Atractylodes rhizome volatile oil extraction method and application of detoxification anti-inflammatory protein polypeptide

Abstract

The present application relates to the technical field of plant extraction, and especially relates to an extraction method of volatile oil of Atractylodes macrocephala and a detoxification and inflammation-eliminating protein polypeptide.The present application discloses an extraction method of volatile oil of Atractylodes macrocephala, which comprises the following steps: firstly, cleaning, air-drying, slicing and crushing Atractylodes macrocephala to obtain Atractylodes macrocephala powder; secondly, extracting volatile oil of Atractylodes macrocephala by using a n-hexane-methanol binary extraction system to obtain the volatile oil; thirdly, carrying out oxidative protection on the volatile oil of Atractylodes macrocephala by using a magnetic enzyme agent, and adding and including an extractant to obtain an oxidized volatile oil-sulfobutyl-beta-cyclodextrin sodium complex solution; fourthly, realizing re-conversion into the original volatile oil by using hydrogen and a palladium-carbon nanofiber membrane to obtain a volatile oil-sulfobutyl-beta-cyclodextrin sodium complex solution; and finally, mixing the volatile oil-sulfobutyl-beta-cyclodextrin sodium complex solution with a lactoferrin-chitosan complex to obtain the detoxification and inflammation-eliminating protein polypeptide.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, and in particular to a method for extracting Atractylodes macrocephala volatile oil and its application as a detoxifying and anti-inflammatory protein polypeptide. Background Technology

[0002] The active ingredients in Atractylodes macrocephala volatile oil are mainly terpenoids, among which atractylone is the main active and drying component. Atractylodes macrocephala volatile oil is highly volatile and sensitive to high temperatures; it easily decomposes when heated for extended periods and is readily oxidized when exposed to air, leading to a darkening of color or polymerization.

[0003] Chinese patent CN100350922C discloses a method for extracting volatile oil from Atractylodes macrocephala. This method involves steps such as raw material crushing, supercritical carbon dioxide extraction, coarse separation using silica gel column chromatography, and purification using C18 silica gel reversed-phase column chromatography. However, this process has certain drawbacks: the supercritical extraction equipment used is expensive and requires specialized technicians to operate; furthermore, during silica gel separation, exposure to light and air may increase the risk of oxidation and deterioration.

[0004] Chinese patent CN110151809A discloses a method for preparing Atractylodes macrocephala essential oil. This method involves sequentially performing pile fermentation, acid extraction, and enzymatic hydrolysis, combined with steam distillation or supercritical carbon dioxide extraction. However, the prolonged pile fermentation and acid extraction processes pose a risk of oxidation of the volatile oils in Atractylodes macrocephala. Steam distillation suffers from drawbacks such as long extraction time and high energy consumption, and its high-temperature environment easily leads to the degradation of heat-sensitive components, resulting in a low yield of essential oil.

[0005] Chinese patent CN109694777A discloses a method for extracting volatile oil from Atractylodes macrocephala. This method involves pulverizing the raw material, then using an ultrasonic-assisted extraction solution of 60-70% ethanol, followed by filtration, vacuum concentration to remove ethanol, purification with macroporous adsorption resin, and drying to obtain the volatile oil. However, the volatile oil obtained by this method exhibits poor stability during storage and use, and is easily oxidized and deteriorated by environmental factors such as high temperature and light.

[0006] Chinese patent CN116059399B discloses a method for preparing coated Atractylodes macrocephala oil. This method involves encapsulating Atractylodes macrocephala oil in a β-cyclodextrin system using a water-cooled ultrasonic technique. After precipitation and separation, the mixture is washed with methanol and dried to obtain the Atractylodes macrocephala oil-β-cyclodextrin inclusion complex. Due to the controllable sustained-release properties of β-cyclodextrin, some Atractylodes macrocephala oil may be carried away during the methanol washing process. Furthermore, during room temperature drying, the Atractylodes macrocephala oil may be exposed to light and air, increasing the risk of oxidative deterioration.

[0007] Chinese patent CN103396890A discloses a method for preparing stable Atractylodes macrocephala volatile oil. This method first extracts the volatile oil using supercritical carbon dioxide extraction, and then uses ultraviolet light irradiation to induce the oxidative decomposition of atractylone, thus obtaining the stable Atractylodes macrocephala volatile oil. According to the invention's conclusion, while ultraviolet light irradiation can increase the content of atractylolactone components, it leads to a decrease in atractylone content, which may weaken its original anti-inflammatory, antioxidant, and anti-tumor biological activities. Furthermore, the thermal effect or prolonged light exposure during ultraviolet irradiation may accelerate the volatilization and loss of low-boiling-point components in the volatile oil, resulting in a reduction in the content of effective ingredients.

[0008] In summary, in the extraction process of Atractylodes macrocephala volatile oil, how to achieve efficient extraction while protecting easily oxidized, photosensitive, and heat-sensitive components, and effectively control adverse reactions caused by the drying components in the volatile oil, has become a key technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0009] To address the aforementioned problems, the present invention aims to provide a method for extracting Atractylodes macrocephala volatile oil and the application of detoxifying and anti-inflammatory protein polypeptides. The method for extracting Atractylodes macrocephala volatile oil specifically includes the following steps:

[0010] S001. Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0011] S002, add extractant, sonicate, filter, collect solution to obtain Atractylodes macrocephala volatile oil;

[0012] S003, add reaction buffer, adjust pH, add magnetic enzyme, treat with enzyme, remove enzyme, and collect solution;

[0013] S004, add the extractant, extract, collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution.

[0014] The volatile oil of oxidized Atractylodes macrocephala was reduced and protected using hydrogen and lactoferrin-chitosan complex to obtain detoxifying and anti-inflammatory protein peptides, specifically including the following steps:

[0015] S101, add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution, and obtain the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0016] S102, Weigh the lactoferrin-chitosan complex, add ultrapure water, and stir gently to dissolve it to obtain the lactoferrin-chitosan complex solution;

[0017] S103, by adjusting the pH of the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and the lactoferrin-chitosan complex solution, detoxifying and anti-inflammatory protein peptides are obtained.

[0018] The specific preparation methods for the above-mentioned solvents are as follows:

[0019] The extractant is composed of n-hexane and methanol in a volume ratio of 4:1.

[0020] The reaction buffer consists of Tris-HCl buffer, NADPH solution, calcium chloride solution, and uridine diphosphate glucose solution. Preparation method of the reaction buffer:

[0021] S201: Weigh 74.54 mg of NADPH powder, dissolve it in 0.05 M Tris-HCl buffer, and dilute to 10 mL in a volumetric flask. Shake well to obtain NADPH stock solution.

[0022] S202: Weigh 11.10 mg of anhydrous calcium chloride powder, dissolve it in 0.05 M Tris-HCl buffer, dilute to 10 mL in a volumetric flask, and shake well to obtain calcium chloride stock solution.

[0023] S203: Weigh 61.03 mg of uridine diphosphate glucose powder, dissolve it in 0.05 M Tris-HCl buffer, and make up to 10 mL in a volumetric flask. Shake well to obtain uridine diphosphate glucose stock solution.

[0024] S204. Transfer 10 mL of NADPH stock solution, calcium chloride stock solution and uridine diphosphate glucose stock solution to 100 mL volumetric flasks respectively, add 0.05 M Tris-HCl buffer to make up to volume, shake well and set aside.

[0025] The magnetic enzyme is composed of a complex enzyme, amino-modified magnetic nanoparticles, and 0.05 M Tris-HCl buffer. The complex enzyme consists of cytochrome P450 enzyme, cyclooxygenase, and uridine diphosphate glycosyltransferase. The amino-modified magnetic nanoparticles are made by introducing amino groups onto the surface of magnetic nanoparticles. The magnetic nanoparticles are made by encapsulating iron(III) oxide (Fe3O4) nanoparticles with silica.

[0026] Preparation method of magnetic enzymes:

[0027] S301: Weigh 50 mg of amino-modified magnetic nanoparticles, 1.15 g of N-hydroxysuccinimide and 1.92 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, add 100 mL of 0.05 M Tris-HCl buffer, vortex mix until homogeneous, and stir at 600 r / min for 1 hour at room temperature to obtain an activated amino-modified magnetic nanoparticle solution.

[0028] S302: Weigh 1 mg of cytochrome P450 enzyme, 1 mg of cyclooxygenase and 1 mg of uridine diphosphate glycosyltransferase into 50 mL of 0.05 M Tris-HCl buffer, vortex until homogeneous, and the complex enzyme solution is obtained.

[0029] S303, the activated amino-modified magnetic nanoparticle solution is mixed with the composite enzyme solution and stirred at 600 r / min for 6 hours at 4~25 ℃;

[0030] S304, placed on a magnetic rack, discarded the liquid, washed three times with twice the volume of 0.05 M Tris-HCl buffer, added 100 mL of 0.05 M Tris-HCl buffer and vortexed until homogeneous, thus obtaining the magnetic enzyme.

[0031] Preparation method of amino-modified magnetic nanoparticles:

[0032] S401, weigh 0.5 g of magnetic nanoparticles, add 50 mL of anhydrous ethanol, and sonicate at 100 W for 20 min;

[0033] S402, add 8 mL of 3-aminopropyltriethoxysilane, purge with nitrogen, and stir at 600 r / min for 12 hours at room temperature;

[0034] S403, place it on a magnetic rack, discard the liquid, wash it three times with twice the volume of ultrapure water, wash it three times with twice the volume of anhydrous ethanol, and collect the magnetic material.

[0035] S404, by placing the magnetic material in a vacuum drying oven at 40~60 ℃ for 12 hours, amino-modified magnetic nanoparticles are obtained.

[0036] Preparation methods of magnetic nanoparticles:

[0037] S501, weigh 1 g of Fe3O4 nanoparticles into a three-necked flask, add 100 mL of ultrapure water and 20 mL of anhydrous ethanol solution, and sonicate at 100 W for 10 min.

[0038] S502, add 5 mL of 25% ammonia and 4 mL of tetraethoxysilane, and stir at 600 r / min for 4 hours at room temperature;

[0039] S503, place it on a magnetic rack, discard the liquid, wash it three times with twice the volume of ultrapure water, wash it three times with twice the volume of anhydrous ethanol, and collect the magnetic material.

[0040] S504: Magnetic nanoparticles are obtained by drying magnetic materials in a vacuum drying oven at 40~60 ℃ for 3 hours.

[0041] Preparation method of Fe3O4 nanoparticles:

[0042] S601, weigh 27.18 g FeCl3・6H2O and 10 g FeCl2・4H2O into a three-necked flask, add 500 mL of ultrapure water, and stir at 600 r / min until dissolved;

[0043] S602, while stirring, introduce nitrogen gas, heat to 70~80 ℃, slowly add 25% ammonia water dropwise, adjust the pH of the solution to 10~11, and continue stirring;

[0044] S603, after 1 hour, stop stirring and heating, let it cool naturally to room temperature, wash 3 times with 2 times the volume of ultrapure water, wash 3 times with 2 times the volume of anhydrous ethanol, and collect the precipitate.

[0045] S604, the precipitate was dried in a vacuum drying oven at 40~60 ℃ for 12 hours to obtain black Fe3O4 nanoparticles.

[0046] The extractant is composed of sodium bisulfite, sodium sulfonyl-β-cyclodextrin, and a 30% aqueous ethanol solution. Preparation method of the extractant:

[0047] S701: Weigh 65 g of sodium bisulfite into a beaker containing 100 mL of ultrapure water, heat to 50 °C, stir at 600 r / min for 1 hour, cool to room temperature, filter to remove undissolved solids, and obtain a saturated sodium bisulfite solution.

[0048] S702: Weigh 20 g of sodium sulfobutyl-β-cyclodextrin powder, add 70 mL of ultrapure water and 30 mL of anhydrous ethanol solution, sonicate at 100 W for 10 min until dissolved, and then add it all to a saturated sodium bisulfite solution. Vortex mix until homogeneous to obtain the extractant.

[0049] Palladium-carbon nanofiber membranes are composed of palladium nanoparticles and carbon nanofibers. Preparation methods for palladium-carbon nanofiber membranes:

[0050] S801: Weigh 1 g of polyacrylonitrile and add it to 10 mL of N,N-dimethylformamide. Sonicate at 100 W for 6 hours at 50~60 ℃ until completely dissolved.

[0051] Add 5 g of palladium nitrate to S802 and continue sonication at 100 W for 3 hours;

[0052] S803 was added to the electrospinning injector, and the parameters were set as follows: voltage 20 kV, feed speed 1 mL / h, receiving distance 15 cm, and continuous spinning for 6 hours.

[0053] S804 was placed in an atmosphere muffle furnace and heated to 200 °C at a heating rate of 4 °C / min under an oxygen atmosphere and held for 3 hours. Then, it was heated to 800 °C at a heating rate of 6 °C / min under a nitrogen atmosphere and held for 2 hours. After cooling to room temperature, it was washed 5 times with 100 mL of ultrapure water.

[0054] S805 is dried in a 70 ℃ oven for 3 hours to obtain palladium carbon nanofiber membrane.

[0055] The lactoferrin-chitosan complex is composed of lactoferrin and chitosan. Preparation method of the lactoferrin-chitosan complex:

[0056] S901, transfer 2.375 mL of glacial acetic acid to a beaker, add an appropriate amount of ultrapure water, vortex mix evenly, transfer to a 250 mL volumetric flask, wash and dilute to volume with ultrapure water, shake well to obtain a 1% acetic acid solution.

[0057] S902: Weigh 1 g of chitosan into 200 mL of 1% acetic acid solution, stir at 600 r / min for 24 hours until completely dissolved to obtain chitosan solution;

[0058] S903: Weigh 0.4 g of lactoferrin into 20 mL of ultrapure water, stir gently until completely dissolved, and you will get a lactoferrin solution.

[0059] S904, stir the chitosan solution at 400 r / min, slowly add the lactoferrin solution, and keep stirring for 2 hours;

[0060] Centrifuge at 15000 r / min for 15 min using S905, collect the precipitate, add the same volume of ultrapure water as the supernatant, and vortex mix thoroughly.

[0061] S906. After repeating step S905 three times, collect the precipitate and dry it in a vacuum environment at 0~20 ℃ for 12 hours to obtain the lactoferrin-chitosan complex.

[0062] In step S002, the extractant is added at a material-to-liquid ratio of 1:3~7, and the ultrasonic treatment conditions are 100 W for 20 min.

[0063] In step S003, the volume-to-mass ratio of reaction buffer to Atractylodes macrocephala volatile oil is 2:1. The pH is adjusted to 6.0-7.0 using 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution. The enzyme treatment conditions are vortexing at 37 °C for 2-6 hours, the amount of magnetic enzyme added is 0.1 times the volume, and the enzyme is removed by placing it in a magnetic rack and letting it stand for 5 minutes.

[0064] In step S004, the amount of extractant added is 1 to 3 times the volume, and the extraction process involves vortexing for 1 hour followed by standing for 20 minutes.

[0065] The reaction process in step S101 involves introducing hydrogen gas, controlling the pressure at 5-6 MPa, stirring at 600 r / min, and controlling the temperature at 40-50 °C. After reacting for 3 hours, heating and stirring are stopped, and the pressure is released to atmospheric pressure.

[0066] In step S102, the mass-to-volume ratio of lactoferrin-chitosan complex to ultrapure water is 0.1:1.

[0067] In step S103, the pH is adjusted to 6.0-7.0 using 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution, and the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and lactoferrin-chitosan complex solution are mixed at a volume ratio of 1:5.

[0068] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0069] This invention constructs a hexane-methanol binary extraction system. Based on the polarity differences and molecular structure characteristics of volatile oil components, it forms an extraction environment with targeted selectivity. This system can efficiently extract different components from volatile oils, achieving deep extraction of all components and thus increasing the efficacy of Atractylodes macrocephala volatile oil. By optimizing the material-liquid ratio, the extraction content of volatile oil is improved.

[0070] This invention utilizes magnetic nanoparticles to immobilize a composite enzyme, resulting in a magnetic enzyme. First, Fe3O4 nanoparticles are encapsulated in silica to form magnetic nanoparticles. This improves the magnetic stability of the Fe3O4 nanoparticles in different environments, extends their lifespan, and increases their dispersibility in solution. Simultaneously, abundant active hydroxyl groups are introduced onto the surface, providing possibilities for subsequent grafting. Second, a silane coupling agent is grafted onto the silica surface through hydrolysis and dehydration condensation reactions, exposing amino groups on the surface. This further enhances the surface reactivity of the magnetic nanoparticles, further increasing their dispersibility in solution, and also imparting a certain adsorption capacity. Finally, the composite enzyme is immobilized on the surface of the magnetic nanoparticles through covalent bonding and adsorption. After the enzyme reaction is complete, under the influence of an external magnetic field, rapid and efficient separation of the enzyme and reaction products can be achieved, improving the enzyme's reusability. Furthermore, the exposed amino groups on the surface can regulate the microenvironment surrounding the enzyme molecules, enhancing the enzyme's catalytic activity.

[0071] This invention employs a composite enzyme catalytic system that can act on the effective components in Atractylodes macrocephala volatile oil. Through a specific oxidation reaction, it can directionally convert easily oxidized and heat-sensitive components in the volatile oil into relatively stable oxidation products.

[0072] This invention constructs a "multi-step synergistic purification-phase transfer enhancement" technical system. First, it utilizes an extractant to achieve deep extraction of volatile oils. Through a nucleophilic addition reaction with a saturated sodium bisulfite solution, components containing double bonds or carbonyl groups in the volatile oil are converted into addition products, further increasing its water solubility and extending its storage and long-term use. Although the water solubility of the oxidized volatile oil is improved through the use of complex enzymes and extractants, it still tends to dissolve in organic reagents. The special structure of sodium sulfobutyl-β-cyclodextrin—a "hydrophobic cavity-hydrophilic shell"—encapsulates the oxidized volatile oil, thereby achieving the transfer of the oxidized volatile oil from the organic phase to the aqueous phase. Simultaneously, the controlled release characteristics of sodium sulfobutyl-β-cyclodextrin achieve a sustained-release effect of the volatile oil, effectively reducing the dryness of Atractylodes macrocephala volatile oil.

[0073] This invention utilizes clean hydrogen as a reducing agent and palladium-carbon nanofiber membranes as a catalyst to reconvert oxidized volatile oils back into their original form. By leveraging the catalyst's specific surface area and highly dispersed palladium active sites, a catalytic mechanism of "hydrogen adsorption-active hydrogen dissociation-substrate reduction" is established, enhancing reaction efficiency. Furthermore, after the reaction, the catalyst can be rapidly separated and recovered through low-pressure filtration based on the physical retention characteristics of the nanofibers. This process eliminates the need for complex post-treatment steps such as acid-base neutralization and extraction, avoiding the damage to volatile oil components caused by residual chemical reagents and high-temperature, high-pressure treatment.

[0074] This invention mixes a lactoferrin-chitosan complex with a volatile oil-sulfobutyl-β-cyclodextrin sodium complex to form a multi-complex solution. First, the composite enhances the antioxidant effect of the complex while retaining active sites on the molecules, providing abundant chemical interfaces for subsequent targeted modification. Second, the composite chitosan alters the microstructure and conformation of sulfobutyl-β-cyclodextrin sodium, enhancing the stability of the contained volatile oil. Due to the bioadhesive and degradable properties of chitosan, the volatile oil of the contained compound can be released slowly, reducing the dryness of the Atractylodes macrocephala volatile oil. Furthermore, the synergistic effect of chitosan and sulfobutyl-β-cyclodextrin sodium may also activate key active components in the volatile oil.

[0075] The preparation method employed in this invention is simple to operate. During the efficient purification process, foreign impurities can be removed through simple magnetic adsorption and filtration. Furthermore, the resulting multi-composite products have a certain synergistic effect, which can enhance the efficacy of the activating product. Detailed Implementation

[0076] The present invention will be further described below with reference to specific embodiments.

[0077] Example 1: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0078] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0079] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:3, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0080] Add reaction buffer to adjust pH, add magnetic enzyme, vortex at 37 °C for 4 hours to remove enzyme, and collect solution;

[0081] Add twice the volume of extraction solvent, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0082] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e., test sample 1.

[0083] Example 2: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0084] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0085] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:5, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0086] Add reaction buffer to adjust pH, add magnetic enzyme, vortex at 37 °C for 4 hours to remove enzyme, and collect solution;

[0087] Add twice the volume of extraction solvent, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0088] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e. test sample 2.

[0089] Example 3: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0090] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0091] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:7, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0092] Add reaction buffer to adjust pH, add magnetic enzyme, vortex at 37 °C for 4 hours to remove enzyme, and collect solution;

[0093] Add twice the volume of extraction solvent, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0094] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e., test sample 3.

[0095] Example 4: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0096] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0097] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:5, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0098] Add reaction buffer, adjust pH, add magnetic enzyme, vortex at 37 °C for 2 hours to remove enzyme, and collect solution;

[0099] Add twice the volume of extraction solvent, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0100] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e., test sample 4.

[0101] Example 5: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0102] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0103] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:5, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0104] Add reaction buffer, adjust pH, add magnetic enzyme, vortex at 37 °C for 6 hours to remove enzyme, and collect solution;

[0105] Add twice the volume of extraction solvent, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0106] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e., test sample 5.

[0107] Example 6: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0108] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0109] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:5, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0110] Add reaction buffer to adjust pH, add magnetic enzyme, vortex at 37 °C for 4 hours to remove enzyme, and collect solution;

[0111] Add an extraction solvent of 1 volume, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0112] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e., test sample 6.

[0113] Example 7: A method for extracting volatile oil from Atractylodes macrocephala, specifically including the following steps:

[0114] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder.

[0115] Weigh 100 g of Atractylodes macrocephala powder, add extractant, sonicate at a material-to-liquid ratio of 1:5, filter, collect the solution, and obtain Atractylodes macrocephala volatile oil.

[0116] Add reaction buffer to adjust pH, add magnetic enzyme, vortex at 37 °C for 4 hours to remove enzyme, and collect solution;

[0117] Add 3 times the volume of extraction solvent, extract, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0118] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, i.e., test sample 7.

[0119] Example 8: Preparation of detoxifying and anti-inflammatory protein polypeptides from test sample 2 obtained in Example 2, the specific steps are as follows:

[0120] Weigh 100 mg of lactoferrin-chitosan complex, add 100 mL of ultrapure water, and stir gently to dissolve it to obtain lactoferrin-chitosan complex solution.

[0121] Test sample 2 was mixed with lactoferrin-chitosan complex solution at a volume ratio of 1:5. The pH was adjusted to 6.0-7.0 with 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution to obtain detoxifying and anti-inflammatory protein polypeptide, which is test sample 8.

[0122] Comparative Example 1

[0123] Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder. Weigh 100 g of Atractylodes macrocephala powder to obtain reference standard 1.

[0124] Comparative Example 2

[0125] Collect the volatile oil obtained from Experiment 1 in Comparative Example 1 to obtain Control 2.

[0126] Comparative Example 3

[0127] The reference standard contains no Atractylodes macrocephala volatile oil, but only sodium sulfobutyl-β-cyclodextrin, lactoferrin, and chitosan. The specific steps include:

[0128] Take the same volume of extractant and reaction buffer as in Example 2, adjust the pH to 6.0-7.0 with 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution, add 0.1 volume of magnetic enzyme, vortex at 37 °C for 4 hours to remove the enzyme, and collect the solution;

[0129] Add twice the volume of extraction solvent, perform extraction, and collect the lower layer solution;

[0130] Add 1 part of palladium carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react and process, filter, collect the solution to obtain solution A;

[0131] Weigh 100 mg of lactoferrin-chitosan complex, add 100 mL of ultrapure water, and stir gently to dissolve it to obtain lactoferrin-chitosan complex solution.

[0132] Solution A was mixed with lactoferrin-chitosan complex solution at a volume ratio of 1:5, and the pH was adjusted to 6.0-7.0 with 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution to obtain reference standard 3.

[0133] Experimental Example 1

[0134] This experimental example uses steam distillation to determine the content of Atractylodes macrocephala volatile oil in the test samples of Examples 1-7 and the control standard of Comparative Example 1. The specific steps are as follows:

[0135] Place the test sample and reference sample in separate flasks, add 500 mL of ultrapure water and a few glass beads, shake to mix, and connect the volatile oil analyzer to the reflux condenser. Add water from the top of the condenser until it fills the graduated section of the volatile oil analyzer and overflows into the flask. Place the flask in a heating mantle and slowly heat to boiling, maintaining a gentle boil for about 5 hours, until the oil volume in the analyzer stops increasing. Stop heating, let it stand for a moment, then open the stopcock at the bottom of the analyzer to slowly release the water until the upper end of the oil layer reaches 5 mm above the 0 mark. Let it stand for at least 1 hour, then open the stopcock again to allow the oil layer to drop until its upper end is exactly level with the 0 mark, and read the volume of the volatile oil. Perform three parallel measurements for each test sample and reference sample, and take the average volume of the volatile oil.

[0136] The content of Atractylodes macrocephala volatile oil in the test sample and reference sample was obtained based on the volume and weight of the volatile oil. The determination results are shown in Table 1.

[0137] Table 1. Content of volatile oil in Atractylodes macrocephala.

[0138]

[0139] Note: * indicates that the examples are significantly different from the comparative examples, p<0.05.

[0140] By comparing with Comparative Example 1, the optimal extractant volume was found in Examples 1-3 to be a material-to-liquid ratio of 1:5; the optimal enzyme reaction time was found in Examples 2, 4, and 5 to be 4 hours; and the optimal extractant volume was found in Examples 2, 6, and 7 to be twice the volume. Table 1 shows that the volume of extractant added, the enzyme reaction time, and the volume of extractant added all affect the extraction of Atractylodes macrocephala volatile oil in the extraction method of the present invention, and the extraction method of the present invention significantly improves the extraction of Atractylodes macrocephala volatile oil.

[0141] Experimental Example 2

[0142] This experimental example determined the contents of atractylone, γ-malinoylene, β-eucalyptol, γ-elemene, atractylenolide I, atractylenolide II, and atractylenolide III in the test samples of Examples 1-7 and the control sample of Comparative Example 2. The analysis was performed by GC-MS, and the specific steps are as follows:

[0143] Preparation of the reference solution:

[0144] Weigh 100 mg of atractylone, γ-malinoylene, β-eucalyptol, γ-elemene, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III reference standards respectively, dissolve them in a small amount of n-hexane, transfer them to 100 mL brown volumetric flasks, and make up to volume with n-hexane to obtain mixed standard stock solution.

[0145] Transfer 0.5 μL, 1 μL, 5 μL, 10 μL, 50 μL, 100 μL, 500 μL, and 1000 μL of the stock solution into 10 mL volumetric flasks, respectively, and dilute to volume with acetic anhydride to obtain standard working solutions with maleic anhydride concentrations of 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL, respectively.

[0146] Preparation of the test sample:

[0147] Transfer 900 mL of ultrapure water and 100 mL of 10X phosphate buffer, vortex to mix, add 0.5 g of β-cyclodextrinase, vortex for 5 min until completely dissolved, filter through a 0.22 μm filter membrane to obtain the enzyme solution.

[0148] Add 0.5 times the volume of the extract to the test samples in Examples 1-7, stir and mix well, react at 30-40 °C for 1 hour, add 2 times the volume of 80% n-hexane-methanol solution, vortex for 10 min, let stand for 5 min, place in a separatory funnel, collect the upper organic phase, blow dry with nitrogen at 40 °C, add 200 μL of n-hexane, vortex for 10 min until dissolved;

[0149] Add 200 μL of n-hexane to the reference standard of Comparative Example 2 and vortex for 10 min until dissolved;

[0150] Instrument conditions: The chromatographic column was a DB-5MS quartz capillary (0.25 mm × 30 m × 0.25 μm). The vaporization temperature was 280℃. The temperature program was as follows: initial temperature 50℃, hold for 2 min; increase to 200℃ at a rate of 5℃ / min, then increase to 250℃ at a rate of 6℃ / min, hold for 10 min. The split ratio was 10:1, and the injection volume was 1 μL. The carrier gas was high-purity helium, the ion source was an EI source, the transfer line temperature was 250℃, the ion source temperature was 200℃, the full scan mode was used, and the mass range m / z was 30~650.

[0151] The analytes were atractylone, γ-malinoside, β-eucalyptol, γ-elemene, atractylenolide I, atractylenolide II, and atractylenolide III. A standard curve was plotted with the concentration of the standard working solution of the analyte as the abscissa and the corresponding peak area as the ordinate. A linear regression equation was obtained, and the content of the analytes in the volatile oil of Atractylodes macrocephala in the test sample and the reference sample was calculated. The determination results are shown in Table 2.

[0152] Table 2. Analytical content of volatile oil from Atractylodes macrocephala.

[0153]

[0154] As can be seen from Table 2, the extraction method of the present invention can extract atractylone, γ-malene, β-eudesmol, γ-elemene, atractylenolide I, atractylenolide II, and atractylenolide III from the volatile oil of Atractylodes macrocephala, and their contents are significantly higher than those extracted by steam distillation method in conventional volatile oil extraction.

[0155] Experimental Example 3

[0156] This experimental example evaluates the detoxification effect of the test sample of Example 8, the control sample of Comparative Example 2, and the control sample of Comparative Example 3 through an in vitro cytotoxin clearance experiment. The specific steps are as follows:

[0157] Preparation of 1X phosphate buffer:

[0158] Transfer 900 mL of sterile water and 100 mL of 10X phosphate buffer, vortex mix to obtain 1X phosphate buffer.

[0159] Preparation of DMEM medium containing 50 μM lead acetate:

[0160] Weigh 189.7 mg of lead acetate trihydrate into a beaker, add 10 mL of 1X phosphate buffer, sonicate at 300 W at 30 °C until completely dissolved, and sterilize through a 0.22 μm filter membrane to obtain 50 mM lead acetate stock solution.

[0161] Transfer 1 mL of 50 mM lead acetate stock solution to a 1 L volumetric flask, add DMEM medium to make up to the final volume, and you will get DMEM medium containing 50 μM lead acetate.

[0162] Preparation of DMEM medium containing 50 μM lead acetate and 1 mM glutathione:

[0163] Weigh 3.07 g of glutathione into a beaker, add 10 mL of 1X phosphate buffer, vortex until completely dissolved, and sterilize through a 0.22 μm filter membrane to obtain 1 M glutathione stock solution.

[0164] Transfer 1 mL of 50 mM lead acetate stock solution and 1 mL of 1 M glutathione stock solution to a 1 L volumetric flask, add DMEM medium to make up to volume, and you will get DMEM medium containing 50 μM lead acetate and 1 mM glutathione.

[0165] Cell preparation:

[0166] Take a concentration of 5×10 4 Logarithmic growth phase HaCaT cells per cell / mL were seeded at 100 μL in 6-well plates and cultured at 37 °C with 5% CO2 for 24 hours until the cells adhered and reached 70-80% confluence.

[0167] Preparation of the test sample:

[0168] In Example 8, 10 mL of DMEM medium was added to the test sample, Comparative Example 2, and Comparative Example 3, respectively. The mixture was incubated at 37 °C with shaking for 2 hours, centrifuged at 4 °C and 3000 r / min for 15 min, and the supernatant was collected and sterilized through a 0.22 μm filter membrane to obtain the DMEM medium containing the test sample.

[0169] nourish:

[0170] Positive control group culture: Discard the original cell culture medium, add 2 mL of DMEM medium containing 50 μM lead acetate and 1 mM glutathione, and incubate at 37 ℃ with shaking for 2 hours; discard the toxin culture medium, wash twice with 1X phosphate buffer, add 2 mL of DMEM medium, and incubate at 37 ℃ with shaking for 24 hours.

[0171] Negative control group culture: Discard the original cell culture medium, add 2 mL of DMEM culture medium, and incubate at 37 ℃ with shaking for 2 hours; discard the original cell culture medium, wash twice with 1X phosphate buffer, add 2 mL of DMEM culture medium, and incubate at 37 ℃ with shaking for 24 hours.

[0172] Toxin-treated group culture: Discard the original cell culture medium, add 2 mL of DMEM medium containing 50 μM lead acetate, and incubate at 37°C with shaking for 2 hours; discard the toxin culture medium, wash twice with 1X phosphate buffer, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 24 hours.

[0173] Blank control group culture: Add 2 mL of DMEM medium containing 50 μM lead acetate to the 6-well plate of blank control group, and incubate at 37°C with shaking for 2 hours; discard the toxin medium, wash twice with 1X phosphate buffer, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 24 hours.

[0174] Culture of test samples: Discard the original cell culture medium, add 2 mL of DMEM medium containing 50 μM lead acetate, and incubate at 37°C with shaking for 2 hours; discard the toxin culture medium, wash twice with 1X phosphate buffer, add 2 mL of DMEM medium containing the test samples, and incubate at 37°C with shaking for 24 hours.

[0175] Cytotoxicity assay:

[0176] Weigh 50 mg of thiazolyl blue powder into a beaker, dissolve it in a small amount of 1X phosphate buffer, transfer it to a 10 mL volumetric flask, and make up to volume with 1X phosphate buffer to obtain a 5 mg / mL thiazolyl blue solution.

[0177] Add 100 μL of fresh DMEM medium to each well, gently pipette the cells to suspend them, add 20 μL of thiazolyl blue solution, incubate at 37 °C for 4 hours, discard the supernatant, add 150 μL of DMSO solution, and shake for 10 min.

[0178] The OD value was detected by an enzyme-linked immunosorbent assay (ELISA) reader: the absorbance at 490 nm was measured and compared with the negative control group, and the cell viability was calculated. The results are shown in Table 3.

[0179] Detection of intracellular toxin residues using inductively coupled plasma mass spectrometry:

[0180] Wash each well three times with 1X phosphate buffer, add 0.5 mL of 0.1% Triton X-100 cell lysis buffer, incubate on ice for 30 min, centrifuge at 4 ℃ and 15000 r / min for 15 min, collect the supernatant to obtain the sample to be tested;

[0181] Add 96.92 mL of ultrapure water to a beaker, slowly add 3.08 mL of 65% nitric acid solution, and stir until homogeneous to obtain a 2% nitric acid solution;

[0182] Transfer 100 μL of 1000 μg / mL 115 indium standard solution to a 10 mL volumetric flask and dilute to volume with 2% nitric acid solution to obtain the internal standard solution;

[0183] Transfer 100 μL of 1000 μg / mL lead standard solution to a 10 mL volumetric flask and dilute to volume with 2% nitric acid solution to obtain a 10 mg / L lead standard solution.

[0184] 1 μL, 5 μL, 10 μL, 50 μL, and 100 μL of 10 mg / L lead standard solution were transferred into 10 mL volumetric flasks and diluted to volume with 2% nitric acid solution to obtain standards with lead concentrations of 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L, respectively.

[0185] Add 0.2 mL of internal standard solution to 10 mL of the test sample, standard, and 2% nitric acid solution respectively. Use the 2% nitric acid solution without internal standard solution as the double blank and the 2% nitric acid solution with internal standard solution as the blank.

[0186] Instrument specifications: RF transmit power 1300 W; high-salt nebulizer; nebulization temperature 2 °C; 2.5 mm central channel rectangular tube; 1.0 mm nickel cone; peristaltic pump speed 0.1 rps; cooling gas flow rate 12 L / min; carrier gas flow rate 1.15 L / min; sampling mode: fully quantitative; scanning mode: peak skipping; residence time per point 0.1–0.5 sec; measurement points: 3; number of repetitions: 3; mass spectrometry counting mode: pulse / simulation; mass resolution: 0.65–0.8 amu.

[0187] A standard curve was plotted with the concentration of the standard as the x-axis and the corresponding signal intensity as the y-axis. A linear regression equation was obtained to determine the lead content in the sample. The clearance rate was calculated by comparing the sample with the toxin treatment group. The results are shown in Table 3.

[0188] Table 3. In vitro cytotoxin clearance status

[0189]

[0190] As can be seen from the test results in Table 3, the protein polypeptide containing Atractylodes macrocephala volatile oil prepared by this invention has a certain detoxification effect.

[0191] Experiment Example 4

[0192] This experimental example evaluates the anti-inflammatory effects of the test sample of Example 8, the control sample of Comparative Example 2, and the control sample of Comparative Example 3 through in vitro inflammatory cell experiments. The specific steps are as follows:

[0193] Preparation of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide:

[0194] Weigh 10 mg of lipopolysaccharide into a beaker, add 10 mL of 1X phosphate buffer, vortex until completely dissolved, and sterilize through a 0.22 μm filter membrane to obtain a 1 mg / mL lipopolysaccharide stock solution.

[0195] Transfer 1 mL of 1 mg / mL lipopolysaccharide stock solution to a 1 L volumetric flask, add RPMI 1640 medium to make up the volume, and you will get RPMI 1640 medium containing 1 μg / mL lipopolysaccharide.

[0196] Preparation of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide and 1 μM dexamethasone:

[0197] Weigh 3.925 mg of dexamethasone into a beaker, add 10 mL of anhydrous ethanol solution, sonicate at 100 W for 20 min until completely dissolved, and sterilize through a 0.22 μm filter membrane to obtain 1 mM dexamethasone stock solution.

[0198] Transfer 1 mL of 1 mg / mL lipopolysaccharide stock solution and 1 mL of 1 mM dexamethasone stock solution to a 1 L volumetric flask, add RPMI 1640 medium to make up to volume, and you will get RPMI 1640 medium containing 1 μg / mL lipopolysaccharide and 1 μM dexamethasone.

[0199] Cell preparation:

[0200] Take a concentration of 1×10 5 Logarithmic growth phase RAW264.7 cells per cell / mL were seeded at 100 μL in 6-well plates and cultured at 37°C with 5% CO2 for 24 hours until the cells adhered and reached 70-80% confluence.

[0201] Preparation of the test sample:

[0202] In Example 8, 10 mL of RPMI 1640 medium was added to the test sample, Comparative Example 2, and Comparative Example 3, respectively. The mixture was incubated at 37 °C with shaking for 2 hours, centrifuged at 4 °C and 3000 r / min for 15 min, and the supernatant was collected and sterilized through a 0.22 μm filter membrane to obtain the RPMI 1640 medium containing the test sample.

[0203] nourish:

[0204] Positive control group culture: Discard the original cell culture medium, add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide and 1 μM dexamethasone, and incubate at 37 ℃ with shaking for 2 hours; discard the culture medium containing the inflammation inducer lipopolysaccharide, wash twice with 1X phosphate buffer, add 2 mL of RPMI 1640 medium, and incubate at 37 ℃ with shaking for 24 hours.

[0205] Negative control group culture: Discard the original cell culture medium, add 2 mL of RPMI 1640 medium, and incubate at 37 ℃ with shaking for 2 hours; discard the original cell culture medium, wash twice with 1X phosphate buffer, add 2 mL of RPMI 1640 medium, and incubate at 37 ℃ with shaking for 24 hours.

[0206] Inflammation-induced group culture: Discard the original cell culture medium, add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide, and incubate at 37 ℃ with shaking for 2 hours; discard the culture medium containing the inflammation inducer lipopolysaccharide, wash twice with 1X phosphate buffer, add 2 mL of RPMI 1640 medium, and incubate at 37 ℃ with shaking for 24 hours.

[0207] Blank control group culture: Add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide to the 6-well plate of blank control group, and incubate at 37 °C with shaking for 2 hours; discard the medium containing the inflammatory inducer lipopolysaccharide, wash twice with 1X phosphate buffer, add 2 mL of DMEM medium, and incubate at 37 °C with shaking for 24 hours.

[0208] Culture of test samples: Discard the original cell culture medium, add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide, and incubate at 37 ℃ with shaking for 2 hours; discard the culture medium containing the inflammation inducer lipopolysaccharide, wash twice with 1X phosphate buffer, add 2 mL of RPMI 1640 medium containing the test samples, and incubate at 37 ℃ with shaking for 24 hours.

[0209] Cytotoxicity assay:

[0210] Weigh 50 mg of thiazolyl blue powder into a beaker, dissolve it in a small amount of 1X phosphate buffer, transfer it to a 10 mL volumetric flask, and make up to volume with 1X phosphate buffer to obtain a 5 mg / mL thiazolyl blue solution.

[0211] Add 100 μL of fresh RPMI 1640 medium to each well, gently pipette the cells to suspend them, add 20 μL of thiazolyl blue solution, incubate at 37 °C for 4 hours, discard the supernatant, add 150 μL of DMSO solution, and shake for 10 min.

[0212] The OD value was detected by an enzyme-linked immunosorbent assay (ELISA) reader: the absorbance at 490 nm was measured and compared with the negative control group, and the cell viability was calculated. The results are shown in Table 4.

[0213] Inflammatory factor detection:

[0214] Cell supernatants from various groups were collected, and the contents of mouse interleukin-6 and mouse tumor necrosis factor-α were measured according to the instructions of the mouse interleukin-6 ELISA kit and mouse tumor necrosis factor-α kit. The anti-inflammatory rate was calculated by comparing with the inflammation-induced group. The results are shown in Table 4.

[0215] Table 4. Status of inflammatory cells in vitro

[0216]

[0217] As can be seen from the test results in Table 4, the protein polypeptide containing Atractylodes macrocephala volatile oil prepared by this invention has a certain anti-inflammatory effect.

[0218] Experimental Example 5

[0219] This experimental example evaluates the dryness of the test sample from Example 8, the control sample from Comparative Example 2, and the control sample from Comparative Example 3. The specific steps are as follows:

[0220] Experimental Methods: 120 healthy volunteers, aged 20-40 years, were selected. These volunteers were randomly divided into 3 groups. After cleansing, the volunteers applied the test product of Example 8, the control product of Comparative Example 2, and the control product of Comparative Example 3, respectively. The control product of Comparative Example 2 was diluted with ultrapure water to the same volume as the test product of Example 8. The objective condition of the volunteers' faces was recorded within 24 hours of application.

[0221] Evaluation Indicators: Dryness is evaluated using objective indicators. The objective evaluation indicators are as follows: the presence of symptoms such as tightness, burning, stinging, peeling, roughness, or redness is used as the evaluation criteria. Scores are given according to the absence of the above symptoms, mild, moderate, and severe, respectively, ranging from 0 to 3 points.

[0222] Experimental results: The objective evaluation scores of each group of volunteers before and after use are shown in Table 5.

[0223] Table 5. Objective evaluation values ​​of volunteers before and after use.

[0224]

[0225] Note: * indicates a significant difference in volunteers before and after use, p<0.05.

[0226] As can be seen from the test results in Table 5, after using the test sample of Example 8 and the control sample of Comparative Example 3, there were no obvious symptoms such as tightness, burning sensation, stinging sensation, peeling, roughness, or redness. However, when using the control sample of Comparative Example 2, which does not contain sodium sulfobutyl-β-cyclodextrin, lactoferrin, or chitosan, there were significant symptoms such as tightness, burning sensation, stinging sensation, peeling, roughness, or redness. This phenomenon may be due to the sustained-release properties of the sodium sulfobutyl-β-cyclodextrin, lactoferrin, and chitosan complex, which reduces the drying properties of Atractylodes macrocephala volatile oil.

[0227] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Equal modifications and variations made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the overall concept of the present invention, shall still fall within the scope of the present invention.

Claims

1. A method for extracting volatile oil from Atractylodes macrocephala, characterized in that: The extraction method for the volatile oil of Atractylodes macrocephala specifically includes the following steps: S001. Wash the harvested Atractylodes macrocephala, let it air dry in a cool place, slice it, and crush it to obtain Atractylodes macrocephala powder. S002, add extractant, sonicate, filter, collect solution to obtain Atractylodes macrocephala volatile oil, wherein the extractant is composed of n-hexane and methanol in a volume ratio of 4:1; S003, add reaction buffer, adjust pH, add magnetic enzyme, perform enzyme treatment, remove enzyme, and collect solution. The reaction buffer consists of Tris-HCl buffer, NADPH solution, calcium chloride solution, and uridine diphosphate glucose solution. The magnetic enzyme consists of a complex enzyme, amino-modified magnetic nanoparticles, and 0.05 M Tris-HCl buffer. The complex enzyme consists of cytochrome P450 enzyme, cyclooxygenase, and uridine diphosphate glycosyltransferase. The amino-modified magnetic nanoparticles are formed by introducing amino groups onto the surface of magnetic nanoparticles. The magnetic nanoparticles are formed by encapsulating iron oxide nanoparticles with silica. During the preparation of the magnetic enzyme, the binding conditions between the complex enzyme and the amino-modified magnetic nanoparticles are: stirring at 600 r / min for 6 hours at 4–25 °C, and washing three times with twice the volume of 0.05 M Tris-HCl buffer after binding. S004, add the extractant, extract, collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, wherein the extractant is composed of sodium bisulfite, sulfobutyl-β-cyclodextrin sodium and 30% ethanol aqueous solution; The detoxifying and anti-inflammatory protein peptides are obtained by reducing and protecting oxidized volatile oils using hydrogen and lactoferrin-chitosan complexes, specifically including the following steps: S101, add 1 part of palladium-carbon nanofiber membrane to the above solution, place it in a high-pressure reactor, react, filter, and collect the solution to obtain volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution. The palladium-carbon nanofiber membrane is composed of palladium nanoparticles and carbon nanofibers. The reaction treatment method is to introduce hydrogen gas, control the pressure at 5-6 MPa, stir at 600 r / min, and heat at 40-50 ℃. After reacting for 3 hours, stop heating and stirring, and release the pressure to atmospheric pressure. S102, Weigh the lactoferrin-chitosan complex, add ultrapure water, and stir gently to dissolve it to obtain a lactoferrin-chitosan complex solution. The lactoferrin-chitosan complex is composed of lactoferrin and chitosan. S103, the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and the lactoferrin-chitosan complex solution are mixed and the pH is adjusted to obtain the detoxifying and anti-inflammatory protein peptide, wherein the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and the lactoferrin-chitosan complex solution are mixed at a volume ratio of 1:

5.

2. The method for extracting Atractylodes macrocephala volatile oil according to claim 1, characterized in that: In step S002, the extractant is added at a material-to-liquid ratio of 1:3~7, and the ultrasonic treatment conditions are 100 W for 20 min. In step S003, the volume-to-mass ratio of the reaction buffer to the Atractylodes macrocephala volatile oil is 2:1, the pH is adjusted to 6.0~7.0, and the enzyme treatment conditions are vortexing at 37 ℃ for 2~6 hours. The enzyme removal method is to place the sample in a magnetic rack and let it stand for 5 min. In step S004, the amount of extractant added is 1~3 times the volume, and the extraction treatment method is to first vortex for 1 hour, and then let it stand for 20 min.

3. The method for extracting Atractylodes macrocephala volatile oil according to claim 1, characterized in that: In step S102, the mass-to-volume ratio of lactoferrin-chitosan complex to ultrapure water is 0.1:

1. In step S103, the pH is adjusted to 6.0-7.0 using 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution.

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