Danfeng peony polypeptide microcapsule, preparation method and application thereof in reducing blood sugar

Abstract

The present application belongs to the field of biological medicine and functional food technology, and discloses a peony polypeptide microcapsule, a preparation method and application thereof in reducing blood sugar. The microcapsule is prepared by using a wrapping material to wrap the peony polypeptide for reducing blood sugar, and is made into a W1 / O / W2 type multiple emulsion, or is further dried to obtain a microcapsule solid. The present application realizes efficient encapsulation and stable release of the peony polypeptide for reducing blood sugar by using the microcapsule wrapping technology for the first time. The obtained microcapsule has uniform particle size, stable structure and good release performance. The product obtained by the technology is suitable for ordinary food, health food, special medical food and drug addition and application of high blood sugar people.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and functional food technology, and in particular to a Danfeng peony polypeptide microcapsule, its preparation method, and its application in lowering blood sugar. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia, with type 2 diabetes accounting for over 90% of all cases worldwide. Long-term hyperglycemia can lead to various complications such as retinopathy, nephrotoxicity, and atherosclerosis, seriously threatening human health. Currently, commonly used antidiabetic drugs such as acarbose and miglitol can lower blood sugar by inhibiting α-glucosidase activity, but long-term use can easily cause side effects such as hepatotoxicity and gastrointestinal discomfort. Therefore, developing naturally derived, highly active antidiabetic ingredients with low toxicity is of significant research importance and practical application value.

[0003] Peony (Peony danfengense) is a plant with both medicinal and edible uses, and has a long history of consumption with good safety. Our research team discovered that peptides obtained from the enzymatic hydrolysis of Peony danfengense petals possess good hypoglycemic activity (CN117568431A). Short peptides are protein fragments formed by 2-20 amino acids linked by peptide bonds, and are classified into two sources: protein synthesis and degradation. They are also known as small peptides or oligopeptides, or collectively as polypeptides. The short peptide proteins described in the above invention are obtained through food-derived enzymatic degradation and belong to the category of polypeptides, hereinafter referred to as polypeptides. Poor oral absorption and low bioavailability of polypeptide drugs and foods have always been bottlenecks and pain points in current research. Natural polypeptides are easily inactivated by factors such as temperature and pH in the in vitro environment; moreover, in the in vivo gastrointestinal environment, various digestive enzymes can specifically cleave peptide bonds, leading to the direct destruction of their active structure. The strong acid environment in the stomach also breaks the internal valence bond structure of polypeptides, causing the specific spatial conformation of polypeptides to be destroyed, losing their ability to bind to target cells, thus failing to effectively exert biological activity. Therefore, using microencapsulation technology to encapsulate active peptide components through wall materials to improve the oral stability of active peptides, prolong their duration of action, and enhance their bioavailability is of great research significance.

[0004] Because peptides are easily degraded and inactivated by external environmental influences, the acid-base properties of the wall material and the high-temperature and high-pressure process conditions during microcapsule preparation can all affect their activity, thus increasing the encapsulation efficiency and the difficulty of stable delivery. The hypoglycemic peptide from *Peony danfengense* petals is a first-time discovery by our team, demonstrating high innovation. Currently, there are no reports on the microencapsulation preparation of short peptides or polypeptides from *Peony danfengense* petals, nor on their application in the anti-diabetic field. This invention is highly innovative. Therefore, this invention uses *Peony danfengense* petals as raw material, prepares hypoglycemic peptides through enzymatic hydrolysis, and uses a two-step emulsification method combined with ultrasonic emulsification technology to prepare microcapsules. The aim is to develop a food-derived microcapsule product with good stability, high bioavailability, and significant anti-diabetic activity, realizing high-value-added utilization of *Peony danfengense* resources, and providing a new direction for the research and development of anti-diabetic functional foods and drugs.

[0005] Through searching, the following published documents related to this invention patent application were found. 1. Patent 1: A short peptide protein from *Peony davidii* petals and its application, Publication No.: CN117568431A. This invention forms the basis of our research team's work. It discloses a method for obtaining protein from *Peony davidii* petals via an alkali-soluble acid-precipitated method, followed by enzymatic hydrolysis with a food-derived hydrolytic enzyme to obtain a short peptide protein. This short peptide protein exhibits good α-glucosidase inhibitory activity, can improve hyperglycemia symptoms in type 2 diabetic mice, and has certain antioxidant effects. This invention only involves the preparation technology of the protein and hypoglycemic peptides and does not involve the subsequent preparation and application of microcapsules, which is fundamentally different from our present invention.

[0006] 2. Patent 2: Wang Feng, et al., A Highly Dispersible Peony Seed Oil Nanoliposome Emulsion and Its Preparation Method and Application, Publication No.: CN120420241A. This patent discloses a highly dispersed nanoliposome emulsion encapsulating peony seed oil. It effectively encapsulates water-insoluble peony seed oil in nanoliposomes through a combination of methods including hot-melt method, thin-film dispersion method, microemulsification method, homogenization ultrasound, and freeze-drying, significantly improving its water solubility and bioavailability. While this invention encapsulates peony seed oil and prepares a liposome emulsion, the encapsulated contents and preparation method are fundamentally different from this invention.

[0007] 3. Journal: Preparation and Characterization of Emulsion-based Peony SeedOil Microcapsule. Journal of Oleo Science. 2023, 69(3), 219-226. This report uses sodium octenyl succinate starch, β-cyclodextrin, and pectin as wall materials and peony seed oil as core material to prepare an oil-in-water (O / W) emulsion. Microcapsules are constructed by spray drying, which improves the water solubility and thermal stability of peony seed oil. The substance encapsulated in this report is peony seed oil, and the dosage form prepared is an oil-in-water emulsion. However, this invention encapsulates peony petal polypeptides and prepares an oil-in-water (W / O / W) emulsion, which is fundamentally different from this invention.

[0008] By comparison, this invention differs fundamentally from the aforementioned published documents. First, the active substance encapsulated in the aforementioned patents is peony seed oil, while the raw material encapsulated in this invention is *Peony peony* petal polypeptide. Due to the different oil / solid forms of the raw materials, the processes are completely different, and there is no precedent or possibility for reference. Second, the dosage form prepared in the aforementioned reports is a liposome emulsion, while this invention prepares a water-in-oil-in-water microcapsule, resulting in a completely different dosage form. Third, this invention relates to *Peony peony* petal polypeptide encapsulated microcapsules exhibiting good hypoglycemic activity and stability, solving the problem of oral polypeptide stability, and demonstrating significant innovation. Therefore, the motivation for this invention is to prepare *Peony peony* petal polypeptide into microcapsules and obtain its application in anti-diabetic treatment, while solving the problems of poor oral stability and low bioavailability of polypeptides. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Danfeng peony polypeptide microcapsule, its preparation method, and its application in lowering blood sugar.

[0010] The technical solution adopted by this invention to solve its technical problem is: A type of peony polypeptide microcapsule, wherein the microcapsule is made by encapsulating the peony hypoglycemic polypeptide with packaging material to form a W1 / O / W2 type double emulsion microcapsule, or by further drying to obtain a solid microcapsule.

[0011] Furthermore, the blood sugar-lowering polypeptide of Peony peony is obtained by hydrolyzing the petals of Peony peony through food-derived enzymes; The packaging material is made of a single wall material or a composite wall material; The W1 / O / W2 type double emulsion refers to a water-in-oil-in-water type double emulsion, which is a liquid microcapsule that can be applied directly or diluted for application.

[0012] Furthermore, the wall material is one or a combination of several of the following: sodium carboxymethyl cellulose, cyclodextrin, xanthan gum, carrageenan, sodium alginate, chitosan, gum arabic, and phospholipids. Alternatively, the drying process refers to vacuum drying, freeze drying, spray drying, or atmospheric pressure heating drying, after which the microcapsules are solid; Alternatively, an emulsifier may be used in the preparation of the W1 / O / W2 type double emulsion, and the emulsifier may be one or more of Span 20, Span 80, lecithin, polyglycerol polyricinoleate PGPR, sucrose fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester (span), and Tween.

[0013] Furthermore, the Danfeng Peony polypeptide microcapsules comprise an inner aqueous phase, an oil phase, and an outer aqueous phase; wherein the inner aqueous phase is a blood sugar-lowering polypeptide from Danfeng Peony petals, the oil phase is composed of medium-chain triglycerides and polyglycerol polyricinoleate, and the outer aqueous phase is composed of sodium carboxymethyl cellulose, cyclodextrin, and Tween 80; a two-step emulsification method is employed, firstly, a water-in-oil primary emulsion is obtained from the inner aqueous phase and the oil phase, then the primary emulsion is mixed with the outer aqueous phase, and combined with ultrasonic emulsification technology to form microcapsules.

[0014] The preparation method of the Danfeng peony polypeptide microcapsules as described above includes the following steps: (1) Preparation of W1 / O primary emulsion: The Danfeng Mudan polypeptide was dissolved in water and fed at a ratio of 1:1 to 1000 g:mL as the inner aqueous phase of W1; the medium-chain triglycerides and lipophilic emulsifier were mixed at a volume ratio of 1:0.01 to 100 as the oil phase O; the inner aqueous phase of W1 was slowly added to the oil phase O at a volume ratio of 1:0.01 to 100, and stirred until the emulsifier was completely hydrated to form an oil-in-water W1 / O type primary emulsion; (2) Preparation of W1 / O / W2 type double emulsion: Dissolve the composite wall material in water at a ratio of 1:1 to 1000 g:mL, then add 0.1% to 10% Tween80 by mass, stir to completely hydrate the emulsifier, and use it as the W2 external aqueous phase for later use; use the above W1 / O primary emulsion as the core material, and add the W2 external aqueous phase to the W1 / O primary emulsion at a volume ratio of 1:0.01 to 100, heat and stir at 40 to 80°C to make the W1 / O primary emulsion and the W2 external aqueous phase evenly mixed, and then use an ultrasonic emulsifier to perform ultrasonic emulsification treatment on the mixed solution to prepare the W1 / O / W2 type double emulsion. The W1 / O / W2 type double emulsion can be used directly or diluted to obtain Danfeng Peony polypeptide microcapsules.

[0015] Furthermore, in step (1), the Danfeng peony polypeptide is a polypeptide obtained by extracting from the petals of the peony, namely the short peptide protein of the peony petals disclosed in Chinese Patent Publication CN117568431A. Alternatively, the lipophilic emulsifier in step (1) is polyglycerol polyricinoleate; Alternatively, in step (2), the composite wall material is a mixture of sodium carboxymethyl cellulose and cyclodextrin in a mass ratio of 4:1; Alternatively, the specific conditions for heating and stirring in step (2) are: heating and stirring at 50°C for 30 to 60 minutes; Alternatively, the specific conditions for ultrasonic emulsification in step (2) are: ultrasonic power of 65 W, ultrasonic for 3 min, ultrasonic for 4 s, and stop for 2 s; Alternatively, the W1 / O / W2 type double emulsion in step (2) also undergoes the following steps: Microcapsule powder: The W1 / O / W2 type double emulsion prepared above is dried to obtain microcapsule solid powder. The drying method is vacuum drying, freeze drying, spray drying or atmospheric pressure heating drying. The conditions for vacuum drying are: 50℃, vacuum degree -0.08~-0.1MPa; the conditions for freeze drying are: temperature < -60℃, vacuum degree < 20MPa; the conditions for spray drying are: inlet air temperature 160~190℃, outlet air temperature 70~90℃, feed rate 5 mL / min; the conditions for atmospheric pressure heating drying are: 50℃, atmospheric pressure.

[0016] Liquid or solid formulations prepared using the Danfeng Mudanjiang polypeptide microcapsules as described above. Preferably, the formulations include powders, granules, powder for injection, tablets, capsules, or liquid formulations.

[0017] The above-mentioned application of Danfeng Mudan polypeptide microcapsules in the preparation of targeted and slow-release drugs in the intestine.

[0018] The application of the Danfeng Peony polypeptide microcapsules as described above in the preparation of hypoglycemic drugs.

[0019] The above-mentioned applications of Danfeng Peony polypeptide microcapsules in the preparation of special medical foods and / or health foods and / or functional foods and / or ordinary foods.

[0020] The advantages and positive effects of this invention are as follows: 1. This invention is the first to utilize microencapsulation technology to achieve efficient encapsulation of hypoglycemic peptides from peony petals and confirm their hypoglycemic activity. Products obtained by this technology are suitable for use in general foods, health foods, special medical foods, and drug additives and applications for people with hyperglycemia.

[0021] 2. This invention employs a two-step emulsification method combined with freeze-drying technology to prepare microcapsules. By optimizing the ratio of wall material and emulsifier, as well as process parameters, the microcapsules exhibit excellent encapsulation efficiency (>85%) and particle size uniformity (average particle size 3–5 μm). Furthermore, the peptides and wall material interact through intermolecular hydrogen bonds, resulting in structural stability. They are stable in strong acid, strong alkali, and strong salt ion environments, and do not undergo thermal decomposition below 200°C, effectively protecting the bioactivity of the *Peony peony* petal peptides. The wall material used protects the peptides from limited release in simulated gastric juice (release rate <30%), reducing degradation in the gastric environment. In simulated intestinal juice, the cumulative release rate is >90%, demonstrating excellent release performance. This allows most of the peptides to penetrate the intestinal mucosa and enter the bloodstream to exert their active effect, enhancing oral bioavailability. This invention is not only suitable for encapsulating oral formulations of *Peony peony* petal peptides, but this technology also provides a good reference for peptides from other medicinal and edible plants.

[0022] 3. The W1 / O / W2 type microcapsules prepared by the two-step emulsification method of this invention can achieve effective bilayer encapsulation of hydrophilic peptides, significantly improving their stability to temperature, salt ions, oxygen, and the gastrointestinal digestive environment. The microcapsules can significantly reduce postprandial blood glucose levels in mice (p < 0.001), and the hypoglycemic effect is superior to that of unencapsulated peptides. Their structural system can improve the absorption efficiency of peptides in vivo, avoid the destruction of microcapsules by acid and enzymatic hydrolysis in the gastric environment, and achieve targeted and slow release of active peptides in the intestine. Compared with unencapsulated peptides, the active duration in the intestinal environment is longer, thus improving bioavailability.

[0023] 4. The Danfeng peony petal polypeptide microcapsules prepared by this invention have significant hypoglycemic activity and are derived from natural plants, with low toxicity and side effects. Compared with chemically synthesized antidiabetic drugs, they are more suitable for long-term use.

[0024] 5. The preparation process of this invention is stable and controllable, and the active peptide microcapsule products have diverse applications. They can be used to prepare various dosage forms of drugs, functional foods / health products / special medical foods, providing new ideas and material basis for the research and development of anti-diabetic products.

[0025] 6. The present invention opens up a new way for the in-depth and high-value utilization of peony resources, avoiding the disposal of some Danfeng peony petals as waste after being stored for more than a year. It fully develops agricultural resources while reducing the burden on farmers on agricultural waste disposal, and realizes the full and effective utilization of Danfeng peony petals.

[0026] 7. The microcapsule products of this invention can be added as functional factors to various functional foods and health foods such as solid beverages, tablets, and capsules, or as raw materials for drug hypoglycemia reduction, and can be used to develop natural preparations that assist in hypoglycemia reduction. They have the characteristics of being safe, natural, and having clear efficacy.

[0027] 8. This invention uses peony petal hypoglycemic polypeptide microcapsules as the active ingredient, prepared into microcapsules using a two-step emulsification method to form a stable microsphere structure. The resulting microcapsules have uniform particle size, stable structure, and good release performance, making them suitable for use in antidiabetic drugs, antidiabetic functional foods, or health products. This invention overcomes the problems of poor oral stability and low bioavailability of natural active peptides, as well as the significant side effects of existing antidiabetic drugs, realizing the high-value utilization of peony resources and providing a food source solution for hypoglycemia.

[0028] 9. The microcapsules prepared by this invention have uniform particle size and dispersion, and exhibit good pH, salt ion, and temperature stability. Simultaneously, they enable the sustained release of peony petal-based hypoglycemic peptides in the intestine and demonstrate significant hypoglycemic activity in vivo. Therefore, this invention provides, for the first time, a peony petal-based peptide microcapsule and its preparation method, as well as its application in hypoglycemia. It aims to improve the oral stability and bioavailability of existing peony petal-based peptides, providing a safe, effective, and long-term hypoglycemic regimen with promising application prospects in the fields of functional food and pharmaceutical research and development. Attached Figure Description

[0029] Figure 1 The appearance changes of W1 / O primary emulsions prepared with different lipophilic emulsifiers in this invention are shown at 0 days and 14 days. Figure 2 The figures show the encapsulation efficiency (a), appearance changes (b), and microscopic morphology changes (c) of W1 / O / W2 composite emulsions prepared with different external aqueous phase composite wall materials in this invention at 0 days and 14 days; wherein the wall materials are sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), xanthan gum (XG), and carrageenan (CARR), which are compounded in pairs in a ratio of 8:1 (w / w, mass ratio); Figure 3 The figures show the encapsulation efficiency (a), appearance changes (b), and microscopic morphology changes (c) of W1 / O / W2 double emulsions prepared with different PGPR concentrations in this invention at 0 days and 14 days. The wall materials are sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), xanthan gum (XG), and carrageenan (CARR), which are compounded in pairs in a ratio of 8:1 (w / w, mass ratio). Figure 4 The figures show the encapsulation efficiency (a), appearance changes (b), and microscopic morphology changes (c) of W1 / O / W2 double emulsions prepared with different oil-water ratios (O:W1) in this invention at 0 days and 14 days. The wall materials are sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), xanthan gum (XG), and carrageenan (CARR), which are compounded in pairs in a ratio of 8:1 (w / w, mass ratio). Figure 5The figures show the encapsulation efficiency (a), appearance changes (b), and microscopic morphology changes (c) of W1 / O / W2 composite emulsions prepared with different W2 phase composite wall material ratios in this invention at 0 days and 14 days; wherein the wall material is sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), and carrageenan (CARR), compounded in pairs at a ratio of 8:1 (w / w, mass ratio); Figure 6 The figures show the encapsulation efficiency (a), appearance changes (b), and microscopic morphology changes (c) of W1 / O / W2 complex emulsions prepared with different total wall material contents in the W2 phases in this invention at 0 days and 14 days. The wall materials are sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), xanthan gum (XG), and carrageenan (CARR), which are compounded in pairs at a ratio of 8:1 (w / w, mass ratio). Figure 7 The figures show the encapsulation efficiency (a), appearance changes (b), and microscopic morphology changes (c) of W1 / O / W2 double emulsions prepared with different emulsion-to-water ratios in this invention at 0 days and 14 days; wherein the wall material is sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), xanthan gum (XG), and carrageenan (CARR), which are compounded in pairs in a ratio of 8:1 (w / w, mass ratio); Figure 8 This is a particle size distribution diagram of the Danfeng peony petal polypeptide microcapsules in this invention; Figure 9 This is a diagram showing the resolubility characteristics of the solid microcapsule powder in this invention; Figure 10 Fourier transform infrared spectra of medium-chain triglycerides, composite wall materials, peony polypeptides, blank microcapsules, and peony petal polypeptide microcapsules in this invention. Figure 11 This is a laser scanning confocal microscope image of the W1 / O / W2 double emulsion in this invention; Figure 12 This is a scanning electron microscope (SEM) image of the Danfeng peony petal polypeptide microcapsules in this invention; Figure 13 This is a pH stability diagram of the Danfeng peony petal polypeptide W1 / O / W2 double emulsion and reconstituted microcapsules in this invention; Figure 14 This is a salt ion stability diagram of the Danfeng peony petal polypeptide W1 / O / W2 double emulsion and reconstituted microcapsules in this invention; Figure 15 This is a graph showing the lipid oxidation stability analysis of the Danfeng peony petal polypeptide microcapsules in this invention; Figure 16 This is a thermogravimetric analysis diagram of the Danfeng peony petal polypeptide microcapsules in this invention; Figure 17 This is a graph showing the effect of the Danfeng Peony petal polypeptide microcapsules of the present invention on postprandial blood glucose in diabetic mice. Figure 18 This is an in vitro release curve of the Danfeng peony petal polypeptide microcapsules of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0031] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0032] A type of peony polypeptide microcapsule, wherein the microcapsule is made by encapsulating the peony hypoglycemic polypeptide with packaging material to form a W1 / O / W2 type double emulsion microcapsule, or by further drying to obtain a solid microcapsule.

[0033] Furthermore, the blood sugar-lowering polypeptide of Peony peony is obtained by hydrolyzing the petals of Peony peony through food-derived enzymes; The packaging material is made of a single wall material or a composite wall material; The W1 / O / W2 type double emulsion refers to a water-in-oil-in-water type double emulsion, which is a liquid microcapsule that can be applied directly or diluted for application.

[0034] Furthermore, the wall material is one or a combination of several of the following: sodium carboxymethyl cellulose, cyclodextrin, xanthan gum, carrageenan, sodium alginate, chitosan, gum arabic, and phospholipids. Alternatively, the drying process refers to vacuum drying, freeze drying, spray drying, or atmospheric pressure heating drying, after which the microcapsules are solid; Alternatively, an emulsifier may be used in the preparation of the W1 / O / W2 type double emulsion, and the emulsifier may be one or more of Span 20, Span 80, lecithin, polyglycerol polyricinoleate PGPR, sucrose fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester (span), and Tween.

[0035] Furthermore, the Danfeng Peony polypeptide microcapsules comprise an inner aqueous phase, an oil phase, and an outer aqueous phase; wherein the inner aqueous phase is a blood sugar-lowering polypeptide from Danfeng Peony petals, the oil phase is composed of medium-chain triglycerides and polyglycerol polyricinoleate, and the outer aqueous phase is composed of sodium carboxymethyl cellulose, cyclodextrin, and Tween 80; a two-step emulsification method is employed, firstly, a water-in-oil primary emulsion is obtained from the inner aqueous phase and the oil phase, then the primary emulsion is mixed with the outer aqueous phase, and combined with ultrasonic emulsification technology to form microcapsules.

[0036] The preparation method of the Danfeng peony polypeptide microcapsules as described above includes the following steps: (1) Preparation of W1 / O primary emulsion: The Danfeng Mudan polypeptide was dissolved in water and fed at a ratio of 1:1 to 1000 g:mL as the inner aqueous phase of W1; the medium-chain triglycerides and lipophilic emulsifier were mixed at a volume ratio of 1:0.01 to 100 as the oil phase O; the inner aqueous phase of W1 was slowly added to the oil phase O at a volume ratio of 1:0.01 to 100, and stirred until the emulsifier was completely hydrated to form an oil-in-water W1 / O type primary emulsion; (2) Preparation of W1 / O / W2 type double emulsion: Dissolve the composite wall material in water at a ratio of 1:1 to 1000 g:mL, then add 0.1% to 10% Tween80 by mass, stir to completely hydrate the emulsifier, and use it as the W2 external aqueous phase for later use; use the above W1 / O primary emulsion as the core material, and add the W2 external aqueous phase to the W1 / O primary emulsion at a volume ratio of 1:0.01 to 100, heat and stir at 40 to 80°C to make the W1 / O primary emulsion and the W2 external aqueous phase evenly mixed, and then use an ultrasonic emulsifier to perform ultrasonic emulsification treatment on the mixed solution to prepare the W1 / O / W2 type double emulsion. The W1 / O / W2 type double emulsion can be used directly or diluted to obtain Danfeng Peony polypeptide microcapsules.

[0037] Furthermore, in step (1), the Danfeng peony polypeptide is a polypeptide obtained by extracting from the petals of the peony, namely the short peptide protein of the peony petals disclosed in Chinese Patent Publication CN117568431A. Alternatively, the lipophilic emulsifier in step (1) is polyglycerol polyricinoleate; Alternatively, in step (2), the composite wall material is a mixture of sodium carboxymethyl cellulose and cyclodextrin in a mass ratio of 4:1; Alternatively, the specific conditions for heating and stirring in step (2) are: heating and stirring at 50°C for 30 to 60 minutes; Alternatively, the specific conditions for ultrasonic emulsification in step (2) are: ultrasonic power of 65 W, ultrasonic for 3 min, ultrasonic for 4 s, and stop for 2 s; Alternatively, the W1 / O / W2 type double emulsion in step (2) also undergoes the following steps: Microcapsule powder: The W1 / O / W2 type double emulsion prepared above is dried to obtain microcapsule solid powder. The drying method is vacuum drying, freeze drying, spray drying or atmospheric pressure heating drying. The conditions for vacuum drying are: 50℃, vacuum degree -0.08~-0.1MPa; the conditions for freeze drying are: temperature < -60℃, vacuum degree < 20MPa; the conditions for spray drying are: inlet air temperature 160~190℃, outlet air temperature 70~90℃, feed rate 5 mL / min; the conditions for atmospheric pressure heating drying are: 50℃, atmospheric pressure.

[0038] Liquid or solid formulations prepared using the Danfeng Mudanjiang polypeptide microcapsules as described above. Preferably, the formulations include powders, granules, powder for injection, tablets, capsules, or liquid formulations.

[0039] The above-mentioned application of Danfeng Mudan polypeptide microcapsules in the preparation of targeted and slow-release drugs in the intestine.

[0040] The application of the Danfeng Peony polypeptide microcapsules as described above in the preparation of hypoglycemic drugs.

[0041] The above-mentioned applications of Danfeng Peony polypeptide microcapsules in the preparation of special medical foods and / or health foods and / or functional foods and / or ordinary foods.

[0042] Specifically, the relevant preparation and testing methods are as follows: The preparation of a microcapsule containing a polypeptide from the petals of Peony peony and its application in antidiabetic activity are described. The microcapsule is prepared by first extracting a polypeptide from the petals of Peony peony, namely a short peptide protein from the petals of Peony peony (for specific technical parameters, please refer to the Chinese patent publication CN117568431A filed by our research team), using a two-step emulsification method, and then drying it.

[0043] Specifically: Example 1: Preparation of a Peony petal polypeptide microcapsule First, the lipophilic emulsifier and composite wall material were optimized, following these steps: (1) Preparation of W1 / O primary emulsion: 100 mg of *Peony peony* petal polypeptide (i.e., the short peptide protein of *Peony peony* petals disclosed in Chinese Patent Publication CN117568431A, hereinafter the same) was weighed and dissolved in 2 mL of deionized water as the inner aqueous phase W1; an appropriate amount of medium-chain triglycerides (MCT) was taken and different lipophilic emulsifiers, including Span20, Span80, lecithin or PGPR (polyglycerol polyricinoleate), were added, and heated and stirred at 50 °C for 15 min to ensure complete hydration of the emulsifiers as the oil phase O, wherein the final mass concentration of the lipophilic emulsifier was 10%; the oil phase O and the inner aqueous phase W1 were mixed at a volume ratio of 4:1, and the inner aqueous phase W1 was slowly added to the oil phase O during stirring, and stirred at room temperature for 30 min to form a W1 / O type primary emulsion; the changes of W1 / O primary emulsions prepared by different types of lipophilic emulsifiers at 0 h and 48 h are as follows. Figure 1As shown, different lipophilic emulsifiers exhibited different emulsifying effects when preparing W1 / O primary emulsions. The W1 / O primary emulsions prepared with Span 40, Span 80, and lecithin were yellowish in color at 0 h, not milky white, and all showed varying degrees of stratification after 48 h. The primary emulsion prepared with PGPR had a uniform color and remained stable after 48 h without stratification. Therefore, to ensure the emulsifier has good emulsifying properties and can prepare a uniform and stable W / O emulsion, PGPR was selected as the optimal emulsifier for preparing W1 / O primary emulsions.

[0044] (2) Preparation of W1 / O / W2 double emulsion: Sodium carboxymethyl cellulose (CMC), cyclodextrin (CD), xanthan gum (XG) and carrageenan (CARR) were selected as wall materials and compounded in pairs to obtain composite wall materials. The composite wall materials were added to 20 mL of deionized water at a ratio of 8:1 (w / w, mass ratio), heated and stirred at 80 °C for 1 h to completely dissolve them. Then, 1% of hydrophilic emulsifier Tween 80 was added and stirred for 30 min to completely hydrate the emulsifier, which was used as the external aqueous phase W2. The total content of the composite wall materials in the external aqueous phase W2 was 12% (w / v, volume percentage). Under heating conditions of 50 ℃, the prepared W1 / O primary emulsion and the external aqueous phase W2 were mixed at a volume ratio of 3:7. The external aqueous phase W2 was slowly added to the W1 / O primary emulsion, and the mixture was heated and stirred at 50 ℃ for 30 min to ensure uniform mixing of the W1 / O primary emulsion and the external aqueous phase W2. The mixture was then ultrasonically emulsified using an ultrasonic emulsifier with an ultrasonic volume of 10 mL, an ultrasonic power of 65 W, and an ultrasonic duration of 3 min (4 s for ultrasonication followed by a 2 s pause) to prepare the W1 / O / W2 composite emulsion. The influence of different types of composite wall materials on the preparation of the W1 / O / W2 composite emulsion is as follows: Figure 2 As shown in (ac). Figure 2 As can be seen from this, the initial W1 / O / W2 encapsulation rates of CD-XG, CMC-CARR, CD-XG, and XG-CARR composite wall materials were low (≤60%). After 7 and 14 days of storage, the encapsulation rates of CMC-XG, CD-CMC, and CD-CARR decreased significantly over time, with all encapsulation rates below 30% at 14 days. Among CMC-CD and CD-CARR, the initial encapsulation rate of CMC-CD showed the smallest decrease after storage, maintaining an encapsulation rate of 67% at 14 days, making it the most stable of all composite wall materials. Figure 2 b shows that all the double emulsions were uniformly milky white at 0 days, with good appearance uniformity and no obvious stratification. After 14 days of storage, the emulsions prepared by CMC-XG and CMC-CARR did not show significant phase stratification, possibly because the double emulsions had higher viscosity. The others showed slight precipitation of the external aqueous phase, but remained stable. Figure 2As shown in Figure c, at 0 days of preparation, CD-XG exhibited poor uniformity, while the droplet distribution of the other composite wall materials was relatively uniform. After 14 days of storage, CMC-CD droplets remained relatively small and uniformly distributed, showing only slight changes and no severe aggregation. CMC-XG droplets became large, CMC-CARR droplets aggregated, and CD-XG, CD-CARR, and XG-CARR droplets showed severe aggregation, even exhibiting a continuous phase. Based on the encapsulation efficiency, stability, and microstructure of W1 / O / W2 multiemulsions prepared with different wall material composites, CMC and CD composites were selected as the optimal wall material for preparing W1 / O / W2 multiemulsions.

[0045] (3) Microcapsule powder: The W1 / O / W2 complex emulsion prepared above is dried to solidify the microcapsule wall material, and then freeze-dried (temperature < -60℃, vacuum degree < 20MPa) to obtain dried microcapsule powder.

[0046] Example 1: Morphological characteristics of Peony petal polypeptide microcapsules (1) Characterization of appearance The prepared W1 / O primary emulsion and W1 / O / W2 secondary emulsion were placed in 5 mL glass bottles, and the color changes of the samples were recorded. The appearance changes of the W1 / O primary emulsion were observed after 0 h, 24 h, and 48 h, and the appearance changes of the W1 / O / W2 secondary emulsion were observed after 0 d and 14 d.

[0047] (2) Characterization of microscopic morphology The prepared W1 / O / W2 double emulsion was diluted 100 times with deionized water. 10 μL of the diluted solution was placed on a glass slide. After the coverslip was slowly placed on the solution from one side, its microstructure was observed under a 40x objective lens using an optical microscope.

[0048] Example 2: Determination of the encapsulation efficiency of Peony petal polypeptide microcapsules (1) Drawing the standard curve Accurately weigh 150 mg of copper sulfate, 0.6 g of potassium sodium tartrate, and 0.5 g of potassium iodide. Add 50 mL of deionized water and stir to dissolve. Then add 30 mL of 10% NaOH solution and dilute to 100 mL. Shake well to obtain the biuret reagent.

[0049] Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of bovine serum albumin standard solution (1 mg / mL) into test tubes, and then add 1.0, 0.8, 0.6, 0.4, 0.2, and 0 mL of distilled water respectively. Add 4 mL of biuret reagent, let stand for 30 min, and measure the absorbance at 540 nm. Plot a standard curve with bovine serum albumin concentration as the x-axis (X) and sample solution absorbance as the y-axis (Y).

[0050] The linear regression equation for the standard curve is Y = 0.2638 x - 0.005, with a linearity factor R² = 0.9993.

[0051] (2) Calculation of encapsulation ratio Take 500 μL of the W1 / O / W2 double emulsion, dilute with 500 μL of distilled water, centrifuge at 500 rpm for 30 min, discard the supernatant, and collect the lower aqueous phase. Mix 200 μL of the lower aqueous phase with 200 μL of organic reagent (ethanol:acetone = 1:1, volume ratio), sonicate for 5 min to demulsify, centrifuge at 13200 rpm for 15 min after demulsification, and collect the supernatant solution for later use. Take 100 μL of the supernatant, add 400 μL of biuret reagent, let stand for 30 min, and measure the absorbance at 540 nm. Calculate the content of Danfeng Mudan polypeptide leaked into the outer aqueous phase W2 according to the standard curve. Calculate the encapsulation efficiency of the microcapsules using the following formula: Wherein, C0 is the content of Danfeng Mudan polypeptide in the external aqueous phase W2 of the sample, and C is the total content of Danfeng Mudan polypeptide in the sample.

[0052] Example 2: Preparation of a Peony petal polypeptide microcapsule (1) Preparation of W1 / O primary emulsion: 100 mg of peony petal polypeptide was weighed and dissolved in 2 mL of deionized water as the inner aqueous phase W1; PGPR was added to MCT and heated and stirred at 50 °C for 15 min to ensure complete hydration of the emulsifier as the oil phase O, wherein the final mass concentration of the lipophilic emulsifier PGPR was 10% and the oil phase O and the inner aqueous phase W1 were mixed at a volume ratio of 4:1. During the stirring process, the inner aqueous phase W1 was slowly added to the oil phase O and stirred at room temperature for 30 min to form a W1 / O type primary emulsion. (2) Preparation of W1 / O / W2 double emulsion: Add 20 mL of deionized water according to the ratio of sodium carboxymethyl cellulose: cyclodextrin 8:1 (w / w, mass ratio), heat and stir at 80 °C for 1 h to completely dissolve it, then add 1% of hydrophilic emulsifier Tween 80 at a final mass concentration, stir for 30 min to completely hydrate the emulsifier, and use it as the external aqueous phase W2, in which the total content of sodium carboxymethyl cellulose and cyclodextrin in the external aqueous phase W2 is 12% (w / v, volume percentage). Under heating conditions of 50 ℃, the prepared W1 / O primary emulsion and the external aqueous phase W2 were mixed at a volume ratio of 3:7. The external aqueous phase W2 was slowly added to the W1 / O primary emulsion, and the mixture was heated and stirred at 50 ℃ for 30 min to make the W1 / O primary emulsion and the external aqueous phase W2 uniformly mixed. The mixed solution was then subjected to ultrasonic emulsification using an ultrasonic emulsifier. The ultrasonic volume was 10 mL, the ultrasonic power was 65 W, and the ultrasonic treatment lasted for 3 min (4 s ultrasonic, 2 s pause) to prepare the W1 / O / W2 double emulsion.

[0053] (3) Microcapsule powder: The W1 / O / W2 complex emulsion prepared above is dried to solidify the microcapsule wall material, and then freeze-dried (temperature < -60℃, vacuum degree < 20MPa) to obtain dried microcapsule powder.

[0054] Example 3 Screening of PGPR concentration in oil phase The difference between this embodiment and embodiment 2 is that the final PGPR concentration in step (1) is 2%, 4%, 6%, 8%, and 10%. Figure 3 Encapsulation efficiency, appearance changes, and micromorphological changes of W1 / O / W2 double emulsions prepared with different PGPR concentrations at 0 days and 14 days.

[0055] The results are as follows Figure 3 As shown in (ac), according to Figure 3 As shown in Figure a, the encapsulation efficiency increases with increasing PGPR concentration; after 14 days of storage, the direct trend of different PGPR concentrations remains unchanged, with a relatively small decrease. Figure 3 As shown in b, at day 0, all double emulsions with different PGPR concentrations exhibited a uniform milky white color. After 14 days of storage, slight precipitation of the external aqueous phase was observed in all cases, but the emulsions remained stable. Figure 3 As shown in c, at low concentrations, the droplet size is uneven, and after 14 days of storage, the number of large droplets increases, while the distribution gradually becomes more uniform with increasing concentration. Based on the encapsulation efficiency, stability, and microstructure of W1 / O / W2 double emulsions prepared with different PGPR concentrations, and considering the appropriate amount of emulsifier, a final mass concentration of 6% PGPR was selected as the optimal concentration for preparing W1 / O / W2 double emulsions.

[0056] Example 4: Screening of the ratio of oil phase O to internal aqueous phase W1 The difference between this embodiment and embodiment 2 is that the volume ratio of oil phase O to internal water phase W1 in step (1) is 1:1, 4:1, 7:1, 10:1, or 13:1. Figure 4 Encapsulation efficiency, appearance, and microstructure changes of W1 / O / W2 double emulsions prepared with different oil-water ratios (O:W1) at 0 and 14 days.

[0057] The results are as follows Figure 4 As shown in (ac), according to Figure 4 As shown in section a, the encapsulation efficiency initially increases and then decreases with decreasing water phase proportion, reaching its highest value at an oil-to-water ratio of 7:1. After 14 days of storage, the encapsulation efficiency decreases across the board, with the highest and smallest decrease observed at an oil-to-water ratio of 4:1. The largest decrease in encapsulation efficiency and weakest stability is observed at an oil-to-water ratio of 7:1 after 14 days of storage. Figure 4 As shown in b, at day 0, all double emulsions with different oil-to-water ratios were uniformly milky white. After 14 days of storage, the precipitation of the external aqueous phase decreased with the increase of the oil phase ratio. Figure 4 As shown in c, the microstructure remained uniform after 14 days of storage for oil-water ratios of 4:1 and 7:1. Based on the encapsulation efficiency, stability, and microstructure of W1 / O / W2 double emulsions prepared with different oil-water ratios, an oil-water ratio of 4:1 was selected as the optimal ratio for preparing W1 / O / W2 double emulsions.

[0058] By comparing Examples 2 to 4, it can be seen that the final mass concentration of PGPR in step (1) of the method of the present invention is 6%, and the volume ratio of oil phase O to inner aqueous phase W1 in step (2) is 4:1. These two conditions have a synergistic effect and can synergistically improve the relevant performance of the prepared Danfeng Peony petal polypeptide microcapsules.

[0059] Example 5: Screening of the ratio of sodium carboxymethyl cellulose and cyclodextrin The difference between this embodiment and embodiment 2 is that the mass ratio of sodium carboxymethyl cellulose and cyclodextrin in step (2) is 10:1, 8:1, 4:1, 1:1, 1:4, 1:8, and 1:10. Figure 5 Encapsulation efficiency, appearance changes, and micromorphological changes of W1 / O / W2 composite emulsions prepared with different W2 phase composite wall material ratios at 0 days and 14 days.

[0060] The results are shown in 5(ac), according to Figure 5 As shown in a, the encapsulation efficiency first increases and then decreases with the decrease of the proportion of sodium carboxymethyl cellulose, reaching its maximum at a sodium carboxymethyl cellulose:cyclodextrin ratio of 4:1. Figure 5As shown in b, all double emulsions with different dosage ratios were uniformly milky white. After 14 days of storage, severe stratification occurred when the ratio of sodium carboxymethyl cellulose to cyclodextrin was below 1:1; the ratios of 10:1, 8:1, and 4:1 still maintained a uniform milky white color. Figure 5 As shown in c, after 14 days of storage, droplet aggregation was severe when the ratio of sodium carboxymethyl cellulose to cyclodextrin was below 1:1. Based on the encapsulation efficiency, stability, and microstructure of W1 / O / W2 double emulsions prepared with different ratios of sodium carboxymethyl cellulose to cyclodextrin, a composite wall material ratio of 4:1 was selected as the optimal ratio for preparing W1 / O / W2 double emulsions.

[0061] Example 6 Screening of sodium carboxymethyl cellulose and cyclodextrin content in external aqueous phase W2 The difference between this embodiment and embodiment 2 is that the total content of sodium carboxymethyl cellulose and cyclodextrin in the external aqueous phase W2 in step (2) is 3%, 5%, 10%, 12%, 15%, and 20% (w / v, volume percentage). Figure 6 The encapsulation efficiency, appearance changes, and micromorphological changes of W1 / O / W2 double emulsions prepared with different total wall material contents in the W2 phase at 0 days and 14 days were studied.

[0062] The results are shown in 6(ac), according to Figure 6 As shown in a, the encapsulation efficiency first increases and then decreases with the increase of wall material content, reaching its maximum when the total wall material content is 15%. Figure 6 As shown in b, the multi-emulsions with different total wall material contents were all uniformly milky white; after 14 days of storage, obvious stratification occurred at low contents, while only slight precipitation of the external aqueous phase occurred as the content increased. Figure 6 c indicates that after 14 days of storage, slight aggregation occurred at a content of 3%, while droplets remained uniformly distributed at other contents. Based on the encapsulation efficiency, stability, and micromorphology of W1 / O / W2 double emulsions prepared with different contents of sodium carboxymethyl cellulose and cyclodextrin, a wall material content of 15% was selected as the optimal content for preparing W1 / O / W2 double emulsions.

[0063] By comparing Examples 2, 5 and 6, it can be seen that the mass ratio of sodium carboxymethyl cellulose to cyclodextrin in step (2) of the method of the present invention is 4:1 and the total volume percentage of sodium carboxymethyl cellulose and cyclodextrin in the external aqueous phase W2 in step (2) is 15%, which have a synergistic effect and can synergistically improve the relevant performance of the prepared peony petal polypeptide microcapsules.

[0064] Example 7 Screening of the ratio of W1 / O primary emulsion to external aqueous phase W2 The difference between this embodiment and embodiment 2 is that the volume ratio of the W1 / O primary emulsion to the external aqueous phase W2 in step (2) is 5:5, 4:6, 3:7, 2:8, 1:9. Figure 7 Encapsulation efficiency, appearance changes, and micromorphological changes of W1 / O / W2 double emulsions prepared with different emulsion-to-water ratios at 0 days and 14 days.

[0065] The results are shown in 7(ac), according to Figure 7 As shown in a, the encapsulation efficiency first increases and then decreases with the increase of the external aqueous phase W2, reaching its maximum at an emulsion-to-water ratio of 4:6. Figure 7 As shown in b, all double emulsions with different emulsion-to-water ratios were uniformly milky white. After 14 days of storage, except for the 1:9 emulsion-to-water ratio which showed significant stratification and emulsion precipitation, the others only showed slight precipitation of the W2 aqueous phase. Figure 7 c indicates that after 14 days of storage, slight aggregation and droplet adhesion occurred at emulsion-to-water ratios of 5:5 and 1:9; at other ratios, the droplets remained uniformly distributed. Based on the encapsulation efficiency, stability, and microstructure of the W1 / O / W2 double emulsions prepared with different emulsion-to-water ratios, an emulsion-to-water ratio of 4:6 was selected as the optimal concentration for preparing the W1 / O / W2 double emulsion.

[0066] according to Figures 3-7 The results show that the preparation parameters of the W1 / O primary emulsion were optimized as follows: PGPR was selected as the lipophilic emulsifier, 6% PGPR was added to the medium-chain triglyceride oil phase, and the ratio of oil phase O to internal aqueous phase W1 was 6:4. The resulting W1 / O primary emulsion was the most stable and could be used as the primary emulsion for preparing the W1 / O / W2 complex emulsion. Construction of the W1 / O / W2 complex emulsion: The composite wall material was sodium carboxymethyl cellulose and cyclodextrin, with a ratio of sodium carboxymethyl cellulose to cyclodextrin of 4:1. The total content of the composite wall material in the external aqueous phase W2 was 15%. The W1 / O / W2 complex emulsion prepared with a ratio of W1 / O primary emulsion to external aqueous phase W2 of 6:4 exhibited the highest encapsulation efficiency and uniform distribution of emulsion droplet microstructure.

[0067] By comparing Examples 2, 6 and 7, it can be seen that the total volume percentage of sodium carboxymethyl cellulose and cyclodextrin in the external aqueous phase W2 in step (2) of the method of the present invention is 15%, and the volume ratio of W1 / O primary emulsion to external aqueous phase W2 in step (2) is 4:6. These two conditions have a synergistic effect and can synergistically improve the relevant performance of the prepared Danfeng peony petal polypeptide microcapsules.

[0068] Example 8: Preparation of a Peony petal polypeptide microcapsule The optimal conditions obtained from the single-factor optimization above were selected for the preparation of microcapsules, as follows: (1) Preparation of W1 / O primary emulsion: 100 mg of peony petal polypeptide was weighed and dissolved in 2 mL of deionized water as the inner aqueous phase W1; polyglycerol polyricinoleate was added to medium chain triglyceride and heated and stirred at 50°C for 15 min to ensure complete hydration of the emulsifier as the oil phase O, wherein the final mass concentration of the lipophilic emulsifier was 6%; the oil phase O and the inner aqueous phase W1 were mixed at a volume ratio of 7:1; during the stirring process, the inner aqueous phase W1 was slowly added to the oil phase O and stirred at room temperature for 30 min to form a W1 / O type primary emulsion; (2) Preparation of W1 / O / W2 double emulsion: Add 20 mL of deionized water according to the ratio of sodium carboxymethyl cellulose: cyclodextrin 4:1 (w / w, mass ratio), heat and stir at 80℃ for 1 h to completely dissolve it, then add 1% of hydrophilic emulsifier Tween80 at a final mass concentration, stir for 30 min to completely hydrate the emulsifier, and use it as the external aqueous phase W2, in which the total content of sodium carboxymethyl cellulose and cyclodextrin in the external aqueous phase W2 is 15% (w / v, volume percentage). Under heating conditions of 50 ℃, the prepared W1 / O primary emulsion and the external aqueous phase W2 were added slowly to the W1 / O primary emulsion at a volume ratio of 4:6. The mixture was heated and stirred at 50 ℃ for 30 min to ensure uniform mixing of the W1 / O primary emulsion and the external aqueous phase W2. The mixture was then subjected to ultrasonic emulsification using an ultrasonic emulsifier with an ultrasonic volume of 10 mL, an ultrasonic power of 65 W, and an ultrasonic duration of 3 min (4 s for ultrasonication followed by a 2 s pause) to prepare the W1 / O / W2 double emulsion.

[0069] (3) Microcapsule powder: The W1 / O / W2 complex emulsion prepared above is dried to solidify the microcapsule wall material, and then freeze-dried (temperature < -60℃, vacuum degree < 20MPa) to obtain solid microcapsule powder.

[0070] Example 9 Preparation of microcapsule reconstituted solution Weigh 100 mg of the microcapsule powder prepared in Example 8, add 2 mL of deionized water to completely disperse it, and use it as a microcapsule reconstitution solution.

[0071] Example 10 Preparation of blank microcapsules (1) Preparation of W1 / O primary emulsion: Measure 2 mL of deionized water as the inner aqueous phase W1; add polyglycerol polyricinoleate to medium chain triglyceride, heat and stir at 50°C for 15 min to ensure complete hydration of the emulsifier, and use it as the oil phase O, wherein the final mass concentration of the lipophilic emulsifier is 6%. The oil phase O and the inner aqueous phase W1 are mixed at a volume ratio of 7:1. During the stirring process, the inner aqueous phase W1 is slowly added to the oil phase O, and stirred at room temperature for 30 min to form a W1 / O type primary emulsion. (2) Preparation of W1 / O / W2 double emulsion: Add 20 mL of deionized water according to the ratio of sodium carboxymethyl cellulose: cyclodextrin 4:1 (w / w, mass ratio), heat and stir at 80℃ for 1 h to completely dissolve it, then add 1% of hydrophilic emulsifier Tween80 at a final mass concentration, stir for 30 min to completely hydrate the emulsifier, and use it as the external aqueous phase W2, in which the total content of sodium carboxymethyl cellulose and cyclodextrin in the external aqueous phase W2 is 15% (w / v, volume percentage). Under heating conditions of 50 ℃, the prepared W1 / O primary emulsion and the external aqueous phase W2 were added slowly to the W1 / O primary emulsion at a volume ratio of 4:6. The mixture was heated and stirred at 50 ℃ for 30 min to ensure uniform mixing of the W1 / O primary emulsion and the external aqueous phase W2. The mixture was then subjected to ultrasonic emulsification using an ultrasonic emulsifier with an ultrasonic volume of 10 mL, an ultrasonic power of 65 W, and an ultrasonic duration of 3 min (4 s for ultrasonication followed by a 2 s pause) to prepare the W1 / O / W2 double emulsion.

[0072] (3) Microcapsule powder: The W1 / O / W2 complex emulsion prepared above is dried to solidify the microcapsule wall material, and then freeze-dried (temperature < -60℃, vacuum degree < 20MPa) to obtain solid microcapsule powder.

[0073] Experimental Example 1: W1 / O / W2 double emulsion and microcapsule powder particle size of Danfeng peony petal polypeptide in Example 8 The W1 / O / W2 double emulsion was diluted 10 times with distilled water. At room temperature (25°C), using a Bettersize 2600 laser particle size analyzer with water as the dispersant and a refractive index of 1.333, the suspension was added dropwise to the sample cell until the opacity of the mixed solution reached 4%–10%. The volume average diameter of the sample was then measured. The microcapsule reconstituted solution was then subjected to particle size determination using the same method.

[0074] Depend on Figure 8 It can be seen that the volume average diameter of the W1 / O / W2 double emulsion is 1.095 μm, and the volume average diameter of the microcapsule powder after reconstitution is 3.925 μm, with a single peak and a bell-shaped particle size distribution. Therefore, the microcapsules prepared in this invention have a uniform particle size distribution.

[0075] Reconstitution characteristics of the Danfeng peony petal polypeptide microcapsules in Example 2 of Example 8 Take an appropriate amount of sample and add 4 mL of deionized water to a glass bottle to prepare microcapsule reconstituted solutions with concentrations of 25, 50, 100, and 200 mg / mL, respectively, and observe their reconstitution characteristics.

[0076] like Figure 9It can be seen that the reconstitution rate decreases with increasing concentration. At 25 mg / mL and 50 mg / mL, the reconstitution rate is relatively fast; only a small number of undispersed particles remain after 60 s, and good clear dispersion is achieved within 120 s. When the reconstitution concentration increases to 100 mg / mL and 200 mg / mL, undispersed particles still exist after 90 s, but no obvious solid particle agglomeration occurs after 120 s. The dispersion uniformity is weaker than that of the low-concentration solution, but the final system homogeneity is still good. Therefore, the microcapsules prepared in this invention exhibit good dispersibility after reconstitution, and no severe irreversible aggregation occurs after lyophilization; they exist in water as a suspension.

[0077] Fourier transform infrared spectroscopy characterization of the Danfeng peony petal polypeptide microcapsules in Example 3 and Example 8. 1 mg of each of the following samples—peony petal peptides, sodium carboxymethyl cellulose, cyclodextrin, blank microcapsules, and peony petal peptide microcapsule powder—were weighed into a quartz mortar. 100 mg of dry potassium bromide was added, and the mixture was thoroughly ground. The samples were then pressed into transparent thin sheets using a tablet press. The samples were then analyzed using a Fourier transform infrared spectrometer with an air background at a 4 cm⁻¹ diameter. -1 At a resolution of [resolution], the sample was scanned 32 times, at a resolution of 4000-400 cm. -1 Scanning is performed within the wavenumber range.

[0078] like Figure 10 It can be seen that both the blank microcapsules and the peony polypeptide microcapsules have a 3200-3600 cm⁻¹ range. -1 OH broad peak, ~2900 cm⁻¹ -1 saturated CH peaks and 1000-1200 cm⁻¹ -1 The CO vibration, corresponding to the wall material used, indicates the successful construction of the microcapsules; simultaneously, the peony petal polypeptide was present at ~1400 cm⁻¹. -1 The characteristic amide peaks clearly appeared in the microcapsules, but no corresponding signals were found in the blank microcapsules, indicating that the *Peony peony petal* peptides in this invention have been successfully encapsulated in the microcapsules. In this invention, the spectrum of the *Peony peony petal* peptide microcapsules is a superposition of the characteristic peaks of the blank microcapsules and the *Peony peony petal* peptides; no new strong characteristic absorption peaks appeared, indicating that the encapsulation process was mainly driven by physical interactions such as hydrogen bonds and van der Waals forces, and no new covalent bonds were formed.

[0079] Microscopic characterization of the Peony petal polypeptide microcapsules in Example 4 and Example 8 (1) Laser scanning confocal microscope Prepare a 1% (w / v, volume percentage) Nile blue isopropanol solution and a 0.1% (w / v, volume percentage) Nile red isopropanol solution. Dilute the freshly prepared W1 / O / W2 double emulsion 200 times. Take 1 mL of the diluted solution, add 50 μL of 0.1% Nile red solution, vortex mix for 15 s, and incubate at room temperature in the dark for 5 min to allow the dye to fully enter the oil phase. Then add 40 μL of 1% Nile blue solution, vortex mix for 15 s, and incubate at room temperature in the dark for 5 min. Take 10 μL of the sample solution and drop it onto a glass slide, cover with a coverslip, and observe using a laser confocal microscope under a 100x oil immersion microscope. The excitation wavelength for Nile blue is 633 nm, and the excitation wavelength for Nile red is 488 nm. The images were processed using NIS-Elements software.

[0080] (2) Scanning electron microscope The conductive double-sided tape and silicon wafer were fixed on the stage. A small amount of peony petal peptides and microcapsule powder were picked up with a toothpick and placed on the conductive tape. The W1 / O / W2 complex emulsion and microcapsule complex solution were respectively drawn up with a dropper and added to the silicon wafer. The liquid samples were then dried. The samples were sputter-coated with gold under vacuum conditions, and then images of the sample's appearance and surface structure were acquired using a scanning electron microscope at a working voltage of 5 kV.

[0081] like Figure 11 It can be seen that the red fluorescent spots in the system correspond to the oil phase region of the system, which are densely distributed and well dispersed. The green fluorescent spots correspond to the aqueous phase region of the system, which also show a dispersed region that overlaps with the distribution region of the red fluorescent spots. At the same time, yellow fluorescence with superposition of red and green fluorescence appears at the co-excitation wavelengths of 488nm and 633nm, which verifies the successful construction of the microcapsule W1-O-W2 emulsion structure of the present invention.

[0082] like Figure 12 It is known that the *Peony danfengense* petal polypeptide of this invention presents as irregular, spherical particles with a relatively smooth surface; the W / O / W double emulsion and the reconstituted microcapsules are spherical; some wall material may adhere to the microcapsules after freeze-drying and reconstituted in water; the freeze-dried microcapsules are in a vacuum environment, and the sublimation of water and shrinkage of the wall material make the outer shell of the microcapsules uneven. This indicates that the *Peony danfengense* petal polypeptide is successfully dispersed in the wall material to form a W1 / O / W2 double emulsion, and the freeze-drying process does not destroy its encapsulation effect. Furthermore, after reconstitution, it can recover from a shrunken solid state to a spherical shape similar to the W / O / W double emulsion, further demonstrating that the freeze-dried microcapsules have good reconstitution properties.

[0083] Experimental Example 5: Stability of Peony Petal Peptide Microcapsules in Experiment 8 (1) pH stability The pH of the W1 / O / W2 complex emulsion and the microcapsule complex solution were adjusted to 2, 3, 4, 5, 6, 7, and 8, respectively, using 1 mol / L HCl and 1 mol / L NaOH. After standing at room temperature (25 °C) for 2 h, the particle size, PDI value, and Zeta potential of the solutions were measured. The pH stability of the microcapsules was investigated by measuring the changes in particle size, PDI value, and Zeta potential.

[0084] (2) Ionic strength stability Different concentrations of NaCl solutions (0, 50, 100, 200, 300, 400, 500 mM) were prepared using deionized water. These solutions were added to the W1 / O / W2 double emulsion and the microcapsule double emulsion at a volume ratio of 1:1. After standing at 25 °C for 2 h, the particle size, PDI value, and Zeta potential of the solutions were measured. The changes in particle size, PDI value, and Zeta potential were used to investigate the salt ion stability of the microcapsules.

[0085] (3) Oxidative stability of oils Take 5 mL of W1 / O / W2 double emulsion into the sample tube of the Metrohm 892 Professional Rancimat instrument, and measure the conductivity-time dynamic curve and rancidity induction period of the sample at 120 °C with an air flow rate of 20 L / h.

[0086] (4) Thermogravimetric analysis Weigh 3-5 mg of microcapsule powder into a crucible, set the temperature range to 40℃-600℃, the nitrogen flow rate to 50 mL / min, and the heating rate to 10℃ / min, and use a thermogravimetric analyzer to determine the thermogravimetric curve of the microcapsule powder.

[0087] like Figure 13-14 It is evident that, under strong acid, strong alkali, and strong salt ion environments, neither the complex emulsion nor the complex solution experienced severe aggregation or rupture, the electrostatic repulsion between droplets was not weakened, and the solution maintained good dispersion uniformity. The Danfeng peony petal polypeptide microcapsules prepared in this invention exhibit good stability under strong acid, strong alkali, and strong salt ion environments.

[0088] like Figure 15 It can be seen that the conductivity curve of soybean oil shows a significant steep increase at around 3.2h, corresponding to a rancidity induction time of 3.25h; while the conductivity of MCT and W / O / W double emulsion changes little during the 8h test, the curves are flat and there is no significant steep increase. Therefore, the rancidity induction period of the oil in the microcapsules is much longer than 8h. The multilayer structure of the microcapsules of this invention has a certain physical barrier effect, which delays the oxidation of MCT oil and the decomposition process of Danfeng peony petal polypeptides.

[0089] like Figure 16It is known that the mass of the microcapsules decreases sharply between 230 and 400°C, and the wall material and active peptides undergo thermal decomposition. After 400°C, the organic components decompose completely, and the mass remains basically stable. At 300°C, the microcapsules undergo preliminary thermal decomposition, and at 350°C, a second stage of thermal decomposition occurs, resulting in deep thermal decomposition. Therefore, the microcapsules of this invention exhibit good thermal stability at 200°C.

[0090] Example 11: In vivo hypoglycemic activity of the polypeptides and their microcapsules from Example 8 in mice. To verify the hypoglycemic activity, the Danfeng peony petal polypeptide and the W1 / O / W2 double emulsion and microcapsules of the representative example 8 were selected and their hypoglycemic activity in type 2 diabetic mice was further evaluated. The drugs were administered by gavage.

[0091] Male SPF-grade mice weighing 18-22 g were purchased from Kunming and housed at 25℃ room temperature, 50% humidity, with alternating light and dark conditions and free access to food and water. After one week of acclimatization, the mice were randomly divided into a control group and a high-fat diet group. The control group was fed a normal diet, while the high-fat diet group was fed a high-fat diet. Both groups had free access to water. Mouse weight was measured weekly, and food and water intake were recorded daily. After four weeks of high-fat diet feeding, mice were fasted for 6 hours with free access to water. Initial blood glucose levels were measured in both groups using tail blood sampling. The high-fat diet group was given an intraperitoneal injection of streptozotocin (STZ) 100 mg / kg, while continuing the high-fat diet. One week later, mouse weight was measured again, and blood glucose levels were measured. A blood glucose level greater than 11.0 mM was considered a successful model. For mice that did not successfully establish a model, STZ was administered intraperitoneally at a dose of 60 mg / kg. One week later, the mice's weight and blood glucose were measured again. During the induction period, the mice's food intake and water intake were recorded.

[0092] After successful modeling, mice fed a high-fat diet were randomly divided into a model group, a positive control group (acarbose), and a treatment group based on body weight and blood glucose levels. Mice fed a normal diet served as the control group. The positive control group was given acarbose 80 mg / kg. The treatment groups were given unencapsulated peptides (peptide group), W1 / O / W1 double emulsion microcapsules (double emulsion group), and dried microcapsule solids (microcapsule group), respectively. The dosage equivalent to peptides was 500 mg / kg for all groups. The control and model groups were given the same volume of physiological saline. The drugs were administered by gavage twice daily for 21 consecutive days. During the drug administration period, mice in the model group, positive control group, and treatment group continued to be fed a high-fat diet, while the control group was fed a normal diet. All mice had free access to water.

[0093] After three consecutive weeks of treatment, mice were fasted for 3 hours but allowed free water intake before administration. Blood was collected from the tail vein 2.5 hours later, and blood glucose levels were measured using a glucometer to detect the postprandial blood glucose concentration in each group of mice.

[0094] Data are expressed as mean ± standard deviation (X ± SEM). Statistical analysis was performed using SPSS software for significance analysis. Within-group comparisons were performed using paired-samples t-tests, and comparisons among multiple groups were performed using one-way ANOVA.

[0095] Based on the postprandial blood glucose level changes during the drug administration period in each group of mice, an area under the curve (AUC) was plotted. Figure 17 As shown in the figure. After three consecutive weeks of administration, postprandial blood glucose levels in mice were measured 2.5 hours later. The model group showed a significant upward trend in blood glucose levels over the three weeks, exhibiting a significant difference compared to the normal control group. The positive control group (80 mg / kg) showed a significant decrease in blood glucose levels compared to the model group (p < 0.001). The peptide group (p < 0.01), W1 / O / W2 double emulsion group (p < 0.001), and microcapsule group (p < 0.01) also showed significant decreases in blood glucose levels compared to the model group. This indicates that the Peony petal peptides, even when encapsulated in microcapsules, can significantly reduce postprandial blood glucose in type 2 diabetes mellitus in vivo.

[0096] Example 10: In vitro release test of the peony petal polypeptide microcapsules from Example 8. The animal experimental data in Example 11 have confirmed that the Danfeng peony petal polypeptide and its microcapsules have good in vivo hypoglycemic activity. In order to verify whether the microcapsules have the characteristic of targeted intestinal release, an in vitro simulated release experiment was also conducted.

[0097] Preparation of simulated gastric juice: Mix 0.5 g NaCl and 1.75 mL 0.1 mol / L HCl in 250 mL deionized water, adjust the pH to 2.0 with concentrated HCl, add 0.2% w / v pepsin to simulate gastric digestion, and prepare simulated gastric juice.

[0098] Preparation of simulated intestinal fluid: Mix 1.7 g KH2PO4 and 47.5 mL 0.1 mol / L NaOH, add to 250 mL deionized water, adjust the pH to 7.4 with NaOH, add 0.2% (w / v) of trypsin and 1.2% (w / v) of bile salts to simulate intestinal digestion, and prepare simulated intestinal fluid.

[0099] One mL of freshly prepared W1 / O / W1 double emulsion, 350 mg of microcapsule powder, and 1 mL of microcapsule reconstituted solution were each mixed with 9 mL of simulated gastric juice and digested on a shaker at 37 ℃ and 100 rpm for 2 h to induce gastric digestion and release. The gastric-digested sample was then adjusted to pH 7 with 1 mol / L NaOH, and an equal volume of simulated intestinal juice was added. Digestion continued at 37 ℃ and 100 rpm for 4 h to induce intestinal digestion and release. Samples from the gastrointestinal digestion stage were taken every 30 min. After sampling, the samples were centrifuged at 4 ℃ and 10,000 rpm for 20 min to stop digestion. The release amount of *Peony peony* petal peptides was then measured, and the cumulative release rate of *Peony peony* petal peptides was calculated.

[0100] like Figure 18 It was found that after 2 hours of simulated gastric juice digestion and release, the in vitro release rates of the W / O / W reconstituted emulsion, solid microcapsules, and microcapsule reconstituted solution were all below 30%, while the Danfeng peony petal polypeptide was rapidly released in gastric juice, with a final digestibility of 96%, indicating that it was essentially completely digested. During the simulated intestinal juice digestion stage, the in vitro release rates of the reconstituted emulsion and microcapsules increased rapidly, with final release rates exceeding 90%. Therefore, the Danfeng peony petal polypeptide microcapsules prepared in this invention release a large amount of polypeptide in the intestinal environment, avoiding the destruction of most of the active peptides in the gastric acid environment, thus enabling the Danfeng peony petal polypeptide to be released into the bloodstream from the intestine to exert its hypoglycemic activity.

[0101] In summary, there are currently no reports on the preparation method of Peony peony petal polypeptide microcapsules or their anti-diabetic effects in the prior art. This invention provides a method for preparing Peony peony petal polypeptide microcapsules and their application in anti-diabetic activity. This invention is the first to encapsulate Peony peony petal polypeptides into microcapsules, improving the stability of the active peptides in the storage environment and the in vivo gastrointestinal environment, enabling the active peptides to be released and exert their activity in the intestine, thus improving bioavailability. Furthermore, the microcapsules have a significant effect in reducing type 2 diabetes. In addition, Peony peony petals are a novel food ingredient in this invention, and the wall material and emulsifier used in the microcapsule preparation process are all food additives, with water as the solvent, ensuring high food safety. Therefore, Peony peony petal polypeptide microcapsules, as a safe and reliable food-derived hypoglycemic substance, have good practical application and market value.

[0102] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A Danfeng peony polypeptide microcapsule, characterized in that: The microcapsules are made by encapsulating the blood sugar-lowering polypeptide of *Phoenix Peony* with packaging materials to form W1 / O / W2 type double emulsion microcapsules, or by further drying to obtain solid microcapsules.

2. The Danfeng Peony polypeptide microcapsule according to claim 1, characterized in that: The blood sugar-lowering polypeptide of Peony peony is obtained by enzymatic hydrolysis of Peony peony petals. The packaging material is made of a single wall material or a composite wall material; The W1 / O / W2 type double emulsion refers to a water-in-oil-in-water type double emulsion, which is a liquid microcapsule that can be applied directly or diluted for application.

3. The Danfeng Peony polypeptide microcapsule according to claim 2, characterized in that: The wall material is one or more of the following: sodium carboxymethyl cellulose, cyclodextrin, xanthan gum, carrageenan, sodium alginate, chitosan, gum arabic, and phospholipids. Alternatively, the drying process refers to vacuum drying, freeze drying, spray drying, or atmospheric pressure heating drying, after which the microcapsules are solid; Alternatively, an emulsifier may be used in the preparation of the W1 / O / W2 type double emulsion, and the emulsifier may be one or more of Span 20, Span 80, lecithin, polyglycerol polyricinoleate PGPR, sucrose fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester (span), and Tween.

4. The Danfeng Peony polypeptide microcapsules according to any one of claims 1 to 3, characterized in that: The Danfeng peony polypeptide microcapsules comprise an inner aqueous phase, an oil phase, and an outer aqueous phase. The inner aqueous phase consists of hypoglycemic polypeptides from Danfeng peony petals, the oil phase is composed of medium-chain triglycerides and polyglycerol polyricinoleate, and the outer aqueous phase is composed of sodium carboxymethyl cellulose, cyclodextrin, and Tween 80. A two-step emulsification method is used: first, a water-in-oil primary emulsion is obtained from the inner aqueous phase and the oil phase; then, the primary emulsion is mixed with the outer aqueous phase, and ultrasonic emulsification technology is used to form microcapsules.

5. The method for preparing Danfeng Peony polypeptide microcapsules according to any one of claims 1 to 4, characterized in that: Includes the following steps: (1) Preparation of W1 / O primary emulsion: The Danfeng Mudan polypeptide was dissolved in water and fed at a ratio of 1:1 to 1000 g:mL as the inner aqueous phase of W1; the medium-chain triglycerides and lipophilic emulsifier were mixed at a volume ratio of 1:0.01 to 100 as the oil phase O; the inner aqueous phase of W1 was slowly added to the oil phase O at a volume ratio of 1:0.01 to 100, and stirred until the emulsifier was completely hydrated to form an oil-in-water W1 / O type primary emulsion; (2) Preparation of W1 / O / W2 type double emulsion: Dissolve the composite wall material in water at a ratio of 1:1 to 1000 g:mL, then add 0.1% to 10% Tween80 by mass, stir to completely hydrate the emulsifier, and use it as the W2 external aqueous phase for later use; use the above W1 / O primary emulsion as the core material, and add the W2 external aqueous phase to the W1 / O primary emulsion at a volume ratio of 1:0.01 to 100, heat and stir at 40 to 80°C to make the W1 / O primary emulsion and the W2 external aqueous phase evenly mixed, and then use an ultrasonic emulsifier to perform ultrasonic emulsification treatment on the mixed solution to prepare the W1 / O / W2 type double emulsion. The W1 / O / W2 type double emulsion can be used directly or diluted to obtain Danfeng Peony polypeptide microcapsules.

6. The preparation method according to claim 5, characterized in that: In step (1), the Danfeng peony polypeptide is a polypeptide obtained by extracting from the petals of the peony, namely the short peptide protein of the petals of the peony disclosed in Chinese patent publication CN117568431A. Alternatively, the lipophilic emulsifier in step (1) is polyglycerol polyricinoleate; Alternatively, in step (2), the composite wall material is a mixture of sodium carboxymethyl cellulose and cyclodextrin in a mass ratio of 4:1; Alternatively, the specific conditions for heating and stirring in step (2) are: heating and stirring at 50°C for 30 to 60 minutes; Alternatively, the specific conditions for ultrasonic emulsification in step (2) are: ultrasonic power of 65 W, ultrasonic for 3 min, ultrasonic for 4 s, and stop for 2 s; Alternatively, the W1 / O / W2 type double emulsion in step (2) also undergoes the following steps: Microcapsule powder: The W1 / O / W2 type double emulsion prepared above is dried to obtain microcapsule solid powder. The drying method is vacuum drying, freeze drying, spray drying or atmospheric pressure heating drying. The conditions for vacuum drying are: 50℃, vacuum degree -0.08~-0.1MPa; the conditions for freeze drying are: temperature < -60℃, vacuum degree < 20MPa; the conditions for spray drying are: inlet air temperature 160~190℃, outlet air temperature 70~90℃, feed rate 5 mL / min; the conditions for atmospheric pressure heating drying are: 50℃, atmospheric pressure.

7. A liquid or solid formulation prepared using the Danfeng Peony polypeptide microcapsules as described in any one of claims 1 to 4.

8. The use of the Danfeng Mudan polypeptide microcapsules as described in any one of claims 1 to 4 in the preparation of a targeted and slow-release medicament in the intestine.

9. The use of the Danfeng Peony polypeptide microcapsules as described in any one of claims 1 to 4 in the preparation of hypoglycemic drugs.

10. The use of the Danfeng Peony polypeptide microcapsules as described in any one of claims 1 to 4 in the preparation of special medical foods and / or health foods and / or functional foods and / or ordinary foods.

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