Extraction method of rhizoma atractylodis macrocephalae volatile oil and toxin-expelling and inflammation-diminishing protein polypeptide applied to extraction method

Abstract

The invention relates to the technical field of plant extraction, in particular to an extraction method of bighead atractylodes rhizome volatile oil and toxin-expelling and inflammation-diminishing protein polypeptide applied to the extraction method. The invention discloses an extraction method of bighead atractylodes rhizome volatile oil, which comprises the following steps: cleaning bighead atractylodes rhizome, drying in the shade, slicing and crushing to obtain bighead atractylodes rhizome powder; secondly, extracting the rhizoma atractylodis macrocephalae volatile oil through an n-hexane-methanol binary extraction system to obtain rhizoma atractylodis macrocephalae volatile oil; then, carrying out oxidation protection on the bighead atractylodes rhizome volatile oil by virtue of a magnetic enzyme agent, and carrying out addition and inclusion by virtue of an extracting agent, so as to obtain an oxidized volatile oil-sulfobutyl-beta-cyclodextrin sodium compound solution; then, through hydrogen and the palladium carbon nanofiber membrane, the volatile oil is converted into the original volatile oil again, and a volatile oil-sulfobutyl-beta-cyclodextrin sodium compound solution is obtained; and finally, mixing with the lactoferrin-chitosan compound to obtain the toxin-expelling and inflammation-diminishing protein polypeptide.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, in particular to a method for extracting Atractylodes macrocephala volatile oil and a detoxifying and anti-inflammatory protein polypeptide used therein. Background Art

[0002] The active ingredients of Atractylodes macrocephala volatile oil are primarily terpenoids, with atractylodesone being the primary active and drying component. Atractylodes macrocephala volatile oil is highly volatile and sensitive to high temperatures, easily decomposing after prolonged heating. It is also susceptible to oxidation upon exposure to air, leading to darkening of color or polymerization.

[0003] Chinese patent publication number CN100350922C discloses a method for extracting essential oil from Atractylodes macrocephala. This method involves raw material pulverization, supercritical carbon dioxide extraction, crude separation on a silica gel column, and separation and purification using a C18 silica gel reverse-phase column. However, this process has certain drawbacks: the supercritical extraction equipment used is expensive and requires specialized technicians to operate. Furthermore, the silica gel separation process may increase the risk of oxidative deterioration due to exposure to light and air.

[0004] Chinese patent publication number CN110151809A discloses a method for preparing Atractylodes macrocephala essential oil, which comprises preparing Atractylodes macrocephala essential oil by sequentially fermenting the oil in a pile, acid extraction, and enzymatic treatment, combined with steam distillation or supercritical carbon dioxide extraction technology. However, during the long fermentation and acid extraction processes, there is a risk of oxidation of the Atractylodes macrocephala volatile oil. Steam distillation has the disadvantages of long extraction time and high energy consumption, and its high temperature environment easily leads to degradation of heat-sensitive components, which in turn results in a low yield of essential oil.

[0005] Chinese patent publication number CN109694777A discloses a method for extracting Atractylodes macrocephala volatile oil. This method involves pulverizing the raw material, followed by ultrasonic-assisted extraction with a 60-70% ethanol solution, followed by filtration, vacuum concentration to remove the ethanol, purification with a macroporous adsorption resin, and drying. However, the Atractylodes macrocephala volatile oil produced by this method exhibits poor stability during storage and use, and is susceptible to oxidation and deterioration due to environmental factors such as high temperature and light.

[0006] Chinese patent publication number CN116059399B discloses a method for preparing coated Atractylodes macrocephala oil. This method involves encapsulating Atractylodes macrocephala oil in a β-cyclodextrin system using water-insulated ultrasonic technology. After precipitation and separation, the product is washed with methanol and dried to produce the Atractylodes macrocephala oil-β-cyclodextrin inclusion complex. Due to the controlled sustained-release properties of β-cyclodextrin, some Atractylodes macrocephala oil may be removed during the methanol washing process. During the room-temperature drying process, the Atractylodes macrocephala oil may be exposed to factors such as light and air, increasing the risk of oxidative deterioration.

[0007] The Chinese patent with publication number CN103396890A discloses a method for preparing a stable Atractylodes macrocephala volatile oil. The method first extracts the Atractylodes macrocephala volatile oil by supercritical carbon dioxide extraction technology, and finally uses ultraviolet light to promote the oxidative decomposition of atractylodes ketone to obtain a stable Atractylodes macrocephala volatile oil. According to the conclusion of the invention, although ultraviolet light can increase the content of Atractylodes macrocephala lactone components, it will cause the content of atractylodes ketone to decrease, which may weaken its original anti-inflammatory, antioxidant, anti-tumor and other biological activities. In addition, the thermal effect or long-term light exposure during the ultraviolet irradiation process 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 taking into account the protection of easily oxidized, photosensitive and heat-sensitive components, and effectively controlling the adverse reactions caused by the drying components in the volatile oil has become a key technical problem that needs to be overcome in this field. Summary of the Invention

[0009] In order to solve the above problems, the purpose of the present invention is to provide a method for extracting Atractylodes macrocephala volatile oil and a detoxifying and anti-inflammatory protein polypeptide used therein. A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0010] S001, washing the harvested Atractylodes macrocephala, placing it in a cool place to dry naturally, slicing it, and crushing it to obtain Atractylodes macrocephala powder;

[0011] S002, adding an extractant, ultrasonically treating, filtering, and collecting the solution to obtain Atractylodes macrocephala volatile oil;

[0012] S003, adding reaction buffer, adjusting pH, adding magnetic enzyme agent, enzyme treatment, removing enzyme, and collecting solution;

[0013] S004, adding an extractant, performing extraction treatment, and collecting the lower layer solution to obtain an oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution.

[0014] The oxidized Atractylodes macrocephala volatile oil is reduced and protected by hydrogen and lactoferrin-chitosan complex to obtain a detoxifying and anti-inflammatory protein polypeptide, which specifically includes the following steps:

[0015] S101, adding 1 part of palladium carbon nanofiber membrane to the above solution, placing the solution in a high-pressure reactor, reacting the solution, filtering the solution, and collecting the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0016] S102, weighing a lactoferrin-chitosan complex, adding ultrapure water, and gently stirring to dissolve it, thereby obtaining a lactoferrin-chitosan complex solution;

[0017] S103, adjusting the pH of the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and the lactoferrin-chitosan complex solution to obtain a detoxifying and anti-inflammatory protein polypeptide.

[0018] The specific preparation methods of the above-mentioned related 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 is composed of Tris-HCl buffer, NADPH solution, calcium chloride solution and uridine diphosphate glucose solution. Preparation method of reaction buffer:

[0021] S201, weigh 74.54 mg of NADPH powder, dissolve it in 0.05 M Tris-HCl buffer, dilute to volume in a 10 mL volumetric flask, and shake well to obtain the 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 volume in a 10 mL volumetric flask, and shake well to obtain a calcium chloride stock solution;

[0023] S203, weigh 61.03 mg of UDP-glucose powder, dissolve it in 0.05 M Tris-HCl buffer, dilute to volume in a 10 mL volumetric flask, and shake well to obtain UDP-glucose stock solution;

[0024] S204, pipette 10 mL of NADPH stock solution, calcium chloride stock solution, and uridine diphosphate glucose stock solution into 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 agent is composed of a complex enzyme, amino-modified magnetic nanoparticles and 0.05M Tris-HCl buffer, wherein the complex enzyme is composed of cytochrome P450 enzyme, cyclooxygenase and uridine diphosphate glycosyltransferase, and the amino-modified magnetic nanoparticles are amino groups introduced on the surface of the magnetic nanoparticles, wherein the magnetic nanoparticles are ferric oxide (Fe3O4) nanoparticles wrapped with silicon dioxide.

[0026] Preparation method of magnetic enzyme agent:

[0027] S301, 50 mg of amino-modified magnetic nanoparticles, 1.15 g of N-hydroxysuccinimide, and 1.92 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were weighed, 100 mL of 0.05 M Tris-HCl buffer was added, vortexed until uniform, and stirred at 600 rpm 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 respectively into 50 mL of 0.05 M Tris-HCl buffer and vortex until homogenized to obtain a complex enzyme solution;

[0029] S303, mixing the activated amino-modified magnetic nanoparticle solution with the complex enzyme solution, and stirring at 600 rpm at 4-25°C for 6 hours;

[0030] S304, place on a magnetic stand, discard the liquid, wash three times with 2 volumes of 0.05M Tris-HCl buffer, add 100 mL of 0.05M Tris-HCl buffer, and vortex mix until uniform, thereby obtaining a magnetic enzyme agent.

[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, introduce nitrogen, and stir at room temperature at 600 rpm for 12 hours;

[0034] S403, placing on a magnetic rack, discarding the liquid, washing three times with 2 volumes of ultrapure water, washing three times with 2 volumes of anhydrous ethanol, and collecting the magnetic material;

[0035] S404, placing the magnetic material in a vacuum drying oven at 40-60°C and drying for 12 hours to obtain amino-modified magnetic nanoparticles.

[0036] Preparation method 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 water and 4 mL of tetraethoxysilane, and stir at room temperature at 600 rpm for 4 hours;

[0039] S503, place on a magnetic rack, discard the liquid, wash three times with 2 volumes of ultrapure water, wash three times with 2 volumes of anhydrous ethanol, and collect the magnetic material;

[0040] S504, placing the magnetic material in a vacuum drying oven at 40-60°C and drying for 3 hours to obtain magnetic nanoparticles.

[0041] Preparation method of Fe3O4 nanoparticles:

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

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

[0044] S603, after 1 hour, stirring and heating were stopped, the mixture was naturally cooled to room temperature, washed three times with 2 volumes of ultrapure water, and washed three times with 2 volumes of anhydrous ethanol, and the precipitate was collected;

[0045] S604, placing the precipitate in a vacuum drying oven at 40-60° C. and drying for 12 hours to obtain black Fe 3 O 4 nanoparticles.

[0046] The extractant is composed of sodium bisulfite, sodium sulfobutyl-β-cyclodextrin and 30% ethanol aqueous 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 rpm for 1 hour, cool to room temperature, and filter to remove undissolved solids to 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, and ultrasonicate at 100 W for 10 min until dissolved. Add the entire amount to a saturated sodium bisulfite solution and vortex to mix evenly to obtain the extractant.

[0049] Palladium carbon nanofiber membrane is composed of palladium nanoparticles and carbon nanofibers. Preparation method of palladium carbon nanofiber membrane:

[0050] S801, weigh 1 g of polyacrylonitrile and add it to 10 mL of N,N-dimethylformamide. Ultrasonicate at 100 W at 50-60 °C for 6 h until completely dissolved.

[0051] S802, add 5 g of palladium nitrate and continue ultrasonication at 100 W for 3 hours;

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

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

[0054] S805, placing the mixture in an oven at 70°C and drying for 3 hours to obtain a palladium carbon nanofiber membrane.

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

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

[0057] S902, weighing 1 g of chitosan into 200 mL of 1% acetic acid solution, stirring at 600 rpm for 24 hours until completely dissolved, to obtain a chitosan solution;

[0058] S903, weighing 0.4 g of lactoferrin into 20 mL of ultrapure water, and gently stirring until completely dissolved to obtain a lactoferrin solution;

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

[0060] S905, centrifuge at 15000 rpm for 15 min, collect the precipitate, add the same volume of ultrapure water as the supernatant, and vortex to mix evenly;

[0061] S906, after repeating step S905 three times, collecting the precipitate, and drying it in a vacuum environment at 0-20°C for 12 hours to obtain a lactoferrin-chitosan complex.

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

[0063] 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 with 0.1M hydrochloric acid solution and 0.1M sodium hydroxide solution, the enzyme treatment conditions are vortexing at 37°C for 2-6 hours, the amount of the magnetic enzyme agent added is 0.1 times the volume, and the method for removing the enzyme is placing it on a magnetic stand and letting it stand for 5 minutes.

[0064] In step S004, the amount of the extractant added is 1 to 3 times the volume, and the extraction treatment method is to first vortex for 1 hour and then let it stand for 20 minutes.

[0065] The reaction treatment method in step S101 is to introduce hydrogen, control the pressure at 5-6 MPa, stir at 600 r / min, heat and control the temperature at 40-50° C., stop heating and stirring after reacting for 3 hours, and release the pressure to normal pressure.

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

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

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] 1. The present invention constructs a n-hexane-methanol binary extraction system. Based on the polarity differences and molecular structural characteristics of volatile oil components, it forms a targeted and selective extraction environment. This system can efficiently extract different components in volatile oil, achieving deep extraction of all volatile oil components, thereby increasing the efficacy of Atractylodes macrocephala volatile oil. By optimizing the material-liquid ratio, the extracted volatile oil content is increased.

[0070] 2. The present invention fixes the complex enzyme by magnetic nanoparticles to obtain a magnetic enzyme. First, Fe3O4 nanoparticles are wrapped with silica to form magnetic nanoparticles, which improves the magnetic performance stability of Fe3O4 nanoparticles in different environments, prolongs their service life, and increases their dispersibility in the solution. At the same time, abundant active groups, such as hydroxyl groups, are introduced on the surface to provide possibilities for subsequent grafting. Secondly, the silane coupling agent is grafted onto the silica surface through hydrolysis and dehydration condensation reactions, so that the surface has exposed amino groups, thereby further improving the surface reaction activity of the magnetic nanoparticles, further increasing their dispersibility in the solution, and at the same time, giving them a certain adsorption capacity. Finally, the complex enzyme is fixed on the surface of the magnetic nanoparticles through covalent bonding and adsorption. After the enzyme reaction is completed, under the action of an external magnetic field, the enzyme and the reaction product can be quickly and efficiently separated, and the reuse rate of the enzyme is increased. At the same time, the exposed amino groups on its surface can regulate the microenvironment around the enzyme molecules and enhance the catalytic activity of the enzyme.

[0071] 3. The present invention adopts a composite enzyme catalytic system, which can act on the effective ingredients in the volatile oil of Atractylodes macrocephala. Through a specific oxidation reaction, the easily oxidized and heat-sensitive components in the volatile oil are directionally converted into relatively stable oxidation products;

[0072] 4. The present invention constructs a "multi-step coordinated purification-phase transfer synergistic" technical system. First, an extractant is used to achieve deep extraction of volatile oils. Through the nucleophilic addition reaction of a saturated sodium bisulfite solution, the components containing double bonds or carbonyl groups in the volatile oils are converted into addition products, further increasing their water solubility and extending the storage and long-term use of the volatile oils. Although the water solubility of the oxidized volatile oils is improved by the composite enzyme and the extractant, it still tends to dissolve in organic reagents as a whole. The special structure of sodium sulfobutyl-β-cyclodextrin, "hydrophobic inner cavity-hydrophilic outer shell", encapsulates the oxidized volatile oils, thereby achieving the transfer of the oxidized volatile oils from the organic phase to the aqueous phase. At the same time, the controlled release characteristics of sodium sulfobutyl-β-cyclodextrin are utilized to achieve a sustained release effect of the volatile oils, thereby effectively reducing the dryness of the Atractylodes macrocephala volatile oils.

[0073] 5. The present invention uses green and clean hydrogen as a reducing agent and palladium-carbon nanofiber membrane as a catalyst to convert oxidized volatile oil back into the original volatile oil. By utilizing the specific surface area of ​​the catalyst and the highly dispersed palladium active sites, a "hydrogen adsorption-active hydrogen dissociation-substrate reduction" catalytic mechanism is formed to improve its reaction efficiency. After the reaction is completed, based on the physical interception characteristics of the nanofibers, the catalyst can be quickly separated and recovered by low-pressure filtration. This process does not require the introduction of complex post-processing steps such as acid-base neutralization and extraction, thus avoiding the damage to the volatile oil components caused by chemical reagent residues and high-temperature and high-pressure treatments.

[0074] 6. The present invention mixes the lactoferrin-chitosan complex with the volatile oil-sulfobutyl-β-cyclodextrin sodium complex to form a multi-complex solution. First, the antioxidant effect of the complex is enhanced by compounding, while retaining the active sites on the molecule, providing a rich chemical interface for subsequent targeted modification; second, the microstructure and conformation of sulfobutyl-β-cyclodextrin sodium are changed by the composite chitosan, thereby enhancing the stability of the contained volatile oil. Due to the bioadhesion and degradability of chitosan, the slow release of the contained volatile oil can be achieved, reducing the dryness of the Atractylodes macrocephala volatile oil. In addition, the synergistic effect of chitosan and sulfobutyl-β-cyclodextrin sodium may also activate the key active ingredients in the volatile oil;

[0075] 7. The preparation method employed in the present invention is simple to operate, and during the efficient purification process, added foreign impurities can be removed by simple magnetic attraction and filtration. Furthermore, the resulting multi-composite product exhibits a certain synergistic effect, which can enhance the efficacy of the activated product. DETAILED DESCRIPTION

[0076] The present invention is further described below in conjunction with specific embodiments.

[0077] Example 1

[0078] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0079] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

[0080] Weigh 100g of Atractylodes macrocephala powder, add the extractant, ultrasonicate at a solid-liquid ratio of 1:3, filter, and collect the solution to obtain Atractylodes macrocephala volatile oil;

[0081] Reaction buffer was added, pH was adjusted, magnetic enzyme reagent was added, vortexed at 37°C for 4 h, enzyme was removed, and the solution was collected;

[0082] Add 2 times the volume of extractant, perform extraction treatment, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0083] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, which is test sample 1.

[0084] Example 2

[0085] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0086] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

[0087] Weigh 100g of Atractylodes macrocephala powder, add the extractant, ultrasonicate at a solid-liquid ratio of 1:5, filter, and collect the solution to obtain Atractylodes macrocephala volatile oil;

[0088] Reaction buffer was added, pH was adjusted, magnetic enzyme reagent was added, vortexed at 37°C for 4 h, enzyme was removed, and the solution was collected;

[0089] Add 2 times the volume of extractant, perform extraction treatment, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0090] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, which is sample 2.

[0091] Example 3

[0092] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0093] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

[0094] Weigh 100g of Atractylodes macrocephala powder, add the extractant, ultrasonicate at a solid-liquid ratio of 1:7, filter, and collect the solution to obtain Atractylodes macrocephala volatile oil;

[0095] Reaction buffer was added, pH was adjusted, magnetic enzyme reagent was added, vortexed at 37°C for 4 h, enzyme was removed, and the solution was collected;

[0096] Add 2 times the volume of extractant, perform extraction treatment, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0097] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, which is sample 3.

[0098] Example 4

[0099] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0100] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

[0101] Weigh 100g of Atractylodes macrocephala powder, add the extractant, ultrasonicate at a solid-liquid ratio of 1:5, filter, and collect the solution to obtain Atractylodes macrocephala volatile oil;

[0102] Reaction buffer was added, pH was adjusted, magnetic enzyme reagent was added, vortexed at 37°C for 2 h, enzyme was removed, and the solution was collected;

[0103] Add 2 times the volume of extractant, perform extraction treatment, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0104] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, namely, test sample 4.

[0105] Example 5

[0106] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0107] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

[0108] Weigh 100g of Atractylodes macrocephala powder, add the extractant, ultrasonicate at a solid-liquid ratio of 1:5, filter, and collect the solution to obtain Atractylodes macrocephala volatile oil;

[0109] Reaction buffer was added, pH was adjusted, magnetic enzyme agent was added, vortexed at 37°C for 6 h, enzyme was removed, and the solution was collected;

[0110] Add 2 times the volume of extractant, perform extraction treatment, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0111] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, namely, test sample 5.

[0112] Example 6

[0113] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0114] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

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

[0116] Reaction buffer was added, pH was adjusted, magnetic enzyme reagent was added, vortexed at 37°C for 4 h, enzyme was removed, and the solution was collected;

[0117] Add 1 volume of extractant, perform extraction, 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, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, namely, test sample 6.

[0119] Example 7

[0120] A method for extracting Atractylodes macrocephala volatile oil specifically comprises the following steps:

[0121] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and grind it to obtain Atractylodes macrocephala powder;

[0122] Weigh 100g of Atractylodes macrocephala powder, add the extractant, ultrasonicate at a solid-liquid ratio of 1:5, filter, and collect the solution to obtain Atractylodes macrocephala volatile oil;

[0123] Reaction buffer was added, pH was adjusted, magnetic enzyme reagent was added, vortexed at 37°C for 4 h, enzyme was removed, and the solution was collected;

[0124] Add 3 times the volume of extractant, perform extraction treatment, and collect the lower layer solution to obtain the oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution;

[0125] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution, namely, sample 7.

[0126] Example 8

[0127] The detoxification and anti-inflammatory protein polypeptide was prepared using the sample 2 prepared in Example 2, and the specific steps were as follows:

[0128] Weigh 100 mg of lactoferrin-chitosan complex, add 100 mL of ultrapure water, and gently stir to dissolve it to obtain a lactoferrin-chitosan complex solution;

[0129] The test sample 2 and the lactoferrin-chitosan complex solution were mixed at a volume ratio of 1:5, and the pH was adjusted to 6.0-7.0 with 0.1M hydrochloric acid solution and 0.1M sodium hydroxide solution to obtain a detoxifying and anti-inflammatory protein polypeptide, namely, the test sample 8.

[0130] Comparative Example 1

[0131] Wash the harvested Atractylodes macrocephala, place it in a cool place to dry naturally, slice it, and crush it to obtain Atractylodes macrocephala powder. Weigh 100g of Atractylodes macrocephala powder to obtain reference substance 1.

[0132] Comparative Example 2

[0133] The volatile oil obtained in Comparative Example 1 through Experimental Example 1 was collected to obtain Reference Sample 2.

[0134] Comparative Example 3

[0135] A control substance containing only sodium sulfobutyl-β-cyclodextrin, lactoferrin, and chitosan without volatile oil of Atractylodes macrocephala specifically comprises the following steps:

[0136] The same volume of extractant and reaction buffer as in Example 2 was transferred, the pH was adjusted to 6.0-7.0 with 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide solution, 0.1 times the volume of magnetic enzyme agent was added, and the mixture was vortexed at 37°C for 4 hours to remove the enzyme, and the solution was collected;

[0137] Add 2 times the volume of extractant, perform extraction, and collect the lower layer solution;

[0138] Add 1 part of palladium carbon nanofiber membrane to the above solution, put it into a high-pressure reactor, react, filter, and collect the solution to obtain solution A;

[0139] Weigh 100 mg of lactoferrin-chitosan complex, add 100 mL of ultrapure water, and gently stir to dissolve it to obtain a lactoferrin-chitosan complex solution;

[0140] Solution A and lactoferrin-chitosan complex solution were mixed 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 substance 3.

[0141] Experimental Example 1

[0142] In this experimental example, the content of Atractylodes macrocephala volatile oil in the test samples of Examples 1 to 7 and the reference sample of Comparative Example 1 was determined by steam distillation. The specific steps are as follows:

[0143] Place the test sample and reference sample in separate flasks, add 500 mL of ultrapure water and several glass beads, shake to mix, and then connect a volatile oil analyzer to a reflux condenser. Add water from the upper end of the condenser until the oil fills the scale on the volatile oil analyzer and overflows into the flask. Place the sample in an electric heating mantle and slowly heat to boiling. Maintain a gentle boil for approximately 5 hours, until the oil level in the analyzer no longer increases. Remove the heat, allow the sample to stand for a moment, open the stopcock at the lower end of the analyzer, and slowly drain the water until the top of the oil layer reaches 5 mm above the zero mark. Allow the sample to stand for at least 1 hour, then open the stopcock again to allow the oil layer to descend until its top is flush with the zero mark. Read the volume of the volatile oil. Perform the measurement three times for each test sample and reference sample, and calculate the average volume of the volatile oil.

[0144] According to the volume and weight of Atractylodes macrocephala volatile oil, the content of Atractylodes macrocephala volatile oil in the test sample and the reference sample was obtained. The determination results are shown in Table 1.

[0145] Table 1 Content of volatile oil in Atractylodes macrocephala

[0146] sample Content of volatile oil in Atractylodes macrocephala (%) Example 1 1.0**** Example 2 1.5**** Example 3 1.5**** Example 4 1.3* Example 5 1.5**** Example 6 1.3 Example 7 1.5**** Comparative Example 1 1.3

[0147] Note: * indicates that there is a significant difference between the examples and the comparative examples, p<0.05.

[0148] By comparison with Comparative Example 1, the optimal extractant volume in Examples 1-3 was a solid-liquid ratio of 1:5; the optimal enzyme reaction time in Examples 2, 4, and 5 was 4 hours; and the optimal extractant volume in Examples 2, 6, and 7 was 2 volumes. As can be seen from Table 1, the volume of extractant added, the enzyme reaction time, and the volume of extractant added in the extraction method of the present invention all affect the extraction of Atractylodes macrocephala volatile oil, and the extraction method of the present invention significantly improves the extraction of Atractylodes macrocephala volatile oil.

[0149] Experimental Example 2

[0150] In this experimental example, the contents of atractylodesone, γ-olemene, β-eudesmol, γ-elemene, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III in the test samples of Examples 1 to 7 and the reference sample of Comparative Example 2 were determined and analyzed by GC-MS. The specific steps are as follows:

[0151] 1. Preparation of reference solution:

[0152] Weigh 100 mg of atractylodesone, γ-malolene, β-eudesmol, γ-elemene, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III reference substances, respectively, dissolve them in a small amount of n-hexane, transfer them to a 100 mL brown volumetric flask, and adjust the volume with n-hexane to obtain a mixed standard stock solution.

[0153] Pipette 0.5 μL, 1 μL, 5 μL, 10 μL, 50 μL, 100 μL, 500 μL, and 1000 μL of the stock solution into a 10 mL volumetric flask, 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;

[0154] 2. Preparation of test samples:

[0155] Pipette 900 mL of ultrapure water and 100 mL of 10X phosphate buffer, vortex mix, add 0.5 g of β-cyclodextrinase, vortex mix for 5 minutes until completely dissolved, and filter through a 0.22 μm filter membrane to obtain the enzyme solution;

[0156] Add 0.5 times the volume of the extract to the test samples of Examples 1 to 7, stir and mix, react at 30-40°C for 1 hour, add 2 times the volume of 80% n-hexane methanol solution, vortex for 10 minutes, let stand for 5 minutes, place in a separatory funnel, collect the upper organic phase, blow dry with nitrogen at 40°C, add 200 μL of n-hexane, and vortex for 10 minutes until dissolved;

[0157] Add 200 μL of n-hexane to the control sample of Comparative Example 2 and vortex for 10 min until dissolved;

[0158] 3. Instrument conditions: The chromatographic column is a DB-5MS quartz capillary (0.25 mm × 30 m × 0.25 μm). The vaporization temperature is 280°C. The temperature program is: initial temperature 50°C, hold for 2 min; increase the temperature to 200°C at a rate of 5°C / min, then increase the temperature to 250°C at a rate of 6°C / min, and hold for 10 min. The split ratio is 10:1, and the injection volume is 1 μL. The carrier gas is high-purity helium, the ion source is an EI source, the transfer line temperature is 250°C, the ion source temperature is 200°C, the full scan mode, and the mass range is m / z 30-650.

[0159] The analytes were atractylodesone, γ-olemene, β-eudesmol, γ-elemene, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III. The concentration of the standard working solution of the analyte was used as the abscissa and the corresponding peak area was used as the ordinate. A standard curve was drawn to obtain a linear regression equation. The content of the analyte in the volatile oil of Atractylodes macrocephala in the test and reference samples was calculated. The determination results are shown in Table 2.

[0160] Table 2 Analyte content of Atractylodes macrocephala volatile oil

[0161]

[0162] As can be seen from Table 2, the extraction method of the present invention can extract atractylodesone, γ-olemene, β-eudesmol, γ-elemene, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III from the volatile oil of Atractylodes macrocephala, and the content thereof is significantly higher than that of the conventional volatile oil extraction by steam distillation.

[0163] Experimental Example 3

[0164] This experimental example evaluated the detoxification effect of the test sample of Example 8, the reference sample of Comparative Example 2, and the reference sample of Comparative Example 3 by an in vitro cytotoxin clearance experiment. The specific steps are as follows:

[0165] 1. Preparation of 1X Phosphate Buffered Saline:

[0166] Pipette 900 mL of sterile water and 100 mL of 10X phosphate buffer and vortex to mix to obtain 1X phosphate buffer.

[0167] 2. Preparation of DMEM medium containing 50 μM lead acetate:

[0168] Weigh 189.7 mg of lead acetate trihydrate into a beaker, add 10 mL of 1X phosphate buffer, and sonicate at 300 W at 30°C until completely dissolved. Sterilize through a 0.22 μm filter to obtain a 50 mM lead acetate stock solution.

[0169] Pipette 1 mL of 50 mM lead acetate stock solution into a 1 L volumetric flask and add DMEM medium to make up to volume to obtain DMEM medium containing 50 μM lead acetate.

[0170] 3. Preparation of DMEM medium containing 50 μM lead acetate and 1 mM glutathione:

[0171] 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 to obtain a 1 M glutathione stock solution.

[0172] Pipette 1 mL of 50 mM lead acetate stock solution and 1 mL of 1 M glutathione stock solution into a 1 L volumetric flask, and add DMEM medium to make up to volume to obtain DMEM medium containing 50 μM lead acetate and 1 mM glutathione.

[0173] 4. Cell Preparation:

[0174] The concentration is 5×10 4 100 μL of HaCaT cells in logarithmic growth phase at 100 μL / mL were seeded into 6-well plates and cultured at 37°C under 5% carbon dioxide for 24 hours until the cells adhered to the wall and the confluence reached 70-80%.

[0175] 5. Preparation of test samples:

[0176] 10 mL of DMEM medium was added to the test sample of Example 8, the control sample of Comparative Example 2, and the control sample of Comparative Example 3, respectively. The mixture was shaken and incubated at 37°C for 2 hours. The mixture was centrifuged at 4°C and 3000 rpm for 15 minutes. The supernatant was taken and sterilized through a 0.22 μm filter membrane to obtain the DMEM medium containing the test sample.

[0177] 6. Cultivation:

[0178] Positive control group culture: discard the original cell culture medium, add 2 mL of DMEM culture medium containing 50 μM lead acetate and 1 mM glutathione, 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 culture medium, and incubate at 37°C with shaking for 24 hours.

[0179] Negative control group culture: discard the original cell culture medium, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 2 hours; discard the original cell culture medium, wash twice with 1X phosphate buffered saline, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 24 hours.

[0180] 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 buffered saline, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 24 hours.

[0181] For blank control group culture, add 2 mL of DMEM medium containing 50 μM lead acetate to the blank wells of the 6-well plate and incubate at 37°C with shaking for 2 hours. Discard the toxin medium, wash twice with 1X phosphate buffered saline, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 24 hours.

[0182] Culture the sample group to be tested: discard the original cell culture medium, add 2 mL of DMEM culture 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 buffered saline, add 2 mL of DMEM culture medium containing the sample to be tested, and incubate at 37°C with shaking for 24 hours.

[0183] 7. Cytotoxicity assay:

[0184] 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 dilute to volume with 1X phosphate buffer to obtain a 5 mg / mL thiazolyl blue solution.

[0185] Add 100 μL of fresh DMEM medium to each well, gently pipette the cells to fully 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 minutes;

[0186] The OD value was detected by enzyme-labeled instrument: the absorbance at 490 nm was measured, compared with the negative control group, and the cell survival rate was calculated. The measurement results are shown in Table 3.

[0187] 8. Detection of endotoxin residues by inductively coupled plasma mass spectrometry:

[0188] Each well was washed three times with 1X phosphate buffer, and 0.5 mL of 0.1% Triton X-100 cell lysis buffer was added. The cells were incubated on ice for 30 min, and centrifuged at 4°C and 15,000 rpm for 15 min. The supernatant was collected to obtain the sample to be tested.

[0189] Add 96.92 mL of ultrapure water to a beaker, slowly add 3.08 mL of 65% nitric acid solution, and stir until uniform to obtain a 2% nitric acid solution.

[0190] Pipette 100 μL of 1000 μg / mL 115 indium standard solution into a 10 mL volumetric flask and dilute to volume with 2% nitric acid solution to obtain the internal standard solution.

[0191] Pipette 100 μL of 1000 μg / mL lead standard solution into a 10 mL volumetric flask and dilute to volume with 2% nitric acid solution to obtain a 10 mg / L lead standard solution.

[0192] Pipette 1 μL, 5 μL, 10 μL, 50 μL, and 100 μL of the 10 mg / L lead standard solution into a 10 mL volumetric flask, and dilute 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;

[0193] Add 0.2 mL of internal standard solution to 10 mL of the sample to be tested, the standard, and the 2% nitric acid solution respectively. Use the 2% nitric acid solution without the internal standard solution as the double blank and the 2% nitric acid solution with the internal standard solution as the blank.

[0194] Instrument conditions: RF transmission power 1300 W; high salt nebulizer; nebulization temperature 2°C; torque tube 2.5 mm center channel; 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 full quantitative; scanning mode peak skipping; dwell time per point 0.1-0.5 sec; measurement points 3; repetition number 3; mass spectrometer counting mode pulse / analog; mass resolution 0.65-0.8 amu;

[0195] The standard curve was drawn with the concentration of the standard as the horizontal axis and the corresponding signal intensity as the vertical axis to obtain the linear regression equation, and the lead content in the sample was obtained. The clearance rate was calculated by comparing with the toxin treatment group. The measurement results are shown in Table 3.

[0196] Table 3 In vitro cytotoxin clearance

[0197] sample Cell survival rate (%) Clearance rate (%) Example 8 86.1 88.3 Comparative Example 2 72.5 74.3 Comparative Example 3 61.8 63.4

[0198] It can be seen from the test results in Table 3 that the protein polypeptide containing Atractylodes macrocephala volatile oil prepared in the present invention has a certain detoxification effect.

[0199] Experimental Example 4

[0200] This experimental example evaluated the anti-inflammatory effects of the test product of Example 8, the reference product of Comparative Example 2, and the reference product of Comparative Example 3 by an in vitro inflammatory cell experiment. The specific steps are as follows:

[0201] 1. Preparation of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide:

[0202] Weigh 10 mg of LPS into a beaker, add 10 mL of 1X phosphate buffer, vortex until completely dissolved, and sterilize through a 0.22 μm filter to obtain a 1 mg / mL LPS stock solution.

[0203] Pipette 1 mL of 1 mg / mL LPS stock solution into a 1 L volumetric flask and add RPMI 1640 medium to make up to volume. This will give RPMI 1640 medium containing 1 μg / mL LPS.

[0204] 2. Preparation of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide and 1 μM dexamethasone:

[0205] Weigh 3.925 mg of dexamethasone into a beaker, add 10 mL of anhydrous ethanol solution, and sonicate at 100 W for 20 min until completely dissolved. Sterilize through a 0.22 μm filter to obtain a 1 mM dexamethasone stock solution.

[0206] Pipette 1 mL of 1 mg / mL lipopolysaccharide stock solution and 1 mL of 1 mM dexamethasone stock solution into a 1 L volumetric flask, and add RPMI 1640 medium to adjust the volume to obtain RPMI 1640 medium containing 1 μg / mL lipopolysaccharide and 1 μM dexamethasone.

[0207] 3. Cell Preparation:

[0208] The concentration is 1×10 5 100 μL of RAW264.7 cells in logarithmic growth phase at 100 μL / mL were seeded into 6-well plates and cultured at 37°C under 5% carbon dioxide for 24 hours until the cells adhered to the wall and the confluence reached 70-80%.

[0209] 4. Preparation of test samples:

[0210] 10 mL of RPMI 1640 medium was added to the test sample of Example 8, the control sample of Comparative Example 2, and the control sample of Comparative Example 3, respectively. The mixture was shaken and incubated at 37°C for 2 hours, centrifuged at 4°C and 3000 rpm for 15 minutes, and the supernatant was taken and sterilized through a 0.22 μm filter membrane to obtain RPMI 1640 medium containing the sample to be tested.

[0211] 5. Cultivation:

[0212] 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°C with shaking for 2 hours; discard the culture medium containing the inflammation-inducing agent lipopolysaccharide, wash twice with 1X phosphate buffered saline, add 2 mL of RPMI 1640 medium, and incubate at 37°C with shaking for 24 hours.

[0213] Negative control group culture: discard the original cell culture medium, add 2 mL of RPMI 1640 medium, and incubate at 37°C with shaking for 2 hours; discard the original cell culture medium, wash twice with 1X phosphate buffered saline, add 2 mL of RPMI 1640 medium, and incubate at 37°C with shaking for 24 hours.

[0214] Inflammation-induced culture: discard the original cell culture medium, add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide, and incubate at 37°C with shaking for 2 hours; discard the culture medium containing the inflammation-inducing agent lipopolysaccharide, wash twice with 1X phosphate buffered saline, add 2 mL of RPMI 1640 medium, and incubate at 37°C with shaking for 24 hours.

[0215] For blank control group culture, add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide to the blank wells of the 6-well plate and incubate at 37°C with shaking for 2 hours. Discard the medium containing the inflammatory inducer lipopolysaccharide, wash twice with 1X phosphate buffered saline, add 2 mL of DMEM medium, and incubate at 37°C with shaking for 24 hours.

[0216] Culture the sample group to be tested: discard the original cell culture medium, add 2 mL of RPMI 1640 medium containing 1 μg / mL lipopolysaccharide, and incubate at 37°C with shaking for 2 hours; discard the culture medium containing the inflammation-inducing agent lipopolysaccharide, wash twice with 1X phosphate buffered saline, add 2 mL of RPMI 1640 medium containing the sample to be tested, and incubate at 37°C with shaking for 24 hours.

[0217] 6. Cytotoxicity assay:

[0218] 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 dilute to volume with 1X phosphate buffer to obtain a 5 mg / mL thiazolyl blue solution.

[0219] Add 100 μL of fresh RPMI 1640 medium to each well, gently pipette the cells to fully 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 minutes;

[0220] The OD value was detected by enzyme-labeled instrument: the absorbance at 490 nm was measured, compared with the negative control group, and the cell survival rate was calculated. The measurement results are shown in Table 4.

[0221] 7. Inflammatory factor detection:

[0222] The cell supernatants of various groups were collected and operated according to the instructions of the mouse interleukin-6 ELISA kit and the mouse tumor necrosis factor-α kit to measure the levels of mouse interleukin-6 and mouse tumor necrosis factor-α. The levels were compared with those of the inflammation-induced group and the anti-inflammatory rate was calculated. The measurement results are shown in Table 4.

[0223] Table 4 In vitro inflammatory cell status

[0224] sample Cell survival rate (%) Anti-inflammatory rate (%) Example 8 87.2 89.0 Comparative Example 2 71.5 70.3 Comparative Example 3 62.6 61.1

[0225] It can be seen from the test results in Table 4 that the protein polypeptide containing Atractylodes macrocephala volatile oil prepared in the present invention has a certain anti-inflammatory effect.

[0226] Experimental Example 5

[0227] This experimental example evaluates the dryness of the test sample of Example 8, the reference sample of Comparative Example 2, and the reference sample of Comparative Example 3. The specific steps are as follows:

[0228] 1. Experimental Method: 120 volunteers with healthy skin, aged 20 to 40 years, were randomly divided into three groups. After cleansing their faces, the volunteers were each smeared with the test product of Example 8, the control product of Comparative Example 2, and the control product of Comparative Example 3. 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 facial condition of the volunteers was recorded within 24 hours of application.

[0229] 2. Evaluation Indicators: Dryness is evaluated using objective indicators. Objective evaluation indicators include the presence of symptoms such as tightness, burning, stinging, scaling, roughness, or redness. Dryness is scored on a scale of 0 to 3, with no symptoms, mild, moderate, or severe.

[0230] 3. Test results: The objective evaluation scores of each group of volunteers before and after use are shown in Table 6.

[0231] Table 5 Objective evaluation values ​​of volunteers before and after use

[0232] sample Before use After use Example 8 0.2±0.4 0.3±0.5 Comparative Example 2 0.2±0.4 1.8±0.5**** Comparative Example 3 0.2±0.4 0.3±0.6

[0233] Note: * indicates significant difference between volunteers before and after use, p<0.05.

[0234] As can be seen from the measurement results in Table 5, after using the test sample of Example 8 and the reference sample of Comparative Example 3, no obvious symptoms such as tightness, burning, stinging, scaling, roughness, or redness were observed. However, using the reference sample of Comparative Example 2, which did not contain sodium sulfobutyl-β-cyclodextrin, lactoferrin, or chitosan, significant symptoms such as tightness, burning, stinging, scaling, roughness, or redness were observed. This phenomenon may be due to the sustained-release properties of the complex of sodium sulfobutyl-β-cyclodextrin, lactoferrin, and chitosan, which reduces the drying properties of the Atractylodes macrocephala volatile oil.

[0235] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any modifications and changes made to the technical solution of the present invention by ordinary persons in the art shall still fall within the scope of the present invention as long as they do not depart from the overall concept of the present invention.

Claims

1. A method for extracting volatile oil from Atractylodes macrocephala, characterized in that: The method for extracting the Atractylodes macrocephala volatile oil specifically comprises the following steps: S001, washing the harvested Atractylodes macrocephala, placing it in a cool place to dry naturally, slicing it, and crushing it to obtain Atractylodes macrocephala powder; S002, adding an extractant, ultrasonically treating, filtering, and collecting the solution to obtain Atractylodes macrocephala volatile oil; S003, adding reaction buffer, adjusting pH, adding magnetic enzyme agent, enzyme treatment, removing enzyme, and collecting solution; S004, adding an extractant, performing extraction treatment, and collecting the lower layer solution to obtain an oxidized volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution.

2. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 1, wherein: The oxidized volatile oil is reduced and protected by hydrogen and lactoferrin-chitosan complex to obtain a detoxifying and anti-inflammatory protein polypeptide, which specifically includes the following steps: S101, adding 1 part of palladium carbon nanofiber membrane to the above solution, placing the solution in a high-pressure reactor, reacting the solution, filtering the solution, and collecting the solution to obtain a volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution; S102, weighing a lactoferrin-chitosan complex, adding ultrapure water, and gently stirring to dissolve it, thereby obtaining a lactoferrin-chitosan complex solution; S103, adjusting the pH of the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and the lactoferrin-chitosan complex solution to obtain a detoxifying and anti-inflammatory protein polypeptide.

3. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 1, wherein: The extractant is composed of n-hexane and methanol in a volume ratio of 4:

1.

4. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 1, wherein: The reaction buffer consists of Tris-HCl buffer, NADPH solution, calcium chloride solution and uridine diphosphate glucose solution.

5. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 1, wherein: The magnetic enzyme agent is composed of a complex enzyme, amino-modified magnetic nanoparticles and 0.05M Tris-HCl buffer, wherein the complex enzyme is composed of cytochrome P450 enzyme, cyclooxygenase and uridine diphosphate glycosyltransferase, and the amino-modified magnetic nanoparticles are amino groups introduced on the surface of the magnetic nanoparticles, wherein the magnetic nanoparticles are ferric oxide nanoparticles wrapped with silicon dioxide.

6. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 1, wherein: The extractant consists of sodium bisulfite, sodium sulfobutyl-β-cyclodextrin and 30% ethanol aqueous solution.

7. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 2, wherein: The palladium-carbon nanofiber membrane is composed of palladium nanoparticles and carbon nanofibers.

8. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 2, wherein: Lactoferrin-chitosan complex is composed of lactoferrin and chitosan.

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

10. The method for extracting the volatile oil of Atractylodes macrocephala according to claim 2, wherein: The reaction treatment method in step S101 is to introduce hydrogen, control the pressure at 5-6 MPa, stir at 600 r / min, control the temperature at 40-50° C., stop heating and stirring after reacting for 3 hours, and release the pressure to normal pressure. In step S102, the mass volume ratio of the lactoferrin-chitosan complex and ultrapure water is 0.1:

1. In step S103, 0.1M hydrochloric acid solution and 0.1M sodium hydroxide solution are used to adjust the pH to 6.0-7.0, and the volatile oil-sulfobutyl-β-cyclodextrin sodium complex solution and the lactoferrin-chitosan complex solution are mixed in a volume ratio of 1:5.

Images