Extraction method of dihydroquercetin, plant extract composition containing dihydroquercetin and application of plant extract composition

Abstract

The invention discloses an extraction method of dihydroquercetin, a plant extract composition containing dihydroquercetin and application of the plant extract composition. The extraction method comprises the following steps: S1, drying and crushing raw materials; s2, raw material pretreatment; s3, raw material extraction; s4, water precipitation and centrifugation; s5, complexing extraction; s6, reverse extraction and concentration; and S7, crystallizing and drying. 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 containing dihydroquercetin disclosed by the invention takes dihydroquercetin as a main component and cooperates with four components at the same time, can play a good antioxidant effect, is applied to a formula and 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 dihydroquercetin extraction method and a plant extract composition containing the dihydroquercetin and application thereof, and belongs to the technical field of cosmetic raw material preparation. Background Art

[0002] Cellular activities that maintain the integrity of skin cells, as well as all immune mechanisms, involve a series of chemical reactions that generate free radicals and reactive oxygen species (ROS). Endogenous and exogenous antioxidant mechanisms work by neutralizing these bioactive molecules, and an imbalance in this neutralization can have a variety of consequences: Excess free radicals and reactive oxygen species (ROS) are associated with the etiology of various skin diseases.

[0003] Antioxidants can produce antioxidant effects by transferring electrons, providing hydrogen, reducing peroxides, quenching singlet oxygen and superoxide, and chelating metal ions. At present, chemically synthesized antioxidants are mostly highly toxic, and some are even carcinogenic, which makes people doubt and reject synthetic antioxidants, and there are great limitations in clinical application. Finding safe, low-toxic and effective antioxidants from natural products with abundant resources has become the focus of people's research. Plant ingredients are an important component of natural antioxidant active ingredients, among which phenols and flavonoids dominate. The structural characteristics of most polyphenols and flavonoids are the presence of phenolic hydroxyl groups or unsaturated conjugated double bonds, which have strong antioxidant activity. Dihydroquercetin has this type of chemical structure, and its chemical molecule has 5 phenolic hydroxyl groups and a strong conjugated double bond structure. The inventor found through the study of the cosmetic antioxidant efficacy model that dihydroquercetin has a strong antioxidant capacity and can be used as a natural antioxidant in cosmetics.

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

[0005] The purpose of the present invention is to overcome the above-mentioned technical defects and develop an extraction method that is stable in process and suitable for industrial large-scale production to obtain dihydroquercetin.

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

[0007] A method for extracting dihydroquercetin comprises the following steps:

[0008] S1. Drying and crushing raw materials: air-drying larch roots, peeling them, crushing them and passing them through a sieve, collecting the sieved materials to obtain crushed larch root powder;

[0009] S2. Raw material pretreatment: Put the crushed larch root powder into a micro-thermal explosion reaction tank, introduce gas, and keep the reaction tank at a certain speed for 1 hour, and blow gas every 15 minutes. After the treatment, the pretreated larch root powder is obtained;

[0010] S3, raw material extraction: the larch root powder pretreated in S2 is mixed with an extraction solvent, placed in a high-voltage pulse electric field for low-temperature extraction, filtered to obtain a first filtrate and a first filter residue, wherein the intensity of the high-voltage pulse electric field is 15-25 kV / cm, the first filter residue is mixed with the extraction solvent for extraction, filtered to obtain a second filtrate and a second filter residue, the second filter residue is mixed with the extraction solvent for extraction, filtered to obtain a third filtrate and a third filter residue, and the first filtrate, the second filtrate and the third filtrate are combined to obtain a larch extract;

[0011] S4, water precipitation centrifugation: the extract obtained in S3 is concentrated under reduced pressure, and deionized water is added to the concentrate after ethanol is recovered, and the concentrate is stirred and allowed to stand, and the water precipitation liquid is centrifuged to obtain a centrifuge and centrifuge residue, and the centrifuge residue is discarded, and the centrifuge liquid is reserved;

[0012] S5, complex extraction: adjust the pH of the centrifuge obtained in S4 and mix it with the complex extractant at a volume ratio of 1:1, transfer it into a separatory funnel, plug it tightly and shake it thoroughly to allow it to fully separate into layers. The upper layer is the complex extraction organic phase loaded with the target solute, and the lower layer is the raffinate phase after extraction. The raffinate phase is discarded;

[0013] S6, stripping and concentration: on the basis of complex extraction, a certain amount of stripping agent is added to the extractant loaded with the extractant, and the mixture is transferred to a separatory funnel, which is plugged tightly and shaken sufficiently to allow the mixture to be fully separated. The obtained upper layer is the organic phase after stripping, and the lower layer is the stripping liquid loaded with the target solute. The upper layer is discarded after the complex extraction solvent is recovered;

[0014] S7, crystallization and drying: after the stripping in S6 is completed, the pH is quickly adjusted to weak acidity with dilute hydrochloric acid, and the insoluble matter is removed by suction. The filtrate is concentrated at a certain temperature until crystals begin to precipitate in the solution, and then the solution is placed under low temperature conditions for crystal growth. After the crystallization is completed, the filter cake is filtered to obtain a filter cake, and the filter cake is top-washed with ice water until the washing liquid is colorless, and the top-washing is stopped. The filter cake is dried and crushed, and then dried to constant weight to obtain pure dihydroquercetin.

[0015] Preferably, the mesh size of the sieve in S1 is 50-100 meshes.

[0016] Preferably, the gas introduced into S2 is SO2, the amount of the gas introduced is 0.1%-5% (w / w), and the reaction tank operating temperature is 40-70°C.

[0017] Preferably, the extraction solvent in S3 is aqueous alcohol containing a surfactant, the surfactant contained in the extraction solvent is cetylpyridinium hydrobromide, 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 amount of the pretreated medicinal material, respectively (mL:g), and the temperature of the low-temperature extraction is 0-5°C.

[0018] Preferably, the amount of water added in S4 is 1 to 3 times the weight of the original medicinal material, and the temperature of the standing temperature after stirring is 2-10° C. and the standing time is 0.5-5 h.

[0019] Preferably, the pH in S5 is adjusted to pH=5-7, the complexing extractant is composed of sodium pyrophosphate, the diluent is sulfonated kerosene, and the volume fraction of the complexing agent sodium pyrophosphate added to the diluent is 1%-10%.

[0020] Preferably, the stripping agent in S6 is a Na2CO3 solution with a mass concentration of 0.1%-5%, and the volume of the stripping agent is 0.5-5 times the volume of the extract.

[0021] Preferably, the dilute hydrochloric acid in S7 adjusts the pH to 4.0-6.0, the temperature for growing the crystal in S7 is 2-10°C, the growing time is 8-24h, and the drying method is vacuum low-temperature drying, spray drying or vacuum freeze drying.

[0022] The present invention also discloses a plant extract composition containing dihydroquercetin obtained according to any of the above extraction methods, which is composed of the following components in parts by weight: 10-20 parts of dihydroquercetin, 2-10 parts of Swertia japonica extract, 2-10 parts of Coleus forskohlii root extract, 2-10 parts of Alpinia serrata extract, and 200-500 parts of fatty alcohol.

[0023] 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.

[0024] Preferably, the cosmetics are one or more of lotion, essence, cream, mask and gel.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The method for extracting and purifying dihydroquercetin from larch disclosed in the present invention is that since larch root is a lignified structure, SO2 is used to perform a micro-thermal explosion treatment on the crushed root powder before extraction. SO2 can combine with water in the larch root powder to form H2SO3, thereby destroying wood cellulose without affecting the dihydroquercetin content in the larch root powder, thereby improving the efficiency of subsequent solvent extraction.

[0027] 2. Using high-voltage pulse electric field and low-temperature extraction can reduce the infiltration of impurities such as pine oil 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 dihydroquercetin.

[0028] 3. The plant extract composition containing dihydroquercetin disclosed in the present invention has dihydroquercetin as the main component and synergistically has four 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.

[0029] 4. The plant extract composition containing dihydroquercetin disclosed in the present invention has better water solubility than dihydroquercetin and is easier to be used as a raw material in cosmetic formulas.

[0030] 5. The plant extract composition containing dihydroquercetin disclosed in the present invention has better antioxidant capacity than using dihydroquercetin alone. Due to the synergistic effect among the components, the efficacy and activity of dihydroquercetin can be maximized.

[0031] 6. The plant extract composition containing 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.

[0032] 7. 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

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0037] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0038] 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.

[0039] 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.

[0040] The reagents and instruments used in the examples and comparative examples of the present invention are all commercially available products.

[0041] 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.

[0042] Preparation example of dihydroquercetin:

[0043] S1. Drying and crushing of raw materials: The larch roots are air-dried, peeled, crushed and passed through a 500-mesh sieve, and the sieved material is collected to obtain crushed larch root powder.

[0044] S2. Raw material pretreatment: Put the crushed larch root powder into a micro-thermal explosion reaction tank, introduce 1% SO2 gas by mass, keep the reaction tank at a certain speed at 50°C for 1 hour, and blow gas every 15 minutes. After the treatment, the pretreated larch root powder is obtained.

[0045] S3. Raw material extraction: Take 10 kg of larch root powder pretreated by the method in S2 and mix it with 4 times the amount of 80% ethanol (mL:g), place it in a high-voltage pulse electric field for low-temperature extraction, filter press to obtain a first filtrate and a first filter residue, the intensity of the high-voltage pulse electric field is 20 kV / cm, the first filter residue is extracted with 3 times the amount of 80% ethanol (mL:g), filter press to obtain a second filtrate and a second filter residue, the second filter residue is mixed with 3 times the amount of 80% ethanol (mL:g) for extraction, 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 larch extract.

[0046] S4, water sediment centrifugation: the extract obtained in S2 is concentrated under reduced pressure, and 20 L of deionized water is added to the concentrate after recovering the ethanol. After stirring, the concentrate is allowed to stand for 1 hour, and the water sediment is centrifuged to obtain the centrifugal liquid and the centrifugal residue. The centrifugal residue is discarded and the centrifugal liquid is reserved.

[0047] S5. Complex extraction: adjust the centrifuge liquid obtained in S4 to pH 5.50, mix it with the complex extractant (the volume fraction of sodium pyrophosphate in sulfonated kerosene is 5%) at a volume ratio of 1:1, transfer it into a separatory funnel, plug it tightly and shake it thoroughly to allow it to fully separate into layers. The upper layer is the complex extraction organic phase loaded with the target solute, and the lower layer is the raffinate phase after extraction. The raffinate phase is discarded.

[0048] S6: Back extraction and concentration: On the basis of complex extraction, add the same volume mass concentration of 5% Na2CO3 solution to the extractant loaded with the extractant, transfer it into a separatory funnel, plug it tightly and shake it thoroughly to allow it to fully separate into layers. The upper layer is the organic phase after back extraction, and the lower layer is the back extraction liquid loaded with the target solute. The upper layer is discarded after recovering the complex extraction solvent.

[0049] S7: Crystallization and drying: After the stripping in S6 is completed, the pH value is quickly adjusted to 5.0 with dilute hydrochloric acid, and the insoluble matter is removed by suction. The filtrate is concentrated at a certain temperature until crystals begin to precipitate in the solution, and the solution is transferred to a low temperature of 4°C and placed for crystal growth for 16 hours. After the crystallization is completed, the filter cake is filtered to obtain a filter cake, and the filter cake is top-washed with ice water until the washing liquid is colorless, and the top-washing is stopped. The filter cake is dried and crushed, and then dried to constant weight to obtain 134.52 g of pure dihydroquercetin. The content measured by HPLC is 96.95%, and the process recovery rate is 91.05%.

[0050] Example 1

[0051] A plant extract composition containing dihydroquercetin is prepared by the following method: 15g of dihydroquercetin, 5g of Swertia japonica extract, 5g of Coleus forskohlii root extract and 6g of Alpinia serrata extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0052] Example 2

[0053] A plant extract composition containing dihydroquercetin is prepared by the following method: 15g of dihydroquercetin, 8g of Swertia japonica extract, 6g of Coleus forskohlii root extract and 5g of Alpinia serrata extract are weighed and added into 420 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0054] Example 3

[0055] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin, 5 g of Swertia japonica extract, 6 g of Coleus forskohlii root extract and 6 g of Alpinia serrata extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0056] Example 4

[0057] A plant extract composition containing dihydroquercetin is prepared by the following method: 18g of dihydroquercetin, 5g of Swertia japonica extract, 6g of Coleus forskohlii root extract and 5g of Alpinia serrata extract are weighed and added into 420 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0058] Example 5

[0059] A plant extract composition containing dihydroquercetin is prepared by the following method: 15g of dihydroquercetin, 8g of Swertia japonica extract, 5g of Coleus forskohlii root extract and 6g of Alpinia serrata extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0060] Comparative Example 1

[0061] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin, 5 g of Coleus forskohlii root extract and 6 g of Alpinia serrata extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0062] Comparative Example 2

[0063] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin, 5 g of Swertia japonica extract and 5 g of Alpinia cerasifera extract are weighed, added into 400 parts of hexyldecanol, and stirred to dissolve to obtain the composition.

[0064] Comparative Example 3

[0065] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin, 8 g of Swertia japonica extract and 6 g of Coleus forskohlii root extract are weighed and added into 400 parts of hexyldecanol, and the mixture is stirred and dissolved to obtain the composition.

[0066] Comparative Example 4

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

[0068] Comparative Example 5

[0069] A plant extract composition containing dihydroquercetin is prepared by the following method: 15 g of dihydroquercetin and 5 g of Coleus forskohlii root extract are weighed, added into 400 parts of hexyldecanol, and stirred to dissolve to obtain the composition.

[0070] 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.

[0071] Test 1: Water solubility test of composition

[0072] 5.00 g of 91% dihydroquercetin, 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. After being fully stirred, the mixture was allowed to stand at room temperature to observe the solubility of the sample. The test results are as follows:

[0073] Table 1. Water solubility test results of Examples 1-5 and Comparative Examples 1-5

[0074]

[0075]

[0076] Compared with single 91% dihydroquercetin, the plant extract composition containing dihydroquercetin disclosed in the present invention has better water solubility and is easier to be used as a raw material in cosmetic formulas, thereby being able to better exert the biological activity of dihydroquercetin.

[0077] Experiment 2: DPPH free radical scavenging experiment

[0078] 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:

[0079] DPPH removal rate (%) = [1-(As-Ar) / A0] × 100%

[0080] 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.

[0081] Table 2. Reaction solution composition

[0082] 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

[0083] 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 five 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.

[0084] Table 3. Results of the experimental study on the scavenging of DPPH free radicals by the composition (0.1% concentration)

[0085] sample DPPH clearance rate (%) Example 1 96.04 Example 2 95.89 Example 3 94.52 Example 4 97.05 Example 5 95.41 Comparative Example 1 74.50 Comparative Example 2 76.91 Comparative Example 3 79.12 Comparative Example 4 74.03 Comparative Example 5 69.16

[0086] Test 3: ABTS free radical scavenging test

[0087] 1.ABTS + Preparation of standard stock solution:

[0088] Accurately weigh 0.0384g ABTS + , dissolve in distilled water and dilute to 10mL volumetric flask to make ABTS + The concentration is 7mmol·L -1 ; Accurately weigh 0.0066g K2S2O8 (potassium sulfate), dissolve it in distilled water and dilute it to a 10mL volumetric flask to make the concentration of K2S2O8 2.45mmol·L -1 Then, mix the two solutions in a brown bottle (1:1 mixture) and place in the dark at room temperature overnight (12-16h) to obtain ABTS. + Solution.

[0089] ABTS + The solution was phosphate buffered saline (10 mmol / L -1 , pH 7.4) (dilution factor 27 or 28) so that its absorbance at 30°C and 734nm wavelength is 0.700±0.020, thus obtaining ABTS + Working fluid.

[0090] 2. Preparation of phosphate buffer (10mmol / L, pH=7.4):

[0091] According to the pharmacopoeia method, take 1.36g of potassium dihydrogen phosphate, add 79mL of 0.1mol / L sodium hydroxide solution, and dilute with water to 200ml.

[0092] 3. Take 1 mL of sample solution diluted appropriately into test tubes, add 3 mL of ABTS+ working solution, mix thoroughly, store at room temperature and away from light for 10 min, measure the absorbance at 734 nm, and use anhydrous ethanol as a blank control. + The clearance rate was calculated as follows:

[0093] Where D is ABTS + Clearance rate (%); Ai is 1mL sample solution + 3mL ABTS + The absorbance value of the mixed solution of the solution; Aj is the absorbance value of the mixed solution of 1mL sample solution + 3mL anhydrous ethanol; Ac is the absorbance value of 1mL anhydrous ethanol + 3mL ABTS + The absorbance value of the solution mixture.

[0094] Table 4. Experimental results of the ability of the composition (0.05% concentration) to scavenge ABTS free radicals

[0095] sample ABTS clearance rate (%) Example 1 95.82 Example 2 95.54 Example 3 95.27 Example 4 94.76 Example 5 95.64 Comparative Example 1 74.15 Comparative Example 2 75.54 Comparative Example 3 76.65 Comparative Example 4 74.84 Comparative Example 5 68.37

[0096] The ABTS free radical scavenging rate reflects its antioxidant capacity to a certain extent. The higher the scavenging rate, the better the antioxidant degree of the sample and the better the antioxidant effect. Therefore, the antioxidant effect of the composition can be judged by studying its ability to scavenge ABTS free radicals. As shown in Table 3, at a test concentration of 0.1%, the scavenging rates of ABTS 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 formula is reasonably compatible and can synergize to exert its good antioxidant effect.

[0097] Test 4: Cellular reactive oxygen species (ROS) inhibition test

[0098] 1. Cytotoxicity Assay

[0099] 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.

[0100] 2. Cellular reactive oxygen species (ROS) inhibition test

[0101] 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.

[0102] 3. Result calculation and judgment

[0103] 3.1 Result calculation

[0104] ROS inhibition rate (%) = 100-(MFI sample-MFI background) / (MFINC-MFI background)*100

[0105] 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.

[0106] 3.2 Experimental Results

[0107] 3.2.1 Cytotoxicity results

[0108] 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 shown in Table 5. 。

[0109] Table 5. Cytotoxicity test results of samples

[0110]

[0111] 3.2.2 Reactive oxygen species (ROS) test results

[0112] 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 0.1014% and 0.0500% had inhibition rates of ROS caused by UVB of 65.34% and 54.34%, respectively, which were significantly different from the negative control (P < 0.0001). The test results are shown in Table 4.

[0113] Table 6. Cellular reactive oxygen species (ROS) test results 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 "****"

[0114] Experiment 5: Human efficacy test

[0115] 1. Test Principle

[0116] 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.

[0117] 2. Test methods

[0118] Test sample: dihydroquercetin plant extract composition skin care lotion (formula as shown in Table 7, sample group*); control sample: blank matrix skin care lotion (formula as shown in Table 7, except that the dihydroquercetin plant extract composition is not added, the rest is the same, control group).

[0119] Table 7. Dihydroquercetin skin care lotion ingredients

[0120]

[0121]

[0122] 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.

[0123] 3. Test equipment and parameters are shown in Table 8

[0124] Table 8. Human efficacy test equipment and parameters

[0125]

[0126] 4. Test results and conclusions

[0127] The following results were obtained by using test samples and control samples for 28 days on 33 healthy Chinese subjects. After 28 days of using the test samples: the water content of the skin stratum corneum improved by 28.07%, which was significantly compared with the control samples; the transepidermal water loss rate improved by 26.67%, which was significantly compared with the control samples; the skin elasticity R2 improved by 35.71%, which was significantly compared with the control samples.

[0128] 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.

[0129] In summary, the present invention scientifically combines dihydroquercetin, Swertia japonica extract, Coleus forskohlii root extract, Alpinia cerasifera 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.

[0130] 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, characterized in that: The following steps are involved: S1. Drying and crushing raw materials: air-drying larch roots, peeling them, crushing them and passing them through a sieve, collecting the sieved materials to obtain crushed larch root powder; S2. Raw material pretreatment: Put the crushed larch root powder into a micro-thermal explosion reaction tank, introduce gas, and keep the reaction tank at a certain speed for 1 hour, and blow gas every 15 minutes. After the treatment, the pretreated larch root powder is obtained; S3, raw material extraction: the larch root powder pretreated in S2 is mixed with an extraction solvent, placed in a high-voltage pulse electric field for low-temperature extraction, filtered to obtain a first filtrate and a first filter residue, wherein the intensity of the high-voltage pulse electric field is 15-25 kV / cm, the first filter residue is mixed with the extraction solvent for extraction, filtered to obtain a second filtrate and a second filter residue, the second filter residue is mixed with the extraction solvent for extraction, filtered to obtain a third filtrate and a third filter residue, and the first filtrate, the second filtrate and the third filtrate are combined to obtain a larch extract; S4, water precipitation centrifugation: the extract obtained in S3 is concentrated under reduced pressure, and deionized water is added to the concentrate after ethanol is recovered, and the concentrate is stirred and allowed to stand, and the water precipitation liquid is centrifuged to obtain a centrifuge and centrifuge residue, and the centrifuge residue is discarded, and the centrifuge liquid is reserved; S5, complex extraction: adjust the pH of the centrifuge obtained in S4 and mix it with the complex extractant at a volume ratio of 1:1, transfer it into a separatory funnel, plug it tightly and shake it thoroughly to allow it to fully separate into layers. The upper layer is the complex extraction organic phase loaded with the target solute, and the lower layer is the raffinate phase after extraction. The raffinate phase is discarded; S6, stripping and concentration: on the basis of complex extraction, a certain amount of stripping agent is added to the extractant loaded with the extractant, and the mixture is transferred to a separatory funnel, which is plugged tightly and shaken sufficiently to allow the mixture to be fully separated. The obtained upper layer is the organic phase after stripping, and the lower layer is the stripping liquid loaded with the target solute. The upper layer is discarded after the complex extraction solvent is recovered; S7, crystallization and drying: after the stripping in S6 is completed, the pH is quickly adjusted to weak acidity with dilute hydrochloric acid, and the insoluble matter is removed by suction. The filtrate is concentrated at a certain temperature until crystals begin to precipitate in the solution, and then the solution is placed under low temperature conditions for crystal growth. After the crystallization is completed, the filter cake is filtered to obtain a filter cake, and the filter cake is top-washed with ice water until the washing liquid is colorless, and the top-washing is stopped. The filter cake is dried and crushed, and then dried to constant weight to obtain pure dihydroquercetin.

2. The extraction method according to claim 1, characterized in that The mesh size of the sieve in S1 is 50-100 meshes.

3. The extraction method according to claim 1, characterized in that The gas introduced into the S2 is SO2, the amount of the gas introduced is 0.1%-5% (w / w), and the working temperature of the reaction tank is 40-70°C.

4. The extraction method according to claim 1, characterized in that The extraction solvent in S3 is aqueous alcohol containing a surfactant, the surfactant contained in the extraction solvent is cetylpyridinium hydrobromide, 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 amount of the pretreated medicinal material (mL: g), and the temperature of the low-temperature extraction is 0-5°C.

5. The extraction method according to claim 1, characterized in that The amount of water added to S4 is 1 to 3 times the weight of the original medicinal material. The temperature of the stirring and standing is 2-10°C and the standing time is 0.5-5h.

6. The extraction method according to claim 1, characterized in that The pH in S5 is adjusted to pH=5-7, the complexing extractant is composed of sodium pyrophosphate, the diluent is sulfonated kerosene, and the volume fraction of the complexing agent sodium pyrophosphate added to the diluent is 1%-10%.

7. The extraction method according to claim 1, characterized in that The stripping agent in S6 is Na2CO3 solution with a mass concentration of 0.1%-5%, and the volume of the stripping agent is 0.5-5 times the volume of the extract.

8. The extraction method according to claim 1, characterized in that The dilute hydrochloric acid in S7 is adjusted to pH=4.0-6.0, the temperature for growing crystals in S7 is 2-10°C, the growing time is 8-24h, and the drying method is vacuum low-temperature drying, spray drying or vacuum freeze drying.

9. A plant extract composition containing dihydroquercetin 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 Swertia japonica extract, 2-10 parts of Coleus forskohlii root extract, 2-10 parts of Alpinia serrata 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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