CN113186065A

CN113186065A - Health wine for enhancing immunity and preparation method thereof

Info

Publication number: CN113186065A
Application number: CN202110451263.4A
Authority: CN
Inventors: 曲风采
Original assignee: Jilin Daqing Luyuan Health Care Technology Co ltd
Current assignee: Jilin Daqing Luyuan Health Care Technology Co ltd
Priority date: 2021-04-26
Filing date: 2021-04-26
Publication date: 2021-07-30

Abstract

The invention relates to health care wine for enhancing immunity and a preparation method thereof, and relates to the technical field of health care products. The formula of the health wine provided by the invention is as follows: 8-12g of American ginseng, 6-10g of pseudo-ginseng, 18-22g of lucid ganoderma, 6-10g of red deer bone, 28-32g of sea buckthorn, 18-22g of rhizoma polygonati and 18-22g of wolfberry fruit; 18-22g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL. The preparation method of the health wine comprises the following steps: weighing radix Panacis Quinquefolii, Notoginseng radix, Ganoderma, Os Cervi, fructus Hippophae, rhizoma Polygonati, and fructus Lycii according to formula proportion, adding 8 times of Chinese liquor, extracting at room temperature, and filtering the extractive solution; weighing Mel according to formula ratio, mixing Mel and the extractive solution, stirring, adding Chinese liquor and purified water, blending until alcoholic strength is (35 + -1)%, adjusting to formula amount, refrigerating, standing, collecting supernatant, standing to room temperature, filtering with plate-and-frame filter, bottling, and sealing. The health care wine has the function of enhancing immunity, and is safe and non-toxic.

Description

Health wine for enhancing immunity and preparation method thereof

Technical Field

The invention relates to the technical field of health care products, in particular to health care wine for enhancing immunity and a preparation method thereof.

Background

Immunity is the body's own defense mechanism, and is the body's ability to recognize and destroy any foreign body (virus, bacteria, etc.) invaded from the outside, to treat aged, damaged, dead, denatured self-cells, and to recognize and treat in vivo mutant cells and virus-infected cells, and is the body's physiological response to recognize and eliminate "heterosis". However, with the improvement of the quality of life, the work rhythm is faster and faster, the mental pressure of people is larger and larger, the body is in a sub-health state, and the immunity of the human body is reduced. The immune function failure such as low immune function, immune dysfunction and the like is a precursor of serious diseases such as cardiovascular and cerebrovascular diseases, diabetes, hypertension, hyperlipidemia, fatty liver, cancer and the like of human beings, is also a root cause of diseases which are difficult to cure, and cannot be ignored. Therefore, improving the autoimmunity of the human body is the basis for preventing and treating sub-health.

Chinese patent application with publication number CN111760002A discloses a traditional Chinese medicine for improving immunity, which is prepared from the following raw materials: the invention relates to a traditional Chinese medicine health-care food, which is prepared from cassia twig, Chinese herbaceous peony, divaricate saposhnikovia root, largehead atractylodes rhizome, astragalus root, ginger, Chinese date, honey-fried licorice root and dangshen and has the advantages of improving the fatigue resistance of an organism, enhancing the immunity of the organism, improving the disease resistance, having lasting effect and no toxic or side effect, exerting the advantage of integral regulation and improving the life quality of suitable people. Compared with other similar health-care foods, the health-care food has certain comprehensive advantages, and provides a safe, effective, reliable and cheap health-care food for suitable people with fatigue symptoms and low immunity. Chinese patent application with publication number CN111329909A discloses an oral liquid for improving human immunity, which comprises the following raw materials by weight: 10-100 parts of ginseng, 10-100 parts of adenophora stricta, 10-100 parts of radix pseudostellariae, 10-100 parts of codonopsis pilosula, 10-100 parts of salvia miltiorrhiza, 10-100 parts of astragalus membranaceus, 10-100 parts of bighead atractylodes rhizome, 10-100 parts of angelica sinensis, 10-100 parts of acanthopanax, 10-100 parts of oldenlandia diffusa, 10-100 parts of hawthorn, 10-100 parts of liquorice, 10-100 parts of cistanche and 10-100 parts of Chinese date. The oral liquid is prepared by matching 14 Chinese herbal medicines such as ginseng, adenophora stricta, radix pseudostellariae, codonopsis pilosula, salvia miltiorrhiza and the like, wherein astragalus, bighead atractylodes rhizome and acanthopanax senticosus are used as qi tonics and can tonify qi and generate yang, Chinese angelica is used as blood tonics and can enrich blood and promote blood circulation, and oldenlandia diffusa shell is used for clearing heat and removing toxicity.

However, the effect of the product for enhancing immunity in the prior art still needs to be improved, so that the search for a more ideal daily health-care product suitable for enhancing immunity and improving health state is still an important task in the field of immunity enhancement and health care development.

Disclosure of Invention

The invention aims to provide health-care wine for enhancing immunity and a preparation method thereof.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the invention provides a health wine for enhancing immunity, which comprises the following components in part by weight:

8-12g of American ginseng, 6-10g of pseudo-ginseng, 18-22g of lucid ganoderma, 6-10g of red deer bone, 28-32g of sea buckthorn, 18-22g of rhizoma polygonati and 18-22g of wolfberry fruit;

18-22g of honey;

adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%;

the total content of the health wine is 1000 mL.

In the above technical solution, preferably, the formula of the health wine is as follows:

8g of American ginseng, 6g of pseudo-ginseng, 18g of lucid ganoderma, 6g of red deer bone, 28g of sea buckthorn, 18g of rhizoma polygonati and 18g of wolfberry fruit;

18g of honey;

the total content of the health wine is 1000 mL.

12g of American ginseng, 10g of pseudo-ginseng, 22g of lucid ganoderma, 10g of red deer bone, 32g of sea buckthorn, 22g of rhizoma polygonati and 22g of wolfberry fruit;

22g of honey;

the total content of the health wine is 1000 mL.

In the above technical solution, most preferably, the formula of the health wine is as follows:

10g of American ginseng, 8g of pseudo-ginseng, 20g of lucid ganoderma, 8g of red deer bone, 30g of sea buckthorn, 20g of rhizoma polygonati and 20g of wolfberry fruit;

20g of honey;

the total content of the health wine is 1000 mL.

The invention also provides a preparation method of the health wine for enhancing immunity, which comprises the following steps:

step 1, weighing American ginseng, pseudo-ginseng, lucid ganoderma, red deer bone, sea buckthorn, rhizoma polygonati and wolfberry fruit according to a formula ratio for later use;

step 2, adding 8 times of white spirit into American ginseng, pseudo-ginseng, lucid ganoderma, red deer bone, sea backthern, sealwort and medlar to carry out normal temperature leaching, and filtering leaching liquor;

step 3, weighing honey according to the formula proportion, mixing the honey and the leaching liquor, stirring uniformly, adding white spirit and purified water, blending until the alcoholic strength is (35 +/-1)%, adjusting to the formula amount, refrigerating and standing, taking supernatant, standing to room temperature, filtering by using a plate-and-frame filter, filling, and sealing.

In the technical scheme, the white spirit used for leaching in the step 2 is 35% white spirit.

In the above technical scheme, the leaching time in the step 2 is 15 days.

In the technical scheme, a 120-mesh stainless steel net is used for filtering in the step 2.

In the technical scheme, the temperature for refrigerating and standing in the step 3 is 0-4 ℃ and the time is 24 hours.

In the above technical scheme, the liquor in step 3 is 65% liquor.

The invention has the beneficial effects that:

the health care wine for enhancing immunity takes American ginseng, pseudo-ginseng, lucid ganoderma, red deer bone, sea buckthorn, rhizoma polygonati and medlar as raw materials, takes honey, white spirit and water as auxiliary materials, and the American ginseng in the formula has the effects of tonifying qi, generating blood, producing sperm and nourishing spirit; the main active ingredients are saponin, polysaccharide and the like; is a good tonic product for enhancing immunity in clinic. Ganoderma lucidum is a precious medicinal fungus in China, and mainly contains ganoderan, triterpenes and the like; has effects in invigorating qi, tranquilizing mind, and relieving cough and asthma; it can be used for treating cough and asthma due to lung deficiency, and asthenia and short breath. The red deer bone has the effects of tonifying deficiency and strengthening bones and muscles. Tang Ben Cao (materia Medica of Tang Dynasty): it can treat consumptive disease, and it can treat wind deficiency and tonify bone marrow. The sea buckthorn is a medicine and food dual-purpose raw material containing rich nutritional ingredients and bioactive substances, is rich in various chemical ingredients such as vitamins, amino acids, flavonoids and the like, and has various physiological functions such as immunity improvement and the like. Rhizoma Polygonati has effects of invigorating spleen and replenishing qi, invigorating qi and nourishing yin, invigorating spleen, moistening lung, invigorating kidney, strengthening tendons and bones, and can be used for treating spleen and stomach qi deficiency, tiredness and debilitation, essence and blood deficiency, soreness of waist and knees, etc. Fructus Lycii has effects of nourishing liver and kidney, replenishing vital essence, and improving eyesight; can be used for treating consumptive disease, essence deficiency, soreness of waist and knees, and sallow complexion due to blood deficiency. The raw materials have the health-care function of enhancing the immunity, and the raw materials cooperate with each other to achieve the health-care function of enhancing the immunity, and are safe and non-toxic.

Detailed Description

18-22g of honey;

the total content of the health wine is 1000 mL.

Preferably, the formula of the health wine is as follows: 8g of American ginseng, 6g of pseudo-ginseng, 18g of lucid ganoderma, 6g of red deer bone, 28g of sea buckthorn, 18g of rhizoma polygonati and 18g of wolfberry fruit; 18g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL.

Preferably, the formula of the health wine is as follows: 12g of American ginseng, 10g of pseudo-ginseng, 22g of lucid ganoderma, 10g of red deer bone, 32g of sea buckthorn, 22g of rhizoma polygonati and 22g of wolfberry fruit; 22g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL.

Most preferably, the formula of the health wine is as follows: 10g of American ginseng, 8g of pseudo-ginseng, 20g of lucid ganoderma, 8g of red deer bone, 30g of sea buckthorn, 20g of rhizoma polygonati and 20g of wolfberry fruit; 20g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL.

step 2, taking American ginseng, pseudo-ginseng, lucid ganoderma, red deer bone, sea buckthorn, sealwort and medlar, adding 8 times of 35 percent white spirit, leaching for 15 days at normal temperature, and filtering the leaching liquor by using a 120-mesh stainless steel net;

step 3, weighing honey according to the formula proportion, mixing the honey and the leaching liquor, stirring uniformly, adding 65% of white spirit and purified water, blending until the alcoholic strength is (35 +/-1)%, adjusting to the formula amount, refrigerating and standing at 0-minus 4 ℃ for 24h, taking supernatant, standing to room temperature, filtering by using a plate-and-frame filter, filling and sealing.

The health wine of the invention has the following raw and auxiliary materials quality requirements:

1. american ginseng, pseudo-ginseng, lucid ganoderma, sea buckthorn, rhizoma polygonati and wolfberry fruit: it should meet the corresponding requirements of pharmacopoeia of the people's republic of China.

2. And (3) red deer bone: it should meet the requirements of deer bone from the first volume of Chinese medicinal materials in the standards of medicine of Ministry of health.

3. Honey: the honey milk powder should meet the regulation of GB14963 national standard honey for food safety.

4. White spirit (alcohol content 65%): the wine should meet the regulation of GB/T10781.2 Qing-Feng-type white spirit.

5. Purifying water: it should meet the regulations of pharmacopoeia of the people's republic of China.

The health wine prepared by the invention meets the following sensory requirements, physicochemical indexes and index of marked components.

TABLE 1-1 sensory requirements

TABLE 2-2 physicochemical indices

TABLE 3-3 index of the marking Components

Example 1

The formula of the health wine for enhancing immunity is as follows: 10g of American ginseng, 8g of pseudo-ginseng, 20g of lucid ganoderma, 8g of red deer bone, 30g of sea buckthorn, 20g of rhizoma polygonati and 20g of wolfberry fruit; 20g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL.

The preparation method of the health wine for enhancing immunity comprises the following steps:

step 3, weighing honey according to the formula proportion, mixing the honey and the leaching liquor, stirring uniformly, adding 65% of white spirit and purified water, blending until the alcoholic strength is (35 +/-1)%, adjusting to the formula amount, refrigerating at-2 ℃, standing for 24h, taking supernatant, standing to room temperature, filtering by using a plate-and-frame filter, filling, each bottle being 100ml, and sealing.

The health wine prepared by the embodiment meets the sensory requirements, physical and chemical indexes and index of marked components.

Example 2

Different from the example 1, the formula of the health wine of the embodiment is as follows: 8g of American ginseng, 6g of pseudo-ginseng, 18g of lucid ganoderma, 6g of red deer bone, 28g of sea buckthorn, 18g of rhizoma polygonati and 18g of wolfberry fruit; 18g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL.

The health wine prepared by the embodiment has the function of enhancing immunity, and is safe and non-toxic.

Example 3

Different from the example 1, the formula of the health wine of the embodiment is as follows: 12g of American ginseng, 10g of pseudo-ginseng, 22g of lucid ganoderma, 10g of red deer bone, 32g of sea buckthorn, 22g of rhizoma polygonati and 22g of wolfberry fruit; 22g of honey; adding Chinese liquor and purified water, and blending until the alcohol content is 35 + -1%; the total content of the health wine is 1000 mL.

The test report of the immunity enhancing function test and the safety toxicology of the health wine prepared in the embodiment 1 of the invention is as follows:

firstly, the health care wine is tested by the centers for disease prevention and control in Tianjin, and the safety toxicology test report is as follows:

1. materials and methods

1.1 sample: the health wine prepared in the embodiment 1 of the invention has the intended dosage of 100ml/60kg BW per day of stock solution, 25 times of concentrated solution of a tested object and brown liquid with the alcoholic strength of 0; the alcohol concentration of the wine base is 50% (v/v). The concentration was 4.0ml/60kg BW daily in terms of the intended dose.

1.2 Experimental animals: SPF-grade Kunming mice and Wistar rats were provided by Beijing Wintonlihua laboratory animal technology, Inc. (for acute oral toxicity test of mice and rats, micronucleus test of mouse bone marrow cells, and teratospermia test of mice). The feed is provided by Beijing Huafukang Biotechnology GmbH. 1.3 oral acute toxicity test:

1.3.1 mice oral acute toxicity test (LD)₅₀): selecting 20 SPF-grade Kunming mice with female and male half at a dose of 15.0g/kg & BW for oral intragastric administration (weighing 150.0g of sample concentrated solution, adding 60ml of 50% wine base, adding distilled water to 200ml, the concentration is 0.75g/ml), the intragastric administration amount is 0.2ml/10g & BW, fasting is carried out for 16h before intragastric administration, and continuous observation is carried out for 14 days after administration. The signs of intoxication and death were recorded.

1.3.2 rat oral acute toxicity test (LD)₅₀): 180-piece 220g SPF-level Wistar rats with half of male and female are selected, and are subjected to oral intragastric lavage (150.0 g of sample concentrated solution is weighed, 60ml of 50% alcohol base is added, distilled water is added to the sample concentrated solution to 200ml, the concentration is 0.75g/ml) once at the dosage of 15.0g/kg & BW, and the lavage amount is 2.0ml/100 g.BW, fasting for 16h before gavage, and continuous observation for 14 days after administration. The signs of intoxication and death were recorded.

1.4 genotoxicity test

1.4.1 Ames test: the test is carried out by four test strains of the salmonella typhimurium histidine-deficient strain TA97, TA98, TA100 and TA102 which are identified to meet the requirements. Liver homogenate prepared after rats are induced by phenobarbital and beta-naphthalenone is used as an in vitro metabolic activation system. Weighing 0.5g of sample concentrated solution, adding sterile distilled water to 10ml, fully and uniformly mixing, carrying out autoclaving at the temperature of 121 ℃ for 20 minutes, and uniformly mixing to obtain the solution I. Taking 2ml of the solution, adding 8ml of sterile distilled water, uniformly mixing to obtain a solution II, taking 2ml of the solution, adding 8ml of the sterile distilled water, uniformly mixing to obtain a solution III, taking 2ml of the solution, adding 8ml of the sterile distilled water, uniformly mixing to obtain a solution IV, taking 2ml of the solution IV, adding 8ml of the sterile distilled water, and uniformly mixing to obtain a solution V (the concentrations of the tested substances are respectively 50, 10, 2, 0.4 and 0.08 mg/ml). The test is provided with 5 doses of 8, 40, 200, 1000 and 5000 mug/dish, and is also provided with a blank control and a positive control. 0.1ml of test strain enrichment liquid, 0.1ml of test substance and 0.5ml of S9 mixed liquid (when metabolic activation is needed) are added into top agar, mixed uniformly and poured onto a bottom culture medium plate, and 3 parallel samples are made for each dose and control. Incubate at 37 ℃ for 48h and count the number of colonies per dish. A test is positive if the number of colonies with the reversion is more than 2 times that of the blank colonies and has a dose-response relationship. The whole set of experiments was repeated once under the same conditions.

1.4.2 mouse bone marrow cell micronucleus assay: the test was performed by oral gavage with two 24h intervals. 60 mice with a weight of 25-30g were randomly divided into 6 groups of 10 mice each, each half of which was male and female. Cyclophosphamide with a dose of 40mg/kg & BW is used as a positive control (40.0 mg cyclophosphamide is weighed and added with sterile physiological saline to 10ml, and the administration is carried out by intraperitoneal injection twice at an interval of 24h, and the administration amount is 0.1ml/10g & BW). The sample dosage is designed to be 2.5, 5.0 and 10.0g/kg & BW respectively, the tested substances are weighed to be 5.0g, 10.0g and 20.0g respectively, each is added with 12ml of 50 percent wine base and then distilled water to be 40ml, the mixture is fully mixed, the concentration is 0.125, 0.250 and 0.500g/ml respectively, the stomach filling amount is 0.2ml/10g & BW, the same amount of distilled water is given to a control group, and the same amount of 15 percent wine base is given to a wine base control group.6h after the last test, the animals were sacrificed by cervical dislocation, and the sternal bone marrow was diluted with calf serum and smeared, fixed with methanol, and stained with Giemsa. Counting 1000 pleochromophilic erythrocytes (PCE) in each animal under an optical microscope, wherein the microkernel occurrence rate is measured by PCE thousandths containing microkernels, and performing statistical treatment by using Chi²Checking; the PCE/NCE ratio is additionally calculated.

1.4.3 mouse teratospermia test: sex-matured male mice weighing 25-35g were used in 30 groups of 5 mice each randomized into 6 groups. Cyclophosphamide at a dose of 40mg/kg & BW was used as a positive control (40 mg cyclophosphamide was weighed and then added to 10ml sterile physiological saline for intraperitoneal injection at a dose of 0.1ml/10g & BW) once a day for 5 consecutive days. The sample dosage is designed to be 2.5, 5.0 and 10.0g/kg & BW respectively, the tested substances are weighed to be 5.0g, 10.0g and 20.0g respectively, each is added with 12ml of 50 percent wine base and then distilled water to be 40ml, the mixture is fully and evenly mixed, the concentration is 0.125, 0.250 and 0.500g/ml respectively, the stomach filling amount is 0.2ml/10g & BW, the stomach filling is carried out once a day for continuous 5 days, the control group is supplied with the same amount of distilled water, and the wine base control group is supplied with the same amount of 15 percent wine base. On day 35 after the first administration of the test substance, the animals were sacrificed by dislocation of cervical vertebrae, bilateral epididymis were taken out and placed in a dish containing 2ml of physiological saline, cut open longitudinally with an ophthalmic scissors, left to stand for 3min, filtered, smeared conventionally, fixed with methanol for 5min, and stained with 1% eosin. Each animal was examined for 1000 sperm cells and each type of teratospermia was counted. The statistical method uses t test, if variance is not uniform or variance is uniform but the coefficient of variation is too large, then uses rank sum test.

2 results

2.1 acute toxicity test: as can be seen from tables 1 and 2, both sexes of large and small mice were gavaged at a dose of 15.0 g/kg. BW for 14 days. No obvious toxic manifestation is seen in the experimental period, no death is caused in the observation period, and no abnormality is found in each main organ in the necropsy. Acute oral toxicity (LD) of test substance to both sexes of large and small mice₅₀) Are all larger than 15.0g/kg BW.

TABLE 1 acute toxicity of health wine (25-fold concentrate) to mice

TABLE 2 acute toxicity of health wine (25-fold concentrate) to rat

2.2 genotoxicity test:

2.2.1Ames test results

As can be seen from tables 3 and 4, the number of the reversion colonies of each dose group of the test substances of the four test strains of the salmonella typhimurium TA97, TA98, TA100 and TA102 does not exceed the number of the blank control colonies by 2 times when S9 is added or not added, and no dose-response relation exists. The test result was negative.

TABLE 3 health wine (25 times concentrated solution) Ames test results (first experiment)

Note: the results above are the mean ± standard deviation of the number of triplicate plate colonies.

TABLE 4 health wine (25 times concentrated solution) Ames test results (repeat experiment)

2.2.2 mouse bone marrow cell micronucleus test results:

as can be seen from Table 5, the difference between the micronucleus rates of the test substance in each dose group and the negative control group is not statistically significant (P > 0.05), while the difference between the cyclophosphamide group and the negative control group is statistically significant (P < 0.05). The proportion of the immature red blood cells in each dose group of the test substance in the total number of the red blood cells is not less than 20 percent of that in the control group, and no obvious cytotoxicity is observed. Therefore, the test substance does not affect micronucleus of mouse bone marrow cells.

TABLE 5 influence of health wine (25-fold concentrate) on the incidence of micronucleus in mouse bone marrow

P < 0.05 compared with negative control group

2.2.3 mouse teratospermia test:

as can be seen from Table 6, the rates of teratospermia in mice in the test substance dose groups were statistically insignificant (P > 0.05) compared to the negative control group, while the differences in cyclophosphamide positive control group were statistically significant (P < 0.05) compared to the negative control group.

Therefore, no effect of the test substance on mouse teratospermia test was observed.

TABLE 6 influence of health wine (25 times concentrated solution) on mouse teratospermia incidence

P < 0.05 compared with negative control group

And (3) summary:

3.1 acute toxicity test: the health wine (25 times concentrated solution) has oral acute toxicity (LD) for big mouse and mouse of two sexes₅₀) The samples are all larger than 15.0g/kg & BW, and belong to non-toxic grade according to the acute toxicity grading standard of health food inspection and evaluation technical specification (2003 edition).

3.2 genotoxicity test:

3.2.1 Ames test: the result was negative.

3.2.2 mouse bone marrow cell micronucleus assay: the result was negative.

3.2.3 mouse teratospermia test: the result was negative.

Secondly, the test report of feeding the health care wine for 30 days by the centers for disease prevention and control in Tianjin city is as follows:

1. materials and methods

1.1 sample: the health wine prepared in example 1 is prepared by using 100ml/60kg BW per day of stock solution, 25 times of concentrated solution of a test object is provided, and the alcohol content is 0; the alcohol concentration of the wine base is 50% (v/v). The concentration was 4.0ml/60kg BW daily in terms of the intended dose.

1.2 Experimental animals: the weight of the rat is 62-92g, 100 SPF Wistar rats with half of male and female are selected and provided by Beijing Wintorlicha laboratory animal technology company Limited.

1.3 Experimental animal feeding Environment

SPF scale experimental animal house, temperature: 20-25 ℃, relative humidity 40-70% RH, certification number: SYXK 2014-. The feed is provided by Beijing Huafukang Biotechnology GmbH.

1.4 test methods

The 25-time concentrated solution for the health care wine has the intended dose of 4.0ml/60kg BW per day, and the specific dose is designed as follows: 1.67, 3.33, 6.67ml/kg & BW (corresponding to 25-fold, 50-fold, 100-fold, respectively, of the intended human dose). Rats were randomly divided into three sample groups, a blank control group and a wine-based control group, 20 per group, each half male and female. Taking 33.3ml of the test substance in a low-dose group, adding 60.0ml of wine base, and adding 106.7ml of distilled water; measuring 66.7ml of the test substance in the medium dosage group, adding 60.0ml of alcohol base, and adding 73.3ml of distilled water; taking 133.3ml of the test substance in a high-dose group, adding 60.0ml of wine base, and adding 6.7ml of distilled water; the alcohol concentration is 15%, mixing well, the stomach filling amount is 1.0ml/100g & BW, setting blank control group and wine base control group, the blank control group is given equal amount of distilled water, the wine base control group is given equal amount of wine base with 15% alcohol concentration (measuring 60.0ml, adding distilled water 140.0 ml). Animals were fed in a single cage, were fed ad libitum, and the food intake and body weight of the rats were recorded once a week for 30 days.

1.5 Observation index

1.5.1 general case observations: animals were observed daily for appearance, behavior, signs of toxicity, and mortality. Weighing body weight and food intake every week, and calculating food utilization rate, total food intake and total weight gain every week.

1.5.2 hematological examination: blood was taken at the end of the experiment to determine hemoglobin content (Hgb), Red Blood Cells (RBC), White Blood Cells (WBC) counts, and white blood cell classifications (lymphoid, monocyte, neutrophil, eosinophilic, basophilic) using a Sysmex XT-2000iv five-differential hemocytometer.

1.5.3 biochemical index determination: blood is taken at the end of the test to measure serum alanine Aminotransferase (ALT), heaven alanine Aminotransferase (AST), urea nitrogen (BUN), Cholesterol (CHO), Triglyceride (TG), blood sugar (GLU), Total Protein (TP), Albumin (ALB) and Creatinine (CRE), the used instrument is a TOSHIBATBA-40FR full-automatic biochemical analyzer, and the kit is provided by Beijing Jiuqiang biotechnology Limited.

1.5.4 gross observations and pathological tissue examinations: animals at the end of the experiment are weighed after being fasted for 16h, the animals are killed by dislocation of cervical vertebrae, and the general pathological changes of all main organs, the chest and the abdominal cavity are observed. The livers, kidneys, spleens, and testicles of all animals were removed, weighed, and organ coefficients calculated. The livers, kidneys, spleens, testicles (or ovaries), stomachs and duodenums of the animals of the blank control group and the high dose group were removed, fixed with 4% formaldehyde, embedded in paraffin, sectioned, HE-stained, and examined histologically under a light microscope.

1.5.5 statistical methods: statistical analysis is carried out on each group of data by SPSS11.5 FORWANDOWS mid-square difference analysis, data conversion is carried out on the people with irregular variances, and non-parameter statistics is carried out if the variance is still irregular after the conversion.

2 results

2.1 general observations of laboratory animals

As can be seen from tables 1-4, the animals did not show food refusal after oral gavage of the rats for 30 days with the test substances of 1.67, 3.33, 6.67 ml/kg.BW. During the test period, compared with the blank control group, the animal weight, weight gain and food utilization rate of the wine-based control group have no statistical significance (P is more than 0.05); compared with the wine-based control group, the animal weight, weight gain and food utilization rate of each dose group have no statistical significance (P is more than 0.05).

TABLE 1 Effect of test substances on rat body weight (g, mean. + -. standard deviation)

TABLE 2 Effect of test substances on weekly food intake in rats (g, mean. + -. standard deviation)

TABLE 3 Effect of test substances on weekly food utilization (%, mean. + -. standard deviation) in rats

TABLE 4 Effect of test substances on Total food utilization in rats (mean. + -. standard deviation)

2.2 Effect of test substances on the hematological indices of rats

As can be seen from tables 5 and 6, the hematological indexes at the end of the experiment are all within the normal value range of the unit of examination. The difference of each index of the wine-based control group and the blank control group has no statistical significance (P is more than 0.05). The percentage of mononuclear cells of the male animal low-dose group is higher than that of the wine-based control group; the percentage of neutrophils in the dose group was higher than the alcohol-based control group and the percentage of lymphocytes was lower than the alcohol-based control group, with the differences being statistically significant (P < 0.05), but not biologically significant. The difference between the other dose groups and the wine-based control group is not statistically significant (P is more than 0.05).

TABLE 5 influence of test substances on the hematological indices of rats (mean. + -. standard deviation)

TABLE 6 Effect of test substances on the white blood cell Classification of rats (%, mean. + -. standard deviation)

Difference compared with wine base control group has statistical significance (P < 0.05)

2.3 Effect of test substances on Biochemical indicators of rats

As can be seen from Table 7, the biochemical indexes of the test animals at the end of the experiment are all within the normal value range of the test unit, and the comparative difference between the indexes of the wine-based control group and the indexes of the blank control group has no statistical significance (P is more than 0.05). The ALB in the low dose group of only male animals was lower than that in the alcohol-based control group, and the difference was statistically significant (P < 0.05), but not biologically significant. The difference between the other dose groups and the wine-based control group is not statistically significant (P is more than 0.05).

TABLE 7 Effect of test substances on Biochemical indices of rats (mean. + -. standard deviation)

2.4 Effect of test substances on rat organs

Gross anatomical observations revealed no abnormalities. Compared with a blank control group, the animal viscera weight and viscera ratio of the wine-based control group have no statistical significance (P is more than 0.05); compared with the wine-based control group, the difference between the weight of the organs and the ratio of the organs of the animals in each dose group is not statistically significant (P is more than 0.05) (tables 8 and 9).

TABLE 8 Effect of test substances on rat organ weight (g, mean. + -. standard deviation)

TABLE 9 Effect of test substances on rat visceral volume ratio (%, mean. + -. standard deviation)

2.5 histopathological examination:

taking each main organ of the high-dose group, the blank control group and the wine-based control group to perform histopathological examination, wherein each group comprises 20 animals and half of animals.

2.5.1 liver: the capsule is complete and has no obvious fibrous tissue proliferation. Liver lobules exist, and connective tissue hyperplasia is not seen in the interstitium. The central hepatic vein, the small hepatic artery and vein are not abnormal. A small number of small rounded inflammation cell infiltrates were seen in some of the animal regions, 4/20 in the blank control group (2 males and 2 females), 5/20 in the wine-based control group (2 males and 3 females), and 4/20 in the high dose group (2 males and 2 females). Three comparisons showed no significant difference.

TABLE 10 Effect of test substances on pathological tissues of rat major organs

2.5.2 spleen: the spleen envelopes of the three groups of animals are complete, the structures of red and white pith are clear, red pith and spleen sinus erythrocytes are full, the white pith hair growing center is active, and the central artery tube wall of the spleen nodule is not thickened or degenerated. Three comparisons showed no significant difference.

2.5.3 Kidney: the three groups of animals have complete kidney envelopes, clear skin and marrow texture structures, and the sizes of glomeruli are not reduced or enlarged, and the number of the glomeruli is not reduced. The renal tubular epithelial cells are not degenerated, necrosed or shed, the tubular shape and calculus are not seen in the lumen, and the fibrous tissue hyperplasia is not seen in the renal interstitium. Small round inflammation cell infiltration can be seen in kidney interstitium of individual animals, 2/20 in blank control group (1 male and 1 female), 2/20 in wine-based control group (1 male and 1 female), and 3/20 in high dose group (2 male and 1 female). Three comparisons showed no significant difference.

2.5.4 stomach: the anterior gastric keratosis is good, the gastric mucosa is complete, bleeding, erosion, ulcer and desquamation are not seen, the gland is not proliferated, metaplasia or atrophy, and the lamina propria, the muscular layer and the serosal layer are not abnormal. There was no obvious abnormality at the junction between the anterior and posterior stomachs. Three comparisons showed no significant difference.

2.5.5 duodenum: the mucous membrane is complete, bleeding, erosion, ulcer and shedding are not seen, the intestinal gland of the inherent membrane is abundant, hyperplasia, metaplasia or atrophy is not seen, and the inherent layer, the muscular layer and the serosa are not abnormal. Three comparisons showed no significant difference.

2.5.6 testis: the testis is complete, seminiferous tubules can show the existence and normal distribution of spermatogenic cells at all levels, and the well-developed spermatids and sperms can be seen in the cavity, and the stroma is not obviously changed. Three comparisons showed no significant difference.

2.5.7 ovary: ovarian follicle cells, corpus luteum and corpus albilineans at different growth and development stages can be seen in the ovary, and no abnormality is found. Three comparisons showed no significant difference.

No histopathological changes associated with the samples were observed by microscopic observation of the high dose groups compared to the blank and wine-based control groups (table 10), so histopathological examination was not performed on the medium and low dose groups.

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The rats were gavaged orally for 30 days with 1.67, 3.33, 6.67ml/kg & BW (25 times, 50 times, 100 times of the intended dose for human respectively) as a health wine (25 times concentrated solution), and during the test period, the weight, weight gain, food utilization rate and viscera ratio of the animals in the wine-based control group were compared with those in the blank control group, and the differences were not statistically significant (P > 0.05); the weight, weight gain, food utilization rate and viscera ratio of animals in each dose group are compared with those in a wine-based control group, and the differences have no statistical significance (P is more than 0.05); the hematology index and the biochemical index are both in the range of the normal value of the detection unit; gross anatomy was not abnormal and no histopathological changes associated with the test samples were found. The product has no obvious toxic effect after being fed to rats for 30 days.

Thirdly, an animal test report of the effect of disease prevention and control center in Tianjin on enhancing immunity of the health care wine:

1. materials and methods

1.2 Experimental animals: 200 female SPF-grade Kunming mice, 18-22g, were provided by Peking Wintonlifa laboratory animal technology, Inc. SPF scale experimental animal house, temperature: 20-25 ℃, relative humidity 40-70% RH, certification number: SYXK 2014-. The feed is provided by Proteus Technical animal Co., Ltd.

1.3 dose selection and subject administration: in the test, three dosage groups of low, medium and high are designed, and are respectively 0.33ml/kg & BW, 0.67ml/kg & BW and 1.34ml/kg & BW, namely, the dosage is 5 times, 10 times and 20 times of the intended dosage for human. Taking 6.7ml of the test substance in a low-dose group, adding 120ml of wine base, and adding distilled water to 400 ml; taking 13.3ml of a test object in the middle-dose group, adding 120ml of wine base, and adding distilled water to 400 ml; the high dose group measures 26.7ml of the test substance, 120ml of wine base and distilled water to 400 ml. Mixing completely, alcohol concentration of 15%, and intragastric amount of 0.2ml/10 g.BW, adding equal amount of distilled water to blank control group, adding equal amount of 15% wine base (120 ml wine base, adding distilled water to 400ml) to wine base control group, intragastric administering once per day,continuously measuring various indexes for 30 days and 24 hours after the test object is given for the last time. The experimental animals were divided into 4 large groups of 50 animals each, and then 50 animals each were divided into 5 small groups of 10 animals each, including a blank control group, a wine-based control group, and three low, medium, and high dose groups. In which 1 group was subjected to HC₅₀Assays, antibody-producing cell assays, delayed type allergy (plantar thickening); 2, carrying out a chicken erythrocyte phagocytosis test of mouse abdominal cavity macrophages and measuring a viscera/body ratio; 3 groups were subjected to carbon clearance test; in group 4, mouse lymphocyte transformation test and NK cell measurement test were performed.

1.4 Main instruments and reagents

1.4.1 main instruments and materials: TU-1810 type ultraviolet visible spectrophotometer, a centrifuge, a constant temperature water bath, a carbon dioxide incubator, a sterile dissector, a microscope, an enzyme-labeling instrument, a clean bench, a slide rack, gauze, a vernier caliper, a micro-injector, a timer, a hemoglobin straw, a 24-hole culture plate, a 96-hole culture plate and the like.

1.4.2 main reagents: sheep Red Blood Cell (SRBC), complement (guinea pig serum), SA buffer, Du's reagent (sodium bicarbonate 1.0g, potassium ferricyanate 0.2g, potassium cyanide 0.05g, and distilled water 1000ml), Hank, s liquid, RPMI0640 cell culture liquid, chicken red blood cell, acetone, methanol, physiological saline, Giemsa staining solution, India ink, Na₂CO₃(analytical grade), calf serum, canavalin A, isopropanol, MTT (thiazole blue), PBS buffer, LDH matrix solution, HCL and the like.

1.4.3 Experimental cells: YAC-1 cells, purchased from Shanghai institute of sciences cell bank, and cultured in cell suspension.

1.5 Experimental methods

1.5.1 half maximal hemolysis value (HC)₅₀) The determination of (1): after 30 days of administration of the test substance, each mouse was intraperitoneally injected with 0.2ml of 2% SRBC, and the test substance was further administered to day 35, blood was collected from the inner canthus, centrifuged at 2000r/min for 10min to collect serum, and diluted 300-fold with SA buffer. 1ml of diluted serum, 0.5ml of 10% SRBC and 1ml of complement were added to the tube. A control tube (with SA buffer instead) without serum was provided, and the reaction was stopped in an ice bath after a water bath at 37 ℃ for 20 min. Centrifuging at 200r/min for 10min, collecting 1ml supernatantAdding 3ml Dushi reagent as sample tube, simultaneously adding 10% SRBC and Dushi reagent to 4ml for measuring optical density value of SRBC at half hemolysis, mixing well, standing for 10min, and measuring optical density value of each tube at 540 nm.

1.5.2 detection of antibody-producing cells: after 30 days of administration of the test substance, each mouse was intraperitoneally injected with 0.2ml of 2% SRBC, and then the test substance was further administered to 35 days, after which the animals were dislocated to be sacrificed, and the spleen was taken out and placed in a dish containing 4 layers of gauze and Hank's solution, and gently pulverized with forceps to prepare a cell suspension. Washing with Hank's solution 3 times, centrifuging at 1000r/min for 10min, suspending the cells in 5ml of RPMI1640 culture solution, and adjusting the cell concentration to 5 × 10⁶One per ml. Heating and dissolving surface layer culture medium, preserving heat in 45 ℃ water bath, mixing with equivalent 2 times concentration Hank's solution, subpackaging into small test tubes, each tube is 0.5ml, adding 50 ul of 10% SRBC and 20 ul of spleen cell suspension, rapidly mixing, inversing, culturing in a carbon dioxide incubator for 1-1.5h, adding complement (1:10) diluted by SA buffer solution, continuing to culture for 1h, and counting the number of hemolytic plaques.

Number of antibody cells produced (10)³Whole spleen, observed plaque number multiplied by dilution factor (400)/1000

1.5.3 delayed-type allergy (plantar thickening): after the test object is given for 30 days, 0.2ml of 2% SRBC is injected into the abdominal cavity of each mouse, the thickness of the left and right plantar parts of the foot is measured when the test object is continuously given to the 34 th day, 20 mul of 20% SRBC is injected into the measurement part, the thickness of the left plantar part of the rear foot is measured after 24h of injection, the thickness of the plantar part of the left rear foot is measured at the same part for three times, and the average value is taken.

Toe thickness difference (mm) before and after attack-attack toe thickness

1.5.4 experiment for phagocytosis of chicken red blood cells by mouse abdominal cavity macrophages (half internal method): after administering the test object for 32 days, injecting 1ml of 20% chicken erythrocyte suspension into the abdominal cavity of each mouse at intervals of 30min, carrying out cervical dislocation to kill the animals, cutting the abdominal wall skin from the center, injecting 2ml of normal saline into the abdominal cavity, slightly massaging the abdominal cavity for 1min with fingers, sucking 1ml of abdominal cavity washing liquid, evenly dripping the abdominal cavity washing liquid on 2 glass slides, putting the glass slides into an enamel tray filled with wet gauze, incubating the glass slides for 30min at 37 ℃, rinsing the normal saline, airing the glass slides, fixing the glass slides by 1:1 acetone-methanol solution, and dyeing 4% Giemsa for 10 min. Macrophages were counted under oil lens, 100 per tablet, and percent phagocytosis and phagocytosis index were calculated as follows.

1.5.5 organ/body weight ratio measurement: after the test subjects were administered for 32 days, the spleen and thymus were removed, and the spleen and thymus were weighed, respectively, to calculate the organ/body ratio.

1.5.6 mouse carbon clearance test: after the test substance was administered for 32 days, 20. mu.l of India ink (0.1ml/10 g. BW) diluted 3.5 times was injected into the tail vein, and the blood was collected from the inner canthus at 2 and 10min, and added to 2.98ml of 0.1% sodium carbonate solution, and the optical density value of each tube was measured at 600 nm. The liver and spleen were weighed, and the phagocytosis index was calculated according to the following formula.

1.5.7 ConA-induced splenic lymphocyte transformation assay in mice (MTT method): after administering the test substance for 33 days, the spleen was aseptically collected and placed in a dish containing 4 layers of gauze and a suitable amount of sterile Hank's solution, and the spleen was gently ground with forceps to prepare a single cell suspension. Washing with Hank's solution 3 times, centrifuging at 1000r/min for 10min, and adjusting cell concentration to 3 × 10⁶One/ml and the cell suspension was added to a 24-well plate in two wells, 1ml per well, 75. mu.l of ConA solution (100. mu.g/ml) in one well and 75. mu.l in the other wellControl, put 5% CO₂And culturing in an incubator at 37 ℃ for 72 hours. 4 hours before the end of the culture, 0.7ml of the supernatant was aspirated from each well, and 0.7ml of calf serum-free RPMI1640 culture medium was added thereto together with 50. mu.l/well of MTT (5mg/ml), and the culture was continued for 4 hours. After the culture is finished, 1ml of acidic isopropanol is added into each hole, and the mixture is uniformly blown and beaten to ensure that the purple crystals are completely dissolved. The optical density value was measured with an ultraviolet spectrophotometer at a wavelength of 570 nm. The difference in optical density was calculated as follows.

Optical Density (ABS) Difference-ConA Aperture optical Density not added

1.5.8NK cell Activity assay (lactate dehydrogenase assay): YAC-1 cells (target cells) were subcultured 24h before the experiment, and the cell concentration was adjusted to 4X 10 with RPMI1640 complete medium⁵One per ml. Aseptically taking the spleen, placing the spleen in a dish containing 4 layers of gauze and a proper amount of aseptic Hank's solution, and gently grinding the spleen by using forceps to prepare a single cell suspension. Centrifuging at 1000r/min for 10min, washing with Hank's solution for 3 times, discarding supernatant, and adjusting cell concentration to 2 × 10⁷One per ml. Taking 100 μ l each of target cell and effector cell (effective target ratio 50:1), adding into 96-well culture plate, adding 100 μ l each of target cell and culture solution into natural release hole of target cell, adding 100 μ l each of target cell and 1% NP40 into maximum release hole of target cell, setting three parallel holes, and culturing at 37 deg.C with 5% CO₂Culturing for 4h in an incubator, sucking 100 μ L of supernatant per well, placing in a 96-well culture plate, simultaneously adding 100 μ L of LDH matrix solution, reacting for 3min, adding 30 μ L of 1mol/L HCl per well, and measuring optical density value at 492nm of an enzyme labeling instrument.

NK cell activity was calculated as follows:

1.6 statistics of test data: the experimental data are statistically tested by SPSS11.5 for Windows, variance analysis is adopted for the control group and the experimental group, if the variance is irregular, data conversion is adopted, and non-parameter statistics is adopted if the conversion is still irregular.

1.7 judging the result:

judging the function of enhancing immunity: the result is positive in any two aspects of cellular immune function, humoral immune function, monocyte-macrophage function and NK cell activity, and the tested sample can be judged to have the function of enhancing the immune function.

Wherein, the results of two experiments in the cellular immune function determination items are both positive, or the results of two dose groups in any experiment are positive, so that the cellular immune function determination results can be judged to be positive. The positive result of the humoral immune function determination result can be judged by the positive results of the two experiments in the humoral immune function determination item or the positive results of the two dosage groups in any experiment. The result of two experiments in the mononuclear-macrophage determination project is positive, or the result of two dosage groups in any experiment is positive, so that the function result of the mononuclear-macrophage can be judged to be positive. More than one dose group of NK cell activity determination experiments have positive results, and the positive result of the NK cell activity can be judged.

2. Results

2.1 Effect of health wine (25-fold concentrate) on mouse body weight

As can be seen from tables 1-4, animals in each group grew well 30 days after oral administration of the test substance at different doses. The animal weight gain of the wine-based control group has no statistical significance (P is more than 0.05) compared with the blank control group, and the animal weight gain of each dosage group has no statistical significance (P is more than 0.05) compared with the wine-based control group.

TABLE 1 Effect of test substances on body weight of immunized 1 group of mice (g, mean. + -. standard deviation)

TABLE 2 Effect of test substances on body weight of immunized 2 groups of mice (g, mean. + -. standard deviation)

TABLE 3 Effect of test substances on body weight of immunized 3 groups of mice (g, mean. + -. standard deviation)

TABLE 4 Effect of test substances on body weight of immunized 4 groups of mice (g, mean. + -. standard deviation)

2.2 Effect of health wine (25-fold concentrate) on the ratio of mouse organ/body weight

As shown in Table 5, the differences between the thymus and spleen/body weight ratios of the mice in the wine-based control group and the blank control group are not statistically significant (P > 0.05), and the differences between the thymus and spleen/body weight ratios of the mice in each dose group and the wine-based control group are not statistically significant (P > 0.05).

TABLE 5 Effect of test substances on mouse organ/body weight ratio (mean. + -. standard deviation)

2.3 Effect of health wine (25-fold concentrate) on humoral immunity

2.3.1 health wine (25 times concentrated solution) for half hemolysis value (HC) of mouse₅₀) Influence of (2)

As can be seen from Table 6, the wine-based control mice had half the hemolysis value (HC)₅₀) Compared with the blank control group, the difference is not statistically significant (P is more than 0.05), and the half hemolysis value (HC) of the mice in the high-dose group₅₀) The difference is higher than that of the wine base control group and has statistical significance (P < 0.05); half maximal Hemolysis (HC) value in middle and low dose group mice₅₀) Compared with the wine-based control group, the difference is not statistically significant (P is more than 0.05).

TABLE 6 test substance-to-mouse median hemolysis value (HC)₅₀) Influence of (mean. + -. standard deviation)

Comparison P < 0.05 with wine base control group.

2.3.2 Effect of health wine (25-fold concentrate) on mouse antibody-producing cell test

As can be seen from Table 7, the difference between the number of mouse antibody-producing cells in the wine-based control group and the number of mouse antibody-producing cells in the blank control group was statistically insignificant (P > 0.05), and the number of mouse antibody-producing cells in the high-dose group was statistically significant (P < 0.05); compared with the wine-based control group, the number of the mouse antibody-producing cells in the medium and low dose groups has no statistical significance (P is more than 0.05).

TABLE 7 Effect of test substances on mouse antibody-producing cells (mean. + -. standard deviation)

Comparison P < 0.05 with wine base control group.

2.4 Effect of health wine (25-fold concentrated solution) on immune function of mouse cells

2.4.1 Effect of health wine (25 times concentrated solution) on delayed allergy to mice (thickening foot)

As can be seen from Table 8, the differences in thickness of the plantar foot before and after challenge in the mice of the wine-based control group were statistically insignificant (P > 0.05) compared to the control group, and the differences in thickness of the plantar foot before and after challenge in the mice of the three dose groups were statistically insignificant (P > 0.05) compared to the wine-based control group.

TABLE 8 Effect of test substances on delayed allergy (plantar thickening) in mice (mean. + -. standard deviation)

2.4.2 Effect of health wine (25-fold concentrated solution) on ConA-induced splenic lymphocyte transformation test (MTT method) in mice

As can be seen from Table 9, the difference between the spleen lymphocyte transformation of mice in the wine-based control group and the blank control group is not statistically significant (P > 0.05), and the difference between the spleen lymphocyte transformation of mice in the three dose groups and the wine-based control group is not statistically significant (P > 0.05).

TABLE 9 Effect of test substances on ConA-induced splenic lymphocyte transformation assay (MTT method) in mice (mean. + -. standard deviation)

2.5 Effect of health wine (25-fold concentrate) on monocyte-macrophage function

2.5.1 Effect of health wine (25 times concentrated solution) on phagocytosis of chicken red blood cells by macrophages in mouse abdominal cavity

As shown in Table 10, the percentage of phagocytosis and the phagocytosis index of the mice in the wine-based control group were statistically insignificant (P > 0.05), and the percentage of phagocytosis and the phagocytosis index of the mice in the three dose groups were statistically insignificant (P > 0.05).

TABLE 10 Effect of test substances on phagocytosis of Chicken erythrocytes by macrophages of the mouse peritoneal cavity (mean. + -. standard deviation)

2.5.2 Effect of health wine (25 times concentrated solution) on mouse carbon clearance test

As can be seen from Table 11, the difference between the phagocytic index of the mice in the wine-based control group and the phagocytic index of the mice in the blank control group is not statistically significant (P is more than 0.05), and the difference between the phagocytic index of the mice in the medium and high dose groups is higher than that of the mice in the wine-based control group and is statistically significant (P is less than 0.05); the phagocytic index of mice in the low dose group was statistically insignificant compared to the control group with alcohol (P > 0.05).

TABLE 11 Effect of test substances on mouse carbon clearance test (mean. + -. standard deviation)

Comparison P < 0.05 with wine base control group.

2.6 Effect of health wine (25-fold concentrate) on NK cell Activity

As can be seen from Table 12, the NK cell activities of the wine-based control group were statistically insignificant (P > 0.05) compared with the blank control group, and the NK cell activities of the three dose groups were statistically insignificant (P > 0.05) compared with the wine-based control group.

TABLE 12 Effect of test substances on NK cell Activity in mice (lactate dehydrogenase assay) (mean. + -. standard deviation)

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After oral administration of various doses of health wine (25 times concentrated solution) for 30 days, the animals in each group grew normally. Half maximal hemolytic value (HC) of mice₅₀) Experiments showed that the half maximal hemolysis value (HC) of the high dose group₅₀) The difference is higher than that of the wine base control group and has statistical significance (P < 0.05); the antibody-producing cell test shows that the number of antibody-producing cells in the high-dose group is higher than that in the wine-based control group, and the difference has statistical significance (P < 0.05); the mouse carbon clearance test shows that the phagocytic index of the mice in the medium and high dose groups is higher than that of the wine-based control group, and the difference has statistical significance (P < 0.05). The other experiments have no immunosuppressive phenomenon. According to the technical specifications of health food inspection and evaluation, the sample is suggested to have the function of enhancing the immunity of animals.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. The health wine for enhancing immunity is characterized by comprising the following components in parts by weight:

18-22g of honey.

the total content of the health wine is 1000 mL.

2. The health wine for enhancing immunity according to claim 1, wherein the formula of the health wine is as follows:

18g of honey;

the total content of the health wine is 1000 mL.

3. The health wine for enhancing immunity according to claim 1, wherein the formula of the health wine is as follows:

22g of honey;

the total content of the health wine is 1000 mL.

4. The health wine for enhancing immunity according to claim 1, wherein the formula of the health wine is as follows:

20g of honey;

the total content of the health wine is 1000 mL.

5. The preparation method of the health care wine for enhancing immunity according to any one of claims 1 to 4 is characterized by comprising the following steps:

6. The method for preparing health-care wine for enhancing immunity according to claim 5, wherein the white wine used for extraction in the step 2 is 35% white wine.

7. The method for preparing health wine for enhancing immunity according to claim 5, wherein the leaching time in step 2 is 15 days.

8. The method for preparing health wine for enhancing immunity according to claim 5, wherein the filtering in step 2 uses a 120 mesh stainless steel net.

9. The preparation method of the health care wine for enhancing immunity according to claim 5, wherein the temperature for refrigerating and standing in the step 3 is 0 to-4 ℃ and the time is 24 hours.

10. The preparation method of the health-care wine for enhancing immunity according to claim 5, wherein the white wine in the step 3 is 65% white wine.