Radix astragali and ganoderma lucidum preparation for enhancing immune response of Newcastle vaccine and preparation method of radix astragali and ganoderma lucidum preparation

Abstract

The invention provides a radix astragali and ganoderma lucidum preparation for enhancing the immune response of a Newcastle vaccine and a preparation method thereof, and belongs to the technical field of veterinary drugs. The preparation method comprises the following steps: immersing pretreated radix astragali and lucid ganoderma raw material powder in an extraction solvent, uniformly mixing, heating to 40-50 DEG C, adjusting the pH value to 3-5, adding pectinase and cellulase, and carrying out first enzymolysis under water bath heat preservation and continuous stirring; adjusting the pH value to be 3-5, controlling the temperature to be 40-60 DEG C, adding acid protease, and carrying out second enzymolysis under water bath heat preservation and continuous stirring; filtering, centrifuging filtrate, and concentrating supernate to obtain a radix astragali and ganoderma lucidum preparation; the extraction solvent comprises a citric acid-sodium citrate solution and at least one monosaccharide. The composite solution composed of the citric acid-sodium citrate solution and the monosaccharide is used as the extraction solvent of the astragalus membranaceus-ganoderma lucidum preparation, the enzymolysis efficiency is greatly improved, the enzymolysis time is shortened, meanwhile, the extraction amount of active ingredients of medicinal materials is increased, and the drug effect of the astragalus membranaceus-ganoderma lucidum preparation in newcastle vaccine immune response is enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drugs, and particularly relates to an Qizhi preparation for enhancing the immune response of a Newcastle vaccine and a preparation method thereof. Background Art

[0002] Newcastle disease is a highly pathogenic infectious disease caused by the Newcastle disease virus, which mainly affects poultry as well as wild and ornamental birds. At present, chickens are the poultry most widely affected by Newcastle disease. Its main symptoms include dyspnea and dysentery, accompanied by neurological symptoms, mucosal bleeding and other symptoms, which can lead to a sharp drop in egg production in laying hens. At present, the most effective measure to prevent and control Newcastle disease is to vaccinate with Newcastle disease vaccines, which are widely administered by intramuscular injection, nasal drops, chicken embryo immunization and spraying. However, traditional vaccine immunization methods have problems such as inaccurate intake dosage, difficult operation, and easy to cause stress response, thereby affecting the immune effect. Among Newcastle disease vaccines, live vaccines are the most widely used, which can produce immunity quickly, but they are highly toxic to chicks, and are prone to symptoms and even death, so adjuvants are often used to improve immune response during vaccination.

[0003] Qizhi oral liquid is a preparation that is currently widely used in the market. Its main ingredients are astragalus extract and ganoderma extract. Since both are plant extracts, they have the advantages of being natural, low in toxicity, easy to metabolize and having low immunogenicity, and have become a type of adjuvant that has attracted much attention among researchers.

[0004] At present, the standard preparation method of Qizhi oral liquid is: add 13 times water to Astragalus and Ganoderma lucidum, heat and reflux to extract 3 times, 3 hours each time, filter and concentrate the filtrate at 60°C to a solid-liquid ratio of 1:1, add ethanol to the alcohol content of 75%, let it stand, separate the precipitate, take the precipitate, add water to dissolve it, add the preservative sodium benzoate to mix, add water to make up the volume, then fill and sterilize to obtain Qizhi oral liquid.

[0005] In the existing Qizhi preparations, astragalus and ganoderma are extracted by reflux water, and their main components are astragalus polysaccharides and ganoderma polysaccharides. In fact, astragalus and ganoderma also contain other components that promote immune response, such as flavonoids, ganoderic acid and other substances. The existing preparation method has caused a large amount of waste of effective active ingredients; or the effective ingredients are extracted by enzymatic hydrolysis. Although this method is safe and green, each enzymatic hydrolysis takes more than several hours, which is inefficient and time-consuming.

[0006] Conventional extraction processes require the assistance of microwaves, ultrasound, etc. combined with organic extraction solvents. Although the extraction efficiency has been improved to a certain extent, microwave and ultrasonic operations are difficult to control and can easily cause ketone components to be destroyed or decomposed, or enzymes to be inactivated. Existing extraction solvents (such as water and alcohols) have limited solubility in the extracts, which inhibits the precipitation efficiency of the effective ingredients in the solvent. In addition, conventional organic solvents (such as methanol and ethanol) will also remain, affecting actual use, and require complex purification steps. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a Qizhi preparation and a preparation method thereof for enhancing the immune response of the Newcastle vaccine, aiming to solve at least one technical problem in the background technology.

[0008] The present invention is achieved in that:

[0009] The first aspect of the present invention provides a method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine, wherein the raw materials of the Qizhi preparation include astragalus and ganoderma lucidum, with a weight ratio of astragalus to ganoderma lucidum = 1:1.5-3;

[0010] The preparation method comprises the following steps:

[0011] The pretreated raw material powder is immersed in the extraction solvent and mixed evenly, and heated to 40°C to 50°C, the pH is adjusted to 3 to 5, pectinase and cellulase are added, and the first enzymolysis is performed under water bath insulation and continuous stirring;

[0012] After the first enzymatic hydrolysis is completed, the pH is adjusted to 3-5, the temperature is controlled at 40°C-60°C, acidic protease is added, and the second enzymatic hydrolysis is performed under water bath insulation and continuous stirring;

[0013] After the second enzymatic hydrolysis is completed, the filtrate is filtered, the filtrate is centrifuged, and the supernatant is concentrated to obtain the Qizhi preparation;

[0014] Wherein, the extraction solvent comprises citric acid-sodium citrate solution and at least one monosaccharide.

[0015] Furthermore, the mass ratio of the pectinase to the cellulase is 1:1-3, and the total dosage is 0.1%-0.5% of the mass of the raw material powder; the first enzymolysis time is 20min-40min;

[0016] The dosage of the acidic protease is 0.1% to 1% of the mass of the raw material powder, and the second enzymolysis time is 30min to 60min.

[0017] Furthermore, the pretreatment steps of the raw material powder are:

[0018] Astragalus and Ganoderma lucidum are made into fine powders of 30 μm to 50 μm;

[0019] Cool down to below -20℃ and keep in the freezer for more than 2h.

[0020] Furthermore, the heating of the first enzymatic hydrolysis adopts microwave-assisted water bath heating, specifically: low-power time-controlled heating is performed during water bath heating, the microwave power is 300W-500W, and the microwave time does not exceed 4 minutes.

[0021] Furthermore, during the second enzymatic hydrolysis, microwave treatment is added, the microwave power is 200W to 300W, and the microwave time is 0.5min to 1min.

[0022] Furthermore, the liquid-to-solid ratio of the extraction solvent to the raw material powder is 10:1 to 30:1.

[0023] Furthermore, the monosaccharide is selected from fructose and / or glucose; the amount of the monosaccharide is 0.1% to 0.5% of the mass of the raw material powder.

[0024] Furthermore, during the first enzymolysis and the second enzymolysis, the pH is adjusted by adding citric acid or sodium citrate.

[0025] The second aspect of the present invention provides a Qizhi preparation for enhancing the immune response of the Newcastle vaccine prepared by the above preparation method.

[0026] Furthermore, the Qizhi preparation comprises extracts of both Astragalus and Ganoderma, citric acid-sodium citrate solution and at least one monosaccharide.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention provides a composite solution composed of citric acid-sodium citrate solution and monosaccharide as an extraction solvent for Qizhi preparations, which replaces conventional extractants such as ethanol, methanol, ethers, etc., greatly improves the enzymatic hydrolysis efficiency, reduces the enzymatic hydrolysis time, and at the same time increases the extraction amount of active ingredients of medicinal materials, thereby enhancing the efficacy of Qizhi preparations in the immune response of Newcastle vaccine.

[0029] 2. The present invention optimizes the medicinal material pretreatment, enzymatic hydrolysis steps and microwave synergistic parameters, thereby promoting the release of active components in the medicinal materials. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation cases described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] A method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine, comprising the following steps:

[0032] S1, pretreatment;

[0033] Weigh the raw materials of astragalus and ganoderma lucidum, according to the weight ratio of astragalus: ganoderma lucidum = 1:1.5-3; for example, it can be 1:1.5, 1:2, 1:3; but it is not limited to the listed values. The following embodiment uses 1:2 as an example, and other values ​​not listed in the numerical range are also applicable.

[0034] Astragalus and Ganoderma lucidum are made into fine powders of 30 μm to 50 μm, which can be ground separately or mixed;

[0035] The fine powder is quickly frozen to below -20°C and kept frozen for more than 2 hours.

[0036] Studies have shown that freezing Astragalus and Ganoderma powder can destroy the cell walls, promote the precipitation of effective ingredients during subsequent enzymatic hydrolysis, and thus reduce the time of subsequent enzymatic hydrolysis.

[0037] S2, first enzymatic hydrolysis;

[0038] A certain amount of monosaccharide is added to a citric acid-sodium citrate buffer solution, and the mixture is mixed and stirred to prepare an extraction solvent;

[0039] The raw material powder pretreated by S1 is immersed in the extraction solvent and mixed evenly, and heated to 40°C to 50°C, and microwave treatment is performed during the heating process; the pH of the reaction system is measured, and if the value is within the range of 3 to 5, no adjustment is required; if it is less than 3, a certain amount of sodium citrate solution is added to adjust the pH to 3 to 5; if it is greater than 5, a certain amount of citric acid solution is added to adjust the pH to 3 to 5, and then pectinase and cellulase are added, and the first enzymolysis is performed under water bath insulation and continuous stirring;

[0040] In a specific implementation, the liquid-to-solid ratio of the extraction solvent to the raw material powder is 10:1 to 30:1, for example, 10:1, 20:1, 30:1; but it is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable;

[0041] The monosaccharide is selected from fructose and / or glucose, and the monosaccharide is 0.1% to 0.5% by mass of the raw material powder, for example, it can be 0.1%, 0.2%, 0.4%, 0.5%; but it is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable;

[0042] The mass ratio of pectinase to cellulase is 1:1-3, and the total amount is 0.1%-0.5% of the mass of the raw material powder;

[0043] The time of the first enzymatic hydrolysis is 20 min to 40 min, for example, 20 min, 30 min, 40 min; but it is not limited to the listed values, and other values ​​not listed in the numerical range are also applicable;

[0044] The microwave power is 300W to 500W, and the microwave time does not exceed 4 minutes (preferably 1 minute to 4 minutes). The effects of microwave assisted water bath heating are: (1) reducing the heating time. Since the raw material powder is a frozen powder below -20°C, after it is mixed with the extraction solvent, the low temperature is not conducive to the dispersion and homogenization of the powder. Short-term microwave treatment can quickly heat up the reaction system; (2) Microwave radiation causes the water molecules in the medicinal material cells to absorb microwave energy, promote the rapid rise in intracellular temperature, break through the cell membrane and cell wall, and further improve the release of effective components in the medicinal material cells; (3) The power of microwave treatment is controlled below 500W to avoid excessively high power microwave treatment that promotes the decomposition of components such as ketones and causes component damage.

[0045] Advantages of setting the extraction solvent: (1) Compared with conventional water, ethanol, methanol, etc., the present invention uses a composite solution of citric acid, sodium citrate and monosaccharides as the extraction solvent. During the extraction process, the polysaccharides, ganoderma acid, flavonoids and other substances precipitated from Astragalus and Ganoderma have better solubility, thereby promoting the precipitation of effective components and improving the extraction capacity; (2) Citric acid, sodium citrate and monosaccharides are green and non-toxic, safer and more environmentally friendly, and their residual storage after extraction is safe and harmless to the efficacy; (3) Citric acid-sodium citrate is a buffer solution. During the enzymatic hydrolysis process, citric acid or sodium citrate can be directly added to adjust the pH without introducing new substances (such as hydrochloric acid, sodium hydroxide, etc.), which is safer and more reliable; (4) Citric acid, sodium citrate and monosaccharides can all be used as food / drug additives, which promote the efficacy of the drug. For example, citric acid can not only play a coordination role, allowing poultry to absorb a variety of essential elements, but also reduce the gastrointestinal pH value. Monosaccharides can improve the taste of the drug. Therefore, the present invention does not need to remove impurities from citric acid, sodium citrate and monosaccharides after the enzymatic hydrolysis, thereby simplifying the process steps.

[0046] In the specific implementation, the enzymatic hydrolysis of pectinase and cellulase can decompose the pectin and cellulose in the cell wall, thereby destroying the cell wall of the medicinal material, which is conducive to the precipitation of the effective ingredients in the subsequent protease hydrolysis. At the same time, sugars and the like will be produced during the first enzymatic hydrolysis process, thereby increasing the monosaccharide content in the extraction solvent, reducing the original amount of monosaccharides used, and reducing costs.

[0047] S3, second enzymatic hydrolysis;

[0048] After the first enzymatic hydrolysis is completed, since new products appear in the reaction system during the first enzymatic hydrolysis, the internal pH may change slightly, and the pH needs to be adjusted; first quickly measure the pH of the reaction system, if the value is within the range of 3 to 5, no adjustment is required; if it is less than 3, add a certain amount of sodium citrate solution to adjust the pH to 3 to 5; if it is greater than 5, add a certain amount of citric acid solution to adjust the pH to 3 to 5; and control the temperature to 40°C to 60°C, add acidic protease, and perform the second enzymatic hydrolysis under water bath insulation and continuous stirring, and microwave treatment for tens of seconds during the second enzymatic hydrolysis;

[0049] In a specific implementation, the amount of acidic protease used is 0.1% to 1% of the mass of the raw material powder, and the second enzymolysis time is 30 min to 60 min;

[0050] The chemical composition of plant cell walls is pectin and cellulose, and the main components of cell membranes are phospholipids and protein molecules, which can be decomposed under the action of hydrolases, thereby destroying the structure of cells; in specific implementation, acidic proteases decompose cell membranes and promote the precipitation of polysaccharides, flavonoids, etc. in cells; this step actually undergoes multi-level enzymolysis, and since the enzyme is not inactivated after the first enzymolysis is completed, and the environment of the second enzymolysis is not much different from that of the first enzymolysis, the enzymolysis of pectinase, cellulase, and acidic protease is performed simultaneously during the second enzymolysis;

[0051] In a specific implementation, the microwave power is 200W to 300W, and the microwave time is 0.5min to 1min. The microwave treatment is to enhance the dispersibility of the enzymatic hydrolysis product and improve its solubility in the extraction solvent. The reason for controlling the microwave power and time is that the structure of the cell has been destroyed during the enzymatic hydrolysis in this step. Microwave treatment exceeding the above range is likely to cause the temperature of the reaction system to be too high, thereby causing decomposition of some polysaccharides. In addition, the extraction solvent of the present invention has better solubility for the active ingredients in the medicinal materials than solvents such as water and ethanol, so there is no need for high-intensity microwave treatment, reducing cost consumption.

[0052] In the present invention, the enzymatic hydrolysis sequence of pectinase+cellulose syrup→acidic protease is set for the following reasons: (1) to maximize the use of the efficacy of each enzyme. Pectinase+cellulose syrup is mainly used to destroy the cell wall, while protease is mainly used to destroy the cell membrane. Their sequential combination can maximize the promotion of effective component precipitation; (2) to simplify the steps. Since different enzymes have different adaptability environments, the enzyme addition sequence of the present invention can greatly simplify the pH adjustment operation. After the final enzymatic hydrolysis is completed, there is no need to adjust the pH and the product can be used directly; (3) after the first stage of enzymatic hydrolysis is completed, the enzyme inactivation operation is not performed, and the enzyme can still play an enzymatic hydrolysis role in the subsequent stage; (4) after two-step enzymatic hydrolysis, the preparation is acidic, which is more conducive to poultry absorption and digestion.

[0053] S4, post-processing;

[0054] After the second enzymatic hydrolysis is completed, the enzyme is inactivated, the filtrate is filtered, the filtrate is centrifuged, and the supernatant is concentrated to obtain the Qizhi preparation;

[0055] The ingredients of the finally prepared Qizhi preparation include extracts of Astragalus and Ganoderma, citric acid-sodium citrate solution and at least one monosaccharide.

[0056] After the enzymatic hydrolysis is completed, the present invention does not need to remove the extraction solvent because: the extraction solvent is a natural solvent, which is green and harmless, and the components such as citric acid and sodium citrate promote the immune response of poultry after vaccination.

[0057] Example 1

[0058] This embodiment is a method for preparing a Qizhi preparation for enhancing the immune response of the Newcastle vaccine, which comprises the following steps:

[0059] S1. Weigh the raw materials of medicinal materials according to the weight ratio of Astragalus: Ganoderma lucidum = 1:2, and grind them into fine powders of 30 μm to 50 μm respectively; quickly freeze the fine powders, cool them to below -20°C, and keep them warm and frozen for more than 2 hours;

[0060] S2, adding 0.1% glucose by weight of the raw material to citric acid-sodium citrate buffer (0.05M, pH=3.5), mixing and stirring to prepare an extraction solvent; immersing the raw material powder pretreated by S1 in the extraction solvent at a liquid-to-solid ratio of 20:1 and mixing evenly, co-treating with water bath heating and microwave treatment (400W, 2min), and keeping warm in a water bath so that the temperature of the reaction system is in the range of 40°C to 50°C; then adding pectinase and cellulase (pectinase: cellulase=1:2, the total amount is 0.1% by weight of the raw material powder), and performing the first enzymolysis for 30min under water bath insulation and continuous stirring;

[0061] S3, measuring the pH of the reaction system, the value is 4.4, controlling the temperature at 40°C to 60°C, adding acidic protease (0.5% of the mass of the raw material powder), and performing the second enzymolysis for 40 minutes under water bath insulation and continuous stirring, and performing microwave treatment (250W, 50s) during the second enzymolysis;

[0062] S4, inactivate the enzyme, filter, centrifuge the filtrate, and concentrate the supernatant to obtain the Qizhi preparation.

[0063] Example 2

[0064] This embodiment is a method for preparing an Qizhi preparation for enhancing the immune response of Newcastle vaccine. On the basis of Example 1, the amount of glucose in step S2 is adjusted to 0.2%, and the other steps and reaction conditions are the same as Example 1.

[0065] Example 3

[0066] This embodiment is a method for preparing an Qizhi preparation for enhancing the immune response of Newcastle vaccine. On the basis of Example 1, the amount of glucose in step S2 is adjusted to 0.4%, and the other steps and reaction conditions are the same as Example 1.

[0067] Example 4

[0068] This embodiment is a method for preparing an Qizhi preparation for enhancing the immune response of Newcastle vaccine. On the basis of Example 1, the amount of glucose in step S2 is adjusted to 0.5%, and the other steps and reaction conditions are the same as Example 1.

[0069] Example 5

[0070] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment power of step S2 is adjusted to 300 W, and the other steps and reaction conditions are the same as those in Example 1.

[0071] Example 6

[0072] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment power of step S2 is adjusted to 500 W, and the other steps and reaction conditions are the same as those in Example 1.

[0073] Example 7

[0074] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment time of step S2 is adjusted to 1 min, and the other steps and reaction conditions are the same as those in Example 1.

[0075] Example 8

[0076] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment time of step S2 is adjusted to 4 minutes, and the other steps and reaction conditions are the same as those in Example 1.

[0077] Example 9

[0078] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the glucose in the extraction solvent in step S2 is changed to fructose, and the other steps and reaction conditions are the same as those in Example 1.

[0079] Example 10

[0080] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the first enzymatic hydrolysis time of step S2 is adjusted to 20 minutes, and the other steps and reaction conditions are the same as those in Example 1.

[0081] Embodiment 11

[0082] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the first enzymatic hydrolysis time of step S2 is adjusted to 40 minutes, and the other steps and reaction conditions are the same as those in Example 1.

[0083] Example 12

[0084] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the liquid-solid ratio of step S2 is adjusted to 10:1, and the other steps and reaction conditions are the same as those in Example 1.

[0085] Embodiment 13

[0086] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the liquid-solid ratio of step S2 is adjusted to 15:1, and the other steps and reaction conditions are the same as those in Example 1.

[0087] Embodiment 14

[0088] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the liquid-solid ratio of step S2 is adjusted to 30:1, and the other steps and reaction conditions are the same as those in Example 1.

[0089] Embodiment 15

[0090] This example is a method for preparing a Qizhi preparation for enhancing the immune response of Newcastle vaccine. On the basis of Example 1, the total amount of pectinase and cellulase in step S2 is adjusted to 0.3% of the mass of the raw material powder.

[0091] Example 16

[0092] This example is a method for preparing a Qizhi preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the total amount of pectinase and cellulase in step S2 is adjusted to 0.5% of the mass of the raw material powder.

[0093] Embodiment 17

[0094] This example is a method for preparing a Qizhi preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the amount of the acidic protease in step S3 is adjusted to 0.1% of the mass of the raw material powder.

[0095] Embodiment 18

[0096] This example is a method for preparing a Qizhi preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the amount of the acidic protease in step S3 is adjusted to 0.8% of the mass of the raw material powder.

[0097] Embodiment 19

[0098] This example is a method for preparing a Qizhi preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the amount of the acidic protease in step S3 is adjusted to 1% of the mass of the raw material powder.

[0099] Embodiment 20

[0100] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the second enzymatic hydrolysis time in step S3 is adjusted to 30 minutes, and the other steps and reaction conditions are the same as those in Example 1.

[0101] Embodiment 21

[0102] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the second enzymatic hydrolysis time of step S3 is adjusted to 60 minutes, and the other steps and reaction conditions are the same as those in Example 1.

[0103] Embodiment 22

[0104] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment power in the second enzymatic hydrolysis process of step S3 is adjusted to 200 W, and the other steps and reaction conditions are the same as those in Example 1.

[0105] Embodiment 23

[0106] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment power in the second enzymatic hydrolysis process of step S3 is adjusted to 300 W, and the other steps and reaction conditions are the same as those in Example 1.

[0107] Embodiment 24

[0108] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment time in the second enzymatic hydrolysis process of step S3 is adjusted to 30 seconds, and the other steps and reaction conditions are the same as those in Example 1.

[0109] Embodiment 25

[0110] This embodiment is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the microwave treatment time in the second enzymatic hydrolysis process of step S3 is adjusted to 60s, and the other steps and reaction conditions are the same as those in Example 1.

[0111] Comparative Example 1

[0112] This comparative example is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the acidic protease in step S3 is replaced with a neutral protease, and the other steps and reaction conditions are the same as in Example 1.

[0113] Comparative Example 2

[0114] This comparative example is a method for preparing a Qizhi preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, steps S2 and S3 are combined, that is, pectinase + cellulase + protease composite treatment, and S2 and S3 are combined as follows:

[0115] 0.1% of the raw material mass of glucose was added to a citric acid-sodium citrate buffer solution (0.05M, pH=3.5), and the mixture was mixed and stirred to prepare an extraction solvent; the raw material powder pretreated by S1 was immersed in the extraction solvent at a liquid-to-solid ratio of 20:1 and mixed evenly, and water bath heating and microwave treatment (400W, 2min) were synergistically treated, and the water bath was kept warm so that the temperature of the reaction system was in the range of 40°C to 50°C; pectinase, cellulase (pectinase: cellulase=1:2, the total amount was 0.1% of the raw material powder mass) and acid protease (0.5% of the raw material powder mass) were added, and enzymolysis was carried out for 80min under water bath insulation and continuous stirring, and microwave treatment (250W, 50s) was carried out during the enzymolysis process.

[0116] Comparative Example 3

[0117] This comparative example is a method for preparing an Astragalus and Ganoderma lucidum preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the freezing treatment of the raw material powder in step S1 is deleted. Step S1 is: weigh the medicinal raw materials according to the weight ratio of Astragalus: Ganoderma lucidum = 1:2, and grind them into fine powders of 30μm to 50μm respectively; the other steps and reaction conditions are the same as those in Example 1.

[0118] Comparative Example 4

[0119] This comparative example is a method for preparing an Qizhi preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, monosaccharides are deleted from the extraction solvent, and the other steps and reaction conditions are the same as those in Example 1.

[0120] Comparative Example 5

[0121] This comparative example is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the citric acid-sodium citrate in the extraction solvent is replaced with acetic acid-sodium acetate, and the other steps and reaction conditions are the same as those in Example 1.

[0122] Comparative Example 6

[0123] This comparative example is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. On the basis of Example 1, the citric acid-sodium citrate in the extraction solvent is replaced with water, and the other steps and reaction conditions are the same as those in Example 1.

[0124] Comparative Example 7

[0125] This comparative example is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. Based on Example 1, the microwave treatment in step S2 is deleted.

[0126] Comparative Example 8

[0127] This comparative example is a method for preparing an Astragalus membranaceus preparation for enhancing the immune response of the Newcastle vaccine. Based on Example 1, the microwave treatment in step S3 is deleted.

[0128] Comparative Example 9

[0129] Conventional water extraction method: add 13 times water to Astragalus and Ganoderma lucidum, heat and reflux for extraction 3 times, 3 hours each time, filter and centrifuge the filtrate, and concentrate the supernatant.

[0130] The UPLC quantitative analysis method was used to measure the contents of polysaccharides, ganoderic acid and total flavonoids in the Qizhi preparations prepared in Examples 1 to 25 and Comparative Examples 1 to 9.

[0131] When determining polysaccharides, analytically pure anhydrous glucose was used as a reference substance. The absorbance was measured at a wavelength of 625 nm according to the UV-visible spectrophotometry method. The standard curve was drawn with the absorbance as the ordinate and the concentration as the abscissa. The linear regression was performed with the mass concentration of the reference substance as the abscissa to obtain the regression equation.

[0132] When determining ganoderic acid, oleanolic acid was used as a reference substance, and the absorbance was measured at a wavelength of 546 nm according to the UV-visible spectrophotometry method. A standard curve was drawn with the absorbance A as the ordinate and the concentration as the abscissa. Linear regression was performed with the mass concentration of the reference substance as the abscissa to obtain a regression equation.

[0133] When determining total flavonoids, rutin was used as a reference substance, and the absorbance was measured at a wavelength of 510 nm. A standard curve was drawn with absorbance as the ordinate and concentration as the abscissa. Linear regression was performed with the mass concentration of the reference substance as the abscissa to obtain the regression equation.

[0134] The Qizhi preparations prepared in Examples 1 to 25 and Comparative Examples 1 to 9 were respectively made into sample solutions, and the absorbance was measured at 625nm, 546nm, and 510nm, respectively. The concentrations of each component in the polysaccharides, ganoderic acid, and total flavonoids in the Qizhi preparations were measured, and the contents of polysaccharides, ganoderic acid, and total flavonoids were calculated. The results are shown in Table 1.

[0135] Polysaccharide content (%) = (C1*V-M0) / M 总 *100%;

[0136] Ganoderic acid content (%) = C2*V / M1*100%;

[0137] Total flavonoid content (%) = C3*V / M 总 *100%;

[0138] Wherein, C1, C2, C3, are the concentrations of polysaccharide, ganoderic acid, and total flavonoids (mg / mL), respectively; V is the volume of the sample solution (mL); M 总 is the total mass of raw materials of Astragalus + Ganoderma lucidum (mg); M0 is the mass of monosaccharide added to the extraction solvent (mg); M1 is the mass of Ganoderma lucidum (mg).

[0139] Table 1

[0140]

[0141]

[0142] It can be seen from the data in Table 1 that the preparation method of the present invention can significantly increase the content of polysaccharides, ganoderic acid and flavonoid compounds extracted compared with the conventional water extraction method.

[0143] It can be seen from Examples 1 to 4 that as the glucose content in the extraction solvent increases, the extracted ganoderic acid and flavonoids content increases, and the polysaccharide content does not change significantly; when the glucose content increases to a certain value, the ganoderic acid and flavonoids content does not increase significantly. It can be seen from Example 9 that the effects of glucose and fructose are not much different.

[0144] It can be seen from Example 1, Example 5 and Example 6, Example 22 and Example 23 that with the increase of microwave power before the first enzymatic hydrolysis and during the second enzymatic hydrolysis, the content of extracted polysaccharides, ganoderic acid and flavonoid compounds increases; when the microwave power increases to a certain value, the content of ganoderic acid and flavonoid compounds does not increase significantly or declines to a small extent. The reason is that excessively high microwave power will cause some active ingredients to precipitate rapidly and decompose at the same time.

[0145] It can be seen from Example 1, Example 7 and Example 8, Example 24 and Example 25 that with the increase of microwave time before the first enzymatic hydrolysis and during the second enzymatic hydrolysis, the content of extracted polysaccharides, ganoderic acid and flavonoid compounds increased significantly; when the microwave time increased to a certain value, the content of ganoderic acid and flavonoid compounds did not increase significantly. The reason is that long-term microwave treatment will cause some active ingredients to precipitate rapidly and decompose at the same time.

[0146] It can be seen from Example 1, Example 10 and Example 11, Example 20 and Example 21 that with the increase of the first enzymatic hydrolysis time and the second enzymatic hydrolysis time, the content of the extracted polysaccharides, ganoderic acid and flavonoid compounds increases.

[0147] It can be seen from Example 1 and Example 12 to Example 14 that as the liquid-to-solid ratio increases, the solubility of the active ingredients of the medicinal materials in the solvent increases, and the content of the extracted polysaccharides, ganoderma acid, and flavonoid compounds increases; but when it increases to a certain value, the content of the active ingredients formed by enzymatic hydrolysis is limited, and the soluble active ingredients will not increase further.

[0148] It can be seen from Examples 1, 15, 16, and 17 to 19 that with the increase in the amount of each enzyme in the first enzymatic hydrolysis and the second enzymatic hydrolysis, the content of the extracted polysaccharides, ganoderic acid, and flavonoid compounds increases rapidly, and with the increase in the enzyme dosage to a certain value, the rate of increase in the active ingredient content slows down.

[0149] It can be seen from Example 1 and Comparative Example 1 that after the protease in the enzymatic hydrolysis process is adjusted from acidic protease to neutral protease, the content of extracted polysaccharides, ganoderma acid and flavonoid compounds is reduced. The reason is that neutral protease is more sensitive to temperature and pH, and is easily inactivated during the enzymatic hydrolysis process, especially after microwave assistance. Combined with the heating effect of the enzymatic hydrolysis itself, the temperature of the reaction system will fluctuate to a certain extent, which will lead to a decrease in the enzymatic hydrolysis efficiency and a decrease in the content of various active ingredients.

[0150] It can be seen from Example 1 and Comparative Example 2 that after adjusting the two-step enzymatic hydrolysis to a one-step composite enzymatic hydrolysis, the contents of the extracted polysaccharides, ganoderic acid and flavonoids decreased, among which the decrease in ganoderic acid and flavonoids was more obvious.

[0151] It can be seen from Example 1 and Comparative Example 3 that by removing the freezing treatment in the medicinal material pretreatment, the contents of the extracted polysaccharides, ganoderic acid and flavonoids are greatly reduced. The reason is that the conventional pretreatment has limited effect on the destruction of the medicinal material cell wall, which is not conducive to the extraction of active components.

[0152] It can be seen from Example 1 and Comparative Examples 4 to 6 that by deleting or replacing the components in the extraction solvent, the contents of polysaccharides, ganoderic acid and flavonoids extracted are greatly reduced, and the effect is only slightly better than the conventional water extraction method in Comparative Example 9. The reason is that the composite system composed of citric acid-sodium citrate-monosaccharide can greatly promote the dissolution of the active ingredient, and the lack of any component greatly reduces its solubility.

[0153] It can be seen from Example 1 and Comparative Examples 7 to 8 that by eliminating the microwave treatment during the enzymatic hydrolysis process, the contents of the extracted polysaccharides, ganoderic acid and flavonoids are reduced, especially the reduction in the contents of ganoderic acid and flavonoids is more obvious.

[0154] Embodiment 26

[0155] This example verifies the application of the oral solution made from the Qizhi preparation in enhancing the immune response to Newcastle disease vaccine.

[0156] The experimental method is as follows: select healthy male chickens aged 20 to 25 days that have not been vaccinated with NDV vaccine, first feed them for 7 days of adaptive breeding and feed them with conventional feed; inject Newcastle disease I live vaccine subcutaneously in the neck of each chicken, feed them with conventional feed, and feed them with 1 ml of different preparations every day for 3 consecutive days according to the following groups, with 2 replicates in each group and 10 chickens in each replicate.

[0157] ①Control group: normal saline

[0158] ② Test Group A: Qizhi preparation prepared in Example 1;

[0159] ③ Experimental Group B: Qizhi preparation prepared in Example 16;

[0160] ④Comparative Group A: Qizhi preparation prepared in Comparative Example 9;

[0161] ⑤ Comparative Group B: The Qizhi preparation prepared in Example 1 was redissolved in water, and a calcium hydroxide solution was added, with which citric acid and sodium citrate reacted to form a calcium citrate precipitate, and then the citric acid and its salt were removed, and the Qizhi preparation was obtained after concentration.

[0162] During the experiment, all experimental chickens had free access to food and water and were kept in daylight for 12 hours a day. The chicken houses were well ventilated and the manure was cleaned and disinfected regularly. The feeding habits and health status of the chickens were observed and recorded every day. The observation period was 4 weeks.

[0163] After vaccination, blood was collected from the wing vein of the experimental chickens every other week. After the serum was separated, the ND antibody titer was detected by β-micro HI test. The ND antibody titer values ​​(log2) before immunization, 7 days, 14 days, 21 days, and 28 days after immunization were recorded, as shown in Table 2.

[0164] Table 2

[0165] 0 days 7th 14th 21st 28th Control group 0.26±0.08 1.22±0.29 2.16±0.26 2.49±0.23 1.86±0.24 Test Group A 0.25±0.08 4.08±0.27 5.48±0.42 6.24±0.29 5.48±0.33 Test Group B 0.24±0.09 4.34±0.19 5.84±0.30 6.57±0.36 5.78±0.31 Compared with Group A 0.26±0.09 2.41±0.26 4.19±0.27 5.26±0.22 4.00±0.24 Compared with Group B 0.26±0.07 2.89±0.24 4.60±0.33 5.58±0.53 4.51±0.47

[0166] From the results in Table 2, it can be seen that before immunization, the antibody titer values ​​of the test chickens all dropped below 2log2, which met the test requirements. During the entire test period of 28 days of immunization, the antibody titer values ​​of the control group, test group A, test group B, comparison group A, and comparison group B all showed a trend of gradually increasing first and then falling back.

[0167] At 7 days of immunization, the test group A, test group B, comparison group A, and comparison group B were significantly higher than the control group, and the test group B>test group A>comparison group B>comparison group A. The test group B used the Qizhi preparation prepared in Example 16, and its active ingredient content was higher than the Qizhi preparation of Example 1 used in the test group A; compared with the test group A, the comparison group B removed citric acid and sodium citric acid, and it can be seen that its synergistic effect on the immune response was reduced, but it was still better than the conventional Qizhi preparation used in the comparison group A.

[0168] At 14 days of immunization, the test group A, test group B, comparison group A, and comparison group B were significantly higher than the control group; test group B > test group A > comparison group B > comparison group A, and the antibody titer values ​​were all higher than 4log2.

[0169] At 21 days of immunization, the test group A, test group B, comparison group A, and comparison group B were significantly higher than the control group; test group B > test group A > comparison group B > comparison group A, and the antibody titer values ​​were all higher than 5log2.

[0170] At 28 days of immunization, the test group A, test group B, comparison group A, and comparison group B were significantly higher than the control group; test group B > test group A > comparison group B > comparison group A, and the downward trend of test group B and test group A was gentler than that of comparison group A and comparison group B.

[0171] This indicates that the Qizhi preparation has different degrees of immune enhancement effect on Newcastle disease vaccine, test group B > test group A > comparison group B > comparison group A.

[0172] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine, characterized in that: The raw materials of the Qizhi preparation include astragalus and ganoderma, with the weight ratio of astragalus to ganoderma being 1:1.5-3; The preparation method comprises the following steps: The pretreated raw material powder is immersed in the extraction solvent and mixed evenly, and heated to 40°C to 50°C, the pH is adjusted to 3 to 5, pectinase and cellulase are added, and the first enzymolysis is performed under water bath insulation and continuous stirring; After the first enzymatic hydrolysis is completed, the pH is adjusted to 3-5, the temperature is controlled at 40°C-60°C, acidic protease is added, and the second enzymatic hydrolysis is performed under water bath insulation and continuous stirring; After the second enzymatic hydrolysis is completed, the filtrate is filtered, the filtrate is centrifuged, and the supernatant is concentrated to obtain the Qizhi preparation; Wherein, the extraction solvent comprises citric acid-sodium citrate solution and at least one monosaccharide.

2. The method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine according to claim 1, characterized in that: The mass ratio of pectinase to cellulase is 1:1-3, and the total dosage is 0.1%-0.5% of the mass of the raw material powder; the first enzymolysis time is 20min-40min; The dosage of the acidic protease is 0.1% to 1% of the mass of the raw material powder, and the second enzymolysis time is 30min to 60min.

3. The method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine according to claim 1, characterized in that: The pretreatment steps of the raw material powder are: Astragalus and Ganoderma lucidum are made into fine powders of 30 μm to 50 μm; Cool down to below -20℃ and keep in the freezer for more than 2h.

4. The method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine according to claim 1, characterized in that: The heating of the first enzymatic hydrolysis adopts microwave-assisted water bath heating, specifically: low-power time-controlled heating is performed during water bath heating, the microwave power is 300W-500W, and the microwave time does not exceed 4 minutes.

5. The method for preparing a Qizhi preparation for enhancing the immune response of Newcastle vaccine according to claim 4, characterized in that: During the second enzymatic hydrolysis, microwave treatment is added, the microwave power is 200W to 300W, and the microwave time is 0.5min to 1min.

6. The method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine according to claim 1, characterized in that: The liquid-to-solid ratio of the extraction solvent to the raw material powder is 10:1 to 30:

1.

7. The method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine according to claim 1, characterized in that: The monosaccharide is selected from fructose and / or glucose; the amount of the monosaccharide is 0.1% to 0.5% of the mass of the raw material powder.

8. The method for preparing a Qizhi preparation for enhancing the immune response of a Newcastle vaccine according to claim 1, characterized in that: During the first enzymolysis and the second enzymolysis, pH is adjusted by adding citric acid or sodium citrate.

9. The Qizhi preparation for enhancing the immune response of Newcastle vaccine prepared by any one of the preparation methods described in claims 1 to 8.

10. The Qizhi preparation for enhancing the immune response of the Newcastle vaccine according to claim 9, characterized in that: The Qizhi preparation comprises extracts of Astragalus and Ganoderma, a citric acid-sodium citrate solution and at least one monosaccharide.

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