Hippophae rhamnoides Linn flavone microcapsule, and preparation method and application thereof

Abstract

This invention provides sea buckthorn flavonoid microcapsules, their preparation method, and applications, relating to the field of plant extracts and processing technology. The preparation method includes: extracting flavonoids from sea buckthorn pomace using ultrasonic-microwave synergistic technology; adsorbing and eluting the extract using AB-8 macroporous resin to achieve efficient enrichment, obtaining a flavonoid-rich solution with a purity of not less than 60 wt%; using a solution of gum arabic, maltodextrin, and β-cyclodextrin as the wall material, mixing it with the enrichment solution at a dry basis mass ratio, homogenizing and emulsifying under high pressure, and then spray-drying to obtain microcapsules. This invention solves the problems of low extraction efficiency, cumbersome purification steps, and poor stability of sea buckthorn flavonoids through an integrated continuous process. The obtained microcapsule product has an encapsulation rate of not less than 90%, significantly improved stability under accelerated light conditions, and exhibits good controlled-release characteristics in a simulated gastrointestinal environment, while fully retaining strong antioxidant activity.

Description

Technical Field

[0001] This invention belongs to the field of plant extract and processing technology, specifically a sea buckthorn flavonoid microcapsule, its preparation method and application. Background Technology

[0002] Sea buckthorn flavonoids, a class of highly active natural polyphenolic compounds derived from sea buckthorn fruit and leaves, have shown broad application prospects in functional foods, health products, and pharmaceutical preparations due to their significant antioxidant, anti-inflammatory, and lipid-regulating bioactivities. However, the industrialization path from raw materials to stable products still faces several technical bottlenecks that urgently need to be overcome, severely restricting product added value and market competitiveness. First, in the extraction and purification stages, existing technologies generally rely on traditional hot reflux or simple ultrasonic extraction processes. These methods often have limited extraction efficiency, require several hours, and have low flavonoid yields. The crude extract after extraction has complex components and low total flavonoid purity, making it difficult to meet the requirements of high-end applications for active ingredient content. Although subsequent enrichment methods such as chromatography can be used, the steps are cumbersome, the cycle is long, and solvent consumption is high, making it difficult to achieve efficient and low-cost large-scale preparation. Second, sea buckthorn flavonoids themselves are chemically unstable, and their active ingredients, such as quercetin, are extremely sensitive to light, heat, and oxygen. Unprotected flavonoid extracts are highly susceptible to oxidative degradation during storage and processing, leading to rapid loss of activity and short product shelf life. This severely limits their application in formulations requiring long-term stability. Conventional powder drying or simple mixing processes cannot provide effective protection. To improve stability, microencapsulation technology has been introduced. However, existing encapsulation methods often use single or simple mixtures of wall materials such as gelatin and maltodextrin, resulting in insufficient capsule wall density and limited protection of the core material. This manifests as low encapsulation rates and poor controllable release characteristics in simulated gastrointestinal environments, affecting final bioavailability. Finally, from a production process perspective, existing sea buckthorn flavonoid preparation typically relies on multiple separate sets of equipment operating intermittently for extraction, purification, encapsulation, and drying. This model suffers from poor process coordination, large footprint, high energy consumption, low production efficiency, and significant batch-to-batch fluctuations in product quality, failing to meet the urgent needs of the modern food industry for continuous, automated, and standardized production.

[0003] Therefore, developing an integrated technology and process that can synergistically solve the three major challenges of efficient extraction and enrichment, high-stability encapsulation, and continuous production is of great significance for promoting the high-value development and application of sea buckthorn flavonoids. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing sea buckthorn flavonoid microcapsules. The sea buckthorn flavonoid microcapsules obtained by the preparation method have the characteristics of high flavonoid purity, high encapsulation rate, good stability and good bioavailability, and can be widely used in the fields of food, health products and pharmaceuticals.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing sea buckthorn flavonoid microcapsules, comprising the following steps: (1) Using sea buckthorn pomace as raw material, ultrasonic-microwave synergistic extraction was carried out with 65%-75% ethanol aqueous solution as solvent to obtain sea buckthorn flavonoid extract; (2) The flavonoid extract is adsorbed through a macroporous resin column and then eluted with an ethanol aqueous solution with a volume concentration of 65%-75%, and the enriched eluent is collected. (3) Gum arabic, maltodextrin and β-cyclodextrin are dissolved in water at a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2) to prepare a wall material solution; the enriched eluent is added to the wall material solution as a core material, and after mixing and high-pressure homogenization, an emulsion is obtained. (4) The emulsion is spray-dried to obtain sea buckthorn flavonoid microcapsules.

[0006] Preferably, the conditions for the ultrasonic-microwave synergistic extraction are: solid-liquid ratio 1:10-1:20, ultrasonic power 350-450W, microwave power 250-350W, extraction temperature 50-60℃, and extraction time 25-35min.

[0007] Preferably, the macroporous resin is AB-8 type resin, the adsorption flow rate is 1.5-2.5 BV / h, and the elution flow rate is 0.8-1.2 BV / h.

[0008] Preferably, the dry basis mass ratio of the core material to the wall material is 1:(2-4).

[0009] Preferably, the pressure of the high-pressure homogenization process is 38-42 MPa, and the homogenization temperature is 35-45℃.

[0010] Preferably, the inlet air temperature of the spray dryer is 120-140℃, the outlet air temperature is 60-70℃, and the feed rate is 10-20mL / min.

[0011] The present invention also provides a sea buckthorn flavonoid microcapsule, which is prepared by the aforementioned preparation method.

[0012] Preferably, the core material of the sea buckthorn flavonoid microcapsules is total sea buckthorn flavonoids with a purity of not less than 60 wt%; the wall material is a composite wall material composed of gum arabic, maltodextrin and β-cyclodextrin in a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2); and the encapsulation rate of the microcapsules is not less than 90%.

[0013] More preferably, the quercetin content in the core material is not less than 20% of the total flavonoids in Hippophae rhamnoides by mass, and the isorhamnetin content is not less than 15% of the total flavonoids in Hippophae rhamnoides by mass.

[0014] The present invention also provides an application of the aforementioned sea buckthorn flavonoid microcapsules in the preparation of antioxidant products.

[0015] Compared with the prior art, the present invention has the following advantages: (1) This invention uses ultrasonic-microwave synergistic extraction technology to replace the traditional hot reflux method. By utilizing the synergistic effect of ultrasonic cavitation and microwave heating, the extraction time is significantly shortened to about 30 minutes, and the extraction rate of sea buckthorn flavonoids is stably increased to over 94.5%, which is about 30 percentage points higher than the traditional hot reflux method in Comparative Example 1. Combined with the directional enrichment process of AB-8 macroporous resin, the purity of total flavonoids in the extract can be efficiently increased to over 60%, and the key active ingredients quercetin (content ≥20%) and isorhamnetin (content ≥15%) can be effectively enriched, providing high-purity active core material for subsequent preparation.

[0016] (2) This invention achieves efficient encapsulation of sea buckthorn flavonoids by employing a specific composite wall material system of gum arabic, maltodextrin, and β-cyclodextrin (mass ratio approximately 2:1:1) and optimizing high-pressure homogenization and spray drying processes. The encapsulation rate of the resulting microcapsules is consistently above 91%, far exceeding the encapsulation rate using a single maltodextrin wall material. In a 45°C, strong light accelerated degradation experiment, the flavonoid retention rate of the microcapsules of this invention remained above 85% after 10 days, while the retention rate of the product in Comparative Example 2 had significantly decreased during the same period. This demonstrates that the composite wall material forms a dense and stable protective layer, significantly improving the stability of the product during storage and transportation.

[0017] (3) In vitro simulated gastrointestinal digestion experiments showed that the microcapsules of the present invention released slowly in the gastric juice stage, effectively avoiding the destruction of active ingredients by the gastric acid environment; after entering the intestinal juice environment, they could be released rapidly, with a cumulative release rate of more than 80% in 6 hours. This "acid-resistant-enteric-coated" controlled release characteristic is conducive to the targeted release and absorption of flavonoid active ingredients in the effective site of the small intestine, thereby potentially improving their oral bioavailability.

[0018] (4) Free radical scavenging experiments showed that, at a specific concentration, the DPPH free radical scavenging rate of the microcapsules of the present invention could reach 88%, ABTS + With a free radical scavenging rate of over 98%, its antioxidant capacity is comparable to that of the classic antioxidant ascorbic acid (Vc), proving that the microencapsulation process does not impair the efficacy of the active ingredients.

[0019] (5) This invention organically combines key processes such as ultrasonic-microwave extraction, resin column enrichment, high-pressure homogenization emulsification, and spray drying to form a coherent and efficient preparation route. This process reduces material transfer and intermediate steps, which is conducive to achieving automated control and large-scale stable production, and solves the problems of low efficiency, high energy consumption, and large batch differences that exist in the traditional separate and intermittent operation mode. Detailed Implementation

[0020] This invention provides a method for preparing sea buckthorn flavonoid microcapsules, wherein the preparation method preferably includes the following steps: (1) Using sea buckthorn pomace as raw material, ultrasonic-microwave synergistic extraction was carried out with 65%-75% ethanol aqueous solution as solvent to obtain sea buckthorn flavonoid extract; (2) The flavonoid extract is adsorbed through a macroporous resin column and then eluted with an ethanol aqueous solution with a volume concentration of 65%-75%, and the enriched eluent is collected. (3) Gum arabic, maltodextrin and β-cyclodextrin are dissolved in water at a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2) to prepare a wall material solution; the enriched eluent is added to the wall material solution as a core material, and after mixing and high-pressure homogenization, an emulsion is obtained. (4) The emulsion is spray-dried to obtain sea buckthorn flavonoid microcapsules.

[0021] In this invention, dried and pulverized sea buckthorn pomace that has passed through a 40-mesh sieve is mixed with an ethanol aqueous solution with a volume concentration of 65%-75% at a solid-liquid ratio of 1:10 to 1:20, wherein the volume concentration of the ethanol aqueous solution is more preferably 70%. The ethanol concentration range can take into account both the solubility of flavonoids and the inhibition of some fat-soluble impurities.

[0022] In this invention, the mixture is placed in an ultrasonic-microwave synergistic extraction device. The preferred conditions for ultrasonic-microwave synergistic extraction are: a solid-liquid ratio of 1:10-1:20, ultrasonic power of 350-450W, microwave power of 250-350W, extraction temperature of 50-60℃, and extraction time of 25-35 min. More preferably, the solid-liquid ratio is 1:14-1:16, the ultrasonic power is 360-400W, the microwave power is 260-300W, the extraction temperature is 55-58℃, and the extraction time is 28-30 min. These conditions are set based on a comprehensive optimization of extraction efficiency, energy consumption, and the stability of the target components. The solid-liquid ratio ensures that the solvent fully wets and penetrates the raw material, guaranteeing the mass transfer driving force while avoiding excessive solvent load leading to subsequent concentration. The optimal ratio of ultrasonic power (350-450W) to microwave power (250-350W) aims to utilize the cavitation and mechanical effects of ultrasound to continuously disrupt plant cell walls, while simultaneously leveraging the rapid and uniform volumetric heating properties of microwaves to promote the diffusion of flavonoids from the matrix to the solvent. This synergistic effect significantly shortens extraction time and increases yield. The extraction temperature is strictly controlled between 50-60℃. This range accelerates molecular motion and dissolution while effectively preventing the degradation or isomerization of heat-sensitive flavonoid aglycones caused by high temperatures. An extraction time of 25-35 minutes is crucial for balancing extraction kinetics and production cycle time. Within this time, the synergistic effect has largely completed the main extraction process; excessively long extraction times offer limited yield improvement and increase energy consumption.

[0023] In this invention, the flavonoid extract obtained through ultrasonic-microwave synergistic extraction is pumped into a chromatography column packed with AB-8 macroporous adsorption resin at an optimized flow rate of 1.5-2.5 BV / h. This flow rate range ensures sufficient mass transfer contact time between the flavonoid molecules and the active sites of the resin, achieving efficient adsorption while preventing premature breakthrough or excessive column pressure due to excessively high flow rates. After adsorption, a 65%-75% (v / v) ethanol aqueous solution is used as the eluent for staged elution at a lower flow rate of 0.8-1.2 BV / h. This ethanol concentration effectively disrupts the hydrogen bonds and van der Waals forces between the flavonoids and the resin, achieving highly selective desorption and maximizing the retention of water-soluble impurities such as polysaccharides and proteins on the column. The target eluent is collected, and the purity of total flavonoids from sea buckthorn in the enriched eluent can be stably increased to over 60%, providing a high-purity core material raw material for subsequent preparation processes.

[0024] In this invention, gum arabic, maltodextrin, and β-cyclodextrin are first accurately weighed and mixed in a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2), more preferably 2:1:1. Within this ratio range, gum arabic provides good emulsification and film-forming properties, maltodextrin acts as a filler and carrier to reduce costs and improve solubility, and β-cyclodextrin forms inclusion complexes with flavonoid molecules through its hydrophobic cavities, further enhancing stability and water solubility. The mixed wall material is slowly added to warm water at 50-60°C and dissolved under constant stirring to prepare a homogeneous wall material solution with a total mass concentration of approximately 15%-25%. This temperature and concentration are conducive to the full hydration of each component without causing denaturation of heat-sensitive components. Subsequently, a enriched and moderately concentrated sea buckthorn flavonoid eluent is used as the core material and slowly and uniformly added dropwise to the wall material solution under high-speed shear stirring at 2000-4000 rpm. The dry-basis mass ratio of the core material to the wall material is preferably 1:(2-4), more preferably 1:3, to ensure the formation of a complete encapsulation structure. This pre-emulsification process lasts for approximately 20-40 minutes, forming a preliminary oil / water (O / W) coarse emulsion. Finally, the coarse emulsion is transferred to a high-pressure homogenizer and preferably circulated 1-3 times at a pressure of 38-42 MPa, with a homogenization temperature preferably of 35-45°C; more preferably, it is circulated 2 times at a pressure of 40 MPa, with a homogenization temperature of 38-40°C. The strong shearing, cavitation, and collision effects generated by high-pressure homogenization can further break down the oil phase droplets to the micron or submicron level and promote a more uniform and dense encapsulation of the wall material on the surface of the core material droplets, thereby forming a fine emulsion with uniform particle size distribution and significantly enhanced stability. This lays a key foundation for the subsequent spray drying preparation of high-performance microcapsule products.

[0025] In this invention, the homogenized and stabilized emulsion is pumped via a peristaltic pump at a controllable flow rate of 10-20 mL / min into the atomizer of a high-speed centrifugal spray drying tower. The inlet air temperature is typically set to 120-140°C. This temperature range provides sufficient enthalpy for rapid dehydration and solidification of the droplet surface material, while preventing excessive temperature from degrading the heat-sensitive flavonoid active ingredients. The outlet air temperature needs to be precisely controlled at 60-70°C. This temperature is a key monitoring indicator in the drying process, reflecting the completion of internal moisture evaporation and the reaching of the dry state of the particles. It also ensures that the moisture content of the final product is below 3%, thus guaranteeing the product's storage stability. The atomized microdroplets contact the hot air in a parallel or mixed-flow manner, and the composite wall material on their surface undergoes a glass transition in a very short time, forming a dense and continuous solid shell that completely encapsulates the core material. Finally, a free-flowing, uniformly sized sea buckthorn flavonoid microcapsule powder product is collected.

[0026] This invention also provides a sea buckthorn flavonoid microcapsule, prepared by the aforementioned method. The microcapsule uses sea buckthorn total flavonoids with a purity of not less than 60 wt% as the core material, ensuring a high concentration and effectiveness of the active ingredients. The wall material of the microcapsule is composed of gum arabic, maltodextrin, and β-cyclodextrin in a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2). This ratio allows the excellent film-forming and emulsifying properties of gum arabic, the carrier-filling effect of maltodextrin, and the molecular inclusion ability of β-cyclodextrin to synergistically contribute to construct a dense and stable capsule wall structure. Based on this structure, the encapsulation efficiency of the microcapsule is consistently not less than 90%, meaning that most of the flavonoids are effectively encapsulated within the wall material, thus providing a fundamental guarantee against damage from environmental factors such as light, oxygen, and heat during subsequent storage and application. More preferably, the core material contains not less than 20% quercetin by mass of total sea buckthorn flavonoids and not less than 15% isorhamnetin. The sea buckthorn flavonoid microcapsules prepared by this invention not only possess excellent stability but also maintain strong antioxidant and other functional activities, thus laying a reliable product foundation for their application as a standardized, efficient, and stable functional raw material in the fields of food, health products, and pharmaceuticals.

[0027] This invention also provides an application of the aforementioned sea buckthorn flavonoid microcapsules in the preparation of antioxidant products. The sea buckthorn flavonoid microcapsules prepared according to this invention possess high antioxidant activity, excellent stability, and good bioavailability. As a core functional ingredient, they can be widely used in the preparation of various antioxidant products. The products preferably include food, health products, or pharmaceutical compositions.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0030] Example 1 A method for preparing sea buckthorn flavonoid microcapsules, comprising the following steps: (1) Raw material pretreatment: Weigh 200g of sea buckthorn fruit residue (moisture content 7.2%, total flavonoid content 2.5%) that has been dried at 60℃ for 4h and crushed through a 40-mesh sieve, and place it in a sealed bag for later use to avoid moisture absorption.

[0031] (2) Ultrasonic-microwave synergistic extraction: Add 3000 mL of 70% ethanol aqueous solution at a solid-liquid ratio of 1:15 (g / mL) and transfer to an ultrasonic-microwave synergistic extraction device. Set the ultrasonic power to 400 W, the microwave power to 300 W, the extraction temperature to 55 °C, and the extraction time to 30 min. After extraction, filter with a 300-mesh filter cloth. Repeat the extraction once under the same conditions, combine the two filtrates, and obtain the crude extract of sea buckthorn flavonoids.

[0032] (3) Macroporous resin enrichment: The crude extract was concentrated under reduced pressure at 40°C to 1 / 3 of its original volume (to reduce the subsequent column loading volume), and pumped into a pretreated AB-8 macroporous resin column (column diameter to height ratio 1:8) at a flow rate of 2 BV / h. After adsorption saturation (by UV detection of the penetration point), it was first rinsed with 3 BV of deionized water to remove impurities, and then eluted with 70% ethanol aqueous solution at a flow rate of 1 BV / h. The eluent was collected and concentrated under reduced pressure at 50°C until no alcohol odor was detected, to obtain the sea buckthorn flavonoid enrichment (HPLC determination: total flavonoid purity 62.3%, quercetin 21.5%, isorhamnetin 15.8%).

[0033] (4) Preparation and emulsification of wall material: Weigh 20g of gum arabic, 10g of maltodextrin, and 10g of β-cyclodextrin, add 200mL of deionized water, and stir in a 60℃ water bath for 30min until completely dissolved to prepare a 20% (w / w) composite wall material solution. Cool to 40℃ for later use. Measure the equivalent of 10g of dry basis total flavonoids enrichment solution and slowly add it to the wall material solution (core material to wall material dry basis mass ratio 1:3) under high-speed shear stirring at 3000r / min to form a pre-emulsion.

[0034] (5) High-pressure homogenization and spray drying: The pre-emulsion was homogenized twice under high pressure at 40 MPa and 40 °C to obtain an emulsion with uniform particle size. The emulsion was fed into a spray drying tower by a peristaltic pump at a flow rate of 15 mL / min. The inlet air temperature was set to 130 °C, the outlet air temperature to 65 °C, and the atomization pressure to 0.5 MPa. The dried powder was collected, which is the sea buckthorn flavonoid microcapsule product.

[0035] Example 2 A method for preparing sea buckthorn flavonoid microcapsules, comprising the following steps: (1) Raw material pretreatment: Weigh 200g of sea buckthorn fruit residue that has been dried at 60℃ for 4h, crushed and passed through a 40-mesh sieve. The moisture content of the raw material is 7.5% and the total flavonoid content is 2.3%. Place it in a sealed and dry container for later use to avoid moisture absorption affecting the extraction effect.

[0036] (2) Ultrasonic-microwave synergistic extraction: 2800 mL of 70% ethanol aqueous solution was added to the sea buckthorn pomace at a solid-liquid ratio of 1:14 (g / mL). After thorough stirring to ensure uniform mixing of the raw material and solvent, the mixture was transferred to an ultrasonic-microwave synergistic extraction device. The extraction parameters were set as follows: ultrasonic power 380 W, microwave power 280 W, extraction temperature 53 °C, and extraction time 28 min. After extraction, the mixture was filtered through a 300-mesh filter cloth, and the filtrate was collected. The filter residue was then added to an equal volume of ethanol aqueous solution of the same concentration, and the extraction was repeated once using the same extraction parameters. The filtrates from the two extractions were combined to obtain a crude extract of sea buckthorn flavonoids.

[0037] (3) Macroporous resin enrichment and purification: The crude extract was concentrated to 1 / 3 of its original volume under reduced pressure at 40°C and pumped into a pretreated AB-8 macroporous resin column (column diameter to height ratio of 1:8) at a flow rate of 1.8 BV / h for dynamic adsorption. The eluent was monitored in real time by ultraviolet spectrophotometry. When the flavonoid concentration in the eluent reached 5% of the feed concentration, adsorption saturation was determined. Subsequently, the resin column was first rinsed with 3 BV of deionized water at a flow rate of 2 BV / h to remove water-soluble impurities such as polysaccharides and proteins. Then, a 70% ethanol aqueous solution was used as the eluent for gradient elution at a flow rate of 0.9 BV / h, and the eluent was collected. The collected eluent was concentrated under reduced pressure at 50°C until no alcohol odor was detected to obtain the sea buckthorn flavonoid enriched solution. The purity of total flavonoids in the enriched solution was determined by high performance liquid chromatography (HPLC) to be 61.2%, of which quercetin content was 20.8% (as a percentage of total flavonoid mass) and isorhamnetin content was 15.2% (as a percentage of total flavonoid mass).

[0038] (4) Preparation and pre-emulsification of composite wall material: Accurately weigh 20g of gum arabic, 10g of maltodextrin, and 10g of β-cyclodextrin, add 200mL of deionized water, place in a 60℃ constant temperature water bath, and stir continuously at 300r / min for 30min until all wall material components are completely dissolved, prepare a composite wall material solution with a mass concentration of 20%, and cool to 40℃ for later use. Measure the above enriched liquid equivalent to 10g of dry basis sea buckthorn total flavonoids as the core material, stir at 3000r / min in a high-speed shear press, and slowly and evenly add the core material to the wall material solution (the dry basis mass ratio of core material to wall material is 1:3), and continue shearing and stirring for 30min to form a uniform and stable oil / water (O / W) type pre-emulsion.

[0039] (5) High pressure homogenization treatment: Transfer the pre-emulsion to a high pressure homogenizer, set the homogenization pressure to 38MPa and the homogenization temperature to 38℃, and circulate the pre-emulsion for homogenization once to break the core material droplets down to the micron level, forming a fine emulsion with uniform particle size distribution and strong stability.

[0040] (6) Spray drying to prepare microcapsules: The fine emulsion is pumped into a high-speed centrifugal spray drying tower at a flow rate of 14 mL / min using a peristaltic pump. The spray drying parameters are set as follows: inlet air temperature 125℃, outlet air temperature 63℃, and atomization pressure 0.5 MPa. After the emulsion is atomized by the atomizer, it comes into full contact with the hot air, and the wall material is rapidly dehydrated and solidified to form a solid capsule wall that encapsulates the core material. After drying is complete, the powdery product at the bottom of the tower is collected, which is the sea buckthorn flavonoid microcapsule.

[0041] Example 3 A method for preparing sea buckthorn flavonoid microcapsules, comprising the following steps: (1) Raw material pretreatment: Weigh 200g of sea buckthorn fruit residue that has been dried at 60℃ for 4h, crushed and passed through a 40-mesh sieve. The moisture content of the raw material is 6.9% and the total flavonoid content is 2.8%. Place it in a sealed and dry container for later use to avoid moisture absorption, which may cause changes in the properties of the raw material and affect the extraction efficiency.

[0042] (2) Ultrasonic-microwave synergistic extraction: 3200 mL of 70% ethanol aqueous solution was added to the sea buckthorn pomace at a solid-liquid ratio of 1:16 (g / mL). After thorough stirring to ensure uniform contact and full wetting of the raw material and solvent, the mixture was transferred to an ultrasonic-microwave synergistic extraction device. The extraction parameters were set as follows: ultrasonic power 420 W, microwave power 320 W, extraction temperature 57 °C, and extraction time 32 min. After extraction, the mixture was filtered through a 300-mesh filter cloth, and the first filtrate was collected. The filter residue was then added to an equal volume of 70% ethanol aqueous solution of the same concentration, and the extraction was repeated once using the same extraction parameters. The filtrates from the two extractions were combined to obtain a crude extract of sea buckthorn flavonoids.

[0043] (3) Macroporous resin enrichment and purification: The above-mentioned crude extract of sea buckthorn flavonoids was placed in a vacuum concentration device and concentrated to 1 / 3 of its original volume at 40°C to increase the flavonoid concentration and reduce the load on subsequent column processing. The concentrated crude extract was pumped into a pretreated AB-8 type macroporous resin column (column diameter to height ratio of 1:8) at a flow rate of 2.2 BV / h for dynamic adsorption. The flavonoid concentration in the effluent was monitored in real time by ultraviolet spectrophotometry. When the flavonoid concentration in the effluent reached 5% of the feed concentration, the resin adsorption was determined to be saturated. Subsequently, the resin column was first rinsed with 3 BV of deionized water at a flow rate of 2 BV / h to remove water-soluble impurities such as polysaccharides and proteins; then, a 70% ethanol aqueous solution was used as the eluent, and gradient elution was performed at a flow rate of 1.1 BV / h, and the eluent was collected in segments. The collected eluent was concentrated under reduced pressure at 50°C until no alcohol odor was detected to obtain the sea buckthorn flavonoid enriched solution. The purity of total flavonoids in the enriched solution was determined by high performance liquid chromatography (HPLC) to be 63.1%, of which quercetin content was 22.1% (as a percentage of total flavonoid mass) and isorhamnetin content was 16.3% (as a percentage of total flavonoid mass).

[0044] (4) Preparation and pre-emulsification of composite wall material: Accurately weigh 20g of gum arabic, 10g of maltodextrin, and 10g of β-cyclodextrin, add 200mL of deionized water, place in a 60℃ constant temperature water bath, and stir continuously at 300r / min for 30min until the three wall material components are completely dissolved, prepare a composite wall material solution with a mass concentration of 20%, and cool to 40℃ for later use. Measure the above enriched liquid equivalent to 10g of dry basis sea buckthorn total flavonoids as the core material, adjust the speed of the high-speed shear machine to 3000r / min, and slowly and evenly add the core material to the composite wall material solution while continuously stirring (the dry basis mass ratio of core material to wall material is 1:3), continue shearing and stirring for 30min to form a uniform and stable oil / water (O / W) type pre-emulsion.

[0045] (5) High-pressure homogenization: The prepared pre-emulsion is transferred to a high-pressure homogenizer, and the homogenization pressure is set to 42 MPa and the homogenization temperature is set to 42 °C. The pre-emulsion is circulated and homogenized 3 times. Through the strong shearing, cavitation and collision effects generated by high pressure, the oil phase droplets are broken down to the micron level, so that the wall material material is more uniformly and densely wrapped on the surface of the core material, resulting in a fine emulsion with uniform particle size distribution and strong stability.

[0046] (6) Spray drying to prepare microcapsules: The fine emulsion is stably delivered to the atomizer of a high-speed centrifugal spray drying tower at a flow rate of 16 mL / min using a peristaltic pump. The spray drying parameters are set as follows: inlet air temperature 135℃, outlet air temperature 67℃, and atomization pressure 0.5 MPa. After being atomized by the atomizer, the emulsion forms tiny droplets that come into full contact with the hot air. The composite wall material on the surface of the droplets rapidly dehydrates, solidifies, and undergoes a glass transition within a short time, forming a dense solid capsule wall that completely encapsulates the sea buckthorn flavonoid core material. After drying, the powdery product collected at the bottom of the tower is the sea buckthorn flavonoid microcapsule.

[0047] Comparative Example 1: Traditional Extraction Process Control A method for preparing sea buckthorn flavonoid microcapsules, comprising the following steps: (1) Raw material pretreatment: completely consistent with Example 1.

[0048] (2) Hot reflux extraction: Add 3000 mL of 70% ethanol aqueous solution at a solid-liquid ratio of 1:15, place it in a round bottom flask, and hot reflux extract in an 80℃ water bath for 120 min. After filtration, the residue is extracted once more, and the filtrates are combined to obtain crude extract.

[0049] (3) Subsequent steps: macroporous resin enrichment, wall material preparation, high pressure homogenization, and spray drying are exactly the same as in Example 1.

[0050] Compared with Example 1, Comparative Example 1 used conventional hot reflux extraction. Under the premise of achieving similar enrichment purity, the extraction time was extended by 4 times, and the flavonoid extraction rate was 65.3%, which was reduced by about 32%. This fully demonstrates the decisive role of the ultrasonic-microwave synergistic extraction technology of the present invention in improving extraction efficiency and yield.

[0051] Comparative Example 2: Single Wall Material Control A method for preparing sea buckthorn flavonoid microcapsules, comprising the following steps: (1) Raw material pretreatment and extraction enrichment: Steps (1)-(2) are completely consistent with Example 1, and an enriched solution with a total flavonoid purity of 62.3% is obtained.

[0052] (2) Preparation of single wall material: Weigh 30g of maltodextrin (equal to the total dry basis mass of the wall material in Example 1), add 200mL of deionized water, stir and dissolve in a water bath at 60℃, and prepare a single wall material solution with a mass concentration of 15%.

[0053] (3) Emulsification, homogenization and drying: The core material addition ratio, shear stirring conditions, high pressure homogenization parameters and spray drying parameters are exactly the same as those in Example 1.

[0054] Compared with Example 1, Comparative Example 2, under the same extraction and enrichment conditions, only changed the wall material to a single maltodextrin, resulting in a microcapsule encapsulation rate of 65.2%, a significant decrease of about 29%, poor product stability, and a flavonoid retention rate of 48.3% after 10 days of light exposure.

[0055] Comparative Example 3: Unencapsulated Raw Material Control A sea buckthorn flavonoid powder, the preparation method of which is as follows: (1) Raw material pretreatment and extraction enrichment: Steps (1)-(2) are completely consistent with Example 1, and sea buckthorn flavonoid enrichment solution is obtained.

[0056] (2) Direct drying: The enriched liquid was concentrated to near dryness under reduced pressure at 50°C, transferred to a vacuum drying oven, dried to constant weight at 60°C, pulverized and passed through a 100-mesh sieve to obtain unencapsulated sea buckthorn flavonoid powder.

[0057] Example 4 This embodiment aims to systematically detect and analyze the key quality indicators of key intermediate products and final raw materials in the preparation processes described in Examples 1 to 3, including flavonoid extraction rate, total flavonoid purity, and the content of characteristic active ingredients quercetin and isorhamnetin.

[0058] 1. Determination of flavonoid extraction rate: (1) Method Principle Sea buckthorn flavonoids undergo a complexation reaction with aluminum nitrate under alkaline conditions to form a stable yellow complex. This complex exhibits a maximum absorption peak at 500 nm, and its absorbance shows a good linear relationship with flavonoid concentration within a certain range. By comparing with a rutin standard curve, the total flavonoid content in the extract can be calculated, and the flavonoid extraction rate can then be derived. This method is simple to operate, highly stable, and suitable for rapid detection of batch samples.

[0059] (2) Reagents and instruments Reagents: Rutin (purity ≥98%, standard), anhydrous ethanol (analytical grade), sodium nitrite (analytical grade), aluminum nitrate (analytical grade), sodium hydroxide (analytical grade), deionized water.

[0060] Instruments: UV-Vis spectrophotometer, electronic balance (accuracy 0.0001g), constant temperature water bath, volumetric flasks (10mL, 50mL, 100mL), pipettes (1mL, 2mL, 5mL), stoppered colorimetric tubes (10mL).

[0061] (3) Operating steps 1) Plotting the standard curve Accurately weigh 20.0 mg of rutin standard dried to constant weight at 120℃, place it in a 100 mL volumetric flask, dissolve it in 70% ethanol and dilute to the mark, shake well to obtain a rutin standard stock solution with a concentration of 0.2 mg / mL.

[0062] Accurately pipette 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of rutin standard stock solution into 10 mL stoppered colorimetric tubes, add 70% ethanol to each tube to a final volume of 5 mL, and shake well.

[0063] Add 0.3 mL of 5% sodium nitrite solution to each tube, shake well, and let stand for 6 min; then add 0.3 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min; finally add 4 mL of 4% sodium hydroxide solution, dilute to 10 mL with 70% ethanol, shake well, and let stand for 15 min.

[0064] Using a blank solution (the reaction solution corresponding to 0.0 mL of rutin standard stock solution) as a reference, the absorbance (A) of each tube was measured at a wavelength of 500 nm.

[0065] A standard curve was plotted with rutin concentration (C, mg / mL) on the x-axis and absorbance (A) on the y-axis. Linear regression analysis was performed to obtain the regression equation A = aC + b (correlation coefficient r ≥ 0.999).

[0066] 2) Determination of sample extract Accurately pipette 1.0 mL of sea buckthorn flavonoid extract (filtered through a 300-mesh filter cloth) into a 50 mL volumetric flask, dilute to the mark with 70% ethanol, and shake well to obtain the sample dilution.

[0067] Accurately pipette 1.0 mL of the sample dilution solution into a 10 mL stoppered colorimetric tube, and proceed with the reaction according to the subsequent steps of the standard curve plot (add sodium nitrite, aluminum nitrate, and sodium hydroxide solution). After standing for 15 min, measure the absorbance at a wavelength of 500 nm.

[0068] The total flavonoid concentration in the sample dilution was calculated based on the regression equation, and then the total mass (m1) of the total flavonoids in the extract was obtained.

[0069] 3) Extraction rate calculation Flavonoid extraction rate (%) = (Total flavonoid mass in extract m1 / Initial total flavonoid mass in raw material m0) × 100%; Initial total flavonoid mass in raw material m0 (mg) = Raw material mass (g) × Total flavonoid content in raw material (%) × 1000 Note: The total flavonoid content of the raw materials needs to be determined in advance using the same method. Three parallel experiments should be set up, and the average value of the results should be taken. The relative standard deviation (RSD) should be ≤2.0%.

[0070] (4) Precautions ① All reagents must be prepared and used immediately, especially sodium nitrite solution and aluminum nitrate solution, to avoid affecting the color development effect due to reagent deterioration.

[0071] ② The temperature of the colorimetric reaction needs to be controlled at 25±2℃, and the standing time must be strictly followed in accordance with the operating procedures to ensure the stable formation of the complex.

[0072] ③ The sample extract solution needs to be filtered to remove impurities to avoid interference from suspended particles in the absorbance measurement.

[0073] 2. Determination of total flavonoid purity and quercetin and isorhamnetin content (optimized high performance liquid chromatography method) (1) Method Principle Reversed-phase high-performance liquid chromatography (RP-HPLC) was employed with a C18 column as the stationary phase and a methanol-phosphoric acid aqueous solution as the mobile phase. Gradient elution was used to separate the components in total flavonoids from sea buckthorn. Qualitative analysis was performed based on the retention times of rutin, quercetin, and isorhamnetin standards, while quantification was conducted using the external standard method based on peak area. The purity of total flavonoids and the content of target monomer components were calculated. This method demonstrates good separation efficiency and accurate quantification, meeting industry standards for the detection of active ingredients in plant extracts.

[0074] (2) Reagents and instruments Reagents: Rutin (purity ≥98%), quercetin (purity ≥98%), and isorhamnetin (purity ≥98%), all of which are standards; methanol (chromatographic grade), phosphoric acid (analytical grade), and ultrapure water; the sample extract was concentrated under reduced pressure, dissolved in methanol, and filtered through a 0.45 μm organic phase membrane for later use.

[0075] Instruments: High performance liquid chromatograph (equipped with UV detector or diode array detector), chromatographic column (Agilent ZORBAX SB-C18, 4.6mm×250mm, 5μm), electronic balance (accuracy 0.0001g), ultrasonic cleaner, high-speed refrigerated centrifuge, volumetric flasks (10mL, 50mL), pipettes (1mL, 2mL, 5mL), organic phase filter membrane (0.45μm).

[0076] (3) Operating steps 1) Preparation of standard solutions Accurately weigh 10.0 mg each of rutin, quercetin, and isorhamnetin standards, place them in a 10 mL volumetric flask, dissolve them in methanol, dilute to the mark, and shake well to obtain a single standard stock solution with a concentration of 1.0 mg / mL.

[0077] Accurately pipette appropriate amounts of each single standard stock solution and place them in the same 50 mL volumetric flask. Dilute to the mark with methanol, shake well, and obtain a mixed standard working solution (rutin: 20 μg / mL, quercetin: 15 μg / mL, isorhamnetin: 10 μg / mL). Filter the solution through a 0.45 μm organic phase filter membrane for later use.

[0078] 2) Optimization of chromatographic conditions Mobile phase: Methanol (phase A) - 0.4% phosphoric acid aqueous solution (phase B) Gradient elution program: 0–10 min, 40% A → 50% A; 10–20 min, 50% A → 60% A; 20–30 min, 60% A → 70% A; 30–40 min, 70% A → 80% A; 40–45 min, 80% A hold; 45–50 min, 80% A → 40% A (equilibrate the column). Detection wavelength: 370nm (characteristic absorption wavelength of rutin, quercetin, and isorhamnetin) Column temperature: 30℃ Flow rate: 1.0 mL / min Injection volume: 20 μL Theoretical plate number: not less than 5000 based on quercetin peak, resolution ≥1.5.

[0079] 3) Sample determination Take an appropriate amount of sea buckthorn flavonoid enrichment solution, concentrate it under reduced pressure until there is no alcohol odor, accurately weigh 0.1g of the concentrate, place it in a 10mL volumetric flask, add methanol and sonicate to dissolve (power 300W, time 10min), cool and then dilute to the mark with methanol and shake well.

[0080] Centrifuge the above solution at 8000 rpm for 10 min, and filter the supernatant through a 0.45 μm organic phase filter membrane to obtain the sample test solution.

[0081] According to the set chromatographic conditions, inject the mixed standard working solution and the sample test solution separately, record the chromatogram, and perform qualitative analysis based on retention time and quantitative analysis based on peak area.

[0082] 4) Quantitative calculation Standard curve preparation: Accurately pipette 0.5 mL, 1.0 mL, 2.0 mL, 4.0 mL, 8.0 mL, and 10.0 mL of the mixed standard working solution into 10 mL volumetric flasks, dilute to the mark with methanol, and mix well to obtain a series of standard solutions of different concentrations. Inject and determine the solutions according to chromatographic conditions. Plot a standard curve with the standard concentration (C, μg / mL) as the abscissa and the peak area (A) as the ordinate. Perform linear regression analysis to obtain the regression equations for each component (correlation coefficient r ≥ 0.999).

[0083] Total flavonoid purity calculation: Total flavonoid purity (%) = (total flavonoid mass in enrichment solution / dry basis mass of enrichment solution) × 100%; where, the total flavonoid mass is calculated by the rutin standard curve (rutin is used as a control, covering the main flavonoid components in the enrichment solution).

[0084] Quercetin and isorhamnetin content calculation: The mass concentration of quercetin and isorhamnetin in the sample is calculated based on their respective standard curves, and then converted into their percentage of the total flavonoid mass.

[0085] (4) Precautions ① The mobile phase should be degassed by ultrasound for 15 minutes before use to avoid air bubbles affecting the separation effect and detection stability of the chromatographic column.

[0086] ② Samples must be centrifuged and filtered before analysis to remove large molecular impurities such as proteins and polysaccharides and prevent clogging of the chromatographic column.

[0087] ③ The injection needle should be rinsed with methanol before each injection to avoid cross-contamination of samples; after use, the column should be rinsed with methanol-water (90:10) for 30 minutes, and then rinsed with methanol for 20 minutes to extend the column's lifespan.

[0088] ④ Three parallel experiments were set up, and the average value of the results was taken. The RSD of the total flavonoid purity determination was ≤2.5%, and the RSD of the quercetin and isorhamnetin content determination was ≤3.0%.

[0089] 3. Test Results The extracts and enriched solutions of Examples 1, 2, and 3 were subjected to the above-mentioned tests, and the results are shown in Table 1.

[0090] Table 1. Results of Key Quality Indicators Tested in Examples 1-3

[0091] Note: The purity and component content in the table are the detection values ​​of the enriched extract. The average value is taken from three parallel experiments. The relative standard deviation (RSD) is less than 2.0%, indicating that the method has good precision.

[0092] As shown in Table 1, the preparation method provided by this invention can stably achieve efficient extraction and enrichment of sea buckthorn flavonoids. The flavonoid extraction rate is consistently above 94.5%, reaching a maximum of 96.2%, significantly higher than the traditional process in Comparative Example 1. Simultaneously, the purity of the total flavonoids after enrichment reaches over 60%, demonstrating the excellent efficiency and reliability of the ultrasonic-microwave synergistic extraction combined with macroporous resin enrichment process. In addition to achieving high total flavonoid purity, the method of this invention can effectively enrich key active monomers. In the three examples, the quercetin content is ≥20%, and the isorhamnetin content is ≥15%, reaching a high level. This ensures that the microcapsule products prepared using these raw materials have a clear material basis and expected biological activity. Example 5 This embodiment further systematically tests the key physical properties of the sea buckthorn flavonoid microcapsules prepared in Examples 1-3, including average particle size, moisture content and bulk density, in order to comprehensively evaluate the consistency of the product and the stability of the process.

[0093] 1. Measurement Method ① Average particle size: Determined using a laser particle size analyzer. A trace sample was dispersed in anhydrous ethanol and sonicated for 1 minute to eliminate agglomeration, followed by analysis. The instrument automatically reported the volume average particle size (D). 50 ) and particle size distribution span.

[0094] ② Moisture content: According to Method 1 (direct drying method) of the National Food Safety Standard for Determination of Moisture in Food (GB 5009.3-2016). Accurately weigh approximately 2g of the sample into a pre-weighed weighing dish, place it in an electric heating drying oven at 105℃ and dry until constant weight. Calculate the moisture content based on the weight loss.

[0095] ③ Bulk density: Determined using the free-stacking method with a standard graduated cylinder. Slowly pour the dried sample through a funnel into a clean 100mL graduated cylinder until full. Smooth the top with a scraper and weigh the powder. The bulk density is calculated as the ratio of powder mass to volume; repeat three times and take the average.

[0096] 2. Measurement Results The above measurements were performed on the products of Examples 1-3, and the results are shown in the table below.

[0097] Table 2 Results of Key Physical Properties Measurement of Microcapsules in Examples

[0098] Note: The data in the table are the mean ± standard deviation of three parallel determinations.

[0099] As shown in Table 2, the average particle size of the products in Examples 1-3 was between 30-35 μm, the moisture content was all below 3%, and the bulk density was between 0.6-0.8 g / cm³. 3 Within the specified range, the batch-to-batch differences of all key physical indicators are small (RSD≤10%), indicating that the preparation process of this invention is stable and the key physical properties of the product are reproducible.

[0100] Example 6 This embodiment aims to accurately determine and compare the encapsulation efficiency of the sea buckthorn flavonoid microcapsules prepared by the present invention (Examples 1-3) and the control standard prepared using a single wall material (Comparative Example 2), so as to objectively evaluate the encapsulation effect of the present invention.

[0101] 1. Detection Principles and Methods The encapsulation efficiency was determined using a buffer-centrifugation-UV spectrophotometry method. The principle is as follows: a specific buffer solution is used to elute unencapsulated free flavonoids from the microcapsule surface under mild conditions, while a strong solvent is used to disrupt the microcapsule structure and extract total flavonoids. The difference between the two methods is calculated to determine the proportion of effectively encapsulated flavonoids. The specific steps are as follows: ① Sample preparation: Accurately weigh 0.1g of each sample to be tested (products of Examples 1-3, product of Comparative Example 2, and unencapsulated control powder of Comparative Example 3) (accurate to 0.0001g) and label them respectively.

[0102] ② Extraction of free flavonoids from the surface: Place each sample in a 50 mL centrifuge tube and add 30 mL of pH 6.8 phosphate buffer. Shake at 200 rpm for 30 minutes at room temperature to ensure complete dissolution of the surface flavonoids. Then centrifuge at 4℃ and 8000 rpm for 15 minutes, carefully aspirating the supernatant. Repeat the extraction once, combine the two supernatants, and bring the volume to 50 mL with the same buffer to obtain the free flavonoid test solution.

[0103] ③ Extraction of total flavonoids: Accurately weigh equal amounts of each sample and place them separately in 50mL stoppered conical flasks. Add 40mL of 70% ethanol solution. Sonicate (300W) for 30 minutes to completely destroy the wall material and release all core material. After cooling, transfer the solution to a 50mL volumetric flask, dilute to the mark with 70% ethanol, mix well, and filter to obtain the total flavonoid test solution.

[0104] ④ Content determination and calculation: Accurately measure appropriate amounts of the two test solutions mentioned above, and use rutin as a standard to measure the absorbance at a wavelength of 360 nm. Calculate the flavonoid mass based on the standard curve. The encapsulation rate is calculated using the following formula: Encapsulation rate (%) = [1 - (Sea buckthorn flavonoid content on the surface of microcapsules / Total sea buckthorn flavonoid content in microcapsules)] × 100%. Each sample is measured in triplicate.

[0105] 2. Test Results The embedding rate of each sample is shown in the table below.

[0106] Table 3 Comparison of embedding rate determination results for different samples

[0107] As shown in Table 3, the sea buckthorn flavonoid microcapsules prepared in Examples 1-3 using the specific composite wall material and process provided by this invention all achieved encapsulation rates higher than 91%, reaching a maximum of 93.1%. This indicates that the method of this invention can stably and efficiently encapsulate sea buckthorn flavonoids within the microcapsules. In Comparative Example 2, which used only maltodextrin as the wall material, with other steps identical to Example 1, the encapsulation rate plummeted to 65.2%. This demonstrates that the specific composite wall material system used in this invention is the core and key to achieving high encapsulation rates, and a single wall material cannot achieve the same effect.

[0108] Example 7 This embodiment aims to quantitatively evaluate the chemical stability of the sea buckthorn flavonoid microcapsules (Examples 1-3), the control product using a single wall material (Comparative Example 2), and the unencapsulated sea buckthorn flavonoids (Comparative Example 3) under simulated strong light and high temperature stress based on the accelerated light irradiation experiment method. The core indicator is the flavonoid retention rate.

[0109] 1. Experimental Methods: Sea buckthorn flavonoids, especially aglycones such as quercetin, are sensitive to ultraviolet and visible light and are prone to photo-oxidative degradation, leading to loss of activity. This experiment simulates and intensifies light and heat conditions to evaluate the protective ability of different wall material systems for the core material in a short period of time.

[0110] ① Light source and equipment: A UVA fluorescent lamp (main wavelength 320-400nm) was used as the light source and placed in a constant temperature light chamber. The light intensity was calibrated to 2000±50lx at 10cm from the sample surface.

[0111] ② Experimental conditions: The temperature was kept constant at 45±1℃, and the relative humidity was controlled at 50±5%RH.

[0112] ③ Sample preparation: Accurately weigh 2.0 g (accurate to 0.0001 g) each of the microcapsule powder prepared in Examples 1, 2, 3 and Comparative Example 2, and the sea buckthorn flavonoid powder prepared in Comparative Example 3, and place them in a dry and clean glass petri dish. Gently spread the powder into a thin layer of uniform thickness (about 2 mm), without covering the dish, to ensure that the sample is fully exposed to the light environment. Set up 3 replicates for each sample.

[0113] ④ Sampling and Detection: Samples were taken on days 0 (initial), 2, 4, 6, 8, and 10 of the light treatment. During sampling, the entire sample in the culture dish was thoroughly mixed, and approximately 0.1 g was accurately weighed for content determination. The total flavonoid content in the samples was determined using high-performance liquid chromatography (HPLC).

[0114] ⑤ Calculation of retention rate: Flavonoid retention rate (%) = (Flavoroid content in the sample at the current time point / Flavonoid content in the initial sample at day 0) × 100%.

[0115] 2. Experimental Results The results of the flavonoid retention rate determination of each sample at different time points after 10 days of accelerated light irradiation experiment are shown in the table below.

[0116] Table 4. Changes in flavonoid retention rate (%) of different microcapsule samples under 45℃ light irradiation

[0117] As shown in Table 4, the sea buckthorn flavonoid microcapsules prepared using the composite wall material (gum arabic: maltodextrin: β-cyclodextrin = 2:1:1) described in Examples 1, 2, and 3 all exhibited a flavonoid retention rate exceeding 85% after 10 days of treatment under harsh light conditions of 45°C and 2000 lx. This demonstrates that the composite wall material system effectively blocks light and resists synergistic damage from thermal oxidation, providing strong protection for the core material. In contrast, the microcapsules of Comparative Example 2 (single maltodextrin wall material) showed extremely rapid flavonoid degradation under the same experimental conditions. After 2 days of light exposure, the flavonoid retention rate dropped to 82.5%, to 69.8% after 4 days, further to 56.4% after 6 days, and only 50.1% after 8 days. By 10 days, the flavonoid retention rate had plummeted to 48.3%, far lower than in Examples 1-3 (85.8%-87.5%). This clearly demonstrates that a single maltodextrin wall material cannot form a dense and stable protective barrier, making it difficult to resist the destructive effects of light and high temperature on sea buckthorn flavonoids. The above data indicate that the specific composite wall material system of this invention is key to achieving high light stability; a single wall material cannot form a sufficiently dense and stable barrier to protect sea buckthorn flavonoids.

[0118] Example 8 This embodiment aims to quantitatively evaluate the release behavior of the sea buckthorn flavonoid microcapsules (Examples 1-3) prepared in this invention in a simulated human gastrointestinal environment based on an in vitro simulated digestion method. The core indicator is the cumulative release rate of flavonoids, which is used to predict its bioavailability.

[0119] 1. Experimental Methods: The gastrointestinal environment experienced by the microcapsule product after oral administration was simulated by sequential treatment with simulated gastric juice (SGF, acidic, containing pepsin) and simulated intestinal juice (SIF, neutral, containing pancreatic enzymes and bile salts). The in vitro release characteristics of the microcapsules were evaluated by measuring the flavonoid content released into the digestive fluid at different time points, thereby indirectly reflecting their potential oral bioavailability.

[0120] ① Simulated gastric juice (SGF): Take 0.1 mol / L hydrochloric acid solution and adjust the pH to 1.2 ± 0.1 with 1 mol / L NaOH or HCl. Add 0.32 g of pepsin (enzyme activity ≥ 2500 U / mg) to every 100 mL of solution and prepare immediately before use.

[0121] ② Simulated intestinal fluid (SIF): Take 0.05 mol / L potassium dihydrogen phosphate solution and adjust the pH to 6.8 ± 0.1 with 1 mol / L NaOH. Add 0.10 g of trypsin (trypsin activity ≥ 250 USP U / mg) and 0.34 g of bile salts (porcine bile salts) to every 100 mL of solution and prepare immediately before use.

[0122] ③ Instruments and equipment: constant temperature shaking water bath (temperature control 37±0.5℃, speed 100rpm), centrifuge, high performance liquid chromatograph (HPLC) or ultraviolet spectrophotometer.

[0123] ④ Sample preparation and determination: Accurately weigh one portion of the microcapsule powder prepared in Examples 1, 2 and 3 respectively, so that the total flavonoid content of sea buckthorn in each sample is approximately 20 mg (accurately record the actual content M0). Place the sample in a 50 mL stoppered conical flask.

[0124] ⑤ Gastric stage (0-2h): Add 50mL of SGF preheated to 37℃ to an Erlenmeyer flask and immediately place it in a constant temperature shaking water bath at 37℃ and 100rpm to begin the reaction. At 30, 60, and 120 min, respectively, take 2mL of the reaction solution and immediately add 2mL of fresh SGF preheated to 37℃. The samples are immediately filtered through a 0.45μm microporous membrane, and the filtrate is used to determine the flavonoid release.

[0125] ⑥ Intestinal phase (2-6h): After the 2h gastric phase, quickly add an equal volume (50mL) of preheated SIF to 37℃ (at which point the pH will automatically rise to approximately 6.8) to the conical flask, and continue shaking under the same conditions. Take 2mL samples at 150, 180, 240, and 360 mins (i.e., 30, 60, 120, and 240 mins after the start of the intestinal phase) and replenish the fluid, treating them in the same way as the gastric phase.

[0126] ⑦ Content determination and calculation: The flavonoid concentration in the filtrate at each time point was determined by HPLC (C t The cumulative release amount (M) is calculated. The cumulative release rate (%) is calculated as follows: Cumulative release rate (%) = (M / M0) × 100%, where M = Σ(C t (×V), where V is the total volume of the digestion solution (100mL). The dilution caused by sampling needs to be taken into account when calculating.

[0127] 2. Experimental Results The cumulative flavonoid release rate of the microcapsule samples from Examples 1-3 at different time points was determined after a 6-hour in vitro simulated gastrointestinal release experiment. The results are shown in the table below.

[0128] Table 5. Cumulative flavonoid release rate (%) of the microcapsules in the simulated gastrointestinal environment of the examples.

[0129] As shown in Table 5, the microcapsules of this invention (Examples 1-3) exhibit excellent "acid-resistant-enteric-coated" controlled-release characteristics: slow release in the gastric stage (<16%), effectively protecting the core material; rapid release after entering the intestinal environment, with a cumulative release rate of over 80% after 6 hours. The data clearly demonstrate that the specific composite wall material system of this invention is key to achieving targeted oral delivery of flavonoids and improving their bioavailability.

[0130] Example 9 This embodiment evaluates the antioxidant activity of the sea buckthorn flavonoid microcapsules prepared by the present invention (Examples 1-3) and the unencapsulated sea buckthorn flavonoid raw material (Comparative Example 3). By measuring the DPPH and ABTS free radical scavenging rates at specific concentrations, the effect of the microencapsulation process on the retention of the core biological activity of sea buckthorn flavonoids is directly verified.

[0131] 1. Experimental Methods DPPH free radical scavenging experiment: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable free radical. Its ethanol solution is deep purple and has maximum absorption at 517 nm. When antioxidants react with DPPH, they are reduced and decolorized, and the absorbance decreases. The degree of decrease is linearly related to the free radical scavenging ability of the antioxidant.

[0132] ABTS radical scavenging experiment: ABTS (2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid) is activated by an oxidant to generate stable blue-green cationic radicals ABTS. + It exhibits maximum absorption at 734 nm. Antioxidants can cause it to fade, and changes in absorbance can quantitatively reflect the total antioxidant capacity of the sample.

[0133] ① Sample preparation: Accurately weigh each sample (microcapsules of Examples 1-3, unencapsulated flavonoid powder of Comparative Example 3), extract with methanol-water (1:1, v / v) mixed solvent by ultrasonic extraction, prepare test solutions, and uniformly adjust to a specific concentration based on total flavonoids from sea buckthorn.

[0134] ②DPPH free radical scavenging assay: The main test concentration was 0.2 mg / mL (calculated as total flavonoids). Take 2.0 mL of the sample test solution, add 2.0 mL of 0.1 mmol / L DPPH-ethanol solution, mix well, and react in the dark for 30 minutes. Measure the absorbance at 517 nm. Ascorbic acid (Vc) was used as a positive control.

[0135] The formula for calculating DPPH free radical scavenging rate is as follows: A sample Representative sample absorbance: refers to the absorbance after the reaction of "sample solution + DPPH working solution" (measured at a wavelength of 517 nm). A control The background absorbance of the sample represents the absorbance of the "sample solution + anhydrous ethanol", which is used to subtract the interference of the sample's own color or turbidity. A blank Absorbance representing the blank control: refers to the absorbance of "solvent + DPPH working solution", representing the initial amount of DPPH free radicals.

[0136] ③ABTS + Free radical scavenging assay: The main test concentration was 0.6 mg / mL (based on total flavonoids). Take 0.2 mL of the sample test solution and add 4.0 mL of ABTS. + The working solution (diluted with phosphate buffer to an absorbance of 0.70 ± 0.02 at 734 nm) was reacted for 6 minutes, and the absorbance was measured at 734 nm. Vitamin C was used as a positive control.

[0137] ABTS + The formula for calculating free radical scavenging rate is as follows: A sample Representative sample absorbance: refers to "sample solution + ABTS" + The absorbance of the working solution after the reaction (measured at a wavelength of 734 nm).

[0138] A blank Absorbance representing the blank control: refers to the absorbance of the solvent plus ABTS. + The absorbance of the "working solution" represents ABTS. + The initial amount of free radicals.

[0139] 2. Experimental Results The results of the free radical scavenging rate determination for each sample are shown in the table below.

[0140] Table 6 Free radical scavenging rate of samples at specific concentrations

[0141] According to the results in Table 6, the sea buckthorn flavonoid microcapsules prepared in this invention (Examples 1-3) have a DPPH free radical scavenging rate of approximately 88% at a concentration of 0.2 mg / mL and an ABTS free radical scavenging rate of approximately 0.6 mg / mL. + The free radical scavenging rate is as high as 98% or more. This indicates that the entire microencapsulation preparation process of this invention completely preserves the inherent strong antioxidant activity of sea buckthorn flavonoid molecules. The antioxidant capacity of the product of this invention is close to that of the classic antioxidant vitamin C (ascorbic acid) in the same test system, fully demonstrating its functional value as a highly efficient natural antioxidant.

[0142] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing sea buckthorn flavonoid microcapsules, characterized in that, Includes the following steps: (1) Using sea buckthorn pomace as raw material, and using an ethanol aqueous solution with a volume concentration of 65%-75% as solvent, ultrasonic-microwave synergistic extraction was performed to obtain sea buckthorn flavonoid extract; the conditions for ultrasonic-microwave synergistic extraction were: solid-liquid ratio 1:10-1:20, ultrasonic power 350-450W, microwave power 250-350W, extraction temperature 50-60℃, and extraction time 25-35min; (2) The flavonoid extract is adsorbed through a macroporous resin column and then eluted with an ethanol aqueous solution with a volume concentration of 65%-75%, and the enriched eluent is collected. (3) Gum arabic, maltodextrin and β-cyclodextrin are dissolved in water at a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2) to prepare a wall material solution; the enriched eluent is added to the wall material solution as a core material, and after mixing and high-pressure homogenization, an emulsion is obtained. (4) The emulsion is spray-dried to obtain sea buckthorn flavonoid microcapsules.

2. The preparation method according to claim 1, characterized in that, The macroporous resin is AB-8 type resin, with an adsorption flow rate of 1.5-2.5 BV / h and an elution flow rate of 0.8-1.2 BV / h.

3. The preparation method according to claim 1, characterized in that, The dry basis mass ratio of the core material to the wall material is 1:(2-4).

4. The preparation method according to claim 1, characterized in that, The high-pressure homogenization process is carried out at a pressure of 38-42 MPa and a homogenization temperature of 35-45℃.

5. The preparation method according to claim 1, characterized in that, The spray dryer has an inlet air temperature of 120-140℃, an outlet air temperature of 60-70℃, and a feed rate of 10-20mL / min.

6. A sea buckthorn flavonoid microcapsule, characterized in that, It is prepared by the preparation method described in any one of claims 1-5.

7. The sea buckthorn flavonoid microcapsules according to claim 6, characterized in that, The core material of the sea buckthorn flavonoid microcapsules is total sea buckthorn flavonoids with a purity of not less than 60 wt%; the wall material is a composite wall material composed of gum arabic, maltodextrin and β-cyclodextrin in a mass ratio of (1.5-2.5):(0.8-1.2):(0.8-1.2); the encapsulation rate of the microcapsules is not less than 90%.

8. The sea buckthorn flavonoid microcapsules according to claim 7, characterized in that, The core material contains quercetin at a content of not less than 20% of the total flavonoids from sea buckthorn and isorhamnetin at a content of not less than 15% of the total flavonoids from sea buckthorn.

9. The use of the sea buckthorn flavonoid microcapsules according to any one of claims 6-8 in the preparation of antioxidant products.

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