CN116459175A - Preparation method of fine components and monomer compounds of hedyotis diversifolia and application of fine components and monomer compounds in cosmetics - Google Patents

Abstract

The invention belongs to the technical field of cosmetics, and particularly discloses an extract of hedysarum gracile and application thereof in preparing cosmetics, wherein the extract refers to an exquisite component of the hedysarum gracile and 7 monomer compounds. The delicate component is the component with the strongest antioxidant activity, tyrosinase activity inhibition and matrix metalloproteinase activity inhibition in the calyx parviflora; the 7 monomer compounds are obtained by macroporous resin column chromatography, methanol-water solution gradient elution, hydroxypropyl sephadex column chromatography, octadecyl silane bonded silica gel column chromatography and high performance liquid chromatography acetonitrile water solution gradient elution of the ethanol extract of the hedysarum calyx. The extract of the calyx parviflora of the invention can be applied to the aspects of skin oxidation resistance, whitening and aging resistance, and is further used for preparing a cosmetic composition containing the extract.

Description

Preparation method of fine components and monomer compounds of hedyotis diversifolia and application of fine components and monomer compounds in cosmetics

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to an delicate component of a hedysarum gracile, a hedysarum gracile monomer compound and application of the delicate component and the monomer compound in preparation of whitening and anti-aging cosmetics.

Background

Age aging, prolonged exposure to adverse conditions, malnutrition, fatigue, etc., can lead to reduced metabolic function of cells and skin tissues, reduced skin blood circulation, loss of skin moisture, accumulation of structural and functional defects, endocrine disorders, reduced ability of the skin to repair itself, and ultimately to changes in the appearance, physiological function and texture of the skin. The most obvious changes in skin aging include appearance of fine lines, wrinkles, loss of skin elasticity, sagging, loss of color, appearance of stains, and the like.

Oxygen radicals generated during cellular metabolism cause cumulative damage to intracellular biomacromolecules, causing cell aging and loss of proliferative capacity, and imbalance of this oxidative and antioxidant system is considered to be an important cause of skin aging. Tyrosinase, also called polyphenol oxidase, is an oxidoreductase widely existing in animals, plants, microorganisms and human bodies, is a key rate-limiting enzyme for melanin synthesis, and is closely related to the occurrence of diseases such as excessive melanin deposition, including freckle, brown spot, and the like of human bodies. In recent years, the application of the modified starch in the fields of medicine, cosmetology, food, environmental protection and the like has attracted wide attention at home and abroad. Most of the whitening agents on the market are based on inhibition of tyrosinase, so as to achieve the whitening effect.

Activation of Matrix Metalloproteinases (MMPs) in vivo by external factors can cause excessive degradation of collagen and elastin supporting skin structures, and thus skin shrinkage, inelastic and other aging symptoms can occur. MMPs are responsible for degrading the extracellular matrix (ECM) of collagen, elastin, gelatin, matrix glycoproteins, and proteoglycans present in bone, cartilage, and skin during normal development. Among them, matrix metalloproteinase-1 (MMP-1) is a kind of vertebrate collagenase subclass dependent on calcium and zinc, is a member of MMP family capable of degrading type I and type III interstitial collagens with high efficiency, and is involved in a series of physiological and pathological processes such as tumor, cardiovascular disease, multiple sclerosis and skin aging. By inhibiting MMP-1 expression or activity, specific degradation of extracellular matrix components can be reduced. Therefore, it is considered that eliminating excessive free radical damage in vital activities, inhibiting collagenase activity and thus reducing degradation loss of collagen is effective in delaying skin aging.

With the increasing demand of consumers for cosmetics for whitening, resisting oxidation and delaying skin aging, plant-derived cosmetic efficacy ingredients have been widely used in the cosmetic industry. These active ingredients originate from various parts of the plant, such as roots, stems, leaves, bark, seeds, fruits, calli, protoplasts and meristems, and are incorporated into the cosmetic in the form of pure or semi-pure components, solid or liquid extracts or derivatives, etc. Plant cosmetics are deeply favored by consumers due to the characteristics of nature, mildness, high safety and the like, and the development of plant source cosmetics has become a necessary trend in the cosmetic industry.

The calyx Seu fructus Physalis (Cuphea hyssopifolia Kunth) is a plant of the genus calyx Seu fructus Physalis of the family Lythraceae, and has advantages of one year or many years, green leaf color, evergreen and luster. The flower is exquisite, the bud is pregnant, the flower bloom is performed, the flowering period is long, and the ornamental value and the landscaping function are high. At present, the provinces of Guangdong, guangxi, yunnan, fujian, beijing and the like in China are introduced and cultivated and widely applied to landscaping. The invention is a further research and development result obtained by the inventor on the basis of the previous research result (see the prior application CN102525860A for details), and mainly relates to an delicate component, a monomer compound of the hedyotis microphylla and application of the compound in the aspects of antioxidation, whitening and anti-aging.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide the effective components of the hedyotis diversifolia with the effects of resisting oxidation, whitening and resisting aging, wherein the effective components are obtained by extracting and separating the hedyotis diversifolia, and the effective components comprise 7 compounds, and the components of the 7 compounds are definite.

To achieve the above object, the present invention firstly obtains an exquisite component of the calyx fine flower (Cuphea hyssopifolia Kunth), and the preparation method of the exquisite component is as follows:

Mixing dried Alternaria alternata and 80% (v/v) ethanol solvent at a feed solution ratio of 1g:5 mL-1 g: mixing 50mL (adding for several times), standing at 20-30deg.C for 9-21 days (preferably 15 days), filtering, separating, concentrating the filtrate at 50deg.C under reduced pressure, and drying to obtain crude extract;

diluting the crude extract of the Pittosporum gracile with water, extracting with petroleum ether and ethyl acetate at 20-30deg.C, concentrating the obtained extract and water solution at 50deg.C under reduced pressure, and drying to obtain extract component and water layer component of the crude extract of the Pittosporum gracile, specifically petroleum ether layer component, ethyl acetate layer component and water layer component.

The method for preparing the fine components of the calyx of fine leaves also comprises the following steps:

(1) Column chromatography primary separation:

extracting the crude extract of the calyx parviflora sequentially by petroleum ether and ethyl acetate to obtain a water layer component, and further purifying by adopting D101 macroporous resin column chromatography: sequentially with methanol-water (v/v) 0: 100. 50: 50. 100:0 gradient elution, and concentrating and drying the eluent under reduced pressure at 50 ℃ to sequentially obtain the component I, the component II and the component III.

(2) Column chromatography secondary separation (further separation relative to prior application CN102525860 a):

Further purifying the component II collected after primary separation by the column chromatography in the step (1) by adopting a hydroxypropyl dextran gel (Sephadex LH-20) column chromatography, eluting by adopting a methanol-water (70:30, v/v) solution and a chloroform-methanol (50:50, v/v) solution in sequence, eluting 3-5 column volumes by adopting the methanol-water (70:30, v/v) solution and the chloroform-methanol (50:50, v/v) solution respectively, and recovering two parts of eluting solutions after the elution is finished. Wherein the methanol-water (70:30, v/v) solution elution part is an active ingredient, and the chloroform-methanol (50:50, v/v) solution elution part is an impurity ingredient;

the eluent with the column volume is used for eluting, so that the effective components can be obtained to the maximum extent and the impurities can be removed.

(3) Three separations by column chromatography:

purifying the methanol-water (70:30, v/v) solution elution part collected after the secondary separation of the column chromatography by adopting octadecylsilane chemically bonded silica column chromatography, wherein the flow rate is 20mL/min, and the methanol-water solution gradient elution is carried out under specific elution conditions: the volume ratio of methanol to water in the methanol-water solution for gradient elution is 0: 100. 20: 80. 40: 60. 60: 40. 80: 20. 100:0, 5 column volumes per volume of eluent. Wherein, the volume ratio of methanol to water is 20: 80. 40:60, respectively collecting and mixing the eluates obtained by eluting with methanol-water solution, concentrating the obtained mixture under reduced pressure at 50deg.C, and drying to obtain refined component of Alternaria minutissima.

The invention also provides 7 monomer compounds contained in the fine components of the hedyotis diversifolia, and the method for obtaining 7 monomer compounds comprises the following steps:

purifying the refined components of the hedyotis diversifolia obtained by three times of separation of the column chromatography by adopting high performance liquid chromatography, performing gradient elution by adopting an acetonitrile water solution with the acetonitrile concentration of 10% (v/v) to 30% (v/v), and collecting 7 monomer compounds in the refined components of the hedyotis diversifolia, wherein the 7 monomer compounds are identified as follows: myricitracin (1), tellimoside (2), myricetin 3-O- (6 "-O-galloyl) -beta-D-glucopyranoside (3), myricetin (4), desmanthin 1 (5), penta-O-galloyl-beta-D-glucopyranoside (6), quercetin 3-O-beta- (2" -O-galloyl-xyloside) (7); the content of the above 7 monomer compounds in the refined component was 1 (8.75%), 2 (7.33%), 3 (6.42%), 4 (18.75%), 5 (7.42%), 6 (7.83%), 7 (8.75%), respectively, and the total amount was 65.25% (more than 50%) of the dry weight of the refined component.

The invention also embodies that the fine component of the hedyotis microphylla and 7 monomer compounds contained in the fine component have remarkable effects in the aspects of antioxidation and inhibition of tyrosinase and matrix metalloproteinase-1 (MMP-1). Wherein, in terms of antioxidation, the fine component of the hedyotis diversifolia and the antioxidation IC of 7 monomer compounds ₅₀ The values are all in the range of 1 mug/mL-5 mug/mL, which shows that the anti-oxidation capability is good; both the fine component of the calyx parviflora and 7 monomer compounds show good activity in inhibiting tyrosinase and matrix metalloproteinase-1, wherein the fine component of the calyx parviflora is better.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the monomeric compounds Myricitracin (1), tellimoside (2), myricetin 3-O- (6 '-O-galloyl) -beta-D-glucopyranoside (3), myricetin (4), desmanthin 1 (5), penta-O-galloyl-beta-D-glucopyranoside (6) and Quercetin 3-O-beta- (2' -O-galloyl-xylopyranoside) (7) are separated and identified for the first time from the extract of Alternaria minutissima.

2. The delicate components of the hedyotis microphylla have remarkable activities in the aspects of antioxidation and inhibition of tyrosinase and matrix metalloproteinase-1, and in-vitro activity screening, animal models and human efficacy research results are obtained.

3. Myricetin 3-O- (6 ' -O-galloyl) -beta-D-glucopyranoside (3) and Quercetin 3-O-beta- (2 ' -O-galloyl-xylopyranoside) (7) of the 7 monomer compounds have remarkable activity on tyrosinase and matrix metalloproteinase-1 inhibition, and Desmonthin 1 (5) has remarkable activity on matrix metalloproteinase-1 inhibition, and Quercetin 3-O-beta- (2 ' -O-galloyl-xylopyranoside) (7) has oxidation resistance.

4. The fine components of the hedysarum gracile reported by the invention have very definite compositions, mainly contain 7 monomer compounds, and the total amount of the 7 monomer compounds exceeds 50% of the dry weight of the fine components of the hedysarum gracile, thus having good development prospect.

5. According to the enzymology experiment of the inventor, the refined components of the hedysarum gracile have stronger tyrosinase and matrix metalloproteinase-1 inhibition activity than the monomer compounds of the hedysarum gracile, so that adverse reactions possibly existing in the medicine can be reduced, and the safety of the product is improved. In addition, the fine components of the calyx of fine leaves have lower manufacturing cost and better cost performance than the monomer compounds.

6. The refined components of the hedyotis microphylla and 7 monomer compounds are extracted, separated and purified from plants, and the whole production process is free of chemical pollution and environment-friendly.

Drawings

FIG. 1 is an HPLC analysis chart of the delicate components of Calyx Seu Calvatia;

FIG. 2 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 1 prepared in example 2;

FIG. 3 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 1 prepared in example 2;

FIG. 4 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 2 prepared in example 2;

FIG. 5 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 2 prepared in example 2;

FIG. 6 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 3 prepared in example 2;

FIG. 7 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 3 prepared in example 2;

FIG. 8 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 4 prepared in example 2;

FIG. 9 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 4 prepared in example 2;

FIG. 10 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 5 prepared in example 2;

FIG. 11 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 5 prepared in example 2;

FIG. 12 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 6 prepared in example 2;

FIG. 13 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 6 prepared in example 2;

FIG. 14 is a hydrogen spectrum (600 MHz, meOD) of the monomer compound 7 prepared in example 2;

FIG. 15 is a graph of the carbon spectrum (150 MHz, meOD) and DEPT of the monomer compound 7 prepared in example 2;

FIG. 16 is a graph depicting the identification of 7 major monomeric compounds and 6 minor components in the fine fraction of Alternaria alternata; FIG. 17 is an ultraviolet absorbance spectrum of 7 major monomer compounds EJH-1-7 in the fine component of Calyx parviflora;

FIG. 18 is an ultraviolet absorbance spectrum of 6 minor ingredients A-F in the fine component of Alternaria alternata;

FIG. 19 shows the scavenging effect of the fine component of Alternaria minutissima and 7 monomeric compounds on ABTS free radicals;

FIG. 20 shows the inhibition of tyrosinase by the fine fraction of Alternaria alternata and alpha-arbutin;

FIG. 21 shows the inhibition of matrix metalloproteinase-1 by the refined components of Alternaria alternata, EJH-4, EJH-5, EJH-6, EJH-7;

FIG. 22 is a graph of guinea pig back skin tissue H & E staining;

FIG. 23 is a chart showing Fontana-Masson silver staining of guinea pig back skin tissue;

FIG. 24 is a skin efficacy experiment of a cream containing 1% fine leaf calyx of fine components;

figure 25 shows the effect of a cream containing 1% fine leaf calyx of hedysarum on various indices of the skin of a subject, with feature scores referring to the percentage of people of the same age.

Detailed Description

For the purpose of making the invention object and summary of the present application more apparent, the invention is further described below with reference to examples.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The materials of the fine calyx of the flowers used in the following examples were: picking up aerial parts of fresh calyx seu fructus physalis from Yunnan Kunming.

The reduced pressure concentration in the examples described below was achieved by means of a rotary evaporator.

Example 1: preparation method of fine components of calyx seu fructus physalis

1. Ethanol extraction of the calyx seu fructus physalis

Taking a fine calyx parviflora raw material, drying, taking 25kg of crushed fine calyx parviflora raw material, soaking and extracting the fine calyx parviflora raw material with 80% v/v ethanol at 20-30 ℃ for three times, wherein the feed-liquid ratio (the feed is 25kg, and the liquid is 80% v/v ethanol volume added each time) is sequentially 1g:10mL, 1g:6mL, 1g:6mL, extracting for 7 days, 5 days and 3 days sequentially, mixing the three filtrates, concentrating the filtrate under reduced pressure at 50deg.C until no ethanol smell exists, and drying to obtain total extract (i.e. crude extract) 1.4kg.

2. Extraction

1.4kg of the total extract obtained in the step 1 is completely dissolved by pure water, the volume of the pure water is 2.5L, and then the pure water is sequentially extracted by petroleum ether and ethyl acetate with equal volumes each time at 20-30 ℃ for 3 times; and concentrating and drying the petroleum ether extract part, the ethyl acetate extract part and the water liquid part at 50 ℃ under reduced pressure to obtain a petroleum ether layer component, an ethyl acetate layer component and a water layer component respectively.

3. Column chromatography primary separation

Carrying out column chromatography on the water layer component obtained in the step 2 by using 5kg of D101 macroporous adsorption resin column, wherein the specific steps are as follows: firstly, filling pretreated macroporous adsorption resin into a glass chromatographic column (with the diameter of 14cm and the height of 100 cm) by wet column packing to ensure uniformity and no bubbles; stopping adding liquid by using a distilled water pressure column until the height of the column is unchanged; closing the liquid outlet when the liquid level is reduced to about 5mm above the cylindrical surface to obtain a D101 macroporous adsorption resin column; slowly adding the water layer component into the column along the column wall after the water layer component is completely dissolved by pure water, opening a liquid outlet, controlling the liquid outlet speed, and avoiding the liquid level from falling too fast; after all samples are added into the chromatographic column, when the liquid level is reduced to about 5mm above the cylindrical surface, the column wall is rinsed with a small amount of distilled water, the rinsing is repeated for 2-3 times until the liquid on the cylindrical surface is clear and colorless, then the column is slowly filled with distilled water, and meanwhile, the liquid outlet is closed, and the column is kept stand for 12 hours until the adsorption equilibrium is reached; then, the liquid outlet is opened, the liquid outlet speed is controlled, and then the methanol-water solution with the concentration of 0% v/v (namely pure water), 50% v/v and 100% v/v (namely anhydrous methanol) is sequentially used for step-by-step elution, the volume of the methanol-water solution used each time is 30L, and the flow rate is 100mL/min; the eluents obtained by eluting the methanol-water solutions of the above concentrations were concentrated and dried under reduced pressure at 50℃to obtain 650g of an eluting component (abbreviated as component I, hereinafter the same) of the methanol-water solution of 0% v/v in methanol, 135g of an eluting component (abbreviated as component II, hereinafter the same) of the methanol-water solution of 50% v/v in methanol, and 142g of an eluting component (abbreviated as component III, hereinafter the same) of the methanol-water solution of 100% v/v in methanol.

4. Column chromatography secondary separation

After dissolving 135g of the component II obtained in the step 3 by using absolute methanol, further purifying by using 500g of hydroxypropyl Sephadex (Sephadex LH-20) column chromatography, wherein the volume ratio of methanol to water is 70:30, the volume ratio of methanol-water solution, chloroform and methanol is 50:50 chloroform-methanol solutions were eluted, each eluting 4 column volumes, and the eluate was recovered after the elution was completed. Wherein, the volume ratio of methanol to water is 70:30, concentrating and drying the eluent of the methanol-water solution at 50 ℃ under reduced pressure to obtain 75g of solid which is taken as an active ingredient, wherein the volume ratio of chloroform to methanol is 50: concentrating and drying the eluate of 50 chloroform-methanol solution at 50deg.C under reduced pressure to obtain impurity component.

The eluent with the column volume is used for eluting, so that the effective components can be obtained to the maximum extent and the impurities can be removed.

5. Three separations by column chromatography

After 75g of the active ingredient obtained in the step 4 is completely dissolved, 500g of octadecylsilane chemically bonded silica (Lichroprep) is adopted for column chromatography purification, and the specific steps are as follows: dissolving the effective components with anhydrous methanol completely, adding into an evaporation dish containing 100g of reverse phase silica gel (Lichroprep RP-18, particle size 40-70 μm, fuji Silysia chemical company, japan), adsorbing sample, standing in a fume hood, air drying, grinding into powder, separating at 20-30deg.C with a flow rate of 20mL/min and a pressure of 15MPa by using a medium pressure glass chromatographic column (Biotage company, sweden, C-615 pump manager, C-605 pump module, C-660 fraction collector, RP-18 chromatographic column); gradient elution is carried out by methanol-water solutions with different concentrations, and specific elution conditions are as follows: the volume ratio of methanol to water is 0: 100. 20: 80. 40: 60. 60: 40. 80: 20. 100:0 in the methanol-water solution gradient elution, wherein each volume ratio of the methanol-water solution used for elution is 2.5L, and the eluent is recovered. The volume ratio of the collected methanol to water is 20:80 and 40:60, and then concentrating and drying under reduced pressure at 50 ℃ to obtain refined components of the calyx Seu fructus Physalis, wherein the quality of the refined components is 45g.

Comparative example 1: comparison of the extraction rates of the refined components obtained by different extraction methods of the Alternaria minutissima

The extraction method comprises the following steps:

100g of the hedyotis diversifolia raw material is dried and crushed, 600mL of ethanol-water solution with the ethanol concentration of 60 percent v/v is used for extraction for 3 times at the temperature of 20-30 ℃, the extraction time length of each time is different, the extraction time is sequentially 5 days, 3 days and 1 day, after the extraction is completed, the subsequent treatment process is sequentially as follows,

1. combining the three filtrates, concentrating the filtrate under reduced pressure at 50deg.C until no ethanol smell exists, and drying to obtain total extract. Adding the total extract into pure water, dissolving completely to 150mL, extracting with petroleum ether for 3 times (150 mL of petroleum ether is added each time), extracting with ethyl acetate for 3 times (150 mL of ethyl acetate is added each time); the obtained extraction parts are respectively combined and then concentrated and dried under reduced pressure at 50 ℃ to obtain the components of the petroleum ether layer, the ethyl acetate layer and the water layer of the calyx seu fructus physalis.

2. The aqueous layer component was further purified by column chromatography using D101 macroporous resin (50 g), gradient eluted with methanol-water (0:100, 50:50, 100:0, v/v) solutions, each gradient eluent volume was 200mL, and the three gradient eluents were collected and concentrated under reduced pressure at 50℃to dry the components I, II and III in that order.

3. Dissolving component II with anhydrous methanol, purifying with hydroxypropyl dextran gel (Sephadex LH-20, 100 g) column chromatography, eluting with methanol-water (70:30, v/v) solution with volume of 300mL, recovering eluate, concentrating under reduced pressure at 50deg.C, and drying to obtain effective component.

4. The active ingredients are purified by adopting octadecylsilane chemically bonded silica (20 g) column chromatography, and are eluted by water-methanol solution gradient, and the specific elution conditions are as follows: the volume ratio of methanol to water in the methanol-water solution is 0: 100. 20: 80. 40: 60. 60: 40. 80: 20. 100:0, wherein the volume of each methanol-water solution with volume ratio concentration is 300mL, and the collection volume ratio is 20:80 and 40:60 (as shown in figure 1, figure 1 is a preliminary HPLC analysis map of the refined component, and is obtained by dissolving the refined component with anhydrous methanol, then injecting the mixture, and then performing gradient elution with 10% v/v acetonitrile aqueous solution to 30% v/v at a constant speed for 15 min).

Repeating the above extraction method, wherein each time of extraction is 7 days, 5 days, and 3 days.

Repeating the extraction method, wherein each time of extraction is 9 days, 7 days, and 5 days.

Repeating the extraction method, wherein the ethanol concentration is 80% v/v, and the extraction time period of each time is 5 days, 3 days and 1 day respectively;

repeating the extraction method, wherein the ethanol concentration is 80% v/v, and the extraction time period of each time is 7 days, 5 days and 3 days respectively;

the extraction method is repeated, wherein the ethanol concentration is 80% v/v, and the extraction time period of each extraction is 9 days, 7 days and 5 days respectively.

Repeating the extraction method, wherein the ethanol concentration is 100% v/v, and the extraction time period of each time is 5 days, 3 days and 1 day respectively;

repeating the extraction method, wherein the ethanol concentration is 100% v/v, and the extraction time period of each time is 7 days, 5 days and 3 days respectively;

the extraction method is repeated, wherein the ethanol concentration is 100% v/v, and the extraction time period of each extraction is 9 days, 7 days and 5 days respectively.

TABLE 1 Fine component extraction Rate of Calophyllum inophyllum with different extraction methods

As shown in Table 1, the extraction yield was highest when an ethanol-water solution having an ethanol concentration of 80% v/v was used as the extraction solvent; the extraction time period is 9 days, 7 days, 5 days (total 21 days) and the extraction rates of 7 days, 5 days and 3 days (total 15 days) are the highest, but the optimal extraction process is that the ethanol concentration is 80% v/v, the extraction time period is 7 days, 5 days and 3 days (total 15 days) in consideration of the extraction time.

Example 2: preparation method of monomer compound in fine components of calyx parviflora

Taking 120mg of fine components of the hedyotis diversifolia (prepared in example 1, the description is omitted below), dissolving the components in absolute methanol, and then performing gradient elution by using an acetonitrile-water solution of a high performance liquid chromatographic column (column: agilent Zorbax SB-C18 column, specification: 9.4mm multiplied by 250 mm), wherein the concentration of acetonitrile is 10-30% v/v, the elution time is 50 minutes, the flow rate of a mobile phase is 4mL/min, and the acetonitrile-water ratio is 0-40min10:90 is changed to 30:70 Acetonitrile-water 30 for 40-50 min: 70, eluting in equal proportion; concentrating and drying the obtained eluent in vacuum to obtain monomer compound 1 (EJH-1 for short, yellow powder, 10.5mg, t) _R =33.5 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 27:73 Monomer Compound 2 (abbreviated as EJH-2, yellow powder, 8.8mg, t) _R =32.6 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 26:74 Monomer Compound 3 (abbreviated as EJH-3, yellow powder, 7.7mg, t) _R =30.4 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 25:75 Monomer Compound 4 (abbreviated as EJH-4, yellow powder, 22.5mg, t) _R =44.6 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 30:70 Monomer Compound 5 (abbreviated as EJH-5, yellow powder, 8.9mg, t) _R =46.3 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 30:70 Monomer Compound 6 (abbreviated as EJH-6, yellow powder, 9.4mg, t) _R =36.2 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 28:72 And monomer Compound 7 (abbreviated as EJH-7, yellow powder, 10.5mg, t) _R =38.5 min, the volume ratio of acetonitrile to water in acetonitrile-water solution is 29: 71).

EJH-1 to 7: the powdery compounds EJH-1 to 7 prepared in example 2 were dissolved in 0.5mL of deuterated methanol, transferred to a nuclear magnetic resonance tube by a 200. Mu.L pipette, and detected for hydrogen and carbon spectra on a nuclear magnetic resonance apparatus (Broker Avance III 600MHz, germany) (see FIGS. 2 to 15). The spectrum information and the nuclear magnetic signal assignment of the above compounds EJH-1 to 7 are as follows:

myricitracin (1): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,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")。 ¹³ C NMR(150MHz,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”)。

terlmoside (2): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,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＝9.7,7.7H-2"),3.80(dd,J＝6.9,6.0H-5"),3.60(dd,J＝9.7,3.7,H-3")。 ¹³ C NMR(150MHz,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”)。

myricetin 3-O- (6' -O-galloyl) -beta-D-glucopyranoside (3): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,δ,ppm,J/Hz)：7.27(s,H-2'/H-6'),7.14(s,H-2"'/6"'),6.32(d,J＝2.2,H-6),6.15(d,J＝2.2,H-8),5.78(d,J＝7.9,H-1"),5.45(dd,J＝9.9,7.9,H-2"),3.96(dd,J＝3.5,1.2,H-4"),3.85(dd,J＝9.9,3.5,H-3"),3.70(d,J＝6.1,H-6"),3.61(td,J＝6.1,1.2,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),156.6(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”)。

myricetin (4): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,δ,ppm,J/Hz)：7.34(s,H-2'/H-6'),6.38(d,J＝2.2,H-6),6.18(d,J＝2.2,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)。

desmonthin 1 (5): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,δ,ppm,J/Hz)：7.08(s,H-2"'/6"'),6.98(s,H-2'/H-6'),6.36(d,J＝2.2,H-6),6.19(d,J＝2.2,H-8),5.63(dd,J＝3.4,1.8H-2"),5.51(d,J＝1.8,H-1"),4.05(dd,J＝9.1,3.4,H-3"),3.52(dq,J＝9.1,5.8,H-5"),3.48(t,J＝9.1,H-4"),1.04(d,J＝5.8,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”)。

Penta-O-ballonyl-beta-D-glucose (6): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,δ,ppm,J/Hz)：7.02,6.96,6.88,6.86,6.80(5×2'/6'),6.14(d,J＝8.3Hz,H-1),5.81(t,J＝9.9Hz,H-3),5.51(t,J＝9.9Hz,H-4),5.48(dd,J＝9.9,8.3Hz,H-2),4.41(dd,J＝12.1,1.9Hz,H-6),4.32(dd,J＝4.3,1.9Hz,H-5),4.28(dd,J＝12.1,4.3Hz,H-6)。 ¹³ C 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'),119.7,119.0,118.9,118.8,118.3(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)。

quercetin 3-O-beta- (2' -O-galloyl-oxypyrranoside) (7): a yellow powder of the pigment was used, ¹ H NMR(600MHz,CD ₃ OD,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”)。

from the results of the above detection, it was confirmed that the structural formula of the obtained compound was as follows in order:

Example 3 analysis of the content of 7 monomer Compounds in the Fine Components of Calophyllum inophyllum and type assignment of micro-ingredients

As can be seen from example 2, 120mg of the fine fraction of Alternaria minutissima was separated by high performance liquid chromatography to give Myricitracin (EJH-1, 10.5 mg), tellimoside (EJH-2, 8.8 mg), myricetin3-O- (6 "-O-galloyl) -beta-D-glucopyranoside (EJH-3, 7.7 mg), myricetin (EJH-4, 22.5 mg), desmanthin 1 (EJH-5, 8.9 mg), penta-O-galloyl-beta-D-glucoside (EJH-6, 9.4 mg), quercetin 3-O-beta- (2" -O-galloyl-xyl-glucoranoside) (EJH-7, 10.5 mg); the content percentages of the 7 monomer compounds in the refined components are respectively as follows: 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%, EJH-7 is 8.75%, and the total mass of 7 monomer compounds together is 65.25% (greater than 50%) of the dry weight of the finished component. Analyzing the ultraviolet absorption spectrum of the product obtained by separating the refined components by HPLC, wherein the ultraviolet absorption spectrum of EJH-1, 2, 3, 4, 5 and 7 is basically consistent, the maximum ultraviolet absorption peak is about 260 and 360nm, and the type of the product is myricetin; the ultraviolet absorption spectrum of EJH-6 is different from that described above, the maximum ultraviolet absorption peaks are 220 and 280nm, and the type is inverse gallitannins. In addition, as shown in fig. 16 and 18, the ultraviolet absorption spectrum of the trace component in the refined component was analyzed, and the maximum ultraviolet absorption peak of the trace component A, B, C, E, F was found to be about 260 and 360nm, which is the same as the ultraviolet absorption of the myricetin compound; the maximum ultraviolet absorption peaks of the trace component D are 220 nm and 280nm, which are the same as the ultraviolet absorption of the inverse gallic tannins compound. Therefore, the trace component A, B, C, E, F was judged to be a myricetin compound, and the trace component D was judged to be a reverse gallin compound.

Example 4: antioxidant Activity study of the extract of Alternaria alternata (Alternaria alternata extract refers to component I, component III, refined component, EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, EJH-7. Hereinafter, the same applies)

7.4mmol/L ABTS solution and 2.6mmol/L K ₂ S ₂ O ₈ The solution was mixed in equal volume and left to stand at room temperature in the dark for 16h to prepare ABTS free radical stock solution. The ABTS free radical stock solution was diluted with phosphate buffer solution (ph=7.4, 10 mmol/L), and the diluted solution was put into a multifunctional microplate reader (Spark 10m, tecan) to have absorbance at wavelength 734nm of 0.70±0.05, to prepare ABTS working solution. 200. Mu.L of ABTS working solution was mixed with 10. Mu.L of phosphate buffer solution (pH=7.4, 10 mmol/L), and the absorbance at 734nm was measured on a multifunctional microplate reader (Spark 10M, tecan) and set to A0. The component I (prepared in example 1, the description is omitted), the component III (prepared in example 1, the description is omitted), the refined component (prepared in example 1, the description is omitted), the component EJH-1-7 (prepared in example 2, the description is omitted), the positive medicines Trolox and alpha-arbutin for comparison are respectively dissolved in dimethyl sulfoxide (DMSO), the mother solution of 10mg/mL is prepared, the mother solution diluted solution is diluted by water, and the mother solution diluted solution with different concentrations is used as the sample solution. Next, 10 μl of the sample solution was mixed with 200 μl of LABTS working fluid: 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. Mu.g/ml, respectively; similarly, the final concentrations of the resulting mixed solutions were 0.78, 1.56, 3.13, 6.25, 12.50 and 25.00. Mu.g/mL for each of component I, component III, elegant component, EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, EJH-7, and positive drug α -arbutin, respectively. The mixed solution was allowed to stand at room temperature for 10min, and then was placed in a multifunctional microplate reader (Spark 10M, tecan) to measure absorbance at a wavelength of 734nm, which was designated as Ai. Meanwhile, 10 μl of the sample solution was mixed with 200 μl of a phosphate buffer solution having ph=7.4 and 10mmol/L, and the mixture was put into a multifunctional microplate reader (Spark 10m, tecan) to measure its background absorbance at a wavelength of 734nm, which was set as Aj. 3 parallel samples were set for each sample concentration.

The formula for calculating the clearance of the sample to ABTS radicals is as follows:

ABTS radical clearance (%) = [1- (Ai-Aj)/A0 ] ×100%

As shown in FIG. 19, the extract of Alternaria alternata showed a good scavenging effect on ABTS free radicals in the tested concentration range, with the scavenging effect of the delicate components being the best, and the scavenging effects of EJH-4 (myricetin) and EJH-5 being the best in EJH-1 to 7. Nonlinear fitting is carried out by using Graphpad Prism 8.0 software, and IC of the extract of the Alternaria minutissima for eliminating ABTS free radicals is calculated ₅₀ (half maximal inhibitory concentration) are listed in Table 2. Except for the component I extracted by pure water, the antioxidant IC of the rest of the extract of the Alternaria minutissima ₅₀ The values were all in the range of 1. Mu.g/mL-5. Mu.g/mL. Meanwhile, IC as a positive drug Trolox for control ₅₀ IC with value of 4.75 mug/mL and alpha-arbutin ₅₀ The value was 1.52. Mu.g/mL. The experimental results show that the components I, III, refined components and EJH-1-7 all have good antioxidant capacity, and most of the activities are superior to Trolox and are equivalent to alpha-arbutin.

Table 2 contains the ability of individual Calotropis gigantea extracts of monomeric compounds to scavenge ABTS free radicals

Example 5: research on tyrosinase activity inhibition of extract of calyx parviflora

1. Solution preparation

Phosphate buffer (0.2 m, ph=6.8): accurately weighing Na ₂ HPO ₄ 2.84g、NaH ₂ PO ₄ 2.4g, respectively dissolving the two solutions to constant volume to 100mL by using purified water, mixing the two solutions in equal volume, and then adjusting the pH to 6.8; preparing an L-dopa solution: 3.95mg of L-dopa was precisely weighed and dissolved in 10mL of phosphate buffer solution (0.2M, pH=6.8) to prepare a 2mM L-dopa solution. Preparing tyrosinase solution: 1mg of tyrosinase (enzyme activity 500U/mg) was precisely weighed and dissolved in 5mL of phosphate buffer solution (0.2)M, ph=6.8), tyrosinase solution with an enzyme activity of 100U/mL was prepared. Sample solution preparation: respectively dissolving the extract of each hedyotis diversifolia with DMSO to prepare a solution of 3 mg/mL; the positive drug kojic acid for control was formulated as a 1mg/mL solution in phosphate buffer (0.2 m, ph=6.8).

2. Tyrosinase activity assay

Four test groups were set up: sample group A ₁ Sample negative control group A ₂ Enzyme standard group B ₁ And enzyme negative control group B ₂ The method comprises the steps of carrying out a first treatment on the surface of the Each sample was set up in 3 replicates, and 200. Mu.L of the reaction system was prepared as in Table 3. To a 96-well plate, a corresponding volume of phosphate buffer solution, a sample solution to be measured (final concentration 0.3 mg/mL), a tyrosinase solution (final concentration 20U/mL) and a reaction substrate L-dopa (final concentration 1.2 mM) were sequentially added, incubated for 30min at 37℃in a microplate thermostatted shaker, and then absorbance at 475nm was measured in a multifunctional microplate reader (SPARK 10M, TECAN). The inhibition ratio of the fine components of the calyx parviflora to tyrosinase is calculated as follows:

Tyrosinase inhibition rate (%) = [1- (a) ₁ -A ₂ )/(B ₁ -B ₂ )]×100％

TABLE 3 tyrosinase activity inhibition assay reaction system formulation (volume/. Mu.L)

TABLE 4 inhibition of tyrosinase by Calyx parviflora extracts

As shown in Table 4, the inhibition rates of 0.3mg/mL positive drug kojic acid and alpha-arbutin on tyrosinase were 97.9.+ -. 1.3% and 54.6.+ -. 2.8%, respectively. Under the same concentration condition, the extracts of the other species except the component I in the extract of the hedyotis microphylla all show good tyrosinase inhibitory activity, and the inhibition rate is in the range of 45-75 percent. Wherein, the inhibition effect of the delicate component on tyrosinase is optimal and reaches 70.5+/-1.9 percent, which is superior to each monomer compound.

To further evaluate the in vitro inhibitory activity of the refined fraction on tyrosinase, the fraction was diluted with phosphate buffer solution to different concentration gradients for the positive drug α -arbutin. With reference to the reaction system formulation of Table 3, final concentrations were set to 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mg/mL, and absorbance of the solution at 475nm (measured in a multifunctional microplate reader (SPARK 10M, TECAN)) after 30min of reaction was measured. As shown in fig. 20, both α -arbutin and the delicate component show a dose-dependent inhibition of tyrosinase activity. Nonlinear fitting is performed by using Graphpad Prism 8.0 software, and then the semi-inhibition concentration IC of refined components and alpha-arbutin to tyrosinase is calculated ₅₀ 0.14mg/mL and 0.27mg/mL, respectively. The experimental result shows that the inhibition effect of the fine component of the hedyotis diversifolia on tyrosinase is nearly twice that of the positive control medicine alpha-arbutin.

Example 6: research on inhibition of matrix metalloproteinase-1 activity by extract of Alternaria minutissima

The inhibition of matrix metalloproteinase-1 activity by the extract of Alternaria minutissima was determined using a matrix metalloproteinase-1 inhibitor screening kit (Abcam, ab 118973). Matrix metalloproteinase-1 hydrolyzes specific Fluorescence Resonance Energy Transfer (FRET) substrates, releasing a fluorescent group whose fluorescent signal is detectable at Em/ex=480/530 nm. While effective MMP-1 inhibitors block hydrolysis of the substrate, reducing the fluorescent signal generated per unit time.

First, an extract of Alternaria alternata was initially screened for inhibition of matrix metalloproteinase-1 activity. A100. Mu.L reaction system was prepared as in Table 5, and three test groups were set up: enzyme control group, group I, group III, refinement and EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6, EJH-7 groups. MMP-1 solution (namely MMP-1 zymogen liquid) with corresponding volume, test buffer (enzyme control group) and final concentration of primary screening of component I, delicate component and component III are all 1mg/mL, final concentration of primary screening of EJH-1, EJH-2, EJH-3, EJH-4, EJH-5, EJH-6 and EJH-7 are all 0.5mg/mL, and each sample concentration is set to 2 parallels. The prepared solution is vibrated and mixed uniformly, substrate is added after the solution is subjected to warm bath for 5min at 37 ℃, then a multifunctional enzyme-labeled instrument (SPARK 10M, TECAN) is used for measuring the initial fluorescence signal F0 of each sample hole, the change condition of the fluorescence signal in 2.5h is monitored, and the fluorescence signal F1 of the sample after 2.5h of reaction is recorded. Calculating the relative enzyme activity after the sample acts according to the following formula, wherein the initial fluorescence signal of the enzyme control group is set as F0e, and the fluorescence signal after 2.5 hours of reaction is set as F1e; the initial fluorescence signal of each sample group was F0s, and the fluorescence signal of the sample solution after 2.5 hours of reaction was F1s.

TABLE 5 preparation of reaction System for determining matrix Metalloprotease-1 Activity (Unit: μL/well)

At a primary screening concentration of 1mg/mL, the inhibition rate of the component I, the component III and the refined component on the activity of the matrix metalloproteinase-1 is more than 50 percent. At a primary screening concentration of 0.5mg/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%.

To further determine the IC of these extracts for inhibition of matrix metalloproteinase-1 activity ₅₀ Different concentration gradients were set. Similarly, 100. Mu.L of the reaction system was prepared according to Table 5 so that the final concentrations of the solutions of the fine components were 0.1, 1, 10, 100 and 1000. Mu.g/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. Mu.g/mL, respectively.

The results are shown in FIG. 21, where the measured refined fractions and EJH-4, 5, 6 and 7 all exhibited dose-dependent inhibition of matrix metalloproteinase-1 activity. At a concentration of 100 μg/mL, the inhibition rate of the delicate component to the matrix metalloproteinase-1 activity reaches 64% (. Times.P < 0.01). Nonlinear fitting is performed by using Graphpad Prism 8.0 software to calculate the essence Semi-inhibitory concentration IC of a pro-component to matrix metalloproteinase-1 ₅₀ 55.0 μg/mL; EJH-4, 5, 6, 7 half inhibitory concentration IC for matrix metalloproteinase-1 ₅₀ 214.6. Mu.g/mL, 155.4. Mu.g/mL, 60.2. Mu.g/mL and 160.7. Mu.g/mL, respectively. The experimental results show that the refined components of the hedyotis diversifolia have better inhibition effect on the matrix metalloproteinase-1 and are better than the monomer compounds.

Example 7: acute toxicity test in mice

Experiments were performed with Kunming mice. 400mg of component I, component III and the delicate components were precisely weighed, dissolved in 0.5mL of DMSO, and the sample was diluted to 200mg/mL with ultrapure water. Blank, DMSO control, group i, group iii, and refinement groups were set up, respectively. One mouse for the blank group and DMSO control group, and two mice (male and female halves) for each experimental group. Mice were fasted for 12h before the experiment and were free to drink water. The weight of fasted mice was weighed, and physiological saline (0.4 mL), 25% DMSO, component I, component III, and refined components were administered orally and gastrically according to the up-down method at 2000mg/kg, and changes in respiratory system, urinary system, digestive system, and nervous system such as tremor, convulsion, salivation, somnolence, and coma were observed, followed by continuous observation for 48 hours. At the end of the observation period, all surviving animals were euthanized after weighing, were observed in general anatomic fashion, and were examined histopathologically.

As can be seen from the above experiments, mice were observed for a period of 48 hours after injection of a larger dose of 2000mg/kg. The mice grow well, the activities are normal, the gloss of the hair is good, and the toxic symptoms such as tremors, salivation, convulsions, leg kicking, sleepiness, coma and the like do not appear. At the end of the experiment, a general anatomic examination was performed and no apparent pathological changes were observed in each organ. The blank group had no significant difference from the experimental group body weight. Acute toxicity experiments show that the components I, III and exquisite components have no obvious animal toxicity at the dose of 2000mg/kg, and the Maximum Tolerance (MTD) of mice is more than 2000mg/kg.

TABLE 6 acute toxicity results in mice

Example 8: the fine component of the calyx parviflora is effective in inhibiting skin melanin deposition of guinea pigs caused by ultraviolet light

20 guinea pigs were selected and weighed 200-250g with large area brown yellow hair on the back and purchased from Liaoning long biotechnology Co. All guinea pigs are bred in an environment with the temperature of 25+/-2 ℃ and the relative humidity of 55+/-15 percent, 12h/12h light and dark alternation is carried out, and all the guinea pigs can be freely eaten with water and special feed for the guinea pigs and are adaptively fed for 7 days. Each guinea pig selects brown hair region before ultraviolet radiation, shaves longer hair with shaver, and then uniformly smears the hair-cutting region with depilatory cream, and washes the depilatory cream with warm water after 5min, and the action is gentle, so that the back skin of guinea pig is exposed by 3 x 3cm ² Is a skin of a subject. The study was approved by the ethical committee of animals at the university of ethnic group in south China.

The 20 guinea pigs were randomly divided into 4 groups of 5. 5 mice were used as blank groups, and the rest groups were irradiated with UVB UV lamp for 1h each: the daily radiation dose is 0.6J/cm on days 1-7 ² On days 6-20, the daily radiation dose is 0.3J/cm ² The skin of the guinea pig test area was depilated using depilatory cream prior to each irradiation for 20 consecutive days.

The specific grouping is as follows:

blank control group: coating the same dose of 0.3% CMC-Na, and not irradiating ultraviolet;

model group: smearing the same dose of 0.3% CMC-Na and irradiating with UVB for 30min;

positive drug group: coating 1mg of beta-arbutin containing 0.3% CMC-Na, and irradiating with UVB for 30min;

experimental group: smearing 2mg of fine components of the Alternaria alternata containing 0.3% CMC-Na, and irradiating with UVB for 30min;

the mold was continuously administered for 20 days, and the degree of damage to the skin of the back was observed by UVB radiation. After the experiment is finished, the guinea pigs are euthanized, back skin tissues are taken and placed into 4% paraformaldehyde fixing solution for fixing, and the subsequent experiment is carried out.

H & E conventional staining: embedding and slicing according to conventional paraffin; dewaxing slices to water; placing into hematoxylin dye for dyeing for 5min, and washing with running water for 1 time; color separation of 1% hydrochloric acid alcohol differentiated solution for 15s, and washing with running water for 2 times; returning 1% ammonia water to blue for 30s;1% eosin dye for 2min, washing with tap water; sequentially dehydrating 70%, 80%, 90%, 95% and 100% (volume percentage) ethanol for two minutes each, and air drying; placing into xylene for transparency, and sealing with neutral resin.

Detection of melanin content in skin layers by Fontana-Masson silver staining: according to a conventional pathological detection method of tissues, the skin tissues of guinea pigs after the experiment are fixed by 4% paraformaldehyde, dehydrated, embedded in paraffin and sliced. Skin sections were stained using the Masson-Fontana silver staining method, and the melanin staining results were observed under a microscope to determine the uv-induced melanin distribution changes of the fine components of the calyx morifolium.

The H & E staining results are shown in FIG. 22, the epidermis of the skin tissue of the blank group is complete, the collagen fibers are closely arranged, and the number of hair follicles is rich. The skin tissue of the model group is obviously thickened, the dermis layer can be infiltrated by lymphocyte, and the hair follicle is reduced. The skin tissue epidermis of the guinea pigs in the positive control group is thicker; the dermis layer is infiltrated by a large number of lymphocytes, collagen fibers are arranged sparsely, and the number of hair follicles is small. The skin tissue of the guinea pig of the experimental group has thicker epidermis and multiple spiny cell water sample denaturation, and loose and light dyeing of cytoplasm; the dermis layer is infiltrated by a small amount of granulocytes, collagen fibers are closely arranged, and hair follicles are abundant. The above results demonstrate that ultraviolet rays cause thickening of guinea pig skin tissue, inflammatory cell infiltration, collagen fiber damage, hair follicle reduction, and the like. Compared with the positive control beta-arbutin, the fine component of the hedyotis diversifolia can inhibit the thickening of skin tissues, promote the proliferation of collagen fibers and promote the proliferation of hair follicles.

As shown in fig. 23, the melanin content of the back skin of guinea pigs in the blank group control group was very small, and the melanin content was mainly concentrated in the basal layer region, and only very small amount of melanin was present in the spinous layer; after ultraviolet irradiation, melanin of the model group is obviously increased, and basal layers and spine layers are expressed in a large amount of melanin, but the model group is mainly concentrated on the basal layers; the positive control group has more melanin content, is mainly concentrated on the basal layer, and has less melanin content in the thorn layer; the melanin distribution of the experimental group is obviously reduced compared with that of the model group, and the melanin distribution is mainly concentrated on the basal layer, and the content of the melanin is less in the acantha layer. The results show that ultraviolet rays can promote the skin tissue of guinea pigs to increase melanin secretion, and the beta-arbutin and the fine components of the fine calyx of the fine leaf calyx can reduce melanin generation, the fine component effect of the calyx parviflora is better than that of beta-arbutin, and the calyx parviflora has wide application prospect in whitening cosmetics.

Example 9 safety test

Human skin closed patch experiments were performed according to cosmetic safety specifications (2015 edition). 30 volunteers were enrolled together, and the spot-attaching test was performed at random for gender and at 18-60 years of age. The experimental method comprises the following steps: mixing 1% of fine calyx of fine components, 2% of ethoxydiglycol, 1% of PEG-40 hydrogenated castor oil, 2% of pentanediol and 94% of pure water (the percentages are all mass percentages), heating for dissolution, and cooling to room temperature to prepare a solution containing 1% of fine components of calyx of fine flowers. 200 mu L of the solution containing 1% of the fine components of the fine calyx of the fine flowers is placed in a spot tester, the control hole is the solution without 1% of the fine components of the fine calyx of the fine flowers, the spot tester is applied to the upper arm of a subject by using a non-stimulated adhesive tape, and the spot tester is uniformly applied to the skin by using palm light pressure for 24 hours. After removing the plaque tester, the skin reaction was observed after the indentation disappeared. Scoring was performed according to the skin reaction ranking table.

TABLE 7 skin reaction grading Table

The test result shows that the refined component of the hedyotis diversifolia with the content of 1% has no adverse reaction on the skin of a human body, only 1 person has weak erythema, and the rest 29 persons have no obvious discomfort.

Table 8 skin safety test results

Example 10 experiments on the skin efficacy of creams containing Fine Components of Alternaria minutissima

A total of 30 volunteers were enrolled, sex randomized, aged 18-60 years, and left and right face control tests were performed. Blank face cream is used on the left side, face cream containing 1% of fine components of the calyx seu fructus physalis is used on the right side, products are used according to usual skin care habits, products are respectively smeared after normal face cleaning, and other skin care products and cosmetics are forbidden to be used in the morning and evening. The device is continuously used for 15 days, before use, and at 7 days and 14 days, VISIA-CR (CANFIELD, america) is adopted for facial image shooting respectively, and the device combines the most advanced digital photographing technology, has various light sources (standard light, UV light, cross polarized light and parallel polarized light), and improves the visual degree of skin analysis.

Surface 9 face cream formulation

And respectively mixing the water phase and the oil phase raw materials, heating to 85 ℃, and then adding the water phase into the oil phase to emulsify for 3min to obtain blank face cream and face cream containing 1% of fine components of the calyx seu calyx.

From the results shown in fig. 24 (a cream skin efficacy experiment containing 1% fine leaf calyx fine flower fine component) and fig. 25 (the effect of the cream containing the fine leaf calyx fine flower fine component on various indexes of the skin of a certain subject), it is known that the prepared cream containing 1% fine leaf calyx fine flower fine component can obviously improve the overall state of the facial skin, such as surface spots, brown spots (deep hidden spots) and other problems; at the same time, the texture (reflecting smoothness and plumpness of skin), ultraviolet color spots (reflecting potential pigment spots under epidermis) and ultraviolet (reflecting skin grease) are obviously improved. The effective improvement rate of the ultraviolet color spot and the brown color spot reaches 60% in 14 days, and the subjects do not have any allergy or discomfort and the like. The delicate components can reduce ultraviolet spots and brown spots in the deep layer of the skin, effectively reduce spots on the surface layer of the skin, and have the whitening effect; and the skin is smooth, the grease is reduced, and the skin care product has good photo-aging resistance.

Claims (10)

1. The structure is thatThe use of a compound having at least one of tyrosinase-inhibiting, matrix metalloproteinase-1-inhibiting and whitening activity in the preparation of a product.

2. The use according to claim 1, characterized in that: the product is a cosmetic.

3. Use according to claim 1 or 2, characterized in that: the product is a face cream.

4. Contains the structural formula ofA product of a compound of (a);

the product has at least one of tyrosinase inhibition, matrix metalloproteinase-1 inhibition and whitening activity.

5. The product according to claim 4, wherein: the product is a cosmetic.

6. The product according to claim 4 or 5, characterized in that: the product is a face cream.

7. A method for extracting 7 monomeric compounds from calyx parviflora, comprising the steps of:

extracting 60-100% v/v ethanol extract of Alternaria tenuifolia sequentially with petroleum ether and ethyl acetate to obtain petroleum ether layer component, ethyl acetate layer component and water layer component;

purifying the water layer component by D101 macroporous resin column chromatography: sequentially using methanol and water with the volume ratio of 0: 100. 50: 50. 100:0 in methanol-water solution, and collecting methanol and water with a volume ratio of 50:50 eluting the obtained eluent by methanol-water solution, concentrating under reduced pressure and drying to obtain a component II;

purifying the component II by hydroxypropyl sephadex column chromatography: the volume ratio of methanol to water is 70:30 eluting with methanol-water solution for 3-5 column volumes to obtain effective components;

Purifying the active ingredients through octadecylsilane chemically bonded silica column chromatography: the volume ratio of methanol to water is 0: 100. 20: 80. 40: 60. 60: 40. 80: 20. 100:0, carrying out gradient elution on methanol-water solution, wherein the volume ratio of methanol to water is 20: 80. 40:60, respectively collecting and mixing the eluents obtained by eluting with methanol-water solution, concentrating and drying the obtained mixed solution under reduced pressure to obtain refined components of the calyx seu fructus physalis;

purifying the refined components of the hedyotis diversifolia by high performance liquid chromatography: gradient elution is carried out by adopting acetonitrile-water solution with the concentration of acetonitrile of 10-30% v/v, eluent is respectively collected and then concentrated and dried under reduced pressure, thus obtaining Myricitrin, tellimoside, myricetin 3-O- (6 '-O-galloyl) -beta-D-glucopyranoside, myricetin, desmanthin 1, penta-O-galloyl-beta-D-glucose and Quercetin 3-O-beta- (2' -O-galloyl-oxypyrnioide) 7 monomer compounds;

the structural formula of the Myricetin 3-O- (6' -O-galloyl) -beta-D-glucopyranoside is

8. The method according to claim 7, characterized in that it comprises in particular the following steps:

s1, drying and crushing a raw material of the hedyotis diversifolia, soaking and extracting for 1-4 times at 20-30 ℃ by adopting 60-100% v/v ethanol for 9-21 days, wherein the feed liquid ratio of the dried raw material of the hedyotis diversifolia to the 60-100% v/v ethanol added during each extraction is 1g:5 mL-1 g:50mL, concentrating and drying the filtrate obtained by solid-liquid separation after extraction under reduced pressure to obtain an ethanol extract of the calyx seu fructus physalis;

S2, dissolving the ethanol extract of the hedyotis diversifolia in water, and sequentially extracting with petroleum ether and ethyl acetate at 20-30 ℃; collecting the petroleum ether extract solution, ethyl acetate extract solution and water solution, concentrating under reduced pressure, and drying to obtain petroleum ether layer component, ethyl acetate layer component and water layer component of the Alternaria tenuifolia extract;

step S3, the water layer component is further purified by adopting D101 macroporous resin column chromatography, and gradient elution is sequentially carried out by using methanol-water solutions with the methanol concentration of 0% v/v,50% v/v and 100% v/v, so that a component I, a component II and a component III are sequentially obtained;

step S4, further purifying the component II by adopting hydroxypropyl sephadex column chromatography, and sequentially adopting methanol and water with the volume ratio of 70:30, the volume ratio of methanol-water solution, chloroform and methanol is 50: eluting with 50 chloroform-methanol solutions, eluting with 3-5 column volumes, and recovering the eluate after eluting; wherein, the volume ratio of methanol to water is 70:30, concentrating and drying the eluent of the methanol-water solution under reduced pressure to obtain an active ingredient, wherein the volume ratio of chloroform to methanol is 50: concentrating and drying the eluent of 50 chloroform-methanol solution under reduced pressure to obtain impurity components;

Step S5, adding methanol into the effective components in the step S4 to dissolve completely, and then adopting octadecylsilane chemically bonded silica gel column chromatography for further purification, wherein the specific mode is as follows: mixing the active ingredients with octadecylsilane chemically bonded silica gel with the particle size of 40-70 mu m after the active ingredients are completely dissolved, standing and air-drying, grinding the octadecylsilane chemically bonded silica gel adsorbed with the sample into powder, and then loading the powder into a medium-pressure glass chromatographic column for separation at room temperature, wherein the flow rate of a mobile phase is 20mL/min, and the pressure is 15MPa; gradient elution is carried out by methanol-water solutions with different concentrations, and specific elution conditions are as follows: the volume ratio of methanol to water is 0: 100. 20: 80. 40: 60. 60: 40. 80: 20. 100:0 in methanol-water solution, wherein the eluent dosage of each volume ratio is 5 column volumes; recovering eluent, wherein the volume ratio of the recovered methanol to water is respectively 20: 80. 40:60, mixing the eluent eluted by the methanol-water solution, concentrating and drying the obtained mixed solution under reduced pressure to obtain an delicate component of the calyx seu fructus physalis;

s6, subjecting the fine components of the hedyotis diversifolia to high performance liquid chromatography column, and performing gradient elution by adopting acetonitrile-water solution with the acetonitrile concentration of 10-30% v/v for 50 minutes, wherein the flow rate of a mobile phase is 4mL/min; wherein the acetonitrile-water volume ratio is from 10:90 is changed to 30:70 Acetonitrile-water 30 at 40-50 min: 70, eluting in equal proportion;

Eluting the monomer compound Myricetin 3-O- (6 '-O-galloyl) -beta-D-glucopyranoside, tellimoside, myricitrin, penta-O-galloyl-beta-D-glucose, quercetin-O-beta- (2' -O-galloyl oxyparanoside) in sequence within 0-40 min;

eluting the monomer compound Myricetin, desmanthin 1 in sequence within 40-50 min;

the eluate of each of the obtained monomer compounds was concentrated and dried in vacuo to obtain the monomer compounds.

9. The method according to claim 8, wherein: in the step S1, 80% v/v ethanol is adopted for soaking and extracting for 3 times at 20-30 ℃ for 15 days.

10. The method according to claim 8 or 9, characterized in that: in the step S1, the feed liquid ratio of the dried fine calyx parviflora raw material to the 80% v/v ethanol added during each extraction is 1g:5 mL-1 g:10mL.