Preparation method of sophora flower bud extract as well as product and application of sophora flower bud extract

Abstract

The invention relates to a preparation method of a sophora flower bud extract as well as a product and application of the sophora flower bud extract. The preparation method comprises the following steps: mixing sophora flower buds with a tartaric acid solution, and performing pretreatment; mixing the pretreated sophora flower buds with an ethanol water solution, and carrying out negative pressure cavitation extraction to obtain a primary extract; and filtering the primary extract, concentrating under reduced pressure, precipitating with water to remove impurities, centrifuging and drying to obtain the traditional Chinese medicine composition. The sophora flower bud extract obtained through a specific preparation method has excellent effects of resisting oxidation, moisturizing, relieving allergy, resisting inflammation and resisting light aging, is safe and non-irritant, does not have sensitization risks, and can be safely applied to the field of skin care products.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and relates to a preparation method of a Sophora japonica bud extract, a product and an application thereof. Background Art

[0002] Sophora japonica buds, also known as Sophora japonica seeds, are dried buds of the genus Sophora japonica in the Leguminosae family. Their Latin name is Sophorajaponica Linn. They are bitter and slightly cold. Their main effects are cooling blood and stopping bleeding, clearing the liver and purging fire. Sophora japonica buds were first recorded in Shennong's Materia Medica. As a traditional Chinese medicine, they have been used in traditional Chinese medicine for more than 2,000 years. They are widely cultivated in all parts of my country, with rich medicinal resources. The main chemical components of Sophora japonica buds are flavonoids, isoflavones, triterpenes, alkaloids and polysaccharides. Among them, rutin is an important active ingredient in Sophora japonica bud extracts. Modern pharmacological studies have shown that rutin has antioxidant, anti-inflammatory, anti-lipogenic, anti-allergic and antibacterial effects. Isoquercetin and quercetin are hydrolysis and desaccharification products of rutin.

[0003] The methods for obtaining Sophora japonica bud extracts are mostly heating reflux, enzyme extraction, fermentation, ultrasonic-assisted extraction, etc. Heating reflux generally takes a long time, and after enzyme extraction or fermentation, rutin is generally converted into isoquercetin or quercetin. These three components cannot be obtained at the same time, and it is easy to cause skin allergies. Ultrasonic extraction is not conducive to industrial production.

[0004] Therefore, there is an urgent need to develop a preparation method for Sophora japonica bud extract that has simple process, high recovery rate, low cost, low pollution, low energy consumption, is suitable for industrial production and has high safety and high activity. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a Sophora japonica bud extract and its product and application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a Sophora japonica bud extract, the preparation method comprising:

[0008] (1) mixing the sophora japonica buds with the fruit acid solution and then pretreating them to obtain a first mixture;

[0009] (2) mixing the first mixture with an ethanol aqueous solution and performing negative pressure cavitation extraction to obtain a primary extract;

[0010] (3) Filter the primary extract, concentrate under reduced pressure, and then precipitate with water to remove impurities. Centrifuge to obtain the precipitate, and dry it to obtain the product.

[0011] The preparation method of the present invention includes high temperature and high pressure combined acid pretreatment, negative pressure cavitation extraction, filtration, reduced pressure concentration, water precipitation impurity removal, centrifugation, and drying. The preparation method is simple, greatly shortens the production cycle, improves efficiency and quality, saves energy to the maximum extent, meets national environmental protection and energy-saving requirements, can achieve rapid extraction of active substances in Sophora japonica buds, and the prepared Sophora japonica bud extract has stable quality, and the content of active ingredients in the extract such as rutin, quercetin and isoquercetin is relatively high, while removing macromolecular impurities and microorganisms to ensure the purity and safety of the extract. The obtained Sophora japonica bud extract has excellent skin moisturizing, anti-oxidation, anti-inflammatory and anti-photoaging abilities, and is friendly to sensitive skin people, and has high stability, is suitable for large-scale production, and can be used as a raw material for the production of large health products.

[0012] Preferably, the pretreatment comprises: treating the mixed solution of Sophora japonica buds and fruit acid at 103-137 kPa and 121-126° C. for 10-30 min.

[0013] The pressure can be selected as 103kPa, 105kPa, 107kPa, 109kPa, 112kPa, 114kPa, 116kPa, 118kPa, 120kPa, 122kPa, 124kPa, 126kPa, 128kPa, 130kPa, 132kPa, 135kPa, 137kPa, etc. The temperature can be selected as 121℃, 122℃, 123℃, 124℃, 125℃, 126℃, etc. The time can be selected as 10min, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min, 28min, 30min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0014] Preferably, the ratio of Sophora japonica buds to fruit acid solution is 1 g: (3-7) mL, and the mixing time is 10-30 min.

[0015] Among them, the specific point values ​​in (3-7) can be selected as 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, etc., and the time can be selected as 10min, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min, 28min, 30min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be listed here one by one.

[0016] Preferably, the mass percentage of fruit acid in the fruit acid solution is 0.8-1.6%, and the fruit acid includes malic acid and / or citric acid.

[0017] The mass percentage of fruit acid can be selected as 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, etc. Other specific points within the above numerical range can be selected, which will not be described here one by one.

[0018] Preferably, the mass ratio of the first mixture to the ethanol aqueous solution is 1:(2-5), and the mass percentage of ethanol in the ethanol aqueous solution is 75-95%.

[0019] Among them, the specific point values ​​in (2-5) can be selected as 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, etc., and the mass percentage of ethanol can be selected as 75%, 78%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described here one by one.

[0020] Preferably, the vacuum degree of the negative pressure cavitation in step (2) is 60-120 mbar, the temperature is 40-70° C., and the time is 1-2 h.

[0021] The vacuum degree can be selected as 60mbar, 65mbar, 70mbar, 75mbar, 80mbar, 85mbar, 90mbar, 95mbar, 100mbar, 105mbar, 110mbar, 115mbar, 120mbar, etc. The temperature can be selected as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc. The time can be selected as 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0022] Preferably, the pressure of the reduced pressure concentration is 100-150 mbar, for example, 100 mbar, 105 mbar, 110 mbar, 115 mbar, 120 mbar, 125 mbar, 130 mbar, 135 mbar, 140 mbar, 145 mbar, 150 mbar, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0023] Preferably, the water precipitation impurity removal specifically comprises: mixing water and the concentrated solution obtained by reduced pressure concentration in a mass ratio of (3-8):1 for 8-12h, wherein the specific point values ​​in (3-8) can be selected from 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, etc., and the time can be selected from 8h, 8.2h, 8.5h, 8.8h, 9h, 9.2h, 9.5h, 9.8h, 10h, 10.2h, 10.5h, 10.8h, 11h, 11.2h, 11.5h, 11.8h, 12h, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0024] Preferably, the centrifugal speed is 3000-5000 rpm and the time is 5-20 min.

[0025] The rotation speed can be selected as 3000rpm, 3200rpm, 3400rpm, 3600rpm, 3800rpm, 4000rpm, 4200rpm, 4400rpm, 4600rpm, 4800rpm, 5000rpm, etc., and the time can be selected as 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described here one by one.

[0026] Preferably, after drying, the process further comprises mixing the product with a 1,2-pentanediol aqueous solution and filtering using a microporous filter membrane.

[0027] Preferably, the mass percentage of 1,2-pentanediol in the 1,2-pentanediol aqueous solution is 5-60%, for example 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc. Other specific points within the above numerical range can be selected, which will not be described here one by one.

[0028] In a second aspect, the present invention provides a Sophora japonica bud extract prepared according to the method for preparing the Sophora japonica bud extract according to the first aspect.

[0029] In a third aspect, the present invention provides a use of the Sophora japonica bud extract according to the second aspect in the preparation of a product with moisturizing, soothing, anti-inflammatory, light protection, antioxidant or anti-aging effects.

[0030] Preferably, the product comprises a skin care product, a cosmetic, a medicine or a health care product.

[0031] Preferably, the amount of the Sophora japonica bud extract added to the product is 0.6-2.4%, for example, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0032] In a fourth aspect, the present invention provides a use of the Sophora japonica bud extract according to the second aspect in the preparation of an inflammatory factor inhibitor.

[0033] Preferably, the inflammatory factors include TNF-α, IL-6 or NO.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present application adopts high temperature and high pressure combined with fruit acid for pretreatment, and then adopts negative pressure cavitation extraction technology and water precipitation and impurity removal purification method to obtain a Sophora japonica bud extract with excellent antioxidant, moisturizing, anti-inflammatory and anti-photoaging effects. The obtained extract is highly safe and provides a new idea for the application of Sophora japonica buds.

[0036] (2) The extraction technology used in the present invention uses edible fruit acid instead of conventional sulfuric acid, which has the characteristics of improving the appearance color, being green and environmentally friendly, and being non-toxic and harmless, and is conducive to sustainable application.

[0037] (3) The extraction technology used in the present invention can obtain an extract with high yield and high content of rutin, isoquercetin and quercetin, wherein the rutin content is greater than 40%, the isoquercetin content is greater than 8%, and the quercetin content is greater than 10%.

[0038] (4) Compared with the extract obtained by conventional heating reflux extraction, the Sophora japonica bud extract obtained by the present invention has significantly improved DPPH free radical inhibition activity and in vitro inhibitory activity against hyaluronidase, and has more excellent inhibitory effects on three inflammatory factors: NO, IL-6 and TNF-α.

[0039] (5) The Sophora japonica bud extract obtained by the present invention has excellent light protection effect and excellent anti-HFF cell aging effect.

[0040] (6) The Sophora japonica bud extract obtained by the present invention is safe and non-irritating, does not have the risk of allergy, and can be safely used in the field of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a graph showing the results of ultra-high performance liquid chromatography (UPLC) analysis of the product prepared in Example 1.

[0042] Figure 2 This is a graph showing the results of ultra-high performance liquid chromatography (UPLC) analysis of the product prepared in Comparative Example 3.

[0043] Figure 3 This is the light protection efficacy test result of the Sophora japonica bud extract prepared in Example 1.

[0044] Figure 4 This is the SA-β-GAL staining picture after HFF cells were treated with the Sophora japonica bud extract prepared in Example 1.

[0045] Figure 5 This is a statistical result graph of the percentage of SA-β-GAL positive cells after HFF cells were treated with the Sophora japonica bud extract prepared in Example 1. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] The sources of the functional ingredients contained in the products involved in the following embodiments and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0048] The raw material of Sophora japonica buds was purchased from Kunming City, Yunnan Province.

[0049] Example 1

[0050] This embodiment provides a method for preparing a Sophora japonica bud extract, the preparation method comprising:

[0051] (1) 10 g of dried raw material of Sophora japonica buds were mixed with a 1.2% malic acid aqueous solution for 20 min, with a solid-liquid ratio of Sophora japonica buds to malic acid aqueous solution of 1 g:5 mL, and pretreated by a high temperature and high pressure method, specifically: the Sophora japonica bud mixture was maintained at a pressure of 103.4 kPa and a temperature of 121.3° C. for 15 min to obtain a pretreated Sophora japonica bud sample;

[0052] (2) mixing the sample obtained in step (1) with 95% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction at an extraction temperature of 55° C., an extraction time of 1 h, and a negative pressure vacuum degree of 90 mbar to obtain a primary extract;

[0053] (3) The primary extract of Sophora japonica buds obtained in step (2) was filtered through a 200-mesh filter bag, and concentrated under reduced pressure to the weight of the raw material under a vacuum degree of 100 mbar to obtain a concentrated solution, which was mixed with water and the concentrated solution in a volume ratio of 5:1 for 8 hours, and the centrifugal speed was 5000 r / min for 10 minutes. The supernatant was removed, and the precipitate was freeze-dried. The Sophora japonica bud freeze-dried powder was mixed with a 30% 1,2-pentanediol aqueous solution in a mass ratio of 1:200, and filtered using a 0.22 μm microporous filter membrane to obtain a Sophora japonica bud extract.

[0054] The obtained Sophora japonica bud extract was measured by ultra-high performance liquid chromatography, and the results were as follows: Figure 1 As shown, the extract also contains high levels of rutin, isoquercetin and quercetin.

[0055] Example 2

[0056] This embodiment provides a method for preparing a Sophora japonica bud extract, the preparation method comprising:

[0057] (1) 10 g of dried raw material of Sophora japonica buds were mixed with 0.8% malic acid aqueous solution for 30 min, the solid-liquid ratio of Sophora japonica buds to malic acid aqueous solution was 1 g:3 mL, and the mixture was pretreated by a high temperature and high pressure method, specifically, the Sophora japonica bud mixture was kept at a pressure of 120 kPa and a temperature of 124° C. for 10 min to obtain a pretreated Sophora japonica bud sample;

[0058] (2) mixing the sample obtained in step (1) with 85% ethanol aqueous solution in a volume ratio of 1:2, performing negative pressure cavitation extraction at an extraction temperature of 40° C., an extraction time of 1.5 h, and a negative pressure vacuum degree of 120 mbar to obtain a primary extract;

[0059] (3) The primary extract of Sophora japonica buds obtained in step (2) was filtered through a 200-mesh filter bag, and concentrated under reduced pressure to the weight of the raw material under a vacuum degree of 120 mbar to obtain a concentrated solution, which was mixed with water and the concentrated solution in a volume ratio of 3:1 for 10 hours, and the centrifugal speed was 5000 r / min for 10 minutes. The supernatant was removed, and the precipitate was freeze-dried. The Sophora japonica bud freeze-dried powder was mixed with a 30% 1,2-pentanediol aqueous solution in a mass ratio of 1:200, and filtered using a 0.22 μm microporous filter membrane to obtain a Sophora japonica bud extract.

[0060] Example 3

[0061] This embodiment provides a method for preparing a Sophora japonica bud extract, the preparation method comprising:

[0062] (1) 10 g of dried raw material of Sophora japonica buds were mixed with a 1.6% malic acid aqueous solution for 10 min, with a solid-liquid ratio of Sophora japonica buds to malic acid aqueous solution of 1 g:7 mL, and pretreated by a high temperature and high pressure method, specifically: the Sophora japonica bud mixture was maintained at a pressure of 137 kPa and a temperature of 126° C. for 30 min to obtain a pretreated Sophora japonica bud sample;

[0063] (2) mixing the sample obtained in step (1) with a 75% ethanol aqueous solution in a volume ratio of 1:5, and performing negative pressure cavitation extraction at a temperature of 70° C., an extraction time of 2 h, and a negative pressure vacuum of 60 mbar to obtain a primary extract;

[0064] (3) The primary extract of Sophora japonica buds obtained in step (2) was filtered through a 200-mesh filter bag, and concentrated under reduced pressure to the weight of the raw material under a vacuum degree of 150 mbar to obtain a concentrated solution, which was mixed with water and the concentrated solution in a volume ratio of 8:1 for 12 hours, and centrifuged at a speed of 5000 r / min for 10 minutes, the supernatant was removed, and the precipitate was freeze-dried to obtain the product.

[0065] Example 4

[0066] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 20% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction at an extraction temperature of 55°C, an extraction time of 1 h, and a negative pressure intensity of 90 mbar to obtain a primary extract", and other operations remain unchanged.

[0067] Example 5

[0068] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 50% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction at an extraction temperature of 55°C, an extraction time of 1 h, and a negative pressure intensity of 90 mbar to obtain a primary extract", and other operations remain unchanged.

[0069] Example 6

[0070] The present embodiment provides a method for preparing a Sophora japonica bud extract, which differs from the embodiment 1 only in that step (1) is "taking 10 g of dried Sophora japonica bud raw material, mixing it with a 1.2% malic acid aqueous solution for 20 min, the solid-liquid ratio of Sophora japonica bud to malic acid aqueous solution being 1 g:5 mL, and pre-treating it by a high temperature and high pressure method, specifically, maintaining the Sophora japonica bud mixture at a pressure of 103.4 kPa and a temperature of 121.3° C. for 5 min to obtain a pre-treated Sophora japonica bud sample", and other operations remain unchanged.

[0071] Example 7

[0072] The present embodiment provides a method for preparing a Sophora japonica bud extract, which differs from the embodiment 1 only in that step (1) is "taking 10 g of dried Sophora japonica bud raw material, mixing it with a 1.2% malic acid aqueous solution for 20 min, the solid-liquid ratio of Sophora japonica bud to malic acid aqueous solution being 1 g:5 mL, and pre-treating it by a high temperature and high pressure method, specifically, maintaining the Sophora japonica bud mixture at a pressure of 103.4 kPa and a temperature of 121.3° C. for 40 min to obtain a pre-treated Sophora japonica bud sample", and other operations remain unchanged.

[0073] Example 8

[0074] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction, the extraction temperature is 55°C, the extraction time is 1h, the negative pressure vacuum is 180mbar, and a primary extract is obtained", and other operations remain unchanged.

[0075] Example 9

[0076] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction, the extraction temperature is 55°C, the extraction time is 1 hour, the negative pressure vacuum is 40 mbar, and a primary extract is obtained", and other operations remain unchanged.

[0077] Example 10

[0078] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction, the extraction temperature is 55°C, the extraction time is 0.5h, the negative pressure vacuum is 90mbar, and a primary extract is obtained", and other operations remain unchanged.

[0079] Embodiment 11

[0080] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:3, performing negative pressure cavitation extraction, the extraction temperature is 55°C, the extraction time is 3 hours, the negative pressure vacuum is 90 mbar, and a primary extract is obtained", and other operations remain unchanged.

[0081] Example 12

[0082] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:1, performing negative pressure cavitation extraction at an extraction temperature of 55°C, an extraction time of 1 h, and a negative pressure vacuum degree of 90 mbar to obtain a primary extract", and other operations remain unchanged.

[0083] Embodiment 13

[0084] This embodiment provides a method for preparing a Sophora japonica bud extract, which differs from Embodiment 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:7, performing negative pressure cavitation extraction at an extraction temperature of 55° C., an extraction time of 1 h, and a negative pressure vacuum degree of 90 mbar to obtain a primary extract", and other operations remain unchanged.

[0085] Comparative Example 1

[0086] This comparative example provides a method for preparing a Sophora japonica bud extract, which differs from Example 1 only in that step (1) is "taking 10 g of dried Sophora japonica bud raw material, mixing it with a 1.2% malic acid aqueous solution for 20 min, with the solid-liquid ratio of Sophora japonica bud to malic acid aqueous solution being 1 g:5 mL, to obtain a Sophora japonica bud sample", and other operations remain unchanged.

[0087] Comparative Example 2

[0088] This comparative example provides a method for preparing a Sophora japonica bud extract, which differs from Example 1 only in that step (1) is "taking 10 g of dried Sophora japonica bud raw material and mixing it with water, with the solid-liquid ratio of Sophora japonica bud to water being 1 g:5 mL, and pretreating it by a high temperature and high pressure method, specifically, maintaining the Sophora japonica bud mixture at a pressure of 103.4 kPa and a temperature of 121.3° C. for 15 minutes to obtain a pretreated Sophora japonica bud sample", and other operations remain unchanged.

[0089] Comparative Example 3

[0090] This comparative example provides a method for preparing a Sophora japonica bud extract, which differs from Example 1 only in that step (1) is "taking 10 g of dried Sophora japonica bud raw material and mixing it with water, with a solid-liquid ratio of Sophora japonica bud to water being 1 g:5 mL, and maintaining it at normal pressure for 15 min to obtain a pretreated Sophora japonica bud sample"; step (2) is "mixing the sample obtained in step (1) with 95% ethanol aqueous solution in a volume ratio of 1:3, extracting it, heating it under reflux at normal pressure for 1 h to obtain a primary extract", and other operations remain unchanged.

[0091] The obtained Sophora japonica bud extract was measured by ultra-high performance liquid chromatography, and the results were as follows: Figure 2 As shown, the extract contained only rutin.

[0092] Comparative Example 4

[0093] This comparative example provides a method for preparing a Sophora japonica bud extract, which differs from Example 1 only in that step (1) is "taking 10 g of dried Sophora japonica bud raw material, mixing it with a 1.2% sulfuric acid aqueous solution for 20 min, the solid-liquid ratio of Sophora japonica bud to sulfuric acid aqueous solution being 1 g:5 mL, and pre-treating it by a high temperature and high pressure method, specifically, maintaining the Sophora japonica bud mixture at a pressure of 103.4 kPa and a temperature of 121.3° C. for 15 min to obtain a pre-treated Sophora japonica bud sample", and other operations remain unchanged.

[0094] Comparative Example 5

[0095] This comparative example provides a method for preparing a Sophora japonica bud extract, which differs from Example 1 only in that step (3) is "filtering the Sophora japonica bud primary extract obtained in step (2) through a 200-mesh filter bag, and concentrating under reduced pressure to the weight of the raw material under a vacuum degree of 100 mbar to obtain a concentrated solution, freeze-drying the concentrated solution, mixing the Sophora japonica bud freeze-dried powder with a 30% 1,2-pentanediol aqueous solution at a mass ratio of 1:200, and filtering using a 0.22 μm microporous filter membrane to obtain a Sophora japonica bud extract", and other operations remain unchanged.

[0096] Comparative Example 6

[0097] This comparative example provides a method for preparing a Sophora japonica bud extract, which differs from Example 1 only in that step (2) is "mixing the sample obtained in step (1) with a 95% ethanol aqueous solution in a volume ratio of 1:3 for extraction, heating and refluxing the extract at normal pressure for 1 hour to obtain a primary extract", and other operations remain unchanged.

[0098] Test Example 1

[0099] This test example provides the effects of different fruit acid concentrations and acid types on the contents of rutin, isoquercetin and quercetin in Sophora japonica bud extract. The preparation method is as follows:

[0100] 10 g of dried raw material of Sophora japonica buds was taken and mixed with acid aqueous solutions of different types and concentrations for 20 min. The solid-liquid ratio of Sophora japonica buds to acid aqueous solution was 1 g:5 mL. The mixture was treated by high temperature and high pressure method. The specific treatment was as follows: the Sophora japonica bud mixture was maintained at a pressure of 103.4 kPa and a temperature of 121.3 ° C for 15 min to obtain a Sophora japonica bud sample. The rutin, isoquercetin and quercetin therein were detected by ultra-high performance liquid chromatography (HPLC). The results are as follows:

[0101] Table 1

[0102]

[0103] From the data results in Table 1, it can be seen that the addition of malic acid and citric acid aqueous solutions can make the Sophora japonica bud extract contain rutin, isoquercetin and quercetin at the same time. Sulfuric acid may be too acidic, resulting in the Sophora japonica bud extract being basically free of rutin; when the malic acid aqueous solution is added at 0.2%, rutin is basically not converted into isoquercetin and quercetin; when the malic acid aqueous solution is added at 2.4%, the rutin content in the Sophora japonica bud extract is extremely low; when the malic acid aqueous solution is added in the range of 0.8%-1.6%, the Sophora japonica bud extract contains rutin, isoquercetin and quercetin at the same time. Therefore, the experimental discussion is carried out for the addition of malic acid aqueous solution in the range of 0.8%-1.6%.

[0104] Test Example 2

[0105] The color, yield, total flavonoids content, rutin content, quercetin content and isoquercetin content of the Sophora japonica bud extracts obtained in Examples 1-3 were tested.

[0106] The calculation formula of yield is:

[0107] Yield (%) = (M1 / M2) × 100%, M1 is the weight of the Sophora japonica bud extract, and M2 is the amount of the Sophora japonica bud extract added.

[0108] Detection method of total flavonoids content: sodium nitrite-aluminum nitrate method.

[0109] Specifically, take a rutin reference substance, prepare a 0.5 mg / mL standard solution, dilute it by a half-dilution method, and obtain rutin standard solutions of different concentrations; prepare the Sophora japonica bud extracts obtained in Examples 1-3 into 0.5 mg / mL solutions, respectively, as the test samples. Take 1.0 mL of the standard solution and the test sample in a 10 mL volumetric flask, add 0.4 mL of 5% sodium nitrite solution to each, mix well, let stand for 6 minutes, then add 0.4 mL of 1% aluminum nitrate solution to each, mix well, let stand for 6 minutes, then add 2.0 mL of 4% sodium hydroxide solution, mix well, make up to volume, let stand at room temperature for 15 minutes, and measure the absorbance at a wavelength of 500 nm.

[0110] The standard curve was drawn according to the absorbance value and concentration, and the standard curve y = 0.2055x + 0.0223, R 2 =0.99;

[0111] The absorbance values ​​of the Sophora japonica bud extracts prepared in Examples 1-3 were substituted into the standard curve to calculate the total flavonoid content in the extracts.

[0112] The content of rutin, quercetin and isoquercetin was determined by high performance liquid chromatography, as follows:

[0113] Rutin, quercetin and isoquercetin standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The detection conditions were the laboratory-built method. The chromatographic column was an Agilent ZORBA×SB-AQ column (3.0×100, 3.5 μm), 0.1% formic acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, the flow rate was 0.4 mL / min, the detection wavelength was 254 nm, the column temperature was 25°C, the elution program was as shown in Table 2, the results were substituted into the corresponding standard curve, and the results were divided by the injection concentration. The results are shown in Table 3.

[0114] Table 2

[0115]

[0116] Rutin standard curve: y = 6517.5x-10.837, R 2 =0.9998

[0117] Quercetin standard curve: y = 2156.4x + 24.401, R 2 =0.999

[0118] Isoquercetin standard curve: y = 2677.7x + 35.321, R 2 =0.999

[0119] The test results are shown in Table 3.

[0120] Table 3

[0121] Example 1 Example 2 Example 3 Yield (%) 36.42 32.61 33.91 Total flavonoids content (%) 81.44 75.45 73.56 color Yellow Green Yellow Green Yellow Green Rutin(%) 52.24 48.25 46.48 Isoquercetin(%) 10.97 9.79 10.56 Quercetin(%) 15.81 10.81 13.52

[0122] It can be seen from the data results in Table 3 that the Sophora japonica bud extract prepared by the preparation method of the present invention has improved appearance color and high yield, and has high contents of active ingredients such as flavonoids, rutin, isoquercetin and quercetin.

[0123] Test Example 3

[0124] DPPH free radical scavenging test

[0125] The antioxidant effects of the Sophora japonica bud extracts obtained in Examples 1-13 and Comparative Examples 1-6 were tested.

[0126] Test method: DPPH free radical scavenging experiment is used to calculate the IC value of DPPH free radical scavenging effect of the sample to be tested through the change of absorbance level. 50 Values, vitamin C was used as a positive control to test the antioxidant capacity of Sophora japonica bud extract.

[0127] DPPH solution: Dissolve DPPH in anhydrous ethanol to make the final concentration of DPPH 1 mg / mL;

[0128] Vitamin C solution: Dissolve vitamin C in water to make the final concentration of vitamin C 1 mg / mL;

[0129] Sophora japonica bud extract solution: Sophora japonica bud extract was dissolved in 75% ethanol to make the final concentration of Sophora japonica bud extract be 1 mg / mL.

[0130] The 96-well plate was divided into a sample group, a control group and a control group; blank controls were set up for both the sample group and the control group, and VC solution was used as a positive control; the reaction system was 200 μL.

[0131] Sample blank group: Sophora japonica bud extract solution + anhydrous ethanol (volume ratio of 1:1)

[0132] Sample group: Sophora japonica bud extract solution + DPPH solution (volume ratio of 1:1)

[0133] Control group: DPPH solution + 75% ethanol (volume ratio 1:1)

[0134] Control blank group: anhydrous ethanol + 75% ethanol (volume ratio of 1:1)

[0135] React at room temperature in the dark for 30 minutes, shake and oscillate, measure the absorbance of each point on the 96-well plate at 517nm, take the average value of the absorbance of each group of samples, and calculate the DPPH scavenging rate of each test sample. With the concentration of the sample to be tested as the X-axis and the scavenging rate as the Y-axis, a scatter plot with smooth lines and data markers is made. After half-dilution, the test samples of different concentrations are obtained, and the upper, middle and lower three points with a scavenging rate of about 50% are selected to draw a straight line, and the linear equation is obtained to preliminarily estimate the DPPH free radical scavenging Ic50 value of the sample and vitamin C.

[0136] Clearance = [1-(Asample-Asampleblank) / (Acontrol-Acontrolblank)] × 100% Where: Asample: absorbance of sample group Asampleblank: absorbance of sample blank group Acontrol: absorbance of control group Acontrolblank: absorbance of control blank group

[0137] Table 4

[0138]

[0139]

[0140] As shown in Table 4, compared with Comparative Examples 1-6, the Sophora japonica bud extracts prepared in Examples 1-13 have stronger inhibitory activity against DPPH free radicals and better antioxidant effects, indicating that the present invention significantly improves the antioxidant capacity of Sophora japonica bud extracts by integrating high temperature and high pressure combined with fruit acid pretreatment technology, negative pressure cavitation technology, and water precipitation and impurity removal technology. And compared with Examples 4-13, the effect of Examples 1-3 is better, indicating that the selection of specific parameters of the preparation process will also affect the efficacy of the product.

[0141] Test Example 4

[0142] Hyaluronidase inhibitory activity assay

[0143] Determination of in vitro hyaluronidase inhibitory activity of the Sophora japonica bud extracts obtained in Examples 1-13 and Comparative Examples 1-6:

[0144] Hyaluronidase activity detection: Hyaluronidase uses sodium hyaluronate as substrate, and the activity of different Sophora japonica bud extracts is determined according to the Elson-Morgan method. The concentration of Sophora japonica bud extracts is 1.2%. Hyaluronidase (HAase) hydrolyzes hyaluronic acid (HA) to generate β-N-acetylglucosamine, which condenses with p-dimethylaminobenzaldehyde under alkaline conditions to generate the chromogen 2-methyl-3-diacetylpyrrole derivative (purple). The hyaluronidase inhibitory activity of different Sophora japonica bud extracts is detected at 585nm.

[0145] The specific groups are shown in Table 5.

[0146] Table 5

[0147]

[0148]

[0149] Hyaluronidase inhibition rate (%) = [(CD) - (AB)] / (CD) × 100% The results are shown in Table 6 (each group of samples was measured 3 times in parallel, and the data are presented as the average value).

[0150] Table 6

[0151]

[0152]

[0153] As shown in Table 6, compared with Comparative Examples 1-6, the Sophora japonica bud extracts prepared in Examples 1-13 have stronger inhibitory activity against hyaluronidase and better moisturizing effect, indicating that the present invention significantly improves the hyaluronidase inhibitory ability of Sophora japonica bud extracts by integrating high temperature and high pressure combined with fruit acid pretreatment technology, negative pressure cavitation technology and water precipitation impurity removal technology. And compared with Examples 4-13, the effect of Examples 1-3 is better, indicating that the selection of specific parameters of the preparation process will also affect the efficacy of the product.

[0154] Test Example 5

[0155] LPS-induced RAW264.7 mouse macrophages were used as the inflammatory factor inhibition effect test model, and a blank control group, an induced stimulation group (1 μg / mL LPS), a positive control group (50 μg / mL dexamethasone, dex) and a sample group were set up. The concentration of the sample group Sophora japonica bud extract was 1.2%. After incubation for 24 hours, the cell supernatant was detected according to the instructions of the ELISA kit and the nitric oxide detection kit, and the contents of TNF-α, IL-6 and NO in the collected cell supernatant were detected respectively. The experimental results were analyzed by data processing software, and the inhibitory effect of the Sophora japonica bud extracts obtained in Examples 1-13 and Comparative Examples 1-6 on inflammatory factors was evaluated, and the results are shown in Table 7.

[0156] Table 7

[0157]

[0158]

[0159] From the data results in Table 7, it can be seen that compared with Comparative Examples 1-6, the Sophora japonica bud extract obtained in Examples 1-13 has a stronger inhibitory effect on the three inflammatory factors NO, TNF-α and IL-6, and its anti-inflammatory activity is more excellent; it shows that the present invention significantly improves the anti-inflammatory activity of the Sophora japonica bud extract by integrating high temperature and high pressure combined with fruit acid pretreatment technology, negative pressure cavitation technology and water precipitation impurity removal technology. And compared with Examples 4-13, the effect of Examples 1-3 is better, indicating that the selection of specific parameters of the preparation process will also affect the efficacy of the product.

[0160] Test Example 6

[0161] The photoprotective activity of the Sophora japonica bud extract prepared in Example 1 was studied in vitro: human immortalized keratinocytes (Hacat cells) irradiated with UVB were used as a photoprotective effect test model.

[0162] Test method:

[0163] Hacat cells with good morphology and in the logarithmic growth phase were selected and inoculated in 96-well plates and incubated in an incubator for 24 hours. Two concentration gradients of Sophora japonica bud extract were set, and the final concentration of Sophora japonica bud extract was 2.4% and 1.2% by dilution with complete culture medium; 3 replicate wells were set in each concentration gradient and incubated in an incubator for 2 hours, then replaced with PBS containing Sophora japonica bud extract (final concentration of 2.4% and 1.2%) for UVB irradiation (0.2mW, 30s), and replaced with DMEM culture medium for incubation for 24 hours after irradiation, the original supernatant was removed, and 20μL of 5mg / mL MTT solution was added to each well. After incubation in the incubator for 2 hours, the absorbance at an absorption wavelength of 490nm was measured with an enzyme marker to calculate the cell activity.

[0164] Cell activity (%) = (absorbance value of control group - absorbance value of experimental group) / (absorbance value of control group) × 100%

[0165] The experimental data were statistically analyzed, the differences in cell viability between different treatment groups were compared, and graphs were drawn to present the results. Figure 3 The results show that when keratinocytes are irradiated by UVB, the cell activity is significantly reduced. The Sophora japonica bud extract prepared by the present invention has significant light protection effect at a concentration of 1.2% and 2.4%, and has the potential for anti-photoaging.

[0166] Test Example 7

[0167] The in vitro anti-aging activity of the Sophora japonica bud extract prepared in Example 1 was studied: β-galactosidase is the most widely used biomarker to highlight the cellular aging process.

[0168] HFF cells came from the laboratory of Beitanni Biotechnology Co., Ltd. and were cultured using DMEM medium. The experiment selected 10-15th generation cells and cultured them in a 24-well plate. The culture plate was then placed in an incubator with a CO2 concentration of 5% and a temperature of 37°C for continuous culture for 24 hours.

[0169] Sample addition: Take out the culture plate from the incubator, remove the original culture medium in the culture wells, add 500μL of new culture medium to each well after rinsing (culture medium composition: 0.5μL Sophora japonica bud extract (30mg / mL) + 120μL D-gal + 379.5μL DMEM), the final concentration of Sophora japonica bud extract is 30μg / mL. Then place the culture plate in an incubator with a CO2 concentration of 5% and a temperature of 37°C for 72 hours. After 72 hours, take out the 24-well plate containing the HFF cells being cultured from the incubator, use a negative pressure aspirator to remove the original cell culture medium in the culture wells, and rinse each culture well with PBS.

[0170] HFF cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, then washed with PBS, and incubated with the SA-β-GAL staining solution mixture at 37°C overnight. Senescent cells (SA-β-GAL staining in blue) were observed and counted under a Leica microscope. Figure 4 As shown, the percentage of SA-β-GAL positive cells was determined, and the results were shown in Figure 5 As shown, blue indicates SA-β-GAL-positive cells.

[0171] Depend on Figure 4 and Figure 5 The results show that the normal control group (NC) has fewer SA-β-GAL positive cells, while the model group (D-gal group) HFF cells are obviously blue, indicating that there are more SA-β-GAL positive cells and the model is established; after the HFF cells were treated with Sophora japonica bud extract, the blue color was significantly reduced compared with the model group. By measuring the percentage of SA-β-GAL positive cells, compared with the D-gal group, its aging marker SA-β-Gal (aging-related β-galactosidase) was reduced by 91.77%, indicating that the Sophora japonica bud extract involved in the present invention has excellent anti-HFF cell aging ability. It can be added to health products as an anti-photoaging ingredient.

[0172] The applicant declares that the present invention illustrates the preparation method of a Sophora japonica bud extract and its product and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0173] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0174] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a Sophora japonica bud extract, It is characterized in that The preparation method comprises: (1) mixing the sophora japonica buds with the fruit acid solution and then pretreating them to obtain a first mixture; (2) mixing the first mixture with an ethanol aqueous solution and performing negative pressure cavitation extraction to obtain a primary extract; (3) Filter the primary extract, concentrate under reduced pressure, and then precipitate with water to remove impurities. Centrifuge to obtain the precipitate, and dry it to obtain the product.

2. The method for preparing the Sophora japonica bud extract according to claim 1, It is characterized in that The pretreatment comprises: treating the mixed solution of Sophora japonica buds and fruit acid at 103-137 kPa and 121-126° C. for 10-30 minutes.

3. The method for preparing the Sophora japonica bud extract according to claim 1, It is characterized in that The ratio of Sophora japonica buds to fruit acid solution is 1 g: (3-7) mL, and the mixing time is 10-30 min.

4. The method for preparing the Sophora japonica bud extract according to claim 1, It is characterized in that The mass percentage of fruit acid in the fruit acid solution is 0.8-1.6%, and the fruit acid includes malic acid and / or citric acid.

5. The method for preparing the Sophora japonica bud extract according to claim 1, It is characterized in that The mass ratio of the first mixture to the ethanol aqueous solution is 1:(2-5), and the mass percentage of ethanol in the ethanol aqueous solution is 75-95%.

6. The method for preparing the Sophora japonica bud extract according to claim 1, It is characterized in that The vacuum degree of the negative pressure cavitation in step (2) is 60-120 mbar, the temperature is 40-70° C., and the time is 1-2 h.

7. The method for preparing the Sophora japonica bud extract according to claim 1, It is characterized in that The water precipitation and impurity removal specifically comprises: mixing water and the concentrated solution obtained by vacuum concentration at a mass ratio of (3-8):1 for 8-12 hours.

8. A sophora japonica bud extract prepared according to the method for preparing the sophora japonica bud extract according to any one of claims 1 to 7.

9. Use of the Sophora japonica bud extract according to claim 8 in preparing products with moisturizing, soothing, anti-inflammatory, light-protective, anti-oxidative or anti-aging effects.

10. Use of the Sophora japonica bud extract according to claim 8 in the preparation of inflammatory factor inhibitors.

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