A Daofu rhododendron extract, its preparation method, composition, and application

Abstract

This invention relates to the field of rhododendron extract technology, and discloses a Daofu rhododendron extract, its preparation method, composition, and application. The method includes the following steps: S1, Daofu rhododendron is vacuum freeze-dried; S2, the freeze-dried Daofu rhododendron is heated, and the volatile components in the Daofu rhododendron are collected by condensation to obtain Daofu rhododendron frozen stem cell stock solution; S3, the Daofu rhododendron processed in the above steps is pulverized and ultrasonically extracted to obtain an extract, and the extract is further extracted to obtain an extract; S4, the extract is subjected to adsorption chromatography with HP20 macroporous adsorption resin in a certain proportion to obtain a primary extract of Daofu rhododendron; S5, the primary extract of Daofu rhododendron is sequentially separated and purified by C18 column and Sephadex LH20 gel chromatography column in a certain proportion to obtain a secondary extract of Daofu rhododendron; S6, the Daofu rhododendron frozen stem cell stock solution obtained in step S2 is mixed with the Daofu rhododendron secondary extract obtained in step S6 to obtain the Daofu rhododendron extract.

Description

Technical Field

[0001] This invention belongs to the technical field of rhododendron extracts, specifically relating to a Daofu rhododendron extract, its preparation method, composition, and application. Background Technology

[0002] During cold, windy, and dry seasons, skin is prone to symptoms such as dryness, roughness, and itching. This is because excessive dryness leads to allergic reactions. However, excessively dry skin is mainly due to a lack of water in the stratum corneum. When the skin becomes so dry that it peels and flakes, the loss of a large number of keratinocytes reduces the skin barrier's protective ability, making it more susceptible to external stimuli. When the skin is frequently stimulated, it easily becomes sensitive. Therefore, developing skincare ingredients with moisturizing and soothing effects has broad application prospects.

[0003] Currently, in the extraction process of plant extracts, heating and stirring are usually required to improve the extraction rate of active ingredients. However, while improving the extraction rate of active ingredients, volatile aroma components are easily dissipated, resulting in a trade-off between efficacy and the original aroma of the plant. This causes the final plant extract product to lose the original fragrance characteristics of the plant and reduces the consumer experience.

[0004] Based on the above description, ensuring that the efficacy and aroma characteristics of plant extracts are combined is an urgent problem to be solved. Summary of the Invention

[0005] This application aims to solve the technical problem that the efficacy and aroma characteristics of plant extracts cannot coexist in the prior art.

[0006] Therefore, the first objective of this invention is to provide a method for preparing Daofu rhododendron extract, which can obtain Daofu rhododendron extract with preserved original aroma, strong biological activity and good solubility from Daofu rhododendron raw materials.

[0007] Specifically, a method for preparing a rhododendron extract from Daofu, the method comprising the following steps:

[0008] S1 involves vacuum freeze-drying of Daofu rhododendrons;

[0009] S2 involves heating the freeze-dried Daofu rhododendron, condensing and collecting the volatile components within the Daofu rhododendron to obtain Daofu rhododendron frozen stem cell stock solution.

[0010] S3 pulverizes the Daofu rhododendron after the above steps and performs ultrasonic-assisted extraction to obtain an extract. The extract is then extracted to obtain an extract.

[0011] S4 The extract is subjected to adsorption chromatography with HP20 macroporous adsorption resin in a certain proportion to obtain the primary extract of Daofu rhododendron.

[0012] S5 The primary extract of Daofu rhododendron is sequentially separated and purified by C18 column and Sephadex LH20 gel chromatography column in a certain proportion to obtain secondary extract of Daofu rhododendron.

[0013] S6. The frozen stem cell stock solution of Daofu rhododendron obtained in step S2 is mixed with the secondary extract of Daofu rhododendron obtained in step S6 to obtain Daofu rhododendron extract.

[0014] Further, the heating conditions set in step S2 are: heating to 30-40°C at a heating rate of 6-8°C / h, and holding at that temperature for 10-12h.

[0015] Furthermore, ultrasonic-assisted extraction was performed using a 70% ethanol aqueous solution, with the mass ratio of the pulverized Daofu rhododendron to the 70% ethanol aqueous solution being 1:5 to 15.

[0016] Further, the extraction includes the following steps:

[0017] S11: The extract is concentrated at 55°C to obtain a paste;

[0018] S21: Dissolve the extract in purified water at a volume ratio of 10-20 to obtain a complex solution;

[0019] S31: The complex solution is shaken with petroleum ether at a volume ratio of 1:0.5 to 1.5, and after standing, the petroleum ether extract is removed to obtain an aqueous solution.

[0020] S41: The aqueous solution and ethyl acetate are shaken at a volume ratio of 1:0.5 to 1.5, the ethyl acetate extract is collected, and the extract is concentrated under reduced pressure at 50°C to a paste-like consistency to obtain the extract.

[0021] Furthermore, the volume ratio of the extract to the HP20 macroporous adsorption resin is 1:40-60, the volume ratio of the primary extract of Daofu rhododendron to the C18 column is 1:50-70, and the volume ratio of the primary extract of Daofu rhododendron after separation and purification by the C18 column to the Sephadex LH20 gel is 1:70-90.

[0022] Furthermore, the vacuum freeze-drying temperature is -50 to -55°C, the vacuum freeze-drying time is 2 to 4 hours, and the vacuum degree of the vacuum freeze-drying is -0.08 to -0.09 MPa.

[0023] A second objective of this invention is to provide a Daofu rhododendron extract, which is prepared by a method for preparing Daofu rhododendron extract.

[0024] A third object of the present invention is to provide a composition comprising a rhododendron extract from Daofu.

[0025] Further, the composition comprises the following components in weight percentage: 0.25-5% Rhododendron dauricum extract, 88-93% water, 1-3% propylene glycol, 0.1-0.3% p-hydroxyacetophenone, 0.1-0.3% 1,2-hexanediol, and 4-6% glycerin.

[0026] A fourth objective of this invention is to provide a composition for use in skincare products.

[0027] The beneficial effects of this application are as follows:

[0028] This application uses *Rhododendron daofuense* as raw material. First, a frozen stem cell stock solution of *Rhododendron daofuense* is obtained using low-temperature vacuum freeze-drying technology. This extract retains the original aroma of *Rhododendron daofuense*. The freeze-dried plant is then pulverized and subjected to ultrasonic-assisted extraction with a 70% ethanol aqueous solution. After filtration and concentration, initial impurity removal is performed using HP20 macroporous adsorption resin, followed by separation and purification using a C18 column and a Sephadex LH20 gel chromatography column to obtain a secondary extract of *Rhododendron daofuense*. The frozen stem cell stock solution and the secondary extract are then mixed to obtain the *Rhododendron daofuense* extract. The *Rhododendron daofuense* extract obtained by this method not only retains the original aroma of *Rhododendron daofuense* but also possesses advantages such as strong biological activity and good solubility.

[0029] In particular, this application shows that the Daofu rhododendron extract has good moisturizing and soothing effects when used in the skin care industry. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] The following is a detailed description of a Daofu rhododendron extract, its preparation method, composition, and application provided by embodiments of the present invention.

[0032] Examples 1-4

[0033] Examples 1-4 provide a composition, the content of each component is shown in Table 1; wherein, the preparation method of the Daofu rhododendron extract in Examples 1-4 includes the following steps:

[0034] S1: Using the whole plant of Daofu Rhododendron as raw material, a vacuum freeze dryer is used to freeze Daofu Rhododendron at -55℃ for 3 hours in the extraction chamber, and then the vacuum degree of the extraction chamber is reduced to -0.09Mpa.

[0035] S2: The temperature of the extraction chamber is increased to 35℃ at a rate of 8℃ / h and maintained at this temperature for 12 hours, allowing the ice crystals in the plant to slowly melt and sublimate. This volatile gas is then frozen and collected at -70℃ using a condenser at the rear, yielding the Daofu rhododendron ice extract. The condenser temperature is then rapidly increased to 40℃ to completely melt the Daofu rhododendron ice extract, and this liquid is collected, yielding the Daofu rhododendron frozen stem cell stock solution.

[0036] S3: The Rhododendron dauricum plant material processed above was removed from the extraction chamber and pulverized into 0.5cm pieces. A 70% ethanol-water solution with a material-to-liquid ratio of 1:10 was added, and ultrasonic-assisted extraction was performed at a frequency of 40kHz, an extraction temperature of 55℃, and an extraction time of 3 hours. The mixture was filtered through 100-mesh gauze, and the filtrate was concentrated under reduced pressure at 55℃ to obtain an extract. The obtained extract was dissolved in 15 times its volume of purified water to obtain a reconstituted solution. An equal volume of petroleum ether was added to the reconstituted solution in a separatory funnel, shaken evenly, and allowed to stand for 4 hours. The petroleum ether extract was removed, and an equal volume of ethyl acetate was added to the aqueous layer, shaken evenly, and allowed to stand for 4 hours. The ethyl acetate extract was collected and concentrated under reduced pressure at 50℃ to obtain an ethyl acetate extract.

[0037] S4: Following the ratio of ethyl acetate extract to HP20 macroporous adsorption resin = 1:50, the ethyl acetate extract was loaded onto an HP20 macroporous adsorption resin chromatography column. The column was flushed with 15 times the volume of purified water to remove impurities. Then, 15 times the volume of 70% ethanol aqueous solution was added to flush the column. The eluent was collected and concentrated under reduced pressure to obtain the primary extract of Rhododendron dauricum.

[0038] S5: Following a ratio of 1:60 for primary extract of *Rhododendron dauricum* to C18 column, the primary extract of *Rhododendron dauricum* was loaded onto a C18 chromatography column and flushed with 30 times the column volume of 20% methanol-water, followed by 30 times the column volume of 70% methanol-water. The eluent from the 70% methanol-water flush was collected and concentrated under reduced pressure to obtain an extract. Following a ratio of 1:80 for the obtained extract to Sephadex LH20 gel, the secondary extract of *Rhododendron dauricum* was loaded onto a Sephadex LH20 gel column and flushed with 30 times the column volume of methanol solution. The first 10 times the column volume of eluent was discarded, and the eluent from 10 to 30 times the column volume was collected and dried under reduced pressure to obtain the secondary extract of *Rhododendron dauricum*.

[0039] S6: Mix the frozen stem cell stock solution of Daofu rhododendron in step 2 with the secondary extract of Daofu rhododendron in step 5 to obtain Daofu rhododendron extract.

[0040] Comparative Example 1

[0041] Example 1 provides a composition whose component contents are shown in Table 1.

[0042] Table 1. Composition and content of the compositions provided in the examples and comparative examples.

[0043]

[0044]

[0045] Experimental Example 1

[0046] Using the effects of the composition samples provided in the examples and comparative examples on the expression of aquaporin 3 (AQP3) in human immortalized keratinocytes (HaCaT) as a model, the moisturizing efficacy of Daofu rhododendron extract was evaluated. The effects of each test sample on the expression level of aquaporin are shown in Table 2.

[0047] Aquaporins (AQPs) are cell membrane surface receptors that transport water in epidermal cells. AQP3 is the most abundant aquaporin in the skin. Increased expression of AQP3 enhances the ability of epidermal cells to transport and absorb water. This experiment used HaCaT cells as a model cell line and treated them with Rhododendron dafuense extract. The effect of Rhododendron dafuense extract on aquaporins was investigated by semi-quantitatively detecting AQP3 expression in the cells. The detection method was immunofluorescence assay.

[0048] Specific experimental methods:

[0049] (1) Cell seeding: HaCaT cells were cultured in a T175 culture flask with 10% FBSDMEM medium. When the cell density was about 80%, the cells were digested with 0.05% trypsin and centrifuged at 1000 rpm / min for 5 min. The cells were then resuspended and seeded into 24-well plates with cell spreaders.

[0050] (2) Drug treatment: After 24 hours, the supernatant was aspirated, and 1 mL of the corresponding concentration of each test concentration was prepared using 10% FBSDMEM. The samples were then incubated at 37°C and 5% CO2 for 72 hours.

[0051] (3) Slide preparation and staining: After incubation, discard the supernatant and wash once with PBS. Add 300 μL of ice-cold methanol to each well and fix at -20℃. After fixation, wash three times with PBS, add 5% BSA and block at room temperature for 1 h. After blocking, discard the supernatant, add AQP3 primary antibody and incubate overnight at 4℃. On the second day, wash three times with PBS, add the corresponding secondary antibody and incubate at room temperature in the dark for 1.5 h. Finally, stain the nuclei with DAPI and mount the slides, then observe and photograph under a fluorescence microscope.

[0052] (4) Statistical analysis: The fluorescence photographs were analyzed using ImageJ software, and the data obtained were statistically analyzed using Graphpad Prism software.

[0053] Table 2. Effects of each test sample on aquaporin expression levels.

[0054] Group AQP3 protein expression level Mean±SD Example 1 1.073±0.051 Example 2 1.136±0.027 Example 3 1.221±0.043 Example 4 1.334±0.035 Example 5 1.321±0.017 Comparative Example 1 1.002±0.038

[0055] As shown in Table 2, Examples 1-5 all promoted the expression of aquaporins, and their effects were superior to those of Comparative Example 1. Example 4 showed the best effect, and the preferred dosage of 2% Daofu rhododendron extract in the composition demonstrated significant moisturizing efficacy.

[0056] Experiment Example 2

[0057] The soothing efficacy of the compositions obtained in the examples and comparative examples was evaluated by comparing changes in the number of spin movements of zebrafish embryos. The effects of each test sample on the relative stimulation intensity of the zebrafish embryos are shown in Table 3.

[0058] Zebrafish embryos are highly sensitive. In the early stages of development, the embryos are completely transparent. When the embryos develop to 24 hpf (Prim-5 stage), they begin to spin. When a fish embryo is exposed to chemical stimulation, the number of spin movements increases; when the test sample is added, the number of spin movements is significantly reduced compared to the model group.

[0059] Specific experimental methods:

[0060] (1) On the afternoon of the day before collecting the eggs, 3-18 month old male and female zebrafish that had already started feeding were transferred to the breeding tank and a partition was inserted. The next morning between 9:00 and 9:30, the partition in the middle of the breeding tank was removed, and the embryos were collected 15 minutes after the partition was removed. The embryos were placed in a constant temperature incubator at (28±0.5℃) and embryo buffer (2000 ml of ultrapure water, 7 g of sodium chloride, 0.7 g of sodium bicarbonate, 0.1 g of potassium chloride, and 0.235 g of calcium chloride) was added. When the embryos reached 24 hpf, the normally developing embryos were selected for the experiment.

[0061] (2) Use 96-well cell culture plates, with 3 replicates per group and 5 embryos per well. Add embryo buffer to the blank group, add sodium dodecyl sulfate (SDS) to the model group, and add a combination of Rhododendron dauricum extract to the test sample group. Record a 30-second video after 15 minutes.

[0062] (3) Count the number of spin movements of each group of embryos and analyze their relative stimulation intensity using the following formula. The results are expressed as mean ± standard error (mean ± SEM).

[0063]

[0064] Table 3. Effects of each test sample on the relative stimulation intensity of zebrafish embryos

[0065]

[0066]

[0067] As can be seen from Table 3, the relative stimulation intensity of the model group was significantly enhanced compared with that of the blank group, indicating that the experimental model was effective. Examples 1-5 all had a weakening effect on the relative stimulation intensity of zebrafish embryos, with Examples 4 and 5 showing the best effects. The preferred dosage of Daofu rhododendron extract was 2%-4%. Since the difference in effect between 2% and 4% was not significant, 2% was preferred as the dosage in the composition, which had a significant soothing effect.

[0068] As demonstrated in Experiment 1 and Experiment 2, the Daofu rhododendron extract obtained in this application exhibits excellent moisturizing and soothing effects when used in the skincare field.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, The composition comprises the following components in weight percentage: 2% Rhododendron dauricum extract, 90.6% water, 2% propylene glycol, 0.2% p-hydroxyacetophenone, 0.2% 1,2-hexanediol, and 5% glycerin; The preparation method of the Daofu rhododendron extract includes the following steps: S1 involves vacuum freeze-drying of Daofu rhododendrons; S2 The Daofu rhododendron, after being vacuum freeze-dried, is heated, and the volatile components inside the Daofu rhododendron are collected by condensation to obtain Daofu rhododendron frozen stem cell stock solution. S3 The Daofu rhododendron processed in the above steps is crushed and ultrasonically extracted to obtain an extract. The extract is then extracted to obtain an extract. S4 The extract is subjected to adsorption chromatography with HP20 macroporous adsorption resin in a certain proportion to obtain the primary extract of Daofu rhododendron. S5 The primary extract of Daofu rhododendron is sequentially separated and purified by C18 column and Sephadex LH20 gel chromatography column in a certain proportion to obtain secondary extract of Daofu rhododendron. S6. The frozen stem cell stock solution of Daofu rhododendron obtained in step S2 is mixed with the secondary extract of Daofu rhododendron obtained in step S6 to obtain Daofu rhododendron extract.

2. The composition according to claim 1, characterized in that, The heating conditions set in step S2 are: heating to 30-40°C at a heating rate of 6-8°C / h and holding at that temperature for 10-12 hours.

3. The composition according to claim 1, characterized in that, Ultrasonic-assisted extraction was performed using a 70% ethanol aqueous solution, with the mass ratio of the pulverized Daofu rhododendron to the 70% ethanol aqueous solution being 1:5~15.

4. The composition according to claim 1, characterized in that, The extraction includes the following steps: S11: The extract is concentrated at 55°C to obtain a paste; S21: Dissolve the extract in purified water at a volume ratio of 10-20 to obtain a complex solution; S31: The complex solution is shaken with petroleum ether at a volume ratio of 1:0.5~1.5, and after standing, the petroleum ether extract is removed to obtain an aqueous solution. S41: The aqueous solution and ethyl acetate are shaken at a volume ratio of 1:0.5~1.5, the ethyl acetate extract is collected, and the extract is concentrated under reduced pressure at 50°C to a paste-like consistency to obtain the extract.

5. The composition according to claim 1, characterized in that, The volume ratio of the extract to the HP20 macroporous adsorption resin is 1:40~60, the volume ratio of the primary extract of Daofu rhododendron to the C18 column is 1:50~70, and the volume ratio of the primary extract of Daofu rhododendron after separation and purification by the C18 column to the Sephadex LH20 gel is 1:70~90.

6. The composition according to claim 1, characterized in that, The vacuum freeze-drying temperature is -50~-55℃, the vacuum freeze-drying time is 2~4h, and the vacuum degree of the vacuum freeze-drying is -0.08~-0.09Mpa.

7. The use of the composition of claim 1 in the preparation of a skin care product.

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