Preparation method, product and application of erigeron breviscapus extract

Abstract

The present invention relates to a preparation method of an Erigeron breviscapus extract, its product and application. The preparation method of the Erigeron breviscapus extract comprises the following steps: mixing an Erigeron breviscapus raw material with an extraction solvent and then carrying out water bath heating extraction, centrifuging and filtering the extraction solution to obtain a filtrate, thus obtaining the extract; the extraction solvent is selected from a deep eutectic solvent or a mixed solvent of a deep eutectic solvent and water. This preparation method creatively uses a deep eutectic solvent or a mixed solvent of a deep eutectic solvent and water to extract the Erigeron breviscapus raw material. On the one hand, it can make the contents of scutellarin and total flavonoids in the obtained extract high, providing an effective strategy for the utilization of the Erigeron breviscapus raw material and the preparation of scutellarin; on the other hand, the obtained extract also has excellent inhibitory activities against hyaluronidase, elastase and collagenase, excellent promoting expression activities of aquaporin, filaggrin and involucrin, excellent anti-free radical activity and excellent anti-aging activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and relates to a preparation method, product and application of an erigeron breviscapus extract, in particular to a preparation method, product and application of an erigeron breviscapus extract with a high scutellarin content. Background Art

[0002] Erigeron breviscapus comes from the dried whole herb of Erigeron breviscapus (Vant.) Hand.-Mazz. of the genus Erigeron in the Compositae family. "Compendium of Materia Medica in Yunnan" records that erigeron breviscapus is cold in nature and bitter in taste, and is used to treat purulent ear in children by instilling the juice into the ear, left hemiplegia and right hemiplegia, and rheumatic pain by decocting and taking with water and rice wine. With a long history of use as a folk medicine, erigeron breviscapus drugs have significant clinical application effects.

[0003] At present, there are still few strategies for extracting scutellarin from erigeron breviscapus. In the existing extraction methods, the extraction solvents used are mostly organic solvents such as ethanol. However, the extensive use of volatile and toxic organic solvents will cause great pollution to the environment. Moreover, the main component scutellarin in erigeron breviscapus is slightly soluble in ethanol, which will result in a large part of scutellarin being unable to fuse with ethanol. And the dissolution of ethanol itself is extensive, so that other impurities will also dissolve into the ethanol solution, which will significantly reduce the scutellarin content and purity in the ethanol solution. In recent years, more and more people pursue green chemistry and the awareness of environmental friendliness is constantly increasing. Therefore, it is very meaningful to develop a method for efficiently, simply and environmentally friendly extracting scutellarin from erigeron breviscapus.

[0004] In addition, there are few reports on applying the active ingredients of erigeron breviscapus to skin anti-aging, repair or moisturization. Aquaporin AQP3 is the most important aquaporin on human skin and has the function of transmembrane transporting small molecules such as water, glycerol and urea. AQP3 is closely related to skin moisturization, barrier repair and anti-aging; Filaggrin FLG is an important molecule connecting keratin fibers in the stratum corneum of human skin. With the assistance of FLG monomers, keratin fibers regularly aggregate to form a solid physical barrier on the outermost layer of the epidermis, which can prevent the loss of epidermal water and the invasion of external allergenic substances; Loricrin LOR is a very important component involved in skin barrier formation and is the main component of the cornified envelope. Its functions mainly include strengthening the cornified envelope, enhancing the permeability barrier, interacting with intermediate filaments, and increasing the flexibility of the cornified envelope structure. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method, product and application of an erigeron breviscapus extract, in particular to a preparation method, product and application of an erigeron breviscapus extract with a high scutellarin content.

[0006] To achieve the object of the present invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing an erigeron breviscapus extract, and the method for preparing the erigeron breviscapus extract comprises the following steps:

[0008] Mix the erigeron breviscapus raw material with an extraction solvent, and then carry out water bath heating extraction. After centrifuging and filtering the extraction solution, a filtrate is obtained, thus obtaining the erigeron breviscapus extract; the extraction solvent is selected from a deep eutectic solvent or a mixed solvent of a deep eutectic solvent and water.

[0009] The method for preparing the erigeron breviscapus extract involved in the present invention creatively uses a deep eutectic solvent or a mixed solvent of a deep eutectic solvent and water to extract the erigeron breviscapus raw material. On the one hand, it can make the contents of scutellarin and total flavonoids in the obtained extract high, providing an effective strategy for the utilization of erigeron breviscapus raw materials and the preparation of scutellarin; on the other hand, the obtained extract also has excellent hyaluronidase, elastase and collagenase inhibitory activities, excellent aquaporin, filaggrin and involucrin promoting expression activities, excellent anti-free radical activity and excellent anti-aging activity. Therefore, the erigeron breviscapus extract prepared by this preparation method can be applied to products with anti-aging, antioxidant, moisturizing and skin barrier repair effects. And this preparation method is green, efficient, simple and easy to operate, and is suitable for large-scale industrial production.

[0010] Preferably, the deep eutectic solvent is obtained by reacting a hydrogen bond acceptor with a hydrogen bond donor, the hydrogen bond acceptor is selected from propylene glycol, glycerol, urea, glycolic acid, citric acid, polyethylene glycol or L-ascorbic acid, and the hydrogen bond donor is selected from choline chloride, acetamide or betaine.

[0011] Preferably, the reaction is carried out at 40-80 °C (such as 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, etc.) until the system becomes a clear and transparent liquid.

[0012] The present invention has found through research that the contents of scutellarin in the products obtained by extracting with deep eutectic solvents obtained by any combination mode of the above hydrogen bond acceptors and hydrogen bond donors are different. More preferably, the following three specific types of deep eutectic solvents are used, so that the contents of scutellarin and total flavonoids in the extract are significantly increased, and are superior to the water extraction method or the alcohol extraction method. At the same time, when the hydrogen bond acceptor is selected as glycerol and the hydrogen bond donor is selected as betaine, the extract has better effects in terms of hyaluronidase, elastase and collagenase inhibitory activities, aquaporin, filaggrin and involucrin promoting expression activities, and anti-free radical activity.

[0013] Preferably, the hydrogen bond acceptor is selected from glycerol, and the hydrogen bond donor is selected from betaine.

[0014] Preferably, the molar ratio of glycerol to betaine is (5 - 7):1, such as 5:1, 5.5:1, 6:1, 6.5:1, 7:1, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0015] When the molar ratio of glycerol to betaine is (5 - 7):1, the yield of scutellarin in the extract is higher. Further decreasing or increasing the relative amount of glycerol will cause the extraction solvent system to contain excessive betaine or glycerol, which will negatively affect the extraction efficiency of scutellarin.

[0016] Preferably, the hydrogen bond acceptor is selected from glycerol, and the hydrogen bond donor is selected from acetamide.

[0017] Preferably, the molar ratio of glycerol to acetamide is (5 - 7):1, such as 5:1, 5.5:1, 6:1, 6.5:1, 7:1, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0018] When the molar ratio of glycerol to acetamide is (5 - 7):1, the yield of scutellarin in the extract is higher. Further decreasing or increasing the relative amount of glycerol will cause the extraction solvent system to contain excessive acetamide or glycerol, which will negatively affect the extraction efficiency of scutellarin.

[0019] Preferably, the hydrogen bond acceptor is selected from glycolic acid, and the hydrogen bond donor is selected from acetamide.

[0020] Preferably, the molar ratio of glycolic acid to acetamide is (3 - 5):1, such as 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0021] When the molar ratio of glycolic acid to acetamide is (3 - 5):1, the yield of scutellarin in the extract is higher. Further decreasing or increasing the relative amount of glycolic acid will cause the extraction solvent system to contain excessive acetamide or glycolic acid, which will negatively affect the extraction efficiency of scutellarin.

[0022] Preferably, the hydrogen bond acceptor is selected from glycerol, and the hydrogen bond donor is selected from choline chloride.

[0023] Preferably, the molar ratio of glycerol to acetamide is (5 - 7):1, such as 5:1, 5.5:1, 6:1, 6.5:1, 7:1, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0024] When the molar ratio of glycerol to choline chloride is (5-7):1, the yield of scutellarin in the extract is higher. Further decreasing or increasing the relative amount of glycerol will result in an excessive amount of choline chloride or glycerol in the extraction solvent system, which will negatively affect the extraction efficiency of scutellarin.

[0025] Preferably, the mass ratio of the deep eutectic solvent to water in the mixed solvent is 10:1-1:1, such as 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 5:2, 2:1, 4:3, 1:1, etc., and more preferably 5:2-4:3.

[0026] The present invention also finds that adding water to the deep eutectic solvent can reduce the viscosity of the deep eutectic extraction solvent, which is beneficial to the extraction process. Moreover, with the increase of the water content, the extraction efficiency increases, and the dissolution efficiency of scutellarin increases. However, when the amount of water exceeds a certain level, it is not conducive to the extraction process, and the extraction efficiency decreases. When the mass ratio of the deep eutectic solvent to water is 5:2-4:3, the effect is better.

[0027] Preferably, the mixing ratio of the Erigeron breviscapus raw material to the extraction solvent is 1:(5-20) g / mL, such as 1:5 g / mL, 1:8 g / mL, 1:10 g / mL, 1:12 g / mL, 1:15 g / mL, 1:18 g / mL, 1:20 g / mL, etc., and more preferably 1:(8-12) g / mL.

[0028] Preferably, the temperature of the water bath heating extraction is 45-85 °C, such as 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, etc., and more preferably 60-70 °C; the time is 10-55 min, such as 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, etc., and more preferably 30-40 min.

[0029] When the extraction temperature is 45-85 °C and the time is 10-55 min, the extraction efficiency of scutellarin is better. Further decreasing or increasing the material-liquid ratio is not conducive to the improvement of the extraction efficiency. Among them, 60-70 °C and 30-40 min are better choices.

[0030] Preferably, the rotation speed of the centrifugation is 2000-5000 r / min, such as 2000 r / min, 3000 r / min, 4000 r / min, 5000 r / min, etc.; the time is 5-20 min, such as 5 min, 8 min, 10 min, 15 min, 20 min, etc.

[0031] Preferably, the filtration adopts a membrane filtration process, and the pore size of the filtration membrane is 0.45 μm.

[0032] Preferably, the Erigeron breviscapus raw material is the dried powder of Erigeron breviscapus after drying and pulverization.

[0033] Preferably, the drying is carried out at 50 - 60 °C (such as 50 °C, 53 °C, 55 °C, 58 °C, 60 °C, etc.), and after pulverization, it is sieved through a 60 - 120 mesh sieve (such as 60 mesh, 70 mesh, 80 mesh, 90 mesh, 100 mesh, 110 mesh, 120 mesh, etc.).

[0034] Preferably, before drying, an enzyme inactivation treatment is carried out at 130 - 155 °C (such as 130 °C, 135 °C, 140 °C, 145 °C, 150 °C, 155 °C, etc.).

[0035] In a second aspect, the present invention provides an Erigeron breviscapus extract prepared by the preparation method of the Erigeron breviscapus extract according to the first aspect.

[0036] In a third aspect, the present invention provides the use of the Erigeron breviscapus extract according to the second aspect in the preparation of a hyaluronidase inhibitor, an elastase inhibitor, or a collagenase inhibitor.

[0037] According to the research results of the present invention, it is known that the Erigeron breviscapus extract prepared by the above - mentioned specific method has the effect of inhibiting hyaluronidase, elastase, or collagenase. Therefore, the Erigeron breviscapus extract prepared by the above - mentioned specific method can be used to prepare a hyaluronidase inhibitor, an elastase inhibitor, or a collagenase inhibitor, and can act as a simple experimental reagent.

[0038] In a fourth aspect, the present invention provides the use of the Erigeron breviscapus extract according to the second aspect in the preparation of an aquaporin expression promoter, a filaggrin expression promoter, or a loricrin expression promoter.

[0039] According to the research results of the present invention, it is known that the Erigeron breviscapus extract prepared by the above - mentioned specific method has the effect of promoting the expression of aquaporin, filaggrin, or loricrin. Therefore, the Erigeron breviscapus extract prepared by the above - mentioned specific method can be used to prepare an aquaporin expression promoter, a filaggrin expression promoter, or a loricrin expression promoter, and can act as a simple experimental reagent.

[0040] In a fifth aspect, the present invention provides the use of the Erigeron breviscapus extract according to the second aspect in the preparation of a product having anti - aging, antioxidant, moisturizing, and skin barrier repair effects.

[0041] Preferably, the mass percentage content of the Erigeron breviscapus extract in the product can be 0.01%, 0.05%, 0.08%, 1%, 1.2%, 1.5%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12%, 13%, 15%, 16%, 17%, 18%, 20%, etc.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The preparation method of the Erigeron breviscapus extract involved in the present invention creatively uses a eutectic solvent or a mixed solvent of a eutectic solvent and water to extract the Erigeron breviscapus raw material. On the one hand, it can make the content of scutellarin and total flavonoids in the obtained extract high, providing an effective strategy for the utilization of Erigeron breviscapus raw materials and the preparation of scutellarin. On the other hand, the obtained extract also has excellent hyaluronidase, elastase and collagenase inhibitory activities, excellent aquaporin, filaggrin and involucrin promoting expression activities, excellent anti-free radical activity and excellent anti-aging activity. Therefore, the Erigeron breviscapus extract prepared by this preparation method can be applied to products with anti-aging, antioxidant, moisturizing and skin barrier repair effects. And this preparation method is green, efficient, simple and easy to operate, and is suitable for large-scale industrial production. Description of the Drawings

[0044] Figure 1 is the standard curve diagram drawn with the standard scutellarin solution;

[0045] Figure 2 is the result diagram of the product prepared in Example 8 measured by an ultra-high performance liquid chromatograph;

[0046] Figure 3 is the result diagram of the product prepared in Comparative Example 3 measured by an ultra-high performance liquid chromatograph;

[0047] Figure 4 is the result diagram of the DPPH free radical scavenging rate determination of each group of Erigeron breviscapus extracts;

[0048] Figure 5 is the result diagram of the ABTS free radical scavenging rate determination of each group of Erigeron breviscapus extracts;

[0049] Figure 6 is the result diagram of the hyaluronidase, elastase and collagenase inhibitory activity determination of each group of Erigeron breviscapus extracts;

[0050] Figure 7 is the result diagram of the determination of the promotion of aquaporin AQP3 expression by each group of Erigeron breviscapus extracts;

[0051] Figure 8 is the result diagram of the determination of the promotion of filaggrin FLG expression by each group of Erigeron breviscapus extracts;

[0052] Figure 9 It is a graph showing the results of measuring the promotion of loricrin (LOR) expression by extracts of each group of Erigeron breviscapus.

[0053] Figure 10 It is a SA-β-GAL staining map after treating HFF cells with samples of each group.

[0054] Figure 11 It is a statistical result graph of the percentage of SA-β-GAL positive cells after treating HFF cells with samples of each group. Detailed implementation manners

[0055] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0056] The Erigeron breviscapus involved in the following examples is the whole plant of Erigeron breviscapus purchased from Honghe Hani and Yi Autonomous Prefecture, Yunnan Province.

[0057] The detection of the content of scutellarin involved below is carried out by an ultra-high performance liquid chromatograph (UPLC), specifically as follows:

[0058] Chromatographic column: Agilent ZORBA×SB-AQ column (3.0×100, 3.5 μm), flow rate: 0.4 mL / min, column temperature: 30 °C, detection wavelength: 327 nm, injection volume: 2 μL, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B), gradient elution conditions: 0 - 5 min, 10% (B), 5 - 20 min, 10% - 40% (B), 20 - 25 min, 40% - 90% (B), 25 - 30 min, 90% (B), 30 - 30.1 min, 90% - 10% (B), 30.1 - 35 min, 10% (B).

[0059] Preparation of standard solution: Weigh an appropriate amount of scutellarin standard product precisely, dissolve it with DMSO-ethanol, make up the volume and shake well to obtain a reference stock solution, refrigerate it at 4 °C, and use the half-dilution method to dilute it to obtain 6 standard scutellarin solutions with different concentrations when in use. Its standard curve is as Figure 1 shown.

[0060] Preparation Examples 1 - 13

[0061] In this preparation example, 13 eutectic solvents are prepared, and their fluidity and appearance are observed.

[0062] Mix different hydrogen bond donors and hydrogen bond acceptors in different molar ratios according to Table 1, react at 50 °C until the system becomes a clear and transparent liquid, and there will be no solid precipitation after standing overnight at 20 °C. It is successfully prepared, and at the same time, their fluidity and appearance state are observed.

[0063] Table 1

[0064] Sample Hydrogen bond donor Hydrogen bond acceptor Molar ratio Fluidity and appearance Preparation Example 1 Choline chloride Glycerol 1:6 Transparent and good fluidity Preparation Example 2 Choline chloride L-Ascorbic acid 1:2 Transparent and good fluidity Preparation Example 3 Choline chloride Citric acid 1:1 Transparent and good fluidity Preparation Example 4 Choline chloride Glycolic acid 1:4 Transparent and good fluidity Preparation Example 5 Betaine Citric acid 1:1 Transparent and good fluidity Preparation Example 6 Betaine Glycolic acid 1:4 Transparent and good fluidity Preparation Example 7 Betaine Urea 1:2 Transparent and good fluidity Preparation Example 8 Betaine Glycerol 1:6 Transparent and good fluidity Preparation Example 9 Betaine L-Ascorbic acid 1:2 Transparent and good fluidity Preparation Example 10 Acetamide Propylene glycol 1:1 Transparent and good fluidity Preparation Example 11 Acetamide Glycerol 1:6 Transparent and good fluidity Preparation Example 12 Acetamide Glycolic acid 1:4 Transparent and good fluidity Preparation Example 13 Acetamide Citric acid 1:1 Transparent and good fluidity

[0065] Example 1

[0066] In this example, a erigeron breviscapus extract was prepared as follows:

[0067] (1) Take erigeron breviscapus, wash it, perform enzyme inactivation treatment at 150 °C for 2 times, 20 s each time, then dry it at 55 °C until the weight remains unchanged, and pass through a 100-mesh sieve to obtain dry erigeron breviscapus powder;

[0068] (2) Mix the dry erigeron breviscapus powder with a deep eutectic solvent aqueous solution (the deep eutectic solvent uses the sample of Preparation Example 1, and the mass ratio of it to water is 2:1) according to a solid-liquid ratio of 1:10 g / mL, perform constant-temperature water bath extraction at 65 °C for 35 min, centrifuge the extract at 3000 r / min for 10 min, and filter it through a membrane with a pore size of 0.45 μm to obtain it.

[0069] Examples 2 - 13

[0070] In this example, twelve erigeron breviscapus extracts were prepared. The difference from Example 1 is only that the deep eutectic solvents were sequentially replaced with the samples of Preparation Examples 2 - 13 respectively. The mass ratio of the deep eutectic solvent to water in the deep eutectic solvent aqueous solution is 2:1, and other preparation conditions remain unchanged.

[0071] Example 14

[0072] In this example, a erigeron breviscapus extract was prepared. The difference from Example 8 is only that the mass ratio of the deep eutectic solvent to water in the deep eutectic solvent aqueous solution is 4:1, the deep eutectic solvent is still the sample of Preparation Example 8, and other preparation conditions remain unchanged.

[0073] Example 15

[0074] In this example, a erigeron breviscapus extract was prepared. The difference from Example 8 is only that the mass ratio of the deep eutectic solvent to water in the deep eutectic solvent aqueous solution is 1:1, the deep eutectic solvent is still the sample of Preparation Example 8, and other preparation conditions remain unchanged.

[0075] Example 16

[0076] In this example, an erigeron breviscapus extract was prepared. The difference from Example 8 was only that the eutectic solvent aqueous solution was replaced with pure eutectic solvent in equal amounts. The eutectic solvent was still the sample of Preparation Example 8, and other preparation conditions remained unchanged.

[0077] Examples 17 - 18

[0078] In this example, two erigeron breviscapus extracts were prepared. The difference from Example 8 was only that the extraction temperature and time were different, specifically, constant temperature water bath extraction was carried out at 55 °C for 55 min and at 75 °C for 25 min, and other preparation conditions remained unchanged.

[0079] Examples 19 - 20

[0080] In this example, two erigeron breviscapus extracts were prepared. The difference from Example 8 was only that the solid - liquid ratio during extraction was different, specifically 1:5 g / mL and 1:15 g / mL, and other preparation conditions remained unchanged.

[0081] Examples 21 - 22

[0082] In this example, two erigeron breviscapus extracts were prepared. The difference from Example 8 was only that the molar ratio of betaine to glycerol during the preparation of the eutectic solvent was different, specifically 1:3 and 1:9, and other preparation conditions remained unchanged.

[0083] Comparative Example 1

[0084] This comparative example provides an erigeron breviscapus extract, and the method is as follows:

[0085] (1) Take erigeron breviscapus, wash it, perform enzyme - inactivation treatment at 150 °C for 2 times, 20 s each time, then dry it at 55 °C until the weight remains unchanged, and pass through a 100 - mesh sieve to obtain erigeron breviscapus dry powder;

[0086] (2) Mix the erigeron breviscapus dry powder with pure water at a solid - liquid ratio of 1:10 g / mL, perform constant temperature water bath extraction at 65 °C for 35 min, centrifuge the extract at 3000 r / min for 10 min, and filter it through a membrane with a pore size of 0.45 μm to obtain the product.

[0087] Comparative Example 2

[0088] This comparative example provides an erigeron breviscapus extract, and the method is as follows:

[0089] (1) Take erigeron breviscapus, wash it, perform enzyme - inactivation treatment at 150 °C for 2 times, 20 s each time, then dry it at 55 °C until the weight remains unchanged, and pass through a 100 - mesh sieve to obtain erigeron breviscapus dry powder;

[0090] (2) Mix the dried Erigeron breviscapus powder with absolute ethanol at a solid-liquid ratio of 1:10 g / mL, perform constant-temperature water bath extraction at 65 °C for 35 min, centrifuge the extract at 3000 r / min for 10 min, and filter it through a membrane with a pore size of 0.45 μm to obtain the product.

[0091] Comparative Example 3

[0092] This comparative example provides an Erigeron breviscapus extract, and the method is as follows:

[0093] (1) Take the Erigeron breviscapus, wash it, perform enzyme inactivation treatment at 150 °C for 2 times, 20 s each time, then dry it at 55 °C until the weight remains unchanged, and pass it through a 100-mesh sieve to obtain the dried Erigeron breviscapus powder;

[0094] (2) Mix the dried Erigeron breviscapus powder with 50% ethanol aqueous solution at a solid-liquid ratio of 1:10 g / mL, perform constant-temperature water bath extraction at 65 °C for 35 min, centrifuge the extract at 3000 r / min for 10 min, and filter it through a membrane with a pore size of 0.45 μm to obtain the product.

[0095] Test Example 1

[0096] Determine the scutellarin content of the Erigeron breviscapus extracts prepared in Examples 1-22 and Comparative Examples 1-3 by using an ultra-high performance liquid chromatograph (UPLC). The results are shown in Table 2. The liquid chromatogram results of Example 8 and Comparative Example 3 are respectively as Figure 2 and Figure 3 shown.

[0097] Table 2

[0098]

[0099]

[0100] It can be seen from the data results in Table 1 that the preparation method of the Erigeron breviscapus extract involved in the present invention can make the yield of scutellarin in the obtained extract at a high level, and there is no need to use organic solvents, which is more environmentally friendly. Among them, the yields of Example 8, Example 11, and Example 12 are higher, and the selection of deep eutectic solvents, solid-liquid ratio, extraction temperature, and time will also affect the yield of scutellarin.

[0101] Test Example 2

[0102] Determine the in vitro DPPH and ABTS free radical scavenging activities of the Erigeron breviscapus extracts prepared in Example 8, Example 11, Example 12, and Comparative Example 3:

[0103] (1) Determination of DPPH free radical scavenging rate:

[0104] Prepare DPPH solutions with concentrations of 0.12 mg / mL and Vc solutions with a concentration of 1 mg / mL using absolute ethanol for later use. Take 30 μL of the 1 mg / mL Vc solution and the Erigeron breviscapus extract into test tubes respectively, and dilute them with absolute ethanol to obtain the test samples. Mix the test samples and the DPPH solution in equal volumes and let them stand in the dark for 30 min. Then measure the absorbance at a wavelength of 517 nm.

[0105] Calculate the free radical scavenging rate according to the following formula.

[0106] DPPH scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0107] Where A0 is the absorbance value of the absolute ethanol replacing the test sample; A1 is the absorbance value after the test sample reacts with the DPPH free radical; A2 is the absorbance value of the absolute ethanol replacing the DPPH solution.

[0108] The results are as Figure 4 shown (each group of samples was measured in parallel 3 times, and the data are presented as the average value).

[0109] (2) Determination of ABTS free radical scavenging rate:

[0110] Accurately weigh 200.0 mg of ABTS and 34.3 mg of potassium persulfate, dissolve them in 50 mL of distilled water, shake well, and let them stand in the dark at room temperature for 16 h to obtain the ABTS stock solution. Take an appropriate amount of the ABTS stock solution and dilute it with 95% ethanol to an absorbance value within 0.70 ± 0.02 (OD734) as the ABTS test solution for later use. Accurately weigh 100 mg of Vc into a volumetric flask and make up the volume to 100 mL with 95% ethanol to obtain a 2 mg / mL Vc solution for later use. Take 20 μL of the Erigeron breviscapus extract and the 2 mg / mL Vc solution into test tubes respectively, and dilute them with a certain amount of 95% ethanol to obtain the test samples. Mix the test samples and the ABTS test solution in a volume ratio of 1:4 and let them stand in the dark for 6 min. Then measure the absorbance at 734 nm.

[0111] Calculate the free radical scavenging rate according to the following formula.

[0112] ABTS scavenging rate (%) = [(A i - A j ) / A i × 100%

[0113] Where A i is the blank control with absolute ethanol replacing the sample; A j is the absorbance value after the test sample reacts with the ABTS test solution.

[0114] The results are asFigure 5 as shown (each group of samples was determined in parallel 3 times, and the data are presented as the average value).

[0115] From Figure 4 and Figure 5 the results, it can be seen that the erigeron breviscapus extract involved in the present invention has excellent anti-free radical activity, and the effect of Example 8 is better.

[0116] Test Example 3

[0117] The erigeron breviscapus extracts prepared in Example 8, Example 11, Example 12, and Comparative Example 3 were subjected to in vitro determination of hyaluronidase, elastase, and collagenase inhibitory activities:

[0118] (1) Detection of hyaluronidase activity: Hyaluronidase uses sodium hyaluronate as a substrate, and the activity of different erigeron breviscapus extracts is determined according to the Elson-Morgan method. Specifically, hyaluronidase (Hyaluronidase, HAase) hydrolyzes hyaluronic acid (Hyaluronic acid, HA) to generate β-N-acetylglucosamine, which condenses with p-dimethylaminobenzaldehyde under alkaline conditions to form a chromogen 2-methyl-3-diacetylpyrrole derivative (purple), and the hyaluronidase inhibitory activity of different erigeron breviscapus extracts is detected at 585 nm.

[0119] (2) Determination of collagenase activity: This method uses N-[3-(2-furyl)acryloyl]-L-leucine-glycine-L-proline-L-alanine, alias FALGPA, as a substrate, and selects type I collagenase from Clostridium histolyticum as the reaction enzyme, and the collagenase inhibitory activity of the erigeron breviscapus extract obtained by extraction with different solvents is detected at 330 nm.

[0120] (3) Determination of elastase activity: Elastase from porcine pancreas is used, and N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline is used as a substrate, and the elastase inhibitory activity of different erigeron breviscapus extracts is detected at 405 nm.

[0121] The results are as Figure 6 shown (each group of samples was determined in parallel 3 times, and the data are presented as the average value).

[0122] From Figure 6 the results, it can be seen that the erigeron breviscapus extract involved in the present invention has excellent hyaluronidase, elastase, and collagenase inhibitory activities, and the effect of Example 8 is better.

[0123] Test Example 4

[0124] Study on the expression activities of aquaporin AQP3, filaggrin FLG, and loricrin LOR in the Erigeron breviscapus extracts prepared in Example 8, Example 11, Example 12, and Comparative Example 3: Determination process: Detection was performed using RT-PCR. 300 μL of Trizol was added to each well of a 12-well cell culture plate, and RNA was extracted according to the operating steps. Then, PCR amplification was performed on the AQP3, FLG, and LOR genes. 10 μL of each reaction product was taken for 1% agarose gel electrophoresis, and the MultilmageTM Light Cabinet ultraviolet gel imager was used to detect the effect of the Erigeron breviscapus extract on the expression levels of the three genes.

[0125] The results are shown respectively in Figure 7 , Figure 8 , Figure 9 (Each sample was measured in parallel 5 times, and the data are presented as the mean. BC represents the blank control group, and PC represents picrinic acid, which is the positive control group).

[0126] It can be seen from the results that the Erigeron breviscapus extract involved in the present invention has excellent activity in promoting the expression of aquaporin AQP3, filaggrin FLG, and loricrin LOR, and the effect of Example 8 is better.

[0127] Test Example 5

[0128] Study on the in vitro anti-aging activity of the Erigeron breviscapus extracts prepared in Example 8, Example 11, Example 12, and Comparative Example 3:

[0129] β-Galactosidase is the most widely used biomarker to highlight the process of cellular senescence.

[0130] (1) HFF cells were from the laboratory of Winona Biotechnology Co., Ltd. and were cultured using DMEM medium. The 10th - 15th generation cells were selected for the experiment and cultured in a 24-well plate. Subsequently, the culture plate was placed in an incubator with a CO2 concentration of 5% and a temperature of 37 °C for continuous culture for 24 hours.

[0131] Sample addition: Weigh a certain amount of the Erigeron breviscapus extract and dilute it with the medium to form a solution. Take out the culture plate in the incubator, suck out the original medium in the culture wells, and add 500 μL of fresh culture medium (culture medium composition: Erigeron breviscapus dilution + D-gal + DMEM) to each well after rinsing. Subsequently, the culture plate was placed in an incubator with a CO2 concentration of 5% and a temperature of 37 °C for continuous culture for 72 hours. After 72 hours, take out the 24-well plate containing the cultured HFF cells from the incubator, use a negative pressure aspirator to suck out the original cell culture medium in the culture wells, and rinse each culture well with PBS.

[0132] (2) The HFF cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, then washed with PBS, and incubated overnight with the SA-β-GAL staining solution mixture at 37°C. Senescent cells (SA-β-GAL staining showed blue) were observed and counted under a Leica microscope, and the results were as Figure 10 shown, and the percentage of SA-β-GAL positive cells was determined, and the results were as Figure 11 shown.

[0133] From Figure 10 and Figure 11 the results, it can be seen that the erigeron breviscapus extract has excellent anti-senescence ability against HFF cells. Compared with the comparative example, the anti-senescence activity of the erigeron breviscapus extract obtained by the preparation method of the present invention is better.

[0134] The applicant declares that the present invention illustrates a preparation method, its products and applications of an erigeron breviscapus extract through the above embodiments, 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 of the present invention, the equivalent replacement of each raw material of the products 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.

[0135] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0136] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A preparation method of a Scutellaria wightiana Wall. ex DC. extract rich in scutellarin, characterized in that, The preparation method of the erigeron breviscapus extract comprises the following steps: Mix the erigeron breviscapus raw material with an extraction solvent at a ratio of 1:(8 - 12) g / mL, and then carry out water bath heating extraction. After centrifuging and filtering the extract, a filtrate is obtained, thus obtaining the erigeron breviscapus extract; the extraction solvent is a mixed solvent of a eutectic solvent and water with a mass ratio of 5:2 - 4:3; The eutectic solvent is obtained by reacting glycerol and betaine at a molar ratio of (5 - 7):1, or by reacting glycerol and acetamide at a molar ratio of (5 - 7):1, or by reacting glycolic acid and acetamide at a molar ratio of (3 - 5):

1.

2. The preparation method of the Erigeron breviscapus extract according to claim 1, wherein, The reaction is carried out at 40 - 80 °C until the system becomes a clear and transparent liquid.

3. The preparation method of the erigeron breviscapus extract according to claim 1, characterized in that, The temperature of the water bath heating extraction is 45 - 85 °C, and the time is 10 - 55 min.

4. The preparation method of the Erigeron breviscapus extract according to claim 1, characterized in that, The rotation speed of the centrifugation is 2000 - 5000 r / min, and the time is 5 - 20 min.

5. The preparation method of the erigeron breviscapus extract according to claim 1, characterized in that, The filtration adopts a membrane filtration process, and the pore size of the filtration membrane is 0.45 μm.

6. The preparation method of the erigeron breviscapus extract according to claim 1, wherein, The erigeron breviscapus raw material is the dried powder of erigeron breviscapus after drying and pulverization.

7. The preparation method of the Erigeron breviscapus extract according to claim 6, characterized in that, The drying is carried out at 50 - 60 °C, and after pulverization, it is sieved through a 60 - 120 mesh sieve.

8. The preparation method of the erigeron breviscapus extract according to claim 6, wherein, Before drying, an enzyme inactivation treatment is carried out at 130 - 155 °C.

9. The erigeron breviscapus extract prepared by the preparation method of the erigeron breviscapus extract according to any one of claims 1 - 8.

10. The application of the erigeron breviscapus extract according to claim 9 in the preparation of a hyaluronidase inhibitor, an elastase inhibitor or a collagenase inhibitor.

11. The application of the erigeron breviscapus extract according to claim 9 in the preparation of an aquaporin expression promoter, a filaggrin expression promoter or a loricrin expression promoter.

12. The application of the erigeron breviscapus extract according to claim 9 in the preparation of a product with anti - aging, antioxidant, moisturizing and skin barrier repair effects.

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