Preparation method of white thorn flower seed extract and its product and application

Abstract

The present invention relates to a preparation method of a white thorn flower seed extract, and its product and application. The preparation method comprises the following steps: subjecting crushed and sieved white thorn flower seeds to ultrasonic-assisted enzymolysis to obtain an enzymolysis product; subjecting the enzymolysis product to subcritical extraction with subcritical water to obtain an extract; filtering and concentrating the extract to obtain a white thorn flower seed crude extract; subjecting the white thorn flower seed crude extract to flocculation and decolorization to obtain the white thorn flower seed extract. The present invention adopts ultrasonic-assisted enzymolysis and subcritical water extraction technology, and further purifies by flocculation and decolorization, so as to obtain a white thorn flower seed extract rich in polysaccharides and oxymatrine, and no organic solvent or toxic and harmful chemical substances are used in the preparation process, and the white thorn flower seed extract is green and environmentally friendly, and the obtained white thorn flower seed extract is safe and non-irritating, has clear biological activity, and can be applied to the field of skin care products to achieve industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and more specifically, relates to a preparation method of a Nitraria tangutorum seed extract, and a product and application thereof. Background Art

[0002] White thorn flower, a plant of the genus Sophora flavescens in the Leguminosae family, is also known as bitter thorn flower, white engraved needle, horse whip, horse hoof needle, and wolf tooth thorn. White thorn flower is bitter and cold in nature, and its roots, flowers, leaves, fruits, and seeds are all used as medicine. White thorn flower is a heat-clearing medicine. It tastes bitter and is cold in nature. It has the effects of clearing heat and detoxifying, promoting dampness and reducing swelling. It can treat sore throat, damp-heat dysentery, hematuria, nosebleeds, skin itching, bloody stools, etc. White thorn flower seeds can regulate qi and eliminate accumulation, and are anti-cancer. They are used for indigestion, stomachache, abdominal pain, epidermal cancer and leukemia, and are listed as one of the Yi medicines. Modern pharmacological studies have found that white thorn flower has anti-inflammatory, anti-allergic, anti-ulcer, anti-tumor, antioxidant, and antibacterial effects. White thorn flower mainly grows in bushes on river valley sand dunes and hillside roadsides. It is distributed in central, southern and western Yunnan. It is a traditional wild vegetable and Chinese medicinal material for ethnic minorities such as the Tibetan, Bai, Miao and Hani in Yunnan. However, there are few reports on the use of white thorn flower as a raw material for skin care products.

[0003] Whitethorn Flower Seeds Sophora The dried seeds (fruits) of the white thorn flower seeds have many and complex chemical components. The currently known chemical components of the white thorn flower seeds mainly include alkaloids, flavonoids, organic acids, glycosides, esters, terpenes, hydrocarbons and other compounds. People usually use the crude extracts of the white thorn flower seeds directly instead of medicinal materials, but this method cannot guarantee the quality of the crude product and cannot achieve large-scale industrial production. At present, the main means for separating and extracting the active ingredients of the white thorn flower seeds are: organic solvent extraction, ultrasonic method, enzymatic method, etc. Among them, the most widely used method is organic solvent extraction, but this separation and extraction method is environmentally unfriendly due to the use of organic solvents. For example: Chinese patent CN108743655B discloses a white thorn flower flavonoid extract and its preparation method and use, which is extracted with 80% ethanol aqueous solution to obtain a crude extract, and enriched with macroporous resin to obtain the total flavonoids of the white thorn flower. Because this method uses a high proportion of ethanol for extraction, ethanol remains in the extract, so that the extract has a certain risk of allergy.

[0004] Subcritical water is water that remains liquid at a temperature between 100°C and 374°C under certain high pressure conditions. As an extraction technology that has not yet been popularized, there are currently no research reports on its application in the preparation of white thorn flower seed extracts. Since the properties of subcritical water under different conditions (such as temperature range) vary greatly, the components of white thorn flower seed extracts extracted using subcritical water under different conditions also vary greatly. Therefore, it is urgent to propose a method for preparing a white thorn flower seed active ingredient extract that is safe, environmentally friendly, and has good extraction effect using subcritical water, as well as the use of white thorn flower seed extracts in the preparation of skin care products with soothing, anti-inflammatory and / or anti-aging effects, so as to solve the technical problems existing in the above-mentioned prior art. Summary of the invention

[0005] In view of the deficiencies in the prior art, the main purpose of the present invention is to propose a preparation method of a Nitraria tangutorum seed extract and its product and application, aiming to solve at least one of the technical problems that the existing crude extraction method of Nitraria tangutorum seed extract has poor safety and environmental protection performance, the quality of the extract cannot be guaranteed, and there is currently no large-scale industrial production of Nitraria tangutorum seed extract for the preparation of skin care products with soothing, anti-inflammatory and / or anti-aging effects.

[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 Nitraria tangutica seed extract, the preparation method comprising the following steps:

[0008] Step S1: subjecting the crushed and sieved white thorn flower seeds to ultrasonic-assisted enzymolysis to obtain an enzymolysis product;

[0009] Step S2: performing subcritical extraction on the enzymatic hydrolysis product using subcritical water to obtain an extract;

[0010] Step S3: filtering and concentrating the extract to obtain a crude extract of white thorn flower seeds;

[0011] Step S4: performing flocculation and decolorization on the crude extract of Nitraria tangutorum seeds to obtain the Nitraria tangutorum seed extract.

[0012] The preparation method of the white thorn flower seed extract involved in the present invention adopts ultrasound-assisted enzymolysis technology and subcritical water extraction technology in a coordinated manner, effectively improving the preparation effect of the white thorn flower extract. At the same time, the subcritical water extraction operation parameters are optimized to further improve the synergistic effect of ultrasound-assisted enzymolysis; in addition, further purification is carried out by flocculation treatment and decolorization treatment, the process is simple, and the operation is convenient. The preparation method described in the present invention does not introduce organic solvents, is non-toxic and harmless, and is green and environmentally friendly. The obtained white thorn flower seed extract is safe and non-irritating, has clear biological activity, has excellent soothing, anti-inflammatory and anti-aging effects, and can be safely applied to the field of skin care products to achieve industrial production.

[0013] Preferably, the enzyme used for the enzymolysis in step S1 is obtained by compounding pectinase, amylase and cellulase, wherein the mass ratio of the pectinase, amylase and cellulase is (0.5-2): (1-3): (0.1-4); the mass of the enzyme used for the enzymolysis accounts for 0.1wt%-2wt% of the mass of the white thorn flower seeds;

[0014] Among them, the above-mentioned "0.5-2" can be specifically taken as: 0.5, 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, etc.;

[0015] Among them, the above-mentioned "1-3" can be specifically taken as: 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, 2.5, 2.6, 2.7, 2.8, 2.9, 3, etc.;

[0016] Among them, the above-mentioned "0.1-4" can be specifically taken as: 0.1, 0.2, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, etc.;

[0017] Among them, the above-mentioned "0.1wt%-2wt%" can be specifically taken as: 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt%, 1.8wt%, 1.9wt%, 2wt%, etc. Other specific point values ​​within the numerical range can be selected, which will not be repeated here one by one;

[0018] Preferably, the step S1 further comprises the step of adding water before the ultrasonic-assisted enzymatic hydrolysis, and the mass ratio of the white thorn flower seeds to water is 1: (5-10);

[0019] Among them, the above-mentioned "1: (5-10)" can be specifically taken as: 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, etc. Other specific point values ​​within this numerical range can be selected, and they will not be elaborated here one by one.

[0020] Preferably, the power of the ultrasound in step S1 is 300-700W; the temperature of the enzymatic hydrolysis is 50-65°C, and the time is 25-40min;

[0021] Among them, the above-mentioned "300-700W" can be specifically taken as: 300W, 325W, 350W, 375W, 400W, 425W, 450W, 475W, 500W, 525W, 550W, 575W, 600W, 625W, 650W, 675W, 700W, etc.;

[0022] Among them, the above-mentioned "50-65°C" can be specifically taken as: 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, etc.;

[0023] Among them, the above-mentioned "25-40min" can be specifically taken as: 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described here one by one.

[0024] Preferably, the subcritical extraction pressure in step S2 is 0.1-1 MPa, the temperature is 130-160°C, the time is 20-60 min, and the mass ratio of the white thorn flower seeds to subcritical water is 1: (4-10);

[0025] The above-mentioned “0.1-1MPa” may be, for example, 0.1MPa, 0.2MPa, 0.3MPa, 0.4MPa, 0.5MPa, 0.6MPa, 0.7MPa, 0.8MPa, 0.9MPa, 1MPa, etc.;

[0026] Wherein, the above-mentioned "130-160°C" can be, for example, 130°C, 132°C, 135°C, 138°C, 140°C, 143°C, 145°C, 147°C, 150°C, 152°C, 155°C, 158°C, 160°C, etc. Preferably, the temperature of the subcritical extraction is 132-158°C; preferably, 135-155°C; preferably, 138-152°C; preferably, 140-150°C; preferably, 143-147°C; preferably, 145°C;

[0027] The preparation method of the Whitethorn Flower Seed Extract involved in the present invention synergistically adopts ultrasound-assisted enzymatic hydrolysis technology and subcritical water extraction technology, and optimizes the subcritical water extraction operating parameters. The applicant unexpectedly found that when the subcritical extraction temperature is within the above-mentioned range of 130-160°C, the biological activity of the Whitethorn Flower Seed Extract prepared by it is better, and the inhibitory activity on inflammatory factors TNF-α, NO and IL-6 is significantly improved, and it has more soothing, anti-inflammatory and anti-aging effects.

[0028] The above “20-60 min” can be, for example, 20 min, 22 min, 25 min, 27 min, 30 min, 33 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min, etc.;

[0029] Among them, the above-mentioned "1: (4-10)" can be, for example, 1:4 g / mL, 1:5 g / mL, 1:6 g / mL, 1:7 g / mL, 1:8g / mL, 1:9 g / mL, 1:10 g / mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0030] Preferably, the extract is cooled before filtering in step S3, and the temperature is lowered to 50-60°C.

[0031] Among them, the above-mentioned "50-60℃" can be, for example, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, etc. Other specific point values ​​within the numerical range can be selected, and they will not be elaborated here one by one.

[0032] Preferably, the flocculation treatment in step S4 is: mixing the crude extract of Nitraria tangutorum seeds with a flocculant to form a mixture, wherein the mass ratio of the flocculant to the mixture is 1wt%-3wt%; and the flocculant comprises a combination of chitosan, gelatin and ZTC-plant extract clarifier.

[0033] In the present invention, a composite flocculant is obtained by compounding chitosan, gelatin and ZTC-plant extract clarifier, wherein chitosan is a natural high molecular weight polysaccharide substance with unique physical and chemical properties and biological activation function, and as a high molecular weight flocculant, it has excellent flocculation performance; gelatin as a natural high molecular weight flocculant has the characteristics of high efficiency, fast reaction and easy biodegradation; ZTC-plant extract clarifier can remove unstable colloid components such as tannins, proteins, waxes, etc., and has no effect on the effective ingredients in Chinese herbal medicines such as flavonoids, polysaccharides, alkaloids, amino acids, etc., and has excellent high efficiency purification performance. The three components cooperate with each other to achieve synergistic synergy, and obtain a better flocculation effect, which is better in improving the soothing, anti-inflammatory and anti-aging effects of the white thorn flower seed extract product.

[0034] Among them, the above-mentioned "1wt%-3wt%" can be specifically taken as: 1wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 2wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3wt%, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0035] Preferably, the mass ratio of chitosan, gelatin and ZTC-plant extract clarifier is (0.1-3):(0.1-5):1;

[0036] Among them, the above-mentioned "0.1-3" can be specifically taken as: 0.1, 0.2, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here;

[0037] Among them, the above-mentioned "0.1-5" can be specifically taken as: 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0038] Preferably, the decolorization treatment in step S4 is followed by filtration and drying; the drying method comprises any one of freeze drying, vacuum drying or spray drying, or a combination of at least two thereof.

[0039] In a second aspect, the present invention provides a White thorn Flower seed extract, which is prepared by the preparation method of the White thorn Flower seed extract described in the first aspect; the content of oxymatrine in the White thorn Flower seed extract is not less than 10wt%, for example, it can be 10wt%, 10.5wt%, 11wt%, 11.5wt%, 12wt%, 12.5wt%, 13wt%, 13.5wt%, 14wt%, 14.5wt%, 15wt%, etc., and other specific point values ​​within the numerical range can be selected, which will not be described one by one here; the total polysaccharide content is not less than 20wt%, for example, it can be 21wt%, 21.5wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, etc., and other specific point values ​​within the numerical range can be selected, which will not be described one by one here.

[0040] The total polysaccharide content of the Nitraria tangutorum seed extract obtained by the present invention is determined by the sulfuric acid-phenol method.

[0041] In a third aspect, the present invention provides a method for preparing the Nitraria tangutorum seed extract described in the first aspect or use of the Nitraria tangutorum seed extract described in the second aspect in preparing a skin care product with soothing, anti-inflammatory and / or anti-aging effects; the skin care product includes an emulsion, a facial cream, a skin care lotion, a skin care essence or a facial mask.

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

[0043] (1) The preparation method of the white thorn flower seed extract of the present invention creatively adopts ultrasound-assisted enzymatic hydrolysis in combination with subcritical extraction and flocculation purification technology, and no organic solvent is used in the entire preparation method, which makes the preparation method not only environmentally friendly, but also more effectively removes impurities in the product through the compound use of three flocculants, namely chitosan, gelatin and ZTC-plant extract clarifier, and can obtain high-quality white thorn flower seed extract with excellent soothing, anti-inflammatory, anti-aging effects and safety, thereby providing a basis for large-scale industrial production and multi-scenario application of white thorn flower seeds.

[0044] (2) The enzyme used for enzymatic hydrolysis in the preparation method of the white thorn flower seed extract of the present invention is obtained by compounding pectinase, amylase and cellulase, wherein the mass ratio of the pectinase, amylase and cellulase is (0.5-2): (1-3): (0.1-4). The enzyme obtained by the compound can better exert the enzymatic hydrolysis effect under the assistance of ultrasound, thereby significantly improving the extraction efficiency of the white thorn flower extract; compared with the extract obtained by the conventional heating reflux extraction process, the obtained white thorn flower seed extract has significantly improved inhibitory activity against inflammatory factors TNF-α, NO and IL-6; and the in vitro inhibitory activity against hyaluronidase, collagenase and elastase is also significantly improved.

[0045] (3) The Whitethorn Seed Extract obtained by the present invention is rich in oxymatrine and polysaccharides, wherein the oxymatrine content is not less than 10wt%; and the total polysaccharide content is not less than 20wt%.

[0046] (4) The Nitraria tangutorum seed extract obtained by the present invention has excellent anti-aging ability of HFF cells (human foreskin fibroblasts). DETAILED DESCRIPTION

[0047] 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.

[0048] The pectinase involved in the following examples is a product with the model number of pectinase 20230223 purchased from Henan Junyi Biotechnology Co., Ltd.;

[0049] Amylase was purchased from Xingtai Wanda Bioengineering Co., Ltd. and was a product of thermostable α-amylase;

[0050] Cellulase was purchased from Henan Junyi Biotechnology Co., Ltd. with the model number of Cellulase 20230319;

[0051] Chitosan was purchased from Yuanye Biotechnology Co., Ltd. with model number S11064-500g;

[0052] Gelatin was purchased from Yuanye Biotechnology Co., Ltd., model number S30952-500g;

[0053] ZTC-plant extract clarifier is a product of model ZTC1+1-Ⅱ purchased from Wuhan Zhengtiancheng Biotechnology Co., Ltd.

[0054] Lipopolysaccharide (LPS, Escherichiacoli055:B5) was purchased from Beijing Solebow Technology Co., Ltd.;

[0055] The HFF cells involved in the following examples are derived from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0056] RAW264.7 mouse macrophages were purchased from the cell bank of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences;

[0057] DMEM medium was purchased from Gibco Advanced DMEM from Thermo Fisher Scientific China Co., Ltd.

[0058] Preparation Example 1

[0059] This preparation example provides a flocculant, which includes the following components in parts by weight: 2 parts of chitosan, 3 parts of gelatin, and 1 part of ZTC-plant extract clarifier.

[0060] Preparation Example 2

[0061] This preparation example provides a flocculant, which includes the following components in parts by weight: 0.5 parts of chitosan, 4.5 parts of gelatin, and 1 part of ZTC-plant extract clarifier.

[0062] Preparation Example 3

[0063] This preparation example provides a flocculant, which includes the following components in parts by weight: 3 parts of chitosan, 2 parts of gelatin, and 1 part of ZTC-plant extract clarifier.

[0064] Preparation Example 4

[0065] This preparation example provides a flocculant, which differs from the preparation example 1 only in that it does not contain chitosan, and the reduced amount of chitosan is proportionally distributed to the gelatin and ZTC-plant extract clarifier components.

[0066] Preparation Example 5

[0067] This preparation example provides a flocculant, which differs from preparation example 1 only in that it does not contain gelatin, and its reduced portion is proportionally distributed to the chitosan and ZTC-plant extract clarifier components.

[0068] Preparation Example 6

[0069] This preparation example provides a flocculant, which differs from the preparation example 1 only in that it does not contain the ZTC-plant extract clarifier, and the reduced amount thereof is proportionally distributed to the chitosan and gelatin components. Example 1

[0070] This embodiment provides a white thorn seed extract, and the preparation method thereof is as follows:

[0071] (1) The seeds of the white thorn flower were crushed and passed through a 50-mesh sieve, mixed with water, and the solid-liquid ratio was 1:8 g / mL, and then subjected to ultrasonic-assisted enzymatic hydrolysis (the enzymes were composed of pectinase, amylase and cellulase in a mass ratio of 1:1:1, and the amount of enzyme was 1 wt% of the mass of the white thorn flower seeds), wherein the ultrasonic power was 500 W, the enzymatic hydrolysis temperature was 58 ° C, and the time was 40 min to obtain an enzymatic hydrolysis product.

[0072] (2) The enzymatic hydrolysate was then subjected to subcritical extraction at 0.7 MPa and 145°C for 40 min using water as the extractant, with the ratio of N. thornia seeds to water being 1:6 g / mL to obtain an extract.

[0073] (3) The extract was cooled to 55°C, filtered, and concentrated under reduced pressure to obtain a crude extract of the seeds of the white thorn tree.

[0074] (4) Take 2% of the mass of the crude extract of Nitraria tangutorum seeds from Preparation Example 1, heat and dissolve the flocculant with water, cool it, and mix it with the crude extract of Nitraria tangutorum seeds. Stir it at 40° C. for 30 min, then add activated carbon decolorizer to the solution treated in the flocculation step, the amount of activated carbon is 1% of the mass of the crude extract of Nitraria tangutorum seeds, continue stirring at 40° C. for 30 min, then filter and freeze-dry to obtain the Nitraria tangutorum seed extract. Example 2

[0075] This embodiment provides a white thorn seed extract, and the preparation method thereof is as follows:

[0076] (1) The seeds of the white thorn flower were crushed and passed through a 40-mesh sieve, mixed with water, and the solid-liquid ratio was 1:5 g / mL, and then subjected to ultrasonic-assisted enzymatic hydrolysis (the enzymes were composed of pectinase, amylase and cellulase in a mass ratio of 0.5:2:0.5, and the amount of enzyme was 2% of the mass of the white thorn flower seeds), wherein the ultrasonic power was 300 W, the enzymatic hydrolysis temperature was 60 ° C, and the time was 35 min to obtain an enzymatic hydrolysis product.

[0077] (2) The enzymatic hydrolysate was then subjected to subcritical extraction at 0.8 MPa and 130°C for 30 min using water as the extractant, with the ratio of N. thornia seeds to water being 1:10 g / mL to obtain an extract.

[0078] (3) The extract was cooled to 50°C, filtered, and concentrated under reduced pressure to obtain a crude extract of the seeds of the white thorn tree.

[0079] (4) Take the flocculant prepared in Preparation Example 2 with a mass of 3% of the crude extract of Nitraria tangutorum seeds, heat and dissolve the flocculant with water, cool it, and mix it with the crude extract of Nitraria tangutorum seeds. Stir it at 40°C for 30 minutes, then add activated carbon decolorizer to the solution treated in the flocculation step, the amount of activated carbon is 0.5% of the mass of the crude extract of Nitraria tangutorum seeds, continue stirring at 40°C for 30 minutes, then filter and vacuum dry to obtain the Nitraria tangutorum seed extract. Example 3

[0080] This embodiment provides a white thorn seed extract, and the preparation method thereof is as follows:

[0081] (1) The seeds of the white thorn flower were crushed and passed through a 30-mesh sieve, mixed with water, and the solid-liquid ratio was 1:10 g / mL, and then subjected to ultrasonic-assisted enzymatic hydrolysis (the enzymes were composed of pectinase, amylase and cellulase in a mass ratio of 1.8:1:0.2, and the amount of enzyme was 0.5% of the mass of the white thorn flower seeds), wherein the ultrasonic power was 600 W, the enzymatic hydrolysis temperature was 50 ° C, and the time was 25 min to obtain an enzymatic hydrolysis product.

[0082] (2) The enzymatic hydrolysate was then subjected to subcritical extraction at 0.5 MPa and 160°C for 20 min with water as the extractant. The ratio of N. thornia seeds to water was 1:4 g / mL to obtain an extract.

[0083] (3) The extract was cooled to 60°C, filtered, and concentrated under reduced pressure to obtain a crude extract of the seeds of the white thorn tree.

[0084] (4) Take the flocculant prepared in Preparation Example 3 with a mass of 1% of the crude extract of Nitraria tangutorum seeds, heat and dissolve the flocculant with water, cool it, and mix it with the crude extract of Nitraria tangutorum seeds. Stir it at 40°C for 30 minutes, then add activated carbon decolorizer to the solution treated in the flocculation step, the amount of activated carbon is 2% of the mass of the crude extract of Nitraria tangutorum seeds, continue stirring at 40°C for 30 minutes, then filter and vacuum dry to obtain the Nitraria tangutorum seed extract. Example 4

[0085] This embodiment provides a Nitraria tangutica seed extract, and the preparation method thereof is different from that of Example 1 only in that an equal amount of the flocculant prepared in Preparation Example 1 is replaced by the flocculant obtained in Preparation Example 4. Example 5

[0086] This embodiment provides a Nitraria tangutica seed extract, and the preparation method thereof is different from that of Example 1 only in that an equal amount of the flocculant prepared in Preparation Example 1 is replaced by the flocculant obtained in Preparation Example 5. Example 6

[0087] This embodiment provides a Nitraria tangutica seed extract, and the preparation method thereof is different from that of Example 1 only in that an equal amount of the flocculant prepared in Preparation Example 1 is replaced by the flocculant obtained in Preparation Example 6. Example 7

[0088] This embodiment provides a Nitraria tangutica seed extract, and the preparation method thereof is different from that of Embodiment 1 only in that the temperature of the subcritical extraction is 100°C. Example 8

[0089] This embodiment provides a Nitraria tangutica seed extract, and the preparation method thereof is different from that of Embodiment 1 only in that the temperature of the subcritical extraction is 180°C.

[0090] Comparative Example 1

[0091] This comparative example provides a kind of Nitraria tangutica seed extract, and its preparation method is different from that of Example 1 only in that the preparation method of the Nitraria tangutica seed crude extract is different: the Nitraria tangutica seeds are crushed and passed through a 50-mesh sieve, and then mixed with 80% ethanol aqueous solution and heated under reflux for extraction, and the ratio of Nitraria tangutica seeds to ethanol aqueous solution is 1:6 g / mL. The extract is cooled to 55°C, filtered, and concentrated under reduced pressure to obtain the Nitraria tangutica seed crude extract.

[0092] Comparative Example 2

[0093] This comparative example provides a White thorn Flower seed extract, and its preparation method is different from that of Example 1 only in that the ultrasonic-assisted enzymatic hydrolysis treatment is replaced by a separate enzymatic hydrolysis treatment, the enzyme is composed of pectinase, amylase and cellulase in a mass ratio of 1:1:1, the amount of the enzyme is 1% of the mass of the White thorn Flower seeds, the enzymatic hydrolysis temperature is 58°C, the time is 40 min, and an enzymatic hydrolysis product is obtained.

[0094] Comparative Example 3

[0095] This comparative example provides a Nitraria tangutica seed extract, the preparation method of which is different from that of Example 1 in that the Nitraria tangutica seeds are crushed through a 50-mesh sieve and then directly subjected to step (2) to obtain an extract.

[0096] Test Example 1

[0097] This test example measures the contents of oxymatrine and total polysaccharides in the Nectarinum sutchuenensis seed extracts obtained in Examples 1-3.

[0098] (1) Determination method of oxymatrine content: ultra-high performance liquid chromatography (UPLC);

[0099] Chromatographic column: Agilent 5 HC-C18 (2) column (250×4.6mm), flow rate: 1mL / min, column temperature: 25℃, detection wavelength: 203nm, injection volume: 2μL, mobile phase: 0.01moL / L ammonium formate solution (pH=8.8) (A) acetonitrile (B), gradient elution conditions: 0-5min, 10% (B), 5-15min, 10%-30% (B), 15-16min, 30%-95% (B), 16-21min, 95% (B), 21-21.1min, 95%-100% (B), 21.1-25min, 100% (B);

[0100] Standard solution: Weigh the oxymatrine standard, dissolve it in DMSO-ethanol solution, make up to volume, shake well, and dilute it by half-fold dilution method to obtain 5 concentrations of standard oxymatrine solution. The standard curve is Y=3411.9039x-32.7757 (r:0.99868, R 2 : 0.99736).

[0101] The Whitethorn Flower Seed Extracts prepared in Examples 1-3 and Comparative Examples 1-3 were respectively prepared into 0.4% solutions using 50% acetonitrile-water solvent for testing. The results are shown in Table 1. From the data in the table, it can be seen that the Whitethorn Flower Seed Extracts prepared in the present invention have a higher content of oxymatrine.

[0102] (2) Determination of total polysaccharide content: determined by sulfuric acid-phenol method;

[0103] ① Preparation of standard solution: prepare 0.1 mg / mL glucose standard solution, pipette 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 2 mL into test tubes in sequence, add ddH2O to make up to 2 mL in each test tube, and set aside.

[0104] ② Preparation of sample solution: Accurately weigh the sample powder, add ddH2O to prepare a 0.1 mg / mL sample solution for testing. Preparation of 5% phenol solution: Accurately weigh 2.5 g of phenol solid powder, add 47.5 mL of ddH2O to dissolve at 75°C, and store in dark.

[0105] ③ Reaction of standard solution and sample solution: 1 mL of the prepared standard solution and sample solution were drawn into a test tube in turn, 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution were added for reaction, and the mixture was gently shaken. ddH2O was used as a blank control, and the absorbance was measured at 490 nm using an enzyme marker. A standard curve was drawn with concentration as the horizontal axis and absorbance as the vertical axis, and the absorbance of the sample solution was substituted into the standard curve for calculation. The results of the polysaccharide content determination are shown in Table 1. It can be seen from the data in the table that the white thorn flower seed extract prepared by the preparation method of the present invention has a higher total polysaccharide content.

[0106] Table 1.

[0107] Oxymatrine content Total polysaccharide content Example 1 14.3% 30% Example 2 12.5% 24% Example 3 13.8% 21% Comparative Example 1 7.2% 5% Comparative Example 2 10.2% 15% Comparative Example 3 8.5% 8%

[0108] Test Example 2

[0109] Anti-inflammatory experiments

[0110] Test method: RAW264.7 mouse macrophages in logarithmic growth phase with good morphology were inoculated in DMEM culture medium containing 10% FBS fetal bovine serum and incubated for 24 h. 5 The cells were seeded at a density of 100 cells / well in a 24-well plate and incubated in an incubator at 37°C and 5% CO2 for 2 hours. The cells were stimulated with 1 μg / mL lipopolysaccharide for 24 hours to establish a cell inflammation model.

[0111] The white thorn flower seed extracts prepared in Examples 1-8 and Comparative Examples 1-3 were respectively prepared into a solution with a concentration of 60 μg / mL using DMEM-water solvent for testing, wherein 50 μg / mL of dexamethasone was added to the positive control group. After incubation at 37°C for 24 hours, the cell supernatant was taken and tested according to the instructions of the ELISA kit and the nitric oxide detection kit, and the contents of TNF-α, NO and IL-6 in the collected cell supernatant were respectively detected, and the experimental results were analyzed by data processing software, and the results are shown in Table 2.

[0112] Table 2.

[0113] TNF-α inhibition rate (%) NO inhibition rate (%) IL-6 inhibition rate (%) Example 1 57.3 64.5 59.7 Example 2 54.7 58.1 56.4 Example 3 53.9 52.3 55.1 Example 4 40.2 29.7 36.1 Example 5 26.4 43.2 35.8 Example 6 38.3 32.5 40.1 Example 7 25.6 18.7 8.5 Example 8 29.8 23.9 12.5 Comparative Example 1 0 12.4 0 Comparative Example 2 22.4 7.3 0 Comparative Example 3 18.9 0 0 Positive control group 55.2 52.6 61.2

[0114] TNF-α, NO and IL-6 are important pro-inflammatory mediators that mediate the occurrence of a variety of inflammatory reactions. By reducing their content, the inflammatory response can be inhibited, thereby achieving an anti-inflammatory effect. As can be seen from the data in Table 2:

[0115] (1) The Whitethorn Flower Seed Extract prepared by the preparation method of the Whitethorn Flower Seed Extract provided in Examples 1 to 3 has a significant inhibitory effect on the three pro-inflammatory mediators when the concentration is only 60 μg / mL, which is far superior to the other examples and comparative examples. Specifically, the Whitethorn Flower Seed Extract obtained in Example 1 (concentration of 60 μg / mL) has an inhibition rate of 57.3% on TNF-α, 64.5% on NO, and 59.7% on IL-6; the Whitethorn Flower Seed Extract obtained in Example 2 (concentration of 60 μg / mL) has an inhibition rate of 54.7% on TNF-α, 58.1% on NO, and 56.4% on IL-6; the Whitethorn Flower Seed Extract obtained in Example 3 (concentration of 60 μg / mL) has an inhibition rate of 53.9% on TNF-α, 52.3% on NO, and 55.1% on IL-6.

[0116] (2) By comparing Example 1 with Examples 4-6, it can be seen that the present invention can more effectively remove impurities in the product by compounding three flocculants, namely chitosan, gelatin and ZTC-plant extract clarifier, to obtain a Nitraria tangutorum seed extract product with better anti-inflammatory activity.

[0117] (3) By comparing Example 1 with Examples 7-8, it can be seen that when the temperature of subcritical extraction is outside the preferred range, the anti-inflammatory effect of the prepared White thorn Flower Seed Extract is not as good as that of Example 1. Therefore, it can be seen that the use of subcritical extraction technology in a specific temperature range can further improve the extraction effect and enhance the biological activity of the extract product.

[0118] (4) By comparing Example 1 with Comparative Example 1, it can be seen that the subcritical extraction technology using water as the extractant adopted by the present invention is far inferior to the anti-inflammatory effect of the Whitethorn Seed Extract prepared in Comparative Example 1 compared with the reflux extraction method using ethanol as the extractant.

[0119] (5) By comparing Example 1 with Comparative Example 2, it can be seen that when enzymatic hydrolysis is not performed with the assistance of ultrasound, even if the enzymatic hydrolysis temperature is increased and the enzymatic hydrolysis time is prolonged, the inhibitory effect of the prepared White thorn Flower seed extract on the three pro-inflammatory mediators TNF-α, NO and IL-6 is not as good as that of Example 1. It can be seen that the present invention further improves the extraction effect of White thorn Flower seeds by ultrasound-assisted enzymatic hydrolysis.

[0120] (6) By comparing Example 1 with Comparative Example 3, it can be seen that when the Whitethorn Seeds without ultrasound-assisted enzymatic hydrolysis are directly subjected to subcritical water extraction, even if the extraction temperature is increased and the extraction time is prolonged, the anti-inflammatory effect of the Whitethorn Seeds extract prepared is not as good as that of Example 1.

[0121] Test Example 3

[0122] a. Anti-aging and allergy relief experiment-1

[0123] Test samples: The extracts of the seeds of the white thorn tree prepared in Examples 1-8 and Comparative Examples 1-3 were respectively prepared with DMSO-H2O solution to make 500 μ g / mL concentration of the solution was tested.

[0124] Test method:

[0125] (1) Hyaluronidase activity detection: According to the Elson Morgan method, sodium hyaluronate is used as the substrate. Hyaluronidase hydrolyzes hyaluronic acid to produce β-N-acetylglucosamine, which condenses with p-dimethylaminobenzaldehyde under alkaline conditions to produce the chromogen 2-methyl-3-diacetylpyrrole derivative (purple). The inhibitory activity of the white thorn seed extract on hyaluronidase is detected at 585 nm.

[0126] (2) Collagenase activity assay: N-[3-(2-furanyl)acryloyl]-L-leucine-glycine-L-proline-L-alanine was used as the substrate, and type I collagenase from Bacillus histolyticus was selected as the reaction enzyme. The inhibitory activity of the extract from the seeds of N. tangutica on collagenase was detected at 330 nm.

[0127] (3) Elastase activity assay: Using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide as a substrate and elastase from porcine pancreas as a reaction enzyme, the inhibitory activity of the white thorn seed extract on elastase was detected at 405 nm. The results are shown in Table 3-1.

[0128] Table 3-1.

[0129] Hyaluronidase inhibition rate Collagenase inhibition rate Elastase inhibition rate Example 1 68.01% 45.31% 20.78% Example 2 54.71% 42.40% 15.92% Example 3 48.73% 43.78% 9.66% Example 4 29.80% 32.34% 0 Example 5 38.91% 28.90% 0 Example 6 32.57% 21.04% 0 Example 7 25.86% 13.01% 0 Example 8 27.73% 16.59% 0 Comparative Example 1 19.21% 6.85% 0 Comparative Example 2 22.72% 11.21% 0 Comparative Example 3 0 9.74% 0

[0130] The in vitro inhibition test of hyaluronidase, collagenase and elastase can be used as a quick screening index for anti-aging and allergy-relieving raw materials. The data in Table 3-1 show that:

[0131] (1) It can be seen from Examples 1 to 3 that the preparation method of the Whitethorn Flower Seed Extract provided by the present invention, the Whitethorn Flower Seed Extract prepared by the method has a very significant inhibitory effect on hyaluronidase and collagenase, and also has a relatively significant inhibitory effect on elastase, which proves that it has excellent anti-aging and soothing effects.

[0132] (2) By comparing Example 1 with Examples 4-6, it can be seen that the present invention can more effectively remove impurities in the product by compounding three flocculants, namely chitosan, gelatin and ZTC-plant extract clarifier, to obtain a White thorn Flower seed extract product with better anti-aging and soothing activity.

[0133] (3) By comparing Example 1 with Examples 7-8, it can be seen that when the temperature of subcritical extraction is outside the preferred range, the anti-aging and soothing effect of the White thorn Flower Seed Extract prepared therefrom is not as good as that of Example 1. Therefore, it can be seen that the use of subcritical extraction technology within a specific temperature range can further improve the extraction effect and enhance the biological activity of the extract product.

[0134] (4) By comparing Example 1 with Comparative Example 1, it can be seen that the inhibitory effect data of the Whitethorn Flower Seed Extract prepared by the subcritical extraction technology using water as the extractant adopted in the present invention on the three enzymes are: hyaluronidase inhibition rate 68.01%, collagenase inhibition rate 45.31%, and elastase inhibition rate 20.78%, respectively. While the reflux extraction method using ethanol as the extractant in Comparative Example 1, the inhibitory effect data of the Whitethorn Flower Seed Extract prepared by the Whitethorn Flower Seed Extract on the three enzymes are: hyaluronidase inhibition rate 19.21%, collagenase inhibition rate 6.85%, and no inhibitory effect on elastase. By comparison, it can be seen that its moisturizing, anti-aging and soothing effects are far inferior to those of Example 1.

[0135] (5) By comparing Example 1 with Comparative Example 2, it can be seen that when enzymatic hydrolysis is not performed with the assistance of ultrasound, even if the enzymatic hydrolysis temperature is increased and the enzymatic hydrolysis time is prolonged, the inhibition rate of the prepared White thorn Flower seed extract on hyaluronidase, collagenase and elastase is not as good as that in Example 1. It can be seen that the present invention further improves the extraction effect of White thorn Flower seeds by ultrasound-assisted enzymatic hydrolysis.

[0136] (6) By comparing Example 1 with Comparative Example 3, it can be seen that when the White thorn Seeds without ultrasound-assisted enzymatic hydrolysis are directly subjected to subcritical water extraction, even if the extraction temperature is increased and the extraction time is prolonged, the anti-aging and soothing effect of the White thorn Seeds extract prepared is not as good as that of Example 1.

[0137] b. Anti-aging experiment-2

[0138] Test sample: the Nitraria tangutorum seed extract prepared in Example 1.

[0139] Test method:

[0140] (1) Culture: HFF cells were cultured using DMEM medium. The 10th generation cells were selected for the experiment and cultured 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 h.

[0141] (2) Sample addition: Weigh 0.1 mg of Nitraria tangutica seed extract and dilute it with 1 mL of culture medium to prepare a solution. Take out the culture plate from the incubator, remove the original culture medium in the culture wells, and rinse with PBS. After rinsing, add 500 μL of new culture medium to each well (the composition of the culture medium in Example 1 group: Nitraria tangutica seed dilution + D-gal + DMEM; the difference between the model group and Example 1 group is that the Nitraria tangutica seed dilution is replaced with an equal amount of water). Then place the culture plate in an incubator with a CO2 concentration of 5% and a temperature of 37°C and continue to culture for 72 hours. After the culture is completed, take out the 24-well plate containing the HFF cells being cultured from the incubator, remove the original cell culture medium in the culture wells, and rinse with PBS.

[0142] (3) The HFF cells in the culture wells were fixed with 4% paraformaldehyde for 15 min, then washed with PBS and incubated with a mixture of SA-β-GAL staining solution at 37°C for 10 h. The number of senescent cells (SA-β-GAL staining appears blue) was observed under a Leica microscope and photographed;

[0143] (4) Remove the staining solution, add 200 μL DMSO, shake at 25°C in the dark for 20-30 min, take 150 μL of the supernatant in a 96-well plate, measure the absorbance at 640 nm and the percentage of SA-β-GAL positive cells. The results are shown in Table 3-2. Table 3-2.

[0144] Proportion of SA-β-GAL positive cells (%) Example 1 7.40% Model Group 98.51%

[0145] It can be seen from the data in Table 3-2 that in the group treated with the White thorn Seed Extract prepared in Example 1, the percentage of SA-β-GAL positive cells decreased by 91.11% compared with the model group, indicating that the preparation method of the White thorn Seed Extract provided by the present invention, and the White thorn Seed Extract prepared by it have excellent anti-HFF cell aging ability.

[0146] Test Example 4

[0147] Allergy testing

[0148] Test sample: the Nitraria tangutorum seed extract prepared in Examples 1-8.

[0149] Test method: The human acute monocytic leukemia cell line THF-1 was used to test the allergenicity of the obtained white thorn flower seed extract through the H-CLAT cell experiment. Among them, CD86 refers to the CD86 protein, which belongs to the immunoglobulin superfamily and is a molecule expressed on antigen presenting cells; CD54 refers to the intercellular adhesion molecule 1, and "-" means negative expression of CD86 / CD54. The expression of surface markers CD86 and CD54 of the white thorn flower seed extract did not exceed the threshold at a concentration of 0.1 mg / mL, and the H-CLAT test was judged to be negative, that is, there is no risk of sensitization. The results are shown in Table 4.

[0150] Table 4.

[0151]

[0152] According to the data in Table 4, the Whitethorn Flower Seed Extract prepared by the preparation method provided by the present invention is safe and non-irritating, has no risk of sensitization, and can be safely used in the field of skin care products. It fully complies with the various indicators in the "Cosmetic Safety Technical Specifications (2015 Edition)" promulgated by the state, is non-irritating to the skin, and is safe and reliable.

[0153] In summary, the present invention uses ultrasound-assisted enzymatic hydrolysis and subcritical water extraction technology, combined with flocculation treatment and decolorization treatment, with a simple process and easy operation. No organic solvent is introduced during the extraction process, and the extraction process is non-toxic, harmless, and environmentally friendly. The obtained Whitethorn Flower Seed Extract is safe and non-irritating, has clear biological activity, has excellent soothing, anti-inflammatory and anti-aging effects, and can be safely used in the field of skin care products.

[0154] The applicant declares that the present invention illustrates the technical solution of the present invention 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.

Claims

1. A method for preparing a white thorn flower seed extract, characterized in that: The preparation method comprises the following steps: (1) subjecting crushed and sieved white thorn flower seeds to ultrasonic-assisted enzymolysis to obtain an enzymolysis product, wherein the enzyme used in the enzymolysis is a compound of pectinase, amylase and cellulase, wherein the mass ratio of the pectinase, amylase and cellulase is (0.5-1.8):(1-2):(0.2-1), the power of the ultrasonic wave is 300-600 W; the temperature of the enzymolysis is 50-60° C., and the time is 25-40 min; (2) performing subcritical extraction on the enzymatic hydrolysis product with subcritical water to obtain an extract, wherein the subcritical extraction pressure is 0.5-0.8 MPa, the temperature is 130-160° C., the time is 20-40 min, and the mass ratio of the white thorn flower seeds to the subcritical water is 1:(4-10); (3) filtering and concentrating the extract to obtain a crude extract of white thorn flower seeds; (4) The crude extract of Nitraria tangutica seeds is subjected to flocculation and decolorization treatment to obtain the Nitraria tangutica seed extract, wherein the flocculation treatment is to mix the crude extract of Nitraria tangutica seeds with a flocculant to form a mixture, wherein the mass ratio of the flocculant to the mixture is 1wt%-3wt%; the flocculant comprises a combination of chitosan, gelatin and ZTC-plant extract clarifier, wherein the mass ratio of the chitosan, gelatin and ZTC-plant extract clarifier is (2-3):(2-4.5):1; wherein, The white thorn flower seed extract comprises oxymatrine, and the content of the oxymatrine is not less than 12.5wt%.

2. The method for preparing the Nitraria tangutica seed extract according to claim 1, characterized in that: The mass of the enzyme used in the enzymolysis in step (1) accounts for 0.1wt%-2wt% of the mass of the white thorn seeds.

3. The method for preparing the Nitraria tangutica seed extract according to claim 1, characterized in that: The step (1) also includes the step of adding water before the ultrasonic-assisted enzymatic hydrolysis, and the mass ratio of the white thorn flower seeds to water is 1: (5-10).

4. The method for preparing the Nitraria tangutica seed extract according to claim 1, characterized in that: In the step (3), the extract is cooled before filtering, and the temperature is lowered to 50-60°C.

5. The method for preparing the Nitraria tangutica seed extract according to claim 1, characterized in that: The decolorization treatment is followed by filtration and drying treatment; the drying treatment includes any one or more of freeze drying, vacuum drying or spray drying.

6. A Whitethorn Seed Extract, characterized in that The Whitethorn Flower Seed Extract is prepared by the preparation method according to any one of claims 1 to 5; the content of oxymatrine in the Whitethorn Flower Seed Extract is not less than 12.5wt%, and the content of total polysaccharides is not less than 20wt%.

7. Use of the White thorn Seed Extract according to claim 6 in the preparation of a skin care product with soothing, anti-inflammatory and / or anti-aging effects; the skin care product comprises an emulsion, a face cream, a skin care lotion, a skin care essence or a face mask.

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