Prinsepia utilis royle extract as well as preparation method and application thereof

Abstract

The invention provides a prinsepia utilis royle extract as well as a preparation method and application thereof, and the preparation method comprises the following steps: picking prinsepia utilis royle seeds, removing epicarp from the prinsepia utilis royle seeds, dehydrating to obtain dried kernels, preparing prinsepia utilis royle crude oil from the kernels by a subcritical extraction method, refining the crude oil by a deodorization vacuum process to obtain refined non-winterized prinsepia utilis royle oil, and drying the refined non-winterized prinsepia utilis royle oil to obtain the prinsepia utilis royle extract. And performing cold tank winterization treatment on the refined non-winterized prinsepia utilis royle oil to obtain the refined winterized prinsepia utilis royle oil. The prinsepia utilis royle extract obtained by the embodiment of the invention has good effects of resisting oxidation, inhibiting tyrosinase, inhibiting hyaluronidase, inhibiting elastinase, repairing zebra fish and the like.

Description

Technical Field

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

[0002] Green prickly ash is the fruit of Prinsepia utilis Royle, a plant of the genus Prinsepia Royle in the Rosaceae family. It is also known as green prickly ash, cannon fruit, milk hammer, egg cake, plum thorn, bullet fruit, Anas, and Mengzi flat-core wood. It grows on hillsides, dry river valley sparse forests and bushes at an altitude of 1400-3200 meters (some data say 1800-3000 meters), especially in areas with an altitude of 2200-2700 meters.

[0003] However, in the past, the use of prickle fruit was mainly limited to folk use, such as cooking oil, treating frostbite and making some simple herbal preparations. Due to the lack of scientific extraction and processing technology, many active ingredients in prickle fruit have not been fully utilized, resulting in limited application value and economic benefits. Summary of the invention

[0004] The purpose of the present invention is to provide a prickle fruit extract and a preparation method and use thereof, so as to solve at least one problem in the background technology.

[0005] In a first aspect, the present invention provides a method for preparing a Prinus prinus extract, the method comprising:

[0006] The seeds of the prickle fruit are picked, the outer peel of the seeds is removed, and the seeds are dehydrated to obtain dried kernels, the kernels are subjected to subcritical extraction to obtain prickle fruit crude oil, the crude oil is refined through a deodorizing vacuum process to obtain refined non-winterized prickle fruit oil, and the refined non-winterized prickle fruit oil is subjected to cold tank winterization treatment to obtain refined winterized prickle fruit oil.

[0007] In a second aspect, the present invention provides a thorn fruit extract prepared by the above-mentioned preparation method, and the refined unwinterized thorn fruit oil is detected by ultra-high performance liquid chromatography. According to mass percentage, the refined unwinterized thorn fruit oil includes 8.15% of thulhodin, 84.97% of 17-octadecynoic acid, 0.86% of 3-O-β-D-glucose-rapeseed sterol, 0.52% of cucurbitacic acid, 0.49% of stevioside D, 0.32% of campesterol glucoside, 0.37% of ipsilon, 0.046% of (E)-4-(1,2,3,6-tetrahydro-2,6-dioxo-1,3-dipropyl-9H-purin-8-yl)cinnamic acid, 0.51% of 3'-N-acetyl-4'-O-(14-methylheptadecanoyl) fusarium chromatin ketone, and 3.78% of linolenic acid.

[0008] In a third aspect, the present invention proposes a thorn fruit extract prepared by the above-mentioned preparation method, and the refined unwinterized thorn fruit oil is detected by ultra-high performance liquid chromatography. According to the mass percentage, the refined winterized thorn fruit oil includes 85.98% of 17-octadecynoic acid, 0.44% of 4β-hydroxysteinazolol, 0.35% of stevioside D, 7.07% of thulhodin, 0.39% of campesterol glucoside, 0.35% of lignoceric acid, 0.80% of resmethrin, 1.19% of acacia acetone, 1.39% of roxatidine acetate, and 2.03% of others.

[0009] In a fourth aspect, the present invention provides a use of the above-mentioned Prinia prinia extract in the preparation of a pharmaceutical composition or a skin care agent with an antioxidant effect, wherein the Prinia prinia extract is refined unwinterized Prinia prinia oil or refined winterized Prinia prinia oil.

[0010] In a fifth aspect, the present invention proposes a use of the above-mentioned Prinia prinia extract in the preparation of a pharmaceutical composition or a skin care agent for inhibiting the action of tyrosinase, wherein the Prinia prinia extract is refined unwinterized Prinia prinia oil or refined winterized Prinia prinia oil.

[0011] In a sixth aspect, the present invention proposes a use of the above-mentioned Prinia prinia extract in the preparation of a pharmaceutical composition or a skin care agent for inhibiting the action of hyaluronidase, wherein the Prinia prinia extract is refined unwinterized Prinia prinia oil or refined winterized Prinia prinia oil.

[0012] In a seventh aspect, the present invention provides a use of the above-mentioned Prinia pritta extract in preparing a pharmaceutical composition or a skin care agent for inhibiting the action of elastase, wherein the Prinia pritta extract is refined unwinterized Prinia pritta oil or refined winterized Prinia pritta oil.

[0013] In an eighth aspect, the present invention proposes a use of the above-mentioned Prinia prinia extract in preparing a pharmaceutical composition or a skin care agent for repairing zebrafish, wherein the Prinia prinia extract is refined unwinterized Prinia prinia oil or refined winterized Prinia prinia oil.

[0014] In a ninth aspect, the present invention provides a pharmaceutical composition, characterized in that it comprises:

[0015] Physiologically or pharmaceutically acceptable excipients;

[0016] and the above-mentioned Principal thorn fruit extract, wherein the Principal thorn fruit extract is refined non-winterized Principal thorn fruit oil or refined winterized Principal thorn fruit oil.

[0017] In a tenth aspect, the present invention provides a skin care agent, characterized in that it comprises:

[0018] Cosmetic base solvents;

[0019] and the above-mentioned Principal thorn fruit extract, wherein the Principal thorn fruit extract is refined non-winterized Principal thorn fruit oil or refined winterized Principal thorn fruit oil.

[0020] Compared with the prior art, the beneficial effects of the embodiments of the present application include:

[0021] 1. In some embodiments, both the refined unwinterized Principal Utility Oil and the refined winterized Principal Utility Oil have good DPPH free radical scavenging ability and ABTS free radical scavenging ability.

[0022] 2. In some embodiments, the refined unwinterized Principal Utility Oil and the refined winterized Principal Utility Oil can inhibit tyrosinase activity to a certain extent and reduce the production of melanin.

[0023] 3. In some embodiments, the inhibition rates of refined unwinterized Prinus utilis oil and refined winterized Prinus utilis oil on hyaluronidase are significantly higher than that of crude oil.

[0024] 4. In some embodiments, low doses of refined unwinterized prickle oil and refined winterized prickle oil can achieve considerable elastase inhibition effects, thereby effectively ensuring the level of elastin in the body and making the skin elastic, and can be used as a natural plant anti-aging additive with considerable application prospects.

[0025] 5. In some embodiments, both the refined unwinterized Prinia utilis oil and the refined winterized Prinia utilis oil have a good promotion rate on the repair of the zebrafish embryo's tail fin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0027] Figure 1 One of the BPI and TIC graphs of the prickle oil crude oil of the present invention;

[0028] Figure 2 The second BPI and TIC graph of the prickle oil crude oil of the present invention;

[0029] Figure 3 The third BPI and TIC graph of the crude prickle oil of the present invention;

[0030] Figure 4 This is a schematic diagram of the DPPH radical scavenging rate of utila prickle oil using different refining technologies as an example of the present invention;

[0031] Figure 5 This is a schematic diagram of the ABTS free radical scavenging rate of prickle oil using different refining technologies exemplified in the present invention;

[0032] Figure 6Schematic diagram of the results of the inhibitory activity of prickle fruit oil with different refining technologies on hyaluronidase in the present invention

[0033] Figure 7 Schematic diagram of the results of the inhibitory activity of prickle fruit oil with different refining technologies on elastase in the present invention

[0034] Figure 8 This is a test result diagram of the zebrafish embryo tail fin repair effect according to the present invention;

[0035] Fig. 9 This is a bar graph of the test results of the zebrafish embryo tail fin repair effect according to the present invention, wherein: Fig. 9 The object of A is crude oil, Fig. 9 B targets refined, unwinterized, Fig. 9 C is targeted for refined winterization. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0037] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0038] Example 1

[0039] In the present embodiment, the thorn fruit seeds belong to the thorn fruit of the genus Prinsepia of the Rosaceae family, and the Latin name is Prinsepiautilis Royle. The thorn fruit seeds were taken from Meiguang Village, Yongsheng County, Lijiang City, Yunnan Province (26°68'N, 100°75'E), and the specific extraction process was as follows: picking thorn fruit seeds, removing the outer skin of the thorn fruit seeds, dehydrating to obtain dried kernels, and using the kernels to obtain thorn fruit crude oil by subcritical extraction, and refining the crude oil by a deodorizing vacuum process to obtain refined non-winterized thorn fruit oil, and performing cold tank winterization on the refined non-winterized thorn fruit oil to obtain refined winterized thorn fruit oil, and the samples were named crude oil (MY), refined non-winterized thorn fruit oil (JZWDH) and refined winterized thorn fruit oil (JZDH).

[0040] Example 2

[0041] In this example, one of the batches of crude oil, refined unwinterized prickle oil and refined winterized prickle oil prepared in Example 1 was sent to Beijing Yingnuo Kaisheng Technology Co., Ltd. for UPLC-QTOF-MS detection.

[0042] The specific process is:

[0043] The detection instruments first used Waters ACQUITY UPLC I-Class ultra-high performance liquid chromatography, Xevo G2-XSQTOF mass spectrometer (Waters Technology (Shanghai) Co., Ltd. (Waters)), low-speed centrifuge (Shanghai Lu Xiangyi Centrifuge Co., Ltd.), etc. The detection steps are: MY, JZWDH, JZDH three groups of samples, repeated three times, a total of 9 samples. Take 100μL of the sample oil, add 200μL of chromatographic grade methanol and 200μL of dichloromethane to dissolve, centrifuge to take the supernatant, and transfer to the injection vial. Set the UPLC-QTOF-MS detection conditions as follows: (1) Liquid phase method: flow rate: 0.4mL / min; A: aqueous solution + 0.1% HCOOH, B: acetonitrile as shown in Table 1; chromatographic column: Waters ACQUITY UPLCr BEH C18 1.7μm; column temperature: 35℃; analysis time: 25.00min. (2) Mass spectrometry conditions: When the electrospray ionization source (ESI) was used for positive source ion mode detection, the capillary voltage was 2.5 kV, the desolvation temperature was 450 °C, and the desolvation gas (N 2 ) was 900 L / h; when the electrospray ion source (ESI) was used for negative ion detection, the capillary voltage was 2.0 kV, the desolvation temperature was 450 °C, and the desolvation gas (N 2 ) was 900 L / h, mass scanning range: m / z 100-1200, acquisition mode was MSE, sensitivity mode. When the peak area was less than 10, it was recorded as NA.

[0044] Table 1 Ultra-high performance liquid chromatography elution gradient

[0045] Time (min) Flow rate (mL / min) A％ B％ Curve 0 0.40 95 5 - 0.50 0.40 95 5 6 21.50 0.40 0 100 6 23.00 0.40 0 100 6 25.00 0.40 95 5 1

[0046] UPLC-QTOF-MS detection results please refer to Figures 1 to 3As shown in Table 2, it can be seen that the components of the different refining processes of the prickle oil in this embodiment include: the peak area difference between the crude oil and the refined unwinterized prickle oil components is: sulphur bee fritillary esters, the percentage in the crude oil is 0, and the percentage in the refined unwinterized prickle oil is 8.15%; 17-octadecynoic acid, the percentage in the crude oil is 100%, and the percentage in the refined unwinterized prickle oil is 84.97%; 3-O-β-D-glucose-rapeseed sterol, the percentage in the crude oil is 0, and the percentage in the refined unwinterized prickle oil is 0.86%; cucurbitacin, the percentage in the crude oil is 0, and the percentage in the refined unwinterized prickle oil is 0.52%; stevioside D, the percentage in the crude oil is 0, and the percentage in the refined unwinterized prickle oil is 0.49%; Oleosteryl glucoside, the percentage in crude oil is 0, and the percentage in refined unwinterized Prinus prinus oil is 0.32%; Ipsalone, the percentage in crude oil is 0, and the percentage in refined unwinterized Prinus prinus oil is 0.37%; (E)-4-(1,2,3,6-tetrahydro-2,6-dioxo-1,3-dipropyl-9H-purin-8-yl)cinnamic acid, the percentage in crude oil is 0, and the percentage in refined unwinterized Prinus prinus oil is 0.046%; 3'-N-acetyl-4'-O-(14-methylheptadecanoyl) fusarium chromatinone, the percentage in crude oil is 0, and the percentage in refined unwinterized Prinus prinus oil is 0.51%; linolenic acid, the percentage in crude oil is 0, and the percentage in refined unwinterized Prinus prinus oil is 3.78%.

[0047] Table 2 Peak difference information of crude oil and refined unwinterized Prinus utilis oil components

[0048]

[0049]

[0050] As shown in Table 3, it can be seen that the components of the different refining processes of the prickle oil in this embodiment include the peak area difference of the crude oil and the refined winterized prickle oil components: MG (20: 4 (6E, 8Z, 11Z, 13E) -2OH (5S, 15S) / 0: 0 / 0: 0), the percentage in the crude oil is 0, and the percentage in the refined winterized prickle oil is 0.51%; 17-octadecynoic acid, the percentage in the crude oil is 95.36%, and the percentage in the refined winterized prickle oil is 85.98%; 4β-hydroxystanozolol, the percentage in the crude oil is 0, and the percentage in the refined winterized prickle oil is 0.44%; 13-O-tetradecanoyl 14-rodol 12-acetate, the percentage in the crude oil is 0, and the percentage in the refined winterized prickle oil is The percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 1.52%; stevioside D, the percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 0.35%; thiocarpone, the percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 7.07%; campesterol glucoside, the percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 0.39%; lignoceric acid, the percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 0.35%; resmethrin, the percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 0.80%; acaciasyl acetone, the percentage in crude oil is 0, and the percentage in refined winterized prickle oil is 1.19%; roxatidine acetate, the percentage in crude oil is 4.64%.

[0051] Table 3 Peak difference information of crude oil and refined winterized Prinia utilis oil components

[0052]

[0053]

[0054] Test Case

[0055] 1. Test content

[0056] 1.1 Antioxidant activity

[0057] 1.2 Tyrosinase inhibitory activity

[0058] 1.3 Hyaluronidase Inhibitory Activity

[0059] 1.4 Elastase inhibitory activity

[0060] 1.5 Zebrafish repair activity

[0061] 2. Experimental Materials

[0062] In the present embodiment, the prickle seed is the oil squeezed from the seeds of Prinsepia utilis Royle, a plant of the genus Prinsepia in the family Rosaceae. Prinsepia utilis Royle oil was taken from Meiguang Village, Yongsheng County, Lijiang City, Yunnan Province (26°68'N, 100°75'E), and the content changes of the material components such as tocopherol, squalene, and sterols in the prickle oil crude oil, refined unwinterized and refined winterized prickle oil were detected. The specific process is as follows: the prickle oil crude oil is obtained by subcritical extraction, the crude oil is subjected to hydration degumming, activated carbon decolorization, alkalization deacidification, and vacuum deodorization refining process to obtain refined unwinterized prickle oil, and the refined unwinterized prickle oil is subjected to cold tank winterization to obtain refined winterized prickle oil, and the samples are named crude oil (MY), refined unwinterized (JZWDH), and refined winterized (JZDH).

[0063] 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.

[0064] 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.

[0065] 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.), zebrafish breeding equipment, spawning tanks, constant temperature boxes, brine shrimp incubators, etc.

[0066] 3. Experimental methods

[0067] 3.1 Antioxidant activity determination method

[0068] (1) Determination of DPPH free radical scavenging ability: Weigh 0.8g of prickle oil and dilute it to 10mL with anhydrous ethanol to obtain prickle oil with a mass concentration of 80mg / mL. Dilute it 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 prickle oil was appropriately modified according to the literature. 100μL of prickle oil sample solutions with different gradients and 100μLDPH standard working solution were aspirated into a 96-well plate. After standing in the dark for 30 minutes, the absorbance was measured at a wavelength of 517nm. Ascorbic acid (VC) was used as a positive control and the experiment was repeated 3 times for parallel experiments. The DPPH free radical scavenging rate of prickle oil sample was calculated as follows:

[0069] DPPH free radical scavenging rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%

[0070] Where: A 1 is the absorbance of 100 μL series gradient prickle oil sample solution + 100 μL DDPPH standard working solution at 517 nm; A 2 A is the absorbance of 100 μL series gradient prickle oil sample solution + 100 μL anhydrous ethanol solution at 517 nm; 0 It is the absorbance of 100 μL anhydrous ethanol solution + 100 μL DDPPH standard working solution at 517 nm.

[0071] (2) ABTS free radical scavenging ability: Prepare 5.00, 10.00, 20.00, 40.00, and 80.00 mg / mL of prickle fruit oil sample solutions. The determination of the ABTS free radical scavenging ability of prickle fruit oil was appropriately modified according to the literature. 40 μL of prickle fruit oil 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 the experiment was repeated 3 times for parallel experiments. The calculation formula for the ABTS free radical scavenging rate of prickle fruit oil samples is as follows:

[0072] ABTS free radical scavenging rate (%) = [1-(A 3 -A 4 ) / A 5 ]×100%

[0073] Where: A 3 A is the absorbance of 40 μL series gradient prickle oil sample solution and 160 μL ABTS standard working solution at 734 nm; 4 A is the absorbance of 40 μL series gradient prickle oil sample solution and 160 μL absolute ethanol solution at 734 nm; 5 The absorbance of 40 μL of anhydrous ethanol solution and 160 μL of ABTS standard working solution at 734 nm.

[0074] 3.2 Tyrosinase inhibitory activity test

[0075] Using DMSO:Tween 20 (1:1) as the solution to promote the dispersion of oil, different mass concentrations of 40.00, 20.00, 10.00, and 5.00 mg / mL of Prinia latifolia oil samples were prepared. 70 μL of Prinia latifolia oil sample 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 had enzyme added and the other well had no enzyme added. 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 Prinia latifolia oil sample. After incubation at (37±2.0℃) for 5 minutes, 110 μL of levodopa solution (2mM) was added, and then reacted at (37±2.0℃) for another 30 minutes, and the absorbance of levodopa was read at 490 nm. Two independent experiments were repeated three times each.

[118] The calculation formula of tyrosinase inhibition rate of prickle oil sample is as follows:

[0076]

[0077] Wherein, A is a negative control containing enzyme, B is a negative control without enzyme, C is a sample of prickle oil containing enzyme or kojic acid, and D is a sample of prickle oil without enzyme or kojic acid.

[0078] 3.3 Hyaluronidase inhibition activity test

[0079] (1) Prinus utilis oil sample: using DMSO as solvent, the concentration gradient was 2.0%, 1.0%, and 0.5%;

[0080] 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, which is used to dissolve hyaluronidase and sodium hyaluronate;

[0081] (2) Acetylacetone solution: weigh 10 mL of sodium carbonate solution (1.0 mol / L) and 0.7 mL of acetylacetone in a beaker and mix well (prepare immediately before use); ④ P-DAB color developer: weigh 0.4 g of p-dimethylaminobenzaldehyde and dissolve it in 7.5 mL of concentrated hydrochloric acid and 7.5 mL of anhydrous ethanol and mix well;

[0082] (3) 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 up in the same group and at the same concentration. The amount of each solution added, the order of addition and the reaction process are shown in Table 4:

[0083] Table 4 Hyaluronidase inhibition activity test reaction process

[0084]

[0085]

[0086] The absorbance was measured at 530 nm, and the hyaluronidase inhibition rate of the prickle oil sample was calculated as follows:

[0087]

[0088] Wherein, A is the average absorbance of the prickle oil sample group, 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 prickle oil sample control group.

[0089] 3.4 Elastase inhibition activity test

[0090] The prickle oil samples were prepared with DMSO as solvent in a concentration gradient of 1.0%, 0.5%, and 0.25%; the remaining solutions were prepared with Tris-HCl buffer as solvent.

[0091] The experiment was divided into a sample group, a positive control group, a blank control group, a model control group and a 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 5:

[0092] Table 5 Elastase inhibition activity test reaction flow

[0093]

[0094] The reaction was allowed to stand at room temperature for 15 minutes, and the absorbance was measured at 420 nm using an ELISA reader. The calculation formula for the inhibition rate of elastase of the prickle oil sample was as follows:

[0095]

[0096] In the formula, A 0 is the average absorbance of the blank control group, A 1 is the average absorbance of the control group of prickle oil samples, A 2 is the average absorbance of the prickle oil sample group, A 3 The absorbance value is the average value of the model control group.

[0097] 3.5 Zebrafish repair activity test

[0098] 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 (Princess prickle oil). 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 a side photo of the fish embryo's tail. The formula for calculating the promotion rate of tail fin repair of the Prinia prickle oil sample is as follows:

[0099]

[0100] Wherein, S is the average tail fin length of fish embryos in the prickle oil treatment group, and C is the average tail fin length of fish embryos in the model control group.

[0101] Test Example 1

[0102] Antioxidant activity

[0103] As shown in Table 6 and Figure 4As shown in the figure, with the increase of the concentration of prickle oil, the DPPH radical scavenging rate continues to increase. The DPPH scavenging ability of prickle oil crude oil is lower than that of traditional pressed prickle oil, which may be due to the different preparation methods. Relevant studies have shown that organic reagents used in subcritical extraction methods are more conducive to the dissolution of antioxidant active ingredients than vegetable oils. The DPPH radical scavenging rate of refined unwinterized prickle oil is significantly higher than that of crude oil, which may be due to the dissolution of phenols and other substances after refining or the formation of Maillard products, which increases the DPPH radical scavenging rate. Winterization has little effect on the DPPH radical scavenging ability.

[0104] Refined unwinterized prickle oil and refined winterized prickle oil IC 50 The values ​​were comparable, namely 8.212 and 8.395 mg / mL respectively. When the concentration was 40 mg / mL, the DPPH radical scavenging rate of both was the highest, reaching about 97%, which was comparable to ascorbic acid (VC), indicating that the refined and winterized Prinia prickle oil had good DPPH radical scavenging ability.

[0105] Table 6 DPPH free radical scavenging rate of prickle oil with different refining processes

[0106]

[0107] As shown in Table 7 and Figure 5 As shown in the figure, the trend of ABTS free radical scavenging ability of prickle oil with different refining methods is roughly the same as that of DPPH. With the increase of prickle oil concentration, the ABTS free radical scavenging rate shows an increasing trend. The ABTS free radical scavenging rate of refined unwinterized prickle oil is higher than that of crude oil, and winterization has little effect on DPPH free radical scavenging ability.

[0108] Refined unwinterized prickle oil and refined winterized prickle oil IC 50 The values ​​were similar, 4.497 and 4.380 mg / mL, respectively. The scavenging ability of prickle oil on ABTS radicals was slightly better than that of DPPH radicals. When the concentration of refined prickle oil was in the range of 5-20 mg / mL, the scavenging ability of prickle oil increased continuously, and then decreased slightly. When the concentration was 20 mg / mL, the ABTS radical scavenging rates of refined unwinterized prickle oil and refined winterized prickle oil were 99.62% and 95.78%, respectively, which could almost completely scavenge ABTS radicals, indicating that prickle oil after refinement and winterization also had good ABTS radical scavenging ability.

[0109] Table 7 ABTS free radical scavenging rate of prickle oil with different processing technologies

[0110]

[0111]

[0112] Test Example 2

[0113] Tyrosinase inhibitory activity of prickle fruit oil

[0114] As shown in Table 8, the three samples all have a certain inhibitory effect on tyrosinase, and the activity inhibition rate shows an increasing trend with the increase of concentration, but the difference between the three is not obvious. When the concentration of the prickle oil sample is 40 mg / mL, the tyrosinase inhibition rate of the refined winterized prickle oil is the highest, reaching 33.41±4.91%. This may be because the whitening ingredients in the prickle oil, such as gallic acid and oleanolic acid, are not high in content and have not been significantly increased after processing, so they do not have outstanding results in whitening. This shows that prickle oil can inhibit tyrosinase activity to a certain extent and reduce the production of melanin. After processing, its activity is not significantly affected. It also has a certain whitening effect, but the effect is not significant.

[0115] Table 8 Inhibitory activity of prickle oils with different refining methods on tyrosinase

[0116]

[0117] Test Example 3

[0118] Hyaluronidase Inhibitory Activity of Principis utilis Oil

[0119] From Table 9 and Figure 6 It can be seen that as the concentration increases, the inhibitory effect of crude prickle oil and refined winterized prickle oil on hyaluronidase continues to increase, and the results are similar to 5.3.1.3. Under the same concentration conditions, the inhibition rate of refined non-winterized prickle oil and refined winterized prickle oil on hyaluronidase is significantly higher than that of crude oil (p<0.05). This may be caused by the enrichment of soothing active substances such as thiophanate-methyl esters and 3-O-β-D-glucose-rapeseed sterol in prickle oil after refining and winterization. Except for the concentration of 1%, the inhibition rate of refined winterized prickle oil on hyaluronidase is significantly higher than that of refined non-winterized prickle oil. The effect of winterization on the inhibition rate of hyaluronidase at other concentrations is not obvious. This shows that the processing can effectively improve the inhibitory activity of hyaluronidase in prickle oil, better maintain the level of hyaluronic acid in the skin, and make the skin moisturized, smooth, delicate and tender.

[0120] Table 9 Inhibitory activity of hyaluronidase in prickle oil with different refining technologies

[0121]

[0122] Note: Different letters at the same concentration indicate significant differences (p<0.05).

[0123] Test Example 4

[0124] Test on the Elastase Inhibition Activity of Prinus utilis Oil

[0125] The experimental results are shown in Table 10 and Figure 7 , with the increase of the concentration of prickle fruit oil, the inhibition rate of elastase showed a downward trend. Under the same concentration conditions, the inhibition rates of refined unwinterized prickle fruit oil and refined winterized prickle fruit oil on elastase were higher than those of crude oil, but the difference was not significant (p>0.05). When the concentration was 0.25%, the average inhibition rate of elastase of prickle fruit oil could reach about 65.65%. The results showed that low-dose prickle fruit oil could achieve considerable elastase inhibition effect before and after processing, thereby effectively ensuring the level of elastin in the body and making the skin elastic. It can be used as a natural plant anti-aging additive with considerable application prospects.

[0126] Table 10 Inhibitory activity of prickle oil with different refining technologies on elastase

[0127]

[0128] Note: Different letters at the same concentration indicate significant differences (p<0.05);

[0129] Test Example 5

[0130] Primarina utilis oil zebrafish repair activity

[0131] When the human body is injured, the body will quickly regenerate the skin to repair the skin barrier. Studies have found that the similarity between the zebrafish and human genomes is as high as 87%. About 70% of human genes have at least one homologous gene in zebrafish. The process of zebrafish repairing wounds is also similar to that of humans. Zebrafish wound skin heals very quickly. After being injured, inflammatory cells will migrate to the wound and form granulation tissue composed of macrophages, fibroblasts and collagen. Therefore, zebrafish can be used as a detection and screening model for the repair efficacy of the human body.

[0132] Results Figure 8 , Fig. 9Table 11 shows that the promotion rate of zebrafish embryo tail fin repair by refined unwinterized prickle oil and refined winterized prickle oil at a volume fraction of 1% was significantly higher than that of the model control group (p<0.05), while there was no significant difference between crude oil and the model control group. Among them, the promotion rate of zebrafish embryo tail fin repair by refined winterized prickle oil reached 15% (p<0.05), which was equivalent to the repair effect of Rehmannia glutinosa extract in the positive control group (18% repair promotion rate). The processed prickle oil at sample concentrations of 1%, 3%, and 5% volume fractions all showed good repair effects, but there was a slight inhibition at a concentration of 7%, and the promotion rate of zebrafish embryo tail fin repair showed a decreasing trend with increasing concentration. In summary, in the zebrafish embryo model test, the processed prickle oil is more conducive to promoting the repair of zebrafish damaged tail fin, and the improvement effect is significantly better than that of the model group (p<0.05), indicating that the processing can effectively enhance the anti-damage and repair ability of prickle oil, which may be related to the increase in the content of red ginsenoside R1.

[0133] Table 11 The repair promotion rate of tail fin of zebrafish embryos by different refining processes of prickle fruit oil

[0134]

[0135] Note: * indicates significant difference compared with the model control group (p<0.05).

[0136] Example 3

[0137] The embodiment of the present application also provides a pharmaceutical composition, comprising the prickle fruit extract extracted in the first embodiment, and the pharmaceutical composition has good antioxidant, tyrosinase inhibition, hyaluronidase inhibition, elastase inhibition, zebrafish repair and other effects.

[0138] Illustratively, the pharmaceutical composition described in the embodiments of the present application may be any one of an ointment and a cream.

[0139] Example 4

[0140] The embodiment of the present application also provides a skin care product, comprising the prickle fruit extract extracted in the first embodiment, and the skin care product has good antioxidant, tyrosinase inhibition, hyaluronidase inhibition, elastase inhibition, zebrafish repair and other effects.

[0141] 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.

[0142] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a Principia utilis extract, characterized in that: The preparation method comprises: The seeds of the prickle fruit are picked, the outer peel of the seeds is removed, and the seeds are dehydrated to obtain dried kernels, the kernels are subjected to subcritical extraction to obtain prickle fruit crude oil, the crude oil is refined through a deodorizing vacuum process to obtain refined non-winterized prickle fruit oil, and the refined non-winterized prickle fruit oil is subjected to cold tank winterization treatment to obtain refined winterized prickle fruit oil.

2. A Principal Purpura Extract prepared by the method for preparing the Principal Purpura Extract according to claim 1, characterized in that: The refined unwinterized prickle oil was detected by ultra-high performance liquid chromatography. According to the mass percentage, the refined unwinterized prickle oil included 8.15% of thulhodin, 84.97% of 17-octadecynoic acid, 0.86% of 3-O-β-D-glucose-rapeseed sterol, 0.52% of cucurbitacic acid, 0.49% of stevioside D, 0.32% of campesterol glucoside, 0.37% of ipsilon, 0.046% of (E)-4-(1,2,3,6-tetrahydro-2,6-dioxo-1,3-dipropyl-9H-purin-8-yl)cinnamic acid, 0.51% of 3'-N-acetyl-4'-O-(14-methylheptadecanoyl) fusarium chromatinone, and 3.78% of linolenic acid.

3. A Principal Purpura Extract prepared by the method for preparing the Principal Purpura Extract according to claim 1, characterized in that: The refined winterized prickle fruit oil was detected by ultra high performance liquid chromatography. According to the mass percentage, the refined winterized prickle fruit oil included 85.98% of 17-octadecynoic acid, 0.44% of 4β-hydroxystanazoline, 0.35% of stevioside D, 7.07% of thulhodin, 0.39% of campesterol glucoside, 0.35% of lignoceric acid, 0.80% of resmethrin, 1.19% of acacia acetone, 1.39% of roxatidine acetate, and 2.03% of others.

4. Use of the prickle fruit extract according to any one of claims 2 to 3 in preparing a pharmaceutical composition or skin care agent with an antioxidant effect, characterized in that: The prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

5. Use of the prickle fruit extract according to any one of claims 2 to 3 in preparing a pharmaceutical composition or skin care agent for inhibiting the action of tyrosinase, characterized in that: The prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

6. Use of the prickle fruit extract according to any one of claims 2 to 3 in preparing a pharmaceutical composition or skin care agent for inhibiting the action of hyaluronidase, characterized in that: The prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

7. Use of the Principia utilis extract according to any one of claims 2 to 3 in preparing a pharmaceutical composition or skin care agent for inhibiting the action of elastase, characterized in that: The prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

8. Use of the extract of Psoriasidium prickle fruit according to any one of claims 2 to 3 in preparing a pharmaceutical composition or skin care agent for repairing zebrafish, characterized in that: The prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

9. A pharmaceutical composition, characterized in that include: Physiologically or pharmaceutically acceptable excipients; The prickle fruit extract according to any one of claims 2 to 3, wherein the prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

10. A skin care agent, characterized in that: include: Cosmetic base solvents; The prickle fruit extract according to any one of claims 2 to 3, wherein the prickle fruit extract is refined non-winterized prickle fruit oil or refined winterized prickle fruit oil.

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