Essential oil composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetics, and discloses an essential oil composition as well as a preparation method and application thereof. The essential oil composition is prepared from chamomile essential oil, magnolia flower essential oil, lavender essential oil and magnolia flower fermentation product freeze-dried powder. Wherein the magnolia flower fermentation product freeze-dried powder is freeze-dried powder of a product obtained by fermenting a magnolia flower water extract with lactic acid bacteria and saccharomyces cerevisiae; the lactic acid bacteria are lactobacillus plantarum with the serial number of CICC (China General Microbiological Culture Collection Center) 20242; the serial number of the saccharomyces cerevisiae is BNCC 186060. The magnolia flower fermentation freeze-dried powder provided by the invention has very excellent anti-allergic ability, the chamomile essential oil, the magnolia flower essential oil and the lavender essential oil are combined to form the essential oil composition, the allergic reaction can be further relieved, the whole essential oil composition is all natural plant extracts, and the magnolia flower fermentation freeze-dried powder has low irritation and multiple functions and is suitable for large-scale production. The compound is suitable for serving as an active substance of anti-allergic products in the field of cosmetics.

Description

Technical Field

[0001] The present application relates to the field of cosmetic technology, and in particular to an essential oil composition, a preparation method, and an application thereof. Background Art

[0002] In recent years, with the continuous improvement of people's living standards and the popularization of cosmetics, skin care has become a hot topic of concern throughout society. However, during the research and development and manufacturing of cosmetics, some chemical ingredients are often added, which have become a major cause of skin allergies. When an allergic reaction occurs, the body achieves an anti-allergic effect by inhibiting the production and release of allergic mediators. Compared with Western medicines such as chlorpheniramine and diphenhydramine, the anti-allergic mechanism of action of natural plant essential oils and extracts has the advantages of multi-layered and multi-target mechanisms. In addition, the anti-allergic active ingredients of natural Chinese herbal products have relatively low toxic side effects, and the probability of drug dependence is very low, which also increases patient acceptance.

[0003] Chinese herbal extracts have the advantages of versatility, low irritation, and multiple targets. Magnolia, a plant of the genus Magnolia in the family Magnoliaceae, has active ingredients with excellent anti-inflammatory, anti-allergic, and antioxidant effects. Studies have shown that magnolia essential oil (magnolia essential oil) can inhibit the production of histamine in inflammatory tissues, thereby achieving an anti-allergic effect. Currently, most magnolia extracts used in cosmetics are magnolia essential oil, magnolia water extract, and magnolia alcohol extract. Compared with traditional extraction techniques, fermentation technology for the extraction of plant active ingredients has gradually become a research hotspot. Biofermentation technology can enrich effective substances, reduce toxicity, and generate new substances under the action of microbial enzymes, thereby producing new effects. Summary of the Invention

[0004] In view of this, the object of the present application is to provide an essential oil composition and a preparation method thereof, so that the essential oil composition can significantly improve the anti-allergic effect;

[0005] Another purpose of the present application is to provide an application of the essential oil composition in the preparation of products with anti-allergic effects.

[0006] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, an essential oil composition is provided, comprising chamomile essential oil, magnolia essential oil, lavender essential oil and magnolia flower fermentation freeze-dried powder;

[0007] The magnolia flower fermentation freeze-dried powder is a freeze-dried powder of a magnolia flower water extract fermented by lactic acid bacteria and brewer's yeast; the lactic acid bacteria is Lactobacillus plantarum numbered CICC 20242; the brewer's yeast numbered BNCC 186060.

[0008] Optionally, the mass ratio of the chamomile essential oil, magnolia essential oil, lavender essential oil and magnolia fermentation freeze-dried powder is 1:1:1:1.

[0009] Optionally, the fermentation product is a product obtained by simultaneous fermentation of magnolia flower water extract by lactic acid bacteria and saccharomyces cerevisiae.

[0010] As a second aspect of the present application, a method for preparing the essential oil composition is provided, comprising:

[0011] S1. Extract magnolia flowers with water, separate the supernatant, and obtain a water extract;

[0012] S2. The aqueous extract was inoculated with Lactobacillus plantarum numbered CICC 20242 and Saccharomyces cerevisiae numbered BNCC 186060 for fermentation, and the fermented magnolia ferment was freeze-dried to obtain a magnolia ferment freeze-dried powder;

[0013] S3. The magnolia fermentation product freeze-dried powder is mixed with chamomile essential oil, magnolia essential oil, and lavender essential oil in proportion to obtain the essential oil composition.

[0014] Optionally, the water extract is inoculated with the Lactobacillus plantarum and Saccharomyces cerevisiae simultaneously for fermentation.

[0015] Further optionally, the fermentation temperature is 30°C±5°C.

[0016] As a third aspect of the present application, provided is the use of the essential oil composition in preparing a product with anti-allergic efficacy.

[0017] Optionally, the product is a cosmetic.

[0018] As a fourth aspect of the present application, a product with anti-allergic efficacy is provided, comprising the essential oil composition described in the present application.

[0019] Optionally, the product is a cosmetic, and further comprises one or more components selected from whitening, anti-wrinkle, moisturizing, and antioxidant substances, and / or adjuvants that can be added to cosmetics.

[0020] This application uses the water extract of dried magnolia buds as the fermentation object, selects suitable fermentation strains to ferment them to obtain magnolia fermentation product, which shows better anti-allergic ability than the fermentation products of single strains and other combined fermentation strains. In particular, the two fermentation strains used in this application have the best effect when they are inoculated with magnolia water extract at the same time for fermentation; combining it with chamomile essential oil, magnolia essential oil, and lavender essential oil to form an essential oil composition can further reduce allergic reactions. The entire essential oil composition is made entirely of natural plant extracts, has low irritation and multifunctionality, and is suitable as an active substance for anti-allergic products in the cosmetics field. DETAILED DESCRIPTION

[0021] The present application discloses an essential oil composition and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0022] It should be noted that, in this document, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the presence of other identical elements in the process, method, article or device comprising the elements. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other in the absence of conflict.

[0023] Magnolia biondii Pamp is an excellent and precious tree species used for medicine, fragrance, timber and ornamental purposes. Its dried flower buds are called "magnolia" in medicine. The flower buds contain volatile oils. The aromatic oil extracted from them is called magnolia essential oil, which is also one of the most common extracts in the cosmetics field. However, its anti-allergic effect is not high.

[0024] Microbial fermentation can not only synthesize easily absorbed small molecules such as amino acids, organic acids, flavonoids, and polyphenols, but also produce macromolecules with important effects such as polysaccharides, peptides, or proteins. The degree of efficacy depends on the choice of fermentation raw materials and fermentation strains. This application accidentally discovered two suitable strains in other research projects. The two were combined to perform microbial fermentation on magnolia water extract. The product after fermentation had a smaller molecular weight, thereby further improving the anti-allergic effect of the product. At the same time, in the study of other essential oil products, it was found that its anti-allergic effect can be further improved.

[0025] Therefore, in a first aspect of the present application, an essential oil composition is provided, consisting of chamomile essential oil, magnolia essential oil, lavender essential oil and magnolia flower fermentation freeze-dried powder;

[0026] The magnolia flower fermentation freeze-dried powder is a freeze-dried powder of a magnolia flower water extract fermented by lactic acid bacteria and brewer's yeast; the lactic acid bacteria is Lactobacillus plantarum numbered CICC 20242; the brewer's yeast numbered BNCC 186060.

[0027] Among them, Lactobacillus plantarum, numbered CICC 20242, was purchased from the China Industrial Microbiology Culture Collection Center. This strain is derived from sauerkraut and was formerly known as Lactobacillus plantarum subspecies plantarum. After Gram staining, the bacteria are medium-rod-shaped; they can perform homolactic fermentation and hydrolyze proteins, and are specifically used in sauerkraut production.

[0028] The brewer's yeast numbered BNCC 186060 was purchased from Beina Biotechnology, and its reference use was apple juice brewing.

[0029] Chamomile essential oil has anti-allergic effects, stabilizing mast cell membranes and preventing the release of histamine and other allergenic mediators. It alleviates or prevents allergic reactions and is effective in treating skin allergies such as urticaria and eczema. German chamomile essential oil is preferred. Lavender essential oil contains linalool, linalyl acetate, and linalool, and genuine lavender essential oil is preferred. Magnolia essential oil can inhibit histamine production in inflamed tissues, achieving anti-allergic effects.

[0030] In certain embodiments of the present application, the mass ratio of the chamomile essential oil, magnolia essential oil, lavender essential oil and magnolia fermentation freeze-dried powder is 1:1:1:1, for example, each component is selected from 75 μg of the essential oil composition.

[0031] In certain embodiments of the present application, the fermentation product is the product of a magnolia flower water extract fermented simultaneously with lactic acid bacteria and saccharomyces cerevisiae. In other embodiments of the present application, the fermentation product is the product of a magnolia flower water extract fermented first with lactic acid bacteria and then with saccharomyces cerevisiae. In certain embodiments of the present application, the magnolia flower water extract is an aqueous extract of dried buds of Magnolia bicinnamon.

[0032] In a second aspect of the present application, a method for preparing the essential oil composition is provided, comprising:

[0033] S1. Extract magnolia flowers with water, separate the supernatant, and obtain a water extract;

[0034] S2. The aqueous extract was inoculated with Lactobacillus plantarum numbered CICC 20242 and Saccharomyces cerevisiae numbered BNCC 186060 for fermentation, and the fermented magnolia ferment was freeze-dried to obtain a magnolia ferment freeze-dried powder;

[0035] S3. The magnolia fermentation product freeze-dried powder is mixed with chamomile essential oil, magnolia essential oil, and lavender essential oil in proportion to obtain the essential oil composition.

[0036] In certain embodiments of the present application, when the magnolia flowers are subjected to water extraction, the mass ratio of magnolia flowers to water is 1:20, the extraction temperature is 90° C., the extraction time is 3 h, and the supernatant obtained by centrifugation after extraction is the water extract.

[0037] In certain embodiments of the present application, the plant lactobacillus and saccharomyces cerevisiae are activated before inoculation and fermentation; wherein the plant lactobacillus is activated by continuous passage in MRS medium until the viable cell count is 1.0×10 8 CFU / mL or more. The brewer's yeast was activated by continuous passage in YPD medium until the viable cell count was 1.0×10 7 In certain embodiments of the present application, the inoculation amount of the activated Lactobacillus plantarum is 5%, and the inoculation amount of the activated Saccharomyces cerevisiae is 1%.

[0038] In certain embodiments of the present application, the magnolia flower water extract is simultaneously inoculated with the plant lactobacillus and saccharomyces cerevisiae for fermentation. The fermentation product obtained by this fermentation method has a significantly improved anti-allergic effect relative to the magnolia flower water extract, as well as the fermentation products obtained by fermentation with a single strain or by fermentation with plant lactobacillus and saccharomyces cerevisiae in sequence. In addition, the present application can also adopt a method of fermentation with plant lactobacillus and saccharomyces cerevisiae in sequence, and the anti-allergic effect of the fermentation product thereof is also significantly improved relative to the fermentation products obtained by fermentation with the magnolia flower water extract or by fermentation with a single strain.

[0039] In some other embodiments of the present application, the temperature for simultaneous inoculation of the Lactobacillus plantarum and Saccharomyces cerevisiae for fermentation is 30°C±5°C, further optionally 30°C±2°C, and the fermentation time is 5-10 days, for example, 7 days.

[0040] In other embodiments of the present application, when Lactobacillus plantarum and Saccharomyces cerevisiae are fermented sequentially, the fermentation temperature of Lactobacillus plantarum is 37° C. and the fermentation time is 7 days; the fermentation temperature of Saccharomyces cerevisiae is 30° C. and the fermentation time is 7 days.

[0041] In a third aspect of the present application, under the premise of maintaining a consistent total concentration of active ingredients, the essential oil composition of the present application exhibits more significant hyaluronidase and histamine inhibition rates compared to other essential oil compositions. Based on this, the present application provides the use of the essential oil composition in the preparation of a product with anti-allergic efficacy, wherein the product may be a cosmetic.

[0042] In a fourth aspect of the present application, a product with anti-allergic efficacy is provided, comprising the essential oil composition described in the present application.

[0043] In certain embodiments of the present application, the product is a cosmetic and further includes one or more components selected from substances with whitening, anti-wrinkle, moisturizing, and antioxidant effects, and / or adjuvants that are permitted to be added to cosmetics. The adjuvants are ingredients used as auxiliary raw materials in cosmetics, primarily to maintain the stability of the cosmetics, enhance their feel, or achieve special functions. They are usually added in relatively small amounts to cosmetics and can add specific functions to the cosmetics, such as fragrance, smoothness, and color. Common cosmetic adjuvants include flavors and fragrances, pigments and pigments, preservatives and antioxidants, surfactants, water-soluble polymers, and the like.

[0044] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials, except for the differences noted in each group, were kept consistent to ensure comparability. In addition, all materials used in this application can be purchased from commercial sources.

[0045] The following further describes an essential oil composition provided in this application, its preparation method, and application.

[0046] Example 1:

[0047] (1) Preparation of Magnolia biondii flower water extract

[0048] Water: Magnolia biondii flower powder = 20:1 ratio (volume mL: mass g), extraction at 90°C for 3 hours, centrifugation and supernatant for index detection.

[0049] (2) Magnolia essential oil extraction

[0050] Magnolia essential oil was obtained by steam distillation with a solid-liquid ratio of 1:4 (g / mL), an ultrasonic power of 200 W, and ultrasonication for 25 min followed by distillation for 4 h.

[0051] (3) German chamomile and real lavender essential oils were purchased from the official website of Huimeishe (https: / / www.prettyvalley.cn / ).

[0052] (4) Bacteria activation

[0053] Lactic acid bacteria: Lactobacillus plantarum (Lactobacillus plantarum subspecies CICC 20242)

[0054] Yeast: Saccharomyces cerevisiae (BNCC 186060, Beina Biotech)

[0055] Control lactic acid bacteria: Lactobacillus plantarum was purchased from China Industrial Microbiology Culture Collection Center CICC 25125

[0056] Control yeast: Saccharomyces cerevisiae was purchased from China Industrial Microbiology Culture Collection Center CICC 1388

[0057] Yeast activation: Inoculate the strain into YPD medium, culture at 30°C for 36-48 hours, and subculture three times to fully activate the strain until the viable count is 1.0×10 7 CFU / mL or above;

[0058] Activation of lactic acid bacteria: inoculate the strain into MRS medium, culture at 35℃ for 24h, and subculture for 3 times to fully activate the strain until the viable count is 1.0×10 8 CFU / mL or above.

[0059] (5) Preparation of fermented products after water extraction of magnolia flowers

[0060] Lactic acid bacteria fermentation alone: ​​Magnolia flower powder was extracted with water at a 1:20 mass-to-volume ratio at 90°C for 3 hours. The supernatant was centrifuged and dispensed into 250mL blue-capped reagent bottles (200mL / bottle). After sterilization (115°C, 30 minutes), fermentation was prepared. First, activated lactic acid bacteria were inoculated at a ratio of 5% of the inoculum, mixed, and fermented in a 37°C incubator for 7 days. The fermented product was then centrifuged at 9000 rpm for 15 minutes to obtain the magnolia flower fermentation product for index testing.

[0061] Yeast Fermentation: Magnolia flower powder was extracted with water at a 1:20 mass-to-volume ratio at 90°C for 3 hours. The supernatant was centrifuged and dispensed into 250mL blue-capped vials (200mL / vial). After sterilization (115°C for 30 minutes), fermentation was prepared. First, a 1% inoculation of Saccharomyces cerevisiae was performed at 30°C for 7 days. The fermented magnolia flower product was then centrifuged at 9000 rpm for 15 minutes to obtain the product for index testing.

[0062] Lactic acid bacteria fermentation followed by yeast fermentation: Magnolia flower powder was extracted with water at a 1:20 mass-to-volume ratio at 90°C for 3 hours. The supernatant was centrifuged and dispensed into 250mL blue-capped reagent bottles (200mL / bottle). After sterilization (115°C, 30 minutes), fermentation was prepared. First, activated lactic acid bacteria were inoculated at a ratio of 5% of the inoculum, mixed, and placed in a 37°C incubator. After fermentation for 7 days, Saccharomyces cerevisiae was inoculated at a ratio of 1%. After fermentation at 30°C for 7 days, the fermented material was centrifuged at 9000 rpm for 15 minutes to obtain the magnolia flower fermentation product for index detection.

[0063] Simultaneous fermentation of lactic acid bacteria and yeast: Magnolia flower powder was extracted by adding water at a 1:20 mass-to-volume ratio at 90°C for 3 hours. The supernatant was centrifuged and dispensed into 250mL blue-capped reagent bottles (200mL / bottle). After sterilization (115°C, 30 minutes), fermentation was prepared. First, activated lactic acid bacteria were inoculated at a ratio of 5% and 1% of Saccharomyces cerevisiae were inoculated. The mixture was mixed and placed in an incubator at 30°C. After fermentation for 7 days, the fermented material was centrifuged at 9000 rpm for 15 minutes to obtain the magnolia flower fermentation product for index detection.

[0064] Example 2:

[0065] (1) Magnolia fermentation freeze-dried powder

[0066] The magnolia flower fermentation liquid obtained from the simultaneous fermentation of lactic acid bacteria and yeast in Example 1 was pre-cooled at -40°C for 6 hours. After pre-cooling, the liquid was placed in a vacuum freeze dryer and frozen for 12 hours at a cold trap temperature of -45°C and a pressure of 30 Pa. The freeze-dried magnolia flower fermentation liquid was pulverized and sieved to obtain a magnolia flower fermentation liquid freeze-dried powder.

[0067] (2) German chamomile essential oil: Add sterilized DMSO and dilute to 0.3 mg / mL -1 In the in vitro hyaluronidase inhibition test, the concentration of chamomile essential oil was 0.3mg.mL -1 In the cell experiment, the concentration of chamomile essential oil was 30 μg mL -1 .

[0068] (3) Magnolia essential oil: Add sterilized DMSO and dilute to 0.3 mg / mL -1In the in vitro hyaluronidase inhibition test, the concentration of magnolia essential oil was 0.3mg.mL -1 In the cell experiment, the concentration of magnolia essential oil was 30 μg mL -1 .

[0069] (4) Real lavender essential oil: Add sterilized DMSO and dilute to 0.3 mg / mL -1 In the in vitro hyaluronidase inhibition test, the concentration of lavender essential oil was 0.3mg.mL -1 In the cell experiment, the concentration of lavender essential oil was 30 μg mL -1 .

[0070] (5) German chamomile essential oil + magnolia essential oil (1:1): 0.15 mg of German chamomile and 0.15 mg of magnolia essential oil were added to 1 mL of sterile DMSO solution for in vitro hyaluronidase inhibition rate experiment; 15 μg of German chamomile and 15 μg of magnolia essential oil were added to 1 mL of sterile DMSO solution for cell experiment.

[0071] (6) German chamomile essential oil + real lavender essential oil (1:1): 0.15 mg of German chamomile and 0.15 mg of lavender essential oil were added to 1 mL of sterile DMSO solution for in vitro hyaluronidase inhibition rate experiment; 15 μg of German chamomile and 15 μg of lavender essential oil were added to 1 mL of sterile DMSO solution for cell experiment.

[0072] (7) Magnolia essential oil + real lavender essential oil (1:1): 0.15 mg magnolia essential oil and 0.15 mg lavender essential oil were added to 1 mL sterile DMSO solution for in vitro hyaluronidase inhibition rate experiment; 15 μg magnolia essential oil and 15 μg lavender essential oil were added to 1 mL sterile DMSO solution for cell experiment.

[0073] (8) German chamomile essential oil + magnolia (magnolia) essential oil + real lavender essential oil (1:1:1): 0.1 mg German chamomile essential oil, 0.1 mg magnolia essential oil and 0.1 mg real lavender essential oil were added to 1 mL of sterile DMSO solution for in vitro hyaluronidase inhibition rate experiment; 10 μg German chamomile, 10 μg magnolia and 10 μg real lavender essential oil were added to 1 mL of sterile DMSO solution for cell experiment.

[0074] (9) German chamomile essential oil + magnolia essential oil + real lavender essential oil (1:1:1) + magnolia fermentation broth freeze-dried powder: 75 μg German chamomile essential oil, 75 μg magnolia essential oil, 75 μg real lavender essential oil and 75 μg magnolia fermentation broth freeze-dried powder were added to 1 mL sterilized DMSO solution for in vitro hyaluronidase inhibition rate experiment; 7.5 μg German chamomile, 7.5 μg magnolia essential oil, 7.5 μg real lavender essential oil and 7.5 μg magnolia fermentation broth freeze-dried powder were added to 1 mL sterilized DMSO solution for cell experiment.

[0075] (10) Magnolia flower fermentation freeze-dried powder: Add sterilized DMSO and dilute to 0.3 mg / mL -1 In the in vitro hyaluronidase inhibition test, the concentration of the fermentation freeze-dried powder was 0.3 mg / mL. -1 In cell experiments, the concentration of the fermentation product freeze-dried powder was 30 μg mL -1 .

[0076] Example 3:

[0077] Hyaluronidase inhibition rate test

[0078] 1. Solution preparation

[0079] Acetate buffer solution (pH = 5.6): Dilute 1155 μL of glacial acetic acid to 100 mL in a volumetric flask. Mix thoroughly and add 4.8 mL to form Solution A. Dissolve 2.72 g of sodium acetate crystals and dilute to 100 mL. Mix thoroughly and add 45.2 mL to form Solution B. Combine Solutions A and B, dilute to 100 mL with deionized water, and mix thoroughly. Accurately determine the pH and adjust to 5.6 with Solution A or B.

[0080] Hyaluronidase solution: Accurately weigh 0.008 g of hyaluronidase and add 9.72 mL of acetate buffer solution to a final concentration of 500 μg mL -1 .

[0081] 0.5mg mL -1 Sodium hyaluronate solution: Accurately weigh 0.005g of sodium hyaluronate, add it to 10mL of acetate buffer solution, and dissolve it fully before use.

[0082] 2.5 mmol / L CaCl2 solution: Add 0.275 g CaCl2 to 1000 mL pure water and mix well.

[0083] Ehrlich reagent: Accurately weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol. Prepare and use immediately.

[0084] Acetylacetone solution: Dissolve 1.4 mL of acetylacetone in 20 mL of 1.0 mol L⁻¹ sodium carbonate solution. Prepare immediately before use.

[0085] 2. Experimental steps

[0086] Add 0.1 mL of 2.5 mmol / L CaCl2 and 0.5 mL of 600 U / mL hyaluronidase to the test tube and treat at 37°C for 20 min;

[0087] Add 0.5 mL of sample and treat at 37°C for 20 min;

[0088] Add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution and treat at 37°C for 30 min;

[0089] Let it stand at room temperature for 5 minutes, add 0.5 mL of acetylacetone solution and 0.1 mL of 0.4 mol / L NaOH solution, boil in water for 15 minutes, and immediately put it in ice bath for 5 minutes;

[0090] Add 1 mL of Ehrlich reagent and color for 20 minutes at room temperature. Measure the absorbance at a wavelength of 530 nm. The inhibition rate is calculated as follows:

[0091]

[0092] Where:

[0093] A: ABS value of the control blank group (acetic acid replaces enzyme solution and sample)

[0094] B: ABS value of control group (acetic acid instead of sample)

[0095] C: ABS value of the experimental blank group (acetic acid solution instead of enzyme solution)

[0096] D: ABS value of experimental group

[0097] 3. Experimental results

[0098] The hyaluronidase inhibition rate was tested according to the grouping in Example 1, as shown in Table 1 below;

[0099] Table 1 Effect of magnolia fermentation on hyaluronidase inhibition rate

[0100]

[0101] Note: Different letters indicate significant differences among different groups

[0102] Hyaluronidase is an enzyme that degrades hyaluronic acid, a key component of skin and connective tissue. During an allergic reaction, hyaluronidase is activated, leading to increased tissue permeability, the spread of inflammatory mediators, and edema. A higher inhibition rate indicates that the substance tested may be inhibiting the spread of the allergic reaction by blocking hyaluronidase activity and reducing the breakdown of hyaluronic acid.

[0103] According to the results in Table 1, it is obvious that the magnolia fermented product obtained by simultaneous fermentation of CICC 20242 and BNCC 186060 in this application has a hyaluronidase inhibition rate of up to 78.3%, which is significantly higher than that of the fermented products of other groups.

[0104] Among them, the effects of simultaneous fermentation of CICC 25125 and CICC 1388, both of which are Lactobacillus plantarum and Saccharomyces cerevisiae, were significantly different from those of simultaneous fermentation of the two strains selected in this application, indicating that a specific combined fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae can achieve a hyaluronidase inhibition rate of nearly 80%;

[0105] There was also a significant difference in the effect of fermenting CICC 20242 first and then BNCC 186060 compared with the simultaneous fermentation of the two strains, indicating that the order of process fermentation can also affect the hyaluronidase inhibition rate.

[0106] The above results fully demonstrate that the magnolia fermented product described in this application has excellent anti-allergic effect.

[0107] Table 2 Effect of essential oil composition on hyaluronidase inhibition rate

[0108]

[0109] Note: Different letters indicate significant differences among different groups

[0110] The hyaluronidase inhibition rate was tested according to the grouping in Example 2, as shown in Table 2 above. According to the results in Table 2, it can be clearly seen that the effective ingredients are all 0.3 mg.mL -1 Under this premise, the hyaluronidase inhibition rate of the essential oil composition of the present application (i.e., German chamomile essential oil + magnolia essential oil + real lavender essential oil + magnolia fermentation freeze-dried powder) is as high as 84.5%, which is significantly higher than that of other groups of essential oil compositions. This fully demonstrates that the essential oil composition described in the present application has excellent anti-allergic effect.

[0111] Example 4:

[0112] Histamine release experiment

[0113] 1. Method for passaging RBL-2H3 cells.

[0114] Discard the culture medium in the culture dish, wash gently with sterile PBS 1-2 times, add 3mL of trypsin and place in a 37℃ cell culture incubator for digestion for 3 minutes to detach the cells, add 6mL of complete culture medium to stop digestion, and gently blow to make the cells completely fall off and form a cell suspension. Transfer it to a sterile centrifuge tube, centrifuge at 1100rpm and 25℃ for 5 minutes, discard the supernatant, add complete culture medium to resuspend the cells, and the specific volume added depends on the growth of the cells. Take 1-1.5mL of cell suspension and add it dropwise to a culture dish containing 10mL of complete culture medium, and place it in a cell culture incubator for culture. When the cells grow to 80-90%, carry out the next passage, which is about once every 2 days. This application uses RBL-2H3 cells within 30 generations.

[0115] 2. Establishment of RBL-2H3 cell degranulation model

[0116] Calcium ionophore A23187 was used to stimulate RBL-2H3 cell degranulation. RBL-2H3 cells were plated at 2×10 5 Cells were seeded at a density of 100 μL / mL in a 24-well plate, with 500 μL per well. After incubation at 37°C for 24 hours, the culture medium was discarded, and cells were washed 1-2 times with PBS. Calcium ionophore A23187 (dissolved in benchtop buffer) was added and incubated at 37°C for 30 minutes. (For testing, 200 μL of benchtop buffer diluted samples were pre-incubated at 37°C for 30 minutes.) The supernatant was collected and stored at -20°C until further use.

[0117] 3. Determination of the inhibitory effect on histamine release in RBL-2H3 cells

[0118] The amount of histamine released is indirectly determined by converting histamine into a fluorescent histamine-OPA product. Take 250 μL of the test substance into a centrifuge tube, add 50 μL of 1M NaOH and 12.5 μL of 1% OPA in sequence to convert the histamine into a fluorescent histamine-OPA product. After reacting at room temperature in the dark for 4 minutes, add 25 μL of 3M HCl to terminate the reaction. Take 200 μL to a completely black 96-well plate and measure the fluorescence intensity in a microplate reader with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. In the maximum release group, the sample diluted with benchtop solution was replaced with benchtop solution, and in the blank group, the sample diluted with benchtop solution and the calcium ion carrier A23187 were replaced with benchtop solution. The histamine content is calculated using the histamine standard curve, and the relative histamine release percentage = extracellular histamine release OD value / (intracellular histamine release OD value + extracellular histamine release OD value) × 100%

[0119] 4. Experimental results

[0120] The histamine release was detected by grouping according to Example 1, as shown in Table 3 below;

[0121] Table 3 Effect of fermentation products on histamine release

[0122]

[0123] Note: Different letters indicate significant differences between different groups; Blank control group: RBL-2H3 cells cultured normally; Model control group: RBL-2H3 cells stimulated with calcium ionophore A23187;

[0124] RBL-2H3 cells are derived from rat basophilic leukemia and have functions similar to mast cells and basophils. They express a high-affinity IgE receptor (FcεRI) on their surface, mimicking a key step in human allergic reactions. When the IgE receptor is activated by an allergen, the cells rapidly degranulate, releasing inflammatory mediators such as histamine and β-hexosaminidase.

[0125] According to the results in Table 3, it is obvious that the magnolia fermented product obtained by simultaneous fermentation of CICC 20242 and BNCC 186060 in this application has the lowest histamine content, which is not significantly different from the blank control group. The histamine inhibition rate is as high as 60.41%, which is significantly higher than that of the fermented products of other groups.

[0126] Among them, the effects of simultaneous fermentation of CICC 25125 and CICC 1388, both of which are Lactobacillus plantarum and Saccharomyces cerevisiae, were significantly different from those of simultaneous fermentation of the two strains selected in this application, indicating that a specific combined fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae can achieve a histamine inhibition rate of about 60%;

[0127] There was also a significant difference in the effect of fermenting CICC 20242 first and then BNCC 186060 compared with the simultaneous fermentation of the two strains, indicating that the order of process fermentation can also affect the histamine inhibition rate.

[0128] The above results fully demonstrate that the magnolia fermented product described in this application has excellent anti-allergic effect.

[0129] Table 4 Effect of essential oil composition on histamine release

[0130]

[0131] Note: Different letters indicate significant differences among different groups

[0132] The histamine release was tested according to the grouping in Example 2, as shown in Table 4 above. According to the results in Table 4, it can be clearly seen that the active ingredient is 0.3 mg.mL -1Under this premise, the essential oil composition of the present application (i.e., German chamomile essential oil + magnolia essential oil + real lavender essential oil + magnolia fermentation freeze-dried powder) has the lowest histamine content and the highest histamine inhibition rate, which is significantly higher than that of other groups of essential oil compositions. This fully demonstrates that the essential oil composition described in the present application has excellent anti-allergic effect.

[0133] Example 5:

[0134] The experiment on the effect of allergic reaction on the expression of pro-inflammatory factors in cells was carried out according to the grouping in Example 1, as shown in Table 5 below;

[0135] RAW264.7 cells were cultured at 1.5 × 10 5 The cells were seeded at a density of 100 μL per well in a 96-well plate at 37°C / mL and incubated in a 37°C incubator. After the cells grew to 90%, the culture medium was aspirated and 100 μL of sample solution diluted with DMEM was added. The cells were incubated at 37°C for 18 hours. The sample solution was aspirated and 100 μL of sample solution containing 1 μg / mL LPS was added. The cells were incubated at 37°C for 6 hours, with 6 replicates per group. The supernatant was collected and centrifuged at 13,000 rpm for 5 minutes. The supernatant after centrifugation was divided into PCR tubes (150 μL / tube). The control group replaced the sample solution with 100 μL of DMEM, and the blank group replaced the sample solution and the sample solution containing LPS with 100 μL of DMEM. According to the instructions, the ELISA kit was used to detect the content of cellular pro-inflammatory factors IL-6, IL-8, IL-1β and TNF-α in the supernatant. The remaining supernatant was stored at -20°C for use and can be stored for one week.

[0136] Table 5 Effects of magnolia fermentation products on the release of pro-inflammatory factors

[0137]

[0138] Note: Different letters indicate significant differences between different groups; Blank control group: RAW264.7 cells cultured normally; Model control group: RAW264.7 cells stimulated with LPS;

[0139] RAW264.7 cells are derived from peritoneal macrophages of BALB / c mice and are established by induction of Abelson murine leukemia virus (A-MuLV). They belong to the mononuclear / macrophage lineage and retain basic macrophage functions, such as phagocytosis, secretion of inflammatory factors (TNF-α, IL-6, NO, etc.), and antigen presentation. These incompletely differentiated macrophages can be further activated to an M1 proinflammatory phenotype by stimuli such as LPS (lipopolysaccharide) and IFN-γ (interferon-γ). The secretion of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 following stimulation is measured.

[0140] According to the results in Table 5, it can be clearly seen that the magnolia fermented product obtained by simultaneous fermentation of CICC 20242 and BNCC 186060 in the present application has the lowest levels of IL-6, IL-8, IL-1β and TNF-α among all groups, and the contents of cellular pro-inflammatory factors in the fermented products of other groups are significantly higher than those of the fermented product of the present application;

[0141] Among them, the effects of simultaneous fermentation of CICC 25125 and CICC 1388, both of which are Lactobacillus plantarum and Saccharomyces cerevisiae, were significantly different from those of simultaneous fermentation of the two strains selected in this application, indicating that the combined fermentation of specific Lactobacillus plantarum and Saccharomyces cerevisiae can maximize the inhibition of the secretion of cellular pro-inflammatory factors;

[0142] There was also a significant difference in the effect of fermenting CICC 20242 first and then BNCC 186060 compared with the simultaneous fermentation of the two strains, indicating that the order of process fermentation can also affect the secretion of cellular pro-inflammatory factors.

[0143] The above results fully demonstrate that the magnolia fermented product described in this application has excellent anti-allergic effect.

[0144] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. An essential oil composition, characterized in that It is composed of chamomile essential oil, magnolia essential oil, lavender essential oil and magnolia flower ferment freeze-dried powder; The magnolia flower fermentation freeze-dried powder is a freeze-dried powder of a magnolia flower water extract fermented by lactic acid bacteria and brewer's yeast; the lactic acid bacteria is Lactobacillus plantarum numbered CICC 20242; the brewer's yeast numbered BNCC 186060.

2. The essential oil composition according to claim 1, wherein The mass ratio of the chamomile essential oil, the magnolia essential oil, the lavender essential oil and the magnolia fermentation freeze-dried powder is 1:1:1:

1.

3. The essential oil composition according to claim 1, wherein The fermented product is a product obtained by simultaneously fermenting the magnolia flower water extract with lactic acid bacteria and brewer's yeast.

4. The method for preparing the essential oil composition according to claim 1, wherein include: S1. Extract magnolia flowers with water, separate the supernatant, and obtain a water extract; S2. The aqueous extract was inoculated with Lactobacillus plantarum numbered CICC 20242 and Saccharomyces cerevisiae numbered BNCC 186060 for fermentation, and the fermented magnolia ferment was freeze-dried to obtain a magnolia ferment freeze-dried powder; S3. The magnolia fermentation product freeze-dried powder is mixed with chamomile essential oil, magnolia essential oil, and lavender essential oil in proportion to obtain the essential oil composition.

5. The preparation method according to claim 4, characterized in that The plant lactobacillus and saccharomyces cerevisiae are simultaneously inoculated into the water extract for fermentation.

6. The preparation method according to claim 4 or 5, characterized in that The fermentation temperature is 30°C ± 5°C.

7. Use of the essential oil composition according to any one of claims 1 to 3 in preparing a product with anti-allergic efficacy.

8. The use according to claim 7, characterized in that The product is a cosmetic.

9. A product with anti-allergic effect, characterized in that: The invention relates to an essential oil composition comprising the essential oil composition according to any one of claims 1 to 3.

10. The product according to claim 9, characterized in that The product is a cosmetic, and further comprises one or more components selected from substances having whitening, anti-wrinkle, moisturizing and anti-oxidation effects, and / or auxiliary agents that can be added to cosmetics.

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