Method for extracting dihydroquercetin and rhamnose from engelhardia roxburghiana and plant extract composition containing dihydroquercetin

Abstract

The invention discloses an extraction method of dihydroquercetin and rhamnose in engelhardia roxburghiana and a plant extract composition containing dihydroquercetin. The extraction method comprises the following steps: S1, extraction; S2, purification of astilbin; S3, hydrolysis of astilbin; the extraction method disclosed by the invention is stable in process and suitable for industrial large-scale production and has a good market prospect, and the plant extract composition of dihydroquercetin disclosed by the invention takes dihydroquercetin extracted from engelhardia roxburghiana leaves as a main component, cooperates with four plant extract components and is applied to a formula, so that the plant extract composition has a good application prospect. The traditional Chinese medicine composition has good effects of removing excessive free radicals of a human body, improving skin laxity and resisting wrinkles, and can effectively relieve symptoms such as dark skin, obvious wrinkles and dry skin.

Description

Technical Field

[0001] The invention discloses a method for extracting dihydroquercetin and rhamnose from wolfberry and a plant extract composition containing dihydroquercetin, belonging to the technical field of cosmetic raw material preparation. Background Art

[0002] Engelhardia roxburghiana Wall, a plant of the genus Engelhardia in the Juglandaceae family, is a commonly used Chinese herbal medicine in my country. It is mainly distributed in Guangdong, Guangxi, Yunnan and other places. It is rich in natural resources and has the effects of clearing heat and relieving pain, promoting qi and removing dampness. It is used to treat colds and fever, damp-heat jaundice, hernia and abdominal pain, etc. Astilbin is one of the main active ingredients obtained from Engelhardia roxburghiana Wall, which has significant antibacterial, anti-cancer and hypoglycemic activities; in terms of the cardiovascular system, it has not only the effects of lowering blood lipids and anti-atherosclerosis, but also myocardial protection.

[0003] At present, astilbin is mainly extracted from Chinese medicinal materials such as Smilax glabra, Astilbe or Fructus Astilbinis leaves, among which the content in Fructus Astilbinis leaves is the highest. Adsorption resin, polyamide column or silica gel column chromatography are often used to extract astilbin from Fructus Astilbinis leaves, and the steps are cumbersome. Dihydroquercetin is the aglycone after the hydrolysis of sugars on the 3-position of astilbin. In cosmetics, it can improve skin moisture content, reduce transepidermal water loss rate, improve skin firmness, elasticity and have a repairing effect on the skin. It can be used as a moisturizer and skin protectant in cosmetics. Due to the low content of dihydroquercetin in plants and the limited main plant source (larch), dihydroquercetin is expensive, which greatly hinders its industrial production and application.

[0004] In view of this, it is necessary to develop an extraction method that is stable and suitable for large-scale industrial production to obtain dihydroquercetin. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned technical defects. It is necessary to develop an extraction method with stable process and suitable for industrial large-scale production to obtain dihydroquercetin. The present invention proposes a method for extracting dihydroquercetin from the leaves of Astilbin, first extracting and obtaining the Astilbin in the leaves of Astilbin, and then hydrolyzing it to obtain dihydroquercetin and rhamnose. The dihydroquercetin extracted from the leaves of Astilbin is used as the main component, and four plant extracts are used in the formula. It has good effects of removing excess free radicals in the human body, improving skin sagging, and fighting wrinkles, and can effectively relieve symptoms such as dull skin, obvious wrinkles, and dry skin.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for extracting dihydroquercetin and rhamnose from Chinese wolfberry, comprising the following steps:

[0008] S1. Extraction

[0009] Mixing the leaves of Astragalus membranaceus with a solvent, placing them in a high-voltage pulse electric field for low-temperature extraction, filtering to obtain a first filtrate and a first filter residue, mixing the first filter residue with an extraction solvent for extraction, filtering to obtain a second filtrate and a second filter residue, adding the second filter residue to an extraction solvent for extraction, filtering to obtain a third filtrate and a third filter residue, combining the first filtrate, the second filtrate and the third filtrate to obtain an Astragalus membranaceus leaf extract, concentrating the Astragalus membranaceus leaf extract and recovering ethanol until there is no alcohol taste, to obtain a concentrated solution;

[0010] S2. Purification of Astilbin

[0011] The concentrated solution obtained in S1 is adjusted to pH and then mixed with the extractant at a volume ratio of 1:1, transferred to a separatory funnel, plugged and fully shaken, and then separated into layers. The upper layer is the extracted organic phase loaded with the target solute, and the lower layer is the raffinate phase (aqueous phase) after extraction. The aqueous phase is discarded, and the extraction operation is repeated several times. The extracted organic phase is concentrated at a certain temperature until dry to obtain an extract. The extract is heated and dissolved with a certain proportion of water, and activated carbon is added and stirred for decolorization. After the decolorization is completed, the filtrate is filtered to obtain a filtrate. The filtrate is placed under certain conditions for crystallization. After the crystallization is completed, the filter cake is filtered to obtain the filter cake, and the filter cake is washed with ice water until the washing liquid is colorless. The washing is stopped, and the filter cake is dried to a constant weight, and then crushed to obtain the pure product of astilbin;

[0012] S3, Astilbin hydrolysis

[0013] Take a certain amount of pure astilbin, add a certain volume of dilute acid, heat to the hydrolysis temperature and keep warm to completely hydrolyze to obtain a hydrolyzate, cool the hydrolyzate and filter and wash the filter cake, the filter cake is dihydroquercetin coarse crystals, the filtrate and the washing liquid are combined to obtain rhamnose feed liquid, the filter cake and the filtrate are stored for later use;

[0014] S4. Dihydroquercetin purification

[0015] The filter cake obtained in S3 is heated and dissolved with an appropriate solvent, activated carbon is added, and the mixture is stirred at a certain temperature. After the stirring is completed, the mixture is filtered to obtain a decolorized solution, and the decolorized solution is placed under low temperature conditions for crystallization. After the crystallization is completed, the mixture is filtered and the filter cake is washed with ice water to obtain a secondary crystallization product. The secondary crystallization product is recrystallized under the same conditions as the secondary crystallization. The crystals obtained by recrystallization are filtered, washed with ice water, and dried to obtain a pure dihydroquercetin product;

[0016] S5. Rhamnose purification

[0017] The rhamnose liquid obtained in S3 is adjusted to pH=3.0 with sodium hydroxide solution, and the liquid after adjusting the pH value is subjected to high-speed centrifugation. The clear liquid obtained by centrifugation is passed through an inorganic ceramic membrane and top-washed with water until no rhamnose is detected in the ceramic membrane interception liquid, and the ceramic membrane is stopped, and the ceramic membrane permeate and the ceramic membrane washing liquid are combined, and the ceramic membrane interception liquid is discarded. The ceramic membrane permeate and the ceramic membrane washing liquid are combined and passed through a nanofiltration membrane and top-washed with water until no rhamnose is detected in the nanofiltration membrane interception liquid, and the nanofiltration is stopped, and the nanofiltration membrane permeate and the nanofiltration membrane washing liquid are combined, and the nanofiltration membrane interception liquid is discarded. The nanofiltration membrane permeate and the nanofiltration membrane washing liquid are combined, and activated carbon is added to stir and decolorize, and the decolorized liquid is filtered to obtain a decolorized liquid. After the decolorized liquid is concentrated, it is cooled and crystallized under certain conditions. After the crystallization is completed, it is filtered and top-washed with an appropriate solvent to obtain wet crystals, and the wet crystals are dried and dehydrated to obtain high-content rhamnose.

[0018] Preferably, the Chinese wolfberry leaves in S1 are fresh Chinese wolfberry leaves that have not been dried or baked.

[0019] Preferably, the extraction solvent in S1 is 40%-95% aqueous alcohol containing a surfactant, the surfactant contained in the extraction solvent is disodium lauryl sulfosuccinate, the amount of the surfactant added to the extraction solvent is 0.1-2%, the amounts of the three extraction solvents are 1-20 times, 1-10 times, and 1-10 times the mass of the wolfberry leaves, respectively (mL:g), and the temperature of the low-temperature extraction is 0-5°C.

[0020] Preferably, the pH in S2 is adjusted to pH=5-7, the extractant is ethyl acetate, and the number of extractions is 3-5 times.

[0021] Preferably, the temperature for growing the crystals in S2 is 2-10° C., the growing time is 8-24 h, and the astilbin crystals are dried by vacuum low-temperature drying or spray drying.

[0022] Preferably, the acid in S3 is hydrochloric acid or sulfuric acid, and the concentration of the acid is 0.1-5 mol / L (H + concentration), the ratio of pure astilbin to dilute acid is 1:100-20:100 (g / mL).

[0023] Preferably, the hydrolysis temperature in S3 is 90-100°C, and the end point of the heat preservation hydrolysis is that no astilbin is detected in the hydrolyzate. The cooling temperature of the hydrolyzate is 2-8°C, and the cooling time is 8-12h. The water used to wash the filter cake is ice water, and the end point of the washing is that the washing liquid is colorless and the pH of the washing liquid is close to neutral.

[0024] Preferably, the appropriate solvent in S4 is 0%-30% aqueous alcohol, filter cake / solvent = 1:5-1:20 (g / mL), the amount of activated carbon added is 0.3%-8% of the filter cake mass, the stirring time is 15-45 min, and the stirring temperature is 65-85°C.

[0025] Preferably, the crystallization temperature in S4 is 5-15° C., the crystallization time is 4-8 h, the end point of the ice water washing is that the washing filtrate is colorless, and the drying is vacuum low-temperature drying or spray drying.

[0026] The present invention also discloses a plant extract composition containing Centella asiatica prepared by any of the above-mentioned extraction methods, which is composed of the following components in parts by weight: 10-20 parts of dihydroquercetin, 2-10 parts of wormwood red algae extract, 2-10 parts of cone stone flower extract, 2-10 parts of elecampane extract, and 200-500 parts of fatty alcohol.

[0027] The invention also discloses the application of the plant extract composition. The composition is applied to the preparation of cosmetics. The cosmetics are external skin preparations prepared by using the effective ingredients as active components and adding conventional pharmaceutical or cosmetic auxiliary materials or auxiliary ingredients.

[0028] Preferably, the cosmetics are one or more of lotion, essence, cream, mask and gel.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. Using high-voltage pulse electric field and low-temperature extraction can reduce the infiltration of impurities such as chlorophyll, oil and wax caused by temperature increase, and reduce the difficulty of subsequent processing. At the same time, the extraction solvent contains surfactants, which can make the solvent fully penetrate into the plant raw materials and fully extract astilbin.

[0031] 2. The plant extract composition containing high-purity dihydroquercetin disclosed in the present invention has dihydroquercetin as the main component and synergistically has 4 other components to exert good antioxidant effects. When applied to formulas, it has good effects of removing excess free radicals from the human body, improving skin sagging, and combating wrinkles. It can effectively alleviate symptoms such as dull skin, obvious wrinkles, and dry skin.

[0032] 3. The plant extract composition containing high-purity dihydroquercetin disclosed in the present invention has better water solubility than dihydroquercetin and is easier to be used as a raw material in cosmetic formulations.

[0033] 4. The plant extract composition containing high-purity dihydroquercetin disclosed in the present invention has better antioxidant capacity than dihydroquercetin used alone. Due to the synergistic effect among the components, the efficacy and activity of dihydroquercetin can be maximized.

[0034] 5. The plant extract composition containing high-purity dihydroquercetin disclosed in the present invention has better antioxidant capacity than dihydroquercetin used alone. This can reduce the amount of dihydroquercetin added during use while ensuring the efficacy, and can greatly reduce the cost of using dihydroquercetin as an antioxidant.

[0035] 6. As a natural flavonoid ingredient, dihydroquercetin is safer than synthetic food antioxidants, and its ability to remove free radicals in the body is also beneficial to human health. When dihydroquercetin is added, the amount of synthetic antioxidants such as BHT and TBHQ can be reduced, which can improve the safety of cosmetic additives. DETAILED DESCRIPTION

[0036] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0039] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0040] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0041] For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0042] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0043] The reagents and instruments used in the examples and comparative examples of the present invention are all commercially available products.

[0044] The following is a further detailed description of the claims of the present invention in conjunction with specific implementation methods, but does not constitute any limitation to the present invention. Any limited modifications made by anyone within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0045] Example of method for extracting dihydroquercetin and rhamnose from Lycium wilfordii leaves:

[0046] S1. Extraction

[0047] 10kg of fresh cyperus rotundus leaves are mixed with 60% ethanol (mL:g) containing 1% disodium lauryl sulfosuccinate, placed in a high-voltage pulse electric field for low-temperature extraction, filtered to obtain a first filtrate and a first filter residue, the intensity of the high-voltage pulse electric field is 20kV / cm, the first filter residue is extracted with 5 times the amount of 60% ethanol (mL:g), filtered to obtain a second filtrate and a second filter residue, the second filter residue is mixed with 5 times the amount of 60% ethanol (mL:g) for extraction, filtered to obtain a third filtrate and a third filter residue, the first filtrate, the second filtrate and the third filtrate are combined to obtain a cyperus rotundus leaf extract. The cyperus rotundus leaf extract is concentrated and ethanol is recovered until there is no alcohol taste to obtain a concentrated solution.

[0048] S2. Purification of Astilbin

[0049] The centrifuge obtained in S1 was adjusted to pH = 5.0 and then mixed with ethyl acetate at a volume ratio of 1:1, transferred to a separatory funnel, plugged and fully shaken, and the upper layer was the ethyl acetate phase loaded with astilbin, and the lower layer was the raffinate phase (aqueous phase) after extraction. After 3 times of concentration and extraction with ethyl acetate, the aqueous phase was discarded. The ethyl acetate phase was concentrated at a temperature of 50°C until dry to obtain an extract. After the extract was heated and dissolved with 15 times the weight of water, 1.0% of the weight of the extract was added to the activated carbon and stirred at a temperature of 70°C for 30 minutes for decolorization. After the decolorization was completed, the filtrate was filtered to obtain a filtrate. The filtrate was placed at a temperature of 10°C for crystallization for 12 hours. After the crystallization was completed, the filter cake was filtered to obtain the filter cake, and the filter cake was washed with ice water until the washing filtrate was colorless, and the washing was stopped. After the filter cake was dispersed, it was placed at 45°C for vacuum drying to constant weight, and then crushed to obtain 743.69g of pure astilbin, and the content was 95.47% determined by HPLC.

[0050] S3, Astilbin hydrolysis

[0051] Take the pure astilbin obtained in S3, add 7.5L of dilute hydrochloric acid with a concentration of 0.6mol / L, heat to 100℃ and keep warm for 35min to completely hydrolyze to obtain a hydrolyzate, cool the hydrolyzate at 5℃ for 12h, filter and wash the filter cake with ice water until the washing liquid is colorless and the pH is close to neutral, the obtained filter cake is the coarse crystal of dihydroquercetin, the filtrate and the washing liquid are combined to obtain the rhamnose liquid, and the filter cake and the filtrate are stored for later use.

[0052] S4. Dihydroquercetin purification

[0053] The filter cake obtained in S3 was heated with water to dissolve, and 4.5 g of activated carbon was added. The mixture was stirred at 70°C for 20 min. After the stirring was completed, the decolorized solution was filtered to obtain a decolorized solution. The decolorized solution was placed at 10°C for 8 h for crystallization. After the crystallization was completed, the filter was filtered and the filter cake was washed with ice water until the washing solution was colorless to obtain a secondary crystallization product. The secondary crystallization product was recrystallized, filtered and washed with ice water under the same conditions as the secondary crystallization. The obtained recrystallized crystals were vacuum dried at 45°C to obtain 487.16 g of pure dihydroquercetin. The content was 95.32% as measured by HPLC.

[0054] S5. Rhamnose purification

[0055] The rhamnose liquid obtained in S3 is adjusted to pH=3.0 with sodium hydroxide solution, and the acidified liquid is centrifuged at high speed. The clear liquid obtained by centrifugation is passed through an inorganic ceramic membrane with a retention pore size of 200nm at a pressure of 0.1MPa and top-washed with water until no rhamnose is detected in the ceramic membrane interception liquid, and the water washing is stopped, the ceramic membrane filtrate and the ceramic membrane water washing liquid are combined, and the ceramic membrane interception liquid is discarded. The ceramic membrane filtrate and the ceramic membrane water washing liquid are combined and passed through a nanofiltration membrane with a retention molecular weight of 400nm at a pressure of 0.8MPa and top-washed with water until no rhamnose is detected in the nanofiltration membrane interception liquid, and the nanofiltration membrane is stopped, and the nanofiltration membrane filtrate and the nanofiltration membrane water washing liquid are combined, and the nanofiltration membrane interception liquid is discarded. 2.42g of activated carbon is added to the combined nanofiltration membrane filtrate and nanofiltration membrane water washing liquid, and the decolorization is stirred at a temperature of 60°C for 30min. After the stirring is completed, the decolorized liquid is filtered to obtain the decolorized liquid, and the decolorized liquid is concentrated to a volume of 710mL to obtain the solution to be crystallized. The initial temperature of the crystallization is 60°C, the end temperature is 5°C, the cooling rate is 2°C / h in the temperature range of 60-20°C, and 5°C / h in the temperature range of 20-5°C. The standing time at 5°C is 2h, and the crystallization stirring speed is 200rpm / min. After the crystallization is completed, the wet crystals are filtered and top washed with 90% ethanol to obtain wet crystals. The wet crystals are vacuum dried and dehydrated at 45°C to obtain 248.91g of rhamnose crystals. The rhamnose content measured by HPLC is 99.12%.

[0056] Example 1

[0057] A plant extract composition containing dihydroquercetin is prepared as follows: 15 g of dihydroquercetin, 5 g of wormwood red algae extract, 5 g of cone stone flower extract, and 6 g of elegans extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0058] Example 2

[0059] A plant extract composition containing dihydroquercetin is prepared as follows: 15 g of dihydroquercetin, 8 g of wormwood red algae extract, 6 g of cone stone flower extract, and 5 g of elecampane extract are weighed and added into 420 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0060] Example 3

[0061] A plant extract composition containing dihydroquercetin is prepared as follows: 15 g of dihydroquercetin, 5 g of wormwood red algae extract, 6 g of cone stone flower extract, and 6 g of elecampane extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0062] Example 4

[0063] A plant extract composition containing dihydroquercetin is prepared as follows: 18 g of dihydroquercetin, 5 g of wormwood red algae extract, 6 g of cone stone flower extract, and 5 g of elecampane extract are weighed and added into 420 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0064] Example 5

[0065] A plant extract composition containing dihydroquercetin is prepared as follows: 15 g of dihydroquercetin, 8 g of wormwood red algae extract, 5 g of cone stone flower extract, and 6 g of elegans extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0066] Comparative Example 1

[0067] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin, 5 g of Glechoma paniculata extract and 6 g of Inula officinalis extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0068] Comparative Example 2

[0069] A plant extract composition containing dihydroquercetin is prepared as follows: 15 g of dihydroquercetin, 5 g of wormwood red algae extract and 5 g of elegans extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0070] Comparative Example 3

[0071] A plant extract composition containing dihydroquercetin is prepared as follows: 15 g of dihydroquercetin, 8 g of wormwood red algae extract, and 6 g of cone stone flower extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0072] Comparative Example 4

[0073] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin and 6 g of elecampane extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0074] Comparative Example 5

[0075] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin and 5 g of a Glechoma paniculata extract are weighed, added into 400 parts of hexyldecanol, and stirred to dissolve.

[0076] In order to better illustrate the advantages of the present invention, the solubility and antioxidant tests of the composition with plant extract efficacy provided by the present invention are given below.

[0077] Test 1: Water solubility test of the composition

[0078] 5.00 g of the dihydroquercetin with a content of 95.47% obtained in the preparation example, the samples prepared in Examples 1-5 and Comparative Examples 1-5 were accurately weighed and added to 95.00 g of deionized water accurately measured in advance, stirred thoroughly and allowed to stand at room temperature, and the solubility of the samples was observed. The test results are as follows:

[0079] Table 1. Water solubility test results of Examples 1-5 and Comparative Examples 1-5

[0080]

[0081]

[0082] The plant extract composition containing high-purity dihydroquercetin disclosed in the present invention has better water solubility than single 95.47% dihydroquercetin, and is more easily used as a raw material in cosmetic formulations, thereby better exerting the biological activity of dihydroquercetin.

[0083] Experiment 2: DPPH free radical scavenging experiment

[0084] Accurately weigh 41.67 mg of DPPH reagent, dilute to 100 mL brown volumetric flask, take 5 mL of it and dilute to 50 mL brown volumetric flask with anhydrous ethanol to obtain DPPH test solution (0.1 mmol / L). Pipette 1 mL of DPPH test solution respectively, and add 0.5 mL of samples prepared in Examples 1-5 and Comparative Examples 1-5 of a certain concentration as shown in Table 2. After fully mixing, let stand in the dark for 30 min, use anhydrous ethanol as a blank control, measure its absorbance at 517 nm, and set vitamin C as a control, and calculate the DPPH clearance rate as follows:

[0085] DPPH removal rate (%) = [1-(As-Ar) / A0] × 100%

[0086] Where: As is the absorbance of the extract and DPPH reaction system; Ar is the absorbance of the extract-anhydrous ethanol; A0 is the absorbance of the blank control anhydrous ethanol.

[0087] Table 2. Reaction solution composition

[0088] Absorbance Sample volume (mL) DPPH test solution (mL) Anhydrous ethanol (mL) A0 0 1 0.5 Ar 0.5 0 1 As 0.5 1 0

[0089] The DPPH free radical scavenging rate reflects its antioxidant capacity to a certain extent. The higher the scavenging rate, the better the antioxidant capacity and the better the antioxidant effect. Therefore, the antioxidant effect of the composition can be judged by studying its ability to scavenging DPPH free radicals. As shown in Table 3, at a test concentration of 0.1%, the scavenging rates of DPPH free radicals of Examples 1-5 and Comparative Examples 1-5 indicate that the antioxidant capacity of the composition containing the four components is better than that of the composition lacking any one or two components. The prescription is reasonably compatible and can synergize to exert its good antioxidant effect.

[0090] Table 3. Results of the experimental study on the scavenging of DPPH free radicals by the composition (0.1% concentration)

[0091]

[0092]

[0093] Test 3: Cellular reactive oxygen species (ROS) inhibition test

[0094] 1. Cytotoxicity assay

[0095] Adjust the concentration of HaCAT cell suspension and add it to 96-well cell plates. Culture overnight at 37°C and 5% CO2 for detection. Dilute the test samples with cell culture medium to different concentrations and add them to the cell plates. Culture them at 37°C and 5% CO2 for 20 to 48 hours. Discard the culture medium, wash with PBS 1-2 times, add MTT working solution and continue to culture. After the culture is completed, discard the culture medium, add DMSO, shake and mix, and measure the absorbance at 570nm (with 630nm as the background). Calculate the cytotoxicity based on the results, and screen the sample concentration without obvious cytotoxicity for the next experiment based on the cell morphology.

[0096] 2. Cellular reactive oxygen species (ROS) inhibition test

[0097] Adjust the concentration of HaCAT cell suspension and add it to 96-well cell plates. Culture overnight at 37°C and 5% CO2 for detection. Dilute the test samples with cell culture medium to different concentrations and add them to the cell plates. Culture them at 37°C and 5% CO2 for 20 to 48 hours. Discard the culture medium, wash with PBS 1-2 times, add DCFH-DA working solution and continue to culture. After the culture is completed, discard the culture medium and then wash twice with PBS. The background group and BC group are protected from light with tin foil, and the other groups will be exposed to UVB0.6J for ultraviolet irradiation. After the irradiation, add 100μl of DMEM culture medium to each well and culture it at 37°C and 5% CO2 for 20 minutes. After the culture is completed, place it on the fluorescence microplate reader detection table, set the incident light wavelength to 525nm, the excitation light wavelength to 488nm, and read the reading.

[0098] 3. Result calculation and judgment

[0099] 3.1. Result calculation

[0100] ROS inhibition rate (%) = 100-(MFI sample-MFI background) / (MFINC-MFI background)*100

[0101] MFI sample is the mean fluorescence intensity value of the sample group; MFI background is the mean fluorescence intensity value of the background group; MFINC refers to the mean fluorescence intensity value of the negative control group. When the fluorescence intensity of the NC group is higher than that of the BC group and there is a significant difference, it means that the model is successfully established and the experiment is established. P < 0.05 indicates a significant difference.

[0102] 3.2 Experimental Results

[0103] 3.2.1 Cytotoxicity results

[0104] The sample obtained in Example 1 was diluted to different concentrations and cytotoxicity experiments were carried out on mouse macrophages. From the cytotoxicity test results, it can be seen that the cell viability of the sample at a concentration of 0.1014 mg / ml is 97.18%, with no obvious cytotoxicity. Therefore, concentrations of 0.1014% and 0.05% were selected for the next test. The results are detailed in Table 4.

[0105] Table 4. Cytotoxicity test results of samples

[0106] concentration(%) 0.0000 0.0016 0.0500 0.1000 0.1500 0.5000 Mean 0.486 0.458 0.423 0.389 0.352 0.145 Cell viability (%) 100.00 99.68 101.62 96.43 78.51 21.13 Vitality standard deviation 2.485 3.783 5.015 7.147 2.417 4.409

[0107] 3.2.2. Reactive oxygen species (ROS) test results

[0108] The samples were diluted to the required concentration and the reactive oxygen species (ROS) test was carried out on keratinocytes. According to the test results, the relative content of ROS in the negative control group was much higher than that in the blank control group, and there was a significant difference between the two (P < 0.0001), indicating that the test was established. The samples at concentrations of 0.1000% and 0.0500% had an inhibition rate of 62.39% and 53.07% on ROS caused by UVB, respectively, which was significantly different from the negative control (P < 0.0001). The test results are shown in Table 5.

[0109] Table 5. Cellular reactive oxygen species (ROS) test results

[0110]

[0111] Note: If P>0.05, it means there is no significant difference, recorded as "ns"; if P<0.05, it means there is a significant difference, and 0.01<P≤0.05, recorded as "*"; 0.001<P≤0.01, recorded as "**"; 0.0001<P≤0.001, recorded as "***"; P≤0.0001, recorded as "****"

[0112] Experiment 4: Human efficacy test

[0113] 1. Test principle

[0114] The subjects tried the test samples and control samples on the left and right half of their faces continuously, tested the skin elasticity R2, transepidermal water loss rate, and skin stratum corneum water content before and after application, and compared the differences between the test samples and the control samples before and after use to verify whether the test samples' firming, moisturizing, and repairing effects were better than those of the control samples.

[0115] 2. Test methods

[0116] Test sample: dihydroquercetin plant extract composition skin care lotion (formula as shown in Table 6, sample group*); control sample: blank matrix skin care lotion (formula as shown in Table 6, except that the high-purity dihydroquercetin plant extract composition is not added, the rest is the same, control group).

[0117] Table 6. Dihydroquercetin skin care lotion ingredients

[0118]

[0119]

[0120] Randomized single-blind test of skin-related indicators before and after use, and comparison of the difference in skin-related indicators before and after use of the test sample with the control sample to see if there is a statistical difference. Test period: 28 days. Before using the sample is recorded as D0, and 28 days after using the sample is recorded as D28.

[0121] 3. Test equipment and parameters

[0122] As shown in Table 7.

[0123] Table 7. Human efficacy test equipment and parameters

[0124]

[0125] 4. Test results and conclusions

[0126] The following results were obtained by using test samples and control samples for 28 days on 30 healthy Chinese subjects. After 28 days of using the test samples: the water content of the skin stratum corneum improved by 30.17%, which was significant compared with the control samples; the transepidermal water loss rate improved by 29.51%, which was significant compared with the control samples; the skin elasticity R2 improved by 36.27%, which was significant compared with the control samples.

[0127] The skin elasticity R2, transepidermal water loss rate, and skin stratum corneum water content of the test samples before and after use were significantly different from those of the control samples (P < 0.05). This indicates that the cosmetic formula with 0.25% dihydroquercetin plant extract composition can increase skin moisture and improve skin firmness, elasticity, and repair effects, and can be used as a moisturizer and skin protectant in cosmetics.

[0128] In summary, the present invention scientifically combines dihydroquercetin, wormwood red algae extract, cone stone flower extract, elecampane extract and fatty alcohol, and the composition has good effects of scavenging free radicals and reactive oxygen species (ROS). The results of human test show that the dihydroquercetin plant extract composition can increase skin moisture and improve skin firmness, elasticity and repair effect. It can effectively relieve skin roughness, obvious wrinkles, spots, etc., and deeply improve skin condition.

[0129] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for extracting dihydroquercetin and rhamnose from wolfberry, characterized in that: The following steps are involved: S1. Extraction Mix the Phellodendron chinense leaves with a solvent, place them in a high-voltage pulse electric field for low-temperature extraction, and filter press to obtain a first filtrate and a first filter residue. The first filter residue is mixed with the extraction solvent for extraction, and filter press to obtain a second filtrate and a second filter residue. The second filter residue is mixed with the extraction solvent for extraction, and filter press to obtain a third filtrate and a third filter residue. The first filtrate, the second filtrate and the third filtrate are combined to obtain a Phellodendron chinense leaf extract. The Phellodendron chinense leaf extract is concentrated and ethanol is recovered until there is no alcohol taste to obtain a concentrated solution. S2. Purification of Astilbin The concentrated solution obtained in S1 is adjusted to pH value and then mixed with the extractant in a volume ratio of 1:1, transferred to a separatory funnel, plugged and fully shaken to separate into layers. The upper layer is the extracted organic phase loaded with the target solute, and the lower layer is the raffinate phase (aqueous phase) after extraction. The aqueous phase is discarded, and the extraction operation is repeated several times. The extracted organic phase is concentrated at a certain temperature until dry to obtain an extract. The extract is heated and dissolved with a certain proportion of water, and activated carbon is added and stirred for decolorization. After the decolorization is completed, the filtrate is filtered to obtain a filtrate. The filtrate is placed under certain conditions for crystallization. After the crystallization is completed, the filter cake is filtered to obtain the filter cake, which is washed with ice water until the washing liquid is colorless. The washing is stopped, and the filter cake is dried to constant weight and crushed to obtain the pure product of astilbin. S3, Astilbin hydrolysis Take a certain amount of pure astilbin, add a certain volume of dilute acid, heat to the hydrolysis temperature and keep warm to completely hydrolyze to obtain a hydrolyzate. After the hydrolyzate is cooled, it is filtered and the filter cake is washed with water. The filter cake is the coarse crystal of dihydroquercetin. The filtrate and the washing liquid are combined to obtain the rhamnose liquid. The filter cake and the filtrate are stored for later use. S4. Dihydroquercetin purification The filter cake obtained in S3 is heated and dissolved with an appropriate solvent, activated carbon is added, and the mixture is stirred at a certain temperature. After the stirring is completed, the mixture is filtered to obtain a decolorized solution, and the decolorized solution is placed under low temperature conditions for crystallization. After the crystallization is completed, the mixture is filtered and the filter cake is washed with ice water to obtain a secondary crystallization product. The secondary crystallization product is recrystallized under the same conditions as the secondary crystallization. The crystals obtained by recrystallization are filtered, washed with ice water, and dried to obtain a pure dihydroquercetin product; S5. Rhamnose purification The rhamnose liquid obtained in S3 is adjusted to pH=3.0 with sodium hydroxide solution, and the liquid after adjusting the pH value is subjected to high-speed centrifugation. The clear liquid obtained by centrifugation is passed through an inorganic ceramic membrane and top-washed with water until no rhamnose is detected in the ceramic membrane interception liquid, and the ceramic membrane is stopped, and the ceramic membrane permeate and the ceramic membrane washing liquid are combined, and the ceramic membrane interception liquid is discarded. The ceramic membrane permeate and the ceramic membrane washing liquid are combined and passed through a nanofiltration membrane and top-washed with water until no rhamnose is detected in the nanofiltration membrane interception liquid, and the nanofiltration is stopped, and the nanofiltration membrane permeate and the nanofiltration membrane washing liquid are combined, and the nanofiltration membrane interception liquid is discarded. The nanofiltration membrane permeate and the nanofiltration membrane washing liquid are combined, and activated carbon is added to stir and decolorize, and the decolorized liquid is filtered to obtain a decolorized liquid. After the decolorized liquid is concentrated, it is cooled and crystallized under certain conditions. After the crystallization is completed, it is filtered and top-washed with an appropriate solvent to obtain wet crystals, and the wet crystals are dried and dehydrated to obtain high-content rhamnose.

2. The extraction method according to claim 1, characterized in that The lycopodiella leaves in S1 are fresh lycopodiella leaves that have not been air-dried or baked.

3. The extraction method according to claim 1, characterized in that The extraction solvent in S1 is 40%-95% aqueous alcohol containing a surfactant, the surfactant contained in the extraction solvent is disodium lauryl sulfosuccinate, the added amount of the surfactant in the extraction solvent is 0.1-2%, the amounts of the three extraction solvents are 1-20 times, 1-10 times, and 1-10 times the mass of the smilax china leaves, respectively (mL:g), and the temperature of the low-temperature extraction is 0-5°C.

4. The extraction method according to claim 1, characterized in that The pH in S2 is adjusted to pH=5-7, the extractant is ethyl acetate, and the number of extractions is 3-5 times.

5. The extraction method according to claim 1, characterized in that The temperature for growing the crystals in S2 is 2-10° C., the growing time is 8-24 hours, and the astilbin crystals are dried by vacuum low-temperature drying or spray drying.

6. The extraction method according to claim 1, characterized in that The acid in S3 is hydrochloric acid or sulfuric acid, and the concentration of the acid is 0.1-5 mol / L (H + concentration), the ratio of pure astilbin to dilute acid is 1:100-20:100 (g / mL).

7. The extraction method according to claim 1, characterized in that The hydrolysis temperature in S3 is 90-100°C, and the end point of the heat preservation hydrolysis is that no astilbin is detected in the hydrolyzate. The cooling temperature of the hydrolyzate is 2-8°C, and the cooling time is 8-12h. The water used for washing the filter cake is ice water, and the end point of the washing is that the washing liquid is colorless and the pH of the washing liquid is close to neutral.

8. The extraction method according to claim 1, characterized in that The appropriate solvent in S4 is 0%-30% aqueous alcohol, filter cake / solvent = 1:5-1:20 (g / mL), the amount of activated carbon added is 0.3%-8% of the filter cake mass, the stirring time is 15-45min, the stirring temperature is 65-85°C, the crystallization temperature is 5-15°C, the crystallization time is 4-8h, the ice water washing end point is that the washing filtrate is colorless, and the drying is vacuum low-temperature drying or spray drying.

9. A plant extract composition containing asiaticoside obtained by the extraction method according to any one of claims 1 to 8, characterized in that: The invention is composed of the following ingredients in parts by weight: 10-20 parts of dihydroquercetin, 2-10 parts of wormwood red algae extract, 2-10 parts of cone stone flower extract, 2-10 parts of safflower extract and 200-500 parts of fatty alcohol.

10. An application of the plant extract composition according to claim 9, characterized in that: The composition is used in the preparation of cosmetics, which are external skin preparations prepared by using the effective ingredients as active components and adding conventional pharmaceutical or cosmetic auxiliary materials or auxiliary ingredients.

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