable bacteria number ratio of Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae in Comparative Example 5 is 0.7:1.5:1. The total viable bacteria number of the composite bacteria in the mixture remains unchanged. Other conditions are the same as those in Example 2.

[0103] Comparative Example 6

[0104] Compared with Example 2, the only difference is that in Comparative Example 6, the viable cell number ratio of Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae is 1.5:1:0.7, and the total viable cell number of the compound bacteria in the mixture remains unchanged, and other conditions are the same as those in Example 2.

[0105] Comparative Example 7

[0106] Compared with Example 2, the only difference is that in Comparative Example 7, Lactobacillus plantarum with the preservation number of CGMCC NO.1.12974 is used to replace Lactobacillus plantarum with the preservation number of CGMCC NO.1.1856, and other conditions are the same as those in Example 2.

[0107] Comparative Example 8

[0108] Based on Example 2, the only difference is that in Comparative Example 8, Saccharomyces cerevisiae with the preservation number of CGMCC NO.2.1527 is used to replace Saccharomyces cerevisiae with the preservation number of CGMCC NO.2.3854, and other conditions are the same as those in Example 2.

[0109] Comparative Example 9

[0110] A compound sleep aid composition based on Aquilaria sinensis, the compound sleep aid composition comprises the following raw materials in parts by weight: 4 parts of Aquilaria sinensis powder, 12 parts of Ziziphus jujuba var. spinosa, 8 parts of Platycladus orientalis seeds, 8 parts of Rosa rugosa, 4 parts of Polygala tenuifolia, 12 parts of Poria cocos, 5 parts of roasted licorice;

[0111] The preparation method of the compound sleep aid composition comprises the following steps: crushing each raw material and passing through a 50-mesh sieve, weighing each raw material according to the formula amount and mixing them to obtain the compound sleep aid composition.

[0112] Effect Example 1 Sleep Test

[0113] Experimental animals: 112 male SPF-grade KM mice, weighing 20-25 g, were fed with ordinary feed under constant temperature conditions without special pathogens, the temperature was 20-24 °C, the environmental relative humidity was 40%-70%, and they were housed individually in cages;

[0114] Grouping: After the mice were adaptively fed in the animal house for one week, they were randomly divided into 14 groups, with 8 mice in each group, namely a blank control group (using normal saline), a positive control group (using Yixin Ningshen tablets), Example 1-3 groups and Comparative Example 1-9 groups (using the compound sleep aid compositions of Example 1-3 groups and Comparative Example 1-9 groups);

[0115] Drug administration:

[0116] Positive control group: Prepare a Yixin Ningshen tablet solution (0.13 g / mL) with normal saline and intragastrically administer the Yixin Ningshen tablet solution at 10 mL / kg;

[0117] Example 1 - 3 groups, Comparative Example 1 - 9 groups: Prepare the corresponding composite sleep - aid composition solutions (0.006 g / mL) for each group with normal saline (75°C) respectively, and dissolve and disperse them under ultrasonic assistance. The ultrasonic frequency is 80 kHz, the ultrasonic power is 120 W. After ultrasonic treatment for 15 min, filter the residue. After the liquid is cooled to room temperature, each group is gavaged with the corresponding composite sleep - aid composition solution at a dose of 10 mL / kg.

[0118] Blank control group: Gavaged with normal saline at a dose of 10 mL / kg.

[0119] Each group is administered the drug once a day.

[0120] Sleep test: The mice in each group are allowed to drink water and eat freely. After continuous gavage for 14 days, 30 minutes after the last administration, each group is intraperitoneally injected with sodium pentobarbital at a dose of 40 mg / kg (fasting but not water - restricting for 12 h before intraperitoneal injection), and the sleep latency (min) and sleep time (min) of the mice are observed and recorded.

[0121] Sleep judgment criterion: Sleep is indicated by the disappearance of the righting reflex. When the mouse is placed in the dorsal recumbent position, it can immediately right itself. If it cannot right itself within 1 minute, it is considered that the righting reflex has disappeared and the mouse has entered sleep. The sleep latency is the time from drug administration to the disappearance of the righting reflex. The restoration of the righting reflex means the mouse wakes up, and the time from the disappearance to the restoration of the righting reflex is the sleep time of the mouse.

[0122] Statistical method: The data are statistically processed using SPSS statistical software, and all measurement data are expressed as mean ± standard deviation. Statistical analysis is performed using the t - test, and P < 0.05 is considered statistically significant; the test results are shown in Table 1.

[0123] Table 1 Test results of sleep latency and sleep time

[0124]

[0125] Note: Compared with the blank group: P < 0.05 is indicated by "*"; compared with the positive control group: P < 0.05 is indicated by " ▲ ".

[0126] Effect Example 2 Neurotransmitter determination

[0127] After the mouse sleep test was completed, the mice were immediately decapitated and sacrificed. The whole brain was quickly dissected on an ice box, and the hippocampus was taken. It was precisely weighed with a ten-thousandth balance. Cold physiological saline was added at a ratio of hippocampal tissue (g): physiological saline (mL) = 1:10. Homogenization was performed with an ultrasonic cell disruptor at low temperature. The homogenate was centrifuged at 4°C and 3000 r / min for 15 min, and the supernatant was aliquoted into 1.5 mL centrifuge tubes to obtain the mouse hippocampal homogenate. The hippocampal homogenate was taken, and an ELISA kit was used to measure the contents of 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) in the brain tissue. All the above kits were purchased from Hefei Branch of Shanghai Keaibo Biotechnology Co., Ltd.

[0128] 5-Hydroxytryptamine (5-HT), also known as serotonin, is an important neurotransmitter that participates in regulating mood, cognition, and sleep and plays a key role in regulating the sleep-wake cycle. An increase in the content level of 5-HT can have a certain sedative and sleep-promoting effect. γ-Aminobutyric acid (GABA) is a natural sleep amino acid present in the human brain. It can soothe the nerves, prevent the nerves from being overly tense and excited, can better relieve mental stress, regulate emotions, and restore physical and mental health. At the same time, it can shorten the sleep latency and prolong the deep sleep time. Therefore, the higher the contents of 5-HT and GABA, the better the sleep improvement effect.

[0129] Statistical method: Data statistics were processed using SPSS statistical software. All measurement data were expressed as mean ± standard deviation. Statistical analysis was performed using a t-test. P < 0.05 was considered statistically significant; the test results are shown in Table 2.

[0130] Table 2 Results of neurotransmitter determination in mice

[0131]

[0132]

[0133] Note: Compared with the blank group: P < 0.05 is indicated by "*"; compared with the positive control group: P < 0.05 is indicated by " ▲ ".

[0134] The test results are shown in Table 1-2. Compared with the blank control group and the positive control group, the sleep latency of the mice in the groups of Examples 1-3 was significantly shortened, the sleep time was significantly prolonged, and the contents of 5-HT and GABA were significantly increased, indicating that the composite sleep aid composition prepared in Examples 1-3 can effectively improve the sleep aid effect.

[0135] The effects of Comparative Example 1 and Comparative Example 9 were both worse than those of the positive control group, indicating that the steps of ultrasonic-assisted enzymatic hydrolysis and mixed bacteria fermentation play an important role in the release of active ingredients in agarwood, making the efficacy of the prepared composite sleep aid composition better.

[0136] Comparative Example 2-4 lacked one of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae respectively. Compared with the composite sleep-aiding compositions prepared in Examples 1-3, the sleep-aiding effect was significantly weakened, indicating that the composition of different strains in the composite bacteria directly affected the fermentation result, and Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae could promote and influence each other during fermentation, effectively improving the therapeutic effect of the composition.

[0137] In Comparative Examples 5-6, the viable cell number ratio of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae exceeded the range defined in the present invention. Compared with the composite sleep-aiding compositions prepared in Examples 1-3, the improvement effect on insomnia was also significantly weakened, indicating that different viable cell number ratios of the same strain would also affect the fermentation result to a certain extent, thereby affecting the therapeutic effect of the composite sleep-aiding composition; and the optimal viable cell number ratio of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae was 1.5:0.7:1.

[0138] In Comparative Examples 7-8, different strains of Lactobacillus plantarum and Saccharomyces cerevisiae were replaced respectively. Compared with the composite sleep-aiding compositions prepared in Examples 1-3, the sleep-aiding effect was weakened, indicating that even for the same strain, the property differences of different strains would also affect the effect of the composite sleep-aiding composition. The strains of Lactobacillus plantarum and Saccharomyces cerevisiae selected in the present invention are the strains with the best synergistic effect with Lactobacillus johnsonii strain obtained by the inventor through a large number of experimental screenings in the early stage.

[0139] In summary, the present invention uses a composite bacteria composed of Lactobacillus plantarum, Lactobacillus johnsonii, and Saccharomyces cerevisiae to perform mixed bacteria fermentation on agarwood after ultrasonic-assisted enzymatic hydrolysis, improving the release rate of active substances in agarwood, and enabling the composite sleep-aiding composition containing the agarwood fermentation product to have a significant regulatory effect on sleep latency and sleep time, and being able to solve the problem of poor therapeutic effect of existing agarwood products in treating insomnia.

[0140] The above is only a preferred embodiment of the present invention, and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or equivalent changes and modifications within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A composite sleep-aiding composition based on agarwood, characterized in that: The composite sleep-aiding composition comprises the following raw materials in parts by weight: 3-5 parts of agarwood fermentation product, 10-15 parts of spiny jujube seeds, 6-10 parts of cypress seeds, 6-9 parts of roses, 3-6 parts of polygala, 10-15 parts of tuckahoe, and 3-6 parts of licorice root; the preparation method of the agarwood fermentation product comprises the following steps: S1. Weigh agarwood powder, add sterile deionized water, mix well, add complex enzyme for ultrasonic-assisted enzymolysis, inactivate the enzyme after the enzymolysis is completed, cool to room temperature, and obtain a mixture; S2, inoculating the mixture with composite bacteria to obtain a mixture, subjecting the mixture to mixed bacterial fermentation, sterilizing and filtering after the fermentation is completed to obtain a filtrate; S3, after reducing pressure and concentrating the filtrate, freeze-drying, obtaining the agarwood fermentation product, and storing it in a sealed state at low temperature and away from light; Wherein, the complex enzyme is composed of amylase and cellulase; The composite bacteria consists of Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae in a live bacteria quantity ratio of 1-2:0.5-1:1, the Lactobacillus plantarum has a preservation number of CGMCC NO.1.1856, the Lactobacillus johnsonii has a preservation number of CGMCC NO.1.3221, and the Saccharomyces cerevisiae has a preservation number of CGMCC NO.2.3854.

2. The composite sleep-aiding composition according to claim 1, characterized in that: The total number of viable bacteria of the composite bacteria in the mixture is 7×10 7 -4×10 8 CFU / mL.

3. The composite sleep-aiding composition according to claim 1, characterized in that: The ratio of the number of live bacteria of Lactobacillus plantarum, Lactobacillus johnsonii and Saccharomyces cerevisiae is 1.5:0.7:

1.

4. The composite sleep-aiding composition according to claim 1, characterized in that: The composite sleep-aiding composition comprises the following raw materials in parts by weight: 4 parts of agarwood fermentation product, 12 parts of spinach seeds, 8 parts of cypress seeds, 8 parts of rose flowers, 4 parts of polygala, 12 parts of poria, and 5 parts of roasted licorice.

5. The composite sleep-aiding composition according to claim 1, characterized in that: In the step S1, the dosage ratio of agarwood powder, sterile deionized water, amylase and cellulase is 100g:1-1.5L:1-2g:0.5-1.5g, the enzymatic activity of the cellulase is 40-60U / mg, and the enzymatic activity of the amylase is 30-40U / mg.

6. The composite sleep-aiding composition according to claim 1, characterized in that: In step S1, the ultrasonic frequency is 20-40 kHz, the ultrasonic power is 100-120 W, the enzymatic hydrolysis temperature is 40-50° C., the enzymatic hydrolysis pH is 5-6, and the enzymatic hydrolysis time is 60-90 min.

7. The composite sleep-aiding composition according to claim 1, characterized in that: The enzyme inactivation temperature in step S1 is 80-90° C. and the enzyme inactivation time is 15-25 min.

8. The composite sleep-aiding composition according to claim 1, characterized in that: The fermentation temperature in step S2 is 30-34° C., and the fermentation time is 48-72 hours.

9. A method for preparing the composite sleep-aiding composition according to any one of claims 1 to 8, characterized in that: The following steps are involved: The raw materials are crushed and passed through a 50-mesh sieve, and the raw materials are weighed according to the formula amount and mixed to obtain the composite sleep-aiding composition.

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