CN112057522A - Composition and preparation method thereof - Google Patents
Composition and preparation method thereof Download PDFInfo
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- CN112057522A CN112057522A CN202010988997.1A CN202010988997A CN112057522A CN 112057522 A CN112057522 A CN 112057522A CN 202010988997 A CN202010988997 A CN 202010988997A CN 112057522 A CN112057522 A CN 112057522A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention provides a composition, which comprises the following components in percentage by weight: 1-8 parts of cistanche, 1-8 parts of astragalus membranaceus, 1-8 parts of glossy privet fruit and 1-8 parts of wolfberry fruit. The composition has various medicinal materials supplementing each other, and can be used as health product for long-term administration. The pharmacodynamic experiment proves that the traditional Chinese medicine composition can obviously enhance the delayed type allergic reaction and the carbon clearance phagocytosis index of a mouse, enhance the phagocytosis function of abdominal cavity macrophages and have the effect of obviously enhancing the immunity.
Description
Technical Field
The invention relates to the technical field of medicines, health-care products and foods, in particular to a composition for enhancing immunity and a preparation method thereof.
Background
Hypoimmunity is divided into primary and secondary categories. The primary is caused by congenital hypoplasia, most of which is related to heredity and mostly occurs in children; secondary symptoms are caused by infection of viruses, bacteria, fungi, etc., or by drugs, tumors, fatigue, insomnia, malnutrition, overstress, etc., and can be seen in people of various ages. The secondary hypoimmunity of the patients with weakness after illness and easy infection.
Modern people have high living and working pressure, many people are in a sub-health state, and some people have symptoms of secondary hypoimmunity such as dizziness, insomnia, fatigue and the like. It is marked by easy illness, and aggravated consumption due to frequent illness, so it is commonly manifested as physical weakness, malnutrition, listlessness, fatigue, weakness, decreased appetite, sleep disorder, etc. It takes a long time to recover each illness and often attacks repeatedly. Besides strengthening exercise, the health food can also improve the immunity of human body through diet conditioning.
Disclosure of Invention
In order to solve the problem of low human immunity, the invention provides a composition and a preparation method thereof.
Specifically, the invention provides a composition which can be directly ground into powder, or can be an extract prepared by a conventional method or other forms, and the composition comprises the following components in parts by weight: 1-8 parts of cistanche, 1-8 parts of astragalus membranaceus, 1-8 parts of glossy privet fruit and 1-8 parts of wolfberry fruit. The raw material medicaments can be directly ground into powder, or can be extracts or other forms prepared by conventional means.
Preferably, the composition comprises: 1-4 parts of cistanche, 1-4 parts of astragalus membranaceus, 1-4 parts of glossy privet fruit and 1-4 parts of wolfberry fruit.
Preferably, the composition comprises: 1-2 parts of cistanche, 1-2 parts of astragalus membranaceus, 1-2 parts of glossy privet fruit and 1-2 parts of wolfberry fruit.
Preferably, the composition comprises 1 part of cistanche, 1 part of astragalus, 1 part of glossy privet fruit and 1 part of medlar.
Preferably, the composition comprises: 2-3 parts of cistanche, 2-3 parts of astragalus membranaceus, 2-3 parts of glossy privet fruit and 2-3 parts of wolfberry fruit.
Preferably, the composition comprises 2 parts of cistanche, 3 parts of astragalus, 2 parts of glossy privet fruit and 3 parts of wolfberry fruit.
The invention also provides a medicament, health-care product or food containing the composition.
The invention also provides application of the composition in preparing a medicine, a health-care product or food for enhancing immunity.
Preferably, the medicine, health product or food further comprises an auxiliary material; the auxiliary materials comprise a diluent, a disintegrating agent and a lubricant.
Preferably, the diluent comprises microcrystalline cellulose and corn starch. The disintegrating agent comprises one or more of carboxymethyl starch sodium and croscarmellose sodium. The lubricant comprises one or more of magnesium stearate, talcum powder and silicon dioxide.
The invention also provides a preparation method of any one of the compositions, which comprises the following steps:
(1) adding 8-12 times of water into the medicinal materials, performing reflux extraction for 1-3 times, each time for 0.5-2.0 hours, filtering, and combining extracting solutions to obtain a filtrate;
(2) concentrating the obtained filtrate under reduced pressure at 55-85 ℃, concentrating to relative density of 1.10-1.30, drying under reduced pressure at 55-85 ℃, crushing, sieving and preparing dry paste powder. The medicine, food or health product is an oral preparation, preferably a capsule.
The invention also provides a preparation method of the medicine, food or health-care product, and when the medicine, health-care product or food is selected from capsules, the preparation method comprises the following steps: (1) adding 8-12 times of water into cistanche, astragalus, glossy privet fruit and wolfberry fruit, carrying out reflux extraction for 1-3 times, and filtering for 0.5-2 hours each time to obtain filtrate;
(2) concentrating the filtrate under reduced pressure until the relative density is 1.20-1.25, drying under reduced pressure, crushing, sieving with a 65-mesh sieve to prepare dry paste powder, adding a diluent and 90-95% ethanol, granulating, drying and finishing granules;
(3) adding disintegrating agent and lubricant into the granules, mixing, and making into capsule.
Preferably, the preparation method comprises the following steps:
1. the extraction, concentration and drying process comprises the following steps: removing impurities from the medicinal materials, weighing the medicinal materials of cistanche, astragalus, glossy privet fruit and barbary wolfberry fruit according to a proportion, adding 8-12 times of water, performing reflux extraction for 1-3 times, each time for 0.5-2 h, combining filtrates, filtering, and performing reduced pressure concentration at 60-70 ℃ to obtain thick paste (60-70 ℃) with the relative density of 1.20-1.25. Drying under reduced pressure at 60-70 ℃, and crushing into fine powder.
2. Mixing: weighing dry paste powder, microcrystalline cellulose and corn starch according to a formula, putting the dry paste powder, the microcrystalline cellulose and the corn starch into a wet mixing granulator, mixing, adding a proper amount of 90-95% ethanol, and stirring to prepare a proper soft material.
3. And (3) granulating: the prepared soft material is granulated by a swing granulator.
4. And (3) drying: spreading the wet granules on a baking pan, placing on a baking vehicle, pushing into a box type drying room, drying at 55 + -5 deg.C, controlling the water content of the granules below 5.0%, and naturally cooling to room temperature.
5. Straightening: and (3) granulating the dry granules by using a swing granulator through a sieve of 15-25 meshes, adding the croscarmellose sodium and silicon dioxide after the granulation, and uniformly mixing.
6. Filling: the dry granules are filled with the adjusted filling amount and then filled into capsules.
Preferably, the preparation method comprises the following steps:
1. the extraction, concentration and drying process comprises the following steps: removing impurities from the medicinal materials, weighing the medicinal materials of the cistanche, the astragalus, the glossy privet fruit and the barbary wolfberry fruit according to a proportion, adding 10 times of water, carrying out reflux extraction for 2 times, wherein each time is 1.0h, combining filtrates, filtering, and carrying out reduced pressure concentration at 60-70 ℃ to obtain thick paste (60-70 ℃) with the relative density of 1.20-1.25. Drying under reduced pressure at 60-70 ℃, and crushing into fine powder.
2. Mixing: weighing dry extract powder, microcrystalline cellulose and corn starch according to a formula, putting into a wet mixing granulator, mixing, adding a proper amount of 90% ethanol, and stirring to prepare a proper soft material.
3. And (3) granulating: the prepared soft material is granulated by a swing granulator through a 20-mesh screen.
4. And (3) drying: spreading the wet granules on a baking pan, placing on a baking vehicle, pushing into a box type drying room, drying at 55 + -5 deg.C, controlling the water content of the granules below 5.0%, and naturally cooling to room temperature.
5. Straightening: and (3) granulating the dry granules by using a swing granulator through a 20-mesh sieve, adding the croscarmellose sodium and the silicon dioxide after the granulation, and uniformly mixing.
6. Filling: the filling amount of the dry granules is adjusted, and then the dry granules are filled into capsules, wherein the capsules are 0.5 g/granule.
The composition is prepared from cistanche, astragalus, glossy privet fruit and barbary wolfberry fruit serving as main raw materials, and has the function of enhancing the immunity of a human body. The term "immunity" is the meaning of "immunity against infection" appearing in "immunity" in Ming dynasty at the earliest time in China. The essence of traditional Chinese medicine "strengthening healthy qi to eliminate pathogenic factors" is based on human, and selects the prescription with the functions of tonifying qi and blood to supplement yin and yang, qi and blood, body fluids and the like of human body, thereby enhancing the immune function of human body.
The composition follows the theory of the traditional Chinese medicine in China, and the cistanche in the formula is warm in nature, enters the kidney channel and the large intestine channel, directly enters the kidney, is warm and moist, can warm and nourish essence and blood to activate yang and qi, nourish five internal organs, strengthen yin and benefit essence and qi, treats both principal and secondary aspects of diseases aiming at the five strains and seven injuries, and is a monarch drug. The astragalus membranaceus is warm in nature, can tonify qi of lung and spleen, benefit defensive qi and strengthen superficies to check sweating, and can tonify qi of skin and superficies, and is particularly suitable for people suffering from fatigue, weakness, listlessness, inappetence and the like caused by spleen-qi deficiency; the glossy privet fruit has mild nature, enters liver and kidney meridians, regulates five internal organs, treats waist and leg pain, and mainly clears meridians, regulates blood and nourishes spirit; wolfberry fruit is sweet, mild and moist, has sexual nourishing effect, and can tonify kidney and qi, which is a medicine for mild tonification; the three medicines are used as assistant medicines to assist the monarch medicine.
The four medicines embody the formula characteristics of nourishing the internal organs, benefiting qi and nourishing blood, and strengthening body resistance and consolidating constitution.
The composition provided by the invention aims at the pathogenesis of low immunity, combines the traditional Chinese medicine and modern medical knowledge, has definite formula function, no incompatibility, is safe and effective to eat, and can play a good health-care role.
Detailed Description
The raw material medicinal materials of the composition are selected from medicinal and edible raw materials or raw materials of available health-care food issued by the national bureau and are scientifically prepared. The addition amount of the auxiliary materials accords with the use standard of food additives and Chinese pharmacopoeia. The operation process and the extraction process are orthogonal, so that the operation is simple, economic and environment-friendly, and the process is stable. The types and the addition amounts of the auxiliary materials are screened and tested, so that the product accords with the characteristics of the product, and the quality of the product can be improved. The product has good safety, and has the effect of enhancing immunity as proved by animal experiments. The product and the process have wide application prospect.
Example 1
1. Selection of extraction Process
Taking 80g of cistanche deserticola, 80g of astragalus mongholicus, 80g of wolfberry fruit and 80g of glossy privet fruit in 9 parts in total, extracting according to the requirements of relevant parameters in a factor level table 1, and determining the transfer rates and the crude polysaccharide contents of echinacoside, verbascoside and astragaloside in an extracting solution. Taking the transfer rates and crude polysaccharide contents of echinacoside, verbascoside and astragaloside IV as investigation indexes, and adopting L9(34) And (4) performing an orthogonal test, and inspecting the amount of the extraction solvent, the extraction time and the extraction frequency.
Table 1 levels of factors
TABLE 2L9(34) Quadrature watch (Echinacoside, verbascoside)
TABLE 3 ANOVA TABLE (Echinacoside, verbascoside)
TABLE 4L9(34) Orthogonal watch (Astragaloside IV)
TABLE 5 ANOVA (Astragaloside IV)
TABLE 6L9(34) Orthogonal watch (crude polysaccharide)
TABLE 7 ANOVA (crude polysaccharide)
As can be seen from tables 2 and 3, the transfer rates of echinacoside and verbascoside in the extracting solution are taken as indexes to be inspected, and the influence of each factor on the extraction process is extremely poor R value; c > B > A, the range of extraction times (factor C) is the largest, and among the factors, C2> C1> C3, B3> B1> B2 and A2> A3> A1, so the optimal extraction process for visual analysis is A2B3C 2. Analysis of variance showed that factor C was significant, so the optimal extraction process was A2B1C2, i.e., 10 times more water each time, and reflux extraction was performed 2 times each for 1.0 hour. As can be seen from tables 4 and 5, the influence of each factor on the extraction process is extremely poor R value by taking the astragaloside transfer rate in the extracting solution as an index for investigation; c > A > B, the range of the extraction times (factor C) is the largest, and among the factors, C1> C2> C3, B3> B2> B1 and A2> A3> A1, so the optimal extraction process for visual analysis is A2B3C 1. Analysis of variance showed that factor C was significant, so the optimal extraction process was A2B1C1, i.e., 10 times more water each time, and reflux extraction was performed 3 times each for 1.0 hour. As can be seen from tables 6 and 7, the influence of each factor on the extraction process is extremely poor R value by taking the content of crude polysaccharide in the extraction solution as an index for investigation; c > A > B, the range of the extraction times (factor C) is the largest, and among the factors, C2> C1> C3, B1> B3> B2 and A3> A2> A1, so the optimal extraction process for visual analysis is A3B1C 2. Analysis of variance showed that factor C was significant, so the optimal extraction process was A3B1C2, i.e., 12 times more water was added each time and the reflux extraction was performed 2 times each time for 1.0 hour.
The three index components are comprehensively considered, the factor C is obvious, and the factor C is determined to be C2 according to the energy-saving principle; the factor B is not obvious and is determined as B1 according to the energy-saving principle; the factor A is not obvious, the transfer rates of all index components are considered, the transfer rates are A2 and A3 respectively, and the transfer rates are determined to be A2 according to the energy-saving principle; therefore, the optimal extraction process is A2B1C2, i.e. 10 times of water is added each time, and the extraction is carried out for 2 times, and each time is 1.0 hour.
2. Extraction Process verification test
According to the orthogonal optimization result, 120g of cistanche deserticola, 120g of astragalus mongholicus, 120g of wolfberry fruit and 120g of glossy privet fruit are taken for each part, 3 parts of cistanche deserticola, 10 times of water are parallelly extracted for 2 times in a refluxing way, each time lasts for 1.0h, the extracting solution is filtered, the filtrate is combined, the content of echinacoside, verbascoside, astragaloside IV and crude polysaccharide in the extracting solution is measured, the transfer rate is used as an index to verify the feasibility and stability of the extraction process parameters, and the result is shown in table 8.
TABLE 8 Experimental chart for extraction process
The results of the verification experiments show that the cistanche and the astragalus are extracted by adopting the optimized extraction process, the transfer rate and the crude polysaccharide content of the echinacoside, verbascoside and astragaloside in the extracting solution are close to those of the orthogonal test results, and the stability and feasibility of the extraction process are proved.
Example 2
1. Medicinal material extracting, concentrating and drying process
Weighing Cistanchis herba 3.0kg, radix astragali 3.0kg, fructus Ligustri Lucidi 3.0kg and fructus Lycii 3.0kg at a certain proportion, adding 1200L water, and extracting under reflux for 2 times, each for 1.0 hr. Filtering, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain concentrated extract with relative density of 1.25. Drying under reduced pressure at 70 deg.C, pulverizing, sieving with 65 mesh sieve, and making into dry extract powder with weight of 7.95 kg.
2. Selection of type and amount of auxiliary materials
2.1 selection of the type of auxiliary Material
Through experimental research, proper auxiliary materials are required to be added so as to solve the problems of caking, poor fluidity, large particle weight difference, difficult disintegration and the like in the preparation process. In the selection process of the diluent, three pharmaceutical common auxiliary materials of microcrystalline cellulose, corn starch and dextrin are selected according to the properties of the intermediate, and the three auxiliary materials are tested, wherein the test results are shown in table 9.
TABLE 9 selection of adjuvants and Experimental results
As can be seen from the experimental results shown in Table 1, different auxiliary materials are selected, so that the material state of the preparation after granulation is influenced to a certain extent, and microcrystalline cellulose is easy to granulate but has longer disintegration time; the corn starch has short disintegration time, but has agglomeration phenomenon during granulation, so the microcrystalline cellulose and the corn starch are simultaneously selected as auxiliary materials.
2.2 selection of the amount of auxiliary materials
The use level of the auxiliary materials can influence the granulation process, so the use levels of the microcrystalline cellulose and the corn starch are inspected, three dry paste powders are weighed, 200g of each dry paste powder is added with the microcrystalline cellulose and the corn starch in different proportions, the bulk density and the angle of repose of the materials are inspected, and the experimental results are shown in table 10.
TABLE 10 selection of adjuvant amounts and test results
As shown in the experimental result table 10, when microcrystalline cellulose accounting for 20% of the dry extract powder and corn starch accounting for 10% of the dry extract powder were added, the granulation was the best, and the disintegration time was acceptable.
2.3 screening of wetting Agents
Respectively preparing soft materials by using ethanol with volume fractions of 70%, 80%, 90% and 95% as wetting agents, extruding and granulating through a 20-mesh sieve, drying at 60 ℃, grading through the 20-mesh sieve, and collecting granules. The granulation effect of ethanol with different volume fractions was examined by using the soft material preparation condition, soft material sieving condition and granule state as indexes, and the results are shown in table 3.
Table 11 wetting agent test results
As can be seen from the experimental results in Table 11, the granulation using 90% ethanol as wetting agent is optimal.
2.4 selection of disintegrating Agents
The traditional Chinese medicine ointment powder becomes sticky when meeting water, which affects the water infiltration in the capsule disintegration process and causes the disintegration difficulty, so a disintegrating agent is needed to be added to improve the disintegration condition, the common disintegrating agents in the preparation production, namely carboxymethyl starch sodium and cross-linked sodium carboxymethyl cellulose, are added with the addition amount of 2-5 percent, 90 percent ethanol is used as a wetting agent to prepare a proper soft material, and the proper soft material is prepared, granulated, dried, granulated, added with the disintegrating agent and the lubricating agent, uniformly mixed, filled into a capsule, and the disintegration time limit is measured, and the result is shown in table 12.
TABLE 12 disintegrant observations
The experimental results table 12 show that, under the condition of the same amount of the auxiliary materials, the croscarmellose sodium has a good disintegration effect on the capsule, so that the influence of the auxiliary materials on filling the capsule and disintegration is integrated, and the croscarmellose sodium with the addition amount of 3% is preferably used as a disintegrant.
2.5 screening of Lubricants
In order to ensure that the filling is carried out smoothly and the filling weight difference meets the specification, a certain amount of lubricant needs to be added into the granules. The commonly used lubricant is magnesium stearate, talcum powder and superfine silica gel powder, and the common dosage is about 1-2%. Other factors in the formulation were fixed, granulated according to the above preferred formulation, and 1% and 2% of magnesium stearate, talc, and silica were added, respectively, with the angle of repose of the granules and the smoothness of the tablets as evaluation indices, and the results are shown in table 13.
TABLE 13 screening of lubricants
As shown in Table 13, the results of the experiments show that 2% silica is preferable as the lubricant.
Therefore, experiments show that the product is added with the following auxiliary materials: microcrystalline cellulose accounting for 20% of the dry extract powder and corn starch accounting for 10% of the dry extract powder are granulated by adopting 90% ethanol as a wetting agent, and 3% of croscarmellose sodium is used as a disintegrating agent and 2% of silicon dioxide is used as a lubricating agent.
Example 3
Weighing Cistanchis herba 54kg, radix astragali 54kg, fructus Ligustri Lucidi 54kg and fructus Lycii 54kg at a certain proportion, adding 2160L water, and reflux extracting for 2 times, each for 1.0 hr. Filtering, and concentrating the filtrate at 70 deg.C under reduced pressure to obtain concentrated extract with relative density of 1.20. And continuously drying for 5 hours under reduced pressure at 70 ℃, crushing, sieving by a 65-mesh sieve to prepare dry paste powder, wherein the weight of the dry paste powder is 47.73kg, then adding auxiliary materials into the dry paste powder according to the proportion, adding 9.82kg of microcrystalline cellulose and 5.04kg of corn starch, uniformly mixing, wetting by 90% ethanol, sieving by a 20-mesh sieve, granulating, drying for 5 hours at 60 ℃, sieving by a 20-mesh sieve, grading, adding 1.92kg of disintegrant croscarmellose sodium and 1.30kg of lubricant into the granules, mixing to obtain the weight of the granules of about 63.24kg, and the yield of the granules of about 96.1%. And (5) filling the mixture into capsules to obtain capsules, wherein each capsule is 0.5 g.
Example 4
Animal experiment for enhancing immunity function
1. Materials and methods
1.1 sample
The recommended dosage of the sample prepared in the third example is 2 times a day, 2 granules are taken each time, and each granule is 0.5g, namely the recommended dosage of a human body is 4 g/person/day. When the weight of the adult is 60kg, the recommended dosage of the sample is 0.067g/kg · bw.
1.2 test animals and conditions
SPF grade ICR mice were purchased from beijing weitonglihua laboratory animal technology ltd, laboratory animal licenses: SCXK (Kyoto) 2016-. The clean grade feed for the experimental animals was purchased from Beijing, Australian cooperative feed Co., Ltd (license number: SCXK (Jing) 2014-0010). Selecting 40 healthy female mice with the weight of 18-22 g, randomly dividing the healthy female mice into 4 groups, and taking 10 healthy female mice in each group as an immune group to perform a carbon clearance test and an animal organ/body ratio determination. Healthy female mice, 40, weighing 18-22 g, were randomly divided into 4 groups of 10 mice each, and half hemolysis value (HC50) measurement and antibody-producing cell detection were performed as two immune groups. 40 healthy female mice with the weight of 18-22 g are randomly divided into 4 groups, and 10 mice in each group are used as three immune groups to perform a delayed allergy test. 40 healthy female mice with the weight of 18-22 g are randomly divided into 4 groups, and each group is 10 mice as four immune groups to perform a test of phagocytosis of chicken erythrocytes by abdominal macrophages of the mice. 40 healthy female mice with the weight of 18-22 g are randomly divided into 4 groups, 10 mice in each group are used as five immune groups, and a ConA-induced mouse spleen lymphocyte transformation test and NK cell activity measurement are carried out.
1.3 dose design
The recommended dosage of the sample is 2g per day, 4 capsules each time, and 0.5g per capsule, namely the recommended dosage of a human body is 4 g/person/day. When the weight of the adult is 60kg, the recommended dosage of the sample is 0.067g/kg · bw. Taking 10 times of the recommended dosage of a human body, namely 0.67g/kg of body weight per day as a medium dosage, setting a dosage group up and down respectively: 2.01g/kg body weight (30 times the human recommended dose is high) and 0.34g/kg body weight (5 times the human recommended dose is low). Respectively weighing 5.025g, 1.675g and 0.85g of samples, dissolving the samples by using distilled water and fixing the volume to 50 mL; meanwhile, a control group (distilled water) is set, the animals are subjected to intragastric administration according to the intragastric administration volume of 2% of the weight, the solvents of the test group are all distilled water, and various immunity indexes are measured after continuous intragastric administration for 30 days.
1.4 instruments and reagents
Animal balance, analytical balance, refrigerator, centrifuge, 722 spectrophotometer, microscope, clean bench, carbon dioxide incubator, microplate reader, etc. Sterile surgical instruments, vernier calipers, microsyringes (25 μ L), cytometers, gauze, test tubes, timers, hemoglobin pipettes, 24-well plates, and the like.
Sheep Red Blood Cells (SRBC), chicken red blood cells, physiological saline, complement (guinea pig serum), SA buffer solution, Du's reagent, India ink, Na2CO3, Hank's solution, RPMI1640 complete culture solution, calf serum, streptomycin, oxidized coenzyme, 0.2mol/L Tris-HCl, YAC-1 cells, 1% NP40, 2-mercaptoethanol (2-ME), penicillin, streptomycin, concanavalin A (ConA), hydrochloric acid, isopropanol, MTT, PBS buffer solution (pH 7.2-7.4), acetone, methanol, and Giemsa staining solution.
1.5 test methods
1.5.1 ConA-induced splenic lymphocyte transformation assay and NK cell Activity assay in mice
1.5.1.1 spleen cell suspension preparation
Animals (five groups of immunizations) were aseptically harvested, placed in small dishes containing appropriate amounts of sterile Hank's solution, and the spleens gently shredded with forceps to make single cell suspensions. Filtering with 200 mesh sieve, washing with Hank's solution for 2 times, centrifuging for 10min (1000r/min) each time, discarding supernatant, bouncing the cell paste, adding 0.5mL of sterilized water for 20s, lysing erythrocytes, adding 0.5mL of 2 times Hank's solution and 8mL of LHank's solution, and centrifuging for 10min at 1000 r/min. Finally, the cells were suspended in 1.0ml LRPMI1640 complete medium and the number of viable cells (which should be above 95%) was counted by staining with Taiwan phenol blue.
1.5.1.2 lymphocyte proliferation reaction (MTT method)
Using an appropriate amount of the stock solution of spleen cells prepared as described above, the cells were diluted with RPMI1640 complete medium to a cell concentration of 3X 106 cells/mL. The diluted cell suspension was added to a 24-well plate in two wells, 1mL per well, 75. mu.L of LCoA solution was added to one well, and 5% CO2 was added to the other well as a control, and the plate was incubated at 37 ℃ for 72 hours. 4h before the culture is finished, gently sucking 0.7mL of supernatant in each well, adding 0.7mL of RPMI1640 complete culture solution without calf serum and MTT (5mg/mL) for complete dissolution, subpackaging the mixture into 96-well culture plates, making 3 parallel wells in each well, and measuring the value by using a microplate reader at a wavelength of 570 nm.
1.5.1.3NK cell Activity assay (LDH assay)
Taking another appropriate amount of the above splenocyte stock solution, diluting with RPMI1640 complete culture solution to make cell concentration be 2 × 107/mL as effector cell concentration, and adjusting target cell (YAC-1) concentration according to the result to make the ratio of effector to target be 50: 1.
Taking 100 mu L of target cells and effector cells, adding 100 mu L of target cells and 1% NP40 into the maximum release holes of the target cells, setting three parallel holes for each, culturing for 4h in a 5% CO2 incubator at 37 ℃, centrifuging a 96-hole plate at 1500rpm for 5 min, sucking 100 mu L of supernatant in each hole, placing the supernatant on an enzyme label plate, adding 100 mu L of LDH matrix solution for reaction for 10min, adding 30 mu L of 1mol/L HCl solution into each hole to stop the reaction, and measuring OD value at 490nm wavelength of an enzyme label instrument to calculate the activity of the NK cells.
NK% ((reaction well OD-natural release well OD)/(maximum release well OD-natural release well OD) × 100%)
1.5.2 delayed type allergy (DTH)
Each mouse of the three immunized groups of animals is intraperitoneally injected with 2% (V/V, prepared by normal saline) to compact 0.2mL of SRBC (2000rpm, 10 minutes), 4 days after sensitization, the thickness of the left spathiphyllum is measured, the thickness is measured three times by using the same part, and an average value is taken. Then, 20% (V/V, prepared with physiological saline) of the compressed SRBC20 μ L was injected subcutaneously into the measurement site, and the thickness of the driver's left and rear foot was measured 24 hours after the injection, and the DTH degree was expressed as the difference in the thickness of the driver's left and rear foot (the degree of swelling in the driver's foot).
1.5.3 half maximal hemolysis value (HC)50) Measurement and antibody-producing cell detection
1.5.3.1 animal immunization
Animal immunization was carried out 4-5 days before the end of the experiment. Taking defibrinated sheep blood, washing with normal saline for 3 times, centrifuging at 2000r/min for 10min each time. The packed SRBC was made into 2% (V/V) cell suspension with physiological saline, and each mouse was injected with 0.2mL of the cell suspension in the abdominal cavity.
1.5.3.2 half maximal hemolysis value (HC)50) Measurement of (2)
After animals (two groups of animals) are immunized for 4-5 days, the eyeballs are removed, blood is taken out of the centrifugal tube and is placed for about 1 hour, coagulated blood is stripped from the tube wall, serum is fully separated out, centrifugation is carried out at 2000rpm for 10 minutes, and the serum is collected. The serum was diluted 250-fold with SA buffer, 1mL was placed in a test tube, and 10% (V/V, prepared with SA buffer) of packed SRBC0.5mL and 1mL of complement were added in this order (diluted 1:10 with SA buffer). A control tube without serum was provided (SA buffer was used instead). After the mixture is placed in a thermostatic water bath at 37 ℃ and kept warm for 30min, the reaction is stopped by ice bath. Centrifuging at 2000rpm for 10min, collecting supernatant 1mL, adding Du's reagent 3mL, simultaneously collecting 10% (V/V, prepared with physiological saline) packed SRBC0.25mL, adding Du's reagent to 4mL, mixing well, standing for 10min, and measuring optical density of each tube with a control tube at 540nm wavelength as blank. The amount of hemolysin is expressed as half the hemolysin value (HC)50) Expressed, it is calculated as follows.
Sample HC50Sample optical density value/optical density value at half SRBC hemolysis × dilution factor
1.5.3.3 antibody producing cell assay
1.5.3.3.1 spleen cell suspension preparation (same as before 1.5.1.1)
1.5.3.3.2 determination of plaques
Heating and dissolving a surface layer culture medium (1g of agarose added with double distilled water to 100mL), placing the mixture in a water bath at 45-50 ℃ for heat preservation, mixing the mixture with an equivalent amount of Hank's solution with the concentration of 7.2-7.42 times, subpackaging small test tubes, wherein each tube is 0.5mL, adding 50 mu L of 10% SRBC (V/V prepared by SA buffer solution) and 20 mu L of spleen cell suspension (5 multiplied by 106/mL) into the tubes, quickly mixing the mixture, pouring the mixture onto a glass sheet with an agarose thin layer, horizontally buckling the glass sheet on a re-rack after the agar is solidified, placing the glass sheet on a carbon dioxide incubator for incubation for 1-1.5 h, adding complement (1:8) diluted by the SA buffer solution into a groove of the glass sheet rack, continuing the incubation for 1-1.5 h, and counting the number of hemolytic plaques.
1.5.4 mouse carbon clearance test and measurement of the dirty/body ratio
1.5.4.1 mouse carbon clearance test
Animals (immunization group) mice were injected with india ink diluted 1:3.5 times into tail vein at 0.1mL/10g body weight, and immediately timed when ink was injected.
2 min and 10min after the injection of ink, 20 μ L of blood was taken from the angular venous plexus, and added to 2 mL0.1% Na2CO3In solution. Weighing mice, dislocating cervical vertebra, killing, removing fascia of liver, spleen and thymus, sucking blood stain on the surface of viscera with filter paper, and weighing.
The optical density value (OD) was measured at a wavelength of 600nm with a 722 spectrophotometer and Na2CO3The solution was used as a blank control and the phagocytic index α was calculated.
1.5.5 mouse peritoneal macrophage phagocytosis of chicken erythrocyte test
1.5.5.1 preparation of chicken red blood cell 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 (2000r/min, 10min), removing supernatant, and preparing 20% (V/V) chicken erythrocyte suspension with normal saline.
1.5.5.2 determination of phagocytic function
Injecting 20% chicken erythrocyte suspension into abdominal cavity of each mouse of animals (immune four groups) at an interval of 30min, dislocating cervical vertebra, killing the animals, fixing the animals on a mouse plate in a supine position, cutting abdominal wall skin at the center, injecting 2mL of normal saline into abdominal cavity, rotating the mouse plate for 1min, sucking 1mL of abdominal cavity washing liquid, averagely dropping the abdominal cavity washing liquid on 2 glass slides, putting the glass slides into an enamel box with wet gauze, moving the glass slide out of an incubator at 37 ℃ for incubation for 30min, rinsing the glass slides in normal saline after incubation is finished to remove non-mounted cells, drying the glass slides in 1:1 acetone methanol solution, staining 4% (V/V) Giemsa-phosphate buffer for 3min, rinsing the glass slides in distilled water, and drying the glass slides in the air. Results the phagocytic function of mouse macrophages is expressed as percent phagocytosis or phagocytic index.
2.1 Effect on mouse body weight
TABLE 14 Effect on mouse body weight (immunization group)
TABLE 15 Effect on mouse body weight (immune groups)
TABLE 16 Effect on mouse body weight (three immunization groups)
TABLE 17 Effect on mouse body weight (four immune groups)
TABLE 18 Effect on mouse body weight (five immunization groups)
As can be seen from the results in tables 14, 15, 16, 17 and 18, in each immunization group, the initial body weight and the final body weight of the low, medium and high three dose groups have no significant difference (P > 0.05) compared with the control group, which indicates that the sample has no influence on the weight gain of the mice under the experimental condition.
TABLE 19 Effect on organ/body weight ratio in mice
When the samples with different doses are orally administered for 30 days, the spleen/body weight ratio and the thymus/body weight ratio have no significant difference (P is more than 0.05) between the low, medium and high dose groups and the control group, which indicates that the visceral/body weight ratio is not influenced.
TABLE 20 Effect on delayed allergy (DTH) in mice
When the samples with different dosages are orally administered for 30 days, the swelling degree of spathula is different before and after SRBC injection, and the comparison between the middle and high dose groups and the control group has very significant difference (P is less than 0.01), which indicates that the delayed type allergic reaction of the mice can be enhanced.
TABLE 21 Effect on mouse spleen lymphocyte transformation
When different doses of samples are orally administered for 30 days, the spleen lymphocyte transformation function of mice in each dose group has no significant difference (P is more than 0.05) compared with that of a control group, and the samples can not enhance the spleen lymphocyte transformation function of the mice.
TABLE 22 hemolysis values (HC) for mouse plate number50) Influence of (2)
Half maximal Hemolysis (HC) of medium and high dose groups administered orally for 30 days with different doses of the samples50) Compared with a control group, the medicine has very significant difference (P is less than 0.01), and the half hemolysis value (HC) of a low-dose group50) Compared with a control group, the sample has significant difference (P is less than 0.05), and the sample can improve the half hemolysis value (HC) of the mice50)。
TABLE 23 Effect on mouse antibody-producing cells
When the samples of different doses are orally administered for 30 days, the number of plaques of each dose group has no significant difference (P is more than 0.05) compared with that of a control group, and the samples can not enhance the functions of the antibody-producing cells of the mice.
TABLE 24 Effect on mouse carbon clearance function
The phagocytosis index of the medium-high dose group is very different from that of the control group (P is less than 0.01), which indicates that the sample can increase the carbon clearance function of the mice.
TABLE 25 Effect on phagocytosis of chicken erythrocytes by mouse macrophages
Orally administering different doses of samples to mice for 30 days, performing homogeneous test of variance and variable transformation on four groups of phagocytosis rates
Wherein P is phagocytosis rate and is represented by decimal fraction. The phagocytosis index of each dose group has a very significant difference P < 0.01 compared with that of a control group. The sample can enhance the phagocytic function of mouse abdominal cavity macrophages.
TABLE 26 Effect on NK cell Activity in mouse
Mice were orally administered with different doses of the samples prepared in example three for 30 days, and the NK cell activity was subjected to variable transformation
Wherein P is NK cell activity and is represented by decimal. Through statistical treatment, the activity of NK cells of each dose has no significant difference (P is more than 0.05) compared with that of a control group, and the result shows that the sample has no influence on the activity function of the NK cells of the mice.
And (4) conclusion: the results of 30 days after orally administering samples of 2.01g/kg · bw, 0.67g/kg · bw and 0.34g/kg · bw (30 times, 10 times and 5 times of the human body recommended dose respectively) to mice show that the samples of the medium and high dose groups can remarkably enhance the delayed type allergic reaction and the carbon clearance phagocytosis index of the mice, and the samples of the low, medium and high dose groups can improve the half hemolysis value of the mice and the high dose group can enhance the phagocytosis function of abdominal cavity macrophages. According to the judgment criteria in "evaluation procedure and test method of health food functionality", the sample is considered to have the function of enhancing immunity.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A composition characterized in that it comprises, by weight: 1-8 parts of cistanche, 1-8 parts of astragalus membranaceus, 1-8 parts of glossy privet fruit and 1-8 parts of wolfberry fruit.
2. The composition according to claim 1, characterized in that it comprises, by weight: 1-4 parts of cistanche, 1-4 parts of astragalus membranaceus, 1-4 parts of glossy privet fruit and 1-4 parts of wolfberry fruit.
3. The composition according to claim 1, characterized in that it comprises, by weight: 1 part of cistanche, 1 part of astragalus, 1 part of glossy privet fruit and 1 part of medlar.
4. The composition of claim 1, wherein the composition comprises, by weight, 2 parts of cistanche, 3 parts of astragalus, 2 parts of fructus ligustri lucidi, and 3 parts of fructus lycii.
5. Use of the composition according to any one of claims 1 to 4 for the preparation of a medicament, health product or food for enhancing immunity.
6. A pharmaceutical, nutraceutical or food product comprising a composition according to any of claims 1 to 4.
7. The medicament, health product or food of claim 6, further comprising an adjuvant; the auxiliary materials comprise a diluent, a disintegrating agent and a lubricant.
8. The drug, health product or food according to claim 7,
the diluent comprises microcrystalline cellulose and corn starch;
the disintegrating agent comprises one or more of carboxymethyl starch sodium and croscarmellose sodium;
the lubricant comprises one or more of magnesium stearate, talcum powder and silicon dioxide.
9. A process for preparing a composition according to any one of claims 1 to 4, comprising:
(1) adding 8-12 times of water into the medicinal materials, performing reflux extraction for 1-3 times, each time for 0.5-2.0 hours, filtering, and combining extracting solutions to obtain a filtrate;
(2) and concentrating the obtained filtrate under reduced pressure at 55-85 ℃ until the relative density is 1.10-1.30, drying under reduced pressure at 55-85 ℃, crushing and sieving.
10. A method for preparing the drug, health product or food according to claim 6, wherein the drug, health product or food is selected from capsules, the method comprising the steps of:
(1) adding 8-12 times of water into cistanche, astragalus, glossy privet fruit and wolfberry fruit, carrying out reflux extraction for 1-3 times, and filtering for 0.5-2 hours each time to obtain filtrate;
(2) concentrating the filtrate under reduced pressure until the relative density is 1.20-1.25, drying under reduced pressure, crushing, sieving with a 65-mesh sieve to prepare dry paste powder, adding a diluent and 90-95% ethanol, granulating, drying and finishing granules;
(3) adding disintegrating agent and lubricant into the granules, mixing, and making into capsule.
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| CN113633719A (en) * | 2021-08-12 | 2021-11-12 | 浙江理工大学绍兴生物医药研究院有限公司 | Antioxidant composition, preparation method and application |
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