Ecdysterone-low molecular weight fructan composite microspheres with fat-reducing and muscle-increasing functions, and preparation method and application thereof

Abstract

The application discloses ecdysterone-low molecular weight fructan composite microspheres with fat-reducing and muscle-increasing functions, and a preparation method and application thereof, and belongs to the technical field of biological medicine and functional food. The composite microspheres have a core-shell structure, the inner core is a calcium alginate gel, the outer shell is chitosan, the inner core co-encapsulates ecdysterone and low molecular weight fructan, and the mass ratio of the two is 0.8-1.2:1. The preparation adopts a liquid drop microfluidic technology combined with an internal gelation method to form the inner core, and adopts a post-crosslinking method to coat the outer shell. The composite microspheres are regular in shape, uniform in particle size, high in encapsulation rate, good in stability, excellent in pH response slow-release and anti-degradation characteristics in the gastrointestinal tract. In-vivo pharmacological experiments prove that the composite microspheres can reduce the body weight and body fat rate of obese mice, increase the lean meat content and muscle strength, realize synergistic effects of fat reduction and muscle increase, and can be used for preparing drugs or functional food for improving obesity-related muscle loss, low-muscle obesity and related metabolic syndrome.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to a ecdysterone-low molecular weight fructan composite microsphere with fat reduction and muscle building functions, its preparation method and application. Background Technology

[0002] Obesity, a complex chronic metabolic disease influenced by multiple factors, is characterized by abnormal or excessive accumulation of adipose tissue. In recent years, with changes in dietary structure and reduced physical activity, the prevalence of obesity in my country has risen sharply. Current obesity intervention strategies all have significant limitations. While exercise therapy has some effect, it is difficult to maintain long-term and is prone to weight rebound; dieting may lead to hormonal imbalances, decreased metabolic levels, and intestinal damage; regarding medications, approved drugs such as orlistat and GLP-1 analogs (e.g., semaglutide) can achieve weight loss goals, but they often have gastrointestinal side effects or cause skeletal muscle loss while reducing fat, severely affecting basal metabolism, and leading to rapid weight rebound after discontinuation. Studies show that weight loss induced by drugs such as semaglutide and tirzepatide is often accompanied by skeletal muscle loss, raising concerns in the academic community about their potential impact on frailty and sarcopenic obesity. Therefore, the core of weight management must shift from simply "weight loss" to optimizing body composition through "fat reduction and muscle gain," which is key to achieving lasting health benefits. Ecdysterone (ECDY) is a naturally occurring phytosterone with significant pharmacological activities, including promoting protein synthesis, lowering blood sugar, regulating blood lipids, and anti-inflammation, showing broad application prospects in functional foods and pharmaceuticals. However, free ecdysterone suffers from drawbacks such as low oral bioavailability, poor stability in the gastrointestinal tract, and rapid metabolism (strong first-pass effect), limiting its further clinical application. Currently, there is limited research on formulations targeting ecdysterone, and conventional dosage forms struggle to address its rapid clearance in the intestines.

[0003] Calcium alginate-chitosan microspheres, due to their pH responsiveness and biocompatibility, have been used in colon-targeted delivery studies of active ingredients. For example, some studies have used sodium alginate and chitosan to form composite microspheres through electrostatic interactions, utilizing the high pH environment and microbial degradation characteristics of the colon to achieve targeted drug release. However, in simulated gastrointestinal environments, the mechanical strength of these microspheres is often insufficient, and the encapsulation efficiency and targeted release performance of active ingredients in the colon still need improvement.

[0004] Furthermore, achieving the dual goals of "fat loss" and "muscle gain" simultaneously in the field of weight management remains a clinical challenge. Therefore, there is an urgent need to develop a novel delivery system that can improve the oral stability of ecdysterone, achieve colon-targeted sustained release, and achieve the dual effects of fat loss and muscle gain through a synergistic mechanism. Summary of the Invention

[0005] The present invention aims to solve the following problems existing in the prior art: low oral bioavailability of free ecdysterone, poor water solubility, poor stability in the gastrointestinal tract, and rapid metabolism; existing weight management strategies are difficult to achieve the dual goals of "fat loss" and "muscle gain" at the same time, and there is a risk of muscle loss.

[0006] To address the aforementioned technical problems, this invention provides a composite microsphere with pH-responsive sustained-release properties, capable of colon-targeted co-delivery, and achieving fat reduction and muscle gain functions through a synergistic mechanism, as well as its preparation method.

[0007] Specifically, the present invention adopts the following technical solution: First, the present invention provides a composite microsphere of ecdysterone-low molecular weight fructan with fat reduction and muscle building functions. The microsphere has a core-shell structure and includes: a core, which is a calcium alginate gel, and the calcium alginate gel is co-encapsulated with ecdysterone and low molecular weight fructan; and a shell, which is a chitosan layer, and the chitosan layer covers the surface of the core.

[0008] The low molecular weight fructans therein serve as both a prebiotic active ingredient and a functional excipient that enhances the density of the microsphere structure; the chitosan shell is cross-linked with the calcium alginate core through electrostatic adsorption between alginate and chitosan ammonium ions; the composite microspheres have pH-responsive and colon-targeted release properties.

[0009] Preferably, the mass ratio of ecdysterone to low molecular weight fructan is 0.8-1.2:1, and more preferably 1:1.

[0010] The ecdysterone mentioned above is also known as β-ecdysone, ecdysone hormone, and ecdysterone in Chinese. Its CAS number is 5289-74-7.

[0011] The low molecular weight fructan is derived from Polygonatum yunnanense (Yunnan Polygonatum sibiricum) Polygonatum kingianum) Its average molecular weight ranges from 1.174 kDa to 1.726 kDa.

[0012] The microspheres also have one or more of the following characteristics: an average particle size of 25-40 μm, more preferably 32 μm; a zeta potential of -25 mV to -15 mV, more preferably -18.78 mV; and an encapsulation efficiency of greater than 99% for both ecdysterone and low molecular weight fructan.

[0013] In the in vitro gut microbiota fermentation system, the complete metabolism time of ecdysterone loaded in the microspheres is not less than 96 hours.

[0014] The present invention also provides a method for preparing the above-mentioned composite microspheres, comprising the following steps: (1) Preparation of aqueous phase: Sodium alginate, calcium ion complexing agent, ecdysterone and low molecular weight fructan are dissolved in water to obtain aqueous working solution; (2) Preparation of the receiving phase: Mix the oil phase with the acid to obtain the receiving phase; (3) Droplet generation and solidification: The aqueous working liquid droplets from step (1) are added to the receiving phase from step (2) using droplet microfluidic technology. Calcium alginate microspheres are formed through an internal gelation reaction and then allowed to solidify. (4) Demulsification and collection: The solidified microspheres were demulsified and centrifuged to collect the core of the calcium alginate microspheres; (5) Chitosan coating: The core of the calcium alginate microspheres obtained in step (4) is brought into contact with the chitosan solution, and a chitosan shell is formed through a post-crosslinking reaction, thus obtaining the composite microspheres.

[0015] Preferably, the mass-volume concentration of sodium alginate in step (1) is 1%-2%, for example, it can be 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%, which will not be listed here; the calcium ion chelating agent is Ca-EDTA, and its concentration is 200-300 mM, for example, it can be 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, or 300 mM, which will not be listed here; the mass ratio of the total mass of ecdysterone and low molecular weight fructan to the mass of sodium alginate is 0.8:1 to 1.2:1.

[0016] Preferably, the Ca-EDTA solution in step (1) is adjusted to pH 7.2 using NaOH.

[0017] Preferably, further, the receiving phase in step (2) is a solution containing 1-3% microdroplet generating oil and 0.5-1% glacial acetic acid; in step (3), the aqueous working solution is added to the receiving phase at a flow rate of 5-7 μL / min using microfluidic technology; the chitosan solution in step (5) is an aqueous solution of chitosan and acetic acid, wherein the mass-volume concentration of chitosan is 0.1%-0.5% and the volume concentration of acetic acid is 0.5%-1%.

[0018] Preferably, the static curing is carried out for about 30-40 minutes.

[0019] This invention also provides the application of the above-mentioned composite microspheres in the preparation of pharmaceuticals or functional foods having any one or more of the following functions: (a) Simultaneously reduce fat and gain muscle; (b) Improve obesity-related muscle loss or oligomuscular obesity; (c) Improve muscle endurance and exercise capacity in obese individuals; (d) Improve metabolic syndrome induced by a high-fat diet, wherein the metabolic syndrome includes one or more of obesity, insulin resistance, hyperlipidemia or osteoporosis.

[0020] Preferably, the drug is administered orally.

[0021] Preferably, the drug is used in combination with diet control and exercise therapy.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The composite microspheres prepared by the present invention using droplet microfluidic technology combined with internal gelation method have regular morphology, smooth surface, average particle size of about 32 μm, uniform particle size distribution, and Zeta potential of -18.78 mV, and the microspheres have good stability; the addition of low molecular weight fructan makes the surface structure of the microspheres more compact than that of ordinary ecdysterone microspheres, significantly improving the encapsulation efficiency, which is beneficial to protecting the drug from easy degradation and leakage in the gastrointestinal tract; (2) The composite microspheres of the present invention have significant pH-responsive colon-targeted release characteristics. In simulated gastric juice (pH 1.2), the composite microspheres take nearly 3 hours to reach the release peak, while ordinary ecdysterone microspheres reach the peak in 2 hours. This indicates that the addition of low molecular weight fructan makes the microsphere structure more compact and can effectively protect the drug from degradation in gastric juice for a long time. In simulated intestinal juice (pH 6.8), the time for the drug release of the composite microspheres to reach the peak is extended by 30 minutes compared with ordinary ecdysterone microspheres, which is beneficial to promoting the absorption of the drug in the colon. (3) The composite microspheres of the present invention have excellent anti-degradation properties. In the in vitro microbial fermentation experiment, free ecdysterone was completely metabolized within 48 hours. Ecdysterone microspheres extended the metabolic time to 96 hours, while the ecdysterone / low molecular weight fructan composite microspheres of the present invention further significantly extended the metabolic time to 112 hours. This indicates that the low molecular weight fructan and the microsphere encapsulation technology produced a synergistic effect, which delayed the degradation of the drug in the intestine to the maximum extent and provided more time for drug absorption. (4) The composite microspheres of this invention showed significantly better fat reduction and muscle gain effects than free drugs, physical mixtures, and single-drug microspheres in a high-fat diet-induced obese mouse model. Specifically, they significantly reduced weight gain and body fat percentage; significantly increased lean meat content and improved muscle strength and exercise endurance; increased wet weight of the gastrocnemius and tibialis anterior muscles, reduced wet weight of inguinal and epididymal fat, improved blood lipid levels, effectively improved glucose tolerance and insulin resistance; and improved skeletal parameters. The comprehensive score showed that the composite microspheres of this invention ranked first in terms of comprehensive fat reduction and muscle gain effects among all test groups, achieving a synergistic effect of "1+1>2". Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the preparation process of the ecdysterone-low molecular weight fructan composite microspheres prepared in Example 2 of the present invention.

[0024] Figure 2 Characterization diagrams of ecdysterone-low molecular weight fructan composite microspheres: A shows the morphology under microscope and scanning electron microscope; B shows the particle size distribution; C shows the zeta potential; and D shows the peak area comparison diagram of encapsulation efficiency measurement.

[0025] Figure 3 The infrared and energy dispersive spectroscopy (EDS) results of the ecdysterone-low molecular weight fructan composite microspheres are shown in Figure 1: A is the infrared spectrum; B is the EDS.

[0026] Figure 4 The in vitro release curves of ecdysterone-low molecular weight fructan composite microspheres are shown: where A is the release curve of simulated gastric juice (pH 1.2); and B is the release curve of simulated intestinal juice (pH 6.8).

[0027] Figure 5 The in vitro microbial metabolic rate diagram of ecdysterone-low molecular weight fructan composite microspheres: where A is the metabolic rate curve; B is the liquid chromatogram at different time points.

[0028] Figure 6 The graph shows the improvement of fat and muscle indices in each group of obese mice: A represents weight change; B represents glucose tolerance test; C represents insulin resistance test; D represents exercise program setting; E represents fatigue time; F represents fatigue distance; G represents rotarod drop time; H represents lean meat content; I represents fat content; J represents bone volume / bone area; K represents trabecular bone thickness; and L represents cortical bone thickness.

[0029] Figure 7 The wet weight of adipose and muscle tissue and serum lipid levels of mice in each group were: A was wet weight of inguinal fat, B was wet weight of epididymal fat, C was weight of gastrocnemius muscle, D was weight of tibialis anterior muscle, and E and H were the levels of cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein in plasma, respectively.

[0030] Figure 8 The images show the morphological features of muscle tissue in each group of obese model mice.

[0031] Figure 9 The images show the morphology of adipose tissue in each group of obese model mice.

[0032] Figure 10 A score chart showing the overall fat loss and muscle gain effects for each group. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0034] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0035] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0036] The following examples contain information on the source and specifications of the main reagent raw materials: Sodium alginate (A9640, Solarbio); Chitosan (CAS: 9012-76-4, Solarbio); Sodium hydroxide (Tianjin Damao Chemical Reagent Factory); Acetic acid (Luon Reagent); Anhydrous calcium chloride (Tianjin Zhiyuan Chemical Reagent Co., Ltd.); Disodium ethylenediaminetetraacetate (CAS: 6381-92-6, Yuanye Biotechnology) dihydrate; Drop-Surf 2% microdroplet generating oil (DGO-20-02-10, Shanghai Pengzan Biotechnology Co., Ltd.); Drop-Surf demulsifier (DB-80-20-10, Shanghai Pengzan Biotechnology Co., Ltd.); PDMS microdroplet generating chip 30 (flow focusing type, hydrophobic) (PDMS-FF-30B, Shanghai Pengzan Biotechnology Co., Ltd.).

[0037] Yunnan Polygonatum ( Polygonatum kingianum The dried rhizomes were collected from Wenshan County, Yunnan Province, China. Ecdysterone (CAS: 5289-74-7), purity 99.23%, was purchased from Chengdu Mansite Biotechnology Co., Ltd.

[0038] The specific conditions for the high-performance liquid chromatography (HPLC) detection of ecdysterone in the following examples are as follows: The chromatographic column was a C18MS-Ⅱ, 4.6 mm I.D. x 250 mm. The mobile phase A was an aqueous formic acid solution (H2O : FoOH = 99.9 : 0.1, v / v), and the mobile phase B was an acetonitrile solution. The flow rate was 0.5 mL / min, the injection volume was 10 μL, and gradient elution was used. The elution program is shown in Table 1.

[0039] Table 1 HPLC gradient elution program

[0040] Example 1: Extraction and separation of low molecular weight fructan (LMWF) from Polygonatum yunnanense Take Yunnan Polygonatum ( Polygonatum kingianum The dried rhizomes were extracted with water and precipitated with alcohol. The supernatant was collected, the extract was concentrated, and the solution was initially purified by passing it through a macroporous resin. Then, it was separated by gel filtration chromatography (such as Sephadex G-25). The low molecular weight components were collected and freeze-dried to obtain the LMWF mixture. According to GPC-MALLS analysis (see Table 2), the average molecular weight of the LMWF used in this example was 1.174-1.726 kDa.

[0041] Table 2. GPC-MALLS results of low molecular weight fructans from Polygonatum odoratum.

[0042] Example 2: Preparation of composite microspheres co-encapsulated with ecdysterone and low molecular weight fructan 1. Preparation of Phase A (Aqueous Phase) Reagent 2 M CaCl2 solution: Weigh 2.2197 g of anhydrous CaCl2 (molecular weight 110.984 g / mol), add ultrapure water and shake to dissolve, then bring the volume to 10 mL for later use.

[0043] 1.5% (wt%) sodium alginate (SA) solution: Weigh 0.15 g of sodium alginate solid powder, add 10 mL of ultrapure water, sonicate and heat at 60℃ to assist dissolution, and set aside.

[0044] 2 M NaOH solution: Weigh 1.6 g NaOH (molecular weight 40.00 g / mol), add ultrapure water and shake to dissolve, then bring the volume to 20 mL for later use.

[0045] 240 mM Ca-EDTA solution (pH=7.2): Take 1.2 mL of 2 M CaCl2 and 4.8 mL of 0.5 M EDTA, adjust the pH to 7.2 with 2 M NaOH, and finally bring the volume to 10 mL for later use.

[0046] Preparation of Phase A working solution: Take an equal volume of 5 mL of 240 mM Ca-EDTA solution and 5 mL of 1.5% sodium alginate solution, shake well to obtain an aqueous phase solution. Add 75 mg of ecdysterone (ECDY) and 75 mg of low molecular weight fructan (LMWF) prepared in Example 1 (i.e., the mass ratio of ecdysterone to LMWF is 1:1), dissolve thoroughly by sonication, and then filter through a 0.45 μm needle filter for later use.

[0047] 2. Preparation of Phase B (receiving phase) reagent Receiver phase solution (1% acetic acid, pH≈4.8): Take 500 μL of 2% Drop-Surf microdroplet-generated oil, add 5 μL of glacial acetic acid, shake well, and set aside.

[0048] 3. Microsphere preparation and curing Droplet generation: Using a microfluidic instrument, phase A (aqueous phase) is added dropwise to phase B (receiving phase) at a set flow rate (e.g., 5-7 μL / min).

[0049] Curing: After stopping collection, gently agitate to accelerate the curing of microdroplets, then allow to stand for about 30 minutes to cure.

[0050] Collection and demulsification: Remove the oil generated from the microdroplets at the bottom of the centrifuge tube to obtain microspheres. Add demulsifier at a ratio of microsphere volume to demulsifier volume of 1:2, and shake to demulsify.

[0051] Centrifugation: Centrifuge at 2500 rpm for 1 minute and discard the demulsifier layer.

[0052] Chitosan coating: The above microspheres were transferred into a 1% (v / v) aqueous solution of 0.5% (w / v) chitosan (CS) and stirred to solidify for 30 minutes, and then washed three times with deionized water.

[0053] Final product: Calcium alginate-chitosan composite microspheres co-encapsulated with ecdysterone and low molecular weight fructan were obtained, named ECDY / LMWF@CS / SA. The preparation process flow diagram is shown below. Figure 1 .

[0054] Comparative Example 1 The difference from Example 2 is that it does not contain low molecular weight fructans, and a single ecdysterone microsphere (ECDY@CS / SA) is prepared. Example 3 Characterization of composite microspheres (ECDY / LMWF@CS / SA) The composite microspheres prepared in Example 2 were characterized as follows: (1) Morphology and particle size The morphology of the microspheres was observed using optical microscopy and scanning electron microscopy. The results are as follows: Figure 2As shown in Figure A, compared to single ecdysterone microspheres (ECDY@CS / SA), ECDY / LMWF@CS / SA microspheres are regularly spherical and have a denser surface structure. The particle size distribution was measured using a laser particle size analyzer, and the results are as follows. Figure 2 As shown in Figure B, the average particle size is approximately 32 μm. The Zeta potential measurement results are as follows... Figure 2 As shown in Figure C, the potential is -18.78 mV, indicating that the microspheres have good colloidal stability.

[0055] (2) Encapsulation efficiency determination The peak area of ​​ecdysterone in the solution before and after encapsulation was determined by high performance liquid chromatography (HPLC). Figure 2 As shown in Figure D, the peak area of ​​the drug before encapsulation was 1848.18, while the peak area after encapsulation was almost undetectable in the supernatant (0.0250). Calculated using the formula (peak area before encapsulation - peak area after encapsulation) / peak area before encapsulation × 100%, the encapsulation efficiency was greater than 99.99%. Simultaneously, the encapsulation efficiency of low molecular weight fructan, determined using the anthrone-sulfuric acid method, was approximately 99.99%, indicating that both drugs were efficiently encapsulated.

[0056] (3) Infrared spectroscopy and energy spectrum analysis The microspheres were scanned using a Fourier transform infrared spectroscopy (FTIR) instrument, and the results are as follows: Figure 3 As shown in Figure A, the characteristic absorption peaks (C=O, COC) of free ecdysterone disappeared after encapsulation, indicating that the drug was successfully encapsulated inside the microspheres. Energy dispersive spectroscopy (EDS) results (see Figure A). Figure 3 Figure B shows that the changes in C and O element content further confirm that ecdysterone and low molecular weight fructan were successfully encapsulated in the microsphere core.

[0057] Example 4 In vitro drug release experiment Single ecdysterone drug-loaded microspheres (ECDY@CS / SA) prepared in Comparative Example 1 and ecdysterone-low molecular weight fructan composite microspheres (ECDY / LMWF@CS / SA) prepared in Example 2, as well as free ecdysterone, were taken. The samples were placed in simulated gastric fluid (pH 1.2) or simulated intestinal fluid (pH 6.8) and shaken at 37°C and 100 rpm. Samples were taken at preset time points, and the ecdysterone concentration was determined by HPLC.

[0058] In gastric juice: the ecdysterone content in the free ecdysterone group, the drug-loaded microsphere group (ECDY@CS / SA), and the ECDY / LMWF@CS / SA group was 1.5±0.3 mg / ml; In intestinal fluid: the ecdysterone content in the free ecdysterone group, the drug-loaded microsphere group (ECDY@CS / SA), and the ECDY / LMWF@CS / SA group was 2.0±0.3 mg / ml.

[0059] The results are as follows Figure 4 As shown: Simulated gastric juice (see) Figure 4 (A) Rapid release of free drug; ECDY@CS / SA reaches peak release in 2 hours; while the release of ECDY / LMWF@CS / SA in this invention is more gradual, reaching peak release in nearly 3 hours, indicating that the microsphere structure is more compact and has stronger gastric acid protection ability after adding LMWF.

[0060] Simulated intestinal fluid (see) Figure 4 (B) ECDY@CS / SA reaches its peak release time in about 60 minutes; the peak release time of ECDY / LMWF@CS / SA in this invention is extended to about 90 minutes, exhibiting a more sustained release characteristic, which is beneficial to drug absorption in the intestine.

[0061] Example 5: In vitro microbial metabolism experiment Intestinal flora were collected from healthy mice (colon contents of healthy adult C57BL / 6 mice were diluted 10-fold with sterile physiological saline, centrifuged, and the supernatant was collected to obtain an intestinal flora suspension), and inoculated into GAM medium. Free ecdysterone, ECDY@CS / SA microspheres (sample prepared in Comparative Example 1), and ECDY / LMWF@CS / SA microspheres (sample prepared in Example 2) were added to each of the three groups, with the same amount of ecdysterone (0.7 ± 0.05 mg / mL).

[0062] Free ecdysterone, ecdysterone microspheres (ECDY@CS / SA), and ecdysterone-low molecular weight fructan composite microspheres (ECDY / LMWF@CS / SA) were cultured anaerobically. Samples were taken at predetermined time points, and drug residue concentrations were determined by HPLC.

[0063] The results are as follows Figure 5 As shown: Free ecdysterone was completely metabolized within 48 hours.

[0064] Ecdysterone microspheres (ECDY@CS / SA) extend the complete drug metabolism time to 96 hours.

[0065] The ecdysterone-low molecular weight fructan composite microspheres (ECDY / LMWF@CS / SA) of this invention further significantly extend the complete drug metabolism time to 112 hours. This result demonstrates that the addition of LMWF and the microsphere encapsulation technology produce a synergistic protective effect, maximally delaying the degradation of ecdysterone by intestinal flora.

[0066] Example 6: In vivo pharmacodynamic experiment in obese mice (1) Animal models and grouping Six-week-old male C57BL / 6J mice were randomly divided into 6 groups of 15 mice each after one week of acclimatization feeding. The grouping and treatment were as follows: Normal control group (CON): low-fat diet + equal volume of solvent administered by gavage; High-fat diet model group (HFD): high-fat diet + equal volume of solvent administered by gavage; Free ecdysterone group (ECDY): high-fat diet + ecdysterone (30 mg / kg / d) by gavage; Ecdysterone microsphere group (ECDY@CS / SA): high-fat diet + gavage administration of microspheres (ECDY@CS / SA, prepared in Comparative Example 1) at a dose of 30 mg / kg / d based on ecdysterone; Physical mixture group (ECDY + LMWF): high-fat diet + ecdysterone (30 mg / kg / d) and low molecular weight fructan (30 mg / kg / d) mixed by gavage (unencapsulated); Ecdysterone-low molecular weight fructan microspheres (ECDY / LMWF@CS / SA): High-fat diet + gavage administration of microspheres (ECDY / LMWF@CS / SA, prepared in Example 2) at a dose of 30 mg / kg / d of ecdysterone.

[0067] (2) Body weight and metabolic indicators Weigh yourself weekly, and the results are as follows Figure 6 As shown in Figure A: After 12 weeks of HFD feeding, the body weight of the HFD group increased significantly; the body weight of each treatment group decreased to varying degrees, among which the body weight of the ecdysterone-low molecular weight fructan microsphere group of the present invention decreased the most significantly, and the effect was better than that of the other groups.

[0068] Twelve weeks after drug administration, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed, and the results were as follows: Figure 6 As shown in Figures B and C: all ecdysterone microsphere groups and ecdysterone-low molecular weight fructan microsphere groups showed better effects than the unencapsulated group, among which the ecdysterone-low molecular weight fructan microsphere group of the present invention showed the best effect in improving glucose tolerance and insulin resistance.

[0069] (3) Motor ability test Exercise endurance test: After 12 weeks of drug administration, the exercise endurance of mice was determined using a treadmill test. The exercise program was set as follows: Figure 6 As shown in Figure D, mice were placed on a treadmill with an initial speed of 10 m / min, increasing by 3 m / min every 5 minutes until a maximum speed of 25 m / min was reached, and the fatigue time and fatigue distance were recorded. The results are shown in [Figure D]. Figure 6 E and F in the middle.

[0070] A rotarod experiment was conducted. Mice were placed on a rotarod apparatus. The rotation speed was initially 5 rpm / min for 5 seconds with a 1-second interval; then 10 rpm / min for 120 seconds with a 5-second interval; followed by 15 rpm / min with a 5-second interval, until the mouse fell. The time it took for the mouse to fall from the rotarod was recorded. The results are shown below. Figure 6 G.

[0071] The results showed that all treatment groups could prolong the exercise capacity of mice. Among them, the fatigue time, fatigue distance and rotarod drop time of mice in the ecdysterone-low molecular weight fructan microsphere group of the present invention were significantly longer than those of other groups, indicating that it had the best effect on improving muscle endurance and balance.

[0072] (4) Body composition analysis The total body fat content and lean meat content of mice were determined using a small animal nuclear magnetic resonance analyzer. The results are as follows: Figure 6 As shown in H and 6I: all treatment groups were able to reduce fat content and increase lean meat content. Among them, the ecdysterone-low molecular weight fructan microsphere group of the present invention achieved the best results in both reducing fat and increasing lean meat, thus achieving the dual goals of fat reduction and muscle gain.

[0073] (5) Skeletal parameter analysis Micro-CT was used to analyze mouse tibial parameters. The results are as follows: Figure 6 As shown in JL, the ecdysterone microsphere group (ECDY@CS / SA) showed the best effect in increasing bone volume / bone area (BV / TV), trabecular bone thickness (Tb.Th), and cortical bone thickness (Ct.Th). The ecdysterone-low molecular weight fructan microsphere group (ECDY / LMWF@CS / SA) of this invention also showed a significant improvement effect, which was better than the unencapsulated group (ECDY+LMWF).

[0074] (6) Wet weight of mouse fat and muscle tissue and serum lipid levels Twelve weeks after drug administration, mice were fasted (e.g., for 4-6 hours), anesthetized, and whole blood was collected through the orbital cavity. Plasma was separated by centrifugation and used to detect the following lipid indicators: total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. Simultaneously, the mice were sacrificed, and the gastrocnemius muscle, tibialis anterior muscle, inguinal fat, and epididymal adipose tissue were separated and weighed separately.

[0075] Figure 7 The results from the Chinese A / D study showed that the administration of drugs to each group increased the wet weight of the gastrocnemius and tibialis anterior muscles in mice, and decreased the wet weight of inguinal and epididymal fat. Figure 7The results from the EH study showed that the blood lipid levels of mice in all groups improved to varying degrees after drug administration. Among them, the ecdysterone-low molecular weight fructan microsphere group (ECDY / LMWF@CS / SA) showed better results than the unencapsulated group (ECDY+LMWF).

[0076] (7) Observation of tissue morphology Muscle tissue: Twelve weeks after drug administration, the gastrocnemius muscle was dissected, stained with hematoxylin and eosin (HE), and observed. Results are as follows: Figure 8 As shown: the muscle fiber bundles in the HFD group were thin, loose, and had a disordered orientation; all drug administration groups could improve muscle morphology, among which the ecdysterone-low molecular weight fructan microsphere group of the present invention had the most compact muscle fiber arrangement, the clearest striations, and the morphology was closest to the normal control group, indicating that it had the best effect in improving obesity-related muscle loss.

[0077] Adipose tissue: After 12 weeks of drug administration, epididymal fat was isolated from mice, stained with hematoxylin and eosin (HE), and observed. Results are as follows: Figure 9 As shown: adipocytes in the HFD group were significantly enlarged; all treatment groups could effectively reduce adipocyte size, among which the ecdysterone-low molecular weight fructan microsphere group of the present invention had the best effect.

[0078] (8) Overall score To quantitatively evaluate the overall effectiveness of each treatment group, the following was conducted: Figure 6 The fat indicators (such as fat content) and muscle indicators (such as lean meat content, fatigue time, and bar rotation time) are weighted and scored. For example... Figure 10 As shown, the scoring system is as follows: fat and muscle indicators each account for 40%, and body weight accounts for 20%. Normalization is performed using the blank group (CON) as 100% baseline.

[0079] The final comprehensive evaluation results showed that the ecdysterone-low molecular weight fructan composite microspheres (ECDY / LMWF@CS / SA) of this invention ranked first in fat reduction and muscle gain among all test groups, verifying its superior synergistic effect.

[0080] Example 7: Group Allocation Ratio Optimization Following the method in Example 2, composite microspheres with ECDY to LMWF mass ratios of 0.8:1, 1:1, and 1.2:1 were prepared respectively.

[0081] Composite microspheres with a mass ratio of 0.8:1: Take equal volumes of 5 mL of 240 mM Ca-EDTA solution and 5 mL of 1.5% sodium alginate solution, shake well, add 66.7 mg of ecdysterone and 83.3 mg of low molecular weight fructan, sonicate thoroughly to dissolve, filter, and prepare composite microspheres according to the method of Example 1.

[0082] Composite microspheres with a mass ratio of 1:1: namely, the composite microspheres prepared in Example 2.

[0083] Composite microspheres with a mass ratio of 1.2:1: Take equal volumes of 5 mL of 240 mM Ca-EDTA solution and 5 mL of 1.5% sodium alginate solution, shake well, add 83.3 mg of ecdysterone and 66.7 mg of low molecular weight fructan, sonicate thoroughly to dissolve, filter, and prepare composite microspheres according to the method of Example 1.

[0084] The encapsulation efficiency of each composite microsphere for ecdysterone was determined by high performance liquid chromatography. The in vitro release curves of each composite microsphere were determined by the method in Example 2. The fat reduction and muscle gain effects of each composite microsphere were determined by the method in Example 4.

[0085] The results showed that the composite microspheres with a mass ratio of 1:1 had the highest encapsulation efficiency, the best release curve, and the best fat-reducing and muscle-building effects. The composite microspheres with a mass ratio of 1.2:1 showed reduced encapsulation efficiency and sustained-release effect due to the relatively insufficient content of low-molecular-weight fructans and insufficient microsphere structural density. The composite microspheres with a mass ratio of 0.8:1 showed a weakened muscle-building effect due to the relatively insufficient content of ecdysterone.

[0086] Overall, a 1:1 ratio of ECDY to LMWF is the optimal blending ratio.

[0087] Example 8 Particle size optimization Following the method in Example 2, composite microspheres were prepared using sodium alginate solutions with concentrations of 1.0%, 1.5%, and 2.0%, respectively.

[0088] 1.0% SA concentration: Weigh 0.10 g of sodium alginate solid powder, add 10 mL of ultrapure water, sonicate and heat at 60℃ to aid dissolution, to obtain a 1.0% sodium alginate solution. Take an equal volume of 5 mL of 240 mM Ca-EDTA solution and 5 mL of 1.0% sodium alginate solution, shake well, add 75 mg of ecdysterone and 75 mg of low molecular weight fructan, sonicate to dissolve completely, filter, and prepare composite microspheres according to the method of Example 1.

[0089] 1.5% SA concentration: i.e., the composite microspheres prepared in Example 2.

[0090] 2.0% SA concentration: Weigh 0.20 g of sodium alginate solid powder, add 10 mL of ultrapure water, sonicate and heat at 60℃ to assist dissolution, to obtain a 2.0% sodium alginate solution. Take an equal volume of 5 mL of 240 mM Ca-EDTA solution and 5 mL of 2.0% sodium alginate solution, shake well, add 75 mg of ecdysterone and 75 mg of low molecular weight fructan, sonicate to dissolve completely, filter, and prepare composite microspheres according to the method of Example 1.

[0091] The average particle size of each composite microsphere was determined using a laser particle size analyzer, and the encapsulation efficiency of each composite microsphere for ecdysterone was determined using high performance liquid chromatography.

[0092] The results showed that the composite microspheres prepared with 1.0% SA concentration had an average particle size of approximately 25 μm and a relatively low encapsulation efficiency. The composite microspheres prepared with 1.5% SA concentration had an average particle size of approximately 32 μm, exhibiting uniform particle size and high encapsulation efficiency. The composite microspheres prepared with 2.0% SA concentration had an average particle size of approximately 40 μm, but the excessively high solution viscosity affected droplet formation, resulting in an insufficiently uniform particle size distribution.

[0093] Overall, a 1.5% SA concentration can achieve an optimal particle size range of 30-35 μm.

[0094] It is understood that the sodium alginate is not limited to commercially available products, but can also be natural sodium alginate extracted from seaweed. The chitosan is not limited to commercially available products, but can also be natural chitosan extracted from the shells of crustaceans. The low molecular weight fructan is not limited to extraction from Polygonatum odoratum, but can also be low molecular weight fructan extracted from other plants such as Jerusalem artichoke, onion, and garlic.

[0095] It is understood that the Ca-EDTA solution is not limited to 240 mM, but can be any value between 200-280 mM. The pH of the Ca-EDTA solution is not limited to 7.2, but can be any value between 6.8-7.4. The concentration of the sodium alginate solution is not limited to 1.5%, but can be any value between 1.0-2.0%. The mass ratio of ecdysterone to low molecular weight fructan is not limited to 1:1, but can be any value between 0.8-1.2:1.

[0096] It is understood that the droplet microfluidic technology is not limited to the specific instrument described in this embodiment, and other types of microfluidic instruments or capillary co-extrusion devices can also be used. The demulsifier is not limited to the specific type described in this embodiment, and can also be other types of demulsifiers such as n-hexane, petroleum ether, etc. The centrifugation conditions are not limited to centrifugation at 2500 rpm for 1 minute, but can also be centrifugation at 2000-3000 rpm for 0.5-2 minutes.

[0097] Obviously, the concentration of the chitosan solution is not limited to 1% (v / v) acetic acid, but can also be 0.5-2% (v / v) acetic acid. The curing time of the chitosan coating is not limited to 30 minutes, but can also be 20-40 minutes. The average particle size of the composite microspheres is not limited to 32 μm, but can also be 20-50 μm, preferably 30-35 μm.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite microsphere containing ecdysterone and low molecular weight fructan with fat-reducing and muscle-building functions, characterized in that, The microspheres have a core-shell structure and include: The core is a calcium alginate gel, which contains ecdysterone and low molecular weight fructan. The outer shell is a chitosan layer that covers the surface of the core.

2. The composite microspheres according to claim 1, characterized in that, The mass ratio of ecdysterone to the low molecular weight fructan is 0.8-1.2:

1.

3. The composite microspheres according to claim 1, characterized in that, The mass ratio of the ecdysterone to the low molecular weight fructan is 1:

1.

4. The composite microspheres according to claim 1, characterized in that, The low molecular weight fructan is derived from Polygonatum yunnanense, with an average molecular weight of 1.174 kDa to 1.726 kDa.

5. The composite microspheres according to claim 1, characterized in that, The microspheres have one or more of the following characteristics: The average particle size is 25-40 μm; The zeta potential ranges from -15 mV to -25 mV. The encapsulation efficiency for both ecdysterone and low molecular weight fructan is greater than 99%.

6. The composite microspheres according to claim 1, characterized in that, In the in vitro gut microbiota fermentation system, the complete metabolism time of ecdysterone loaded in the microspheres is not less than 96 hours.

7. A method for preparing the composite microspheres according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of aqueous phase: Sodium alginate, calcium ion complexing agent, ecdysterone and low molecular weight fructan are dissolved in water to obtain aqueous working solution; (2) Preparation of the receiving phase: Mix the oil phase with the acid to obtain the receiving phase; (3) Droplet generation and solidification: The aqueous working liquid droplets from step (1) are added to the receiving phase from step (2) using droplet microfluidic technology. Calcium alginate microspheres are formed through an internal gelation reaction and then allowed to solidify. (4) Demulsification and collection: The solidified microspheres were demulsified and centrifuged to collect the core of the calcium alginate microspheres; (5) Chitosan coating: The core of the calcium alginate microspheres obtained in step (4) is brought into contact with the chitosan solution, and a chitosan shell is formed through a post-crosslinking reaction, thus obtaining the composite microspheres.

8. The method according to claim 7, characterized in that, The sodium alginate in step (1) has a mass-volume concentration of 1%-2%; the calcium ion chelating agent is Ca-EDTA with a concentration of 200-300 mM; and the mass ratio of the total mass of ecdysterone and low molecular weight fructan to sodium alginate is 0.8-1.2:

1.

9. The method according to claim 7, characterized in that, The receiving phase in step (2) is a solution containing 1-3% droplet-generated oil and 0.5-1% glacial acetic acid; In step (3), microfluidic technology is used to drop the aqueous working solution into the receiving phase at a flow rate of 5-7 μL / min; The chitosan solution mentioned in step (5) is an aqueous solution of chitosan and acetic acid, wherein the mass volume concentration of chitosan is 0.1%-0.5% and the volume concentration of acetic acid is 0.5%-1%.

10. The use of the composite microspheres according to any one of claims 1-6 or the composite microspheres prepared by the method according to any one of claims 7-9 in the preparation of pharmaceuticals or functional foods having any one or more of the following functions: (a) Simultaneously reduce fat and gain muscle; (b) Improve obesity-related muscle loss or oligomuscular obesity; (c) Improve muscle endurance and exercise capacity in obese individuals; (d) Improve metabolic syndrome induced by a high-fat diet, wherein the metabolic syndrome includes one or more of obesity, insulin resistance, hyperlipidemia or osteoporosis.

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