Rose and cubilose oral liquid and preparation method thereof
By modifying and loading the bird's nest acid glycoprotein on quaternary ammonium salt chitosan, the problem of insufficient absorption and retention time of bird's nest acid in the body is solved, and more efficient bird's nest acid absorption and utilization is achieved.
Patent Information
- Application Number
- CN202510392288.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-05-23
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The absorption and retention time of sialic acid in bird's nest in the body is insufficient, making it difficult to be effectively utilized.
The rose flavonoid extract obtained by ethanol extraction was modified to the bird's nest acid glycoprotein and produced more free bird's nest acid and bird's nest acid bound to oligosaccharides or glycan peptides during trypsin digestion. Bird's nest extract is loaded on quaternary ammonium salted chitosan, and it is used to break the intestinal absorption barrier nonspecifically and improve the absorption rate of bird's nest acid.
It improves the absorption rate and utilization efficiency of bird's nest acid in the body, extends its retention time in the body, and enhances the absorption capacity of protein-bound bird's nest acid.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of health care products, and particularly relates to a rose bird's nest oral liquid and a preparation method thereof. Background Art
[0002] Both rose and bird's nest contain many ingredients that are beneficial to the human body. They are commonly used raw materials in modern health products, and bird's nest is even more popular. Sialic acid is widely present in cell membrane glycoproteins and lipoproteins in organisms. By binding with positively charged ions, it can act as a recognition site to serve as a biological target, and can also mask the recognition site of cytokines, thereby participating in many important processes of the organism. The natural substance with the highest sialic acid content is bird's nest, so sialic acid is also called bird's nest acid.
[0003] However, the sialic acid in bird's nests is mainly bound to the end of the sugar chain of glycoproteins, and a small amount exists in the form of free and polysaccharides. After the bird's nest is eaten directly, after digestion in the stomach, the content of free bird's nest acid increases, which is beneficial to human absorption, but the retention time in the body is too short, while the sialic acid bound to oligosaccharides or polysaccharide peptides has a suitable retention time in the body, which is also beneficial to absorption and utilization, but the sialic acid bound to oligosaccharides or polysaccharide peptides obtained after gastrointestinal digestion is less, and most of it is protein-bound sialic acid that is difficult to be converted and absorbed. Summary of the invention
[0004] The object of the present invention is to provide a rose bird's nest oral liquid and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology.
[0005] In order to achieve the above technical objectives, the technical solution of the present invention is: A method for preparing rose bird's nest oral liquid comprises the following steps: S1, extracting rose flavonoids extract by organic solvent extraction method; S2, extracting the dry bird's nest powder with distilled water for 3 times to obtain a supernatant, mixing the supernatants, freeze-drying to obtain a bird's nest extract powder, dissolving the bird's nest extract powder in a sodium nitrate buffer solution, separating with a G-150 chromatography column, and then eluting with a sodium nitrate solution, ultrafiltering the eluate, and freeze-drying to obtain bird's nest acid glycoprotein; S3, adding bird's nest acid glycoprotein to the rose flavonoid extract, adjusting the pH to 8.5 with sodium hydroxide solution, stirring and reacting at 50°C for 2 hours, ultrafiltration and freeze-drying after the reaction to obtain modified bird's nest acid glycoprotein, and then digesting with trypsin for 40 minutes, separating the supernatant to obtain the bird's nest extract; S4, adding bird's nest extract to the quaternary ammonium chitosan aqueous solution, stirring evenly, adding sodium tripolyphosphate aqueous solution, stirring and reacting for 1 hour, obtaining a suspension by high-speed centrifugation to obtain a white precipitate, and freeze-drying the white precipitate to obtain a quaternary ammonium chitosan loaded with bird's nest extract; S5. Mix the rose flavonoid extract, quaternary ammonium chitosan loaded with bird's nest extract, xanthan gum and distilled water, stir at 26° C. for 2 h, sterilize and cool to obtain the rose bird's nest oral liquid.
[0006] As a further improvement, in step S5, the preparation method of the quaternary ammonium chitosan aqueous solution is: disperse chitosan in deionized water, heat it in an oil bath to 80°C, then dissolve 2,3-epoxypropyltrimethylammonium chloride in deionized water, add it to the chitosan aqueous solution twice, each time at an interval of 3 hours, stir and react for 2 hours after all are dropped, cool to room temperature, precipitate the reaction solution with acetone, dissolve the precipitate in deionized water and then filter to remove insoluble matter, then dialyze the filtrate with deionized water for 2 days, the molecular weight cutoff is 8000Da, the retentate is freeze-dried to obtain quaternary ammonium chitosan, and the quaternary ammonium chitosan is dissolved in deionized water to obtain a quaternary ammonium chitosan aqueous solution.
[0007] As a further improvement, the mass ratio of the chitosan to the 2,3-epoxypropyltrimethylammonium chloride is 1:3.
[0008] As a further improvement, in step S1, the dried rose pollen is extracted twice with a 70% ethanol solution, the material-liquid ratio of the first extraction is 1:10, and the material-liquid ratio of the second extraction is 1:5. The filtrates after the two extractions are mixed and passed through an AB-8 macroporous resin column, the loading flow rate is 1.0 mL / min, and the elution flow rate is 1.0 mL / min.
[0009] As a further improvement, in step S2, during the extraction of the dry bird's nest powder, the solid-liquid ratio of dry bird's nest powder to distilled water is 1:5, the extraction is carried out at 37°C for 2h, the concentration of the sodium nitrate buffer is 0.05mol / L, the flow rate of the eluent is 0.8mL / min, and the molecular weight cutoff of the ultrafiltration process is 10kDa.
[0010] As a further improvement, in step S3, 50 mg of sialic acid glycoprotein is added to every 100 mL of rose flavonoid extract, and the molecular weight cutoff of the ultrafiltration process is 10 kDa.
[0011] As a further improvement, in step S4, the mass ratio of quaternary ammonium salt chitosan, bird's nest extract and sodium tripolyphosphate is 3:1:1.
[0012] The present invention also provides a rose bird's nest oral liquid, wherein the weight proportions of the components are: 30-35 parts of rose flavonoid extract, 30-35 parts of quaternary ammonium salt chitosan loaded with bird's nest extract, 8-10 parts of xanthan gum, and 17-20 parts of distilled water.
[0013] As a further improvement, the weight proportions of each component are: 30 parts of rose flavonoid extract, 30 parts of quaternary ammonium chitosan loaded with bird's nest extract, 8 parts of xanthan gum, and 17 parts of distilled water.
[0014] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: The rose bird's nest oral liquid provided by the present invention is prepared by modifying bird's nest acid glycoprotein with rose flavonoid extract obtained by ethanol extraction, and then decomposing the modified bird's nest acid glycoprotein with trypsin to produce more free bird's nest acid and bird's nest acid combined with oligosaccharides or polyglycopeptides, which is more conducive to being absorbed by the human body after entering the human body.
[0015] By loading the obtained bird's nest extract on quaternary ammonium chitosan, after entering the human intestine, the quaternary ammonium chitosan can non-specifically and temporarily break the intestinal absorption barrier, adjust the cell gap, improve the paracellular transport effect of protein-bound bird's nest acid in the bird's nest extract, and improve the human body's absorption rate of protein-bound bird's nest acid.
[0016] At the same time, in the oral liquid of the present invention, after entering the intestine, the rose flavonoids can chelate with extracellular calcium ions under the weak alkaline conditions in the intestine, reduce the concentration of extracellular calcium ions, and are also beneficial to the expansion of cell gaps, thereby increasing the absorption rate of protein-bound bird's nest acid. DETAILED DESCRIPTION
[0017] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0018] Example 1 A rose bird's nest oral liquid comprises the following components by weight: 30 parts of rose flavonoid extract, 30 parts of quaternary ammonium salt chitosan loaded with bird's nest extract, 8 parts of xanthan gum, and 17 parts of distilled water.
[0019] This embodiment also provides a method for preparing the rose bird's nest oral liquid, comprising the following steps: S1. Weigh the dried rose pollen, add 70% ethanol solution at a solid-liquid ratio of 1:10, extract in a water bath at 40°C for 2h, then filter with four layers of gauze, extract the residue twice with 70% ethanol solution at a solid-liquid ratio of 1:5 for 2h, filter with four layers of gauze, and mix the filtrates obtained from the two extractions; The filtrate was centrifuged at 5000 rpm for 20 min, and then the supernatant was passed through an AB-8 macroporous resin column at a loading flow rate of 1.0 mL / min, eluted with 70% ethanol at an elution flow rate of 1.0 mL / min, and the eluted eluate was distilled and concentrated to obtain a rose flavonoid extract; S2, take bird's nest dry powder, add it to distilled water at a solid-liquid ratio of 1:5, shake and extract in a shaker at 37°C for 2 hours, centrifuge at a speed of 10000 rpm for 10 minutes, collect the supernatant, and continue to extract the precipitate for 3 times. The supernatants obtained from the 3 extractions are mixed and freeze-dried to obtain the extract powder; The extract powder was dissolved in a 0.05 mol / L sodium nitrate buffer solution, loaded on a G-150 chromatography column, and then eluted with 0.05 mol / L sodium nitrate at a flow rate of 0.8 mL / min, the eluate was collected, and then the eluate was ultrafiltered and freeze-dried to obtain santol glycoprotein, and the molecular weight cutoff during the ultrafiltration process was 10 kDa; S3, adding saccharide glycoprotein to the rose flavonoid extract, wherein 50 mg of saccharide glycoprotein was added to every 100 mL of rose flavonoid extract, the pH was adjusted to 8.5 with sodium hydroxide solution, and the reaction was stirred at 50° C. for 2 h. After the reaction was completed, ultrafiltration and freeze-drying were performed to obtain modified saccharide glycoprotein, and the molecular weight cutoff during the ultrafiltration process was 10 kDa; Add potassium chloride and sodium bicarbonate to distilled water, stir evenly, then add potassium hydrogen phosphate thereto, wherein the concentration of potassium chloride is 6.8 mmol / L, the concentration of sodium bicarbonate is 85 mmol / L, and the concentration of potassium hydrogen phosphate is 0.8 mmol / L, and adjust the pH to 7.5 with sodium hydroxide to obtain a digestion solvent; Take 10 mL of digestion solvent, add 5 mg of modified bird's nest acid glycoprotein, and add 5 mg of trypsin, the enzyme activity of trypsin is 40 U / mg, digest for 40 minutes, add 30 μL of acetic acid solution to terminate the digestion, separate the supernatant to obtain the bird's nest extract; S4, adding the bird's nest extract to the quaternary ammonium chitosan aqueous solution, stirring evenly, adding the sodium tripolyphosphate aqueous solution, stirring and reacting at room temperature for 1 hour, obtaining a suspension, high-speed centrifuging at a speed of 10000 rpm to obtain a white precipitate, and freeze-drying the white precipitate to obtain the quaternary ammonium chitosan loaded with the bird's nest extract, the mass ratio of the quaternary ammonium chitosan, the bird's nest extract and the sodium tripolyphosphate is 3:1:1; S5. Mix the rose flavonoid extract, quaternary ammonium chitosan loaded with bird's nest extract, xanthan gum and distilled water, stir at 26° C. for 2 h, sterilize and cool to obtain the rose bird's nest oral liquid.
[0020] In this embodiment, the preparation method of quaternary ammonium chitosan is as follows: chitosan is dispersed in deionized water, heated to 80°C in an oil bath, and then 2,3-epoxypropyltrimethylammonium chloride is dissolved in deionized water, and then added to the chitosan aqueous solution twice, each time at an interval of 3 hours, and stirred for reaction for 2 hours after all are added dropwise, and then cooled to room temperature, and the reaction solution is precipitated with acetone, and the precipitate is dissolved in deionized water and then filtered to remove insoluble matter, and then the filtrate is dialyzed with deionized water for 2 days, and the molecular weight cut-off is 8000Da. The retentate is freeze-dried to obtain quaternary ammonium chitosan, and the mass ratio of chitosan to 2,3-epoxypropyltrimethylammonium chloride is 1:3.
[0021] Example 2 This example provides a rose bird's nest oral liquid, which comprises the following components by weight: 35 parts of rose flavonoid extract, 35 parts of quaternary ammonium salt chitosan loaded with bird's nest extract, 10 parts of xanthan gum, and 20 parts of distilled water.
[0022] The preparation method of the rose bird's nest oral liquid of this embodiment is the same as that of Example 1.
[0023] Example 3 This example provides a rose bird's nest oral liquid, which comprises the following components by weight: 32 parts of rose flavonoid extract, 32 parts of quaternary ammonium salt chitosan loaded with bird's nest extract, 9 parts of xanthan gum, and 19 parts of distilled water.
[0024] The preparation method of the rose bird's nest oral liquid of this embodiment is the same as that of Example 1.
[0025] Example 3 The bird's nest acid glycoprotein obtained in step S2 of Example 1 was directly digested with trypsin, and the specific digestion process was the same as that of Example 1 to obtain an unmodified bird's nest extract.
[0026] The bird's nest extract obtained in step S3 of Example 1 and the above-mentioned unmodified bird's nest extract were respectively measured, and the contents of free bird's nest acid, polysaccharide bird's nest acid and protein bird's nest acid were determined by OPD pre-column derivatization method. The bird's nest extract obtained in step S3 of Example 1 was recorded as the experimental group, and the unmodified bird's nest extract was recorded as the control group. The experimental results are shown in Table 1.
[0027] The determination method of free sialic acid is as follows: take 1 mL of sample, add 1 mL of 20 mg / mL o-phenylenediamine hydrochloride solution, place in a light-proof water bath at 50°C for 2.5 hours, change the temperature to 4°C and continue to derivatize for 48 hours, filter with a water filter membrane and then enter the OPD column.
[0028] Determination method of polysaccharide-state sialic acid: Take 1 mL of sample, add Sevage reagent (V (chloroform): V (n-butanol) = 4:1) at a volume ratio of 1:1, extract repeatedly to remove protein, then add 1 mL of 20 mg / mL o-phenylenediamine hydrochloride solution, place in a light-proof water bath at 80°C for 40 minutes, filter with a water filter membrane and then enter the OPD column.
[0029] For determination of protein-based sialic acid, take 1 mL of sample for acid hydrolysis and determine the total amount of sialic acid in the clear solution.
[0030] Table 1 Determination results of bird's nest acid in modified and unmodified bird's nest extracts It can be seen from Table 1 that after the modified bird's nest glycoprotein was digested and hydrolyzed by trypsin, the contents of free bird's nest acid and polysaccharide bird's nest acid in the supernatant were increased.
[0031] Example 4 Take 2 mg of the quaternary ammonium salt chitosan loaded with bird's nest extract obtained in step S4 of Example 1, and dilute it to 10 mL with HBSS balanced salt solution as the sample solution of the experimental group.
[0032] The bird's nest extract in step S3 of Example 1 was freeze-dried to obtain a bird's nest extract powder, and 2 mg of the bird's nest extract powder was diluted to 10 mL with HBSS balanced salt solution as a control group sample solution.
[0033] The culture medium in a 24-well plate inoculated with a 21-day Caco-2 monolayer was used, 0.4 mL of sample solution was added to the AP side, and 0.6 mL of HBSS balanced salt solution was added to the BL side. The TEER values were measured at 0 min, 60 min, and 120 min, respectively. The experimental results are shown in Table 2.
[0034] Table 2 Transmembrane absorption results of quaternary ammonium salt chitosan loaded and unloaded bird's nest extract It can be seen from Table 2 that by loading the bird's nest extract onto quaternary ammonium chitosan, the quaternary ammonium chitosan can temporarily break the intestinal cell absorption barrier, change the integrity of the cell tight junctions, and expand the paracellular transport absorption of protein-state bird's nest acid.
[0035] Example 5 Take 30 parts of quaternary ammonium chitosan loaded with bird's nest extract prepared in Example 1, 8 parts of xanthan gum and 17 parts of distilled water, mix them and stir them at 26°C for 2 hours, sterilize and cool to obtain rose bird's nest oral liquid as the control group; take the rose bird's nest oral liquid prepared in Example 1 as the experimental group.
[0036] Take 2 mL of the rose bird's nest oral liquid from the control group and dilute it to 10 mL with HBSS balanced salt solution as the control group sample solution; take 2 mL of the rose bird's nest oral liquid from the experimental group and dilute it to 10 mL with HBSS balanced salt solution as the experimental group sample solution.
[0037] The experimental method in Example 4 was followed to measure the TEER values at 0 min, 60 min and 120 min. The experimental results are shown in Table 3.
[0038] Table 3 TEER value results of samples in the control group and experimental group As can be seen from Table 3, the rose flavonoids extract also has a certain effect on the change of the integrity of cell tight junctions.
[0039] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.
Claims
1. A method for preparing rose bird's nest oral liquid, characterized in that: The following steps are involved: S1, extracting rose flavonoids extract by organic solvent extraction method; S2, extracting the dry bird's nest powder with distilled water for 3 times to obtain a supernatant, mixing the supernatants, freeze-drying to obtain a bird's nest extract powder, dissolving the bird's nest extract powder in a sodium nitrate buffer solution, separating with a G-150 chromatography column, and then eluting with a sodium nitrate solution, ultrafiltering the eluate, and freeze-drying to obtain bird's nest acid glycoprotein; S3, adding bird's nest acid glycoprotein to the rose flavonoid extract, adjusting the pH to 8.5 with sodium hydroxide solution, stirring and reacting at 50°C for 2 hours, ultrafiltration and freeze-drying after the reaction to obtain modified bird's nest acid glycoprotein, and then digesting with trypsin for 40 minutes, separating the supernatant to obtain the bird's nest extract; S4, adding bird's nest extract to the quaternary ammonium chitosan aqueous solution, stirring evenly, adding sodium tripolyphosphate aqueous solution, stirring and reacting for 1 hour, obtaining a suspension by high-speed centrifugation to obtain a white precipitate, and freeze-drying the white precipitate to obtain a quaternary ammonium chitosan loaded with bird's nest extract; S5. Mix the rose flavonoid extract, quaternary ammonium chitosan loaded with bird's nest extract, xanthan gum and distilled water, stir at 26° C. for 2 h, sterilize and cool to obtain the rose bird's nest oral liquid.
2. The method for preparing the rose bird's nest oral liquid according to claim 1, characterized in that: In step S5, the preparation method of the quaternary ammonium chitosan aqueous solution is as follows: disperse chitosan in deionized water, heat the oil bath to 80°C, then dissolve 2,3-epoxypropyltrimethylammonium chloride in deionized water, add it to the chitosan aqueous solution twice, each time at an interval of 3 hours, stir and react for 2 hours after all are added dropwise, cool to room temperature, precipitate the reaction solution with acetone, dissolve the precipitate in deionized water and then filter to remove insoluble matter, then dialyze the filtrate with deionized water for 2 days, the molecular weight cutoff is 8000Da, the retentate is freeze-dried to obtain quaternary ammonium chitosan, and the quaternary ammonium chitosan is dissolved in deionized water to obtain a quaternary ammonium chitosan aqueous solution.
3. The method for preparing the rose bird's nest oral liquid according to claim 2, characterized in that: The mass ratio of the chitosan to the 2,3-epoxypropyltrimethylammonium chloride is 1:
3.
4. The method for preparing the rose bird's nest oral liquid according to claim 1, characterized in that: In step S1, the dried rose pollen is extracted twice with a 70% ethanol solution, the material-liquid ratio of the first extraction is 1:10, and the material-liquid ratio of the second extraction is 1:
5. The filtrates after the two extractions are mixed and passed through an AB-8 macroporous resin column, the loading flow rate is 1.0 mL / min, and the elution flow rate is 1.0 mL / min.
5. The method for preparing the rose bird's nest oral liquid according to claim 1, characterized in that: In step S2, during the extraction process of the dry bird's nest powder, the solid-liquid ratio of dry bird's nest powder to distilled water is 1:5, and the extraction is carried out at 37°C for 2 hours. The concentration of the sodium nitrate buffer is 0.05 mol / L, the flow rate of the eluent is 0.8 mL / min, and the molecular weight cutoff of the ultrafiltration process is 10 kDa.
6. The method for preparing the rose bird's nest oral liquid according to claim 1, characterized in that: In step S3, 50 mg of syringic acid glycoprotein is added to every 100 mL of rose flavonoid extract, and the molecular weight cutoff during ultrafiltration is 10 kDa.
7. The method for preparing the rose bird's nest oral liquid according to claim 1, characterized in that: In step S4, the mass ratio of quaternized chitosan, bird's nest extract and sodium tripolyphosphate is 3:1:
1.
8. A rose bird's nest oral liquid obtained by the preparation method of the rose bird's nest oral liquid according to claim 1, characterized in that: The weight proportions of the components are: 30-35 parts of rose flavonoid extract, 30-35 parts of quaternary ammonium salt chitosan loaded with bird's nest extract, 8-10 parts of xanthan gum, and 17-20 parts of distilled water.
9. The rose bird's nest oral liquid obtained by the preparation method of the rose bird's nest oral liquid according to claim 8, characterized in that: The weight proportions of the components are: 30 parts of rose flavonoid extract, 30 parts of quaternary ammonium salt chitosan loaded with bird's nest extract, 8 parts of xanthan gum, and 17 parts of distilled water.