Method for extracting curcuma oil body from fresh curcuma, curcuma oil body microcapsule and application

Abstract

The present application relates to the technical fields of natural product extraction and biomimetic preparation, and provides a method for extracting curcuma oil body from fresh turmeric, curcuma oil body microcapsules and application thereof.The method comprises the following steps: S11, adding the pretreated fresh turmeric into an extraction solution and shearing and crushing; S21, adjusting the pH of the extraction solution, adding a composite enzyme system, and then performing ultrasonic enzymolysis; S31, adjusting the pH of the enzymolysis solution, and then performing inactivation and filtration to obtain a first filtrate and a filter residue; S41, adding a density gradient medium to the first filtrate, and then performing low-temperature centrifugal separation to collect the upper cream to resuspend and obtain an initial emulsion containing curcuma oil body; S51, adding a surfactant to the initial emulsion, adjusting the pH, and then filtering to obtain a second filtrate; S61, centrifuging the second filtrate at low temperature, collecting the upper cream to resuspend and obtaining curcuma oil body emulsion.The curcuma oil body obtained by the present application has the advantages of high solubility, high stability and high bioavailability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product extraction and biomimetic preparation, and in particular relates to a method for extracting curcuma oil bodies from fresh turmeric, curcuma oil body microcapsules and applications. BACKGROUND

[0002] Turmeric (Curcuma longa) is the dried rhizome of the perennial herbaceous plant Curcuma longa of the Zingiberaceae family, and is considered as one of the most valuable herbs in the ancient Ayurvedic therapy, with the reputation of "plant gold" and "Indian solid gold". It is an indispensable important healing spice. Modern medicine has proved that curcumin compounds (also known as curcuminoids) extracted from turmeric mainly include curcumin, demethoxycurcumin and bisdemethoxycurcumin, which have the effects of antioxidant, anti-inflammatory, liver protection, blood lipid reduction, blood sugar reduction, anti-ulcer, cardiovascular protection, anti-depression, antibacterial, antiviral, antifungal, anti-radiation and anti-tumor, and are increasingly widely used in the field of health in recent years.

[0003] Due to the low solubility of curcumin compounds (for example, the solubility of curcumin is only 0.011 mg / mL), the existing extraction technology usually uses organic solvents (ethanol, acetone, ethyl acetate) to extract curcumin compounds from dried turmeric powder. However, there are many limitations in the organic solvent extraction method. 1) The organic solvent has toxicity and environmental pollution problems, which may cause harm to the human body and the environment; 2) The organic solvent is flammable and explosive, which increases the safety risk in the operation process; 3) The cost of the organic solvent is high, which increases the overall cost of the extraction process; 4) The organic solvent may exist in the form of residue, which affects the safety of the turmeric extraction product.

[0004] In recent years, some new extraction technologies have also been applied to the extraction of curcumin compounds, such as supercritical CO2 extraction technology and ionic liquid extraction technology. These technologies have the advantages of green, high efficiency and high selectivity, but the production cost is also significantly increased.

[0005] More and more studies have shown that demethoxycurcumin and bisdemethoxycurcumin have advantages in stability, water solubility, antioxidant, anti-inflammatory and anti-tumor activity. The demethoxycurcumin molecule has improved coplanarity, is easier to embed into the hydrophobic pocket of DNA / protein, and the phenolic hydroxyl group in the molecular structure is exposed, which enhances the antioxidant performance. The combined application of curcumin, demethoxycurcumin and bisdemethoxycurcumin is expected to achieve synergistic effect through multi-target coverage, metabolic complementation and epigenetic regulation, and has great potential in disease treatment and health products. It has been reported that the combination of the three has stronger inhibitory effect on human osteosarcoma HOS cells than the use of single or two of them, which is reflected in reducing cell viability, colony formation and promoting apoptosis. However, the existing extraction technology is difficult to ensure the sufficient extraction of the three curcumin compounds.

[0006] In practical applications, curcumin compounds have the problems of low water solubility, poor physical and chemical stability, fast degradation in the gastrointestinal tract, poor intestinal permeability, fast metabolism and elimination, etc., which have been the application barriers in the fields of beverages, flour products, compound seasonings and medicines. At present, the solubilization technologies of curcumin compounds include cyclodextrin inclusion solubilization, micellar solubilization, microemulsion solubilization, solid dispersion solubilization, adsorption solubilization, etc. However, these methods still have their own problems. The addition amount of β-cyclodextrin is limited when used as a stabilizer and a processing aid in beverages, dairy products, baked foods and special diet foods, and the release of curcumin compounds in the body by decomposition or by competing for cavity positions may affect their physiological activity. The use of a large amount of surfactants or amphiphilic polymers in micellar and microemulsion solubilization does not meet the market demand for "all natural". Other methods also have a series of problems such as low stability of the complex, difficulty in storage, complex preparation process and high preparation cost.

[0007] Therefore, it is an important challenge for curcumin production and application to explore green, safe and efficient extraction technologies for three curcumin compounds and solubilization methods for curcumin compounds that can be applied in the food, cosmetic and pharmaceutical industries, so as to improve the bioavailability of curcumin as a food efficacy factor or a therapeutic agent. SUMMARY

[0008] In view of the above deficiencies in the prior art, the purpose of the present application is to provide a method for extracting curcumin oil from fresh turmeric, curcumin oil microcapsules and applications. The method uses fresh turmeric as a raw material and adopts a combination of water enzymatic method, ultrasonic cell wall breaking, density gradient centrifugal separation, interfacial protein reconstitution and other synergistic effects to extract oil containing curcumin, demethoxycurcumin and bisdemethoxycurcumin, three active ingredients. The oil is coated with a high molecular capsule material to prepare curcumin oil microcapsules and their derivative products, which have the advantages of high solubility, high stability and high bioavailability.

[0009] The present application is realized by the following technical solutions:

[0010] The first purpose of the present application is to provide a method for extracting curcumin oil from fresh turmeric, which comprises the following steps:

[0011] S11, adding the pretreated fresh turmeric to an extraction solution and performing shearing and crushing to obtain an extraction solution; the extraction solution is a phosphate buffer, a glycine-sodium hydroxide buffer, a sodium bicarbonate buffer or a tris-hydrochloride buffer;

[0012] S21, adjust the pH of the extract to 4.0-5.0, and add a complex enzyme system, then perform ultrasonic enzymolysis to obtain an enzymolysis liquid; the complex enzyme system comprises one or more of cellulase, xylanase, arabinoxylanase, pectinase, laccase, and snail enzyme;

[0013] S31, adjust the pH of the enzymolysis liquid to 7.0-8.0, then perform inactivation and filtration to obtain a first filtrate and a filter residue;

[0014] S41, add a density gradient medium to the first filtrate, then perform low-temperature centrifugal separation to form an isodensity zone, and collect the upper cream to resuspend to obtain a primary emulsion containing curcuma oil bodies; the density gradient medium comprises one or more of sucrose, glucose, dextran, hyaluronic acid, glycerol, polyethylene glycol, and a cyclodextrin derivative;

[0015] S51, add a surfactant to the primary emulsion, adjust the pH to 3.0-3.5, then perform stirring to reconfigure the interfacial proteins on the surface of the curcuma oil bodies, and perform filtration to obtain a second filtrate;

[0016] S61, perform centrifugation on the second filtrate at low temperature, and collect the upper cream to resuspend to obtain a curcuma oil body emulsion.

[0017] Plant oil bodies are subcellular organelles of micron or submicron size (0.5-2 μm), and are a solution to the problem of handling two immiscible phases in nature. Plant oil bodies are O / W emulsion droplets formed by a single layer of phospholipid-interfacial proteins wrapping triacylglycerol, and play important roles in energy storage, oxidative buffering, signal transduction, pathogen resistance, and low-temperature protection in the growth and metabolism of plants. Extraction of plant oil bodies in a natural form can directly obtain O / W emulsions, which have great application space in the fields of food, cosmetics, and medicine. The advantages of extracting plant oil bodies to prepare derivative products are: usually using water extraction or aqueous enzymatic method, without using organic solvents, which is green and safe; the extraction process is simple, and a large number of extraction processes in traditional processes are saved; as natural droplets, oil bodies can stably exist in the water phase without the need for additional emulsifiers or homogenization process steps; active ingredients existing in or encapsulated in oil bodies usually have good water solubility, stability, and bioavailability. It can be seen that the characteristic structure of plant oil bodies formed through thousands of years of evolution opens up a new way for their advanced applications.

[0018] The present application first discovers that curcumin compounds including curcumin, demethoxycurcumin and bisdemethoxycurcumin exist in oil bodies of Curcuma longa. Therefore, the present application provides a method for directly extracting curcumin-containing oil bodies from fresh Curcuma longa and application. The method provided by the present application extracts oil bodies containing curcumin, demethoxycurcumin and bisdemethoxycurcumin by using a combination of enzymatic hydrolysis, ultrasonic assisted cell wall breaking, density gradient zonal centrifugation and interfacial protein reconstitution, and then coats the oil bodies with a high molecular weight capsule material to prepare Curcuma longa oil body microcapsules and derivative products thereof.

[0019] Specifically, the fresh Curcuma longa is first pretreated, washed to remove impurities such as sand and drained. This step is to prepare for subsequent extraction, and removing impurities can avoid interference with the extraction process and ensure the purity of the extraction. Then the treated fresh Curcuma longa is added to a specific extraction solution, which can be selected from phosphate buffer, glycine-sodium hydroxide buffer, sodium bicarbonate buffer or Tris hydrochloride buffer. Among them, the phosphate buffer is preferred because it can ensure a certain osmotic pressure and maintain the stability of the oil body membrane. The fresh Curcuma longa and the extraction solution are mixed at a weight ratio of 1:1 to 1:5 (preferably 1:3), and shearing is performed at a speed of 20,000 to 40,000 rpm for 25 to 35 min. High-speed shearing can quickly break the fresh Curcuma longa, causing the cell structure to be initially damaged, which creates conditions for the subsequent extraction solution to enter the cell interior and contact the Curcuma longa oil body.

[0020] The extraction solution obtained after breaking needs to be adjusted to a pH of 4.0 to 5.0, which is suitable for the action of a complex enzyme system. The complex enzyme system contains one or more of cellulase, xylanase, arabinanase, pectinase, laccase and snailase, preferably a complex system of 0.5 to 2.0% cellulase and 0.1 to 0.5% pectinase, and the final concentration in the extraction solution is 0.5 to 2.5%. Subsequently, enzymatic hydrolysis is performed under ultrasonic assistance, and the ultrasonic parameters are set as follows: power 100 W / cm 2 , frequency 18 kHz, pulse mode open 3 s / close 2 s, enzymatic hydrolysis time 20 to 40 min, and temperature control at 50 to 60℃. The penetration and cavitation effect of ultrasonic waves can accelerate the enzymatic hydrolysis process, allowing the enzyme to act more efficiently on the cell wall structure, greatly shortening the enzymatic hydrolysis and cell wall breaking time, and achieving efficient cell wall breaking in about 30 min, releasing the Curcuma longa oil body in the cell into the extraction solution, while ensuring the integrity of the oil body structure and function.

[0021] After the enzymatic hydrolysis is completed, the enzymatic hydrolysis solution needs to be adjusted to pH 7.0-8.0, and then inactivated at a temperature of 70-90°C for 5-10 min to make the complex enzyme system lose activity, so as to avoid unnecessary effects of the complex enzyme system on the components in the system. After inactivation, the solid residues in the enzymatic hydrolysis solution are separated from the liquid by filtering through a 200-mesh gauze to obtain a first filtrate and a filter residue, respectively. The filter residue is subsequently dried, crushed and sieved to process and prepare turmeric powder, so as to realize high-value utilization of the turmeric medicinal material; and the first filtrate is used for extracting turmeric oil bodies in the next step.

[0022] A density gradient medium is added to the first filtrate, which includes one or more of sucrose, glucose, dextran, hyaluronic acid, glycerol, polyethylene glycol and cyclodextrin derivatives, and the final concentration is controlled at 1-20%, preferably a mixed system of 10% sucrose and 10% hydroxypropyl-β-cyclodextrin. Hydroxypropyl-β-cyclodextrin can precisely control the density gradient of sucrose and play a role as a "lubricant" to prevent the oil bodies from being damaged and non-specific adsorption on the surface thereof during centrifugal separation. After uniform mixing, low-temperature centrifugation is performed at a speed of 3000-15000 rpm for 30-60 min to make the solution form an isodensity zone. Since the density of the turmeric oil bodies is different from that of other components, the turmeric oil bodies will be gathered in a specific area, so that the upper cream is collected and resuspended in an aqueous solution to obtain a primary emulsion containing the turmeric oil bodies.

[0023] A surfactant is added to the primary emulsion, which can be selected from one or more of sodium deoxycholate, rhamnolipid, sodium stearoyl lactylate, sucrose fatty acid ester, Tween 20, Tween 40, Tween 60, Tween 80, poloxamer F68 and alkyl polyglycoside, and the final concentration in the primary emulsion is 0.5-1.5%, preferably Tween 80. At the same time, the pH is adjusted to 3.0-3.5, and stirring is performed for 10-30 min. In this process, the surfactant precipitates impurities on one hand and competitively replaces the interfacial proteins on the surface of the oil body phospholipid membrane on the other hand, so as to form a more hydrophilic phospholipid-surfactant-interfacial protein mixed membrane, play a role in stabilizing the oil bodies and adsorbing the capsule materials, and reconfigure the interfacial proteins on the surface of the turmeric oil bodies. Then, filtration is performed to remove the protein precipitate to obtain a second filtrate.

[0024] The second filtrate is subjected to low-temperature centrifugation at a speed of 1000-5000 rpm for 30-60 min, and since the high-purity turmeric oil bodies have a different density from other impurities, the turmeric oil bodies will be gathered in the upper layer to form a cream. The upper cream is collected and resuspended in water to finally obtain a high-purity turmeric oil body emulsion. The emulsion is a yellow O / W thick emulsion, and after dilution with an aqueous solution, a solution with a slight opalescence is obtained, and the concentration of curcumin compounds in the turmeric oil body emulsion is 10-50 mg / mL, which retains the natural ratio of three active ingredients of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the turmeric medicinal material.

[0025] Further, the rotation speed of the shearing and crushing is 20000-40000 rpm, and the shearing and crushing time is 25-35 min; and / or,

[0026] The ultrasonic enzymolysis comprises the following parameters: time is 20-40 min, temperature is 50-60 DEG C, ultrasonic power is 100 W / cm 2 , ultrasonic frequency is 18 kHz, and ultrasonic pulse mode is open 3 s / close 2 s; and / or,

[0027] The rotation speed of the low-temperature centrifugal separation is 3000-15000 rpm, and the low-temperature centrifugal separation time is 30-60 min.

[0028] Further, the extraction solution is a phosphate buffer solution, and the weight ratio of the fresh turmeric to the extraction solution is 1:3; and / or,

[0029] The composite enzyme system is a composite system of cellulase with a final concentration of 0.5-2.0% in the extraction liquid and pectinase with a final concentration of 0.1-0.5% in the extraction liquid;

[0030] The density gradient medium is a mixed system of sucrose with a final concentration of 10% in the first filtrate and hydroxypropyl-beta-cyclodextrin with a final concentration of 10% in the first filtrate.

[0031] Further, the inactivation temperature is 70-90 DEG C, and the inactivation time is 5-10 min; and / or,

[0032] The stirring time is 10-30 min; and / or,

[0033] The rotation speed of the low-temperature centrifugal separation is 3000-15000 rpm, and the low-temperature centrifugal separation time is 30-60 min.

[0034] Further, the surfactant comprises one or more of deoxycholic acid sodium, rhamnolipid, sodium stearoyl lactylate, sucrose fatty acid ester, Tween 20, Tween 40, Tween 60, Tween 80, poloxamer F68 and alkyl polyglycoside, and the final concentration of the surfactant in the primary emulsion is 0.5-1.5%.

[0035] A third object of the application is to provide an application of the turmeric oil body emulsion or the turmeric oil body microcapsule, which is used for preparing a dietary supplement, an edible and feed product and a supplement thereof, a nutritional supplement, a flavoring agent, a condiment, a preparation component of brewing or cosmetics, or a preparation containing a drug component in the form of a natural carrier of a hydrophobic drug and an active molecule.

[0036] The curcumin-containing turmeric oil body microcapsule solution prepared by the extraction method has a yellow O / W thick emulsion appearance, the concentration of the curcumin is 10-50 mg / mL, and the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric can be maintained, and the solution is slightly opalescent after dilution with water.

[0037] The curcumin-containing turmeric oil body microcapsule solution prepared by the extraction method has a yellow O / W thick emulsion appearance, the concentration of the curcumin is 10-50 mg / mL, and the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric can be maintained, and the solution is slightly opalescent after dilution with water.

[0038] The curcumin-containing turmeric oil body microcapsule solution prepared by the extraction method has a yellow O / W thick emulsion appearance, the concentration of the curcumin is 10-50 mg / mL, and the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric can be maintained, and the solution is slightly opalescent after dilution with water.

[0039] The curcumin-containing turcumin oil body microcapsule solution prepared by the extraction method has a yellow O / W thick emulsion appearance, the concentration of the curcumin is 10-50 mg / mL, and the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric can be maintained, and the solution is slightly opalescent after dilution with water.

[0040] (1) The water enzyme method is used to extract the turmeric oil body from fresh turmeric medicinal materials, which can avoid the harm to the environment and human body caused by the use of organic solvents in the traditional extraction process, and has the advantages of green environmental protection.

[0041] (2) The process combined with ultrasonic crushing can greatly shorten the enzymatic wall breaking time, and high-efficiency wall breaking can be realized in 30 min to release the structure and function of the turmeric oil body.

[0042] (3) The advantages of using sucrose and hydroxypropyl-β-cyclodextrin mixed medium for density gradient zonal centrifugation to extract the turmeric oil body include: 1) good separation effect, and relatively pure turmeric oil body can be obtained; 2) the oil body will not be extruded and deformed during centrifugation, and the integrity of the oil body morphology and function can be maintained, which also helps to prevent the mixing of the formed zones caused by convection.

[0043] (4) The surfactant is used for precipitating impure proteins, and competitively replacing the interface proteins on the phospholipid membrane surface of the oil body to form a more hydrophilic phospholipid-surfactant-interface protein mixed membrane. This process of reconstituting the membrane protein of the turmeric oil body can significantly improve the stability of the turmeric oil body, and is also helpful for subsequent encapsulation.

[0044] (5) It can ensure that curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric medicinal materials are extracted in a natural proportion, so as to exert the synergistic effect of the three curcuminoid compounds.

[0045] (6) The surface of turmeric oil is coated with polymer capsules, which helps to improve the stability of curcuminoids and their resistance to harsh gastrointestinal conditions, thereby effectively ensuring the effective performance of the efficacy of curcuminoids.

[0046] (7) Turmeric oil microcapsules appear as yellow emulsions in aqueous solution, and the solubility of curcuminoids can reach 2-10 mg / mL, which is 150-900 times the solubility of curcumin (0.011 mg / mL), which is conducive to the promotion and application of subsequent products.

[0047] (8) The oral bioavailability of curcuminoids in turmeric oil microcapsules is 20 to 30 times that of the same dose of curcumin.

[0048] (9) Compared with the same dose of curcumin, the three curcuminoid compounds in turmeric oil microcapsules have stronger antioxidant, anti-inflammatory, antibacterial, and anti-tumor effects.

[0049] (10) It has a wide range of applications, including various uses in food, cosmetics and biomedicine.

[0050] (11) The process is easy to control and suitable for industrial production.

[0051] (12) The filter residue after enzymatic hydrolysis is dried, crushed and sieved, and processed into turmeric powder, which can be used as a seasoning and dietary fiber, realizing the high-value utilization of turmeric medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0053] Figure 1 A physical image of the turmeric extract after enzymatic hydrolysis provided in Example 1 of the present invention;

[0054] Figure 2 A physical image of the turmeric extract after Tween 80 treatment provided in Example 1 of the present invention;

[0055] Figure 3 A physical picture of the creamy turmeric oil collected by gradient centrifugation provided in Example 1 of the present invention;

[0056] Figure 4 A physical picture of the concentrated solution of curcumin oil body microcapsules provided for Example 1 of the present application;

[0057] Figure 5 A picture of the Tyndall phenomenon of the curcumin oil body microcapsule solution provided for Example 2 of the present application after dilution;

[0058] Figure 6 A column chart of the particle size distribution of the curcumin oil body microcapsules provided for Example 2 of the present application;

[0059] Figure 7 A picture of the morphology of the curcumin oil body microcapsules observed under a microscope provided for Example 2 of the present application;

[0060] Figure 8 A fluorescence microscope picture of the curcumin oil body microcapsules after Nile red staining provided for Example 2 of the present application;

[0061] Figure 9 A picture of the morphology of the curcumin oil body microcapsules observed under a freeze electron microscope provided for Example 2 of the present application;

[0062] Figure 10 A high performance liquid chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the curcumin oil body microcapsules prepared from fresh curcumin provided for Example 2 of the present application;

[0063] Figure 11 A high performance liquid chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the curcumin oil body microcapsules prepared from fresh curcumin provided for Example 2 of the present application;

[0064] Figure 12 A high performance liquid chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the curcumin oil body microcapsules prepared from fresh curcumin provided for Example 3 of the present application;

[0065] Figure 13 A high performance liquid chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the curcumin oil body microcapsules prepared from fresh curcumin provided for Example 3 of the present application;

[0066] Figure 14 A picture of the protein bands of the curcumin oil body microcapsules provided for Example 3 of the present application after gel electrophoresis and Coomassie brilliant blue staining treatment;

[0067] Figure 15 A physical picture of the spray-dried solid powder of the curcumin oil body microcapsules provided for Example 4 of the present application;

[0068] Figure 16 Pictures of the solution of the curcumin oil body microcapsule solid powder provided for Example 4 of the present application after resuspension at different concentrations;

[0069] Figure 17Figure of scavenging performance of DPPH free radicals by curcumin oil body microcapsules provided in Example 5 of the present application;

[0070] Figure 18 Figure of scavenging performance of ABTS free radicals by curcumin oil body microcapsules provided in Example 5 of the present application;

[0071] Figure 19 Figure of scavenging performance of reactive oxygen free radicals in NIH / 3T3 cells by curcumin oil body microcapsules provided in Example 6 of the present application;

[0072] Figure 20 Figure of resistance to oxidative damage in NIH / 3T3 cells by curcumin oil body microcapsules provided in Example 6 of the present application;

[0073] Figure 21 Figure of inhibitory effect of interleukin-6 secretion by proinflammatory RAW264.7 cells by curcumin oil body microcapsules provided in Example 7 of the present application;

[0074] Figure 22 Figure of inhibitory effect of interleukin-1β secretion by proinflammatory RAW264.7 cells by curcumin oil body microcapsules provided in Example 7 of the present application;

[0075] Figure 23 Figure of inhibitory effect of tumor necrosis factor-α secretion by proinflammatory RAW264.7 cells by curcumin oil body microcapsules provided in Example 7 of the present application;

[0076] Figure 24 Figure of inhibitory effect of nitric oxide (NO) secretion by proinflammatory RAW264.7 cells by curcumin oil body microcapsules provided in Example 7 of the present application;

[0077] Figure 25 Figure of inhibitory effect of growth of Propionibacterium acnes by curcumin oil body microcapsules provided in Example 8 of the present application;

[0078] Figure 26 Figure of ability of growth of Propionibacterium acnes to form biofilm provided in Example 8 of the present application;

[0079] Figure 27 Figure of inhibitory effect of biofilm of Propionibacterium acnes by curcumin oil body microcapsules provided in Example 8 of the present application;

[0080] Figure 28 Figure of inhibitory effect of growth of mouse squamous carcinoma SCC-7 cells by curcumin oil body microcapsules provided in Example 9 of the present application;

[0081] Figure 29 Figure of inhibitory effect of growth of human hepatoma HepG2 cells by curcumin oil body microcapsules provided in Example 9 of the present application;

[0082] Figure 30 The blood concentration-time curve of the curcuma oil body microcapsule provided by the embodiment 10 of the present application in rats after intragastric administration is shown in the following figure. DETAILED DESCRIPTION

[0083] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the reagents or instruments used are not indicated by the manufacturers, they are all conventional products that can be purchased in the market.

[0084] Embodiment 1. Preparation of curcuma oil body microcapsule solution

[0085] Fresh curcuma harvested in February 2024 in Daxing Town, Qianwei County was washed with clean water to remove mud and surface impurities, drained and cut into pieces. 100 g of the curcuma was added into a previously prepared tris-hydroxymethyl aminomethane hydrochloride buffer solution (0.05 mol / L, pH 7.8) 300 mL, and broken in a JIUYANG cell disruptor at a speed of 20000 rpm. Cellulase (Dongheng Huadao Biological Technology Co., Ltd.) and pectinase (Dongheng Huadao Biological Technology Co., Ltd.) were added at concentrations of 1.5% and 0.2%, respectively. The pH was adjusted to 4.0-5.0 with dilute hydrochloric acid. The broken cells were subjected to enzymatic hydrolysis at a constant temperature of 50°C under ultrasonic waves (power 100 W / cm², frequency 18 kHz; pulse mode on 3 s / off 2 s) for 30 min. After cooling to room temperature, the filtrate was collected by filtering with 200 mesh gauze, and the filtrate was a deep yellow emulsion. Figure 1 ).

[0086] Tween 80 was added to the emulsion to a final concentration of 1.0%, and then the pH was adjusted to about 3.5. The mixture was stirred at 4°C for 2 h. After standing, a small amount of white flocculent precipitate was observed at the bottom of the container, Figure 2 which was due to the competitive displacement of oil body interface proteins by Tween 80. The precipitate was removed by filtration, and 10% sucrose and 10% hydroxypropyl-β-cyclodextrin were added to the filtrate to form a density gradient. The mixture was then centrifuged at a speed of 8000 rpm for 30 min, and a yellow cream appeared on the top, Figure 3). The cream was collected, washed with distilled water, and centrifuged at 3000 rpm for 30 min. The upper layer of the purified curcumin oil was dispersed in 50 mL of distilled water, and then maltodextrin, gum arabic, and β-cyclodextrin (mass ratio 3:1:1) were added as the capsule material at a mass concentration of about 5%. Ascorbic acid palmitate ethanol solution was added as an antioxidant at a mass concentration of 0.05%, and high-pressure homogenization was performed to prepare the capsule. The high-pressure homogenization parameters were as follows: HL2000 high-pressure homogenizer (Shanghai Hongli Biological Technology Co., Ltd.); pressure 50 MPa; 2 min each time, intermittent treatment 3 times; temperature ≤ 25°C. The treated curcumin oil microcapsule solution was a yellow thick emulsion ( Figure 4 ).

[0087] Example 2. Characterization of curcumin oil microcapsule solution

[0088] The curcumin oil microcapsule solution was diluted 20 times with distilled water, and obvious Tyndall phenomenon was observed after irradiation with a laser pen ( Figure 5 ), confirming the colloidal dispersed phase of the curcumin oil microcapsule. The particle size and distribution of the curcumin oil microcapsule were detected by dynamic laser scattering method, and the average particle size was 1.19 ± 0.146 μm, and the polydispersity coefficient was 0.173 ± 0.013 ( Figure 6 ), proving that the curcumin oil microcapsule was uniformly distributed. Under an optical microscope, the curcumin oil microcapsule showed a regular spherical morphology and was uniformly distributed ( Figure 7 ). The lipophilic fluorescent dye Nile red was used to label the curcumin oil microcapsule, and under laser confocal microscopy, uniformly distributed red emulsion droplets were observed ( Figure 8 ), confirming the oil hydrophobic core of the curcumin oil microcapsule. Under a freeze scanning electron microscope, the surface morphology of the curcumin oil microcapsule was observed, and it was found that the capsule material uniformly coated the surface of the curcumin oil to form a protective layer ( Figure 9 ).

[0089] The following experiments were carried out after freeze-drying of the curcumin oil body microcapsule solution. The content of triacylglycerol in the curcumin oil body microcapsule was quantitatively determined by enzyme colorimetry (kit purchased from Beijing Solaybao Technology Co., Ltd.), and the average content of three measurements was 42.3 ± 8.25%. Then the curcumin compounds were extracted with methanol, and the content of curcumin compounds in the curcumin oil body microcapsule was detected by high performance liquid chromatography (HPLC). The HPLC detection parameters are as follows: Agilent 1260 high performance liquid chromatograph; Diamonsil C18 chromatographic column (5 μm; 250 x 4.6 mm); column temperature 35 °C; flow rate 1 mL / min; detection wavelength 430 nm; injection volume 10 μL; mobile phase: acetonitrile: 0.3% glacial acetic acid (47:53). The HPLC detection results show that the content of curcumin compounds is 15.0 ± 3.2%, and the ratio of curcumin: demethoxycurcumin: bisdemethoxycurcumin is 55:21:24 ( Figure 10 ). At the same time, the content of curcumin compounds in dried fresh curcumin was detected according to the method for determining the content of curcumin in Chinese Pharmacopoeia 2020 edition, and the HPLC detection result was 3.52 ± 0.25%, and the ratio of curcumin: demethoxycurcumin: bisdemethoxycurcumin was 55:22:23 ( Figure 11 ). It can be seen that the curcumin oil body microcapsule product prepared by the extraction method in the application can maintain the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in curcuma rhizome.

[0090] Example 3. Preparation and characterization of curcumin oil body microcapsule solution

[0091] Fresh curcumin rhizomes collected in February 2025 in Daxing Town, Qianwei were used to prepare curcumin oil body microcapsule solution by the method of Example 1, and then the content and ratio of three curcumin compounds in curcumin oil body microcapsule and dried fresh curcumin were detected by the method of Example 2. The HPLC detection results show that the content of curcumin compounds in curcumin oil body microcapsule is 12.8 ± 1.9%, and the ratio of curcumin: demethoxycurcumin: bisdemethoxycurcumin is 62:20:18 ( Figure 12 ); the content of curcumin compounds in dried fresh curcumin is 3.28 ± 0.36%, and the ratio of curcumin: demethoxycurcumin: bisdemethoxycurcumin is 62:21:17 ( Figure 13It can be seen that the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric is still maintained in the curcuminoid microcapsules. In addition, compared with the fresh turmeric harvested in 2024, the content of curcuminoids in the fresh turmeric harvested in 2025 is slightly reduced, and the ratio of the three curcuminoids has changed, mainly in the significant decrease of bisdemethoxycurcumin. Compared with turmeric from other producing areas, the ratio of demethoxycurcumin and bisdemethoxycurcumin in turmeric from Qianwei is higher. For example, the average contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric extract are 65.0%, 16.0% and 12.3%, respectively, as reported in the literature (Determination of the contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric extract, Chinese Journal of Chinese Medicine, 2015, 30(205): 853-855), and the ratio of the three is 70:17:13.

[0092] A dry sample of curcumin oil body microcapsules was precisely weighed, and a Cocktail / SDS lysis solution (Sigma-Aldrich, USA) was used for lysis according to the instructions. The supernatant protein solution was collected and the protein concentration was detected. The protein sample was heated at 97°C in a water bath for 10 min, and after cooling, SDS-PAGE gel electrophoresis separation was performed, and protein Marker was added as a reference. After electrophoresis, the gel was taken out and treated with Coomassie Brilliant Blue R250 staining solution for protein staining until the protein bands were clearly visible. Figure 14 As shown in FIG. 6, the protein bands of the curcumin oil body microcapsule sample are located in the molecular weight range of 10-35 kDa, and the main proteins are lipid transfer proteins (molecular weight about 10 kDa, promoting the interfacial transfer of phosphatidylcholine), a small amount of oil body proteins (molecular weight 15-20 kDa, maintaining the structure and stability of lipid droplets in plant cells, and participating in the dynamic regulation of lipid metabolism), and oil body calcium proteins (molecular weight 25-35 kDa, participating in calcium signal transduction and dynamic regulation of lipid droplets). These proteins located at the "oil-water" interface are related to the structure, stability and function of lipid droplets in curcumin cells, and are successfully retained on the surface of curcumin oil body microcapsules to form a protein shell, which will help to stabilize the microcapsule structure. In addition, the main proteins in the precipitated protein are those with a molecular weight greater than 60 kDa, among which the protein bands with a molecular weight of 60-75 kDa are cellulase, and the proteins with a molecular weight of more than 100 kDa include transmembrane transport proteins, key enzymes and skeleton proteins in curcumin cells.

[0093] Example 4. Preparation and characterization of curcumin oil body microcapsule spray-dried solid powder

[0094] Fresh Curcuma longa L. harvested in February 2025 in Daxing Town, Qianwei County was used to prepare curcuminoid microcapsule solution by the method of Example 1, then lactose and microcrystalline cellulose were added, the final concentration was ≤ 5%; at the same time, silicon dioxide and hydroxypropyl methylcellulose were added, the amount was ≤ 1. The amount of microcrystalline cellulose added was much lower than that of lactose, otherwise it would affect the solubility of the spray-dried curcuminoid microcapsule. The curcuminoid microcapsule solution was processed by B-290 small-scale spray dryer (BUCHI Labortechnik AG, Switzerland) to obtain curcuminoid microcapsule solid powder. Figure 15 The spray drying parameters were as follows: inlet temperature 150℃; outlet temperature 90℃; power 85 W; nozzle pressure 2 MPa; sample feeding speed 8-10 mL / min; atomizing gas pressure 3-4 MPa. The curcuminoid microcapsule powder was accurately weighed, and the curcuminoids were extracted with methanol. The content of curcuminoids was detected by the HPLC method in Example 2, which was 9.6±1.1%. The curcuminoid microcapsule solid powder was dispersed in distilled water to prepare microcapsule solutions of different concentrations. As shown in Figure 16 , the solution showed clear orange yellow, and no obvious precipitation was observed, which confirmed that the curcuminoid microcapsule solid powder prepared by the present application had good water solubility and was a solid beverage nutritional supplement with excellent properties.

[0095] Example 5. Antioxidant performance of curcuminoid microcapsule

[0096] The antioxidant performance of the curcuminoid microcapsule solution prepared according to the method in Example 3 was investigated by in vitro free radical scavenging experiment. 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a free radical with deep purple color, which is converted to a colorless stable form after reaction with antioxidant substances, so it is widely used for antioxidant performance detection. In a 1 mL reaction system, 0.5 mL of sample solution (curcumin and curcuminoid microcapsule solution) of different concentrations and 0.5 mL of DPPH ethanol solution were added, and the reaction was carried out in the dark for about 10 min. Then the absorbance of DPPH at 517 nm wavelength was measured by ultraviolet-visible spectrophotometer, and the DPPH free radical scavenging rate was calculated according to the following formula.

[0097] Scavenging rate% = (A0- A x + A d ) ×100% / A0(A0: absorbance of the system when the same volume of deionized water instead of sample solution (blank control); A d : absorbance of the system when the same volume of deionized water instead of DPPH solution (background control); A x : absorbance of the sample solution and DPPH reaction system)

[0098] The detection results are shown in Figure 17As shown, the scavenging rate of turmeric oil microcapsules on DPPH free radicals was concentration-dependent, confirming their significant antioxidant activity. Furthermore, at the same concentration, turmeric oil microcapsules exhibited significantly higher antioxidant properties than curcumin, which is attributed to their good water solubility and the presence of demethoxycurcumin and bisdemethoxycurcumin.

[0099] 2, 2'-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is also a stable free radical. It is oxidized to green ABTS by potassium persulfate. ·+ , with a characteristic absorption peak at 734 nm. When antioxidants are present, ABTS ·+ The production of ABTS is inhibited, causing the reaction system to fade and the absorbance to decrease. Since ABTS is applicable to both water-soluble and fat-soluble antioxidants, it is widely used in the evaluation of antioxidant properties. The present invention further uses an ABTS scavenging experiment to investigate the antioxidant properties of turmeric oil microcapsules. In a 1 mL reaction system, 0.5 mL of sample solutions (curcumin and turmeric oil microcapsules) of different concentrations and 0.5 mL of ABTS + potassium persulfate mixed solution were added. The reaction was in the dark for 10 minutes, and the absorbance at a wavelength of 734 nm was detected. The ABTS free radical scavenging rate was calculated according to the following formula.

[0100] Clearance % = (A0- A x + A d ) × 100% / A0 (A0: absorbance of the system when an equal volume of deionized water replaces the sample solution (blank control); A d : Absorbance of the system when an equal volume of deionized water replaces the ABTS+potassium persulfate mixed solution (background control); A x : absorbance of the reaction system of sample solution and ABTS+potassium persulfate mixed solution)

[0101] The test results are basically consistent with the DPPH clearance test results. Figure 18 As shown in the results, the scavenging rate of ABTS free radicals by turmeric oil microcapsules was concentration-dependent, and at the same concentration, it showed significantly higher antioxidant properties than curcumin, further confirming its excellent antioxidant properties.

[0102] Example 6. ROS scavenging performance of turmeric oil microcapsules at the cellular level and their ability to resist oxidative damage

[0103] Mouse embryonic fibroblast NIH / 3T3 cells (American Type Culture Collection) were cultured in RPMI-1640 complete medium (Dalian Melon Biotech Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin / streptomycin solution at 37°C in a cell incubator with 5% CO2. The cells were stimulated with 1 mmol / L H2O2 to construct an oxidative stress cell model, which was used to evaluate the ROS scavenging performance of curcumin oil body microcapsules and their ability to resist cell oxidative damage.

[0104] First, the ROS fluorescence probe 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to detect the ROS level in the cells, and the antioxidant performance was evaluated at the cell level. The experimental method is as follows: the cells were seeded in a 12-well culture plate at a density of 3×10 5 After 24 h of incubation in the cell incubator, the cells were stimulated with RPMI-1640 medium containing H2O2 for 1 h. Then, different concentrations of curcumin oil body microcapsules (0-25 μmol / L) were added for overnight incubation. The next day, 1 mL of DCFH-DA working solution was added to each well, incubated for 30 min, and the medium was discarded. The cells were collected by centrifugation, washed with pH 7.4 phosphate buffer, and finally resuspended in 300 μL of phosphate buffer. The cell pellets were filtered through a 200-mesh nylon screen, and the fluorescence signal intensity of 2,7-dichlorofluorescein (DCF) generated in the cells was detected using an Accuri™ C6 flow cytometer (BD, USA) to evaluate the intracellular ROS level. The detection results are shown in Fig. 2. The stimulation of H2O2 greatly increased the ROS level in NIH / 3T3 cells, indicating the successful construction of the oxidative stress cell model. Curcumin oil body microcapsules could down-regulate the ROS level in oxidative stress model cells in a concentration-dependent manner, indicating that curcumin oil body microcapsules had strong intracellular ROS scavenging ability. Due to the hydrophobicity of curcumin, it is difficult to penetrate the cell membrane and enter the cells, so the ROS scavenging effect in oxidative stress model cells is not significant (no detailed data is provided). It can be seen that the excellent antioxidant performance of curcumin oil body microcapsules is due to the water solubility of curcumin compounds, as well as the stronger antioxidant capacity of bisdemethoxycurcumin and demethoxycurcumin than curcumin. Figure 19

[0105] Next, the CCK8 experiment was used to evaluate the resistance of NIH / 3T3 cells to H2O2 oxidative stimulation after the addition of curcumin oil body microcapsules. The experimental method is as follows: the cells were seeded in a 96-well culture plate at a density of 1×10 4 ​The cells were seeded in 96-well plates at a density of 1 cell / well, and incubated in an incubator for 24 h, and then stimulated with H2O2-containing RPMI-1640 medium for 1 h. Subsequently, complete medium containing different concentrations of curcumin oil body microcapsules was added, with the concentration of curcumin ranging from 0 to 100 μmol / L, and incubation was continued for 48 h. The medium was discarded, and the cells were treated with CCK-8 reagent-containing medium for 1.5 h, and finally the absorbance value of each well was detected at 450 nm using a Multiskan FC microplate reader (Thermo Scientific, USA). The survival rate of the cells in the control well without H2O2 stimulation and curcumin oil body microcapsule administration was taken as 100%, and the survival rate of the cells in other groups was calculated based on the ratio of the absorbance value to that of the control group. The detection results are shown in Table 1. Figure 20 H2O2 stimulation significantly inhibited the in vitro growth of NIH / 3T3 cells, indicating that it caused oxidative damage to the cells. Curcumin oil body microcapsules showed a concentration-dependent promotion of cell growth in the concentration range of 2.5-15 μmol / L of curcumin, which was attributed to the successful elimination of intracellular ROS. When the concentration of curcumin was greater than 20 μmol / L, the promotion of in vitro growth of the oxidative damaged cells by curcumin microcapsules began to weaken. This is because curcumin has a multi-target effect and exhibits bidirectional regulation activity in cells, with low concentrations promoting cell growth and repair, and high concentrations possibly inhibiting cell proliferation.

[0106] Example 7. Anti-inflammatory effect of curcumin oil body microcapsules at the cellular level

[0107] Mouse monocyte macrophage RAW264.7 (adherent cells) was obtained from the China Plasmid Bank, and cultured in a cell incubator at 37°C and 5% CO2 using DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin solution. The cells were stimulated with DMEM basal medium (Dalian Melin Biological Technology Co., Ltd.) containing 1 μg / mL lipopolysaccharide (LPS) to construct a pro-inflammatory macrophage model for evaluating the anti-inflammatory effect of curcumin oil body microcapsules.

[0108] The experimental method was as follows: RAW264.7 cells were seeded in 96-well plates at a density of 3.0×10 5The density of 1 cell / hole was inoculated in 12-hole culture plates, incubated in a cell culture incubator for 24 h, and then LPS stimulation was given to add sample solutions (curcumin, demethoxycurcumin, bisdemethoxycurcumin and curcumin oil body microcapsules), and the final concentration of curcumin compounds was 7.5 μmol / L, and then incubated for 24 h. The next day, the cell culture medium was collected and centrifuged at 1000xg for 10 min, and then the content of inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture medium was detected by using an ELISA kit (Wuhan Aibiotek Biological Co., Ltd.) and referring to the method in the instruction manual, and the anti-inflammatory effect of the sample solution was evaluated, and the detection results are shown in Figure 21 LPS stimulation greatly promoted the secretion of IL-6, IL-1β and TNF-α by RAW264.7 cells, which confirmed the successful construction of the pro-inflammatory macrophage model. Curcumin, demethoxycurcumin, bisdemethoxycurcumin and curcumin oil body microcapsules all significantly down-regulated the secretion levels of the three pro-inflammatory cytokines by pro-inflammatory RAW264.7 cells, which confirmed the anti-inflammatory activity of curcumin compounds. At the same concentration, the anti-inflammatory activity of curcumin was slightly stronger than that of demethoxycurcumin and bisdemethoxycurcumin, and the anti-inflammatory effect of curcumin oil body microcapsules was the strongest.

[0109] Next, the Griess method was used to detect the secretion level of NO in the above-mentioned cell culture medium by using a NO kit (Dalian Milen Biotechnology Co., Ltd.). The specific steps are as follows: 50 μL of gradient-diluted standard and sample were added to a 96-well plate, 50 μL of room-temperature Griess Reagent I was added to each well, and then 50 μL of room-temperature Griess Reagent II was added after shaking and mixing, and then the absorbance value was measured at a wavelength of 540 nm, and the content of NO was calculated. The detection results are shown in Figure 24 As shown in the table, curcumin oil body microcapsules still showed the strongest anti-inflammatory effect and effectively inhibited the production of inflammatory mediator NO by pro-inflammatory RAW264.7 cells.

[0110] Example 8. Inhibition effect of curcumin oil body microcapsules on Propionibacterium acnes

[0111] Curcuma longa harvested in February 2024 in Daxing Town, Qianwei County was used as raw material, and curcumin oil body microcapsules with chitosan as the capsule material were prepared according to the method in Example 1, which were used to investigate the inhibition effect of curcumin oil body microcapsules prepared in the present application on Propionibacterium acnes.

[0112] Firstly, the bacteriostatic performance of curcumin oil body microcapsules was evaluated. Propionibacterium acnes freeze-dried powder was purchased from Beina Creative Link Biotechnology Co., Ltd. (strain number: BNCC330605), and 0.5 mL of brain heart infusion broth (BHI) was used to fully dissolve it, which was inoculated on a Columbia blood agar plate (Hibio Biotechnology Co., Ltd.) and incubated in a 37°C anaerobic incubator for 24 h. Then, 3-4 single colonies of Propionibacterium acnes on the blood plate were picked and inoculated in a culture bottle containing BHI liquid medium, and cultured in a shaking incubator under anaerobic conditions at 37°C. When the culture reached the mid-log phase, the bacterial turbidity meter was used to adjust the bacterial liquid concentration to 0.5 McF, which was used in the following experiments. The bacterial liquid was diluted to a certain concentration and inoculated in a 96-well plate, 100 μL of bacterial liquid per well, then different concentrations of curcumin oil body microcapsule solution (10-200 μmol / L) were added, and incubated in an incubator for 48 h. The CCK8 experiment in Example 6 was used to detect the survival rate of bacterial cells. The detection results are shown in Figure 25 Figure 6, the bacteriostatic effect of curcumin is limited, the minimum bacteriostatic concentration is 12.5 μmmol / L, and when the concentration is as high as 200 μmmol / L, there are still 70% of bacteria alive; in contrast, curcumin compounds (curcumin / demethoxycurcumin / bisdemethoxycurcumin = 60 / 20 / 20) have stronger bacteriostatic performance; the bacteriostatic effect of curcumin oil body microcapsules is significantly stronger than that of curcumin and curcumin compounds, and when the concentration is 200 μmmol / L, the bacteriostatic efficiency is as high as 80%. The bacteriostatic performance of curcumin oil body microcapsules is due to its good water solubility and the bacteriostatic effect of the surface capsule material chitosan.

[0113] The crystal violet method was used to investigate the inhibition of curcumin oil body microcapsules on Propionibacterium acnes biofilm. The crystal violet method is a commonly used staining method for measuring and evaluating the formation and adhesion of biofilm, which reflects the formation and adhesion of biofilm by evaluating the number of bacteria on the adhesion surface of the biofilm. First, we investigated the ability of Propionibacterium acnes to form biofilm. The detection results showed that the bacterial liquid with OD value of 0.5 at 600 nm could form a complete biofilm after 92 h of culture in a 96-well plate Figure 26 ). Subsequently, we investigated the inhibition of curcumin oil body microcapsules on Propionibacterium acnes biofilm. The bacterial liquid was inoculated in a 96-well plate, 100 μL of bacterial liquid per well, then different concentrations of curcumin oil body microcapsule solution (0.01-1.0 mmol / L) were added, and incubated for 92 h before crystal violet staining. Finally, 200 μL of 33% glacial acetic acid solution was added to each well at 37°C for 20 min to decolorize, and the absorbance at 595 nm was measured. The bacterial inhibition rate was calculated according to the following formula.

[0114] Biofilm survival rate % = (OD 样品 / OD 空白 ) ×100% (OD空白 OD value of blank control; OD 样品 OD value of sample

[0115] The results are shown in Figure 27 Curcumin could not effectively inhibit biofilm formation, due to the fact that curcumin itself has weak antibacterial property and has limited permeability to biofilm. Curcuminoids (curcumin / demethoxycurcumin / bisdemethoxycurcumin = 60 / 20 / 20) exerted significant biofilm formation inhibition at higher concentrations (≥ 0.5 mmol / L). In contrast, curcumin oil body microcapsules have the strongest biofilm inhibition activity, showing obvious concentration dependence, with a half-inhibitory concentration of about 0.62 mmol / L curcuminoids. The experimental results further demonstrate that the antibacterial property of curcumin oil body microcapsules is due to its good water solubility and the fact that the surface capsule material chitosan also exerts antibacterial effect.

[0116] Example 9. Inhibition of tumor cell growth by curcumin oil body microcapsules

[0117] The CCK8 experiment was used to investigate the in vitro growth inhibition activity of curcumin oil body microcapsules on tumor cells. Two kinds of tumor cells were selected for the experiment, mouse squamous cell carcinoma SCC-7 cells (purchased from Shenzhen Haodihuatuo Biological Technology Co., Ltd.) and human hepatoma HepG2 cells (from the American Type Culture Collection). Among them, the culture conditions of SCC-7 cells were RPMI-1640 complete culture medium (Dalian Milen Biotechnology Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin / streptomycin solution, and the culture conditions of HepG2 cells were high-glucose DMEM culture medium (Dalian Milen Biotechnology Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin / streptomycin solution, and were cultured in an incubator at 37°C and 5% CO2. The experimental method is as follows: the tumor cells were resuspended with culture medium after trypsin digestion, and the cell density was adjusted to 2.5×10 4 3 cells) in a 96-well culture plate, and placed in an incubator for 24 h. Subsequently, the culture medium was replaced with culture medium containing different concentrations of curcumin oil body microcapsules, and the concentration of curcuminoids ranged from 0 to 200 μmol / L. Continue to culture for 48 h, and process according to the instructions of CCK-8 reagent, and finally detect the absorbance of each well at 450 nm wavelength with a microplate reader. Taking the cell survival rate of the control well as 100%, the cell survival rate of each group was calculated (at least three duplicate wells at each concentration, and the average value was calculated), and the column chart was drawn with concentration as the abscissa and cell survival rate as the ordinate. The detection results of SCC-7 cells and HepG2 cells are shown in Figure 27 and Figure 28 ​As shown, the curcumin oil body microcapsules all showed concentration-dependent tumor cell growth inhibition, but the inhibition on SCC-7 cells was significantly stronger than that on HepG2 cells. For example, the half inhibitory concentration of curcumin oil body microcapsules on SCC-7 cells was about 65 μmol / L, while the half inhibitory concentration on HepG2 cells was about 108 μmol / L, which may be due to the drug resistance of liver cancer HepG2 cells.

[0118] Example 10. Oral bioavailability of curcumin oil body microcapsules in rats

[0119] Curcuminoids, as an important active ingredient in turmeric, have various pharmacological effects. However, due to its low water solubility, poor intestinal absorption and rapid metabolism in vivo, the bioavailability is extremely low. References report that there are significant differences in the oral bioavailability of different curcumin preparations (Pharmacokinetics and pharmacodynamics of three oral formulations of curcumin in rats, J. Pharmacokinet. Pharmacodyn., 2020, 47(2): 131-144; Study on the differences in blood drug concentration of different dosage forms of curcumin in rats and the influence on SOD activity, 2018, Master's thesis of Beijing University of Chinese Medicine), and the bioavailability of commercially available curcumin preparations also varies greatly.

[0120] The present application adopts ultra performance liquid chromatography-mass spectrometry (UPLC-MS / MS) to determine curcumin in rat plasma, and investigates the oral bioavailability of curcumin oil body microcapsules. The specific steps are as follows: healthy female SD rats (body weight 250±20 g) are purchased from Beijing Vantoll Life-Science Experimental Animals Technology Co., Ltd., and are adaptively fed for 6 days with free food and water. Then, the rats are divided into 3 groups (6 rats / group), namely, a curcumin compound (control) group, a curcumin oil body microcapsule solution group prepared according to the method in Example 1, and a curcumin oil body microcapsule solid powder group prepared according to the method in Example 4. In the control group, curcumin, demethoxycurcumin and bisdemethoxycurcumin are mixed in a ratio of 60 / 20 / 20, dissolved in dimethyl sulfoxide (DMSO), diluted with distilled water to a final DMSO concentration ≤ 5%, and then administered intragastrically; the curcumin oil body microcapsule solution is administered intragastrically directly, and the curcumin oil body microcapsule solid powder is suspended in distilled water and then administered intragastrically; the dose of curcumin compounds in the three preparations is 250 mg / kg. At 0 min, 10 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h and 12 h after intragastric administration, 0.3 mL of whole blood is collected, the whole blood sample is placed in an EP tube containing heparin, centrifuged at 4℃ and a speed of 5000 rpm for 10 min, and the upper plasma sample is taken and stored in a -80℃ ultra-low temperature freezer. The plasma sample is extracted with methanol to extract curcuminoids, salbutamol is used as an internal standard, and then the sample is detected on the machine. The chromatographic conditions are as follows: an Agilent G6460C UPLC-MS / MS triple quadrupole liquid chromatography tandem system (Agilent Technologies, USA); the chromatographic column is an Agilent Extend-C18 analytical column (RRHD 2.1 mm×50 mm, 1.8 μm); the mobile phase is acetonitrile (A) and 0.1% formic acid water (B), and gradient elution is performed (0 min, 5% A; 0.5 min, 5% A; 1.5 min, 95% A; 5 min, 95% A; 6 min, 5% A; 10 min, 5% A) at a flow rate of 0.2 mL / min; the injection volume is 5 μL; and the column temperature is 30℃. The mass spectrometry conditions are as follows: the ion source is ESI; the mode is MRM-Positive; the spray drying gas temperature is 350℃; the spray gas pressure is 275.8 kPa; the drying gas flow rate is 10 L / min; the capillary voltage is 4 kV; and the retention times of curcuminoids and salbutamol are 3.5-4.3 min and 1.7 min, respectively. The DAS software is used to analyze the blood drug concentration ( C ) and time ( t ), the area under the curve of the drug-time curve ( AUC (0-∞) ) is calculated by the trapezoidal method, and the peak concentration ( C max) for measured values, pharmacokinetic parameters (Table 1) were calculated, mean plasma concentration-time curves (Fig. C - t curves) are shown in Figure 30 .

[0121] Table 1. Pharmacokinetic parameters of turmeric samples (n = 6)

[0122]

[0123] Bioavailability of turmeric oleosome microcapsule solution and turmeric oleosome microcapsule solid powder were 27.0 and 20.4 times that of curcuminoids, respectively, and blood elimination half-life t 1 / 2 was significantly prolonged.

[0124] Example 11. Preparation and application of a hangover gelatin drink of turmeric oleosome microcapsules

[0125] The hangover gelatin drink formula is as follows:

[0126]

[0127] Method: Turmeric oleosome microcapsule solution, pueraria extract, jujube extract, green plum juice, and honey are uniformly mixed, distilled water is added to 50 mL, and finally chia seeds are sprinkled in, and the gelatin is formed after standing for 5 minutes. The formula components are simple, 1 pack is drunk 10 minutes before drinking, and the effect of increasing the amount of alcohol is very obvious.

[0128] Example 12. Preparation and application of a Yizhi (prevention of Alzheimer's disease) solid beverage of turmeric oleosome microcapsules

[0129] The Yizhi (prevention of Alzheimer's disease) solid beverage formula is as follows:

[0130]

[0131] Method: First, mix the microorganism B12 with the plant hydrolyzed peptide according to the equal addition method, and then mix with other ingredients. This formula can prevent Alzheimer's disease through multiple mechanisms, multiple pathways, and multiple targets.

[0132] Example 13. Preparation and application of a acne medical mask of turmeric oleosome microcapsules

[0133] The mask base formula is as follows:

[0134]

[0135] Method: Carbomer was dispersed in deionized water, hyaluronic acid, ceramide, laurocapram were added in turn, homogenized emulsification for 5 min at 3000 rpm, pH was adjusted to about 5.5-6.0 with triethanolamine. Turmeric oil microcapsule solution, salicylic acid-tea tree oil complex, comfrey liposome were added into the above-mentioned matrix in batches, and mixed thoroughly under stirring at a speed of ≤800 rpm (avoiding high-speed shear to destroy the structure), thus the anti-acne mask matrix was obtained.

[0136] Example 14. Turmeric oil microcapsule hepatoprotective tablets

[0137] The formula of the oral care chewable tablets is as follows:

[0138]

[0139] Method: The main drugs (turmeric oil microcapsule solid powder, glycyrrhizic acid ethanol extract, vitamin C, N-acetylcysteine, piperine, inulin) were respectively passed through an 80-mesh sieve, mixed with microcrystalline cellulose, and 5% PVP ethanol solution was added to prepare a soft material, which was granulated through a 14-mesh sieve, dried at 70-80℃, and then the granules were sieved through a 12-mesh sieve. After talc and magnesium stearate were added and mixed, the tablets were pressed, thus the turmeric oil microcapsule hepatoprotective tablets were obtained.

[0140] The above only for the preferred embodiments of the present application, and not for limiting the present application, for those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method of extracting curcuma oleoresin from fresh turmeric, characterized by, The method comprises the following steps: S11, adding the pretreated fresh turmeric to an extraction solution, and performing shearing and crushing to obtain an extraction liquid; the extraction solution is a phosphate buffer solution, a glycine-sodium hydroxide buffer solution, a sodium bicarbonate buffer solution, or a tris-hydrochloride buffer solution; S21, adjusting the pH of the extraction liquid to 4.0-5.0, and adding a composite enzyme system, followed by ultrasonic enzymolysis to obtain an enzymolysis liquid; The composite enzyme system comprises cellulase and pectinase; S31, adjusting the pH of the enzymolysis liquid to 7.0-8.0, followed by inactivation and filtration to obtain a first filtrate and a filter residue; S41, adding a density gradient medium to the first filtrate, followed by low-temperature centrifugal separation to form an isodensity zone, and collecting the upper cream to resuspend to obtain a primary emulsion containing curcuma oil bodies; the density gradient medium comprises sucrose and hydroxypropyl-β-cyclodextrin; S51, adding a surfactant to the primary emulsion, adjusting the pH to 3.0-3.5, and then stirring to reconfigure the interfacial proteins on the surface of the curcuma oil bodies, and then filtering to obtain a second filtrate; S61, centrifuging the second filtrate at low temperature, and collecting the upper cream to resuspend to obtain a curcuma oil body emulsion.

2. The method of extracting curcuma oleoresin from fresh turmeric as claimed in claim 1 wherein, The rotation speed of the shearing and crushing is 20,000-40,000 rpm, and the shearing and crushing time is 25-35 min; and / or, The ultrasonic enzymolysis comprises the following parameters: time is 20-40 min, temperature is 50-60 DEG C, ultrasonic power is 100 W / cm 2 , ultrasonic frequency is 18 kHz, ultrasonic pulse mode is open 3s / close 2s; and / or, The rotation speed of the low-temperature centrifugal separation is 3,000-15,000 rpm, and the low-temperature centrifugal separation time is 30-60 min.

3. The method of extracting curcuma oleoresin from fresh turmeric as claimed in claim 1 wherein, The weight ratio of the fresh turmeric to the extraction solution is 1:1-1:5; and / or, The final concentration of the composite enzyme system in the extraction liquid is 0.5-2.5%; and / or, The final concentration of the density gradient medium in the first filtrate is 1-20%.

4. The method of extracting curcuma oleoresin from fresh turmeric as claimed in claim 3 wherein, The extraction solution is a tris-hydrochloride buffer solution, and the weight ratio of the fresh turmeric to the extraction solution is 1:3; and / or, The composite enzyme system is a composite system of cellulase with a final concentration of 0.5-2.0% in the extraction liquid and pectinase with a final concentration of 0.1-0.5% in the extraction liquid; The density gradient medium is a mixed system of sucrose with a final concentration of 10% in the first filtrate and hydroxypropyl-β-cyclodextrin with a final concentration of 10% in the first filtrate.

5. The method of extracting curcuma oleoresin from fresh turmeric as claimed in claim 1 wherein, The inactivation temperature is 70-90 ℃, and the inactivation time is 5-10 min; and / or, The stirring time is 10-30 min; and / or, The rotation speed of the centrifugation at low temperature is 1,000-5,000 rpm, and the centrifugation time at low temperature is 30-60 min.

6. The method of extracting curcuma oleoresin from fresh turmeric as claimed in claim 1 wherein, The surfactant comprises one or more of sodium deoxycholate, rhamnolipid, sodium stearoyl lactylate, sucrose fatty acid ester, Tween 20, Tween 40, Tween 60, Tween 80, poloxamer F68, and alkyl polyglycoside, and the final concentration of the surfactant in the primary emulsion is 0.5-1.5%.

7. A process for the preparation of curcumin oleobodies microcapsules, characterized by, The method comprises the following steps: S12, adding a capsule material and an antioxidant to the curcumin oil body emulsion prepared by the method of any one of claims 1-6, and obtaining a curcumin oil body microcapsule solution by high-pressure homogenization; the high-pressure homogenization comprises the following parameters: a pressure of 30-80 MPa, a time of 1-5 min, and a number of repetitions of 3-5 times; S22, adding an anti-sticking agent and a flow agent to the curcumin oil body microcapsule solution, and obtaining a curcumin oil body microcapsule solid powder by spray drying.

8. The process for the preparation of curcumin oleoresin microcapsules as claimed in claim 7 wherein, The capsule material comprises one or more of sodium alginate, malt dextrin, gum arabic, isomalto-oligosaccharide, cyclodextrin, inulin, pullulan, chitosan, gelatin, whey protein, casein phosphopeptide, sodium carboxymethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, hydroxypropyl starch, sodium octenyl succinate starch, and polyvinylpyrrolidone, and the final concentration of the capsule material in the curcumin oil body emulsion is 5-20%; and / or, The antioxidant comprises one or more of ascorbic acid, ascorbic acid palmitate, rosemary extract, glutathione, vitamin E, and propyl gallate, and the final concentration of the antioxidant in the curcumin oil body emulsion is ≤0.05%; and / or, The anti-sticking agent comprises one or more of silicon dioxide, magnesium silicate, glyceryl monostearate, sodium tripolyphosphate, hydroxypropyl methyl cellulose, and polyvinylpyrrolidone, and the final concentration of the anti-sticking agent in the curcumin oil body microcapsule solution is ≤1%; and / or, The flow agent comprises one or more of glucose, lactose, fructose, fructooligosaccharide, xylitol, sorbitol, mannitol, trehalose, and microcrystalline cellulose, and the final concentration of the flow agent in the curcumin oil body microcapsule solution is ≤5%.

9. The process for the preparation of curcumin oleoresin microcapsules as claimed in claim 7 wherein, The concentration of curcuminoids in the curcumin oil body microcapsule solution is 10-50 mg / mL; The mass content of curcuminoids in the curcumin oil body microcapsule solid powder is 5-10%.

10. Use of a curcumin oil body emulsion prepared by the method of any one of claims 1 to 6 or a curcumin oil body microcapsule prepared by the method of any one of claims 7 to 9, characterized in that, The curcumin oil body microcapsule solid powder is used as a formulation ingredient for preparing edible and feed products and supplements or cosmetics, or as a natural carrier of hydrophobic drugs and active molecules to prepare a drug-containing formulation.

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