CN113069510A - Composition for enhancing immunity and preparation method and application thereof - Google Patents
Composition for enhancing immunity and preparation method and application thereof Download PDFInfo
- Publication number
- CN113069510A CN113069510A CN202110377444.7A CN202110377444A CN113069510A CN 113069510 A CN113069510 A CN 113069510A CN 202110377444 A CN202110377444 A CN 202110377444A CN 113069510 A CN113069510 A CN 113069510A
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- composition
- spore powder
- ganoderma lucidum
- lucidum spore
- chinese medicine
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Abstract
The invention provides a composition for enhancing immunity, which comprises ganoderma lucidum spore powder and a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises astragalus mongholicus, wolfberry, wild jujube and dendrobium officinale. According to the invention, the ganoderma lucidum spore powder is combined with the traditional Chinese medicine composition, so that the medicine composition can improve the immunity of a human body, and has high safety and no toxic or side effect. The production process is simple and convenient, is easy to operate, can realize industrial continuous production, and has low production cost and good product market prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a composition for enhancing immunity as well as a preparation method and application 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 cells infected with mutant nuclear viruses in the body, and is the body's physiological response to recognize and eliminate "cross-sexing". The immune system of the human body plays a decisive role because of the healthy survival of humans in various environments. With the continuous development of social economy and the faster and faster pace of life, the health of everyone is vulnerable to a plurality of external threats, including environmental pollution, working pressure, economic pressure, bacteria and viruses, improper diet and the like. In addition, due to the reduction of irregular living habits and eating habits, exercise amount and sleeping time, the body is continuously damaged, and the immunity is reduced. The decline of immunity directly leads to the decline of disease resistance of organism, is manifested as symptoms such as easy cold, slow wound healing, weakened gastrointestinal tract function, easy fatigue, etc. Therefore, the probability of infecting pathogenic microorganisms can be effectively reduced by improving the immunity of the human body. It follows that improving immunity is crucial to self-health.
The traditional Chinese medicine has wide medicine sources, and has the advantages of small toxic and side effects, obvious efficacy and the like for enhancing immunity. The Ganoderma spore powder contains a large amount of effective components for enhancing immunity, such as Ganoderma polysaccharides and Ganoderma triterpenes. The Ganoderma spore powder can activate phagocytic function of macrophage, antagonize glucocorticoid, enhance phagocytic and serum agglutination function of mouse abdominal cavity macrophage, enhance nonspecific immunity, and improve humoral immunity level and cellular immunity level. The effective components of the wall-broken ganoderma lucidum spore powder are easier to absorb, and have a certain protection effect on relevant functional viscera of an immune system. The astragalus has the effects of strengthening heart, reducing blood pressure, resisting bacteria, improving immunity and the like, can promote the increase of white blood cells in human blood, resist the reduction of the white blood cells of the human body caused by chemical substances, radiation or other reasons, and obviously improve the phagocytic function of a mononuclear macrophage system and the white blood cells. The lycium barbarum polysaccharides in the lycium barbarum can reduce the activity of oxide dismutase and oxidase of an organism and reduce the content of malondialdehyde in a heart, is very important for resisting fatigue and recovering fatigue of a human body, and has the effects of promoting immunity, resisting aging and tumors, removing free radicals, protecting the liver and the like. The wild jujube has the effects of nourishing the liver, calming the heart, soothing the nerves and the like, can be used for treating insomnia and other diseases, mainly contains saponins, flavonoids, triterpenes, alkaloids and the like as chemical components, can relieve stress, keep a relaxed state, improve attention, has an immunoregulation function on organisms, can enhance the immune function of human bodies, can inhibit a central nervous system, has the effects of calming and hypnosis, and can obtain a remarkable and ideal curative effect on mild or severe insomnia. The dendrobium officinale granules can promote the phagocytic function of macrophages of tumor-bearing animals, enhance the proliferation and differentiation of T lymphocytes and the activity of NK cells, and obviously improve the serum hemolysin value of the tumor-bearing animals, and prompt that the dendrobium officinale granules have certain improvement effect on nonspecific immune function or specific cellular immune and humoral immune function.
In the face of world wide abuse of new crown epidemic in 2020, the immunity enhancement of human bodies can effectively prevent the invasion of new crown viruses and reduce the infection probability, so the immunity enhancement of human bodies has important significance for the life and health of the masses. By utilizing the advantages of high safety of the traditional Chinese medicine, synergistic cooperation of components and the like, a high-efficiency and safe pharmaceutical composition for enhancing the immunity of the human body is developed, and related functional health products are developed, so that the pharmaceutical composition has remarkable social and economic benefit prospects for the human society.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a composition for enhancing immunity, a preparation method and an application thereof, wherein the composition provided by the present invention has an effect of enhancing immunity, a good curative effect and no toxic or side effect.
The invention provides a composition for enhancing immunity, which comprises ganoderma lucidum spore powder and a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises astragalus mongholicus, wolfberry, wild jujube and dendrobium officinale.
Preferably, the feed additive is prepared from the following raw materials in parts by mass:
6-8 parts of ganoderma lucidum spore powder, wherein the ganoderma lucidum spore powder is 300-400 meshes of wall-broken ganoderma lucidum spore powder;
2-4 parts of a traditional Chinese medicine composition, wherein the mass ratio of the astragalus root, the medlar, the wild jujube and the dendrobium officinale in the traditional Chinese medicine composition is (5-8): 7-10): 3-6): 2-5.
Preferably, the feed additive is prepared from the following raw materials in parts by mass:
7 parts of ganoderma lucidum spore powder, wherein the ganoderma lucidum spore powder is 350-mesh wall-broken ganoderma lucidum spore powder;
3 parts of a traditional Chinese medicine composition, wherein the mass ratio of the astragalus to the Chinese wolfberry to the wild jujube to the dendrobium officinale is 6:9:4: 3.
The invention also provides a preparation method of the composition, which comprises the following steps:
A) breaking the wall of the ganoderma lucidum spore powder to obtain wall-broken ganoderma lucidum spore powder;
B) extracting the Chinese medicinal composition with alcohol, and filtering to obtain filtrate;
C) and mixing the filtrate with the wall-broken ganoderma lucidum spore powder to obtain the composition.
Preferably, the wall breaking method is to break the wall of the ganoderma lucidum spore powder by adopting an ultralow-temperature airflow wall breaking technology.
Preferably, the alcohol extraction is ethanol reflux extraction, and the ethanol reflux extraction is performed for 2-3 times by 60-90% (v/v) ethanol with the weight 6-9 times of the total weight of the traditional Chinese medicine composition, and each time is 60-90 min.
Preferably, the method also comprises a step of concentrating after the filtrate is obtained; concentrating the filtrate at 50-60 ℃ to obtain thick paste, wherein the relative density of the thick paste is 1.10-1.15 g/mL.
The invention also provides an application of the composition in preparing products for enhancing immune function.
Preferably, the product is health food or medicine;
the dosage form of the product is tablets, granules, capsules, powder, pills or oral liquid.
Compared with the prior art, the invention provides a composition for enhancing immunity, which comprises ganoderma lucidum spore powder and a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises astragalus mongholicus, wolfberry, wild jujube and dendrobium officinale. According to the invention, the ganoderma lucidum spore powder is combined with the traditional Chinese medicine composition, so that the medicine composition can improve the immunity of a human body, and has high safety and no toxic or side effect. The production process is simple and convenient, is easy to operate, can realize industrial continuous production, and has low production cost and good product market prospect.
The results show that the composition can obviously improve the delayed type allergic reaction capacity and the NK cell activity of a mouse, the half hemolysis value of the mouse and the number of antibody-producing cells with lymphocyte transformation capacity in animal experiments taking the pharmaceutical composition, and has no obvious influence on the weight gain, the thymus/body ratio, spleen/body wallpaper, mononuclear-macrophage carbon clearance capacity and the ability of macrophages to phagocytize chicken erythrocytes of the mouse, which indicates that the pharmaceutical composition has the function of enhancing the immunity. In addition, acute toxicity and long-term toxicity experiments of the pharmaceutical composition show that the pharmaceutical composition has no toxicity to mice, belongs to a non-toxic level and can be safely used.
Detailed Description
The invention provides a composition for enhancing immunity, which comprises ganoderma lucidum spore powder and a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises astragalus mongholicus, wolfberry, wild jujube and dendrobium officinale.
According to the invention, the ganoderma lucidum spore powder and the traditional Chinese medicine are combined, so that the pharmaceutical composition can enhance the immunity of a human body, and has high safety and no toxic or side effect.
In the invention, the composition for enhancing immunity is prepared from the following raw materials in parts by mass:
6-8 parts of ganoderma lucidum spore powder, wherein the ganoderma lucidum spore powder is 300-400 meshes of wall-broken ganoderma lucidum spore powder;
2-4 parts of a traditional Chinese medicine composition, wherein the mass ratio of the astragalus root, the medlar, the wild jujube and the dendrobium officinale in the traditional Chinese medicine composition is (5-8): 7-10): 3-6): 2-5.
In some embodiments of the invention, the composition for enhancing immunity is prepared from the following raw materials in parts by mass:
7 parts of ganoderma lucidum spore powder, wherein the ganoderma lucidum spore powder is 350-mesh wall-broken ganoderma lucidum spore powder;
3 parts of a traditional Chinese medicine composition, wherein the mass ratio of the astragalus to the Chinese wolfberry to the wild jujube to the dendrobium officinale is 6:9:4: 3.
The invention also provides a preparation method of the composition, which comprises the following steps:
A) breaking the wall of the ganoderma lucidum spore powder to obtain wall-broken ganoderma lucidum spore powder;
B) extracting the Chinese medicinal composition with alcohol, and filtering to obtain filtrate;
C) and mixing the filtrate with the wall-broken ganoderma lucidum spore powder to obtain the composition.
Specifically, the wall breaking method is to break the wall of ganoderma lucidum spore powder to obtain the wall-broken ganoderma lucidum spore powder, wherein the wall breaking method is preferably to break the wall of ganoderma lucidum spore powder by adopting an ultralow-temperature airflow wall breaking technology.
The traditional Chinese medicine composition is subjected to alcohol extraction and then is filtered to obtain a filtrate. The alcohol extraction method of the traditional Chinese medicine composition is not particularly limited, and the alcohol extraction method known to the skilled person in the art can be used. Preferably, the percolation method, the immersion method or the reflux extraction method is adopted, and further, the traditional Chinese medicine composition is extracted by ethanol reflux. The specific method comprises the following steps:
wherein the mass ratio of the ethanol to the traditional Chinese medicine composition is (6-9): 1, preferably (7-8): 1. the ethanol is an ethanol extract with the volume percentage of 60-90%. The reflux extraction frequency is 2-3 times, and the extraction time is 60-90 min each time.
In addition, in order to facilitate the preparation of the pharmaceutical composition of the present invention into various medicaments, the preparation method of the present invention further comprises:
in some embodiments of the invention, the step of concentrating is further included after collecting the filtrate; and concentrating the filtrate at the concentration temperature of 50-60 ℃ to obtain thick paste, wherein the relative density of the thick paste is 1.10-1.15 g/mL.
In the invention, the ganoderma lucidum spore powder and the raw material medicines in the traditional Chinese medicine composition are mutually compatible and cooperate, so that the immunity of a human body can be effectively improved. The pharmaceutical composition has no toxicity or drug dependence, and can be applied to preparation of various functional products for improving immunity.
In the application of preparing functional products for improving immunity, the pharmaceutical composition can be prepared into various functional foods or clinical conventional preparations according to conventional preparation methods in the field, for example, pharmaceutically acceptable auxiliary materials can be added into the pharmaceutical composition with effective dose, and the type of the auxiliary materials is not particularly limited, and the type of the auxiliary materials known by persons skilled in the art can be any. In the invention, the auxiliary material can be one or more of a disintegrating agent, a lubricating agent, a binding agent and a dispersing agent, and can be prepared into various oral medicaments, such as tablets, granules, capsules, powder, pills or oral liquid.
Various pharmaceutical products can be easily prepared by the conventional preparation method knowing the active ingredients, and the auxiliary materials used in the preparation are well known to those skilled in the art, such as sodium carboxymethyl starch, sucrose powder, honey, sesame oil, etc.
The animal experiment that the composition can obviously improve the delayed type allergic reaction capacity and the NK cell activity of a mouse, the half hemolysis value of the mouse and the number of antibody-producing cells with lymphocyte transformation capacity has no obvious influence on the weight increase, the thymus/body ratio, spleen/body weight wallpaper, the mononuclear-macrophage carbon clearance capacity and the ability of macrophages to phagocytize chicken erythrocytes of the mouse shows that the pharmaceutical composition has the function of enhancing the immunity. In addition, acute toxicity and long-term toxicity experiments of the pharmaceutical composition show that the pharmaceutical composition has no toxicity to mice, belongs to a non-toxic level and can be safely used.
The invention reasonably optimizes the formula, adopts the ganoderma lucidum spore powder and the extract of the traditional Chinese medicine composition to prepare the high-efficiency and safe medicine composition, has the effect of enhancing immunity, has no toxic or side effect, and can be widely applied to the preparation of functional products for enhancing immunity.
For further understanding of the present invention, the composition for enhancing immunity and the preparation method and application thereof provided by the present invention are described below with reference to the following examples, and the scope of the present invention is not limited by the following examples.
The pharmaceutical composition and the raw materials and reagents used in the application thereof provided by the invention are all available in the market.
Example 1: capsules for preparing the pharmaceutical composition of the invention
Weighing raw materials (unit: g) including wall-broken Ganoderma spore powder 70 and Chinese medicinal composition extract 30;
taking the traditional Chinese medicine composition (the mixture ratio is 6 parts of astragalus, 9 parts of medlar, 4 parts of wild jujube and 3 parts of dendrobium officinale), respectively adding 70% ethanol with the weight being 8 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to form thick paste with the relative density of 1.10-1.15 g/mL, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma lucidum spore powder and the Chinese medicinal composition extract raw material medicine according to the weight, adding the carboxymethyl starch sodium, fully and uniformly mixing, preparing into granules, and filling into No. 1 capsules to obtain capsules.
Example 2: tablets for preparing the pharmaceutical composition of the present invention
Weighing raw materials (unit: g) including wall-broken Ganoderma spore powder 60 and Chinese medicinal composition extract 40;
taking the traditional Chinese medicine composition (the mixture ratio is that astragalus membranaceus 7, medlar 8, wild jujube 5 and dendrobium officinale 4), respectively adding 80% ethanol with the weight 6 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to form thick paste with the relative density of 1.10-1.15, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma lucidum spore powder and the Chinese medicinal composition extract raw material medicine according to the weight, adding the carboxymethyl starch sodium, fully and uniformly mixing, and feeding into a tablet machine to prepare the tablet.
Example 3: granules for preparing the pharmaceutical composition of the invention
Weighing raw materials (unit: g) such as wall-broken Ganoderma spore powder 65 and Chinese medicinal composition extract 35;
taking the traditional Chinese medicine composition (the mixture ratio is 5 astragalus mongholicus, 9 lycium barbarum, 5 wild jujube and 3 dendrobium officinale), respectively adding 85% ethanol with the weight 8 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to form thick paste with the relative density of 1.10-1.15, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma spore powder and the Chinese medicinal composition extract raw material medicine according to the weight, adding the mixture of sucrose powder and carboxymethyl starch sodium, mixing uniformly, and preparing into granules.
Example 4: pills for preparing the pharmaceutical composition of the invention
Weighing raw materials (unit: g) including wall-broken Ganoderma spore powder 75 and Chinese medicinal composition extract 25;
taking the traditional Chinese medicine composition (the mixture ratio is 8 of astragalus, 9 of medlar, 5 of wild jujube and 4 of dendrobium officinale), respectively adding 75 percent ethanol with the weight 7 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to form thick paste with the relative density of 1.10-1.15, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma spore powder and the Chinese medicinal composition extract raw material medicines according to the weight, adding pure sesame oil or honey, mixing uniformly, and conveying to a centrifugal pill making machine to prepare pills.
Comparative example 1
Taking the traditional Chinese medicine composition (the mixture ratio is 6 parts of astragalus, 9 parts of medlar, 4 parts of wild jujube and 3 parts of dendrobium officinale), respectively adding 70% ethanol with the weight being 8 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to form thick paste with the relative density of 1.10-1.15 g/mL, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma lucidum spore powder and the Chinese medicinal composition extract raw material medicine according to the weight, adding the carboxymethyl starch sodium, fully and uniformly mixing, preparing into granules, and filling into No. 1 capsules to obtain capsules.
Comparative example 2
Weighing raw materials (unit: g) including wall-broken Ganoderma spore powder 70 and Chinese medicinal composition extract 30;
taking the traditional Chinese medicine composition (the mixture ratio is that wild jujube 4 and dendrobium officinale 3), respectively adding 70% ethanol with the weight 8 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to obtain thick paste with the relative density of 1.10-1.15 g/mL, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma lucidum spore powder and the Chinese medicinal composition extract raw material medicine according to the weight, adding the carboxymethyl starch sodium, fully and uniformly mixing, preparing into granules, and filling into No. 1 capsules to obtain capsules.
Comparative example 3
Weighing raw materials (unit: g) including wall-broken Ganoderma spore powder 70 and Chinese medicinal composition extract 30;
taking the traditional Chinese medicine composition (the mixture ratio is 6 astragalus mongholicus and 9 wolfberry fruit), respectively adding 70% ethanol which is 8 times of the total weight of the extract to carry out reflux extraction for 2 times, 60min each time, filtering, concentrating the filtrate at the temperature of below 55 ℃ under reduced pressure to obtain thick paste with the relative density of 1.10-1.15 g/mL, and carrying out spray drying to obtain the crude drug of the traditional Chinese medicine composition extract. Weighing the ganoderma lucidum spore powder and the Chinese medicinal composition extract raw material medicine according to the weight, adding the carboxymethyl starch sodium, fully and uniformly mixing, preparing into granules, and filling into No. 1 capsules to obtain capsules.
Example 5: animal experiments with pharmaceutical compositions
To verify the efficacy of the pharmaceutical composition of the present invention in enhancing immunity, a mouse functional test was performed on the pharmaceutical composition.
1. Test materials and methods
1.1 test materials
1.1.1 sample: the pharmaceutical composition of the present invention (the pharmaceutical composition granules prepared in example 1)
1.1.2 test animals and groups: 350 Kunming mice are purchased from the center of laboratory animals of Hainan medical college, and have male and female halves and weight of 18-22 g. The feed is provided by the same unit.
Each 70 mice was in one large group, for a total of five large groups. Immunization group 1 was subjected to carbon clearance test; immunizing the group 2, and performing ConA-induced mouse lymphocyte transformation experiment and activity determination of NK cells; immunization 3 groups for visceral ratio determination, median hemolysis value (HC)50) And the number of antibody-producing cells; the immunization 4 groups were subjected to delayed type allergy (DTH) experiments; immunization group 5 mice were subjected to the experiment of macrophages in the abdominal cavity. 70 mice in each large group are randomly divided into 7 groups according to body weight, namely a control group and a comparison 1 to c3 groups and low, medium and high dose groups.
1.1.3 laboratory site: the laboratory site is isolated in a common laboratory, the sunlight is sufficient, the ventilation is good, the temperature is controlled at 25 ℃, the relative humidity is 40-70%, and an ultraviolet disinfection lamp is installed indoors.
1.1.4 dose selection: the low, medium and high doses are respectively 0.180g/kg.bw, 0.360g/kg.bw and 1.080g/kg.bw (corresponding to 5/10/30 times of the recommended dose of a human body); selecting 0.360g/kg.bw compared with the groups 1-3; the control group was gavaged with deionized water.
1.1.5 statistical treatment: the data were transformed and statistically analyzed using Excel, Spss software.
1.2 test methods:
1.2.1 organ/body weight ratio measurement
Each group was weighed 7 days after administration, and spleen and thymus of each group were weighed, and spleen index and thymus index of each group were calculated according to the following formulas.
1.2.2 delayed type allergic reaction
Sheep blood was collected and washed 3 times with physiological saline, and each mouse was immunized by intraperitoneal injection of 0.2mL of 2% (v/v, prepared with physiological saline) SRBC. After 4 days of sensitization, the thickness of the plantar region of the left hind foot was measured, and then 20% (v/v) of SRBC (0.2. mu.L/mouse) was injected under the measurement site, and the thickness of the plantar region of the left hind foot was measured 24h after the injection, and the same site was measured 3 times, and the average value was taken. The extent of DTH is expressed as the difference in thickness between the plantar aspect of the foot before and after the attack.
1.2.3 ConA-induced mouse lymphocyte transformation experiment (MTT method)
The spleens were aseptically removed, ground with forceps in a dish containing sterile Hank's solution, made into a cell suspension, and filtered through a 200 mesh screen. Two washes with Hank's solution were performed, each time for 10min (1000 rpm). The pellet was then suspended in 1mL of complete medium and counted stained with Tulip blue (above 95%). Cell concentration was adjusted to 3X 10 by RPMI1640 medium6Per mL, thenThe cell suspension was added to a 24-well plate in two wells, 1mL per well, and 75. mu.L of ConA solution (equivalent to 7.5. mu.g/mL) was added to one well, and the other well was used as a control, to 5% CO2Incubate at 37 ℃ for 4 days. 4 hours before the end of the culture, 0.7mL of the supernatant was gently aspirated from each well, and 0.7mL of RMPI1640 medium without calf serum was added thereto together with 50. mu.L/well of MTT (5mg/mL), and the culture was continued for 4 hours. After the culture, 1mL of acidic isopropanol was added to each well to completely dissolve the crystals, which were then dispensed into 96-well culture plates, each well was prepared as 3 parallel wells, and the optical density was measured using a microplate reader at a wavelength of 570 nm. The proliferation capacity of lymphocytes was expressed as the optical density value of the ConA plus wells minus the optical density value of the ConA not plus wells.
1.2.4 antibody-producing cell assay
Washing sheep blood with normal saline for 3 times, centrifuging for 10min (2000r/min, preparing 2% (v/v) cell suspension from packed SRBC with normal saline, injecting 0.2mL into abdominal cavity of mouse, dislocating cervical vertebra of mouse immunized for 5-half days, taking out spleen, grinding spleen, making into cell suspension, filtering with 200 mesh screen, centrifuging for 10min (1000r/min), washing with Hank's solution twice, suspending cells in 5mL RMPI1640 culture solution, staining and counting (above 95%), adjusting cell concentration to 5 × 10 with RPMI1640 culture medium6And (4) preparing a spleen cell suspension. Preparing agarose into 1% water solution, decocting in water bath for 30min, mixing with equal amount of double concentration Hank's solution, subpackaging into small tubes 0.5 mL/tube, adding 10% packed SRBC 50 μ L (v/v) prepared with SA buffer solution, and 20 μ L spleen cell suspension (5 × 10)6one/mL) of the slide glass, quickly mixing the two samples, pouring the mixture on an agarose thin-layer glass slide, horizontally buckling the glass slide on a glass slide rack after agarose is solidified, and putting CO into the glass slide rack2Incubate in the incubator for 1.5h, then add complement to the slide well, continue incubation for 1.5h, count the number of hemolytic plaques. The complement preparation comprises collecting guinea pig blood, separating serum (at least 5 guinea pig mixed serum), adding 1mL packed SRBC into 5mL guinea pig serum, standing in refrigerator at 4 deg.C for 30min, shaking frequently, centrifuging to obtain supernatant, packaging, and storing at-70 deg.C. When in use, the ratio of 1: and 15, diluting.
1.2.5 determination of the median hemolysis value (HC50)
At the 3 rd time of administration, each group of mice was injected with 0.2mL of 5% chicken red blood cell suspension intraperitoneally, continuously for 5 days, and administered for 24h at the last time, each group of mice was removed from the eyeball and blood was collected, and serum was obtained by centrifugation at 3500rpm and refrigerated at 4 ℃ for future use. A part of serum was diluted 100-fold with physiological saline, and 1mL of the diluted serum was mixed with 0.5mL of 5% chicken red blood cell suspension and 0.5mL of 10% guinea pig complement, and the control group was replaced with 1mL of physiological saline, and the rest steps were as described above. The sample is placed in a constant temperature water bath at 37 ℃ for heat preservation for 30min, and the reaction is stopped in a refrigerator at 4 ℃. And finally centrifuging at 2000rpm for 10min, taking 1mL of supernatant, standing for 10min, and blank the control tube at 540nm to respectively determine the optical density value of each tube. The amount of hemolysin is expressed as half the hemolysin value (HC)50) Expressed, calculated as:
1.2.6 mouse carbon clearance test
Injecting India ink diluted 5 times with physiological saline into tail vein of mouse, timing immediately after injecting ink 0.1mL/10g, collecting 20 μ L blood from spleen venous plexus 2 and 10min after injecting ink, respectively, adding 2 mL0.1% Na2CO3Shaking up in the solution. With 0.1% Na2CO3The solution was used as a blank control and the optical density values were measured in a spectrophotometer at 600 nm. After blood sampling, mice were sacrificed, livers and spleens were weighed, and phagocytic index was calculated.
1.2.7 phagocytic function of mouse abdominal macrophage
Preparing a chicken BRC suspension: under aseptic operation, heparin anticoagulation is performed on the blood collected from the inferior vein of chicken wings, the blood is washed for 3 times by using permanent physiological saline, the centrifugation speed of the first 2 times is 1500rmp, the centrifugation is performed for 5min, the supernatant and the leukocyte layer of the interface are discarded, finally the centrifugation is continuously performed for 2 times at the speed of 2000rmp and the centrifugation is performed for 5min until the chicken erythrocyte volume value is constant, and the physiological saline is used for preparing 5 percent of chicken erythrocyte suspension.
Each group of mice was weighed 7 days after administration, injected with 0.5mL of 5% chicken red blood cell suspension intraperitoneally, killed by dislocation of cervical vertebrae after 8h, fixed on a mouse plate in an upward position, injected with 2.0mL of PBS2 into abdominal cavity, gently applied to the abdomen of the mice for 1min, absorbed with 0.5mL of abdominal liquid, dropped on a glass slide, placed in an enamel box with wet gauze, placed at 37 ℃, and incubated in an incubator for 30 min. The method comprises the following steps of mixing methanol: acetone (1:1) fixation, staining with 4% Giemsa-phosphate buffer, rinsing with distilled water and air drying. 100 phagocytes were counted per plate under oil lens, and the phagocytic rate and phagocytic index were calculated as follows:
1.2.8 measurement of NK cell Activity
Subjecting test mouse to cervical dislocation, aseptically taking spleen, making into spleen cell suspension, washing with Hank's lotion for 2 times, centrifuging for 10min (1000rpm) each time, discarding supernatant to bounce cell pulp, adding 0.5mL sterile water for 20s, lysing erythrocytes, adding 0.5mL2 times of Hank's solution and 8 mLHank's solution, centrifuging for 10min at 1000rpm, resuspending with 1mL RPMI1640 complete culture solution containing 10% calf serum, diluting with 1% glacial acetic acid, counting, staining with talofulen blue, adjusting cell concentration to 2 × 107each/mL, which is effector cell, YAC-1 cell with good growth after passage for 24h is adjusted to 4 × 10 cell concentration by RPMI1640 complete culture solution5one/mL, this is the target cell. Taking 100 mu L of each of target cells and effector cells, and adding each of the target cells and 100 mu L of 2.5% Triton into the maximum release hole of the target cells; all the above items are provided with three parallel holes at 37 ℃ and 5% CO2After culturing for 4h in an incubator, the 96-well culture plate is centrifuged at 1500rpm for 5min, 100. mu.L of supernatant is aspirated from each well and placed in a flat-bottomed 96-well culture plate, 100. mu.L of LDH matrix solution is added simultaneously, 1mol/L of HCl 30. mu.L is added to each well according to a greenhouse reaction for 3-10min, and the optical density is measured at 490nm of a microplate reader.
2. Results
2.1 Effect of the composition on mouse body weight
The results of the effect of the pharmaceutical composition on the body weight of mice in each immunization group are shown in tables 1 to 5. The weight of mice in the early stage, the middle stage and the final stage of the experiment of each dose group and the comparison group and the weight increase of the mice in the experiment period are compared with the control group, and the difference is not significant (P is more than 0.05).
Table 1 immunization 1 group of mice body weights
Table 2 immunization 2 groups of mice body weights
TABLE 3 immunization of 3 groups of mice body weights
Table 4 immunization of 4 groups of mice body weights
Table 5 immunization of 5 groups of mice body weights
| Group of | Number of animals | Initial body weight (g) | Middle-term body weight (g) | Terminal body weight (g) | Weight gain (g) |
| Control group | 10 | 19.79±1.14 | 27.34±1.91 | 34.69±2.62 | 14.90±2.90 |
| Low dose group | 10 | 20.71±1.12 | 27.50±1.48 | 33.85±3.53 | 13.15±4.03 |
| Middle dose group | 10 | 20.49±0.84 | 27.09±1.37 | 33.97±1.73 | 13.48±2.18 |
| High dose group | 10 | 20.62±0.85 | 27.26±1.67 | 34.01±2.68 | 13.40±3.23 |
| Comparative 1 group | 10 | 20.64±1.08 | 28.07±1.05 | 33.34±2.48 | 12.71±2.87 |
| Comparative 2 group | 10 | 20.15±1.02 | 27.84±0.97 | 33.61±2.36 | 13.45±2.19 |
| For you 3 groups | 10 | 20.26±1.06 | 28.11±0.89 | 33.28±2.17 | 13.03±2.56 |
2.2 Effect of the composition on the organ/body weight ratio of the immune organs of mice
The results of the effect of the pharmaceutical composition on the organ/body weight ratio of the mouse immune organs are shown in table 6. Each dose of the composition had no significant effect on the spleen/body weight ratio and thymus/body weight ratio (P > 0.05) in mice.
TABLE 6 Effect of compositions on the organ/body weight ratio of the immune organs of mice
2.3 Effect of compositions on mouse cellular immune function
2.3.1 Effect of the composition on delayed hypersensitivity (DTH) in mice
See table 7. Compared with a control group, the delayed type allergic reaction capability of the mice in the medium and high dose groups is remarkably different (P is less than 0.05).
TABLE 7 Effect of compositions on delayed allergy (DTH) in mice
2.3.2 Effect of the composition on the ConA-induced lymphocyte transformation Capacity in mice
See table 8. The delayed type allergic reaction capability of the mice of the low and medium dose groups and the control group is not obviously different (P is more than 0.05), and the lymphocyte transformation capability of the mice of the high dose group is obviously different (P is less than 0.05) compared with the control group.
TABLE 8 Effect of compositions on the transforming ability of mouse lymphocytes
2.4 Effect of the composition on humoral immunity
2.4.1 Effect of the composition on the number of mouse antibody-producing cells
See table 9. High dose effect on the number of mouse antibody-producing cells significantly increased (P < 0.01) compared to control Table 9 compositions
2.4.2 composition on half maximal hemolysis value (HC) of mice50) Influence of (2)
See table 10. Half maximal hemolysis value (HC) in high dose group mice50) Compared with a control group, the content of the compound is remarkably improved (P is less than 0.05).
TABLE 10 composition vs. half maximal hemolysis value (HC) in mice50) Influence of (2)
2.5 Effect of the composition on phagocytic function of mouse monocyte-macrophages
See table 11. Each dose had no significant effect on mouse carbon clearance capacity (P > 0.05).
TABLE 11 Effect of compositions on mouse monocyte-macrophage carbon clearance
| Group of | Number of animals | Half maximal hemolysis value | P value |
| Control group | 10 | 4.57±1.23 | - |
| Low dose group | 10 | 4.89±1.17 | 0.404 |
| Middle dose group | 10 | 5.16±0.76 | 0.351 |
| High dose group | 10 | 5.14±1.07 | 0.384 |
| Comparative 1 group | 10 | 4.72±1.02 | 0.514 |
| Comparative 2 group | 10 | 4.85±0.81 | 0.461 |
| For you 3 groups | 10 | 4.93±1.12 | 0.428 |
2.6 Effect of the composition on the ability of mouse macrophages to phagocytose chicken erythrocytes
See Table 12-1/12-2. Each dose of the composition had no significant effect on the ability of mouse macrophages to phagocytose chicken erythrocytes (P > 0.05).
TABLE 12-1 Effect of compositions on the phagocytosis of chicken erythrocytes by mouse macrophages
TABLE 12-2 Effect of compositions on the phagocytosis index of chicken erythrocytes by mouse macrophages
| Group of | Number of animals | Phagocytic index | P value |
| Control group | 10 | 0.79±0.16 | - |
| Low dose group | 10 | 0.78±0.19 | 0.507 |
| Middle dose group | 10 | 0.88±0.24 | 0.314 |
| High dose group | 10 | 0.92±0.14 | 0.276 |
| Comparative 1 group | 10 | 0.81±0.11 | 0.427 |
| Comparative 2 group | 10 | 0.84±0.13 | 0.332 |
| For you 3 groups | 10 | 0.82±0.16 | 0.366 |
2.7 Effect of the composition on NK cell Activity in mice
See table 13. Compared with a control group, the NK cell activity of the mice is obviously improved by the medium and high dose groups (P is less than 0.05).
TABLE 13 Effect of compositions on NK cell Activity in mice
Under the experimental condition, the composition of the invention is orally administrated to mice by gavage for 30 days at doses of 0.180g/Kg.bw, 0.360g/Kg.bw and 1.080g/Kg.bw, compared with a control group, the doses of 0.360g/Kg.bw and 1.080g/Kg.bw can obviously improve the delayed type allergic reaction capability and the NK cell activity of the mice, the dose of 1.080g/Kg.bw can obviously improve half of the value of the mice and the number of antibody generating cells with lymphocyte transformation capability (P is less than 0.05), the lymphocyte transformation capability of the mice in low and medium dose groups has no obvious influence on the weight increase, the thymus/hemolytic body ratio, spleen/body weight wallpaper, the carbon clearance capability of mononuclear-macrophage and the red blood cell phagocytic capability of macrophage chicken (P is more than 0.05). The composition has the function of enhancing immunity.
Example 6: toxicity study of the pharmaceutical composition of the present invention
1. Mouse in vitro fibroblast L929 toxicity test
Taking mouse fibroblast L929 cells in a logarithmic growth phase, digesting the mouse fibroblast L929 cells into single cell suspension, and then respectively inoculating the single cell suspension into 6 pieces of 96-well plates. Each plate was seeded with a 6 row by 5 column cell matrix, and the remaining wells were filled with D-Hank's solution alone without cells to maintain humidity. 4000 cells were added to each well and incubated overnight at 37 ℃. The supernatant was discarded, and only the ethanol-cell maintenance solution (ethanol: volume fraction of 2% fetal bovine serum 1:10) was added to column 1 (C1) of the 96-well plate, and the ethanol-cell maintenance solution having a final concentration of cytarabine of 2 μ g/mL was added to column 2 (C2), and the ethanol-cell maintenance solutions having respective concentrations of 100 μ L were added to columns 3 to 5 (C3 to 5) of each pharmaceutical composition (particles of the composition of example 1). After 3d of culture, the growth of the cells was measured by MTT method, and the effect on the relative proliferation rate (RGR) of mouse fibroblast L929 was calculated, and the results are shown in Table 14.
TABLE 14 pharmaceutical compositions mouse L929 toxicity test
Note: p <0.05, P <0.01, compared to positive control group.
The experimental result shows that the pharmaceutical composition has no toxicity to mouse L929 cells at low concentration and high concentration, and belongs to a non-toxic level.
The test results of the pharmaceutical compositions prepared in examples 2-4 are not significantly different from those of the pharmaceutical composition prepared in example 1 (P > 0.05). The pharmaceutical composition provided by the invention has no toxicity to mouse L929 cells at low concentration and high concentration, and belongs to a nontoxic grade.
2. Acute toxicity study
Acute toxicity studies were performed on mice using the maximum tolerated dose method. Kunming mice were fasted for 16h before the experiment without limiting drinking water. 20 mice are selected, half of each mouse is male and female, and the weight of each mouse is 18.0-22.0 g. The gavage capacity of the mice was 20mL/kg, the concentration of the drug of the present invention (the drug composition granule prepared in example 1) tested was 500mg/mL, and the mice were gavaged with the test substance 2 times within 24 hours. After the infection, the animals were observed for general status, toxic manifestations and death for a period of 14 days. At the end of the experiment, the animals were weighed, sacrificed for dissection and pathological examination of the various organs. The Maximum Tolerated Dose (MTD) of the male and female mice was determined by the maximum tolerated dose method and subjected to acute toxicity classification. The results are shown in Table 15.
TABLE 15 test results of acute toxicity of mice with the pharmaceutical composition of the present invention
| Sex | Dosage (g/kg) | Quantity (only) | Initial body weight (g) | Final body weight (g) | Number of deaths | MTD(g/kg) |
| Male part | 20.0 | 10 | 18.7±0.6 | 34.2±1.1 | 0 | ＞20.0 |
| Female part | 20.0 | 10 | 19.8±0.7 | 33.9±1.6 | 0 | ＞20.0 |
After the administration, all animals appeared a little diarrhea in the first day, recovered in the second day, and grew well in other times, the activity was normal, the hair color glossiness was good, and no obvious poisoning symptom and death were seen. The animal was examined for pathological changes in the organs, and no pathological changes in the organs were observed. Therefore, the maximum oral tolerance dose (MTD) of the pharmaceutical composition is more than 20.0g/kg of body weight of male and female mice, and the pharmaceutical composition belongs to a nontoxic grade.
The test results of the pharmaceutical preparations prepared in examples 2 to 4 were not significantly different from those of the pharmaceutical preparation prepared in example 1 (P > 0.05). The pharmaceutical composition and the pharmaceutical preparation provided by the invention have the maximum oral tolerance dose (MTD) of more than 20.0g/kg body weight for male and female mice, and belong to a nontoxic grade.
3. Long term toxicity study
The drug (the drug composition granules prepared in example 1) of the invention is taken from Kunming mice and administered into the stomach according to the middle and large dose groups (4g/kg, 8g/kg), 1 time a day, after 14 days of continuous administration, the indexes of the weight, the cardiac function, the liver function, the kidney function, the electrocardiogram and the like of the mice are measured, and the administration groups have no obvious abnormality compared with the control group. The pathological examination of heart, liver, kidney, spleen, lung, stomach, duodenum, large intestine, small intestine, adrenal gland and genital organs, and the administration group has no obvious toxic change compared with the control group. The results show that the pharmaceutical composition is safe and nontoxic, and can be used clinically.
The test results of the pharmaceutical preparations prepared in examples 2 to 4 were not significantly different from those of the pharmaceutical preparation prepared in example 1 (P > 0.05). The pharmaceutical composition and the pharmaceutical preparation provided by the invention are used for measuring the indexes of the mouse such as weight, cardiac function, liver function, kidney function, electrocardiogram and the like, and the administration group has no obvious abnormality compared with a control group. The pathological examination of heart, liver, kidney, spleen, lung, stomach, duodenum, large intestine, small intestine, adrenal gland and genital organs, and the administration group has no obvious toxic change compared with the control group. The results show that the pharmaceutical composition is safe and nontoxic, and can be used clinically.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A composition for enhancing immunity is characterized by comprising ganoderma lucidum spore powder and a traditional Chinese medicine composition, wherein the traditional Chinese medicine composition comprises astragalus mongholicus, wolfberry, wild jujube and dendrobium officinale.
2. The composition according to claim 1, which is prepared from the following raw materials in parts by mass:
6-8 parts of ganoderma lucidum spore powder, wherein the ganoderma lucidum spore powder is 300-400 meshes of wall-broken ganoderma lucidum spore powder;
2-4 parts of a traditional Chinese medicine composition, wherein the mass ratio of the astragalus root, the medlar, the wild jujube and the dendrobium officinale in the traditional Chinese medicine composition is (5-8): 7-10): 3-6): 2-5.
3. The composition according to claim 1, which is prepared from the following raw materials in parts by mass:
7 parts of ganoderma lucidum spore powder, wherein the ganoderma lucidum spore powder is 350-mesh wall-broken ganoderma lucidum spore powder;
3 parts of a traditional Chinese medicine composition, wherein the mass ratio of the astragalus to the Chinese wolfberry to the wild jujube to the dendrobium officinale is 6:9:4: 3.
4. A process for the preparation of a composition according to any one of claims 1 to 3, comprising the steps of:
A) breaking the wall of the ganoderma lucidum spore powder to obtain wall-broken ganoderma lucidum spore powder;
B) extracting the Chinese medicinal composition with alcohol, and filtering to obtain filtrate;
C) and mixing the filtrate with the wall-broken ganoderma lucidum spore powder to obtain the composition.
5. The preparation method according to claim 4, wherein the wall breaking method is to break the wall of the Ganoderma spore powder by ultra-low temperature air flow wall breaking technology.
6. The preparation method of claim 4, wherein the alcohol extraction is ethanol reflux extraction, and the ethanol reflux extraction is performed for 2-3 times with 60-90% (v/v) ethanol with the weight 6-9 times of the total weight of the traditional Chinese medicine composition, and each time is 60-90 min.
7. The method according to claim 4, further comprising a step of concentrating after obtaining the filtrate; concentrating the filtrate at 50-60 ℃ to obtain thick paste, wherein the relative density of the thick paste is 1.10-1.15 g/mL.
8. Use of a composition according to any one of claims 1 to 3 for the preparation of a product for enhancing immune function.
9. The use according to claim 8, wherein the product is a health food or a pharmaceutical product;
the dosage form of the product is tablets, granules, capsules, powder, pills or oral liquid.
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| CN113197954A (en) * | 2021-04-15 | 2021-08-03 | 广州逸仙生命健康科技有限公司 | Antioxidant wall-broken ganoderma lucidum spore powder and preparation method thereof |
| CN113599461A (en) * | 2021-09-24 | 2021-11-05 | 广州颐寿达生物科技有限公司 | Ganoderma lucidum composition for improving immunity and preparation method thereof |
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