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

Abstract

The invention relates to the technical field of natural product extraction and bionic preparations, and provides a method for extracting curcuma oil from fresh turmeric, a curcuma oil microcapsule and application. The method comprises the following steps: S11, adding pretreated fresh turmeric into an extraction solution, and shearing and crushing; s21, adjusting the pH value of the extracting solution, adding a compound enzyme system, and then carrying out ultrasonic enzymolysis; s31, adjusting the pH value of the enzymatic hydrolysate, and then inactivating and filtering to obtain a first filtrate and filter residues; s41, adding a density gradient medium into the first filtrate, then carrying out low-temperature centrifugal separation, collecting upper-layer cream, and resuspending to obtain primary emulsion containing curcuma oil; s51, adding a surfactant into the primary emulsion, adjusting the pH value, and filtering to obtain a second filtrate; and S61, centrifuging the second filtrate at low temperature, collecting the upper-layer cream, and resuspending to obtain the curcuma oil emulsion. The curcuma oil obtained by the invention has the advantages of high solubility, high stability, high bioavailability and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction and biomimetic preparation, and in particular relates to a method for extracting turmeric oleosome from fresh turmeric, a turmeric oleosome microcapsule and an application thereof. Background Art

[0002] Turmeric (Curcuma longa) is the dried rhizome of the perennial herb Curcuma longa, a member of the Zingiberaceae family. Regarded as one of the most medicinally valuable herbs in ancient Ayurvedic medicine, it is known as "plant gold" and "Indian solid gold" and is an indispensable healing spice. Modern medicine has proven that curcuminoids (also known as curcuminoids) extracted from turmeric, primarily including curcumin, demethoxycurcumin, and bisdemethoxycurcumin, possess antioxidant, anti-inflammatory, liver-protective, lipid-lowering, blood-sugar-lowering, anti-ulcer, cardiovascular-protective, antidepressant, antibacterial, antiviral, antifungal, anti-radiation, and anti-tumor properties. Their application in the healthcare sector has been increasingly widespread in recent years.

[0003] Due to the low solubility of curcuminoids (for example, the solubility of curcumin is only 0.011 mg / mL), existing extraction techniques typically use organic solvents (ethanol, acetone, and ethyl acetate) to extract curcuminoids from dried turmeric powder. However, organic solvent extraction methods have numerous limitations. 1) Organic solvents are toxic and potentially harmful to humans and the environment; 2) they are flammable and explosive, increasing safety risks during operation; 3) organic solvents are expensive, increasing the overall cost of the extraction process; and 4) organic solvent residues may remain, affecting the safety of turmeric extract products.

[0004] In recent years, new extraction technologies have been applied to the extraction of curcuminoids, such as supercritical CO2 extraction and ionic liquid extraction. These technologies offer advantages such as being environmentally friendly, efficient, and highly selective, but they also significantly increase production costs.

[0005] A growing body of research indicates that demethoxycurcumin and bisdemethoxycurcumin offer advantages in stability, water solubility, antioxidant, anti-inflammatory, and anti-tumor activity. This is due to the improved coplanarity of the curcumin molecule after demethoxylation, which allows for easier integration into hydrophobic pockets in DNA and proteins, as well as the exposure of phenolic hydroxyl groups in the molecular structure, which enhances antioxidant properties. The combined application of curcumin, demethoxycurcumin, and bisdemethoxycurcumin is expected to achieve synergistic effects through multi-target coverage, metabolic complementarity, and epigenetic regulation, and holds great potential in disease treatment and health products. Literature reports indicate that the combined use of these three compounds has a stronger inhibitory effect on human osteosarcoma HOS cells than either of them alone, as demonstrated by reduced cell viability, colony formation, and the promotion of apoptosis. However, existing extraction techniques struggle to fully extract the three curcumin compounds.

[0006] In practical applications, curcuminoids suffer from low water solubility, poor physical and chemical stability, rapid gastrointestinal degradation, poor intestinal permeability, and rapid metabolism and elimination. These issues have hindered their application in beverages, pasta products, compound seasonings, and pharmaceuticals. Currently, curcuminoid solubilization techniques include cyclodextrin inclusion solubilization, micellar solubilization, microemulsion solubilization, solid dispersion solubilization, and adsorption solubilization. However, each of these approaches still presents its own challenges. The use of β-cyclodextrin as a stabilizer and processing aid in beverages, dairy products, baked goods, and specialty dietary foods is subject to dosage limits. Release of curcuminoids through in vivo decomposition or competition for cavities may affect their physiological activity. Micellar and microemulsion solubilization require large amounts of surfactants or amphiphilic polymers, which do not meet market demands for "all-natural" formulations. Other approaches also suffer from low complex stability, storage difficulties, complex preparation processes, and high costs.

[0007] Therefore, exploring green, environmentally friendly, safe and efficient extraction technologies that can effectively obtain the three curcuminoids, as well as solubilization methods for curcuminoids that can be applied to the food, cosmetics and pharmaceutical industries, and improving the bioavailability of curcuminoids as food efficacy factors or therapeutic agents are important challenges facing the production and application of turmeric. Summary of the Invention

[0008] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for extracting turmeric oil bodies from fresh turmeric, turmeric oil body microcapsules, and applications thereof. Using fresh turmeric as raw material, an aqueous enzymatic method is employed in combination with ultrasonic cell wall disruption, density gradient centrifugation, and interfacial protein reconstruction to extract oil bodies containing three active ingredients: curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Turmeric oil body microcapsules and their derivatives are then coated with a polymer encapsulation material, exhibiting advantages such as high solubility, high stability, and high bioavailability.

[0009] The present invention is achieved through the following technical solutions: A first object of the present invention is to provide a method for extracting turmeric oil from fresh turmeric, comprising the following steps: S11, adding the pretreated fresh turmeric to an extraction solution, and shearing and crushing it to obtain an extract; the extraction solution is phosphate buffer, glycine-sodium hydroxide buffer, sodium bicarbonate buffer or Tris hydrochloride buffer; S21, adjusting the pH of the extract to 4.0-5.0, adding a complex enzyme system, and then performing ultrasonic enzymatic hydrolysis to obtain an enzymatic solution; the complex enzyme system comprises: one or more of cellulase, xylanase, arabinanase, pectinase, laccase and snailase; S31, adjusting the pH of the enzymatic hydrolyzate to 7.0-8.0, then inactivating and filtering to obtain a first filtrate and a filter residue; S41, adding a density gradient medium to the first filtrate, and then performing low-temperature centrifugation to form isopycnic zones, collecting the upper layer of cream and resuspending it to obtain a primary emulsion containing turmeric oil, wherein the density gradient medium comprises one or more of sucrose, glucose, polysucrose, dextran, hyaluronic acid, glycerol, ethylene glycol, isopropyl alcohol, polyethylene glycol, and cyclodextrin derivatives; S51, adding a surfactant to the primary emulsion, adjusting the pH to 3.0-3.5, and then stirring to reconstruct the interfacial protein on the surface of the turmeric oil body, and filtering to obtain a second filtrate; S61. Centrifuge the second filtrate at low temperature, collect the upper layer of cream, and resuspend it to obtain turmeric oil emulsion.

[0010] Plant oil bodies are micron- or submicron-sized (0.5-2 μm) subcellular organelles that represent nature's solution for managing immiscible two-phase solutions. Oil bodies are oil-in-water (O / W) emulsions composed of triglycerides encapsulated by a monolayer of phospholipid-interfacial protein membranes. They play important roles in plant growth, development, and metabolism, including energy storage, oxidative buffering, signal transduction, pathogen resistance, and cryoprotection. Extracting plant oil bodies in their natural form directly yields O / W emulsions, offering promising applications in food, cosmetics, and pharmaceuticals. The advantages of extracting plant oil bodies to produce derivatives include: They typically utilize aqueous or aqueous enzymatic methods, eliminating the use of organic solvents and are environmentally friendly and safe. The extraction process is simple, eliminating numerous traditional extraction steps. As natural emulsions, oil bodies are stable in aqueous phases, eliminating the need for additional emulsifiers or homogenization. Active ingredients encapsulated within or encapsulated within these emulsions typically exhibit excellent water solubility, stability, and bioavailability. The unique structural characteristics of plant oil bodies, shaped over millennia of evolution, have opened up new avenues for their advanced applications.

[0011] The present invention discovers for the first time that curcuminoids, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin, are present in turmeric oil bodies. Therefore, the present invention provides a method and application for directly extracting turmeric oil bodies containing curcuminoids from fresh turmeric. The method provided herein utilizes an aqueous enzymatic method combined with ultrasonic cell wall disruption, density gradient zonal centrifugation, and interfacial protein reconstruction to extract oil bodies containing the three active ingredients, curcumin, demethoxycurcumin, and bisdemethoxycurcumin. These oil bodies are then coated with polymer encapsulation materials to prepare turmeric oil body microcapsules and their derivatives.

[0012] Specifically, the present invention first pre-treats fresh turmeric, washing it to remove impurities such as mud and sand, and draining it. This step prepares for subsequent extraction. Removing impurities prevents interference with the extraction process and ensures the purity of the extract. The treated fresh turmeric is then added to a specific extraction solution, which can be selected from phosphate buffer, glycine-sodium hydroxide buffer, sodium bicarbonate buffer, or Tris-HCl buffer. Phosphate buffer is preferred because it maintains a certain osmotic pressure and maintains the stability of the oil body membrane. After mixing the fresh turmeric and the extraction solution in a weight ratio of 1:1 to 1:5 (preferably 1:3), the mixture is sheared and crushed at a speed of 20,000 to 40,000 rpm for 25 to 35 minutes. The high speed shearing and crushing quickly breaks up the fresh turmeric, initially disrupting the cellular structure and facilitating the subsequent extraction solution's entry into the cells and contact with the turmeric oil body.

[0013] The pH of the extract obtained after crushing needs to be adjusted to 4.0-5.0. This acidic environment is suitable for the complex enzyme system to work. The complex enzyme system contains one or more of cellulase, xylanase, arabinanase, pectinase, laccase and snailase, preferably 0.5-2.0% cellulase and 0.1-0.5% pectinase complex system, and the final concentration in the extract is 0.5-2.5%. Subsequently, enzymatic hydrolysis is carried out with the assistance of ultrasound, and the ultrasonic parameters are set to a power of 100W / cm 2 The frequency is 18kHz, the pulse mode is on for 3s / off for 2s, the enzymatic hydrolysis time is 20-40 minutes, and the temperature is controlled at 50-60°C. The ultrasonic penetration and cavitation effect can accelerate the enzymatic hydrolysis process, allowing the enzyme to act more efficiently on structures such as cell walls, greatly shortening the enzymatic hydrolysis time. Efficient cell wall breakdown can be achieved in about 30 minutes, releasing the turmeric oil bodies inside the cells into the extract while ensuring the integrity of the oil body structure and function.

[0014] After enzymatic hydrolysis is complete, the pH of the hydrolyzate is adjusted to 7.0-8.0, then inactivated at 70-90°C for 5-10 minutes to inactivate the complex enzyme system and prevent it from further affecting the components. After inactivation, the solid residue in the hydrolyzate is filtered through 200-mesh gauze to separate the liquid from the solid residue, yielding the first filtrate and the filter residue. The filter residue can then be dried, crushed, and sieved to produce turmeric powder, achieving high-value utilization of turmeric. The first filtrate is then used to extract turmeric oil.

[0015] A density gradient medium is added to the first filtrate, comprising one or more of sucrose, glucose, polysucrose, dextran, hyaluronic acid, glycerol, ethylene glycol, isopropyl alcohol, polyethylene glycol, and cyclodextrin derivatives, with the final concentration controlled at 1-20%. A mixture of 10% sucrose and 10% hydroxypropyl-β-cyclodextrin is preferred. Hydroxypropyl-β-cyclodextrin can precisely control the sucrose density gradient, acting as a lubricant to prevent the oil bodies from being destroyed during centrifugation and nonspecific adsorption on their surfaces. After uniform mixing, the mixture is centrifuged at low temperature for 30-60 minutes at a speed of 3000-15000 rpm to form isopycnic zones. Because the density of turmeric oil bodies differs from that of other components, they aggregate in specific areas, thereby collecting the upper layer of cream and resuspending it in an aqueous solution to obtain a primary emulsion containing turmeric oil bodies.

[0016] A surfactant is added to the primary emulsion. The surfactant can be selected from one or more of sodium deoxycholate, rhamnolipid, sodium stearoyl lactylate, sucrose fatty acid esters, Tween 20, Tween 40, Tween 60, Tween 80, poloxamer F68, and alkyl polyglycosides. The final concentration in the primary emulsion is 0.5-1.5%, preferably Tween 80. Simultaneously, the pH is adjusted to 3.0-3.5, and the mixture is stirred for 10-30 minutes. During this process, the surfactant not only precipitates impurities but also competitively displaces interfacial proteins on the surface of the oil body phospholipid membrane, forming a more hydrophilic phospholipid-surfactant-interfacial protein hybrid membrane. This membrane stabilizes the oil bodies and acts as an adsorbent material, thereby reconstructing the interfacial proteins on the surface of the turmeric oil body. The mixture is then filtered to remove the protein precipitate, yielding a second filtrate.

[0017] The second filtrate is centrifuged at low temperature for 30 to 60 minutes at a speed of 1000 to 5000 rpm. Due to its different density from other impurities, the highly purified turmeric oil will accumulate in the upper layer to form a creamy paste. The upper creamy layer is collected and resuspended in water to obtain a high-purity turmeric oil emulsion. This emulsion appears as a thick yellow O / W emulsion, which becomes a slightly opalescent solution upon dilution with an aqueous solution. The concentration of curcuminoids in the turmeric oil emulsion is 10 to 50 mg / mL, preserving the natural ratio of the three active ingredients in turmeric: curcumin, demethoxycurcumin, and bisdemethoxycurcumin.

[0018] Furthermore, the shearing and crushing speed is 20000-40000 rpm, and the shearing and crushing time is 25-35 min; and / or, The ultrasonic enzymatic hydrolysis parameters include: time of 20 to 40 min, temperature of 50 to 60°C, ultrasonic power of 100 W / cm 2 , the ultrasonic frequency is 18 kHz, the ultrasonic pulse mode is on 3s / off 2s; and / or, The rotation speed of the low-temperature centrifugal separation is 3000-15000 rpm, and the time of the low-temperature centrifugal separation is 30-60 minutes.

[0019] Furthermore, 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, The complex enzyme system is a complex system of cellulase with a final concentration of 0.5-2.0% in the extract and pectinase with a final concentration of 0.1-0.5% in the extract; 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.

[0020] Furthermore, the inactivation temperature is 70-90° C., and the inactivation time is 5-10 minutes; and / or, The stirring time is 10 to 30 minutes; and / or, The rotation speed of the centrifugation at low temperature is 1000-5000 rpm, and the time of the centrifugation at low temperature is 30-60 minutes.

[0021] Furthermore, the surfactant includes 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 polysaccharide, and the final concentration of the surfactant in the primary emulsion is 0.5-1.5%.

[0022] The third object of the present invention is to provide an application of a turmeric oil emulsion or turmeric oil microcapsule for preparing dietary supplements, edible and feed products and their supplements, nutritional supplements, spices, condiments, brewing or cosmetic preparation ingredients, or as a natural carrier form of hydrophobic drugs and active molecules for preparing preparations containing drug ingredients.

[0023] The present invention also provides a turmeric oil microcapsule solution containing curcuminoids prepared by the above extraction method, which has the appearance of a yellow O / W thick emulsion, the concentration of the curcuminoids is 10 to 50 mg / mL, and can maintain the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric. After dilution with water, it becomes a solution with a slight opalescence.

[0024] The present invention also provides a spray-dried product of turmeric oil microcapsules containing curcuminoids prepared by the extraction method. The product is a yellow solid powder, and the content of curcuminoids is 5-10%. The product can maintain the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric, is well soluble in warm water or cold water, and presents a solution with a slight opalescence.

[0025] The present invention also provides applications of turmeric oil, its microcapsule solution, and spray-dried solid powder, which can be used as ingredients in human or animal diets or edible and feed products and supplements (functional food preparations, i.e., food, beverages, feed, or pet food, or supplements of food, beverages, feed, or pet food), nutritional supplements, spices or condiments, winemaking, or cosmetic preparations. They can also be used as a natural carrier form for hydrophobic drugs and active molecules, exerting effects such as solubilization and improving bioavailability, and preparing preparations containing drug / veterinary drug ingredients.

[0026] The beneficial effects of the present invention are: (1) The aqueous enzymatic method is used to extract turmeric oil from fresh turmeric, which can avoid the harm to the environment and human body caused by the use of organic solvents in traditional extraction processes and has the advantage of being green and environmentally friendly.

[0027] (2) The combined ultrasonic crushing process can greatly shorten the enzymatic wall breaking time, and efficient wall breaking can be achieved in 30 minutes, releasing turmeric oleosomes with complete structure and function.

[0028] (3) The advantages of using a mixed medium of sucrose and hydroxypropyl-β-cyclodextrin for density gradient zonal centrifugation to extract turmeric oil bodies include: 1) good separation effect, and relatively pure turmeric oil bodies can be obtained; 2) the oil bodies will not be squeezed and deformed during the centrifugation process, which can maintain the integrity of the oil body morphology and function, and also help prevent the formed zones from mixing due to convection.

[0029] (4) Surfactants are used to precipitate foreign proteins and competitively displace the interfacial proteins on the surface of the oil body phospholipid membrane, forming a more hydrophilic phospholipid-surfactant-interfacial protein mixed membrane. This process of reconstructing turmeric oil body membrane proteins can significantly improve the stability of turmeric oil bodies and facilitate subsequent encapsulation.

[0030] (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.

[0031] (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.

[0032] (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.

[0033] (8) The oral bioavailability of curcuminoids in turmeric oil microcapsules is 20 to 30 times that of the same dose of curcumin.

[0034] (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.

[0035] (10) It has a wide range of applications, including various uses in food, cosmetics and biomedicine.

[0036] (11) The process is easy to control and suitable for industrial production.

[0037] (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

[0038] 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.

[0039] Figure 1 A physical image of the turmeric extract after enzymatic hydrolysis provided in Example 1 of the present invention; Figure 2 A physical image of the turmeric extract after Tween 80 treatment provided in Example 1 of the present invention; Figure 3 A physical picture of the creamy turmeric oil collected by gradient centrifugation provided in Example 1 of the present invention; Figure 4 A physical picture of the turmeric oil microcapsule concentrated solution provided in Example 1 of the present invention; Figure 5 This is a diagram of the Tyndall phenomenon of the turmeric oil microcapsule solution after dilution provided in Example 2 of the present invention; Figure 6 A histogram showing the particle size distribution of turmeric oil microcapsules provided in Example 2 of the present invention; Figure 7 This is a morphology image of turmeric oil microcapsules observed under a microscope provided in Example 2 of the present invention; Figure 8 This is a fluorescence microscopy image of turmeric oil microcapsules after Nile red staining provided in Example 2 of the present invention; Figure 9 This is a morphology of turmeric oleosome microcapsules observed under a cryo-electron microscope provided in Example 2 of the present invention; Figure 10 HPLC chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric oil microcapsules prepared from fresh turmeric provided in Example 2 of the present invention; Figure 11 HPLC chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin after drying of fresh turmeric provided in Example 2 of the present invention; Figure 12 HPLC chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric oil microcapsules prepared from fresh turmeric provided in Example 3 of the present invention; Figure 13 HPLC chromatogram of curcumin, demethoxycurcumin and bisdemethoxycurcumin after drying of fresh turmeric provided in Example 3 of the present invention; Figure 14 This is a protein band diagram of the turmeric oil microcapsules provided in Example 3 of the present invention after gel electrophoresis and Coomassie Brilliant Blue staining; Figure 15 This is a photo of the spray-dried solid powder of turmeric oil microcapsules provided in Example 4 of the present invention; Figure 16 These are photos of solutions at different concentrations after resuspension of the turmeric oil microcapsule solid powder provided in Example 4 of the present invention; Figure 17 This is a graph showing the scavenging performance of turmeric oil microcapsules on DPPH free radicals provided in Example 5 of the present invention; Figure 18 This is a graph showing the scavenging performance of turmeric oil microcapsules on ABTS free radicals provided in Example 5 of the present invention; Figure 19 This is a graph showing the scavenging performance of turmeric oil microcapsules on reactive oxygen species free radicals in NIH / 3T3 cells provided by Example 6 of the present invention; Figure 20 This is a graph showing the resistance of turmeric oil microcapsules to oxidative damage in NIH / 3T3 cells provided in Example 6 of the present invention; Figure 21 This is a graph showing the inhibitory effect of turmeric oil microcapsules on interleukin-6 secretion by pro-inflammatory RAW264.7 cells provided in Example 7 of the present invention; Figure 22 This is a graph showing the inhibitory effect of turmeric oil microcapsules on interleukin-1β secretion by pro-inflammatory RAW264.7 cells provided in Example 7 of the present invention; Figure 23 This is a graph showing the inhibitory effect of turmeric oil microcapsules on the secretion of tumor necrosis factor-α by pro-inflammatory RAW264.7 cells provided in Example 7 of the present invention; Figure 24This is a graph showing the inhibitory effect of turmeric oil microcapsules on the secretion of nitric oxide (NO) by pro-inflammatory RAW264.7 cells provided in Example 7 of the present invention; Figure 25 This is a graph showing the inhibitory effect of turmeric oil microcapsules on the growth of Propionibacterium acnes provided in Example 8 of the present invention; Figure 26 This is a graph showing the ability of Propionibacterium acnes to grow and form biofilms, as provided in Example 8 of the present invention; Figure 27 This is a graph showing the inhibitory effect of turmeric oil microcapsules on Propionibacterium acnes biofilm provided in Example 8 of the present invention; Figure 28 This is a graph showing the inhibitory effect of turmeric oil microcapsules on the growth of mouse squamous cell carcinoma SCC-7 cells provided in Example 9 of the present invention; Figure 29 This is a graph showing the inhibitory effect of turmeric oil microcapsules on the growth of human liver cancer HepG2 cells provided in Example 9 of the present invention; Figure 30 This is a blood drug concentration-time curve of the turmeric oil microcapsules provided in Example 10 of the present invention in rats after oral administration. DETAILED DESCRIPTION

[0040] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0041] Example 1. Preparation of turmeric oil microcapsule solution Fresh turmeric harvested in Daxing Town, Qianwei in February 2024 was rinsed with clean water to remove sediment and surface impurities, drained, and cut into pieces. 100 g was weighed and added to 300 mL of pre-prepared tris(hydroxymethyl)aminomethane hydrochloride buffer (0.05 mol / L, pH 7.8). The pieces were then broken at 20,000 rpm in a Joyoung blender. Cellulase (Dongheng Huadao Biotechnology Co., Ltd.) and pectinase (Dongheng Huadao Biotechnology 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 pieces were enzymatically broken at 50°C for 30 min under ultrasonic conditions (power 100 W / cm², frequency 18 kHz; pulse mode on 3 s / off 2 s). After cooling to room temperature, the pieces were filtered through 200-mesh gauze, the residue discarded, and the filtrate collected, which was a dark yellow emulsion ( Figure 1 ).

[0042] Add surfactant Tween 80 to the emulsion to a final concentration of 1.0%, then adjust the pH to about 3.5, stir at 4°C for 2 h, and after standing, a small amount of white flocculent precipitate can be clearly observed at the bottom of the container ( Figure 2 ), which is 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 fully dissolve and form a density gradient. The mixture was then centrifuged at 8000 rpm for 30 minutes, and a yellow cream layer appeared on the upper layer ( Figure 3 ). The cream was collected, washed with distilled water and centrifuged at 3000 rpm for 30 min. The upper purified turmeric oil was dispersed in 50 mL of distilled water. Maltodextrin, gum arabic and β-cyclodextrin (mass ratio of 3:1:1) were added as capsule materials with a mass concentration of about 5%. At the same time, ascorbyl palmitate ethanol solution was added as an antioxidant with a mass concentration of 0.05%. The capsules were prepared by high-pressure homogenization. High-pressure homogenization parameters: HL2000 high-pressure homogenizer (Shanghai Hongli Biotechnology Co., Ltd.); pressure 50 MPa; 2 min each time, intermittent treatment 3 times; temperature ≤ 25°C. The treated turmeric oil microcapsule solution was a thick yellow emulsion ( Figure 4 ).

[0043] Example 2. Characterization of turmeric oil microcapsule solution Take the turmeric oil microcapsule solution, dilute it 20 times with distilled water, and irradiate it with a laser pen to observe the obvious Tyndall phenomenon ( Figure 5 ), confirming the colloidal dispersion of turmeric oil microcapsules. The particle size and distribution of turmeric oil microcapsules were detected by dynamic laser scattering method. The average particle size was 1.19±0.146 μm and the polydispersity coefficient was 0.173±0.013 ( Figure 6 ), proving that the turmeric oil microcapsules are evenly distributed. Under an optical microscope, the turmeric oil microcapsules showed regular spherical morphology and were evenly distributed ( Figure 7 Turmeric oil microcapsules were labeled with the lipophilic fluorescent dye Nile red, and uniformly distributed red emulsion droplets were observed under a laser confocal microscope ( Figure 8 ), confirming the hydrophobic core of the turmeric oil microcapsules. The surface morphology of the turmeric oil microcapsules was observed under a cryo-scanning electron microscope, and it was found that the capsule material was evenly coated on the surface of the turmeric oil to form a protective layer ( Figure 9 ).

[0044] The turmeric oil microcapsule solution was freeze-dried and then subjected to the following experiments. The triglyceride content in the turmeric oil microcapsules was quantitatively determined using an enzymatic colorimetric method (the kit was purchased from Beijing Solaibao Technology Co., Ltd.). The average content of the three measurements was 42.3 ± 8.25%. Curcuminoids were then extracted with methanol, and the content of curcuminoids in the turmeric oil microcapsules was determined by high-performance liquid chromatography (HPLC). HPLC parameters included an Agilent 1260 HPLC instrument, a Diamonsil C18 column (5 μm; 250 × 4.6 mm), a column temperature of 35°C, a flow rate of 1 mL / min, a detection wavelength of 430 nm, an injection volume of 10 μL, and a mobile phase of acetonitrile:0.3% glacial acetic acid (47:53). HPLC analysis revealed a curcuminoid content of 15.0 ± 3.2%, with a curcumin:demethoxycurcumin:bisdemethoxycurcumin ratio of 55:21:24 ( Figure 10 At the same time, the content of curcuminoid compounds in fresh turmeric after drying was detected according to the turmeric content determination method of the 2020 edition of the Chinese Pharmacopoeia. The HPLC test result was 3.52±0.25%, of which the ratio of curcumin: demethoxycurcumin: bisdemethoxycurcumin was 55:22:23 ( Figure 11 ). It can be seen that the turmeric oil microcapsule product prepared according to the extraction method of the present invention can maintain the natural ratio of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the turmeric medicinal material.

[0045] Example 3. Preparation and characterization of turmeric oil microcapsule solution Fresh turmeric harvested in Daxing Town, Qianwei in February 2025 was used to prepare a turmeric oil microcapsule solution using the method of Example 1. The content and ratio of the three curcuminoid compounds in the turmeric oil microcapsules and the dried fresh turmeric were then detected using the method of Example 2. HPLC test results showed that the content of curcuminoid compounds in the turmeric oil microcapsules was 12.8±1.9%, with the ratio of curcumin:demethoxycurcumin:bisdemethoxycurcumin being 62:20:18 ( Figure 12 The content of curcuminoids in dried fresh turmeric was 3.28±0.36%, of which the ratio of curcumin: demethoxycurcumin: bisdemethoxycurcumin was 62:21:17 ( Figure 13). It can be seen that the turmeric oil microcapsules still maintain the natural ratio of curcumin, demethoxycurcumin, and bisdemethoxycurcumin in turmeric medicinal materials. In addition, compared with fresh turmeric harvested in 2024, the content of curcuminoids in fresh turmeric medicinal materials harvested in 2025 was slightly lower, and the ratio of the three curcuminoids changed, mainly reflected in a significant decrease in the proportion of bisdemethoxycurcumin. Compared with turmeric medicinal materials from other origins, the ratio of demethoxycurcumin to bisdemethoxycurcumin in Qianwei turmeric is higher. For example, the literature (Determination of the contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric extract, Journal of Traditional Chinese Medicine, 2015, 30(205): 853-855) reported that the average contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric extract were 65.0%, 16.0% and 12.3%, respectively, and the calculated ratio of the three was 70:17:13.

[0046] Accurately weigh the dried sample of turmeric oleosome microcapsules and lyse them with Cocktail / SDS lysis buffer (Sigma-Aldrich, USA) according to the instructions. Collect the supernatant protein solution and test the protein concentration. Heat the protein sample in a water bath at 97°C for 10 min. After cooling, separate it by SDS-PAGE gel electrophoresis. Be sure to add a protein marker as a reference. After the electrophoresis is completed, remove the gel and treat it with Coomassie Brilliant Blue R250 staining solution for protein staining until the protein bands are clearly visible. Figure 14 As shown, protein bands within the turmeric oil microcapsule sample are located in the molecular weight range of 10-35 kDa. The primary proteins are lipid transporters (approximately 10 kDa, which facilitate the interfacial transfer of phosphatidylcholine). Small amounts of oleosins (15-20 kDa, which maintain the structure and stability of lipid droplets in plant cells and participate in the dynamic regulation of lipid metabolism) and calcooleosins (25-35 kDa, which participate in calcium signaling and lipid droplet dynamics) are also present. These proteins, located at the oil-water interface, are associated with the structure, stability, and function of lipid droplets in turmeric cells. Their successful retention on the surface of the turmeric oil microcapsules, forming a protein shell, contributes to the stability of the microcapsule structure. Furthermore, the precipitated proteins are primarily proteins with molecular weights greater than 60 kDa, including cellulases with bands between 60 and 75 kDa. Proteins above 100 kDa include transmembrane transporters, key enzymes, and cytoskeletal proteins in turmeric cells.

[0047] Example 4. Preparation and characterization of spray-dried solid powder of turmeric oil microcapsules Fresh turmeric harvested in Daxing Town, Qianwei in February 2025 was used to prepare a turmeric oil microcapsule solution using the method of Example 1. Lactose and microcrystalline cellulose were then added to a final concentration of ≤ 5%. Silicon dioxide and hydroxypropyl methylcellulose were also added at a dosage of ≤ 1. The amount of microcrystalline cellulose added was much lower than that of lactose, otherwise it would affect the solubility of the spray-dried turmeric oil microcapsules. The turmeric oil microcapsule solution was spray-dried using a B-290 mini spray dryer (BUCHI Labortechnik AG, Switzerland) to obtain a solid powder of turmeric oil microcapsules ( Figure 15 ). The spray drying parameters are as follows: inlet temperature 150℃; outlet temperature 90℃; power 85 W; nozzle pressure 2 MPa; injection speed 8-10 mL / min; atomization pressure 3-4 MPa. Accurately weigh the turmeric oil microcapsule powder, extract the curcuminoids with methanol, and use the HPLC method in Example 2 to detect the content of curcuminoids, which is 9.6±1.1%. Disperse the turmeric oil microcapsule solid powder in distilled water to prepare microcapsule solutions of different concentrations. Figure 16 As shown, the solution exhibited a clear orange-yellow color and no obvious precipitation was observed, which confirmed that the turmeric oil microcapsule solid powder prepared by the present invention had good water solubility and was a solid beverage nutritional supplement with excellent properties.

[0048] Example 5. Antioxidant properties of turmeric oil microcapsules An in vitro free radical scavenging experiment was conducted to investigate the antioxidant properties of the turmeric oil microcapsule solution prepared according to the method described in Example 3. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a deep purple free radical that converts to a colorless, stable form upon reaction with antioxidants. Therefore, it is widely used in antioxidant testing. To a 1 mL reaction system, 0.5 mL of sample solution (curcumin and turmeric oil microcapsule solution) at varying concentrations and 0.5 mL of DPPH ethanol solution were added. The reaction was incubated in the dark for approximately 10 minutes. The absorbance of DPPH at a wavelength of 517 nm was then measured using a UV-visible spectrophotometer, and the DPPH radical scavenging rate was calculated according to the following formula.

[0049] 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 DPPH solution (background control); A x : absorbance of the sample solution and DPPH reaction system) Test results such as 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, due to their good water solubility and the presence of demethoxycurcumin and bisdemethoxycurcumin.

[0050] 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 carried out 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.

[0051] 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) 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.

[0052] Example 6. ROS scavenging performance of turmeric oil microcapsules at the cellular level and their ability to resist oxidative damage Mouse embryonic fibroblast NIH / 3T3 cells (American Type Culture Collection) were cultured in RPMI-1640 complete medium (Dalian Meilun Biotechnology Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin / streptomycin solution at 37°C in a cell culture incubator with 5% CO2. The cells were stimulated with 1 mmol / L H2O2 to construct an oxidative stress cell model to evaluate the ROS scavenging performance of turmeric oil microcapsules and their ability to resist cell oxidative damage.

[0053] First, the ROS fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to detect the ROS level in cells and evaluate the antioxidant activity at the cellular level. The experimental method was as follows: 3×10 5 The cells were seeded in a 12-well culture plate at a density of 100 cells / well, incubated in an incubator for 24 h, and then stimulated with RPMI-1640 medium containing H2O2 for 1 h. Subsequently, different concentrations of turmeric oil microcapsules (0-25 μmol / L) were added and incubated overnight. The next day, 1 mL of DCFH-DA working solution was added to each well and incubated for 30 minutes. The culture medium was discarded, the cells were collected by centrifugation, washed with pH 7.4 phosphate buffer, and finally 300 μL of phosphate buffer was added to resuspend the cell pellet, filtered through a 200-mesh nylon mesh, and finally an Accuri™ C6 flow cytometer (BD Company, USA) was used to detect the fluorescence signal intensity of 2,7-dichlorofluorescein (DCF) generated in the cells to evaluate the level of intracellular ROS. The test results are shown in Figure 19 As shown. H2O2 stimulation greatly increased the ROS level in NIH / 3T3 cells, indicating the successful construction of the oxidative stress cell model. Turmeric oil microcapsules were able to downregulate ROS levels in oxidative stress model cells in a concentration-dependent manner, indicating that turmeric oil microcapsules have a strong ability to scavenge intracellular ROS. Due to the hydrophobicity of curcumin, it is difficult to penetrate the cell membrane and enter the cell, so its ROS scavenging effect in oxidative stress model cells is not significant (detailed data not provided). It can be seen that the excellent antioxidant properties of turmeric oil microcapsules are due to the water solubility of curcuminoids and the fact that bisdemethoxycurcumin and demethoxycurcumin have stronger antioxidant capacity than curcumin.

[0054] Next, CCK8 assay was used to evaluate the ability of turmeric oil microcapsules to protect NIH / 3T3 cells from H2O2 oxidative stimulation. 4The cells were seeded into 96-well culture plates at a density of cells / well, placed in an incubator and cultured for 24 hours, and then stimulated with RPMI-1640 medium containing H2O2 for 1 hour. Subsequently, complete culture medium containing different concentrations of turmeric oil microcapsules was added, in which the concentration of turmeric compounds ranged from 0-100 μmol / L, and incubation continued for 48 hours. The culture medium was discarded and treated with culture medium containing CCK-8 reagent for 1.5 hours. Finally, the absorbance value of each well was detected at a wavelength of 450 nm using a Multiskan FC microplate reader (ThermoScientific, USA). The cell survival rate in the control wells without H2O2 stimulation and turmeric oil microcapsule administration was 100%, and the cell survival rate was calculated by the ratio of the absorbance values ​​of other groups to the control group. The test results are shown in Figure 20 As shown. H2O2 stimulation significantly inhibited the in vitro growth of NIH / 3T3 cells, indicating that it caused oxidative damage to the cells. Turmeric oil microcapsules showed a concentration-dependent cell growth-promoting effect in the curcuminoid concentration range of 2.5-15 μmol / L, which was attributed to the successful removal of intracellular ROS. When the concentration of curcuminoids was greater than 20 μmol / L, the promoting effect of curcumin microcapsules on the in vitro growth of oxidatively damaged cells began to weaken. This is because curcuminoids have multi-target effects and exhibit bidirectional regulatory activity in cells. Low concentrations promote cell growth and repair, while high concentrations may inhibit cell proliferation.

[0055] Example 7. Anti-inflammatory effects of turmeric oil microcapsules at the cellular level Mouse mononuclear macrophage RAW264.7 cells (adherent cells) were obtained from the China Plasmid Vector, Bacteria, Cell Protein, and Antibody Gene Collection and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin solution in a cell culture incubator at 37°C and 5% CO₂. A pro-inflammatory macrophage model was constructed by stimulating the cells with DMEM basal medium (Dalian Meilun Biotechnology Co., Ltd.) supplemented with 1 µg / mL lipopolysaccharide (LPS) to evaluate the anti-inflammatory effects of turmeric oil microcapsules.

[0056] The experimental method is as follows: RAW264.7 cells were cultured at 3.0×10 5Cells were seeded at a density of 1000 μmol / well in a 12-well culture plate and incubated in a cell culture incubator for 24 h. After LPS stimulation, sample solutions (curcumin, demethoxycurcumin, bisdemethoxycurcumin, and turmeric oil microcapsules) were added. The final concentration of curcuminoids was 7.5 μmol / L, and the culture was continued for 24 h. The next day, the cell culture medium was collected and centrifuged at 1000 × g for 10 min. The inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the cell culture medium were then detected using an ELISA kit (Wuhan Aibote Biotechnology Co., Ltd.) according to the instructions. The anti-inflammatory effect of the sample solution was evaluated. The test results are shown in Figure 2. Figure 21 As shown in Figure 23, LPS stimulation significantly promoted the secretion of IL-6, IL-1β, and TNF-α by RAW264.7 cells, confirming the successful establishment of a proinflammatory macrophage model. Curcumin, demethoxycurcumin, bisdemethoxycurcumin, and turmeric oil microcapsules all significantly downregulated the secretion of these three proinflammatory cytokines by proinflammatory RAW264.7 cells, confirming the anti-inflammatory activity of curcuminoids. At the same concentration, curcumin exhibited slightly stronger anti-inflammatory activity than demethoxycurcumin and bisdemethoxycurcumin, while turmeric oil microcapsules exhibited the strongest anti-inflammatory effect.

[0057] Next, the Griess method was used to detect the NO secretion level in the above cell culture medium using a NO kit (Dalian Meilun Biotechnology Co., Ltd.). The specific steps are as follows: 50 μL of the serially 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 after vortexing and mixing, 50 μL of room temperature Griess Reagent II was added. The absorbance value was then measured at a wavelength of 540 nm to calculate the NO content. The test results are shown in Figure 2. Figure 24 As shown in the results, turmeric oil microcapsules still showed the strongest anti-inflammatory effect and effectively inhibited the production of inflammatory mediator NO in pro-inflammatory RAW264.7 cells.

[0058] Example 8. Inhibitory effect of turmeric oil microcapsules on Propionibacterium acnes Turmeric harvested in Daxing Town, Qianwei in February 2024 was used as raw material, and turmeric oil microcapsules with chitosan as the capsule material were prepared according to the method in Example 1 to investigate the inhibitory effect of the turmeric oil microcapsules prepared by the present invention on Propionibacterium acnes.

[0059] First, the antibacterial properties of turmeric oil microcapsules were evaluated. Propionibacterium acnes freeze-dried powder was purchased from Beina Chuanglian Biotechnology Co., Ltd. (strain number: BNCC330605), fully dissolved with 0.5 mL brain heart infusion broth (BHI), inoculated on Columbia blood agar plates (Haibo Biotechnology Co., Ltd.), and placed in an incubator at 37°C for anaerobically incubated for 24 h. Then, 3-4 single colonies of Propionibacterium acnes from the blood plate were picked and inoculated into culture bottles containing BHI liquid culture medium, and cultured on a shaker under anaerobic conditions at 37°C. When cultured to the mid-logarithmic phase, the bacterial solution concentration was adjusted to 0.5 McF using a bacterial turbidimeter for the following experiments. The bacterial solution was diluted to a certain concentration and inoculated into a 96-well plate, with 100 μL of bacterial solution per well, and then different concentrations of turmeric oil microcapsule solution (10-200 μmol / L) were added and placed in an incubator for 48 h. The survival rate of bacterial cells was detected using the CCK8 experiment in Example 6. The test results are as follows. Figure 25 As shown, curcumin has limited antibacterial activity, with a minimum inhibitory concentration of 12.5 μmmol / L. Even at concentrations as high as 200 μmmol / L, 70% of bacteria still survived. In contrast, curcuminoids (curcumin / demethoxycurcumin / bisdemethoxycurcumin = 60 / 20 / 20) exhibited stronger antibacterial properties. Turmeric oil microcapsules exhibited significantly stronger antibacterial activity than curcumin and curcuminoids, achieving an antibacterial efficiency of 80% at a concentration of 200 μmmol / L. The antibacterial activity of turmeric oil microcapsules is attributed to their good water solubility and the antibacterial effect of the chitosan surface material.

[0060] The crystal violet method was used to investigate the inhibitory effect of turmeric oil microcapsules on Propionibacterium acnes biofilm. The crystal violet method is a commonly used staining method for measuring and evaluating the formation and adhesion of biofilms. It reflects the formation and adhesion of biofilms by evaluating the number of bacteria on the biofilm attachment surface. First, we investigated the ability of Propionibacterium acnes to form biofilms. The test results showed that a bacterial solution with an OD value of 0.5 at a wavelength of 600 nm was able to form a complete biofilm after 92 hours of culture in a 96-well plate ( Figure 26 Subsequently, we investigated the inhibitory effect of turmeric oil microcapsules on P. acnes biofilms. 100 μL of bacterial suspension was inoculated into each well of a 96-well plate. Then, various concentrations of turmeric oil microcapsule solution (0.01-1.0 mmol / L) were added. After incubation for 92 hours, the plates were stained with crystal violet. Finally, 200 μL of 33% glacial acetic acid solution was added to each well and decolorized at 37°C for 20 minutes. The absorbance at 595 nm was measured, and the bacterial inhibition rate was calculated according to the following formula.

[0061] Biofilm survival rate % = (OD 样品 / OD 空白 ) × 100% (OD空白 : OD value of blank control; OD 样品 : OD value of the sample) Test results such as Figure 27 As shown. Curcumin was ineffective in inhibiting biofilm formation due to its inherently weak antibacterial properties and limited permeability into biofilms. Curcuminoids (curcumin / demethoxycurcumin / bisdemethoxycurcumin = 60 / 20 / 20) exhibited significant inhibition of biofilm formation at higher concentrations (≥0.5 mmol / L). In contrast, turmeric oil microcapsules exhibited the strongest biofilm inhibition activity, showing a clear concentration-dependent pattern, with a median inhibitory concentration of approximately 0.62 mmol / L of curcuminoids. These results further demonstrate that the antibacterial activity of turmeric oil microcapsules is due to their good water solubility and the antibacterial effect of the chitosan surface material.

[0062] Example 9. Inhibitory effect of turmeric oil microcapsules on tumor cell growth The CCK8 assay was used to investigate the inhibitory activity of turmeric oil microcapsules on the growth of tumor cells in vitro. Two tumor cells were used for the experiment: mouse squamous cell carcinoma SCC-7 cells (purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.) and human hepatocellular carcinoma HepG2 cells (from the American Type Culture Collection). SCC-7 cells were cultured in RPMI-1640 complete medium (Dalian Meilun Biotechnology Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin / streptomycin solution, while HepG2 cells were cultured in high-glucose DMEM medium (Dalian Meilun Biotechnology Co., Ltd.) containing 10% fetal bovine serum and 1% penicillin / streptomycin solution. They were cultured in an incubator at 37°C and 5% CO2. The experimental method was as follows: the tumor cells were trypsinized and resuspended in culture medium, and the cell density was adjusted to 2.5×10 4 cells / mL, and inoculated into 96-well culture plates (5×10 3 cells), placed in an incubator and cultured for 24 hours. Subsequently, the culture medium was replaced with a culture medium containing turmeric oil microcapsules at different concentrations, and the concentration range of curcumin compounds was 0-200 μmol / L. Culture was continued for 48 hours, treated with CCK-8 reagent according to the instructions, and finally the absorbance of each well at a wavelength of 450 nm was detected by a microplate reader. The cell survival rate of the control well was 100%, and the survival rate of each group of cells was calculated (at least three replicate wells at each concentration were used to calculate the average value), and a bar graph was drawn with the concentration as the horizontal axis and the cell survival rate as the vertical axis. The detection results of SCC-7 cells and HepG2 cells are shown below. Figure 27 and Figure 28As shown in the results, turmeric oil microcapsules exhibited a concentration-dependent inhibitory effect on tumor cell growth, but the inhibitory effect on SCC-7 cells was significantly stronger than that on HepG2 cells. For example, the 50% inhibitory concentration of turmeric oil microcapsules on SCC-7 cells was approximately 65 μmol / L, while that on HepG2 cells was approximately 108 μmol / L, possibly because HepG2 liver cancer cells have a certain degree of drug resistance.

[0063] Example 10. Oral bioavailability of turmeric oil microcapsules in rats Curcuminoids, as important active ingredients in turmeric, possess a variety of pharmacological effects. However, due to their low water solubility, poor intestinal absorption, and rapid metabolism in the body, their bioavailability is extremely low. References report 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 plasma concentrations of curcumin in rats and their effects on SOD activity in different dosage forms, 2018, Master's thesis, Beijing University of Chinese Medicine). The bioavailability of commercially available curcumin preparations also varies greatly.

[0064] This study used ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) to determine curcumin in rat plasma and investigate the oral bioavailability of turmeric oil microcapsules. The specific steps were as follows: Healthy female SD rats (weighing 250±20 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and acclimated for 6 days with free access to food and water. The rats were then divided into three groups (6 rats / group): a curcuminoid compound (control) group, a turmeric oil microcapsule solution group prepared according to Example 1, and a turmeric oil microcapsule solid powder group prepared according to Example 4. In the control group, curcumin, demethoxycurcumin, and bisdemethoxycurcumin were mixed in a 60 / 20 / 20 ratio, dissolved in dimethyl sulfoxide (DMSO), diluted with distilled water to a final DMSO concentration of ≤ 5%, and administered orally. The turmeric oil microcapsule solution was administered orally directly, while the turmeric oil microcapsule solid powder was suspended in distilled water and administered orally. The dose of curcuminoid compounds in all three formulations was 250 mg / kg. At 0, 10, 30, 1, 1.5, 2, 4, 8, and 12 hours after oral administration, 0.3 mL of whole blood was collected. The whole blood samples were placed in heparinized EP tubes and centrifuged at 5000 rpm at 4°C for 10 minutes. The upper plasma layer was collected and stored in a -80°C ultra-low temperature freezer. The plasma samples were extracted with methanol for curcuminoids, using salbutamol as an internal standard, and then analyzed by flow cytometry. The chromatographic conditions were as follows: an Agilent G6460C UPLC-MS / MS triple quadrupole tandem mass spectrometer (Agilent Technologies, Inc., USA); an Agilent Extend-C18 analytical column (RRHD 2.1 mm × 50 mm, 1.8 μm); a mobile phase consisting of acetonitrile (A) and 0.1% formic acid in water (B), with a gradient elution (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; an injection volume of 5 μL; and a column temperature of 30°C. The mass spectrometry conditions were as follows: ESI source; MRM-Positive mode; spray drying temperature of 350°C; spray pressure of 275.8 kPa; drying gas flow rate of 10 L / min; capillary voltage of 4 kV; retention times of curcuminoids and salbutamol were 3.5-4.3 min and 1.7 min, respectively. C ) and time ( t ) were analyzed, and the area under the drug-time curve ( AUC (0-∞) ) was calculated using the trapezoidal method, and the peak concentration ( C max) are measured values, and the pharmacokinetic parameters (Table 1) and the average blood drug concentration-time curve ( C - t curve) see Figure 30 .

[0065] Table 1. Pharmacokinetic parameters of turmeric samples (n = 6)

[0066] The bioavailability of turmeric oil microcapsule solution and turmeric oil microcapsule solid powder is 27.0 times and 20.4 times that of turmeric compounds, respectively. t 1 / 2 Significantly extended.

[0067] Example 11. Preparation and application of a hangover-relieving gel beverage containing turmeric oil microcapsules The recipe for the hangover gel drink is as follows:

[0068] Method: Evenly mix turmeric oil microcapsule solution, kudzu root extract, Hovenia dulcis extract, green plum juice, and honey, add distilled water to 50 mL, and finally sprinkle in chia seeds. Let stand for 5 minutes to form a gel. This simple formula significantly improves alcohol tolerance by consuming one bottle 10 minutes before drinking alcohol.

[0069] Example 12. Preparation and application of a turmeric oil microcapsule-containing solid beverage for improving intelligence (preventing Alzheimer's disease) The formula of the solid beverage for improving intelligence (preventing Alzheimer's disease) is as follows:

[0070] Method: First, mix the microbial B12 with the plant hydrolyzed peptide in equal amounts, then mix thoroughly with the other ingredients. This formula can prevent Alzheimer's disease through multiple mechanisms, pathways, and targets.

[0071] Example 13. Preparation and application of turmeric oil microcapsules as an anti-acne medical beauty mask The formula of the mask base is as follows:

[0072] Methods: Carbomer was dispersed in deionized water, and hyaluronic acid, ceramide, and laurocapram were added in sequence. Homogenization and emulsification were performed at 3000 rpm for 5 minutes. The pH was adjusted to approximately 5.5-6.0 with triethanolamine. Turmeric oil microcapsule solution, salicylic acid-tea tree oil complex, and Centella asiatica liposomes were added to the matrix in portions and thoroughly mixed at ≤800 rpm (to avoid high-speed shear damage). This yielded the anti-acne mask matrix.

[0073] Example 14. Turmeric Oil Microcapsule Liver Protection Tablets The formula of oral care chewable tablets is as follows:

[0074] Methods: The main drugs (turmeric oil microcapsule solid powder, glycyrrhizic acid ethanol extract, vitamin C, N-acetylcysteine, black piperine, inulin) were sieved with 80 meshes respectively, mixed with microcrystalline cellulose, added with 5% PVP ethanol solution to prepare a soft material, sieved with 14 meshes to granulate, dried at 70-80℃, sieved with 12 meshes to size the granules, added with talc powder and magnesium stearate, mixed and then compressed into tablets to obtain turmeric oil microcapsule liver protection tablets.

[0075] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for extracting turmeric oil from fresh turmeric, characterized in that, The steps include: S11, adding the pretreated fresh turmeric to an extraction solution, and shearing and crushing it to obtain an extract; the extraction solution is phosphate buffer, glycine-sodium hydroxide buffer, sodium bicarbonate buffer or Tris hydrochloride buffer; S21, adjusting the pH of the extract to 4.0-5.0, adding a complex enzyme system, and then performing ultrasonic enzymatic hydrolysis to obtain an enzymatic solution; the complex enzyme system comprises: one or more of cellulase, xylanase, arabinanase, pectinase, laccase and snailase; S31, adjusting the pH of the enzymatic hydrolyzate to 7.0-8.0, then inactivating and filtering to obtain a first filtrate and a filter residue; S41, adding a density gradient medium to the first filtrate, and then performing low-temperature centrifugation to form isopycnic zones, collecting the upper layer of cream and resuspending it to obtain a primary emulsion containing turmeric oil, wherein the density gradient medium comprises one or more of sucrose, glucose, polysucrose, dextran, hyaluronic acid, glycerol, ethylene glycol, isopropyl alcohol, polyethylene glycol, and cyclodextrin derivatives; S51, adding a surfactant to the primary emulsion, adjusting the pH to 3.0-3.5, and then stirring to reconstruct the interfacial protein on the surface of the turmeric oil body, and filtering to obtain a second filtrate; S61. Centrifuge the second filtrate at low temperature, collect the upper layer of cream, and resuspend it to obtain turmeric oil emulsion.

2. The method for extracting turmeric oil from fresh turmeric according to claim 1, wherein The shearing and crushing rotation speed is 20000-40000 rpm, and the shearing and crushing time is 25-35 minutes; and / or, The ultrasonic enzymatic hydrolysis parameters include: time of 20 to 40 min, temperature of 50 to 60°C, ultrasonic power of 100 W / cm 2 , the ultrasonic frequency is 18 kHz, the ultrasonic pulse mode is on 3s / off 2s; and / or, The rotation speed of the low-temperature centrifugal separation is 3000-15000 rpm, and the time of the low-temperature centrifugal separation is 30-60 minutes.

3. The method for extracting turmeric oil from fresh turmeric according to claim 1, wherein The weight ratio of the fresh turmeric to the extraction solution is 1:1 to 1:5; and / or, The final concentration of the complex enzyme system in the extract 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 for extracting turmeric oil from fresh turmeric according to claim 3, wherein The extraction solution is tris hydrochloride buffer, and the weight ratio of the fresh turmeric to the extraction solution is 1:3; and / or, The complex enzyme system is a complex system of cellulase with a final concentration of 0.5-2.0% in the extract and pectinase with a final concentration of 0.1-0.5% in the extract; 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 for extracting turmeric oil from fresh turmeric according to claim 1, wherein The inactivation temperature is 70-90° C., and the inactivation time is 5-10 minutes; and / or, The stirring time is 10 to 30 minutes; and / or, The rotation speed of the centrifugation at low temperature is 1000-5000 rpm, and the time of the centrifugation at low temperature is 30-60 minutes.

6. The method for extracting turmeric oil from fresh turmeric according to 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 polysaccharide, and the final concentration of the surfactant in the primary emulsion is 0.5-1.5%.

7. A method for preparing turmeric oil microcapsules, characterized in that: The steps include: S12. Add capsule material and antioxidant to the turmeric oil emulsion prepared by the method according to any one of claims 1 to 6, and obtain a turmeric oil microcapsule solution by high-pressure homogenization; the high-pressure homogenization comprises the following parameters: pressure of 30 to 80 MPa, time of 1 to 5 min, and repetition number of 3 to 5 times; S22, adding an anti-adhesive agent and a glidant to the turmeric oil microcapsule solution, and spray-drying to obtain a turmeric oil microcapsule solid powder.

8. The method for preparing turmeric oil microcapsules according to claim 7, wherein: The capsule material comprises one or more of sodium alginate, maltodextrin, gum arabic, oligomaltodextrose, cyclodextrin, inulin, pullulan, chitosan, gelatin, whey protein, casein phosphopeptide, sodium carboxymethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, hydroxypropyl starch, sodium starch octenylsuccinate, polyethylene glycol and polyvinyl pyrrolidone, and the final concentration of the capsule material in the turmeric oil emulsion is 5-20%; and / or, The antioxidant comprises: one or more of ascorbic acid, ascorbyl palmitate, rosemary extract, glutathione, vitamin E and propyl gallate, and the final concentration of the antioxidant in the turmeric body lotion is ≤0.05%; and / or, The anti-adhesive agent comprises one or more of silicon dioxide, magnesium silicate, glyceryl monostearate, sodium tripolyphosphate, hydroxypropyl methylcellulose, polyvinyl pyrrolidone and polyethylene glycol, and the final concentration of the anti-adhesive agent in the turmeric oil microcapsule solution is ≤1%; and / or, The glidant comprises one or more of glucose, lactose, fructose, oligofructose, xylitol, sorbitol, mannitol, trehalose and microcrystalline cellulose, and the final concentration of the glidant in the turmeric oleosome microcapsule solution is ≤5%.

9. The method for preparing turmeric oil microcapsules according to claim 7, wherein: The concentration of curcuminoids in the turmeric oil microcapsule solution is 10 to 50 mg / mL; The mass content of curcuminoid compounds in the turmeric oil microcapsule solid powder is 5-10%.

10. Use of the turmeric oil emulsion prepared by the method according to any one of claims 1 to 6 or the turmeric oil microcapsules prepared by the method according to any one of claims 7 to 9, characterized in that: Used as a preparation ingredient for dietary supplements, edible and feed products and their supplements, nutritional supplements, spices, condiments, winemaking or cosmetics, or as a natural carrier for hydrophobic drugs and active molecules to prepare preparations containing drug ingredients.

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