Propolis low-temperature gradient extraction method based on lysine aqueous solution as well as product and application thereof

Abstract

The invention discloses a propolis low-temperature gradient extraction method based on a lysine aqueous solution as well as a product and application of the propolis low-temperature gradient extraction method. According to the invention, the lysine aqueous solution is cooperated with the low-temperature ultrasonic-centrifugal technology to realize efficient reservation of active components of propolis and essential improvement of water solubility, and the obtained product can be applied to development of immunomodulators, functional foods and anti-inflammatory and anti-oxidation pharmaceutical preparations. Through a low-temperature (less than or equal to 60 DEG C) ultrasonic-assisted gradient centrifugation technology, the total flavone extraction rate is greater than or equal to 13%, and the relative dissolution rate of total phenols is greater than or equal to 120%; extraction, dewaxing and purification are completed through a one-step method, and the working time is shortened to 1 / 3 of that of a traditional process; the propolis extract with the solubility greater than or equal to 830 mg / mL is obtained, and the medical-grade requirement of an immunomodulator (the dilution ratio is 1: 1000) is met.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a low-temperature gradient extraction method for propolis based on lysine aqueous solution, its products, and applications. Background Technology

[0002] Current propolis extraction technologies primarily rely on organic solvents (such as ethanol and acetone) as the core extraction medium. For example, patent CN1839890A uses a mixed solution of ethanol and acetone for reflux extraction. While this can partially dissolve the active components of propolis (such as flavonoids and phenolic acids), it has the following significant drawbacks: poor solvent residue and biocompatibility: when the ethanol extract (70% concentration) is mixed with the aqueous phase, flocculation and precipitation occur (turbidity > 100 NTU), making it unsuitable for direct use in injectable formulations; the residual acetone content (GC detection > 500 ppm) exceeds the ICH Q3C standard limit (300 ppm). Loss of active ingredients: traditional processes require multiple reflux dewaxing with petroleum ether (as in patent CN1839890A), but some fat-soluble active components (such as terpenes) may be removed along with the beeswax during the beeswax removal process, resulting in insufficient extraction rate of flavonoids (total flavonoid extraction rate is usually below 11.5%).

[0003] To improve the water solubility of propolis, existing technologies mostly rely on chemical modification or complex excipient systems, but these present issues of biosafety and process efficiency: Polyethylene glycol (PEG) dependence: Patent CN118079442 A uses PEG as a carrier, mixing it with propolis extract through heating and ultrasound to form a solid dispersion. While this improves water solubility, PEG may introduce non-natural components, leading to metabolic burden with long-term use. Furthermore, high-temperature treatment can easily destroy heat-sensitive active ingredients (such as some terpenes). Acid-base modification risks: Some technologies (such as patent CN113855617A) adjust the pH of propolis extract with strong acids / bases to enhance solubility. However, strong acid and alkali environments can easily lead to the oxidative degradation of flavonoids and may leave harmful ions, increasing the risk of formulation toxicity.

[0004] High beeswax content (approximately 30%) is a key factor limiting the purification efficiency of propolis. Existing dewaxing technologies, such as patent CN1839890A, use high-temperature reflux extraction with petroleum ether, which can remove some beeswax. However, the process requires high temperatures (60-70℃) and multiple repeated operations, leading to thermal degradation of flavonoids and significant solvent residue issues. Newer room-temperature dewaxing methods (such as patent CN106213387A) utilize stepwise extraction with petroleum ether at 25℃, but still require multiple ultrasonic vibrations and filtrations, making the process cumbersome and time-consuming, and limiting the retention rate of phenolic acids in propolis.

[0005] Some technologies attempt to combine multiple extraction methods (such as CO2 supercritical extraction with ethanol leaching and eutectic solvent extraction). For example, patent CN118807253A uses ethanol aqueous solution for initial leaching, followed by supercritical extraction combined with eutectic solvent for secondary extraction. Although this yields a high recovery rate of effective components, the equipment cost is high and the process is complex (requiring freeze pulverization and homogenization), making it difficult to scale up and failing to solve the water solubility compatibility problem.

[0006] Existing propolis preparations (such as soft capsules and drops) mostly rely on oil-based systems (such as soybean oil mixed with ethanol extract, patent CN102940648A). Although this masks the unpleasant odor of propolis, its poor water solubility leads to low bioavailability, and there is a risk of excessive heavy metals (such as lead residue), which limits its application in high-purity pharmaceutical fields such as immunomodulators.

[0007] Existing propolis extraction and modification technologies suffer from the following core defects: solvent dependence and safety issues: residual organic solvents such as ethanol and acetone, as well as strong acid / alkali modification, lead to poor biocompatibility; loss of active ingredients: active ingredients (flavonoids, phenolic acids) are severely lost during dewaxing and extraction processes; insufficient solubility: reliance on non-natural excipients (such as PEG) or complex processes makes it difficult to meet the requirements of pharmaceutical-grade applications; process complexity and cost: multi-step, high-temperature / high-pressure operations limit large-scale production. Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide a low-temperature gradient extraction method for propolis based on lysine aqueous solution.

[0009] Another technical problem that this invention aims to solve is to provide a high-purity water-soluble propolis powder obtained by the above extraction method.

[0010] Another technical problem that this invention aims to solve is to provide the application of propolis water-soluble powder in immunomodulators, growth promoters, or anti-inflammatory agents.

[0011] The final technical problem to be solved by this invention is to provide an immunomodulator or anti-inflammatory agent.

[0012] Technical Solution: To solve the above-mentioned technical problems, this invention provides a low-temperature gradient extraction method for propolis based on lysine aqueous solution, comprising the following steps:

[0013] (1) Raw material pretreatment and sample extraction: The propolis raw material was frozen and embrittled at low temperature, pulverized and sieved to obtain uniform propolis fine powder; the propolis fine powder was added to lysine aqueous solution and ultrasonically extracted to obtain propolis extract A;

[0014] (2) Dewaxing and impurity removal: Cool the propolis extract A, centrifuge to remove the bottom precipitate and surface wax components, collect the intermediate clear phase, and then filter it under reduced pressure through qualitative filter paper to finally obtain clear and transparent propolis extract B.

[0015] (3) Preparation of water-soluble propolis powder: Propolis extract B was freeze-dried to remove moisture, and finally water-soluble propolis powder was obtained.

[0016] The low temperature mentioned in step (1) is -20 to 0℃.

[0017] In step (1), the concentration of the lysine aqueous solution is 2-15% (w / v). Preferably, the concentration of the lysine aqueous solution is 4-8% (w / v).

[0018] In step (1), the solid-liquid ratio of the propolis powder to the lysine aqueous solution is 1:2 to 1:50 (g / mL). Preferably, the solid-liquid ratio of the propolis powder to the lysine aqueous solution is 1:8 to 1:25 (g / mL).

[0019] In step (1), the ultrasonic temperature is 20–60°C and the ultrasonic power is 240–400W. Preferably, the ultrasonic temperature is 25–60°C and the ultrasonic power is 240–320W.

[0020] In step (2), the centrifugation temperature is 1–10°C, the centrifugation speed is 10,000–15,000 rpm, and the centrifugation time is 10–20 min.

[0021] The specific steps of freeze drying in step (3) are as follows: First, the product is pre-frozen by rapid freezing at -45℃ to -35℃ for 1.5 to 3 hours, and then transferred to a freeze dryer. The first drying is carried out under the following conditions: vacuum pressure 15 to 30 Pa, cold trap temperature ≤ -55℃, shelf temperature starts at -40℃ to -35℃ and slowly rises to -25℃ to -15℃, and is maintained for 10 to 15 hours. Then, a second drying is carried out: vacuum pressure 5 to 15 Pa, shelf temperature at 20℃ to 55℃, for 6 to 10 hours, in order to fully remove residual moisture and finally obtain high-purity propolis water-soluble powder with lysine as a co-solvent.

[0022] Preferably, the freeze-drying process involves the following steps: First, pre-freezing is performed by rapid freezing at -40°C for 2 hours. Then, the product is transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours. A second drying process is then performed: vacuum pressure 8 Pa, shelf temperature between 20°C and 50°C, for 8 hours, to thoroughly remove residual moisture, ultimately obtaining high-purity propolis water-soluble powder with lysine as a co-solvent.

[0023] This composite system increases the total flavonoid dissolution rate of propolis to 127.31% ± 1.19% (while the dissolution rate of traditional ethanol reflux extraction is 100%).

[0024] The present invention also includes a water-soluble propolis powder prepared by the extraction method described above, using lysine as a co-solvent.

[0025] The present invention also includes the application of the propolis water-soluble powder with lysine as a co-solvent in the preparation of immunomodulators or anti-inflammatory agents.

[0026] The present invention also includes an immunomodulator or anti-inflammatory agent, wherein the immunomodulator or anti-inflammatory agent comprises the propolis water-soluble powder with lysine as a cosolvent.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention achieves efficient retention of propolis active ingredients and essential improvement of water solubility by using lysine aqueous solution in combination with low temperature ultrasonic-centrifugation technology. The resulting product can be applied to the development of immunomodulators, functional foods and anti-inflammatory and antioxidant pharmaceutical preparations.

[0028] 1. Enhanced safety: FDA GRAS certified lysine aqueous solution is used to replace organic solvents, eliminating the risk of solvent residue while achieving targeted dissolution of multiple components of propolis;

[0029] 2. Component preservation: Low-temperature (≤60℃) ultrasonic-assisted gradient centrifugation technology is used to ensure the high extraction rate of total flavonoids.

[0030] ≥13%, relative dissolution rate of total phenols ≥120%;

[0031] 3. Technological Innovation: One-step simultaneous extraction-dewaxing-purification. This process achieves a breakthrough by simultaneously extracting, dewaxing, and purifying propolis: (1) Simultaneous extraction and dewaxing: While extracting the effective components of propolis with lysine solution, the dewaxing process is completed simultaneously, completely eliminating the step of using organic solvents such as petroleum ether for dewaxing in the traditional process. (2) Direct extraction, avoiding loss: Propolis is directly extracted with lysine solution, eliminating the pre-extraction step with solvents such as ethanol, thus effectively avoiding component loss caused by this step. (3) Significantly improved efficiency: The overall process time is greatly reduced to 1 / 3 of the traditional method.

[0032] 4. Application Expansion: Obtain a water-soluble propolis extract with a solubility ≥830mg / mL using lysine as a cosolvent, and obtain a propolis extract that meets the standards for water for injection in the Chinese Pharmacopoeia (bacterial endotoxin <0.25EU / mg), meeting the needs of immunomodulators or anti-inflammatory agents. Detailed Implementation

[0033] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained through commercial purchases.

[0034] Example 1

[0035] A low-temperature gradient high-efficiency extraction process for propolis based on lysine aqueous solution includes the following steps:

[0036] (1) Pretreatment and sample extraction:

[0037] The propolis raw material (purchased from Linqing Lvdu Bee Products Co., Ltd., item number: FJ20241225) was frozen and brittled at 0℃, then pulverized and passed through an 80-mesh sieve to obtain uniform propolis fine powder. Subsequently, 1.0 g of the propolis fine powder was added to a 2% (w / v) lysine aqueous solution, controlling the solid-liquid ratio at 1:2 g / mL, and ultrasonic-assisted extraction was performed at 20℃ (ultrasonic power 240W, time 30 min) to finally obtain propolis extract A.

[0038] (2) Dewaxing and impurity removal:

[0039] Propolis extract A was cooled to 4°C and then centrifuged at 10,000 rpm for 10 minutes at 4°C. After centrifugation, the bottom precipitate and surface waxy components were carefully removed, and the intermediate clear phase was collected. This was then filtered under reduced pressure through qualitative filter paper to obtain a clear and transparent propolis extract B.

[0040] (3) Propolis water-soluble powder:

[0041] Propolis extract B was freeze-dried. First, it was pre-frozen by rapid freezing at -40°C for 2 hours; then it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours; then a second drying was performed: vacuum pressure 8 Pa, shelf temperature in the range of 20°C to 50°C for 8 hours to fully remove residual moisture, finally obtaining high-purity water-soluble propolis powder with lysine as a co-solvent.

[0042] Example 2

[0043] A low-temperature gradient high-efficiency extraction process for propolis based on lysine aqueous solution includes the following steps:

[0044] (1) Pretreatment and sample extraction:

[0045] The propolis raw material was frozen and brittled at -20℃, then pulverized and passed through an 80-mesh sieve to obtain uniform propolis powder. Subsequently, 0.5g of the propolis powder was added to an 8% (w / v) lysine aqueous solution, with a solid-liquid ratio of 1:25g / mL, and ultrasonic-assisted extraction was performed at 40℃ (ultrasonic power 320W, time 60min) to finally obtain propolis extract A.

[0046] (2) Dewaxing and impurity removal:

[0047] Propolis extract A was cooled to 1°C and then centrifuged at 12,000 rpm for 15 minutes at 1°C. After centrifugation, the bottom precipitate and surface waxy components were carefully removed, and the intermediate clear phase was collected. Then, it was filtered under reduced pressure through qualitative filter paper to finally obtain clear and transparent propolis extract B.

[0048] (3) Propolis water-soluble powder:

[0049] Propolis extract B was freeze-dried. First, it was pre-frozen by rapid freezing at -40°C for 2 hours; then it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours; then a second drying was performed: vacuum pressure 8 Pa, shelf temperature in the range of 20°C to 50°C for 8 hours to fully remove residual moisture, finally obtaining high-purity water-soluble propolis powder with lysine as a co-solvent.

[0050] Example 3

[0051] A low-temperature gradient high-efficiency extraction process for propolis based on lysine aqueous solution includes the following steps:

[0052] (1) Pretreatment and sample extraction:

[0053] The propolis raw material was frozen and brittled at -4℃, then pulverized and passed through an 80-mesh sieve to obtain uniform propolis powder. Subsequently, 0.5g of the propolis powder was added to a 15% (w / v) lysine aqueous solution, with a solid-liquid ratio of 1:50g / mL, and ultrasonic-assisted extraction was performed at 60℃ (ultrasonic power 400W, time 60min) to finally obtain propolis extract A.

[0054] (2) Dewaxing and impurity removal:

[0055] Propolis extract A was cooled to 8°C and then centrifuged at 10°C and 15,000 rpm for 20 min. After centrifugation, the bottom precipitate and surface waxy components were carefully removed, and the intermediate clear phase was collected. This was then filtered under reduced pressure through qualitative filter paper to obtain a clear and transparent propolis extract B.

[0056] (3) Preparation of water-soluble propolis powder:

[0057] Propolis extract B was freeze-dried. First, it was pre-frozen by rapid freezing at -40°C for 2 hours; then it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours; then a second drying was performed: vacuum pressure 8 Pa, shelf temperature in the range of 20°C to 50°C for 8 hours to fully remove residual moisture, finally obtaining high-purity water-soluble propolis powder with lysine as a co-solvent.

[0058] Example 4

[0059] A low-temperature gradient high-efficiency extraction process for propolis based on lysine aqueous solution includes the following steps:

[0060] (1) The propolis raw material was frozen and brittled in a low temperature environment of 0℃, pulverized and sieved through an 80-mesh sieve to obtain uniform propolis fine powder. Then, it was added to a 4% (w / v) lysine aqueous solution at a solid-liquid ratio of 1:8 (g / mL) of propolis fine powder: lysine, stirred evenly, and ultrasonically extracted at 240W power and 60℃ for 60 min to obtain propolis extract A.

[0061] (2) Cool the propolis extract A to 4°C, then centrifuge at 4°C and 10,000 rpm for 15 min. After centrifugation, carefully remove the bottom precipitate and the surface waxy components, collect the intermediate clear phase, and then filter it under reduced pressure through qualitative filter paper to finally obtain clear and transparent propolis extract B.

[0062] (3) The propolis extract B was freeze-dried to remove moisture. The specific freeze-drying steps were as follows: First, it was pre-frozen by rapid freezing at -40℃ for 2 hours; then it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20Pa, cold trap temperature -60℃, shelf temperature starting at -40℃ and slowly rising to -20℃, and maintained for 12 hours; then a second drying was carried out: vacuum pressure 8Pa, shelf temperature in the range of 20℃ to 50℃, for 8 hours, to fully remove residual moisture and finally obtain high-purity propolis water-soluble powder with lysine as a co-solvent.

[0063] Comparative Example 1: Propolis Extraction Process Based on Petroleum Ether Dewaxing

[0064] (1) Petroleum ether dewaxing

[0065] The propolis raw material was frozen at low temperature and then pulverized, passing through an 80-mesh sieve to obtain propolis powder. The propolis powder was then wrapped in filter paper and placed in a Soxhlet extractor for siphon extraction using petroleum ether as a solvent (3 cycles). After dewaxing, the filter paper packet was removed, and residual petroleum ether was evaporated to obtain dewaxed fine propolis powder.

[0066] (2) Sample extraction

[0067] Dewaxed propolis powder was added to an 8 w / v % lysine aqueous solution (solid-liquid ratio 1:25 g / mL) and subjected to ultrasonic-assisted extraction at 40℃ (ultrasonic power 120 W, time 30 min) to obtain propolis extract A.

[0068] (3) Impurity removal and purification

[0069] The propolis extract A was cooled to 1°C and centrifuged at 12,000 rpm for 15 minutes. After centrifugation, the bottom precipitate was carefully removed, and the supernatant was collected. Then, it was filtered under reduced pressure through qualitative filter paper to obtain a clear and transparent propolis extract B.

[0070] (4) Preparation of water-soluble propolis powder

[0071] Propolis extract B was freeze-dried. First, it was pre-frozen by rapid freezing at -40°C for 2 hours; then it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours; then a second drying was performed: vacuum pressure 8 Pa, shelf temperature in the range of 20°C to 50°C for 8 hours to fully remove residual moisture, finally obtaining high-purity water-soluble propolis powder with lysine as a co-solvent.

[0072] Comparative Example 2: Propolis Extraction Process Based on Ethanol Reflux Extraction

[0073] (1) Raw material pretreatment and extraction

[0074] The propolis raw material was frozen at low temperature and then pulverized and passed through an 80-mesh sieve to obtain fine propolis powder. Subsequently, the fine propolis powder was added to a 70% ethanol solution (solid-liquid ratio 1:25 g / mL) and extracted by reflux at 90℃ for 60 min to obtain propolis extract A.

[0075] (2) Purification

[0076] Propolis extract A was centrifuged at 12,000 rpm for 15 minutes at 24°C to remove insoluble impurities, and the supernatant was collected. The supernatant was then filtered through qualitative filter paper to obtain clear propolis extract B.

[0077] (3) Preparation of propolis powder

[0078] Propolis extract B was dried under reduced pressure at 65℃ and -0.085 kPa. First, it was pre-frozen by rapid freezing at -40℃ for 2 hours. Then, it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60℃, shelf temperature initially at -40℃ and slowly increased to -20℃, maintained for 12 hours. A second drying process was then performed: vacuum pressure 8 Pa, shelf temperature within the range of 20℃ to 50℃, for 8 hours to thoroughly remove residual moisture, ultimately obtaining high-purity water-soluble propolis powder with lysine as a co-solvent.

[0079] Comparative Example 3: A process for preparing water-soluble propolis based on petroleum ether-ethanol synergistic extraction

[0080] (1) Dewaxing and alcohol extraction of propolis

[0081] After the propolis raw material was cryogenically embrittled at -20℃, it was pulverized using a liquid nitrogen cryogenic pulverizer and passed through an 80-mesh standard sieve to obtain propolis powder. A measured amount of propolis powder was wrapped in special filter paper and placed in a Soxhlet extractor. Petroleum ether with a boiling range of 30-60℃ was used as the solvent (the amount of petroleum ether added was 50 mL / g propolis), and extraction was carried out under continuous reflux at 85℃ for 6 hours. After dewaxing, the filter paper package was removed, and the residual solvent was allowed to evaporate naturally in a fume hood to obtain dewaxed fine propolis powder.

[0082] Accurately weigh 0.5 g of dewaxed propolis powder and add 12.5 mL of 70% (v / v) ethanol solution (solid-liquid ratio 1:25 g / mL). Extract by reflux at 90℃ for 12 hours. After extraction, concentrate the extract under reduced pressure at 45℃ and 0.09 MPa to obtain refined propolis extract.

[0083] (2) Water-soluble modification treatment

[0084] Accurately weigh 20 mg of refined propolis extract and add 2 mL of 3% (w / v) L-lysine aqueous solution. Place the solution in a 50°C constant temperature water bath with a magnetic stirrer and stir at 600 rpm until completely dissolved (approximately 30 minutes). Freeze-dry the resulting solution. First, pre-freeze by rapid freezing at -40°C for 2 hours. Then, transfer the solution to a freeze dryer and perform a first drying under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours. A second drying is then performed: vacuum pressure 8 Pa, shelf temperature within the range of 20°C to 50°C, for 8 hours to thoroughly remove residual moisture, ultimately obtaining a water-soluble propolis powder with lysine as a co-solvent.

[0085] Comparative Example 4: Propolis Extraction Process Based on Other Amino Acids

[0086] A propolis extraction process based on arginine aqueous solution includes the following steps:

[0087] (1) Pretreatment and sample extraction:

[0088] The propolis raw material (purchased from Linqing Lvdu Bee Products Co., Ltd., item number: FJ20241225) was frozen and brittled at 0℃, then pulverized and passed through an 80-mesh sieve to obtain uniform propolis powder. Subsequently, 1.0g of the propolis powder was added to an 8% (w / v) arginine aqueous solution, controlling the solid-liquid ratio at 1:5g / mL, and ultrasonic-assisted extraction was performed at 20℃ (ultrasonic power 240W, time 30min) to finally obtain propolis extract A.

[0089] (2) Dewaxing and impurity removal:

[0090] Propolis extract A was cooled to 4°C and then centrifuged at 10,000 rpm for 10 minutes at 4°C. After centrifugation, the bottom precipitate and surface waxy components were carefully removed, and the middle layer was collected. Then, it was filtered under reduced pressure through qualitative filter paper to obtain propolis extract B.

[0091] (3) Propolis water-soluble powder:

[0092] Propolis extract B was freeze-dried. First, it was pre-frozen by rapid freezing at -40°C for 2 hours; then it was transferred to a freeze dryer and dried under the following conditions: vacuum pressure 20 Pa, cold trap temperature -60°C, shelf temperature initially at -40°C and slowly increased to -20°C, maintained for 12 hours; then a second drying was performed: vacuum pressure 8 Pa, shelf temperature in the range of 20°C to 50°C for 8 hours to fully remove residual moisture, finally obtaining arginine-extracted propolis powder.

[0093] Experimental example:

[0094] 1. Determination of total flavonoid content

[0095] The total flavonoid content in the water-soluble propolis powder samples or propolis powder samples prepared in Examples 1-4 and Comparative Examples 1-4 using lysine as a co-solvent was determined by ultraviolet-visible spectrophotometry. A series of standard solutions of varying concentrations were prepared using rutin as a standard. Aluminum nitrate solution and potassium acetate solution were added, and after reacting for 1 hour, the absorbance was measured at 415 nm to establish a standard curve. 20 mg of each extracted propolis powder sample was accurately weighed, dissolved in water, and then aluminum nitrate solution and potassium acetate solution were added. The solution was diluted to volume with 95% ethanol, reacted for 1 hour, and the absorbance was measured at 415 nm. The regression equation for the rutin standard curve was: Y = 0.0033X + 0.0087(R²). 2 =0.999), where Y is absorbance and X is the concentration of rutin standard (μg / mL), with a linear range of 0 to 350.4 μg / mL, showing good linearity.

[0096] The total flavonoid content was calculated based on the standard curve, and the total flavonoid extraction rate (W,%) was calculated according to formula (1-1). The results are shown in Table 1. The total flavonoid extraction rate of the traditional ethanol reflux extraction method was used as the benchmark value (set as "1"). The extraction rates of other groups were compared with this benchmark value, and the relative dissolution rates of their total flavonoids were calculated according to formula (1-2). The results are shown in Table 1.

[0097]

[0098] In formula (1), W: total flavonoid extraction rate, %; C: concentration on the standard curve corresponding to the absorbance value measured by the sample solution, g / mL; V1: volume of the test solution, mL; V2: volume of the sample to be tested after dilution, mL; V3: volume of the test solution taken when measuring the absorbance value, mL; M: mass of propolis, g.

[0099]

[0100] 2. Determination of total phenols

[0101] The total phenol content in the water-soluble propolis powder samples or propolis powder samples prepared in Examples 1-4 and Comparative Examples 1-4 using lysine as a co-solvent was determined by ultraviolet-visible spectrophotometry, and the relative dissolution rate of total phenols was calculated. Gallic acid was used as a standard to prepare a series of standard solutions of different concentrations. Folin-Ciocalteu reagent and 20% Na₂CO₃ aqueous solution were added, and after reacting for 2 hours, the absorbance was measured at 765 nm to establish a standard curve. 20 mg of each propolis sample was accurately weighed, dissolved in water, and then Folin-Ciocalteu reagent and 20% Na₂CO₃ aqueous solution were added. The solution was then diluted to volume with distilled water, reacted for 2 hours, and the absorbance was measured at 765 nm. The regression equation for the gallic acid standard curve was: Y = 0.0107X + 0.0624(R²). 2=0.9993), where Y is the absorbance and X is the concentration of gallic acid standard (μg / mL). The linear range is 6.9 to 207 μg / mL, showing good linearity.

[0102] The total phenol content was calculated based on the standard curve, and the total phenol yield (W,%) was calculated according to formula (2-1). The results are shown in Table 1. The total phenol extraction rate of the traditional ethanol reflux extraction method was used as the benchmark value (set as "1"). The extraction rates of other groups were compared with this benchmark value (see formula 2-2) to calculate their relative dissolution rates of total phenols. The results are shown in Table 1.

[0103]

[0104] In formula (2), W: total phenol yield, %; C: concentration on the standard curve corresponding to the absorbance value of the sample solution, g / mL; V1: volume of the test solution, mL; V2: volume of the sample solution to be tested after dilution, mL; V3: volume of the test solution taken when measuring the absorbance value, mL; M: mass of propolis, g.

[0105]

[0106] 3. Determination of solubility in water

[0107] The water solubility of the propolis water-soluble powder samples or propolis powder prepared in Examples 1-4 and Comparative Examples 1-4 with lysine as a co-solvent was determined (as per the General Rules of Part I of the Chinese Pharmacopoeia). The sample was weighed and placed in 100 mL of water at 25℃±2℃. The mixture was vigorously shaken for 30 seconds every 5 minutes. The dissolution was observed over 30 minutes. Complete dissolution was considered complete when no visible solute particles or droplets were observed. See formula (3). The results are shown in Table 1.

[0108]

[0109] In the formula: M: weight of the completely dissolved test sample (g)

[0110] 4. DPPH free radical scavenging activity assay

[0111] Prepare a 0.1 mM DPPH anhydrous ethanol solution. Accurately weigh 10 mg of the propolis water-soluble powder sample or propolis powder sample prepared in Examples 1-4 and Comparative Examples 1-4, dissolve in anhydrous ethanol and dilute to 10 mL. Mix 2 mL of the sample solution with 2 mL of DPPH solution, react in the dark for 30 minutes, and then measure the absorbance at 517 nm. Use an equal volume of anhydrous ethanol instead of the sample solution as a blank control, and calculate the free radical scavenging rate according to formula (4). The results are shown in Table 1.

[0112] in:

[0113] A 样品 : Absorbance of the sample solution and DPPH mixture;

[0114] A 背景 : Absorbance of the sample solution mixed with anhydrous ethanol;

[0115] A 溶剂 : Absorbance of the sample extraction solvent and DPPH mixture;

[0116] A 对照 : Absorbance of the mixture of DPPH solution and anhydrous ethanol.

[0117] Table 1. Results of the impact of the examples and comparative examples on various indicators.

[0118]

[0119] Note: Comparative Example 1 vs. Example 2: * P<0.05, P<0.001; Comparative Example 2 vs. Example: # P<0.05, ## P<0.001; Comparative Example 3 vs. Example 1: ※ P<0.05, ※※ P<0.001; Comparative Example 4 vs. Example 1: P<0.05, P<0.001. Regarding the total flavonoid extraction rate, the extraction rates of Examples 1-4 were significantly higher than those of Comparative Examples 1-4 (P<0.001). The relative dissolution rate of total phenols also showed the same result, with the relative dissolution rates of Examples 1-4 significantly higher than those of Comparative Examples 1-4 (P<0.001), indicating that the method of the present invention has a highly significant advantage in extraction efficiency. As for solubility, the water solubility of Examples 1-4 was not significantly different from that of Comparative Examples 1 and 3 (P>0.05), indicating they were easily soluble. The water solubility of Comparative Example 2 was the lowest among all groups, showing a highly significant difference from that of Examples 1-4. ## P<0.001), classifying it as extremely slightly soluble; the water solubility of Comparative Example 4 was improved compared to Comparative Example 2, but was significantly lower than that of Example 4 (P<0.001). P<0.001). Regarding free radical scavenging activity, the activities of Examples 2, 3, and 4 were not significantly different from Comparative Examples 3 and 4, but were significantly higher than Comparative Example 1. * P<0.05), and was significantly higher than that of comparative example 2 (P<0.05). ## P<0.001); the activity of Example 1 was also significantly better than that of Comparative Example 2 (P<0.001); #P<0.05). The total flavonoid extraction rate of propolis in water-soluble powder with lysine as a co-solvent was increased to 127.31% ± 1.19% (the extraction rate of traditional ethanol reflux extraction was 100%). The process of this invention achieves a higher total flavonoid extraction rate (significantly improved) by optimizing extraction parameters; the improved water solubility is also significantly advantageous; and excellent free radical scavenging activity is maintained (Examples 2, 3, and 4 are significantly better than Comparative Examples 1-4).

[0120] In summary, this method is significantly superior to traditional processes in terms of extraction efficiency, product performance, and retention of bioactivity. Application Examples:

[0121] (1) Application of water-soluble propolis powder as an immune enhancer

[0122] The lysine-based water-soluble propolis powder prepared in Example 3 was dissolved in PBS and administered to SPF-grade BALB / c male mice via gavage at doses of 0.125 g / kg, 0.5 g / kg, and 1.25 g / kg body weight (0.2 mL per mouse). The experiment was divided into the following groups: propolis dosage groups: low-dose propolis group (0.125 g / kg); medium-dose propolis group (0.5 g / kg); high-dose propolis group (1.25 g / kg); blank control group and model group: administered an equal volume of PBS via gavage. All groups were administered via gavage once daily for 30 consecutive days. Except for the blank control group, all propolis dosage groups and the model group underwent immunosuppression modeling by intramuscular injection of hydrocortisone (40 mg / kg) on ​​days 22, 24, 26, 28, and 30 (a total of 5 times).

[0123] On day 31 of the experiment, blood was collected from the mice and the animals were euthanized. The test indicators included: spleen index, calculated according to formula (5); peripheral blood leukocyte count; spleen lymphocyte activity index (Cellviabilty): determined by the concanavalin A (ConA) induction method, calculated according to formula (6).

[0124]

[0125] Among them, OD 450nm 1 represents the absorbance of the stimulated cell, OD. 450nm 2 represents the absorbance of unstimulated cells.

[0126] Results Analysis: Compared with the blank control group, the spleen index of mice in the model group (hydrocortisone-induced model group) was significantly reduced (mean value in the model group: (4.38±0.74) (mg / g); mean value in the blank group: (5.51±0.91) (mg / g)), indicating that the model successfully induced immune organ damage. The spleen index of each propolis dosage group was higher than that in the blank control group (range: 5.66–5.84 mg / g). In terms of spleen lymphocyte activity index and peripheral blood leukocyte count, the immunomodulatory activity of the three propolis dosage groups was significantly higher than that in the blank control group and the model group (see Table 2 for details).

[0127] Table 2. Results of the immune-enhancing effect of propolis water-soluble powder with lysine as a co-solvent.

[0128]

[0129] Note: Comparison between the model group and the blank control group. # p<0.05, ## p<0.01; Comparison between each propolis dosage group and the model group, * p<0.05, ** p < 0.01, n = 8.

[0130] (2) Study on the antibacterial effect of water-soluble propolis powder

[0131] The propolis water-soluble powder (10% wt) prepared in Example 1 with lysine as a co-solvent was mixed with glycerol (5% wt) and pure water (85% wt) to prepare a spray containing 10% (w / v) propolis powder (equivalent to 100 mg / mL) for in vitro antibacterial experiments.

[0132] The bacterial strains used in the experiment were *Escherichia coli* (ATCC 25922), *Staphylococcus aureus* (ATCC 6538), and *Streptococcus pneumoniae* (ATCC 49619). Before the experiment, each strain was inoculated into ordinary broth medium and incubated at 37°C for 24 hours for activation. Typical colonies were inoculated into 5 mL of ordinary nutrient broth and incubated at 37°C for 16-18 hours, followed by two subcultures. The third-generation culture was transferred to a 37°C shaker and incubated for approximately 4 hours, adjusting the turbidity to 1×10⁻⁶. 6 CFU / mL, for later use.

[0133] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microbroth dilution method. The specific procedure was as follows: 100 μL of sterile MH broth was added to wells 2 through 9 and well 12 of a 96-well plate. 100 μL of the test drug solution (spray) was added to wells 1, 2, and 10. Starting from well 2, the drug solution was serially diluted up to well 9. Subsequently, 100 μL of the above bacterial solution was added to wells 1 through 9 and well 11. At this point, the drug concentrations in wells 1 through 9 were 50, 25, 12.5, 6.25, 3.125, 1.563, 0.78, 0.39, and 0.195 mg / mL, respectively; well 10 served as the drug control (no bacteria), well 11 as the bacterial control (no drug), and well 12 as the culture medium control (blank). Each experiment was performed in triplicate. The 96-well plate was incubated at 37°C for 24 hours.

[0134] After incubation, 100 μL of liquid was taken from each well and evenly spread onto MH agar plates. The plates were incubated at 37°C for 24 hours, and the results were observed. Judgment criteria: the lowest drug concentration at which colonies showed dispersed growth (not confluent) on the plate was defined as the MIC value; the lowest drug concentration at which no colonies were observed to grow visually was defined as the MBC value. Referring to the CLSIM07-A10 standard: MIC < 4 mg / ml was highly sensitive; 4 mg / ml ≤ MIC ≤ 32 mg / ml was moderately sensitive; and MIC > 32 mg / ml was insensitive.

[0135] The MIC (micron activity) assay results of the propolis water-soluble extract against three bacteria showed that its MIC and MBC (micron-to-micron cross-linking) values ​​were the same against *Escherichia coli* and *Staphylococcus aureus*, at 12.5 mg / mL and 25 mg / mL, respectively, indicating moderate sensitivity. For *Streptococcus pneumoniae*, the MIC and MBC values ​​were 3.125 mg / mL and 6.25 mg / mL, respectively, indicating high sensitivity. In summary, the propolis water-soluble extract exhibited significant in vitro antibacterial effects against *Escherichia coli*, *Staphylococcus aureus*, and *Streptococcus pneumoniae*.

[0136] (3) Evaluation of the therapeutic effect of water-soluble propolis powder on dermatitis in mice

[0137] The propolis water-soluble powder (10% wt) prepared in Example 1 with lysine as a co-solvent was mixed with glycerin (5% wt) and pure water (85% wt) to prepare a spray containing 10% (w / v) propolis powder (equivalent to 100 mg / mL) for use in anti-dermatitis experiments.

[0138] Establishment of a mouse dermatitis model: One day prior to the experiment, mice in each group underwent hair removal on their backs using a hair removal cream, covering an area of ​​approximately 2cm × 2cm. Except for the normal control group, mice in the other groups were sensitized on the day of the experiment by applying 100μL of a 7% 2,4-dinitrochlorobenzene solution in acetone to the shaving area on their backs. Five days after sensitization, 5μL of a 0.5% 2,4-dinitrochlorobenzene solution was applied topically to the inner and outer sides of the right ear to stimulate stimulation. Simultaneously, acetone was applied to the left ear. Stimulation was repeated every 3 days for a total of 4 times. The normal control group mice received the same amount and method of acetone solution applied to their backs and right ears as a control. Successful model establishment was indicated by the appearance of dry, rough, and thickened skin, as well as restlessness.

[0139] Dosage regimen: Forty SPF-grade male BALB / c mice were randomly divided into four groups (n=10 per group): normal control group, model group, Eucerin group, and propolis water-soluble powder spray group. All groups began administration on the day of challenge. The normal control group and model group received distilled water applied to the shaved skin on their backs and the inner and outer sides of their right ears; the Eucerin group received 1.5 mg / cm² propolis powder spray at the same locations. 2 The dosage of Eucerin; the drug group was administered at 1.5 mg / cm². 2 The drug dosage is evenly sprayed with propolis water-soluble powder spray twice a day for 10 consecutive days; if 2,4-dinitrochlorobenzene activation is required on the day of administration, the time interval between administration and activation is 2 hours.

[0140] Detection indicators: Twenty-four hours after the last stimulation, tissue samples were taken. The thickness of the middle part of the right ear was measured using micrometer calipers, and the thickness difference of the right ear before and after stimulation was calculated. A 0.6 cm diameter metal punch was used to drill a hole in the middle of the ear. The mass of each tissue block was weighed using an electronic balance, and the mass difference between the left and right ears was calculated. Twenty-four hours after each stimulation, the right ear of the mice was scored based on edema, erythema, and scratch marks (see Table 3). The average value of each group was taken. One mL of blood was collected from the eyeballs of the mice, allowed to stand at room temperature for 1 hour, centrifuged at 1500 r / min for 20 min, and the serum was placed in a microcentrifuge tube and stored at -80℃ for later use. The serum levels of cytokines such as IL-1β, IL-4, and TNF-α were measured using ELISA kits (ELISA kits purchased from ABclonal), and the procedures were performed according to the instructions of the respective kits.

[0141] Table 3 Ear allergy scoring criteria for mice

[0142]

[0143]

[0144] Results: The weight and thickness of the right ear of mice were measured 24 hours after the last stimulation. The ear swelling in the propolis group (0.26±0.08mm) was significantly lower than that in the model group (0.38±0.08mm, p<0.05), and the therapeutic effect was comparable to that of Eucerin (0.26±0.09mm). The results are shown in Table 4.

[0145] Table 4. Effects of propolis spray on ear weight and thickness in mice with dermatitis

[0146] Group <![CDATA[Dose / mg / cm 2 > Poor ear quality / mg Ear thickness difference / mm normal - 2.00±0.11 0.09±0.05 Model - <![CDATA[8.54±0.78 ## ]]> <![CDATA[0.38±0.08 ## ]]> Yu Zhuoer 1.5 <![CDATA[3.80±0.61 * ]]> <![CDATA[0.26±0.09 * ]]> Propolis spray 1.5 <![CDATA[3.78±0.77 * ]]> <![CDATA[0.26±0.08 * ]]>

[0147] Note: Comparison between the model group and the blank control group. # p<0.05, ## p<0.01; Compared with the model group, the propolis spray group... * p<0.05, ** p<0.01, n=10.

[0148] Effects of propolis spray on ear allergy scores in mice with dermatitis: After four provocations, the right ear of the dermatitis model group showed significant redness, swelling, and crusting. Treatment with propolis spray significantly improved the symptoms. Compared with the normal group, ear allergy scores increased to varying degrees after the first provocation, while the ear allergy score of the model group increased significantly from the first to the last provocation (P<0.01). Compared with the model group, both the propolis spray group and the Eucerin group significantly reduced the swelling of the right ear in mice with dermatitis (P<0.05). The results are shown in Table 5.

[0149] Table 5. Effects of propolis spray on ear allergy scores in mice with dermatitis

[0150]

[0151] Note: Comparison between the model group and the blank control group. # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01, n=10.

[0152] Effects of propolis spray on serum inflammatory factors in mice with dermatitis: Compared with the normal group, the model group showed significantly increased serum levels of inflammatory factors IL-1β and TNF-α (P<0.01), significantly decreased levels of anti-inflammatory factor IL-4 (P<0.01), and a significantly increased IFN-γ / IL-4 ratio (P<0.01), indicating disruption of immune homeostasis. Compared with the model group, the propolis spray group and the Eucerin group showed significantly decreased serum levels of IL-1β and TNF-α (P<0.05), significantly increased levels of anti-inflammatory factor IL-4 (P<0.05), and a significantly increased IFN-γ / IL-4 ratio (P<0.01), indicating some recovery of immune homeostasis. See Table 6.

[0153] Table 6. Effects of propolis spray on serum cytokines in mice with dermatitis

[0154] Note: Comparison between the model group and the blank control group. # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01, n=10.

[0155] Propolis spray has a certain therapeutic effect on dermatitis. It can effectively relieve swelling of the right ear tissue and improve symptoms of inflammation and edema. It may work by downregulating the expression of pro-inflammatory factors, upregulating the expression of anti-inflammatory factors, and correcting immune homeostasis.

Claims

1. A method for low-temperature gradient extraction of propolis based on lysine aqueous solution, characterized in that, Includes the following steps: (1) Raw material pretreatment and sample extraction: The propolis raw material was frozen and brittled at low temperature, pulverized and sieved to obtain uniform propolis fine powder; the propolis fine powder was added to lysine aqueous solution and ultrasonically extracted to obtain propolis extract A; (2) Dewaxing and impurity removal: Cool the propolis extract A, centrifuge to remove the bottom precipitate and surface wax components, collect the intermediate clear phase, and then filter it through qualitative filter paper or nylon mesh to finally obtain clear and transparent propolis extract B; (3) Preparation of water-soluble propolis powder: Propolis extract B was freeze-dried to remove moisture and finally water-soluble propolis powder was obtained.

2. The method for low-temperature gradient extraction of propolis based on lysine aqueous solution according to claim 1, characterized in that, The low temperature mentioned in step (1) is -20~0℃.

3. The method for low-temperature gradient extraction of propolis based on lysine aqueous solution according to claim 1, characterized in that, The concentration of the lysine aqueous solution in step (1) is 2-15% (w / v).

4. The method for low-temperature gradient extraction of propolis based on lysine aqueous solution according to claim 1, characterized in that, The solid-liquid ratio of the propolis powder to the lysine aqueous solution in step (1) is 1:2 to 1:50 (g / mL).

5. The method for low-temperature gradient extraction of propolis based on lysine aqueous solution according to claim 1, characterized in that, The ultrasonic temperature in step (1) is 20~60℃ and the ultrasonic power is 240~400W.

6. The method for low-temperature gradient extraction of propolis based on lysine aqueous solution according to claim 1, characterized in that, The centrifugation temperature in step (2) is 1~10℃ and the centrifugation speed is 10,000~15,000 rpm.

7. The method for low-temperature gradient extraction of propolis based on lysine aqueous solution according to claim 1, characterized in that, The specific steps of freeze drying in step (3) are as follows: First, the product is pre-frozen by rapid freezing at -45℃ to -35℃ for 1.5 to 3 hours, and then transferred to a freeze dryer. The first drying is carried out under the following conditions: vacuum pressure 15 to 30 Pa, cold trap temperature ≤ -55℃, shelf temperature starts at -40℃ to -35℃ and slowly rises to -25℃ to -15℃, and is maintained for 10 to 15 hours. Then, a second drying is carried out: vacuum pressure 5 to 15 Pa, shelf temperature at 20℃ to 55℃, for 6 to 10 hours, in order to fully remove residual moisture and finally obtain high-purity propolis water-soluble powder with lysine as a co-solvent.

8. The water-soluble propolis powder obtained by the extraction method according to any one of claims 1 to 7.

9. The use of the propolis water-soluble powder according to claim 8 in the preparation of immunomodulators, functional foods or anti-inflammatory and antioxidant pharmaceutical preparations.

10. An immunomodulatory agent or anti-inflammatory preparation, characterized in that, The immunomodulator or anti-inflammatory agent includes the propolis water-soluble powder according to any one of claims 1 to 7.

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