Hawthorn condensed tannin polymers, methods of making and uses thereof

Abstract

The application provides a hawthorn condensed tannin polymer and a preparation method and application thereof. The hawthorn condensed tannin polymer is extracted from hawthorn pulp, and the hawthorn condensed tannin polymer comprises a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers of epicatechin shown in formula (I). Wherein, n is a positive integer between 2 and 18, and at least one of the dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers comprises a B-type procyanidin polymer obtained by connecting epicatechin structural units through C4-C8 or C4-C6.

Description

[0001] This application claims priority to the Chinese Patent Application No. 202210291037.9, filed on March 23, 2022, entitled “Tyrosinase Inhibitors and Methods of Making and Using the Same”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of food and medicine technology, in particular to a hawthorn condensed tannin polymer and a preparation method and use thereof. BACKGROUND

[0003] Hawthorn (Crataegus pinnatifida Bunge), also known as “Shanlihong”, “Xiangguo”, and “Mufuli”. Its producing areas mainly include Heilongjiang, Jilin, Liaoning, Inner Mongolia, Hebei, Henan, Shandong, Shanxi, Shaanxi, Jiangsu, and Jiangxi. Hawthorn is a unique fruit and medicine tree species in China with large yield. The flesh can be eaten raw or made into preserved fruits and cakes, and dried fruits can also be used as medicine. Hawthorn contains various vitamins, hawthorn acid, tartaric acid, citric acid, malic acid, flavonoids, lactones, sugars, proteins, fats, and other substances. Modern pharmacological studies have shown that hawthorn has the effects of lowering blood lipids, lowering blood pressure, strengthening the heart, and preventing arrhythmia. It is also a good medicine for invigorating the spleen, stimulating appetite, promoting digestion, removing food stagnation, promoting blood circulation, and reducing phlegm. It has good effects on chest and diaphragm fullness, hernia, blood stasis, and amenorrhea.

[0004] Tyrosinase, also known as polyphenol oxidase, is a copper-containing oxidoreductase of about 75 ku, which exists widely in animals, plants, microorganisms and human bodies, is a rate-limiting enzyme for melanin synthesis, and directly affects the synthesis of melanin. Tyrosinase is composed of multiple subunits, each of which contains two metal copper ions, and the two copper ions are covalently bound to the imino groups of three histidine residues in the active center, and in addition, one endogenous bridge connects the two copper ions together to form the active center of tyrosinase. If copper is oxidized, the enzyme will be inactivated and can be reactivated by an electron donor, such as L-3,4 dihydroxyphenylalanine, ascorbic acid, superoxide anion, etc. In plants, tyrosinase is called polyphenol oxidase; in insects, it is called phenol oxidase; in microorganisms and human bodies, it is called tyrosinase. The tyrosinase gene family has three common effects in catalyzing melanin production, namely TYR, TYRP1 and TYRP2, of which TYRP1 and TYRP2 play a catalytic role in the last few steps of melanin production in melanocytes. TYR is a key rate-limiting enzyme in the initial process of melanin synthesis, and has both tyrosine hydroxylase and dopa oxidase activities, and is involved in the oxidation and transport of tyrosinase in the process of melanin synthesis. Tyrosine generates dopa under the action of tyrosinase, and finally generates melanin through a series of steps. Melanin makes the skin of animals appear darker, especially black and brown, and sometimes yellow.

[0005] Tyrosinase inhibitors can inhibit melanin formation by inhibiting the formation of tyrosinase, thereby reducing the deposition of skin melanin to play a role in the treatment of skin diseases and whitening. However, existing tyrosinase inhibitors are limited by weak inhibition effect, poor solubility, low yield, high cost and many other problems, and cannot be applied on a large scale. Hawthorn is rich in phenolic compounds, which have a similar structure to the substrate of tyrosinase, and therefore have extremely high tyrosinase inhibition potential. SUMMARY

[0006] The present application provides a hawthorn condensed tannin polymer and a preparation method and use thereof, which has high yield, good water solubility and excellent inhibition capacity for tyrosinase.

[0007] In a first aspect, the present application provides a hawthorn condensed tannin polymer, which is extracted from hawthorn pulp, and comprises a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers and heptadecamers of epicatechin represented by the structural unit of formula (I);

[0008]

[0009] wherein n is a positive integer between 2 and 18, at least one of the dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, dodecamer, tridecamer, tetradecamer, pentadecamer, hexadecamer, heptadecamer and octadecamer comprises a B-type proanthocyanidin polymer obtained by C4-C8 or C4-C6 linkage of epicatechin structural units.

[0010] In combination with the first aspect, the hawthorn condensed tannin polymer has characteristic peaks at wavelengths of 215 nm to 220 nm and 270 nm to 280 nm in an ultraviolet spectrum.

[0011] In combination with the first aspect, the hawthorn condensed tannin polymer has characteristic peaks at wavelengths of 215 nm to 220 nm and 270 nm to 280 nm in an ultraviolet spectrum.

[0012] In combination with the first aspect, the hawthorn condensed tannin polymer has characteristic peaks at wavelengths of 215 nm to 220 nm and 270 nm to 280 nm in an ultraviolet spectrum.

[0013] In combination with the first aspect, the hawthorn condensed tannin polymer has characteristic peaks at wavelengths of 215 nm to 220 nm and 270 nm to 280 nm in an ultraviolet spectrum.

[0014] In combination with the first aspect, the hawthorn condensed tannin polymer has characteristic peaks at wavelengths of 215 nm to 220 nm and 270 nm to 280 nm in an ultraviolet spectrum.

[0015] In combination with the second aspect, the method comprises at least one of the following features (1) to (12):

[0016] (1) the hawthorn pulp dry powder is obtained by performing first vacuum freeze-drying treatment, grinding and sieving on hawthorn pulp;

[0017] (2) the hawthorn pulp dry powder is obtained by performing first vacuum freeze-drying treatment, grinding and sieving on hawthorn pulp, wherein the temperature of the first vacuum freeze-drying treatment is -85℃ to -55℃;

[0018] (3) the hawthorn pulp dry powder is obtained by performing first vacuum freeze-drying treatment, grinding and sieving on hawthorn pulp, wherein the vacuum degree of the first vacuum freeze-drying treatment is less than 9 Pa;

[0019] (4) the hawthorn pulp dry powder is obtained by performing first vacuum freeze-drying treatment, grinding and sieving on hawthorn pulp, wherein the time of the first vacuum freeze-drying treatment is 24 h to 48 h;

[0020] (5) the hawthorn pulp dry powder is obtained by performing first vacuum freeze-drying treatment, grinding and sieving on hawthorn pulp, wherein the mesh number of the sieving is 40 mesh;

[0021] (6) the solvent concentration of the first extraction is 60% to 70% acetone;

[0022] (7) the time of the first extraction is 15 min to 30 min;

[0023] (8) the mass ratio of the dried hawthorn pulp to the solvent of the first extraction is 1 : (5-20) ;

[0024] (9) the first extraction is further followed by a step of performing a first rotary evaporation treatment on the aqueous extract;

[0025] (10) the first extraction is further followed by a step of performing a first rotary evaporation treatment on the aqueous extract, and the temperature of the first rotary evaporation is less than 40°C;

[0026] (11) the first extraction is further followed by a step of performing a first rotary evaporation treatment on the aqueous extract, and the time of the first rotary evaporation is 1 h to 2 h;

[0027] (12) the first extraction is further followed by a step of performing a first rotary evaporation treatment on the aqueous extract, and the rotation speed of the first rotary evaporation is 50 r / h to 80 r / h.

[0028] In combination with the second aspect, the organic solvent extraction treatment on the aqueous extract to obtain the crude extract specifically includes: sequentially performing petroleum ether extraction and ethyl acetate extraction on the aqueous extract, and then performing a second rotary evaporation treatment and a second freeze-drying treatment to obtain the crude extract, and the method includes at least one of the following features (1) to (9):

[0029] (1) the volume ratio of the aqueous extract to the petroleum ether is (2-3) : 1;

[0030] (2) the volume ratio of the aqueous extract to the ethyl acetate is (2-3) : 1;

[0031] (3) the number of times of the ethyl acetate extraction is 1 to 3 times;

[0032] (4) the temperature of the second rotary evaporation treatment is less than 40°C;

[0033] (5) the time of the second rotary evaporation treatment is 30 min to 60 min;

[0034] (6) the rotation speed of the second rotary evaporation treatment is 50 r / h to 80 r / h;

[0035] (7) the time of the second freeze-drying treatment is 24 h to 48 h;

[0036] (8) the vacuum degree of the second freeze-drying treatment is less than 9 Pa;

[0037] (9) the temperature of the third freeze-drying treatment is -85℃ to -55℃.

[0038] In combination with the second aspect, the Sephadex LH 20 column chromatography elution treatment of the crude extract specifically includes: passing the crude extract through a Sephadex LH 20 column, and then eluting with 50% to 60% methanol and 70% acetone, respectively, followed by third rotary evaporation treatment and third freeze-drying treatment to obtain the hawthorn condensed tannin polymer, and the method includes at least one of the following features (1) to (9):

[0039] (1) the height-diameter ratio of the packing of the Sephadex LH 20 column is 6:1;

[0040] (2) the flow rate of the Sephadex LH 20 column is 0.2 drop / s to 0.5 drop / s;

[0041] (3) the amount of the crude extract loaded on the Sephadex LH 20 column is 100 mg to 200 mg per 1 mL of the packing by wet loading;

[0042] (4) the temperature of the third rotary evaporation treatment is less than 40℃;

[0043] (5) the time of the third rotary evaporation treatment is 30 min to 60 min;

[0044] (6) the rotation speed of the third rotary evaporation treatment is 50 r / h to 80 r / h;

[0045] (7) the time of the third freeze-drying treatment is 24 h to 48 h;

[0046] (8) the vacuum degree of the third freeze-drying is less than 9 Pa;

[0047] (9) the temperature of the third freeze-drying is -85℃ to -55℃.

[0048] In a third aspect, the present application provides the use of the hawthorn condensed tannin polymer of the first aspect or the hawthorn condensed tannin polymer prepared by the method of the second aspect in a medicament for treating pigmentation diseases.

[0049] In a fourth aspect, the present application provides the use of the hawthorn condensed tannin polymer of the first aspect or the hawthorn condensed tannin polymer prepared by the method of the second aspect in a cosmetic product for whitening.

[0050] Compared with the prior art, the present application has at least the following advantages:

[0051] (1) The application selects fresh organs of hawthorn pulp as raw materials, which are widely available and easy to obtain.

[0052] (2) The application uses hawthorn pulp as raw materials to obtain a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers represented by Formula I through separation and purification, which are mainly composed of flavan-3-ol basic structural units connected by C4-C8 or C4-C6, and the structural unit is epicatechin, which has a similar ortho-hydroxyl structure as the substrate of tyrosinase, which makes the hawthorn condensed tannin polymers of the application have obvious enzyme inhibition effect, and the water solubility and purity are high, the purity can reach 98%, and the inhibition effect on the activity of tyrosinase is obviously better than most of the existing hawthorn condensed tannin polymers, and it is safe and non-toxic.

[0053] (3) The hawthorn condensed tannin polymers of the application can be used to prepare drug polymers for whitening and fruit and vegetable preservation, etc., and have good application prospect.

[0054] Other features and advantages of the embodiments of the application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the embodiments of the application. The objects and other advantages of the embodiments of the application will be realized and achieved by the structure particularly pointed out in the specification and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0055] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can be obtained from these drawings without creative labor.

[0056] Figure 1 The matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) diagram of the hawthorn condensed tannin polymers prepared in Example 1 in positive ion mode (Cs + ) is shown in Figure 1.

[0057] Figure 2 The preparation flow chart of the hawthorn condensed tannin polymers of the application is shown in Figure 2.

[0058] Figure 3 The NMR diagram of the hawthorn condensed tannin polymers prepared in Example 1 is shown in Figure 3.

[0059] Figure 4 The HPLC diagram of the hawthorn condensed tannin polymers prepared in Example 1 after thiolysis is shown in Figure 4.

[0060] Figure 5 UV spectrum of the hawthorn condensed tannin polymer prepared in Example 1;

[0061] Figure 6 Comparison chart of the polyphenol oxidase activity of fresh-cut apples with the hawthorn condensed tannin polymer of Example 1;

[0062] Figure 7 Comparison chart of the total phenol activity of fresh-cut apples with the hawthorn condensed tannin polymer of Example 1.

[0063] The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate embodiments consistent with the application and serve to explain the principles of the application. DETAILED DESCRIPTION

[0064] For a better understanding of the technical solutions of the present application, the embodiments of the present application are described in detail below with reference to the drawings.

[0065] It should be clear that the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0066] The terms used in the embodiments of the present application are only for the purpose of describing the specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0067] It should be understood that the term "and / or" used herein only describes the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B, which can represent the three cases of A alone, A and B together, and B alone. In addition, the character " / " herein generally represents an "or" relationship between the front and rear associated objects.

[0068] The Sephadex LH 20 in the present application is purchased from GE Healthcare, and acetone, petroleum ether, ethyl acetate and methanol are all domestic analytical pure.

[0069] The existing tyrosinase inhibitors are limited by poor safety, weak inhibitory effect, poor solubility, low yield, high cost and many other problems, and cannot be applied on a large scale. Based on the understanding of the structure-activity relationship (tannin polymer has a similar structure to the substrate of tyrosinase), the applicant found that compared with most existing tyrosinase inhibitors, the hawthorn pulp hawthorn condensed tannin polymer has high tyrosinase inhibitory activity, easy solubility in water, high yield and many other advantages. Therefore, the hawthorn pulp hawthorn condensed tannin polymer has the potential to develop into a skin melanin disease drug, a whitening agent, and a fruit and vegetable preservative.

[0070] The present application provides a hawthorn condensed tannin polymer, which is extracted from hawthorn pulp. The hawthorn condensed tannin polymer includes a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers of epicatechin as shown in formula (I):

[0071]

[0072] wherein n is a positive integer between 2 and 18, and at least one of the dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers includes a B-type procyanidin polymer obtained by connecting C4-C8 or C4-C6 using an epicatechin structural unit.

[0073] In the above technical solution, the present application takes hawthorn pulp as raw material, and a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers represented by formula I is obtained through separation and purification extraction. The hawthorn condensed tannin polymer with high polymerization degree can more effectively block the pocket of the active center of tyrosinase, reduce the catalytic ability of the enzyme and the activity of the substrate to the active center of the enzyme, thereby preventing the oxidation process of the substrate, which makes the hawthorn condensed tannin polymer of the present application have better anti-melanin production and anti-browning effects. The basic structural unit in the hawthorn condensed tannin polymer of the present application is epicatechin, a polyhydroxy phenolic compound containing an ortho-hydroxyl structure, which can chelate copper ions in the active center of tyrosinase, and at the same time produce hydrogen bonds, hydrophobic interactions and other non-covalent forces with amino acid residues of acids, thereby inhibiting the activity of tyrosinase. At least one of the dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers of the present application includes a B-type proanthocyanidin polymer composed of C4-C8 or C4-C6 connection of epicatechin structural units, as shown in formula (II), which is a structural formula of the polymer marker C atom connection site of the hawthorn condensed tannin polymer of the present application. In addition, the hawthorn condensed tannin polymer of the present application has high water solubility and purity, and the purity can reach 98% after determination, which is conducive to its further application in the fields of medicine, cosmetics and food.

[0074]

[0075] In the present application, the structural unit of the hawthorn condensed tannin polymer contains two connection modes, C4-C6 and C4-C8, respectively. In the same polymerization degree polymer, C4-C8 or C4-C6 connection obtains compounds with different spatial configurations, i.e. the dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers of the hawthorn condensed tannin polymer of the present application can be each independently a pure substance, or a mixture of a plurality of compounds with different configurations. For example, as shown in formula (III) and formula (IV), formula (III) is one configuration of the dimer, and formula (IV) is another configuration of the dimer. The molecular weights of the two configurations are the same, and they can be distinguished by nuclear magnetic resonance hydrogen spectrum.

[0076]

[0077] In some alternative technical solutions, the hawthorn condensed tannin polymer of this application is composed of dimers, trimers, tetramers, pentamers, hexamers, heptomers, octamers, nonamers, decamers, undecamerrers, dodecamerrers, thiamers, tetradecamerrers, decamers, hexadecamerrers, heptadecamerrers, and octadecamerrers as described in Formula I, comprising a mixture of seventeen polymers. Mass spectrometry analysis of the above-mentioned mixture of seventeen polymers is performed, such as... Figure 1 The image shown is a MALDI-TOF MS plot of the hawthorn condensed tannin polymer of this application. Figure 1 It can be seen that the MS spectrum contains characteristic peaks of polymers from 2 to 18, specifically at molecular weights of 711.07, 999.15, 1287.31, 1575.44, 1863.52, 2152.60, 2440.66, 2728.69, 3016.54, 3303.54, 3592.33, 3880.48, 4168.04, 4456.03, 4744.23, 5031.88, and 532. The presence of a mass spectrometry peak at position 0.39 indicates that the hawthorn condensed tannin polymer of this application comprises a mixture of seventeen polymers, all of which are detectable. This highly polymerized polymer extracted in this application can more effectively block the pockets of the enzyme's active site, reduce the enzyme's catalytic ability and competition with the substrate for the enzyme's active site, thereby preventing the substrate's oxidation process. This makes the hawthorn condensed tannin polymer of this application have superior anti-melanin production and anti-browning effects.

[0078] In some alternative technical solutions, the ultraviolet spectrum of the hawthorn condensed tannin polymer of this application has characteristic peaks at wavelengths of 215nm~220nm and 270nm~280nm.

[0079] In some alternative technical solutions, the hawthorn condensed tannin polymer of this application is an amorphous powder.

[0080] In some alternative technical solutions, the hawthorn condensed tannin polymer of this application is at least one of yellow, brown, and yellowish-brown.

[0081] This application also provides a method for preparing hawthorn condensed tannin polymer, such as... Figure 2 The diagram shown is a flowchart of the preparation process of the hawthorn condensed tannin polymer of this application, including the following steps:

[0082] Step S100: The hawthorn pulp powder is subjected to a single extraction process to obtain an aqueous extract;

[0083] Step S200: The aqueous extract is subjected to organic solvent extraction to obtain a crude extract;

[0084] Step S300, the crude extract is subjected to Sephadex LH 20 column chromatography elution treatment to obtain the hawthorn condensed tannin polymer.

[0085] In the above technical solution, the present application takes hawthorn pulp as raw material, and obtains a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers of epicatechin shown in formula I by selective separation and extraction. Specifically, the present application removes small molecular impurities and fat-soluble substances by one purification treatment, extraction treatment and column chromatography treatment, and obtains the hawthorn condensed tannin polymer with high purity and high yield. The preparation method of the present application has a simple process route, high yield and obvious inhibition effect on tyrosinase activity.

[0086] Specifically, the hawthorn condensed tannin polymer is extracted from hawthorn pulp according to the following steps:

[0087] Step 100, the hawthorn pulp dry powder is subjected to one extraction to obtain the water phase extract.

[0088] In some optional embodiments, the temperature of the first vacuum freeze-drying treatment is -85℃ to -55℃, for example, -85℃, -75℃, -65℃ and -55℃, and of course can be other values within the above range, which are not limited herein. The freeze-drying treatment within the above temperature range is beneficial to maximumly maintaining the structure and activity of the tannin polymer.

[0089] In some optional embodiments, the vacuum degree of the first vacuum freeze-drying treatment is less than 9Pa, for example, 1Pa, 2Pa, 3Pa, 4Pa, 5Pa, 6Pa, 7Pa and 8Pa, and of course can be other values within the above range, which are not limited herein.

[0090] In some optional embodiments, the time of the first vacuum freeze-drying treatment is 24h to 48h, for example, 24h, 28h, 30h, 36h, 40h, 44h and 48h, and of course can be other values within the above range, which are not limited herein.

[0091] In some optional embodiments, the mesh number of the screening is 40 mesh.

[0092] In some optional embodiments, the solvent for the first extraction includes acetone with a concentration of 60% to 70%, for example, the concentration of acetone can be 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, and the like, and of course, other values within the above range are also possible, which are not limited herein.

[0093] In some optional embodiments, the time for the first extraction is 15 min to 30 min, for example, the time for the first extraction can be 15 min, 20 min, 25 min, 30 min, and the like, and of course, other values within the above range are also possible, which are not limited herein.

[0094] In some optional embodiments, the ratio of the hawthorn pulp dry powder to the solvent for the first extraction is 1:(5-20), for example, the ratio of the hawthorn pulp dry powder to the solvent for the first extraction can be 1:5, 1:6, 1:8, 1:10, 1:13, 1:15, 1:20, and the like, and of course, other values within the above range are also possible, which are not limited herein.

[0095] In some optional embodiments, the first extraction is further followed by a step of performing a first rotary evaporation treatment on the aqueous phase extract.

[0096] In some optional embodiments, the temperature for the first rotary evaporation treatment is lower than 40°C, for example, the temperature for the first rotary evaporation treatment can be 20°C, 25°C, 30°C, 35°C, 38°C, and the like, and of course, other values within the above range are also possible, which are not limited herein. Limiting the temperature for the first rotary evaporation treatment within the above range is beneficial to maintaining the biological activity of the condensed tannin polymers of hawthorn.

[0097] In some optional embodiments, the time for the first rotary evaporation treatment is 1 h to 2 h, for example, the time for the first rotary evaporation treatment can be 1 h, 1.33 h, 1.5 h, 2 h, and the like, and of course, other values within the above range are also possible, which are not limited herein.

[0098] In some optional embodiments, the rotation speed for the first rotary evaporation treatment is 50 r / h to 80 r / h, for example, the rotation speed for the first rotary evaporation treatment can be 50 r / h, 55 r / h, 60 r / h, 65 r / h, 70 r / h, 75 r / h, 80 r / h, and the like, and of course, other values within the above range are also possible, which are not limited herein.

[0099] In step S200, the aqueous phase extract is subjected to an organic solvent extraction treatment to obtain a crude extract, specifically, the aqueous phase extract is sequentially subjected to petroleum ether extraction and ethyl acetate extraction, and then subjected to a second rotary evaporation treatment and a second vacuum freeze-drying treatment to obtain the crude extract.

[0100] In the above step, the petroleum ether extraction can effectively remove the fat-soluble substances in the crude extract, and the ethyl acetate extraction can remove the small molecular phenolic substances in the crude extract.

[0101] In some alternative embodiments, the volume ratio of the water phase extract to petroleum ether is (2-3): 1, for example, the volume ratio of the water phase extract to petroleum ether can be 2:1 and 3:1, and of course can also be other values within the above range, which is not limited herein.

[0102] In some alternative embodiments, the volume ratio of the water phase extract to ethyl acetate is (2-3): 1, for example, the volume ratio of the water phase extract to ethyl acetate can be 2:1 and 3:1, and of course can also be other values within the above range, which is not limited herein.

[0103] In some alternative embodiments, the number of times of ethyl acetate and petroleum ether extraction is 1-3 times.

[0104] In some alternative embodiments, the temperature of the second rotary evaporation treatment is less than 40°C, for example, the temperature of the second rotary evaporation treatment can be 20°C, 25°C, 30°C, 35°C and 38°C, and of course can also be other values within the above range, which is not limited herein.

[0105] In some alternative embodiments, the time of the second rotary evaporation treatment is 30-60 min, for example, the time of the second rotary evaporation treatment can be 30 min, 40 min, 50 min and 60 min, and of course can also be other values within the above range, which is not limited herein.

[0106] In some alternative embodiments, the rotation speed of the second rotary evaporation treatment is 50-80 r / h, for example, the rotation speed of the second rotary evaporation treatment can be 50 r / h, 55 r / h, 60 r / h, 65 r / h, 70 r / h, 75 r / h and 80 r / h, and of course can also be other values within the above range, which is not limited herein.

[0107] In some alternative embodiments, the time of the second vacuum freeze-drying treatment is 24-48 h, for example, the time of the second vacuum freeze-drying treatment can be 24 h, 30 h, 36 h and 48 h, and of course can also be other values within the above range, which is not limited herein.

[0108] In some alternative embodiments, the vacuum degree of the second vacuum freeze-drying treatment is less than 9 Pa, for example, the vacuum degree of the second vacuum freeze-drying treatment can be 1 Pa, 2 Pa, 3 Pa, 4 Pa, 5 Pa, 6 Pa, 7 Pa, 8 Pa, or the like, and of course, other values within the above range are also possible, which are not limited herein.

[0109] In some alternative embodiments, the temperature of the second vacuum freeze-drying treatment is -85℃ to -55℃, for example, the temperature of the second vacuum freeze-drying treatment can be -85℃, -75℃, -65℃, -55℃, or the like, and of course, other values within the above range are also possible, which are not limited herein.

[0110] In step S300, the crude extract is subjected to Sephadex LH 20 column chromatography elution treatment to obtain the refined hawthorn condensed tannin polymer. Specifically:

[0111] The crude extract is subjected to column chromatography using Sephadex LH 20 column, and after column chromatography, 50% to 60% methanol elution and 60% to 70% acetone elution are sequentially performed, and finally 60% to 70% acetone is subjected to third rotary evaporation treatment and third vacuum freeze-drying to obtain the refined hawthorn condensed tannin polymer.

[0112] In the above steps, the methanol elution can remove small molecule polyphenols and sugars in the crude extract, and the hawthorn condensed tannin polymer-containing part is extracted by acetone elution and removed by third rotary evaporation treatment to remove acetone in the hawthorn condensed tannin polymer. The refined hawthorn condensed tannin polymer obtained by column chromatography and elution treatment in the present application has high purity, high yield, and obvious inhibition effect on tyrosinase activity.

[0113] In some alternative embodiments, the height-to-diameter ratio of the filler loading is 6:1.

[0114] In some alternative embodiments, the column loading flow rate is 0.2 drop / s to 0.5 drop / s, for example, the column loading flow rate can be 0.2 drop / s, 0.3 drop / s, 0.4 drop / s, 0.5 drop / s, or the like, and of course, other values within the above range are also possible, which are not limited herein.

[0115] In some alternative embodiments, wet loading is used, and the loading amount of the filler and the crude extract is: 100 mg to 200 mg of the crude extract per 1 mL of the filler.

[0116] In some alternative embodiments, the temperature of the third rotary evaporation treatment is less than 40℃, for example, the temperature of the third rotary evaporation treatment can be 20℃, 25℃, 30℃, 35℃, 38℃, and the like, and of course, can also be other values within the above range, which are not limited herein.

[0117] In some alternative embodiments, the time of the third rotary evaporation treatment is 30min-60min, for example, the time of the third rotary evaporation treatment can be 30min, 40min, 50min, 60min, and the like, and of course, can also be other values within the above range, which are not limited herein.

[0118] In some alternative embodiments, the rotation speed of the third rotary evaporation treatment is 50r / h-80r / h, for example, the rotation speed of the third rotary evaporation treatment can be 50r / h, 55r / h, 60r / h, 65r / h, 70r / h, 75r / h, 80r / h, and the like, and of course, can also be other values within the above range, which are not limited herein.

[0119] In some alternative embodiments, the time of the third freeze-drying treatment is 24h-48h, for example, the time of the third freeze-drying treatment can be 24h, 30h, 36h, 48h, and the like, and of course, can also be other values within the above range, which are not limited herein.

[0120] In some alternative embodiments, the vacuum degree of the third freeze-drying treatment is less than 9Pa, for example, the vacuum degree of the third freeze-drying treatment can be 1Pa, 2Pa, 3Pa, 4Pa, 5Pa, 6Pa, 7Pa, 8Pa, and the like, and of course, can also be other values within the above range, which are not limited herein.

[0121] In some alternative embodiments, the temperature of the third freeze-drying treatment is -85℃--55℃, for example, the temperature of the third freeze-drying treatment can be -85℃, -75℃, -65℃, -55℃, and the like, and of course, can also be other values within the above range, which are not limited herein.

[0122] The embodiments of the present application are further described in the following examples. The embodiments of the present application are not limited to the following specific examples. Within the scope of the same rights, appropriate changes can be made.

[0123] Example 1

[0124] Step S100, the fresh hawthorn pulp is subjected to first vacuum freeze-drying treatment, grinding and 40 mesh screen to obtain 10 g of hawthorn pulp dry powder, the hawthorn pulp dry powder is subjected to ultrasonic extraction with 70% acetone for 25 min, and the water layer obtained by rotary evaporation at 70 r / min and 35°C for 60 min is the aqueous phase extract.

[0125] Step S200, the aqueous phase extract is first extracted with petroleum ether, and the volume ratio of the aqueous phase extract to petroleum ether is 3:1. Then the aqueous phase extract is extracted with ethyl acetate, and the volume ratio of the aqueous phase extract to ethyl acetate is 3:1. Finally, the rotary evaporator is used at 70 r / min and 35°C for 60 min and freeze-dried to obtain the crude extract.

[0126] Step S300, the aqueous phase extract is subjected to column chromatography with Sephadex LH 20 column, and then eluted with 50% methanol and 70% acetone in sequence, and then subjected to rotary evaporation at 70 r / min and 35°C for 60 min, and then freeze-dried at -55°C to obtain 1.108 mg of refined tannin extract, which is the hawthorn condensed tannin polymer of the present application.

[0127] In this embodiment, the yield of the prepared refined tannin extract is 11.08%, and the purity of the refined hawthorn condensed tannin polymer is calculated to be 99.05% based on condensed tannin dimer as standard.

[0128] The mass spectrum analysis of the refined tannin extract prepared in this embodiment is carried out, Figure 1 The MALDI-TOF MS spectrum of the refined hawthorn condensed tannin polymer of Example 1 of the present application in positive ion (Cs + ) mode is shown in Figure 1. Figure 1 It can be seen that the MS spectrum has characteristic peaks of 2-18 polymers, and has mass spectrum peaks at molecular weights of 711.07, 999.15, 1287.31, 1575.44, 1863.52, 2152.60, 2440.66, 2728.69, 3016.54, 3303.54, 3592.33, 3880.48, 4168.04, 4456.03, 4744.23, 5031.88, 5320.39, indicating that the hawthorn condensed tannin polymer of the present application includes seventeen kinds of polymers.

[0129] The nuclear magnetic resonance (NMR) analysis of the refined hawthorn condensed tannin polymer of this embodiment is carried out, Figure 3 as shown in Figure 2. Figure 3 The spectrum shown in Figure 2 has all the typical peaks of catechin C atoms, indicating that the hawthorn condensed tannin polymer of the present application is a procyanidin polymer composed of catechin, and has very high homogeneity.

[0130] The refined haw condensed tannin polymer of the present example was subjected to high performance liquid chromatography analysis after thiol degradation and compared with commercially available epicatechin (Sigma-Aldrich, St. Louis, MO, USA). As shown in FIG. 1, the results showed that the haw pulp haw condensed tannin polymer was composed of epicatechin composed of procyanidin polymer, and the purity of the haw condensed tannin polymer of the present application was further determined to be close to 100% (almost no impurity peak) from the HPLC spectrum. Figure 4

[0131] The refined haw condensed tannin polymer of the present example was weighed to prepare a haw condensed tannin polymer solution with a concentration of 1.5 mg / mL, 100 μL of the tannin solution was taken as the sample to be tested, and a UV-Vis spectrophotometer with a wavelength scanning range of 190 nm to 600 nm was used to record the change between the absorbance and wavelength of the haw condensed tannin polymer. As shown in FIG. 2, the full wavelength scanning of the haw pulp tannin can find that there are two obvious characteristic peaks at 217 nm and 276 nm. Figure 5

[0132] From the above structure analysis, it can be known that the haw condensed tannin polymer of the present example is a mixture composed of B-type procyanidin polymers with 2-18 polymerization of epicatechin. The B-type procyanidin is a polyhydroxy phenolic compound with ortho-hydroxyl structure (tyrosinase substrate structure analog), which can chelate copper ions in the active center of the enzyme, and at the same time produce hydrogen bonds, hydrophobic interactions and other non-covalent forces with amino acid residues of acids. Therefore, the haw condensed tannin polymer extracted and purified by the present application can efficiently inhibit the activity of tyrosinase.

[0133] The calculated half-inhibition rate (IC 50 ) of the haw condensed tannin polymer prepared in the present example for tyrosine was 0.16 ± 0.02 mg / mL.

[0134] Comparative Example 1

[0135] Different from Example 1, arbutin was used in step S100.

[0136] The half-inhibition rate (IC 50 ) of the sample prepared in the present comparative example for tyrosine was (1.45 ± 0.03 mg / mL), which was significantly weaker than the sample prepared by using haw pulp in Example 1.

[0137] Performance test:

[0138] I. Test of tyrosinase inhibiting ability of haw condensed tannin polymer

[0139] ​​The specific test procedure is as follows: the hawthorn condensed tannin polymer prepared in Example 1 and the sample prepared in Comparative Example 1 are respectively dissolved in double distilled water to prepare tannin solutions with different concentrations. L-DOPA is used as a substrate, and in a 3 mL active system, 0.3 mL of the substrate solution, 0.75 mL of a phosphate buffer (0.05 mol / L, pH 6.8), 1.8 mL of double distilled water, and 0.1 mL of the tannin solution with different concentrations are sequentially added to a colorimetric cup. The colorimetric cup is incubated in a 30°C constant temperature water bath, then 0.05 mL of a mushroom tyrosinase aqueous solution is added and immediately mixed, and the linear increase in light absorption value with time is measured (the wavelength is set to 475 nm), and the slope of the straight line is the enzyme activity. The relative residual activity of the enzyme is plotted against the concentration of the hawthorn condensed tannin polymer, and the enzyme activity inhibition (IC 50 ) of the hawthorn condensed tannin polymer is obtained. The test results are shown in Table 1.

[0140] Table 1. Inhibition of tyrosinase by hawthorn condensed tannin polymer in hawthorn pulp (IC 50 The smaller the value, the better the inhibition effect)

[0141]

[0142] As can be seen from the data in Table 1, the hawthorn condensed tannin polymer extracted from the hawthorn pulp has a strong tyrosinase inhibition ability, which is significantly stronger than that of the positive control arbutin. Tyrosinase is a key rate-limiting enzyme for melanin production, which catalyzes the oxidation of tyrosine in melanocytes to melanin. Therefore, the hawthorn condensed tannin polymer of the present application can inhibit the production of melanin, and thus can be added to drugs for skin whitening, drugs for treating pigmentation diseases, etc., and thus has a wide application in skin whitening and treatment of pigmentation diseases (chloasma, freckles, melanoma, etc.).

[0143] II. Antioxidant ability test of hawthorn condensed tannin polymer

[0144] The antioxidant ability of the hawthorn condensed tannin polymer is tested according to the following method, with ascorbic acid and butylated hydroxyanisole as positive controls.

[0145] (1) DPPH method: 0.1 mL of samples with different concentrations (15.63 μg / mL, 31.25 μg / mL, 62.5 μg / mL, and 125 μg / mL) are taken, and 3 mL of 0.004% DPPH methanol solution is added. After mixing well, the absorbance value is measured at 517 nm after standing for 30 min (the blank sample uses methanol).

[0146] (2) ABTS method: 0.0384 g of ABTS and 0.0134 g of potassium persulfate were dissolved in 10 mL of distilled water, respectively, and then mixed 1:1, to obtain an ABTS cation radical mother liquor after 16 hours of reaction in the dark. The above mother liquor was diluted with 80% ethanol aqueous solution before use, and the absorbance was adjusted to 0.700 ± 0.050 at a wavelength of 734 nm, to thereby obtain an ABTS radical working solution. 0.1 mL of a sample or standard solution of a suitable concentration was mixed uniformly with 3.9 mL of the ABTS cation radical working solution, and after 6 min of standing, the absorbance was detected at 734 nm. The blank control used 80% ethanol aqueous solution. The test results are shown in Table 2.

[0147] Table 2. Comparison of radical scavenging ability (IC50) of examples and positive controls 50 The smaller the value, the better the inhibitory effect

[0148]

[0149] From the data in Table 2, it can be seen that the hawthorn condensed tannin polymer of Example 1 of the present application can efficiently scavenge DPPH and ABTS radicals compared to the two positive controls of ascorbic acid and butylated hydroxyanisole, indicating that the hawthorn condensed tannin polymer of the present application has excellent antioxidant capacity, and can therefore be applied to whitening-related cosmetics or personal care products.

[0150] It can be understood that the hawthorn condensed tannin polymer of the present application can also be used as a skin care product. Exemplarily, the polymer can be a cosmetic or personal care skin care product, which comprises the hawthorn condensed tannin polymer of the present application, a pharmaceutically or cosmetically acceptable carrier and other additives. The carrier can be a polyhydric alcohol, an oil component and a water component, and mixtures thereof. The polyhydric alcohol can be, for example, ethanol, isopropyl alcohol, ethylene glycol and glycerol, etc. The oil component is a component of an O / W emulsion, which can be, for example, a C6-C20 fatty acid, a linear or branched C6-C20 aliphatic alcohol or an ester of a C6-C20 aliphatic alcohol, such as myristyl stearate, myristyl oleate, myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate and cetyl stearate, etc. The other additives can be alkylbenzenesulfonate, glyceryl ether sulfate and sulfoglycerol triester, etc. The polymer of the present application can be applied as a toner, an emulsion, a cream, an essence, a sunscreen product and a mask, etc.

[0151] III. Preservation ability test of hawthorn condensed tannin polymer

[0152] (1) Polyphenol oxidase (PPO) activity determination procedure: 1 mL of sodium phosphate buffer (pH 6.8), 1 mL of catechol and 1 mL of fresh-cut apple enzyme solution were mixed, and the absorbance value was measured at 420 nm every 1 min.

[0153] (2) Total phenol content determination procedure: The total phenol content of red beans was determined by Folin-Ciocalteu method. 200 μL of sample initial extract, 300 μL of distilled water, 500 μL of FC reagent (0.5 mol / L) and 2500 μL of saturated Na2CO3 (20%) were sequentially added to the test tube, shaken and mixed, reacted for 30 min, and then the absorbance value of the solution was measured at 725 nm.

[0154] Polyphenol oxidase (PPO) is the key enzyme of enzymatic browning of fruits and vegetables, and inhibiting its activity can prevent the oxidation of polyphenolic substances and play a role in preservation. In this application, fresh-cut apples were used as the control group, and different concentrations (0.5 mg / mL, 1 mg / mL and 2 mg / mL) of hawthorn condensed tannin polymers of Example 1 were used for activity determination. The results are shown in Figure 6 The activity in fresh-cut apples increased during the whole storage period, however, with the increase of hawthorn pulp tannin concentration, the PPO activity decreased. Statistical analysis showed that the PPO activity of fresh-cut apples treated with different concentrations of hawthorn pulp tannin was significantly lower than that of the control group (CK). Compared with the control group of apples, at 72 hours, the PPO activity of fresh-cut apples treated with different concentrations of hawthorn pulp tannin was reduced by 69.6%, 77.6% and 88%, respectively, and the inhibition effect was 9.6%, 17.6% and 28% stronger than that of the control group. This shows that the hawthorn condensed tannin polymer of hawthorn pulp has excellent preservation ability.

[0155] As shown in Figure 7 Compared with the control, the total phenol content increased with the increase of the concentration of hawthorn condensed tannin polymer, indicating that the hawthorn condensed tannin polymer of this application can prevent the oxidation of total phenols in fresh-cut apples. This shows that the hawthorn condensed tannin polymer prepared in this application can effectively prevent the oxidation of phenolic substances, thereby playing a role in preservation, and the hawthorn condensed tannin polymer of this application can be used as a fruit and vegetable preservative.

[0156] The above only describes the preferred embodiments of the present application patent, and does not limit the present application patent. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application patent shall be included in the protection scope of the present application patent.

Claims

1. A hawthorn condensed tannin polymer, characterized by, The condensed tannin polymer of hawthorn is extracted from hawthorn pulp, and the condensed tannin polymer of hawthorn comprises a mixture of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers of epicatechin shown in formula (I); （Ⅰ） wherein n is a positive integer between 2 and 18, and at least one of the dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, undecamers, dodecamers, tridecamers, tetradecamers, pentadecamers, hexadecamers, heptadecamers and octadecamers comprises a B-type proanthocyanidin polymer obtained by connecting epicatechin structural units through C4-C8 or C4-C6; The condensed tannin polymer of hawthorn is prepared by the following steps: The hawthorn pulp dry powder is subjected to one-time extraction to obtain a water phase extract, and the solvent concentration of the one-time extraction is 60%-70% acetone; The water phase extract is subjected to petroleum ether extraction and ethyl acetate extraction in sequence to obtain a crude extract; The crude extract is subjected to column chromatography using a Sephadex LH-20 column, and after column chromatography, the condensed tannin polymer of hawthorn is eluted in sequence using 50%-60% methanol and 70% acetone.

2. The hawthorn condensed tannin polymer according to claim 1, characterized by, The condensed tannin polymer of hawthorn has characteristic peaks at wavelengths of 215 nm-220 nm and 270 nm-280 nm in the ultraviolet spectrum.

3. A method for preparing the condensed tannin polymer of hawthorn according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: The hawthorn pulp dry powder is subjected to one-time extraction to obtain a water phase extract, and the solvent concentration of the one-time extraction is 60%-70% acetone; The water phase extract is subjected to petroleum ether extraction and ethyl acetate extraction in sequence to obtain a crude extract; The crude extract is subjected to column chromatography using a Sephadex LH-20 column, and after column chromatography, the condensed tannin polymer of hawthorn is eluted in sequence using 50%-60% methanol and 70% acetone.

4. The production method according to claim 3, characterized by, The method comprises at least one of the following features (1)-(11): (1) The hawthorn pulp dry powder is obtained by subjecting hawthorn pulp to first-time vacuum freeze-drying treatment, grinding and sieving; (2) The hawthorn pulp dry powder is obtained by subjecting hawthorn pulp to first-time vacuum freeze-drying treatment, grinding and sieving, and the temperature of the first-time vacuum freeze-drying treatment is -85 ℃ to -55 ℃; (3) The hawthorn pulp dry powder is obtained by subjecting hawthorn pulp to first-time vacuum freeze-drying treatment, grinding and sieving, and the vacuum degree of the first-time vacuum freeze-drying treatment is less than 9 Pa; (4) The hawthorn pulp dry powder is obtained by subjecting hawthorn pulp to first-time vacuum freeze-drying treatment, grinding and sieving, and the time of the first-time vacuum freeze-drying treatment is 24 h-48 h; (5) The hawthorn pulp dry powder is obtained by subjecting hawthorn to first-time vacuum freeze-drying treatment, grinding and sieving, and the mesh number of the sieving is 40 mesh; (6) The time of the one-time extraction is 15 min-30 min; (7) the mass ratio of the said hawthorn pulp dry powder to the said solvent of the first extraction is 1: (10-20); (8) the said first extraction is further followed by a step of performing first rotary evaporation treatment on the said water phase extract; (9) the said first extraction is further followed by a step of performing first rotary evaporation treatment on the said water phase extract, and the temperature of the said first rotary evaporation is less than 40 ℃; (10) the said first extraction is further followed by a step of performing first rotary evaporation treatment on the said water phase extract, and the time of the said first rotary evaporation is 1 h-2 h; (11) the said first extraction is further followed by a step of performing first rotary evaporation treatment on the said water phase extract, and the rotation speed of the said first rotary evaporation is 50 r / h-80 r / h.

5. The production method according to claim 3, wherein The said water phase extract is further followed by petroleum ether extraction and ethyl acetate extraction, and then further followed by: performing second rotary evaporation treatment and second freeze drying treatment on the product of the ethyl acetate extraction to obtain a crude extract, and the said method comprises at least one of the following features (1)-(9): (1) the static ratio of the said water phase extract to the said petroleum ether is (2-3): 1; (2) the volume ratio of the said water phase extract to the said ethyl acetate is (2-3): 1; (3) the number of times of the said ethyl acetate extraction is 2-3 times; (4) the temperature of the said second rotary evaporation treatment is less than 40 ℃; (5) the time of the said second rotary evaporation treatment is 30 min-60 min; (6) the rotation speed of the said second rotary evaporation treatment is 50 r / h-80 r / h; (7) the time of the said second freeze drying treatment is 24 h-48 h; (8) the vacuum degree of the said second freeze drying treatment is less than 9 Pa; (9) the temperature of the said second freeze drying treatment is -55 ℃--85 ℃.

6. The production method according to claim 3 or 5, characterized by, The said washing treatment with methanol of a concentration of 50%-60% and acetone of a concentration of 70% is further followed by: performing third rotary evaporation treatment and third freeze drying treatment on the product of the said washing treatment to obtain hawthorn condensed tannin polymer, and the said method comprises at least one of the following features (1)-(9): (1) the height-diameter ratio of the packing of the said Sephadex LH 20 column is 6:1; (2) the flow rate of column loading of the said Sephadex LH 20 column is 0.2 drop / s-0.5 drop / s; (3) the loading amount of the said packing of the Sephadex LH 20 column to the said crude extract is: wet loading is adopted, and 1 mL of the said packing corresponds to 100 mg-200 mg of the said crude extract; (4) the temperature of the said third rotary evaporation treatment is less than 40 ℃; (5) the time of the said third rotary evaporation treatment is 30 min-60 min; (6) the rotation speed of the said third rotary evaporation treatment is 50 r / h-80 r / h; (7) the time of the said third freeze drying treatment is 24 h-48 h; (8) the vacuum degree of the said third freeze drying treatment is less than 9 Pa; (9) the third freeze-drying temperature is -85℃ to -55℃.

7. Use of the hawthorn condensed tannin polymer according to any one of claims 1 to 2 or prepared by the method according to any one of claims 3 to 6 in the preparation of a medicament for treating a pigmentation disorder.

8. Use of the hawthorn condensed tannin polymer according to any one of claims 1 to 2 or prepared by the method according to any one of claims 3 to 6 in the preparation of a cosmetic product for whitening.

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