Angelica sinensis essential oil, preparation method thereof and soothing anti-allergic protein polypeptide applied to angelica sinensis essential oil

Abstract

The invention relates to the technical field of plant essential oil extraction, in particular to angelica sinensis essential oil, a preparation method thereof and soothing anti-allergic protein polypeptide applied to the angelica sinensis essential oil. The invention discloses a preparation method of angelica sinensis essential oil, which comprises the following steps: firstly, improving the solubility of the angelica sinensis essential oil through a cosolvent; secondly, activating polyethylene glycol in the cosolvent through an activating agent; then, forming a triblock copolymer with a polylactic acid-glycolic acid copolymer in the extracting agent, and rinsing and purifying through ultrapure water and absolute ethyl alcohol; and finally, redissolving the triblock copolymer through a redissolving agent to obtain the soothing anti-allergic protein polypeptide liquid containing the angelica sinensis essential oil. Meanwhile, through the multi-mechanism synergistic effect of the protective agent, UVA and UVB full-wave band protection and weather resistance are provided for the angelica sinensis essential oil and the soothing anti-allergy protein polypeptide liquid containing the angelica sinensis essential oil.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant essential oil extraction, in particular to a soothing anti-allergic protein polypeptide of angelica essential oil and a preparation method and application thereof. BACKGROUND

[0002] Essential oils have characteristics such as high concentration, high volatility and strong penetration, and contain components such as terpenes, alcohols, esters, aldehydes and ketones, which together give essential oils unique aroma and efficacy.

[0003] Essential oils are volatile aromatic substances extracted by distillation, pressing, extraction and other methods. In the distillation process, some heat-sensitive components may be destroyed, such as ligustrazine, thereby affecting the quality of essential oils, and the extraction efficiency of some less volatile components, such as ferulic acid, may be low. The extraction efficiency of the pressing method is relatively low, a large amount of raw materials is needed to obtain a certain amount of essential oil, and the essential oil obtained by pressing may contain more impurities, which needs to be further filtered and refined. In the extraction method, organic solvents may be left in the essential oil, which needs to be strictly removed to ensure the safety of the essential oil.

[0004] A preparation method of angelica essential oil for repairing facial acne is disclosed in Chinese patent CN116870082A, which extracts angelica essential oil through steps such as washing, drying, deionized water soaking, high-pressure cell wall breaking, and carbon dioxide supercritical fluid extraction. Although it is extracted by carbon dioxide supercritical fluid extraction at a lower temperature, which effectively protects heat-sensitive components and active substances in essential oils, the equipment and site investment is large, the operating conditions are harsh, and high-pressure equipment and professional technicians are needed for operation.

[0005] A preparation method of angelica essential oil is disclosed in Chinese patent CN114989891A, which extracts angelica essential oil through steps such as screening, washing, drying, crushing and sieving, mixing with a eutectic solvent, microwave heating, centrifugation, ethyl acetate extraction, removing the solvent, and molecular distillation. The eutectic solvent is composed of an alcohol hydrogen bond donor, choline chloride and water, and an excess of choline chloride may interfere with cell metabolism. The use of microwave heating may have risks such as scale limitation and poor temperature uniformity in industrial scale-up. Ethyl acetate extraction has the problem of residual organic solvents. Compared with traditional distillation, molecular distillation has complex equipment, high cost, small processing capacity, and may also have the loss of light components such as limonene.

[0006] The Chinese patent with publication number CN114209722B discloses a kind of angelica essential oil emulsification by mixing with surfactant and co-surfactant. The surfactant used is Tween-80, and the co-surfactant is ethanol. However, due to the high volatility, heat sensitivity, easy oxidation and photosensitivity of angelica essential oil, the environmental conditions for its storage need to be strictly controlled to prevent failure.

[0007] The Chinese patent with publication number CN118286162A discloses a preparation method of angelica essential oil-loaded pea protein and ipomoea pectin composite nanoparticles. The angelica essential oil is uniformly stirred with a pea protein solution to form angelica essential oil-protein composite nanoparticles, which are further mixed with an ipomoea pectin solution, freeze-dried to obtain angelica essential oil-loaded pea protein-ipomoea pectin composite nanoparticle solution. This method protects angelica essential oil from oxidative damage through pea protein and ipomoea pectin composite nanoparticles. However, the stability of protein solution and pectin is easily affected by many external factors, such as temperature change, pH change, interfacial adsorption and enzymatic hydrolysis.

[0008] In summary, the key technical problems to be solved at present are: in the process of efficient extraction of angelica essential oil, it is necessary to effectively inhibit the loss of volatility, thermal degradation, oxidation deterioration and photolysis reaction, and to ensure the stability of the final essential oil and the efficient use of bioactive ingredients. SUMMARY

[0009] To solve the above problems, the purpose of the present application is to provide a soothing anti-allergic protein polypeptide of angelica essential oil and its preparation method and application. The preparation method can prepare a kind of angelica essential oil, which specifically includes the following steps:

[0010] S001, screen, wash, dry, crush and sieve angelica in sequence to obtain angelica powder;

[0011] S002, add cosolvent, vortex for 20 min at a solid-liquid ratio of 1:1-5, centrifuge at 15000 r / min for 15 min, discard the precipitate and collect the solution;

[0012] S003, add the same volume of activator, vortex for 6 hours to obtain the original solution;

[0013] S004, slowly add the original solution to 2 times the volume of extraction agent, stir while adding, vortex at room temperature for 24 hours;

[0014] S005, add 3 times the volume of ultrapure water, slowly add the same volume of anhydrous ethanol, stir while adding;

[0015] S006, centrifuge at 15000 r / min for 20 min, discard the impurity solution and collect the precipitate;

[0016] S007, add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution and collect the precipitate;

[0017] S008, add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, collect the precipitate, and dry it with nitrogen to obtain the copolymer of essential oil-polyethylene glycol (PEG)-polylactic acid / glycolic acid copolymer (PLGA), denoted as essential oil-PEG-PLGA copolymer.

[0018] The soothing and anti-allergic protein polypeptide solution containing angelica essential oil was obtained by dissolving the essential oil-PEG-PLGA copolymer with a complex solvent. The specific steps are as follows:

[0019] Take the essential oil-PEG-PLGA copolymer, add the resolvent, and vortex for 10 minutes at a material-liquid ratio of 1:3.

[0020] The specific preparation methods for the above-mentioned solvents are as follows:

[0021] The cosolvent is composed of 10% PEG, anhydrous ethanol and 5% protective agent. Preparation method: Weigh 10g of PEG powder, add 100mL of anhydrous ethanol, sonicate until dissolved, add 5mL of protective agent, and vortex for 5min.

[0022] The activator consists of 0.1M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.1M N-hydroxysuccinimide (NHS), and 0.1M citrate buffer. Preparation method: Weigh 19.17g of EDC powder and 11.509g of NHS powder, add an appropriate amount of citrate buffer, stir until dissolved, and bring the volume to 1L.

[0023] The extractant is composed of PLGA and caprylic / capric glyceride. Preparation method: Weigh 5g of PLGA powder, add 100mL of caprylic / capric glyceride, heat to 50℃, and sonicate until dissolved.

[0024] The reconstitution solvent consists of a complex protein peptide solution, Tween-80, a complex enzyme solution, and a protective agent in a volume ratio of 95:3:2:1. The complex protein peptide solution comprises 0.1% acetyl heptapeptide-4, 0.5% casein peptide, 3% collagen peptide, and 2% citric acid solution. The complex enzyme solution comprises 1% coenzyme Q10, 0.1% vitamin B complex coenzyme, 0.01% biotin, and anhydrous ethanol.

[0025] The protective agent is composed of a titanium dioxide-zinc oxide-stearic acid complex and anhydrous ethanol, and is prepared as follows:

[0026] S101. Weigh 5g of zinc acetate into 30mL of ultrapure water and stir until dissolved to obtain a zinc source solution.

[0027] S102, take 10 mL of tetrabutyl titanate and slowly add it to 50 mL of anhydrous ethanol, stir well to obtain a titanium source solution;

[0028] S103, the zinc source solution is slowly added dropwise to the titanium source solution while stirring. Sodium hydroxide solution is added to adjust the pH to 8. Stir for 2 hours to obtain a mixed solution of precipitate.

[0029] S104, weigh 15.53g of stearic acid into the precipitate mixture, stir well, transfer to a hydrothermal reactor, seal, place in an oven, and react at 140℃ for 24 hours;

[0030] S105. After the reaction is complete, allow it to cool naturally to room temperature. Transfer the reaction product to a centrifuge tube, centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate.

[0031] S106, add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution and collect the precipitate;

[0032] S107, add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0033] S108, the precipitate was dried in a vacuum drying oven at 100℃ to obtain the titanium dioxide-zinc oxide-stearic acid complex.

[0034] S109, weigh 10g of titanium dioxide-zinc oxide-stearic acid complex into 100mL of anhydrous ethanol, and sonicate until homogeneous.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] 1. This invention solves the problems of high volatility, low water solubility, and crystallization / coagulation of Angelica sinensis essential oil during the extraction process by using a co-solvent. Through the hydrogen bonds and hydrophobic interactions formed between the PEG in the co-solvent and the essential oil molecules, an amorphous dispersion system is constructed. Its lipophilic groups encapsulate the essential oil molecules to form a micelle structure, improving the solubility of the originally water-insoluble essential oil. Simultaneously, the steric hindrance effect inhibits the accumulation of essential oil molecules, prolonging the crystallization induction period.

[0037] 2. This invention activates PEG in a cosolvent using an activator. At room temperature, a PEG-NHS active ester is efficiently constructed via a two-step EDC / NHS method. First, EDC rapidly activates the hydroxyl groups of PEG under neutral conditions to generate an O-acylurea intermediate. Subsequently, NHS captures the intermediate to form a stable succinimide ester bond. The room-temperature reaction not only avoids the oxidative degradation of PEG and essential oils caused by high temperatures but also yields an active ester with pH-responsive properties, while preserving the biocompatibility of PEG and the effective components of the essential oils.

[0038] 3. This invention constructs an essential oil-PEG-PLGA copolymer using an extractant. When PEG-NHS is mixed with PLGA in the extractant, the active ester groups on PEG-NHS undergo a nucleophilic substitution reaction with the hydroxyl groups on the PLGA molecular chain. The ester bonds in the active ester are opened, and NHS, as a leaving group, detaches, forming new ester bonds. This connects PEG to the PLGA molecular chain, forming the essential oil-PEG-PLGA copolymer. This reaction is carried out at room temperature, effectively avoiding the degradation of the active components of Angelica sinensis essential oil by high temperatures. Utilizing the hydrophilicity of the PEG segments and the hydrophobicity of the PLGA segments, the polarity of the solution is gradually reduced by adding ethanol to the aqueous solution. This weakens the hydration of PEG, while the hydrophobicity of PLGA promotes aggregation between copolymer molecules through hydrophobic interactions, thereby reducing the solubility of the essential oil-PEG-PLGA copolymer and causing it to gradually precipitate from the solution. The essential oil-PEG-PLGA copolymer also exhibits certain temperature-sensitive and slow-release characteristics. At low temperatures or room temperature, the PEG segments are in a highly hydrated extended conformation, while the PLGA segments are in a glassy state, forming a low-viscosity sol. When the temperature reaches approximately 37°C (body temperature), the hydration level of the PEG segment decreases slightly, and its conformation changes from extended to slightly contracted. The PLGA segment changes from a glassy state to a rubbery state, causing the copolymer molecules to aggregate and form a three-dimensional network structure, thus exhibiting a certain degree of thermosensitivity. This thermosensitive property allows the essential oil-PEG-PLGA copolymer to quickly form a hydration film upon contact with the skin, retaining moisture in the stratum corneum and giving the essential oil a certain retention effect. Due to the viscoelasticity and network pore structure of this three-dimensional network structure, the diffusion rate of the essential oil can be relatively slowed down, achieving a sustained release of the essential oil components.

[0039] 4. The PEG, PLGA, NHS and EDC used in this invention can all terminate the coupling reaction of essential oil-PEG-PLGA copolymer with appropriate amounts of ultrapure water and anhydrous ethanol. Then, the reaction product can be purified by centrifugation to remove unreacted PEG, PLGA, NHS, EDC and other impurities.

[0040] 5. This invention synthesizes a titanium dioxide-zinc oxide-stearic acid complex via a hydrothermal method, which serves as a protective agent capable of absorbing and scattering UVA and UVB ultraviolet rays. First, a stearic acid surface coating technology is employed to form a lipophilic layer on the surface of nanoparticles through esterification. This significantly reduces particle aggregation and poor dispersibility, and more importantly, it utilizes the conjugated structure of fatty acid chains to form a dual sun protection barrier, achieving a synergistic effect of ultraviolet scattering and absorption, thus enhancing sun protection efficacy. Second, it achieves surface wettability reversal, maintaining sun protection performance while obtaining excellent water resistance and durability, solving the problem of traditional water-induced failure. Furthermore, the addition of Angelica sinensis essential oil not only inhibits the photolysis of Angelica sinensis essential oil through steric hindrance but also allows the stearic acid layer to capture phototoxic intermediates, thereby improving the photostability of the essential oil. Therefore, through the multi-mechanism synergistic effect of this protective agent, it can provide full-band UVA / UVB protection and weather resistance for essential oils and soothing and anti-allergic protein peptide liquids containing Angelica sinensis essential oil.

[0041] 6. This invention resolvates the essential oil-PEG-PLGA copolymer using a resolvent. The adsorption of complex protein peptides in the resolvent creates an amphiphilic coating on the PLGA surface, improving the dispersion efficiency of the essential oil-PEG-PLGA copolymer and solving the problem of increasing copolymer solubility. Simultaneously, the addition of a complex enzyme to the resolvent not only accelerates cell metabolism and increases the penetration rate of the active ingredients in the essential oil, but its antioxidant properties also synergistically enhance the photostability of the essential oil components and reduce photodegradation.

[0042] 7. The soothing and anti-allergic protein polypeptide liquid containing angelica essential oil in this invention replenishes the skin with nutrients through a triple mechanism of delivery, repair and enhancement, while also building a cell repair microenvironment. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments.

[0044] The specific preparation methods of the solvents used in the examples and comparative examples are as follows:

[0045] Cosolvent: Weigh 10g of PEG powder, add 100mL of anhydrous ethanol, sonicate until dissolved, add 5mL of protective agent, and vortex for 5min.

[0046] Activator: Weigh 19.17g of EDC powder and 11.509g of NHS powder, add an appropriate amount of citrate buffer, stir until dissolved, and bring the volume to 1L.

[0047] Extraction agent: Weigh 5g of PLGA powder, add 100mL of caprylic / capric glyceride, heat to 50℃, and sonicate until dissolved.

[0048] The reconstitution solvent consists of a complex protein peptide solution, Tween-80, a complex enzyme solution, and a protective agent in a volume ratio of 95:3:2:1. The complex protein peptide solution comprises 0.1% acetyl heptapeptide-4, 0.5% casein peptide, 3% collagen peptide, and 2% citric acid solution. The complex enzyme solution comprises 1% coenzyme Q10, 0.1% vitamin B complex coenzyme, 0.01% biotin, and anhydrous ethanol.

[0049] The preparation method of the protective agent is as follows:

[0050] 1. Weigh 5g of zinc acetate into 30mL of ultrapure water and stir until dissolved to obtain a zinc source solution;

[0051] 2. Transfer 10 mL of tetrabutyl titanate and slowly add it to 50 mL of anhydrous ethanol. Stir until homogeneous to obtain a titanium source solution.

[0052] 3. Slowly add the zinc source solution dropwise to the titanium source solution while stirring. Add sodium hydroxide solution to adjust the pH to 8, and stir for 2 hours to obtain a mixed solution of precipitate.

[0053] 4. Weigh 15.53g of stearic acid into the precipitate mixture, stir well, transfer to a hydrothermal reactor, seal, and place in an oven at 140℃ for 24 hours;

[0054] 5. After the reaction is complete, allow it to cool naturally to room temperature. Transfer the reaction product to a centrifuge tube, centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate.

[0055] 6. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0056] 7. Add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0057] 8. The precipitate was dried in a vacuum drying oven at 100°C to obtain the titanium dioxide-zinc oxide-stearic acid complex.

[0058] 9. Weigh 10g of titanium dioxide-zinc oxide-stearic acid complex into 100mL of anhydrous ethanol and sonicate until homogeneous.

[0059] Example 1

[0060] The specific steps for preparing Angelica sinensis essential oil are as follows:

[0061] 1. Angelica sinensis is subjected to screening, washing, air-drying, crushing and sieving processes in sequence to obtain Angelica sinensis powder;

[0062] 2. Weigh 200g of Angelica sinensis powder, add a solubilizer, vortex at a 1:1 material-to-liquid ratio for 20min, centrifuge at 15000r / min for 15min, discard the precipitate, and collect the solution;

[0063] 3. Add the same volume of activator and vortex for 6 hours to obtain the stock solution;

[0064] 4. Slowly add the stock solution dropwise to twice the volume of the extractant while stirring, and vortex at room temperature for 24 hours;

[0065] 5. Add 3 times the volume of ultrapure water, and slowly add the same volume of anhydrous ethanol dropwise while stirring.

[0066] Centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate;

[0067] 7. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0068] 8. Add anhydrous ethanol of the same volume as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, collect the precipitate, and dry it with nitrogen to obtain the essential oil-PEG-PLGA copolymer.

[0069] Example 2

[0070] The specific steps for preparing Angelica sinensis essential oil are as follows:

[0071] 1. Angelica sinensis is subjected to screening, washing, air-drying, crushing and sieving processes in sequence to obtain Angelica sinensis powder;

[0072] 2. Weigh 200g of Angelica sinensis powder, add a solubilizing agent, vortex at a material-to-liquid ratio of 1:3 for 20 minutes, centrifuge at 15000r / min for 15 minutes, discard the precipitate, and collect the solution;

[0073] 3. Add the same volume of activator and vortex for 6 hours to obtain the stock solution;

[0074] 4. Slowly add the stock solution dropwise to twice the volume of the extractant while stirring, and vortex at room temperature for 24 hours;

[0075] 5. Add 3 times the volume of ultrapure water, and slowly add the same volume of anhydrous ethanol dropwise while stirring.

[0076] Centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate;

[0077] 7. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0078] 8. Add anhydrous ethanol of the same volume as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, collect the precipitate, and dry it with nitrogen to obtain the essential oil-PEG-PLGA copolymer.

[0079] Example 3

[0080] The specific steps for preparing Angelica sinensis essential oil are as follows:

[0081] 1. Angelica sinensis is subjected to screening, washing, air-drying, crushing and sieving processes in sequence to obtain Angelica sinensis powder;

[0082] 2. Weigh 200g of Angelica sinensis powder, add a solubilizing agent, vortex at a material-to-liquid ratio of 1:5 for 20 minutes, centrifuge at 15000r / min for 15 minutes, discard the precipitate, and collect the solution;

[0083] 3. Add the same volume of activator and vortex for 6 hours to obtain the stock solution;

[0084] 4. Slowly add the stock solution dropwise to twice the volume of the extractant while stirring, and vortex at room temperature for 24 hours;

[0085] 5. Add 3 times the volume of ultrapure water, and slowly add the same volume of anhydrous ethanol dropwise while stirring.

[0086] Centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate;

[0087] 7. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0088] 8. Add anhydrous ethanol of the same volume as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, collect the precipitate, and dry it with nitrogen to obtain the essential oil-PEG-PLGA copolymer.

[0089] Example 4

[0090] The essential oil was released by dialysis filtration of the PEG-PLGA copolymer, wherein the essential oil-PEG-PLGA copolymer in this embodiment was obtained through the steps of Example 2, the specific steps of which are as follows:

[0091] 1. Place the 5 kDa regenerated cellulose dialysis bag into ultrapure water and incubate at 60°C for 1 hour;

[0092] 2. Add the essential oil-PEG-PLGA copolymer to 15mL of ultrapure water, vortex until homogeneous, and prepare a suspension. Place the suspension into a dialysis bag and tie the bag tightly.

[0093] 3. Place the dialysis bag into an Erlenmeyer flask containing PBS, ensuring the bag is completely submerged in the buffer solution;

[0094] 4. Place the conical flask in a constant temperature incubator and incubate with shaking at a set temperature of 37℃;

[0095] 5. Take out PBS at different time points (1h, 3h, 6h, 12h, 24h, 48h) and replenish with an equal amount of PBS.

[0096] Example 5

[0097] The specific steps for preparing protective agent 1 are as follows:

[0098] 1. Weigh 5g of zinc acetate into 30mL of ultrapure water and stir until dissolved to obtain a zinc source solution;

[0099] 2. Transfer 10 mL of tetrabutyl titanate and slowly add it to 50 mL of anhydrous ethanol. Stir until homogeneous to obtain a titanium source solution.

[0100] 3. Slowly add the zinc source solution dropwise to the titanium source solution while stirring. Add sodium hydroxide solution to adjust the pH to 8, and stir for 2 hours to obtain a mixed solution of precipitate.

[0101] 4. Weigh 15.53g of stearic acid into the precipitate mixture, stir well, transfer to a hydrothermal reactor, seal, and place in an oven at 140℃ for 24 hours;

[0102] 5. After the reaction is complete, allow it to cool naturally to room temperature. Transfer the reaction product to a centrifuge tube, centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate.

[0103] 6. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0104] 7. Add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0105] 8. The precipitate was dried in a vacuum drying oven at 100°C to obtain the titanium dioxide-zinc oxide-stearic acid complex.

[0106] 9. Weigh 0.1g of titanium dioxide-zinc oxide-stearic acid complex into a 50mL volumetric flask, add an appropriate amount of anhydrous ethanol, sonicate until homogeneous, and finally dilute to volume with anhydrous ethanol to obtain protective agent 1.

[0107] Example 6

[0108] The specific steps for preparing the soothing and anti-allergic protein polypeptide liquid containing Angelica sinensis essential oil-PEG-PLGA copolymer are as follows:

[0109] Take the essential oil-PEG-PLGA copolymer prepared according to Example 2, add a complex solvent, and vortex for 10 min at a material-liquid ratio of 1:3.

[0110] Comparative Example 1

[0111] The specific steps for preparing Angelica sinensis essential oil by steam distillation are as follows:

[0112] 1. Angelica sinensis is subjected to screening, washing, air-drying, crushing and sieving processes in sequence to obtain Angelica sinensis powder;

[0113] 2. Weigh 200g of Angelica sinensis powder, place it in a 2000mL distillation flask, add 1000mL of ultrapure water, and soak for 3 hours;

[0114] 3. Turn on the cooling water, set the heating mantle to 150℃, and control the distillation rate at 2-3 drops per second until no more oily substances are added. Transfer the oil-water mixture in the receiving bottle to the separatory funnel, let it stand to separate into layers, and remove the lower aqueous phase.

[0115] 4. Add 2g of anhydrous sodium sulfate, let stand for 30 minutes, and then filter to obtain angelica essential oil.

[0116] Comparative Example 2

[0117] The specific steps for preparing PEG-PLGA copolymers without essential oils are as follows:

[0118] 1. Take the same volume of cosolvent as in Example 2, add the same volume of activator, and vortex for 6 hours to obtain the stock solution;

[0119] 2. Slowly add the stock solution dropwise to twice the volume of the extractant while stirring, and vortex at room temperature for 24 hours;

[0120] 3. Add 3 times the volume of ultrapure water, and slowly add the same volume of anhydrous ethanol dropwise while stirring.

[0121] 4. Centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate;

[0122] 5. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0123] 6. Add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, collect the precipitate, and dry it with nitrogen to obtain the PEG-PLGA copolymer.

[0124] The specific steps for further dialysis and filtration of the PEG-PLGA copolymer are as follows:

[0125] 1. Place the 5 kDa regenerated cellulose dialysis bag into ultrapure water and incubate at 60°C for 1 hour;

[0126] 2. Add PEG-PLGA copolymer to 15 mL of ultrapure water, vortex until homogeneous, and prepare a suspension. Place the suspension into a dialysis bag and tie the bag tightly.

[0127] 3. Place the dialysis bag into an Erlenmeyer flask containing PBS, ensuring the bag is completely submerged in the buffer solution;

[0128] 4. Place the conical flask in a constant temperature incubator and incubate with shaking at a set temperature of 37℃;

[0129] 5. Take out PBS at different time points (1h, 3h, 6h, 12h, 24h, 48h) and replenish with an equal amount of PBS.

[0130] Comparative Example 3

[0131] The specific steps for preparing essential oils without PEG-PLGA copolymers are as follows:

[0132] 1. Place the 5 kDa regenerated cellulose dialysis bag into ultrapure water and incubate at 60°C for 1 hour;

[0133] 2. Add the Angelica sinensis essential oil obtained by Comparative Example 1 to 15 mL of ultrapure water, vortex until homogeneous, prepare a layered solution, put it into a dialysis bag, and tie the bag tightly.

[0134] 3. Place the dialysis bag into an Erlenmeyer flask containing PBS, ensuring the bag is completely submerged in the buffer solution;

[0135] 4. Place the conical flask in a constant temperature incubator and incubate with shaking at a set temperature of 37℃;

[0136] 5. Take out PBS at different time points (1h, 3h, 6h, 12h, 24h, 48h) and replenish with an equal amount of PBS.

[0137] Comparative Example 4

[0138] The specific steps for preparing protectant 2 are as follows:

[0139] 1. Weigh 5g of zinc acetate into 30mL of ultrapure water and stir until dissolved to obtain a zinc source solution;

[0140] 2. Transfer 10 mL of tetrabutyl titanate and slowly add it to 50 mL of anhydrous ethanol. Stir until homogeneous to obtain a titanium source solution.

[0141] 3. Slowly add the zinc source solution dropwise to the titanium source solution while stirring. Add sodium hydroxide solution to adjust the pH to 8, and stir for 2 hours to obtain a mixed solution of precipitate.

[0142] 4. Transfer the precipitated mixed solution to a hydrothermal reactor, seal it, and place it in an oven at 140°C for 24 hours;

[0143] 5. After the reaction is complete, allow it to cool naturally to room temperature. Transfer the reaction product to a centrifuge tube, centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate.

[0144] 6. Add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0145] 7. Add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate;

[0146] 8. The precipitate was dried in a vacuum drying oven at 100°C to obtain the titanium dioxide-zinc oxide composite.

[0147] 9. Weigh 0.1g of titanium dioxide-zinc oxide complex into a 50mL volumetric flask, add an appropriate amount of anhydrous ethanol, sonicate until homogeneous, and finally dilute to volume with anhydrous ethanol to obtain protective agent 2.

[0148] Comparative Example 5

[0149] The specific steps for preparing protective agent 3 are as follows:

[0150] Weigh 0.1g of titanium dioxide into a 50mL volumetric flask, add an appropriate amount of anhydrous ethanol, sonicate until homogeneous, and finally dilute to volume with anhydrous ethanol to obtain protective agent 3.

[0151] Comparative Example 6

[0152] The preparation of protective agent 4 is carried out through the following steps:

[0153] Weigh 0.1g of zinc oxide into a 50mL volumetric flask, add an appropriate amount of anhydrous ethanol, sonicate until homogeneous, and finally dilute to volume with anhydrous ethanol to obtain protective agent 4.

[0154] Comparative Example 7

[0155] The specific steps for preparing the soothing and anti-allergic protein peptide solution containing PEG-PLGA copolymer are as follows:

[0156] Take the PEG-PLGA copolymer prepared according to Comparative Example 2, add the complex solvent, and vortex for 10 min at a feed-to-liquid ratio of 1:3.

[0157] Comparative Example 8

[0158] The specific steps for preparing a soothing and anti-allergic protein polypeptide liquid without Angelica sinensis essential oil-PEG-PLGA copolymer are as follows:

[0159] Take ultrapure water, add a redissolver, and vortex at a feed-to-liquid ratio of 1:3 for 10 minutes.

[0160] Experimental Example 1

[0161] This experiment determined the content of Angelica sinensis essential oil extracted in Examples 1-3 and Comparative Example 1. The essential oil-PEG-PLGA copolymers of Examples 1-3 and the Angelica sinensis essential oil of Comparative Example 1 were used as test samples. The specific steps are as follows:

[0162] 1. Place 300 mL of ultrapure water and several glass beads in a flask and connect it to a volatile oil analyzer. Add ultrapure water from the top of the analyzer until it fills the graduated section and overflows into the flask. Then, add 1 mL of xylene using a pipette and connect the reflux condenser. Heat the contents of the flask to boiling and continue distillation at a rate that keeps the middle of the condenser cool. After 30 minutes, stop heating, let it stand for at least 15 minutes, and then read the volume of xylene.

[0163] 2. Place the test sample, 300 mL of ultrapure water, and several glass beads in a flask. Shake to mix, then 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 level 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 part 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 part is exactly level with the 0 mark. Read the total volume of the volatile oil and xylene mixture.

[0164] The content of Angelica sinensis essential oil prepared by the extraction method and steam distillation method of this invention is obtained by subtracting the volume of xylene from the total volume of the mixture of volatile oil and xylene, i.e. the volume of volatile oil, and dividing by the weight of Angelica sinensis. The results are shown in Table 1.

[0165] Table 1. Content of Angelica sinensis essential oil

[0166] Sample Angelica essential oil content (%) Example 1 1.2 Example 2 1.3 Example 3 1.3 Comparative Example 1 0.9

[0167] As can be seen from Table 1, the extraction method of the present invention can significantly improve the extraction of Angelica sinensis essential oil.

[0168] Experiment Example 2

[0169] This experimental example determined the ligustilide content in the Angelica sinensis essential oil extracted in Examples 1-3 and Comparative Example 1, according to the method for determining ligustilide content in WM / T 4-2004. The specific steps are as follows:

[0170] 1. Weigh 2 mg of ligustilide reference standard, place it in a 20 mL brown volumetric flask, add methanol solution to dissolve and dilute to volume, shake well to obtain the reference solution.

[0171] 2. Take the essential oil-PEG-PLGA copolymers from Examples 1-3 and the Angelica sinensis essential oil from Comparative Example 1, place them in a 20 mL brown volumetric flask, add 5 mL of ultrapure water, and sonicate for 5 min until all solids are dissolved, forming a milky white solution. Then add 40 mL of methanol solution, sonicate for 10 min, and remove. Let it stand at room temperature, add methanol solution to make up to volume, and shake well. Filter through a 0.45 μm microporous membrane, and place the filtrate in a brown sample bottle to obtain the test solution.

[0172] 3. Inject the control solution and the test solution into the liquid chromatograph at an injection volume of 5 μL, respectively.

[0173] 4. Chromatographic conditions: Column: Octadecylsilane-bonded silica gel column. Mobile phase: Phase A was methanol, and Phase B was water. Phase A decreased linearly from 40% to 100% within 20 min. Flow rate: 0.8 mL / min. Column temperature: 25℃. Detection wavelength: 320 nm.

[0174] The content was determined by the external standard method, and the results are shown in Table 2.

[0175] Table 2. Ligustilide Content

[0176] Sample Ligustilide content (%) Example 1 1.7 Example 2 1.8 Example 3 1.8 Comparative Example 1 1.1

[0177] As can be seen from the results in Table 2, the extraction method of the present invention can efficiently extract ligustilide from the index component of Angelica sinensis.

[0178] Experimental Example 3

[0179] This experiment investigated the ferulic acid content of Angelica sinensis essential oil extracted in Examples 1-3 and Comparative Example 1. The determination was performed according to the method for determining ferulic acid content in WM / T 4-2004. The specific steps are as follows:

[0180] 1. Weigh 8 mg of ligustilide reference standard, place it in a 100 mL volumetric flask, add methanol-formic acid solution with a volume ratio of 95:5 to dissolve and dilute to volume, shake well to obtain the reference solution.

[0181] 2. Take the essential oil-PEG-PLGA copolymers from Examples 1-3 and the Angelica sinensis essential oil from Comparative Example 1, place them in a 25 mL volumetric flask, add 20 mL of a methanol-formic acid solution with a volume ratio of 95:5, sonicate for 15 min, let stand at room temperature, add a methanol-formic acid solution with a volume ratio of 95:5 to make up to volume, and shake well. Filter through a 0.45 μm microporous membrane to obtain the test solution.

[0182] 3. Inject the control solution and the test solution into the high-performance liquid chromatograph at an injection volume of 5 μL, respectively.

[0183] 4. Chromatographic conditions: Column: Octadecylsilane-bonded silica gel column. Mobile phase: Methanol-0.5% acetic acid solution (v / v) at a ratio of 20:80. Detection wavelength: 313 nm.

[0184] The content was determined by the external standard method, and the results are shown in Table 3.

[0185] Table 3 Ferulic Acid Content

[0186] Sample Ferulic acid content (%) Example 1 0.25 Example 2 0.28 Example 3 0.28 Comparative Example 1 0.01

[0187] As can be seen from the measurement results in Table 3, the extraction method of the present invention can efficiently extract ferulic acid from the indicator components of Angelica sinensis.

[0188] Experiment Example 4

[0189] In this experiment, the essential oil content of PBS collected in Example 4, Comparative Example 2 and Comparative Example 3 was determined. The determination method was the same as in Experiment 1. By measuring the essential oil content of PBS at different time points, the release capacity of PEG-PLGA copolymer for essential oil was indirectly determined. The results are shown in Table 4.

[0190] Table 4. Angelica oil content released from PEG-PLGA copolymer

[0191]

[0192] As can be seen from Table 4, the PEG-PLGA copolymer can protect the Angelica sinensis essential oil, forming a tri-chimeric copolymer of essential oil and PEG-PLGA, which allows the Angelica sinensis essential oil to be released slowly.

[0193] Experimental Example 5

[0194] This experimental example measures the ultraviolet absorption of the protective agents in Example 5 and Comparative Examples 4-6. The specific steps are as follows:

[0195] 1. Turn on the UV-Vis spectrophotometer, preheat for 30 minutes and perform calibration;

[0196] 2. Pour the suspensions of protective agents 1 to 4 into quartz cuvettes respectively, and place them into the sample cell. Use anhydrous ethanol as a blank control.

[0197] 3. The transmittance of protective agents 1-4 under UVA and UVB at wavelengths of 365nm and 305nm, respectively.

[0198] The measurement results are shown in Table 5.

[0199] Table 5. Light transmittance of protective agents 1-4 under UVA and UVB ultraviolet light.

[0200]

[0201] As can be seen from the measurement results in Table 5, the transmittance of the titanium dioxide-zinc oxide composite is lower than that of titanium dioxide and zinc oxide alone, while the transmittance of the titanium dioxide-zinc oxide-stearic acid composite is even lower than that of the titanium dioxide-zinc oxide composite.

[0202] Experimental Example 6

[0203] This experimental example evaluates the soothing and anti-allergic effects of the protein polypeptide solutions prepared in Example 6, Comparative Example 7, and Comparative Example 8. The specific steps are as follows:

[0204] 1. Experimental Methods: 120 volunteers with sensitive and allergic skin, aged 30-40 years, were selected. These volunteers were randomly divided into three groups. After cleansing their faces morning and evening, they applied the soothing and anti-allergic protein peptide liquid of Example 6 (containing Angelica essential oil-PEG-PLGA copolymer), Comparative Example 7 (containing PEG-PLGA copolymer), and Comparative Example 8 (not containing Angelica essential oil-PEG-PLGA copolymer) twice daily for four weeks, once in the morning and once in the evening. The objective condition of the volunteers' faces before and after use was recorded.

[0205] 2. Evaluation Indicators: The soothing and anti-allergic effects are evaluated using objective indicators. The objective evaluation indicators are as follows: Symptoms such as redness, dryness, itching, and peeling are used as evaluation criteria, with scores ranging from 0 to 3 points for no symptoms, mild, moderate, and severe symptoms, respectively.

[0206] 3. Experimental results: The objective evaluation scores of volunteers in each group before and after use are shown in Table 6.

[0207] Table 6. Objective evaluation values ​​of volunteers before and after use.

[0208] Sample Before use After use Example 6 2.00±0.68 0.80±0.65**** Comparative Example 7 1.65±1.14 1.43±0.96 Comparative Example 8 2.05±1.04 1.90±0.87

[0209] Note: * indicates a significant difference in volunteers before and after use, p<0.01.

[0210] As can be seen from the test results in Table 6, after using the soothing and anti-allergic protein polypeptide liquid containing Angelica essential oil-PEG-PLGA copolymer, containing PEG-PLGA copolymer, and not containing Angelica essential oil-PEG-PLGA copolymer for 4 weeks, the objective evaluation scores all decreased. In particular, there was a significant difference before and after using the protein polypeptide liquid containing Angelica essential oil-PEG-PLGA copolymer, indicating that the protein polypeptide liquid can play a certain role in soothing and anti-allergy.

[0211] 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 preparing Angelica sinensis essential oil, characterized in that: The preparation method of the Angelica sinensis essential oil specifically includes the following steps: S001, Angelica sinensis is subjected to screening, washing, air drying, crushing and sieving processes in sequence to obtain Angelica sinensis powder; S002, add co-solvent, vortex for 20 min at a material-to-liquid ratio of 1:1 to 5, centrifuge at 15000 r / min for 15 min, discard the precipitate and collect the solution; Add the same volume of activator to S003 and vortex for 6 hours to obtain the stock solution; S004, slowly add the stock solution dropwise to twice the volume of the extractant while stirring, and vortex at room temperature for 24 hours; S005, add 3 times the volume of ultrapure water, and slowly add the same volume of anhydrous ethanol dropwise while stirring. S006, centrifuge at 15000r / min for 20min, discard the impurity solution, and collect the precipitate; S007, add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution and collect the precipitate; S008, add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, collect the precipitate, and dry it with nitrogen to obtain the copolymer of essential oil-polyethylene glycol (PEG)-polylactic acid / glycolic acid copolymer (PLGA), denoted as essential oil-PEG-PLGA copolymer.

2. The method for preparing Angelica sinensis essential oil according to claim 1, characterized in that: The soothing and anti-allergic protein polypeptide solution containing angelica essential oil was obtained by dissolving the essential oil-PEG-PLGA copolymer with a complex solvent. The specific steps are as follows: Take the essential oil-PEG-PLGA copolymer, add the resolvent, and vortex for 10 minutes at a material-liquid ratio of 1:

3.

3. The method for preparing Angelica sinensis essential oil according to claim 1, characterized in that: The cosolvent consists of 10% PEG, anhydrous ethanol, and 5% protective agent.

4. The method for preparing Angelica sinensis essential oil according to claim 1, characterized in that: The activator consists of 0.1M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.1M N-hydroxysuccinimide, and 0.1M citrate buffer.

5. The method for preparing Angelica sinensis essential oil according to claim 1, characterized in that: The extractant consists of PLGA and caprylic / capric glyceride.

6. The preparation method according to claim 2, characterized in that: The reconstitution solvent consists of a complex protein / peptide solution, Tween-80, a complex enzyme solution, and a protective agent, in a volume ratio of 95:3:2:

1. The complex protein peptide solution consists of 0.1% acetyl heptapeptide-4, 0.5% casein peptide, 3% collagen peptide and 2% citric acid solution, and the complex enzyme solution consists of 1% coenzyme Q10, 0.1% vitamin B coenzyme, 0.01% biotin and anhydrous ethanol.

7. The preparation method according to claim 3 or claim 6, characterized in that: The protective agent is composed of a titanium dioxide-zinc oxide-stearic acid complex and anhydrous ethanol, and is prepared as follows: S101. Weigh 5g of zinc acetate into 30mL of ultrapure water and stir until dissolved to obtain a zinc source solution. S102, take 10 mL of tetrabutyl titanate and slowly add it to 50 mL of anhydrous ethanol, stir well to obtain a titanium source solution; S103, the zinc source solution is slowly added dropwise to the titanium source solution while stirring. Sodium hydroxide solution is added to adjust the pH to 8. Stir for 2 hours to obtain a mixed solution of precipitate. S104, weigh 15.53g of stearic acid into the precipitate mixture, stir well, transfer to a hydrothermal reactor, seal, place in an oven, and react at 140℃ for 24 hours; S105. After the reaction is complete, allow it to cool naturally to room temperature. Transfer the reaction product to a centrifuge tube, centrifuge at 15000 r / min for 20 min, discard the impurity solution, and collect the precipitate. S106, add the same volume of ultrapure water as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution and collect the precipitate; S107, add the same volume of anhydrous ethanol as the impurity solution, vortex for 5 min, centrifuge at 15000 r / min for 20 min, discard the solution, and collect the precipitate; S108, the precipitate was dried in a vacuum drying oven at 100℃ to obtain the titanium dioxide-zinc oxide-stearic acid complex. S109, weigh 10g of titanium dioxide-zinc oxide-stearic acid complex into 100mL of anhydrous ethanol, and sonicate until homogeneous.

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