Preparation method of nephrolepis chinensis extract, nephrolepis chinensis extract and application thereof

Abstract

This application discloses a method for preparing a nephrolepis chinensis extract, a nephrolepis chinensis extract, and its applications. The method comprises obtaining a crude polyphenol extract using an acidic enzyme; purifying the polyphenol extract using an acidic supramolecular solution; forming a complex by a polyphenol coordination reaction; obtaining a crude flavonoid extract from the residue using an alkaline enzyme extraction method; purifying the flavonoid extract using an alkaline supramolecular solution; forming an inclusion complex by a flavonoid inclusion reaction; and compounding the complex with the inclusion complex to obtain the nephrolepis chinensis extract. The method utilizes an acid-base biphasic enzymatic hydrolysis technique to efficiently extract the active ingredients, combined with a dual supramolecular elution technique to improve recovery, and employs a zinc salt chelate-cyclodextrin inclusion dual stabilization technique to enhance stability. This method allows the nephrolepis chinensis extract to maintain its oil-control and soothing effects while achieving long-lasting color and physical properties.

Description

Technical Field

[0001] The present application belongs to the technical field of daily cosmetics, and in particular relates to a preparation method of a kidney tea extract, a kidney tea extract and applications thereof. Background Art

[0002] Kidney tea, commonly known as cat's whisker grass, fossil grass, cat's whisker man, and Yanushao, is a plant of the genus Kidney Tea in the Lamiaceae family. It is a commonly used herbal medicine among the Dai people in my country. The plant contains many types of chemical components, including flavonoids, diterpenes, lignans, steroidal saponins, chromones, phenolic acids, etc.

[0003] It is difficult to extract highly stable kidney tea extract using traditional technology. Summary of the Invention

[0004] The present application provides a preparation method of a kidney tea extract, a kidney tea extract and applications thereof, which can maintain the long-term stability of the color and physical properties of the kidney tea extract while maintaining the oil-controlling and soothing effects of the kidney tea extract.

[0005] In a first aspect, the present application provides a method for preparing a nephrolepis chinensis extract, comprising: mixing nephrolepis chinensis powder with an acidic enzyme aqueous solution and performing an extraction treatment to obtain a crude polyphenol extract and a filter residue, wherein the acidic enzymes are acidic protease and pectinase; enriching and purifying the crude polyphenol extract and performing an acidic supramolecular solvent elution treatment to obtain a polyphenol refined extract; mixing the polyphenol refined extract with a complexing agent and performing a coordination reaction to obtain a complex; mixing the filter residue with an alkaline enzyme aqueous solution and performing an extraction treatment to obtain a crude flavonoid extract, wherein the alkaline enzyme is an alkaline protease; enriching and purifying the crude flavonoid extract and performing an alkaline supramolecular solvent elution treatment to obtain a flavonoid refined extract; mixing the flavonoid refined extract with an inclusion agent and performing an inclusion reaction to obtain an inclusion complex; mixing the complex with the inclusion complex, and performing post-processing to obtain the nephrolepis chinensis extract.

[0006] In any embodiment of the present application, in the step of mixing the Herba Cyperi spathulae powder with the acidic enzyme aqueous solution and performing the extraction treatment, the temperature of the acidic enzyme aqueous solution is maintained at 35° C. to 45° C.; and / or the pH of the acidic enzyme is maintained at 3-4.

[0007] In any embodiment of the present application, in the step of mixing the nephrodisiac tea powder with the alkaline enzyme aqueous solution and performing the extraction treatment, the temperature of the alkaline enzyme is maintained at 50° C. to 60° C.; and / or the pH of the alkaline enzyme is maintained at 8 to 10.

[0008] In any embodiment of the present application, the acidic supramolecular solvent comprises at least polyol, citric acid and water; and / or the mass percentage of the polyol in the acidic supramolecular solvent is 50% to 70%.

[0009] In any embodiment of the present application, the alkaline supramolecular solvent comprises at least a polyol, choline chloride and water; and / or the mass percentage of the polyol in the alkaline supramolecular solvent is 50% to 70%.

[0010] In any embodiment of the present application, the polyol includes one or more of 1,3-propylene glycol, glycerol, 1,2-propylene glycol, methylpropylene glycol, dipropylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 1,2-pentanediol, isoprene glycol or 1,2-hexanediol.

[0011] In any embodiment of the present application, during the process of mixing the polyphenol extract with the complexing agent and performing the coordination reaction, the pH of the polyphenol extract is 6.0-7.0.

[0012] In any embodiment of the present application, the complexing agent includes a zinc salt; and / or the inclusion agent includes cyclodextrin or a cyclodextrin derivative.

[0013] In a second aspect, the present application provides a nephrolepis chinensis extract, which is prepared by the preparation method described in the first aspect.

[0014] In a third aspect, the present application provides a use of the Camellia ternata extract prepared by the method described in the first aspect or the Camellia ternata extract described in the second aspect in preparing a product with oil-controlling and / or soothing effects, the product including a cosmetic or skin care preparation; and / or, the mass percentage of the Camellia ternata extract in the product is 1% to 5%.

[0015] Compared with the prior art, the preparation method of the tea extract of the present application includes the following steps: mixing tea powder with an acidic enzyme and subjecting it to an acidic enzyme extraction treatment to obtain a crude polyphenol extract and a filter residue; enriching and purifying the crude polyphenol extract and subjecting it to an acidic supramolecular solvent elution treatment to obtain a polyphenol refined extract; mixing the polyphenol refined extract with a complexing agent and subjecting it to a coordination reaction to obtain a complex; mixing the filter residue with an alkaline enzyme and subjecting it to an alkaline enzyme extraction treatment to obtain a crude flavonoid extract; enriching and purifying the crude flavonoid extract and subjecting it to an alkaline supramolecular solvent elution treatment to obtain a flavonoid refined extract; mixing the flavonoid refined extract with an inclusion agent and subjecting it to an inclusion reaction to obtain an inclusion complex; mixing the complex with the inclusion complex, and subjecting it to a post-processing step to obtain the tea extract. Acid-base biphasic enzymatic hydrolysis technology is used to efficiently extract active ingredients such as polyphenols and flavonoids, combined with dual supramolecular elution technology to improve the recovery rate of target ingredients, and zinc salt chelation-cyclodextrin inclusion dual stabilization technology is used to enhance its stability, thereby maintaining the oil-control and soothing effects of kidney tea extract while achieving long-term stability of color and physical properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 This is a schematic diagram of the cytotoxic effects of different concentrations of nephrolepis chinensis extract on sebaceous gland cells in Example 1 of the present application.

[0018] Figure 2 Schematic diagram of the oil control results of the tea extracts of Example 1, Example 2, Comparative Examples 1-2, Comparative Example 6, Comparative Example 8, Comparative Example 10, Comparative Example 13 and Comparative Example 15 of the present application on inhibiting oil secretion of sebaceous gland cells in vitro (scale bar is 10000 μm).

[0019] Figure 3 This is a schematic diagram of the oil control results of IOD values ​​of different concentrations of kidney tea extracts in Example 1, Example 2, Comparative Examples 1-2, Comparative Example 6, Comparative Example 8, Comparative Example 10, Comparative Example 13 and Comparative Example 15 of the present application for inhibiting oil secretion in sebaceous gland cells in vitro.

[0020] Figure 4 Schematic diagram of the effects of the nephrodisiac tea extracts of Example 1, Example 2, Comparative Examples 1-2, Comparative Example 6, Comparative Example 10 and Comparative Example 15 of the present application on the content of the inflammatory factor NO (### represents: blank group vs. modeling group, P < 0.001; ** represents: modeling group vs. sample group, P < 0.01; *** represents: modeling group vs. sample group, P < 0.001).

[0021] Figure 5 It is a schematic diagram of the effects of the kidney tea extracts of Example 1, Example 2, Comparative Examples 1-2, Comparative Example 6, Comparative Example 10 and Comparative Example 15 of the present application on the increase rate of skin oil in human experiments.

[0022] Figure 6 Schematic diagram of the clinical soothing effects of the kidney tea extracts of Example 1, Example 2, Comparative Examples 1-2, Comparative Example 6, Comparative Example 10 and Comparative Example 15 of the present application. DETAILED DESCRIPTION

[0023] In order to make the application purpose, technical solutions and beneficial technical effects of this application clearer, the application is further described in detail below with reference to the embodiments. It should be understood that the implementation regulations described in this specification are only for the purpose of explaining this application and are not intended to limit this application.

[0024] For simplicity, this application only explicitly discloses certain numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and similarly, any upper limit can be combined with any other upper limit to form an unspecified range. In addition, although not explicitly stated, each point or individual value between the endpoints of a range is included in the range. Thus, each point or individual value can serve as its own lower limit or upper limit and be combined with any other point or individual value, or with other lower limits or upper limits, to form an unspecified range.

[0025] It should be noted that the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, the elements defined by the phrase "comprises..." do not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the elements.

[0026] Unless otherwise stated, the numerical values ​​of the various parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the examples of this application). Unless otherwise stated, the test temperature of each parameter mentioned in this application is 25°C and the test pressure is standard atmospheric pressure.

[0027] The above disclosure of the present application is not intended to describe every disclosed embodiment or every implementation in the present application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided through a series of examples, and these implementation regulations can be used in various combinations. In each example, the enumeration is only intended to be representative and should not be construed as exhaustive.

[0028] Kidney tea extract is rich in polyphenols, chlorophyll and tannic acid active ingredients. Although it has excellent oil-control and soothing effects, it is extremely sensitive to light and heat. In daily chemical applications, it is easy to cause color deepening and precipitation due to light and high temperature, which seriously affects product stability.

[0029] In view of the above problems, the inventors propose a preparation method of a nephrolepis chinensis extract, a nephrolepis chinensis extract and applications thereof.

[0030] The first aspect of the embodiment of the present application provides a method for preparing a nephrolepis chinensis extract, comprising: mixing nephrolepis chinensis powder with an acidic enzyme aqueous solution and performing an extraction treatment to obtain a crude polyphenol extract and a filter residue, wherein the acidic enzymes are acidic protease and pectinase; enriching and purifying the crude polyphenol extract and performing an acidic supramolecular solvent elution treatment to obtain a polyphenol refined extract; mixing the polyphenol refined extract with a complexing agent and performing a coordination reaction to obtain a complex; mixing the filter residue with an alkaline enzyme aqueous solution and performing an extraction treatment to obtain a crude flavonoid extract, wherein the alkaline enzyme is alkaline protease; enriching and purifying the crude flavonoid extract and performing an alkaline supramolecular solvent elution treatment to obtain a flavonoid refined extract; mixing the flavonoid refined extract with an inclusion agent and performing an inclusion reaction to obtain an inclusion complex; mixing the complex with the inclusion complex, and performing post-processing to obtain the nephrolepis chinensis extract.

[0031] Optionally, the preparation method further includes the steps of raw material pretreatment and powder post-treatment.

[0032] Optionally, the raw material pretreatment includes pulverizing the dried kidney tea raw material, and the pulverization treatment includes coarse crushing, pre-pulverization, ultrafine pulverization, etc.

[0033] Exemplarily, ultrafine grinding includes grinding the kidney tea using a grinder such as a turbine grinder or a jet grinder.

[0034] Optionally, the powder post-processing includes sieving the kidney tea powder, such as using an ultrasonic vibration screen (60 mesh) to remove coarse fiber particles to ensure powder uniformity.

[0035] Optionally, enriching and purifying the crude polyphenol extract comprises adding resin to the crude polyphenol extract and stirring for adsorption, collecting the adsorbed resin by filtration, and then selectively eluting the resin adsorption phase with an acidic supramolecular solvent.

[0036] Alternatively, the resin may be a macroporous adsorption resin, an ion exchange resin, or the like.

[0037] For example, the resin may be a non-polar / weakly polar resin such as AB-8, XAD-16 or D101.

[0038] This application uses the aforementioned resin to enrich and purify crude polyphenol extracts. Through hydrophobic interactions, hydrogen bonding, or π-π interactions, it preferentially adsorbs polyphenols (such as phenolic acids) while ignoring impurities such as sugars, proteins, and pigments in the crude extract. Through agitation and adsorption, the resin concentrates the dispersed polyphenols in the crude extract, significantly increasing their local concentration and facilitating subsequent purification.

[0039] Acidic supramolecular solvents efficiently release polyphenols through chemical reactions (breaking adsorption bonds) and physical interactions (solubilization) while ensuring their stability and purity. In acidic supramolecular solvents, citric acid, containing multiple carboxyl and hydroxyl groups, acts as hydrogen bond donors, providing hydrogen ions. The polyols, containing hydroxyl groups, act as hydrogen bond acceptors, forming a supramolecular complex through strong hydrogen bonds. Furthermore, the presence of a small amount of water reduces viscosity and improves fluidity. First, the acidic conditions inhibit the oxidation of polyphenols (which are susceptible to oxidation under alkaline conditions). Furthermore, the mild nature of the supramolecular solvents minimizes structural damage, ensuring that the active components of the polyphenols are not lost during elution. Second, in the acidic supramolecular solvent elution system, acidic solvents such as citric acid can adjust the pH value, promoting polyphenol elution through the following mechanisms: If the resin adsorbs polyphenols via ion exchange (e.g., the phenolic hydroxyl anions released by the dissociated polyphenols bind to the resin cations), the acidic conditions can displace the polyphenol ions, disrupting the ionic bonds. Furthermore, the acidic environment can weaken the hydrogen bonds between the polyphenols and the resin, causing the polyphenols to fall off the resin surface.

[0040] Optionally, enriching and purifying the crude flavonoid extract includes adding resin to the crude flavonoid extract and stirring to adsorb, collecting the adsorbed resin by filtration, and then selectively eluting the resin adsorption phase with an alkaline supramolecular solvent.

[0041] Alternatively, the resin may be a macroporous adsorption resin, an ion exchange resin, or the like.

[0042] For example, the resin may be a macroporous adsorption resin such as AB-8, D101, or XAD-16.

[0043] The above-mentioned resin selectively captures flavonoids through hydrogen bonding, π-π conjugated adsorption or hydrophobic interaction. Impurities such as pigments, polysaccharides, proteins, etc. in the crude extract are difficult to be effectively adsorbed by the resin due to their large structural differences with flavonoids, thus achieving preliminary separation.

[0044] The phenolic hydroxyl groups of flavonoids readily dissociate into phenoloxy anions in alkaline solutions, weakening hydrogen bonding or hydrophobic interactions between them and the resin. Some flavonoids, such as flavonoid glycosides, are more stable under a certain range of alkaline conditions than under acidic conditions, reducing hydrolysis or oxidation losses during elution. Alkaline supramolecular solvents utilize pH specificity and supramolecular selectivity to inhibit impurity elution, ultimately achieving high-yield enrichment and high-purity separation of flavonoids. In alkaline supramolecular solvents, choline chloride acts as a hydrogen bond acceptor, while polyols act as hydrogen bond donors, forming a strong hydrogen bond network. A small amount of water regulates viscosity and stabilizes the supramolecular structure. This process is particularly suitable for the purification of systems containing flavonoid glycosides or polyhydroxyflavones as primary components.

[0045] Optionally, the post-processing includes using membrane filtration technology to separate and purify the mixed system of the complex and the inclusion compound.

[0046] In some embodiments, during the step of mixing the Herba Cyperi spathulae powder with the acidic enzyme aqueous solution and performing the extraction treatment, the temperature of the acidic enzyme aqueous solution is maintained at 35° C. to 45° C.; and / or the pH of the acidic enzyme is maintained at 3-4.

[0047] Optionally, the mass ratio of acid protease to pectinase is 1:(1-5), for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5. Preferably, the mass ratio of acid protease to pectinase is 1:2.

[0048] Optionally, the mass concentration of acid protease in the acid enzyme aqueous solution is 0.01% to 0.1%, for example, it can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, and preferably 0.05%.

[0049] Optionally, the mass concentration of pectinase in the acidic enzyme aqueous solution is 0.05% to 0.5%, for example, it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, and preferably 0.1%.

[0050] Illustratively, the temperature of the acidic enzyme may be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.

[0051] Illustratively, the pH of the acidic enzyme can be 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.

[0052] At 35°C to 45°C and a pH of 3 to 4, the catalytic efficiency of acidic enzymes reaches a cumulative effect, significantly increasing the enzymatic hydrolysis rate. At the same time, low temperatures inhibit thermal degradation, increasing the retention of active ingredients in the extract, such as total flavonoids.

[0053] Optionally, after the acidic enzyme extraction treatment, a filtration step is further included, and the filtrate is the crude polyphenol extract.

[0054] In some embodiments, during the step of mixing the nephrodisiac tea powder with the alkaline enzyme aqueous solution and performing the extraction treatment, the temperature of the alkaline enzyme is maintained at 50° C. to 60° C.; and / or the pH of the alkaline enzyme is maintained at 8 to 10.

[0055] Optionally, the alkaline enzyme comprises alkaline protease.

[0056] Optionally, the mass concentration of alkaline protease in the alkaline enzyme aqueous solution is 0.01% to 0.1%, for example, it can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, and preferably 0.05%.

[0057] Illustratively, the temperature of the alkaline enzyme may be 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0058] Illustratively, the pH of the alkaline enzyme can be 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.

[0059] The above-mentioned suitable pH and temperature can activate the optimal catalytic activity of alkaline enzyme and maintain the active conformation of alkaline enzyme. At the same time, the alkaline environment can also inhibit the degradation of easily oxidized components such as flavonoids.

[0060] Optionally, after the alkaline enzyme extraction treatment, a filtration step is further included, and the filtrate is the crude flavonoid extract.

[0061] In some embodiments, the acidic supramolecular solvent comprises at least polyol, citric acid and water; and / or the mass percentage of the polyol in the acidic supramolecular solvent is 50% to 70%.

[0062] Illustratively, the mass percentage of the polyol in the acidic supramolecular solvent is 50%, 52%, 54%, 55%, 56%, 58%, 60%, 62%, 64%, 65%, 66%, 68% or 70%.

[0063] For example, the mass ratio of polyol, citric acid and water in the acidic supramolecular solvent can be (55-65):20:20, preferably 60:20:20.

[0064] In some embodiments, the alkaline supramolecular solvent comprises at least a polyol, choline chloride, and water; and / or the mass percentage of the polyol in the alkaline supramolecular solvent is 50% to 70%.

[0065] Illustratively, the mass percentage of the polyol in the alkaline supramolecular solvent is 50%, 52%, 54%, 55%, 56%, 58%, 60%, 62%, 64%, 65%, 66%, 68% or 70%.

[0066] When the mass percentage of polyols in acidic or alkaline supramolecular solvents reaches 50%~70%, their high osmotic pressure and solvent binding ability can effectively inhibit the growth of microorganisms, and the antiseptic function can be achieved without the addition of additional chemical preservatives.

[0067] For example, the mass ratio of the polyol, choline chloride, and water in the alkaline supramolecular solvent may be (55-65):20:20, preferably 60:20:20.

[0068] In some embodiments, the polyol includes one or more of 1,3-propylene glycol, glycerol, 1,2-propylene glycol, methylpropylene glycol, dipropylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 1,2-pentanediol, isopentylene glycol, or 1,2-hexanediol, preferably 1,3-propylene glycol or glycerol.

[0069] Optionally, the temperature during the acidic supramolecular solvent elution treatment is 38° C. to 48° C.

[0070] Illustratively, the temperature during the acidic supramolecular solvent elution treatment can be 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, or 48°C.

[0071] When the temperature is too low, the solvent molecules lack kinetic energy, resulting in low desorption efficiency. When the temperature is too high, the solvent's ability to dissolve impurities increases, resulting in a decrease in the purity of the purified product. Under acidic conditions, polyphenols tend to exist in molecular form. Low temperatures of 38°C to 48°C can reduce the risk of oxidation or degradation. Low-temperature elution can maintain the structural integrity of the compounds and improve the recovery rate of polyphenols.

[0072] Optionally, the temperature during the alkaline supramolecular solvent elution treatment is 70° C. to 80° C.

[0073] Illustratively, the temperature during the alkaline supramolecular solvent elution treatment may be 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C.

[0074] High temperatures of 70°C to 80°C further accelerate the dissociation of flavonoids from the resin adsorption sites, destroying intermolecular forces (such as van der Waals forces) through thermal motion, thereby improving desorption efficiency. Under alkaline conditions, flavonoids exist in an ionic state (phenol oxide anions), and their water solubility increases significantly with increasing temperature. High temperatures can promote the separation of impurities (such as polysaccharides and proteins) from flavonoids: some impurities are easily denatured and precipitated under alkaline and high temperatures, or retained on the resin due to weak interactions with the solvent, thereby improving the purity of flavonoids.

[0075] In some embodiments, during the process of mixing the polyphenol extract with the complexing agent and performing the coordination reaction, the pH of the polyphenol extract is 6.0-7.0.

[0076] Illustratively, the pH of the polyphenol extract may be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0077] In the coordination reaction between polyphenol extracts and complexing agents, controlling the pH within the neutral range of 6.0-7.0 maximizes coordination efficiency and complex stability by optimizing the dissociation state of the polyphenols, the activity of the complexing agent, and the stability of the reaction environment. This pH promotes the selective exposure of coordination sites and ensures the appropriate generation of phenol oxide anions, which can form coordination bonds with metal ions such as zinc ions or other zinc salt complexing agents.

[0078] In some embodiments, the complexing agent comprises a zinc salt; and / or the inclusion agent comprises a cyclodextrin or a cyclodextrin derivative.

[0079] Optionally, the zinc salt includes at least one of zinc hyaluronate, zinc DNA, zinc PCA, zinc phenolsulfonate, zinc ricinoleate, zinc glycinate, zinc glycyrrhetinate, zinc sulfide, zinc sulfate, zinc chloride, zinc gluconate, zinc myristate, zinc lactate, zinc undecylenate, zinc salicylate, zinc carbonate, zinc aspartate, zinc acetate, zinc stearate, zinc laurate or zinc palmitate; more preferably, the zinc salt includes at least one of zinc hyaluronate or zinc chloride.

[0080] Polyphenols contain multiple phenolic hydroxyl groups, such as gallic acid. These compounds are highly water-soluble and exhibit a slightly acidic nature due to their presence. However, these phenolic hydroxyl groups are highly temperature-sensitive, and exposure to high temperatures can darken the color of polyphenols, hindering their application. Polyphenols possess unique physicochemical properties, allowing them to bind to a variety of substances through hydrogen bonds, hydrophobic interactions, π-π interactions, and cation-π interactions. Based on these characteristics, polyphenols can form dynamic covalent bonds with zinc ions, enhancing their stability.

[0081] Optionally, the cyclodextrin or cyclodextrin derivative includes at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or hydroxypropyl-β-cyclodextrin, preferably hydroxypropyl-β-cyclodextrin.

[0082] The cyclodextrin molecule is shaped like a slightly conical annular cavity. The central cavity of the molecule contains the carbon skeleton and ether oxygen of glucose residues, making the cavity hydrophobic. The hydroxyl groups are oriented toward the outer edge of the cone, with the primary hydroxyl groups of the sugar residues located at the narrow edge and the secondary hydroxyl groups at the wider edge. This gives the cyclodextrin molecule excellent hydrophilicity. The hydrophilic exterior and hydrophobic interior of cyclodextrins act as molecular containers, trapping poorly soluble guest molecules such as flavonoids within the cavity to form inclusion complexes, reducing the effects of light, heat, and oxygen on the guest molecules and their degradation, thereby improving their stability.

[0083] The preparation method of the kidney tea extract in this application has a safe, green and environmentally friendly extraction process, can extract a wide range of different required components with a high extraction rate, and the stability of the extract is improved after treatment, especially the color is moderate, and the application restrictions are reduced, because if the color is heavy, it is not suitable for use in spray products, facial mask products and other aqueous products.

[0084] A second aspect of the embodiments of the present application provides a nephrolepis chinensis extract, which is prepared by the preparation method described in the first aspect.

[0085] A third aspect of the embodiments of the present application provides a use of the Camellia ternata extract prepared by the method described in the first aspect or the Camellia ternata extract described in the second aspect in preparing a product with oil-controlling and / or soothing effects, the product including a cosmetic or skin care preparation; and / or, the mass percentage of the Camellia ternata extract in the product is 1% to 5%.

[0086] Illustratively, the mass percentage of the Herba Cynanchifoliae extract in the product may be 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8% or 5%.

[0087] Optionally, the product includes at least one of essence, skin care oil, makeup remover oil, facial cleanser, lotion, cream, shampoo and hair care or liquid foundation, pressed powder, lip gloss, lipstick and mascara.

[0088] Example

[0089] The following examples describe the present disclosure in more detail. These examples are intended for illustrative purposes only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by mass, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing. The instruments used in the examples are commercially available.

[0090] (1) Acidic protease: Ningxia Xiasheng Industrial Group Co., Ltd., protease (acidic APRL type), production batch number: 12311030.

[0091] (2) Pectinase: Ningxia Xiasheng Industrial Group Co., Ltd., pectinase (special for plant extraction SPE-006 type), production batch number: 12401041.

[0092] (3) Alkaline protease: Ningxia Xiasheng Industrial Group Co., Ltd., protease (alkaline), production batch number: 22301008.

[0093] (4) Dried kidney tea: Yunnan Xianghui Pharmaceutical Co., Ltd., origin: Xishuangbanna, Yunnan.

[0094] (5) 1,3-Propanediol: Zemea® Select Propanediol produced by Primient Covation LLC, with a mass concentration of 99.8-100%, production batch number: LP22G000111.

[0095] (6) Citric acid: Shanghai Lijing Industrial Co., Ltd., citric acid monohydrate, mass concentration of 99.5-100.5%.

[0096] (7) Choline chloride: Zhengzhou Yukong Biotechnology Co., Ltd., production batch number: YC20240302.

[0097] (8) Zinc hyaluronate: Bloomage Biotech Co., Ltd., hydrolyzed zinc hyaluronate, production batch number: J201240308.

[0098] (9) Hydroxypropyl-β-cyclodextrin: Shandong Binzhou Zhiyuan Biotechnology Co., Ltd., production batch number: HP20230213.

[0099] (10) γ-cyclodextrin: Shandong Binzhou Zhiyuan Biotechnology Co., Ltd., production batch number: GCD20230318.

[0100] (11) Acidic supramolecular solvent: Grind citric acid into a fine powder to increase the reaction surface area. Add the polyol and citric acid in proportion to the reaction vessel. Raise the temperature to 80°C and stir (300-500 rpm) until a transparent, homogeneous liquid is formed. After cooling to 60°C, slowly add deionized water with stirring. Stir for 30 minutes. Cool to room temperature to obtain the acidic supramolecular solvent.

[0101] (12) Alkaline supramolecular solvent: Place choline chloride in a vacuum drying oven at 60°C for 24 hours to remove moisture. Add choline chloride and polyol into a reactor and stir at 75°C (500 rpm) until a transparent uniform liquid is formed. Cool to 60°C and slowly add deionized water with stirring. Stir for 30 minutes. After cooling to room temperature, it becomes an alkaline supramolecular solvent.

[0102] In the Examples and Comparative Examples of this application, the enzyme solutions containing acidic proteases, pectinase, and alkaline proteases are used, and the type of solvent used in the dilution process is not particularly limited. Those skilled in the art can select appropriate enzyme diluents based on conventional technical requirements, such as, but not limited to: buffer solutions (such as phosphate buffer, Tris-HCl buffer, etc.); deionized water or ultrapure water; dilution systems containing stabilizers (such as glycerol, calcium chloride); and other inert solvents that meet the requirements for maintaining enzyme activity. It should be understood that the above-mentioned solvents are merely illustrative; any equivalent diluent that can achieve enzyme dilution without affecting its activity falls within the scope of protection of this application.

[0103] Example 1

[0104] Example 1 of the present application provides a method for preparing a nephrolepis chinensis extract, comprising:

[0105] 10 kg of dried kidney tea was crushed, passed through a 60-mesh sieve and put into a tank, 50 kg of an enzyme solution containing 0.1% wt of acidic protease and 50 kg of an enzyme solution containing 0.2% wt of pectinase were prepared respectively, and the two solutions were mixed to form an acidic enzyme aqueous solution with a pH of 3.5, a mass concentration of 0.05% of acidic protease in the acidic enzyme aqueous solution, and a mass concentration of 0.1% of pectinase in the acidic enzyme aqueous solution. The solution was stirred and enzymatically extracted at a temperature of 40° C. for 2.0 h; the solution was filtered using a 300-mesh filter bag, and the filtrate was the crude polyphenol extract, and the filter residue was set aside;

[0106] An appropriate amount of resin was added to the crude polyphenol extract, and the mixture was stirred and adsorbed for 2 hours. The mixture was filtered through a 100-mesh filter bag. The resin was removed and eluted using an acid-washing supramolecular solvent formed by 1,3-propylene glycol, citric acid, and water (mass ratio = 60:20:20) at 40°C for 2 hours. The mixture was filtered through a 100-mesh filter bag, and the filtrate was obtained as the polyphenol extract. The pH value of the polyphenol extract was adjusted to 6.5, and an appropriate amount of zinc hyaluronate was added. The mixture was stirred for 1 hour to form a polyphenol-zinc ion complex. The amount of zinc hyaluronate added was 0.05% of the mass of the polyphenol extract.

[0107] The filter residue was put into a tank, and 100 kg of enzyme solution containing 0.05% wt alkaline protease and pH = 9.0 was added. The mixture was stirred and extracted at a temperature of 58 ° C for 2 hours. The mixture was filtered using a 300-mesh filter bag. The filtrate was the crude flavonoid extract, and the filter residue was discarded. An appropriate amount of resin was added to the crude flavonoid extract, stirred and adsorbed for 2 hours, and filtered using a 100-mesh filter bag. The resin was taken and eluted using an alkaline supramolecular solvent formed by 1,3-propylene glycol, choline chloride, and water (mass ratio = 60:20:20) at a temperature of 75 ° C for 2 hours. The filtrate was taken using a 100-mesh filter bag, and the filtrate was the flavonoid extract. An appropriate amount of hydroxypropyl-β-cyclodextrin was added and stirred for inclusion for 2 hours to form a flavonoid-cyclodextrin inclusion complex. The amount of hydroxypropyl-β-cyclodextrin added was 0.05% of the mass of the flavonoid extract.

[0108] The polyphenol-zinc ion complex and the flavonoid-cyclodextrin inclusion complex were mixed in a mass ratio of 1:1, stirred for 30 minutes, and filtered through a 0.45 μm nylon membrane to obtain a nephrolepis extract.

[0109] Example 2 to Example 5

[0110] Examples 2 to 5 were prepared using a method substantially the same as that of Example 1, with the only differences from Example 1 being the amount of the compounding agent, inclusion agent, polyol, pH during the acidic or alkaline enzyme extraction process, pH of the polyphenol extract, and temperature during the acidic or alkaline supramolecular solvent elution process. For details, see the corresponding items in Table 1.

[0111] Comparative Example 1

[0112] Comparative Example 1 of the present application provides a method for preparing a nephrolepis chinensis extract, comprising:

[0113] 10 kg of dried kidney tea was crushed, passed through a 60-mesh sieve and put into a tank, 100 kg of pure water was added, and the mixture was stirred and extracted at 40°C for 2 h; filtered using a 300-mesh filter bag to obtain filtrate 1; the filter residue was put into a tank, 100 kg of pure water was added, and the mixture was stirred and extracted at 58°C for 2 h, and filtered using a 300-mesh filter bag to obtain filtrate 2.

[0114] The filtrate 1 and the filtrate 2 were combined, filtered using a plate and frame filter until clear and transparent, and a 60 wt % 1,3-propylene glycol aqueous solution as an eluent was added, ultrasonically homogenized, and concentrated until the liquid volume was consistent with that of Example 1 to obtain the product.

[0115] Comparative Example 2

[0116] The difference between Comparative Example 2 and Comparative Example 1 is that the steps of enrichment and purification and dual supramolecular elution are added:

[0117] Combine filtrates 1 and 2, filter using a plate-and-frame filter until clear and transparent, concentrate to an appropriate volume, add resin for adsorption for 2 hours, and filter to remove the resin. Elute first with a supramolecular solvent composed of 1,3-propylene glycol, citric acid, and water (mass ratio = 60:20:20) at 40°C for 2 hours, then elute with a supramolecular solvent composed of 1,3-propylene glycol, choline chloride, and water (mass ratio = 60:20:20) at 75°C for 2 hours. Combine the two eluates and filter until clear and transparent to obtain the product.

[0118] Comparative Example 3

[0119] The difference between Comparative Example 3 and Comparative Example 1 is that the steps of enrichment and purification, dual supramolecular elution, coordination, and inclusion are added:

[0120] Combine filtrates 1 and 2, filter using a plate-and-frame filter until clear and transparent, concentrate to an appropriate volume, add resin for adsorption for 2 hours, and filter to remove the resin. Elute with a supramolecular solvent composed of 1,3-propylene glycol, citric acid, and water (mass ratio = 60:20:20) at 40°C for 2 hours, and filter to obtain a phenol solution. Adjust the pH of the polyphenol solution to 6.5, add an appropriate amount of zinc hyaluronate, and stir for 1 hour to form a polyphenol-zinc ion complex.

[0121] Then, the solution was eluted with a supramolecular solvent composed of 1,3-propylene glycol, choline chloride, and water (mass ratio = 60:20:20) at 75°C for 2 h. An appropriate amount of hydroxypropyl-β-cyclodextrin was added to the filtered filtrate and stirred for 2 h to form a flavonoid-cyclodextrin inclusion complex.

[0122] The polyphenol complex and the flavonoid inclusion complex are combined.

[0123] Comparative Example 4

[0124] The only difference between Comparative Example 4 and Comparative Example 1 is that the extractant is changed to 50% wt ethanol aqueous solution:

[0125] 10 kg of dried kidney tea was crushed, passed through a 60-mesh sieve and placed in a tank. 100 kg of a 50% wt ethanol aqueous solution was added and stirred at room temperature for 2 hours. The mixture was filtered through a 300-mesh filter bag to obtain filtrate 1. The filter residue was placed in a tank, 100 kg of a 50% wt ethanol aqueous solution was added, and the mixture was stirred and extracted for 2 hours. The mixture was filtered through a 300-mesh filter bag to obtain filtrate 2.

[0126] Combine filtrate 1 and filtrate 2, filter using a plate and frame filter until clear and transparent, and concentrate until there is no ethanol smell. Add 60% wt 1,3-propylene glycol aqueous solution as eluent to dissolve, and sonicate to obtain the product.

[0127] Comparative Example 5

[0128] The only difference between Comparative Example 5 and Comparative Example 1 is that the extraction agent is changed to 50% wt ethanol aqueous solution, and the steps of enrichment, purification and dual supramolecular elution are added:

[0129] 10 kg of shade-dried kidney tea was crushed, passed through a 60-mesh sieve and placed in a tank. 100 kg of 50% wt ethanol aqueous solution was added, and the mixture was stirred and extracted at room temperature for 2 hours. The mixture was filtered using a 300-mesh filter bag to obtain filtrate 1. The filter residue was placed in a tank, 100 kg of 50% wt ethanol aqueous solution was added, and the mixture was stirred and extracted for 2 hours. The mixture was filtered using a 300-mesh filter bag to obtain filtrate 2.

[0130] Combine filtrates 1 and 2, filter using a plate-and-frame filter until clear and transparent, concentrate to an appropriate volume, add resin for adsorption for 2 h, and filter to remove the resin. Elute first with a supramolecular solvent composed of 1,3-propylene glycol, citric acid, and water (mass ratio = 60:20:20) at 40°C for 1-32 h. Then, elute with a supramolecular solvent composed of 1,3-propylene glycol, choline chloride, and water (mass ratio = 60:20:20) at 75°C for 2 h. Combine the two eluates and filter until clear and transparent to obtain the product.

[0131] Comparative Example 6

[0132] The only difference between Comparative Example 6 and Comparative Example 1 is that the extractant is changed to 50% wt ethanol aqueous solution, and the steps of enrichment and purification, dual supramolecular elution, coordination and inclusion are added:

[0133] 10 kg of shade-dried kidney tea was crushed, passed through a 60-mesh sieve and placed in a tank. 100 kg of a 50% wt ethanol aqueous solution was added, and the mixture was stirred and extracted at room temperature for 2 hours. The mixture was filtered through a 300-mesh filter bag to obtain filtrate 1. The filter residue was placed in a tank, 100 kg of a 50% wt ethanol aqueous solution was added, and the mixture was stirred and extracted for 2 hours. The mixture was filtered through a 300-mesh filter bag to obtain filtrate 2.

[0134] Combine filtrates 1 and 2, filter using a plate-and-frame filter until clear and transparent, concentrate to an appropriate volume, add resin for adsorption for 2 hours, and filter to remove the resin. Elute with a supramolecular solvent composed of 1,3-propylene glycol, citric acid, and water (mass ratio = 60:20:20) at 40°C for 2 hours. Filter to obtain a phenol solution. Adjust the pH of the polyphenol solution to 6.5, add an appropriate amount of zinc hyaluronate, and stir for 1 hour to form a polyphenol-zinc ion complex.

[0135] The solution was then eluted with a supramolecular solvent consisting of 1,3-propylene glycol, choline chloride, and water (mass ratio = 60:20:20) at 75°C for 2 h. An appropriate amount of hydroxypropyl-β-cyclodextrin was added to the filtrate to form a flavonoid-cyclodextrin inclusion complex.

[0136] The polyphenol complex and the flavonoid inclusion complex are combined.

[0137] Comparative Example 7

[0138] The difference between Comparative Example 7 and Comparative Example 1 is that the traditional high-temperature water extraction is used, and the steps of enrichment and purification, dual supramolecular elution, coordination and inclusion are added:

[0139] 10 kg of dried kidney tea was crushed, passed through a 60-mesh sieve and put into a tank, 100 kg of pure water was added, and the mixture was stirred and extracted at 60°C for 2 hours; filtered using a 300-mesh filter bag to obtain filtrate 1; the filter residue was put into a tank, 100 kg of pure water was added, and the mixture was stirred and extracted at 80°C for 2 hours, and filtered using a 300-mesh filter bag to obtain filtrate 2.

[0140] Combine filtrates 1 and 2, filter using a plate-and-frame filter until clear and transparent, concentrate to an appropriate volume, add resin for adsorption for 2 hours, and filter to remove the resin. Elute with a supramolecular solvent composed of 1,3-propylene glycol, citric acid, and water (mass ratio = 60:20:20) at 40°C for 2 hours, and filter to obtain a phenol solution. Adjust the pH of the polyphenol solution to 6.5, add an appropriate amount of zinc hyaluronate, and stir for 1 hour to form a polyphenol-zinc ion complex.

[0141] Then, a supramolecular solvent formed by 1,3-propylene glycol, choline chloride and water (mass ratio = 60:20:20) was used for elution at a temperature of 75°C and an elution time of 2 hours. An appropriate amount of hydroxypropyl-β-cyclodextrin was added to the filtered filtrate and stirred for 2 hours to form a flavonoid-cyclodextrin inclusion complex; the above-mentioned polyphenol complex and flavonoid inclusion complex were combined.

[0142] Comparative Example 8

[0143] Comparative Example 8: The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, except that the extractant was entirely replaced with water.

[0144] Comparative Example 9

[0145] Comparative Example 9 The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, except that the extractant was adjusted to a 50% wt ethanol aqueous solution.

[0146] Comparative Example 10

[0147] Comparative Example 10 The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, except that the supramolecular elution procedure was completely adjusted to use only a 60% wt 1,3-propylene glycol aqueous solution for ordinary elution.

[0148] Comparative Example 11

[0149] Comparative Example 11: The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, with the only difference being that the pH of the polyphenol extract was adjusted to 5.5.

[0150] Comparative Example 12

[0151] Comparative Example 12: The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, with the only difference being that the pH of the polyphenol extract was adjusted to 7.5.

[0152] Comparative Example 13

[0153] Comparative Example 13: The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, except that the pH of the extractant was adjusted to 2.0 when extracting polyphenols and to 11.0 when extracting flavonoids.

[0154] Comparative Example 14

[0155] Comparative Example 14 The Herba Cynanchifoliae extract was prepared by a method substantially the same as that of Example 1, with the only difference being that the extraction temperature for extracting polyphenols was adjusted to 50° C., and the extraction temperature for extracting flavonoids was adjusted to 60° C.

[0156] Comparative Example 15

[0157] Comparative Example 15: The kidney tea extract was prepared by a method basically the same as that in Example 1, except that the mass proportion of the polyol in the acidic supramolecular solvent was adjusted to 40%, and the mass proportion of the polyol in the alkaline supramolecular solvent was adjusted to 80%.

[0158] The relevant parameters of Examples 1 to 5 and Comparative Examples 1 to 15 are shown in Table 1 and Table 2.

[0159] Table 1

[0160]

[0161] Table 2

[0162]

[0163] Detection

[0164] Test Method

[0165] 1. Polyphenol and flavonoid content

[0166] (1) Total polyphenols detection

[0167] 1 The total polyphenol content in plant extracts was determined by the Folin-phenol spectrophotometric method.

[0168] 2 Reagents and consumables

[0169] 2.1 Gallic acid stock solution (0.1 mg / mL) (gallic acid content ≥ 98%): Prepare freshly prepared gallic acid by weighing 0.0110 g ± 0.001 g (W = 188.14) into a 100 mL volumetric flask, dissolve in water, and make up to volume (based on the actual weight).

[0170] 2.2 Preparation of 10% Folin phenol (1 mol / L): Place 10 mL of Folin phenol in a 100 mL volumetric flask and dilute to 100 mL with water.

[0171] 2.3 Na2CO3 solution (7.5%): Weigh 37.5 g ± 0.01 g, dissolve in water and dilute to 500 mL.

[0172] 3 Instruments and Equipment

[0173] 3.1 UV spectrophotometer (UV-1900i); volumetric flask, analytical balance, sensitivity 0.1 mg

[0174] 3.2 Colorimetric tube 10 mL;

[0175] 3.3 Cuvette 10 mm.

[0176] 4 Analysis steps

[0177] 4.1 To establish a standard curve for gallic acid, take 6 10 mL colorimetric tubes and add the reagents in the order shown in Table 3 below.

[0178] Table 3

[0179]

[0180] 4.2 Detection: After standing for 60 min, use 0# as reference and adjust the blank to zero. Measure the absorbance at a wavelength of 765 nm and fit a standard curve with absorbance as the ordinate and concentration as the abscissa.

[0181] 4.3 Sample test: Take 1 mL of sample and measure the absorbance according to step 5.0. Substitute it into the standard curve to calculate its concentration.

[0182] 5 Calculation formula

[0183] Concentration mark calculation formula:

[0184] (1)

[0185] (2)

[0186] c: final sample concentration in mg / mL;

[0187] y: sample concentration mg / mL (sample concentration in the system);

[0188] x: absorbance;

[0189] f: sample dilution factor;

[0190] v 体系 :10mL;

[0191] v 取 :1mL;

[0192] The results are shown in Table 5.

[0193] (2) Determination of total flavonoid content

[0194] 1 Instruments and equipment

[0195] UV-visible spectrophotometer, analytical balance with a sensitivity of 0.1 mg, volumetric flasks of 25 ml and 100 ml, quartz cuvettes (1 cm), pipettes of 1 ml, 5 ml, and 200 μl, centrifuge tubes of 15 ml, alcohol meter, and graduated cylinder of 500 ml.

[0196] 2 Reagents and Materials

[0197] 2.1 Rutin standard, CAS number: 58-96-8, purity ≥ 98.0%.

[0198] 2.2 Grade 3 pure water.

[0199] 2.3 Sodium hydroxide.

[0200] 2.4 Aluminum nitrate.

[0201] 2.5 Sodium nitrite.

[0202] 2.6 Ethanol.

[0203] 3 Analysis steps

[0204] 3.1 Preparation of Rutin Standard Stock Solution

[0205] Weigh 10 mg (accurate to 0.1 mg) of rutin reference substance into a 100 ml volumetric flask, dissolve it in ethanol and dilute to the mark, shake well, and use it on the same day it is prepared.

[0206] 3.2 Preparation of reagent solution

[0207] Weigh 5.0 g of sodium nitrite (accurate to 0.1 g), place it in a 100 ml volumetric flask, dissolve it in pure water and dilute to the mark, shake well to obtain a 5% sodium nitrite aqueous solution.

[0208] Weigh 10.0 g (accurate to 0.1 g) of aluminum nitrate into a 100 ml volumetric flask, dissolve it in pure water and dilute to the mark, then shake well to obtain a 10% aluminum nitrate aqueous solution.

[0209] Weigh 4.0 g of sodium hydroxide (accurate to 0.1 g) and place it in a 100 ml volumetric flask. Dissolve it in pure water and dilute to the mark. Shake well to obtain a 1 mol / L NaOH aqueous solution.

[0210] Use a 500 ml graduated cylinder to measure an appropriate amount of ethanol, add an appropriate amount of pure water, and use an alcohol meter to adjust the ethanol content to 30% (volume percentage (v / v)).

[0211] 3.3 Analysis steps

[0212] Pipette 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 ml of rutin standard stock solution into a 15 ml centrifuge tube. Add 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, and 2.0 ml of 30% ethanol / water solution, respectively. Shake well. Add 0.3 ml of 5% sodium nitrite solution and 0.3 ml of 10% aluminum nitrate solution, shake well. Add 3.0 ml of 1 mol / L sodium hydroxide solution and 1.4 ml of 30% ethanol / water solution, shake well, and let the mixture react for 50 minutes. Measure the absorbance of each standard solution at 530 nm. Plot a standard curve with the absorbance as the y-axis and the corresponding rutin concentration (mg / ml) as the x-axis. The standard curve is Y = aX + b.

[0213] 3.4 Sample testing steps

[0214] Dilute the sample to be tested by half with pure water. Pipette 0.2 ml of the diluted sample into a 15 ml centrifuge tube. Add 4.8 ml of 30% ethanol aqueous solution and shake well. Then add 0.3 ml of 5% sodium nitrite aqueous solution and 0.3 ml of 10% aluminum nitrate aqueous solution and shake well. Then add 3.0 ml of 1 mol / L sodium hydroxide aqueous solution and 1.4 ml of 30% ethanol aqueous solution and shake well. Let the mixture react for 50 minutes. Measure the absorbance (A0) at 530 nm. Calculate the total flavonoid content (W) (ppm) according to the method in 3.5.

[0215] 3.5 Presentation of analysis results

[0216] The total flavonoid content of the sample to be tested (calculated as rutin) is calculated according to formula (1).

[0217] Formula (1):

[0218]

[0219] In formula (1):

[0220] W (ppm)——the content of total flavonoids, ppm;

[0221] b, a——correlation coefficient of standard curve;

[0222] A0——absorbance value of the diluted sample after reaction;

[0223] The arithmetic mean of two parallel determinations was taken as the final result, and the result was retained to two significant figures;

[0224] The absolute difference between the results of two parallel measurements shall not exceed 3% of the arithmetic mean.

[0225] The results are shown in Table 5.

[0226] 2. Stability Test

[0227] (1) The product is placed in a high temperature environment of 45°C for 30 consecutive days. During this period, its appearance, color, clarity, transparency, and whether there is precipitation and other physical properties are regularly observed.

[0228] (2) Color Gardner Chroma Test

[0229] Plug the Lovibond instrument into an outlet and press the power button. The instrument will start and warm up. Perform a self-check. After the self-check is complete, perform a baseline calibration. Set the optical path to 50 mm, take a clean 50 mm cuvette, add deionized water, and close the lid to perform the baseline calibration. Remove the cuvette and pour out the deionized water. Rinse the cuvette three times with the sample to be measured, then add the sample to be measured and begin measurement.

[0230] The results are shown in Tables 5 and 6.

[0231] 3. Sebaceous gland cell oil control experiment

[0232] 1. Sebaceous gland cells were selected to simulate the sebum secretion of human skin. Nile red dye was used to perform fluorescent staining on neutral lipids. Fluorescence microscopy and fluorescence quantitative methods were used to qualitatively and quantitatively detect the oil content of sebaceous gland cells, thereby evaluating the oil control effect of the test samples.

[0233] 2 Experimental steps

[0234] 2.1 Cell viability test

[0235] Sebaceous gland cells were seeded in 96-well plates ( Each well was treated with 100 μL of culture medium diluted with the tea extract prepared in Example 1 (volume percentages of 1%, 0.5%, 0.25%, and 0.1%). Control and blank groups were set up and incubated for 24 hours after the last stimulation. Cell morphology was observed under an inverted microscope. The supernatant was discarded and washed twice with PBS. The cells were stained according to the MTT kit instructions. The optical density (OD) was measured at 490 nm using a microplate reader. Cell viability = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0236] 2.2 Neutral Lipid Fluorescence Quantification: Cell suspensions were plated in 96-well cell culture plates. After incubation, the supernatant was discarded. Maintenance medium was added to the negative control group, maintenance medium was added to the model control group, and 0.25% wt concentration of the test sample and positive control were added to the remaining groups. After incubation, the supernatant was discarded. After washing with PBS, Nile red dye was added to the experimental group, and FDA was added to the control group. The released fluorescence was measured on a multifunctional microplate reader. Results were determined using IOD values. Statistical analysis was performed using the SPSS software package, and significance tests were performed. A P < 0.05 was considered statistically significant.

[0237] 2.3 Neutral lipid fluorescence microscopy: Cells were routinely cultured and seeded in culture dishes. The test sample and positive control were added at a concentration of 0.25% by weight. After incubation, the supernatant was discarded, and the cells were quickly washed with PBS. The cells were fixed with paraformaldehyde at room temperature, and Nile red dye was added. The cells were observed under a fluorescence microscope.

[0238] The results are as follows Figure 1 、 Figure 2 、 Figure 3 shown.

[0239] IV. In vitro soothing test

[0240] The experiment was divided into four groups: blank, model, positive control, and sample. The model group received 1 μg / mL LPS plus culture medium; the positive control group received 1 μg / mL LPS plus 50 μmol / L dexamethasone plus culture medium; the blank group received culture medium alone; and the sample group received 1 μg / mL LPS plus 1% and 0.5% nephritis extract plus culture medium. For evaluation, RAW264.7 cells in the logarithmic growth phase were seeded into 6-well plates at various concentrations and incubated overnight at 37°C, 5% CO2. When the cell confluence reached 50%-60%, samples were collected and the cells were incubated in a 37°C, 5% CO2 incubator. After completion of the cell culture, the cell supernatant was collected and the NO content was determined using a NO detection kit. Each experiment was repeated three times.

[0241] The results are as follows Figure 4shown.

[0242] 5. Clinical oil control and soothing test

[0243] 1 The above plant extract - Example 1 was made into essence according to the formula in Table 4 below, and clinical oil control and soothing tests were carried out. The test results are as follows Figure 5 、 Figure 6 shown.

[0244] Table 4

[0245]

[0246] 2. Reagents and instruments:

[0247] Sodium lauryl sulfate (99% purity, SIGMA) was prepared with distilled water to make a 1% SLS aqueous solution, which was set aside. Also included were distilled water (Watsons), a patch applicator (Finn Chambers, SmartPractice R), a Microscope™ adhesive tape (3M hypoallergenic topical tape), a Sebumeter SM815 skin oil test probe (Courage-Khazaka, Germany), and a VISIA 7 skin analyzer (Canfield, USA).

[0248] 3 Experimental steps

[0249] In a constant temperature and humidity environment, the tester's face was cleaned and dried with a standard facial cleanser, and the initial value of the skin oil content (μg / cm ２ The area without any sample was designated as the blank group, the area evenly coated with (2.0 ± 0.5) g of sample was designated as the experimental group, and the area treated only with purified water was designated as the pure water group within the experimental group. Skin oil content was measured 6 hours after the test began.

[0250] In a constant temperature and humidity environment, cleanse the subject's forearm with a standard cleanser and wipe dry. Select a flat area on the forearm for patching. Apply a 1% SLS aqueous solution for 24 hours. Remove the patch tester and apply the sample to the corresponding well. Then measure the skin's redness value (a).

[0251] Experimental results

[0252] Table 5

[0253]

[0254] Table 6

[0255]

[0256] As shown in Tables 5 and 6, the flavonoid contents obtained using conventional water extraction methods in Comparative Examples 1, 2, and 3 are relatively low. This is primarily due to the poor water solubility of most flavonoids. Based on the principle of like dissolves like, conventional water extraction methods yield relatively low flavonoid yields. Furthermore, Comparative Example 2, based on Comparative Example 1, employed a supramolecular solvent for elution and enrichment, which, to a certain extent, made the extract more clear and transparent, with no precipitation produced upon initial preparation. Comparative Example 3, based on Comparative Example 2, employed zinc salts for the discoloration-prone polyphenols and cyclodextrin encapsulation for the poorly water-soluble flavonoids. Possibly due to ratio issues, the extracts became turbid after one month, but no precipitation was produced, demonstrating that the encapsulation and elution of the water-extracted enriched solution can indeed improve the stability of the liquid. Comparative Examples 4, 5 and 6 all use the organic solvent ethanol for extraction. Because ethanol has strong penetrability, more flavonoids and polyphenols can be extracted. However, high levels of active substances are more likely to aggregate and precipitate, increasing instability. The use of ethanol for extraction is not green, environmentally friendly, energy-saving, safe, or Halal. Comparative Example 7 uses high-temperature water extraction. High temperatures can increase the dissolution of active substances, but high temperatures also tend to deepen the color of the active substances. Using supramolecular solvents to elute and enrich the active substances, zinc salts are coordinated and cyclodextrins are encapsulated to improve stability during initial preparation. The liquid color is clear and transparent with no precipitation. After one month, the color deepens and it is impossible to tell whether it is still clear and transparent, but shaking and observing the liquid produces no precipitation. Although this process has good stability, the color is too dark to affect back-end applications. Comparative Examples 8 and 9 employed acid-base aqueous extraction and acid-base ethanol extraction, respectively. Ethanol extraction yielded a higher content of active ingredients, but also carried the risk of poor stability. Acid-base aqueous extraction yielded a higher content of polyphenols, but the polyphenols had poor stability. After using zinc salts, the prepared liquid was initially clear and transparent, free of precipitation, and remained clear and transparent after one month. However, precipitation may have occurred due to issues such as improper ratios. Furthermore, due to the higher polyphenol content, the Gardnor change value was higher over one month, and the color became darker. Comparative Example 10, based on the extraction of Example 1, used a conventional polyol aqueous solution for elution instead of a supramolecular solvent. The results in Table 2 show that conventional polyol aqueous solutions have poor elution effects and are unable to form strong hydrogen bonds with the target eluted molecules. Consequently, the final extract had a lower content of active ingredients and, consequently, a lighter color.

[0257] Figure 1 The results showed that the extracts of Herba Schizonepetae at mass concentrations of 1%, 0.5%, 0.25% and 0.1% had no cytotoxicity to sebaceous gland cells.

[0258] Figure 2 and Figure 3The results showed that when sebaceous gland cells are stimulated by linoleic acid, they secrete large amounts of oil (red fluorescence). When retinoic acid, the positive control, is used, it inhibits sebaceous gland cell oil secretion. Examples 1 and 2 both effectively inhibit sebaceous gland cell oil secretion. Comparative Examples 6, 8, 13, and 15 are less effective than Examples 1 and 2 in inhibiting sebaceous gland cell oil secretion. Comparative Examples 1, 2, and 10 are not able to inhibit sebaceous gland cell oil secretion.

[0259] Figure 4 The results showed that when macrophages were stimulated by LPS, they could produce a large amount of inflammatory NO. When the positive control dexamethasone was used, the inflammatory NO content caused by stimulation could be reduced. Figure 4 It can be seen that Examples 1 and 2 can effectively inhibit the inflammatory secretion of macrophages induced by LPS stimulation, while Comparative Examples 6 and 15 have weaker inhibitory effects than Examples 1 and 2. The inhibitory effects of Comparative Examples 1, 2, and 10 are relatively poor.

[0260] Figure 5 、 Figure 6 The results showed that Example 1, Example 2, Comparative Example 6, and Comparative Example 15 all had good skin oil secretion inhibition and redness reduction effects. Among them, Comparative Examples 6 and 15 had weaker skin oil secretion inhibition and redness reduction abilities than Example 1 and Example 2. Comparative Examples 1, 2, and 10 had weaker skin oil secretion inhibition and redness reduction abilities, with little change compared to the blank matrix.

[0261] The above description is only a specific embodiment of the present application. Those skilled in the art will clearly understand that for the convenience and brevity of description, the specific working process described above can refer to the corresponding process in the aforementioned method embodiment, and will not be repeated here. It should be understood that the scope of protection of the present application is not limited to this. Any person skilled in the art can easily think of various equivalent modifications or replacements within the technical scope disclosed in this application, and these modifications or replacements should be included in the scope of protection of this application.

Claims

1. A method for preparing a kidney tea extract, characterized in that: include, Mixing the tea powder with an acidic enzyme aqueous solution and performing an extraction treatment to obtain a polyphenol crude extract and a filter residue, wherein the acidic enzyme is an acidic protease and a pectinase; The crude polyphenol extract is enriched and purified and eluted with an acidic supramolecular solvent to obtain a polyphenol refined extract; mixing the polyphenol extract with a complexing agent and performing a coordination reaction to obtain a complex; The filter residue is mixed with an alkaline enzyme aqueous solution and subjected to extraction treatment to obtain a flavonoid crude extract, wherein the alkaline enzyme is alkaline protease; The crude flavonoid extract is enriched and purified and eluted with an alkaline supramolecular solvent to obtain a flavonoid refined extract; mixing the flavonoid extract with an inclusion agent and performing an inclusion reaction to obtain an inclusion compound; The complex is mixed with the inclusion compound, and then processed in a subsequent step to obtain a nephrolepis chinensis extract; In the step of mixing the kidney tea powder with the acidic enzyme aqueous solution and performing the extraction treatment, the temperature of the acidic enzyme aqueous solution is maintained at 35° C. to 45° C.; the pH of the acidic enzyme is maintained at 3 to 4; In the step of mixing the kidney tea powder with the alkaline enzyme aqueous solution and performing the extraction treatment, the temperature of the alkaline enzyme is maintained at 50° C. to 60° C.; the pH of the alkaline enzyme is maintained at 8 to 10; The acidic supramolecular solvent comprises at least a polyol, citric acid and water; the mass percentage of the polyol in the acidic supramolecular solvent is 50% to 70%; The alkaline supramolecular solvent comprises at least a polyol, choline chloride and water; the mass percentage of the polyol in the alkaline supramolecular solvent is 50% to 70%; During the process of mixing the polyphenol extract with the complexing agent and performing the coordination reaction, the pH of the polyphenol extract is 6.0-7.0; The complexing agent includes zinc salt; the inclusion agent includes cyclodextrin or a cyclodextrin derivative.

2. The preparation method according to claim 1, characterized in that The polyol includes one or more of 1,3-propylene glycol, glycerol, 1,2-propylene glycol, methylpropylene glycol, dipropylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 1,2-pentanediol, isoprene glycol or 1,2-hexanediol.

3. A Herba Cynanchifoliae extract, characterized in that: The tea extract is prepared by the preparation method according to any one of claims 1 to 2.

4. Use of the tea extract prepared by the method according to any one of claims 1 to 2 or the tea extract according to claim 3 in preparing a product with oil-controlling and / or soothing effects, characterized in that: The product comprises a cosmetic; and / or, The mass percentage of the tea extract in the product is 1% to 5%.

5. The use according to claim 4, characterized in that The products include skin care formulations.

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