Trichosanthes sinensis seed extract as well as preparation method and application thereof

Abstract

The invention provides a flat-core wood seed extract as well as a preparation method and application thereof, and the method comprises the following steps: picking dry flat-core wood seeds without pulp and seed coats, and crushing the flat-core wood seeds; the method comprises the following steps of: adding petroleum ether into crushed flat walnut seeds, soaking for multiple times, soaking for 20-28 hours each time, filtering a soaking solution obtained after soaking each time by using gauze, cotton and filter paper in sequence, and then combining all the filtered soaking solutions to obtain an extracting solution; and concentrating the extracting solution into an oily suspension by adopting a rotary evaporator to obtain the abutilon indicum seed extract. The abalone walnut seed extract obtained by the embodiment of the invention has a plurality of biological activities of resisting oxidation, inhibiting tyrosinase, hyaluronidase, elastase and the like.

Description

Technical Field

[0001] The invention relates to the technical field of elasmocarpus seed extraction, and in particular to an elasmocarpus seed extract, a preparation method and application thereof. Background Art

[0002] As global attention to natural products and their health benefits continues to increase, plant extracts, as an important type of natural resources, have shown great application potential in many fields such as biomedicine, food supplements, cosmetics and personal care products.

[0003] Prinsepia utilis, a traditional Chinese medicinal material rich in a variety of bioactive ingredients, has attracted widespread attention from the scientific research and industry in recent years. With the advancement of modern science and technology, especially the continuous innovation of separation, purification and bioactivity evaluation technology, discovering and optimizing the active ingredients from Prinsepia utilis seeds and developing new functional products or drugs have become a hot topic in the field of natural product research and development. Summary of the invention

[0004] The purpose of the present invention is to provide a Pleurotus eryngii seed extract and a preparation method and application thereof, so as to provide a new Pleurotus eryngii seed extraction method, and the obtained extract has significant effects in anti-oxidation, inhibiting tyrosinase, inhibiting hyaluronidase, inhibiting elastase, repairing zebrafish, etc.

[0005] In a first aspect, the present invention provides a method for preparing a Psoralea corylifolia seed extract, the preparation method comprising:

[0006] Picking and removing the dried walnut seeds with the pulp and seed coat, and crushing the walnut seeds;

[0007] The crushed flat walnut seeds are added to petroleum ether for multiple soaking, each soaking time is 20-28 hours, and the soaking liquid after each soaking is filtered through gauze, cotton, and filter paper in turn, and then all the soaking liquids after filtering are combined to obtain an extract;

[0008] The extract was concentrated into an oily suspension by a rotary evaporator to obtain the Juglans regia seed extract.

[0009] Furthermore, the rotary evaporator is set at a water temperature of 35-45° C., a rotation speed of 70-90 rpm / min, and the time required to concentrate each 300 mL of the extract to an oily suspension is 25 to 30 minutes.

[0010] In a second aspect, the present invention provides a Juglans regia seed extract, which is prepared according to the above-mentioned method for preparing the Juglans regia seed extract.

[0011] In a third aspect, the present invention provides a use of the above-mentioned Juglans regia seed extract in the preparation of a pharmaceutical composition or a skin care agent with an antioxidant effect.

[0012] In a fourth aspect, the present invention provides a use of the above-mentioned Juglans regia seed extract in the preparation of a pharmaceutical composition or a skin care agent for inhibiting the action of tyrosinase.

[0013] In a fifth aspect, the present invention provides a use of the above-mentioned Juglans regia seed extract in the preparation of a pharmaceutical composition or a skin care agent for inhibiting the action of hyaluronidase.

[0014] In a sixth aspect, the present invention provides a use of the above-mentioned Juglans regia seed extract in the preparation of a pharmaceutical composition or a skin care agent for inhibiting the action of elastase.

[0015] In a seventh aspect, the present invention provides a use of the above-mentioned Juglans regia seed extract in the preparation of a pharmaceutical composition or a skin care agent for repairing zebrafish.

[0016] In an eighth aspect, the present invention provides a pharmaceutical composition comprising:

[0017] Physiologically or pharmaceutically acceptable excipients;

[0018] and Juglans regia seed extract as described above.

[0019] In a ninth aspect, the present invention provides a skin care agent comprising:

[0020] Cosmetic base solvents;

[0021] and Juglans regia seed extract as described above.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The present invention proposes a method for preparing a flat walnut seed extract, first picking and drying flat walnut seeds, removing the pulp and seed coat, and then crushing, then soaking in petroleum ether for a long time and fine filtering for many times, and finally concentrating under specific conditions using a rotary evaporator to obtain an extract in the form of an oily suspension. The preparation method not only ensures the purity and quality of the extract, but also effectively extracts the active ingredients in the flat walnut seeds.

[0024] 2. The flat walnut seed extract obtained by the specific method of the present invention has shown excellent application value in many fields. Through a series of experimental verifications, the flat walnut seed extract has been confirmed to have multiple biological activities such as anti-oxidation, inhibition of tyrosinase, hyaluronidase and elastase, etc. These properties make it an ideal ingredient for preparing drugs or skin care products with anti-aging, whitening and lightening, moisturizing and firming effects. In addition, the extract can also repair zebrafish, suggesting that it may have a potential role in promoting skin repair and regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the DPPH free radical scavenging rate test results of a petroleum ether extract according to an embodiment of the present invention;

[0026] Figure 2 This is a schematic diagram of the test results of the ABTS free radical scavenging rate of the petroleum ether extract in one embodiment of the present invention;

[0027] Figure 3 This is a schematic diagram of the test results of the hydroxyl radical scavenging rate of the petroleum ether extract in one embodiment of the present invention;

[0028] Figure 4 This is a schematic diagram of the tyrosinase inhibition rate test results of a petroleum ether extract in one embodiment of the present invention;

[0029] Figure 5 This is a schematic diagram of the hyaluronidase inhibition rate test results of the petroleum ether extract in one embodiment of the present invention;

[0030] Figure 6 This is a schematic diagram of the test results of the elastase inhibition rate of the petroleum ether extract in one embodiment of the present invention;

[0031] Figure 7 This is a schematic diagram of the zebrafish repair activity test results of the petroleum ether extract in one embodiment of the present invention.

[0032] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0033] In order to make the purpose, 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. Obviously, the described embodiments are 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. Unless otherwise defined, the technical terms or scientific terms used herein should be understood by people with general skills in the field to which the present invention belongs. "Including" and similar words used in this article mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0034] Embodiment 1

[0035] Sample information: The dried flat-core wood seeds with the pulp and seed coat removed were collected on June 1, 2024 at Fanren Town, Zhuarong Village, Hongqiao Town, Ninglang County, Lijiang City, Yunnan Province, located at 100.81742° east longitude and 27.51562° north latitude.

[0036] Sample processing: Use a grinder to properly crush the dry flat-core wood seeds with seed coat (small pieces with a diameter of about 0.1 to 0.5 cm), and then bag them for later use.

[0037] Preparation of polar parts of petroleum ether: Weigh 1 kg of crushed flat-core wood seed sample and place it in a conical flask, add an appropriate amount of petroleum ether to soak (petroleum ether dosage: 3 to 5 cm above the sample surface), soak 5 times (soak until the color of the petroleum ether solution becomes lighter), soak for 24 hours each time, filter the soaking liquid (generally filter three times, the first time with gauze, the second time with cotton, and the third time with filter paper), combine the soaking liquid after filtration to obtain an extract, and then use a rotary evaporator to concentrate the extract to an oily suspension (rotary evaporator conditions: water temperature of about 40°C, speed of 80rpm / min. It takes 25 to 30 minutes to concentrate 300 mL of extract to an oily suspension. The petroleum ether can be reused after recovery.), combine the extracts, and finally obtain a petroleum ether extract (flat-core wood seed extract) with a weight of 279.3 g.

[0038] The following examples will conduct experimental verification on the petroleum ether extract prepared in Example 1, and the materials and reagents used are described as follows:

[0039] Tyrosinase (≥500U / mg, CAS: 9002-10-2), elastase from porcine pancreas (≥30U / mg, CAS: 39445-21-1), 1,1-diphenyl-2-picrylhydrazylradical (DPPH·) (CAS: 1898-66-4), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, ABTS + )(CAS: 30931-67-0), etc., Shanghai McLean Biochemical Technology Co., Ltd.; hyaluronidase (300IU / mg, CAS: 37259-53-3), sodium hyaluronate (≥95%, CAS: 9067-32-7), etc., Sichuan Weikeqi Biotechnology Co., Ltd.

[0040] Sodium acetate, levodopa, acetylacetone, AAAPVN, etc., Shanghai McLean Biochemical Technology Co., Ltd.; Tween 20, etc., Shanghai McLean Biochemical Technology Co., Ltd.; DMSO, etc., Chengdu Cologne Chemical Co., Ltd.; Folin, etc., Beijing Solebold Technology Co., Ltd.; Tris-HCl (0.01 mol / L, pH = 7.0-9.0), etc., Shanghai Yuanye Biotechnology Co., Ltd. Sea salt; brine shrimp eggs; anhydrous calcium chloride, magnesium sulfate heptahydrate, sodium bicarbonate, potassium chloride (analytical grade); tricaine (analytical grade); Rehmannia glutinosa extract solution. Unless otherwise specified, all reagents used in this test were analytically pure or chromatographically pure, and the water was grade 3 water specified in GB / T 6682.

[0041] The instruments and equipment used are described as follows:

[0042] UV-5500PC spectrophotometer (Shanghai Yuanxi Instrument Co., Ltd.), ME204 / 02 electronic balance (Toledo (Shanghai) Co., Ltd.), Cence Xiangyi centrifuge (Changsha High-tech Industrial Development Zone Xiangyi Centrifuge Instrument Co., Ltd.), KQ-500E ultrasonic cleaner (Kunshan Ultrasonic Instrument), HH-4 digital display constant temperature water bath (Changzhou Aohua Instrument Co., Ltd.), ZQZY-CGST oscillating incubator (Shanghai Zhichu Instrument Co., Ltd.), R-1001VN rotary evaporator (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.), etc.

[0043] Example 2: Antioxidant Activity Test

[0044] 2.1 Test Method

[0045] The determination process of DPPH free radical scavenging ability: weigh 0.8g of petroleum ether extract, dilute to 10mL with anhydrous ethanol, obtain a petroleum ether extract sample with a mass concentration of 80mg / mL, and dilute with anhydrous ethanol to obtain sample solutions with different mass concentrations of 5.00, 10.00, 20.00, 40.00, and 80.00mg / mL. The determination of the DPPH free radical scavenging ability of petroleum ether extract was appropriately modified with references. 100μL of petroleum ether extract sample solutions with different gradients and 100μL of DPPH standard working solution were aspirated into a 96-well plate, and the absorbance was determined at a wavelength of 517nm after standing in the dark for 30min. Ascorbic acid (VC) was used as a positive control, and parallel experiments were performed in duplicate 3 times. The calculation formula for the DPPH free radical scavenging rate of petroleum ether extract samples is as follows:

[0046] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%

[0047] Wherein: A1 is the absorbance of 100 μL series of gradient petroleum ether extract sample solution + 100 μL DPPH standard working solution at 517 nm; A2 is the absorbance of 100 μL series of gradient petroleum ether extract sample solution + 100 μL anhydrous ethanol solution at 517 nm; A0 is the absorbance of 100 μL anhydrous ethanol solution + 100 μL DPPH standard working solution at 517 nm.

[0048] Determination of ABTS free radical scavenging ability: Prepare petroleum ether extract sample solutions with different mass concentrations of 5.00, 10.00, 20.00, 40.00, and 80.00 mg / mL. The determination of the ability of petroleum ether extract to scavenge ABTS free radicals was appropriately modified with reference to the literature. 40 μL of petroleum ether extract sample solutions of different concentrations and 160 μL ABTS standard working solution were aspirated into a 96-well plate, kept in the dark for 30 minutes, and the absorbance was measured at a wavelength of 734 nm. Ascorbic acid (VC) was used as a positive control, and parallel experiments were performed in duplicate 3 times. The formula for calculating the ABTS free radical scavenging rate of petroleum ether extract samples is as follows:

[0049] ABTS free radical scavenging rate (%) = [1-(A3-A4) / A5] × 100%

[0050] Wherein: A3 is the absorbance of 40 μL series of gradient petroleum ether extract sample solution and 160 μL ABTS standard working solution at 734 nm; A4 is the absorbance of 40 μL series of gradient petroleum ether extract sample solution and 160 μL anhydrous ethanol solution at 734 nm; A5 is the absorbance of 40 μL anhydrous ethanol solution and 160 μL ABTS standard working solution at 734 nm.

[0051] Determination of hydroxyl radical scavenging ability:

[0052] ①6mmol / L FeSO4 solution: 0.9114gFeSO4+1000mL distilled water

[0053] ②6mmol / L salicylic acid-ethanol solution: 0.8287g salicylic acid + 1000mL anhydrous ethanol

[0054] ③6mmol / L30%H2O2 solution: 0.204g30%H202+1000mL distilled water

[0055] Sample group: sample solution 1mL + FeSO4 solution 1mL + salicylic acid-ethanol solution 1mL + 30% H202 solution 1mL

[0056] Sample control group: 1 mL sample solution + 2 mL distilled water + 1 ml anhydrous ethanol solution

[0057] Blank control: 1 mL of sample solvent + 1 mL of FeSO4 solution + 1 mL of salicylic acid-ethanol solution + 1 mL of 30% H202 solution

[0058] Mix well and incubate in a water bath at 37°C for 1 h. After the reaction is complete, take 200 μL of each well in a 96-well plate and measure the absorbance at 510 nm.

[0059] The calculation formula of hydroxyl free radical scavenging rate is:

[0060] I=(A0-A S +A C ) / A 0× 100%

[0061] In the formula, As is the absorbance of the sample group, A0 is the absorbance of the blank control, and Ac is the absorbance of the sample control. The hydroxyl radical scavenging rate of each group is calculated, and each experiment is repeated three times. Prepare petroleum ether extract sample solutions with different mass concentrations of 5.00, 10.00, 20.00, 40.00, and 80.00 mg / mL.

[0062] 2.2 Test results:

[0063] As shown in Table 1 and Figure 1 As shown, in the DPPH free radical scavenging experiment, when the sample concentration was 80 mg / mL, the DPPH free radical scavenging rate of the petroleum ether extract was 21.90%.

[0064] Table 1 Experimental data of DPPH free radical scavenging rate of petroleum ether extract

[0065] Sample concentration 5mg / mL 10mg / mL 20mg / mL 40mg / mL 80mg / mL <![CDATA[A1]]> 0.7819 0.7877 0.7068 0.6495 0.7136 <![CDATA[A2]]> 0.0474 0.0488 0.0473 0.0828 0.1782 <![CDATA[A0]]> 0.6516 0.6881 0.6816 0.6716 0.6855 Clearance -12.73％ -7.38％ 3.25％ 15.61％ 21.90％

[0066] As shown in Table 2 and Figure 2As shown, in the ABTS free radical scavenging experiment, when the sample concentration was 80 mg / mL, the ABTS free radical scavenging rate of the petroleum ether extract was 22.71%.

[0067] Table 2 Experimental data of ABTS free radical scavenging rate of petroleum ether extract

[0068] Sample concentration 5mg / mL 10mg / mL 20mg / mL 40mg / mL 80mg / mL A3 0.6357 0.5987 0.5464 0.5166 0.5078 A4 0.1154 0.0766 0.0438 0.0449 0.0454 A5 0.5965 0.5916 0.5921 0.5959 0.5983 Clearance 12.78％ 11.75％ 15.12％ 20.84％ 22.71％

[0069] As shown in Table 3 and Figure 3 As shown, in the hydroxyl radical scavenging experiment, when the sample concentration was 40 mg / mL, the hydroxyl radical scavenging rate of the petroleum ether extract was 89.38%, and when the sample concentration was 80 mg / mL, the hydroxyl radical scavenging rate of the petroleum ether extract was 88.93%.

[0070] Table 3 Experimental data of hydroxyl radical scavenging rate of petroleum ether extract

[0071] Sample concentration 5mg / mL 10mg / mL 20mg / mL 40mg / mL 80mg / mL As 0.8157 0.8219 0.8268 0.8063 1.0713 Ac 0.0981 0.1735 0.2837 0.7015 0.9526 A0 1.0609 1.0715 1.0355 0.9874 1.0715 Clearance 32.36％ 39.49％ 47.55％ 89.38％ 88.93％

[0072] Example 3: Tyrosinase Inhibitory Activity Test

[0073] 3.2 Test Method

[0074] DMSO: Tween 20 (1:1) was used as a solution to promote the dispersion of oil. Samples of petroleum ether extracts of Pleurotus eryngii seeds were prepared at different mass concentrations of 80.00, 40.00, 20.00, 10.00, and 5.00 mg / mL. 70 μL of the petroleum ether extract of Pleurotus eryngii seeds was aspirated and 30 μL of tyrosinase (500 U / mL) was added to a 96-well plate. Each concentration of sample was set in two different wells, one of which was added with enzyme and the other was not. All volume defects were compensated by adding phosphate buffer. The negative control was DMSO: Tween 20 solution and the positive control was kojic acid solution. The treatment method was the same as that of the petroleum ether extract of Pleurotus eryngii seeds. After incubation at (37±2.0℃) for 5 minutes, 110 μL of L-DOPA solution (2mM) was added, and then reacted at (37±2.0℃) for another 30 minutes. The absorbance of L-DOPA was read at 490 nm. Two independent experiments were performed, each repeated three times. The tyrosinase inhibition rate of the petroleum ether extract of the seeds of the flat-core wood was calculated as follows:

[0075]

[0076] Wherein, A is a negative control containing enzyme, B is a negative control without enzyme, C is a petroleum ether extract sample of Pleurotus eryngii seeds containing enzyme or kojic acid, and D is a petroleum ether extract sample of Pleurotus eryngii seeds without enzyme or kojic acid.

[0077] 3.2 Test Results

[0078] As shown in Table 4. Table 5 and Figure 4 As shown, in the tyrosinase inhibition test, when the sample concentration was 80 mg / mL, the tyrosinase inhibition rate of the petroleum ether extract was 33.95%, reaching 35% of the positive kojic acid.

[0079] Table 4 Experimental data of tyrosinase inhibition rate of petroleum ether extract

[0080] Sample concentration 5mg / mL 10mg / mL 20mg / mL 40mg / mL 80mg / mL C 0.4678 0.4825 0.5442 0.6082 0.6804 D 0.0716 0.1167 0.1783 0.2517 0.3542 A 0.5670 0.5670 0.5670 0.5670 0.5670 B 0.0732 0.0732 0.0732 0.0732 0.0732 Inhibition rate 19.75％ 25.92％ 25.90％ 27.80％ 33.95％

[0081] Table 5 Experimental data of tyrosinase inhibition rate of positive control (kojic acid)

[0082] Sample concentration 5mg / mL 10mg / mL 20mg / mL 40mg / mL 80mg / mL C 0.0956 0.0726 0.0679 0.0663 0.0715 D 0.0510 0.0520 0.0533 0.0556 0.0608 A 0.4740 0.5051 0.4720 0.4864 0.4757 B 0.0534 0.0513 0.0522 0.0503 0.0506 Inhibition rate 89.41％ 95.47％ 96.51％ 97.54％ 97.49％

[0083] Example 4: Hyaluronidase inhibition activity test of petroleum ether extract of Psoralea corylifolia seeds

[0084] 4.1 Test Method

[0085] ① Petroleum ether extract of Pleurotus eryngii seeds: DMSO was used as solvent and the concentration gradient was 2.0%, 1.0%, and 0.5%;

[0086] ② Bufer: Measure 4.8 mL of acetic acid solution (0.2 mol / L) and 45.2 mL of sodium acetate solution (0.2 mol / L) into a beaker, mix well and dilute to 100 mL with distilled water to prepare an acetate buffer solution with a pH of 5.6 for dissolving hyaluronidase and sodium hyaluronate;

[0087] ③ Acetylacetone solution: Measure 10mL of sodium carbonate solution (1.0mol / L) and 0.7mL of acetylacetone into a beaker and mix well (prepare before use);

[0088] ④P-DAB color developer: weigh 0.4g of p-dimethylaminobenzaldehyde and dissolve it in 7.5mL of concentrated hydrochloric acid and 7.5mL of anhydrous ethanol, and mix well;

[0089] The experiment was divided into sample group, sample control group, positive control group, blank control group and model control group. Three replicate wells were set at the same concentration in the same group. The amount of each solution added, the order of addition and the reaction process are shown in Table 6.

[0090] Table 6 Hyaluronidase inhibition activity test reaction flow

[0091]

[0092] The absorbance was measured at 530 nm, and the hyaluronidase inhibition rate of the petroleum ether extract of the seeds of the genus Pinnata was calculated as follows:

[0093]

[0094] Wherein, A is the average absorbance of the sample group of the petroleum ether extract of the seeds of the flat-core wood, B is the average absorbance of the model control group, C is the average absorbance of the blank control group, and D is the average absorbance of the sample control group of the petroleum ether extract of the seeds of the flat-core wood.

[0095] 4.2 Test Results

[0096] As shown in Table 7 and Figure 5 As shown, in the hyaluronidase inhibition activity test, when the sample concentration was 2.00%, the hyaluronidase inhibition rate of the petroleum ether extract reached 88.51%. When the concentration of positive dipotassium glycyrrhizinate was 1 mg / mL, i.e., the content was 0.10%, the hyaluronidase inhibition rate was 53.37%, which was 1 / 20 of the amount of petroleum ether extract of the seeds of the flat-core wood.

[0097] Table 7 Experimental data of hyaluronidase inhibition rate of petroleum ether extract

[0098] Sample concentration 0.50％ 1.00％ 2.00％ A 0.1174 0.1280 0.1365 D 0.1138 0.1260 0.1354 B 0.0878 0.0878 0.0878 C 0.0782 0.0782 0.0782 Inhibition rate 62.35％ 79.79％ 88.51％

[0099] Example 5: Elastase Inhibition Activity Test

[0100] 5.1 Test Method

[0101] The petroleum ether extract of the seeds of the Pleurotus eryngii was prepared with DMSO as the solvent in a concentration gradient of 1.0%, 0.5%, and 0.25%; the remaining solutions were prepared with Tris-HCl buffer as the solvent.

[0102] The experiment was divided into sample group, positive control group, blank control group, model control group and sample control group. Three replicate wells were set up in the same group and at the same concentration. The amount of each solution added is shown in Table 1.2.

[0103] Table 8 Elastase inhibition activity test reaction flow

[0104]

[0105]

[0106] The reaction was allowed to stand at room temperature for 15 minutes, and the absorbance was measured at 420 nm using an enzyme marker. The formula for calculating the inhibition rate of elastase in the petroleum ether extract of the seeds of the genus Pinnata is as follows:

[0107]

[0108] Wherein, A0 is the average absorbance of the blank control group, A1 is the average absorbance of the control group of the petroleum ether extract of the seeds of the flat-core wood, A2 is the average absorbance of the petroleum ether extract of the seeds of the flat-core wood, and A3 is the average absorbance of the model control group.

[0109] 5.2 Test Results

[0110] As shown in Table 9, Table 10 and Figure 6 As shown, in the elastase inhibition activity test, when the sample concentration was 1.00%, the elastase inhibition rate of the petroleum ether extract reached 94.29%. When the positive control (EGCE) was at the same concentration, the elastase inhibition rate was 92.67%.

[0111] Table 9 Experimental data of elastase inhibition rate of petroleum ether extract

[0112] Sample concentration 0.25％ 0.50％ 1.00％ A2 0.0895 0.1177 0.1544 A1 0.0879 0.1171 0.1541 A0 0.0527 0.0527 0.0527 A3 0.0594 0.0594 0.0594 Inhibition rate 75.66％ 91.72％ 94.29％

[0113] Table 10 Experimental data of elastase inhibition rate of positive control (EGCE)

[0114]

[0115]

[0116] Example 6. Zebrafish repair activity test

[0117] 6.1 Test Method

[0118] Refer to T / SHRH 036-2021 "Cosmetic Melanin Inhibition-Zebrafish Embryo Test" and T / GDST1-2021 "Zebrafish Embryo Acute Toxicity Test Method A and Method B". Use tricaine solution to anesthetize the zebrafish embryos, and use a scalpel to remove the zebrafish embryo tail fin under a microscope to establish an injury model. The test requires the establishment of a model control group (containing only zebrafish embryo culture medium), a positive control group (Rehmannia glutinosa extract), and a test group (petroleum ether extract of Pleurotus eryngii seeds). Each well contains one fish embryo and 0.2mL of culture medium or test substance. It is placed in a 28±1℃ incubator and cultured for 48±1h. The zebrafish is anesthetized with tricaine, and then placed under a stereo microscope to take side photos of the fish embryo's tail. The formula for calculating the tail fin repair promotion rate of the Pleurotus eryngii seed petroleum ether extract sample is as follows:

[0119]

[0120] Wherein, S is the average tail fin length of fish embryos in the group treated with petroleum ether extract of Psoralea corylifolia seeds, and C is the average tail fin length of fish embryos in the model control group.

[0121] 6.2 Test Results

[0122] As shown in Table 11 and Figure 7 As shown, in the zebrafish repair activity test, when the sample concentration was 0.2% to 5%, the skin barrier repair rate of the petroleum ether extract was ≥28%, with significant differences.

[0123] Table 1.11 Experimental data on the repair activity of petroleum ether extract in zebrafish

[0124]

[0125] In summary, the flat walnut seed extract obtained by a specific method has shown excellent application value in many fields. Through a series of experimental verifications, the flat walnut seed extract has been confirmed to have multiple biological activities such as anti-oxidation, inhibition of tyrosinase, hyaluronidase and elastase. These properties make it an ideal ingredient for the preparation of drugs or skin care products with anti-aging, whitening and freckle removal, moisturizing and firming effects. In addition, the extract can also repair zebrafish, suggesting that it may have a potential role in promoting skin repair and regeneration.

[0126] Embodiment 7

[0127] The present application also provides a pharmaceutical composition comprising the Pleurotus eryngii seed extract obtained in Example 1. The pharmaceutical composition has good antioxidant, tyrosinase inhibition, hyaluronidase inhibition, elastase inhibition, zebrafish repair and other effects.

[0128] Illustratively, the pharmaceutical composition described in the embodiments of the present application may be any one of an ointment and a cream.

[0129] Embodiment 8

[0130] The embodiment of the present application also provides a skin care product, comprising the Pleurotus eryngii seed extract extracted in Example 1, and the skin care product has good antioxidant, tyrosinase inhibition, hyaluronidase inhibition, elastase inhibition, zebrafish repair and other effects.

[0131] Skin care products can be understood as liquid external skin preparations. For example, the skin care products described in the embodiments of the present application can be any one of lotion, essence, spray, emulsion, cream, mask, gel, sunscreen, and isolation.

[0132] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A method for preparing a Psoralea corylifolia seed extract, characterized in that: The preparation method comprises: Picking and removing the dried walnut seeds with the pulp and seed coat, and crushing the walnut seeds; The crushed flat walnut seeds are added to petroleum ether for multiple soaking, each soaking time is 20-28 hours, and the soaking liquid after each soaking is filtered through gauze, cotton, and filter paper in turn, and then all the soaking liquids after filtering are combined to obtain an extract; The extract was concentrated into an oily suspension by a rotary evaporator to obtain the Juglans regia seed extract.

2. The method for preparing the flat walnut wood extract according to claim 1, characterized in that: The conditions for setting the rotary evaporator are water temperature 35-45°C, rotation speed 70-90rpm / min, and the time for concentrating each 300mL extract to an oily suspension is 25-30min.

3. A flat walnut seed extract, characterized in that The Juglans regia seed extract is prepared according to the preparation method of the Juglans regia seed extract according to any one of claims 1-2.

4. Use of the Juglans regia seed extract as claimed in claim 3 in the preparation of a pharmaceutical composition or skin care agent with antioxidant effect.

5. Use of the Juglans regia seed extract as claimed in claim 3 in the preparation of a pharmaceutical composition or skin care agent for inhibiting the action of tyrosinase.

6. Use of the Juglans regia seed extract as claimed in claim 3 in the preparation of a pharmaceutical composition or skin care agent for inhibiting the action of hyaluronidase.

7. Use of the Juglans regia seed extract as claimed in claim 3 in the preparation of a pharmaceutical composition or skin care agent for inhibiting the action of elastase.

8. Use of the Juglans regia seed extract as claimed in claim 3 in preparing a pharmaceutical composition or skin care agent for repairing zebrafish.

9. A pharmaceutical composition, characterized in that include: Physiologically or pharmaceutically acceptable excipients; And the flat walnut seed extract as described in claim 3.

10. A skin care agent, characterized in that: include: Cosmetic base solvents; And the flat walnut seed extract as described in claim 3.

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