Hippophae rhamnoides leaf extract, preparation method and application of hippophae rhamnoides leaf extract in anti-allergic soothing products

Abstract

The invention belongs to the technical field of cosmetics, and discloses a hippophae rhamnoides leaf extract, a preparation method and application of the hippophae rhamnoides leaf extract in anti-allergic soothing products. The hippophae rhamnoides leaf extract is obtained by crushing hippophae rhamnoides leaves, decocting the crushed hippophae rhamnoides leaves with water to obtain an extracting solution, and centrifuging, concentrating and spray-drying the extracting solution, and is applied to the anti-allergic soothing product. The hippophae rhamnoides leaf extract disclosed by the invention has a good anti-allergic soothing effect, is mild and non-irritant, and has the economic advantages of sustainability and low cost.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and more particularly to a seabuckthorn leaf extract, a preparation method and application thereof in anti-allergic soothing products. Background Art

[0002] As the largest organ in the human body, the skin is our first barrier against the external environment. However, many factors in modern life, such as improper skin care methods, long-term use of hormone drugs, and lack of proper care after laser treatment, can cause skin sensitivity. Most of the traditional skin care products commonly seen on the market are mixed with chemical raw materials, artificial synthetic raw materials and chemical additives. Some of these chemical synthetic additives, preservatives, and even heavy metal elements are easy to cause skin problems such as redness, allergies, and peeling. They are especially not suitable for people with sensitive skin or skin problems such as acne and inflammation. In view of these problems, there is an urgent need to develop an additive derived from natural plants, non-irritating and soothing, to effectively solve the existing problems.

[0003] Seabuckthorn (Hippophae rhamnoides L.) is a plant of the genus Hippophae in the family Elaeagnaceae. Seabuckthorn leaves are an important byproduct of the fruit planting and management process. They have the advantages of high yield, easy collection and storage. They contain polyphenols, flavonoids, polysaccharides, carotenoids, saponins and other bioactive ingredients with strong anti-inflammatory, antioxidant and antibacterial properties.

[0004] Currently, the application scope of sea buckthorn leaves is still limited to the production of sea buckthorn leaf tea or animal feed, and there is no related application of adding sea buckthorn leaf extract as an anti-soothing skin care product. Summary of the invention

[0005] The object of the present invention is to provide an application of a seabuckthorn leaf extract in the preparation of an anti-allergic soothing product. This natural plant extract has good anti-allergic soothing effects, is mild and non-irritating, and has the economic advantages of sustainability and low cost. In view of this, the present invention provides a seabuckthorn leaf extract, a preparation method, and an application thereof in an anti-allergic soothing product.

[0006] One of the purposes of the present invention is to provide a method for preparing a seabuckthorn leaf extract, comprising the following steps:

[0007] (1) Pretreatment: Take seabuckthorn leaves, wash them to remove impurities and dust, and dry them in the sun;

[0008] (2) Separation and extraction: Add seabuckthorn leaves to distilled water, soak at room temperature for 12 hours, decoct for 1 hour, and separate the solid and liquid; add distilled water to the solid separation, continue to decoct for 1 hour, separate the solid and liquid, combine the liquids obtained from the two separations, filter, centrifuge, and take the supernatant for later use;

[0009] (3) Concentration and drying: The supernatant is concentrated by rotary evaporation and spray-dried at high temperature to obtain seabuckthorn leaf extract powder.

[0010] Preferably, in step (2), the material-liquid ratio of the initial decoction is 1:10, the volume ratio of the solid separator to distilled water is 1:5, and the decoction temperature is 80°C-100°C.

[0011] Preferably, in step (2), the pore size of the filtration is 200 nm, and the centrifugation is 3000 r / min for 10 min.

[0012] Preferably, in step (3), the temperature of the rotary evaporation concentration is 75-80°C.

[0013] Preferably, in step (3), the temperature of the high temperature spray drying is 100°C.

[0014] The second object of the present invention is to provide a seabuckthorn leaf extract, which has the effect of inhibiting the activity of hyaluronidase; the seabuckthorn leaf extract can inhibit the activation of mast cells; the seabuckthorn leaf extract can reduce local allergic reactions in the toes of mice; the seabuckthorn leaf extract can reduce capillary dilation; the seabuckthorn leaf extract can inhibit the release of inflammatory factors; the seabuckthorn leaf extract includes other functional active ingredients (i.e., gallic acid, ellagic acid, isoquercetin and isorhamnetin-3O-neohesperidin); the cream made from the seabuckthorn leaf extract has a soothing and fading effect.

[0015] The third object of the present invention is to provide an application of seabuckthorn leaf extract in an anti-allergic soothing product.

[0016] Preferably, the seabuckthorn leaf extract is used in a cream.

[0017] Preferably, the cream comprises the following raw materials in parts by weight: 79.87 parts of water, 3-5 parts of 1,3-propylene glycol, 0.03-0.5 parts of ethylenediaminetetraacetic acid, 2-6 parts of glycerin, 1-1.2 parts of cetearyl alcohol, 1-2 parts of dimethicone, 1.5-2.5 parts of methyl glucose sesquistearate, 0.6-0.8 parts of hydroxypropyl methylcellulose, 0.5-1 parts of 1,2-pentanediol, 0.3-0.8 parts of phenoxyethanol, and 1-3 parts of sea buckthorn leaf extract.

[0018] It can be seen from the above technical scheme that compared with the prior art, the present invention evaluates the anti-allergic soothing effect of seabuckthorn leaf extract by measuring the release of allergic mediator tryptase in zebrafish induced by C48 / 80, the degree of swelling of mouse toes, the dilation of capillaries and the release of inflammatory factor TNF-α. The soothing and redness-removing effect of the cream added with seabuckthorn leaf extract is evaluated by the human red zone, the main components of seabuckthorn leaf extract are identified, and the hyaluronidase inhibition experiment is used for evaluation.

[0019] Through experiments, it was found that seabuckthorn leaf extract can regulate the expression of trypsin in zebrafish; effectively reduce the symptoms of local and systemic anaphylactic reactions in mice; and significantly improve the symptoms caused by anaphylactic reactions, confirming that seabuckthorn leaf extract can be used as an anti-allergic soothing cosmetic. At the same time, the results of hyaluronidase inhibition experiments showed that gallic acid, ellagic acid, and isoquercetin in seabuckthorn leaf extract may be the key ingredients for its function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 This is the effect of different temperatures on the hyaluronidase inhibition rate of seabuckthorn leaf extract.

[0022] Figure 2 The effects of different concentrations of seabuckthorn leaf extract on the hatching rate and survival rate of zebrafish embryos: A is the 72h hatching rate, and B is the 96h survival rate.

[0023] Figure 3 Effect of seabuckthorn leaf extract on trypsin expression in zebrafish.

[0024] Figure 4 The effect of seabuckthorn leaf extract on C48 / 80-induced paw swelling in Balb / c mice.

[0025] Figure 5 The effect of seabuckthorn leaf extract on C48 / 80-induced capillary dilation in Balb / c mice.

[0026] Figure 6 To investigate the effect of seabuckthorn leaf extract on histamine release induced by C48 / 80 in Balb / c mice.

[0027] Figure 7The effect of seabuckthorn leaf extract on C48 / 80-induced TNF-α release in Balb / c mice.

[0028] Figure 8 The cream with sea buckthorn leaf extract has a de-reddening effect on sensitive red skin on the human face.

[0029] Fig. 9 These are the qualitative and quantitative analysis results of seabuckthorn leaf extract: A and B are UPLC-QExactive-Orbitrap-MS results, and C and D are HPLC results.

[0030] Fig.10 This is the result of the inhibition of hyaluronidase by seabuckthorn leaf extract and its main components. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Embodiment 1

[0033] Preparation of seabuckthorn leaf extract, seabuckthorn leaf (provided by Huahao Testing, Jiamusi University Science and Technology Park):

[0034] (1) Pretreatment: Take seabuckthorn leaves, wash them to remove impurities and dust, and dry them in the sun;

[0035] (2) Separation and extraction: Add seabuckthorn leaves to distilled water at a solid-liquid ratio of 1:10, soak at room temperature for 12 hours; decoct at 80°C for 1 hour, separate the solid and liquid; add 1:5 volume of distilled water to the solid separation and continue to decoct for 1 hour, separate the solid and liquid, combine the liquids obtained from the two separations, pass through a 200nm filter paper, centrifuge at 3000r / min for 10 minutes, and take the supernatant for later use;

[0036] (3) Concentration and drying: The filtrate is concentrated by rotary evaporation at 30 r / min and 75-80°C, and then spray-dried at 100°C to obtain seabuckthorn leaf water extract powder.

[0037] Embodiment 2

[0038] (1) Pretreatment: same as in Example 1;

[0039] (2) Separation and extraction: Add seabuckthorn leaves to distilled water at a solid-liquid ratio of 1:10, soak at room temperature for 12 hours; decoct at 90°C for 1 hour to separate the solid and liquid; add 1:5 volume of distilled water to the solid separation and continue to decoct for 1 hour to separate the solid and liquid; combine the liquids obtained from the two separations, pass through a 200nm filter paper, centrifuge at 3000r / min for 10 minutes, and take the supernatant for later use;

[0040] (3) Concentration and drying: Same as in Example 1.

[0041] Embodiment 3

[0042] (1) Pretreatment: same as in Example 1;

[0043] (2) Separation and extraction: Add seabuckthorn leaves to distilled water at a solid-liquid ratio of 1:10, soak at room temperature for 12 hours; boil at 100°C for 1 hour to separate the solid and liquid; add 1:5 volume of distilled water to the solid separation and continue boiling for 1 hour to separate the solid and liquid. Combine the liquids obtained from the two separations, pass through a 200nm filter paper, centrifuge at 3000r / min for 10 minutes, and take the supernatant for later use;

[0044] (3) Concentration and drying: Same as in Example 1.

[0045] Experimental Example 1

[0046] Hyaluronidase inhibition assay

[0047] Experimental materials: hyaluronidase, sodium dihydrogen phosphate, sodium dihydrogen phosphate, sodium hyaluronate and bovine serum albumin (purchased from Shanghai Yuanye Biotechnology Co., Ltd.)

[0048] Experimental method: In a 96-well plate, 50 μL of sample solution and 50 μL of hyaluronidase solution (0.3 mg / mL) were shaken and mixed, and pre-incubated at 37°C for 10 minutes. 12 μL of phosphate buffer (0.3 mol / L, pH = 5.35) was added, shaken and mixed, and pre-incubated at 37°C for 10 minutes. Then 50 μL of sodium hyaluronate solution was added, shaken and mixed, and incubated at 37°C for 45 minutes, and then 100 μL of acidic albumin solution was added. Undigested sodium hyaluronate and acidic albumin formed a precipitate. The mixture was placed at room temperature for 10 minutes, shaken and mixed, and the absorbance was measured at a wavelength of 600 nm using an enzyme reader. Each sample was repeated three times. The hyaluronidase inhibition rate was calculated according to the following formula

[0049] Hyaluronidase inhibition rate % = (OD 样品组 -OD 对照组 ) / OD 对照组 ×100%

[0050] In the formula: OD sample group is the sample to be tested; OD control group is the treatment group containing all solvents but not the sample to be tested.

[0051] The in vitro inhibition method of hyaluronidase is one of the main test methods for determining the anti-allergic properties of substances at present. The effect of different temperatures on the inhibition rate of hyaluronidase in seabuckthorn leaf extract is shown in the following table. Figure 1 As shown, the hyaluronidase inhibition rate of the seabuckthorn leaf extracts extracted in Example 1 (extraction temperature 80°C), Example 2 (extraction temperature 90°C), and Example 3 (extraction temperature 100°C) can reach about 90% at a concentration of 10 mg / mL, indicating that the seabuckthorn leaf extract has a strong anti-allergic potential. In addition, the seabuckthorn leaf extract obtained in Example 1 has a higher hyaluronidase inhibition rate than that of Examples 2 and 3 at a low concentration of 0.31 mg / mL, so the seabuckthorn leaf extract powder obtained in Example 1 is used for subsequent experiments.

[0052] The following experimental examples and application examples are operated using the product of Example 1.

[0053] Experiment 2

[0054] Zebrafish embryo toxicity assay

[0055] Experimental materials: AB wild zebrafish purchased from Huante Company

[0056] Experimental method: In the experiment, 7 groups of seabuckthorn leaf extract treatment groups (0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, and 1mg / mL) were set up, and the blank control was embryos cultured in zebrafish embryo culture medium without the test substance. A 24-well culture plate was used as the experimental container, 10 zebrafish embryos were placed in each well, 2000μL of embryo culture medium corresponding to the test substance concentration was added to each well, and 3 duplicate wells were set up to observe and record the morphology of zebrafish embryos in each group. The hatching rate at 72h and the survival rate at 96h of seabuckthorn leaf extract treatment were recorded to evaluate the safe dosage range of seabuckthorn leaf extract.

[0057] like Figure 2 As shown in the figure, as the concentration of seabuckthorn leaf extract increased from 0.05 mg / mL to 1 mg / mL, the hatching rate and survival rate of zebrafish decreased significantly. At the concentration (0.4 mg / mL) for 72 hours of exposure, there was no significant difference with the blank group.

[0058] Experiment 3

[0059] Determination of trypsin expression in zebrafish

[0060] Experimental materials: C48 / 80 (purchased from Sigma), BAPNA (purchased from Shanghai Yuanye Technology Co., Ltd.)

[0061] Experimental method: 1 mL of seabuckthorn leaf extract of different concentrations was added to the drug treatment group, and 500 μL of distilled water was added to the model group and the solvent control group, and incubated for 24 hours. Then 500 μL of C48 / 80 containing 1 μg / mL was added to each well, and 500 μL BAPNA (500 μg / mL) was added at the same time and incubated at 28°C in the dark for 3 hours. The absorbance value was measured at 405 nm using an ELISA reader.

[0062] The expression level of trypsin was calculated as follows:

[0063] Trypsin expression level / % = (OD 药物处理组 -OD 溶剂组 ) / OD 溶剂组 ×100%

[0064] Wherein: OD drug treatment group is seabuckthorn leaf extract and model group; OD solvent group is the treatment group containing all solvents but without zebrafish.

[0065] like Figure 3 As shown, the expression level of trypsin in the model group was significantly increased, indicating that the model was successful. With the increase of the concentration of seabuckthorn leaf extract, the expression level of trypsin in zebrafish decreased, indicating that seabuckthorn leaf extract can effectively inhibit the release of allergic mediator trypsin during allergic reaction.

[0066] Experiment 4

[0067] Experimental materials: Evans blue (purchased from Shanghai MacLean Technology Co., Ltd.), other

[0068] Experimental Methods: Effects of Seabuckthorn Leaf Extract on C48 / 80-induced Paw Swelling in Balb / c Mice

[0069] Balb / c mice were randomly divided into control group, model group, low-dose seabuckthorn leaf extract group (25mg / kg), medium-dose seabuckthorn leaf extract group (50mg / kg), and high-dose seabuckthorn leaf extract group (100mg / kg), with 6 mice in each group. The drug administration group was orally administered with the corresponding dose of drug 0.2mL / 20g according to the weight of the mice, and the control group and model group were only administered with the corresponding volume of normal saline. One hour after the end of the drug administration, 5μL C48 / 80 (30ug / mL) was intradermally injected into the right toes of the mice in the model group and the drug administration group for sensitization. The mice in the normal group were only given the corresponding volume of normal saline. The mice were killed 30 minutes later, the foot skin was fixed with 4% paraformaldehyde, and the toe thickness before and after sensitization was recorded.

[0070] The tissue swelling rate was calculated as follows:

[0071] Toe swelling rate (%) = right foot thickness after sensitization - right foot thickness before sensitization / right foot thickness before sensitization × 100%

[0072] like Figure 4 As shown in the figure, the degree of toe swelling of the mice in the model group increased significantly, indicating that the model was successfully established. The degree of toe swelling of Balb / c mice induced by C48 / 80 decreased with the increase of the concentration of seabuckthorn leaf extract. This shows that seabuckthorn leaf extract can effectively inhibit the edema phenomenon during the anaphylactic reaction.

[0073] Experiment 5

[0074] Effects of Seabuckthorn Leaf Extract on C48 / 80-Induced Capillary Dilation in Balb / c Mice

[0075] 1. Fixation: Immediately place the sampled toes in 4% paraformaldehyde for fixation for no less than 24 hours;

[0076] 2. Dehydration: Place the mouse toes in an embedding box and dehydrate them in 70%, 80%, 90%, and 95% ethanol solutions for 2 hours each;

[0077] 3. Transparency: After dehydration, the toe skin is immersed in xylene I and xylene II for 40 minutes to make it transparent;

[0078] 4. Wax dipping and embedding: Place the toes and back skin after transparent treatment in 56℃ and 65℃ molten paraffin for 1 hour each, and after cooling at room temperature, place them upright in a mold for paraffin embedding;

[0079] 5. Slice: Place the wax block on a microtome and slice it, place the slices on a glass slide, and dry them at 60°C;

[0080] 6. Dewaxing: Dewax the sections in xylene I, xylene II for 20 min each, 95% anhydrous ethanol, 80% ethanol for 5 min each, and 70% ethanol (5 min).

[0081] 7. HE staining: After washing with running water, stain with hematoxylin for 5 minutes, and then wash the dye with running water for 2 minutes. Dehydrate the slices with 70%, 80%, 90%, and 95% ethanol solutions for 5 minutes respectively, and finally soak the slices in anhydrous ethanol solution for dehydration for 10 minutes. Soak the slices in xylene I and II for 5 minutes each for transparency, and seal the slices with neutral gum after the liquid around the slices evaporates completely.

[0082] like Figure 5 As shown, the capillaries in the foot skin of the mice in the model group were significantly dilated, indicating that the model was successfully established. The seabuckthorn leaf extract inhibited the dilation of the mouse foot skin capillaries induced by C48 / 80 in a dose-dependent manner. This indicates that the seabuckthorn leaf extract can inhibit edema by inhibiting the dilation of capillaries.

[0083] Experiment 6

[0084] Effects of Seabuckthorn Leaf Extract on Histamine Release Induced by C48 / 80 in Balb / c Mice

[0085] Experimental materials: Same as Experiment 3, histamine kit purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.

[0086] Experimental methods: Balb / c mice were randomly divided into control group, model group, low-dose seabuckthorn leaf extract group (25 mg / kg), medium-dose seabuckthorn leaf extract group (50 mg / kg), and high-dose seabuckthorn leaf extract group (100 mg / kg), with 6 mice in each group. The drug administration group was orally administered with the corresponding dose of drug 0.2 mL / 20 g according to the weight of the mice, and the control group and model group were only administered with the corresponding volume of normal saline. One hour after administration, normal saline was injected into the tail vein of the control group mice, and C48 / 80 (0.3 mg / kg) was injected into the tail vein of the model group and the drug administration group. After 6 hours of injection of C48 / 80 (0.3 mg / kg), blood was collected from the eyeball and centrifuged, and the supernatant was taken as the experimental sample. The histamine detection in each group of experimental samples was carried out according to the instructions of the mouse histamine kit.

[0087] like Figure 6 As shown in the figure, compared with the control group, 30 μg / mL of C48 / 80 in the model group can significantly increase the serum histamine concentration of mice, indicating that the model was successfully established. The seabuckthorn leaf extract can inhibit the release of histamine in mice in a dose-dependent manner.

[0088] Experiment 7

[0089] Effects of Seabuckthorn Leaf Extract on TNF-α Release Induced by C48 / 80 in Balb / c Mice

[0090] Experimental materials: Same as Experiment 3, TNF-α kit purchased from Jiangsu ELISA Industrial Co., Ltd.

[0091] Experimental methods: Balb / c mice were randomly divided into control group, model group, low-dose seabuckthorn leaf extract group (25 mg / kg), medium-dose seabuckthorn leaf extract group (50 mg / kg), and high-dose seabuckthorn leaf extract group (100 mg / kg), with 6 mice in each group. The drug administration group was orally administered with the corresponding dose of drug 0.2 mL / 20 g according to the weight of the mice, and the control group and model group were only administered with the corresponding volume of normal saline. One hour after administration, normal saline was injected into the tail vein of the control group mice, and C48 / 80 (0.3 mg / kg) was injected into the tail vein of the model group and the drug administration group. After 6 hours of injection of C48 / 80 (0.3 mg / kg), blood was collected from the eyeball and centrifuged, and the supernatant was taken as the experimental sample. The TNF-α detection in the experimental samples of each group was carried out according to the instructions of the mouse TNF-α kit.

[0092] like Figure 7As shown in the figure, compared with the control group, 30 μg / mL of C48 / 80 in the model group can significantly increase the serum TNF-α concentration of mice, indicating that the model was successfully established. The seabuckthorn leaf extract can inhibit the release of TNF-α in mice in a dose-dependent manner.

[0093] Application Example 1

[0094] Preparation of Skin Anti-allergic Soothing Cream with Seabuckthorn Leaf Extract:

[0095] Table 1 Seabuckthorn leaf cream formula

[0096]

[0097] Experiment 8

[0098] Human evaluation test

[0099] The subjects' skin redness scores after using the cream containing seabuckthorn leaf extract

[0100] The evaluation method is as follows: 20 subjects aged 35-40 were selected using a skin tester. All subjects had skin redness, etc. The subjects were in good health, had no history of skin diseases, and were able to use skin care products according to the specifications. The subjects applied the cream prepared from the seabuckthorn leaf water extract to their faces every morning and evening, once a day, for one week, and compared the skin red zone scores. The test was repeated 5 times, and the average value calculated after removing the highest and lowest values ​​was the data for each volunteer's test. Before each test, the volunteers were required to clean their faces uniformly with a facial cleanser, sit quietly for 30 minutes, and then measure again. The red zone scores of the subjects in each group were counted. The smaller the red zone, the better the skin soothing effect.

[0101] The experimental results are as follows Figure 8 As shown in the figure, the red area of ​​the subjects improved in the second week compared with the initial period, and the improvement was more significant in the fourth week.

[0102] Experiment 9

[0103] Analysis of components of seabuckthorn leaf extract

[0104] UPLC-MS detection conditions: Hypersil GOLD C18 chromatographic column (1.9 μm, 100×2.1 mm, ThermoFisher Scientific, MA, USA), mobile phase 0.1% formic acid water (A)-acetonitrile (B) gradient elution (0-20 min, 5%-100% B; 20-25 min, 100% B; 25-26 min, 100%-5% B; 26-30 min, 5% B), column temperature 30°C, flow rate 0.25 mL / min, detection wavelength 220 nm, injection volume 3 μL, and 30 min chromatogram recording. A heated electrospray ion source (HESI) was used, with full scan mode of positive and negative ion switching, spray voltage: 3.8 kV (+) / 3.2 kV (-), capillary temperature: 320 ° C, heater temperature: 350 ° C, sheath gas flow rate: 35 arb, auxiliary gas flow rate: 5 arb, backflush gas flow rate: 0 arb, S-lens RF level: 50 V. Rapid data-dependent acquisition (ddMS2) mode was used, mass scan range: m / z 80-1200, primary mass spectrometry resolution: 70000, maximum ion accumulation time: auto; secondary mass spectrometry scanning resolution: 17500: number of cycles: 1 (TOP1), collision voltage (NCE): 20, 40, 60, dynamic exclusion: 5s, maximum ion accumulation time: auto, isolation window: 3m / z.

[0105] HPLC chromatographic conditions: HPLC analysis was performed using a Shimadzu system. All separations were performed on a C18 column (4.6×250 mm, 5 μm). Mobile phase: Phase A was 0.1% formic acid in water, and phase B was acetonitrile. Elution gradient: (0-5 min, 5% B; 5-15 min, 5%-10% B; 15-25 min, 10%-20% B; 25-35 min, 20% B; 35-36 min, 20%-5% B; 36-40 min, 5% B). After each run, the equilibrium time was 5 min. The injection volume of each sample was 10 μL, the flow rate was 0.8 mL / min, and the column temperature was maintained at 30°C. The chromatogram was acquired at 370 nm.

[0106] like Fig. 9As shown in Table 2, UPLC-Q Exactive-Orbitrap-MS and HPLC were used to analyze the main components of seabuckthorn leaf extract. The results showed that a total of 61 components were identified in the seabuckthorn leaf extract, with the highest content of polyphenolic compounds, of which gallic acid was 16.73±0.30 mg / g and ellagic acid was 2.03±0.19 mg / g. The largest number of flavonoids were mainly isorhamnetin, kaempferol, and quercetin aglycones, of which isorhamnetin was 0.61±0.01 mg / g and isorhamnetin-3O-neohesperidin was 0.42±0.03 mg / g.

[0107] Table 2 Chemical components of seabuckthorn leaf extract

[0108]

[0109]

[0110] Experiment 10

[0111] Hyaluronidase inhibition experiment Experimental materials: Same as Experiment 1 Experimental methods: Same as Experiment 1.

[0112] like Fig.10 As shown in the figure, in order to clarify the active ingredients of seabuckthorn leaf extract, the hyaluronidase inhibition test was used for evaluation. The seabuckthorn leaf extract and its main ingredients gallic acid, ellagic acid, isoquercetin and isorhamnetin-3O-neohesperidin inhibited the hyaluronidase activity in a dose-dependent manner. Except for isorhamnetin-3O-neohesperidin, the hyaluronidase inhibition rate of seabuckthorn leaf extract and the other three ingredients at a concentration of 10 mg / mL can reach about 90%. This shows that seabuckthorn leaf extract and its three main ingredients have strong potential for anti-allergic reactions.

[0113] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a seabuckthorn leaf extract, characterized in that: The following steps are involved: Add sea buckthorn leaves with a solid-liquid ratio of 1:10 into distilled water, soak at room temperature for 12 hours, boil at 80°C-100°C for 1 hour, and separate the solid and liquid; add distilled water with a volume ratio of 1:5 into the solid separation, boil at 80°C-100°C for 1 hour, separate the solid and liquid, combine the liquids obtained from the two separations, filter, centrifuge, take the supernatant, concentrate and dry, and obtain sea buckthorn leaf extract powder.

2. The method for preparing a seabuckthorn leaf extract according to claim 1, characterized in that: The pore size of the filtration is 200 nm, and the centrifugation is 3000 r / min for 10 min.

3. The method for preparing a seabuckthorn leaf extract according to claim 1, characterized in that: The concentration and drying is to concentrate the supernatant at 75° C.-80° C. by rotary evaporation and high-temperature spray drying.

4. A seabuckthorn leaf extract, characterized in that A seabuckthorn leaf extract prepared according to any one of claims 1 to 3.

5. Application of seabuckthorn leaf extract in anti-allergic soothing products, characterized in that: The use of seabuckthorn leaf extract in cosmetics as claimed in claim 4.

6. The use according to claim 5, characterized in that: Application of the seabuckthorn leaf extract in cream.

7. The use according to claim 6, characterized in that: The cream comprises the following raw materials in parts by weight: 79.87 parts of water, 3-5 parts of 1,3-propylene glycol, 0.03-0.5 parts of ethylenediaminetetraacetic acid, 2-6 parts of glycerin, 1-1.2 parts of cetearyl alcohol, 1-2 parts of dimethicone, 1.5-2.5 parts of methyl glucose sesquistearate, 0.6-0.8 parts of hydroxypropyl methylcellulose, 0.5-1 parts of 1,2-pentanediol, 0.3-0.8 parts of phenoxyethanol, and 1-3 parts of seabuckthorn leaf extract.

8. The use of a seabuckthorn leaf extract in an anti-allergic soothing product according to claim 7, characterized in that: The invention comprises the following raw materials in parts by weight: 79.87 parts of water, 5 parts of 1,3-propylene glycol, 0.03 parts of ethylenediaminetetraacetic acid, 6 parts of glycerin, 1.2 parts of cetearyl alcohol, 2 parts of dimethicone, 2.5 parts of methyl glucose sesquistearate, 0.6 parts of hydroxypropyl methylcellulose, 1 part of 1,2-pentanediol, 0.8 parts of phenoxyethanol and 1 part of sea buckthorn leaf extract.

Images