CN116515585A - Sanbao wine and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Sanbao wine which comprises the following components in parts by volume: 40-50 parts of gastrodia tuber extract, 25-35 parts of ganoderma extract and 15-25 parts of eucommia bark extract, wherein the alcohol content of the Sanbao wine is 37% vol. The optimal Sanbao wine with the sauce fragrance and the wine fragrance, harmonious fragrance, mellow taste, complete wine body and good color is prepared, is rich in nutrition, has stronger in-vivo antioxidant activity, reduces the damage of alcohol to the organism, and maintains the normal defensive ability of the organism, thereby achieving the aim of delaying aging, and has the effects of improving sleep and improving the health performance of intestinal tracts.

Description

Sanbao wine and preparation method and application thereof

Technical Field

The invention relates to health care wine, in particular to Sanbao wine and a preparation method and application thereof.

Background

The Chinese has the habit of drinking and preserving health from ancient times, and wine culture is increasingly valued along with the development of society, the improvement of living standard of people and the gradual enhancement of health consciousness. The compound wine is used as a branch of the traditional wine in China, and has a wide consumption basis in China. The mixed wine is also called liqueur and blended wine, and belongs to a special mixed wine product. Is prepared from fermented wine, distilled wine and edible alcohol through adding edible auxiliary materials or food additives including edible flowers, grasses, animals and plants, chinese herbal medicines, etc. and immersing, boiling, re-steaming, etc. The wine is rich in mineral, polysaccharide, amino acid, lipoid, organic acid, vitamin, flavone, soap, alkaloid and other nutritious and effective components.

Insomnia is a sleep disorder characterized by frequent and persistent difficulty falling asleep or difficulty maintaining sleep and resulting in inadequate sleep satisfaction. More and more people suffer from insomnia. Prolonged insomnia can lead to numerous mental disorders and physical diseases in the human body. Current clinical barbiturates and benzodiazepines are currently common chemicals for sleep disorders. Such as the android, the non-benzodiazepines and the like, has rapid curative effect and definite action mechanism. But the long-term use of the medicine can easily produce adverse reactions such as residual effect, forgetting effect, medicine stopping effect and the like. The disadvantage of using common traditional Chinese medicine to treat intractable insomnia is slow effect and long treatment course.

In the Chinese patent literature, the application number is CN200410064361.9, the application date is 2004-08-20, and the medicinal liquor for treating insomnia is prepared from the following components in percentage by weight: 10 parts of glossy privet fruit, 6 parts of cortex moutan, 8 parts of eucommia ulmoides, 15 parts of radix rehmanniae, 5 parts of tortoise plastron, 6 parts of mistletoe, 5 parts of cistanche salsa, 10 parts of clematis stem, 7 parts of uncaria, 3 parts of pericarpium granati, 3 parts of fructus cnidii, 5 parts of fritillaria cirrhosa, 10 parts of rhizoma corydalis and 700 parts of 30-degree white spirit, and soaking the traditional Chinese medicine components in the white spirit for 70-80 days. However, the medicinal liquor has the effects of tranquilizing and allaying excitement by improving the blood microcirculation of human bodies, dredging heart and cerebral vessels, and treating various insomnia, and has no definite effect of improving sleep function and influence on the anti-aging capability of organisms proved by animal or clinical experiments at present.

According to the findings of literature and current patent authorization, other similar medicinal liquor is in researching the anti-aging and insomnia regulating effects, the step of in-vivo investigation and verification is lacking, the research on anti-aging effect is only remained in the common measurement research of the anti-oxidation index, but the data support is lacking for the deep research of taking the medicinal liquor into the body; at present, most of medicinal liquor with the effect of improving sleep has the effect of improving the sleep time, but the influence relationship between the main action body of the medicinal liquor and the base liquor is not supported by the data.

Disclosure of Invention

The invention aims to provide a Sanbao wine and a preparation method and application thereof. The Sanbao wine of the invention has rich fragrance and nutrition, and has the characteristics of excellent aging delaying and sleep improving.

The technical scheme of the invention is as follows: the Sanbao wine comprises the following components in parts by volume: 40-50 parts of gastrodia tuber extract, 25-35 parts of ganoderma extract and 15-25 parts of eucommia bark extract, wherein the alcohol content of the Sanbao wine is 37% vol.

The three-treasure wine comprises the following components in percentage by volume: 49% of gastrodia elata leaching liquor, 30% of ganoderma lucidum leaching liquor and 21% of eucommia ulmoides leaching liquor, wherein the alcoholic strength of the Sanbao liquor is 37% vol.

A preparation method of Sanbao wine comprises mixing rhizoma Gastrodiae extract, ganoderma extract and Eucommiae cortex extract at a volume ratio of 49:30:21, adding honey to obtain a mixed liquor with the alcohol content of 37%vol.

In the preparation method of the Sanbao wine, the preparation method of the gastrodia elata leaching solution comprises the following steps: crushing the dried gastrodia elata, adding base wine according to the feed liquid ratio of 1:10, uniformly mixing, sealing, performing ultrasonic power 220w, performing ultrasonic treatment for 25-35 min, shaking for 1-3 times a day, soaking for 5 days, and filtering to obtain gastrodia elata leaching solution.

In the preparation method of the three-treasure liquor, the preparation method of the ganoderma lucidum leaching liquor comprises the following steps: pulverizing dried ganoderma lucidum, adding base liquor according to a feed liquid ratio of 1:10, mixing uniformly, sealing, performing ultrasonic power of 220w, performing ultrasonic treatment for 25-35 min, shaking for 1-3 times a day, soaking for 30 days, and filtering to obtain ganoderma lucidum leaching liquor.

In the preparation method of the Sanbao liquor, the preparation method of the eucommia ulmoides leaching liquor comprises the following steps: crushing the dried eucommia ulmoides, adding base liquor according to the feed liquid ratio of 1:10, uniformly mixing, sealing, carrying out ultrasonic power 220w, ultrasonic time 25-35 min, sealing, shaking for 1-3 times a day, soaking for 15 days, and filtering to obtain Du Zhongjin extract.

In the preparation method of the three-treasure wine, the base wine adopts the soy sauce base wine with the alcohol content of 50% vol.

The Sanbao wine is applied to the preparation of health-care food or medicine capable of increasing GSH-Px, SOD and GSH content and activity, reducing MDA content and down regulating IL-4, IL-6 and TNF-alpha level.

The Sanbao wine is applied to the preparation of hypnotic health-care food or medicine capable of increasing the content of 5-HT and GABA, reducing the content of GIU and NE and improving pressure type insomnia.

The application of Sanbao wine in preparing health food or medicine for improving intestinal flora disturbance caused by insomnia.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts gastrodia elata leaching liquor: glossy ganoderma leaching liquor: the volume ratio of the eucommia ulmoides leaching solution is 49:30:21, and preparing the optimal three-treasure wine with soy sauce flavor, bouquet, harmony flavor, mellow taste, complete wine body and good color, wherein GABA, T-AOC and sensory scores are respectively 0.121mg/mL, 5.78umol/mL and 92.02 minutes.

The solid-phase microextraction-gas chromatography-mass spectrometry combined technology and an amino acid automatic analyzer are adopted to detect that 64 aroma components, mainly esters and alcohols exist in the Sanbao liquor, 21 kinds of free amino acids are detected totally, the content of the total free amino acids is 318.7mg/L, and the content of the essential amino acids of a human body is 71mg/L.

Experiments show that the Sanbao wine group can slow down the appetite, weight and organ index reduction of the aged mice caused by D-galactose, and can not cause damage to organs of the mice; can remarkably increase GSH-Px, SOD and GSH content and activity in serum and brain tissues, reduce MDA content, lower the level of inflammatory factors such as IL-4, IL-6, TNF-alpha and the like, reduce inflammatory factors, relieve the damage degree of D-galactose and base wine to brain tissues and liver tissues of mice, and inhibit the development of symptoms of aging mice. The three-treasure wine has stronger in-vivo antioxidant activity, reduces the damage of alcohol to the organism, and maintains the normal defensive ability of the organism, thereby achieving the aim of deferring senility.

Experiments show that the Sanbao wine and the pentobarbital sodium have synergistic effect, can effectively shorten the sleep latency of mice, increase the sleep time, improve the sleep rate, have no direct sleep effect, and have the effects of improving the sleep and improving the sleep quality of the mice. Secondly, p-chlorophenylalanine is injected into the abdominal cavity to prepare an insomnia mouse model, and the Sanbao wine is found to be capable of effectively improving symptoms of neurotransmitter disorder of the insomnia mouse, increasing the contents of 5-HT and GABA in cerebral cortex and serum, simultaneously reducing the contents of GIU and NE, inhibiting excitatory neurotransmitters, recovering 5-HT in brain tissue and serum of the insomnia mouse and protecting nerve cells; meanwhile, pathological sections are carried out on the hippocampal tissue of the insomnia mouse, and the Sanbao wine can relieve the damage of the hippocampal tissue of the mouse caused by insomnia.

Fecal 16S rRNA sequencing is carried out on insomnia mice, and the fact that after the insomnia mice are subjected to the intervention of Sanbao liquor, the types and the relative abundance of microorganisms are increased at the levels of phylum and genus, the intestinal flora structure is improved, and the diversity, the richness, the flora quantity and the flora structure of intestinal flora of a model group are restored; the intestinal flora diversity of the insomnia mice is effectively recovered, the proportion of beneficial bacteria to pathogenic bacteria is changed, the intestinal flora of the insomnia mice is recovered to a normal state to a certain extent, and the bacteria closely related to the level of neurotransmitters and inflammatory factors in the brain of the insomnia mice are regulated, so that the effect of regulating insomnia is achieved.

The experiment sets up a plurality of target groups, in original normal blank mice, insomnia model mice, administration group mice and positive group mice, the base wine group and alcohol-free group mice are added simultaneously, and a plurality of groups of discussion and analysis on the study on the effect of the three-treasure wine on improving sleep is carried out.

Drawings

FIG. 1 is a graph showing the trend of the content of flavor compounds in Sanbao liquor and the type of flavor compounds;

FIG. 2 is a graph showing the effect (x.+ -. S) of Sanbao liquor on the body weight of aged mice caused by D-galactose;

FIG. 3 is a graph showing the effects of Sanbao liquor on the SOD activity (A), MDA concentration (B), GSH (C) and GSH-PX activity (D) of mouse serum;

FIG. 4 is a graph showing the effects of Sanbao wine on SOD activity (A), MDA concentration (B), GSH (C) and GSH-PX activity (D) of mouse brain tissue;

fig. 5 is a graph of liver histomorphometric observations (HE staining, 20×20) of mice;

FIG. 6 is a graph showing the content of 5-HT, GIU, GABA, NE in brain tissue of each group of mice;

FIG. 7 is a graph showing the content of 5-HT, GIU, GABA, NE in serum of each group of mice;

FIG. 8 is a graph of the results of HE staining experiments with hippocampal neuronal cells from each group of mice;

FIG. 9 is a petal plot of intestinal flora of mice of each group;

FIG. 10 is a graph showing the trend of the intestinal flora diversity index of each group of mice;

FIG. 11 is a graph of the relative abundance of gut flora gate levels for each group of mice;

FIG. 12 is a graph of relative abundance of gut flora levels for each group of mice;

FIG. 13 is a graph of genus levels with significant differences in relative abundance in the feces of each group of mice;

FIG. 14 is a heat map analysis of species dependence at the intestinal flora level for each group of mice.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

Examples:

the Sanbao wine comprises the following components in parts by volume: 49 parts of gastrodia tuber extract, 30 parts of ganoderma extract and 21 parts of eucommia bark extract, wherein the alcohol degree of the Sanbao wine is 37 percent vol.

The preparation method of the Sanbao wine comprises the following steps: the gastrodia tuber leaching solution, the ganoderma lucidum leaching solution and the eucommia bark leaching solution are mixed according to the volume ratio of 49:30:21, mixing to obtain highly mixed liquor with consistent sauce fragrance and liquor fragrance, and transparent color without precipitate; then adding honey to obtain mixed liquor with the alcohol content of 37 percent vol.

The preparation method of the gastrodia elata leaching solution comprises the following steps: pulverizing dried rhizoma Gastrodiae, adding sauce base liquor with alcohol content of 53% vol according to the ratio of 1:10, mixing, sealing, ultrasonic power of 220w, ultrasonic time of 30min, placing in shade and dry place, shaking for 1 time every day, soaking for 5 days, standing, settling, filtering, and collecting supernatant to obtain rhizoma Gastrodiae extractive solution.

The preparation method of the ganoderma lucidum leaching solution comprises the following steps: pulverizing dried Ganoderma, adding sauce base liquor with alcohol content of 53% vol according to the ratio of 1:10, mixing, sealing, ultrasonic power of 220w, ultrasonic time of 30min, placing in shade and dry place, shaking for 1 time every day, soaking for 30 days, standing, settling, filtering, and collecting supernatant to obtain Ganoderma extractive solution.

The preparation method of the eucommia ulmoides leaching solution comprises the following steps: pulverizing dried Eucommiae cortex, adding sauce base liquor with alcohol content of 53% vol according to the ratio of 1:10, mixing, sealing, ultrasonic power of 220w, ultrasonic time of 30min, placing in shade and dry place, shaking for 1 time daily, soaking for 15 days, standing, settling, filtering to obtain clear liquid, and collecting Eucommiae cortex leaching solution.

The soluble solid content of rhizoma Gastrodiae extract, ganoderma extract and cortex Eucommiae extract is maximized in 5 days, 30 days and 15 days respectively.

According to the test of gamma-aminobutyric acid (GABA), the rhizoma Gastrodiae extract has a large influence on GABA content. According to the test of total antioxidant capacity (T-AOC), the influence of the ganoderma lucidum extract on the antioxidant capacity is larger. However, too much ganoderma lucidum extract can cause bitter taste, and the leaching time of ganoderma lucidum and eucommia is longer than that of gastrodia elata, so that the sauce flavor is not retained enough.

Therefore, the invention selects the gastrodia tuber extract, the ganoderma extract and the eucommia bark extract according to the volume ratio of 49:30:21, the soaking time of the gastrodia elata leaching solution is 5 days, the soaking time of the ganoderma lucidum leaching solution is 30 days, and the soaking time of the eucommia ulmoides leaching solution is 15 days to obtain the Sanbao wine. Proved by tests, the gamma-aminobutyric acid (GABA) of the Sanbao wine is 0.121mg/mL, the total antioxidant capacity (T-AOC) is 5.78umol/mL, and the sensory evaluation is 92.02 (sensory evaluation is combined with the sensory requirement formulation of GB/T27588-2011 'liqueur'). The Sanbao wine has the advantages of sauced fragrance, wine fragrance, harmony of all the fragrances, mellow taste and complete wine body, and has good color and luster and typical style.

1. Determination and analysis of volatile substances of the product:

the extraction method comprises the following steps: 2mL of the uniformly mixed sample is measured, the uniformly mixed sample is placed in a 10mL solid phase microextraction instrument sampling bottle, a manual sample injector with a 2cm-50/30 mu mDVB/CAR/PDMS Stableflex fiber head is inserted, the head is removed after headspace extraction is performed for 60min under the condition of magnetic stirring and heating on a heating flat plate at 60 ℃, and the head is immediately inserted into a gas chromatograph sample inlet (at the temperature of 230 ℃) for thermal analysis sample injection.

Gas chromatography conditions: the chromatographic column is HP-5MS (30 m×0.25mm×0.25 μm) elastic quartz capillary column, the column temperature is 40deg.C, the temperature is kept for 2min, the temperature is raised to 220deg.C at 4 deg.C for-1, the temperature is kept for 15min, and the running time is 62min; the temperature of the vaporization chamber is 230 ℃; the carrier gas is high purity He (99.999%); the pre-column pressure is 7.06psi, and the carrier gas flow rate is 1.0mL/min; split sample injection, split ratio: 10:1; solvent delay time: 1min;

mass spectrometry conditions: the ion source is an EI source; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; electron energy 70eV; emission current 34.6 μa; multiplier voltage 1823V; interface temperature 240 ℃; the mass range is 29-500 amu.

The contribution degree ROAV value calculation formula of the detected volatile components to the six base wine aroma systems is evaluated by adopting the relative smell activity value (ROAV) as follows:

Wherein: V-ROAV value; c (C) _i -relative percentage,%; c (C) _max -the relative percentage,%; t (T) _i -a fragrance threshold for the component; t (T) _max -a fragrance threshold for the largest component in the wine fragrance system. The greater the ROAV value, the greater its effect on the aroma system of the wine. The compound with the ROAV value more than or equal to 1 is used as a core aroma component, the ROAV value less than or equal to 0.1 is used as a modified aroma component, and the ROAV value less than 0.1 is used as a potential aroma component.

The qualitative analysis of the main volatile compounds is carried out on the Sanbao wine by adopting SPME-GC-MS, the detection results are shown in figure 1 and table 1, 64 main volatile components (aroma components) are detected, and the main volatile components mainly comprise esters (22), alcohols (14), acids (7) aldehydes (5), alkanes (5), aromatics (3), pyrazines (2) and ketones (2) and other types (4). As is known from the analysis of the ROAV value, the ester substance is one of the main aroma components of the three-treasure wine.

TABLE 1 key aroma ROAV value analysis of Sanbao wine

Threshold (μg/L) (x.zhang, 2019); description of the fragrance type (Xia, wang, 2019)

As is clear from the detection results, the three-treasure wine of the present invention has 2 kinds of alcohols with ROAV greater than 1, i.e., isopentyl alcohol and phenethyl alcohol. Isoamyl alcohol has the spicy fragrance of apple brandy; the phenethyl alcohol has rose honey fragrance.

The types and contents of the ester compounds are most abundant, wherein the typical ester compounds are ethyl n-caproate (olibanum, raspberry fruit), ethyl palmitate (fruit), ethyl valerate (flower, fruit, sweet), ethyl octanoate (grape, lemon), isoamyl acetate (pineapple, pear, flower), ethyl butyrate (fruit with bouquet) and the like, so that the wine body has the characteristics of sweet, soft and quiet and long aftertaste.

4 organic acids with ROAV more than 1 are acetic acid, isovaleric acid, butyric acid and caproic acid respectively, which can keep the fragrance of esters and adjust the flavor of wine.

2. Determination and analysis of free amino acid of the product:

sample treatment: detecting by using an S-433D amino acid automatic analyzer, wherein the column temperature is as follows: 37 ℃ reactor: 130 ℃; accurately weighing 1.022g of sulfosalicylic acid into a 100mL volumetric flask, and fixing the volume to prepare 1% sulfosalicylic acid solution; taking 1mL of different samples, respectively adding 9mL of sulfosalicylic acid solution, oscillating uniformly, centrifuging at 10000r/min for 15min, filtering the supernatant with a 0.22 μm water-based filter membrane, and detecting on a machine.

Integrating and extracting the peak area of the gas chromatography by using Dataanalysis software, selecting the matching degree to be more than 80, setting X volatile chemical components, and determining the relative content of each chemical component by using a peak area normalization method.

The measurement of free amino acids was performed using a fully automatic amino acid analyzer, and the detection results are shown in Table 2. 21 free amino acid components were detected in Sanbao liquor, and the total free amino acid content was 318.7mg/L. The Sanbao wine contains 7 human essential amino acids of threonine, valine, isoleucine, phenylalanine, leucine, lysine and methionine, and the total content is 71mg/L. The content of amino acids such as glutamic acid, alanine, aspartic acid, asparagine, etc. is higher, 53.2 mg/L, 46.7 mg/L, 23.4 mg/L, 30.2mg/L, respectively.

TABLE 2 free amino acid content of Sanbao wine

Note that: EAA (essential amino acids) essential amino acids, marked in the upper right hand corner of the amino acid abbreviation, TFAA (total free amino acids) total free amino acids; ND represents not measured

3. The physical and chemical indexes of the product are measured and analyzed:

the physicochemical index of the Sanbao wine was measured with reference to GB/T27588-2011, cordial, and the measurement results are shown in Table 3. The indexes of alcohol degree, total sugar, total acid and the like of the Sanbao wine meet the national standard requirement, and the indexes of methanol, cyanide, lead and the like are lower than the standard limit. According to the determination method of Chinese pharmacopoeia, the content of the gastrodin is 1.45mg/mL, the total saponins are 274.56ug/mL, the total flavonoids are 1.613mg/mL, and the soluble solid content is 23.165g/L.

TABLE 3 physical and chemical index analysis of Sanbao wine

4. The antioxidation measurement and analysis of the product:

experimental animals: SPF class eight week old male KM mice (production license number: SCXK Beijing 2019-0010); the animal is fed to clean animal houses at the temperature of 22+/-2 ℃ and relative humidity of 55+/-5% on the biotechnological stock company of Chongqing Tengxin, the lighting time is controlled to be 12 hours per day, and free drinking and eating are performed, so that the experiment is operated by strictly referring to the ethical requirements of animals in Guizhou medical university and the instruction of using animals.

Experimental raw materials: the finished product of the Sanbao wine is subjected to gastric lavage administration of mice.

Model building and experimental grouping:

(1) Establishing a subacute aging mouse model: KM mice were randomized into 6 groups after 1 week acclimation. The mice of each group were intraperitoneally injected with 300 mg/kg/BW of D-galactose solution, and the mice of the blank group were intraperitoneally injected with an equal dose of physiological saline, and the subacute aging mice were modeled after continuous injection for 42 days.

(2) Experimental grouping: KM mice were acclimatized for one week, randomly divided into 6 groups of 10 animals each, and administered continuously for 42 days in a blank group (equivalent distilled water), a model group (equivalent distilled water), a base wine group, a low dose group, a medium dose group, and a high dose group of the sanbao wine. According to the report of nutrition and chronic disease status of Chinese residents (2020), the daily alcohol intake of each person is less than or equal to 25g, the daily recommended daily human body dosage of Sanbao liquor is less than or equal to 1.1mL/kg, the low, medium and high dosage groups are respectively set to be 5, 10, 15 mL/(kg.d), and the gastric lavage dosage of the mice is 0.1mL/10g and is 1 time per day.

And (3) acquisition of an anatomical sample:

(1) Mouse serum collection: collecting serum by orbital blood sampling after last gastric lavage for 2h, centrifuging at 4deg.C for 10min at 3500r/min, collecting upper serum sample, and storing in a refrigerator at-80deg.C.

(2) Tissue material selection: after killing the mice by neck breakage, putting the heads of the mice on ice, taking out brain, liver, spleen and heart tissues, washing away blood and dirt by using physiological saline, weighing, preparing 10% tissue homogenate by using the physiological saline, the liver tissues and the brain tissues, centrifuging at the temperature of 4 ℃, centrifuging at the speed of 3500r/min for 10min, taking the supernatant, and storing in a refrigerator at the temperature of minus 80 ℃ for testing.

(3) HE staining material: mouse liver tissue was soaked in 4% paraformaldehyde and fixed overnight in a refrigerator at 4 ℃.

Liver histopathological examination:

(1) Soaking liver tissue soaked by paraformaldehyde in paraffin, dewaxing paraffin sections to water, sequentially placing the sections into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, cleaning, and drying the sections;

(2) Hematoxylin is impregnated for 3 minutes, hydrochloric acid alcohol differentiation liquid is differentiated for 2s after washing, and drying is carried out after washing;

(3) Eosin dyeing dip dyeing for 1 second, washing for 3s, and dehydrating with 100% alcohol for 2 seconds;

(4) After drying, sealing the neutral resin;

(5) The results were observed with an optical microscope and images were collected.

SOD, MDA, GSH-PX and GSH determination of mouse serum and brain tissue:

after the last gastric lavage for 2 hours, serum is collected by an orbital blood collection method, centrifuged for 10min at 4 ℃ and 300 mu L of serum is taken out from a centrifuge tube at 2500 r/min. GSH-Px, GSH, SOD activity and MDA concentration in serum of each group of mice are strictly determined according to the method of the kit instruction. Taking 0.1g of brain tissue, adding 0.9mL of ice physiological saline to prepare 10% brain tissue homogenate, centrifuging at 3500r/min for 10min, taking supernatant, and measuring MDA level, SOD, GSH-PX and GSH activity according to the requirements of the kit. The calculation results were averaged for statistical analysis.

Determination of IL-4, IL-6, TNF- α content of mouse liver tissue:

the 10% mouse liver tissue supernatant was prepared according to the above method, and the IL-4, IL-6 and TNF-alpha contents of the mice were measured by a double antibody sandwich method using an Elisa kit.

Mouse organ index determination:

organ index = organ mass (mg)/mouse mass (g) ×100%

Recording and calculating experimental data by using Excel 2018; processing by using SPSS 13.0 software, wherein the result is expressed as mean ± standard deviation, the comparison of the mean between groups adopts t test, and the chart is drawn by Origin 2018; the difference shown by P < 0.05 is statistically significant, and the difference shown by P < 0.01 is significant.

Experimental results and analysis:

the results of the statistical analysis of the mice mass data are shown in figure 2. After 2 weeks of intraperitoneal injection of D-galactose solution (300 mg/kg), the body mass of the mice in the model group was remarkably reduced compared with that of the blank group. After 4 weeks of modeling and administration, the weight of mice in the low, medium and high dose groups of Sanbao wine was significantly increased. After 6 weeks of modeling and dosing, the weight mass of mice in each dosing group was significantly increased compared to the model group, except for the base alcohol group. Experimental observation shows that the model group mice have aging symptoms such as slow action, reduced food intake, slow mechanical reaction and the like; the three-treasure wine dosage group has the effect of relieving the symptoms such as weight reduction, appetite reduction and the like of aged mice.

The heart, liver, spleen and kidney of each group of mice were subjected to organ index calculation and analysis, and the results are shown in table 4. Compared with the blank control group, the index of liver, kidney and spleen of the mice in the model group is obviously reduced (P is less than 0.01), and other administration groups have no statistical difference. The Sanbao wine administration group can relieve the reduction condition of the organ index of the mice to a certain extent, can not damage the organs of the mice, and meanwhile, the base wine group can not change the organ index change of the D-galactose to the mice.

TABLE 4 influence of Sanbao wine on organ index of D-galactose aging mice

Note that: p < 0.05, P < 0.01 compared to the placebo group; compared with the model group

The D-galactose induced aging model of mice can promote the generation of a large amount of free radicals in the mice, so that the level of lipid peroxide malondialdehyde in the mice is increased, and the level of antioxidant enzymes in the bodies such as superoxide dismutase, catalase, glutathione peroxidase and the like is obviously reduced. The results of analysis of SOD, GSH-PX, GSH activity and MDA content in the serum of mice are shown in figure 3. Compared with a blank group, the activity of SOD, GSH-PX and GSH in the serum of a model group is obviously reduced (P < 0.01), and the MDA content is obviously increased (P < 0.01), which indicates that the model modeling of an aging mouse model is successful; the activity of SOD, GSH-PX and GSH in the low, medium and high dose groups of the Sanbao liquor is obviously enhanced (P < 0.01) compared with the model group, wherein the improvement in the medium and high dose groups of the Sanbao liquor is obvious, and the concentration of MDA in the medium and high dose groups of the Sanbao liquor is obviously reduced (P < 0.01) compared with the model group. The mice in the basal alcohol group had no statistical difference in SOD, GSH-PX, GSH compared with the model group, and the MDA concentration was decreased (P < 0.05). The three-treasure wine can effectively improve the activity of SOD, GSH-PX and GSH in serum of the aging mice, reduce the MDA content and inhibit the development of symptoms of the aging mice.

The results of analysis of SOD, GSH-PX, GSH activity and MDA content in mouse brain tissue are shown in figure 4. Compared with the normal blank mice, the brain tissue of the model mice has significantly reduced activity of SOD, GSH, GSH-PX (P < 0.01); the concentration of MDA increased significantly (P < 0.01). Compared with mice in the model group, the comparison analysis shows that the activity of SOD, GSH, GSH-PX in the middle and high doses of Sanbao liquor is obviously increased (P < 0.01), and the activity of SOD and GSH in the low dose group has statistical difference (P < 0.05); the MDA content of the medium-dose group and the high-dose group of the Sanbao wine is obviously reduced (P < 0.01) compared with that of the model group, and the MDA concentration of the low-dose group and the base wine group is reduced (P < 0.05). The three-treasure wine can obviously improve the SOD, GSH, GSH-PX activity of brain tissues of the aging mice, and lighten the damage of oxidation reaction to the brain tissues, thereby achieving the anti-aging effect.

The effects of IL-4, IL-6 and TNF- α levels in mouse liver tissue were analyzed and the results are shown in Table 5. As shown in Table 5, the levels of IL-4, IL-6 and TNF-alpha in liver tissues of the model control group and the base wine control group were significantly increased (P < 0.01) compared with the blank control group, which indicates that D-galactose and base wine can destroy the enzyme defense system of the organism, up-regulate the expression of inflammatory factors IL-4, IL-6 and TNF-alpha, cause damage to the organism and accelerate aging. Compared with the model control group, the contents of IL-4, IL-6 and TNF-alpha in liver tissues of each dosage group of the Sanbao liquor are obviously reduced (P is less than 0.05 or P is less than 0.01), which proves that the Sanbao liquor not only can increase the activity and the content of antioxidant substances of organisms, but also can reduce the expression of inflammatory factors and delay the aging degree of the organisms.

TABLE 5 Effect of Sanbao wine on mouse liver tissue IL-4, IL-6 and TNF-alpha levels

The effect of the liver tissue pathological morphology of the aged mice caused by D-galactose was analyzed, and the analysis result is shown in FIG. 5. The antioxidant defense system is an important reason for preventing toxic reactions of oxygen and metabolic products, and maintaining cell balance is an important reason for maintaining cell survival. As can be seen from fig. 5, the liver tissues of mice in the blank group are clear, arranged radially, and orderly, without obvious cavitation, and the gaps around the cell nuclei are uniform; the cell arrangement of the liver tissue of the mice in the model control group is disordered, and most liver cells are in edema phenomenon, inflammatory cell infiltration and liver cell fibrosis; fat drop cavitation bubbles with different sizes appear in the liver tissue structure of mice in the base wine control group, liver cells are blurred, and the arrangement is disordered. Compared with the model control group and the base wine control group, the three-treasure wine mice in low, medium and high dose groups have clear liver tissue structure and orderly liver cell arrangement, and the phenomena of cell vacuoles and the like are obviously reduced. This demonstrates that the Sanbao wine can alleviate the damage of the base wine and the D-galactose to the liver tissue of the mice to a certain extent.

5. The measurement and analysis of insomnia treatment by the product:

experimental animals: SPF grade KM male mice, weighing 20-22g, license number: SCXK (Beijing) 2019-0010, available from Si Bei Fu (Beijing) Biotechnology Co., ltd.) was performed according to the requirements of the university of Guizhou medical science on ethics and the instruction of animal use.

Feeding conditions: the temperature (22+/-2) DEG C and the relative humidity (55+/-5)% clean animal houses, the lighting time is controlled to be 12 hours per day, the free drinking water and eating are realized, the natural light is alternated day and night, and the animal houses are adaptively fed for one week.

Experimental raw materials: the finished product of the three-treasure wine in the embodiment; alcohol-free group: the base wine is subjected to rotary evaporation to remove alcohol, and distilled water of equal amount is added.

Experimental grouping and dosing: 200 KM male mice were randomly divided into 2 batches according to body weight, experiment one: the sodium pentobarbital-induced mice sleep experiments were performed 60 times. Experiment II: neurotransmitter index and immunofluorescence, HE staining, intestinal microbiota assay were performed 140 alone. The experiment one group is as follows: blank group, model group, base wine group, positive drug group (esmolam), sanbao wine, alcohol-free group. The second group of experiments is: blank, model, base, positive (eszolam), low dose of treasures (5 mL/(kg.d), medium dose of treasures (10 mL/(kg.d)), high dose of treasures (15 mL/(kg.d)), low dose of alcohol-free (5 mL/(kg.d)), medium dose of alcohol-free (10 mL/(kg.d), high dose of alcohol-free (15 mL/(kg.d)), and administration time of stomach lavage for both experiment one and experiment two was 10d.

Establishing an insomnia mouse model: except for the blank group, PCPA suspension (400 mg/Kg, dissolved in normal saline) is injected intraperitoneally, the mice are continuously injected for 3 days 1 time a day, the mice behavior is observed, and compared with the blank group, the rest mice have the advantages of reduced sleeping time, frequent daytime activity, disordered hair, aggressiveness increase and weight reduction, which indicates that the insomnia mice model is successfully prepared. The administration was continued for 10d at a dose of 0.1mL/10g,1 time/d.

Direct sleep experiment: the animals of the test group were observed for sleep after administration of 3 doses of the test samples and the control group was administered the same volume of solvent. Sleep is indicated by the disappearance of the eversion and the reflection. When the mouse is placed in the dorsum position, the body position can be immediately turned over. If the person cannot turn over for more than 30-60 seconds, the person considers that the turning over reflection disappears and goes to sleep. The recovery of the regular reflection is the arousal of the animal, the time from the disappearance of the regular reflection to the recovery is the sleep time of the animal, and the number of animals falling asleep and the sleep time of the negative control group and the tested sample group are recorded.

Pentobarbital sodium subthreshold dose hypnotic test: the synergy of the test agent with sodium pentobarbital (or sodium barbiturate) was observed. Since sodium pentobarbital is metabolized by liver enzymes and drugs having an inhibitory effect on the enzymes also prolong the sleep time of sodium pentobarbital, to exclude this effect, subthreshold dose experiments should be performed. Preliminary experiments were performed prior to the formal experiments to determine the sub-threshold hypnotic dose of sodium pentobarbital (or sodium barbiturate) (pentobarbital 16-30mg/kg/BW or sodium barbiturate 100-150 mg/kg/BW), i.e., the maximum sub-threshold dose of sodium pentobarbital where 80-90% of mice do not have lost everlasting reflection. Animals were given solvent and samples at different concentrations last 10-15 minutes before peak effect, and each group was given their maximum sub-threshold hypnotic dose of pentobarbital sodium intraperitoneally, and the number of animals falling asleep (those with disappearance of the everlasting reflection for more than 1 minute) was recorded within 30 minutes. The experiment is preferably carried out in a quiet environment at 24-25 ℃.

Extended pentobarbital sodium sleep time trial: based on the hypnotic effect of the pentobarbital sodium, whether the test object can prolong the sleep time is observed, and if the sleep time is prolonged, the test object and the pentobarbital sodium have a synergistic effect. Preliminary experiments were performed prior to the formal experiments, and the dose of pentobarbital sodium (30-60 mg/kg/BW) was determined to make the animals fall asleep 100% but not for too long a period of time. After animals are given solvent and tested samples with different dosages at last time, 10-15 minutes before peak effect appears, the pentobarbital sodium is injected into the abdominal cavity of each group of animals, the injection amount is 0.2mL/20g, the disappearance of the eversion and the regular reflection is taken as an index, and whether the tested samples can prolong the sleeping time of the pentobarbital sodium is observed.

Barbital sodium sleep latency experiment: based on the hypnosis of barbital sodium, whether the test substance can shorten the sleep latency period is observed, and if the sleep latency period is shortened, the test substance and the barbital sodium have a synergistic effect. Pre-experiments were performed prior to the formal experiments, and the dose of barbituric sodium (200-300 mg/kg/BW) was determined to make the animals fall asleep 100% but not for an excessive period of time. After the animals are given with the solvent and the tested sample for 10-20 minutes at last time, the animals are intraperitoneally injected with the barbital sodium with the injection amount of 0.2mL/20g, the disappearance of the regular reflection is taken as an index, and the influence of the tested sample on the sleeping latency of the barbital sodium is observed.

The brain tissue of the mice is obtained and fixed: last time of gastric lavage administration for 1h, taking eyeball to obtain blood, killing the mice by breaking the ends, taking down the whole brain tissue of the mice, cleaning blood stain by using normal saline, fixing the brain tissue of the mice by using 4% paraformaldehyde, and storing in a refrigerator at 4 ℃.

HE stained sections:

(1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water.

(2) Hematoxylin staining: the slices are stained with hematoxylin dye solution for 3-5min, washed with running water, differentiated with differentiation solution, washed with running water, returned to blue, and washed with running water.

(3) Eosin staining: the slices are dehydrated in gradient alcohol of 85% and 95% for 5min respectively, and then are dyed in eosin dye solution for 5min.

(4) And (3) removing the water sealing piece: sequentially slicing, adding absolute ethyl alcohol I5 min-absolute ethyl alcohol II5 min-absolute ethyl alcohol III 5 min-dimethyl I5 min-dimethyl II5min, and sealing with neutral resin.

(5) Microscopic examination, image acquisition and analysis.

After the last gastric lavage administration for 1h, the mice are sacrificed, the cerebral cortex tissues of the mice are taken out after the broken ends, the cerebral tissues are washed off with normal saline to remove blood stains, 10% of tissue homogenate is prepared by the normal saline, the supernatant is sucked after centrifugation for 10min at 3500r/min at 4 ℃, and the supernatant is stored in a refrigerator at-80 ℃. The neurotransmitter changes in the mouse cerebral cortex were measured using enzyme-linked immunosorbent assay.

Experimental data results are expressed in mean±sd (standard deviation), plotted by GraphPad Prism 5 software, and significant differences compared to the blank group are expressed as #p <0.05, #p <0.01, # # p <0.001; compared to the model group, significant differences are expressed as p <0.05, p <0.01, p <0.001.

The analysis results of the direct sleep experiments are shown in Table 6, and after 7d of administration, the mice of each dose of experiment group are subjected to experiment observation that the mice are mostly represented as calm and reduced in activity after the administration of samples, the administration group has the person without regular reflection and the base alcohol group has partial direct sleep phenomenon, and the drunk condition is caused. Animals in the blank control group were all awake, indicating that none of the sample groups had a direct sleep effect on the mice.

TABLE 6 direct sleep experiments in mice

The results of the pentobarbital sodium subthreshold dose hypnotic test are shown in Table 7, and the pre-experiment shows that the experiment adopts 25mg/kg pentobarbital sodium for intraperitoneal injection, and the number of animals falling asleep within 30 minutes (the situation that the eversion disappears for more than 1 minute) is recorded. The mice in the blank group had a sleep rate of 40%, while the positive and sanbao groups had a sleep rate of 100%, and the base and alcohol-free groups had sleep rates of 70% and 50%, respectively. It shows that the Sanbao wine group can produce synergistic effect with pentobarbital sodium to raise the sleep rate of mouse. Compared with the comparative analysis of the falling asleep rate of mice in a blank group, the base wine group and the alcohol-free group can have a certain influence on the falling asleep rate of the mice to a certain extent.

TABLE 7 sub-threshold dose mouse hypnotic experiments with sodium pentobarbital

The effect of pentobarbital sodium on the sleep time and sleep latency of mice is shown in Table 8, and the pre-experiment results show that the pre-determined dose of pentobarbital sodium with hypnotic on the threshold of 50mg/kg (i.e. the dose of mice with the effect of losing the eversion) is selected, and the observation index is the sleep maintenance time. As can be seen from table 8, the sleep time of the drug administration group was significantly increased (P < 0.01) compared to the blank group, and the sleep time of the base wine group and the positive drug group was significantly increased (P < 0.01) compared to the blank group. Even the base wine group can prolong the sleeping time of the mice, but compared with the blank group and the Sanbao wine group, the sleep latency is obviously increased, which is not beneficial to effectively improving the sleeping quality of the mice. Compared with the blank mice, the three-treasure wine group can remarkably prolong the sleep time of the mice induced by pentobarbital sodium and reduce the sleep latency time of the mice, and compared with the base wine group, the three-treasure wine greatly shortens the sleep latency time of the mice and improves the sleep quality condition of the mice.

TABLE 8 sodium pentobarbital sleep synergy experiment mice sleep latency, sleep duration

The effect of Sanbao wine on the body weight level of mice is analyzed, and the analysis results are shown in Table 9. The weight of each group of mice changed, and the molding time was 3d in total. Mice in the blank group had normal sleep, diet and activity and smooth coat. After modeling, the mice in each group, except the blank group, showed continuous exercise around the day and night and frequently developed aggressive behavior; abnormal excitation, diet decrease and circadian rhythm disorder are caused by external sound and light thorn. The body weight of each of the remaining mice was significantly reduced (P < 0.01) compared to the mice in the blank group. Compared with the mice in the model group, the mice in the administration group have normal sleep-wake circulation, normal eating, white hair and weight recovery (P < 0.01). However, the hair dilution, appetite reduction and weight loss of the mice in the wine group were not significantly recovered, and it was presumed that alcohol intake could lead to appetite reduction. Indicating that after the intervention of administration, the sleep-lost mice recover the body weight, the food intake is increased and the behavior tends to be normal.

Table 9 influence of Sanbao wine on weight of mice suffering from insomnia caused by PCPA

Note that: in comparison with the set of models, ^# P<0.05, ^## P<0.01; comparison with blank group ^* P<0.05， ^** P<0.01

The results of analysis of the effect of Sanbao wine on the level of neurotransmitters in the cerebral cortex of insomnia mice are shown in FIG. 6. When insomnia occurs, the cerebral cortex is in an excited state, resulting in changes in some neurotransmitters in the body, especially in the decrease of inhibitory neurotransmitters such as GABA, and in the increase of excitatory neurotransmitters such as 5-HT. Compared with a blank group, the comparison analysis shows that the content of 5-HT, GIU, GABA, NE in the cerebral cortex tissue of the mice in the model group has a significant difference (P < 0.01), and positive drugs (esmolam), sanbao wine (low, medium and high dose groups) and alcohol-free groups (low, medium and high dose groups) can increase the content of 5-HT and GABA in the cerebral cortex compared with the model group, inhibit neurotransmitters and significantly inhibit central neuron excitation; simultaneously reduces the content of GIU and NE, and reduces the release of the arousal neurotransmitter. Compared with a blank group, the base wine group and the model group can not generate obvious change on the change of the inhibitory neurotransmitter of the cerebral cortex of the insomnia mouse, but increase the content of the excitatory neurotransmitter, so that the poor sleep quality of the insomnia mouse can be aggravated, and the mice can be subjected to more frequent behavioural changes such as circadian rhythm disorder, aggressiveness increase and the like.

The results of analysis of the effect of Sanbao wine on serum neurotransmitter levels in insomnia mice are shown in FIG. 7. The serum of the insomnia mice is tested, and compared with a blank group, the serum of the model group mice shows a remarkable reduction trend of the content of 5-HT and the content of GABA, which shows that the model formation of the insomnia by using p-phenylalanine (PCPA) is successful. A significant reduction in 5-HT also occurred in the serum of mice from the basal alcohol group relative to the blank group; however, there is no statistical significance compared to the model set. This suggests that the base alcohol group had no significant improvement in the symptoms of neurotransmitter dysfunction in insomnia mice caused by PCPA. The Sanbao wine (low, medium and high dose groups) and the alcohol-free group (low, medium and high dose groups) can increase the content of 5-HT and GABA in serum compared with a model group, inhibit neurotransmitters and obviously inhibit central nerve cell excitation; simultaneously reduces the content of GIU and NE, and reduces the release of the arousal neurotransmitter.

The results of the HE staining experiments are shown in FIG. 8. The boundary of the hippocampal nerve cells of the mice in the blank group is clear, the cells are dyed blue, the cells are larger and round, the phenomenon of shrinkage does not occur, the nuclei and cytoplasm are clear and visible, compared with the blank group, the model group and the base wine group both have the effects of neuronal cytopenia, disordered arrangement and the defect of cone cell integrity, and the phenomenon of shrinkage occurs. The positive medicine group has clear cell structure, can see nucleus and cytoplasm, and the Sanbao wine and alcohol-free group have obvious improvement compared with the model group, have clear cell structure, have improved cell morphology and reduced cell shrinkage phenomenon, and indicate that the administration group can relieve the symptoms of insomnia mice caused by PCPA by improving nerve cells in the brain tissues of the mice and simultaneously relieve the damage of alcohol to the nerve cells of the brain tissues of the mice to a certain extent.

In conclusion, the invention adopts the intraperitoneal injection of 350mg/kg PCPA to induce and prepare the insomnia mouse model, so as to examine the research on the action mechanism of Guizhou Sanbao liquor on the insomnia mouse. When insomnia occurs, the cerebral cortex is in an excited state, resulting in changes in some neurotransmitters in the body, especially in the decrease of inhibitory neurotransmitters such as 5-HT, GABA, etc., while the increase of excitatory neurotransmitters such as GIU, NE, etc. According to studies, it was found that the 5-HT content in the brain of mice was greatly reduced after the administration of chloroalanine by means of modelling. After the intervention of Sanbao liquor, the neurotransmitter changes of brain tissues and serum of the insomnia mice are measured by adopting an enzyme-linked immunosorbent assay. The experimental result shows that the brain tissue and serum of the insomnia mice are obviously increased (P < 0.01), and simultaneously, the inhibitory neurotransmitter GABA is increased, and the excitatory neurotransmitters such as GIU, NE and the like are reduced (P < 0.01). In addition, it was found that after alcohol intervention in insomnia mice, it was found that the mice in the group of base alcohol significantly reduced the GIU content (P < 0.01) in the brain tissue of the mice, but did not recover the symptoms of reduced 5-HT content in the brain tissue and serum of insomnia mice caused by PCPA. The intervention of the alcohol-free group also restored 5-HT in the brain tissue and serum of the hypnotic mice. In a pathological experiment, the HE staining of the nerve cells in the CAI region of the hippocampus of the mouse shows that the nerve cells of the mice in the model group and the base wine group undergo large-area apoptosis, the cell arrangement is disordered, and the cells are stained in dark color; the mouse hippocampal nerve cells of the Sanbao alcohol group and the alcohol-free group are arranged in relatively orderly and clear cell structures. The administration group is shown to have a protective effect on the damage of hippocampal nerve cells of the insomnia mice.

6. The influence of the product on intestinal microorganisms of insomnia mice is measured and analyzed:

sample preparation: before dissection, collecting the mouse faeces of each group, putting the collected mouse faeces samples into sterile 10mL centrifuge tubes, loading 200mg of each tube, placing the tubes in a refrigerator at the temperature of minus 80 ℃ for preservation, and sending the tubes to Shanghai Meiji biological medicine technology Co., ltd for detection under the dry ice state.

Total DNA extraction: DNA extraction kit is adopted first, and DNA extraction is carried out on 60 mouse faeces samples of different groups. Next, the concentration and purity were measured by using Nanodrop2000, and the integrity of DNA was measured by gel electrophoresis using 1% agarose gel electrophoresis at a voltage of 5V/cm for 20 min.

And (3) PCR amplification: the hypervariable region of the bacterial 16S rRNA gene V3-V4 is amplified by adopting a thermal cycle PCR system. Primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT), amplification system: transGenAP221-02 was used: transStart Fastpfu DNAPolymerase, 20. Mu.l reaction, i.e.4. Mu.l of 5 XFastpfu Buffer, 2. Mu.l of 2.5mM dNTPs, 0.8. Mu.l of upstream primer (5. Mu.M), 0.8. Mu.l of downstream primer (5. Mu.M), 0.4. Mu. LFastPfu DNAPolymerase, 0.2. Mu.l of BSA,10ng of genomic DNA, and ddH2O to 20. Mu.l. PCR reaction parameters: pre-denaturation: 1× (3 minutes at 95 ℃); denaturation: cycle number x (30 seconds at 95 °) annealing: 30seconds at annealing temperature ℃; extension (45 seconds at 72 ℃); stable extension (10 minutes at 72 ℃), preservation at 10 ℃ until the reaction is completed.

Sequencing by using a Miseq PE300 platform, calculating Alpha diversity knowledge Chao 1, shannon indexes and the like by using mothur software, and performing inter-group difference analysis of Alpha diversity by using Wilxocon rank sum test; PCoA analysis (principal coordinate analysis) based on a bray-curtis distance algorithm is used for detecting the similarity of the microbial community structures among samples, and PERMANOVA non-parametric detection is combined for analyzing whether the difference of the microbial community structures among sample groups is obvious or not; bacterial populations with significant differences in abundance from portal to genus level between the different groups were determined using LEfSe analysis (LDA >2, p < 0.05). Distance-based redundancy analysis was used to investigate the impact of soil physicochemical/clinical indicators on soil/intestinal bacterial community structure. Linear regression analysis was used to evaluate the effect of the primary biochemical index determined in the db-RDA analysis on the Alpha diversity index of the microorganism. Species were selected for correlation network map analysis based on the spaman correlation |r| >0.6p < 0.05. The results of this study are all expressed in x+ -s, and based on SPSS (v 19.0) and Graphpad prism (v 8.02) data processing, single-factor analysis of variance is used between sets of averages, and a minimum significant difference test is used for pairwise comparison of sample averages.

OTU classification result analysis: based on the sequencing results using the Miseq PE300 platform, a total of 70 samples were taken from 10 groups, 3304044 sequences were measured, and the sequences were screened to classify the sequences into a number of panels according to their similarity to each other, one panel being one OTU. OTU partitioning can be performed for all sequences according to different similarity levels, typically with a biometric analysis of OTUs at 97% similarity level. The obtained effective sequence is 1659910, and then the effective sequence is subjected to OTU classification, wherein each group of 7 samples is analyzed to obtain the result, wherein the result is shown in figure 9, the total number of the 10 groups is 327 OTUs, the total number of the blank groups is 4 OTUs, the model groups are 3 OTUs, the base wine groups are 1 OTU, the positive groups are 1 OTU, the three-treasure wine low-dose groups are 3 OTUs, the three-treasure wine is 4 OTUs, the high-dose groups are 4 OTUs, the alcohol-free groups are 9 OTUs, the medium-dose groups are 2 OTUs, and the high-dose groups are 5 OTUs.

Alpha diversity analysis of insomnia mice intestinal flora by Sanbao wine:

the information of abundance, coverage, diversity and the like of the species in the intestinal community can be obtained through diversity index analysis, and the common measurement standards include sob, shannon, ace, simpson and the like. The general sob, ace index is used to represent the richness of the community of intestinal microorganisms of the sample. shannon, simpson and the like are used to reflect the diversity of the community of intestinal microorganisms of the sample, the higher the shannon value, the higher the community diversity, and the higher the simpson index value, the lower the community diversity. As shown in fig. 10, the shannon values of the model group were significantly reduced and the simpson index values were increased relative to the blank group; the sob and ace indexes of the model group are obviously reduced. The intestinal microorganism diversity and the richness of the mice in the model group are greatly reduced. However, after the insomnia mice ingest the Sanbao liquor, the intestinal flora structure of the insomnia mice is improved to a certain extent. The sob and ace indexes of the administration group are remarkably increased relative to the model group; the shannon value increases and the simpson index value becomes smaller. The simultaneous base wine group is not twisted about the symptoms of flora diversity, richness reduction and the like of the intestinal flora of the insomnia mice no matter the richness and the diversity of the intestinal flora.

Effect of sanbao on intestinal flora level species composition of insomnia mice:

the high-throughput sequencing technology is adopted in the research to analyze the composition structure of the insomnia and mouse fecal microorganism caused by PCPA by Sanbao wine. The OTU representative sequences at 97% similarity level were subjected to taxonomic analysis using RDP classifer bayesian algorithm and at each taxonomic level: domain, kingdom, phylum, class, order, family, genus, specie, and the like. Statistical analysis was performed on the composition of 10 groups, namely blank, model, base, positive, treasured low, medium, high, no alcohol medium, no alcohol high, exclusively at the levels of their intestinal flora Phylum (Phylum) and Genus (Genus). As shown in fig. 11, the intestinal flora of the blank group, model group, base alcohol group, positive group and two dosing groups is mainly divided into 10 phylum, namely, firmicutes, bacteroides (bacterioidota), actinomycetes (actionobacteria), (campylobacter), desulphus (desulphus), proteus (proteus), verruca (verrucomicrobiosa), deferens (defericillium), patella bacteria (patascibera), cyanobacteria (Cyanobacteria), and other species (other) for determining classification, at the phylum level. The thick-walled mycota is the most abundant mycota in each group, and is the absolute dominance mycota of the 10 groups. The relative abundance of the firmicutes in each group was 50% for the blank group, 47% for the model group, 57% for the base wine group, 61% for the positive group, 56% for the low dose group of the treasures wine, 56% for the medium dose group of the treasures wine, and 58% for the high dose group of the treasures wine; the alcohol-free low dose group is 45%, the alcohol-free medium dose group is 48%, and the high dose group is 49%; the bacteroides is the second dominant bacteria in each group, and the relative abundance of the bacteria group and the absolute dominant bacteria group on the portal level of the insomnia mice can be increased in the three-treasure dosing group relative to the blank group and the model group, as shown by the analysis that the blank group is 48%, the model group is 43%, the base wine group is 32%, the positive group is 32%, the three-treasure low dose is 37%, the three-treasure dosing group is 38%, the three-treasure dosing group is 36%, the alcohol-free low dose group is 45%, the alcohol-free medium dose group is 47% and the alcohol-free high dose is 43%.

As shown in fig. 12, the composition and kind analysis of intestinal flora microorganisms was performed on fecal samples of each group of mice based on genus level. The mice in the blank group were in a plurality of bacteria such as Lactobacillus (33%), norank_f __ Muribaculaceae (31%), bacteroides (9.8%), staphylococcus (1.8%), and Bacillus (allobaculo) (0.76%) at the genus level. The main species contained at the model group genus level were Lactobacillus (8.3%), nrank_f __ Muribacuerae (32%), bacteroides (1.8%), staphylococcus (6.1%), isopobacterium (allobacum) (0.1%), respectively; the low, medium and high dose groups of the Sanbao wine mainly comprise Lactobacillus (35%, 38%, 28%), (norank_f __ Muribaculaceae) (29%, 31%, 27%), bacteroides (3.0%, 2.7%, 2.9%), staphylococcus (Staphylococcus) (0.56%, 3.2%, 15%), and heterologous bacillus (allobaculom) (1.3%, 2.6%, 2.7%); the alcohol-free group was low, medium and high doses of Lactobacillus (18%, 25%), (nonrank_f __ Muribamulac) (30%, 35%, 32%), bacteroides (Bacteroides) (7.3%, 4.5%, 6.9%), staphylococcus (Staphylococcus) (0.66%, 0.95%, 0.74%), and Bacillus (Alobacus) (2.1%, 1.5%, 1.8%). The relative abundance of lactobacillus in the mice of the model group is obviously reduced compared with the blank group, the relative abundance of lactobacillus in the three-treasure wine administration group is obviously increased compared with the mice of the model group, other strains are also added, and compared with the mice of the model group, the dominant bacteria of the intestinal flora of the mice of the alcohol-free group are also added.

Differential analysis of intestinal flora of insomnia mice by Sanbao wine:

as shown in fig. 13, 8 species of bacteria with statistical difference in relative abundance in each group of samples were selected from the subordinate horizontal, wherein the bacteria with statistical difference (P < 0.5) in the intestinal flora of the model group mice were respectively unknown genus f-type gastroenterology (nrank_f __ peptococea), unknown genus f-type Ruminococcaceae (nrank_f __ ruminococcales), lachnospirace_nk 4a136_group (trichosporon), deisobacterium (defericillium), lactobacillus (lactobacilli), nrank_f __ eubacterium_copostanoligenes_group, bacteroides (Bacteroides), and butyric acid monads (buricoides), as compared with the blank group.

Compared with the model group, the low-dose group of the Sanbao liquor administration group can call back Lactobacillus, butyricimonas of the fecal matter of the insomnia mice; the low, medium and high dose groups of the Sanbao wine can down regulate unclassified_f __ Erysipelotrichaceae, deferribacterota of the mouse faeces with insomnia; the middle dose group of Sanbao wine extremely significantly down-regulates unclassified_f __ Erysipelotorich (P < 0.05), and the normal_f __ Peptococcaceae, norank _f __ Ruminococaceae (P < 0.01); the high dose group of Sanbao wine can be down-regulated to Deferribacterota, norank _f __ Peptococcaceae, norank _f __ Ruminococaceae (P < 0.01); the low dose group of the alcohol free group may call back Lactobacillus, butyricimonas, norank _f __ eubacterium_co-procostanoligens_ group, bacteroides;

The middle and high dose groups of the alcohol-free group can call back the normal_f __ Eubacterium_co-procostanoligens_group; the high dose group of the alcohol free group may call back to bacterioides. Simultaneously, the low dose groups of the alcohol-free groups can down-regulate the Norank_f __ Peptococcales and the Norank_f __ Ruminococcales; the high dose group can significantly down-regulate the norank_f __ Ruminococcea and the norank_f __ Peptococcea; the medium dose group can down-regulate unclassified_f __ Erysipelototrichace and norank_f __ Peptocochace. Therefore, both the Sanbao wine and the alcohol-free group can call back or down regulate the relative abundance of the mouse intestinal flora disturbed by insomnia. By analyzing the changes of the phylum level and the flora composition of the genus level of different groups of intestinal microorganisms and the relative abundance of the flora, the three-treasure wine administration group and the alcohol-free group can have a certain influence on the intestinal microorganisms of the insomnia mice. When insomnia occurs, the relative abundance of beneficial flora in the intestinal tract of the mice is reduced, and the abundance of harmful bacteria and pathogenic bacteria is increased. After the administration intervention of the Sanbao wine and the alcohol-free group is given to the insomnia mice, the intestinal flora diversity of the insomnia mice can be effectively recovered, the proportion of beneficial bacteria to pathogenic bacteria flora is changed, the intestinal flora of the insomnia mice is recovered to a normal state to a certain extent, and the effect of regulating the insomnia is achieved.

Correlation analysis of intestinal microorganisms and cerebral cortex indexes of insomnia mice:

based on the research theory of flora-gut-brain axis, intestinal flora can affect the central nervous system by modulating the serotonin system of the brain, e.g. by modulating the secretion of serotonin and tryptophan, cortisol; or functional metabolites such as 5-HT, GABA, etc. are produced which affect the circadian rhythm variation of the brain. As shown in fig. 14, the correlation of microorganisms with environmental variables was evaluated by showing the relationship between different species in the sample and the environmental variables. On the basis of the genus level classification, the relative abundance of the colonies of the intestinal flora of the mice among different groups is obviously different, the abundance of the bacteria is represented by red and blue, the higher the abundance is, the closer to red, and the lower the abundance is, the closer to blue is. As can be seen from the figure, the microbial flora positively correlated with the difference of 5-HT is Lactobacillus, phascolarctobacterium (P < 0.05), and the microbial flora negatively correlated with the difference of 5-HT is unclassified_f __ Lachnospiraceae, norank _f __ Oscillospiraceae, desulfovibrio (P < 0.05); the microbial flora which is extremely obviously and positively correlated with GABA is Candida-Saccharimonas (P < 0.05), family_XIII_AD3011_group (P < 0.05), christensenenlace_R-7_group (P < 0.05), blauthia (P < 0.01), NK4A214_group (0.05 < P < 0.01), and extremely and obviously and negatively correlated with GABA is allocupvotela; microbial flora which is extremely and positively correlated with NE is Pharmacolarbacterium (P < 0.01), family_XIII_AD3011_group (P < 0.01), christenssellaceae_R-7_group (0.05 < 0.01), blautia, NK4A214_group (P < 0.01), lachnoclostrichum (P < 0.01), non_f __ non_o __ Clostridia_UCG-014 (P < 0.05), ruminocus_gnav_ group, bacteroides (P < 0.05), UCG-005 (P < 0.05), prevoltellaceae_NK3B31_group (P < 0.05), non_ __ _tissue_UCG-014 (P < 0.05), and Rumincoccum_35_gnus_ group, bacteroides (P < 0.05); the microbial flora that is extremely significantly inversely related to the presence of NE is Bifidobacterium (P < 0.05); the microbial flora positively related to the difference of the GIU is Romboustia (P < 0.05), dubosiella (P < 0.05), faecalibaculum (P < 0.05), bifidobacterium (P < 0.05). In summary, the intestinal flora has a certain correlation with neurotransmitters of cerebral cortex tissues of insomnia mice, and the mainly related bacteria are classified into Phascolarctobacterium, christensenellaceae _R-7_group and Blauthia bacteria.

Therefore, the Sanbao wine can influence the structural change of intestinal microorganisms of the insomnia mice and the genus bacteria closely related to the levels of neurotransmitters and inflammatory factors in the brain of the insomnia mice, thereby improving the insomnia condition of the mice.

Claims (10)

1. A Sanbao wine is characterized in that: comprises the following components in parts by volume: 40-50 parts of gastrodia tuber extract, 25-35 parts of ganoderma extract and 15-25 parts of eucommia bark extract, wherein the alcohol content of the Sanbao wine is 37% vol.

2. A Sanbao wine is characterized in that: comprises the following components in percentage by volume: 49% of gastrodia elata leaching liquor, 30% of ganoderma lucidum leaching liquor and 21% of eucommia ulmoides leaching liquor, wherein the alcoholic strength of the Sanbao liquor is 37% vol.

3. A preparation method of Sanbao wine is characterized in that: comprises the steps of extracting gastrodia tuber, ganoderma lucidum and eucommia bark according to the volume ratio of 49:30:21, adding honey to obtain a mixed liquor with the alcohol content of 37%vol.

4. The method for preparing the Sanbao wine according to claim 2, wherein: the preparation method of the gastrodia elata leaching solution comprises the following steps: crushing the dried gastrodia elata, adding base wine according to the feed liquid ratio of 1:10, uniformly mixing, sealing, performing ultrasonic power 220w, performing ultrasonic treatment for 25-35 min, shaking for 1-3 times a day, soaking for 5 days, and filtering to obtain gastrodia elata leaching solution.

5. The method for preparing the Sanbao wine according to claim 2, wherein: the preparation method of the ganoderma lucidum leaching solution comprises the following steps: pulverizing dried Ganoderma, adding base liquor according to a feed liquid ratio of 1:10, mixing, sealing, ultrasonic power of 220w, ultrasonic time of 25-35 min, shaking for 1-3 times a day, soaking for 30 days, and filtering to obtain Ganoderma leaching solution.

6. The method for preparing the Sanbao wine according to claim 2, wherein: the preparation method of the eucommia ulmoides leaching solution comprises the following steps: crushing the dried eucommia ulmoides, adding base liquor according to the feed liquid ratio of 1:10, uniformly mixing, sealing, performing ultrasonic power of 220w, performing ultrasonic treatment for 25-35 min, shaking for 1-3 times a day, soaking for 15 days, and filtering to obtain Du Zhongjin extract.

7. The method for producing a sanbao wine according to any one of claims 2 to 6, wherein: the base wine adopts a sauce-flavor base wine with the alcoholic strength of 50%vol.

8. The use of the sanbao liquor according to any one of claims 1-2 in the preparation of an anti-oxidative aging health food or drug capable of increasing GSH-Px, SOD and GSH content and activity, decreasing MDA content and down-regulating IL-4, IL-6, TNF- α levels.

9. The use of the sanbao liquor according to any one of claims 1-2 in the preparation of hypnotic health-care food or medicine capable of increasing the content of 5-HT and GABA, reducing the content of GIU and NE, and improving pressure type insomnia.

10. Use of the sanbao wine according to any one of claims 1-2 for the preparation of a health food or a medicine for improving intestinal flora disorder caused by insomnia.