CN113181231A - Composition with function of enhancing phagocytic activity of macrophages, application thereof and immune drug - Google Patents
Composition with function of enhancing phagocytic activity of macrophages, application thereof and immune drug Download PDFInfo
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Abstract
The invention provides a composition with a function of enhancing phagocytic activity of macrophages, application thereof and an immune medicament, and relates to the technical field of medicines. The composition with the function of enhancing the phagocytic activity of the macrophages comprises the brown alga polysaccharide and the elderberry, and the inventor finds that the brown alga polysaccharide and the elderberry both have an immunoregulation function, can enhance the phagocytic activity of the macrophages and inhibit lipopolysaccharide from inducing the macrophages to secrete nitric oxide.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a composition with a function of enhancing phagocytic activity of macrophages, application thereof and an immune medicament.
Background
Macrophages (Macrophages) are leukocytes located in tissues, derived from monocytes, and play an important role in body development, immunity, inflammatory response, and the development of various diseases by phagocytosis of autologous damaged cells or exogenous pathogens. That is, almost every human disease involves them, which are also the primary therapeutic targets, as their function can be potentiated or inhibited to alter the outcome of the disease. Therefore, increasing the phagocytic activity of macrophages contributes to an increase in the immune level of the body and the treatment of various diseases.
The existing research shows that macrophages in a living organism can change the phenotype and the immune function of the macrophages along with the change of the microenvironment, and continuously change the physiological state and the immune activity of the macrophages according to the stimulation of the macrophages, so that the health of organisms and tissues of the living organism is better ensured. The different stimulating substances comprise body self protein molecules, small molecule substances, extracts of fungi and bacteria and the like, and can stimulate and activate macrophages, so that the immune response and phagocytosis capacity of the macrophages are enhanced. However, the existing stimulating substances are limited, and have priority to the enhancement effect of the phagocytic capacity of macrophages, so that other substances capable of improving the phagocytic activity of the macrophages are necessarily searched.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first objective of the invention is to provide a composition with a function of enhancing phagocytic activity of macrophages, which can enhance the phagocytic activity of macrophages.
The second purpose of the invention is to provide the application of the composition with the function of enhancing the phagocytic activity of macrophages in preparing immune medicaments.
The third purpose of the invention is to provide an immune medicament, which comprises the composition with the function of enhancing the phagocytic activity of macrophages.
In a first aspect, the present invention provides a composition having a function of enhancing phagocytic activity of macrophages, which comprises fucoidan and elderberry.
As a further technical scheme, the composition also comprises a pine pollen extract;
preferably, the pine pollen extract comprises a pine pollen aqueous extract;
preferably, the pine pollen extract is pine pollen polysaccharide.
As a further technical scheme, the preparation method of the pine pollen aqueous extract comprises the following steps:
mixing pollen Pini with water, and ultrasonically treating under heating to obtain the aqueous extract;
preferably, the mass ratio of the pollen pini to the water is 1 (8-12), preferably 1: 10;
preferably, the heating temperature is 25-45 ℃, and preferably 37 ℃;
preferably, the time of the ultrasonic treatment is 10-30 min, and preferably 20 min.
According to a further technical scheme, the mass ratio of the brown algae polysaccharide to the elderberry in the composition is (1-4): 1.
As a further technical scheme, the mass ratio of the brown algae polysaccharide to the elderberry in the composition is 2: 1.
According to a further technical scheme, the mass ratio of brown algae polysaccharide to elderberry to pine pollen extract in the composition is (1-4) to 1 (3-6).
Preferably, the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen extract in the composition is 2:1 (3-6).
According to a further technical scheme, the mass ratio of brown algae polysaccharide to elderberry to pine pollen extract in the composition is 2:1: 3;
the pine pollen extract is a pine pollen aqueous extract.
According to a further technical scheme, the mass ratio of brown algae polysaccharide to elderberry to pine pollen extract in the composition is 2:1: 6;
the pine pollen extract is pine pollen polysaccharide.
In a second aspect, the invention provides the use of a composition having the function of enhancing phagocytic activity of macrophages in the preparation of an immune medicament.
In a third aspect, the present invention provides an immunopharmaceutical comprising a composition having a function of enhancing phagocytic activity of macrophages and an adjuvant.
Compared with the prior art, the invention has the following beneficial effects:
the composition with the function of enhancing the phagocytic activity of the macrophages comprises the brown alga polysaccharide and the elderberry, and the inventor finds that the brown alga polysaccharide and the elderberry both have an immunoregulation function, can enhance the phagocytic activity of the macrophages and inhibit lipopolysaccharide from inducing the macrophages to secrete nitric oxide.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a technical research route;
FIG. 2 is a graph of the effect of monocomponent on the proliferative activity of RAW264.7 cells;
FIG. 3 is a graph of the effect of pterostilbene on RAW264.7 cell proliferation activity;
FIG. 4 is a graph of the effect of monocomponent on phagocytic activity of RAW264.7 cells;
FIG. 5 is a graph of the effect of pterostilbene on phagocytic activity of RAW264.7 cells;
FIG. 6 is a graph showing the effect of single components on the amount of lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, with P < 0.05 indicating significant differences from the model group and P < 0.01 indicating significant differences from the model group;
fig. 7 shows the effect of different combinations on the amount of nitric oxide production induced by lipopolysaccharide in RAW264.7 cells, P < 0.05 indicated significant difference from the model group, and P < 0.01 indicated very significant difference from the model group;
fig. 8 is a graph showing the effect of multiple components on the amount of lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, with P < 0.05 indicating significant differences from the model group and P < 0.01 indicating significant differences from the model group;
Fig. 9 shows the effect of single components on NK92 cell natural killer activity, with P < 0.05 indicating significant difference from the normal group and P < 0.01 indicating significant difference from the normal group.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the present invention provides a composition having a function of enhancing phagocytic activity of macrophages, which comprises fucoidan and elderberry.
The brown algae polysaccharide is mainly from brown algae such as herba Zosterae Marinae, thallus laminariae, Macrocystis, Sargassum, Ascophyllum nodosum, and Fucus vesiculosus. Unlike general polysaccharides, fucoidan is also called "fucoidan sulfate" because some of the hydroxyl groups of fucoidan are esterified with sulfuric acid and the "sulfate groups" are connected to each other via "ester bonds". Research has shown that brown algae polysaccharide has various biological activities, such as immunoregulation, anticoagulation, antioxidation, antitumor, blood sugar lowering, blood fat lowering, antivirus, etc. The research of the invention finds that the brown algae polysaccharide has an immunoregulation effect, can enhance the phagocytic activity of macrophages and reduce the secretion of Nitric Oxide (NO) by Lipopolysaccharide (LPS) induced cells.
The elderberry is deciduous shrub, belongs to the family of Caprifoliaceae, contains abundant anthocyanin, flavone and polyphenol in fruits, has strong antioxidation, has antiviral effect on various influenza viruses, but has less research on immunoregulation. The research of the invention shows that the elderberry has immunoregulation capability, can enhance the phagocytic activity of macrophages and reduce the secretion of nitric oxide by lipopolysaccharide-induced cells.
The composition with the function of enhancing the phagocytic activity of the macrophages comprises the brown alga polysaccharide and the elderberry, and the inventor finds that the brown alga polysaccharide and the elderberry both have an immunoregulation function, can enhance the phagocytic activity of the macrophages and inhibit lipopolysaccharide from inducing the macrophages to secrete nitric oxide, the enhancing effect of the phagocytic activity of the macrophages by using the brown alga polysaccharide and the elderberry together is obviously superior to that of a single component, and the brown alga polysaccharide and the elderberry have a synergistic effect and are safe and reliable.
In a preferred embodiment, the composition further comprises pine pollen extract;
preferably, the pine pollen extract comprises a pine pollen aqueous extract;
preferably, the pine pollen extract is pine pollen polysaccharide.
The pollen Pini is pollen of Pinus massoniana lamb, Pinus tabulaeformis or congeneric plant of Pinaceae, and is a Chinese medicinal and edible variety in Chinese medical treasury. The pine pollen contains most of nutrient components required by a human body, including various active components such as protein, amino acid, monosaccharide, polysaccharide, nucleic acid, mineral substances and the like, is reasonable in matching, can supplement and balance the nutrition required by the human body, is called as green gold, and has various functional activity effects of enhancing immunity, resisting fatigue, resisting aging, protecting cardiovascular and the like.
The pine pollen extract is obtained by extracting pine pollen serving as a raw material in a manner known by persons skilled in the art, and comprises but is not limited to a water extraction method.
The pine pollen aqueous extract is extracted from pine pollen by a water extraction method.
The pollen Pini polysaccharide is extracted from pollen Pini.
In a preferred embodiment, the preparation method of the pine pollen aqueous extract comprises the following steps:
the preparation method of the pine pollen aqueous extract comprises the following steps:
mixing pollen Pini with water, and ultrasonically treating under heating to obtain the aqueous extract;
preferably, the mass ratio of the pine pollen to the water is 1 (8-12), and the pine pollen to the water can be, but is not limited to, 1:8, 1:9, 1:10, 1:11 or 1:12, and is preferably 1: 10;
Preferably, the heating temperature is 25-45 ℃, for example, but not limited to, 25 ℃, 30 ℃, 35 ℃, 40 ℃ or 45 ℃, preferably 37 ℃;
preferably, the time of the ultrasonic treatment is 10-30 min, for example, but not limited to, 10min, 15min, 20min, 25min or 30min, preferably 20 min.
In the invention, the method for preparing the pine pollen aqueous extract is further optimized and adjusted, so that the extraction of effective substances in the pine pollen is simply and efficiently realized.
In a preferred embodiment, the mass ratio of the brown algae polysaccharide to the elderberry in the composition is (1-4): 1, and can be, but is not limited to, 1:1, 2:1, 3:1 or 4: 1.
In a preferred embodiment, the mass ratio of brown algae polysaccharide to elderberry in the composition is 2: 1.
In the invention, the combination of brown algae polysaccharide and elderberry is fully developed by further optimizing and adjusting the mass ratio of the brown algae polysaccharide to the elderberry in the composition, and the enhancing effect of the composition on the phagocytosis activity of macrophages is improved. And when the brown algae polysaccharide and the elderberry are combined according to the mass ratio of 2:1, the composition has the strongest effect of enhancing the phagocytic activity of macrophages.
According to the influence of the brown alga polysaccharide and the elderberry on the phagocytic activity of macrophages, the brown alga polysaccharide and the elderberry are further combined with the pine pollen extract, and the influence of multiple components on the phagocytic activity of the macrophages is researched.
In a preferred embodiment, the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen extract in the composition is (1-4) to 1 (3-6), and may be, but not limited to, 1:1:6, 2:1:5, 3:1:4 or 4:1: 3.
Preferably, the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen extract in the composition is 2:1 (3-6), and the mass ratio can be, but is not limited to, 2:1:3, 2:1:4, 2:1:5 or 2:1: 6.
In a preferred embodiment, the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen extract in the composition is 2:1: 3;
the pine pollen extract is a pine pollen aqueous extract.
When the pine pollen extract is a pine pollen aqueous extract, the cooperation among the brown algae polysaccharide, the elderberry and the pine pollen aqueous extract in the composition is fully exerted by further optimizing and adjusting the brown algae polysaccharide, the elderberry and the pine pollen aqueous extract, and the phagocytic effect of macrophages is enhanced. And when the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen aqueous extract is 2:1:3, the composition has the strongest enhancing effect on phagocytic activity of phagocytes.
In a preferred embodiment, the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen extract in the composition is 2:1: 6;
the pine pollen extract is pine pollen polysaccharide.
When the pine pollen extract is the pine pollen polysaccharide, the brown alga polysaccharide, the elderberry and the pine pollen polysaccharide in the composition are further optimized and adjusted, the cooperation among the brown alga polysaccharide, the elderberry and the pine pollen polysaccharide is fully exerted, the phagocytic effect of macrophages is enhanced, and compared with the brown alga polysaccharide, the elderberry and the pine pollen aqueous extract, the phagocytic effect of the macrophages is further enhanced. And when the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen polysaccharide is 2:1:6, the composition has the strongest enhancing effect on phagocytic activity of phagocytes. Moreover, the composition with the mass ratio of the brown alga polysaccharide to the elderberry to the pine pollen polysaccharide of 2:1:6 has better effect of enhancing phagocytic activity of phagocytes than the composition with the mass ratio of the brown alga polysaccharide to the elderberry to the pine pollen aqueous extract of 2:1: 3.
In a second aspect, the invention provides the use of a composition having the function of enhancing phagocytic activity of macrophages in the preparation of an immune medicament.
The inventor finds that the brown alga polysaccharide and the elderberry have an immunoregulation function, can enhance phagocytosis activity of macrophages and inhibit lipopolysaccharide from inducing the macrophages to secrete nitric oxide, the enhancing effect of the phagocytosis activity of the macrophages by using the brown alga polysaccharide and the elderberry together is obviously superior to that of a single component, and the brown alga polysaccharide and the elderberry have a synergistic effect, are safe and reliable and can be used for preparing an immune medicament.
In a third aspect, the present invention provides an immunopharmaceutical comprising a composition having a function of enhancing phagocytic activity of macrophages and an adjuvant. The adjuvant used in the present invention is a pharmaceutically acceptable adjuvant, including but not limited to starch, dextrin, ethanol, syrup, hydrogenated vegetable oil, sodium carboxymethyl starch, and the like, which are described in the art.
Because the components of the immunopharmaceutical comprise the composition with the function of enhancing the phagocytic activity of macrophages, the immunopharmaceutical can also enhance the phagocytic activity of the macrophages and improve the immunity of the organism.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
The main idea of the research is to screen single-component raw materials which have no influence on cell activity or have proliferation effect, then to screen the best combination in pairs by 5 proportions, and finally to evaluate the immunoregulation effect of the combination in pairs and the pine pollen and to determine the best proportion of multiple components.
The specific research technical route is shown in figure 1.
Example 1
1. Preparation of the starting materials
(1) Brown algae polysaccharide: dissolving 200mg brown algae polysaccharide in 2ml hot water by ultrasonic treatment for 10min, filtering with 0.22 μm filter membrane to obtain 100mg/ml brown algae polysaccharide stock solution, and storing in refrigerator at 5 deg.C.
(2) Bone-knitting raspberry: youda international corporation, containing 10% anthocyanin, preparing 40mg/ml elderberry stock, dissolving 200mg extract in 5ml water, sterilizing with 0.22 μm filter membrane, and storing at-20 deg.C for use.
(3) Pterostilbene: dissolving 200mg of pterostilbene in 2ml of 50% alcohol, filtering with 0.22 μm filter membrane to obtain 100mg/ml pterostilbene stock solution, and storing in a refrigerator at 5 deg.C for use.
(4) The pine pollen aqueous extract comprises the following components: taking 500mg of pollen Pini in 5ml of water, performing ultrasonic treatment at 37 deg.C in water bath for 20min, filtering with 0.22 μm filter membrane to obtain 100mg/ml flos Pini water extract stock solution, and placing in refrigerator at 5 deg.C for use.
(5) And (3) preparing pine pollen polysaccharide: dissolving 100mg of self-made flos Pini polysaccharide in 1ml of hot water, centrifuging at 10000r/min for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, making into 100mg/ml flos Pini polysaccharide stock solution, and storing in refrigerator at 5 deg.C.
2. Cell culture
2.1 macrophage RAW264.7
(1) Cell growth characteristics: semi-adherent/adherent, the growth is fast;
(2) culture medium: 90% DMEM (high-sugar, double antibody-containing) + 10-12% FBS;
(3) And (3) resuscitation: water bath at 37 deg.C, culture medium containing 15% fetal bovine serum (preparation method: 85% DMEM (high sugar, containing double antibody) + 15% FBS);
(4) liquid changing: once every 1 day;
(5) passage: digesting with 0.25% pancreatin (containing EDTA) for 3-5 minutes at a passage ratio of 1: 4-6;
(6) freezing and storing: 50% complete medium + 40% serum + 10% DMSO or 90% serum + 10% DMSO.
2.2NK92 cells
(1) Cell growth characteristics: suspension, extremely slow growth;
(2) culture medium: 90% 1640 medium (containing double antibiotics) + 10-12% FBS;
(3) and (3) resuscitation: water bath at 37 deg.C, containing 15% culture medium;
(4) liquid changing: taking half of the culture medium to a new bottle once every 2 days, supplementing half of the culture medium respectively, or collecting and centrifuging (1500r, 5min), and then re-suspending;
(5) passage: changing the liquid at the passage ratio of 1: 2;
(6) freezing and storing: 90% serum + 10% DMSO;
(7) note that: the whole process is mild in operation.
3. RAW264.7 cell proliferation Rate assay
(1) Recovering and culturing RAW264.7 cells.
(2) 2.0 to 3.0 x 105The cells were seeded at a density of/ml in 96-well plates and cultured for 24 h.
(3) Diluting each raw material with culture medium to obtain sample solutions containing raw materials with different concentrations.
(4) After the culture is finished, discarding cell supernatant, adding the fucoidan, elderberry, pine pollen polysaccharide, pine pollen aqueous extract and pterostilbene sample solutions with the concentrations of 0 mu g/ml, 50 mu g/ml, 100 mu g/ml, 200 mu g/ml, 400 mu g/ml, 800 mu g/ml and 1000 mu g/ml into each hole, wherein each hole has 100 mu l and each concentration has 4 multiple holes, setting a normal control, setting 4 multiple holes in the same way, adding 100 mu l of culture medium into each hole, and culturing for 24 h.
(5) Preparing CCK8 incubation liquid, adding 100 mu l of CCK8 solution into 1ml of culture medium according to the instructions of the Biyuntian kit, uniformly mixing, finishing the culture, discarding the supernatant, adding PBS for washing once, adding 110 mu l of CCK8 incubation liquid into each hole, and incubating for 2 hours.
(6) After the incubation, OD450 was measured, and the cell proliferation rate was calculated according to the following equation.
Cell proliferation rate (%) [ (sample well OD value-normal well OD value)/normal well OD value ] × 100%.
The experimental results are shown in fig. 2 and fig. 3, within the range of 0-1mg/ml, the brown algae polysaccharide, elderberry, pine pollen polysaccharide and pine pollen aqueous extract have no influence on the proliferation activity of RAW264.7 cells, and the pterostilbene has an anti-proliferation effect on RAW264.7 cells, further research shows that the pterostilbene has no influence on the proliferation activity only within the range of 0-4 mug/ml.
4. Method for detecting phagocytic activity of RAW264.7 cells by using neutral red method
(1) Recovering and culturing RAW264.7 cells.
(2) 2.0 to 3.0 x 105The cells are inoculated into a 96-well plate at a density of/ml, cultured for 2h and attached to the wall.
(3) After adherence, the supernatant was discarded and divided into a blank group (containing only the culture medium), a normal control group (cells + culture medium), a lipopolysaccharide control group (cells + culture medium containing 1. mu.g/ml lipopolysaccharide), and a sample group (cells + sample solution), each group was provided with 4 duplicate wells, and after administration, the cells were cultured in an incubator for 24 hours.
(4) Discarding the supernatant, washing each well with PBS once, adding 50 μ l/well of Biyunyan neutral red solution, incubating for 10min in the dark, discarding neutral red, washing with PBS 3 times, 200 μ l/well each time, adding 100 μ l/well of prepared cell lysate (acetic acid: absolute ethyl alcohol ═ 1:1), and incubating for 30min in the dark.
(5) Immediately after incubation, OD values were measured at 570nm and the phagocytic activity of the sample groups was calculated as follows:
relative phagocytosis activity (%) - [ (sample well OD value-blank well OD value)/(normal well OD value-blank well OD value) -1] × 100%.
The influence of brown algae polysaccharide, elderberry, pine pollen aqueous extract and pterostilbene with the concentrations of 0 mug/ml, 50 mug/ml, 100 mug/ml, 200 mug/ml, 400 mug/ml, 800 mug/ml and 1000 mug/ml respectively on the phagocytic activity of RAW264.7 cells is researched by taking the brown algae polysaccharide, the elderberry, the pine pollen aqueous extract and the pterostilbene as sample groups, and the experimental results are shown in fig. 4 and fig. 5. Within the range of 1-1mg/ml, the brown algae polysaccharide, the pine pollen polysaccharide, the elderberry and the pine pollen aqueous extract can enhance the phagocytic activity of RAW264.7 cells, and the enhancement effect is in positive correlation with the dosage. The pterostilbene is only in the range of 0-0.5 mu g/ml, has no obvious influence, and the attenuation of the phagocytic activity of the RAW264.7 cells by the pterostilbene is increased along with the increase of the dosage.
According to the results of the proliferation activity and phagocytic activity test of each monocomponent, the influence of the combination of fucoidan and elderberry on the phagocytic activity of RAW264.7 cells was examined by using different ratios (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1) as a sample group, and the total mass concentration of the monocomponent (1:0, 0:1) and the composition (4:1, 2:1, 1:1, 1:2, 1:4) was 1mg/ml, and the results of the test are shown in Table 1.
As can be seen from Table 1, the brown algae polysaccharide and the elderberry have obvious combined synergistic action, wherein the 2:1 combined ratio has the strongest action, and the relative phagocytosis activity reaches 171.69%.
TABLE 1 percentage relative phagocytic Activity of different combinations on RAW264.7 cells (%, mean + -SD)
According to the influence of the two components on the phagocytic activity of RAW264.7 cells, the combination with the best enhancement effect (brown alga polysaccharide: elderberry: 2:1) is taken as the combination 1. The influence of the combination 1, the aqueous extract of pine pollen and the polysaccharide of pine pollen on the phagocytic activity of RAW264.7 cells was studied by using the combination as a sample group at different ratios (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1), wherein the total mass concentrations of the single component (1:0, 0:1) and the combination (4:1, 2:1, 1:1, 1:2, 1:4) were 1mg/ml, and the experimental results are shown in Table 2.
As can be seen from table 2, the enhancement effect of combination 1 with pine pollen polysaccharides is greater than that of combination 1 with pine pollen aqueous extract; the multi-component combination has a proportional enhancing effect which is larger than that of the two-component combination; the combination 1 and the pine pollen polysaccharide are mixed according to the proportion of 1:2, the phagocytic activity enhancement effect on RAW264.7 cells is strongest and is stronger than that of the combination of the two components.
TABLE 2% relative percentage phagocytosis activity (%, mean + -SD) of multicomponent on RAW264.7 cells
5. Griess method for detecting content of nitric oxide secreted by RAW264.7 cells
(1) Taking logarithmic phase cells RAW264.7 with density of 2 × 105And (3) inoculating each cell/ml to a 24-well plate, adding 1ml of cell suspension to each well, arranging a normal control well (1ml of cell suspension), a lipopolysaccharide-induced model well (1ml of cell suspension), a sample well (1ml of cell suspension + sample) and a blank well (only containing cell culture medium), and culturing for 24 hours.
(2) The supernatant was carefully discarded, washed once with PBS, 1ml of medium was added to the normal control and blank wells, 1ml of medium containing 1. mu.g/ml lipopolysaccharide was added to the model and sample wells, incubated for 24h, and the cell culture medium was collected from each well.
(3) Centrifuging the culture medium at 5000r/min for 5min, and collecting the supernatant to be tested.
(4) The nitric oxide concentration in each well was determined and calculated according to the instructions of the nitric oxide kit (beijing bi yunnan, S0021S).
The influence of brown algae polysaccharide, elderberry, pine pollen aqueous extract and pine pollen polysaccharide with the concentration of 1mg/ml on nitric oxide secretion of RAW264.7 cells is researched by taking the brown algae polysaccharide, the elderberry, the pine pollen aqueous extract and the pine pollen polysaccharide as samples, and the result is shown in figure 6.
As can be seen from FIG. 6, before lipopolysaccharide induces inflammatory reaction of RAW264.7 cells, the cells are pre-incubated for 24 hours by using 1mg/ml of brown alga polysaccharide, elderberry, pine pollen aqueous extract and pine pollen polysaccharide respectively, so that the generation amount of nitric oxide of RAW264.7 cells induced by lipopolysaccharide can be remarkably reduced, and the damage of high-concentration nitric oxide environment to the cells can be reduced. Among them, elderberry, pine pollen polysaccharide and brown algae polysaccharide have the most powerful reducing effect, but the effect is the weakest among 5 single components.
The influence of the combination of brown algae polysaccharide and elderberry in different ratios (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1) on the secretion of nitric oxide by RAW264.7 cells was studied by using samples, the total mass concentration of the single component (1:0, 0:1) and the composition (4:1, 2:1, 1:1, 1:2, 1:4) was 1mg/ml, and the experimental results are shown in FIG. 7.
As can be seen from fig. 7, each combination can reduce the content of nitric oxide secreted by the lipopolysaccharide-induced RAW264.7 cells, but the brown alga polysaccharide and elderberry composition has no obvious coordination effect on reducing the content of nitric oxide secreted by the lipopolysaccharide-induced RAW264.7 cells.
According to the results of reducing the level of nitric oxide secreted by lipopolysaccharide-induced RAW264.7 cells with two components, the combination 1 (brown alga polysaccharide: elderberry ═ 2:1) and the pine pollen aqueous extract and the pine pollen polysaccharide are combined in different ratios (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1) respectively to serve as a sample group to study the influence on the nitric oxide secreted by RAW264.7 cells, and the total mass concentration of the single component (1:0, 0:1) and the combination (4:1, 2:1, 1:1, 1:2, 1:4) is 1mg/ml, and the experimental results are shown in FIG. 8.
According to fig. 8, each combination can reduce the content of nitric oxide secreted by RAW264.7 cells induced by lipopolysaccharide, but the combination 1, the aqueous extract of pine pollen and the pine pollen polysaccharide have no obvious coordination effect on reducing the content of nitric oxide secreted by RAW264.7 cells induced by lipopolysaccharide.
6. Measurement of Natural killer cell (NK92 cell) killing Activity by CCK8 method
(1) Recovering and culturing NK92 cells (effector cells);
(2) recovering and culturing HepG2 cells (target cells);
(3) respectively preparing cell suspensions, and detecting the cell density;
(4) cells were seeded into 96-well plates, grouped: effector cell well, target cell well, normal control well [ also known as effective target cell well, effective target ratio 2:1, i.e. 2X 10 effector cells4(per well), 5X 105(per well), target cells 1X 104(each hole)]Sample wells (normal control wells + sample) and blank control wells (no cells). Slightly shaking and uniformly mixing 200 mu L of each pore system, and placing the mixture in a cell culture box for culturing for 24 hours;
(5) after the completion of the culture, the medium was discarded, and 100. mu.l of PBS buffer was added to each well and washed twice, and 110. mu.L of a reagent solution (medium: CCK8 reagent: 100:10) was added to each well, mixed well, and incubated for 2 hours.
(6) After the incubation is finished, measuring OD 450;
(7) the NK cell killing activity was calculated according to the following formula: natural killing activity (%) [ 1- (effective target cell pore OD value-effective cell pore OD value)/target cell pore OD value ] × 100%;
(8) the relative killing activity was calculated according to the following formula: relative killing activity (%) × 100% (sample well natural killing activity/normal well natural killing activity).
Brown algae polysaccharide and elderberry with a concentration of 0.5mg/ml were used as samples. NK92 cells have natural killing power on cancer cells mainly through granzyme-perforin mediated cytotoxicity, the survival rate of liver cancer cells HepG2 without and after treatment of NK92 cells is detected by a CCK8 method, and the natural killing activity of NK92 cells on the liver cancer cells is judged. Meanwhile, a part of NK92 cells are incubated in culture media containing different functional components for 24 hours in advance, the natural killing activity of the cells on HepG2 cells is measured, and the influence of each component on the natural killing activity of the NK92 cells is judged according to the result. The results are shown in FIG. 9.
As can be seen from fig. 9, brown algae polysaccharides had no effect on NK92 cell natural killer activity, compared to the normal group; elderberry significantly reduced the natural killing activity of NK92 cells against HegG2 cells.
The influence of the combination of fucoidan and elderberry in different ratios (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1) on NK92 cell natural killing activity was studied by using as samples, the total mass concentration of the single component (1:0, 0:1) and the composition (4:1, 2:1, 1:1, 1:2, 1:4) was 0.5mg/ml, and the experimental results are shown in Table 3.
As can be seen from Table 3, the combination of brown algae polysaccharide and elderberry can not improve the relative killing activity, and the brown algae polysaccharide and elderberry have no obvious coordination effect on the natural killing activity of NK92 cells.
TABLE 3 relative killing activity (%)
The influence of combination 1 (brown algae polysaccharide: elderberry ═ 2:1) and pine pollen aqueous extract and pine pollen polysaccharide in different ratios (1:0, 4:1, 2:1, 1:1, 1:2, 1:4, 0:1) respectively were taken as a sample group to study the natural killing activity of NK92 cells, the total mass concentration of monocomponent (1:0, 0:1) and combination (4:1, 2:1, 1:1, 1:2, 1:4) was 0.5mg/ml, and the experimental results are shown in Table 4.
From table 4, it can be seen that the pine pollen aqueous extract and the pine pollen polysaccharide have inhibitory effects on killing HepG2 cells by NK92 cells, and a multi-component combination formed by matching with other combinations still has inhibitory effects, but the inhibitory effects of the combinations are weakened compared with the inhibitory effects of the pine pollen aqueous extract and the pine pollen polysaccharide.
TABLE 4 relative killing activity (%)
According to the analysis results, the brown algae polysaccharide and the elderberry are in synergistic cooperation, and the phagocytic activity of macrophages can be remarkably enhanced by using the brown algae polysaccharide and the elderberry together; the brown algae polysaccharide, the elderberry and the pine pollen extract have the same synergistic cooperation, and the effect of enhancing macrophage phagocytosis activity is better when the brown algae polysaccharide, the elderberry and the pine pollen extract are used together compared with the brown algae polysaccharide and the elderberry.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A composition with the function of enhancing the phagocytic activity of macrophages comprises brown alga polysaccharides and elderberries.
2. The composition for enhancing phagocytic activity of macrophages according to claim 1, wherein the composition further comprises pine pollen extract;
preferably, the pine pollen extract comprises a pine pollen aqueous extract;
preferably, the pine pollen extract is pine pollen polysaccharide.
3. The composition for enhancing phagocytic activity of macrophages according to claim 2, wherein the preparation method of said aqueous extract of pine pollen comprises the following steps:
Mixing pollen Pini with water, and ultrasonically treating under heating to obtain the aqueous extract;
preferably, the mass ratio of the pollen pini to the water is 1 (8-12), preferably 1: 10;
preferably, the heating temperature is 25-45 ℃, and preferably 37 ℃;
preferably, the time of the ultrasonic treatment is 10-30 min, and preferably 20 min.
4. The composition with the function of enhancing the phagocytic activity of macrophages as claimed in claim 1, wherein the mass ratio of brown algae polysaccharide to elderberry in the composition is (1-4): 1.
5. The composition with the function of enhancing the phagocytic activity of macrophages according to claim 4, wherein the mass ratio of brown alga polysaccharides to elderberry in the composition is 2: 1.
6. The composition with the function of enhancing the phagocytic activity of macrophages as claimed in claim 2 or 3, wherein the mass ratio of brown algae polysaccharide to elderberry to pine pollen extract in the composition is (1-4) to 1 (3-6);
preferably, the mass ratio of the brown algae polysaccharide to the elderberry to the pine pollen extract in the composition is 2:1 (3-6).
7. The composition with the function of enhancing the phagocytic activity of macrophages according to claim 6, wherein the mass ratio of brown alga polysaccharides, elderberry and pine pollen extract in the composition is 2:1: 3;
The pine pollen extract is a pine pollen aqueous extract.
8. The composition with the function of enhancing the phagocytic activity of macrophages according to claim 6, wherein the mass ratio of brown alga polysaccharides, elderberry and pine pollen extract in the composition is 2:1: 6;
the pine pollen extract is pine pollen polysaccharide.
9. Use of the composition according to any one of claims 1 to 8 for enhancing phagocytic activity of macrophages in the preparation of an immunopharmaceutical.
10. An immunopharmaceutical comprising the composition of any one of claims 1 to 8 having a function of enhancing phagocytic activity of macrophages and an adjuvant.
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