Anti-aging and Anti-allergic skin topical composition comprising elaeagnus angustifolia l. fruit extract and preparation method therefor

Abstract

Use of an Elaeagnus angustifolia L. fruit extract in preparing an anti-aging skin topical composition and a method for preparing the Elaeagnus angustifolia L. fruit extract. The Elaeagnus angustifolia L. fruit extract has the related effects of promoting skin type I collagen proliferation and skin fibroblast proliferation, inhibiting type I metalloproteinase activity, down-regulating tumor necrosis factor-α, and inhibiting the expression of the allergic medium β-hexosaminidase, and has an allergic medium-inhibiting effect or use for soothing sensitive skin.

Description

Anti-aging and anti-allergic skin external composition containing extract of elaeagnus angustifolia fruit and method for preparing the same TECHNICAL FIELD

[0001] The present invention provides an extract of Elaeagnus angustifolia fruit for preparing an anti-aging and anti-allergic skin external composition and a method for preparing the same. BACKGROUND

[0002] Elaeagnus angustifolia L. is a deciduous tree belonging to the Elaeagnaceae family. It is considered a safe and delicious medicinal food and has been used for thousands of years. Due to the fact that the fruit of Elaeagnus angustifolia is rich in (-)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, kaempferol, quercetin, apigenin, isorhamnetin, and isorhamnetin-3-O-β-D-galactoside, and polysaccharides, which have biological and pharmacological activities, Elaeagnus angustifolia has been confirmed to have various medicinal effects including potent antioxidant, anti-inflammatory, and analgesic effects, and treatment of fever, jaundice, asthma, tetanus, anti-mutagenesis, nausea, cough, diarrhea, kidney disease and long-term dysfunction, burns, hemostasis, and has been used to treat various diseases in East Asia for thousands of years.

[0003] There are many scientific papers, patent applications, and publications on the use of Elaeagnus angustifolia. CN 112641709 describes a combination of Elaeagnus angustifolia pollen, Rhus verniciflua, Rhus verniciflua essential oil, pumpkin seeds, lavender, soapnut extract, and bamboo leaf as a skin care cosmetic, emphasizing its tyrosinase inhibitory, free radical scavenging ability, and skin water retention efficacy. CN1065792 and CN101084872 describe the use of Elaeagnus angustifolia polysaccharide and a mixture of Elaeagnus angustifolia polysaccharide and various plant extracts in hair styling liquids and hair creams, respectively.

[0004] CN115645397 discloses a method for inhibiting free radicals and skin tyrosinase activity for skin whitening and inhibition of Staphylococcus aureus and Escherichia coli; and CN110354154 discloses that oral administration of an extract of Elaeagnus angustifolia L. gum can reduce transepidermal water loss (TEWL) and inhibit IL-6 inflammatory cytokines in mice, and can be used for cosmetic purposes to alleviate atopic dermatitis caused by dry skin. Other documents also report that Elaeagnus angustifolia L. polysaccharide enhances the barrier water retention function of the stratum corneum of mice in a dry skin model, while inhibiting the levels of inflammatory cytokines including IL-6, IL-17 and TNF-a, and up-regulating the mRNA expression of water channel-related proteins such as Aquaporin 3 (AQP-3), Filaggrin (FLG) and Loricrin (LOR), suggesting that Elaeagnus angustifolia L. gum polysaccharide can be used as a skin care cosmetic and to treat atopic dermatitis and skin barrier dysfunction caused by dry skin.

[0005] Elaeagnus angustifolia L. also has specific efficacy reports in the field of wound healing. For example, CN 109331051 describes a pharmaceutical composition comprising an alcohol extract of Elaeagnus angustifolia L. leaves, and states that they are effective for treating the healing of mechanical skin wounds in mice. Tavangar discloses in a published document that a methanol extract of Elaeagnus angustifolia L. fruit has a potential effect on accelerating the healing of injured skin in a rat model. Another in vitro experimental study on human fibroblasts showed that a methanol extract of Elaeagnus angustifolia L. fruit can activate human leukocyte antigen-G (HLA-G) and vascular endothelial growth factor-A (VEGF-A), promote the proliferation of 10% human fibroblasts, and in an in vitro scratch test, it was confirmed that a methanol aqueous extract of Elaeagnus angustifolia L. fruit can promote complete wound healing within 24 hours. In addition, Taheri reports that a 10-19% aqueous gel of Elaeagnus angustifolia L. fruit extract has the effect of relieving pain, reducing inflammation and promoting healing in the symptoms of oral lichen planus and oral mucositis, and this method provides a safer and more effective new treatment approach for relieving pain and promoting healing in oral lichen planus and oral mucositis. These effects on skin wound healing can be attributed to the ability of quercetin and kaempferol, which are abundant in the extract, to inhibit inflammatory factors such as TNF-a and COX-2 to promote anti-inflammatory responses.

[0006] However, there is still a need to develop skin external compositions with anti-aging and anti-allergic effects using natural plants.

[0007] SUMMARY

[0008] The present invention discloses that the extract of Elaeagnus angustifolia L. fruit has the effects of anti-aging and anti-allergy, and can be developed as a skin and cosmetic composition. Therefore, the present invention provides a use of the extract of Elaeagnus angustifolia L. fruit for preparing an anti-aging and anti-allergic skin external composition.

[0009] In the examples of the present invention, the extract of Elaeagnus angustifolia L. fruit has the effects of promoting the proliferation of human skin fibroblasts, promoting the proliferation of collagen type I, or inhibiting the activity of collagenase type I. It can be applied to the skin to increase collagen proliferation, efficiently inhibit collagenase, and promote the proliferation of skin fibroblasts, thereby providing the effects of anti-aging, protection, and repair of the skin.

[0010] In the examples of the present invention, the extract of Elaeagnus angustifolia L. fruit also has the effects of inhibiting allergic mediators or soothing sensitive skin. Therefore, the extract of Elaeagnus angustifolia L. fruit can be used for the effects of anti-aging, protection, and repair of the skin.

[0011] In another aspect, the present invention provides a method for preparing the extract of Elaeagnus angustifolia L. fruit, which comprises the following steps:

[0012] (1) drying Elaeagnus angustifolia L. fruit tissue containing a fruit core and grinding the tissue into powder with a grinder;

[0013] (2) covering and soaking the Elaeagnus angustifolia L. fruit tissue with a solvent system to form an Elaeagnus angustifolia L. fruit tissue soaking solution;

[0014] (3) heating the Elaeagnus angustifolia L. fruit tissue soaking solution at a temperature below 100°C, and then heating to 100°C for extraction, and obtaining an Elaeagnus angustifolia L. fruit tissue extract after the solution is left to stand at room temperature;

[0015] (4) centrifuging the Elaeagnus angustifolia L. fruit tissue extract to obtain a supernatant and a precipitate;

[0016] (5) filtering the precipitate under reduced pressure and collecting the supernatant, and removing volatile substances in the supernatant to obtain the extract of Elaeagnus angustifolia L. fruit.

[0017] In the examples of the present invention, the solvent system is pure water, methanol, or a 70% ethanol aqueous solution. The solvent system is preferably pure water.

[0018] According to the present invention, the extract of Elaeagnus angustifolia L. fruit can be further prepared into a solid powder of the extract of Elaeagnus angustifolia L. fruit, and the preparation method comprises the following steps:

[0019] (1) continuing to filter and sterilize the extract of Elaeagnus angustifolia L. fruit to obtain a sterilized clear filtrate;

[0020] (2) freeze-drying the sterilized clear filtrate to obtain a solid powder of the extract of Elaeagnus angustifolia L. fruit.

[0021] In still another aspect, the present application provides an anti-aging skin external composition comprising the extract of Elaeagnus angustifolia L. fruit prepared by the method of the present application and a skin-external acceptable carrier.

[0022] In the examples of the present application, the effective dose of the extract of Elaeagnus angustifolia L. fruit ranges from 310 to 630 μg / mL.

[0023] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application, as claimed. BRIEF DESCRIPTION OF DRAWINGS

[0024] The foregoing summary, as well as the following detailed description of the application, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the application, there is shown in the drawings a presently preferred embodiment. It should be understood, however, that the application is not limited to the embodiment shown in the drawings.

[0025] FIG. 1 shows a high performance liquid chromatogram of the effective component analysis of the extract of Elaeagnus angustifolia L. fruit.

[0026] FIG. 2 shows the effect of the extract of Elaeagnus angustifolia L. fruit on the cell viability of human keratinocytes. 0: 0 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 80: 80 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 160: 160 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 310: 310 μg / ml; 560: 560 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 630: 630 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 1250: 1250 μg / ml of the extract of Elaeagnus angustifolia L. fruit.

[0027] FIG. 3 shows the effect of the extract of Elaeagnus angustifolia L. fruit on the proliferation and cell viability of human fibroblasts. 0: 0 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 80: 80 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 160: 160 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 310: 310 μg / ml; 560: 560 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 630: 630 μg / ml of the extract of Elaeagnus angustifolia L. fruit; 1250: 1250 μg / ml of the extract of Elaeagnus angustifolia L. fruit.

[0028] Figure 4 shows the effect of Elaeagnus angustifolia L. fruit extract on the proliferation of collagen type I. Control: DMEM medium; TGF-β1 : DMEM medium containing TGF-β1 positive control; 160 μg / ml: DMEM medium containing 160 μg / ml of Elaeagnus angustifolia L. fruit extract; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 560 μg / ml: DMEM medium containing 560 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0029] Figure 5 shows the effect of Elaeagnus angustifolia L. fruit extract on the inhibition of collagenase type I. Control: DMEM medium; TGF-β1 : DMEM medium containing TGF-β1 positive control; 160 μg / ml: DMEM medium containing 160 μg / ml of Elaeagnus angustifolia L. fruit extract; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 560 μg / ml: DMEM medium containing 560 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0030] Figure 6 shows the effect of Elaeagnus angustifolia L. fruit extract on the viability of mouse RAW264.7 cells. Control: DMEM medium; LPS: DMEM medium containing 0.1 μg / ml LPS; Dex (1 μM): DMEM medium containing 1 μM Dex; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 500 μg / ml: DMEM medium containing 500 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0031] Figure 7 shows the effect of Elaeagnus angustifolia L. fruit extract on IL-6 inhibition rate in mice. Control group: DMEM medium; LPS: DMEM medium containing 0.1 μg / ml LPS; Dex (1 μM): DMEM medium containing 1 μM Dex; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 500 μg / ml: DMEM medium containing 500 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0032] Figure 8 shows the effect of Elaeagnus angustifolia L. fruit extract on TNF-α inhibition rate. Control group: DMEM medium; LPS: DMEM medium containing 0.1 μg / ml LPS; Dex (1 μM): DMEM medium containing 1 μM Dex; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 500 μg / ml: DMEM medium containing 500 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0033] Figure 9 shows the effect of Elaeagnus angustifolia L. fruit extract on COX-2 inhibition rate. Control group: DMEM medium; LPS: DMEM medium containing 0.1 μg / ml LPS; Dex (1 μM): DMEM medium containing 1 μM Dex; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 500 μg / ml: DMEM medium containing 500 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0034] Figure 10 shows the effect of Elaeagnus angustifolia L. fruit extract on PGE-2 inhibition rate. Control: DMEM medium; LPS: DMEM medium containing 0.1 μg / ml LPS; Dex (1 μM): DMEM medium containing 1 μM Dex; 310 μg / ml: DMEM medium containing 310 μg / ml of Elaeagnus angustifolia L. fruit extract; 500 μg / ml: DMEM medium containing 500 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 1250 μg / ml of Elaeagnus angustifolia L. fruit extract.

[0035] Figure 11 shows the effect of Elaeagnus angustifolia L. fruit extract on β- hexosaminidase inhibition rate. Control: DMEM medium; DPN-DSA: DMEM medium containing 0.45 μg / mL anti-DPN IgE and 10 μg / mL DPN-BSA as a negative control; Quercetin 10 μM: DMEM medium containing 0.45 μg / mL anti-DPN IgE, 10 μg / mL DPN-BSA and 10 μM quercetin as a positive control; 130 μg / ml: DMEM medium containing 0.45 μg / mL anti-DPN IgE, 10 μg / mL DPN-BSA and 130 μg / ml of Elaeagnus angustifolia L. fruit extract; 250 μg / ml: DMEM medium containing 0.45 μg / mL anti-DPN IgE, 10 μg / mL DPN-BSA and 250 μg / ml of Elaeagnus angustifolia L. fruit extract; 500 μg / ml: DMEM medium containing 0.45 μg / mL anti-DPN IgE, 10 μg / mL DPN-BSA and 500 μg / ml of Elaeagnus angustifolia L. fruit extract; 630 μg / ml: DMEM medium containing 0.45 μg / mL anti-DPN IgE, 10 μg / mL DPN-BSA and 630 μg / ml of Elaeagnus angustifolia L. fruit extract; 1250 μg / ml: DMEM medium containing 0.45 μg / mL anti-DPN IgE, 10 μg / mL DPN-BSA and 1250 μl / ml of Elaeagnus angustifolia L. fruit extract.

[0036] The details of one or more embodiments of the application are set forth in the description below. Other features or advantages of the application will be apparent from the following detailed description of several embodiments, and from the appended claims. DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0038] The present application is further illustrated by the following examples, which are provided by way of illustration and not limitation.

[0039] As used herein, the indefinite article "a" refers to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "a component" refers to one component or more than one component.

[0040] As used herein, the term "Elaeagnus pungens" refers to a deciduous tree of the Elaeagnaceae family. It is rich in bioactive and pharmacological active ingredients such as (-)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, kaempferol, quercetin, apigenin, isorhamnetin, and isorhamnetin-3-O-β-D-galactoside, as well as polysaccharides. It has various medical effects including potent antioxidant, anti-inflammatory, and analgesic effects, as well as therapeutic effects for fever, jaundice, asthma, tetanus, anti-mutagenesis, nausea, cough, diarrhea, kidney disease and long-term dysfunction, burns, hemostasis, etc. In traditional Chinese medicine, Elaeagnus pungens is also believed to have the effects of regulating the spleen and stomach, tonifying blood and calming the nerves, and is often used in traditional Chinese medicine formulations.

[0041] As used herein, the term "extraction" refers to the process of separating a mixture using solubility in a solvent. It is mainly divided into liquid extraction and solid-liquid extraction, both of which use liquid solvents to separate certain components from liquid and solid mixtures, respectively. The solvent itself must be insoluble in the target mixture and have selective solubility, good thermal and chemical stability, low toxicity and corrosion, and meet the extraction standards.

[0042] As used herein, the term "collagen" refers to a long-chain protein composed of amino acids, mainly composed of glycine, hydroxyproline, and proline. Collagen plays an important role in the human body as a supporting structure, increasing tissue strength and elasticity, maintaining skin elasticity and moisture, promoting wound healing, etc. It mainly exists in tissues such as skin, bones, muscles, tendons, ligaments, blood vessels, teeth, cornea, etc. Collagen has wide application value in the fields of beauty, health care, and medicine, and is often used in beauty products to improve skin elasticity and texture, reduce wrinkles, promote joint health, and regenerate skeletal tissue.

[0043] The present application provides the use of Elaeagnus pungens fruit extract for the preparation of an anti-aging and anti-allergic skin external composition. According to the present application, the Elaeagnus pungens fruit extract has the effects of promoting human skin fibroblast proliferation, promoting type I collagen proliferation, or inhibiting type I collagenase activity. It also has the effects of inhibiting allergic mediators or soothing sensitive skin. Therefore, the Elaeagnus pungens fruit extract can achieve the purposes of skin anti-aging, protection, soothing, repair, and anti-allergy.

[0044] According to the present application, the Elaeagnus pungens Thunb. fruit extract also has excellent inhibitory activity against inflammatory factors TNF-α, COX-2, IL-6 and inflammatory mediator PGE-2.

[0045] The present application further provides a method for preparing the Elaeagnus pungens Thunb. fruit extract, which comprises the following steps:

[0046] (1) drying Elaeagnus pungens Thunb. fruit tissues containing fruit core and grinding the Elaeagnus pungens Thunb. fruit tissues into powder with a grinder;

[0047] (2) covering and soaking the Elaeagnus pungens Thunb. fruit tissues with a solvent system to form an Elaeagnus pungens Thunb. fruit tissue soaking solution;

[0048] (3) heating the Elaeagnus pungens Thunb. fruit tissue soaking solution at a temperature lower than 100°C, and then heating to 100°C extraction, and then standing the solution at room temperature to obtain an Elaeagnus pungens Thunb. fruit tissue extract solution;

[0049] (4) centrifuging the Elaeagnus pungens Thunb. fruit tissue extract solution to obtain a supernatant and a precipitate;

[0050] (5) filtering the precipitate under reduced pressure and collecting the supernatant, and removing volatile substances in the supernatant to obtain the Elaeagnus pungens Thunb. fruit extract.

[0051] According to the present application, the solvent system is pure water, methanol or 70% ethanol aqueous solution, and the best one is pure water.

[0052] According to the present application, the Elaeagnus pungens Thunb. fruit extract can be further prepared into a solid powder of the Elaeagnus pungens Thunb. fruit extract, and the preparation method comprises the following steps:

[0053] (1) continuing to filter the Elaeagnus pungens Thunb. fruit extract to remove bacteria to obtain a sterilized clear filtrate;

[0054] (2) freeze-drying the sterilized clear filtrate to obtain the solid powder of the Elaeagnus pungens Thunb. fruit extract.

[0055] According to the present application, the Elaeagnus pungens Thunb. fruit extract prepared by the above method of the present application can be combined with a skin-externally acceptable carrier to prepare an anti-aging skin-externally applied composition according to general existing methods. The above skin-externally acceptable carrier can be an acceptable carrier or adjuvant known or commonly known in the field of cosmetics or dermatology, including aqueous and non-aqueous liquid media and various solid and semi-solid dosage forms. The carrier can also include different ingredients and dosage forms, such as microemulsions, microcapsules, microparticles, and any other ingredients and dosage forms commonly used in cosmetics.

[0056] For the preparation of the skin external composition of the present application, the Elaeagnus angustifolia L. fruit extract can be further comprised of any cosmetically or dermatologically known or well-known acceptable carriers or adjuvants, such as, but not limited to, fatty substances, organic solvents, dissolving agents, condensing agents, gelling agents, softening agents, antioxidants, stabilizers, foaming agents, surfactants, diluents, preservatives, fillers, flow control agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, fragrances, lubricants, dispersing agents, chelating agents, and hydrophilic or lipophilic active agents, etc., depending on the mode of administration and the nature of the dosage form.

[0057] The skin external composition is formulated according to many factors within the present or commonly used range by those having ordinary knowledge in the art to which the present application pertains, which can vary depending on the type and nature of the Elaeagnus angustifolia L. fruit extract to be formulated, the individual to be administered, the intended route of administration, and the therapeutic indication. The Elaeagnus angustifolia L. fruit extract of the present application, having a high content of physiologically active substances and free radical scavenging ability, can be effectively used to prepare a skin external composition or cosmetic for skin anti-aging, which promotes collagen proliferation, inhibits collagenase type I, and inhibits inflammatory factors such as TNF-α, IL-6, COX-2, PGE-2, and β-hexosaminidase, and promotes fibroblast proliferation.

[0058] The technical features of the present application are further illustrated by the following examples, but are not intended to limit the scope of the present application.

[0059] Example

[0060] Example 1 Preparation of Elaeagnus angustifolia L. fruit extract

[0061] The dried Elaeagnus angustifolia L. fruit tissues containing the core were ground into powder using a grinder. The Elaeagnus angustifolia L. fruit tissues were covered and soaked with a solvent system to form an Elaeagnus angustifolia L. fruit tissue soaking solution. The Elaeagnus angustifolia L. fruit tissue soaking solution was heated to 70°C for 1 hour, and then heated to 100°C for 10 minutes, and then allowed to stand at room temperature for 4 hours to obtain an Elaeagnus angustifolia L. fruit tissue extract. The Elaeagnus angustifolia L. fruit tissue extract was centrifuged to obtain an Elaeagnus angustifolia L. fruit extract and a precipitate. The precipitate was filtered under reduced pressure using a Buchner funnel covered with a 0.7 μm glass filter paper, and the Elaeagnus angustifolia L. fruit extract was collected, and the filtrate was dried under vacuum and reduced pressure to remove volatile substances.

[0062] As described above, the Elaeagnus angustifolia L. fruit extract was further filtered using a 0.45 μm filter membrane filter and a 0.20 μm Millipore sterilizing filter in series to obtain a sterilized and clear filtrate. The clear and sterilized Elaeagnus angustifolia L. filtrate was further freeze-dried at a gradient of -45°C to -20°C and 0.1 torr for 16 hours to obtain an Elaeagnus angustifolia L. fruit extract solid powder.

[0063] Example 2 Determination of Total phenolic content (TPC) of Extracts of Elaeagnus angustifolia L. fruit

[0064] To confirm the active ingredients that might be present in the extracts of Elaeagnus angustifolia L. fruit, High performance liquid chromatography (HPLC) was used for analysis. The contents of catechin, quercetin, rutin and kaempferol in the extracts were determined by reverse phase high performance liquid chromatography (JASCO LC2000). A C18 column was used and UV detector was set at 365 nm (λmax) for kaempferol determination. The mobile phase was 1 M H3PO4, acetonitrile, water and methanol (1:10:30:60) with pH adjustment to 4. The flow rate was 0.5 mL / min. Gallic acid (0.01 mg / mL) was added to the sample as an internal standard and the injection volume was 10 μL. To determine the content of kaempferol in the extracts, a calibration curve was established for different concentrations of kaempferol in methanol (0.25, 0.5, 0.6, 0.8, 1.0 and 2.0 ng / mL). The results are shown in Figure 1. The extracts of Elaeagnus angustifolia L. fruit contained catechin, kaempferol, quercetin and rutin, all of which have antioxidant and anti-inflammatory functions. The content of kaempferol was 17 μg / mL.

[0065] The total phenolic content was quantified using Folin-Ciocalteau reagent. The Folin-Ciocalteau reagent was diluted 10 times with distilled water. 50 μl of the extract of Elaeagnus angustifolia L. fruit was mixed with 1 ml of the diluted Folin-Ciocalteau reagent, 1 ml of sodium bicarbonate solution (7.5%) and 2 ml of distilled water. The mixture was allowed to react at room temperature in the dark for 15 minutes. The absorbance of the solution was determined at a wavelength of 730 nm using a UV spectrophotometer and compared with the standard curve of gallic acid to calculate the regression. The unit was expressed as gallic acid equivalent per gram (GAE). The results of the total phenolic content are shown in Table 1. The results showed that the total phenolic content of the extract of Elaeagnus angustifolia L. fruit with 70% ethanol was higher than that of the extracts with pure water and methanol.

[0066] Table 1. Total phenolic content of Elaeagnus angustifolia L. fruit

[0067] Example 3 Safety evaluation of extracts of Elaeagnus angustifolia L. fruit - human keratinocyte cell viability test

[0068] Human skin cell viability test was performed using human keratinocyte (HaCaT) cells. HaCaT cells were cultured in DMEM medium (purchased from Gibco) containing 10% fetal bovine serum (FBS; purchased from Sigma) and 1% penicillin / streptomycin (pen / strep; purchased from Sigma) at 37°C with 5% CO2, and the cell culture medium was replaced every 2 days. Before the experiment, the density of the human keratinocyte cell suspension was adjusted to 10 5 cells and inoculated uniformly in a 96-well culture plate, and incubated at 37°C with 5% CO2 for at least 24 hours. The cytotoxicity of the pomegranate fruit extract on human keratinocyte cells was evaluated by the MTT method. The specific implementation method is as follows: human keratinocyte cells were inoculated in a 96-well plate at a density of 10 5 cells / well and incubated for at least 24 hours. Seven concentrations were designed for the experiment, and a blank control group was also designed. Different concentrations of pomegranate fruit extract (0, 80, 160, 310, 560, 630, and 1250 μg / ml) were added to each well, and three repeated tests were set for each concentration. Incubation was continued for 24 hours. Then 20 μl of MTT (5 mg / mL) was added to each well, and incubation was continued at 37°C in the dark for 4 hours. The culture supernatant was carefully aspirated, 100 μl of DMSO was added to each well, and oscillation was performed for 10 minutes to ensure that the precipitated Formazan was completely dissolved. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay instrument (BioTek, SynergyTM2, USA).

[0069] The test results are shown in Table 2 and Figure 2. After the human keratinocyte cells (HaCaT cells) were treated with the water extract of pomegranate fruit and the 70% methanol and ethanol extracts (80, 160, 310, 560, 630, and 1250 μg / mL) for 24 hours, the cell viability of the human keratinocyte cells treated with the water extract of pomegranate fruit was 102.5, 101, 95, 83, 77, and 48%, respectively; the cell viability of the human keratinocyte cells treated with the methanol extract of pomegranate fruit was 93, 84, 78, 72, 65, and 43%, respectively; and the cell viability of the human keratinocyte cells treated with the 70% ethanol extract of pomegranate fruit was 91, 80, 74, 62, 51, and 29%, respectively. Therefore, the water extract of pomegranate fruit has safety at a concentration of <630 μg / mL (cell viability >80%) for human keratinocyte cells. However, the cell safety concentration of the methanol and 70% ethanol extracts of pomegranate fruit decreased to 160 μg / mL, indicating that the use of alcohol with higher lipophilicity as the extraction solvent has 3 times higher cytotoxicity to keratinocytes than the water extract of pomegranate fruit.

[0070] Table 2. Human keratinocyte cell viability

[0071] Cell viability was measured as 100 ± 2.7% of the control group. (Mean ± SD, N = 3, n = 3)

[0072] Example 4 Proliferation test of skin fibroblast cells by Elaeagnus angustifolia L. fruit extract

[0073] The deep reason for skin aging and wrinkling is the decrease in the activity of fibroblast cells and the decrease in the secretion of collagen and elastin. Therefore, the evaluation of the effect of cosmetics on the proliferation of fibroblast cells can evaluate the wrinkle-removing ability to some extent. The proliferation of fibroblast cells can be detected by MTT method of cell viability. The specific experimental method is as follows: The skin fibroblast cells (Hs68) were uniformly inoculated in the 96-well culture plate at a density of 10 5 cells / well, 100 μl was added to each well, the edge wells were filled with sterile phosphate buffer, and then the plate was placed in a CO2incubator for 24 hours. The culture supernatant was aspirated, and the medium containing different concentrations of Elaeagnus angustifolia L. extract was added to each well. Seven concentrations (0, 80, 160, 310, 560, 630, 1250 μg / mL) were set for each sample, and three replicate wells were set for each concentration. A blank control group without sample was also set. After 24 hours of culture, the culture supernatant was aspirated, 100 μl of DMEM medium was added to each well, and 20 μl of MTT solution with a concentration of 5 mg / ml was added. After incubation in a CO2incubator for 4 hours, the culture supernatant was carefully aspirated, 100 μl of DMSO was added to each well, and the plate was shaken for 10 minutes to ensure that the Formazan precipitate was completely dissolved. The absorbance value was measured at 570 nm using an enzyme-linked immunosorbent instrument (BioTek, SynergyTM2, USA).

[0074] The results are shown in Table 3 and Figure 3. After 24 hours of treatment with the aqueous extract and the 70% ethanol extract of Elaeagnus angustifolia L. fruit at concentrations of 80, 160, 310, 560, 630, and 1250 pg / mL, the cell viability of human fibroblasts treated with the aqueous extract was 111, 117, 119, 120, 120, and 81%, respectively. The cell viability of human fibroblasts treated with the methanol extract of Elaeagnus angustifolia L. fruit was 107, 104, 96, 79, 69, and 49%, respectively. The cell viability of human fibroblasts treated with the ethanol extract of Elaeagnus angustifolia L. fruit was 102, 96, 84, 73, 63, and 41%, respectively. Thus, the aqueous extract of Elaeagnus angustifolia L. fruit was safe for human fibroblasts at a safe concentration of 160-630 pg / mL and promoted the proliferation of 20% of human fibroblasts, confirming its efficacy in wound healing. However, the safety of the methanol and 70% ethanol extracts of Elaeagnus angustifolia L. fruit for human fibroblasts was lower than that of the aqueous extract, and the safe concentration was <560 pg / mL, at which there was no significant proliferation of human fibroblasts. Thus, the concentration required for safety and efficacy was considered in the present application, and the aqueous extract of Elaeagnus angustifolia L. fruit, which was safer, was selected for the following tests of the claimed physiological efficacy.

[0075] Table 3. Cell viability of human fibroblasts

[0076] Cell viability was determined with reference to the control group, which was 100 ± 1.7%. (Mean ± SD, N = 3, n = 3)

[0077] Example 5. In vitro test of collagen type I production

[0078] Collagen is an extracellular matrix protein that is essential for the three-dimensional structure of the skin, and increased synthesis of collagen improves the firmness and elasticity of the skin. The following test experiment shows the ability of the extract of Elaeagnus angustifolia L. fruit to stimulate the production of collagen type I by human dermal fibroblasts. Human fibroblasts (Hs68) were used, and the ability of the extract of Elaeagnus angustifolia L. fruit to stimulate collagen type I production was measured using a human collagen type I alpha Simple Step Kit (Abcam, lot number: ab210966), which evaluates the release of human collagen type I alpha N-terminal propeptide (PINP). The principle is that after collagen is synthesized, the PINP peptide is cleaved from the procollagen molecule to facilitate the formation of a triple helix from procollagen, and thus the amount of free PINP propeptide cleaved can be calculated on the basis of stoichiometry.

[0079] The specific steps of the collagen proliferation test are as follows: human fibroblasts were cultured in modified DMEM medium (containing 5% fetal bovine serum and 1% penicillin and streptomycin) and 2 x 10 4 Cells were transplanted into 96-well plates at a concentration of 100 μL per well and incubated at 37°C in a humidified 5% CO2 atmosphere for 24 hours. Each well was then treated with 100 μL of culture medium containing an Elaeagnus angustifolia fruit extract, resulting in final concentrations of 160, 310, 500, 630, and 1250 μg / mL. Three replicates were set up for each concentration. A negative control containing no sample and a positive control containing 10 ng / mL TGF-β1 were also set up. The plates were incubated at 37°C for 24 hours. The ELISA module for type I collagen concentration was performed according to the manufacturer's (Abcam) recommended method: 50 μL of the suspension supernatant was transferred to a 96-well plate, and 50 μL of the antibody cocktail provided in the reagent module was added to each well, mixed evenly, and incubated at room temperature for 1 hour. After incubation, each well was washed three times with PBS buffer, and 100 μL of tetramethylbenzidine (TMB) solution was added to each well and incubated for 10 minutes, and then 100 μL of stop solution was added to stop the reaction. The 96-well plate was then evenly shaken to mix, and the absorbance value was read at 450 nm using an enzyme-linked immunosorbent assay reader (BioTek, SynergyTM2, USA) within 15 minutes after adding the stop solution, and the collagen concentration was calculated using a standard linear regression line. The results of the collagen proliferation test are shown in Table 4 and Figure 4. The water extract of Elaeagnus angustifolia can promote the proliferation of type I procollagen, and has a collagen proliferation promoting effect of 15-25% at a safe concentration below 0.63 μg / mL.

[0080] Table 4. Type I collagen proliferation

[0081] Example 6 In vitro type I collagenase inhibition test

[0082] The primary cause of skin wrinkles is an imbalance between collagen synthesis and degradation. In normal, healthy skin, the synthesis of type I collagen and its type I matrix metalloproteinase (MMP-1) is balanced and regulated. However, in aging skin, the synthesis of type I and type III collagen decreases, while the activity of MMP-1 increases. MMP-1 is an enzyme protein that degrades the extracellular matrix, particularly type I collagen. Under normal conditions, it is typically associated with wound healing and tissue regeneration. Therefore, inhibiting the activity of MMP-1 is an important goal in anti-aging cosmetic research.

[0083] The following assay shows that the Elaeagnus angustifolia L. fruit extract has excellent ability to inhibit the expression of matrix metalloproteinase-1 (MMP-1) in human fibroblast cells. The assay uses human fibroblast cells (Hs68) and measures the inhibitory ability of the Elaeagnus angustifolia L. fruit extract on MMP-1 by using a human matrix metalloproteinase-1 (MMP1) ELISA kit (purchased from Abeam). The kit is designed to detect the expression of mmp-1 in human epidermal fibroblast cells. The assay uses a monoclonal antibody specific for MMP-1 to bind quantitatively to a sandwich enzyme immunoassay technique, and finally measures the concentration of MMP-1 and cell viability by ultraviolet absorption spectrum. The specific implementation steps are as follows: human fibroblast cells are cultured using a modified DMEM medium (containing 5% fetal bovine serum and 1% penicillin and streptomycin) and transplanted into a 96-well plate at a concentration of 2 x 10 4 The cells are incubated at 37°C in a humidified 5% CO2 atmosphere for 24 hours. Then, each well is applied with different concentrations of 100 μL of culture solution containing the Elaeagnus angustifolia L. fruit extract, so that the final concentration of the Elaeagnus angustifolia L. fruit extract is 0, 80, 150, 310, 500, 630, 1250 μg / mL, and 10 ng / mL of TNF-α is also added to each well to stimulate the expression of MMP-1, and three replicate wells are set for each concentration. A negative control without samples and TNF-α and a positive control without samples and with only 10 ng / mL of TNF-α are also set, and incubation is continued at 37°C for 24 hours. The ELISA module detection of Mmp-1 concentration is carried out according to the manufacturer's (Abeam) recommended method without modification: 50 μL of the supernatant is transferred to a 96-well plate, 50 μL of the MMP-1 antibody cocktail provided by the reagent module is added to each well, mixed uniformly, and incubated at room temperature for 1 hour. After incubation, each well is washed with PBS buffer three times, 100 μL of tetramethylbenzidine (TMB) solution is added to each well, incubated for 10 minutes, and then 100 μL of stop solution is added to stop the reaction. Then, the 96-well plate is mixed uniformly, the absorbance value is read at 450 nm within 15 minutes after the addition of the stop solution using an enzyme-linked immunosorbent assay reader (BioTek, SynergyTM2, USA), and the concentration of MMP-1 is calculated by standard linear regression line. The results of the Elaeagnus angustifolia L. fruit extract inhibition test of MMP-1 are shown in Table 5 and Figure 5, and the Elaeagnus angustifolia L. fruit extract has excellent MMP-1 inhibition effect, and the average inhibition rate of MMP-1 is greater than 87% when the concentration of the Elaeagnus angustifolia L. fruit extract applied is less than the safe cytotoxicity dose (<630 μg / mL). Therefore, the high MMP-1 inhibition effect of the Elaeagnus angustifolia L. fruit extract indicates its great application ability in anti-aging cosmetics.

[0084] Table 5. Inhibition of matrix metalloproteinase-1

[0085] Average % inhibition of mmp-1 relative to positive control group (3 replicates)

[0086] *Addition of 10 ng / mL TNF-α stimulates mmp-1 expression

[0087] Example 7 Mouse RAW 264.7 cell toxicity test

[0088] This preliminary mouse RAW 264.7 cell toxicity assay determined the safe concentration range for all test materials and was used for subsequent cytokine tests. The specific experimental procedure was as follows: 1. Mouse RAW 264.7 cells were cultured at a density of 1.0 x 10 4 cells / 0.1 mL and dispensed into 96-well plates and then incubated for 18-24 hours. 2. After removing the old culture medium by aspiration, 100 μL of various concentrations of Elaeagnus angustifolia L. fruit extract or the anti-inflammatory drug Dexamethasone was added to each well, and an additional inflammatory condition test group stimulated by ester polysaccharides was set up, and each group was further incubated for 24 hours. 3. 3 μL of MTT solution (5 mg / mL) was added to each well and incubated at 37°C for 3 hours. 4. The MTT solution was then removed, 100 μL of DMSO was added to each well and shaken evenly for 10 minutes to ensure complete dissolution of the precipitated tetrazolium salt. 5. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay reader (BioTek, Synergy™2, USA). The relative cell survival rate of each group was calculated by setting the control group as 100% survival, and the appropriate safe concentration was selected for subsequent IL-6, TNF-α, COX-2 and PGE-2 tests to ensure that the effect of the test material on the survival of RAW 264.7 cells could be reliably evaluated. After adding different concentration ratios of Elaeagnus angustifolia L. fruit extract to Raw 264.7 cells and incubating for 24 hours, the cell survival rate was as shown in Table 6 and Figure 6. The cell viability was >80% at concentrations below 1250 μg / mL, so Elaeagnus angustifolia L. fruit extract at less than 1250 μg / mL was selected as the experimental concentration for subsequent TNF-α, IL-6, COX-2 and PGE-2 inhibition tests.

[0089] Table 6. Cell survival rate of mouse macrophages (RAW 264.7) to Elaeagnus angustifolia L. fruit extract

[0090] Cell survival rate was determined with reference to 100 ± 1.0% of the control group. (Mean ± SD, N = 3, n = 3); p < 0.05.

[0091] Example 8 Anti-inflammatory test - Interleukin-6 inhibition test

[0092] The experimental procedure for Interleukin-6 inhibition was as follows: Mouse RAW 264.7 cells were cultured at a density of 3.0 x 10 5 / mL and seeded in a 24-well plate and incubated in an incubator for 24 hours. After removing the old culture medium, 10 μL of stimulating lipopolysaccharide (LPS, 100 μg / mL) and 4 concentrations (310, 500, 630, 1250 μg / mL) of the extract solution of the Elaeagnus conferta fruit were added to each well, with 3 replicates for each concentration, and negative control groups without samples and ester polysaccharides and a positive control group containing 1 μM of the anti-inflammatory drug dexamethasone were set up. After 24 hours of continued incubation, the concentration of interleukin-6 was measured and calculated using a mouse IL-6 ELISA kit (Abeam, batch number: ab178013, USA) according to the manufacturer's recommended procedure. The effect of the Elaeagnus conferta fruit extract at different concentrations on the expression level of interleukin-6 after 24 hours of co-culture with mouse macrophages (Raw 264.7) was evaluated, and the results are shown in Table 7 and Figure 7. The expression level of interleukin-6 increased when the cells were induced to be inflamed by the addition of LPS (positive control group, P-value < 0.05), and the anti-inflammatory drug dexamethasone (Dex) effectively reduced the expression of interleukin-6 (negative control group, P-value < 0.05), confirming the effectiveness of the experimental system. As shown in Table 7 and Figure 7, the water extract of the Elaeagnus conferta fruit had no inhibitory effect at higher concentrations (>630 μg / mL), but at concentrations lower than 630 μg / mL, it had a significant inhibitory effect on interleukin-6 compared to the positive control group (LPS group), and the inhibitory effect was similar to or higher than that of the negative control group (Dex group), with an interleukin-6 inhibition rate of up to 43-58%, indicating that the water extract of the Elaeagnus conferta fruit at a concentration of 0.31-0.63% had a higher interleukin-6 inhibition efficiency than the commercially available anti-inflammatory drug dexamethasone.

[0093] Table 7. Interleukin-6 inhibition rate

[0094] *: 0.1 μg / mL LPS was added to the negative control group, the positive control group, and the experimental group; (N = 2, n = 3; P < 0.05).

[0095] Example 9 Soothing efficacy & efficacy verification test - Tumor necrosis factor-α inhibition test

[0096] The experimental procedure for tumor necrosis factor-α (TNF-α) inhibition was as follows: Mouse RAW 264.7 cells were cultured at a density of 3.0 x 10 5The density culture was carried out at 1 x 106 / mL and seeded in 24-well plates and incubated in an incubator for 24 hours. After removing the old culture, 10 μL of stimulating lipopolysaccharide (LPS, 100 μg / mL) and 4 concentrations (310, 500, 630, 1250 μg / mL) of the extract containing the fruits of Ziziphus jujuba Mill. were added to each well, 3 replicate wells were set for each concentration, and a negative control group without sample and a positive control group containing 1 μM anti-inflammatory drug dexamethasone were set. After 24 hours of continuous culture, the concentration of tumor necrosis factor-α was measured and calculated using the mouse TNF alpha ELISA Kit (Lot: ab178013, Abeam, USA) kit according to the manufacturer's recommended procedure.

[0097] The results are shown in Table 8 and Figure 8, and the data show that the extract of the fruits of Ziziphus jujuba Mill. at concentrations of 1250, 630, 500, and 310 μg / mL can significantly inhibit the expression of tumor necrosis factor-α compared with the negative control group (LPS group), with inhibition rates of 21, 33, 35, and 39%, respectively, which are comparable to the inhibition rate of the positive control group (Dex group); while the extract of the fruits of Ziziphus jujuba Mill. only at lower concentrations of 310 and 500 μg / mL has tumor necrosis factor-α inhibition performance of 39 and 35% compared with the negative control group (LPS group) (P-value <0.05). The results of this experiment show that the tumor necrosis factor-α content of the negative control group (LPS group) increased by ≧5 times, the tumor necrosis factor-α inhibition rate of the positive control group (Dex group) compared with the negative control group (LPS group) was 32% (≧25%), and the C.V value of each group was <20% and the cell viability was >90%, confirming that the test system is effective. Moreover, the tumor necrosis factor-α content of the extract of the fruits of Ziziphus jujuba Mill. compared with the negative control group (LPS group) decreased and had a significant difference (P<0.05), indicating that the test substance in this experiment has the effect of inhibiting the content of tumor necrosis factor-α and skin soothing effect at the tested concentration, and can be used as supporting evidence for the soothing claim of cosmetic raw materials.

[0098] Table 8. Tumor necrosis factor-α inhibition rate

[0099] *0.1 μg / mL LPS was added to the negative control group, the positive control group, and the experimental group; (N=2, n=3; P<0.05).

[0100] Example 10 Anti-inflammatory efficacy test - cyclooxygenase-2 inhibition test

[0101] The main role of cyclooxygenase-2 (COX-2) is to metabolize arachidonic acid (AA1) to produce prostaglandin (PG) products, which can induce vascular responses and induce inflammatory symptoms such as pain. Therefore, inhibiting the activity of cyclooxygenase-2 reduces the biosynthesis of prostaglandin-2 (PGE-2), which is the main direction of anti-inflammatory drug design. The experimental procedure for cyclooxygenase-2 inhibition is as follows: mouse RAW 264.7 cells were cultured at a density of 3.0 x 10 5 / mL and seeded in a 24-well plate and incubated in an incubator for 24 hours. After removing the old culture, 10 μL of stimulating lipopolysaccharide (LPS, 100 μg / mL) was added to each well, and 4 concentrations (310, 500, 630, 1250 μg / mL) of extracts containing Alnus glutinosa (L.) Gaertn. fruit were added, with 3 replicate wells for each concentration, and a negative control group without sample and a positive control group containing 1 μM anti-inflammatory drug dexamethasone were set. After 24 hours of continuous culture, the concentration of COX-2 was measured and calculated using the mouse COX-2 ELISA Kit (Lot: 2101036350, Abeam, USA) kit according to the manufacturer's recommended procedure. The results are shown in Table 9 and Figure 9 data, and the Alnus glutinosa (L.) Gaertn. water extract at concentrations of 1250, 630, 500, and 310 μg / mL all have a significant trend of inhibiting cyclooxygenase-2 compared to the negative control group (LPS group), with inhibition rates of 29, 17, 18, and 14%, respectively. Therefore, the results of this experiment show that the Alnus glutinosa (L.) Gaertn. water extract has anti-inflammatory efficacy in inhibiting cyclooxygenase-2.

[0102] Table 9. Cyclooxygenase-2 inhibition

[0103] * 0.1 μg / mL LPS was added to the negative control group, the positive control group, and the experimental group; (N = 2, n = 3; P < 0.05).

[0104] Example 11 Anti-inflammatory efficacy test - prostaglandin E2 inhibition test

[0105] The experimental procedure for prostaglandin E2 inhibition is as follows: mouse RAW 264.7 cells were cultured at a density of 3.0 x 10 5The density culture of 0.1 mL / mL was seeded in 24-well plates and incubated in an incubator for 24 hours. After removing the old culture, 10 μL of stimulating lipopolysaccharide (LPS, 100 μg / mL) and 5 concentrations (50, 100, 250, 500 μg / mL) of the extract containing the Elaeagnus angustifolia L. fruit were added to each well, with 3 replicate wells for each concentration, and a negative control group without samples and a positive control group containing 1 μM of the anti-inflammatory drug dexamethasone were set. After 24 hours of continuous culture, the concentration of PGE-2 was measured and calculated using the mouse PGE-2 ELISA Kit (Lot: 2101036350, Abeam, USA) kit according to the manufacturer's recommended procedure. The results are shown in Table 10 and Figure 10. The different concentrations of the Elaeagnus angustifolia L. fruit water extract had a significant inhibitory effect on the expression of the inflammatory cytokine prostaglandin E2 compared to the negative control group (LPS group), and the inhibitory effect was better than that of the positive control group (Dex group), with an inhibition rate of 66% at a safe concentration of 630 μg / mL. Therefore, the prostaglandin E2 inhibitory efficiency of the Elaeagnus angustifolia L. fruit water extract was more than 1 times higher than that of the anti-inflammatory drug dexamethasone, proving its extremely high skin anti-inflammatory and pain-relieving purposes.

[0106] Table 10. Prostaglandin E2 inhibition rate

[0107] * 0.1 μg / mL LPS was added to the negative control group, the positive control group, and the experimental group; (N = 2, n = 3; P < 0.05).

[0108] Example 12 Rat basophil (RBL-2H3) cell degranulation test

[0109] This preliminary rat basophil (RBL-2H3) cell toxicity analysis was used to determine the safe concentration range of all test materials and was used for subsequent cell degranulation tests. RBL-2H3 cells suspended in DMEM containing 10% FBS were seeded at 2 x 10 5The cells were then incubated in a 37°C 5% CO2 incubator for 24 hours. Then 20 μL (1 μg / ml) of anti-DNP IgE (purchased from Sigma) was added to each well and co-cultured for 24 hours to sensitize the RBL-2H3 cells. After washing the cells twice with extracellular buffer (5 mM KCl, 12.5 mM NaCl, 20 mM HEPES, 1.5 mM CaCl2, 1 mM glucose, pH 7.4), 160 μL of siraganian buffer solution (5.6 mM glucose, 1 mM CaCl2, 0.1% BSA) was added. Different concentrations of Alnus glutinosa fruit extract dissolved in DME culture solution and 10 μM of positive control quercetin (purchased from Sigma) were added to the reaction wells and incubated for 1 hour. Then 20 μL (100 ng / ml) of DNP-BSA (purchased from Sigma) was added and incubated for 30 minutes to stimulate cell degranulation. The cell degranulation reaction was then terminated by freezing in an ice bath for 10 minutes. The supernatant was transferred to a 96-well plate, and an equal volume of 4-nitrophenyl-N-β-D-glucoside (p-NAG, 1 μM, pH = 4.5, 0.1 M citric acid, purchased from Sigma) was added to each well and reacted at 37°C for 1 hour, and then 200 μL of solution (0.1 M aqueous Na2CO3: NaHCO3, pH = 10, Sigma) was added to terminate the reaction. The absorbance was measured at 405 nm using an enzyme-linked immunosorbent assay reader (BioTek, Synergy™2, USA). The test results are shown in Table 11 and Figure 11. When the DNP-BSA was added to induce the cells, the cell survival rate was ≥ 100%, and the positive control group (DNP-BSA) could increase the expression of β-hexosaminidase, and the negative control group with 10 μM quercetin could effectively reduce the expression, confirming that the experimental system was functioning normally. The experimental results show that as the concentration of Alnus glutinosa fruit extract increases, the release of β-hexosaminidase gradually decreases. In the safe concentration range of 130, 250, 500, and 630 μg / mL, the Alnus glutinosa fruit extract has a significant ability to inhibit the expression of β-hexosaminidase compared to the negative control group (DNP-BSA), with inhibition rates of 16, 22, 32, and 35%, respectively, indicating that the Alnus glutinosa fruit extract has the effect of inhibiting allergy and soothing the skin.

[0110] Table 11. β-hexosaminidase inhibition rate

[0111] * Each group was added with 0.45 μg / mL anti-DPN IgE; 10 μg / mL DPN-BSA was added to the negative control group, the positive control group and the experimental groups.

[0112] Although the present application provides the above preferred embodiments, it is not intended to limit the present application. Any person skilled in the art of the present application can make modifications and modifications without departing from the spirit and scope of the present application. Therefore, the scope of protection of the present application should be defined by the appended claims of the present application.

Claims

1. A use of an Elaeagnus angustifolia fruit extract for preparing an anti-aging skin external composition.

2. The use according to claim 1, wherein the Elaeagnus angustifolia fruit extract has the effect of promoting the proliferation of human skin fibroblasts, promoting the proliferation of type I collagen or inhibiting the activity of type I collagenase.

3. The use according to claim 1, wherein the Elaeagnus angustifolia fruit extract has the effects of providing anti-aging, protection and repairing effects on the skin.

4. The use according to claim 1, wherein the Elaeagnus angustifolia fruit extract has the effect of inhibiting allergic mediators or soothing sensitive skin.

5. The use according to claim 1, wherein the Elaeagnus angustifolia fruit extract has skin soothing, anti-allergic and anti-inflammatory effects.

6. A method for preparing an extract of an Elaeagnus angustifolia fruit, the preparation method comprising the following steps: (1) The dried fruit tissue containing the pit of the Elaeagnus angustifolia fruit was ground into powder using a grinder; (2) Covering and soaking the Elaeagnus angustifolia fruit tissue with a solvent system to form an Elaeagnus angustifolia fruit tissue soaking solution; (3) The Elaeagnus angustifolia fruit tissue soak was heated at a temperature below 100°C and then heated to 100°C for extraction. The solution was allowed to stand and return to room temperature to obtain an Elaeagnus angustifolia fruit tissue extract; (4) Centrifuging the Elaeagnus angustifolia fruit tissue extract to obtain a supernatant and a precipitate; (5) Filtering the precipitate under reduced pressure and collecting the supernatant, removing volatile substances in the supernatant to obtain the Elaeagnus angustifolia fruit extract.

7. The method of claim 6, wherein the solvent system is pure water, methanol or 70% ethanol aqueous solution. The method of claim 7 , wherein the solvent system is pure water.

9. The method according to claim 6, wherein the Elaeagnus angustifolia fruit extract can be further prepared into a solid powder of Elaeagnus angustifolia fruit extract, the preparation method comprising the following steps: (1) Continue to filter and sterilize the Elaeagnus angustifolia fruit extract to obtain a sterilized clarified filtrate; (2) freeze-drying the sterilized clarified filtrate to obtain a solid powder of the Elaeagnus angustifolia fruit extract.

10. An anti-aging composition for external use on the skin, comprising an extract of Elaeagnus angustifolia fruit prepared by the method according to any one of claims 6 to 9 and a carrier acceptable for external use on the skin. The anti-aging composition for external use as claimed in claim 10 , wherein the effective dosage of the Elaeagnus angustifolia fruit extract is in the range of 310 to 630 μg / mL.

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