A method for preparing fine components and monomer compounds of Calyx tenuifolia and application thereof in cosmetics

Abstract

The present invention belongs to the technical field of cosmetics, and specifically discloses a fine-leaved calyx flower extract and its application in the preparation of cosmetics, wherein the extract refers to fine-leaved calyx flower refined components and 7 monomer compounds. The refined components are the components with the strongest antioxidant activity, tyrosinase inhibition activity and matrix metalloproteinase inhibition activity in fine-leaved calyx flower; the 7 monomer compounds are obtained by macroporous resin column chromatography, methanol-water gradient elution, hydroxypropyl dextran gel column chromatography, octadecylsilane bonded silica gel column chromatography, and high performance liquid chromatography acetonitrile water gradient elution of the ethanol extract of fine-leaved calyx flower. The fine-leaved calyx flower extract of the present invention can be applied to skin anti-oxidation, whitening and anti-aging, and is further used to prepare a cosmetic composition containing the extract.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and specifically relates to refined components of Calyx spathulata, Calyx spathulata monomer compounds, and applications of the two in preparing whitening and anti-aging cosmetics. Background Art

[0002] Aging, long-term exposure to adverse environments, malnutrition, fatigue and other factors can lead to reduced metabolic function of cells and skin tissues, reduced blood circulation in the skin, loss of skin moisture, accumulation of structural and functional defects, endocrine disorders, and reduced skin's ability to repair itself, ultimately causing changes in skin appearance, physiological function and texture. The most obvious changes of skin aging include the appearance of fine lines and wrinkles, loss of skin elasticity, sagging, loosening, discoloration, and the appearance of spots.

[0003] Oxygen free radicals produced during cell metabolism can cause cumulative damage to intracellular biomacromolecules, causing cell aging and loss of proliferation capacity. This imbalance of oxidation and antioxidant systems is considered to be an important cause of skin aging. Tyrosinase, also known as polyphenol oxidase, is an oxidoreductase widely found in animals, plants, microorganisms and the human body. It is a key rate-limiting enzyme in melanin synthesis and is closely related to the occurrence of diseases such as excessive melanin deposition such as freckles and brown spots in the human body. In recent years, its application in the fields of medicine, beauty, food and environmental protection has attracted widespread attention at home and abroad. Most of the whitening agents on the market are based on the inhibition of tyrosinase to achieve whitening effects.

[0004] The activation of matrix metalloproteinases (MMPs) in the body caused by external factors can lead to excessive degradation of collagen and elastin that support the skin structure, resulting in aging symptoms such as skin wrinkling and inelasticity. During normal development, MMPs are responsible for degrading extracellular matrices (ECM) such as collagen, elastin, gelatin, matrix glycoproteins and proteoglycans present in bones, cartilage and skin. Among them, matrix metalloproteinase-1 (MMP-1) is a calcium and zinc-dependent vertebrate collagenase subclass. It is a member of the MMP family that can efficiently degrade type I and type III interstitial collagens and is involved in a series of physiological and pathological processes such as tumors, cardiovascular diseases, multiple sclerosis and skin aging. By inhibiting the expression or activity of MMP-1, the specific degradation of extracellular matrix components can be reduced. Therefore, eliminating excessive free radical damage in life activities, inhibiting collagenase activity and thus reducing the degradation and loss of collagen are considered to be effective in delaying skin aging.

[0005] With the growing demand for cosmetics for whitening, anti-oxidation and anti-aging, plant-derived cosmetics have been widely used in the cosmetics industry. These active ingredients come from various parts of plants, such as roots, stems, leaves, bark, seeds, fruits, callus, protoplasts and meristems, and are added to cosmetics in different forms such as pure or semi-pure components, solid or liquid extracts or derivatives. Plant cosmetics are deeply loved by consumers for their natural, mild and high safety characteristics. Creating plant-derived cosmetics has become an inevitable trend in the development of the cosmetics industry.

[0006] Cuphea hyssopifolia Kunth is a plant of the genus Cuphea of ​​the Lythraceae family. It is one-year or perennial, with opposite leaves, dark green leaves, evergreen and shiny all year round. The flowers are exquisite, blooming while budding, with a long flowering period, and have strong ornamental value and landscaping functions. At present, it has been introduced and cultivated in Guangdong, Guangxi, Yunnan, Fujian, Beijing and other provinces (autonomous regions, municipalities directly under the central government) in China and widely used in landscaping. The present invention is a further research and development result obtained by the inventor on the basis of his previous research results (for details, please refer to the prior application CN102525860A), mainly involving the fine components and monomer compounds of Cuphea hyssopifolia Kunth and its application in anti-oxidation, whitening and anti-aging. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide an effective ingredient of Calyx spatholobifolia with antioxidant, whitening and anti-aging effects. The effective ingredient is obtained by extraction and separation of Calyx spatholobifolia, and includes 7 compounds, and the components of the 7 compounds are clear.

[0008] To achieve the above object, the present invention first obtains the refined components of Cuphea hyssopifolia Kunth, and the preparation method of the refined components is as follows:

[0009] The dried spatholobifolia and 80% (v / v) ethanol solvent are mixed at a solid-liquid ratio of 1 g: 5 mL to 1 g: 50 mL (can be added in multiple times), and the mixture is allowed to stand at 20 to 30° C. for a total of 9 to 21 days (preferably 15 days). After the extraction, the mixture is separated by filtration, and the filtrate is concentrated and dried under reduced pressure at 50° C. to obtain a crude extract of the spatholobifolia;

[0010] The crude extract of Calyx spurredii is diluted with water, extracted with petroleum ether and ethyl acetate solvents in sequence at 20-30°C, and the obtained extract part and aqueous liquid part are concentrated and dried under reduced pressure at 50°C to obtain the extraction components and water layer components of the crude extract of Calyx spurredii, specifically the petroleum ether layer component, the ethyl acetate layer component and the water layer component.

[0011] The method for preparing the refined components of Calyx tenuifolia also includes the following steps:

[0012] (1) Column chromatography primary separation:

[0013] The aqueous layer components obtained by extracting the crude extract of Calyx spathulata with petroleum ether and ethyl acetate were further purified by D101 macroporous resin column chromatography: gradient elution with methanol-water (v / v) 0:100, 50:50, and 100:0 was performed in sequence, and the eluate was concentrated and dried under reduced pressure at 50°C to obtain component I, component II and component III in sequence.

[0014] (2) Secondary separation by column chromatography (relative to the further separation in the prior application CN102525860A):

[0015] The component II collected after the first separation by column chromatography in step (1) is further purified by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography, and eluted with methanol-water (70:30, v / v) solution and chloroform-methanol (50:50, v / v) solution in sequence, and the methanol-water (70:30, v / v) solution and chloroform-methanol (50:50, v / v) solution are eluted for 3-5 column volumes respectively. After the elution is completed, the two parts of elution solution are respectively recovered. The part eluted by methanol-water (70:30, v / v) solution is the effective component, and the part eluted by chloroform-methanol (50:50, v / v) solution is the impurity component;

[0016] Elution with the above column volume of eluent can maximize the acquisition of effective components and remove impurities.

[0017] (3) Column chromatography three times separation:

[0018] The methanol-water (70:30, v / v) solution eluted portion collected after the secondary separation of the column chromatography was purified by octadecylsilane bonded silica gel column chromatography, with a flow rate of 20 mL / min, and gradient elution with methanol-water solution. The specific elution conditions were as follows: the volume ratios of methanol and water in the methanol-water solution used for gradient elution were 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0, respectively, and the eluent of each volume ratio was 5 column volumes. Among them, the eluents obtained by eluting the methanol-water solution with a volume ratio of methanol to water of 20:80 and 40:60 were collected and mixed respectively, and the obtained mixed solution was concentrated and dried under reduced pressure at 50°C to obtain the fine components of the fine-leaved calyx flower.

[0019] The present invention also provides 7 monomer compounds contained in the fine components of the fine-leaved calyx spurred flower, and the method for obtaining the 7 monomer compounds is as follows:

[0020] The fine components of the fine-leaved spurred flower obtained by the three separations of the column chromatography were purified by high performance liquid chromatography, and acetonitrile aqueous solution with an acetonitrile concentration of 10% (v / v) to 30% (v / v) was used for gradient elution, and the flow rate of the mobile phase was 4 mL / min, and 7 monomer compounds in the fine components of the fine-leaved spurred flower were collected and identified as: Myricitrin (1), Tellimoside (2), Myricetin 3-O-(6"-O-galloyl)-β-D-glucopyranoside (3), Myricetin (4), Desmanthin 1 (5), Penta-O-galloyl-β-D-glucose (6), Quercetin 3-O-β-(2"-O-galloylxylopyranoside)(7); the contents of the above 7 monomer compounds in the refined fraction are 1 (8.75%), 2 (7.33%), 3 (6.42%), 4 (18.75%), 5 (7.42%), 6 (7.83%), and 7 (8.75%), respectively, and the total amount accounts for 65.25% (greater than 50%) of the dry weight of the refined fraction.

[0021] The present invention also shows that the fine components of the fine-leaved calyx and the seven monomeric compounds contained therein have significant effects in anti-oxidation and inhibition of tyrosinase and matrix metalloproteinase-1 (MMP-1). Among them, in terms of anti-oxidation, the antioxidant IC 50 The values ​​were all in the range of 1μg / mL-5μg / mL, indicating that they all had good antioxidant capacity; in terms of inhibiting tyrosinase and matrix metalloproteinase-1, the refined components and 7 monomer compounds of the fine-leaved calyx showed good activity, among which the refined components of the fine-leaved calyx were better.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. For the first time, the monomer compounds Myricitrin(1), Tellimoside(2), Myricetin 3-O-(6”-O-galloyl)-β-D-glucopyranoside(3), Myricetin(4), Desmanthin 1(5), Penta-O-galloyl-β-D-glucose(6), and Quercetin 3-O-β-(2”-O-galloylxylopyranoside)(7) were isolated and identified from the extract of the flower of the genus Calyx spathulata.

[0024] 2. For the first time, it was disclosed that the refined components of the fine-leaved calyx flower have significant activity in anti-oxidation and inhibition of tyrosinase and matrix metalloproteinase-1, and the results of in vitro activity screening, animal models and human efficacy research were obtained.

[0025] 3. For the first time, it was disclosed that among the seven monomer compounds, Myricetin 3-O-(6”-O-galloyl)-β-D-glucopyranoside(3) and Quercetin 3-O-β-(2”-O-galloylxylopyranoside)(7) have significant activity in inhibiting tyrosinase and matrix metalloproteinase-1, Desmanthin 1(5) has significant activity in inhibiting matrix metalloproteinase-1, and Quercetin 3-O-β-(2”-O-galloylxylopyranoside)(7) has antioxidant properties.

[0026] 4. The refined components of the fine-leaved calyx spurred flower reported in the present invention have a very clear composition, mainly containing 7 monomer compounds, and the total amount of the 7 monomer compounds exceeds 50% of the dry weight of the refined components of the fine-leaved calyx spurred flower, and has good development prospects.

[0027] 5. According to the enzymatic experiments of the inventors, the refined components of the fine-leaved calyx flower show stronger tyrosinase and matrix metalloproteinase-1 inhibitory activity than the monomeric compounds of the fine-leaved calyx flower, which can reduce the possible adverse reactions of the drug and improve the safety of the product. In addition, the manufacturing cost of the refined components of the fine-leaved calyx flower is low, and the cost performance is superior to the monomeric compounds.

[0028] 6. The refined components and seven monomer compounds of the fine-leaved calyx spurredii of the present invention are all obtained by extraction, separation and purification from plants. The entire production process is chemical-free and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the HPLC analysis spectrum of the fine components of the flower of Calyx tenuifolia;

[0030] Figure 2 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 1 prepared in Example 2;

[0031] Figure 3 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 1 prepared in Example 2;

[0032] Figure 4 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 2 prepared in Example 2;

[0033] Figure 5 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 2 prepared in Example 2;

[0034] Figure 6 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 3 prepared in Example 2;

[0035] Figure 7 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 3 prepared in Example 2;

[0036] Figure 8 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 4 prepared in Example 2;

[0037] Fig. 9 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 4 prepared in Example 2;

[0038] Fig.10 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 5 prepared in Example 2;

[0039] Fig.11 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 5 prepared in Example 2;

[0040] Fig.12 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 6 prepared in Example 2;

[0041] Fig.13 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 6 prepared in Example 2;

[0042] Fig.14 This is the hydrogen spectrum (600 MHz, MeOD) of the monomer compound 7 prepared in Example 2;

[0043] Fig.15 The carbon spectrum (150 MHz, MeOD) and DEPT diagram of the monomer compound 7 prepared in Example 2;

[0044] Fig.16 To identify 7 major monomer compounds and 6 trace components in the refined components of the flower of the tenuifolia calyx; Fig.17 This is the UV absorption spectrum of 7 main monomer compounds EJH-1~7 in the refined components of the flower of Calyx tenuifolia;

[0045] Fig.18 This is the UV absorption spectrum of 6 trace components A to F in the fine components of the flower of the tenuifolia calyx;

[0046] Fig.19 The scavenging effects of the refined components and seven monomer compounds of the flower of Calyx tenuifolia on ABTS free radicals;

[0047] Fig. 20 The inhibitory effects of the refined components of the flower of the genus Calyx tenuifolia and α-arbutin on tyrosinase;

[0048] Fig.21 The inhibitory effects of refined components of Semen spathulata and EJH-4, EJH-5, EJH-6 and EJH-7 on matrix metalloproteinase-1;

[0049] Fig. 22 This is the H&E staining picture of the guinea pig back skin tissue;

[0050] Fig.23 This is the Fontana-Masson silver staining of the guinea pig back skin tissue;

[0051] Fig.24 A skin efficacy test of a facial cream containing 1% of the fine components of the flower of the genus Calyx;

[0052] Fig.25 The effect of a face cream containing 1% of the fine components of Calyx spurredans on various skin indicators of a certain subject. The characteristic score refers to the percentage that exceeds that of people of the same age. DETAILED DESCRIPTION

[0053] In order to make the purpose and content of the invention of this application more clear, the present invention is further described below in conjunction with embodiments.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] The raw materials of Calyx spathulata used in the following examples are all the above-ground parts of fresh Calyx spathulata picked in Kunming, Yunnan.

[0057] The reduced pressure concentration in the following examples was all achieved by a rotary evaporator.

[0058] Example 1: Preparation method of fine components of Calyx tenuifolia

[0059] 1. Ethanol extraction of Sesamum indica

[0060] Take the raw material of Calyx spurredii, dry it, take 25 kg and crush it, use 80% v / v ethanol to soak and extract three times at 20-30°C, the material-liquid ratio (the material is 25 kg, the liquid is the volume of 80% v / v ethanol added each time) is 1g:10mL, 1g:6mL, 1g:6mL, the extraction time is 7 days, 5 days, 3 days, the three filtrates are combined, the filtrate is concentrated under reduced pressure at 50°C until there is no ethanol taste, and dried to obtain 1.4 kg of total extract (i.e. crude extract).

[0061] 2. Extraction

[0062] The 1.4 kg total extract obtained in step 1 was completely dissolved in pure water with a volume of 2.5 L, and then extracted with equal volumes of petroleum ether and ethyl acetate at 20-30° C., and the petroleum ether and ethyl acetate were extracted 3 times each; the combined petroleum ether extraction part, ethyl acetate extraction part and aqueous part were concentrated and dried under reduced pressure at 50° C. to obtain a petroleum ether layer component, an ethyl acetate layer component and an aqueous layer component, respectively.

[0063] 3. Column chromatography primary separation

[0064] The water layer component obtained in step 2 is subjected to column chromatography using a 5kg D101 macroporous adsorption resin column, and the specific steps are as follows: first, the pre-treated macroporous adsorption resin is filled into a glass chromatography column (diameter 14cm, height 100cm) by wet packing to ensure uniformity and absence of bubbles; the column is pressed with distilled water until the column height remains unchanged, and the addition of liquid is stopped; when the liquid level drops to about 5mm above the column surface, the liquid outlet is closed to obtain a D101 macroporous adsorption resin column; the water layer component is completely dissolved with pure water, and then slowly added to the column along the column wall, and the liquid outlet is opened to control the liquid outlet rate to prevent the liquid level from dropping too fast; when all samples are added to the chromatography column, when the liquid level drops to about 5mm above the column surface, the column wall is rinsed with a small amount of distilled water, and the rinsing is repeated 2-3 times until the liquid on the column surface is clear and colorless, and then the column is slowly filled with distilled water, and the liquid outlet is closed at the same time, and it is allowed to stand for 12h. Reach adsorption equilibrium; then, open the liquid outlet, control the liquid outlet rate, and then elute step by step with methanol-water solutions with methanol concentrations of 0% v / v (i.e. pure water), 50% v / v and 100% v / v (i.e. anhydrous methanol), the volume of the methanol-water solution used each time is 30L, and the flow rate is 100mL / min; the eluates obtained by eluting with the above-mentioned methanol-water solutions of various concentrations are concentrated and dried under reduced pressure at 50°C to obtain 650g of an elution component of a methanol-water solution with a methanol concentration of 0% v / v (referred to as component I, the same below), 135g of an elution component of a methanol-water solution with a methanol concentration of 50% v / v (referred to as component II, the same below), and 142g of an elution component of a methanol-water solution with a methanol concentration of 100% v / v (referred to as component III, the same below).

[0065] 4. Secondary separation by column chromatography

[0066] After dissolving 135g of component II obtained in step 3 in anhydrous methanol, further purify it by column chromatography on 500g of hydroxypropyl dextran gel (Sephadex LH-20), and eluting it in turn with a methanol-water solution with a volume ratio of methanol to water of 70:30 and a chloroform-methanol solution with a volume ratio of chloroform to methanol of 50:50, each of which is eluted with 4 column volumes, and the eluent is recovered after the elution is completed. Among them, the eluent of the methanol-water solution with a volume ratio of methanol to water of 70:30 is concentrated and dried under reduced pressure at 50°C to obtain 75g of solid, which is the effective component, and the eluent of the chloroform-methanol solution with a volume ratio of chloroform to methanol of 50:50 is concentrated and dried under reduced pressure at 50°C to obtain the impurity component.

[0067] Elution with the above column volume of eluent can maximize the acquisition of effective components and remove impurities.

[0068] 5. Column chromatography three-time separation

[0069] After the 75 g of active ingredient obtained in step 4 was completely dissolved, it was purified by column chromatography using 500 g of octadecylsilane bonded silica gel (Lichroprep), specifically, the active ingredient was completely dissolved in anhydrous methanol and then added to a 100 g reverse phase silica gel (Lichroprep RP-18, particle size 40-70 μm, Fuji, Japan) column. The sample was adsorbed in an evaporating dish of Silysia Chemical Company, air-dried in a fume hood, ground into powder, and then separated at 20-30°C using a medium-pressure glass column (Sweden Biotage Company, C-615 pump manager, C-605 pump module, C-660 fraction collector, RP-18 column) at a flow rate of 20 mL / min and a pressure of 15 MPa; gradient elution with methanol-water solution of different concentrations, the specific elution conditions: the volume ratio of methanol to water was 0:100, 20:80, 40:60, 60:40, 80:20, 100:0, and the volume ratio of methanol-water solution used for elution was 2.5 L, and the eluent was recovered. The eluents collected with the volume ratio of methanol to water of 20:80 and 40:60 were mixed, and then concentrated and dried under reduced pressure at 50°C to obtain the refined components of the fine-leaved calyx spur flower, with a mass of 45 g.

[0070] Comparative Example 1: Comparison of extraction rates of refined components obtained from the flower of the genus Calyx tenuifolia by different extraction methods

[0071] The extraction method is:

[0072] 100 g of the raw material of Calyx spathulata was dried and crushed, and extracted with 600 mL of 60% v / v ethanol-water solution at 20-30° C. The extraction was performed 3 times, and the extraction time of each time was different, which was 5 days, 3 days, and 1 day respectively. After the extraction was completed, the subsequent processing process was as follows:

[0073] 1. Combine the three filtrates, concentrate the filtrates under reduced pressure at 50°C until there is no ethanol smell, and dry to obtain the total extract. Add the total extract into pure water and dissolve it completely to 150mL, extract it with petroleum ether 3 times (add 150mL petroleum ether each time) and ethyl acetate 3 times (add 150mL ethyl acetate each time); combine the obtained extracts, concentrate and dry them under reduced pressure at 50°C to obtain the petroleum ether layer, ethyl acetate layer and water layer components of the fine-leaved calyx.

[0074] 2. The aqueous layer component was further purified by D101 macroporous resin (50 g) column chromatography and gradient eluted with methanol-water (0:100, 50:50, 100:0, v / v) solution. The volume of each gradient eluent was 200 mL. The eluents of three gradients were collected and concentrated and dried under reduced pressure at 50°C to obtain component I, component II and component III, respectively.

[0075] 3. Dissolve component II in anhydrous methanol and further purify it by Sephadex LH-20 (100 g) column chromatography. Elution is performed using a methanol-water (70:30, v / v) solution with a volume of 300 mL. After recovering the eluate, concentrate and dry it under reduced pressure at 50°C to obtain the effective ingredient.

[0076] 4. The active ingredient was purified by octadecylsilane bonded silica gel (20 g) column chromatography and eluted with a water-methanol solution gradient. The specific elution conditions were as follows: the volume ratio of methanol to water in the methanol-water solution was 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0, respectively. The volume of each volume ratio concentration of methanol-water solution was 300 mL. The eluted parts of the methanol-water solution with a volume ratio of 20:80 and 40:60 were collected and combined, and concentrated and dried under reduced pressure at 50°C to obtain the fine components of the fine-leaved calyx flower (such as Figure 1 , Figure 1 This is the preliminary HPLC analysis spectrum of the refined component, which was obtained by dissolving the refined component in anhydrous methanol and then injecting it, and eluting it with a gradient of 10% v / v acetonitrile aqueous solution at a constant speed to 30% v / v for 15 min).

[0077] The above extraction method was repeated, wherein the extraction time each time was 7 days, 5 days, and 3 days respectively.

[0078] The above extraction method was repeated, wherein the extraction time each time was 9 days, 7 days, and 5 days respectively.

[0079] Repeat the above extraction method, wherein the ethanol concentration is 80% v / v, and the extraction time of each time is 5 days, 3 days, and 1 day respectively;

[0080] Repeat the above extraction method, wherein the ethanol concentration is 80% v / v, and the extraction time of each time is 7 days, 5 days, and 3 days respectively;

[0081] The above extraction method was repeated, wherein the ethanol concentration was 80% v / v, and the extraction time for each time was 9 days, 7 days, and 5 days, respectively.

[0082] Repeat the above extraction method, wherein the ethanol concentration is 100% v / v, and the extraction time of each time is 5 days, 3 days, and 1 day respectively;

[0083] Repeat the above extraction method, wherein the ethanol concentration is 100% v / v, and the extraction time of each time is 7 days, 5 days, and 3 days respectively;

[0084] The above extraction method was repeated, wherein the ethanol concentration was 100% v / v, and the extraction time for each time was 9 days, 7 days, and 5 days, respectively.

[0085] Table 1 Extraction rate of fine components of Calyx spathulata by different extraction methods

[0086]

[0087] As shown in Table 1, when the ethanol-water solution with an ethanol concentration of 80% v / v was used as the extraction solvent, the extraction rate was the highest; the extraction rates were the highest when the extraction time was 9 days, 7 days, 5 days (21 days in total) and 7 days, 5 days, 3 days (15 days in total), but considering the extraction time, the optimal extraction process was 80% v / v ethanol concentration and 7 days, 5 days, 3 days (15 days in total).

[0088] Example 2: Preparation method of monomeric compounds in the refined components of Calyx tenuifolia

[0089] 120 mg of fine components of Calyx spurredii (prepared in Example 1, the same below, no further description) were taken, dissolved in anhydrous methanol and then eluted with acetonitrile-water solution in a high performance liquid chromatography column (column: Agilent Zorbax SB-C18 column, specification: 9.4 mm×250 mm), the concentration of acetonitrile was 10-30% v / v, the elution time was 50 minutes, the flow rate of the mobile phase was 4 mL / min, wherein the acetonitrile-water ratio was uniformly changed from 10:90 to 30:70 from 0 to 40 min, and the acetonitrile-water ratio was 30:70 from 40 to 50 min; the eluate was concentrated and dried in vacuum to prepare monomer compound 1 (abbreviated as EJH-1, yellow powder, 10.5 mg, t R=33.5min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 27:73), monomer compound 2 (abbreviated as EJH-2, yellow powder, 8.8mg, t R =32.6min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 26:74), monomer compound 3 (abbreviated as EJH-3, yellow powder, 7.7mg, t R =30.4min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 25:75), monomer compound 4 (abbreviated as EJH-4, yellow powder, 22.5mg, t R =44.6min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 30:70), monomer compound 5 (abbreviated as EJH-5, yellow powder, 8.9mg, t R =46.3min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 30:70), monomer compound 6 (abbreviated as EJH-6, yellow powder, 9.4mg, t R =36.2min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 28:72) and monomer compound 7 (abbreviated as EJH-7, yellow powder, 10.5mg, t R =38.5min, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 29:71).

[0090] Structural identification of EJH-1-7: The powdered compounds EJH-1-7 prepared in Example 2 were dissolved in 0.5 mL of deuterated methanol, respectively, and transferred to a nuclear magnetic resonance tube using a 200 μL pipette. The hydrogen and carbon spectra (e.g., 600 MHz, Bruker, Germany) were detected on a nuclear magnetic resonance instrument. Figure 2-15 ). The spectral information and NMR signal attribution of the above compounds EJH-1 to 7 are as follows:

[0091] Myricitrin(1): yellow powder, 1 H NMR (600MHz, CD3OD, δ, ppm, J / Hz): 6.95 (s, H-2' / H-6'), 6.36 (d, J=2.1, H-8), 6.20 (d, J=2.1, H-6), 5.32 (d, J=1.6, H-1"), 4.22 (dd,J=3.4,1.6,H-2"), 3.79(dd,J=9.6,3.4,H-3"), 3.34(t,J=9.6,H-4"), 3.52(dd,J=9.6,6.2,H-5"), 0.96(d,J=6.2,H-6"). 1313C NMR (150 MHz, δ, ppm): 178.3 (C-4), 164.5 (C-7), 161.7 (C-5), 158.0 (C-2), 157.1 (C-9), 145.5 (C-3' / 5'), 136.5 (C-4'), 134.9 (C-3), 120.5 (C-1'), 108.2 (C-2' / 6'), 104.5 (C-10), 102.2 (C-1”), 98.4 (C-6), 93.3 (C-8), 71.9 (C-4”), 70.7 (C-3”), 70.6 (C-5”), 70.5 (C-2”), 16.3 (C-6”).

[0092] Tellimoside (2): Yellow powder, 1 1H NMR (600 MHz, CD3OD, δ, ppm, J / Hz): 7.78 (d, J = 2.2, H-2'), 7.56 (dd, J = 8.4, 2.2, H-6'), 6.89 (s, H-2"' / 6"'), 6.81 (d, J = 8.4, H-5'), 6.37 (d, J = 2.0, H-8), 6.17 (d, J = 2.0, H-6), 5.10 (d, J = 7.7, H-1"), 4.32 (dd, J = 11.0, 6.9, H-6"), 4.20 (dd, J = 11.0, 6.0, H-6"), 3.88 (d, J = 3.7, H-4"), 3.85 (m, dd, J = 9.7, 7.7 H-2"), 3.80 (dd, J = 6.9, 6.0 H-5"), 3.60 (dd, J = 9.7, 3.7, H-3"). 13 13C NMR (150 MHz, δ, ppm): 178.1 (C-4), 166.6 (C-7”'), 164.6 (C-7), 161.4 (C-5), 157.6 (C-2), 156.9 (C-9), 148.5 (C-4'), 144.9 (C-3”' / 5”'), 144.3 (C-3'), 138.4 (C-4”'), 134.3 (C-3), 121.6 (C-1'), 121.4 (C-6'), 119.6 (C-1”'), 116.4 (C-2'), 114.7 (C-5'), 108.7 (C-2”' / 6”'), 104.1 (C-10), 104.1 (C-1”), 98.6 (C-6), 93.4 (C-8), 73.6 (C-5”), 73.1 (C-3”), 71.6 (C-2”), 68.6 (C-4”), 62.3 (C-6”).

[0093] Myricetin 3-O-(6”-O-galloyl)-β-D-glucopyranoside(3): 1 H NMR(600MHz,CD3OD,δ,ppm,J / Hz):7.27(s,H-2′ / H-6′),7.14(s,H-2" ' / 6"'),6.32(d,JS2.2,H-6),6.15(d,JS2.2,H-8),5.78(d,J7.9,H-1 "),5.45(dd,J9.9,7.9,H-2"),3.96(dd,J3.5,1.2,H-4"),3.85(dd, J9.9,3.5,H-3"),3.70(d,J6.1,H-6"),3.61(td,J6.1,1.2,H-5'). 13 C NMR(150MHz,δ,ppm): 177.5(C-4), 166.9(C-7”'), 164.3(C-7), 161.7(C-9), 156.8(C-5),15 6.6(C-2), 144.9(C-3' / 5'), 144.8(C-3”' / 5”'), 138.4(C-4”'), 136.5(C-4'), 133.9(C-3), 120.6(C-1'), 120.2(C-1”'), 109.2(C-2”' / 6”'), 108.4(C-2' / 6'), 104.5(C-10), 99.9(C-1). ”), 98.2(C-8), 93.1(C-6), 76.1(C-5”), 73.2(C-2”), 72.1(C-3”), 69.1(C-4”), 60.6(C-6”).

[0094] Myricetin(4):Acrylic, 1 H NMR (600MHz,CD3OD,δ,ppm,J / Hz):7.34(s,H-2' / H-6'),6.38(d,J2.2,H-6),6.18(d,J2.2,H-8). 13 C NMR (150MHz, δ, ppm): 175.9(C-4), 164.2(C-7), 161.1(C-5), 156.8(C-9), 146.6(C-2), 145.3(C-3' / 5). '), 136.0(C-3), 135.5(C-4'), 121.7(C-1'), 107.1(C-2' / 6'), 103.1(C-10), 97.8(C-8), 93.0(C-6).

[0095] Desmanthin 1(5):Entertainment, 1 H NMR(600MHz,CD3OD,δ,ppm,J / Hz):7.08(s,H-2"' / 6"'),6.98(s,H-2' / H-6'),6.36(d,JC2.2,H-6),6.19(d,JC2.2,H-8),5.63(dd,J)3.4 ,1.8H-2"),5.51(d,JC1.8,H-1"),4.05(dd,J9.1,3.4,H-3"),3 (dq,JC9.1,5.8,H-5"),3.48(t,JC9.1,H-4"),1.04(d,J5.8,H-6"). 13 C NMR(150MHz,δ,ppm): 177.9(C-4), 166.1(C-7”'), 164.5(C-7), 161.8(C-9), 158.1(C -2), 157.1(C-5), 145.5(C-3' / 5'), 145.1(C-3”' / 5”'), 138.5(C-4”'), 136.6(C-4'), 134.2(C-3), 120.4(C-1'), 119.8(C-1”'), 108.9(C-2”' / 6”'), 108.2(C-2' / 6'),104. 5(C-10), 99.1(C-1”), 98.2(C-8), 93.3(C-6), 72.5(C-4”), 72.1(C-2”), 70.8(C-5”), 69.3(C-3”) ,16.4(C-6”).

[0096] Penta-O-galloyl-β-D-glucose(6); 1 H NMR(600MHz,CD3OD,δ,ppm,J / Hz):7.02,6.96,6.88,6.86,6.80(5×2' / 6'),6.14(d,JS8.3Hz,H-1),5.81(t,JS9.9Hz,H-3),5.51(t,J6). 9.9Hz,H-4),5.48(dd,J9.9,8.3Hz,H-2),4.41(dd,J12.1,1.9Hz,H-6),4.32(dd,J4.3,1.9Hz,H-5),4.28(dd,J12.1,4.3Hz,H-6). 13C NMR (150MHz, δ, ppm): 166.6, 165.9, 165.6, 165.5, 164.8 (5×C-7'), 145.2, 145.1, 145.1,145.0,144.9(5×C-3' / 5'),139.4,139.0,138.9,138.7,138.6(5×C-4'),11 9.7,119.0,118.9,118.8,118.3(5×C-1'),109.3,109.1,109.1,109.0,109.0(5× C-2' / 6'),92.4(C-1),73.0(C-5),72.7(C-3),70.8(C-2),68.4(C-4),61.8(C-6).

[0097] Quercetin 3-O-β-(2”-O-galloylxylopyranoside)(7): yellow powder, 1 H NMR (600MHz, CD3OD, δ, ppm, J / Hz): 7.61 (d, J=2.3Hz, H-2'), 7.53 (dd, J=8.4, 2.3Hz, H-6'), 7.13 (s, H-2"', H-6"'), 6.84 (d, J=8.4Hz, H-5' ), 6.18 (d, J = 2.2Hz, H-6), 6.36 (d, J = 2.2Hz, H-8), 5.55 (d, J = 5.9Hz, H-1”), 5.46 (dd, J = 7.6, 5.9Hz, H-2”), 3.50 (m, H-4”), 3.89 (m, H-5”). 13 C NMR (150MHz, δ, ppm): 177.8 (C-4), 166.3 (C-7"'), 164.4 (C-7), 161.7 (C-5), 157.1 (C-2), 156 .9(C-9),148.4(C-4'),145.0(C-3”' / 5”’),144.7(C-3’),138.6(C-4”’),133.7(C-3),121.9( C-1'),121.5(C-6'),120.0(C-1"'),115.7(C-2'),114.9(C-5'),109.1(C-2 / 6"'),104.4(C- 10),99.5(C-6),98.4(C-1”),93.2(C-8),72.5(C-2”),70.4(C-3”),67.5(C-4”),64.8(C-5”).

[0098] According to the results of the above tests, the structural formulas of the obtained compounds are confirmed as follows:

[0099] Example 3 Analysis of the contents of seven monomeric compounds in the fine components of Calyx tenuifolia and identification of the types of trace components

[0100] As shown in Example 2, 120 mg of fine components of Calyx spathulata were separated by HPLC to obtain Myricitrin (EJH-1, 10.5 mg), Tellimoside (EJH-2, 8.8 mg), Myricetin3-O-(6"-O-galloyl)-β-D-glucopyranoside (EJH-3, 7.7 mg), Myricetin (EJH-4, 22.5 mg), Desmanthin1 (EJH-5, 8.9 mg), Penta-O-galloyl-β-D-glucose (EJH-6, 9.4 mg), Quercetin 3-O-β-(2"-O-galloylxylopyranoside) (EJH-7, 10.5 mg); the content percentages of the above 7 monomer compounds in the refined fraction are: EJH-1 is 8.75%, EJH-2 is 7.33%, EJH-3 is 6.42%, EJH-4 is 18.75%, EJH-5 is 7.42%, EJH-6 is 7.83%, and EJH-7 is 8.75%. The total mass of the 7 monomer compounds accounts for 65.25% (greater than 50%) of the dry weight of the refined fraction. The ultraviolet absorption spectrum of the product obtained after HPLC separation of the refined fraction was analyzed, as shown in FIG. Fig.16 and 17 As shown in the figure, the UV absorption spectra of EJH-1, 2, 3, 4, 5, and 7 are basically the same, with the maximum UV absorption peaks at around 260 and 360 nm, and their types are myricetin; the UV absorption spectrum of EJH-6 is different from the above, with the maximum UV absorption peaks at 220 and 280 nm, and its type is retrogallic tannin. In addition, the UV absorption spectra of trace components in the refined fractions were analyzed, such as Fig.16 and 18 As shown, it was found that the maximum ultraviolet absorption peaks of trace components A, B, C, E, and F were basically around 260 and 360 nm, which was the same as the ultraviolet absorption of the above-mentioned myricetin compounds; it was found that the maximum ultraviolet absorption peak of trace component D was 220 and 280 nm, which was the same as the ultraviolet absorption of the above-mentioned retrogallantan compounds. Therefore, it was determined that trace components A, B, C, E, and F were myricetin compounds, and trace component D was a retrogallantan compound.

[0101] Example 4: Study on the Antioxidant Activity of the Extract of Calyx julibrissin (the extract of Calyx julibrissin refers to component I, component III, refined component, EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, and EJH-7. The same below)

[0102] 7.4mmol / L ABTS solution and 2.6mmol / L K2S2O8 solution were mixed in equal volumes and kept at room temperature in the dark for 16 hours to prepare ABTS free radical stock solution. The ABTS free radical stock solution was diluted with phosphate buffer solution (pH=7.4, 10mmol / L), and the dilution was placed in a multifunctional microplate reader (Spark 10M, Tecan) and the absorbance at a wavelength of 734nm reached 0.70±0.05 to prepare ABTS working solution. 200μL ABTS working solution was mixed with 10μL phosphate buffer solution (pH=7.4, 10mmol / L), and the absorbance at 734nm was measured on a multifunctional microplate reader (Spark10M, Tecan) and set as A0. Component I (prepared in Example 1, the same below, no further description), component III (prepared in Example 1, the same below, no further description), refined component (prepared in Example 1, the same below, no further description), EJH-1-7 (prepared in Example 2, the same below, no further description), positive control drug Trolox and α-arbutin were dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL mother solution, which was then diluted with water to mother solution dilution solutions of different concentrations, and the mother solution dilution solutions of different concentrations were used as sample solutions. Next, 10 μL of the sample solution was mixed with 200 μL of ABTS working solution: for the positive drug Trolox, the final concentrations of the obtained mixed solutions were 0.9, 1.79, 3.58, 7.15, 10.73 and 14.30 μg / ml, respectively; similarly, for each of component I, component III, refined component, EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, EJH-7, and positive drug α-arbutin, the final concentrations of the obtained mixed solutions were 0.78, 1.56, 3.13, 6.25, 12.50 and 25.00 μg / mL, respectively. The mixed solution was allowed to stand at room temperature for 10 minutes, and then placed in a multifunctional microplate reader (Spark 10M, Tecan) to measure the absorbance at a wavelength of 734 nm, which was set as Ai. At the same time, 10 μL of the sample solution was mixed with 200 μL of pH=7.4, 10 mmol / L phosphate buffer solution, and placed in a multifunctional microplate reader (Spark 10M, Tecan) to measure the background absorbance at a wavelength of 734 nm as Aj. Three parallel samples were set for each sample concentration.

[0103] The formula for calculating the scavenging rate of the sample to ABTS free radicals is as follows:

[0104] ABTS free radical scavenging rate (%) = [1-(Ai-Aj) / A0] × 100%

[0105] like Fig.19 As shown in the figure, within the test concentration range, the extract of the fine-leaved calendula showed good scavenging effect on ABTS free radicals, among which the refined fraction had the best scavenging effect, and among EJH-1 to 7, EJH-4 (myricetin) and EJH-5 had the best scavenging effect. Graphpad Prism 8.0 software was used for nonlinear fitting to calculate the IC value of the extract of the fine-leaved calendula for scavenging ABTS free radicals. 50 The half maximal inhibitory concentration is shown in Table 2. Except for component I obtained by pure water extraction, the antioxidant IC values ​​of the other extracts of the spatholobifolia were 50 The values ​​were all in the range of 1μg / mL-5μg / mL. At the same time, the IC 50 The IC value of α-arbutin is 4.75μg / mL. 50 The value was 1.52 μg / mL. The above experimental results show that component I, component III, refined components and EJH-1-7 all have good antioxidant capacity, and most of them are better than Trolox and comparable to α-arbutin.

[0106] Table 2 The scavenging ability of various extracts of Calyx spathulata including monomeric compounds on ABTS free radicals

[0107]

[0108]

[0109] Example 5: Study on the inhibition of tyrosinase activity by the extract of Cuphea spathulata

[0110] 1. Solution preparation

[0111] Preparation of phosphate buffer solution (0.2M, pH=6.8): Accurately weigh 2.84g of Na2HPO4 and 2.4g of NaH2PO4, dissolve them in purified water to 100mL respectively, mix the two solutions in equal volumes and adjust the pH to 6.8; Preparation of L-DOPA solution: Accurately weigh 3.95mg of L-DOPA, dissolve it in 10mL of phosphate buffer solution (0.2M, pH=6.8) to prepare a 2mM L-DOPA solution. Preparation of tyrosinase solution: Accurately weigh 1mg of tyrosinase (enzyme activity is 500U / mg), dissolve it in 5mL of phosphate buffer solution (0.2M, pH=6.8) to prepare a tyrosinase solution with an enzyme activity of 100U / mL. Sample solution preparation: Dissolve each of the extracts of Calyx spathulata in DMSO to prepare a 3 mg / mL solution; the positive drug kojic acid for control was prepared in a phosphate buffer solution (0.2 M, pH = 6.8) to prepare a 1 mg / mL solution.

[0112] 2. Tyrosinase activity assay

[0113] Four test groups were set up: sample group A1, sample negative control group A2, enzyme standard group B1 and enzyme negative control group B2; 3 parallels were set for each sample, and 200 μL reaction system was prepared according to Table 3. The corresponding volume of phosphate buffer solution, sample solution to be tested (final concentration 0.3 mg / mL), tyrosinase solution (final concentration 20 U / mL) and reaction substrate L-DOPA (final concentration 1.2 mM) were added to the 96-well plate in sequence, incubated in a microplate constant temperature oscillator at 37 ° C for 30 minutes, and then the absorbance at 475 nm was measured in a multifunctional microplate reader (SPARK10M, TECAN). The inhibition rate of the fine components of the fine-leaved calyx flower on tyrosinase was calculated as follows:

[0114] Tyrosinase inhibition rate (%) = [1-(A1-A2) / (B1-B2)] × 100%

[0115] Table 3 Tyrosinase activity inhibition test reaction system preparation (volume / μL)

[0116]

[0117] Table 4 Inhibition rate of tyrosinase by extract of Calyx spathulata

[0118]

[0119] As shown in Table 4, the inhibition rates of 0.3 mg / mL positive drugs kojic acid and α-arbutin on tyrosinase were 97.9±1.3% and 54.6±2.8%, respectively. Under the same concentration conditions, except for component I, the other types of extracts in the extract of Calyx spathulata showed good tyrosinase inhibition activity, and the inhibition rates were in the range of 45%-75%. Among them, the refined component had the best inhibitory effect on tyrosinase, reaching 70.5±1.9%, which was better than each monomer compound.

[0120] In order to further evaluate the in vitro inhibitory activity of the refined fraction on tyrosinase, the fraction and the positive drug α-arbutin were diluted with phosphate buffer solution to different concentration gradients. The reaction system was prepared with reference to Table 3, and the final concentrations were 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg / mL. The absorbance of the solution at 475 nm was measured after 30 minutes of reaction (measured in a multifunctional microplate reader (SPARK 10M, TECAN)). Fig. 20 As shown in the figure, α-arbutin and refined fractions showed dose-dependent inhibitory effects on tyrosinase activity. The half-inhibitory concentration IC of refined fractions and α-arbutin on tyrosinase was calculated by nonlinear fitting using Graphpad Prism 8.0 software. 50 The above experimental results show that the inhibitory effect of the fine components of the flower of Calyx tenuifolia on tyrosinase is nearly twice that of the positive control drug α-arbutin.

[0121] Example 6: Study on the inhibition of matrix metalloproteinase-1 activity by the extract of Cuphea spathulata

[0122] The inhibitory effect of the extract of Calyx spathulata on the activity of matrix metalloproteinase-1 was determined using a matrix metalloproteinase-1 inhibitor screening kit (Abcam, ab118973). Matrix metalloproteinase-1 can hydrolyze specific fluorescence resonance energy transfer (FRET) substrates to release fluorescent groups, and its fluorescence signal can be detected at Em / Ex=480 / 530nm. Effective MMP-1 inhibitors can block the hydrolysis of substrates and reduce the fluorescence signal generated per unit time.

[0123] First, the inhibitory activity of the extract of the fine-leaved calyx flower on matrix metalloproteinase-1 was preliminarily screened. According to Table 5, a 100 μL reaction system was prepared, and three test groups were set up: enzyme control group, component group I, component group III, refined component, and EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, and EJH-7 groups. The corresponding volumes of MMP-1 solution (i.e., MMP-1 enzyme stock solution) and test buffer (enzyme control group) were added to the black 96-well plate in sequence. The final concentrations of component I, refined component, and component III were all 1 mg / mL, and the final concentrations of EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, and EJH-7 were all 0.5 mg / mL. Two parallels were set for each sample concentration. The prepared solution was shaken and mixed, and the substrate was added after incubation at 37°C for 5 minutes. The initial fluorescence signal F0 of each sample well was then measured using a multifunctional microplate reader (SPARK 10M, TECAN), and the change of the fluorescence signal within 2.5 hours was monitored, and the fluorescence signal F1 of the sample after 2.5 hours of reaction was recorded. The relative enzyme activity after the sample was acted on was calculated according to the following formula, where the initial fluorescence signal of the enzyme control group was set to F0e, and the fluorescence signal after 2.5 hours of reaction was set to F1e; the initial fluorescence signal of each sample group was set to F0s, and the fluorescence signal of the sample solution after 2.5 hours of reaction was set to F1s.

[0124]

[0125] Table 5 Preparation of the reaction system for determining the activity of matrix metalloproteinase-1 (unit: μL / well)

[0126]

[0127] At the initial screening concentration of 1 mg / mL, the inhibition rates of component I, component III, and refined components on the activity of matrix metalloproteinase-1 were greater than 50%. At the initial screening concentration of 0.5 mg / mL, the inhibition rates of EJH-4, 5, 6, and 7 on the activity of matrix metalloproteinase-1 were greater than 50%, while the inhibition rates of EJH-1 to 3 on the activity of matrix metalloproteinase-1 were less than 50%.

[0128] To further determine the IC values ​​of these extracts for inhibiting MMP-1 activity 50 , set different concentration gradients. Similarly, according to Table 5, 100 μL of the reaction system was prepared so that the final concentrations of the refined component solutions were 0.1, 1, 10, 100 and 1000 μg / mL, respectively, and the final concentrations of the solutions of each of EJH-4, 5, 6, and 7 were 31.2, 62.5, 125, 250 and 500 μg / mL, respectively.

[0129] The results are as follows Fig.21As shown in the figure, the refined fractions and EJH-4, 5, 6 and 7 showed a dose-dependent inhibitory effect on the activity of matrix metalloproteinase-1. At a concentration of 100 μg / mL, the inhibition rate of the refined fraction on the activity of matrix metalloproteinase-1 reached 64% (**P < 0.01). The half-inhibitory concentration IC of the refined fraction on matrix metalloproteinase-1 was calculated by nonlinear fitting using Graphpad Prism 8.0 software. 50 The half-inhibitory concentration IC of EJH-4, 5, 6, and 7 on matrix metalloproteinase-1 was 55.0 μg / mL. 50 The results of the above experiments showed that the refined components of the flower of Calyx tenuifolia had a better inhibitory effect on matrix metalloproteinase-1 than the monomer compounds.

[0130] Example 7: Acute toxicity test in mice

[0131] The experiment was conducted with Kunming mice. 400 mg of component I, component III, and refined component were accurately weighed, 0.5 mL of DMSO was added to dissolve, and the sample was diluted to 200 mg / mL with ultrapure water. Blank group, DMSO control group, component I group, component III group, and refined group group were set up respectively. There was one mouse in the blank group and DMSO control group, and two mice in each experimental group (half male and half female). The mice were fasted for 12 hours before the experiment and had free access to water. The weight of the fasted mice was weighed, and physiological saline (0.4 mL), 25% DMSO, component I, component III, and refined component were given by oral gavage at 2000 mg / kg according to the upper-lower method. The changes in the respiratory system, urinary system, digestive system, and nervous system such as tremor, convulsion, salivation, drowsiness, and coma were observed for 48 hours. At the end of the observation period, all surviving animals were weighed and euthanized, and gross anatomical observations and histopathological examinations were performed.

[0132] Through the above experiments, it can be obtained that mice were continuously observed for 48 hours after being injected with a larger dose of 2000mg / kg. The mice grew well, moved normally, had good fur gloss, and did not show symptoms of poisoning such as tremor, salivation, convulsion, kicking legs, drowsiness, and coma. At the end of the experiment, a gross anatomical examination was performed, and no obvious pathological changes were observed in the organs. There was no significant difference in body weight between the blank group and the experimental group. The acute toxicity experiment showed that component I, component III, and refined component had no obvious animal toxicity at a dose of 2000mg / kg, and the maximum tolerated dose (MTD) of mice was greater than 2000mg / kg.

[0133] Table 6 Acute toxicity results in mice

[0134]

[0135] Example 8: The refined components of the flower of the herb effectively inhibit the melanin deposition in guinea pig skin caused by ultraviolet light

[0136] 20 guinea pigs weighing 200-250g with a large area of ​​brown-yellow hair on their backs were selected and purchased from Liaoning Changsheng Biotechnology Co., Ltd. All guinea pigs were kept in an environment with a temperature of 25℃±2℃ and a relative humidity of 55%±15%, with 12h / 12h alternating light and dark. All guinea pigs were free to consume water and guinea pig-specific feed, and were adaptively fed for 7 days. One day before ultraviolet radiation, each guinea pig had its brown hair area selected, and the longer hair was shaved with a razor, and then the depilatory cream was evenly applied to the hair-cut area. After 5 minutes, the depilatory cream was washed off with warm water, and the action was gentle, leaving 3*3cm of the guinea pig's back skin exposed. 2 This study was approved by the Animal Ethics Committee of South-Central University for Nationalities.

[0137] 20 guinea pigs were randomly divided into 4 groups, 5 in each group. 5 were used as blank group, and the rest of the groups were irradiated with UVB light for 1 hour each time. The radiation dose was 0.6 J / cm on the 1st to 7th day. 2 , Day 6-20, daily radiation dose 0.3J / cm 2 , irradiation was continued for 20 days, and the guinea pig test area skin was depilated with depilatory cream before each irradiation.

[0138] The specific groups are as follows:

[0139] Blank control group: the same dose of 0.3% CMC-Na was applied but no UV irradiation was performed;

[0140] Model group: the same dose of 0.3% CMC-Na was applied and UVB was irradiated for 30 min;

[0141] Positive drug group: 1 mg of β-arbutin containing 0.3% CMC-Na was applied and irradiated with UVB for 30 min;

[0142] Experimental group: 2 mg of fine fraction of Calyx tenuifolia containing 0.3% CMC-Na was applied and irradiated with UVB for 30 min;

[0143] The model was established and the drugs were administered for 20 consecutive days, and the degree of damage to the back skin caused by UVB radiation was observed. After the experiment, the guinea pigs were euthanized, and the back skin tissue was taken and fixed in 4% paraformaldehyde fixative for subsequent experiments.

[0144] H&E routine staining: embed and slice in paraffin as usual; dewax the slices to water; stain in hematoxylin dye for 5 minutes, rinse once with running water; separate with 1% hydrochloric acid alcohol differentiation solution for 15 seconds, rinse twice with running water; blue with 1% ammonia water for 30 seconds; stain with 1% eosin dye for 2 minutes, rinse with tap water; dehydrate with 70%, 80%, 90%, 95%, 100% (all volume percentages) ethanol in turn, two minutes each, and air dry; put in xylene to make it transparent, and seal with neutral gum.

[0145] Fontana-Masson silver staining method was used to detect the melanin content in each layer of the skin: According to the conventional tissue pathology detection method, the guinea pig skin tissue was fixed with 4% paraformaldehyde after the experiment, dehydrated, paraffin-embedded, and sliced. The skin sections were stained with Masson-Fontana silver staining method, and the melanin staining results were observed under a microscope to determine the changes in the distribution of melanin induced by ultraviolet light in the fine components of the flower of the spathula.

[0146] The results of H&E staining are as follows Fig. 22 As shown in the figure, the epidermis of the skin tissue of the blank group was intact, the collagen fibers were closely arranged, and the number of hair follicles was abundant. The skin tissue of the model group was significantly thickened, the dermis could be infiltrated with lymphocytes, and the number of hair follicles was reduced. The epidermis of the skin tissue of guinea pigs in the positive control group was thicker; there were a large number of lymphocytes infiltrating in the dermis, the collagen fibers were sparsely arranged, and the number of hair follicles was small. The epidermis of the skin tissue of guinea pigs in the experimental group was thicker, with a large number of spinous cells with watery degeneration, loose and lightly stained cytoplasm; there were a small number of granulocytes infiltrating in the dermis, the collagen fibers were closely arranged, and the hair follicles were abundant. The above results show that ultraviolet rays can cause thickening of guinea pig skin tissue, infiltration of inflammatory cells, damage to collagen fibers, and reduction of hair follicles. Compared with the positive control β-arbutin, the fine components of the fine-leaved calyx can inhibit the thickening of skin tissue, promote the proliferation of collagen fibers, and promote the proliferation of hair follicles.

[0147] Melanin content test results Fig.23 As shown, the melanin content of the back skin of guinea pigs in the blank control group was very low, mainly concentrated in the basal layer, and only a small amount of melanin existed in the spinous layer; the melanin content of the model group increased significantly after ultraviolet irradiation, and a large amount of melanin was expressed in the basal layer and spinous layer, but mainly concentrated in the basal layer; the melanin content of the positive control group was relatively high, mainly concentrated in the basal layer, and the content in the spinous layer was relatively low; the distribution of melanin in the experimental group was significantly reduced compared with the model group, mainly concentrated in the basal layer, and the content in the spinous layer was relatively low. The above results show that ultraviolet rays can promote the increase of melanin secretion in guinea pig skin tissue, and both β-arbutin and fine components of calyx spathulata can reduce melanin production. The fine components of calyx spathulata are better than β-arbutin, and have broad application prospects in whitening cosmetics.

[0148] Example 9 Safety Test

[0149] According to the "Technical Specifications for Safety of Cosmetics" (2015 edition), a human skin closed patch test was conducted. A total of 30 volunteers were recruited, with random gender and age ranging from 18 to 60 years old, for patch tests. Experimental method: 1% of fine components of calyx spurredii, 2% ethoxydiglycol, 1% PEG-40 hydrogenated castor oil, 2% pentanediol and 94% pure water were mixed (the above percentages are all mass percentages), heated to dissolve, cooled to room temperature, and prepared into a solution containing 1% of fine components of calyx spurredii. 200 μL of the above solution containing 1% of fine components of calyx spurredii was placed in the patch tester, and the control well was a solution without 1% of fine components of calyx spurredii. The patch tester was applied to the upper arm of the subject with non-irritating tape, and gently pressed with the palm of the hand to evenly apply it to the skin for 24 hours. After removing the patch tester, observe the skin reaction after the indentation disappears. Score according to the skin reaction grading table.

[0150] Table 7 Skin reaction grading table

[0151]

[0152] The test results showed that 1% of the fine components of the spatholobifolia had no adverse reactions on human skin. Only one person developed slight erythema, while the other 29 people had no obvious discomfort.

[0153] Table 8 Skin safety test results

[0154]

[0155] Example 10 Skin efficacy test of facial cream containing fine components of Calyx spathulata

[0156] A total of 30 volunteers were recruited, with random gender and age ranging from 18 to 60 years old, and left and right face comparison tests were performed. A blank cream was used on the left side, and a cream containing 1% fine components of Calyx spurredii was used on the right side. The products were used according to the usual skin care habits, and the products were applied after normal cleansing, once in the morning and once in the evening. Other skin care products and cosmetics were prohibited during this period. The products were used continuously for 15 days, and facial images were taken using VISIA-CR (CANFIELD, America) before use, on the 7th day of use, and on the 14th day of use. The instrument combines the most advanced digital photography technology and has multiple light sources (standard light, UV light, cross-polarized light, parallel polarized light), which improves the visibility of skin analysis.

[0157] Table 9 Cream formula

[0158]

[0159] The aqueous phase and oil phase raw materials were mixed separately and heated to 85° C., and then the aqueous phase was added to the oil phase and emulsified for 3 minutes to obtain a blank cream and a cream containing 1% of the fine components of Calyx tenuifolia.

[0160] Depend on Fig.24 (Skin efficacy test of face cream containing 1% fine components of Calyx spathulata) and Fig.25 The results shown in (The effect of a face cream containing the fine components of Calyx spurredii on various skin indicators of a subject) show that the face cream containing 1% of the fine components of Calyx spurredii can significantly improve the overall condition of facial skin, such as surface spots, brown spots (deep hidden spots) and other problems; at the same time, the texture (reflecting the smoothness and fullness of the skin), UV spots (reflecting the potential pigment spots under the epidermis) and porphyrin (reflecting skin oil) are significantly improved. The effective improvement rate for UV spots and brown spots in 14 days reached 60%, and none of the subjects showed any allergies or discomfort. This shows that the fine components can reduce UV spots and brown spots in the deep layer of the skin, effectively reduce spots on the surface of the skin, and have a whitening effect; and promote skin smoothness, reduce oil, and have a good anti-photoaging effect.

Claims

1. A method for extracting a mixture from the flower of the herbaceous plant, characterized in that: The steps include: Step (1), mixing dried Calyx spatholobifolia with 60-100% v / v ethanol at a solid-liquid ratio of 1 g:5 mL to 1 g:50 mL, extracting at 20-30° C. for a total of 9-21 days, separating by filtration after extraction, and concentrating the filtrate under reduced pressure and drying at 50° C. to obtain a crude extract of Calyx spatholobifolia; Step (2), diluting the crude extract of Calyx spatholobifolia with water, extracting with petroleum ether and ethyl acetate solvents in sequence at 20-30° C., concentrating the obtained extract and aqueous liquid portion under reduced pressure and drying at 50° C., to obtain an extraction component and an aqueous layer component of the crude extract of Calyx spatholobifoliafolia, specifically a petroleum ether layer component, an ethyl acetate layer component and an aqueous layer component; Step (3), primary separation by column chromatography: extracting the crude extract of Calyx tenuifolia with petroleum ether and ethyl acetate in turn to obtain the aqueous layer component, further purifying it by D101 macroporous resin column chromatography: gradient elution was performed with a methanol-water solution having a volume ratio of methanol to water of 0:100, 50:50, and 100:0, and the eluate was concentrated and dried under reduced pressure at 50° C. to obtain component I, component II, and component III in turn; Step (4), secondary separation by column chromatography: further purify the component II collected after the primary separation by column chromatography in step (3) by hydroxypropyl dextran gel column chromatography, and sequentially elute with a methanol-water solution with a volume ratio of methanol to water of 70:30 and a chloroform-methanol solution with a volume ratio of chloroform to methanol of 50:50, and elute with 3-5 column volumes of the methanol-water solution with a volume ratio of methanol to water of 70:30 and the chloroform-methanol solution with a volume ratio of chloroform to methanol of 50:50, respectively. After the elution is completed, the two parts of the elution solution are respectively recovered; wherein, the part eluted with the methanol-water solution with a volume ratio of methanol to water of 70:30 is the effective component, and the part eluted with the chloroform-methanol solution with a volume ratio of chloroform to methanol of 50:50 is the impurity component; Step (5), three-time separation by column chromatography: the methanol-water solution eluted with a volume ratio of methanol to water of 70:30 collected after the secondary separation by column chromatography in step (4) is purified by octadecylsilane bonded silica gel column chromatography at a flow rate of 20 mL / min, with a methanol-water solution gradient elution, and the specific elution conditions are as follows: the volume ratios of methanol to water in the methanol-water solution used for gradient elution are 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0, respectively, and the eluent of each volume ratio is 5 column volumes; wherein the eluents obtained by eluting with a methanol-water solution with a volume ratio of methanol to water of 20:80 and 40:60 are collected separately and then mixed, and the obtained mixed solution is concentrated and dried under reduced pressure at 50°C to obtain the refined components of the fine-leaved calyx spur flower, which is the mixture; The mixture comprises 7 monomer compounds, which are Myricitrin, Tellimoside, Myricetin 3-O-(6"-O-galloyl)-β-D-glucopyranoside, Myricetin, Desmanthin 1, Penta-O-galloyl-β-D-glucose, Quercetin 3-O-β-(2"-O-galloylxylopyranoside), the structural formulas of the obtained 7 monomer compounds are as follows:

2. The method according to claim 1, characterized in that: In the step (1), 80% v / v ethanol is used for immersion extraction at 20-30° C. for 3 times for a total of 15 days.

3. The method according to claim 2, characterized in that: In the step (1), the material-liquid ratio of the dried Calyx spathulata raw material and the 80% v / v ethanol added during each extraction is 1 g:5 mL to 1 g:10 mL.

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