CN114848711A - Pharmaceutical composition containing astragalus and glossy privet immunopotentiating particle material basis and preparation method and application thereof - Google Patents

Pharmaceutical composition containing astragalus and glossy privet immunopotentiating particle material basis and preparation method and application thereof Download PDF

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CN114848711A
CN114848711A CN202210662336.9A CN202210662336A CN114848711A CN 114848711 A CN114848711 A CN 114848711A CN 202210662336 A CN202210662336 A CN 202210662336A CN 114848711 A CN114848711 A CN 114848711A
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pharmaceutical composition
glossy privet
immunity
astragalus membranaceus
enhancing
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闫琰
卢迪
王方
孙悦
赵宝凯
齐六卫
王大成
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Shenyang Weijia Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of traditional Chinese veterinary medicines, and particularly relates to a pharmaceutical composition containing astragalus membranaceus and glossy privet immunopotentiating particles based on medicinal substances, and a preparation method and application thereof. The pharmaceutical composition comprises one or more of the following components: astragaloside IV, quercetin, lupeol, rutin, apigenin-7-O-beta-D-glucoside, salidroside or ligustric acid. The medicinal composition has definite medicinal effect substance basis, definite chemical structure, fixed components and controllable quality for enhancing the immunity of livestock and poultry, and lays a foundation for the safety and the effectiveness of the Chinese medicinal active ingredient composition.

Description

Pharmaceutical composition containing astragalus and glossy privet immunopotentiating particle material basis and preparation method and application thereof
Technical Field
The invention belongs to the technical field of traditional Chinese veterinary medicines, and particularly relates to a pharmaceutical composition containing astragalus membranaceus and glossy privet immunopotentiating particles based on medicinal substances, and a preparation method and application thereof.
Background
The astragalus membranaceus and glossy privet fruit immunity-enhancing granules are accepted in national standards, are veterinary drugs commonly used for clinically treating low immunity of livestock and poultry, and mainly comprise astragalus membranaceus, herba epimedii and glossy privet fruit, wherein the astragalus membranaceus is used as a main drug for tonifying spleen and lung, tonifying qi and strengthening exterior, the herba epimedii is used for tonifying kidney and yang, the glossy privet fruit is used for nourishing yin and tonifying kidney, nourishing liver and improving eyesight, and the astragalus membranaceus and glossy privet fruit immunity-enhancing granules are used as auxiliary drugs.
Currently, researchers have investigated the regulation effect of astragalus ligustrum immunopotentiating particles on normal mouse immune cytokines, and the results show that the astragalus ligustrum immunopotentiating particles can significantly improve the secretion of IL-2, IL-4, IFN-gamma and TNF-alpha in the mouse school district (see, for example, Lenzhui et al, "research on the regulation effect of two traditional Chinese medicine granules on normal mouse immune cytokines", Chinese veterinary medicine impurities, Vol 53, No 7, 7 months in 2019, p 56-60). That is, the function of qi zhen immunopotentiating granule for enhancing immunity has been confirmed by modern pharmacological methods.
However, at present, no relevant research aiming at the material basis of the immunopotentiation drug effect of the astragalus membranaceus and glossy privet immunopotentiation particles exists. The screening of the 'drug effect substance basis' removes ineffective and even harmful components in the traditional Chinese medicine and the compound thereof through the 'processes of coarse removal, fine extraction and false removal and false preservation', so that the traditional Chinese medicine has more prominent curative effect and lower toxic and side effects. The discovery of 'pharmacodynamic substance basis' ensures that the traditional Chinese medicine and the compound thereof can be safer, more effective and controllable in quality, ensures the medication safety of patients and maximizes the benefit of the medicine to the patients.
Therefore, it is very necessary to research the material basis of the immunopotentiation drug effect of the classical veterinary drug 'Qizhen immunopotentiation granule'.
Disclosure of Invention
The invention fills up the blank of the related technology in the prior art, and develops a pharmaceutical composition containing astragalus ligustrum lucidum immunopotentiating particles and a material basis of the immunopotentiating drug effect, a preparation method and application thereof.
Specifically, the invention is realized by the following technical schemes:
in a first aspect, the invention provides a pharmaceutical composition for enhancing livestock immunity, which contains a pharmaceutical effect substance basis of astragalus membranaceus-glossy privet immunity-enhancing particles, wherein the astragalus membranaceus-glossy privet immunity-enhancing particles are prepared from 190 parts by weight of astragalus membranaceus, 80-100 parts by weight of epimedium and 80-100 parts by weight of glossy privet fruit.
Optionally, in the pharmaceutical composition, the astragalus membranaceus and glossy privet fruit immunopotentiating particles are prepared from 180 parts by weight of astragalus membranaceus, 90 parts by weight of epimedium and 90 parts by weight of glossy privet fruit.
Optionally, in the above pharmaceutical composition, the pharmaceutical composition is an 80% ethanol elution part concentrate obtained by performing reflux extraction on the astragalus membranaceus and glossy privet fruit immunopotentiating particles with a 75% ethanol solution, performing vacuum concentration to obtain a thick extract, dispersing the total extract in water to a fully soluble state, passing through a D101 macroporous resin, and eluting with an 80% ethanol solution.
Alternatively, in the above pharmaceutical composition, the pharmaceutical composition comprises one or more of the following components: astragaloside IV, quercetin, lupeol, rutin, apigenin-7-O-beta-D-glucoside, salidroside or privet acid.
Alternatively, in the above pharmaceutical composition, the pharmaceutical composition consists of astragaloside, quercetin, lupeol, rutin, apigenin-7-O- β -D-glucoside, salidroside, and ligustric acid.
Optionally, in the above pharmaceutical composition, the pharmaceutical composition comprises, by weight, 1 to 20 parts of astragaloside iv, 0.5 to 5 parts of quercetin, 0.05 to 0.05 part of lupeol, 0.5 to 5 parts of rutin, 0.1 to 0.5 part of apigenin-7-O- β -D-glucoside, 3 to 10 parts of salidroside, and 0.1 to 0.5 part of ligustric acid.
In a second aspect, the present invention provides a process for preparing a pharmaceutical composition according to the first aspect, comprising the steps of:
and (2) carrying out reflux extraction on the astragalus membranaceus and glossy privet immunity-enhancing particles by using a 75% ethanol solution, carrying out reduced pressure concentration to obtain a thick extract, dispersing the total extract in water to a fully-dissolved state, passing through D101 macroporous resin, and eluting by using an 80% ethanol solution to obtain an 80% ethanol elution part concentrate, namely the pharmaceutical composition.
Alternatively, in the above production method, the reflux extraction is carried out three times, each for 2 hours, and the amount of the alcohol added in the step of dispersing water to a completely dissolved state is not more than 10% of the total system.
As an alternative mode, in the above preparation method, the feed-liquid ratio of the three reflux extractions is 1: 10. 1: 12. 1: 12.
in a third aspect, the present invention provides a use of the pharmaceutical composition according to the first aspect or the pharmaceutical composition prepared by the preparation method according to the second aspect in preparing a medicament for enhancing immunity of livestock and poultry.
Alternatively, in the above use, the dosage form of the drug is selected from: granule, oral liquid, powder or powder.
Preferably, the dosage form of the medicament is granules.
Alternatively, in the above use, the medicament is used for preventing or treating low immunity of the following livestock and poultry: chicken, duck, goose, pig, cattle or sheep.
Preferably, the medicine is used for preventing or treating low immunity of chickens.
Compared with the prior art, the invention has the following beneficial effects:
the invention firstly researches the material basis of the immunopotentiation drug effect of the 'Qizhen immunopotentiation particles', fills the technical blank of the related field, and develops a pharmaceutical composition containing the material basis of the immunopotentiation drug effect of the Qizhen immunopotentiation particles, a preparation method and application thereof. The medicinal composition has definite medicinal effect substance basis, definite chemical structure, fixed components and controllable quality for enhancing the immunity of livestock and poultry, and lays a foundation for the safety and the effectiveness of the Chinese medicinal active ingredient composition.
Drawings
FIG. 1 thin layer chromatography identification of control.
FIG. 2 dichloromethane: methanol 3: 1 part of the control was developed.
FIG. 3 dichloromethane: methanol 20: 1 part of the control was developed.
FIG. 4: the contrast medicinal materials of each extraction section and astragalus membranaceus and glossy privet immunity-enhancing particles are prepared by the following steps of mixing dichloromethane: methanol 3: 1 is unfolded.
In fig. 5, the ratio of each extraction section to the astragalus ligustrum lucidum immunopotentiating granule control medicinal material is as follows: methanol 5: 1 is unfolded.
In fig. 6, the ratio of each extraction section to the astragalus ligustrum lucidum immunopotentiating granule control medicinal material is as follows: methanol 3: 1 is unfolded.
FIG. 7 ear swelling rates of mice in each group.
FIG. 8 shows the contents of IL-2, IgG, IgM and IFN-. gamma.cytokines in the sera of the groups of mice.
FIG. 9MTT method for determining macrophage safety concentration of astragalus membranaceus-glossy privet immunopotentiating granule complete component.
FIG. 10 effect of different doses of LPS on the production of RAW264.7 macrophage inflammatory factor.
FIG. 11 shows the effect of the fraction and fraction of Qizhen immunopotentiating granule on the production of macrophage inflammatory factor induced by LPS.
Detailed Description
The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.
Example (b):
preparation of effective parts of various extracts of astragalus membranaceus and glossy privet fruit immunopotentiating particles
Taking 10g of 'Qizhen Zengyun granule' sample, carrying out reflux extraction with 75% ethanol solution for three times (material-liquid ratio is 1: 10, 1: 12, extraction time is 2h each time), decompressing, concentrating to obtain a thick extract, and weighing. Dispersing the total extract in water to completely dissolved state (the amount of alcohol added can not exceed 10% of the total system). Passing through D101 macroporous resin, eluting with ethanol solutions of different concentrations, collecting water fractions, respectively, concentrating 10%, 30%, 50%, 80%, and 95% of the eluted parts, volatilizing, and weighing for use.
The 'Qizhen immunity-enhancing granule' is produced by Shenyang Weijia biotechnology limited, and consists of 180kg of astragalus mongholicus and 90kg of glossy privet fruit, and the preparation process comprises the following steps of adding water, decocting for 3 times, merging decoction, filtering, concentrating filtrate into clear paste with the relative density of 1.30-1.35(80 ℃), adding 3 parts of cane sugar and 1 part of dextrin according to the amount of the clear paste, uniformly mixing, granulating, and drying at low temperature to prepare 300kg of granules.
2. Thin-layer chromatographic analysis of chemical components of astragalus membranaceus and glossy privet immunity-enhancing particle split component
2.1 Experimental methods
According to the data, the main components of the astragaloside triterpenoid saponins are astragaloside IV and oleanolic acid, and the astragaloside triterpenoid saponins contain quercetin and acetyl oleanolic acid, but not the main components. The main components of fructus Ligustri Lucidi triterpenes are oleanolic acid and ursolic acid, the main components of iridoid are salidroside, and rutin, luteolin, privet acid, lupeol, and apigenin-7-O-beta-D-glucoside are all non-main components of fructus Ligustri Lucidi.
Respectively sucking the reference medicinal material solution and each section of sample solution, respectively dropping on the same self-made silica gel G thin-layer chromatography plate, developing with dichloromethane-methanol as developing agent, taking out, air drying, inspecting under 254nm ultraviolet lamp, and spraying 5% vanillin sulfuric acid solution for observation.
2.2 results of the experiment
The total extract obtained by alcohol extraction is 7.4023g, and the extraction rate is 74%. 10%, 30%, 50%, 80% and 95% ethanol eluate, concentrating under reduced pressure, and volatilizing to obtain 309.2mg, 273.6mg, 369.9mg, 222.8mg and 170.2mg respectively, wherein water is not volatilized.
The ratio of developing agent is dichloromethane: methanol 5: thin-layer identification was performed on 1 of the 11 controls, and the results are shown in FIG. 1. As can be seen from the figure, the Rf values of apigenin-7-O-beta-D-glucoside, salidroside and ligustric acid are between 0.3 and 0.5, so the proportion of the selected developing agent is that dichloromethane: methanol 5: 1. the results of multiple trials of other control samples to find the optimum ratio of developing solvent are shown in FIGS. 2-3. Astragaloside, quercetin, rutin in dichloromethane: methanol 3: 1, and Rf is about 0.4. Ursolic acid, oleanolic acid, acetyl oleanolic acid, lupeol, luteolin in the ratio of dichloromethane: methanol 20: 1, and Rf is about 0.5.
Astragaloside, quercetin, rutin and the extraction stages are mixed with dichloromethane: methanol 3: 1 spreading agent ratio. As is presumed from FIG. 4, the ethyl acetate layer, 80% ethanol stage, may contain astragaloside IV; the ethyl acetate layer, n-butanol layer, and 80% section may contain quercetin and rutin. apigenin-7-O-beta-D-glucoside, salidroside, ligustric acid and all extraction segments are mixed with dichloromethane: methanol 5: 1 spreading agent ratio. As is presumed from FIG. 5, the n-butanol layer, the 30% ethanol layer, the 50% ethanol layer, and the 80% ethanol layer may contain apigenin-7-O-beta-D-glucoside, salidroside, and ligustric acid. Ursolic acid, oleanolic acid, acetyl oleanolic acid, lupeol, luteolin and the respective extraction fractions are extracted with dichloromethane: methanol 20: 1 spreading agent ratio. It is speculated from FIG. 6 that the ethyl acetate layer, 95% ethanol stage, may contain acetyl oleanolic acid; the n-butanol layer and 80% ethanol section may contain lupeol; the ethyl acetate layer may contain oleanolic acid and ursolic acid; the 95% ethanol fraction and n-butanol layer may contain luteolin.
The thin-layer chromatography analysis result of chemical components of each split component shows that: the 30% ethanol section and the 50% ethanol section may contain apigenin-7-O-beta-D-glucoside, salidroside, and ligustric acid; the 80% ethanol phase may contain astragaloside IV, quercetin, lupeol, rutin, apigenin-7-O-beta-D-glucoside, salidroside and privet acid. The 95% ethanol stage may contain acetyl oleanolic acid.
3. Experiment for influence of astragalus membranaceus and glossy privet immunopotentiating particle split component on immune function of mice with low immunity
3.1 materials
3.1.1 drugs and reagents
Cyclophosphamide, sterile normal saline, india ink, 1% sodium carbonate solution; 2, 4-dinitrofluorobenzene; IL-2, IgG, IgM, IFN-gamma cytokine kit.
Astragalus membranaceus-glossy privet immunopotentiating granule extract and split components (30%, 50% and 80% which are prepared by the method in the part 1).
3.1.2 test animals
ICR mice 84, 20. + -.2 g, male and female halves.
3.2 methods
3.2.1 Effect on non-specific immune function in mice (carbon particle size clearance method)
Grouping: the 48 mice were randomly divided into 6 groups of 8 mice each, each group being: blank control group (CT), model control group (M), full component group (Q), 30% group (Q30), 50% group (Q50), 80% group (Q80).
Administration and molding: except for the blank control group and the model control group, the other groups were administered by gavage daily at the corresponding doses in table 1 (table 1), and were administered for 4 days continuously. Except for the blank control group, 5 groups were intraperitoneally administered with 100mg/kg of cyclophosphamide on the 2 nd and 4 th days of the experiment.
On test day 5, 200 μ L of 25% India ink was injected into the tail vein, 2min (t) after injection 1 )、15min(t 2 ) 20 μ L of blood was drawn from the angular (orbital) venous plexus by capillary puncture and immediately added to 2.0mL of 0.1% sodium carbonate solution, and after thoroughly shaking it was measured for Optical Density (OD) at 600 nm. Neck of mouseAnd (4) performing dislocation and killing, and weighing the mass of the liver and the mass of the spleen respectively. The clearance index K and the corrected clearance index (phagocytosis index) alpha are calculated according to the following formulas.
Figure BDA0003691342040000081
TABLE 1 CARBON CLEANING TEST ANIMAL ADMINISTRATION DOMETER
Figure BDA0003691342040000082
Figure BDA0003691342040000091
3.2.2 detection of the Effect on mouse cellular Immunity (delayed hypersensitivity DTH)
The 36 mice were randomly divided into 6 groups of 6 mice each, which were: blank Control (CT), model control (M), full component (Q), 30% group (Q30), 50% group (Q50), 80% (Q80). The dosage administered is shown in table 2. The administration was continued for 7 days. Except for the blank control group, 5 groups were intraperitoneally administered with 100mg/kg of cyclophosphamide on the 4 th and 6 th days of the test.
TABLE 2 animal dosage table for cellular immunity test
Figure BDA0003691342040000092
Each group of mice was evenly smeared with 50. mu.L of 1% Dinitrofluorobenzene (DNFB) on the abdominal skin from the 2 nd day of the experiment for 2 consecutive days. On the 7 th day of the test, 10 mu L of 1% dinitrofluorobenzene is uniformly coated on the back side of the right ear of the mouse for antigen excitation, the eyeball is removed after 24h to take blood, the neck is removed to be killed, the left ear and the right ear are cut off, the ear pieces are punched at the same positions of the two ears by a 6mm puncher and weighed, and the weight difference of the left ear and the right ear is the swelling degree of the ears. And (3) measuring the contents of IL-2, IgG, IgM and IFN-gamma in the serum by an ELISA method. Meanwhile, thymus and spleen of the mice are picked, accurately weighed, and spleen index and thymus index are calculated according to the following formulas.
Swelling degree-right ear quality-left ear quality
Swelling rate ═ right ear-left ear)/left ear × 100%
3.3 results
3.3.1 Effect of drugs on mouse organ index
The organ index changes of each group of mice are shown in table 3. Compared with the blank control group, the spleen index and the thymus index of the model group are extremely lower than those of the blank control group, which indicates that the cyclophosphamide-induced mice are successfully immunized in the low-immunity model. Compared with the model group, the organ indexes of each group administrated after the model building have no obvious difference with the model control group, which indicates that the medicines of each group can not improve the mice immune organ atrophy caused by cyclophosphamide.
TABLE 3 Effect of drugs on organ index in immunosuppressed mice
Figure BDA0003691342040000101
Figure BDA0003691342040000102
Note: compared with the blank control group, the composition of the composition, # P＜0.05， ## p is less than 0.01; p < 0.05, P < 0.01 compared to model control.
3.3.2 Effect of drugs on non-specific immune function in mice
The results of the drug on mouse nonspecific immune function (carbon clearance test) are shown in table 4. The results show that compared with the blank control group, the clearance index and the phagocytosis index of the model control group are extremely obviously lower than those of the blank control group, which indicates that the cyclophosphamide molding is successful. Compared with the model control group, the clearance index and the phagocytic index of the astragalus ligustrum lucidum immunopotentiating particle complete component group are higher than those of the model control group, which indicates that the astragalus ligustrum immunopotentiating particle complete component can improve the phagocytic function of the body of the hypoimmunity mouse. Among the three split fractions, the clearance index and phagocytosis index were significantly higher for 50% and 80% of the fractions than for 30% of the fractions and were close to the full fraction.
TABLE 4 drug pair immunosuppressionInfluence of mouse phagocytic function
Figure BDA0003691342040000103
Figure BDA0003691342040000104
Figure BDA0003691342040000111
Note: compared with the blank control group, the composition of the composition, # P＜0.05， ## p is less than 0.01; p < 0.05, P < 0.01 compared to model control.
3.3.3 Effect of drugs on immune function of mouse cells
The ear swelling rate of each group of mice is shown in fig. 7. Compared with the blank group (CT), the swelling rate of the model group (M) is obviously lower than that of the blank group (CT), which indicates that the cyclophosphamide caused immune hypofunction model is successful. Compared with the model group, the astragalus ligustrum immunopotentiating granule complete component group (Q) significantly reduces the ear swelling rate of mice, while 30% of the three split components (Q30) and 50% of the three split components (Q50) both showed the effect of reducing the ear swelling rate of mice, wherein the ear swelling rate of 50% of the three split components is close to that of the complete component group.
The serum contents of IL-2, IgG, IgM and IFN-gamma cytokines in each group of mice are shown in FIG. 8. The content of IL-2 and IgM is not obviously different among groups; the IgG and IFN-gamma model groups are lower than the normal group, which shows that the hypoimmunity caused by the cyclophosphamide can reduce the levels of IgG and IFN-gamma cytokines of the body; compared with the model group, IgG and IFN-gamma of the complete component group of the astragalus glossy privet immunity-enhancing particles are higher than those of the model group, which indicates that the astragalus glossy privet immunity-enhancing particles can improve the levels of the two cytokines of the mice with low immunity; among the fractions, IFN-. gamma.showed no significant difference in the results for all three fractions and no effect of increasing IFN-. gamma.whereas IgG showed no effect of increasing IgG levels in all three fractions, with 50% and 80% of the IgG levels being higher than 30% of the fractions.
3.4 conclusion
The astragalus membranaceus and glossy privet immunity-enhancing particle full components can improve the phagocytic function of a low-immunity mouse body, and correspond to the early-stage test result. Of the resolved fractions, 50% and 80% had higher phagocytic indices. The astragalus membranaceus and glossy privet immunity-enhancing particle full components can relieve the ear swelling degree of a mouse in a cell immune function test, and correspond to an early test result. The ear swelling rate in 50% of the split fractions was closest to the full range of the fractions, indicating that 50% of the fractions may contain active ingredients that inhibit the inflammatory response. The astragalus membranaceus and glossy privet immunopotentiating particles can improve the levels of IgG and IFN-gamma cytokines of an immunocompromised organism, and correspond to early results. Of the resolved fractions, 50% and 80% of the groups were able to significantly increase IgG levels.
4. Influence of astragalus membranaceus and glossy privet immunopotentiating particle split component on LPS-induced TNF-alpha inflammatory factor production of RAW macrophage
4.1 materials
RAW264.7 macrophage, LPS, TNF-alpha cytokine ELISA kit, DMEM culture solution and fetal calf serum. Astragalus membranaceus-glossy privet immunity-enhancing granules are divided into components (30%, 50% and 80%, and the astragalus membranaceus-glossy privet immunity-enhancing granules are prepared by the method in the part 1).
4.2 methods
4.2.1 determination of the safe concentration of drug on RAW246.7 macrophage
Adjusting RAW246.7 macrophage to 1 × 10 5 Adding 100 mu L of each 96-well plate per well, adding 100 mu L of 9-concentration astragalus membranaceus immunopotentiating particles (7.81-2000 mu g/mL) per well after overnight adherence, repeating the steps of 4-well each concentration, 37 ℃ and 5% CO 2 After 20h of culture, adding 20 mu L of MTT into each hole, after continuing to culture for 4h, adding 100 mu L of lysate DMSO into each hole, placing the cell plate on a micro-oscillator to shake so as to completely dissolve the cell plate, and detecting the light absorption value of the cell plate at 490nm on an enzyme-linked immunosorbent assay. The maximum safe concentration of the drug was selected as the maximum concentration at which the OD was not significantly lower than that of the control group of cells.
4.2.2 Effect of different doses of LPS on RAW246.7 macrophage inflammatory factor production
Adjusting RAW246.7 macrophage to 3 × 10 5 Per/mL, 100. mu.L per well in a 96-well plate, 4 concentrations of LPS (1, 2, 5, 10. mu.g/mL), 10. mu.L/well, 2 wells at 37 ℃ in duplicate, 5% CO per concentration 2 After 24h of culture, the supernatant was collected, and TNF-. alpha.content was measured using ELISA kit to compare the effect of LPS stimulation with different gradients.
4.2.3 Effect of drugs and resolved fractions on LPS-induced TNF- α inflammatory factor production from RAW macrophages
Adjusting RAW246.7 macrophage to 5 × 10 5 And each of the particles is added with the total component of the astragalus ligustrum immunopotentiating particles with the concentration of 1000 mug/mL and 100 mug/hole of the split component (30%, 50% and 80%) with the corresponding dose after the particles are attached to the wall, each concentration is repeated for 4 holes, LPS with the final concentration of 1 mug/mL is added for induction for 24 hours after incubation for 4 hours, and a blank group and a model group are set. The supernatants were collected and assayed for TNF-. alpha.content using an ELISA kit.
4.3 results
4.3.1 determination of the safe concentration of RAW246.7 macrophage by drug
The results of the drug versus safe concentration of RAW246.7 macrophages are shown in fig. 9. The results show that the total component of the astragalus membranaceus immunopotentiating particles has low cytotoxicity in the concentration range of 7.81-2000 mu g/mL, and has a certain stimulation effect on the proliferation of macrophages when the concentration is 500-1000 mu g/mL. The maximum safe concentration of the astragalus membranaceus and glossy privet immunity-enhancing particles is 2000 mug/mL.
4.3.2 Effect of different doses of LPS on the production of RAW264.7 macrophage inflammatory factor
The effect of different doses of LPS on RAW264.7 macrophage inflammatory factor production results are shown in figure 10. As a result, there was no significant difference in the TNF-. alpha.content in the cell supernatants after stimulation with four concentrations of LPS.
4.3.3 Effect of Qizhen immunopotentiating granule and resolved fraction on LPS-induced TNF-alpha inflammatory factor production from RAW macrophage
The effect of the astragalus membranaceus immunopotentiating granules and the fraction thereof on the production of macrophage inflammatory factor induced by LPS is shown in fig. 11. Compared with the blank group (CT), the content of TNF-alpha in the model group (MT) can be obviously improved after 24 LPS stimulation, and the total components of the astragalus glossy privet immunopotentiating particles can further improve the level of TNF-alpha in cell supernatant. Wherein 80% of the astragalus membranaceus immunopotentiating granule split component has higher TNF-alpha level than the other two split components and is close to the complete component group.
4.4 conclusion
The maximum safe concentration of the astragalus membranaceus immune enhancing particles on RAW macrophages obtained by the test result is 2000 mug/mL, and the astragalus membranaceus immune enhancing particles have a certain stimulation effect on the proliferation of the macrophages within the concentration range of 500-1000 mug/mL, so that 1000 mug/mL is selected as the reference dose of the whole components of the astragalus membranaceus immune enhancing particles in the next test, and the corresponding split component dose is calculated on the basis of the reference dose. Finally, the test result shows that 80% of the astragalus ligustrum immunopotentiating particle split components can obviously improve the TNF-alpha expression of cells after being stimulated by LPS. In order to find out the optimal stimulation concentration of LPS on RAW264.7 macrophage within 24h, the experiment carries out gradient investigation on the stimulation dose of LPS before the experiment for splitting the components, and as a result, the content of TNF-alpha in cell supernatant can not be obviously influenced by increasing the concentration of LPS, and finally 1 mug/mL is selected as the stimulation concentration of LPS in the formal experiment. The 80% of the astragalus membranaceus immunopotentiating granule split component has higher TNF-alpha level than the other two split components, and is close to the complete component.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (10)

1. A pharmaceutical composition for enhancing immunity of livestock and poultry is characterized in that: the pharmaceutical composition contains a material basis of an immunopotentiation drug effect of astragalus membranaceus-glossy privet fruit immunity-enhancing particle, wherein the astragalus membranaceus-glossy privet fruit immunity-enhancing particle is prepared from 190 parts of astragalus membranaceus, 80-100 parts of epimedium and 80-100 parts of glossy privet fruit by weight.
2. The pharmaceutical composition of claim 1, wherein: the astragalus membranaceus and glossy privet immunity-enhancing particle is prepared from 180 parts of astragalus membranaceus, 90 parts of epimedium and 90 parts of glossy privet fruit in parts by weight.
3. The pharmaceutical composition according to claim 1 or claim 2, wherein: the pharmaceutical composition is 80% ethanol elution part concentrate obtained by performing reflux extraction on the astragalus membranaceus and glossy privet immunity-enhancing particles by using a 75% ethanol solution, performing reduced pressure concentration to obtain a thick extract, dispersing the total extract in water to a full-dissolved state, passing through D101 macroporous resin, and eluting by using an 80% ethanol solution.
4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that: the pharmaceutical composition comprises one or more of the following components: astragaloside IV, quercetin, lupeol, rutin, apigenin-7-O-beta-D-glucoside, salidroside or privet acid.
5. A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 4, characterized in that: the method comprises the following steps:
and (2) carrying out reflux extraction on the astragalus membranaceus and glossy privet immunity-enhancing particles by using a 75% ethanol solution, carrying out reduced pressure concentration to obtain a thick extract, dispersing the total extract in water to a fully-dissolved state, passing through D101 macroporous resin, and eluting by using an 80% ethanol solution to obtain an 80% ethanol elution part concentrate, namely the pharmaceutical composition.
6. The method of claim 5, wherein: the reflux extraction is carried out three times, each time for 2 hours, and the amount of alcohol added in the step of dispersing water to a full-dissolved state is not more than 10 percent of the total system.
7. The method of claim 6, wherein: the material-liquid ratio of the three reflux extractions is 1: 10. 1: 12. 1: 12.
8. use of the pharmaceutical composition of any one of claims 1 to 4 or the pharmaceutical composition prepared by the preparation method of any one of claims 5 to 7 in the preparation of a medicament for enhancing the immunity of livestock and poultry.
9. Use according to claim 8, characterized in that: the dosage form of the medicament is selected from: granule, oral liquid, powder or powder.
10. The use according to claim 8 or claim 9, the medicament is for preventing or treating hypoimmunity in livestock and poultry: chicken, duck, goose, pig, cattle or sheep.
CN202210662336.9A 2022-06-13 2022-06-13 Pharmaceutical composition containing astragalus and glossy privet immunopotentiating particle material basis and preparation method and application thereof Pending CN114848711A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101647852A (en) * 2009-08-28 2010-02-17 缪雪荣 Veterinary medicine for enhancing immunologic function
CN101884788A (en) * 2010-06-28 2010-11-17 南京农业大学 Traditional Chinese medicine astragalus polysaccharide immunopotentiator
CN107041902A (en) * 2016-10-31 2017-08-15 四川美嘉龙生物科技有限公司 A kind of stilbene chastity, which increases, exempts from particle and preparation technology
CN110882304A (en) * 2019-12-02 2020-03-17 广东容大生物股份有限公司 Method for preparing astragalus root and glossy privet fruit medicine for livestock and poultry

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101647852A (en) * 2009-08-28 2010-02-17 缪雪荣 Veterinary medicine for enhancing immunologic function
CN101884788A (en) * 2010-06-28 2010-11-17 南京农业大学 Traditional Chinese medicine astragalus polysaccharide immunopotentiator
CN107041902A (en) * 2016-10-31 2017-08-15 四川美嘉龙生物科技有限公司 A kind of stilbene chastity, which increases, exempts from particle and preparation technology
CN110882304A (en) * 2019-12-02 2020-03-17 广东容大生物股份有限公司 Method for preparing astragalus root and glossy privet fruit medicine for livestock and poultry

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