Selenized cinnamon polysaccharide as well as preparation method and application thereof

Abstract

The invention provides selenized cinnamon polysaccharide as well as a preparation method and application thereof, and relates to the technical field of preparation of natural medicine modified polysaccharide. The selenized cinnamon polysaccharide comprises a cinnamon polysaccharide chain and selenium bound to the cinnamon polysaccharide chain in the form of a covalent bond. The preparation method of the selenized cinnamon polysaccharide comprises the following steps: sequentially carrying out enzymolysis, decoloration, deproteinization, purification and selenylation on cinnamon to obtain the selenized cinnamon polysaccharide. Inorganic selenium is introduced into cinnamon polysaccharide (CCP1) to form selenized cinnamon polysaccharide (Se-CCP1), and the selenized cinnamon polysaccharide shows higher activity than non-selenized cinnamon polysaccharide in whitening effect, which indicates that selenized modification can enhance the whitening effect of cinnamon polysaccharide, and selenized modification can change molecular conformation, physical properties and physiological functions of polysaccharide, so that the skin whitening effect of the cinnamon polysaccharide is improved. Therefore, the biological activity is improved.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of natural medicine modified polysaccharides, and in particular to selenized cinnamon polysaccharide and a preparation method and application thereof. Background Art

[0002] Whitening cosmetics are very popular among consumers, and cosmetics companies are also very optimistic about this market and have launched their own whitening products. Cosmetics that help to reduce or slow down skin pigmentation and achieve skin whitening and brightening effects are called whitening cosmetics. At present, the mechanism of action of whitening cosmetics, in addition to the physical coverage required for the product to produce a whitening effect, mostly includes the following aspects: inhibiting melanin production, inhibiting melanin transport, reducing melanin, and promoting the shedding of epidermal melanin. Arbutin, niacinamide, glabridin, phenylethyl resorcinol, ascorbic acid, etc. are commonly used whitening agents in cosmetics. Traditional whitening skin care active ingredients, such as retinoic acid, arbutin and niacinamide, although the effect is very good, their adverse reactions are becoming increasingly apparent, such as contact dermatitis, irritation, and even genotoxicity and carcinogenicity.

[0003] Due to the growing demand for safe and effective natural cosmetic ingredients, natural products are widely used in skin care products due to their safety, low toxicity and low irritation. The practice of extracting active ingredients from natural products and adding them to cosmetics has gradually become widely sought after. Chinese herbal active ingredients have great potential value in removing spots and whitening the skin. Many Chinese herbal ingredients can whiten the skin by reducing or inhibiting the activity of intracellular tyrosinase and reducing the formation of melanin. For example, dendrobium polysaccharides, purslane polysaccharides, ganoderma lucidum polysaccharides, etc., have been reported in relevant literature to have whitening effects, and are widely used as cosmetic raw materials in moisturizing whitening creams or facial masks and other cosmetic products.

[0004] Cinnamon is the dried bark of Cinnamomum cassia Preesl. (CCP), a plant of the Lauraceae family. It is widely used around the world for its fragrance and spicy taste. It can be used not only as a daily condiment, but also as a raw material for medical products, with high economic value. Cinnamon is one of the essential medicinal and edible species stipulated by the state, and has a variety of natural effects. Cinnamon is a common traditional Chinese medicine. There are more than 500 recipes containing cinnamon for the treatment of various diseases, such as cardiovascular disease, chronic gastrointestinal disease, gynecological diseases and inflammatory diseases. Ancient books such as Shennong's Herbal Classic and Compendium of Materia Medica have recorded the medicinal value of cinnamon. In modern Chinese medicine pharmacology research, it was found that cinnamon has pharmacological effects such as antibacterial, antioxidant, analgesic, anti-inflammatory, anti-tumor, anti-diabetic, anti-obesity, cardiovascular protection, neuroprotection, and inhibition of tyrosinase activity. Cinnamon has a wide variety of chemical components, mainly including volatile oils, polysaccharides, polyphenols, flavonoids, etc. Cinnamon is one of the medicinal and edible varieties stipulated by the state. It has many efficacy activities and low toxicity, which makes cinnamon widely used. Among them, there are many cinnamon-related products on the market, such as cinnamon rolls, cinnamon tea and other foods; cinnamon toothpaste, cinnamon hair care essential oil and other cosmetics. Many studies have shown that a large number of polysaccharide compounds isolated from natural products have many biological activities, such as scavenging free radicals, inhibiting lipid peroxidation and linoleic acid oxidation. Cinnamon has a variety of chemical components. In addition to volatile oils, there are polysaccharides, polyphenols, etc. Polysaccharides are a research hotspot after proteins and nucleic acids. The consumption of polysaccharides in various industries is increasing, which requires improving their structural and functional properties. Therefore, appropriate modification of the structure of polysaccharides has become one of the research focuses in the field of polysaccharides.

[0005] Structural modification of polysaccharides refers to the derivation of the structure of carbohydrates and carbohydrate complexes through physical, chemical and biological modification, so that the active or toxic sites and functional groups of carbohydrates and carbohydrate complexes are optimized to reduce toxicity and improve biological activity, giving polysaccharides many new medicinal values. Chemical modification using carboxylation, sulfation, phosphorylation and selenization is an effective way to improve the safety of polysaccharides. After chemical modification, the biological activity of polysaccharides is significantly improved. These polysaccharides with improved biological activity after chemical modification will be widely used in food, medicine and cosmetics to improve the health of consumers.

[0006] Selenium is an essential trace element for life. In humans and animals, selenium is an important trace element that participates in many important physiological activities and has superior antioxidant and detoxification effects on the skin. Selenium is one of the four internationally recognized antioxidants. It can not only improve the antioxidant level of animals through glutathione peroxidase, but also improve immune function by maintaining the integrity of membrane structure. Inorganic selenium and polysaccharides synthesize organic selenium compounds-selenopolysaccharides, which have higher biological activity than selenium and polysaccharides. It is also easily absorbed and utilized by organisms. Selenium polysaccharides have been increasingly widely used in antioxidant and anti-inflammatory aspects.

[0007] Inorganic selenium and organic selenium exist in nature. Inorganic selenium is toxic to the human body and needs to be converted into organic selenium to be used by the human body; organic selenium is widely used because of its higher biological activity and low toxicity. Selenium can help improve the problem of uneven skin color. It helps to inhibit the production of melanin and reduce the appearance of spots and freckles. Organic selenium exists mainly in the form of selenopolysaccharides and selenoproteins in organisms. Selenium polysaccharides play an important role in the selection of selenium supplements, but the selenium content of selenium polysaccharides is generally low, so there are certain regional limitations in obtaining selenium polysaccharides. Therefore, efficient selenium-enriched treatment of polysaccharides has become a research hotspot. Selenized polysaccharides are made by mixing inorganic selenides and organic polysaccharides together, and introducing selenium into the polymer chain of polysaccharides in the presence of a catalyst. In selenopolysaccharides, selenium and polysaccharides are combined through special selenium-oxygen bonds. The commonly used selenization modification methods for polysaccharides include the nitric acid-sodium selenite method, the glacial acetic acid-sodium selenite method, and the selenium oxychloride method.

[0008] However, the existing methods for cinnamon polysaccharides and their modification still have the following major defects: 1. At present, the application of cinnamon in cosmetics is mainly focused on the use of cinnamon volatile oil, and the main components of cinnamon volatile oil, such as cinnamaldehyde and cinnamyl alcohol, have certain skin irritation, while there are few research reports on other milder components of cinnamon, such as polysaccharides. Polysaccharide selenization modification has potential in whitening, but it has not been fully developed and utilized. 2. Selenized modified polysaccharides have certain potential in whitening, but have not been fully developed and utilized. The currently disclosed extraction and / or purification methods of cinnamon polysaccharides cannot obtain polysaccharide products with high whitening activity. 3. Safety and irritation issues: Although the addition of traditional whitening skin care active ingredients, such as retinoic acid, arbutin and niacinamide, has a good effect, its adverse reactions are becoming increasingly apparent, such as contact dermatitis, irritation, and even genotoxicity and carcinogenicity.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The purpose of the present invention is to provide a selenized cinnamon polysaccharide and a preparation method and application thereof. The selenized cinnamon polysaccharide of the present invention enhances the whitening activity of cinnamon polysaccharide by selenization modification of cinnamon polysaccharide, and at the same time improves the safety and functionality of cosmetics by using natural extracts. The systematic and comprehensive study of the selenization modification and whitening effect of cinnamon polysaccharide is helpful to promote the full development and utilization of cinnamon resources, and provides a reference for the development and application of cinnamon polysaccharide in cosmetics.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides a selenized cinnamon polysaccharide, wherein the selenized cinnamon polysaccharide comprises a cinnamon polysaccharide chain and selenium covalently bonded to the cinnamon polysaccharide chain.

[0013] The present invention provides a selenized cinnamon polysaccharide: the purpose is to enhance the whitening activity of cinnamon polysaccharide by modifying the cinnamon polysaccharide. The present invention explores the application of cinnamon polysaccharide in cosmetics: by systematically and comprehensively studying the extraction method, structural identification and cosmetic efficacy of cinnamon polysaccharide, the full development and utilization of cinnamon resources are promoted, and a reference is provided for the development and application of cinnamon polysaccharide in cosmetics, especially in whitening. The present invention improves the safety and functionality of cosmetics: by using natural extracts, the safety and functionality of cosmetics are improved, general cosmetics are improved and innovated, and the market demand for safe and effective natural cosmetic ingredients is met.

[0014] Preferably, the molecular weight of the selenized cinnamon polysaccharide is 6000-7000Da.

[0015] Preferably, the selenium content in the selenized cinnamon polysaccharide is 3100-3300 mg / kg.

[0016] Preferably, the covalent bond includes a Se=O bond, a selenium bond and a CO-Se bond.

[0017] Preferably, the monosaccharide composition of the selenized cinnamon polysaccharide includes xylose, mannose and glucose, and the molar ratio of xylose, mannose and glucose is (17.050-17.055):(6.950-6.955):(75.990-75.995).

[0018] Preferably, the particle size of the selenized cinnamon polysaccharide is 221.1±5.5 nm, and the Zeta potential of the selenized cinnamon polysaccharide is -11.6±1.4 mV.

[0019] In a second aspect, the present invention provides a method for preparing the selenized cinnamon polysaccharide as described in the first aspect. The method for preparing the selenized cinnamon polysaccharide comprises: sequentially performing enzymatic hydrolysis, decolorization, protein removal, purification and selenization on cinnamon to obtain the selenized cinnamon polysaccharide.

[0020] Preferably, the enzymatic hydrolysis specifically comprises the following steps:

[0021] The cinnamon powder and water are mixed, and after adjusting the pH, cellulase is added to perform enzymolysis to obtain an enzymolysis solution; the enzymolysis solution is subjected to ultrasonication, enzyme inactivation, filtration and concentration in sequence to obtain a crude polysaccharide solution.

[0022] Preferably, the mass ratio of the cinnamon powder to water is 1:(10-30).

[0023] Preferably, the reagents used to adjust the pH are citric acid and sodium citrate.

[0024] Preferably, the pH of the enzymatic hydrolysis is 5.0±0.05.

[0025] Preferably, the added amount of the cellulase is 1-5% of the total mass of the cinnamon powder and water.

[0026] Preferably, the enzymatic hydrolysis temperature is 45-55° C., and the enzymatic hydrolysis time is 30-35 min.

[0027] Preferably, the power of the ultrasound is 100-110 W, and the time of the ultrasound is 60-70 min.

[0028] Preferably, the enzyme inactivation is carried out under the condition of water bath heating, the temperature of the enzyme inactivation is 85-95° C., and the time of the enzyme inactivation is 1-10 min.

[0029] Preferably, the decolorization specifically comprises the following steps:

[0030] The crude polysaccharide solution obtained by enzymatic hydrolysis is mixed with a decolorizing resin for decolorization; after the decolorization is completed, the resin is washed, the decolorized solution is collected and combined, and the decolorized solution is subjected to a first centrifugation, concentration, alcohol precipitation and a second centrifugation in sequence to obtain decolorized cinnamon polysaccharide.

[0031] Preferably, the decolorizing resin is a weakly basic anion exchange resin with a polystyrene macroporous structure, preferably D354FD resin.

[0032] Preferably, the volume ratio of the crude polysaccharide solution to the decolorizing resin is 1:(0.5-2).

[0033] Preferably, the decolorization is carried out under the condition of water bath heating, the decolorization temperature is 50-60° C., and the decolorization time is 2-4 hours.

[0034] Preferably, during the decolorization process, stirring is performed once every 10 to 30 minutes.

[0035] Preferably, the rotation speed of the first centrifugation is 3000-5000 rpm, the time of the first centrifugation is 5-15 min; and after the first centrifugation, the supernatant is collected for subsequent concentration.

[0036] Preferably, the concentration temperature is 50-55° C.; and the solid content of the concentrated solution obtained by the concentration is 70-80%.

[0037] Preferably, during the alcohol precipitation process, the volume ratio of the concentrated solution obtained by concentration to ethanol is 1:(4-5).

[0038] Preferably, the temperature of the alcohol precipitation is 1 to 10° C., and the time of the alcohol precipitation is 6 to 18 hours.

[0039] Preferably, the rotation speed of the second centrifugation is 7000-9000 rpm, the time of the second centrifugation is 5-15 min; and after the second centrifugation, the precipitate is collected to obtain decolorized cinnamon polysaccharide.

[0040] Preferably, the protein removal specifically comprises the following steps:

[0041] The decolorized cinnamon polysaccharide is mixed with water to obtain an aqueous solution, which is then mixed with a Sevage reagent, shaken, allowed to stand and separated into layers, and the aqueous layer solution is collected; after the protein is removed, the aqueous layer solution is sequentially concentrated, alcohol precipitated, centrifuged and dried to obtain refined cinnamon polysaccharide.

[0042] Preferably, the mass ratio of the decolorized cinnamon polysaccharide to water is 1:(20-25).

[0043] Preferably, the volume ratio of the aqueous solution to the Sevage reagent is (3-5):1.

[0044] Preferably, the standing and stratifying time is 30 to 50 minutes.

[0045] Preferably, in the protein removal process, the steps of mixing with the Sevage reagent, shaking and then standing to separate layers are repeated at least twice until no denatured protein is produced at the interface of the solution.

[0046] Preferably, the concentration temperature is 50-55° C.; and the solid content of the concentrated solution obtained by the concentration is 70-80%.

[0047] Preferably, during the alcohol precipitation process, the volume ratio of the concentrated solution obtained by concentration to ethanol is 1:(4-5).

[0048] Preferably, the centrifugal speed is 7000-9000 rpm, the centrifugal time is 5-15 min; and the precipitate is collected after the centrifugation.

[0049] Preferably, the purification specifically comprises the following steps:

[0050] The cinnamon refined polysaccharide obtained by removing protein is mixed with water to obtain a cinnamon refined polysaccharide solution; the cinnamon refined polysaccharide solution is purified once by using a chromatography column, eluted with pure water, and the polysaccharide component of the pure water elution peak is collected; the polysaccharide component of the pure water elution peak is purified twice by using a gel column, eluted with pure water, the main peak eluate is collected, and concentrated and dried in sequence to obtain cinnamon polysaccharide CCP1.

[0051] Preferably, the concentration of the cinnamon refined polysaccharide solution is 10-30 mg / mL.

[0052] Preferably, the chromatography column is a DEAE ion exchange chromatography column.

[0053] Preferably, the one-time purification process further comprises eluting with different salt solutions, and the concentrations of the different salt solutions are: 0.05 mol / L, 0.1 mol / L, 0.3 mol / L and 0.5 mol / L.

[0054] Preferably, the gel column is Sephadex G-100 gel.

[0055] Preferably, the monosaccharide composition of the cinnamon polysaccharide CCP1 includes xylose, mannose and glucose, and the molar ratio of xylose, mannose and glucose is (4.810-4.815):(16.190-16.195):(78.995-79.000).

[0056] Preferably, the particle size of the cinnamon polysaccharide CCP1 is 472.2±51.4 nm, and the Zeta potential of the cinnamon polysaccharide CCP1 is -8.9±0.9 mV.

[0057] Preferably, the selenization specifically comprises the following steps:

[0058] The purified cinnamon polysaccharide is dissolved in nitric acid, stirred, and then sodium selenite and barium chloride are added to carry out selenization reaction to obtain a selenized solution; the selenized solution is subjected to pH adjustment, barium chloride is removed, centrifuged and dialyzed to obtain selenized cinnamon polysaccharide.

[0059] Preferably, the mass ratio of cinnamon polysaccharide, nitric acid, sodium selenite and barium chloride is (1-3):(0.5-1.5):(1-2):(6-8).

[0060] Preferably, the concentration of the nitric acid is 0.1-1% v / v.

[0061] Preferably, the stirring time is 0.1 to 1 h.

[0062] Preferably, the temperature of the selenization reaction is 65-75° C., and the time of the selenization reaction is 6-8 hours.

[0063] Preferably, the pH of the selenization solution is adjusted to 7-8.

[0064] Preferably, the pH is adjusted using a 0.5-2 mol / L sodium hydroxide aqueous solution.

[0065] Preferably, the reagent used to remove barium chloride is sodium sulfate.

[0066] Preferably, the centrifugal force of the centrifugation is 600-650 xg, and the centrifugation time is 5-15 min.

[0067] Preferably, the dialysis is performed using a 3 kDa dialysis bag with distilled water, and the dialysis time is 66 to 78 hours.

[0068] In a third aspect, the present invention provides a use of the selenized cinnamon polysaccharide as described in the first aspect in preparing cosmetics with whitening effect.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] (1) The present invention introduces inorganic selenium into cinnamon polysaccharide (CCP1) to form selenized cinnamon polysaccharide (Se-CCP1). The selenized cinnamon polysaccharide shows stronger activity in whitening effect than non-selenized cinnamon polysaccharide, which indicates that selenization modification can enhance the whitening effect of cinnamon polysaccharide, and selenization modification can change the molecular conformation, physical properties and physiological functions of polysaccharides, thereby improving their biological activity.

[0071] (2) The present invention adopts the HNO3-NaSeO3 method to prepare selenized cinnamon polysaccharide, which is simpler and more efficient than other methods for synthesizing selenium polysaccharides.

[0072] (3) The selenized cinnamon polysaccharide (Se-CCP1) prepared by the present invention has a high selenium content, which is crucial for exerting the biological activity of selenium.

[0073] (4) The selenized cinnamon polysaccharide prepared by the efficient selenization method provided by the present invention has the characteristics of small toxic and side effects, high biological activity and high bioavailability, and these characteristics make it have wide application potential in the fields of biology, physiology, pharmacology, nutrition, food science, etc.

[0074] (5) The selenized cinnamon polysaccharide prepared by the efficient selenization method provided by the present invention shows stronger activity in whitening effect than unselenized cinnamon polysaccharide, which indicates that selenization modification can enhance the whitening effect of cinnamon polysaccharide.

[0075] (6) The present invention provides an efficient selenization method, which can change the molecular conformation, physical properties and physiological functions of polysaccharides through selenization modification, thereby improving their biological activity.

[0076] (7) The preparation method of selenized cinnamon polysaccharide of the present invention adopts a biosynthesis method, which is greener and healthier, meets clinical and production requirements, and is currently a widely used method.

[0077] (8) The present invention can effectively analyze the chemical bonds, configuration and other structural characteristics of the synthesized selenium polysaccharides through infrared spectroscopy, monosaccharide composition, scanning electron microscopy, particle size and zeta potential, which is helpful for studying the mechanism of pharmacological effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0079] Figure 1 This is the elution pattern of the chromatography column during the separation and purification of cinnamon polysaccharides provided in Example 1.

[0080] Figure 2 This is the elution pattern of the gel column during the separation and purification of cinnamon polysaccharides provided in Example 1.

[0081] Figure 3A This is the infrared spectrum analysis diagram of CCP1 provided in Example 3.

[0082] Figure 3B This is the infrared spectrum analysis diagram of Se-CCP1 provided in Example 3.

[0083] Figure 4A This is the monosaccharide composition analysis diagram of CCP1 provided in Example 4.

[0084] Figure 4B This is the monosaccharide composition analysis diagram of Se-CCP1 provided in Example 4.

[0085] Figure 5 Scanning electron microscopy images of CCP1 and Se-CCP1 provided in Example 5.

[0086] Figure 6 This is a graph showing the effects of CCP1 and Se-CCP1 on zebrafish survival rate provided in Example 7.

[0087] Figure 7This is a diagram showing the effects of CCP1 and Se-CCP1 on the distribution and content of zebrafish melanin provided in Example 7.

[0088] Figure 8 This is a comparison chart of the effects of CCP1 and Se-CCP1 on the survival rate of B16 cells provided in Example 8.

[0089] Fig. 9 This is a comparison chart of the effects of CCP1 and Se-CCP1 provided in Example 9 on the tyrosinase activity of B16 cells.

[0090] Fig.10 This is a comparison chart of the effects of CCP1 and Se-CCP1 provided in Example 10 on the melanin content of B16 cells.

[0091] Fig.11 This is a comparison chart of the effects of CCP1 and Se-CCP1 provided in Example 11 on related genes in the melanin synthesis pathway of B16 cells.

[0092] Fig.12 This is a comparison chart of the effects of CCP1 and Se-CCP1 provided in Example 12 on related proteins in the melanin synthesis pathway of B16 cells. DETAILED DESCRIPTION

[0093] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0094] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0095] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0096] In a first aspect, the present invention provides a selenized cinnamon polysaccharide, wherein the selenized cinnamon polysaccharide comprises a cinnamon polysaccharide chain and selenium covalently bonded to the cinnamon polysaccharide chain.

[0097] In the present invention, inorganic selenium is introduced into cinnamon polysaccharide (CCP1) to form selenized cinnamon polysaccharide (Se-CCP1), and selenized cinnamon polysaccharide shows stronger activity in whitening effect than non-selenized cinnamon polysaccharide, which indicates that selenization modification can enhance the whitening effect of cinnamon polysaccharide, and selenization modification can change the molecular conformation, physical properties and physiological functions of polysaccharides, thereby improving its biological activity.

[0098] As an optional embodiment, the molecular weight of the selenized cinnamon polysaccharide is 6000-7000Da, for example, it can be 6000Da, 6100Da, 6200Da, 6300Da, 6400Da, 6500Da, 6600Da, 6700Da, 6800Da, 6900Da, 7000Da, etc.

[0099] As an optional embodiment, the selenium content in the selenized cinnamon polysaccharide is 3100-3300 mg / kg, for example, it can be 3100 mg / kg, 3110 mg / kg, 3120 mg / kg, 3210 mg / kg, 3140 mg / kg, 3150 mg / kg, 3160 mg / kg, 3170 mg / kg, 3180 mg / kg, 3190 mg / kg, 3200 mg / kg, 3210 mg / kg, 3220 mg / kg, 3230 mg / kg, 3240 mg / kg, 3250 mg / kg, 3260 mg / kg, 3270 mg / kg, 3280 mg / kg, 3290 mg / kg, 3300 mg / kg and the like.

[0100] As an optional embodiment, in the selenized cinnamon polysaccharide (Se-CCP1) of the present invention, the covalent bond includes a Se=O bond, a selenium bond and a CO-Se bond.

[0101] It should be noted that the present invention adopts the HNO3-NaSeO3 method to prepare the above-mentioned selenized cinnamon polysaccharide (Se-CCP1), and its selenization principle is to connect the selenium-containing groups to the polysaccharide molecules through the catalyst HNO3, and finally exist in the form of selenoester on the polysaccharide skeleton.

[0102] As an optional embodiment, the monosaccharide composition of the selenized cinnamon polysaccharide includes xylose, mannose and glucose, and the molar ratio of xylose, mannose and glucose is (17.050-17.055):(6.950-6.955):(75.990-75.995).

[0103] As an optional embodiment, the particle size of the selenized cinnamon polysaccharide is 221.1±5.5nm, for example, it can be 215.6nm, 216.1nm, 216.6nm, 217.1nm, 217.6nm, 218.1nm, 218.6nm, 219.1nm, 219.6nm, 220.1nm, 220.6nm, 221.1nm, 221.6nm, 222.1nm, 222.6nm, 223.1nm, 223.6nm, 224.1nm, 224.6nm, 225.1nm, 225.6nm, 226.1nm, 226.6nm, etc.

[0104] As an optional embodiment, the Zeta potential of the selenized cinnamon polysaccharide is -11.6±1.4 mV, for example, it can be -13 mV, -12.8 mV, -12.6 mV, -12.4 mV, -12.2 mV, -12 mV, -11.8 mV, -11.6 mV, -11.4 mV, -11.2 mV, -11 mV, -10.8 mV, -10.6 mV, -10.4 mV, -10.2 mV, etc.

[0105] In a second aspect, the present invention provides a method for preparing the selenized cinnamon polysaccharide as described in the first aspect, wherein the method for preparing the selenized cinnamon polysaccharide comprises: sequentially performing enzymatic hydrolysis, decolorization, protein removal, purification and selenization on cinnamon to obtain the selenized cinnamon polysaccharide.

[0106] In the present invention, cinnamon is sequentially enzymatically hydrolyzed, decolorized, deproteinized, and purified to obtain a high-purity cinnamon polysaccharide sample (CCP1), and inorganic selenium is introduced into cinnamon polysaccharide (CCP1) by selenization, and selenium is covalently bonded to the cinnamon polysaccharide chain to prepare selenized cinnamon polysaccharide (Se-CCP1). This method is simpler and more efficient than other methods for synthesizing selenopolysaccharides, and the biosynthesis method is greener and healthier, meeting clinical and production requirements. At the same time, the selenized cinnamon polysaccharide (Se-CCP1) prepared by the preparation method of the present invention has high purity and high selenium content, which is crucial for exerting the biological activity of selenium. Furthermore, the selenized cinnamon polysaccharide (Se-CCP1) prepared by the preparation method of the present invention also has the characteristics of small toxic and side effects, high biological activity and high bioavailability; and the selenized cinnamon polysaccharide (Se-CCP1) shows stronger activity in whitening effect than non-selenized cinnamon polysaccharide (CCP1), which indicates that selenization modification can significantly enhance the whitening effect of cinnamon polysaccharide.

[0107] As an optional embodiment, the preparation method of selenized cinnamon polysaccharide comprises the following steps:

[0108] (1) Enzymatic hydrolysis: using cellulase to enzymatically hydrolyze the cinnamon raw material to obtain a crude polysaccharide solution;

[0109] (2) Decolorization: Decolorizing the crude polysaccharide solution with a decolorizing resin to obtain decolorized cinnamon polysaccharide;

[0110] (3) removing protein: using Sevage reagent to decolorize the decolorized cinnamon polysaccharide to obtain refined cinnamon polysaccharide;

[0111] (4) Purification: first purifying the cinnamon refined polysaccharide using a chromatography column, and then purifying the cinnamon refined polysaccharide using a gel column to obtain cinnamon polysaccharide (CCP1);

[0112] (5) Selenization: Inorganic selenium is introduced into the polysaccharide chain of the cinnamon polysaccharide CCP1 by the HNO3-NaSe2O3 method to obtain the selenized cinnamon polysaccharide (Se-CCP1).

[0113] As an optional embodiment, the enzymatic hydrolysis specifically comprises the following steps:

[0114] The cinnamon powder and water are mixed, and after adjusting the pH, cellulase is added to perform enzymolysis to obtain an enzymolysis solution; the enzymolysis solution is subjected to ultrasonication, enzyme inactivation, filtration and concentration in sequence to obtain a crude polysaccharide solution.

[0115] As an optional embodiment, the mass ratio of the cinnamon powder to water is 1:(10-30), for example, it can be 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20, 1:22, 2:14, 1:25, 1:26, 1:28, 1:30, etc.

[0116] As an optional embodiment, the reagents used to adjust the pH are citric acid and sodium citrate.

[0117] As an optional embodiment, the pH of the enzymatic hydrolysis is 5.0±0.05, for example, it can be 4.95, 4.96, 4.98, 5, 5.02, 5.04, 5.05, etc.

[0118] As an optional embodiment, the added amount of the cellulase is 1-5% of the total mass of cinnamon powder and water, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.

[0119] As an optional embodiment, the enzymatic hydrolysis temperature is 45-55°C, for example, it can be 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, 55°C, etc., and the enzymatic hydrolysis time is 30-35min, for example, it can be 30min, 31min, 32min, 33min, 34min, 35min, etc.

[0120] As an optional embodiment, the power of the ultrasound is 100-110 W, for example, it can be 100 W, 102 W, 104 W, 105 W, 106 W, 108 W, 110 W, etc., and the time of the ultrasound is 60-70 min, for example, it can be 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, 70 min, etc.

[0121] As an optional embodiment, the enzyme inactivation is carried out under the condition of heating in a water bath.

[0122] As an optional embodiment, the temperature for inactivating the enzyme is 85-95°C, for example, it can be 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, etc.

[0123] As an optional embodiment, the time for inactivating the enzyme is 1 to 10 minutes, for example, it can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.

[0124] As an optional embodiment, the enzymatic hydrolysis specifically comprises the following steps:

[0125] The method comprises mixing cinnamon powder and water in a mass ratio of 1:(10-30), adding citric acid and sodium citrate to adjust the pH of the aqueous solution of the cinnamon powder to 5.0±0.05, adding 1-5wt% of cellulase, performing enzymolysis at 45-55°C for 30-35min, and obtaining an enzymolysis solution; ultrasonically treating the enzymolysis solution, heating it in a water bath to inactivate the enzyme, filtering it while hot and collecting the filtrate, and concentrating the filtrate under reduced pressure to obtain a crude polysaccharide solution.

[0126] As an optional embodiment, the decolorization specifically comprises the following steps:

[0127] The crude polysaccharide solution obtained by enzymatic hydrolysis is mixed with a decolorizing resin for decolorization; after the decolorization is completed, the resin is washed, the decolorized solution is collected and combined, and the decolorized solution is subjected to a first centrifugation, concentration, alcohol precipitation and a second centrifugation in sequence to obtain decolorized cinnamon polysaccharide.

[0128] As an optional embodiment, the decolorization resin is a weakly alkaline anion exchange resin with a polystyrene macroporous structure, preferably D354FD resin.

[0129] As an optional embodiment, the volume ratio of the crude polysaccharide solution to the decolorization resin is 1:(0.5-2), for example, it can be 1:0.5, 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, etc.

[0130] As an optional embodiment, the decolorization is carried out under the condition of heating in a water bath.

[0131] As an optional embodiment, the decolorization temperature is 50-60°C, for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.

[0132] As an optional embodiment, the decolorization time is 2 to 4 hours, for example, it can be 2h, 2.2h, 2.4h, 2.5h, 2.6h, 2.8h, 3h, 3.2h, 3.4h, 3.6h, 3.8h, 4h, etc.

[0133] As an optional embodiment, during the decolorization process, stirring is performed once every 10 to 30 minutes (for example, 10 minutes, 12 minutes, 14 minutes, 15 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 25 minutes, 26 minutes, 28 minutes, 30 minutes, etc.).

[0134] As an optional embodiment, during the post-treatment after decolorization, the rotation speed of the first centrifugation is 3000-5000rpm, for example, it can be 3000rpm, 3200rpm, 3400rpm, 3500rpm, 3600rpm, 3800rpm, 4000rpm, 4200rpm, 4400rpm, 4500rpm, 4600rpm, 4800rpm, 5000rpm, etc.

[0135] As an optional embodiment, during the post-treatment after decolorization, the time of the first centrifugation is 5 to 15 minutes, for example, it can be 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, etc.

[0136] As an optional embodiment, the supernatant is collected after the first centrifugation for subsequent concentration.

[0137] As an optional embodiment, during the post-treatment after decolorization, the concentration temperature is 50-55°C, for example, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.

[0138] As an optional embodiment, during the post-treatment after decolorization, the solid content of the concentrated solution obtained by concentration is 70-80%, for example, it can be 70%, 72%, 74%, 75%, 76%, 78%, 80%, etc.

[0139] As an optional embodiment, in the process of post-treatment after decolorization, in the process of alcohol precipitation, the volume ratio of the concentrated solution and ethanol is 1:(4-5), for example, it can be 1:4, 1:4.2, 1:4.4, 1:4.5, 1:4.6, 1:4.8, 1:5, etc.

[0140] As an optional embodiment, during the post-treatment after bleaching, the temperature of the alcohol precipitation is 1-10°C, for example, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, etc.

[0141] As an optional embodiment, in the process of post-treatment after decolorization, the alcohol precipitation time is 6 to 18 hours, for example, it can be 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc.

[0142] As an optional embodiment, during the post-treatment after decolorization, the second centrifugation speed is 7000-9000rpm, for example, it can be 7000rpm, 7200rpm, 7400rpm, 7500rpm, 7600rpm, 7800rpm, 8000rpm, 8200rpm, 8400rpm, 8500rpm, 8600rpm, 8800rpm, 9000rpm, etc.

[0143] As an optional embodiment, during the post-treatment after decolorization, the second centrifugation time is 5 to 15 minutes, for example, it can be 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, etc.

[0144] As an optional embodiment, the precipitate is collected after the second centrifugation to obtain decolorized cinnamon polysaccharide.

[0145] As an optional embodiment, the decolorization specifically comprises the following steps:

[0146] The crude polysaccharide solution obtained by enzymatic hydrolysis and the decolorizing resin are mixed in a volume ratio of 1:(0.5-2), stirred evenly, heated in a water bath at 50-60°C for 2-4 hours for decolorization, and stirred once every 10-30 minutes during the decolorization process; after the decolorization is completed, the resin is rinsed with water, the decolorizing solution is collected and combined, the decolorizing solution is centrifuged at a speed of 3000-5000 rpm, the supernatant is collected, the supernatant is concentrated under reduced pressure to obtain a concentrated solution, ethanol is added to the concentrated solution for alcohol precipitation, centrifuged at a speed of 7000-9000 rpm, the precipitate is collected, and the decolorized cinnamon polysaccharide is obtained.

[0147] As an optional embodiment, the protein removal specifically comprises the following steps:

[0148] The decolorized cinnamon polysaccharide is mixed with water to obtain an aqueous solution of cinnamon polysaccharide, which is then mixed with a Sevage reagent, shaken, allowed to stand for stratification, and the aqueous layer solution is collected; after protein removal, the aqueous layer solution is sequentially concentrated, alcohol precipitated, centrifuged and dried to obtain refined cinnamon polysaccharide.

[0149] As an optional embodiment, the Sevage reagent is a mixture of chloroform and n-butanol in a volume ratio of 5:1.

[0150] As an optional embodiment, the Sevage reagent is prepared by the following steps: chloroform and n-butanol are uniformly mixed in a volume ratio of 5:1, and stored in a brown volumetric flask for later use, thereby obtaining the Sevage reagent.

[0151] As an optional embodiment, the mass ratio of the decolorized cinnamon polysaccharide to water is 1:(20-25), for example, it can be 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, etc.

[0152] As an optional embodiment, the volume ratio of the aqueous solution of cinnamon polysaccharide and Sevage reagent is (3-5):1, for example, it can be 3:1, 3.2:1, 3.4:1, 3.5:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.5:1, 4.6:1, 4.8:1, 5:1, etc.

[0153] As an optional embodiment, the standing stratification time is 30 to 50 min, for example, it can be 30 min, 32 min, 34 min, 35 min, 36 min, 38 min, 40 min, 42 min, 44 min, 45 min, 46 min, 48 min, 50 min, etc.

[0154] As an optional embodiment, during the protein removal process, the steps of mixing with Sevage reagent, shaking and then standing for stratification are repeated at least twice (for example, 2 times, 3 times, 4 times, 5 times, etc.) until no denatured protein is produced at the interface of the solution.

[0155] As an optional embodiment, during the post-treatment after protein removal, the concentration temperature is 50-55°C, for example, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, etc.

[0156] As an optional embodiment, during the post-treatment after protein removal, the solid content of the concentrated solution obtained by concentration is 70-80%, for example, it can be 70%, 72%, 74%, 75%, 76%, 78%, 80%, etc.

[0157] As an optional embodiment, during the post-treatment after protein removal, during the alcohol precipitation process, the volume ratio of the concentrated solution and ethanol is 1:(4-5), for example, it can be 1:4, 1:4.2, 1:4.4, 1:4.5, 1:4.6, 1:4.8, 1:5, etc.

[0158] As an optional embodiment, during the post-treatment process after protein removal, the centrifugal rotation speed is 7000-9000rpm, for example, it can be 7000rpm, 7200rpm, 7400rpm, 7500rpm, 7600rpm, 7800rpm, 8000rpm, 8200rpm, 8400rpm, 8500rpm, 8600rpm, 8800rpm, 9000rpm, etc.

[0159] As an optional embodiment, during the post-treatment after protein removal, the centrifugation time is 5 to 15 minutes, for example, it can be 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, etc.

[0160] As an optional embodiment, during post-treatment after protein removal, the precipitate is collected after the centrifugation.

[0161] As an optional embodiment, the protein removal specifically comprises the following steps:

[0162] The decolorized cinnamon polysaccharide and water are mixed at a mass ratio of 1:(20-25) to obtain an aqueous solution of cinnamon polysaccharide, and then the aqueous solution of cinnamon polysaccharide is mixed with a Sevage reagent at a volume ratio of (3-5):1, and after shaking, the mixture is allowed to stand for 30-50 minutes to separate layers, and the aqueous layer solution is collected, and the steps are repeated until no denatured protein is generated at the interface of the solution; after the protein is removed, the aqueous layer solution is successively concentrated under reduced pressure to obtain a concentrated solution, ethanol is added to the concentrated solution for alcohol precipitation, the solution is centrifuged at a speed of 7000-9000 rpm, the precipitate is collected, and the solution is dried to obtain refined cinnamon polysaccharide.

[0163] As an optional embodiment, the purification specifically comprises the following steps:

[0164] The cinnamon refined polysaccharide obtained by removing protein is mixed with water to obtain a cinnamon refined polysaccharide solution; the cinnamon refined polysaccharide solution is purified once by using a chromatography column, eluted with pure water, and the polysaccharide component of the pure water elution peak is collected; the polysaccharide component of the pure water elution peak is purified twice by using a gel column, eluted with pure water, the main peak eluate is collected, and concentrated and dried in sequence to obtain cinnamon polysaccharide CCP1.

[0165] As an optional embodiment, the concentration of the cinnamon refined polysaccharide solution is 10-30 mg / mL, for example, it can be 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL, 22 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, etc.

[0166] As an optional embodiment, the chromatography column is a DEAE ion exchange chromatography column.

[0167] As an optional embodiment, the one-time purification process further includes eluting with different salt solutions, and the concentrations of the different salt solutions are: 0.05 mol / L, 0.1 mol / L, 0.3 mol / L and 0.5 mol / L.

[0168] As an optional embodiment, the gel column is Sephadex G-100 gel.

[0169] As an optional embodiment, the purification specifically comprises the following steps:

[0170] The cinnamon refined polysaccharide obtained by removing protein is mixed with water to obtain a cinnamon refined polysaccharide solution with a concentration of 10 to 30 mg / mL; the cinnamon refined polysaccharide solution is purified once using a DEAE ion exchange chromatography column, eluted with pure water, and the polysaccharide component of the pure water elution peak is collected; the polysaccharide component of the pure water elution peak is purified twice using Sephadex G-100 gel, eluted with pure water, the main peak eluate is collected, and concentrated and dried in sequence to obtain cinnamon polysaccharide CCP1.

[0171] As an optional embodiment, the monosaccharide composition of the cinnamon polysaccharide CCP1 includes xylose, mannose and glucose, and the molar ratio of xylose, mannose and glucose is (4.810-4.815):(16.190-16.195):(78.995-79.000).

[0172] As an optional embodiment, the particle size of the cinnamon polysaccharide CCP1 is 472.2±51.4 nm, for example, it can be 420.8 nm, 430.8 nm, 440.8 nm, 450.8 nm, 460.8 nm, 470.8 nm, 472.2 nm, 482.2 nm, 492.2 nm, 502.2 nm, 512.2 nm, 522.2 nm, 523.6 nm, etc.

[0173] As an optional embodiment, the Zeta potential of the cinnamon polysaccharide CCP1 is -8.9±0.9 mV, for example, it can be -9.8 mV, -9.6 mV, -9.4 mV, -9.2 mV, -9.0 mV, -8.9 mV, -8.8 mV, -8.6 mV, -8.4 mV, -8.2 mV, -8 mV, etc.

[0174] As an optional implementation, the selenization specifically includes the following steps:

[0175] The purified cinnamon polysaccharide is dissolved in nitric acid, stirred, and then sodium selenite and barium chloride are added to carry out selenization reaction to obtain a selenized solution; the selenized solution is subjected to pH adjustment, barium chloride is removed, centrifuged and dialyzed to obtain selenized cinnamon polysaccharide.

[0176] As an optional embodiment, the mass ratio of cinnamon polysaccharide CCP1, nitric acid HNO3, sodium selenite Na2SeO3 and barium chloride BaCl2 is (1-3):(0.5-1.5):(1-2):(6-8);

[0177] Among them, “1-3” can be, for example, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.5, 2.6, 2.8, 3, etc.; “0.5-1.5” can be, for example, 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.5, etc.; “1-2” can be, for example, 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, etc.; “6-8” can be, for example, 6, 6.2, 6.4, 6.5, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8, etc.

[0178] As an optional embodiment, the concentration of nitric acid is 0.1-1% v / v, for example, it can be 0.1% v / v, 0.2% v / v, 0.3% v / v, 0.4% v / v, 0.5% v / v, 0.6% v / v, 0.7% v / v, 0.8% v / v, 0.9% v / v, 1% v / v, etc.

[0179] As an optional embodiment, the stirring time is 0.1 to 1 h, for example, it can be 0.1 h, 0.2 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1 h, etc.

[0180] As an optional embodiment, the temperature of the selenization reaction is 65-75°C, for example, it can be 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, etc.

[0181] As an optional embodiment, the selenization reaction time is 6 to 8 hours, for example, it can be 6 hours, 6.2 hours, 6.4 hours, 6.5 hours, 6.6 hours, 6.8 hours, 7 hours, 7.2 hours, 7.4 hours, 7.6 hours, 7.8 hours, 8 hours, etc.

[0182] As an optional embodiment, the selenization reaction is carried out in the presence of a protective gas atmosphere.

[0183] As an optional embodiment, the protective gas is nitrogen.

[0184] As an optional embodiment, the pH of the selenization solution is adjusted to 7-8, for example, 7, 7.2, 7.4, 7.5, 7.6, 7.8, 8, etc.

[0185] As an optional embodiment, the pH is adjusted using 0.5-2 mol / L sodium hydroxide aqueous solution, for example, 0.5 mol / L, 0.6 mol / L, 0.8 mol / L, 1 mol / L, 1.2 mol / L, 1.4 mol / L, 1.6 mol / L, 1.8 mol / L, 2 mol / L, etc.

[0186] As an optional embodiment, the reagent used to remove barium chloride is sodium sulfate.

[0187] As an optional embodiment, during post-treatment after selenization, the centrifugal force of the centrifuge is 600-650xg, for example, it can be 600xg, 605xg, 610xg, 615xg, 620xg, 625xg, 630xg, 635xg, 640xg, 645xg, 650xg, etc.

[0188] As an optional embodiment, during the post-treatment after selenization, the centrifugation time is 5 to 15 minutes, for example, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, etc.

[0189] As an optional embodiment, the dialysis is performed using a 3kDa dialysis bag with distilled water.

[0190] As an optional embodiment, the dialysis time is 66 to 78 hours, for example, it can be 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 76 hours, 77 hours, 78 hours, etc.

[0191] As an optional implementation, the selenization specifically includes the following steps:

[0192] The purified cinnamon polysaccharide was dissolved in nitric acid and stirred for 0.1-1h, and then sodium selenite and barium chloride were added, and selenization reaction was carried out at 65-75°C for 6-8h under a protective gas atmosphere to obtain a selenized solution; after the reaction was completed, the solution was cooled, and the pH of the selenized solution was adjusted to 7-8, anhydrous Na2SO4 was added to remove the barium chloride, and the solution was centrifuged at a centrifugal force of 600-650xg, and dialyzed using a 3kDa dialysis bag until the ascorbic acid solution did not turn red when dropped, thereby obtaining selenized cinnamon polysaccharide.

[0193] In a third aspect, the present invention provides a use of the selenized cinnamon polysaccharide as described in the first aspect in preparing cosmetics with whitening effect.

[0194] In the present invention, the selenium content is determined by inductively coupled plasma-mass spectrometry, and the changes in structural characteristics before and after selenization are further analyzed by infrared spectroscopy, monosaccharide composition analysis, scanning electron microscopy, particle size and Zeta potential. The changes in the whitening effects on zebrafish and B16 cells are compared, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and protein blotting (Western blot) are used to preliminarily explore its whitening mechanism.

[0195] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0196] Example 1

[0197] Preparation of Selenized Cinnamon Polysaccharide

[0198] This embodiment provides a selenized cinnamon polysaccharide (Se-CCP1), and the selenized cinnamon polysaccharide (Se-CCP1) is prepared by the following steps:

[0199] (1) Enzymatic hydrolysis of cinnamon crude polysaccharides:

[0200] 100.0 g of cinnamon powder was weighed, mixed with 2000 mL of distilled water in a 5000 mL conical flask, an appropriate amount of citric acid-sodium citrate was added to adjust the pH value to 5.0±0.05, 3.4 wt % of cellulase was added for enzymolysis, and the enzymolysis was performed at 50° C. for 30 min; after the enzymolysis was completed, ultrasonic treatment was performed at a power of 100 W for 64 min, and then heated in a constant temperature water bath at 90° C. for 5 min to inactivate the enzyme, and suction filtration was performed while hot, and the filtrate was collected, and the filtrate was concentrated under reduced pressure using a rotary evaporator at 55° C. to obtain a crude polysaccharide solution.

[0201] (2) Decolorization of cinnamon polysaccharide:

[0202] The crude polysaccharide solution was added to the pretreated D354FD resin at a volume ratio of 1:1, stirred evenly with a glass rod, heated in a water bath at 55°C for 3h, and stirred once every 20min; after decolorization, the resin was rinsed with deionized water 3 times, the crude polysaccharide solution was collected and combined, centrifuged at 4000rpm for 10min, the supernatant was taken, and concentrated under reduced pressure at 50-55°C to obtain 200mL of concentrated solution (solid content of the concentrated solution was 70%), and ethanol was added at a volume ratio of 1:4 to the concentrated solution. Stirring while adding, precipitated at 4°C for 10h, centrifuged at 8000rpm for 10min, and the precipitate was collected to obtain decolorized cinnamon polysaccharide.

[0203] (3) Protein removal from cinnamon polysaccharides:

[0204] Chloroform and n-butanol were mixed evenly at a volume ratio of 5:1, stored in a brown volumetric flask for standby use, and a Sevage reagent solution was obtained; cinnamon polysaccharide powder and distilled water were mixed at a mass ratio of 1:20, dissolved, and an aqueous solution of cinnamon polysaccharide powder was obtained; the aqueous solution of cinnamon polysaccharide powder and the above-mentioned Sevage reagent solution were mixed at a volume ratio of 4:1, and fully oscillated in a separatory funnel, left to stand for 40 minutes, and the aqueous layer solution was collected, and repeated twice until no denatured protein was produced at the interface of the solution; the polysaccharide solution after protein removal was concentrated at 65°C using a rotary evaporator to obtain 200mL of a concentrated solution (the solid content of the concentrated solution was 70%), and ethanol was added at a volume ratio of 1:4 to the concentrated solution. Stirring while adding, ethanol precipitation was carried out at 4°C for 10 hours, centrifuged at 8000rpm for 10 minutes, the precipitate was collected, placed in an oven for drying, and weighed to obtain cinnamon refined polysaccharide.

[0205] (4) Separation and purification of cinnamon polysaccharides:

[0206] Weigh 500 mg of cinnamon refined polysaccharide sample, add 25 mL of distilled water, stir evenly with magnetic force at room temperature to fully dissolve it, and obtain a 20 mg / mL cinnamon refined polysaccharide solution; the separation and purification of cinnamon refined polysaccharide usually adopts DEAE ion exchange chromatography column for preliminary separation, and uses distilled water and different salt concentrations (0.05 mol / L, 0.1 mol / L, 0.3 mol / L and 0.5 mol / L) for gradient elution, and collects the polysaccharide components (such as Figure 1 As shown, the polysaccharide component CCP1 of the distilled water elution peak was collected; subsequently, a Sephadex G-100 gel column was used for further purification, eluted with distilled water, and the polysaccharide elution curve was determined by measuring the absorbance, and finally the main peak eluate was collected and concentrated and freeze-dried to obtain a high-purity cinnamon polysaccharide sample (as shown Figure 2 As shown, the main peak of cinnamon polysaccharide sample CCP1 was collected.

[0207] (5) Preparation of Se-CCP1 using HNO3-NaSeO3 method:

[0208] Accurately weigh 200 mg of CCP1 component, dissolve it in 100 mL HNO3 (0.5%, v / v) at room temperature, stir at room temperature for 0.5 h, then add Na2SeO3 (160 mg) and BaCl2 (700 mg), and react with magnetic stirring in an oil bath at 70°C for 7 h under a nitrogen atmosphere. After the reaction, cool to room temperature, adjust the pH to 7-8 with 1 mol / L NaOH solution, add anhydrous Na2SO4 to remove BaCl2, centrifuge at 625 x g for 10 min, and dialyze against distilled water with a 3 kDa dialysis bag for 72 h until it does not turn red when dropped into ascorbic acid solution. This is the prepared selenized CCP1 (Se-CCP1).

[0209] Example 2

[0210] Detection of selenium content in selenized polysaccharides

[0211] Test method:

[0212] 10.0264 g of Se-CCP1 prepared in Example 1 was accurately weighed and transferred to a digestion tube; acid was added to the digestion tube and the sample was heated to digest; after the digestion was completed, the selenium content of Se-CCP1 was detected using an Agilent ICPMS 7700 inductively coupled plasma-mass spectrometer.

[0213] Result analysis:

[0214] According to the ICP-MS results, the selenium content of the selenized cinnamon polysaccharide (Se-CCP1) prepared by the present invention is 3268.27 mg / kg, indicating that the initial selenization is successful. In addition, the selenized cinnamon polysaccharide (Se-CCP1) prepared by the efficient selenization method provided by the present invention has a high selenium content, which is crucial for exerting the biological activity of selenium.

[0215] Example 3

[0216] Infrared Spectroscopic Analysis of CCP1 and Se-CCP1

[0217] Test method:

[0218] Cinnamon polysaccharide CCP1, selenized cinnamon polysaccharide Se-CCP1 and KBr were dried under an infrared lamp for about 2 h. 2 mg CCP1 and Se-CCP1 were ground evenly with 200 mg pure KBr and pressed into thin slices on a tablet press. The samples were analyzed by Fourier transform infrared spectrophotometer (Thermo Nicolet iS50 FT-IR) at 4000-400 cm -1 Infrared scanning is performed over a range of wavenumbers.

[0219] Result analysis:

[0220] like Figure 3A and Figure 3B Compared with the original CCP1, Se-CCP1 has higher FTIR at 917, 847, and 764 cm -1 There are three obvious characteristic absorption peaks at the site, indicating the formation of Se=O bond, selenium bond and CO-Se bond, which all indicate that Se-CCP1 has been successfully selenized. The main sugar functional groups have not changed, indicating that the synthesis method is mild and has not changed the typical sugar groups.

[0221] Example 4

[0222] Analysis of monosaccharide composition of CCP1 and Se-CCP1

[0223] Test method:

[0224] Polysaccharide hydrolysis: Weigh 10 mg of CCP1 and Se-CCP1 and place them in a 5 mL ampoule tube. Add 4 mL of 2 M trifluoroacetic acid solution and seal it with an alcohol burner. After hydrolysis at 110 °C for 6 h, concentrate to dryness under reduced pressure on a rotary evaporator. Add methanol and concentrate to dryness again. Repeat this process 5 times until the sample is neutral.

[0225] Derivatization and GC analysis of monosaccharide standards and hydrolysis products of CCP1 and Se-CCP1: Take 10 mg of glucose, erythritol, fucose, rhamnose, xylose, mannose, arabinose, galactose standards and hydrolysis products of CCP1 and Se-CCP1 in 10 mL stoppered bottles, add 10 mg of hydroxylamine hydrochloride and 1 mL of pyridine, incubate at 90°C in a water bath for 30 min, shake once every 5 min, cool to room temperature, add 1 mL of acetic anhydride, and react in a 90°C water bath for 30 min, shake once every 5 min, take out and cool to room temperature, and finally generate volatile sugar nitrile acetate derivatives. After passing through a membrane (0.22 μm), GC analysis is performed. According to the retention time comparison between the sample and the standard, the monosaccharide composition of various sample polysaccharides is obtained, and the molar ratio of various monosaccharides is calculated according to the area ratio of each peak.

[0226] Chromatographic conditions: Thermo TRACE 1300 equipped with single quadrupole gas chromatograph ISQ, AI / AS1310 autosampler series GC-MS system, equipped with SHIMADZUSH-1701 capillary column analysis (30m×0.25mm×0.25μm) and ion trap mass spectrometer detector. Program temperature: initial 180℃ (hold for 2min), then increase to 220℃ (hold for 1min) at 5℃ / min, and increase to 250℃ (hold for 2min) at 5℃ / min; no split flow, carrier gas flow rate 1mL / min.

[0227] Result analysis:

[0228] like Figure 4A As shown, by comparing the retention time with that of each monosaccharide standard, it was determined that CCP1 was mainly composed of xylose, mannose and glucose; the molar ratio of xylose, mannose and glucose in CCP1 was 4.811:16.195:78.994; Figure 4B As shown in the comparison, it was determined that the monosaccharide composition of Se-CCP1 and CCP1 was consistent, mainly composed of xylose, mannose and glucose; the molar ratio of xylose, mannose and glucose in Se-CCP1 was 17.054:6.955:75.991.

[0229] Example 5

[0230] SEM analysis of CCP1 and Se-CCP1

[0231] Test method:

[0232] Take an appropriate amount of dried CCP1 and Se-CCP1 and adhere them to the sample stage with conductive glue, and cover them with a conductive gold film in an ion sputtering instrument. Observe them under a FEIQuanta 400FEG scanning electron microscope. The experimental conditions are set as follows: the acceleration voltage is 20kV, the appropriate magnification is selected, and a representative field of view is selected for photographing and recording.

[0233] Result analysis:

[0234] like Figure 5 As shown in the figure, the scanning electron microscopy results of CCP1 and Se-CCP1 show that selenization modification significantly changes the apparent morphology of cinnamon polysaccharides, which is consistent with previous studies on selenized polysaccharides. The surface of Se-CCP1 mainly shows some irregular spherical or aggregated structures, which may be caused by the strong attraction between molecules. The selenization process can effectively change the structure of CCP1 and make the interaction between polysaccharides and aqueous solvents more sufficient. Compared with CCP1, the surface of Se-CCP1 is relatively smooth, which may be due to the strong attraction between surface functional groups, which leads to an increase in the number of cross-links of polysaccharides, and then causes the aggregation of polysaccharide chains. Therefore, during the process of selenization, the surface morphology of their aggregates changes, which may be caused by changing the intramolecular and intermolecular hydrogen bonds and van der Waals forces.

[0235] Example 6

[0236] Analysis of particle size and zeta potential of CCP1 and Se-CCP1

[0237] Test method:

[0238] CCP1 and Se-CCP1 were prepared into solutions with a concentration of 0.1 mg / mL, and the particle size and Zeta potential of CCP1 and Se-CCP1 solutions were measured using a Malvern laser particle size analyzer.

[0239] The test results are shown in Table 1 below:

[0240] Table 1

[0241]

[0242] Result analysis:

[0243] The size of particle size and Zeta potential can reflect the stability of the solution. The smaller the particle size, the larger the absolute value of Zeta potential, and the higher the stability of the solution. The results of particle size and Zeta potential of 0.1 mg / mL CCP1 and Se-CCP1 are shown in Table 1. The particle size of Se-CCP1 (221.1±5.5nm) is only half of the particle size of CCP1 molecule (472.2±51.4nm). It shows that the reduction of polysaccharide particle size after selenization modification may be related to the change of spatial conformational characteristics of polysaccharide. The Zeta potential of CCP1 and Se-CCP1 are both negative, indicating that they are anionic polysaccharides. And the absolute value of Zeta potential of Se-CCP1 (11.6±1.4mV) is higher than the absolute value of Zeta potential of CCP1 (8.9±0.9mV). The change of Zeta potential of cinnamon polysaccharide after selenization modification may be related to the access of selenium groups. The higher the absolute value of the Zeta potential, the greater the repulsion between molecules and the more stable the solution, that is, the better the solubility and stability, indicating that selenization modification is beneficial to improving the stability and dispersibility of cinnamon polysaccharides in solution.

[0244] Example 7

[0245] Comparison of the effects of CCP1 and Se-CCP1 on the distribution and content of melanin in zebrafish

[0246] Test method:

[0247] Zebrafish rearing conditions and embryo reproduction and culture: Zebrafish were provided by the Zebrafish Experimental Platform of the Central Laboratory of Southern Medical University. Strictly abide by the rules and regulations and zebrafish breeding and breeding experimental requirements. In the breeding and special breeding system for adult wild-type (AB type) zebrafish, the pH range is 7.0-8.0, the room temperature range of the fish room is (26℃±1℃), the water temperature range is (28℃±1℃), and the photoperiod is 14h light and 10h dark. The adult male and female zebrafish in the spawning period were caught on both sides of the partition of the breeding tank. After a ten-hour dark period, the partition in the middle of the breeding tank was removed. After light stimulation, the zebrafish began to mate, wait for spawning and collect embryos. The embryos were transferred to a 10cm culture dish and cultured at 28℃ with system water containing 1% methylene blue for 24h. The embryos used in the experiment were all normal embryos.

[0248] Effects of CCP1 and Se-CCP1 on the distribution and content of zebrafish melanin: 24hpf embryos of wild-type zebrafish of AB strain were collected and placed in 24-well plates with 8 embryos per well for grouping into blank group, positive arbutin (AR) group, melanin-free group (PTU) group and sample group. The blank group was fresh system water containing 1% methylene blue, the AR group was arbutin solution with a final concentration of 200μg / mL prepared with fresh 1% methylene blue system water, and the melanin-free group was phenylthiourea solution with a final concentration of 30μg / mL prepared with fresh 1% methylene blue system water. The sample group was CCP1 and Se-CCP1 solutions with different concentrations prepared with fresh 1% methylene blue system water. 1mL of the corresponding solution was added to each well, and 6 replicate wells were set. The cells were cultured in a 28℃ incubator for 72h, and the distribution of melanin was observed and photographed with a stereo microscope.

[0249] NaOH lysis method to detect melanin content: PBS solution containing 1% Triton X-100 was added to each group and fully broken using an ultrasonic disruptor. The precipitate was taken out by centrifugation, and 400 μL of 1 mol / L NaOH (containing 10% DMSO) solution was added. The mixture was incubated in a water bath at 90°C for 2 h, and then aspirated onto an ELISA plate. The absorbance was measured at 405 nm using an ELISA reader.

[0250] Result analysis:

[0251] Effects of CCP1 and Se-CCP1 on zebrafish survival rate Figure 6 As shown. The results showed that after incubation with 200μg / mL of arbutin in the positive group and 30μg / mL of PTU, the survival rate of zebrafish was not affected compared with the blank group. The safe concentration of CCP1 was 125μg / mL. As in previous studies, the safe concentration of Se-CCP1 was 31.25μg / mL. Therefore, the above concentration was used as the maximum concentration for subsequent experiments. The safe concentrations of CCP1 and Se-CCP1 are different. The reason may be that the solubility and dispersibility of Se-CCP1 is better than that of CCP1. Excessive selenium will produce certain biological selenium toxicity on the embryonic liver development of zebrafish, so Se-CCP1 needs to be used within a reasonable range.

[0252] Effects of CCP1 and Se-CCP1 on the distribution and content of melanin in zebrafish Figure 7As shown. The results showed that after incubation with PTU, zebrafish did not produce melanin. After incubation with the positive groups AR, CCP1 and Se-CCP1, zebrafish had different degrees of inhibition of melanin production compared with the blank control group, indicating that CCP1 and Se-CCP1 may have the effect of inhibiting melanin production in zebrafish. Among them, Se-CCP1 at 15.63μg / mL had a similar inhibitory effect on zebrafish melanin production as CCP1 at 31.25μg / mL, indicating that selenized polysaccharides have the effect of enhancing their whitening activity.

[0253] Example 8

[0254] Comparison of the effects of CCP1 and Se-CCP1 on the survival rate of B16 cells

[0255] Test method:

[0256] Toxicity test of CCP1 and Se-CCP1 on B16 cells: After digesting the cells in the logarithmic phase with trypsin, pipette and mix them evenly. Pipette 100 μL into a 96-well plate, and the inoculation density is 5×10 per well. 3 Cells were transferred to a CO2 incubator and cultured until the cells adhered to the wall. The old complete culture medium was aspirated and the cells were divided into groups. 100 μL of complete culture medium containing samples of different concentrations was added to the wells of the sample group, 100 μL of complete culture medium was added to the blank cell group, and 100 μL of complete culture medium was added to the zero group (without cells). Six replicate wells were set and placed in a 5% CO2, 37°C incubator for further culture for 24 hours. After the incubation, according to the instructions of the CCK8 cell kit, the old culture medium was discarded and 100 μL of complete culture medium containing 10% CCK-8 solution was added. The cells were cultured in the incubator for 4 hours, and the absorbance value (at a wavelength of 450 nm) was measured using an ELISA reader to calculate the cell viability.

[0257] Result analysis:

[0258] Comparison of the effects of CCP1 and Se-CCP1 on the survival rate of B16 cells Figure 8 As shown, the results showed that the B16 cell survival rates of CCP1 and Se-CCP1 were both greater than 80% below 125 μg / mL, and the effects on B16 cell survival rates were consistent at the same concentration. Therefore, 125 μg / mL was used as the maximum safe concentration for subsequent experiments.

[0259] Example 9

[0260] Comparison of the effects of CCP1 and Se-CCP1 on tyrosinase activity in B16 cells

[0261] Test method:

[0262] B16 cells were cultured at 2×10 5The cells were inoculated in a 6-well plate at a density of 10 cells / well. After adhesion, the old culture medium was removed and grouped into a blank group (complete culture medium containing DEME basal culture medium), a positive group (200 μg / mL arbutin solution prepared with complete culture medium of DEME basal culture medium), and a sample group (CCP1 and Se-CCP1 solutions of different concentrations prepared with complete culture medium of DEME basal culture medium). 2 mL of the corresponding solution was added to each well, and 3 replicates were set. After 48 hours of incubation in the incubator, the old culture medium was removed and the cells were washed twice with PBS. The experimental results were observed and photographed using an inverted microscope. After that, a cell scraper was used to gently collect the drug-intervention B16 cells into an ep tube. After the cells were broken, the supernatant was centrifuged into an ELISA plate, and an equal amount of 1 mg / mL levodopa solution was added. After gently shaking and mixing, it was placed in a 37°C incubator for reaction for 2 hours. The inhibition rate of intracellular tyrosinase activity was measured at 475 nm using an ELISA reader.

[0263] Result analysis:

[0264] Comparison of the effects of CCP1 and Se-CCP1 on tyrosinase activity in B16 cells Fig. 9 As shown, the results showed that at the same concentration, the tyrosinase activity of Se-CCP1 was lower than that of CCP1, and the ability of 125μg / mL CCP1 to inhibit tyrosinase activity was equivalent to that of 200μg / mL AR, which means that Se-CCP1 may be a promising new skin whitening ingredient.

[0265] Example 10

[0266] Comparison of the effects of CCP1 and Se-CCP1 on melanin content in B16 cells

[0267] Test method:

[0268] The supernatant after the cells of each group were crushed was taken and the melanin content of B16 cells was determined by the NaOH lysis method in Example 7.

[0269] Result analysis:

[0270] Comparison of the effects of CCP1 and Se-CCP1 on melanin content in B16 cells Fig.10As shown, the results showed that at the same concentration, compared with CCP1, the relative melanin content of Se-CCP1 was lower, and the melanin production was inhibited in a dose-dependent manner. The ability of 31.25μg / mL Se-CCP1 to inhibit melanin production was similar to that of 125μg / mL CCP1. Se-CCP1 could achieve similar whitening effects at lower concentrations. In summary, it was shown that selenized cinnamon polysaccharides enhanced its whitening effect, which may be through increasing the ability to inhibit tyrosinase activity and melanin production. Its whitening mechanism needs further study.

[0271] Embodiment 11

[0272] Comparison of the effects of CCP1 and Se-CCP1 on genes related to melanin synthesis pathway in B16 cells

[0273] Test method:

[0274] Cell treatment and total RNA extraction: When B16 cells were in the logarithmic growth phase and the confluence in the culture dish reached about 80%, the cells were digested and collected, and B16 cells were plated at 5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / well. After adhesion, the old culture medium was removed and the cells were divided into a blank group (complete culture medium containing DEME basal medium), a CCP1 group, and a Se-CCP1 group (diluted to 125 μg / mL, 62.5 μg / mL, and 31.25 μg / mL with complete culture medium containing DEME basal medium). 2 mL of the corresponding solution was added to each well, and 3 replicates were set. After incubation for 48 hours, the cell supernatant was discarded, PBS was added to wash the cells twice, 1 mL of Trizol RNA extraction reagent was added to each well, and the cells were allowed to stand at room temperature for 5 minutes, and the cells were blown and collected into ep tubes. 200 μL of chloroform was added to each tube, shaken vigorously for about 15 seconds, mixed to make it milky white, and allowed to stand for 10 minutes. Centrifuge at 4℃ and 12000rpm for 15min. After centrifugation, the solution is divided into three layers (supernatant water layer, middle layer, bright red organic layer). Transfer the supernatant water layer to a pre-cooled new centrifuge tube. Do not touch the middle layer (generally take 400-500μL). Add 500μL / tube of isopropanol, mix upside down, and let stand on ice for 15 min. Centrifuge at 4℃ and 12000rpm for 10 min, discard the supernatant, and do not touch the precipitate. Add 1mL75% ethanol (prepared with anhydrous ethanol and DEPC water), mix well, centrifuge at 4℃ and 7500rpm for 5 min, carefully discard the solution, add 1mL of 75% ethanol solution to wash again, and centrifuge. Place the centrifuge tube in a fume hood to dry until the precipitate in the tube changes from white to colorless and transparent. Add 20-40μL of DEPC water to each tube and heat it in a 60℃ metal bath for 10 min to fully dissolve the precipitate in the tube.

[0275] Determination of RNA concentration and purity: using NanoDrop TM One / OneC micro-volume UV-Vis spectrophotometer was used to measure RNA concentration and purity. The tip of the instrument was rinsed with DEPC water, and the sample was added to measure the RNA concentration and purity of the sample. The purity of the extracted RNA was determined by the OD260 / OD280 value, and the OD260 / OD280 value should be maintained between 1.8 and 2.1. When the OD260 / OD280 value is 2, the RNA purity is the highest. When it is greater than 2.2, the RNA is degraded into single nucleotides. When it is less than 1.8, the content of impurities is too high. In the latter two cases, the RNA needs to be re-extracted.

[0276] RNA reverse transcription: Perform reverse transcription on RNA with required purity and concentration according to the kit instructions. The first step is to react at 65°C for 10 min with a solution volume of 13 μL. Then add the remaining reagents to complete the second step (25°C for 10 min, 55°C for 30 min, and 85°C for 5 min). After the reaction, store the cDNA sample obtained by reverse transcription at -20°C.

[0277] Design primers: The sequences of relevant genes were obtained by querying the NCBI gene library and the Primer Bank website was used for auxiliary design. The obtained primer sequences were compared on the blast website, and finally the appropriate primer sequences of the target gene were obtained and synthesized. The primer sequences used in the RT-qPCR reaction are shown in Table 2.

[0278] Table 2

[0279]

[0280] The 20 μL RT-qPCR amplification reaction system in Table 3 below was used to detect the effects of CCP1 and Se-CCP1 on genes related to the melanin synthesis pathway of B16 cells.

[0281] Table 3

[0282]

[0283] Structural analysis:

[0284] like Fig.11 As shown, excessive melanin deposition stimulates the upregulation of cAMP response element binding protein (CREB), which in turn induces the expression of melanogenesis-related genes such as tyrosinase (TYR), tyrosinase-related protein 1 (TYR1), and dopachrome tautomerase (TYR2) through the expression of microphthalmia-associated transcription factor (MITF).

[0285] It has been reported that extracellular signal-regulated kinase (ERK) is activated by many extracellular stimuli and plays a key role in the synthesis of melanin. For example, phosphorylated ERK can inhibit the expression of MITF, thereby reducing the synthesis of melanin. The effects of CCP1 and Se-CCP1 on the expression of CREB1, MITF, ERK1, ERK2, TYR, TYR1 and TYR2 mRNA in B16 cells are shown in Figure 2. Fig.11 shown.

[0286] Compared with the blank control group, CCP1 and Se-CCP1 could significantly reduce the expression levels of CREB1, MITF, TYR, TYR1 and TYR2 mRNA in a dose-dependent manner. At the same concentration, Se-CCP1 was more capable of reducing the expression levels of CREB1, MITF, ERK1, ERK2, TYR, TYR1 and TYR2 mRNA in B16 cells than CCP1, indicating that Se-CCP1 enhanced the whitening activity of CCP1. However, there was no effect on the expression levels of ERK1 and ERK2 mRNA, which may be because the inhibition of melanin synthesis in B16 cells by CCP1 and Se-CCP1 is not related to the ERK pathway.

[0287] Example 12

[0288] Comparison of the effects of CCP1 and Se-CCP1 on related proteins in the melanin synthesis pathway of B16 cells

[0289] Test method:

[0290] Cell culture and protein extraction: Culture and collect cells according to the method of “Effects of crude and purified cinnamon polysaccharides on melanin production in B16 cells”, add RIPA lysis buffer containing protease inhibitors, grind thoroughly, and let stand on ice for 40 min. Vortex once every 10 min for 15 s each time, then centrifuge at 12000 rpm for 20 min, and carefully aspirate the supernatant into a centrifuge tube.

[0291] Protein concentration determination and denaturation: According to the instructions of the kit, the protein concentration was measured by BCA method. After the measurement, the volume of protein solution and ultrapure water required when the protein loading amount was 30 μg and the total volume of each portion was 20 μL were calculated. 5 μL of 5× protein loading buffer was added to each tube, mixed thoroughly, and heated in a 100℃ metal bath for 10 minutes. Store in a -80℃ refrigerator.

[0292] SDS-PAGE electrophoresis: According to the molecular weight of the protein, select 10% separation gel and 5% concentration gel to separate the target protein. Connect the electrophoresis device, pour in the electrophoresis solution, carefully pull out the comb, and use the sample loading gun tip to add the protein solution to each comb hole. First set the voltage to 60V, and the protein sample runs through the concentration gel part. Adjust the voltage to 80V, and after 10 minutes, adjust it to 100V. After about 90 minutes, when the bromophenol blue indicator runs to about 1cm from the bottom of the separation gel, turn off the power and end the electrophoresis.

[0293] Transfer: 5 minutes in advance, immerse the PVDF membrane in methanol to activate it, and 30 minutes in advance, place the sponge, clamps, filter paper, etc. in the transfer working solution for pre-balance. Make the clamps in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge" from the negative electrode to the positive electrode, remove bubbles with a glass rod, clamp the transfer clamp, place it in the transfer tank, and fill it with transfer solution. Transfer the membrane at a constant voltage of 100V for 100 minutes under ice bath conditions.

[0294] Blocking: After the transfer is completed, turn off the power, take out the transfer clip, carefully open the PVDF membrane, cut it appropriately, put the PVDF membrane into an incubation box containing 5% skim milk, shake it at a constant speed on a horizontal shaker, and block it at room temperature for 1.5 hours.

[0295] Incubation of primary and secondary antibodies: After blocking, wash the PVDF membrane three times with 1×TBST buffer, shake on a horizontal shaker for 10 min each time. Add the corresponding primary antibody and incubate overnight in a 4°C refrigerator. After the primary antibody incubation is completed, recover the primary antibody, wash the PVDF membrane three times with 1×TBST buffer, shake on a horizontal shaker for 10 min each time. Add the secondary antibody, shake on a horizontal shaker at a constant speed, and incubate on ice for 2 h.

[0296] Development: After the secondary antibody incubation is completed, the secondary antibody is recovered and the PVDF membrane is washed 3 times with 1×TBST buffer and shaken on a horizontal shaker for 10 minutes each time. In a dark room environment, the ECL color development solution is evenly added to the membrane, and after reacting for 1 minute, a chemical gel luminescence imager is used to take pictures.

[0297] Result analysis:

[0298] The effects of CCP1 and Se-CCP1 on the expression of p-CREB, MITF, and TYR proteins in B16 cells are shown in the example Fig.12As shown. The results showed that the blank group had high protein expression of p-CREB, MITF, and TYR. After the intervention of different concentrations of CCP1 and Se-CCP1, the protein expression of p-CREB, MITF, and TYR changed significantly. Under the action of CCP1, the protein expression levels of p-CREB and TYR decreased in a dose-dependent manner, and Se-CCP1 had a higher ability to reduce the protein expression of p-CREB, MITF, and TYR. Therefore, the above results indicate that CCP1 and Se-CCP1 may inhibit tyrosinase activity and melanin production through the CREB / MITF / TYR pathway, and selenized polysaccharides can enhance the whitening activity of the original polysaccharides.

[0299] In summary, the present invention provides a selenized cinnamon polysaccharide and a preparation method and application thereof, which have at least the following key points:

[0300] (1) Preparation method of selenized cinnamon polysaccharide: Inorganic selenium was introduced into cinnamon polysaccharide by HNO3-NaSe2O3 method to form selenized cinnamon polysaccharide (Se-CCP1). The selenium content of Se-CCP1 was determined by inductively coupled plasma-mass spectrometry (ICP-MS) to ensure successful selenization.

[0301] (2) Analysis of structural property changes: The structural property changes of cinnamon polysaccharides before and after selenization were compared and analyzed using infrared spectroscopy (FTIR), monosaccharide composition analysis, scanning electron microscopy (SEM), particle size and Zeta potential analysis.

[0302] (3) Evaluation of whitening effect: The whitening effect of selenized cinnamon polysaccharides on zebrafish and B16 cells was compared to evaluate the whitening activity of selenized polysaccharides.

[0303] (4) Investigation of the whitening mechanism: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and protein blotting (Western blot) were used to preliminarily explore the whitening mechanism of selenized cinnamon polysaccharides, especially the effects on related genes and proteins in the melanin synthesis pathway.

[0304] (5) Safety and efficacy of selenized cinnamon polysaccharides: Determine the safe concentration of selenized cinnamon polysaccharides and evaluate its effects on melanin production at this concentration, including the inhibitory effects on tyrosinase activity and melanin content.

[0305] (6) Enhanced whitening effect of selenized cinnamon polysaccharide: It was demonstrated that selenized cinnamon polysaccharide can achieve a similar whitening effect as unselenized cinnamon polysaccharide at a lower concentration, showing an enhanced whitening effect.

[0306] (7) Application of selenized cinnamon polysaccharide: Selenized cinnamon polysaccharide is used in the field of whitening, especially in improving skin whitening and pigmentation.

[0307] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A selenized cinnamon polysaccharide, characterized in that: The selenized cinnamon polysaccharide comprises cinnamon polysaccharide chains and selenium covalently bonded to the cinnamon polysaccharide chains.

2. The selenized cinnamon polysaccharide according to claim 1, characterized in that The molecular weight of the selenized cinnamon polysaccharide is 6000-7000Da; Preferably, the selenium content in the selenized cinnamon polysaccharide is 3100-3300 mg / kg.

3. The selenized cinnamon polysaccharide according to claim 1, characterized in that The covalent bonds include Se=O bonds, selenium bonds and CO-Se bonds; Preferably, the monosaccharide composition of the selenized cinnamon polysaccharide includes xylose, mannose and glucose, and the molar ratio of xylose, mannose and glucose is (17.050-17.055):(6.950-6.955):(75.990-75.995); Preferably, the particle size of the selenized cinnamon polysaccharide is 221.1±5.5 nm, and the Zeta potential of the selenized cinnamon polysaccharide is -11.6±1.4 mV.

4. A method for preparing selenized cinnamon polysaccharide according to any one of claims 1 to 3, characterized in that: The preparation method of the selenized cinnamon polysaccharide comprises: sequentially performing enzymatic hydrolysis, decolorization, protein removal, purification and selenization on cinnamon to obtain the selenized cinnamon polysaccharide.

5. The method for preparing selenized cinnamon polysaccharide according to claim 4, characterized in that: The enzymolysis specifically comprises the following steps: The cinnamon powder and water are mixed, the pH is adjusted, and then cellulase is added to perform enzymolysis to obtain an enzymolysis solution; the enzymolysis solution is subjected to ultrasonication, enzyme inactivation, filtration, and concentration in sequence to obtain a crude polysaccharide solution; Preferably, the mass ratio of the cinnamon powder to water is 1:(10-30); Preferably, the reagents used to adjust the pH are citric acid and sodium citrate; Preferably, the pH of the enzymatic hydrolysis is 5.0±0.05; Preferably, the added amount of the cellulase is 1 to 5% of the total mass of the cinnamon powder and water; Preferably, the enzymatic hydrolysis temperature is 45-55°C, and the enzymatic hydrolysis time is 30-35 min; Preferably, the power of the ultrasound is 100-110W, and the time of the ultrasound is 60-70min; Preferably, the enzyme inactivation is carried out under the condition of water bath heating, the temperature of the enzyme inactivation is 85-95° C., and the time of the enzyme inactivation is 1-10 min.

6. The method for preparing selenized cinnamon polysaccharide according to claim 4, characterized in that: Described decolorization specifically comprises the following steps: The crude polysaccharide solution obtained by enzymatic hydrolysis is mixed with a decolorizing resin to perform decolorization; after the decolorization is completed, the resin is rinsed, the decolorized solution is collected and combined, and the decolorized solution is subjected to a first centrifugation, concentration, alcohol precipitation and a second centrifugation in sequence to obtain decolorized cinnamon polysaccharide; Preferably, the decolorizing resin is a weakly basic anion exchange resin with a polystyrene macroporous structure, preferably D354FD resin; Preferably, the volume ratio of the crude polysaccharide solution to the decolorizing resin is 1:(0.5-2); Preferably, the decolorization is carried out under the condition of water bath heating, the decolorization temperature is 50-60°C, and the decolorization time is 2-4h; Preferably, during the decolorization process, stirring is performed once every 10 to 30 minutes; Preferably, the speed of the first centrifugation is 3000-5000 rpm, the time of the first centrifugation is 5-15 min; and after the first centrifugation, the supernatant is collected for subsequent concentration; Preferably, the concentration temperature is 50-55°C; and the solid content of the concentrated solution obtained by the concentration is 70-80%; Preferably, during the alcohol precipitation process, the volume ratio of the concentrated solution to ethanol is 1:(4-5); Preferably, the temperature of the alcohol precipitation is 1 to 10°C, and the time of the alcohol precipitation is 6 to 18 hours; Preferably, the rotation speed of the second centrifugation is 7000-9000 rpm, and the time of the second centrifugation is 5-15 min; and after the second centrifugation, the precipitate is collected to obtain decolorized cinnamon polysaccharide.

7. The method for preparing selenized cinnamon polysaccharide according to claim 4, characterized in that: The protein removal specifically comprises the following steps: The decolorized cinnamon polysaccharide is mixed with water to obtain an aqueous solution, which is then mixed with a Sevage reagent, shaken, allowed to stand and separated into layers, and the aqueous layer solution is collected; after the protein is removed, the aqueous layer solution is sequentially concentrated, alcohol precipitated, centrifuged and dried to obtain refined cinnamon polysaccharide; Preferably, the mass ratio of the decolorized cinnamon polysaccharide to water is 1:(20-25); Preferably, the volume ratio of the aqueous solution to the Sevage reagent is (3-5):1; Preferably, the standing and stratifying time is 30 to 50 minutes; Preferably, in the protein removal process, the steps of mixing with Sevage reagent, shaking and then standing for stratification are repeated at least twice until no denatured protein is produced at the interface of the solution; Preferably, the concentration temperature is 50-55°C; and the solid content of the concentrated solution obtained by the concentration is 70-80%; Preferably, during the alcohol precipitation process, the volume ratio of the concentrated solution to ethanol is 1:(4-5); Preferably, the centrifugal speed is 7000-9000 rpm, the centrifugal time is 5-15 min; and the precipitate is collected after the centrifugation.

8. The method for preparing selenized cinnamon polysaccharide according to claim 4, characterized in that: The purification specifically comprises the following steps: The cinnamon refined polysaccharide obtained by removing protein is mixed with water to obtain a cinnamon refined polysaccharide solution; the cinnamon refined polysaccharide solution is purified once by using a chromatography column, eluted with pure water, and the polysaccharide component of the pure water elution peak is collected; the polysaccharide component of the pure water elution peak is purified twice by using a gel column, eluted with pure water, the main peak eluate is collected, and concentrated and dried in sequence to obtain cinnamon polysaccharide CCP1; Preferably, the concentration of the cinnamon refined polysaccharide solution is 10 to 30 mg / mL; Preferably, the chromatography column is a DEAE ion exchange chromatography column; Preferably, in the process of the primary purification, elution is further performed using different salt solutions, and the concentrations of the different salt solutions are: 0.05 mol / L, 0.1 mol / L, 0.3 mol / L and 0.5 mol / L; Preferably, the gel column is Sephadex G-100 gel; Preferably, the monosaccharide composition of the cinnamon polysaccharide CCP1 includes xylose, mannose and glucose, and the molar ratio of xylose, mannose and glucose is (4.810-4.815):(16.190-16.195):(78.995-79.000); Preferably, the particle size of the cinnamon polysaccharide CCP1 is 472.2±51.4 nm, and the Zeta potential of the cinnamon polysaccharide CCP1 is -8.9±0.9 mV.

9. The method for preparing selenized cinnamon polysaccharide according to claim 4, characterized in that: The selenization specifically comprises the following steps: The purified cinnamon polysaccharide is dissolved in nitric acid, stirred, and then sodium selenite and barium chloride are added to perform a selenization reaction to obtain a selenized solution; the selenized solution is subjected to pH adjustment, barium chloride is removed, centrifuged and dialyzed to obtain selenized cinnamon polysaccharide; Preferably, the mass ratio of cinnamon polysaccharide, nitric acid, sodium selenite and barium chloride is (1-3):(0.5-1.5):(1-2):(6-8); Preferably, the concentration of the nitric acid is 0.1 to 1% v / v; Preferably, the stirring time is 0.1 to 1 h; Preferably, the temperature of the selenization reaction is 65-75°C, and the time of the selenization reaction is 6-8h; Preferably, the pH of the selenization solution is adjusted to 7-8; Preferably, the pH is adjusted using a 0.5-2 mol / L sodium hydroxide aqueous solution; Preferably, the reagent used to remove barium chloride is sodium sulfate; Preferably, the centrifugal force of the centrifugation is 600-650 xg, and the centrifugation time is 5-15 min; Preferably, the dialysis is performed using a 3 kDa dialysis bag with distilled water, and the dialysis time is 66 to 78 hours.

10. Use of the selenized cinnamon polysaccharide according to any one of claims 1 to 3 in preparing cosmetics with whitening effect.

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