Extraction process of rape pollen and application of rape pollen in oral liquid of Kuyuan

Abstract

The invention discloses an extraction process of rape pollen and application of the rape pollen in oral liquid of Kuyuan, and belongs to the technical field of traditional Chinese medicines. The process provided by the invention comprises the following steps: freezing and breaking walls of dried rape pollen, crushing, dipping with ethanol to extract alcohol-soluble substances such as flavone, decocting filter residues with water to extract water-soluble substances such as polysaccharide, concentrating a water extract, adding ethanol and a dual-modified chitosan derivative, settling to remove impurities, and finally combining the alcohol extract and alcohol precipitation supernatant to obtain the full-component extract. Compared with the prior art, the method has the advantages that the alcohol-soluble and water-soluble substances in the rape pollen are synchronously and efficiently extracted, the utilization rate of effective components is increased, the stability and safety of the extract are enhanced, a high-activity, low-cost and high-stability raw material solution is provided for the functional oral liquid, and the requirements of the modern health-care product market are met.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a rapeseed pollen extraction process and its application in Meiyuanchun oral liquid. Background Technology

[0002] Rapeseed pollen, an important raw material in traditional Chinese medicine, has attracted much attention due to its rich content of flavonoids, proteins, polysaccharides, and trace elements, among other bioactive substances. These components exhibit significant synergistic effects in regulating endocrine balance, enhancing immune function, resisting radiation, protecting the liver, and treating prostate diseases. In the modern health product and pharmaceutical fields, rapeseed pollen extract is widely used in oral liquid preparations, such as Meiyuan Chun oral liquid, to enhance the overall efficacy of the product.

[0003] However, despite the promising application prospects of rapeseed pollen, existing extraction processes still face numerous challenges in maximizing the utilization of all pollen components, leading to significant losses of effective ingredients and resource waste. Currently, rapeseed pollen extraction mainly employs single solvents or stepwise processes, but these methods all have inherent drawbacks. For example, in Guo Xuewei and Lu Ting's study on the extraction process of flavonoids and other bioactive substances from rapeseed bee pollen, the direct extraction of total flavonoids using 95% ethanol can achieve a high flavonoid content, but this method can only enrich alcohol-soluble substances, such as flavonoids, and cannot extract water-soluble polysaccharides or trace elements, resulting in the discarding of water-soluble components in the pollen and an overall pollen utilization rate of less than 50%. In addition, the utilization rates of proteins and polysaccharides are also low, failing to fully realize the comprehensive value of pollen.

[0004] On the other hand, CN105326880A discloses a water-soluble extract of rapeseed pollen, its preparation method, and its application, which obtains polysaccharides and other substances through water extraction. For example, Yang Xiaoping, Luo Zuyou, Wu Moucheng, and others optimized the water extraction conditions (material-to-liquid ratio 1:4, water extraction at 90℃) in their study "Research on Extraction Process Conditions of Rapeseed Pollen Polysaccharides," achieving a polysaccharide yield of 1.45%. However, this method neglects alcohol-soluble components, resulting in flavonoids remaining in the filter residue.

[0005] While existing technologies employ stepwise processing methods (such as alcohol extraction followed by discarding the filter residue), none achieve complete component recovery. For instance, CN114129647A discloses an oral liquid and its preparation method. Although rapeseed pollen is used in the oral liquid, it is only one of the raw materials, and the extraction process is not optimized. This results in a large amount of active substances in the pollen (such as trace elements and flavonoids) being discarded in the filter residue, leading to resource waste. Furthermore, most existing technologies target only single substances (such as flavonoids or polysaccharides), neglecting the synergistic effect of alcohol-soluble and water-soluble components in pollen. This not only leads to unstable efficacy of the oral liquid product but also affects the reproducibility of product quality.

[0006] From a process flow perspective, existing technologies mostly employ independent steps (such as alcohol extraction or water extraction) without integrating the process, resulting in wasted solvents and energy. For example, water extraction residue is not reused, and ethanol cannot be recovered, further exacerbating resource waste. In summary, existing rapeseed pollen extraction processes have significant shortcomings in component utilization, process integration, and product quality stability. Therefore, an innovative process is urgently needed to achieve simultaneous and efficient recovery of alcohol-soluble and water-soluble components through segmented extraction and merging, while optimizing the process to reduce costs and energy consumption. This would provide a high-activity, low-cost, stable, and controllable raw material base for functional oral liquids, meeting the demands of the modern health product market. Summary of the Invention

[0007] In order to overcome the shortcomings of the existing technology, the present invention aims to provide a rapeseed pollen extraction process and its application in Meiyuanchun oral liquid.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] The following is a process for extracting rapeseed pollen:

[0010] Take dried rapeseed pollen, freeze it, crush it, put the broken-cell pollen into a multi-functional extraction tank, add ethanol aqueous solution and seal it for soaking, collect the soaking liquid; transfer the filter residue to the next process, add water to the filter residue and reflux to boil, stir continuously, filter while hot, collect the filtrate, concentrate the filtrate, add ethanol aqueous solution and double-modified chitosan derivative to the concentrate and let it stand to settle, filter to remove the precipitate, collect the alcohol precipitation supernatant, combine the soaking liquid and the alcohol precipitation supernatant to obtain rapeseed pollen extract.

[0011] The preparation method of the dual-modified chitosan derivative is as follows:

[0012] 5-Fluoro-2-methoxynicotinic acid and 2,4-butyrosulactone were reacted in N,N-dimethylformamide, and the mixture was washed and dried to obtain a zwitterionic compound. Next, chitosan was dispersed in n-propanol, and an epoxide compound was added to react with it. After washing, dialysis, and drying, modified chitosan was obtained. Finally, the modified chitosan was reacted with a carbonyl-containing compound in dimethyl sulfoxide, and an aqueous solution of the zwitterionic compound was added to continue the reaction. After dialysis and drying, a double-modified chitosan derivative was obtained.

[0013] The dried rapeseed pollen was frozen at -10 to -20°C for 24-72 hours.

[0014] The pulverization refers to pulverizing to a particle size of 10-200 μm.

[0015] The ethanol-water solution is used for sealed impregnation by adding 3-8 times the amount of 70-80wt% ethanol-water solution and impregnating for 24-96 hours.

[0016] The process of adding water and refluxing involves adding 2-6 times the amount of water and refluxing at 85-95℃ for 1-3 hours.

[0017] The filtrate is concentrated to a concentration-to-raprose pollen production feed ratio of 0.5-2:0.5-2.

[0018] The amount of ethanol aqueous solution and dual-modified chitosan derivative added during the static sedimentation process is 1-3 times the mass of the concentrated solution, which is 85-95 wt% ethanol aqueous solution; the amount of dual-modified chitosan derivative added is 0.05-0.1 wt% of the concentrated solution, and the mixture is allowed to settle for 24-72 hours after stirring.

[0019] Preferably, the preparation method of the dual-modified chitosan derivative is as follows, in parts by weight:

[0020] S1. Add 1-3 parts of 5-fluoro-2-methoxynicotinic acid to 30-50 parts of N,N-dimethylformamide, add 8-12 parts of 2,4-butanolactone, stir at 60-70℃ for 10-30 h, cool to room temperature, filter to collect the solid product, wash with N,N-dimethylformamide, transfer the solid to 40-80 parts of anhydrous ethanol, stir at room temperature for 0.5-2 h, filter, wash with anhydrous ethanol 1-3 times, and vacuum dry to obtain a zwitterionic compound;

[0021] S2. Add 8-12 parts of chitosan to 180-220 parts of n-propanol and stir at 50-60℃ for 1-3 hours to obtain a chitosan suspension. Add 18-22 parts of epoxide to 20-30 parts of water and add it dropwise to the chitosan suspension. React at 60-80℃ for 10-30 hours. Collect the precipitate and wash it with anhydrous ethanol 1-3 times to remove impurities. Dialyze the sample with a molecular weight cutoff of 0.4-0.6 kDa to remove small molecule residues. Freeze-dry to obtain modified chitosan.

[0022] S3. Add 0.2-0.4 parts of modified chitosan to 15-25 parts of dimethyl sulfoxide, add 0.3-0.5 parts of carbonyl compound, stir at room temperature for 1-5 hours, heat to 50-60℃, add 2-4 parts of 10-20wt% aqueous solution of zwitterionic compound, continue the reaction at 50-60℃ for 10-30 hours, dialyze the reaction solution with a molecular weight cutoff of 1-1.5kDa to remove unreacted substances, freeze dry to obtain the double-modified chitosan derivative.

[0023] The epoxy compound is at least one of (2,3-epoxypropyl)ethylbis(2-hydroxyethyl)ammonium chloride and 1,2-epoxy-3-(diethylamino)propane.

[0024] The carbonyl-containing compound is at least one of N-hydroxysuccinimide and 1,1'-carbonylbis(2-methylimidazole).

[0025] This invention also discloses the application of the above-mentioned rapeseed pollen extract in Meiyuanchun oral liquid. Specifically, the Meiyuanchun oral liquid comprises 50g of black-boned chicken, 20g of rapeseed pollen extract, 150g of astragalus, 30g of donkey-hide gelatin, 50g of privet fruit, 50g of chuanxiong rhizome, 50g of cyperus rhizome, 50g of wolfberry fruit, 30g of jujube, 2.2g of ferrous lactate, and 180g of sucrose, with water added to make up to 1000mL, thus preparing the Meiyuanchun oral liquid.

[0026] This invention utilizes a segmented extraction process to obtain rapeseed pollen substances, flavonoids, polysaccharides, proteins, and trace elements. These substances synergistically exert their core functions in oral liquid, including regulating endocrine balance, enhancing immune function, improving blood circulation, and providing nutritional support. Specifically, flavonoids play a leading role in hormone regulation and anti-radiation, polysaccharides enhance immunity and protect the liver, and proteins and trace elements work together to repair tissues and alleviate related symptoms.

[0027] The functions of each substance are as follows:

[0028] 5-Fluoro-2-methoxynicotinic acid provides fluorine atoms and methoxy groups, enhancing antioxidant activity (free radical scavenging).

[0029] 2,2,4-Butylsulfonyl lactone introduces a sulfonic acid group through a ring-opening reaction to construct an amphoteric structure.

[0030] N,N-Dimethylformamide, as an anhydrous solvent, promotes the nucleophilic ring-opening reaction between carboxyl groups and sulfonyl lactones.

[0031] Anhydrous ethanol is used for washing and purification to remove unreacted substances and solvent residues.

[0032] Chitosan serves as the basic framework, providing primary amino groups (-NH2) for quaternization modification.

[0033] (2,3-Epoxypropyl)ethylbis(2-hydroxyethyl)ammonium chloride epoxy group ring-opening linker connects quaternary ammonium salt and bishydroxyethyl chain to enhance hydrophilicity and antibacterial properties.

[0034] n-Propanol forms a suspension medium, preventing chitosan from dissolving and promoting a uniform reaction.

[0035] Dissolve the quaternary ammonium salt monomer in water and ensure homogeneous dropwise addition to the chitosan suspension.

[0036] Anhydrous ethanol precipitation and washing remove byproducts and unreacted monomers.

[0037] N-hydroxysuccinimide activates the carboxyl group to generate highly reactive NHS esters, promoting mild esterification.

[0038] Dimethyl sulfoxide (DMSO) is an anhydrous solvent that ensures the stability of NHS esters and prevents hydrolysis failure.

[0039] Compared with existing technologies, it has the following advantages:

[0040] 1) This invention achieves simultaneous extraction of alcohol-soluble and water-soluble substances from rapeseed pollen through a segmented process of "freezing and cell wall disruption, ethanol impregnation, water extraction of filter residue, alcohol precipitation purification, and merging of extracts," overcoming the limitations of existing technologies that only target single components. Compared with existing technologies, the effective utilization rate of pollen is improved, and the residual active substances in the filter residue are significantly reduced, achieving efficient utilization of all pollen components and solving the problems of insufficient pollen utilization and waste of a large number of components in existing technologies.

[0041] 2) This invention employs a segmented extraction and merging process combined with alcohol precipitation purification, effectively concentrating active substances and significantly increasing the content of key components. The high flavonoid content directly improves product quality, and synergistically enhances the effects of regulating endocrine function, resisting radiation, and protecting the liver, along with polysaccharides and proteins. Furthermore, the total flavonoid content exhibits minimal fluctuation, and the process has good reproducibility, ensuring the stability of the product's efficacy.

[0042] 3) This invention optimizes the process flow, integrates the ethanol and water extraction steps, and introduces alcohol precipitation purification, achieving green and efficient production. Freezing-cell disruption reduces mechanical energy consumption, and ethanol impregnation and water extraction can be performed in parallel. Ethanol can be recycled and reused, reducing solvent costs, and the water extraction residue can be converted into feed raw materials, realizing waste resource utilization. The extract can be directly integrated into the Meiyuanchun oral liquid formula without additional purification, making it suitable for large-scale production and meeting the demands of the modern health product market.

[0043] 4) The dual-modified chitosan derivative used in this invention significantly improves the efficiency and product quality of rapeseed pollen extraction through its unique molecular structure design. Its dihydroxyethyl structure not only enhances the hydrophilicity and flexibility of the molecule and optimizes the balance between impurity removal and active ingredient retention, but also protects the active ingredients from oxidative degradation by forming a hydrogen bond network, thus extending the storage stability of the extract. Detailed Implementation

[0044] Main source of materials:

[0045] Rapeseed pollen, grade: food grade, mesh size: 80-120, Lanzhou Waterles Biotechnology Co., Ltd.

[0046] Chitosan, Product No.: BR-024, Grade: Food Grade, Henan Bangrun Chemical Products Co., Ltd.

[0047] All other raw materials used in the embodiments and comparative examples of this invention are commercially available products.

[0048] Example 1

[0049] The following is a process for extracting rapeseed pollen:

[0050] Dry rapeseed pollen was placed in a -15℃ environment and frozen for 48 hours. It was then pulverized in a low-temperature pulverizer to a particle size of 10-100μm. The pulverized pollen was then placed in a multi-functional extraction tank, and 5 times the amount of 75wt% ethanol aqueous solution was added. The mixture was sealed and soaked for 72 hours. The soaking liquid was collected. The filter residue was transferred to the next process. 4 times the amount of water was added to the filter residue, and the mixture was refluxed at 90℃ for 2 hours with continuous stirring. The mixture was filtered while hot, and the filtrate was collected. The filtrate was concentrated to a mass ratio of 1:1 with the rapeseed pollen production feed. 2 times the mass of 90wt% ethanol aqueous solution and 0.08wt% of the mass of the concentrated solution of double-modified chitosan derivative were added to the concentrated solution. After stirring, the mixture was allowed to stand for 48 hours to settle. The precipitate was removed by filtration, and the supernatant of the ethanol precipitation was collected. The ethanol soaking liquid and the supernatant of the ethanol precipitation were combined to obtain the rapeseed pollen extract.

[0051] The preparation method of the dual-modified chitosan derivative is as follows:

[0052] S1. Add 2g of 5-fluoro-2-methoxynicotinic acid to 40g of N,N-dimethylformamide, add 10g of 2,4-butanolactone, stir at 65℃ for 20h, cool to room temperature, filter to collect solid product, wash with N,N-dimethylformamide, transfer solid to 60g of anhydrous ethanol, stir at room temperature for 1h, filter, wash 3 times with anhydrous ethanol, and vacuum dry to obtain zwitterionic compound;

[0053] S2. Add 10g of chitosan to 200g of n-propanol and stir at 55℃ for 2h to obtain a chitosan suspension. Add 20g of (2,3-epoxypropyl)ethylbis(2-hydroxyethyl)ammonium chloride to 25g of water and add it dropwise to the chitosan suspension. React at 70℃ for 24h, collect the precipitate, wash with anhydrous ethanol three times to remove impurities, dialyze with a molecular weight cutoff of 0.5kDa to remove small molecule residues, and freeze-dry to obtain modified chitosan.

[0054] S3. Add 0.3g of modified chitosan to 20g of dimethyl sulfoxide, add 0.4g of N-hydroxysuccinimide, stir at room temperature for 3h, raise the temperature to 55℃, slowly add 3g of 16wt% zwitterionic compound aqueous solution, continue the reaction at 55℃ for 24h, dialyze the reaction solution to remove unreacted substances with a molecular weight cutoff of 1.2kDa, freeze dry to obtain the double-modified chitosan derivative.

[0055] Example 2

[0056] A rapeseed pollen extraction process is basically the same as that in Example 1, except that the preparation method of the double-modified chitosan derivative is different.

[0057] The preparation method of the dual-modified chitosan derivative is as follows:

[0058] S1. Add 2g of 5-fluoro-2-methoxynicotinic acid to 40g of N,N-dimethylformamide, add 10g of 2,4-butanolactone, stir at 65℃ for 20h, cool to room temperature, filter to collect solid product, wash with N,N-dimethylformamide, transfer solid to 60g of anhydrous ethanol, stir at room temperature for 1h, filter, wash 3 times with anhydrous ethanol, and vacuum dry to obtain zwitterionic compound;

[0059] S2. Add 10g of chitosan to 200g of n-propanol and stir at 55℃ for 2h to obtain a chitosan suspension. Add 20g of 1,2-epoxy-3-(diethylamino)propane to 25g of water and add it dropwise to the chitosan suspension. React at 70℃ for 24h, collect the precipitate, wash three times with anhydrous ethanol to remove impurities, dialyze with a molecular weight cutoff of 0.5kDa to remove small molecule residues, and freeze-dry to obtain modified chitosan.

[0060] S3. Add 0.3g of modified chitosan to 20g of dimethyl sulfoxide, add 0.4g of N-hydroxysuccinimide, stir at room temperature for 3h, raise the temperature to 55℃, slowly add 3g of 16wt% zwitterionic compound aqueous solution, continue the reaction at 55℃ for 24h, dialyze the reaction solution to remove unreacted substances with a molecular weight cutoff of 1.2kDa, freeze dry to obtain the double-modified chitosan derivative.

[0061] Example 3

[0062] A rapeseed pollen extraction process is basically the same as that in Example 1, except that the preparation method of the double-modified chitosan derivative is different.

[0063] The preparation method of the dual-modified chitosan derivative is as follows:

[0064] S1. Add 2g of 5-fluoro-2-methoxynicotinic acid to 40g of N,N-dimethylformamide, add 10g of 2,4-butanolactone, stir at 65℃ for 20h, cool to room temperature, filter to collect solid product, wash with N,N-dimethylformamide, transfer solid to 60g of anhydrous ethanol, stir at room temperature for 1h, filter, wash 3 times with anhydrous ethanol, and vacuum dry to obtain zwitterionic compound;

[0065] S2. Add 10g of chitosan to 200g of n-propanol and stir at 55℃ for 2h to obtain a chitosan suspension. Add 20g of (2,3-epoxypropyl)ethylbis(2-hydroxyethyl)ammonium chloride to 25g of water and add it dropwise to the chitosan suspension. React at 70℃ for 24h, collect the precipitate, wash three times with anhydrous ethanol to remove impurities, dialyze with a molecular weight cutoff of 0.5kDa to remove small molecule residues, and freeze-dry to obtain modified chitosan.

[0066] S3. Add 0.3g of modified chitosan to 20g of dimethyl sulfoxide, add 0.4g of 1,1'-carbonylbis(2-methylimidazole), stir at room temperature for 3h, raise the temperature to 55℃, slowly add 3g of 16wt% zwitterionic compound aqueous solution, continue the reaction at 55℃ for 24h, dialyze the reaction solution to remove unreacted substances with a molecular weight cutoff of 1.2kDa, freeze dry to obtain the double-modified chitosan derivative.

[0067] Comparative Example 1

[0068] A rapeseed pollen extraction process is basically the same as that in Example 1, except that the preparation method of the double-modified chitosan derivative is different.

[0069] The preparation method of the dual-modified chitosan derivative is as follows:

[0070] S1. Add 2g of 5-fluoro-2-methoxynicotinic acid to 40g of N,N-dimethylformamide, add 10g of 2,4-butanolactone, stir at 65℃ for 20h, cool to room temperature, filter to collect solid product, wash with N,N-dimethylformamide, transfer solid to 60g of anhydrous ethanol, stir at room temperature for 1h, filter, wash 3 times with anhydrous ethanol, and vacuum dry to obtain zwitterionic compound;

[0071] S2. Add 10g of chitosan to 200g of n-propanol and stir at 55℃ for 2h to obtain a chitosan suspension. Add 20g of 2,3-epoxypropyltrimethylammonium chloride to 25g of water and add it dropwise to the chitosan suspension. React at 70℃ for 24h, collect the precipitate, wash three times with anhydrous ethanol to remove impurities, dialyze with a molecular weight cutoff of 0.5kDa to remove small molecule residues, and freeze-dry to obtain modified chitosan.

[0072] S3. Add 0.3g of modified chitosan to 20g of dimethyl sulfoxide, add 0.4g of N-hydroxysuccinimide, stir at room temperature for 3h, raise the temperature to 55℃, slowly add 3g of 16wt% zwitterionic compound aqueous solution, continue the reaction at 55℃ for 24h, dialyze the reaction solution to remove unreacted substances with a molecular weight cutoff of 1.2kDa, freeze dry to obtain the double-modified chitosan derivative.

[0073] Comparative Example 2

[0074] A rapeseed pollen extraction process is basically the same as that in Example 1, except that the preparation method of the double-modified chitosan derivative is different.

[0075] The preparation method of the dual-modified chitosan derivative is as follows:

[0076] S1. Add 2g of 5-fluoro-2-methoxynicotinic acid to 40g of N,N-dimethylformamide, add 10g of 2,4-butanolactone, stir at 65℃ for 20h, cool to room temperature, filter to collect solid product, wash with N,N-dimethylformamide, transfer solid to 60g of anhydrous ethanol, stir at room temperature for 1h, filter, wash 3 times with anhydrous ethanol, and vacuum dry to obtain zwitterionic compound;

[0077] S2. Add 10g of chitosan to 200g of n-propanol and stir at 55℃ for 2h to obtain a chitosan suspension. Add 20g of (2,3-epoxypropyl)ethylbis(2-hydroxyethyl)ammonium chloride to 25g of water and add it dropwise to the chitosan suspension. React at 70℃ for 24h, collect the precipitate, wash three times with anhydrous ethanol to remove impurities, dialyze with a molecular weight cutoff of 0.5kDa to remove small molecule residues, and freeze-dry to obtain modified chitosan.

[0078] S3. Add 0.3g of modified chitosan to 20g of dimethyl sulfoxide, add 0.4g of N,N'-carbonyldiimidazole, stir at room temperature for 3h, heat to 55℃, slowly add 3g of 16wt% zwitterionic compound aqueous solution, continue the reaction at 55℃ for 24h, dialyze the reaction solution to remove unreacted substances with a molecular weight cutoff of 1.2kDa, freeze dry to obtain the double-modified chitosan derivative.

[0079] Comparative Example 3

[0080] A rapeseed pollen extraction process is basically the same as that in Example 1, except that the double-modified chitosan derivative is replaced with an equal amount of chitosan.

[0081] Comparative Example 4

[0082] A rapeseed pollen extraction process is basically the same as that in Example 1, except that the double-modified chitosan derivative is not added.

[0083] Test Example 1

[0084] Determination of total flavonoids and protein:

[0085] Reagents: Rutin reference standard, sodium nitrite (analytical grade), aluminum nitrate (analytical grade), anhydrous ethanol (analytical grade), sodium hydroxide (analytical grade), methanol (analytical grade).

[0086] Instruments: Ultraviolet spectrophotometer, ultrasonic cleaner.

[0087] Preparation of reference solution: Accurately weigh 15.0 mg of rutin reference standard dried to constant weight at 105℃, add methanol and dilute to 100 mL in a volumetric flask to obtain 150 μg / mL.

[0088] Preparation of the standard curve: Accurately pipette 0, 0.50, 1.00, 2.00, 3.00, and 4.00 mL of rutin standard solution (equivalent to 0.75, 150, 300, 450, and 600 μg of rutin) into 25 mL graduated colorimetric tubes. Add 30% ethanol solution to a final volume of 5 mL, add 1.0 mL of 5% nitrous acid solution, shake, and let stand for 6 min. Add 1.0 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Add 10 mL of 1.0 mol / L sodium hydroxide solution, and dilute to the mark with 30% ethanol. Using the zero tube as a blank, shake well and measure the absorbance at 510 nm using a 1 cm cuvette. Plot the standard curve of rutin content (μg) versus absorbance.

[0089] Determination: Accurately pipette 5 mL of the rapeseed pollen extract prepared in this invention into a 50 mL volumetric flask, add approximately 80% methanol solution, sonicate for 0.5 h, remove, cool, add methanol to the mark, shake well, filter, pipette 3.0 mL of the filtrate, transfer to a 25 mL graduated colorimetric tube, add 30% ethanol to 5 mL, add 1.0 mL of 5% sodium nitrite solution, shake and let stand for 6 min, add 1.0 mL of 10% aluminum nitrate solution, shake well and let stand for 6 min, add 10 mL of 1.0 mol / L sodium hydroxide solution, dilute to the mark with 30% ethanol, shake well, and measure the absorbance at 510 nm using a 1 cm cuvette. Take another 3.0 mL and transfer to a 25 mL graduated colorimetric tube, dilute to the mark with 30% ethanol as a blank control. Calculate the flavonoid content in the sample solution using the standard curve.

[0090] Protein levels were tested according to GB5009.5-2016, "National Food Safety Standard - Determination of Protein in Food".

[0091] Stability test: The rapeseed pollen extracts of the examples and comparative examples were placed at 25°C for 60 days, and then the flavonoid content in the sample solution was determined using the above method.

[0092] The results are shown in Table 1.

[0093] Table 1

[0094]

[0095] The higher the total flavonoid content, the better the quality and the better the extraction of effective components from rapeseed flowers.

[0096] Test Example 2

[0097] Stability test: The rapeseed pollen extracts of the examples and comparative examples were placed at 35°C for 60 days, and then the contents of flavonoids and proteins in the sample solutions were determined using the method of Test Example 1.

[0098] The results are shown in Table 2.

[0099] Table 2

[0100]

[0101]

[0102] Test Example 3

[0103] Safety toxicology assessment

[0104] The experiments were conducted in accordance with the "Procedures and Methods for Food Safety Toxicology Evaluation" (2015 revised edition) issued by the National Health Commission. The specific experimental methods and results are summarized below:

[0105] a. Acute toxicity test: Experimental animals were given a single dose of rapeseed pollen extract prepared in this invention, equivalent to 150 times the recommended human dose. No significant symptoms of poisoning or adverse reactions were observed during the observation period. No animal deaths were recorded during two weeks of continuous monitoring, indicating that the beverage belongs to the low toxicity category.

[0106] b. Genetic toxicity assessment: A series of genetic toxicity tests were conducted, including the modified Ames test, mouse peripheral blood cell micronucleus test, and male mouse sperm cell morphology observation experiment. All three test results were negative, indicating that the beverage does not have genetic toxicity and will not cause gene mutations.

[0107] c. Subchronic feeding study: A 35-day continuous feeding experiment was conducted on rats, and the overall health status, blood biochemical indicators, and histopathological changes of important organs of the rats were closely monitored. No significant adverse effects or abnormal indicators were found during the experiment, indicating that long-term intake of this rapeseed pollen extract has no negative impact on the health and physiological function of rats.

[0108] The (2,3-epoxypropyl)ethyl bis(2-hydroxyethyl)ammonium chloride used in Example 1 significantly improves the performance of the modified chitosan derivative in rapeseed pollen extraction due to the synergistic effect of its bishydroxyethyl structure, enhanced hydrophilicity, protection of active ingredients, and optimized structural flexibility. This structure increases molecular hydrophilicity, allowing for uniform dispersion in the alcohol precipitation system. It efficiently adsorbs colloidal impurities through charge neutralization, reducing the encapsulation of flavonoids and polysaccharides by impurities, thereby increasing the initial yield. Simultaneously, the hydroxyl groups form a hydrogen bond network with the pollen's active ingredients, creating a spatial barrier during sedimentation, reducing the loss of active ingredients with the precipitate, and blocking oxygen permeation during storage, inhibiting oxidative degradation. Furthermore, the bishydroxyethyl chain imparts higher flexibility to the molecule, avoiding excessive flocculation caused by excessive rigidity, thus balancing impurity removal and active ingredient retention.

[0109] In the preparation of dual-modified chitosan derivatives, the core advantage of N-hydroxysuccinimide lies in the synergistic effect of its highly reactive intermediate and molecular symmetry. The intermediate generated by the reaction of N-hydroxysuccinimide with the carboxyl group has extremely strong electrophilicity, which can precisely target the primary hydroxyl groups of modified chitosan and efficiently form stable ester bonds under mild conditions, reducing the damage of side reactions to the active sites of flavonoids / proteins. Its molecular symmetry ensures the uniformity of modification, avoids molecular chain distortion caused by local over-substitution, thereby maintaining structural integrity and improving thermal stability.

Claims

1. A rapeseed pollen extraction process, characterized in that, The process is as follows: Take dried rapeseed pollen, freeze it, crush it, put the broken pollen into a multi-functional extraction tank, add ethanol aqueous solution and seal it for soaking, collect the soaking liquid; transfer the filter residue to the next process, add water to the filter residue and reflux to boil, stir continuously, filter while hot, collect the filtrate, concentrate the filtrate, add ethanol aqueous solution and double-modified chitosan derivative to the concentrate and let it stand to settle, filter to remove the precipitate, collect the alcohol precipitation supernatant, combine the soaking liquid and the alcohol precipitation supernatant to obtain rapeseed pollen extract; The preparation method of the dual-modified chitosan derivative is as follows: 5-Fluoro-2-methoxynicotinic acid and 2,4-butyrosulactone were reacted in N,N-dimethylformamide, and the mixture was washed and dried to obtain a zwitterionic compound. Next, chitosan was dispersed in n-propanol, and an epoxide compound was added to react with it. After washing, dialysis, and drying, modified chitosan was obtained. Finally, the modified chitosan was reacted with a carbonyl-containing compound in dimethyl sulfoxide, and an aqueous solution of the zwitterionic compound was added to continue the reaction. After dialysis and drying, a double-modified chitosan derivative was obtained.

2. The rapeseed pollen extraction process as described in claim 1, characterized in that, The dried rapeseed pollen was frozen at -10 to -20°C for 24-72 hours.

3. The rapeseed pollen extraction process as described in claim 1, characterized in that, The pulverization refers to pulverizing to a particle size of 10-200 μm.

4. The rapeseed pollen extraction process as described in claim 1, characterized in that, The ethanol-water solution is used for sealed impregnation by adding 3-8 times the amount of 70-80wt% ethanol-water solution and impregnating for 24-96 hours.

5. The rapeseed pollen extraction process as described in claim 1, characterized in that, The process of adding water and refluxing involves adding 2-6 times the amount of water and refluxing at 85-95℃ for 1-3 hours.

6. The rapeseed pollen extraction process as described in claim 1, characterized in that, The filtrate is concentrated to a concentration-to-raprose pollen production feed ratio of 0.5-2:0.5-2.

7. The rapeseed pollen extraction process as described in claim 1, characterized in that, The amount of ethanol aqueous solution and dual-modified chitosan derivative added during the static sedimentation process is 1-3 times the mass of the concentrated solution, which is 85-95 wt% ethanol aqueous solution; the amount of dual-modified chitosan derivative added is 0.05-0.1 wt% of the concentrated solution, and the mixture is allowed to settle for 24-72 hours after stirring.

8. The rapeseed pollen extraction process as described in claim 1, characterized in that, The preparation method of the dual-modified chitosan derivative is as follows, in parts by weight: S1. Add 1-3 parts of 5-fluoro-2-methoxynicotinic acid to 30-50 parts of N,N-dimethylformamide, add 8-12 parts of 2,4-butanolactone, stir at 60-70℃ for 10-30 h, cool to room temperature, filter to collect the solid product, wash with N,N-dimethylformamide, transfer the solid to 40-80 parts of anhydrous ethanol, stir at room temperature for 0.5-2 h, filter, wash with anhydrous ethanol 1-3 times, and vacuum dry to obtain a zwitterionic compound; S2. Add 8-12 parts of chitosan to 180-220 parts of n-propanol and stir at 50-60℃ for 1-3 hours to obtain a chitosan suspension. Add 18-22 parts of epoxide to 20-30 parts of water and add it dropwise to the chitosan suspension. React at 60-80℃ for 10-30 hours. Collect the precipitate and wash it with anhydrous ethanol 1-3 times to remove impurities. Dialyze the sample with a molecular weight cutoff of 0.4-0.6 kDa to remove small molecule residues. Freeze-dry to obtain modified chitosan. S3. Add 0.2-0.4 parts of modified chitosan to 15-25 parts of dimethyl sulfoxide, add 0.3-0.5 parts of carbonyl compound, stir at room temperature for 1-5 hours, heat to 50-60℃, add 2-4 parts of 10-20wt% aqueous solution of zwitterionic compound, continue reaction at 50-60℃ for 10-30 hours, dialyze the reaction solution with a molecular weight cutoff of 1-1.5kDa to remove unreacted substances, freeze dry to obtain double-modified chitosan derivative; The epoxy compound is at least one of (2,3-epoxypropyl)ethylbis(2-hydroxyethyl)ammonium chloride and 1,2-epoxy-3-(diethylamino)propane; The carbonyl-containing compound is at least one of N-hydroxysuccinimide and 1,1'-carbonylbis(2-methylimidazole).

9. A rapeseed pollen extract, characterized in that, It is prepared using the extraction process described in any one of claims 1-8.

10. The application of the rapeseed pollen extract as described in claim 9 in Meiyuanchun oral liquid.

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