CN113181235A - Preparation method and application of chestnut pollen extract - Google Patents
Preparation method and application of chestnut pollen extract Download PDFInfo
- Publication number
- CN113181235A CN113181235A CN202110498320.4A CN202110498320A CN113181235A CN 113181235 A CN113181235 A CN 113181235A CN 202110498320 A CN202110498320 A CN 202110498320A CN 113181235 A CN113181235 A CN 113181235A
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- extraction
- chestnut
- chestnut pollen
- extract
- flavone
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Abstract
The invention relates to the technical field of pollen extraction, and discloses a preparation method and application of a chestnut pollen extract. The chestnut pollen extract contains flavone and active peptide in preparing medicine or food for regulating immunity, promoting macrophage phagocytic activity, promoting spleen lymphocyte proliferation with Con A, or activating and strengthening NK cell killing activity. The preparation method of chestnut pollen extract comprises extracting flavone with water as solvent; then, taking water as a solvent, and carrying out enzymolysis extraction on active peptide in filter residue; combining the two extracted products. According to the preparation method provided by the application, the flavone and the active peptide in the chestnut pollen are respectively extracted in a separate extraction mode, the active substances such as the flavone and the active peptide in the pollen can be fully extracted, the effect is better when the preparation method is applied to immunity regulation and the promotion of the activity of immune cells, and the effective dose of the chestnut pollen is greatly reduced.
Description
Technical Field
The invention relates to the technical field of pollen extraction, in particular to a preparation method and application of a chestnut pollen extract.
Background
Chinese chestnut, also known as chestnut, chestnut and dried plum, is native to China and has a history of cultivation for more than two thousand years. At present, the planting area in China is 2700 more than ten thousand mu, the yield is 260 more than ten thousand tons (accounting for 84 percent of the world), and the area and the yield are the first place in the world. The chestnut trees are isogenic, cross pollinated, the number ratio of male flowers to female flowers can reach 1:2349, and researches show that only a small amount of male flowers can meet the pollination requirement, a large amount of male flowers are redundant, and the nutrition of trees is consumed by white flowers. Therefore, in order to improve the yield of the Chinese chestnut, about 90% of male inflorescences need to be manually removed, most of the Chinese chestnut male flowers are discarded as waste, and huge waste of resources is caused.
According to the record of materia medica compendium, the chestnut flower is sweet in nature and taste, flat and nontoxic. Chestnut flower can be used for treating laryngitis, toxic swelling, scrofula, red and white dysentery, etc., or used externally, and pounding and juicing for treating sore due to infection. Modern pharmacological research shows that the chestnut flower male inflorescence has the functions of enhancing vascular tension, reducing vascular fragility, improving vascular permeability and reducing blood fat and cholesterol; reduce the aggregation of red blood cells and platelets and reduce the formation of thrombus; protecting liver, removing toxic substances, and improving immunity; antibacterial, antiviral, antioxidant, antiaging, antifungal, and antitumor effects. The research at home and abroad shows that the chestnut flower has development and application values in various fields of food, medicine, health care products and the like.
Pollen is a male gametophyte of sexual propagation of plants, is a 'sperm' of a plant multiplication progeny, exists in a male anther, contains various vitamins, enzymes, trace elements, bioactive substances and other nutritional ingredients necessary for human bodies, has the characteristics of low fat and high protein, and is known as a 'micro nutrient bank'. Recent scientific research and clinical application prove the medical health-care function of the pollen. Pollen contains a lot of bioactive substances such as phenols, flavonoids, lipids, proteins, vitamins, trace elements, etc., provides potential energy sources and functional material sources for human consumption, is considered as natural health food, and is called a 'concentrated nutrient bank'. Wherein, the flavone substance in the chestnut pollen is the highest content in all pollen, and the chestnut pollen aggregates the characteristics of nature, green, nutrition, health care and the like, thereby gradually receiving wide attention at home and abroad. In addition, the chestnut pollen is rich in bioactive substances such as active calcium, phosphorus, vitamin E, estradiol, flavone, protein and the like. However, there are few patents on chestnut pollen. The specification with the application number of 201310071627.1 discloses a chestnut pollen processing method, which is characterized in that vacuum-packaged chestnut pollen is obtained by processing through the procedures of collection, airing, drying, powder screening and packaging, and the nutritional ingredients and the effects of the chestnut pollen are not researched. The specification with application number CN201210351320.2 discloses a method for extracting flavone mixture from chestnut flower, wherein the extraction process needs alcohol solution, alkali liquor and mixed solution of sodium sulfite or potassium sulfite, and the extract has the problem of residue of organic solvent and inorganic salt, which is not beneficial to subsequent practical application. At present, the existing research on other plant pollen takes a certain single substance in the pollen as a target product. Although the extracted components have better activity, other active components in the pollen are lost in the extraction process, so that the pollen only becomes a raw material of a certain component, and the characteristic of a full-functional nutrient bank of the pollen cannot be embodied.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide application of a chestnut pollen extract in preparing immunity-regulating medicines or foods and a preparation method of the chestnut pollen extract.
The invention is realized by the following steps:
in a first aspect, the present invention provides the use of a chestnut pollen extract comprising flavonoids and active peptides for the preparation of a medicament or food for regulating immunity.
In a second aspect, the invention provides an application of a chestnut pollen extract in preparing a medicament or food for promoting macrophage phagocytosis activity, promoting spleen lymphocyte proliferation in cooperation with Con A, or activating and enhancing NK cell killing activity, wherein the chestnut pollen extract contains flavone and active peptide.
In an alternative embodiment, the dosage form of the medicament comprises: powders, tablets, capsules, soft capsules, syrups, tinctures, pills, powders, granules, aqueous pharmaceuticals, emulsions, suspensions, gels, creams, ointments or emulsions.
In a third aspect, the present invention provides a method for preparing a chestnut pollen extract, comprising:
a method for preparing chestnut pollen extract comprises:
mixing chestnut pollen with water by using water as a solvent, and performing flavone extraction by adopting a reflux extraction method, an ultrasonic extraction method or a microwave extraction method to obtain a first extracting solution and first filter residue;
mixing the first filter residue with water by taking water as a solvent, and sequentially extracting active polypeptide in the first filter residue by adopting cellulase and protease to obtain a second extracting solution and a second filter residue;
combining the first extract and the second extract;
in an alternative embodiment, the first and second extracts are combined to obtain a chestnut pollen extract, and the chestnut pollen extract is dried to obtain a chestnut pollen extract.
In alternative embodiments, the flavone extraction is: mixing chestnut pollen with water, extracting at 30-60 deg.C, and performing solid-liquid separation to obtain first extractive solution and first filter residue, wherein the extraction method comprises reflux extraction, ultrasonic extraction or microwave extraction;
in alternative embodiments, the active peptide extraction is: mixing the first filter residue with water again to obtain mixed slurry, adjusting the pH of the mixed slurry to 4.0-7.0, adding cellulase into the mixed slurry, and performing primary enzymolysis at 30-60 ℃;
adjusting the pH of the mixed slurry to 4.0-7.0 after the primary enzymolysis is finished, adding protease into the mixed slurry, and performing secondary enzymolysis at 30-60 ℃;
after the secondary enzymolysis, the mixed slurry is subjected to enzyme deactivation;
after enzyme deactivation, carrying out solid-liquid separation to obtain a second extracting solution and second filter residue;
in an alternative embodiment, the active peptide extraction step is repeated at least once and the extracts obtained each time are combined to obtain a second extract.
In an optional embodiment, the flavone is extracted by a reflux extraction method for 2-6 h;
preferably, the extraction is repeated 1-2 times, and the extractive solutions obtained from each extraction are combined to obtain the first extractive solution.
In an optional embodiment, the flavone is extracted by an ultrasonic extraction method, wherein the ultrasonic frequency is 10-50KHz, and the extraction time is 20-120 min;
preferably, the extraction is repeated 1-2 times, and the extractive solutions obtained from each extraction are combined to obtain the first extractive solution.
In an optional embodiment, the flavone is extracted by a microwave extraction method, wherein the microwave power is 350-;
preferably, the extraction is repeated 1-2 times, and the extractive solutions obtained from each extraction are combined to obtain the first extractive solution.
In an optional embodiment, the enzymolysis time of the first enzymolysis is 1-6 h;
preferably, the addition amount of the cellulase is 0.1-2% of the mass of the chestnut pollen.
In an optional embodiment, the enzymolysis time of the secondary enzymolysis is 1-6 h;
preferably, the addition amount of the protease is 0.1-2% of the mass of the chestnut pollen.
In alternative embodiments, the protease is selected from at least one of trypsin, pepsin, bromelain, papain, flavourzyme and neutral protease.
In an alternative embodiment, the enzyme deactivation is carried out by raising the temperature of the reaction system to 85-95 ℃ and keeping the temperature for 8-12 min.
In an optional embodiment, the chestnut powder and water are mixed in a material-liquid ratio of 1: 5-50;
preferably, the first filter residue is mixed with water again in a material-liquid ratio of 1: 2-10.
The invention has the following beneficial effects:
the chestnut pollen extract containing flavone and active peptide can promote phagocytic activity of macrophage, promote proliferation of spleen lymphocyte and activate and enhance killing activity of NK cell by cooperating with Con A, so that it can be used for preparing medicine and food for regulating or exciting the above cell activity; the chestnut pollen extract containing flavone and active peptide has remarkable effect on regulating immunity, and can be applied to preparation of medicines or foods for regulating immunity. Particularly, the preparation method provided by the application maintains the biological activity of the chestnut pollen to the maximum extent, and adopts a separate extraction mode to extract the flavone and the active peptide in the chestnut pollen respectively, so that the flavone and the active peptide in the pollen and other active substances can be fully extracted, the effect is better when the method is applied to regulating or exciting the cell activity or regulating the immunity, and the effective dose of the chestnut pollen is greatly reduced.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The preparation method and application of the chestnut pollen extract provided by the invention are specifically described below.
The application of the chestnut pollen extract in preparing the medicine or food for regulating immunity, wherein the chestnut pollen extract contains flavone and active peptide.
According to the disclosure of the prior art, it is generally suggested that the substance in pollen that plays a role in regulating immunity is flavone. The inventors have made extensive and inventive work to find that a mixture containing flavone and active peptide extracted from chestnut pollen extract has a significant effect on immune modulation, and that when the extract is used in equal amounts, the effect on immune modulation is better when the extract contains both flavone and active peptide than when the extract contains flavone alone.
The application of the chestnut pollen extract in preparing the medicines or the foods for promoting macrophage phagocytosis activity, promoting spleen lymphocyte proliferation in cooperation with Con A or activating and enhancing NK cell killing activity, wherein the chestnut pollen extract contains flavone and active peptide.
The inventor conducts extensive creative work and finds that the mixture containing flavone and active peptide extracted from chestnut pollen extract has significant effects on promoting macrophage phagocytosis activity, promoting spleen lymphocyte proliferation in cooperation with Con A, or activating and enhancing NK cell killing activity.
Preferably, when applied to the preparation of a medicament, the dosage form of the medicament comprises: powders, tablets, capsules, soft capsules, syrups, tinctures, pills, powders, granules, aqueous pharmaceuticals, emulsions, suspensions, gels, creams, ointments or emulsions.
A method for preparing chestnut pollen extract comprises:
s1, extracting flavone
Mixing chestnut pollen and water according to a material-liquid ratio of 1: 5-50 (such as 1:5, 1:10, 1:20, 1:40 or 1:50), extracting at 30-60 ℃ (such as 30 ℃, 40 ℃, 50 ℃ or 60 ℃), and performing solid-liquid separation after extraction to obtain a first extracting solution and a first filter residue, wherein the extracting method comprises a reflux extraction method, an ultrasonic extraction method or a microwave extraction method.
Preferably, when the flavone is extracted by reflux (agitation) extraction, the extraction time is 2-6h (e.g. 2h, 4h or 6h) to ensure sufficient extraction.
More preferably, in order to further ensure sufficient extraction, multiple extractions can be carried out, the extraction is repeated for 1-2 times after the first extraction is finished, and the extracting solutions obtained from each extraction are combined to obtain a first extracting solution.
Preferably, when the flavone is extracted by ultrasonic extraction, the ultrasonic frequency is 10-50KHz (such as 10KHz, 20KHz, 30KHz or 50KHz), and the extraction time is 20-120min (such as 20min, 40min, 60min, 80min, 100min or 120min) to ensure sufficient extraction.
More preferably, in order to further ensure sufficient extraction, multiple extractions can be carried out, the extraction is repeated for 1-2 times after the first extraction is finished, and the extracting solutions obtained from each extraction are combined to obtain a first extracting solution.
Preferably, when the microwave extraction method is used for extracting the flavone, the microwave power is 350-600W (for example 350W, 450W, 500W or 600W), and the extraction time is 20-60min (for example 20min, 30min, 40min, 50min or 60min) to ensure sufficient extraction.
More preferably, in order to further ensure sufficient extraction, multiple extractions can be carried out, the extraction is repeated for 1-2 times after the first extraction is finished, and the extracting solutions obtained from each extraction are combined to obtain a first extracting solution.
Preferably, the solid-liquid separation is by conventional filtration or centrifugation.
Through the extraction in the step, the flavone in the chestnut pollen is fully extracted into the first extracting solution. The active substances extracted into the first extract solution may contain other substances beneficial to health in addition to the flavones.
S2 extraction of active peptide
(1) One-time enzymolysis
Mixing the first filter residue with water again in a material-to-liquid ratio of 1: 2-10 (such as 1:2, 1:4, 1:6, 1:8 or 1:10) to obtain a mixed slurry, adjusting the pH of the mixed slurry to 4.0-7.0, adding cellulase into the mixed slurry, and performing primary enzymolysis at 30-60 ℃ (such as 30 ℃, 40 ℃, 50 ℃ or 60 ℃).
Preferably, the amount of cellulase added is 0.1-2% (e.g., 0.1%, 0.5%, 1%, 1.5% or 2%) of the mass of chestnut pollen, in order to ensure sufficient enzymolysis. More preferably, the time of one enzymolysis is 1-6h (e.g., 1h, 2h, 4h or 6 h).
(2) Secondary enzymolysis
Adjusting pH of the mixed slurry to 4.0-7.0 after the primary enzymolysis, adding protease into the mixed slurry, and performing secondary enzymolysis at 30-60 deg.C (such as 30 deg.C, 40 deg.C, 50 deg.C or 60 deg.C).
Preferably, in order to ensure sufficient enzymolysis, the addition amount of the protease is 0.1-2% (e.g. 0.1%, 0.5%, 1%, 1.5% or 2%) of the mass of the chestnut pollen; more preferably, the enzymolysis time of the second enzymolysis is 1-6h (for example: 1h, 2h, 4h or 6 h).
Specifically, the protease may be at least one selected from trypsin, pepsin, bromelain, papain, flavourzyme and neutral protease.
(3) Enzyme deactivation
Raising the temperature of the reaction system to 85-95 ℃ (for example, 85 ℃, 90 ℃ or 95 ℃), and keeping the temperature for 8-12 min (for example, 8min, 10min or 12min) for enzyme deactivation.
(4) Solid-liquid separation
And performing solid-liquid separation by adopting a filtering or centrifuging mode to obtain a second filter residue and a second extracting solution.
Preferably, the S3 active peptide extraction step is repeated at least once, specifically, the first silicone oil extraction step is repeated with the second filter residue as the first filter residue for extraction, and the obtained extracts are combined to obtain the second extract.
The active peptide in the chestnut pollen can be fully extracted into the extracting solution by the step.
S3, mixing the extractive solutions
And (3) combining the first extracting solution finally obtained in the step S1 and the second extracting solution finally obtained in the step S2 to obtain the chestnut flower powder extracting solution.
S4, drying
Drying the chestnut pollen extracting solution to obtain the chestnut pollen extract. Preferably, the drying mode adopts spray drying, and an air inlet 165 ℃ and an air outlet are arranged during spray drying: 85 ℃.
The method comprises the following steps of firstly, adopting stirring extraction or microwave or ultrasonic extraction to mainly obtain a flavonoid compound extracting solution, secondly, adopting biological enzymolysis to extract the chestnut pollen active peptide, wherein the acidic enzymolysis is divided into cellulase enzymolysis and protease enzymolysis, the environment with the optimal enzymolysis efficiency is acidic regardless of cellulase or pepsin, and the active peptide in the chestnut pollen is extracted by fully utilizing the characteristics of the cellulase and the pepsin. The effective components in the chestnut flower pollen are extracted separately to the maximum extent, and the application shows that the chestnut flower pollen extract has obvious effect on the immune regulation of mice.
The chestnut flower pollen extract obtained by extraction can be directly used as an edible chestnut flower pollen extract, namely, no additive or organic solvent extractant is added, a recovery process of adding the organic solvent extractant is not needed, and the extraction process is safe and efficient.
The preparation method of the chestnut flower pollen extract provided by the application is simple in process, mild in condition, safe and environment-friendly, easy to popularize on a large scale, low in requirements on process parameter control, high in equipment recycling rate and capable of reducing the investment scale of enterprises. The whole extraction process does not use solvents or reagents with high toxicity.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Mixing chestnut pollen and water according to a material-liquid ratio of 1:10, and extracting for 4h at 40 ℃ under reflux and stirring. Separating the extract and the filter residue after extraction, repeatedly extracting for 1 time by using the filter residue as a raw material, and combining the extract obtained by the two-time extraction after extraction to obtain a first extract and a first filter residue.
Mixing the first filter residue with water again at a material-liquid ratio of 1:5 to obtain mixed slurry, adjusting pH of the mixed slurry to 5.0, adding cellulase accounting for 1% of chestnut pollen mass into the mixed slurry, and performing primary enzymolysis at 40 deg.C for 6 h.
Adjusting pH of the mixed slurry to 7.0 after the first enzymolysis, adding trypsin which accounts for 0.5% of the mass of the chestnut pollen into the mixed slurry, and performing second enzymolysis at 40 ℃ for 6 h.
After the second enzymolysis, the temperature of the reaction system is raised to 90 ℃ to inactivate the enzyme for 10 min.
Filtering to obtain extractive solution and residue, repeating the active peptide extraction step with the residue as raw material for 1 time, and mixing the extractive solutions to obtain second extractive solution and second residue.
And mixing the first extracting solution and the second extracting solution to obtain the chestnut pollen extracting solution.
Transferring the chestnut pollen extracting solution to a spray dryer for spray drying, wherein an air inlet 165 ℃ and an air outlet are arranged during spray drying: 85 ℃. To obtain chestnut pollen extract A.
Example 2
Mixing chestnut pollen and water at a ratio of 1:20, and performing ultrasonic extraction at 30 deg.C and 20KHz for 60 min. Separating the extract and the filter residue after extraction, repeatedly extracting for 1 time by using the filter residue as a raw material, and combining the extract obtained by the two-time extraction after extraction to obtain a first extract and a first filter residue.
Mixing the first filter residue with water again at a material-liquid ratio of 1:10 to obtain mixed slurry, adjusting pH of the mixed slurry to 4.5, adding cellulase accounting for 2% of the mass of the chestnut pollen into the mixed slurry, and carrying out primary enzymolysis at 30 ℃ for 4 h.
Adjusting pH of the mixed slurry to 6.5 after the first enzymolysis, adding neutral protease 1% of chestnut pollen mass into the mixed slurry, and performing second enzymolysis at 30 deg.C for 4 hr.
After the second enzymolysis, the temperature of the reaction system is raised to 90 ℃ to inactivate the enzyme for 10 min.
Filtering to obtain extractive solution and residue, repeating the active peptide extraction step with the residue as raw material for 1 time, and mixing the extractive solutions to obtain second extractive solution and second residue.
And mixing the first extracting solution and the second extracting solution to obtain the chestnut pollen extracting solution.
Transferring the chestnut pollen extracting solution to a spray dryer for spray drying, wherein an air inlet 165 ℃ and an air outlet are arranged during spray drying: 85 ℃. Obtaining chestnut pollen extract B.
Example 3
Mixing chestnut pollen and water at a ratio of 1:5, and performing microwave extraction at 50 deg.C with power of 400W for 20 min. Separating the extract and the filter residue after extraction, repeatedly extracting for 1 time by using the filter residue as a raw material, and combining the extract obtained by the two-time extraction after extraction to obtain a first extract and a first filter residue.
Mixing the first filter residue with water again at a material-liquid ratio of 1:2 to obtain mixed slurry, adjusting pH of the mixed slurry to 5.5, adding cellulase accounting for 0.5% of chestnut pollen mass into the mixed slurry, and performing primary enzymolysis at 50 deg.C for 2 h.
Adjusting pH of the mixed slurry to 6.0 after the first enzymolysis, adding papain with a content of 1% of chestnut pollen, and performing second enzymolysis at 50 deg.C for 2 hr.
After the second enzymolysis, the temperature of the reaction system is raised to 90 ℃ to inactivate the enzyme for 10 min.
Filtering to obtain extractive solution and residue, repeating the active peptide extraction step with the residue as raw material for 1 time, and mixing the extractive solutions to obtain second extractive solution and second residue.
And mixing the first extracting solution and the second extracting solution to obtain the chestnut pollen extracting solution.
Transferring the chestnut pollen extracting solution to a spray dryer for spray drying, wherein an air inlet 165 ℃ and an air outlet are arranged during spray drying: 85 ℃. To obtain chestnut pollen extract C.
Comparative example 1
This comparative example is substantially the same as example 1 except that no extraction of active peptide was performed as compared to example 1. To obtain chestnut pollen extract D.
Examples of the experiments
Dissolving the chestnut pollen extract A, B, C, D with distilled water respectively to obtain extract solutions with respective concentrations of 50, 100, 200, 400, and 800 μ g/mL for use.
Influence of chestnut pollen extract on immune cell activity
(1) Effect on RAW264.7 macrophage immune activity: RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody for 2d passages. Culturing RAW264.7 macrophage in logarithmic growth phase with DMEM high-glucose culture solution containing 10% fetal calf serum and 1% double antibody, and adjusting cell concentration to 1 × 105And (2) inoculating 200 mu L of the extract per mL into a 96-well culture plate, adaptively culturing for 4h, setting the cells into extract sample groups with different concentrations (50, 100, 200, 400 and 800 mu g/mL), adding extracts with different concentrations, an LPS (1 mu g/mL) positive control group and a blank group (adding equal volume of culture solution) into the sample groups, setting 3 parallel wells in each group, and culturing for 24h under the same culture condition. Gently removing 20 μ L of supernatant 2h before experiment, adding neutral red reagent, culturing, slowly sucking away the culture solution, washing with PBS preheated to 37 deg.C for 3 times, discarding the washing solution, and addingThe cells were lysed with 200. mu.L of cell lysate, and then placed on a shaker for 10min, and absorbance was measured with a microplate reader at 540 nm. Phagocytosis was calculated as follows.
Macrophage phagocytosis rate (OD sample-OD blank)/OD blank × 100%
Macrophage phagocytosis rates were recorded for each experimental group in the table below.
TABLE 1 statistics of macrophage phagocytosis ratio in each experimental group
Note: indicates significant difference compared to LPS group (p < 0.05); indicates very significant differences compared to LPS group (p < 0.01).
Macrophages are phagocytic cells of the human body, are widely distributed in tissues, and have the effects of transmitting immune information, coordinating and phagocytosing and processing antigens. After entering tissues and organs along with blood circulation, monocytes can further differentiate and develop into macrophages, and become cells with the strongest phagocytic capacity in organisms. The phagocytic ability of macrophages is the main effect, the phagocytic ability depends on the activity of cells, dead cells and cells with poor states cannot phagocytize, and the phagocytic ability of the macrophages to phagocytose neutral red can indicate the activity of the macrophages and indirectly reflect the intensity of the immune function of the organism.
The results of the experiment of phagocytosis of neutral red by RAW264.7 macrophages are shown in table 1. Within the range of experimental concentration, the phagocytosis rate of RAW264.7 macrophages is more than 0, which indicates that the extract has the function of promoting phagocytosis and can enhance the ability of the macrophages to phagocytize neutral red. The extract has dose-dependent promoting effect in lower concentration range, the neutral red phagocytosis rate increases with the increase of the extract concentration, and the four extracts promote to reach the maximum value at 200 μ g/mL. The maximum promotion effect of the three extracts obtained by the method provided by the application on the phagocytic activity of macrophages is higher than that of a positive control LPS, and the chestnut flower flavone is higher than that of the positive control only at 100 mu g/mL and 200 mu g/mL. The promotion effect of the chestnut flower powder extract obtained by the method provided by the application on phagocytosis of neutral red by RAW264.7 macrophages is stronger than that of the chestnut flower flavone extract under the same concentration.
(2) Effect of extracts on Con a-induced splenic lymphocyte proliferation in mice: the mouse is killed by removing the cervical vertebra, the spleen is picked up under the aseptic condition, the spleen capsule is torn off, and the spleen is cut into small pieces by an ophthalmic scissors. Washing with D-Hanks solution containing double antibodies, then placing into a sterile culture dish, adding 2mL of D-Hanks solution, slowly rotating a grinding rod, and grinding until homogenate is obtained; sieving with 200 mesh sieve, collecting washing solution, centrifuging at 1000rpm for 10min, washing with cell cleaning solution for 3 times, and centrifuging to obtain precipitate. Adding a small amount of RPMI 1640 culture solution, and counting by trypan blue staining, wherein the survival rate is required to be more than 95%. Cell concentration was adjusted to 1X 10 with 1% double antibody, 10% fetal bovine serum in RPMI 1640 complete medium6one/mL. 100 μ L of the cell suspension was added to a 96-well plate cell culture, and a set of extract samples at different concentrations (0, 50, 100, 200, 400, 800 μ g/mL) was set with a final concentration of 4.5 μ g/mL concanavalin (Con A) per well. Culturing in a cell culture box for 72h, adding the WST-1 reagent 4h before the experiment is finished, and measuring the cell proliferation condition according to the requirements of the kit after the culture is finished. The group with zero addition of the extract and Con A was a blank group, and the absorbance was measured at 520nm with an enzyme reader, and the proliferation rate was calculated according to the following formula.
Spleen lymphocyte proliferation rate (OD sample-OD blank)/OD blank × 100%
The rate of proliferation of splenic lymphocytes of the mice for each experimental group is recorded in the following table.
TABLE 2 statistics of splenic lymphocyte proliferation rates by each experimental group
Note: indicates significant difference compared to the representation versus blank group (p < 0.05); indicates very significant difference compared to blank group (p < 0.01).
Spleen is an important immune apparatus of human body, and the state of spleen can reflect the immune function of the body to a certain extent. A large number of immune cells are located in the spleen, are the sites for lymphocyte maturation and differentiation, and can regulate the immune function of the body through cellular immunity and humoral immunity. Lymphocytes are an important part of immunity, and the spleen has 25 percent of T lymphocytes of the whole body, is directly involved in cellular immunity and is a key cell of adaptive immune response of an organism. The lymphocyte transformation test is that T cells are converted into lymphoblasts under the action of specific antigens (specific soluble antigens, cell surface antigens, tuberculin) or mitogens (Con A, LPS and the like), so that the volume is larger, the metabolism is vigorous, different stimulators can stimulate different lymphocytes to differentiate and proliferate, and the functional states of different lymphocyte subsets are reflected.
The results of experiments on the mouse spleen lymphocyte transformation induced by Con A synergistic extract are shown in Table 2, and it can be seen from the data in the table that the effect of the extract on lymphocyte activity is enhanced with the increase of the sample concentration, but after the maximum value is reached, the concentration is continuously increased, and the activity of immunocytes is weakened. The four extracts have the strongest proliferation promoting effect when the concentration of the four extracts is 200 mu g/mL; the chestnut flower powder extract obtained by the method provided by the application has stronger proliferation effect on splenic lymphocytes of mice than the chestnut flower flavone extract under the same concentration.
(3) And (3) detecting the killing activity of NK cells of the mice: the mouse is killed by removing the cervical vertebra, the spleen is picked up under the aseptic condition, the spleen capsule is torn off, and the spleen is cut into small pieces by an ophthalmic scissors. Washing with D-Hanks solution containing double antibodies, then placing into a sterile culture dish, adding 2mL of D-Hanks solution, slowly rotating a grinding rod, and grinding until homogenate is obtained; sieving with 200 mesh sieve, collecting washing solution, and centrifuging at 1000rpm for 10 min. Collecting precipitate, adding 2mL cell lysate to lyse red blood cells for 30s, adding 8mL Hanks solution immediately to stop reaction, centrifuging at 1000rpm for 10min to wash cells, collecting precipitate, adding small amount of RPMI 1640 culture solution, staining trypan blue to count, requiring survival rate of more than 95%, and adjusting cell concentration to 1 × 10 with RPMI 1640 culture solution containing 1% double antibody and 10% fetal calf serum6one/mL. Adding 100 mu L of cell culture solution into a 96-well cell culture plate, adaptively culturing for 4h, dividing the cells into a natural release group and an extract sample group with different concentrations (50, 100, 200, 400 and 800 mu g/mL), adding extracts with different concentrations into the sample group, adding equal volume of complete culture solution into the natural release hole, acting for 12h, adding 50 mu L of YAC-1 (effective target ratio 50:1), and mixing and culturing for 4 h. According to the requirements of the lactate dehydrogenase kitThe absorbance at 590nm was calculated using the following formula.
NK cell killing activity ═ 100% (OD experimental group-OD natural release group)/(OD maximum release group-OD natural release group) ×
The NK cell killing activity of each experimental group is recorded in the table below.
TABLE 3 statistics of NK cell killing Activity by various groups
Note: indicates significant difference compared to blank group (p < 0.05); indicates very significant difference compared to blank group (p < 0.01).
There are a number of natural killer cells (NK cells) in the spleen, which are an important subset of lymphocytes in the body, and like macrophages, are also important immune cells in the body. The NK cell is an innate immune cell, has functions of recognizing a target cell, secreting perforin, and the like as a first line of immune defense, and actively participates in an intracellular immune reaction. Under normal conditions, lactate dehydrogenase in cytoplasm of living cells cannot permeate cell membranes, but the lactate dehydrogenase is released out of the cells after the cell membranes are damaged, the amount of the released lactate dehydrogenase can be measured by a lactate dehydrogenase kit after target cells are killed by NK cells, and the content of the lactate dehydrogenase in a cell culture solution can reflect the killing activity of the NK cells.
As can be seen from Table 3, the four extracts can activate and enhance the NK cell killing activity, the promotion effect of the extract C is the best, the NK cell killing activity is 32.5% at 200 mu g/mL, which is far greater than that of the blank group (18.1%), and the difference is very significant (p is less than 0.01). The chestnut flower flavone extract (D) can enhance the killing activity of NK cells to a certain extent, but has no significance.
Secondly, the influence of the extract on the immune function of mice
1. Mice were randomly divided into 8 groups of 10 animals each in half male and female. Respectively a blank group, a model group, a C high dose group, a C medium dose group, a C low dose group, a D high dose group, a D medium dose group and a D low dose group; the blank group and the model group are subjected to intragastric administration by 0.1mL/10g of physiological saline every day, the chestnut pollen extract C and D are dissolved and intragastric administered according to the weight of each 10g of mouse and 0.1mL of physiological saline for 21 days, wherein the high dose group is 600mg/kg, the medium dose group is 400mg/kg, and the low dose group is 200 mg/kg. The model group, high and low dose groups were administered at day 18, 19, 20, 21 with cyclophosphamide 80mg/kg dissolved in saline 0.1mL per 10g of mouse body weight for intraperitoneal injection, and the blank group was administered with mice body weight per 10g of saline 0.1mL for intraperitoneal injection.
2. Determination of thymus index and spleen index
Fasting for 12h after the last administration, weighing, removing neck and killing, taking spleen and thymus, washing with cold physiological saline, weighing, and calculating the spleen index and the thymus index of the mice according to the following formulas.
Thymus index ═ weight of thymus (mg)/weight (10g)
Spleen index spleen weight (mg)/body weight (10g)
The thymus and spleen indices were recorded for each experimental group in the following table:
TABLE 4 statistics of thymus index and spleen index for each experimental group
Note: comparison with blank group: p <0.05, P <0.01, P < 0.001; comparison with model groups: # P <0.05, # P <0.01, # P < 0.001.
Spleen and thymus shrink as immune function decreases. The thymus index and the spleen index reflect the functions of two major immune organs, namely thymus and spleen. As can be seen from table 4, spleen and thymus indices were significantly decreased in the model group compared to the blank group (P < 0.001); sample C three dose groups increased spleen and thymus indices and were dose dependent compared to the model group; the spleen index and the thymus index of the mice are obviously increased in the high-dose group and the medium-dose group (P <0.05 and P <0.01), and the thymus index of the mice can be increased in the low-dose group, but the low-dose group has no statistical difference compared with the model group. The result shows that the chestnut pollen extract prepared by the method provided by the application can improve the atrophy of immune organs (spleen and thymus) caused by cyclophosphamide. Both thymus and spleen indices were statistically different in mice with the D high dose group (P <0.05), but not as elevated as in group a.
3. Determination of the proliferative Capacity of splenic lymphocytes
Removing neck of mouse after fasting for 12h after the last administration, killing the mouse, soaking in 75% alcohol for 3min, taking spleen of the mouse in an ultra-clean bench, cleaning with cold PBS, shearing, grinding piston rubber head of a disposable syringe, sieving with a 200 mesh sieve, centrifuging at 1500r/min for 10min, discarding supernatant, adding erythrocyte lysate, blowing to blow uniformly, placing in ice for reaction for 15min to lyse erythrocyte, centrifuging at 1500r/min for 10min, discarding supernatant, adding 1640 culture medium to stop lysis, washing lysate, centrifuging at 1500r/min for 10min, discarding supernatant, resuspending with 1640 culture medium of 10% fetal calf serum, 37 ℃, 5% CO2And removing adherent cells after 24h of culture to obtain primary splenocytes, staining trypan blue, counting the cells, and ensuring that the viable cell rate is 90-93%.
1×105Spleen primary cells were inoculated per well, induced by addition of LPS (final concentration 1. mu.g/mL) to each group, and then incubated at 37 ℃ with 5% CO2Adding 10 μ L CCK-8 into each well after culturing for 68h, continuously culturing for 4h, measuring absorbance (OD) value at 450nm, and calculating B lymphocyte proliferation rate; con A (final concentration 5. mu.g/mL) was added for induction, and the proliferation rate of T lymphocytes was calculated in the same manner as in the above other steps.
Cell proliferation rate (OD induction group-OD control group)/OD induction × 100%
TABLE 5 Effect of the groups on the proliferative Capacity of splenic lymphocytes from mice
| Group of | T lymphocyte (%) | B lymphocyte (%) |
| Blank group | 46.14±2.37 | 33.45±1.15 |
| Model set | 24.72±3.43*** | 22.25±1.12*** |
| C Low dose group | 26.54±2.51*** | 23.13±1.37*** |
| C Medium dose group | 29.73±4.66***# | 26.32±1.79***# |
| C high dose group | 31.26±3.89***## | 28.57±2.36***## |
| D Low dose group | 24.42±2.23*** | 22.36±2.54*** |
| D middle dose group | 26.59±3.62***# | 24.13±1.43***# |
| D high dose group | 27.88±3.67***# | 25.48±2.14***## |
Note: compared to the blank group: p <0.05, P <0.01, P < 0.001; compared to the model set: # P <0.05, # P <0.01, # P < 0.001.
Lymphocyte proliferation is an important index reflecting the immune state of an organism, T lymphocytes and B lymphocytes are activated when stimulated by antigen or mitogen, and LPS and Con A can respectively induce the proliferation of the B lymphocytes and the T lymphocytes. As can be seen from table 5, after LPS and Con a induction, the B lymphocyte proliferation capacity and T lymphocyte proliferation capacity of the model group were significantly lower than those of the blank group (P < 0.001); compared with a model group, the chestnut flower extract C obtained by the method provided by the application has the advantages that the lymphocyte proliferation capacity of high-dose and medium-dose groups is remarkably improved (P <0.05 and P <0.01), and the lymphocyte proliferation capacity of low-dose groups is improved but has no statistical significance difference. The result shows that the chestnut pollen extract is beneficial to the proliferation and activation of lymphocytes and enhances the immune function of mice. At the same concentration, the proliferation capacity of the chestnut flower flavone group D on B lymphocytes and the proliferation capacity of T lymphocytes are lower than that of the chestnut flower powder extract C obtained by the method provided by the application.
In conclusion, the chestnut pollen extract containing flavone and active peptide can promote phagocytic activity of macrophages, promote proliferation of spleen lymphocytes by cooperating with Con A, activate and enhance killing activity of NK cells, so that the chestnut pollen extract can be applied to preparation of medicines and foods for regulating or exciting the cell activity; the chestnut pollen extract containing flavone and active peptide has remarkable effect on regulating immunity, and can be applied to preparation of medicines or foods for regulating immunity. Particularly, the preparation method provided by the application maintains the biological activity of the chestnut pollen to the maximum extent, and adopts a separate extraction mode to extract the flavone and the active peptide in the chestnut pollen respectively, so that the flavone and the active peptide in the pollen and other active substances can be fully extracted, the effect is better when the method is applied to regulating or exciting the cell activity or regulating the immunity, and the effective dose of the chestnut pollen is greatly reduced.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The application of the chestnut pollen extract in preparing the medicine or food for regulating immunity is disclosed, wherein the chestnut pollen extract contains flavone and active peptide.
2. The application of the chestnut pollen extract in preparing the medicines or the foods for promoting macrophage phagocytosis activity, promoting spleen lymphocyte proliferation in cooperation with Con A or activating and enhancing NK cell killing activity, wherein the chestnut pollen extract contains flavone and active peptide.
3. The use according to claim 1 or 2, wherein the medicament is in a dosage form comprising: powders, tablets, capsules, soft capsules, syrups, tinctures, pills, powders, granules, aqueous pharmaceuticals, emulsions, suspensions, gels, creams, ointments or emulsions.
4. The preparation method of the chestnut pollen extract is characterized by comprising the following steps:
mixing chestnut pollen with water by using water as a solvent, and performing flavone extraction by adopting a reflux extraction method, an ultrasonic extraction method or a microwave extraction method to obtain a first extracting solution and first filter residue;
mixing the first filter residue with water by taking water as a solvent, and sequentially extracting active polypeptide in the first filter residue by adopting cellulase and protease to obtain a second extracting solution and a second filter residue;
combining the first extract and the second extract;
preferably, the first extracting solution and the second extracting solution are combined to obtain a chestnut pollen extracting solution, and the chestnut pollen extracting solution is dried to obtain the chestnut pollen extract.
5. The method for preparing chestnut pollen extract according to claim 4, characterized in that the flavone extraction is: mixing chestnut pollen with water, extracting at 30-60 deg.C, and performing solid-liquid separation to obtain first extractive solution and first filter residue, wherein the extraction method comprises reflux extraction, ultrasonic extraction or microwave extraction.
6. The method for preparing chestnut pollen extract according to claim 4, characterized in that the active peptide extraction is: mixing the first filter residue with water again to obtain mixed slurry, adjusting the pH of the mixed slurry to 4.0-7.0, adding cellulase into the mixed slurry, and performing primary enzymolysis at 30-60 ℃;
adjusting the pH of the mixed slurry to 4.0-7.0 after the primary enzymolysis is finished, adding protease into the mixed slurry, and performing secondary enzymolysis at 30-60 ℃;
after the secondary enzymolysis, the mixed slurry is subjected to enzyme deactivation;
after enzyme deactivation, carrying out solid-liquid separation to obtain a second extracting solution and second filter residue;
preferably, the enzyme deactivation is carried out by raising the temperature of the reaction system to 85-95 ℃ and keeping the temperature for 8-12 min.
7. The method for preparing chestnut pollen extract according to claim 5, wherein the second extract is obtained by combining the extracts obtained each time by repeating the extraction step at least once with active peptide.
8. The method for preparing chestnut pollen extract according to claim 5, wherein the extraction of flavone is carried out by reflux extraction for 2-6 h; preferably, the extraction is repeated for 1-2 times, and the extracting solution obtained from each extraction is combined to obtain the first extracting solution;
or extracting flavone with ultrasonic frequency of 10-50KHz for 20-120 min; preferably, the extraction is repeated for 1-2 times, and the extracting solution obtained from each extraction is combined to obtain the first extracting solution;
or extracting flavone by microwave extraction method with microwave power of 350-; preferably, the extraction is repeated 1-2 times, and the extractive solutions obtained from each extraction are combined to obtain the first extractive solution.
9. The method for preparing chestnut pollen extract according to claim 5, characterized in that the enzymolysis time of one enzymolysis is 1-6 h;
preferably, the addition amount of the cellulase is 0.1-2% of the mass of the chestnut pollen;
preferably, the enzymolysis time of the secondary enzymolysis is 1-6 h;
preferably, the addition amount of the protease is 0.1-2% of the mass of the chestnut pollen;
preferably, the protease is selected from at least one of trypsin, pepsin, bromelain, papain, flavourzyme and neutral protease.
10. The method for preparing chestnut pollen extract according to claim 5, wherein the chestnut pollen is mixed with water in a feed-liquid ratio of 1: 5-50;
preferably, the first filter residue is mixed with water again in a material-liquid ratio of 1: 2-10.
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| CN114681500A (en) * | 2022-03-14 | 2022-07-01 | 西藏藏医药大学 | Low-toxicity and high-efficiency plena total flavone extract and application thereof |
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| CN104546980A (en) * | 2013-10-21 | 2015-04-29 | 中国科学院大连化学物理研究所 | Bee pollen extract as well as preparation and application thereof |
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| CN104546980A (en) * | 2013-10-21 | 2015-04-29 | 中国科学院大连化学物理研究所 | Bee pollen extract as well as preparation and application thereof |
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Application publication date: 20210730 |