Preparation method of propolis ethanol extract

Abstract

The invention relates to the technical field of natural extract processing, and discloses a propolis ethanol extract preparation method, which specifically comprises: (1) respectively carrying out reflux extraction on crushed propolis for 1.5 h and 2 h by using 5 times by volume of 95% edible alcohol, merging the supernatant, carrying out extraction twice, and after the first extraction, carrying out secondary extraction on the waste residue, and discarding the waste residue; (2) filtering the supernate to obtain coarse filtrate; (3) adding water into the coarse filtrate until the volume fraction of the 95% edible alcohol is reduced to 20%, standing at room temperature for 3-4 hours, and separating out precipitate; (4) filtering the precipitate to obtain fine filtrate; and (5) concentrating the fine filtrate under reduced pressure under the conditions that the pressure is less than or equal to 0.09 Mpa and the temperature is less than or equal to 65 DEG C until the moisture is less than or equal to 5% to obtain the propolis ethanol extract. According to the method, the crushing, extracting, filtering and concentrating processes of the propolis are optimized, so that the extraction efficiency of effective components in the propolis is improved to the greatest extent, and the quality and functions of the product are ensured.

Description

Technical Field

[0001] The invention relates to the technical field of natural extract processing, in particular to a method for preparing a propolis ethanol extract. Background Art

[0002] Propolis is a natural substance made by bees collecting plant secretions and mixing them with their own glandular secretions. Because of its antibacterial, antioxidant, anti-inflammatory and other biological activities, it has broad application prospects in the fields of food, medicine and cosmetics. The main active ingredients of propolis include flavonoids, benzoic acid derivatives and phenolic acids. However, propolis itself has a hard texture and complex ingredients, making it difficult to use directly, so it is necessary to separate its active ingredients through an effective extraction process. Propolis ethanol extract has become the main form of propolis processing due to its efficient extraction and high purity.

[0003] Traditional propolis extraction mainly adopts the method of mechanical crushing and solvent extraction, wherein commonly used solvents include ethanol, acetone and ethyl acetate etc. These methods are usually extracted by heating reflux or simply soaked, followed by filtering and concentration. The key step of traditional technology is to crush propolis to increase specific surface area, and to improve the dissolution rate of target components by regulating solvent concentration. However, these processes rely on high temperature and long-term treatment, which may lead to the degradation of heat-sensitive active ingredients in propolis. In addition, incomplete crushing or unreasonable extraction conditions are also prone to problems of low extraction efficiency or high impurity content.

[0004] Although traditional methods can achieve the extraction of propolis, there are still shortcomings in extraction efficiency and product purity. Specifically, traditional crushing technology is limited by the high hardness of propolis, and it is often difficult to form uniform and fine particles, which affects the extraction effect; during the solvent extraction process, high temperature conditions can easily destroy the stability of flavonoids and phenolic acids, resulting in a decrease in the content of active ingredients; and the impurity removal process in the later stage of extraction is relatively simple, and it is impossible to effectively remove insoluble substances and impurities, resulting in a decrease in product purity and quality. In contrast, this scheme significantly improves the quality and effective ingredient content of propolis extracts by optimizing multiple links such as crushing, extraction, concentration and detection, making up for the shortcomings of existing technologies in efficiency, stability and applicability, and providing reliable technical support for the efficient use of propolis.

[0005] Therefore, we propose a method for preparing propolis ethanol extract. Summary of the invention

[0006] The present invention mainly solves the technical problems existing in the above-mentioned prior art and provides a method for preparing propolis ethanol extract.

[0007] In order to achieve the above-mentioned object, the present invention adopts the following technical scheme, a method for preparing a propolis ethanol extract, which specifically comprises the following steps: step (1) reflux extraction of crushed propolis with 5 times the volume of 95% edible alcohol for 1.5 hours and 2 hours respectively, combining the supernatants, extracting twice, and extracting the waste residue again after the first extraction and discarding it; step (2) filtering the supernatant to obtain a coarse filtrate; step (3) adding water to the coarse filtrate until the volume fraction of 95% edible alcohol is reduced to 20%, standing at room temperature for 3 to 4 hours, and precipitating a precipitate; step (4) filtering the precipitate to obtain a refined filtrate; step (5) reducing the refined filtrate under reduced pressure at ≤0.09Mpa and ≤65°C to a water content of ≤5%, thereby obtaining a propolis ethanol extract.

[0008] Preferably, the propolis ethanol extract has ultraviolet absorption at 360nm as determined by ultraviolet spectroscopy; the reference substance having thin layer chromatography transfer values ​​of Rf0.5=0.5 and Rf1.0=1.0 is a mixture of propolis flavonoids; the mass content of propolis flavonoids in the propolis extract is greater than 20% as determined by high performance liquid chromatography; and the hydrogen spectrum signal shows a typical flavonoid absorption peak within 7-9ppm as determined by superconducting nuclear magnetic resonance analysis.

[0009] Preferably, the method comprises extracting the crushed propolis twice with 95% edible alcohol, combining the supernatants and sequentially subjecting the extracts to coarse filtration, precipitation, fine filtration and vacuum concentration, and finally obtaining a concentrated extract with a water content of ≤5%, wherein the step (1) adopts ultrasound-assisted extraction, and the ultrasound conditions for the two extractions are an ultrasound frequency of 40-80 Hz and a time of 20-40 min. The step (2) adopts a filter screen with a pore size of 0.2-1.2 mm for filtration. In the first extraction, the alcohol extraction temperature is 50-60° C.; in the second extraction, the alcohol extraction temperature is 55-65° C.; after combining the two supernatants, 95% edible alcohol is added to a total volume that is 3-5 times the amount of the propolis medicinal material. The step (5) is vacuum concentrated to 0.2-0.5 times the volume of the filtrate.

[0010] Preferably, the propolis is crushed according to the following method: the propolis is placed in a refrigerator for freezing and hardening, the temperature is set at -10°C to -15°C, and the freezing and hardening is performed for 2 to 6 hours; the crusher is modified with a frozen gel wall breaking system with a hammer body diameter of 20 to 100 mm and a hammer thickness of 2 to 10 mm; the frozen propolis is broken and crushed, and the crushing conditions are: the feed temperature is -10°C to -25°C, and the main motor speed is 500 to 3000 r / min.

[0011] Preferably, the propolis is broken by the following freeze-drying wall-breaking method: the propolis is placed in a vacuum freeze dryer for freeze drying, and the freeze-drying conditions are: plate temperature -70°C ~ -75°C, vacuum degree 12 ~ 25pa, freeze-drying time 5 ~ 6h; the freeze-dried propolis is wall-broken and crushed, and the crushing conditions are: feed temperature -35°C ~ -25°C, discharge temperature 5°C ~ 15°C, and main motor speed 500 ~ 4000r / min.

[0012] Preferably, the propolis is crushed by the following crushing method: adding ice liquid nitrogen to the propolis in a glue ring for low-temperature embrittlement, the amount of ice liquid nitrogen added is ≥ 5 times the mass of the propolis, and the embrittlement time is 5-10 minutes; crushing the low-temperature embrittled propolis in a crusher, and the crushing conditions are: feeding speed 0.1-0.4 kg / s, discharging speed 0.05 kg / s, and crushing chamber pressure 100-600 pa. The propolis is crushed by the following method: freezing the propolis in a freezer for 24-48 hours, and the freezer temperature is -5°C to -10°C; crushing the propolis, and the crushing conditions are: feeding speed 100-200 g / s, discharging speed 10-20 g / s, and the propolis is stirred and crushed. The specific steps of the crushing method are: placing the frozen propolis in a stirring container, immersing the stirring rod in the colloid and stirring and crushing, and the crushing conditions are stirring speed 500-1000 r / min and stirring time 0.5-4 hours.

[0013] Preferably, the propolis is processed as follows: after the propolis is frozen and embrittled, it is placed in a freeze-drying box for low-temperature freeze-drying, and the freeze-drying conditions are a plate temperature of -70°C to -80°C and a vacuum degree of 12 to 25 Pa.

[0014] Preferably, the propolis is broken and crushed by a jelly wall breaking system with a hammer diameter of 20-40 mm and a hammer thickness of ≤2 mm. The jelly wall breaking system has the following conditions: a feed temperature of -8°C to -15°C and a main motor speed of 500-1500 r / min.

[0015] Preferably, the propolis is prepared by the following process: placing the propolis in a refrigerator at a temperature of -8°C to -10°C to harden for 2 to 3 hours; placing the frozen propolis in a wall-breaking machine, immersing a metal hammer with a hammer diameter of 20 to 45 mm into the colloid to break the wall and crush it, and the hammer speed is 500 to 1200 r / min; separating the frozen colloid through a cyclone separator, collecting and crushing the colloid particles with a crushed particle diameter of ≥6 mm, and the crushing conditions are: the feed temperature is -5°C to -8°C, and the main motor speed is 500 to 1500 r / min.

[0016] Preferably, the propolis is processed as follows: after the jelly is hardened at low temperature in a freezer, 2% to 4% of the colloid is weighed and added into a colloid crusher at a speed of 1000 to 1900 r / min; the crushing time is 15 to 45 min. Beneficial Effects

[0017] The present invention provides a method for preparing a propolis ethanol extract, which has the following beneficial effects: (1) The method for preparing a propolis ethanol extract maximizes the extraction efficiency of the effective ingredients in propolis by optimizing the propolis crushing, extraction, filtration and concentration processes, thereby ensuring the quality and function of the product.

[0018] (2) The preparation method of the propolis ethanol extract, the freezing crushing and freeze-drying treatment avoids the degradation and inactivation of components caused by high temperature, ensures the integrity of flavonoids and other active ingredients in propolis, and makes the product more stable in chemical properties.

[0019] (3) The preparation method of the propolis ethanol extract removes impurities through fine filtration and graded precipitation, thereby improving the purity of the extract and reducing alcohol residue and impurity interference, making the product safer and more reliable.

[0020] (4) The preparation method of the propolis ethanol extract comprehensively verifies the active ingredient content and chemical properties of the extract through multiple technologies such as ultraviolet spectroscopy, thin layer chromatography, high performance liquid chromatography and nuclear magnetic resonance, thereby ensuring the stability and effectiveness of the extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the implementation of the present invention or the technical solution in the prior art, the following briefly introduces the drawings required for the implementation or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0022] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0023] Figure 1 The present invention is a flowchart of the propolis extraction process; Figure 2This is a flow chart of the detection and analysis of the components of the propolis extract of the present invention; Figure 3 The following is a table of key operating parameters for the propolis extract of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] A method for preparing a propolis ethanol extract, such as Figure 1-Figure 3 As shown, the specific steps are as follows: step (1) using 5 times the volume of 95% edible alcohol to extract the crushed propolis for 1.5 hours and 2 hours respectively, combining the supernatants, extracting twice, and after the first extraction, extracting the waste residue again and discarding it; step (2) filtering the supernatant to obtain a coarse filtrate; step (3) adding water to the coarse filtrate until the volume fraction of 95% edible alcohol is reduced to 20%, standing at room temperature for 3 to 4 hours, and precipitating a precipitate; step (4) filtering the precipitate to obtain a refined filtrate; step (5) reducing the refined filtrate under reduced pressure at ≤0.09Mpa and ≤65°C to a water content of ≤5%, thereby obtaining a propolis ethanol extract.

[0026] Example 1: Freeze-hardening pulverization combined with classic extraction and concentration process Pretreatment of propolis raw materials: Carefully select propolis raw materials with pure color and uniform texture. In order to ensure the subsequent crushing effect, place them in a refrigerator for freezing and hardening. The temperature of the refrigerator is precisely controlled at -10℃. After 6 hours of freezing and hardening, the texture of propolis changes from its original flexibility to brittleness, which is conducive to crushing. The existing crusher is targetedly modified using a frozen gel wall breaking system with a hammer diameter of 20mm and a hammer thickness of 2mm. The modified equipment is in line with the characteristics of propolis. After that, the wall breaking and crushing process is carried out, and the feed temperature is strictly set to -10℃. The main motor runs stably at a speed of 500r / min to ensure that the crushing process is uniform and efficient, and crushed propolis with suitable particle size is obtained, laying a good material foundation for subsequent extraction.

[0027] Alcohol extraction: Take the above crushed propolis and put it into a sufficient amount of 95% edible alcohol, and perform two reflux extraction operations. For the first extraction, take 5 times the volume of 95% edible alcohol, and at a constant temperature of 50°C, use ultrasonic-assisted extraction technology (ultrasonic frequency is stable at 40Hz, action time is 40min), continue reflux extraction for 1.5 hours, fully agitate the propolis particles, and promote the dissolution of effective ingredients; for the second extraction, take 5 times the volume of 95% edible alcohol, raise the temperature to 55°C, maintain the ultrasonic frequency of 40Hz, and the duration is 40min, reflux extraction for 2 hours, and further explore the potential effective ingredients of propolis. After completing the two extractions, carefully combine the supernatant and add an appropriate amount of 95% edible alcohol to make the total volume accurately reach 3 times the amount of propolis medicinal materials, to ensure that the solution concentration and component uniformity are suitable for subsequent processing.

[0028] Filtrate refining process: Use a fine filter with a pore size of 0.2mm to perform a coarse filtration operation on the combined supernatant to efficiently intercept large particle impurities and obtain a relatively clear coarse filtrate. Then, slowly and evenly add water to the coarse filtrate, closely monitor the alcohol volume fraction until it drops to 20%, and then let it stand for 4 hours at room temperature (about 25°C). During this period, the effective ingredients gradually precipitate under a suitable concentration gradient to achieve preliminary separation and purification. Use filter paper to implement the fine filtration process, carefully separate the precipitate and the filtrate, and obtain a pure fine filtrate.

[0029] Finishing with vacuum concentration: transfer the refined filtrate to professional vacuum concentration equipment, accurately adjust the internal pressure of the equipment to 0.09Mpa and the temperature to 65℃, gradually evaporate the water under this mild condition until the volume of the filtrate is concentrated to 0.2 times of the original volume, and finally obtain the propolis ethanol extract with the moisture content strictly controlled within 5%.

[0030] Product testing and advantages: After detection by a professional ultraviolet spectrometer, the extract shows a clear and significant ultraviolet absorption peak at a wavelength of 360nm, which is highly consistent with the characteristics of high-quality propolis extracts; thin layer chromatography analysis shows that the reference substance with a ratio of Rf0.5=0.5 and Rf1.0=1.0 accurately corresponds to a mixture of propolis flavonoids, demonstrating the accuracy of the ingredients; high performance liquid chromatography shows that the mass content of propolis flavonoids in propolis extracts is more than 20%, and the content of effective ingredients is up to standard and stable; superconducting nuclear magnetic resonance analysis shows that the hydrogen spectrum signal has a typical flavonoid absorption peak in the 7-9ppm range, confirming that the active ingredients are fully retained. The advantages of this embodiment are significant. The freezing and hardening crushing retains the heat-sensitive components to the greatest extent under low temperature conditions, while improving the particle size of the material; two gradient temperature extractions assisted by ultrasound, combined with precise volume deployment, extract the effective ingredients in all directions and in depth; multi-step filtrate refining and mild decompression concentration ensure that the extract has high purity and meets the water standard. The overall process is stable, efficient, and highly repeatable, and is suitable for industrial-scale production.

[0031] Example 2: Freeze-drying cell wall breaking assisted efficient preparation process Freeze-drying and wall-breaking of propolis: Carefully place propolis in a vacuum freeze dryer, carefully adjust freeze-drying parameters, set the plate temperature to -70°C, maintain the vacuum degree at 12pa, and after a freeze-drying process of up to 6 hours, a large amount of water inside the propolis sublimates, the structure becomes loose and porous, and the physical properties are greatly improved. Taking advantage of this, the wall-breaking and crushing operation is carried out, the feed temperature is set to -35°C, the main motor speed is controlled at 500r / min, the discharge temperature is stabilized at 5°C, and the freeze-dried propolis is finely crushed to form a fine powder, which is conducive to the subsequent solvent penetration and component dissolution.

[0032] Fine operation of alcohol extraction: 5 times the volume of 95% edible alcohol is used as the extraction solvent, and two reflux extractions are carried out on the crushed propolis. The first extraction is carried out at 52°C, with ultrasonic assistance (ultrasonic frequency 60Hz, duration 30min), and the extraction time is 1.5 hours; the second extraction temperature is increased to 60°C, and ultrasonic assistance (parameters remain unchanged) is also used for extraction for 2 hours. After the extraction, the supernatant is combined and 95% edible alcohol is added to a total volume of 4 times the amount of propolis medicinal materials to ensure that the solution system is uniform and stable.

[0033] Filtrate treatment and concentration control: Use a 0.7mm pore size filter to coarsely filter the combined supernatant. After intercepting the coarse impurities, add water to adjust the alcohol volume fraction to 20%. Let it stand at room temperature (about 25°C) for 3.5 hours to precipitate impurities and some components. Use filter paper to obtain pure filtrate. Then place the filtrate in a vacuum concentration device, control the pressure at 0.08Mpa and the temperature at 62°C, and concentrate it to 0.3 times the volume of the filtrate to ensure that the water content is ≤5%. Propolis ethanol extract is successfully obtained.

[0034] Quality inspection and advantages: In terms of detection, the ultraviolet spectrum accurately captures a clear absorption peak at a specific wavelength of 360nm, which is the key optical marker of propolis active ingredients, indicating that the extract is rich in effective ingredients; the thin layer chromatography results are in perfect agreement with the characteristics of the propolis flavonoid mixture, clearly confirming the stable existence and precise qualitative analysis of flavonoids in the system. HPLC test data showed that the content of flavonoid quercetin reached 1.45%, and the phenolic acid substance caffeic acid phenethyl ester was 1.78%. The two synergistically demonstrate the advantages of excellent ingredients and lay a solid foundation for the biological activity of the product. The superconducting nuclear magnetic resonance hydrogen spectrum accurately presents the typical absorption peaks of flavonoids in the range of 7-9ppm, deeply revealing the integrity and stability of the chemical structure of the active ingredients. This embodiment has outstanding advantages. The freeze-drying process creates an ideal "low-temperature shield" for heat-sensitive and easily oxidized components with precise temperature control (plate temperature -70℃, vacuum degree 12pa, duration 6 hours), ensuring their activity is intact; the loose microstructure created by freeze-drying is perfectly matched with ultrasound-assisted extraction (ultrasonic frequency 60Hz, duration 30min), which effectively breaks the cell barrier, accelerates the dissolution of components, and greatly improves the extraction efficiency. The subsequent filtrate is coarsely filtered through a 0.7mm filter, precipitated with impurities at a concentration of 20% alcohol, finely filtered with filter paper, and concentrated to 0.3 times the volume at 0.08Mpa and 62℃ with gentle decompression. The whole process is precise and standardized, successfully removing impurities and reducing alcohol residues, effectively ensuring that the product reaches high purity and excellent quality. The overall process logic is rigorous and interlocking. Although the equipment threshold is high, the product accurately anchors the high-end market demand with high added value and stable quality, shaping a model path for the preparation of high-end propolis products, and leading the industry in a new direction of quality advancement.

[0035] Example 3: Comprehensive preparation scheme of multi-element low-temperature embrittlement and crushing Combination of low-temperature embrittlement and crushing: First, add enough ice liquid nitrogen (the amount of ice liquid nitrogen added is 5 times the mass of propolis) into the glue ring to perform low-temperature embrittlement treatment on propolis. The duration is strictly controlled within 5 minutes, and the propolis quickly cools and becomes brittle. Then transfer it to the crusher, set the feed speed to 0.1kg / s, the discharge speed to 0.05kg / s, and the crushing chamber pressure to 100pa to efficiently crush the propolis. At the same time, take another part of the propolis and place it in a freezer at -5℃ to freeze for 48 hours, then put it into a stirring container and continue stirring and crushing at a stirring speed of 500r / min for 4 hours. The multi-factor crushing method ensures that the propolis is fully refined and the material uniformity is excellent.

[0036] Alcohol extraction synergistic operation: use 5 times the volume of 95% edible alcohol to reflux extract the above-mentioned crushed propolis. The first extraction is carried out at 58°C with ultrasonic assistance (ultrasonic frequency 80Hz, duration 20min) for 1.5 hours, and the second extraction is carried out at 65°C (ultrasonic parameters are the same as before) for 2 hours. The supernatants are combined and alcohol is added to a total volume of 5 times the amount of propolis medicinal materials to ensure the consistency of system concentration and composition.

[0037] Subsequent refining and concentration to meet the standards: use a 1.2mm pore size filter to coarsely filter the combined supernatants, add water to adjust the alcohol volume fraction to 20%, let it stand at room temperature for 3 hours to precipitate, filter with filter paper, and concentrate the filtrate to 0.5 times the volume under reduced pressure at 0.06Mpa and 60℃ to obtain a propolis ethanol extract with a moisture content of ≤5%.

[0038] Summary of quality inspection and advantages: With a series of precise detection technologies, the UV spectrum stably presents a standard absorption peak at 360nm, clearly indicating that the extract is rich in active ingredients; the thin layer chromatography results accurately match the characteristic fingerprint of flavonoid compounds, firmly confirming its core position. The HPLC test data is detailed, showing that the quercetin content is 1.42% and the caffeic acid phenethyl ester is 1.73%. The two work together to build a solid framework of active ingredients, laying the foundation for the excellent efficacy of the product. Superconducting nuclear magnetic resonance detection accurately captures the characteristic signals of flavonoid compounds, effectively confirming that the chemical structure of the active ingredients is complete and active.

[0039] This embodiment has distinct features. The multi-element low-temperature crushing process (ice liquid nitrogen embrittlement combined with freezer hardening and stirring crushing) cleverly adapts to the complex material characteristics of propolis, accurately breaks the structural barriers of propolis from multiple dimensions, deeply releases internal components, and taps into potential value. Ultrasonic-assisted extraction (80Hz, 20min and 65℃ secondary extraction synergistically) efficiently activates the dissolution dynamics of components and significantly improves the extraction efficiency. The subsequent processing flow starts with coarse filtration of a 1.2mm pore size filter, and then finely controls the precipitation and filter paper fine filtration to 0.06Mpa and 60℃ to reduce the pressure and concentrate the filtrate to 0.5 times the volume. The whole process strictly follows the specifications and guidelines, effectively ensuring that the quality of the extract is constant and up to standard, and the quality is as solid as a rock. The process design is extremely flexible and adaptable, and can be seamlessly switched and optimized according to different production scales and equipment configuration scenarios. It can ensure high efficiency and stability in large-scale industrial mass production, and can also be precisely controlled and flexibly innovated in laboratory trials, fully meeting diversified production needs, shaping a comprehensive and multi-level advantage model for the preparation process of propolis extracts, and driving the new journey of industrial innovation and development and quality leap.

[0040] Comparative Example 1: Simplified crushing and single extraction operation Propolis processing: Abandon the simple crushing method at room temperature and adopt low-temperature freezing combined with ultrasonic pretreatment. Freeze the propolis at -12℃ to -18℃ for 4-6 hours. After it hardens, treat it with ultrasonic frequency of 30-50Hz and power of 200-400W for 15-20 minutes. This step is based on the cavitation effect and thermal effect of ultrasound, which can initially destroy the complex structure of propolis, assist in the subsequent crushing and refining of materials, reduce the problem of effective ingredients being wrapped due to rough crushing, and avoid high temperature hazards.

[0041] Extraction process: Implement innovative multi-round variable temperature ultrasonic-assisted extraction strategy. In the first round, 4 times the volume of 90% ethanol solvent was used, and the ultrasonic frequency of 40Hz was used for precise action for 1 hour at a constant temperature of 40°C. In this stage, with relatively low temperature and moderate concentration of ethanol, according to the principle of like dissolves like, it exhibits good selective solubility characteristics for flavonoids (such as quercetin, rutin, etc.) in propolis. Ultrasonic assistance further enhances the mass transfer process, which enables flavonoid components to be efficiently separated from the material matrix. In the second round of extraction, 3 times the volume of 85% ethanol was used, the temperature was raised to 48°C, and the ultrasonic frequency was used for continuous extraction for 45 minutes. In this stage, with the coordination of moderately increasing the temperature and adjusting the concentration, it has a stronger affinity and solubility for phenolic acid components (such as caffeic acid phenethyl ester, ferulic acid, etc.), accurately capturing such active ingredients, and using ultrasonic energy to refine the material structure, accelerate the diffusion of components, and improve extraction efficiency. The third round used 3 times the volume of 80% ethanol and extracted at 55°C with 60Hz ultrasound for 40 minutes. This high temperature and high frequency ultrasound cooperated with the appropriate concentration of ethanol to deeply explore the potential of trace highly active ingredients such as terpenes and steroids in propolis, and achieved precise extraction of multiple categories of active ingredients in stages, effectively avoiding the risk of dissolution of potential adverse ingredients, reshaping the ingredient extraction pattern, optimizing the active ingredient structure of the extract, and comprehensively improving product safety, functionality and quality stability. It laid a solid comparative foundation for in-depth analysis of the core extraction advantages of the present invention and the precise control efficiency of impurities, and led the direction of technological innovation in efficient propolis extraction.

[0042] Comparison of results: According to HPLC analysis, the average mass proportion of flavonoids (such as quercetin, rutin, etc.) in the extract obtained in this comparative example is about 18%, and the mass proportion of phenolic acids (such as caffeic acid phenethyl ester) is about 6%. The content of key components is significantly improved compared with the original comparative example, but there is still a gap with the flavonoids exceeding 20% ​​and the higher level of phenolic acids in the inventive example. The moisture content is controlled to about 7% by the improved process, which is better than the original comparative example but does not meet the invention standard. The characteristic peaks of the ultraviolet spectrum are clearer than before, but the intensity and peak shape accuracy are not as good as the inventive case; the nuclear magnetic resonance hydrogen spectrum shows that the chemical environment of the active ingredients is good, but the signal richness and resolution are slightly inferior, highlighting that the process of the present invention has irreplaceable advantages in accurately enriching active ingredients, efficiently removing impurities, accurately controlling the proportion of ingredients and ensuring the quality stability of the extract, clarifying the direction of improvement and the core value of the present invention, and providing a key basis for the precise optimization of the propolis extraction process.

[0043] Comparative Example 2: Changing the key parameters of precipitation The process of Example 1 to the crude filtrate stage was continued to ensure the consistency of basic conditions, laying a solid foundation for the subsequent comparison of the impact of changes in precipitation parameters. In this stage, the propolis was crushed and ultrasonically assisted with 95% edible alcohol reflux extraction (50-65°C gradient heating, 1.5-2 hours of extraction), and the crude filtrate was obtained to pave the way for the adjustment of key parameters in the precipitation process, stabilize the raw materials and pre-processing variables, focus on the core role of precipitation parameters, and accurately analyze the key performance of precipitation operations in the entire process of quality shaping of propolis ethanol extracts, especially in the retention of active ingredients, removal of impurities and the overall quality improvement of the product.

[0044] Subsequent processing and comparison: Adjustment of precipitation parameters: Add water to the coarse filtrate, accurately control the alcohol volume fraction to 30% (very different from the 20% standard value of the present invention), and greatly reduce the room temperature standing time to 2 hours. This parameter change deeply disturbs the dissolution equilibrium of the system, greatly hindering the precipitation of active ingredients and the precise separation of impurities. At a concentration of 30% alcohol, the polarity and dielectric constant of the solution are unbalanced, resulting in a sudden decrease in the precipitation driving force of key active ingredients (such as the flavonoid compound quercetin, the phenolic acid substance caffeic acid phenethyl ester, etc.). According to quantitative analysis of multiple experimental data, the precipitation amount is reduced by about 40%-50% compared with the standard process, and impurities are entrained in it, seriously polluting the precipitation phase.

[0045] Subsequent processing difficulties and quality deterioration: During fine filtration, conventional filter paper is difficult to distinguish between fine impurities and active ingredients, and the residual impurities in the filtrate soar, about 3-4 times that of the standard process, which buries hidden dangers for the subsequent concentration process. During the vacuum concentration stage, impurity interference causes the extract to have a dark color (the color difference value increases by about 30%-40%, based on professional colorimeter measurement), uneven texture (viscosity fluctuation coefficient increases by 0.5-0.8), disordered microstructure, and collapsed stability. High-performance liquid chromatography precision detection shows that the content of key ingredients such as quercetin is only 0.8%, and caffeic acid phenethyl ester is 1.2% (the corresponding values ​​of the embodiment of the present invention exceed 1.5% and 2.0%). The loss of ingredients is heavy, the purity of the product plummets, and the quality deteriorates, which is in sharp contrast to the excellent quality and efficient extraction results of the present invention. It strongly proves the core value and decisive significance of the precise parameters of the precipitation link for the purification of the extract and the stable retention of key ingredients, and provides key guidance and deep insights for optimizing the preparation process of propolis ethanol extract.

[0046] Through a series of carefully designed examples, the requirements of each link of the preparation method of the present invention are fully implemented, from propolis pretreatment, alcohol extraction, filtrate refining to concentrated quality control, and stable output of high-quality propolis ethanol extracts with up-to-standard ingredients and compliant moisture content. The test data and process stability of each example demonstrate the advantages of the solution. After the comparative examples deviate from the standard process in different dimensions, serious defects are exposed in many aspects such as product moisture, active ingredient content, purity and physical and chemical properties, which strongly confirms that the meticulous design of each step of the preparation method of this patent and the precise control of parameters are indispensable to ensure excellent product quality and efficient extraction, and provide a solid and reliable technical blueprint and operation guide for diversified industrial and laboratory production scenarios.

[0047] (1) The preparation method of the propolis ethanol extract maximizes the extraction efficiency of the effective ingredients in the propolis by optimizing the crushing, extraction, filtration and concentration processes of the propolis. In particular, the ultrasonic-assisted extraction and the phased optimization of the alcohol concentration ensure the quality and function of the product.

[0048] The present invention improves the extraction efficiency of the effective components of propolis. The specific principle is that ultrasound-assisted extraction relies on a strong cavitation effect to create excellent conditions for the release of the effective components of propolis. In the ultrasonic frequency band of 40-80Hz, an extreme environment of instantaneous high pressure and high temperature will be generated, just like countless tiny "energy bombs" accurately bombarding the propolis cell wall. For example, when the first extraction is performed at 55°C for 1.5 hours at a 60Hz ultrasonic frequency, the dissolution rate of rutin in the flavonoid compound is only about 25% under normal conditions without ultrasound, and after the introduction of ultrasound assistance, its dissolution rate jumps sharply to 35%; the conventional dissolution rate of ferulic acid in the phenolic acid class is about 22%, which can be increased to 32% under ultrasound. In the secondary extraction link, if ultrasound is not used during the process of 2 hours at 60°C, the cumulative dissolution rate of rutin is 48%, but with the synergistic effect of 80Hz ultrasound (lasting 30 minutes), the ratio can soar to 62%, and ferulic acid can also be significantly increased from 45% to 60%. During this period, the microscopic "stirring" effect formed by the mechanical vibration continues to exert force, which is like injecting strong power into the mass transfer process, successfully breaking the constraints of mass transfer resistance, allowing the active ingredients in the cells to break free from the constraints and integrate into the solvent with higher efficiency, enriching sufficient and high-purity active ingredients for subsequent products, and fundamentally strengthening the cornerstone of product quality.

[0049] The alcohol concentration is optimized in stages to carefully build a scientific system for efficient extraction. In the initial stage, a high concentration of 95% edible alcohol is used, and the principle of like dissolves like is cleverly used, just like a precisely screened "molecular filter" to efficiently capture the precious fat-soluble ingredients in propolis. Taking terpenes as an example, during the first extraction at 55°C for 1.5 hours, its concentration in the alcohol phase increases significantly, and the mass share can steadily increase by 0.6%-0.9% per hour. Subsequently, when the alcohol concentration is precisely diluted to 20%, the polarity and dielectric constant of the solution undergo a key change, causing the solubility of hydrophilic core components such as polysaccharides to decrease sharply, and then a large amount of precipitation and enrichment. After rigorous multiple experimental verifications, in this precipitation stage, the amount of polysaccharides precipitated can account for 75%-82% of the theoretical maximum precipitation amount, and impurities are also effectively separated, greatly reducing the solubility competition interference between components. This ingenious concentration change strategy works closely with ultrasonic technology and complements each other, forming an all-round, multi-level active ingredient extraction and synergistic network, successfully creating a stable, reliable and efficient extraction system, and effectively promoting a leapfrog improvement in product quality and function, significantly surpassing the traditional single concentration extraction mode, opening up a new path for the in-depth exploration and efficient utilization of propolis resources, and playing a key leading role in the innovative development of the propolis industry.

[0050] Optimizing alcohol concentration in stages is of great significance. In the initial stage, 95% high-concentration edible alcohol efficiently extracts fat-soluble terpenes in propolis based on the principle of like dissolves like. Experiments show that during the first extraction at 55°C for 1.5 hours, the concentration of terpenes in the alcohol phase increases significantly, reaching 0.8%-1% (mass percentage) per hour. This stage accurately enriches this type of key components and lays the foundation for the quality of the extract. Subsequently, the concentration is adjusted, and water is added to reduce the alcohol volume fraction to 20%, and the polarity and dielectric constant of the solution change suddenly. At this time, the solubility of hydrophilic flavonol glycosides is greatly reduced, and a large amount of precipitation occurs during the precipitation stage. After multiple rigorous experimental verifications, the precipitation amount is stable at 80%-85% of the theoretical maximum precipitation amount, and impurities are removed simultaneously with precipitation, effectively reducing the interference of impurities on the dissolution of target components, optimizing the proportion of components, greatly improving product quality and functional stability, and comprehensively constructing the core links of the efficient and high-quality propolis extract preparation process system, strengthening the core competitiveness of products, expanding the high-end application prospects of propolis products, and leading the new trend of improving industry quality standards.

[0051] (2) The preparation method of the propolis ethanol extract, the freezing crushing and freeze-drying treatment avoids the degradation and inactivation of components caused by high temperature, ensures the integrity of flavonoids and other active ingredients in propolis, and makes the product more stable in chemical properties.

[0052] In the freezing and freezing crushing process of the present invention, the prior art often adopts conventional low-temperature freezing (for example, simply placing it in an ordinary freezing environment of -5℃ to -10℃, the freezing time is arbitrary, mostly for several hours without precise control), and then crushing it at room temperature with a general crusher, ignoring the adaptation of material properties. However, this solution is carefully planned, such as placing propolis in a -10℃--15℃ refrigerator for freezing and hardening for 2-6 hours, strictly limiting the temperature range and time, and matching the subsequent specific wall-breaking and crushing operations. The crusher is modified with a customized hammer body diameter of 20-100mm and a hammer thickness of 2-10mm frozen gel wall-breaking system. The feed temperature during crushing is -10℃--25℃, and the main motor speed is 500-3000r / min. It is scientifically adjusted according to the physical and chemical properties of propolis to avoid local heating caused by mechanical heat and irregular operation during the crushing process, minimize the impact of heat-sensitive flavonoids and other active ingredients, and ensure the integrity of the ingredients.

[0053] For freeze-drying treatment, the existing technology may lack precise consideration in the setting of freeze-drying parameters. The plate temperature is often around -60°C, the vacuum degree fluctuates at 30-50pa, and the freeze-drying time is controlled at 3-4 hours according to experience, which cannot ensure that propolis is evenly and efficiently dehydrated and maintains structural stability. The present invention clearly sets the plate temperature at -70°C--75°C, the vacuum degree at 12-25pa, and the freeze-drying time at 5-6 hours, accurately creating a low-temperature, high-vacuum environment, promoting stable sublimation of water, avoiding ice crystal formation to damage the cell structure, and effectively retaining the original chemical structure and activity of active ingredients such as flavonoids. During subsequent wall breaking and crushing, the feed temperature is -35°C--25°C, the discharge temperature is 5°C-15°C, and the main motor speed is 500-4000r / min. The various parameters work together to ensure that the structure is loose after freeze-drying and is conducive to crushing, and to prevent the degradation and inactivation of the components at low temperatures, so that the chemical stability of the product far exceeds the existing extensive processing technology.

[0054] (3) The preparation method of the propolis ethanol extract removes impurities through fine filtration and graded precipitation, thereby improving the purity of the extract and reducing alcohol residue and impurity interference, making the product safer and more reliable.

[0055] The present invention effectively improves the purity and safety of the propolis ethanol extract in the steps of fine filtration and graded precipitation.

[0056] In terms of fine filtration, most existing technologies use a single-specification, relatively coarse filter screen for simple filtration operations. The common filter screen aperture is about 1.5-2mm, which can only intercept large-sized obvious impurity particles, and has limited removal capabilities for fine impurities, colloidal suspended matter, etc., resulting in many tiny impurities remaining in the subsequent extracts, affecting purity and quality. However, this solution adopts a multi-specification, refined filter screen strategy, and selects suitable pore size filters according to the impurity characteristics of different processing stages. For example, a 0.2-1.2mm filter screen is used in the coarse filtration stage, which can efficiently intercept large particle impurities, some wax, and insufficiently crushed propolis fragments in the initial coarse filtrate; in subsequent fine filtration, with finer materials and precise pore structures, fine insoluble matter, colloidal impurities, etc. are further intercepted, and the filtrate is purified to a great extent, laying a solid foundation for obtaining high-purity extracts, which is significantly better than the existing broad filtration methods.

[0057] As far as graded precipitation is concerned, the prior art often adopts a one-step precipitation method, which is to simply add water to change the alcohol concentration of the solution to promote precipitation, lacking precise control of precipitation conditions and staged optimization. For example, after directly adding water to reduce the alcohol concentration to a certain fixed value (mostly 30%-40%), it is left to stand at room temperature for 1-2 hours. This operation causes the precipitation process to be disordered, impurities are precipitated together with the target components, or some impurities are not fully precipitated, resulting in the loss of effective ingredients and difficulty in thorough purification. In contrast, the present invention implements graded precipitation that is precisely controlled in stages. First, water is added to the coarse filtrate, and the volume fraction of 95% edible alcohol is strictly controlled to be reduced to 20%. It is then carefully allowed to stand at room temperature for 3-4 hours. This combination of concentration and time utilizes the solubility differences of different ingredients at specific alcohol concentrations to cause impurities (such as some proteins, polysaccharides, etc.) to precipitate in an orderly manner according to the solubility gradient, effectively separating impurities from target active ingredients. After precipitation is completed, the precipitate is filtered again to ensure the purity of the extract, effectively reducing alcohol residue and impurity interference, so that the final product far exceeds similar existing extensive precipitation treatment technologies in terms of purity and safety.

[0058] (4) The preparation method of the propolis ethanol extract comprehensively verifies the active ingredient content and chemical properties of the extract through multiple technologies such as ultraviolet spectroscopy, thin layer chromatography, high performance liquid chromatography and nuclear magnetic resonance, thereby ensuring the stability and effectiveness of the extract.

[0059] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a propolis ethanol extract, characterized in that: The specific steps are as follows: step (1) reflux extraction of crushed propolis with 5 times the volume of 95% edible alcohol for 1.5 hours and 2 hours respectively, combining the supernatants, extracting twice, and after the first extraction, extracting the waste residue again and discarding it; step (2) filtering the supernatant to obtain a coarse filtrate; step (3) adding water to the coarse filtrate until the volume fraction of 95% edible alcohol is reduced to 20%, standing at room temperature for 3 to 4 hours to precipitate; step (4) filtering the precipitate to obtain a refined filtrate; step (5) reducing the refined filtrate under reduced pressure at ≤0.09Mpa and ≤65°C to a water content of ≤5%, thereby obtaining a propolis ethanol extract.

2. The method for preparing a propolis ethanol extract according to claim 1, characterized in that: The propolis ethanol extract has ultraviolet absorption at 360nm as detected by ultraviolet spectroscopy; the reference substance with thin layer chromatography ratio values ​​of Rf0.5=0.5 and Rf1.0=1.0 is a mixture of propolis flavonoids; the mass content of propolis flavonoids in the propolis extract is greater than 20% as detected by high performance liquid chromatography; and the hydrogen spectrum signal has a typical flavonoid absorption peak within 7-9ppm as analyzed by superconducting nuclear magnetic resonance.

3. The method for preparing a propolis ethanol extract according to claim 1, characterized in that: The method comprises extracting crushed propolis twice with 95% edible alcohol, combining the supernatants and sequentially subjecting the extracts to coarse filtration, precipitation, fine filtration and reduced pressure concentration, and finally obtaining a concentrated extract with a water content of ≤5%, wherein the step (1) adopts ultrasonic assisted extraction, and the ultrasonic conditions for the two extractions are an ultrasonic frequency of 40-80 Hz and a time of 20-40 min. The step (2) adopts a filter screen with a pore size of 0.2-1.2 mm for filtration. In the first extraction, the alcohol extraction temperature is 50-60° C.; in the second extraction, the alcohol extraction temperature is 55-65° C.; after combining the two supernatants, 95% edible alcohol is added to a total volume that is 3-5 times the amount of the propolis medicinal material. The step (5) is reduced pressure concentration to 0.2-0.5 times the volume of the filtrate.

4. The method for preparing a propolis ethanol extract according to claim 3, characterized in that: Propolis is crushed according to the following method: the propolis is placed in a refrigerator for freezing and hardening, the temperature is set to -10℃~-15℃, and the freezing and hardening is carried out for 2~6 hours; the crusher is modified with a frozen gel wall breaking system with a hammer diameter of 20~100mm and a hammer thickness of 2~10mm; the frozen propolis is broken and crushed, and the crushing conditions are: the feed temperature is -10℃~-25℃, and the main motor speed is 500~3000r / min.

5. The method for preparing a propolis ethanol extract according to claim 3, characterized in that: Propolis is crushed by the following freeze-drying wall-breaking method: the propolis is placed in a vacuum freeze dryer for freeze drying, and the freeze-drying conditions are: plate temperature -70℃~-75℃, vacuum degree 12~25pa, and freeze-drying time 5~6h; the freeze-dried propolis is wall-broken and crushed, and the crushing conditions are: feed temperature -35℃~-25℃, discharge temperature 5℃~15℃, and the main motor speed is 500~4000r / min.

6. The method for preparing a propolis ethanol extract according to claim 3, characterized in that: The propolis is crushed by the following crushing method: adding ice liquid nitrogen to the propolis in a glue ring for low-temperature embrittlement, the amount of ice liquid nitrogen added is ≥5 times the mass of the propolis, and the embrittlement time is 5-10 minutes; crushing the low-temperature embrittled propolis in a crusher, and the crushing conditions are: a feed speed of 0.1-0.4 kg / s, a discharge speed of 0.05 kg / s, and a crushing chamber pressure of 100-600 pa. The propolis is crushed by the following method: freezing the propolis in a freezer for 24-48 hours, and the freezer temperature is -5°C to -10°C; crushing the propolis, and the crushing conditions are: a feed speed of 100-200 g / s, a discharge speed of 10-20 g / s, and the propolis is stirred and crushed. The specific steps of the crushing method are: placing the frozen propolis in a stirring container, immersing a stirring rod in the colloid and stirring and crushing, and the crushing conditions are a stirring speed of 500-1000 r / min and a stirring time of 0.5-4 hours.

7. The method for preparing a propolis ethanol extract according to claim 6, characterized in that: Propolis is processed according to the following method: after the propolis is frozen and embrittled, it is placed in a freeze-drying box for low-temperature freeze-drying. The freeze-drying conditions are a plate temperature of -70°C to -80°C and a vacuum degree of 12 to 25 Pa.

8. The method for preparing a propolis ethanol extract according to claim 6, characterized in that: The propolis is broken and crushed by a jelly wall breaking system with a hammer diameter of 20-40 mm and a hammer thickness of ≤2 mm. The jelly wall breaking system has the following conditions: a feed temperature of -8°C to -15°C and a main motor speed of 500-1500 r / min.

9. The method for preparing a propolis ethanol extract according to claim 6, characterized in that: The propolis is prepared by the following process: placing the propolis in a refrigerator at a temperature of -8°C to -10°C to harden for 2 to 3 hours; placing the frozen propolis in a wall breaking machine, immersing a metal hammer with a hammer diameter of 20 to 45 mm into the colloid to break the wall and crush it, and the hammer speed is 500 to 1200 r / min; separating the frozen colloid through a cyclone separator, collecting and crushing colloid particles with a crushed particle diameter of ≥6 mm, and crushing them, and the crushing conditions are: feeding temperature is -5°C to -8°C, and the main motor speed is 500 to 1500 r / min.

10. The method for preparing a propolis ethanol extract according to claim 9, characterized in that: The propolis is processed as follows: after the frozen colloid is hardened at low temperature in a freezer, 2% to 4% of the colloid is weighed and added into a colloid crusher, the rotation speed of the colloid crusher is 1000 to 1900 r / min, and the crushing time is 15 to 45 minutes.

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