Saffron flower extract as well as preparation method and application thereof

Abstract

The invention belongs to the field of cosmetics, and particularly relates to a saffron crocus flower extract as well as a preparation method and application thereof. The preparation method of the saffron crocus flower extract comprises the following steps: (1) crushing and sieving saffron crocus flowers to obtain saffron crocus powder, adding deionized water, adjusting the pH value, adding a nonionic surfactant and a compound enzyme for enzymolysis, inactivating the enzyme, and centrifuging to obtain supernate A and saffron crocus residues; (2) adding a green eutectic solvent into the saffron crocus residues, solubilizing, extracting and centrifuging to obtain supernate B and the saffron crocus residues; and (3) mixing the supernatant A and the supernatant B to obtain a crude extract, separating and purifying the crude extract, eluting, removing ethanol by a vacuum membrane distillation method, and drying to obtain the traditional Chinese medicine composition. According to the method disclosed by the invention, more active ingredients in saffron crocus can be more fully solubilized and extracted, and the content of effective ingredients is obviously increased. When the saffron crocus flower extract is added into cosmetics, the effects of resisting oxidation and delaying senescence can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics, and particularly relates to a saffron flower extract, a preparation method thereof, and an application thereof. Background Art

[0002] Saffron (scientific name: Crocus sativus L.), also known as saffron crocus and saffron, is a perennial flower of the genus Crocus in the iris family. It is a precious medicinal material, a precious food flavor, and a valuable raw material for beauty cosmetics and dyes. Saffron enjoys three world records: first, it is the most expensive medicinal plant in the world; second, it is the best dye in the world; third, it is the most upscale spice in the world. The main component of saffron is picrocrocin (C 16 H 26 O7). The coloring substance is crocin (C 44 H 64 O 26 ·H2O). The chemical components include crocin-1, 2, 3, 4, picrocrocin, crocetin dimethyl ester, α-crocetin, safranal, volatile oil, etc. Saffron mainly has the effects of promoting blood circulation to remove blood stasis, cooling blood and detoxifying, relieving depression and calming the nerves, and beautifying the skin. Therefore, fully extracting the effective components of saffron is a very crucial step to utilize and exert the true use value of saffron.

[0003] Chinese Patent Application CN110251596A discloses a method for extracting active ingredients from saffron, which includes the following steps: 1) Pretreatment before extraction: Place saffron in an oven at 45 °C for drying, crush it with a high-speed crusher, and sieve to obtain saffron powder; 2) Primary extraction: Place the saffron powder obtained in step 1) in a beaker, add an ethanol solution for ultrasonic extraction, filter out the liquid residue to obtain extract 1; 3) Fine extraction: Add an equal amount of ethanol solution as in step 2) to the liquid residue in step 2) for ultrasonic extraction, filter out the liquid residue to obtain extract 2, and mix extract 1 and extract 2; 4) Centrifugation: Centrifuge the mixed extract obtained in step 3) at high speed and freeze to obtain a saffron extract with higher purity; 5) Concentration: Concentrate the extract obtained in step 4) using a rotary evaporator so that the mass of the concentrated extract is 10 times the mass of the initial saffron to obtain a saffron extract with a higher concentration; 6) Add a certain amount of bacteriostatic agent to the extract obtained in step 5). Although the saffron extraction process of this invention is simple and easy to operate, the extraction rate of crocin is relatively low. CN115028668A discloses a crocin extraction process, which mainly includes the following steps: Grind and crush saffron, add an extraction solvent, place it in a microwave extraction tank for extraction, collect the extract after extraction, purify and adsorb the extract using a chromatography column filled with crocin molecularly imprinted polymer microspheres, collect the eluate, concentrate and dry it to finally obtain crocin. The molecularly imprinted polymer microspheres used in this invention have good selective adsorption for crocin and strong specificity, but it requires crocin molecularly imprinted polymer microspheres prepared by a specific method.

[0004] Currently, the existing saffron extraction processes have drawbacks such as the inability to fully extract the active ingredients in saffron and low content of effective ingredients, which limit their applications. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a saffron flower extract, its preparation method and application. The preparation method of the saffron flower extract provided by the present invention is simple, the raw materials used are easily available, and it is easy to implement industrially. The method of the present invention can solubilize and extract more active ingredients in saffron more fully, significantly increasing the content of effective ingredients. Adding the saffron flower extract prepared by the method of the present invention to cosmetics can achieve the effects of antioxidant and anti-aging, and has broad application prospects in the field of cosmetics.

[0006] A method for preparing a saffron flower extract, which includes the following steps:

[0007] (1) Crush and sieve the saffron flowers to obtain saffron powder. Add water to the saffron powder, adjust the pH, add a non-ionic surfactant and a composite enzyme at a certain temperature for enzymatic hydrolysis to break the cell walls of the saffron flowers, inactivate the enzyme activity, and centrifuge to obtain supernatant A and saffron residue;

[0008] (2) Add a green deep eutectic solvent to the saffron residue obtained in step (1), and carry out solubilization extraction with the green deep eutectic solvent at a certain temperature, then centrifuge to obtain supernatant B;

[0009] (3) Mix the supernatant A obtained in step (1) and the supernatant B obtained in step (2) to obtain a crude extract. Separate and purify the crude extract through AB-8 macroporous adsorption resin. First, wash it with deionized water until it is colorless to remove the extractant, and then elute it with an ethanol aqueous solution with a volume fraction of 70-90%. Collect the eluate, remove ethanol by vacuum membrane distillation, and dry it to obtain the saffron flower extract.

[0010] Further, in step (1), the pH is adjusted to 4.5-7.5.

[0011] Further, in step (1), the non-ionic surfactant and the composite enzyme are added at a temperature of 45-65 °C for enzymatic hydrolysis, and the enzymatic hydrolysis time is 1-2 h.

[0012] Further, the non-ionic surfactant in step (1) is one or more of rhamnolipid, trehalose lipid, sucrose ester, Tween 20, and Tween 80.

[0013] Further, the composite enzyme in step (1) includes hemicellulase and bromelain.

[0014] Preferably, the mass ratio of hemicellulase to bromelain in the composite enzyme is 1-3:1.

[0015] Further, the green deep eutectic solvent in step (2) includes an organic acid and a zwitterion of the betaine type.

[0016] Further, the organic acid is one or more of citric acid, lactic acid, malic acid, and oxalic acid.

[0017] Further, the zwitterion of the betaine type is one or more of betaine, alkyl betaine, alkyl amidopropyl betaine, and phosphate ester betaine.

[0018] Further, in step (2), the solubilization extraction with the green deep eutectic solvent is carried out at a temperature of 60-80 °C, and the extraction time is 30-60 min.

[0019] Another object of the present invention is to provide the saffron flower extract prepared by the preparation method of the saffron flower extract described above.

[0020] Another object of the present invention is to provide the application of the saffron flower extract prepared by the preparation method of the saffron flower extract in cosmetics.

[0021] Through a large amount of creative research, the present invention discovers that using a specific non-ionic surfactant for saffron can synergistically promote biological enzymatic hydrolysis to release the active ingredients with cell wall broken in saffron flowers. Then, using an organic acid as a hydrogen bond donor can protect and stabilize saffron pigment substances, and using a zwitterionic surfactant of the betaine type as a hydrogen bond acceptor can fully exert the solubilization and dispersion effects of the surfactant. In addition, adding a certain amount of water to reduce the viscosity can promote dissolution. The organic acid, the zwitterionic surfactant of the betaine type, and water are mixed together to form a green eutectic solvent with properties similar to those of a novel ionic liquid. The green eutectic solvent solubilizes and extracts the remaining saffron residues to extract the active ingredients that are insoluble or poorly soluble in water, and then the effective ingredients are separated and purified to obtain a saffron flower extract with increased effective ingredients and enhanced efficacy. Adding the saffron flower extract prepared by the method of the present invention to cosmetics can achieve the effects of antioxidant and anti-aging, and has broad application prospects in the cosmetics field.

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

[0023] (1) The preparation method of the saffron flower extract provided by the present invention is simple, the raw materials used are easily available, and it is easy to implement industrially.

[0024] (2) The method of the present invention can solubilize and extract more active ingredients in saffron more fully, significantly increasing the content of effective ingredients. It has been experimentally confirmed that in the saffron flower extract prepared by the preparation method of the saffron flower extract provided by the present invention, the content of the effective ingredient crocin is high.

[0025] (3) The saffron flower extract prepared by the method of the present invention can significantly improve the ability to scavenge free radicals and can achieve good antioxidant effects.

[0026] (4) Adding the saffron flower extract prepared by the method of the present invention to cosmetics can achieve the effects of antioxidant and anti-aging, and has broad application prospects in the cosmetics field. Description of the Drawings

[0027] Figure 1 It is a process flow chart of the saffron flower extraction of the present invention.

[0028] Figure 2 It is a picture of preparing the green eutectic solvent in step (2) of Example 1 of the present invention, where Figure 2 A is a picture of citric acid and betaine mixed in proportion; Figure 2Picture of the green eutectic solvent before dilution with water, which is stirred and mixed evenly until clear and transparent at 80 °C.

[0029] Figure 3 Sample pictures of supernatant A obtained after centrifugation in step (1) of Example 1, supernatant B obtained after centrifugation in step (2), saffron residue obtained after centrifugation in step (1), saffron waste residue obtained after centrifugation in step (2), and saffron flower extract liquid obtained without drying after removing ethanol by vacuum membrane distillation in step (3). Among them, Figure 3 A is a picture of supernatant A obtained after centrifugation in step (1) of Example 1 of the present invention; Figure 3 B is a picture of supernatant B obtained after centrifugation in step (2) of Example 1 of the present invention; Figure 3 C is a picture of the saffron residue obtained after centrifugation in step (1) of Example 1 of the present invention; Figure 3 D is a picture of the saffron waste residue obtained after centrifugation in step (2) of Example 1 of the present invention; Figure 3 E is a sample picture of the saffron flower extract liquid obtained without drying after removing ethanol by vacuum membrane distillation in step (3) of Example 1 of the present invention. Detailed implementation manners

[0030] To make the technical solutions of the present invention clearer and more understandable to those skilled in the art, the typical but non-limiting embodiments of the present invention are described as follows. The hemicellulase (model 50,000 U / g) used in the present invention was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and bromelain (specification 1,200,000 U / g) was purchased from Nanning Donghenghuadao Biotechnology Co., Ltd. The purity of the non-ionic surfactant > 95%, the purity of the organic acid > 95%, and the purity of the zwitterion of the betaine type > 95%. There is no special limitation on its source, and it can be purchased on the market or prepared according to the conventional methods well-known to those skilled in the art.

[0031] The production process flow chart of the saffron flower extraction of the present invention is as Figure 1 shown.

[0032] Example 1. A preparation method of saffron flower extract

[0033] The preparation method of the saffron flower extract includes the following steps:

[0034] (1) Crush the dried saffron flowers, pass through a 150-mesh sieve to obtain saffron powder. Add deionized water to the saffron powder, and the addition amount of deionized water is 10 times the mass of the saffron powder. Adjust the pH to 5.5, add a non-ionic surfactant and a composite enzyme for enzymatic hydrolysis at a temperature of 55 °C for 2 h to break the cell wall of the saffron flowers, inactivate the enzyme at 85 °C, and centrifuge at 5000 rpm for 10 min to obtain supernatant A and saffron residue;

[0035] The non-ionic surfactant is rhamnolipid, and the mass ratio of rhamnolipid to deionized water is 0.8:100. The addition amount of the composite enzyme is 0.5% of the mass of saffron powder. The composite enzyme is hemicellulase and bromelain, and the mass ratio of hemicellulase to bromelain in the composite enzyme is 1:1.

[0036] (2) Add a green deep eutectic solvent to the saffron residue obtained in step (1). The addition amount of the green deep eutectic solvent is 10 times the mass of the saffron residue. Perform solubilization extraction with the green deep eutectic solvent at 70 °C for 50 min, and centrifuge at 5000 rpm for 10 min to obtain supernatant B and saffron waste residue.

[0037] The green deep eutectic solvent includes citric acid and betaine. The preparation method of the green deep eutectic solvent is as follows: Take citric acid and betaine with a molar ratio of 2:1, stir and mix evenly at 80 °C until clear and transparent to obtain a mixture, then add water to reduce the viscosity. The addition amount of water is 30% of the total mass of citric acid and betaine, and adjust the pH value to 4.0.

[0038] (3) Mix the supernatant A obtained in step (1) and the supernatant B obtained in step (2) to obtain a crude extract. Separate and purify the crude extract through AB-8 macroporous adsorption resin. First, elute with deionized water until colorless, and then elute with an ethanol aqueous solution with a volume fraction of 85%. Collect the eluate, remove ethanol by vacuum membrane distillation, and dry to obtain saffron flower extract.

[0039] The picture of preparing the green deep eutectic solvent in step (2) of Example 1 of the present invention is as Figure 2 shown, where Figure 2 A is a picture of citric acid and betaine mixed in proportion; Figure 2 B is a picture of the green deep eutectic solvent before dilution with water after being stirred and mixed evenly at 80 °C until clear and transparent.

[0040] The sample pictures of supernatant A obtained after centrifugation in step (1) of Example 1 of the present invention, supernatant B obtained after centrifugation in step (2), saffron residue obtained after centrifugation in step (1), saffron waste residue obtained after centrifugation in step (2), and the liquid sample of saffron flower extract before drying after removing ethanol by vacuum membrane distillation in step (3) are as Figure 3 shown, where the picture of supernatant A obtained after centrifugation in step (1) of Example 1 of the present invention is as Figure 3 shown in A. It can be seen from Figure 3 A that supernatant A obtained after centrifugation in step (1) is slightly turbid because the non-ionic surfactant synergistically promotes the dissolution of various different active ingredients after enzymatic hydrolysis and cell wall breaking. The picture of supernatant B obtained after centrifugation in step (2) of Example 1 of the present invention is as Figure 3 shown in B. It can be seen fromFigure 3 It can be seen that the supernatant B obtained after centrifugation in step (2) is slightly turbid because the green deep eutectic solvent formed with an organic acid as the hydrogen bond donor and a zwitterion of the betaine type as the hydrogen bond acceptor solubilizes and extracts the active ingredients that are insoluble or poorly soluble in water; the picture of the saffron residue obtained after centrifugation in step (1) of Example 1 of the present invention is as Figure 3 shown in Figure 3 C. It can be seen from Figure 3 C that there are still active ingredients of saffron that have not been dissolved; the picture of the saffron waste residue obtained after centrifugation in step (2) of Example 1 of the present invention is as Figure 3 shown in Figure 3 D. It can be seen from

[0041] Example 2. A preparation method of saffron flower extract

[0042] The preparation method of the saffron flower extract includes the following steps:

[0043] (1) Crush the dried saffron flowers, pass through a 200-mesh sieve to obtain saffron powder, add deionized water to the saffron powder, the addition amount of deionized water is 15 times the mass of the saffron powder, adjust the pH to 7.5, add a non-ionic surfactant and a composite enzyme for enzymatic hydrolysis at a temperature of 45 °C, the enzymatic hydrolysis time is 1 h to break the cell wall of the saffron flowers, inactivate the enzyme at 90 °C, and centrifuge at 3000 rpm for 20 min to obtain supernatant A and saffron residue;

[0044] The non-ionic surfactant is rhamnolipid, the mass ratio of rhamnolipid to deionized water is 0.5:100, and the addition amount of the composite enzyme is 0.8% of the mass of the saffron powder; the composite enzyme is hemicellulase and bromelain, and the mass ratio of hemicellulase to bromelain in the composite enzyme is 2:1;

[0045] (2) Add a green deep eutectic solvent to the saffron residue obtained in step (1), the addition amount of the green deep eutectic solvent is 15 times the mass of the saffron residue, carry out green deep eutectic solvent solubilization extraction at a temperature of 80 °C, the extraction time is 30 min, and centrifuge at 5000 rpm for 10 min to obtain supernatant B and saffron waste residue;

[0046] The green deep eutectic solvent includes citric acid and betaine, and the preparation method of the green deep eutectic solvent is: take citric acid and betaine with a molar ratio of 1:1, stir and mix evenly at 80 °C until clear and transparent to obtain a mixture, then add water to reduce the viscosity, the addition amount of water is 40% of the total mass of citric acid and betaine, and adjust the pH value to 6.0;

[0047] (3) Mix the supernatant A obtained in step (1) and the supernatant B obtained in step (2) to obtain a crude extract. Separate and purify the crude extract through AB-8 macroporous adsorption resin. First, wash it with deionized water until it becomes colorless, and then elute it with an ethanol aqueous solution with a volume fraction of 75%. Collect the eluate, remove ethanol by vacuum membrane distillation, and dry it to obtain the saffron flower extract.

[0048] Example 3. A preparation method of saffron flower extract

[0049] The preparation method of the saffron flower extract includes the following steps:

[0050] (1) Crush the dried saffron flowers, pass them through a 150-mesh sieve to obtain saffron powder. Add deionized water to the saffron powder, and the addition amount of deionized water is 5 times the mass of the saffron powder. Adjust the pH to 4.5, add a non-ionic surfactant and a composite enzyme at 65 °C for enzymatic hydrolysis for 2 h to break the cell wall of the saffron flowers. Inactivate the enzyme at 85 °C, and centrifuge at 5000 rpm for 10 min to obtain supernatant A and saffron residue;

[0051] The non-ionic surfactant is Tween 80, and the mass ratio of Tween 80 to deionized water is 1:100. The addition amount of the composite enzyme is 0.5% of the mass of the saffron powder; the composite enzyme is hemicellulase and bromelain, and the mass ratio of hemicellulase to bromelain in the composite enzyme is 1:1;

[0052] (2) Add a green deep eutectic solvent to the saffron residue obtained in step (1). The addition amount of the green deep eutectic solvent is 15 times the mass of the saffron residue. Carry out solubilization extraction with the green deep eutectic solvent at 60 °C for 60 min, and centrifuge at 5000 rpm for 10 min to obtain supernatant B and saffron waste residue;

[0053] The green deep eutectic solvent includes lactic acid and betaine. The preparation method of the green deep eutectic solvent is as follows: Take lactic acid and betaine with a molar ratio of 2:1, stir and mix them evenly at 80 °C until they are clear and transparent to obtain a mixture, and then add water to reduce the viscosity. The addition amount of water is 20% of the total mass of lactic acid and betaine, and adjust the pH value to 4.0;

[0054] (3) Mix the supernatant A obtained in step (1) and the supernatant B obtained in step (2) to obtain a crude extract. Separate and purify the crude extract through AB-8 macroporous adsorption resin. First, wash it with deionized water until it becomes colorless, and then elute it with an ethanol aqueous solution with a volume fraction of 85%. Collect the eluate, remove ethanol by vacuum membrane distillation, and dry it to obtain the saffron flower extract.

[0055] Comparative Example 1. A preparation method of saffron flower extract

[0056] The preparation method of the saffron flower extract is similar to that of Example 1;

[0057] The difference from Example 1 is only that no non-ionic surfactant is added in step (1).

[0058] Comparative Example 2. A preparation method of saffron flower extract

[0059] The preparation method of the saffron flower extract is similar to that of Example 1;

[0060] The difference from Example 1 is only that no composite enzyme (hemicellulase and bromelain) is added in step (1).

[0061] Comparative Example 3. A preparation method of saffron flower extract

[0062] The preparation method of the saffron flower extract is similar to that of Example 1;

[0063] The difference from Example 1 is only that the green deep eutectic solvent in step (2) is replaced by water.

[0064] Comparative Example 4. A preparation method of saffron flower extract

[0065] The preparation method of the saffron flower extract is similar to that of Example 1;

[0066] The difference from Example 1 is only that the green deep eutectic solvent in step (2) is replaced by 75% aqueous ethanol solution.

[0067] Comparative Example 5. A preparation method of saffron flower extract

[0068] The preparation method of the saffron flower extract is similar to that of Example 1;

[0069] The difference from Example 1 is only that step (1) is deleted (saffron is not enzymolyzed with the coordination and promotion of non-ionic surfactant), and the saffron powder obtained after crushing and sieving the saffron flowers is directly added to the green deep eutectic solvent for solubilization extraction.

[0070] Comparative Example 6. A preparation method of saffron flower extract

[0071] The preparation method of the saffron flower extract is similar to that of Example 1;

[0072] The difference from Example 1 is only that step (2) is deleted (the saffron residue is not solubilized and extracted with the green deep eutectic solvent to recover the active ingredients), and after the saffron powder in step (1) is enzymolyzed with the coordination and promotion of non-ionic surfactant, it directly goes to step (3) for separation and purification.

[0073] Application Example 1

[0074] This application example provides a skin cream formula containing saffron flower extract, which is made of components with the mass percentages shown in Table 1. That is, the numbers in the table represent mass percentages. For example, 5.00 represents 5.00%.

[0075] Table 1 Skin Cream Formula Containing Saffron Flower Extract

[0076]

[0077]

[0078] Preparation method of the skin cream in this application example: Dissolve and mix the components of Group A evenly at 80°C, dissolve and mix the components of Group B evenly at 80°C. When emulsifying, add Group B to Group A and homogenize for 5 minutes. When the temperature is lowered to 50°C, add Group C and homogenize for 5 minutes. After aging for 24 hours, package it to obtain the product.

[0079] Application Example 2

[0080] Same as Application Example 1, the difference is that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Example 2, and other components and preparation methods remain unchanged.

[0081] Application Example 3

[0082] Same as Application Example 1, the difference is that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Example 3, and other components and preparation methods remain unchanged.

[0083] Application Comparative Example 1

[0084] Same as Application Example 1, the difference is that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Comparative Example 1, and other components and preparation methods remain unchanged.

[0085] Application Comparative Example 2

[0086] Same as Application Example 1, the difference is that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Comparative Example 2, and other components and preparation methods remain unchanged.

[0087] Application Comparative Example 3

[0088] Same as Application Example 1, the difference is that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Comparative Example 3, and other components and preparation methods remain unchanged.

[0089] Application Comparative Example 4

[0090] Same as Application Example 1, except that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Comparative Example 4, and other components and preparation methods remain unchanged.

[0091] Application of Comparative Example 5

[0092] Same as Application Example 1, except that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Comparative Example 5, and other components and preparation methods remain unchanged.

[0093] Application of Comparative Example 6

[0094] Same as Application Example 1, except that the saffron flower extract prepared in Example 1 in Phase B is replaced with the saffron flower extract prepared in Comparative Example 6, and other components and preparation methods remain unchanged.

[0095] Test Example 1: Determination of the content of crocin

[0096] Take the saffron flower extracts obtained in Examples 1 - 3 and Comparative Examples 1 - 6, and determine the content of crocin according to the method in the Chinese Pharmacopoeia. The test results are shown in Table 2.

[0097] Table 2 Test results of crocin content

[0098]

[0099]

[0100] According to the test results of crocin content in Table 2, it can be seen that the content of crocin in the saffron flower extracts prepared by the methods of Examples 1 - 3 of the present invention is significantly higher than that in the saffron flower extracts prepared by the methods of Comparative Examples 1 - 6. This shows that the non - ionic surfactant coordinately promotes the enzymatic hydrolysis effect, and through the green deep eutectic solvent, the remaining saffron residues are solubilized and extracted, so that more active ingredients of saffron are fully solubilized and extracted, increasing the content of active ingredients.

[0101] Test Example 2: Evaluation of antioxidant effect by free radical scavenging ability test

[0102] Dissolve the saffron flower extract samples prepared in Examples 1 - 3 and Comparative Examples 1 - 6 in water to prepare a sample solution with a mass concentration of 1%, and conduct the following tests:

[0103] (1) DPPH free radical scavenging ability evaluation test

[0104] Take an equal volume (3 mL) of the sample solution + 2×10 -4In a stoppered test tube, add DPPH solution at -4 mol / L, mix well, react for 30 min in the dark, and measure the absorbance value A1 at a wavelength of 517 nm; take an equal volume (3 mL) of the sample solution + 95% absolute ethanol in a stoppered test tube, mix well, react for 30 min in the dark, and measure the absorbance value A2 at a wavelength of 517 nm; take an equal volume (3 mL) of 2×10

[0105] mol / L DPPH solution + 95% absolute ethanol in a stoppered test tube, mix well, react for 30 min in the dark, and measure the absorbance value A0 at a wavelength of 517 nm;

[0106] The calculation formula for the DPPH radical scavenging rate is: DPPH radical scavenging rate (%) = [1 - (A1 + A2) / A0] × 100%

[0107] In a 25 mL colorimetric cell, add 3 mL of 2 mmol / L FeSO4, 3 mL of 1 mmol / L H2O2 in sequence, shake well, then add 3 mL of 6 mmol / L salicylic acid, shake well, take it out after heating in a water bath at 37 °C for 15 min, and measure its absorbance; add a sample solution with a certain concentration respectively, shake well, continue to heat in a water bath for 15 min, and take it out to measure its absorbance.

[0108] The calculation formula for the hydroxyl radical scavenging rate is as follows:

[0109] Hydroxyl radical scavenging rate (%) = [A0 - A x - (A0 - A x0 )] / A0 × 100%

[0110] Among them, A0, A x , A x0 are the absorbance values of the reaction system before adding the sample, the absorbance value of the system after the sample scavenges hydroxyl radicals, and the absorbance value of the system after the sample solvent scavenges hydroxyl radicals respectively.

[0111] The test results of the DPPH radical scavenging rate and hydroxyl radical scavenging rate of the samples prepared in Examples 1 - 3 and Comparative Examples 1 - 6 are shown in Table 3.

[0112] Table 3 Test results of DPPH radical scavenging rate and hydroxyl radical scavenging rate

[0113] Test sample DPPH radical scavenging rate (%) Hydroxyl radical scavenging rate (%) Example 1 89.16 77.19 Example 2 86.90 75.51 Example 3 88.01 76.35 Comparative example 1 75.59 60.21 Comparative example 2 63.91 50.98 Comparative example 3 68.95 51.65 Comparative example 4 70.65 55.24 Comparative example 5 55.97 40.57 Comparative example 6 60.54 48.68

[0114] According to the test results in Table 3, the free radical scavenging rates of the saffron flower extract samples prepared by the methods of Examples 1-3 of the present invention are significantly better than those of the saffron flower extract samples prepared by the methods of Comparative Examples 1-6. This shows that the non-ionic surfactant coordinately promotes the enzymatic hydrolysis effect, and the remaining saffron residues are solubilized and extracted through the green eutectic solvent, so that more active ingredients of saffron can be fully solubilized and extracted, which can improve the ability to scavenge free radicals, enhance the efficacy, and achieve a good antioxidant effect.

[0115] Test Example 3: Skin elasticity test

[0116] Evaluation method: Use the skin elasticity tester MPA580 of CK Company in Germany. Select the skin care creams prepared in Application Examples 1-3 and Application Comparative Examples 1-6. By measuring the skin surface before and after using the product, a negative pressure is generated to suck the skin into a specific test probe. The depth of the skin sucked into the test probe is measured by a non-contact optical test system. The test probe includes a light emitter and a receiver, and the ratio of light is proportional to the depth of the skin sucked in. Then, the elastic properties of the skin are determined through the analysis of MPA software. The ratio of the skin rebound amount without negative pressure to the maximum stretching amount with negative pressure is closer to 1, indicating better skin elasticity.

[0117] Select 54 people with dry skin (accumulated stinging sensation ≥ 3 points), aged 20-45 years old, with half men and half women. Randomly divide them into 9 groups, with 3 men and 3 women in each group. The test area cannot use any products (cosmetics, topical drugs, oral health products) for the first 15 days. Before the test, the subjects are required to wash their faces and enter the climate control room (22 ± 1°C, relative humidity 50%) 2 hours after washing and sit still for more than 20 minutes, and keep a relaxed state.

[0118] Test method: In the test, half-face tests are carried out according to the random table. 9 groups of volunteers use the skin care creams prepared in Application Examples 1-3 and Application Comparative Examples 1-6 respectively. The subjects use it once in the morning and evening every day for 28 days. During this period, follow-up visits are carried out on the 0th day (D0), 14th day (D14), and 28th day (D28). After sitting still for 30 minutes in a constant temperature and humidity environment, the elasticity test is carried out. Then, the average elasticity data of the subjects at different time intervals are calculated, and the results are shown in Table 4.

[0119] Table 4 Test results of skin elasticity test

[0120]

[0121] According to the test results in Table 4, after using the above samples, the skin elasticity of each experimental group was improved, but the skin care creams prepared using Examples 1-3 had a more significant effect on improving skin elasticity than the skin care creams prepared using Comparative Examples 1-6, which indicates that the saffron flower extract prepared using the method of the present invention has a significant effect of enhancing skin elasticity.

[0122] Test Example 4: Anti-aging efficacy test

[0123] 27 healthy volunteers were selected and randomly divided into groups of 3. 9 groups of volunteers used the skin cream prepared by application examples 1-3 and application comparative examples 1-6 respectively. The subjects used it once a day in the morning and evening for 4 consecutive weeks. Through questionnaires, the evaluation of the skin condition of the volunteers after using the samples was collected to analyze the use effect of the products. The main dimensions of collecting feedback include: skin elasticity, skin firmness, reduction of fine lines, and light lines to evaluate the anti-aging effect. The specific evaluation is reflected in the form of scoring, 1-10 points, and the higher the score, the more popular the sample is with the volunteers and the better the effect. The average score of each group of people is shown in Table 5.

[0124] Table 5 Anti-aging efficacy analysis results

[0125]

[0126] According to the results in Table 5, after self-evaluation by the subjects, it was found that the anti-aging effect of the skin cream prepared by using Examples 1-3 was better than the anti-aging effect of the skin cream prepared by using Comparative Examples 1-6. At the same time, this also shows that the anti-aging effect of the saffron flower extract prepared by the method of Examples 1-3 of the present invention is better than the saffron flower extract prepared by the method of Comparative Examples 1-6. This also further illustrates that the non-ionic surfactant coordinates and promotes enzymolysis, and more active ingredients of the remaining saffron residue are fully solubilized and extracted through the green deep eutectic solvent, thereby increasing the anti-aging effect.

[0127] In summary, combined with the crocin content of saffron flower extract, the antioxidant efficacy test results and the efficacy test results of the product made by adding saffron flower extract to the formula, it is explained that the use of the specific non-ionic surfactant of the present invention has the effect of synergistically promoting enzymatic hydrolysis. First, the non-ionic surfactant is used to synergistically promote biological enzymatic hydrolysis, which can release the active ingredients of the saffron flower cell wall breaking, and then the green low eutectic solvent is used to solubilize and extract the remaining saffron residue, so that more active ingredients of saffron can be fully solubilized and extracted, and then separation and purification are performed to obtain saffron flower extract with increased effective ingredients and enhanced efficacy.

[0128] The applicant declares that the present invention uses the above embodiments to illustrate the saffron flower extract, its preparation method and application, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for preparing a saffron flower extract, characterized in that, It includes the following steps: (1) Crush and sieve the saffron flowers to obtain saffron powder. Add water to the saffron powder, adjust the pH, add a non-ionic surfactant and a composite enzyme for enzymatic hydrolysis at a certain temperature to break the cell walls of the saffron flowers, inactivate the enzyme activity, and centrifuge to obtain supernatant A and saffron residues; (2) Add a green deep eutectic solvent to the saffron residues obtained in step (1), and perform solubilization extraction with the green deep eutectic solvent at a certain temperature, then centrifuge to obtain supernatant B; (3) Mix the supernatant A obtained in step (1) and the supernatant B obtained in step (2) to obtain a crude extract. Separate and purify the crude extract through AB-8 macroporous adsorption resin. First, elute with deionized water until colorless, then elute with an ethanol aqueous solution with a volume fraction of 70-90%. Collect the eluate, remove ethanol by vacuum membrane distillation, and dry to obtain the saffron flower extract.

2. The preparation method of the saffron flower extract according to claim 1, characterized in that, In step (1), the non-ionic surfactant and the composite enzyme are added for enzymatic hydrolysis at a temperature of 45-65 °C, and the enzymatic hydrolysis time is 1-2 h.

3. The preparation method of the saffron flower extract according to claim 1, characterized in that, The non-ionic surfactant in step (1) is one or more of rhamnolipid, trehalolipid, sucrose ester, Tween 20, and Tween 80.

4. The preparation method of the saffron flower extract according to claim 1, wherein, The composite enzyme in step (1) includes hemicellulase and bromelain.

5. The preparation method of the saffron flower extract according to claim 1, wherein, The green deep eutectic solvent in step (2) includes an organic acid and a zwitterion of the betaine type.

6. The preparation method of the saffron flower extract according to claim 5, wherein, The organic acid is one or more of citric acid, lactic acid, malic acid, and oxalic acid.

7. The preparation method of the saffron flower extract according to claim 5, characterized in that, The zwitterion of the betaine type is one or more of betaine, alkyl betaine, alkyl amidopropyl betaine, and phosphate ester betaine.

8. The preparation method of the saffron flower extract according to claim 1, characterized in that, In step (2), the solubilization extraction with the green deep eutectic solvent is carried out at a temperature of 60-80 °C, and the extraction time is 30-60 min.

9. The saffron flower extract prepared by the method for preparing a saffron flower extract according to any one of claims 1-8.

10. The application of the saffron flower extract prepared by the method for preparing a saffron flower extract according to any one of claims 1-8 in cosmetics.

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