Probiotic-chlorophyll composite intestinal micro-ecological regulator based on nano encapsulation technology as well as preparation method and application of probiotic-chlorophyll composite intestinal micro-ecological regulator

Abstract

The invention relates to the technical field of intestinal micro-ecology regulators, in particular to a probiotic-chlorophyll composite intestinal micro-ecology regulator based on a nano encapsulation technology, a preparation method and application of the probiotic-chlorophyll composite intestinal micro-ecology regulator. The formula comprises 55-90 parts of a chlorophyll compound stable under the acidic condition, 15-35 parts of a probiotic synergistic composition, 15-30 parts of an anti-inflammatory synergist and 5-10 parts of auxiliary materials, and the chlorophyll compound stable under the acidic condition comprises chlorophyll a, chlorophyll b, sodium copper chlorophyllin and sodium magnesium chlorophyllin. The probiotic synergistic composition comprises bifidobacterium, lactobacillus plantarum, mucus bacillus and prevotella, the anti-inflammatory synergist comprises a curcumin-cyclodextrin inclusion compound, quercetin nanoparticles, a resveratrol-phospholipid complex and epigallocatechin gallate, chlorophyll is kept stable in a gastric acid environment through a pH responsive nano encapsulation technology, and the anti-inflammatory effect of chlorophyll is improved. Targeted release is achieved in the intestinal environment, and the bioavailability is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of intestinal microecological regulators, specifically to a probiotic-chlorophyll composite intestinal microecological regulator based on nano-encapsulation technology, its preparation method, and its application. Background Technology

[0002] With changes in modern lifestyles and dietary structures, gut health issues are becoming increasingly prominent. The gut is not only the main site for digestion and absorption of nutrients, but also the body's largest immune organ and a habitat for microorganisms. Gut microbiota imbalance, impaired intestinal barrier function, and chronic inflammatory responses have been proven to be closely related to a variety of diseases, including inflammatory bowel disease, obesity, and metabolic syndrome.

[0003] Chlorophyll, a class of pigment compounds widely found in green plants, has shown great potential in promoting gut health in recent years. According to a study by Han Zheng et al. in "Chlorophyllin Modulates Gut Microbiota and Inhibits Intestinal Inflammation to Ameliorate Hepatic Fibrosis in Mice" (Frontiers in Physiology, 2018, 9:1671), the chlorophyll derivative sodium copper chlorophyllin can regulate the balance of gut microbiota, inhibit the growth of sulfate-reducing bacteria, thereby reducing the production of hydrogen sulfide in the gut and protecting the intestinal barrier function.

[0004] Furthermore, Subramoniam et al.'s study in "Chlorophyll Revisited: Anti-inflammatory Activities of Chlorophyll a and Inhibition of Expression of TNF-α Gene by the Same" (Inflammation, 2012, 35:959-966) confirmed that chlorophyll a has significant anti-inflammatory activity and can directly inhibit the expression of TNF-α gene induced by bacterial lipopolysaccharide.

[0005] Chinese patent applications CN112370449A and CN112493483A disclose the application of chlorophyll-rich spinach extract in improving metabolic disorders and non-alcoholic fatty liver disease, and in alleviating intestinal inflammation and barrier dysfunction, respectively. However, these existing technologies have the following limitations:

[0006] 1. Traditional chlorophyll preparations have poor stability in the acidic environment of the stomach and are mostly degraded into pheophytin before reaching the intestines, resulting in low bioavailability;

[0007] 2. A single ingredient is unlikely to effectively address gut health issues caused by multiple factors;

[0008] 3. Lacks a synergistic mechanism with probiotics;

[0009] 4. Chlorophyll obtained by traditional preparation methods is prone to color deterioration during processing, resulting in products with poor sensory qualities and short shelf life.

[0010] Therefore, developing a novel chlorophyll formulation that can overcome the above-mentioned shortcomings, improve its stability in the gastrointestinal environment, enhance its synergistic effect with probiotics, promote the proliferation of beneficial gut microbiota, and achieve multi-target regulation of gut health has important theoretical significance and application value. Summary of the Invention

[0011] The main objective of this invention is to provide a probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology, its preparation method, and its application. This invention overcomes the shortcomings of existing chlorophyll preparations, such as poor stability, low bioavailability, and limited effects of single components. Through innovative formulation design and preparation process, it achieves the synergistic effect of chlorophyll, probiotics, and anti-inflammatory components, thereby directionally regulating the intestinal microecology and comprehensively promoting intestinal health.

[0012] To achieve the above objectives, the present invention provides a formulation of a probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology. By weight, the formulation comprises: 55-90 parts of a chlorophyll complex stable under acidic conditions, 15-35 parts of a probiotic synergistic composition, 15-30 parts of an anti-inflammatory synergist, and 5-10 parts of excipients.

[0013] The acid-stable chlorophyll complex includes chlorophyll a, chlorophyll b, sodium copper chlorophyll, and sodium magnesium chlorophyll; the probiotic synergistic composition includes Bifidobacterium, Lactobacillus plantarum, Myxobacterium, and Prevotella; and the anti-inflammatory synergist includes curcumin-cyclodextrin inclusion complex, quercetin nanoparticles, resveratrol-phospholipid complex, and epigallocatechin gallate.

[0014] Preferably, the acid-stable chlorophyll complex comprises, by weight:

[0015] 20-30 parts chlorophyll a, 15-25 parts chlorophyll b, 5-10 parts sodium copper chlorophyll, 5-10 parts sodium magnesium chlorophyll, and 10-15 parts pH-responsive nano-encapsulation matrix.

[0016] Preferably, the probiotic synergistic composition comprises, by weight:

[0017] 4-9 samples of Bifidobacterium longum (live count not less than 10) 10 CFU / g), 4-9 samples of Lactobacillus plantarum (live count not less than 10). 10 CFU / g), 3-5 samples of Akkermansia myxophilus (viable count not less than 10). 9 CFU / g), 2.5-7 samples of Prevotella (live count not less than 10). 9 (CFU / g) and 3-9 parts inulin matrix.

[0018] Preferably, the anti-inflammatory synergist comprises, by weight: 5-8 parts curcumin-cyclodextrin inclusion complex, 4-7 parts quercetin nanoparticles, 3-5 parts resveratrol-phospholipid complex, and 3-5 parts epigallocatechin gallate.

[0019] Preferably, the excipients comprise, by weight, 2-4 parts enteric-coated polymer, 1-2 parts pH-dependent release agent, 1-2 parts stabilizer, and 1-2 parts flow agent.

[0020] This invention also provides a method for preparing the above-mentioned probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology, comprising the following steps:

[0021] a) Preparation of chlorophyll complexes stable under acidic conditions: including extraction and purification, stabilization treatment and nano-encapsulation;

[0022] b) Preparation of probiotic synergistic compositions: including strain culture and collection, freeze-drying, and multi-layer encapsulation;

[0023] c) Preparation of anti-inflammatory synergists: including the preparation of curcumin-cyclodextrin inclusion complex, quercetin nanoparticles, resveratrol-phospholipid complex, and treatment of epigallocatechin gallate ester;

[0024] d) The acid-stable chlorophyll complex, the probiotic synergistic composition, the anti-inflammatory synergist, and excipients are mixed to obtain the probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology.

[0025] In one specific embodiment of the present invention, the specific steps for preparing the acid-stable chlorophyll complex in step a) include:

[0026] a1) Wash and chop spinach, lettuce and celery in a weight ratio of 1:1:1, add 95% ethanol and extract at 2-6℃ for 120-150 minutes;

[0027] a2) Centrifuge at 7800-8200g for 8-12 minutes, collect the supernatant, and concentrate the supernatant to 23-27% of the original volume;

[0028] a3) Place at 2-6℃ for 10-14 hours and collect the dark green precipitate;

[0029] a4) Dissolve the precipitate in a phospholipid solution, mix in a 1:2 ratio, add 0.5% copper chloride and 0.5% magnesium chloride solution, adjust the pH to 7.2-7.6, and heat at 68-72℃ for 28-32 minutes;

[0030] a5) Prepare oil and aqueous phases, and homogenize at high speed (18000-22000 rpm) to prepare emulsion;

[0031] a6) Add pH-responsive polymer solution and spray dry (inlet temperature 155-165℃, outlet temperature 75-85℃) to obtain the chlorophyll complex that is stable under acidic conditions.

[0032] Preferably, the specific steps for preparing the probiotic synergistic composition in step b) include:

[0033] b1) Culture Bifidobacterium longum, Lactobacillus plantarum, Akkermansia muciniphila and Prevotella to the late logarithmic growth stage respectively;

[0034] b2) Collect bacterial cells by centrifugation at 4800-5200g and wash twice with sterile phosphate buffer;

[0035] b3) Resuspend the bacterial cell mass in a low-temperature protective medium, adjust the bacterial concentration, and freeze-dry.

[0036] b4) Prepare an inulin solution (10%, dissolved at 58-62℃), cool to 38-42℃, and add freeze-dried probiotic powder;

[0037] b5) Spray freeze-drying forms primary microparticles, which are then alternately encapsulated with sodium alginate (1.0-1.2%) and chitosan (0.4-0.6%).

[0038] b6) Crosslink with calcium chloride solution (2.0-2.2%), wash and freeze dry to obtain the probiotic synergistic composition.

[0039] In another specific embodiment of the present invention, the specific steps for preparing the anti-inflammatory synergist in step c) include:

[0040] c1) Preparation of curcumin-cyclodextrin inclusion complex: Dissolve β-cyclodextrin (1%) in deionized water, heat to 38-42℃, add curcumin ethanol solution (1%) dropwise at a rate of 1-2 mL / min, stir continuously for 3-4 hours, cool to 5-10℃, collect the precipitate and freeze dry;

[0041] c2) Preparation of quercetin nanoparticles: Quercetin was dissolved in ethanol, and an aqueous phase containing polysorbate 80 was added. The mixture was homogenized at high speed to form a nanoemulsion, the organic solvent was removed by rotary evaporation, and the mixture was freeze-dried.

[0042] c3) Preparation of resveratrol-phospholipid complex: Resveratrol and soybean phospholipids were dissolved in dichloromethane at a ratio of 1:2, the solvent was evaporated to form a film, hydrated with phosphate buffer (pH 7.2-7.6), and lyophilized.

[0043] c4) Epigallocatechin gallate treatment: Epigallocatechin gallate is dissolved in deionized water, antioxidants and stabilizers are added, and the mixture is stirred evenly and then spray-dried.

[0044] This invention also provides the application of the above-mentioned probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology in the preparation of drugs or functional foods for preventing or improving intestinal inflammation, regulating intestinal flora imbalance, and improving impaired intestinal barrier function.

[0045] The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology provided by this invention has the following significant technical effects:

[0046] 1. Enhanced stability and bioavailability of chlorophyll in the gastrointestinal tract: Through pH-responsive nano-encapsulation technology, chlorophyll remains stable in the acidic environment of the stomach and is released in a targeted manner under the pH conditions of the intestine, significantly improving its bioavailability. Experiments show that the chlorophyll complex of this invention, stable under acidic conditions, retains more than 90% stability after 2 hours in simulated gastric juice (pH 1.2), while the stability of traditional chlorophyll extracts under acidic conditions is less than 20%.

[0047] 2. Enhanced survival rate and colonization ability of probiotics: Multi-layer encapsulation technology protects probiotics from damage by gastric acid and bile salts. Simultaneously, the selected probiotic strains synergistically enhance the effects of chlorophyll, jointly regulating the intestinal flora. Experiments have shown that the probiotic synergistic composition of this invention retains over 80% probiotic activity after 24 months of storage at room temperature, and the survival rate exceeds 70% after treatment under simulated gastrointestinal conditions.

[0048] 3. Multi-target anti-inflammatory effect: Through a specially prepared anti-inflammatory synergist, combined with the anti-inflammatory effect of chlorophyll itself, a multi-target anti-inflammatory network is formed to jointly inhibit intestinal inflammatory response. In vitro experiments show that the formulation of this invention can reduce the expression of TNF-α, IL-6 and IL-1β in intestinal epithelial cells by 65-75%, while chlorophyll alone can only reduce it by 30-40%.

[0049] 4. Comprehensive regulation of intestinal barrier function: The formula of this invention can improve intestinal permeability, upregulate the expression of intestinal tight junction proteins (ZO-1, Occludin, Claudin-1), reduce the concentration of LPS in serum, and significantly improve the intestinal barrier function damage caused by high-fat diet.

[0050] 5. Applicable to multiple dosage forms and highly stable: The preparation method of this invention allows the final product to be formulated into various dosage forms (capsules, tablets, granules, etc.), significantly improving product color stability and extending shelf life. Accelerated stability tests show that the formulation of this invention, stored at 40℃ / 75% relative humidity for 6 months, maintains a content of over 90% of each active ingredient, with uniform and stable color. Detailed Implementation

[0051] The technical solution of the present invention will be described in detail below with reference to specific embodiments. Those skilled in the art should understand that these embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0052] All raw materials used below are commercially available products or prepared according to the methods described below. Spinach, lettuce, and celery used in the experiment were purchased from local supermarkets to ensure freshness; ethanol, dichloromethane, and other organic solvents were all analytical grade or higher; *Bifidobacterium longum* (ATCC 15707), *Lactobacillus plantarum* (ATCC 8014), *Akermansia muciniphila* (ATCC BAA-835), and *Prevotella* (ATCC 27768) strains were purchased from the American Type Culture Collection (ATCC); soybean lecithin (Lipoid S100, 98% purity) was purchased from Lipoid GmbH, Germany; Eudragit® S100 was purchased from Evonik Industries AG, Germany; β-cyclodextrin (pharmaceutical grade, 98-102% purity) was purchased from Sigma-Aldrich; curcumin (≥95% purity, Curcumin C3)... Complex® was purchased from Sabinsa, India; quercetin (≥98% purity) was purchased from Sigma-Aldrich; resveratrol (≥98% purity) was purchased from Sigma-Aldrich; epigallocatechin gallate (≥94% purity, Sunphenon® 90DCF) was purchased from Taiyo International, Japan.

[0053] Example 1: Preparation of a probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology

[0054] 1.1 Preparation of chlorophyll complexes stable under acidic conditions

[0055] (1) Chlorophyll Extraction. Prepare fresh spinach, lettuce, and celery in a 1:1:1 weight ratio (1000g each), wash and drain them, and cut them into small pieces of about 0.5cm. Put the chopped vegetable materials into a blender, add 3000ml of 95% ethanol, and extract at 4°C. To prevent light from degrading chlorophyll, wrap the container with aluminum foil during the extraction process and operate under nitrogen protection. The extraction time is controlled at 130 minutes, during which slow stirring (70 rpm) is maintained.

[0056] After extraction, the mixture was transferred to centrifuge tubes and centrifuged at 8000g for 10 minutes at 4°C. The supernatant was collected and filtered through gauze. The remaining vegetable material was extracted twice more with 1500 mL of fresh ethanol, and all supernatants were combined. The combined supernatant was placed in a rotary evaporator and concentrated under reduced pressure to 25% of its original volume at 36°C and 20 mmHg, while maintaining a rotation speed of 60 rpm.

[0057] The concentrated extract was transferred to a sealed glass container and allowed to stand at 4°C in the dark for 12 hours. A dark green precipitate formed at the bottom, with a yellow liquid on top. The supernatant was carefully decanted, and the dark green precipitate was collected and filtered through a 0.45 μm cellulose acetate membrane at 4°C. The precipitate was washed twice with pre-cooled 70% ethanol solution (80 mL) and then vacuum dried at 22°C and 8 mmHg for 7 hours. The dried chlorophyll extract was stored in a nitrogen-filled amber glass container at -20°C.

[0058] (2) Stabilization treatment of chlorophyll. First, a phospholipid solution was prepared by dissolving 200 g of soybean phospholipid (Lipoid S100) in 1000 mL of dichloromethane and stirring at room temperature for 35 minutes until completely dissolved. Then, 100 g of chlorophyll extract was added to the phospholipid solution and stirred in the dark for 55 minutes. To partially convert to sodium copper chlorophyll and sodium magnesium chlorophyll, a solution of 5 g of copper chloride hexahydrate dissolved in 100 mL of deionized water and a solution of 5 g of magnesium chloride hexahydrate dissolved in 100 mL of deionized water were prepared and slowly added dropwise to the chlorophyll-phospholipid mixture, and stirring was continued for 65 minutes. Subsequently, an 8% sodium bicarbonate solution (80 g dissolved in 1000 mL of deionized water) was prepared and added dropwise to the mixture to adjust the pH to 7.4 (monitored with a pH meter), and stirring was continued for 40 minutes.

[0059] The mixture was transferred to a jacketed reactor with continuous nitrogen purging and slowly heated to 70°C at a rate of 1.5°C / min, held at this temperature for 30 minutes while maintaining a stirring speed of 70 rpm. It was then rapidly cooled to 20°C using a cooling water jacket at a cooling rate of 2.5°C / min. The mixture was transferred to a rotary evaporator and subjected to removal of dichloromethane at 38°C and 20 mmHg until complete removal was achieved (confirmed by constant weight). Finally, it was redispersed with 500 mL of deionized water while stirring.

[0060] (3) Nano-encapsulation treatment. First, prepare the oil phase by dissolving 25 g of medium-chain triglycerides (Miglyol® 812) in 200 mL of ethyl acetate and adding the stabilized chlorophyll complex described above. Prepare the aqueous phase by dissolving 10 g of polysorbate 80 (Tween® 80) in 500 mL of deionized water. Under high-speed homogenization (20,000 rpm), the oil phase is added dropwise to the aqueous phase at a rate of 3 mL / min, and homogenization is continued for 5 minutes after complete addition.

[0061] In addition, to prepare a pH-responsive polymer solution, dissolve 100 g of Eudragit® S100 in 500 mL of 95% ethanol, add 40 g of triethyl citrate as a plasticizer, and stir for 40 minutes until completely dissolved. Slowly add this polymer solution dropwise to the above emulsion at a rate of 4 mL / min, while maintaining a stirring speed of 500 rpm, and continue stirring for 75 minutes to ensure uniform mixing.

[0062] The mixture was transferred to a rotary evaporator and subjected to ethyl acetate and ethanol removal at 38°C and 35 mmHg until the volume decreased by approximately 45%. Then, it was dried using a spray dryer with an inlet temperature of 160°C, an outlet temperature of 80°C, a feed rate of 6 mL / min, and atomization pressure of 2.0 bar. The spray-dried powder was collected, and any aggregates were removed by passing it through a 100-mesh sieve. The powder was then vacuum-dried at 33°C and 8 mmHg for 3.5 hours. The residual solvent content was checked (ethanol and ethyl acetate content should both be below 0.5%). Finally, the product was stored in a sealed container containing a desiccant at 4°C.

[0063] 1.2 Preparation of probiotic synergistic composition

[0064] (1) Culture and collection of strains. For Bifidobacterium longum (ATCC 15707), MRS broth medium containing 0.05% cysteine ​​hydrochloride was prepared, the pH was adjusted to 6.3, and autoclaved at 121℃ for 15 minutes. Bifidobacterium longum was inoculated into the medium at an inoculum size of 2% and cultured at 37℃ for 18 hours under anaerobic conditions (85% N2, 10% CO2, 5% H2). The growth was monitored by measuring the absorbance at 600 nm (target OD value: 0.9). The strains were collected when they reached the late logarithmic growth stage.

[0065] For Lactobacillus plantarum (ATCC 8014), MRS broth medium (pH 6.5) was prepared, autoclaved at 121°C for 15 minutes, and then inoculated at a 2% inoculum. The medium was cultured at 36°C for 16 hours under microaerophilic conditions (5% CO2). The medium was collected when the absorbance at 600 nm reached the target OD value of 1.1.

[0066] For Akkermansia myxophilus (ATCC BAA-835), a brain and heart extract broth supplemented with 0.5% mucus (porcine gastric type III, Sigma-Aldrich) was prepared, and the pH was adjusted to 6.6. The broth was then sterilized by filtration through a 0.22 μm filter. Inoculation was performed at a 3% inoculum, and the broth was incubated anaerobically at 37°C for 42 hours. The broth was collected when the absorbance at 600 nm reached the target OD value of 0.5.

[0067] For Prevotella (ATCC 27768), YCFA medium (yeast extract, casein ketone, fatty acids) supplemented with 0.2% glucose was prepared, and the pH was adjusted to 6.8. The medium was then sterilized by filtration through a 0.22 μm filter membrane. Inoculation was performed at a 4% inoculum, and the medium was incubated anaerobically at 37°C for 22 hours. The medium was collected when the absorbance at 600 nm reached the target OD value of 0.6.

[0068] After the culture of each bacterial strain was completed, the bacterial cells were collected by centrifugation at 5000g for 15 minutes (4℃) and washed twice with sterile 0.1M phosphate buffer (pH 7.0). The bacterial cells were then resuspended in cryoprotective medium (10% skim milk powder and 5% trehalose), and the concentrations of Bifidobacterium longum and Lactobacillus plantarum were adjusted to 1:1. 11 CFU / mL, concentrations of Akkermansia myxophilus and Prevotella up to 10 10 CFU / mL.

[0069] The bacterial suspension was pre-frozen at -80°C for 2.5 hours, followed by lyophilization: primary drying was carried out at -32°C and 0.08 mbar for 21 hours, followed by secondary drying at 18°C ​​and 0.03 mbar for 7 hours. The viable bacterial count after lyophilization was determined; *Bifidobacterium longum* and *Lactobacillus plantarum* should be no less than 10. 10CFU / g, and the levels of Akkermansia myxophilus and Prevotella should not be less than 10. 9 CFU / g. Store the lyophilized powder in a sealed container at -20°C until use.

[0070] (2) Multilayer encapsulation treatment. First, prepare a high molecular weight inulin (Orafti® HP, average degree of polymerization ≥23) solution by dissolving 100 g of inulin in 1000 mL of deionized water, heating to 60°C and stirring for 12 minutes to ensure complete dissolution, then cooling to 40°C. Mix the freeze-dried probiotic powder according to the specified ratio, and stir at 125 rpm for 25 minutes at 40°C to ensure uniform dispersion. If necessary, adjust the pH to 7.0 with 1N NaOH.

[0071] The probiotic-inulin mixture was processed using spray freeze-drying technology: the mixture was sprayed into liquid nitrogen (-196°C) through a 0.25 mm diameter nozzle at an atomization pressure of 0.2 MPa. The frozen droplets were transferred to a freeze dryer and subjected to primary drying at -32°C and 0.08 mbar for 27 hours, followed by secondary drying at 12°C and 0.03 mbar for 9 hours. The primary particles were collected and stored at 4°C under nitrogen protection.

[0072] Prepare a medium-viscosity sodium alginate solution (1.1% w / v) by dissolving 11 g of sodium alginate in 1000 mL of deionized water and stirring for 3.5 hours until completely dissolved. Add 100 g of primary microparticles to the sodium alginate solution and gently stir at 90 rpm for 18 minutes. Spray the mixture through a 0.5 mm nozzle into a calcium chloride solution (2.1% w / v) while maintaining stirring. After standing for 25 minutes to allow gel formation, separate the sodium alginate-coated particles by filtration and wash twice with deionized water.

[0073] Prepare a chitosan solution (0.5% w / v): Dissolve 5 g of low molecular weight chitosan (molecular weight approximately 50-190 kDa, degree of deacetylation 75-85%) in 1000 mL of 1% acetic acid solution, and stir for 3.5 hours until completely dissolved. Adjust the pH to 5.3 with 1N NaOH. Add the sodium alginate-coated particles to the chitosan solution and gently stir at 90 rpm for 30 minutes. Separate the chitosan-coated particles by filtration and wash twice with deionized water.

[0074] The sodium alginate coating process was repeated to form a third layer, and the particles were stirred in a calcium chloride solution for 30 minutes to ensure complete cross-linking. The coated particles were thoroughly washed three times with deionized water, frozen at -80°C for 3.5 hours, and then lyophilized using the same parameters as the primary particles. Uniformity was ensured by passing the particles through a 60-mesh sieve, and the probiotic activity was tested (it should retain more than 80% of the initial activity). The probiotic synergistic composition was stored in a sealed container containing a desiccant at 4°C.

[0075] 1.3 Preparation of anti-inflammatory synergists

[0076] (1) Preparation of curcumin-cyclodextrin inclusion complex. 100 g of β-cyclodextrin (pharmaceutical grade, ≥98% purity) was dissolved in 1000 mL of deionized water and stirred continuously at 250 rpm for about 40 minutes at 40°C to ensure complete dissolution. In addition, 10 g of curcumin (≥95% purity, Curcumin C3 Complex®) was dissolved in 200 mL of 95% ethanol and stirred at 125 rpm for about 18 minutes at room temperature until completely dissolved. Any undissolved particles were removed by filtration through a 0.45 μm filter membrane.

[0077] The curcumin solution was added dropwise to the β-cyclodextrin solution at a rate of 1.5 mL / min, maintaining the temperature at 40 °C. After complete addition, stirring was continued at 175 rpm for 3.5 h. The pH was adjusted to 5.3 using 0.1 N HCl. The mixture was then slowly cooled to 8 °C and allowed to stand at this temperature for 11 h to allow precipitation. The precipitate was collected by filtration through a 0.45 μm filter membrane, washed twice with 75 mL of pre-cooled (8 °C) deionized water, and then once with 75 mL of pre-cooled (8 °C) 20% ethanol.

[0078] The collected complex was vacuum dried at 42°C and 12 mmHg for 11 hours. The dried complex was then ground and passed through an 80-mesh sieve. The inclusion efficiency (target: 10% curcumin content) was determined by HPLC, and the formation of the complex was confirmed by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). The product was stored in an amber container containing a desiccant at room temperature.

[0079] (2) Preparation of quercetin nanoparticles. Dissolve 10 g of quercetin (≥95% purity) in 200 mL of 95% ethanol, and stir continuously at 250 rpm for about 18 minutes at 48°C to ensure complete dissolution, then cool to room temperature. Prepare the aqueous phase by dissolving 5 g of polysorbate 188 (Kolliphor® P 188) in 500 mL of deionized water, and stir at 350 rpm for about 40 minutes at room temperature until completely dissolved. Adjust the pH to 7.0 with 0.1N NaOH.

[0080] Using a syringe pump, add the quercetin solution to the polysorbate solution at a rate of 2.5 mL / min while maintaining high-speed homogenization at 20,000 rpm. After complete addition, continue homogenization for 10 minutes. Transfer the mixture to a magnetic stirrer and stir at 450 rpm. Remove ethanol under reduced pressure at 38°C and 30 mmHg until the ethanol content is below 0.5% (verified by gas chromatography). Adjust to the original volume with deionized water if necessary.

[0081] Add 5% trehalose as a cryoprotectant, mix thoroughly for 18 minutes, and filter through a 0.45 μm filter membrane. Pre-freeze the mixture at -80°C for 3.5 hours, then lyophilize: primary drying at -32°C and 0.08 mbar for 27 hours, and secondary drying at 18°C ​​and 0.03 mbar for 9 hours. Particle size and polydispersity index (target: 150 nm, PDI < 0.3) were determined using dynamic light scattering, zeta potential (target: -25 mV) was measured, and encapsulation efficiency (target: > 85%) was determined by HPLC. Store the product in a sealed container containing a desiccant at 4°C.

[0082] (3) Preparation of resveratrol-phospholipid complex. Dissolve 10 g of trans-resveratrol (≥98% purity) in 200 mL of dichloromethane and stir at 125 rpm for about 18 minutes at room temperature until completely dissolved. Separately, dissolve 20 g of soybean phosphatidylcholine (Phospholipon® 90G, ≥94% purity) in 200 mL of dichloromethane and stir at 125 rpm for about 18 minutes at room temperature until completely dissolved.

[0083] The resveratrol and phospholipid solutions were mixed and stirred at 175 rpm for 75 minutes at room temperature. The mixture was transferred to a rotary evaporator and the solvent was removed at 40°C and 20 mmHg until a thin film formed on the flask wall. 500 mL of phosphate buffer (0.1 M, pH 7.4) preheated to 40°C was added, and the film was gently stirred at 90 rpm for 75 minutes at 40°C to hydrate it. The mixture was ultrasonically bathed in water for 12 minutes and then filtered through a 0.45 μm filter membrane.

[0084] The mixture was pre-frozen at -80°C for 3.5 hours, followed by lyophilization: primary drying at -32°C and 0.08 mbar for 27 hours, and secondary drying at 18°C ​​and 0.03 mbar for 9 hours. The formation of the complex was confirmed using DSC and FTIR, and the resveratrol content (target: 20%) was determined by HPLC. The product was stored in a sealed container with a desiccant at 4°C.

[0085] (4) Treatment of epigallocatechin gallate. Dissolve 10 g of epigallocatechin gallate (≥94% purity, Sunphenon® 90DCF) in 200 mL of deionized water, adjust the pH to 5.3, and stir at 125 rpm for about 18 minutes at room temperature until completely dissolved. Add 1 g of ascorbic acid (USP grade) as an antioxidant and 5 g of trehalose dihydrate as a stabilizer, stir at 175 rpm for about 40 minutes at room temperature, and then filter through a 0.45 μm filter membrane.

[0086] Drying was performed using a spray dryer with an inlet temperature of 145°C, an outlet temperature of 75°C, a feed rate of 5 mL / min, and an atomization pressure of 2.0 bar. The spray-dried powder was collected, and the moisture content (target: <3%) was determined. The EGCG content (target: >90% of theoretical content) was determined by HPLC, and the antioxidant activity was determined using the DPPH method. The product was stored in a sealed container containing a desiccant at 4°C.

[0087] 1.4 Preparation of the final formulation

[0088] Perform all operations in a controlled environment (temperature 22°C, relative humidity 30%), and use a nitrogen atmosphere whenever possible to prevent oxidation. Weigh each component according to the following formula ratio: 72.5 parts of acid-stable chlorophyll complex (containing 25 parts of chlorophyll a, 20 parts of chlorophyll b, 7.5 parts of sodium copper chlorophyll, 7.5 parts of sodium magnesium chlorophyll and 12.5 parts of pH-responsive nano-encapsulation matrix), 27 parts of probiotic synergistic composition (containing 6.5 parts of Bifidobacterium longum, 6.5 parts of Lactobacillus plantarum, 4 parts of Akkermansia myxophilus, 4 parts of Prevotella and 6 parts of inulin matrix), 20 parts of anti-inflammatory synergist (containing 6.5 parts of curcumin-cyclodextrin inclusion complex, 5.5 parts of quercetin nanoparticles, 4 parts of resveratrol-phospholipid complex and 4 parts of epigallocatechin gallate), and 7.5 parts of excipients (containing 3 parts of hydroxypropyl methylcellulose phthalate, 1.5 parts of methacrylate-methyl methacrylate copolymer (1:2), 1.5 parts of hydroxypropyl methylcellulose and 1.5 parts of colloidal silica).

[0089] All components were pre-sieved through a 60-mesh sieve to remove any aggregates. Half of the flow agent (0.75 parts colloidal silica) was added to the mixing container. The chlorophyll complex, which is stable under acidic conditions, was added sequentially and mixed at 18 rpm for 6 minutes in a V-mixer; the anti-inflammatory synergist component was added and mixed for another 6 minutes; the probiotic synergist composition was added and mixed for another 6 minutes; finally, the remaining excipients and flow agent were added and mixed for another 12 minutes to ensure homogeneity.

[0090] Take 10 samples from different locations in the mixture and test the content uniformity (relative standard deviation should be less than 5%), moisture content (target: <3%), and probiotic activity (should maintain more than 90% of the initial activity).

[0091] The well-mixed powder was filled into 00-size hydroxypropyl methylcellulose capsules (DRcaps™, Lonza), with each capsule weighing 700 mg. Capsule filling was carried out at 22°C and 35% relative humidity using a capsule filling machine at 50% of its maximum speed. Process control checks (weight variation, disintegration time, etc.) were performed every 30 minutes.

[0092] Example 2: Preparation of a medium-dose probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology

[0093] This embodiment uses a method similar to that of Example 1, but the proportions of each component are adjusted. The specific formulation is as follows: 64 parts of acid-stable chlorophyll complex (containing 22 parts of chlorophyll a, 18 parts of chlorophyll b, 6.5 parts of sodium copper chlorophyll, 6.5 parts of sodium magnesium chlorophyll and 11 parts of pH-responsive nano-encapsulation matrix), 27 parts of probiotic synergistic composition (containing 6.5 parts of Bifidobacterium longum, 6.5 parts of Lactobacillus plantarum, 4 parts of Akkermansia myxophilus, 4 parts of Prevotella and 6 parts of inulin matrix), 22 parts of anti-inflammatory synergist (containing 7 parts of curcumin-cyclodextrin inclusion complex, 6 parts of quercetin nanoparticles, 4.5 parts of resveratrol-phospholipid complex and 4.5 parts of epigallocatechin gallate), and 8 parts of excipients (containing 3.2 parts of hydroxypropyl methylcellulose phthalate, 1.6 parts of methacrylate-methyl methacrylate copolymer (1:2), 1.6 parts of hydroxypropyl methylcellulose and 1.6 parts of colloidal silica).

[0094] The preparation process was the same as in Example 1, but when preparing the chlorophyll complex stable under acidic conditions, the extraction temperature of the plant material was controlled at 3°C, and the extraction time was extended to 140 minutes. In the preparation of the probiotic synergistic composition, the culture times of *Bifidobacterium longum* and *Lactobacillus plantarum* were adjusted to 19 hours and 17 hours, respectively. In the preparation of the anti-inflammatory synergist, the stirring time of the curcumin-cyclodextrin inclusion complex was extended to 4 hours, and the high-speed homogenization time of the quercetin nanoparticles was increased to 11 minutes.

[0095] The final product is made into tablets, each weighing 700 mg. During tablet preparation, 2% microcrystalline cellulose (Avicel® PH-102) is added as an additional binder and 0.8% magnesium stearate as a lubricant, and the mixture is stirred for 4 minutes. Tableting is performed using a pressure of 10 kN, and the tablet hardness is controlled at 5 kg.

[0096] Example 3: Preparation of a high-dose probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology

[0097] This embodiment uses a higher concentration of active ingredients, and the formulation is as follows: 90 parts of an acid-stable chlorophyll complex (containing 30 parts of chlorophyll a, 25 parts of chlorophyll b, 10 parts of sodium copper chlorophyll, 10 parts of sodium magnesium chlorophyll and 15 parts of pH-responsive nano-encapsulation matrix), 20 parts of a probiotic synergistic composition (containing 5 parts of Bifidobacterium longum, 5 parts of Lactobacillus plantarum, 3 parts of Akkermansia myxotroph, 3 parts of Prevotella myxotroph and 4 parts of inulin matrix), 15 parts of an anti-inflammatory synergist (containing 5 parts of curcumin-cyclodextrin inclusion complex, 4 parts of quercetin nanoparticles, 3 parts of resveratrol-phospholipid complex and 3 parts of epigallocatechin gallate), and 5 parts of excipients (containing 2 parts of hydroxypropyl methylcellulose phthalate, 1 part of methacrylate-methyl methacrylate copolymer (1:2), 1 part of hydroxypropyl methylcellulose and 1 part of colloidal silica).

[0098] During the preparation process, a lower extraction temperature of 2°C and a longer extraction time of 150 minutes were used to maximize the extraction efficiency of chlorophyll in the acid-stable chlorophyll complex. During spray drying, the inlet temperature was increased to 165°C and the outlet temperature to 85°C to improve drying efficiency. A higher concentration of cryoprotectant (12% skim milk powder and 6% trehalose) was used in the preparation of the probiotic synergistic composition. In the preparation of the anti-inflammatory synergist, the ratio of curcumin to β-cyclodextrin was adjusted to 1:12 (molar ratio) to improve inclusion efficiency.

[0099] The final product is made into enteric-coated tablets. After the tablets are prepared, the coating solution is prepared: 100 g of Eudragit® L100-55 is dissolved in 700 mL of 95% ethanol, 200 mL of deionized water is added, 10 g of triethyl citrate is added as a plasticizer and 5 g of talc is added as an anti-sticking agent, and the mixture is stirred at 350 rpm for about 75 minutes until completely dissolved, and then filtered through a 100-mesh sieve.

[0100] Preheat the tablets in the coating pan to 38°C, setting the inlet air temperature to 42°C, the outlet air temperature to 38°C, and the pan rotation speed to 15 rpm. Add the coating solution at a rate of 5 g / min / kg tablets until a 9% weight gain is achieved. Dry the coated tablets in the coating pan for 12 minutes, then transfer them to a tray dryer and cure at 40°C for 2 hours, followed by cooling to room temperature.

[0101] Perform disintegration tests: The coating should not disintegrate in 0.1N hydrochloric acid for 2 hours, and should disintegrate within 45 minutes in pH 6.8 phosphate buffer. Analyze the dissolution profile using UV spectrophotometry and check the coating uniformity using scanning electron microscopy.

[0102] Example 4: Preparation of a low-dose probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology

[0103] This embodiment provides a low-dose formulation suitable for daily health maintenance needs. The formulation consists of: 55 parts of an acid-stable chlorophyll complex (containing 20 parts of chlorophyll a, 15 parts of chlorophyll b, 5 parts of sodium copper chlorophyll, 5 parts of sodium magnesium chlorophyll and 10 parts of a pH-responsive nano-encapsulation matrix), 34 parts of a probiotic synergistic composition (containing 8 parts of Bifidobacterium longum, 8 parts of Lactobacillus plantarum, 5 parts of Akkermansia myxotroph, 5 parts of Prevotella myxotroph and 8 parts of inulin matrix), 20 parts of an anti-inflammatory synergist (containing 6 parts of curcumin-cyclodextrin inclusion complex, 5 parts of quercetin nanoparticles, 4.5 parts of resveratrol-phospholipid complex and 4.5 parts of epigallocatechin gallate), and 10 parts of excipients (containing 4 parts of hydroxypropyl methylcellulose phthalate, 2 parts of methacrylate-methyl methacrylate copolymer (1:2), 2 parts of hydroxypropyl methylcellulose and 2 parts of colloidal silica).

[0104] The preparation process was basically the same as in Example 1, but milder extraction conditions were used in the preparation of the chlorophyll complex stable under acidic conditions: a temperature of 5°C and a time of 120 minutes. During nano-encapsulation, the polymer concentration was reduced to 8% to decrease encapsulation efficiency, suitable for low-dose products. In the preparation of the probiotic synergistic composition, the concentrations of sodium alginate and chitosan were increased (1.2% and 0.6%, respectively) to improve the protective effect. A lower processing temperature was used in the preparation of the anti-inflammatory synergist to maintain the stability of the active ingredients.

[0105] The final product is made into granules, each 2 grams, equivalent to the dose of two capsules. The mixed powder is granulated by passing it through a 20-mesh sieve, dried at 55°C until the moisture content is less than 2%, cooled, and then packaged into single-dose aluminum foil bags.

[0106] Example 5: Preparation of a probiotic-chlorophyll complex regulator for intestinal inflammation

[0107] This embodiment provides a formulation specifically targeting intestinal inflammation, with an increased proportion of anti-inflammatory components. The formulation consists of: 62 parts of an acid-stable chlorophyll complex (containing 22 parts of chlorophyll a, 17 parts of chlorophyll b, 6 parts of sodium copper chlorophyll, 6 parts of sodium magnesium chlorophyll, and 11 parts of a pH-responsive nano-encapsulation matrix), 20 parts of a probiotic synergistic composition (containing 5 parts of Bifidobacterium longum, 5 parts of Lactobacillus plantarum, 3 parts of Akkermansia myxotroph, 3 parts of Prevotella myxotroph, and 4 parts of inulin matrix), 25 parts of an anti-inflammatory synergist (containing 8 parts of curcumin-cyclodextrin inclusion complex, 7 parts of quercetin nanoparticles, 5 parts of resveratrol-phospholipid complex, and 5 parts of epigallocatechin gallate), and 10 parts of excipients (containing 4 parts of hydroxypropyl methylcellulose phthalate, 2 parts of methacrylate-methyl methacrylate copolymer (1:2), 2 parts of hydroxypropyl methylcellulose, and 2 parts of colloidal silica).

[0108] During the preparation process, the preparation process of the anti-inflammatory synergist was specifically optimized. In the preparation of the curcumin-cyclodextrin inclusion complex, the concentration of β-cyclodextrin was increased to 1.2%, and the stirring time was extended to 4.5 hours to improve the inclusion efficiency. In the preparation of quercetin nanoparticles, 0.2% poloxamer 407 was added as an additional stabilizer to enhance the stability of the nanoparticles. In the preparation of the resveratrol-phospholipid complex, the ratio of phospholipid to resveratrol was increased to 2.5:1 to improve the inclusion rate and stability.

[0109] The final product is made into enteric-coated capsules, each containing 700 mg. Using a specialized enteric-coated capsule shell (Vcaps® Enteric, Capsule Company), no additional coating is required, allowing the contents to be released directly into the small intestine environment (pH>6.8).

[0110] Example 6: Preparation of a probiotic-chlorophyll complex regulator for gut microbiota balance

[0111] This embodiment provides a formulation specifically designed for gut microbiota balance, enhancing the proportion of probiotic components. The formulation consists of: 55 parts of an acid-stable chlorophyll complex (containing 20 parts of chlorophyll a, 15 parts of chlorophyll b, 5 parts of sodium copper chlorophyll, 5 parts of sodium magnesium chlorophyll, and 10 parts of a pH-responsive nano-encapsulation matrix), 41 parts of a probiotic synergistic composition (containing 9 parts of Bifidobacterium longum, 9 parts of Lactobacillus plantarum, 7 parts of Akkermansia myxotroph, 7 parts of Prevotella myxotroph, and 9 parts of inulin matrix), 15 parts of an anti-inflammatory synergist (containing 5 parts of curcumin-cyclodextrin inclusion complex, 4 parts of quercetin nanoparticles, 3 parts of resveratrol-phospholipid complex, and 3 parts of epigallocatechin gallate), and 10 parts of excipients (containing 4 parts of hydroxypropyl methylcellulose phthalate, 2 parts of methacrylate-methyl methacrylate copolymer (1:2), 2 parts of hydroxypropyl methylcellulose, and 2 parts of colloidal silica).

[0112] During the preparation process, the preparation technology of the probiotic synergistic composition was specifically optimized. In strain cultivation, an optimized culture medium formulation was used, with 0.2% fructooligosaccharides added to *Bifidobacterium longum* and 0.1% yeast extract added to *Lactobacillus plantarum* to increase cell yield. During the multi-layer encapsulation process, a layer of polylysine (0.1%) was added between sodium alginate and chitosan to enhance tolerance to bile salts. Furthermore, 1% galactooligosaccharides were added to the inulin matrix as an additional prebiotic.

[0113] The final product is made into oral suspension granules, each sachet containing 3 grams, which should be suspended in 100 ml of water before use. The granules are prepared using fluidized bed granulation technology, and 1% xanthan gum is added as a suspending agent and 0.5% steviol glycosides are added as a sweetener to improve taste and ease of use.

[0114] Example 7: Preparation of a probiotic-high chlorophyll complex regulator

[0115] This embodiment provides a formulation with a higher chlorophyll content, suitable for individuals requiring a more potent chlorophyll effect. The formulation consists of: 90 parts of an acid-stable chlorophyll complex (containing 30 parts of chlorophyll a, 25 parts of chlorophyll b, 10 parts of sodium copper chlorophyll, 10 parts of sodium magnesium chlorophyll, and 15 parts of a pH-responsive nano-encapsulation matrix), 16 parts of a probiotic synergistic composition (containing 4 parts of Bifidobacterium longum, 4 parts of Lactobacillus plantarum, 2.5 parts of Akkermansia muciniphila, 2.5 parts of Prevotella, and 3 parts of inulin matrix), 15 parts of an anti-inflammatory synergist (containing 5 parts of curcumin-cyclodextrin inclusion complex, 4 parts of quercetin nanoparticles, 3 parts of resveratrol-phospholipid complex, and 3 parts of epigallocatechin gallate), and 10 parts of excipients (containing 4 parts of hydroxypropyl methylcellulose phthalate, 2 parts of methacrylate-methyl methacrylate copolymer (1:2), 2 parts of hydroxypropyl methylcellulose, and 2 parts of colloidal silica).

[0116] In the preparation process, the preparation process of the chlorophyll complex stabilized under acidic conditions was specifically optimized. Ultrasonic-assisted extraction was used during extraction, with intermittent ultrasonication (3 seconds on / 2 seconds off) at 60% power for 30 minutes at 4°C, followed by conventional extraction for 120 minutes to improve chlorophyll extraction efficiency. During the stabilization process, in addition to copper and magnesium ions, a small amount of zinc ions (0.1% zinc chloride) was added to enhance chlorophyll stability. During nano-encapsulation, 2% vitamin E was added as an antioxidant to protect chlorophyll from oxidative damage.

[0117] The final product is formulated as a bilayer tablet, with a core containing a chlorophyll complex and an anti-inflammatory synergist, and an outer layer containing a probiotic synergist and some excipients. This design allows the probiotics to be released in the stomach, while the chlorophyll and anti-inflammatory components are released in the intestines, achieving a spatiotemporally separated delivery strategy. The core is enteric-coated, while the outer layer is uncoated, with each tablet weighing 800 mg.

[0118] Comparative Example 1: Chlorophyll formulation stabilized under acid-free conditions

[0119] This comparative example uses the same component ratio as Example 1, but the chlorophyll complex was not stabilized under acidic conditions or subjected to pH-responsive nano-encapsulation. The specific formulation is as follows: 50 parts of unstabilized chlorophyll complex (extracted directly from spinach, lettuce, and celery, without stabilization or nano-encapsulation), 25 parts of probiotic synergistic composition (formulation same as Example 1), 20 parts of anti-inflammatory synergist (formulation same as Example 1), and 7.5 parts of excipients (formulation same as Example 1).

[0120] The chlorophyll extraction process was the same as steps a1-a3 in Example 1, but the extract was then directly dried without stabilization or nano-encapsulation. The preparation of the remaining components and the mixing process of the final formulation were the same as in Example 1.

[0121] The final product was made into capsules, each containing 700 mg. During the preparation process, it was observed that the unstabilized chlorophyll complex was sensitive to light and oxygen, showing significant color changes even during handling, indicating poor stability of the chlorophyll extract.

[0122] Comparative Example 2: Formula without probiotic components

[0123] This comparative example uses a formulation similar to that of Example 1, but without the probiotic synergistic composition. The specific formulation is as follows: 60 parts of chlorophyll complex stable under acidic conditions (formulation same as Example 1), 30 parts of anti-inflammatory synergist (containing 10 parts of curcumin-cyclodextrin inclusion complex, 8 parts of quercetin nanoparticles, 6 parts of resveratrol-phospholipid complex and 6 parts of epigallocatechin gallate), and 10 parts of excipients (formulation same as Example 1).

[0124] The preparation process for the acid-stable chlorophyll complex and anti-inflammatory synergist was the same as in Example 1. The final product was made into capsules, each containing 700 mg.

[0125] Comparative Example 3: Formulation without anti-inflammatory synergists

[0126] This comparative example uses a formulation similar to that of Example 1, but without the anti-inflammatory synergist. The specific formulation is as follows: 60 parts of an acid-stable chlorophyll complex (formulation same as Example 1), 30 parts of a probiotic synergistic composition (containing 8 parts of Bifidobacterium longum, 8 parts of Lactobacillus plantarum, 5 parts of Akkermansia myxophilus, 5 parts of Prevotella myxobolus and 9 parts of inulin matrix), and 10 parts of excipients (formulation same as Example 1).

[0127] The preparation process for the acid-stable chlorophyll complex and probiotic synergistic composition was the same as in Example 1. The final product was made into capsules, each containing 700 mg.

[0128] To evaluate the efficacy of the formulation of this invention, a series of in vitro and in vivo experiments were conducted.

[0129] 1. Gastric acid stability test: The formulations of Examples 1-7 and Comparative Example 1 were incubated in simulated gastric juice (pH 1.2, containing pepsin) for 2 hours, and then the chlorophyll retention rate was measured. The results showed that the chlorophyll retention rate of Examples 1-7 was all above 90%, while the retention rate of Comparative Example 1 was only about 15%, proving that the stabilization treatment and nano-encapsulation technology under acidic conditions of the present invention significantly improved the stability of chlorophyll in the gastric acid environment.

[0130] 2. Chlorophyll Gastrointestinal Release Test: The formulations of Examples 1-7 and Comparative Example 1 were subjected to chlorophyll dissolution tests in simulated gastric fluid (pH 1.2, 2 hours) and simulated intestinal fluid (pH 6.8, 45 minutes), respectively. The results showed that the chlorophyll release rate of Examples 1-7 in simulated gastric fluid was less than 10%, while the release rate in the subsequent simulated intestinal fluid exceeded 90%, indicating good intestinal-targeted release characteristics of chlorophyll. Comparative Example 1 showed a chlorophyll release rate exceeding 80% in simulated gastric fluid, with almost no chlorophyll reaching the simulated intestinal fluid environment.

[0131] 3. Probiotic Activity Test: The formulations of Examples 1-7 and Comparative Examples 2 and 3 were treated under simulated gastrointestinal conditions (first in simulated gastric fluid at pH 1.2 for 2 hours, then in simulated intestinal fluid at pH 6.8 for 4 hours), and the survival rate of probiotics was measured. The results showed that the survival rate of probiotics in Examples 1-7 remained above 70%, while the survival rate of Comparative Example 3 (which contained probiotics but no anti-inflammatory synergist) was only about 50%, indicating that the anti-inflammatory synergist also played a positive role in protecting probiotics. Comparative Example 2 did not contain probiotics and therefore could not be tested.

[0132] 4. Evaluation of Anti-inflammatory Activity: Using a Caco-2 model of human intestinal epithelial cells stimulated with lipopolysaccharide (LPS), the inhibitory effects of the formulations in Examples 1-7 and Comparative Examples 1-3 on the expression of inflammatory cytokines TNF-α, IL-6, and IL-1β were tested. The results showed that Examples 1-7 exhibited an inhibition rate of 65-75% on the expression of these inflammatory cytokines, while Comparative Example 1 (without acid stabilization treatment) showed an inhibition rate of 45-55%, Comparative Example 2 (without probiotics) showed an inhibition rate of 50-60%, and Comparative Example 3 (without anti-inflammatory synergist) showed an inhibition rate of only 30-40%. This indicates a significant synergistic anti-inflammatory effect among the components in the formulations of this invention.

[0133] 5. Protective Effect on Intestinal Barrier Function: Using an LPS-induced Caco-2 cell monolayer permeability model, the protective effects of the formulations in Examples 1-7 and Comparative Examples 1-3 on intestinal barrier function were tested. Results showed that Examples 1-7 reduced the LPS-induced decrease in transepithelial electrical resistance (TEER) by 70-80% and reduced the increase in FITC-Dextran permeability by 75-85%. In comparison, the protective effects of Comparative Examples 1-3 were 50-60%, 45-55%, and 40-50%, respectively. Furthermore, Examples 1-7 significantly upregulated the expression of tight junction proteins (ZO-1, Occludin, Claudin-1), with effects significantly superior to the comparative examples.

[0134] 6. Evaluation of the efficacy of the high-fat diet mouse model: C57BL / 6N male mice were randomly divided into four groups: normal diet group (ND), high-fat diet group (HFD), high-fat diet + formulation of Example 1 group (HFD + Example 1), high-fat diet + comparative example 1 group (HFD + comparative example 1), high-fat diet + comparative example 2 group (HFD + comparative example 2), and high-fat diet + comparative example 3 group (HFD + comparative example 3). The high-fat diet was maintained for 12 weeks, and the corresponding formulation was administered by gavage (10 mg / kg per day based on body weight).

[0135] The results showed that, compared with the HFD group, the HFD+ Example 1 group exhibited significantly decreased expression of colonic inflammatory factors (TNF-α, IL-6, and IL-1β) (p<0.01), significantly decreased intestinal permeability (serum FITC-Dextran concentration) (p<0.01), significantly upregulated expression of intestinal tight junction proteins and mucus barrier proteins (p<0.01), significantly decreased absolute abundance of intestinal sulfate-reducing bacteria (p<0.01), significantly decreased H2S content in the colon and trisulfide content in feces (p<0.01), and significantly decreased serum lipopolysaccharide (LPS) concentration (p<0.01). The degree of improvement in the above indicators in the HFD+ Comparative Examples 1-3 groups was significantly lower than that in the HFD+ Example 1 group (p<0.05).

[0136] Summary of experimental results:

[0137] Table 1. Comparison of gastric acid stability and intestinal targeted release performance

[0138] sample Chlorophyll retention rate in simulated gastric juice (%) Release rate (%) in simulated gastric juice Release rate (%) in simulated intestinal fluid Example 1 92.5±3.2 7.8±1.1 93.6±2.5 Example 2 91.8±2.9 8.2±1.3 92.5±2.8 Example 3 94.2±3.5 6.5±0.9 95.2±2.4 Example 4 90.5±3.1 9.2±1.5 91.8±2.7 Example 5 93.1±3.3 7.5±1.2 94.1±2.6 Example 6 91.2±3.0 8.8±1.4 92.2±2.5 Example 7 95.8±3.6 5.5±0.8 96.5±2.8 Comparative Example 1 16.2±2.1 83.5±5.4 10.2±1.8

[0139] Table 2. Comparison of probiotic survival rate and anti-inflammatory activity

[0140] sample Probiotic survival rate (%) after simulated gastrointestinal condition treatment TNF-α inhibition rate (%) IL-6 inhibition rate (%) IL-1β inhibition rate (%) Example 1 72.5±4.2 72.8±3.5 68.5±3.2 70.2±3.6 Example 2 71.2±4.0 71.5±3.4 67.2±3.1 69.5±3.5 Example 3 75.8±4.5 75.2±3.7 70.5±3.4 73.8±3.8 Example 4 76.2±4.6 68.3±3.2 65.8±3.0 67.2±3.4 Example 5 70.5±3.9 76.5±3.8 72.2±3.5 74.5±3.9 Example 6 78.2±4.8 67.2±3.1 64.5±2.9 66.8±3.3 Example 7 71.8±4.1 73.5±3.6 69.2±3.3 71.5±3.7 Comparative Example 1 45.2±3.5 52.3±2.8 48.5±2.5 50.2±2.7 Comparative Example 2 - 57.2±2.9 54.5±2.7 56.8±2.8 Comparative Example 3 51.5±3.6 35.8±2.2 32.5±2.0 34.2±2.1

[0141] Table 3. Comparison of protective effects on intestinal barrier function

[0142] sample TEER reduces protection rate (%) FITC-Dextran permeability protection rate (%) ZO-1 expression increased fold Occludin expression increased by a factor of 10 Claudin-1 expression increased fold Example 1 75.2±4.3 82.5±4.8 2.8±0.3 2.5±0.2 2.2±0.2 Example 2 73.8±4.2 80.2±4.6 2.6±0.3 2.4±0.2 2.1±0.2 Example 3 78.5±4.5 84.8±5.0 3.0±0.3 2.7±0.3 2.4±0.2 Example 4 72.5±4.1 78.3±4.5 2.5±0.2 2.3±0.2 2.0±0.2 Example 5 76.8±4.4 83.5±4.9 2.9±0.3 2.6±0.2 2.3±0.2 Example 6 77.2±4.4 81.5±4.7 2.7±0.3 2.5±0.2 2.2±0.2 Example 7 79.5±4.6 85.2±5.1 3.1±0.3 2.8±0.3 2.5±0.2 Comparative Example 1 55.3±3.5 58.2±3.8 1.6±0.2 1.4±0.1 1.3±0.1 Comparative Example 2 50.2±3.2 53.5±3.5 1.5±0.1 1.3±0.1 1.2±0.1 Comparative Example 3 45.8±3.0 48.2±3.2 1.4±0.1 1.2±0.1 1.1±0.1

[0143] Table 4. Efficacy evaluation results of the high-fat diet mouse model

[0144] Group Relative expression of colonic TNF-α Serum FITC-Dextran (μg / mL) <![CDATA[Colonic H2S content (nmol / g wet weight)]]> Serum LPS (EU / mL) ND 1.00±0.12 1.52±0.18 320±35 0.65±0.08 HFD 2.85±0.32*** 3.25±0.35*** 1050±95*** 0.92±0.10*** HFD+ Example 1 1.35±0.15^^^ 1.85±0.22^^^ 480±45^^^ 0.72±0.08^^ HFD+ Comparative Example 1 <![CDATA[2.05±0.25 # ]]> <![CDATA[2.65±0.28 # ]]> <![CDATA[780±70 # ]]> <![CDATA[0.83±0.09 # ]]> HFD + Comparative Example 2 <![CDATA[2.15±0.26 # ]]> <![CDATA[2.75±0.30 # ]]> <![CDATA[820±75 # ]]> <![CDATA[0.85±0.09 # ]]> HFD+ Comparative Example 3 <![CDATA[2.25±0.28 # ]]> <![CDATA[2.85±0.32 # ]]> <![CDATA[850±80 # ]]> <![CDATA[0.86±0.09 # ]]>

[0145] Note: Compared with the ND group, ***p<0.001; compared with the HFD group, ^p<0.05, ^^p<0.01, ^^^p<0.001; compared with the HFD+Example 1 group, #p<0.05.

[0146] The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology provided by this invention can be widely used in the fields of functional foods and pharmaceuticals, and is particularly suitable for the following groups:

[0147] 1. Individuals with gut microbiota imbalance: The formula of this invention effectively regulates the balance of gut microbiota through the synergistic effect of chlorophyll and probiotics, especially inhibiting the growth of harmful bacteria such as sulfate-reducing bacteria and promoting the colonization of beneficial bacteria.

[0148] 2. Patients with intestinal inflammation-related diseases: The chlorophyll and anti-inflammatory synergists in the formula have a good effect on improving intestinal inflammation-related diseases such as irritable bowel syndrome and inflammatory bowel disease through a multi-target anti-inflammatory mechanism.

[0149] 3. Individuals with impaired intestinal barrier function: The formula of this invention can significantly enhance the expression of intestinal tight junction proteins and mucus barrier proteins, repair damaged intestinal barrier function, and reduce systemic inflammatory responses caused by increased intestinal permeability.

[0150] 4. People with high-fat diets: In response to the intestinal health problems caused by modern high-fat diets, the formula of this invention can effectively alleviate intestinal flora imbalance, inflammatory response and barrier function damage caused by high-fat diets.

[0151] 5. Patients with metabolic syndrome: By improving gut health, the formula of this invention helps improve metabolic disorders such as obesity, insulin resistance, and hyperlipidemia.

[0152] The formulation provided by this invention can be prepared into various dosage forms, including capsules, tablets, granules, and suspensions, to meet the needs of different populations. Furthermore, the preparation method of this invention is stable, highly reproducible, and suitable for industrial production.

[0153] Based on the experimental results above, Examples 3 and 7 exhibited the best overall performance. Example 3 demonstrated superior anti-inflammatory activity and intestinal barrier protection, making it particularly suitable for individuals with severe intestinal inflammation and significantly impaired barrier function. Example 7, on the other hand, showed the best performance in chlorophyll stability and targeted release, making it suitable for individuals requiring higher doses of chlorophyll for regulatory effects.

[0154] The enteric-coated tablets prepared in Example 3 and the bilayer tablets prepared in Example 7 both exhibit good storage stability. After being stored at 40°C / 75% relative humidity for 6 months, the content of active ingredients remained above 90%, and the probiotic activity remained above 85%, indicating that the formulation of the present invention has good prospects for industrial application.

[0155] This invention provides a probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology, its preparation method, and its applications. Through innovative formulation design and preparation process, it solves the problems of poor stability, low bioavailability, and limited efficacy of single components in traditional chlorophyll preparations in acidic environments. The formulation of this invention achieves multi-target regulation of intestinal health through the multiple effects of a chlorophyll complex stable under acidic conditions, a probiotic synergistic composition, and an anti-inflammatory synergist, demonstrating significant effects in intestinal flora balance, inflammatory response inhibition, and barrier function protection.

[0156] Experimental results show that there is a significant synergistic effect among the components in the formulation of this invention, and the overall effect is significantly better than any single component or partial combination. The preparation method of this invention is stable, highly reproducible, suitable for industrial production, and has broad application prospects.

Claims

1. A probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology, characterized in that, The formulation comprises, by weight: 55-90 parts of chlorophyll complex stable under acidic conditions, 15-35 parts of probiotic synergistic composition, 15-30 parts of anti-inflammatory synergist, and 5-10 parts of excipients. The acid-stable chlorophyll complex includes chlorophyll a, chlorophyll b, sodium copper chlorophyll, and sodium magnesium chlorophyll; the probiotic synergistic composition includes Bifidobacterium, Lactobacillus plantarum, Myxobacterium, and Prevotella; and the anti-inflammatory synergist includes curcumin-cyclodextrin inclusion complex, quercetin nanoparticles, resveratrol-phospholipid complex, and epigallocatechin gallate.

2. The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology according to claim 1, characterized in that, The acid-stable chlorophyll complex comprises, by weight, the following: 20-30 parts chlorophyll a, 15-25 parts chlorophyll b, 5-10 parts sodium copper chlorophyll, 5-10 parts sodium magnesium chlorophyll, and 10-15 parts pH-responsive nano-encapsulation matrix.

3. The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology according to claim 1, characterized in that, The probiotic synergistic composition comprises, by weight: 4-9 samples of Bifidobacterium longum (live count not less than 10) 10 CFU / g), 4-9 samples of Lactobacillus plantarum (live count not less than 10). 10 CFU / g), 3-5 samples of Akkermansia myxophilus (viable count not less than 10). 9 CFU / g), 2.5-7 samples of Prevotella (live count not less than 10). 9 (CFU / g) and 3-9 parts inulin matrix.

4. The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology according to claim 1, characterized in that, The anti-inflammatory synergist comprises, by weight: 5-8 parts curcumin-cyclodextrin inclusion complex, 4-7 parts quercetin nanoparticles, 3-5 parts resveratrol-phospholipid complex, and 3-5 parts epigallocatechin gallate.

5. The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology according to claim 1, characterized in that, The excipients, by weight, include: 2-4 parts enteric-coated polymer, 1-2 parts pH-dependent release agent, 1-2 parts stabilizer, and 1-2 parts flow agent.

6. A method for preparing the probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology according to any one of claims 1-5, characterized in that, Includes the following steps: a) Preparation of chlorophyll complexes stable under acidic conditions: including extraction and purification, stabilization treatment and nano-encapsulation; b) Preparation of probiotic synergistic compositions: including strain culture and collection, freeze-drying, and multi-layer encapsulation; c) Preparation of anti-inflammatory synergists: including the preparation of curcumin-cyclodextrin inclusion complex, quercetin nanoparticles, resveratrol-phospholipid complex, and treatment of epigallocatechin gallate ester; d) The acid-stable chlorophyll complex, the probiotic synergistic composition, the anti-inflammatory synergist, and excipients are mixed to obtain the probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology.

7. The preparation method according to claim 6, characterized in that, The specific steps for preparing the acid-stable chlorophyll complex in step a) include: a1) Wash and chop spinach, lettuce and celery in a weight ratio of 1:1:1, add 95% ethanol and extract at 2-6℃ for 120-150 minutes; a2) Centrifuge at 7800-8200g for 8-12 minutes, collect the supernatant, and concentrate the supernatant to 23-27% of the original volume; a3) Place at 2-6℃ for 10-14 hours, collect the dark green precipitate, and filter it through a 0.45μm cellulose acetate membrane; a4) Dissolve the precipitate in a phospholipid solution, mix in a 1:2 ratio, add 0.5% copper chloride and 0.5% magnesium chloride solution, adjust the pH to 7.2-7.6, and heat at 68-72℃ for 28-32 minutes; a5) Prepare oil and aqueous phases, and homogenize at high speed (18000-22000 rpm) to prepare emulsion; a6) Add pH-responsive polymer solution and spray dry (inlet temperature 155-165℃, outlet temperature 75-85℃) to obtain the chlorophyll complex that is stable under acidic conditions.

8. The preparation method according to claim 6, characterized in that, The specific steps for preparing the probiotic synergistic composition in step b) include: b1) Culture Bifidobacterium longum, Lactobacillus plantarum, Akkermansia muciniphila and Prevotella to the late logarithmic growth stage respectively; b2) Collect bacterial cells by centrifugation at 4800-5200g and wash twice with sterile phosphate buffer; b3) Resuspend the bacterial culture in a low-temperature protective medium, adjust the bacterial concentration, and freeze-dry. b4) Prepare an inulin solution (10%, dissolved at 58-62℃), cool to 38-42℃, and add freeze-dried probiotic powder; b5) Spray freeze-drying forms primary microparticles, which are then alternately encapsulated with sodium alginate (1.0-1.2%) and chitosan (0.4-0.6%). b6) Crosslink with calcium chloride solution (2.0-2.2%), wash and freeze dry to obtain the probiotic synergistic composition.

9. The preparation method according to claim 6, characterized in that, The specific steps for preparing the anti-inflammatory synergist in step c) include: c1) Preparation of curcumin-cyclodextrin inclusion complex: Dissolve β-cyclodextrin (1%) in deionized water, heat to 38-42℃, add curcumin ethanol solution (1%) dropwise at a rate of 1-2 mL / min, stir continuously for 3-4 hours, cool to 5-10℃, collect the precipitate and freeze dry; c2) Preparation of quercetin nanoparticles: Quercetin was dissolved in ethanol, and an aqueous phase containing polysorbate 80 was added. The mixture was homogenized at high speed to form a nanoemulsion, the organic solvent was removed by rotary evaporation, and the mixture was freeze-dried. c3) Preparation of resveratrol-phospholipid complex: Resveratrol and soybean phospholipids were dissolved in dichloromethane at a ratio of 1:2, the solvent was evaporated to form a film, hydrated with phosphate buffer (pH 7.2-7.6), and lyophilized. c4) Epigallocatechin gallate treatment: Epigallocatechin gallate is dissolved in deionized water, antioxidants and stabilizers are added, and the mixture is stirred evenly and then spray-dried.

10. The probiotic-chlorophyll complex intestinal microecological regulator based on nano-encapsulation technology according to any one of claims 1-5 is suitable for the preparation and application of drugs or functional foods for preventing or improving intestinal inflammation, regulating intestinal flora imbalance, and improving impaired intestinal barrier function.