High-purity tea saponin and application thereof in prevention and treatment of poultry virus diseases

Abstract

The invention relates to the technical field of natural product separation, and provides high-purity tea saponin and application thereof in poultry virus disease prevention and treatment, and a preparation method of the high-purity tea saponin, the preparation method comprises the following steps: S1, adding tea seed meal powder into an extracting agent, mixing, and filtering to obtain an extracting solution; s2, purifying the extracting solution to obtain tea saponin; the extracting agent comprises a component A and a component B in a molar ratio of 1: (1-8); the component A comprises one or two of alanine and histidine; the component B comprises methylurea. According to the technical scheme, the problem that the purity and the yield are low when tea saponin is extracted from plants in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of natural product separation, and in particular to high-purity tea saponin and application thereof in the prevention and treatment of poultry viral diseases. Background Art

[0002] Tea saponin is a natural saponin compound extracted from the Camellia family. The production process of tea saponin faces the problems of low purity and yield: low-purity tea saponin may contain more impurities, which may affect its application effect in specific fields. For example, in the pharmaceutical field, high-purity tea saponin is crucial to the safety and effectiveness of drugs. If the purity is not high, it may introduce unnecessary side effects or reduce the therapeutic effect of the drug. In addition, the low yield of tea saponin also means that more raw materials and energy are needed to produce the same amount of tea saponin, which increases the cost to a certain extent.

[0003] Therefore, it is necessary to obtain a high-purity and high-yield tea saponin to reduce production costs, expand its application range, and promote the further development and application of tea saponin. Summary of the invention

[0004] The invention provides a high-purity tea saponin and application thereof in the prevention and treatment of poultry viral diseases, solving the problems of low purity and yield of tea saponin extracted from plants in the related art.

[0005] The technical solution of the present invention is as follows: The present invention provides a method for preparing high-purity tea saponin, comprising the following steps: S1, adding tea seed meal powder into an extractant, mixing, filtering, and obtaining an extract; S2, purifying the extract to obtain tea saponin; The extractant comprises component A and component B in a molar ratio of 1:1-8; The component A includes one or two of alanine and histidine; The component B includes methyl urea.

[0006] As a further technical solution, when the component A is alanine, the molar ratio of the alanine to the component B is 1:3-5.

[0007] In the present invention, when component A is alanine, and the molar ratio of alanine to component B is 1:3-5, the purity and yield of tea saponin are further improved.

[0008] As a further technical solution, the component A also includes betaine, and the molar ratio of the betaine, alanine and component B is 1-2:1:4.

[0009] In the present invention, component A also includes betaine, and betaine, alanine and component B are used as extractants. When the molar ratio of betaine, alanine and component B is 1-2:1:4, the purity and yield of tea saponin are further improved.

[0010] As a further technical solution, the water content of the extractant is 20wt%~40wt%.

[0011] As a further technical solution, the method for preparing the extractant comprises the following steps: stirring and mixing the component A and the component B at 70-80° C., and diluting with water to obtain the extractant.

[0012] As a further technical solution, the purification includes the following steps: adding a flocculant to the extract to mix, filtering the filtrate, adsorbing and eluting the filtrate with a macroporous resin, collecting the eluate, concentrating it, and drying it to obtain tea saponin.

[0013] As a further technical solution, the model of the macroporous resin is one of AB-8, D-101 and HPD-100.

[0014] As a further technical solution, the volume ratio of the flocculant to the extract is 0.01-0.03:1; The preparation method of the flocculant comprises the following steps: mixing chitosan, xylan and sodium carboxymethyl cellulose with water in a mass ratio of 1:2:1-3 to obtain a flocculant; The total mass of the chitosan, xylan and sodium carboxymethyl cellulose is 2% to 3% of the mass of water; During the elution, water and ethanol are used in sequence; the volume fraction of the ethanol is 70% to 90%.

[0015] As a further technical solution, the preparation method of the tea seed meal powder comprises the following steps: crushing, defatting and drying the tea seed meal to obtain the tea seed meal powder; The crushing step is to crush the powder into 60-80 mesh.

[0016] As a further technical solution, in step S1, the mixing temperature is 65-75°C and the mixing time is 60-80 minutes; The mass volume ratio of the tea seed meal powder and the extractant is 1g:20-30mL.

[0017] The invention also proposes the application of the tea saponin prepared by the high-purity tea saponin preparation method in the prevention and treatment of poultry viral diseases.

[0018] In the present invention, the prepared high-purity tea saponin has more significant antiviral effect, better stability and lower risk of side effects, is beneficial to improving the immunity of poultry, reduces the risk of viral infection, has excellent effect in the prevention and treatment of poultry viral diseases, and provides strong support for the healthy development of poultry farming.

[0019] The working principle and beneficial effects of the present invention are: In the present invention, methyl urea is used as a hydrogen bond donor, alanine and / or histidine is used as a hydrogen bond acceptor, and the two are mixed as an extractant to extract tea saponin from tea seed meal, which is fast and efficient, and significantly improves the purity and yield of tea saponin. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] In the following examples and comparative examples, alanine is L-alanine; histidine is L-histidine.

[0022] Example 1 The preparation method of high-purity tea saponin comprises the following steps: S1. After the tea seed meal is crushed to 80 meshes, defatted and dried, tea seed meal powder is obtained, and the tea seed meal powder is added to the extractant and mixed at 75° C. for 60 minutes, and filtered to obtain an extract; wherein the mass volume ratio of the tea seed meal powder to the extractant is 1 g:20 mL; The extractant includes alanine and methyl urea in a molar ratio of 1:8; The preparation method of the extractant comprises the following steps: stirring and mixing alanine and methyl urea at 70° C., adding water to dilute, and obtaining an extractant with a water content of 40 wt %; S2, the extract and the flocculant are mixed in a volume ratio of 1:0.01, and the filtrate is filtered to obtain the filtrate, the filtrate is adsorbed by a macroporous resin of model AB-8, and deionized water and 70% ethanol are used to elute in sequence, and the eluate is collected, concentrated, and dried to obtain tea saponin; The preparation method of the flocculant comprises the following steps: mixing chitosan, xylan and sodium carboxymethyl cellulose with water in a mass ratio of 1:2:1 to obtain the flocculant; the total mass of chitosan, xylan and sodium carboxymethyl cellulose is 2% of the mass of water.

[0023] Example 2 The preparation method of high-purity tea saponin comprises the following steps: S1. After the tea seed meal is crushed to 60 mesh, defatted and dried, tea seed meal powder is obtained, and the tea seed meal powder is added to the extractant and mixed at 65° C. for 80 minutes, and filtered to obtain an extract; wherein the mass volume ratio of the tea seed meal powder to the extractant is 1 g:30 mL; The extractant comprises component A and methyl urea in a molar ratio of 1:6; component A comprises alanine and histidine in a molar ratio of 1:1; The preparation method of the extractant comprises the following steps: stirring and mixing alanine, histidine and methyl urea at 80° C., adding water to dilute, and obtaining an extractant with a water content of 20 wt %; S2, the extract and the flocculant are mixed in a volume ratio of 1:0.03, and the filtrate is filtered to obtain the filtrate, the filtrate is adsorbed by a macroporous resin of model HPD-100, and deionized water and 90% ethanol by volume are used for elution in sequence, and the eluate is collected, concentrated, and dried to obtain tea saponin; The preparation method of the flocculant comprises the following steps: mixing chitosan, xylan and sodium carboxymethyl cellulose with water in a mass ratio of 1:2:3 to obtain a flocculant; the total mass of chitosan, xylan and sodium carboxymethyl cellulose is 3% of the mass of water.

[0024] Example 3 The preparation method of high-purity tea saponin comprises the following steps: S1. After the tea seed meal is crushed to 75 mesh, defatted and dried, tea seed meal powder is obtained, and the tea seed meal powder is added to the extractant and mixed at 70° C. for 75 minutes, and filtered to obtain an extract; wherein the mass volume ratio of the tea seed meal powder to the extractant is 1 g:25 mL; The extractant includes histidine and methyl urea in a molar ratio of 1:1; The preparation method of the extractant comprises the following steps: stirring and mixing histidine and methyl urea at 80° C., adding water to dilute, and obtaining an extractant with a water content of 30 wt %; S2, mixing the extract and the flocculant in a volume ratio of 1:0.02, filtering and collecting the filtrate, adsorbing the filtrate with a macroporous resin of model AB-8, eluting with deionized water and 90% ethanol by volume, collecting the eluate, concentrating, and drying to obtain tea saponin; The preparation method of the flocculant comprises the following steps: chitosan, xylan and sodium carboxymethyl cellulose are mixed with water in a mass ratio of 1:2:2 to obtain the flocculant; the total mass of chitosan, xylan and sodium carboxymethyl cellulose is 2% of the mass of water.

[0025] Example 4 The difference between this example and Example 3 is that the histidine in the extractant is replaced by alanine.

[0026] Example 5 The only difference between this embodiment and embodiment 4 is that the molar ratio of alanine to methyl urea in the extractant is 1:8.

[0027] Example 6 The only difference between this embodiment and embodiment 4 is that the molar ratio of alanine to methyl urea in the extractant is 1:3.

[0028] Example 7 The only difference between this embodiment and embodiment 4 is that the molar ratio of alanine to methyl urea in the extractant is 1:5.

[0029] Example 8 The difference between this embodiment and embodiment 4 is that the molar ratio of alanine to methyl urea in the extractant is 1:4.

[0030] Example 9 The only difference between this example and Example 8 is that the extractant includes betaine, alanine and methyl urea in a molar ratio of 0.5:1:4; The preparation method of the extractant comprises the following steps: stirring and mixing betaine, alanine and methyl urea at 80° C., adding water to dilute, and obtaining an extractant with a water content of 30 wt %.

[0031] Example 10 The only difference between this example and Example 9 is that the extractant includes betaine, alanine and methyl urea in a molar ratio of 3:1:4.

[0032] Embodiment 11 The only difference between this example and Example 9 is that the extractant includes betaine, alanine and methyl urea in a molar ratio of 2:1:4.

[0033] Example 12 The only difference between this example and Example 9 is that the extractant includes betaine, alanine and methyl urea in a molar ratio of 1:1:4.

[0034] Comparative Example 1 The difference between this comparative example and Example 3 is that histidine is replaced by choline chloride, and methyl urea is replaced by glycerol.

[0035] Comparative Example 2 The difference between this comparative example and Example 3 is that histidine is replaced by choline chloride.

[0036] Comparative Example 3 The difference between this comparative example and Example 3 is that histidine is replaced by L-proline.

[0037] The purity and yield of tea saponin obtained in Examples 1 to 12 and Comparative Examples 1 to 3 were calculated and tested respectively, and the results are shown in Table 1 below.

[0038] Table 1 Purity and yield of tea saponin

[0039] By comparing the data of Examples 1 to 12 and Comparative Examples 1 to 3, it is found that compared with Comparative Examples 1 to 3, the purity and yield of the tea saponin extracted in Examples 1 to 12 are higher, indicating that using methyl urea as a hydrogen bond donor and alanine and / or histidine as a hydrogen bond acceptor, and mixing the two as an extractant to extract tea saponin from tea seed meal, significantly improves the purity and yield of tea saponin.

[0040] By comparing the data of Examples 3 to 8, it is found that compared with Examples 3 to 5, the purity and yield of the tea saponin extracted in Examples 6 to 8 are higher, indicating that when component A is alanine and the molar ratio of alanine to component B is 1:3 to 5, the purity and yield of the tea saponin are further improved, especially when the molar ratio of alanine to component B is 1:4, the purity and yield of the tea saponin are higher.

[0041] By comparing the data of Examples 8 to 12, it is found that compared with Examples 8 to 10, the purity and yield of the tea saponin extracted in Examples 11 to 12 are higher, indicating that component A also includes betaine, and when betaine, alanine and component B are used as extractants, and the molar ratio of betaine, alanine and component B is 1 to 2:1:4, the purity and yield of the tea saponin can be further improved.

[0042] Poultry Bursal Disease Test Select chicks and divide them into 5 groups, 10 in each group: Group 1: The tea saponin prepared in Example 12 was prepared into a dosage group of 30 mg / kg; Group 2: The tea saponin prepared in Example 12 was prepared into a dosage group of 10 mg / kg; Group 3: High immune yolk antibody group; Group 4: Control group - no medication, only challenge with poison; Group 5: Blank group - no medication or challenge.

[0043] On the third day of administration, 100 BID of bursal disease Beijing strain BC6 / 85 was used. 50 / 0.1mL was used to challenge the poison in groups 1 to 4, and groups 1 to 3 were given medication for three consecutive days after the challenge, and groups 4 to 5 were given an equal amount of normal saline. The observation lasted for 14 days. During the observation period, the clinical symptoms of each group were observed, and the number of dead chickens was recorded to calculate the survival rate. The results are shown in Table 2 below.

[0044] Table 2 Test results of tea saponin on poultry bursal disease

[0045] The results in Table 2 show that the survival rate of the 30 mg / kg tea saponin dosage group prepared in Example 12 can reach 80%, and the preventive effect is significantly higher than that of other groups, and is equivalent to that of Group 3, indicating that tea saponin has a significant antiviral effect, better stability and lower risk of side effects, which is beneficial to improving the immunity of poultry and reducing the risk of viral infection.

[0046] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing high-purity tea saponin, characterized in that: The following steps are involved: S1, adding tea seed meal powder into an extractant, mixing, filtering, and obtaining an extract; S2, purifying the extract to obtain tea saponin; The extractant comprises component A and component B in a molar ratio of 1:1-8; The component A includes one or two of alanine and histidine; The component B includes methyl urea.

2. The method for preparing high-purity tea saponin according to claim 1, characterized in that: When the component A is alanine, the molar ratio of the alanine to the component B is 1:3-5.

3. The method for preparing high-purity tea saponin according to claim 2, characterized in that: The component A also includes betaine, and the molar ratio of the betaine, alanine and component B is 1-2:1:

4.

4. The method for preparing high-purity tea saponin according to claim 1, characterized in that: The water content of the extractant is 20wt% to 40wt%.

5. The method for preparing high-purity tea saponin according to claim 4, characterized in that: The preparation method of the extractant comprises the following steps: stirring and mixing the component A and the component B at 70-80° C., and diluting with water to obtain the extractant.

6. The method for preparing high-purity tea saponin according to claim 1, characterized in that: The purification comprises the following steps: adding a flocculant to the extract to mix, filtering to obtain a filtrate, adsorbing and eluting the filtrate with a macroporous resin, collecting the eluate, concentrating it, and drying it to obtain tea saponin.

7. The method for preparing high-purity tea saponin according to claim 6, characterized in that: The preparation method of the flocculant comprises the following steps: mixing chitosan, xylan and sodium carboxymethyl cellulose with water in a mass ratio of 1:2:1-3 to obtain a flocculant; During the elution, water and ethanol are used in sequence; the volume fraction of the ethanol is 70% to 90%.

8. The method for preparing high-purity tea saponin according to claim 1, characterized in that: The preparation method of tea seed meal powder comprises the following steps: crushing, defatting and drying tea seed meal to obtain tea seed meal powder; The crushing step is to crush the powder into 60-80 mesh.

9. The method for preparing high-purity tea saponin according to claim 1, characterized in that: In step S1, the mixing temperature is 65-75°C and the mixing time is 60-80 minutes; The mass volume ratio of the tea seed meal powder and the extractant is 1g:20-30mL.

10. Use of the tea saponin obtained according to the method for preparing high-purity tea saponin according to any one of claims 1 to 9 in the prevention and treatment of poultry viral diseases.

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