阅读说明：本技术 一种复方菌草鹿角灵芝粉冲剂及其制备方法 (Compound Juncao antler lucid ganoderma powder electuary and preparation method thereof ) 是由 张桂清 刘仁玉 梁志豪 于 2020-06-16 设计创作，主要内容包括：本发明公开了一种复方菌草鹿角灵芝粉冲剂及其制备方法,将菌草鹿角灵芝、富硒茶、紫苏采用水提法制备粉冲剂,配方配伍科学,能提高机体免疫力,具有防癌、抗衰老,减肥、护肤等功效。制备方法简便快捷,有效保留活性物质,保健效果显著。(The invention discloses a compound fungus grass antler lucid ganoderma powder electuary and a preparation method thereof. The preparation method is simple, convenient and quick, effectively retains active substances, and has remarkable health promotion effect.)

1. A compound fungus grass antler lucid ganoderma powder electuary is characterized in that: the main raw materials are calculated according to the parts by weight: 15-20 parts of grass-cultivated antler lucid ganoderma, 10-15 parts of selenium-rich tea, 1-2 parts of purple perilla and 1-5 parts of maltodextrin.

2. The powder according to claim 1, wherein: the main raw materials are calculated according to the parts by weight: 17.7 parts of grass of Chinese juncus, antler and glossy ganoderma, 12.38 parts of selenium-rich tea, 1.27 parts of purple perilla and 3 parts of maltodextrin.

3. A process for preparing the powder formulation of claim 1 or 2, wherein: the method comprises the following steps:

1) oven drying Ganoderma, selenium-rich tea and Perillae herba, adding 20 times of water, soaking for 6 hr, performing ultrasonic treatment at 80 deg.C for 4 hr, and filtering; adding 20 times of water into the residue, performing ultrasonic treatment at 80 deg.C for 4 hr, and mixing filtrates; vacuum concentrating at 60 deg.C under reduced pressure, and vacuum freeze drying to obtain dry extract powder;

2) adding maltodextrin, mixing uniformly, sterilizing for 15-20 min at 70-75 ℃, homogenizing, spray drying and packaging.

4. The method of claim 3, wherein: spray drying parameters: the air inlet temperature is 190-.

Technical Field

The invention belongs to the technical field of health care products, and particularly relates to compound Juncao antler lucid ganoderma powder electuary and a preparation method thereof.

Background

The ganoderma lucidum is a fungus with medicinal value, contains bioactive components such as polysaccharide, triterpenoids, organic germanium and the like, has proved to have the effects of immunoregulation, blood fat reduction, fatigue resistance, tumor resistance, liver protection and the like, has higher pharmacological activity on cardiovascular and cerebrovascular diseases, is a good natural raw material for developing health-care beverages, has positive effects on health care of middle-aged and old people, has higher effective component content of the grass-cultivated antler ganoderma lucidum by 3 percent than that of the common ganoderma lucidum, has high safety compared with the common ganoderma lucidum, and has health-care effects of improving immunity, improving insomnia, assisting in treating rheumatic arthritis and the like.

The perilla contains precious alpha-linolenic acid which is a basic substance forming cell membranes and biological enzymes, can be converted into DHA, DPA, EPA and the like in vivo and is a mother of human health and longevity. Human beings lack of alpha-linolenic acid, which can cause a series of diseases such as hyperlipidemia, hypertension, hyperglycemia, cerebral thrombosis, arteriosclerosis, dementia, hypomnesis, rheumatism, diabetes and the like, and particularly has adverse effects on the growth and development of brain tissues. Clinical medicine proves that the alpha-linolenic acid can prevent and cure more than 50 diseases, can also make human bodies become young and delay senility by supplementing a proper amount of alpha-linolenic acid every day. Perilla leaf has the effects of dispelling exterior cold and inducing sweating, and can be used for treating wind-cold exterior syndrome with symptoms of aversion to cold, fever, anhidrosis, etc. Recently, a great deal of research shows that the mixture of perillaldehyde and volatile oil has the effects of antibiosis, anticancer, anisakis resistance, antioxidation, vasodilation and the like.

The selenium-rich tea contains selenium element which is one of essential nutrient elements for human body and has important action on physiological function of human body, and it has been proved that more than 40 diseases of human body, such as cardiovascular and cerebrovascular diseases, diabetes, cancer and tumor, etc. are all related to selenium deficiency, and the supplement of selenium has obvious preventing and curing effect and peculiar action, so that selenium is called as the microelement mainly for life. Tea with far higher selenium content than other tea leaves is called selenium-enriched tea. The selenium-rich tea has effects of reducing blood lipid, reducing weight, preventing and treating cardiovascular and cerebrovascular diseases, resisting toxic substance, sterilizing, preventing cancer, and resisting aging.

At present, ganoderma lucidum and perilla are mainly used for making tea or making wine in the market, so that the tea is inconvenient to eat and has single effect.

Disclosure of Invention

The invention aims to provide the compound Juncao antler lucid ganoderma powder electuary and the preparation method thereof, the formula compatibility is scientific, the immunity of the organism can be improved, and the compound Juncao antler lucid ganoderma powder electuary has the effects of cancer prevention, aging resistance, weight reduction, skin care and the like. The preparation method is simple, convenient and quick, effectively retains active substances, and has remarkable health promotion effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

a compound fungus grass antler lucid ganoderma powder electuary comprises the following main raw materials in parts by weight: 15-20 parts of grass-cultivated antler lucid ganoderma, 10-15 parts of selenium-rich tea, 1-2 parts of purple perilla and 1-5 parts of maltodextrin.

Preferably, the main raw materials are calculated according to the parts by weight: 17.7 parts of grass of Chinese juncus, antler and glossy ganoderma, 12.38 parts of selenium-rich tea, 1.27 parts of purple perilla and 3 parts of maltodextrin.

The preparation method comprises the following steps:

1) oven drying Ganoderma, selenium-rich tea and Perillae herba, adding 20 times of water, soaking for 6 hr, performing ultrasonic treatment at 80 deg.C for 4 hr, and filtering; adding 20 times of water into the residue, performing ultrasonic treatment at 80 deg.C for 4 hr, and mixing filtrates; vacuum concentrating at 60 deg.C under reduced pressure, and vacuum freeze drying to obtain dry extract powder;

2) adding maltodextrin, mixing uniformly, and sterilizing at the temperature of: and (3) at 70-75 ℃, time: and (3) homogenizing the sterilized materials for 15-20 min, controlling the flow rate by using a high-pressure pump, carrying out spray drying at the feeding speed of 25 r/min, the air inlet temperature of 190-200 ℃, the air exhaust temperature of 90-95 ℃ and the negative pressure of 150 Pa, detecting and packaging to obtain the compound Juncao, antler and lucid ganoderma powder granules.

The invention has the beneficial effects that: the formula is scientific in compatibility, can improve the immunity of the organism, and has the effects of preventing cancer, resisting aging, losing weight, protecting skin and the like. The preparation method is simple, convenient and quick, effectively retains active substances, and has remarkable health promotion effect.

Drawings

FIG. 1 shows the effect of compatibility of each component of compound Ganoderma amboinense extract on phagocytosis rate of macrophage.

FIG. 2 shows the cytokine contents of IFN-. gamma.IL-2 and IL-10 in mice.

FIG. 3 shows the effect of compound grass Kazuno Ganoderma extract on hemolytic plaque in mice.

FIG. 4 shows the effect of compound grass Kazuno Ganoderma extract on tumor volume increase in mice.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1

The main raw materials are calculated according to the parts by weight: 15 parts of grass, antler and lucid ganoderma, 10 parts of selenium-rich tea, 1 part of purple perilla and 1 part of maltodextrin.

The preparation method comprises the following steps:

1) oven drying Ganoderma, selenium-rich tea and Perillae herba, adding 20 times of water, soaking for 6 hr, performing ultrasonic treatment at 80 deg.C for 4 hr, and filtering; adding 20 times of water into the residue, performing ultrasonic treatment at 80 deg.C for 4 hr, and mixing filtrates; vacuum concentrating at 60 deg.C under reduced pressure, and vacuum freeze drying to obtain dry extract powder;

2) adding maltodextrin, mixing, sterilizing at 70 deg.C for 20 min, homogenizing, spray drying, and packaging.

Spray drying parameters: the air inlet temperature is 190 ℃, the air outlet temperature is 90 ℃, the negative pressure is 150 Pa, and the feeding speed is 25 r/min.

Example 2

The main raw materials are calculated according to the parts by weight: 17.7 parts of grass of Chinese juncus, antler and glossy ganoderma, 12.38 parts of selenium-rich tea, 1.27 parts of purple perilla and 3 parts of maltodextrin.

The preparation method comprises the following steps:

1) oven drying Ganoderma, selenium-rich tea and Perillae herba, adding 20 times of water, soaking for 6 hr, performing ultrasonic treatment at 80 deg.C for 4 hr, and filtering; adding 20 times of water into the residue, performing ultrasonic treatment at 80 deg.C for 4 hr, and mixing filtrates; vacuum concentrating at 60 deg.C under reduced pressure, and vacuum freeze drying to obtain dry extract powder;

2) adding maltodextrin, mixing, sterilizing at 72 deg.C for 18 min, homogenizing, spray drying, and packaging.

Spray drying parameters: the air inlet temperature is 195 ℃, the air outlet temperature is 93 ℃, the negative pressure is 150 Pa, and the feeding speed is 25 r/min.

Example 3

The main raw materials are calculated according to the parts by weight: 20 parts of grass, antler and lucid ganoderma, 15 parts of selenium-rich tea, 2 parts of purple perilla and 5 parts of maltodextrin.

The preparation method comprises the following steps:

1) oven drying Ganoderma, selenium-rich tea and Perillae herba, adding 20 times of water, soaking for 6 hr, performing ultrasonic treatment at 80 deg.C for 4 hr, and filtering; adding 20 times of water into the residue, performing ultrasonic treatment at 80 deg.C for 4 hr, and mixing filtrates; vacuum concentrating at 60 deg.C under reduced pressure, and vacuum freeze drying to obtain dry extract powder;

2) adding maltodextrin, mixing, sterilizing at 75 deg.C for 15 min, homogenizing, spray drying, and packaging.

Spray drying parameters: the air inlet temperature is 200 ℃, the air exhaust temperature is 90-95 ℃, the negative pressure is 150 Pa, and the feeding speed is 25 r/min.

1 influence of ultrasonic extraction process and traditional water extraction process on extract yield

The extract yield and polysaccharide content obtained by the ultrasonic water extraction method are higher than those obtained by the traditional water extraction process, which is probably because the ultrasonic extraction utilizes the mechanical effect, cavitation effect and heat effect of the ultrasonic extraction method to extract the biological effective components by increasing the movement speed of medium molecules and the penetrating power of the medium, thereby accelerating the dissolution of polysaccharide molecules, improving the dissolution effect of polysaccharide, simultaneously ensuring that the structure of polysaccharide is not damaged, and playing a good protection effect on the activity of polysaccharide in human bodies and animals.

2 influence of compatibility relationship of each component of compound Juncao antler ganoderma lucidum extract on macrophage function

2.1 animal pretreatment

Male, 4-5 week old SPF grade BALB/c mice were randomly divided into 9 groups of 10 mice each. The experiment takes the low dose of the compound fungus grass, antler and ganoderma lucidum extract as an example, and analyzes whether the single medicinal materials are related or not under the same dose of compatibility, and whether the compound has influence or not. The following experimental groups were designed: 4.065G/kg of compound grass antler lucid ganoderma extract low dose group (group A), 2.3G/kg of grass antler lucid ganoderma water extract group (group B), 0.165G/kg of perilla water extract group (group C), 1.6G/kg of selenium-rich tea water extract group (group D), 3.9G/kg of grass antler lucid ganoderma and selenium-rich tea water extract group (group E), 2.465G/kg of grass antler lucid ganoderma and perilla water extract group (group F), 1.765G/kg of perilla and selenium-rich tea water extract group (group G), 1 time of administration and 0.3 mL of physiological saline per day for mice in the white control group, 1 time of administration and 0.3 mL of levamisole per day for mice in the positive control group, the dose is 10 mg/kg, and 28 days of continuous administration are carried out.

2.2 preparation of chicken erythrocyte suspension

The chicken blood was taken and placed in a conical flask with glass beads and shaken thoroughly in one direction to defibrate. Washing with normal saline for 2-3 times, centrifuging at 2000 r/min for 10 min, removing supernatant, and preparing into 1% V/V chicken erythrocyte suspension with normal saline.

2.3 phagocytic function assay

(1) Three days before drug withdrawal, 1 mL of 5% starch broth solution was injected intraperitoneally into each mouse 1 time a day for 3 consecutive days. Stopping taking the medicine the next day, injecting 1 mL of 1% chicken red blood cell suspension into the abdominal cavity of each mouse at intervals of 30 min, killing the animal by dislocation of cervical vertebra, fixing the animal on a mouse plate in an upward position, cutting the abdominal wall skin at the center, injecting 2 mL of physiological saline into the abdominal cavity, rotating the mouse plate for 1 min, and sucking out 1 mL of abdominal cavity washing liquid. (2) And injecting the recovered abdominal cavity washing liquid into a clean centrifugal tube, centrifuging for 10 min at 1200 r/min, removing supernatant, and adding PBS (phosphate buffer solution) to wash cells for 2 times. Washing the obtained cell precipitate with pre-cooled RPMI1640 culture solution, adding the precipitated cells into the pre-cooled RPMI1640 culture solution for resuspension, counting, and adjusting the concentration to 2 × 10⁶one/mL. Cells were seeded in 96-well plates at 100. mu.L per well, 3 replicates per sample, incubated at 37 ℃ in 5% CO₂Culturing in an incubator to allow macrophages to adhere to the wall, incubating for 3 h, slightly shaking the culture plate, discarding culture supernatant, slightly flushing the culture hole for 1-2 times by RPMI1640 culture solution pre-warmed at 37 ℃, and fully discarding non-adherent lymphocytes and other cells to obtain adherent cells, namely monolayer macrophages. The percent phagocytosis and phagocytosis index were calculated as follows:

percent phagocytosis (%) = number of macrophages engulfing chicken red blood cells/number of macrophages counted × 100%

Phagocytosis index = total number of phagocytosed chicken erythrocytes/count of macrophages.

2.4 analysis of the results

As shown in figure 1, compared with the blank control group, the phagocytic rate of macrophages of the positive control group is very significant (P <0.01), the phagocytic rate of macrophages of the compound grass antler ganoderma lucidum extract low dose group, the grass antler ganoderma lucidum water extract group (B group), the ganoderma lucidum + selenium-rich tea water extract group (E group), the grass antler ganoderma lucidum + perilla water extract group (F group) and the perilla herb + selenium-rich tea water extract group (G group) are significant (P <0.05), the perilla water extract group (C group) and the selenium-rich tea water extract group (D group) have no significant difference compared with the blank control group, the phagocytic rate of macrophages of the E group and the F group is obviously higher than that of the B group and the F group after the selenium-rich tea and perilla components are respectively added in the B group and the E group, and the single-taste medicinal material selenium-rich tea water extract and perilla water extract have no significant difference compared with the blank control group, the result shows that the phagocytic rate of mouse macrophage can be enhanced after the selenium-rich tea and the perilla are added. Through analysis of the perilla water extract group (group C), the selenium-rich tea water extract group (group D) and the perilla + selenium-rich tea water extract group (group G), the effect of improving the phagocytosis rate of mouse macrophages after combination of the perilla and the selenium-rich tea components is superior to the effect of independent action of the components, compared with the group E, the group F and the group G, the phagocytosis rate of mouse macrophages of the compound grass antler ganoderma lucidum extract low-dose group is obviously higher than the phagocytosis rate of the mouse macrophages of the group E, the group F and the group G, the effect of improving the phagocytosis rate of mouse macrophages after combination of the perilla, the selenium-rich tea and the grass antler ganoderma lucidum is better than the effect of combining the two components, and the analysis shows that the perilla, the selenium-rich tea and the grass antler ganoderma lucidum have a synergistic effect in the aspect of improving the phagocytosis rate of the mouse macrophages.

3 Effect of Compound Juncao Cornus Cervi Ganoderma aqueous extract on immune function of mouse cells

3.1 animal pretreatment

Male, 4-5 week old SPF grade BALB/c mice were randomly divided into 5 groups of 10 mice each. The compound grass-cultivated ganoderma lucidum antler extract is administrated by gavage for 1 time (0.3 mL each time) for mice according to low dose of 4.065 g/kg, medium dose of 8.13 g/kg and high dose of 16.26 g/kg respectively every day, gavage for 1 time (0.3 mL physiological saline) for empty control group mice every day, gavage for 1 time (0.3 mL) levamisole for positive control group mice every day, and the dose is 10 mg/kg, and the administration is continuously carried out for 28 days.

3.2 preparation of spleen cell suspension

Killing mice immunized with SRBC for 4-5 days after dislocation of cervical vertebrae, soaking mice in 75% ethanol for 3 min, cutting open abdominal cavity along midline of the abdominal cavity, taking out spleen, placing in culture dish, dissecting, taking out spleen, removing surface envelope and adipose tissue, and placing on a patientIn a large petri dish with sterile PBS, the spleen was rolled with two sterile frosted slides, the slides were washed with PBS, and finally the cytosol was passed through a 200 mesh screen. The spleen cells were washed 2 times and centrifuged at 1000 r/min for 10 min each time. The cells were counted and the cell concentration was adjusted to 5X 10⁶Number of plaque-forming cells in spleen/mL was measured.

3.3 cytokine assay

Taking eyeball blood sample, centrifuging at 3000 r/min for 5 min, and collecting supernatant. Taking out INF-gamma, IL-2 and IL-10 kit 20 min in advance, balancing to room temperature, diluting 20 × concentrated washing solution with ultrapure water to 1 × working solution, adding standard product&Adding 1.5 mL of specimen universal diluent into the freeze-dried sample, standing for 15 min, mixing gently, diluting to 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0 pg/mL, adding standard substance into blank hole in 96-well plate&Adding general diluent for the specimen, adding specimen or standard substances (100 mu/hole) with different concentrations into the other holes, sealing the reaction holes with sealing plate adhesive paper, incubating at 37 ℃ for 120 min, washing the plate for 5 times, adding enzyme conjugate diluent into the blank holes, adding enzyme conjugate working solution (100 mu/hole) into the other holes, sealing the reaction holes with sealing plate adhesive paper, and incubating at 37 ℃ for 60 min in the dark. The plate was washed 5 times. Chromogenic substrate (TMB) was added at 100. mu.l/well and incubated at 37 ℃ in the absence of light for 20 min. Adding 100 mu/well of stop solution, shaking for 1 min, and immediately measuring OD₄₅₀The value is obtained.

3.4 analysis of test results

As can be seen from FIG. 2, the IFN-. gamma., IL-2 and IL-10 contents in the mice of the low and medium dose groups were increased compared to the blank control group, and the IFN-. gamma., IL-2 and IL-10 contents in the mice of the high dose group were not significantly different from those of the blank control group. IFN-gamma and IL-2 are two main positive immune regulation factors generated by Th1, IFN-gamma not only can enhance T cell function, but also can induce bone marrow macrophage to polarize to M1 type macrophage to play a killing role, IL-2 can promote T cell proliferation activation and NK cell killing activity, IL-10 is an anti-inflammatory cytokine generated by T cell, natural killer cell and macrophage, IL-10 can prevent and inhibit strong specific and non-specific immune reaction and tissue damage caused by the strong specific and non-specific immune reaction, IL-10 content increase can enhance the capability of eliminating pathogen, thereby achieving the immune regulation role, therefore, IFN-gamma, IL-2 and IL-10 content increase shows that low-dose and medium-dose compound grass ganoderma lucidum extract can improve IFN-gamma, IL-gamma, gamma-2 and IL-10 content in mice, IL-2 and IL-10 cytokines have promoting effects, the compound grass Kazuno Ganoderma extract can enhance the immune efficacy of mice, the IFN-gamma, IL-2 and IL-10 contents in the mice of a high-dose group have no significant difference compared with those of a blank group, probably because the high-dose compound grass Kazuno Ganoderma extract causes the metabolism and function abnormality of the liver and kidney of the mice, thereby affecting the immune function of the mice, and the side effects of the mice need to be further researched by experiments.

Influence of compound grass, antler and ganoderma lucidum aqueous extract on humoral immunity function of mice

4.1 animal pretreatment

Male, 4-5 week old SPF grade BALB/c mice were randomly divided into 5 groups of 10 mice each. Respectively performing intragastric administration on the compound grass-cultivated antler-glossy ganoderma extract for 1 time (0.3 mL each time) for low dose 4.065 g/kg, medium dose 8.13 g/kg and high dose 16.26 g/kg for mice every day, performing intragastric administration on the mice of an empty control group for 1 time (0.3 mL each time) for empty control group for mice every day, and performing intragastric administration on the mice of a positive control group for 1 time (0.3 mL each time) for levamisole for 10 mg/kg for 28 days.

4.2 preparation of spleen cell suspension

Killing mice immunized by SRBC for 4-5 days after dislocation of cervical vertebrae, soaking the mice in 75% ethanol for 3 min, cutting off the abdominal cavity of the mice along the midline of the abdominal cavity, taking out the spleen, placing the spleen in a culture dish, completely stripping surface envelopes and adipose tissues, placing the spleen in the culture dish containing sterile PBS, rolling the spleen by using two sterile frosted glass slides, cleaning the glass slides by using PBS, and passing cell sap through a 200-mesh screen. The spleen cells were washed 2 times and centrifuged at 1000 r/min for 10 min each time. Counting, and adjusting the cell concentration to 5X 10⁶One cell/mL, spleen cell suspension was prepared, and the number of plaque-forming cells in the spleen was measured.

4.3 plaque assay

Weighing 1g of agarose, adding double distilled water to 100 mL to prepare a surface layer culture medium for later use, heating and dissolving the surface layer culture medium, placing the surface layer culture medium in a 45 ℃ water bath for heat preservation, mixing the surface layer culture medium with Hanks liquid with the same amount of pH 7.2-7.4 and 2-fold concentration, subpackaging small test tubes with 0.5 mL of each tube, adding 50 mu L of 10% SRBC (V/V) in the tube to prepare with SA liquid, adding 20 mu L of spleen cell suspension, quickly mixing the suspension uniformly, pouring the suspension on a glass slide with an agarose thin layer, making parallel sheets, horizontally buckling the glass slide on a sheet frame after the agar is solidified, placing the glass slide in a carbon dioxide incubator for incubation for 1-1.5 h, adding complement diluted by SA buffer solution 1: 8 to the upper layer of the culture medium, continuing incubation for 1-1.5 h, and counting the number of hemolytic plaques.

As can be seen from the analysis of FIG. 3, compared with the blank control group, the numbers of hemolytic plaques of the low dose group and the middle dose group are significantly different (P <0.05), the numbers of hemolytic plaques of the high dose group mice are not significantly different from the blank control group, the plaques are formed by B lymphocytes releasing hemolytic antibodies which can dissolve surrounding sheep red blood cells under the participation of complement, a visible plaque is formed around the B lymphocytes, and one plaque represents an antibody forming cell, so the number of plaques reflects the humoral immunity function of the organism and the number of specific antibody generating cells in the mice.

5 Effect of Compound Juncao cornu Cervi and Ganoderma aqueous extract on mouse monocyte-macrophage function

5.1 animal pretreatment

Male, 4-5 week old SPF grade Balb/c mice were randomly divided into 5 groups of 10 mice each. Respectively performing intragastric administration on the compound grass-cultivated antler-glossy ganoderma extract for 1 time (0.3 mL each time) for low dose 4.065 g/kg, medium dose 8.13 g/kg and high dose 16.26 g/kg for mice every day, performing intragastric administration on the mice of an empty control group for 1 time (0.3 mL each time) for empty control group for mice every day, and performing intragastric administration on the mice of a positive control group for 1 time (0.3 mL each time) for levamisole for 10 mg/kg for 28 days.

5.2 preparation of Chicken erythrocyte suspension

The chicken blood was taken and placed in a conical flask with glass beads and shaken thoroughly in one direction to defibrate. Washing with normal saline for 2-3 times, centrifuging at 2000 r/min for 10 min, removing supernatant, and preparing into 1% V/V chicken erythrocyte suspension with normal saline.

5.3 phagocytic function assay

(1) Three days before drug withdrawal, 1 mL of 5% starch broth solution was injected intraperitoneally into each mouse 1 time a day for 3 consecutive days. Stopping taking the medicine the next day, injecting 1 mL of 1% chicken red blood cell suspension into the abdominal cavity of each mouse at intervals of 30 min, killing the animal by dislocation of cervical vertebra, fixing the animal on a mouse plate in an upward position, cutting the abdominal wall skin at the center, injecting 2 mL of physiological saline into the abdominal cavity, rotating the mouse plate for 1 min, and sucking out 1 mL of abdominal cavity washing liquid.

(2) And injecting the recovered abdominal cavity washing liquid into a clean centrifugal tube, centrifuging for 10 min at 1200 r/min, removing supernatant, and adding PBS (phosphate buffer solution) to wash cells for 2 times. Washing the obtained cell precipitate with pre-cooled RPMI1640 culture solution, adding the precipitated cells into the pre-cooled RPMI1640 culture solution for resuspension, counting, and adjusting the concentration to 2 × 10⁶one/mL. Cells were seeded in 96-well plates at 100. mu.L per well, 3 replicates per sample, incubated at 37 ℃ in 5% CO₂Culturing in an incubator to allow macrophages to adhere to the wall, incubating for 3 h, slightly shaking the culture plate, discarding culture supernatant, slightly flushing the culture hole for 1-2 times by RPMI1640 culture solution pre-warmed at 37 ℃, and fully discarding non-adherent lymphocytes and other cells to obtain adherent cells, namely monolayer macrophages. The percent phagocytosis and phagocytosis index were calculated as follows:

percent phagocytosis (%) = number of macrophages engulfing chicken red blood cells/number of macrophages counted × 100%

Phagocytosis index = total number of phagocytosed chicken erythrocytes/count of macrophages.

5.4 analysis of the results of the experiment

TABLE 1 spleen and thymus indices in macrophage-group mice

Note: compared with blank control group, # means P <0.01, # means P <0.05

As can be seen from table 1, the spleen indexes of the mice in the low dose group and the medium dose group are significantly different (P <0.05) compared with those in the blank control group, and the spleen indexes of the mice in the high dose group and the positive control group are not significantly different. Compared with the blank control group, the thymus index of the mice in the experimental group has no significant difference. The spleen index and thymus gland index of mice in the low-dose group, the medium-dose group and the positive control group are all higher than those of mice in the blank control group, while the spleen index and thymus gland index of mice in the high-dose group are lower than those of mice in the blank control group, and no significant difference exists, which indicates that the compound grass-antler-ganoderma lucidum extract in the low-dose and medium-dose has a promoting effect on the growth and development of the spleen and thymus gland of the mice.

TABLE 2 Effect of Compound Juncao Kazuno Ganoderma extract on phagocytic potency of mouse macrophages

Note: in comparison with blank control group, P <0.01, 0.01< P <0.05, and Δ <0.05 in comparison with medium dose group

Statistical one-way anova was performed on the phagocytic rate and phagocytic index data of mouse peritoneal macrophages in table 2, with the following results: 1. the phagocytosis rate and phagocytosis index of the macrophages of the mice in the positive control group, the low dose group, the medium dose group and the high dose group are all higher than those of the mice in the blank control group, statistical analysis shows that the phagocytosis rate of the macrophages of the mice in the positive control group, the low dose group and the medium dose group is very significant (P <0.01) compared with that of the mice in the blank control group, the phagocytosis index of the macrophages of the mice in the positive control group and the medium dose group is very significant (P <0.01) compared with that of the mice in the blank control group, and the phagocytosis index of the macrophages of the mice in the low dose group and the blank control group is significant (P <0.05), the positive control group mice have the highest phagocytic rate and phagocytic index, and the results show that the compound grass-antler-ganoderma lucidum extract with low and medium doses has obvious effect on improving the phagocytic capacity of macrophages, so that the immunity of the organism is improved. 2. Compared with the blank control group, the phagocytic rate and the phagocytic index of the macrophages in the high-dose group have no significant difference, which shows that the phagocytic effect of the macrophages is enhanced by the high-dose group is poorer than that of the low-dose group and the middle-dose group, and the statistical analysis shows that the significant difference (P > 0.05) is not generated 3. compared with the middle-dose group, the high-dose group presents significant difference (P <0.05), which shows that the appropriate dose has promotion effect on the phagocytic effect of the macrophages, and the high-dose compound grass degrana extract can affect the immune system of mice due to the negative effect on the liver, the kidney and the like, so that the phagocytic rate and the phagocytic index of the mice are reduced on the contrary.

6 Effect of Compound Juncao, cornu Cervi and Ganoderma aqueous extract on anti-tumor function of mice

6.1 animal model establishment and treatment

40 male, 4-5 week old SPF grade ICR mice were prepared at a concentration of 1.0X 10⁷S180 mouse sarcoma cell suspension/mL. The right anterior axillary region of ICR mice was surface-sterilized with alcohol, and then 0.2 mLS180 mouse sarcoma cells were injected subcutaneously into the right anterior axillary region. Dividing the inoculated mice into 4 groups randomly every other day, namely 3 dose groups of a blank control group (normal saline group), a low dose group (4.065 g/kg), a medium dose group (8.13 g/kg) and a high dose group (16.26 g/kg), wherein after one week of inoculation, the inoculated part grows to 80-90 mm³When the size of the tumor indicates that the model is successfully made, the intragastric administration is started according to the body weight of the mouse, and the administration is carried out once a day for 10 days continuously. Tumor size and ICR mouse body weight were recorded daily after dosing.

6.2 measurement of tumor suppression Rate, spleen index, and thymus index

After 10 days, groups were sacrificed ICR mice, 8 hours before sacrifice, and mice were unable to contact food. Mice are killed by dislocation of cervical vertebrae, tumors, thymus and spleen are stripped, weighed respectively, and the inhibition rate of the compound grass Kazuno Ganoderma extract on S180 tumors of the mice, the spleen index and the thymus index of the mice are calculated. Calculation of spleen and thymus indices the spleen and thymus indices were calculated according to the following formulas: spleen index = spleen weight (mg)/body weight at death (g); thymus index = thymus weight (mg)/body weight at death (g).

6.3 Life cycle test

Sterilizing the right anterior axillary part of the right anterior limb of a SPF-grade ICR mouse with alcohol, aseptically collecting tumor cell suspension of S180 mouse inoculated for 7 days, staining with trypan blue, counting, diluting with PBS to 1 × 10⁷Perml, 0.2 mL of mouse ascites tumor cells were injected into the right forelimb axilla. The day after inoculation, mice were randomly divided into 4 groups, each group10, female and male. The compound grass-cultivated antler ganoderma lucidum extract is respectively administrated by stomach irrigation for 1 time per day according to low dose of 4.065 g/kg, medium dose of 8.13 g/kg and high dose of 16.26 g/kg, a blank control group is administrated by stomach irrigation for 1 time per day with 0.3 mL of physiological saline, the drug is stopped and observed after being administrated for 18 days, the death time of mice is observed and recorded, the survival days are recorded, and the life prolonging rate of each group of mice is calculated according to a formula.

Life prolonging rate = (average survival days in treatment group-average survival days in control group)/average survival days in control group) × 100%

TABLE 3 Effect of Compound Juncao Kazuno Ganoderma extract on spleen index and thymus index of mouse tumor groups

Note: compared with blank control group, # means P <0.01, # means P <0.05

TABLE 4 Effect of Compound Juncao Kazuno Ganoderma extract on mouse tumors

Note: compared with blank control group, # means P <0.01, # means P <0.05

Through statistical analysis of the data in table 4, compared with the blank control group, the tumor weights of the mice in the low dose group, the middle dose group and the high dose group are all lower than that in the blank control group, the average tumor weights of the mice in the low dose group and the middle dose group are significantly different from that in the blank control group (P <0.05), and the tumor weight of the mice in the high dose group is not significantly different from that in the blank control group (P > 0.05), which indicates that the tumor growth can be inhibited to some extent after continuous drug feeding, the tumor inhibition rate of the mice in the middle dose group is 40.30%, the tumor inhibition rate of the mice in the low dose group is 36%, and the tumor inhibition rate of the mice in the high dose group is 12.49%. The tumor inhibition rate of the high-dose group mice is lower than that of the low-dose group and the medium-dose group, and by combining the previous experiment and the thymus index and the spleen index, the mice can be preliminarily judged to have certain side effects on the immune function of the mice under the high dose, so that the anti-tumor effect of the mice is poorer than that of the low-dose group and the medium-dose group.

7 comparison of influence of compound Juncao antler lucid ganoderma powder electuary and compound Juncao antler lucid ganoderma tea powder on hemolytic plaque and tumor inhibition rate

Referring to the preparation process and experimental flow of compound Juncao antler ganoderma lucidum tea powder of patent No. CN201910220780.3, under the same experimental conditions, taking the dosage of the compound Juncao antler ganoderma lucidum tea powder as an example: the formula combination is evaluated by researching the difference of the effect of the ganoderma lucidum crude polysaccharide 100mg/kg, the selenium-rich black tea polyphenol 320mg/kg and the perilla ethanol extract 8.54mg/kg on hemolysis blank and tumor inhibition rate with the compound Juncao antler ganoderma lucidum powder electuary in low dosage. In order to carry out comparison at the same level, the dosage of the compound fungus grass antler ganoderma lucidum tea powder is obtained by converting the extraction rate: 2.403 g/kg of grass, antler and glossy ganoderma, 3.596 g/kg of selenium-enriched tea and 0.166 g/kg of perilla. The compound granule of Juncao, antler and lucid ganoderma has the following low dose: 2.3g/kg of grass, antler and glossy ganoderma, 1.6g/kg of selenium-rich black tea and 0.165g/kg of purple perilla.

TABLE 5 influence of compound granule, powder and tea on hemolytic plaque and tumor inhibition

From table 5, it can be seen that the compound grass antler ganoderma powder medicinal granules in the low-dose group are higher than the compound grass antler ganoderma powder in the number of hemolytic plaques and the tumor inhibition rate on mouse S180 sarcoma, and the dosage of the raw materials of the compound grass antler ganoderma powder in the low-dose group is higher than the dosage of the compound grass antler ganoderma powder medicinal granules in the low-dose group, which indicates that the compound grass antler ganoderma powder medicinal granules in the compatibility proportion have better effects on improving hemolytic plaques and the tumor inhibition rate of mice, and the effective component dissolution effects in grass antler ganoderma, selenium-rich tea and perilla are enhanced through response surface optimization due to an ultrasonic-assisted water extraction method, and the biological activity of the effective components is retained, so that the compound grass antler ganoderma powder medicinal granules have important significance on saving raw materials in production and application.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.