Preparation method of camellia nitidissima extract, camellia nitidissima extract and application thereof

Abstract

The invention discloses a preparation method of a camellia nitidissima extract, the camellia nitidissima extract and application of the camellia nitidissima extract. The preparation method comprises the following steps: crushing camellia nitidissima flowers, mixing with water, and extracting to obtain a first camellia nitidissima crude extract; treating with an enzyme agent or an alcohol agent to obtain a second camellia nitidissima crude extract; combining centrifugation and membrane filtration to obtain a camellia nitidissima refined extract, concentrating to prepare an extract, and mixing the extract with a complex solvent to obtain a camellia nitidissima concentrated solution; adding auxiliary materials to prepare a golden camellia-containing suspension, and performing spray drying to prepare the golden camellia extract. According to the camellia nitidissima extract and the preparation method thereof, the camellia nitidissima extract is subjected to composite synergistic treatment by adopting a multi-enzyme agent or an alcohol agent, so that the camellia nitidissima extract which is rich in high-content flavone and saponin active ingredients can be obtained, the application effect of the camellia nitidissima extract is improved, and meanwhile, the camellia nitidissima extract has relatively good comprehensive effects of resisting oxidation, relieving, controlling oil and the like and can be used as a functional raw material.

Description

Technical Field

[0001] The present application belongs to the field of cosmetic technology, and in particular relates to a preparation method of a camellia chrysantha extract, a camellia chrysantha extract and applications thereof. Background Art

[0002] Camellia nitidissima (CWChi), a member of the Camellia family of the Theaceae family, is a relic of the primitive camellias from the Quaternary glacial period. It ranks among the rare "living fossils" of plants, including Cathaya, Alsophila spinulosa, and Davidia involucrata. Due to its distinctive yellow petals and extremely narrow growth range, it is known as the "Queen of Teas." It is primarily found in Guangxi, Yunnan, and Guangdong. The "Guangxi Zhuang Autonomous Region Zhuang Medicine Quality Standards" (Volume 2) states that CWChi tea has heat-clearing and detoxifying properties, as well as diuretic and swelling-reducing properties. It is primarily used to treat nephritis, edema, pharyngitis, dysentery, and hypertension.

[0003] Camellia chrysantha contains a variety of chemical components, such as carbohydrates, fats, fiber, ash, flavonoids, polyphenols, saponins, amino acid-grade proteins, minerals and various trace elements. It is widely used in food and health products, but rarely used in cosmetics.

[0004] To date, the traditional extraction of Camellia chrysantha ingredients lies in the extraction and purification of single ingredients, and most of them are processed with organic solvents. The extraction process contains many impurities and the steps are complicated. In addition, the Camellia chrysantha extract prepared by traditional extraction methods cannot simultaneously take into account the high content of saponins and flavonoids, and its oil control and antioxidant activity are relatively low. Summary of the Invention

[0005] The embodiments of the present application provide a preparation method of a camellia chrysantha extract, a camellia chrysantha extract and its application, which can simultaneously take into account the efficient extraction of saponin and flavonoid effective ingredients, and the prepared camellia chrysantha extract has good oil control and antioxidant effects.

[0006] In the first aspect, the present application provides a method for preparing a camellia chrysantha extract, comprising the steps of mixing camellia chrysantha pollen with water and performing an extraction treatment, filtering, and obtaining a first crude extract of camellia chrysantha; treating the first crude extract of camellia chrysantha with an enzyme or an alcohol agent to obtain a second crude extract of camellia chrysantha; centrifuging and membrane filtering the second crude extract of camellia chrysantha to obtain a camellia chrysantha extract; concentrating the camellia chrysantha extract to obtain a camellia chrysantha extract; mixing the camellia chrysantha extract with a resolvent to obtain a camellia chrysantha concentrate; mixing the camellia chrysantha concentrate with an auxiliary material to obtain a camellia chrysantha suspension; spray-drying the camellia chrysantha suspension to obtain a camellia chrysantha extract; wherein the enzyme treatment comprises using a first complex enzyme and a second complex enzyme to treat the camellia chrysantha extract. The first crude tea extract is treated with an enzyme, wherein the first complex enzyme includes at least tannase, pectinase and acid protease, and the second complex enzyme includes at least cellulase and α-amylase, the mass ratio of tannase, pectinase and acid protease is (0.1-0.3): (0.01-0.4): (0.01-1), and the mass ratio of cellulase and α-amylase is (0.1-1): (0.25-1); the alcohol treatment includes using a complex alcohol to treat the first crude camellia chrysantha extract, the complex alcohol includes a combination of ethanol and isopropanol, or a combination of ethanol and n-propanol; the mass ratio of ethanol to isopropanol is 5: (2-5); and the mass ratio of ethanol to n-propanol is 5: (2-5).

[0007] In any embodiment of the present application, the extraction process includes more than two extraction processes; the extraction process includes two extraction processes, wherein the solid-liquid ratio in the first extraction process is 1:(10-20) (g / g), and the solid-liquid ratio in the second extraction process is 1:(5-15) (g / g); or, the extraction process includes three extraction processes, wherein the solid-liquid ratio in the first extraction process is 1:(10-20) (g / g), the solid-liquid ratio in the second extraction process is 1:(5-15) (g / g), and the solid-liquid ratio in the third extraction process is 1:(1-10) (g / g). In any embodiment of the present application, the volume fraction of the complex alcohol agent in the second crude extract of Camellia chrysantha is 20% to 70%, preferably, the volume fraction of the complex alcohol agent in the second crude extract of Camellia chrysantha is 50% to 70%.

[0008] In any embodiment of the present application, during the enzymatic extraction of the Camellia chrysantha crude extract using the first and second complex enzymes, the temperature of the first complex enzyme is 40°C to 50°C; and / or the temperature of the second complex enzyme is 65°C to 75°C.

[0009] In any embodiment of the present application, the membrane filtration is microfiltration membrane filtration and / or reverse osmosis membrane filtration.

[0010] In any embodiment of the present application, the complex solvent includes at least one of water, ethanol, isopropanol, glycerol, propylene glycol, butylene glycol, 1,3-propanediol, 1,3-butylene glycol and dipropylene glycol; and / or, the excipient includes maltodextrin, resistant dextrin, cyclodextrin and its derivatives, trehalose, starch and its derivatives.

[0011] In a second aspect, the present application provides a Camellia chrysantha extract, which is prepared according to the preparation method described in the first aspect, and the Camellia chrysantha extract includes saponins and flavonoids.

[0012] In any embodiment of the present application, the mass proportion of saponins in the Camellia chrysantha extract is 25% to 40%; and / or the mass proportion of flavonoids in the Camellia chrysantha extract is 10% to 20%.

[0013] In the third aspect, the present application provides a method for preparing a Camellia chrysantha extract according to the first aspect or the Camellia chrysantha extract according to the second aspect in the preparation of an external care composition, wherein the mass proportion of the Camellia chrysantha extract in the external care composition is 0.001% to 5%.

[0014] In any embodiment of the present application, the dosage form of the external care composition includes at least one of liquid, powder, semisolid or solid.

[0015] The preparation method of the camellia chrysantha extract of the embodiment of the present application includes the following steps: crushing camellia chrysantha flowers, mixing them with water, performing extraction treatment, and filtering to obtain a first crude extract of camellia chrysantha; treating the first crude extract of camellia chrysantha with an enzyme or an alcohol agent to obtain a second crude extract of camellia chrysantha; centrifuging and membrane filtering the second crude extract of camellia chrysantha to obtain a camellia chrysantha extract; concentrating the camellia chrysantha extract to obtain a camellia chrysantha extract; mixing the camellia chrysantha extract with a resolvent to obtain a camellia chrysantha concentrate; mixing the camellia chrysantha concentrate with an auxiliary material to obtain a camellia chrysantha suspension; spray-drying the camellia chrysantha suspension to obtain a camellia chrysantha extract; wherein the enzyme treatment includes using a first complex enzyme and a second complex enzyme to treat the first crude extract of camellia chrysantha; A crude extract is treated with an enzyme, wherein a first complex enzyme comprises at least tannase, pectinase and acid protease, and a second complex enzyme comprises at least cellulase and α-amylase, the mass ratio of tannase, pectinase and acid protease is (0.1-0.3):(0.01-0.4):(0.01-1), and the mass ratio of cellulase and α-amylase is (0.1-1):(0.25-1); the alcohol treatment comprises treating the first crude extract of Camellia chrysantha with a complex alcohol, wherein the complex alcohol comprises a combination of ethanol and isopropanol, or a combination of ethanol and n-propanol; the mass ratio of ethanol to isopropanol is 5:(2-5); and the mass ratio of ethanol to n-propanol is 5:(2-5). By adjusting and optimizing the formula of the first complex enzyme, the second complex enzyme and the complex alcohol agent, specific types of complex enzymes or complex alcohol agents are combined and used in specific mass ratios, which has a synergistic effect. It can not only increase the content of active ingredients in the camellia chrysantha extract, especially saponins and flavonoids, but also improve the efficacy of the camellia chrysantha extract in anti-oxidation, soothing, oil control and other aspects. It can be used as a functional raw material to improve the application effect of the product. 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 effect of the Camellia chrysantha extract prepared in Example 1 of the present application on the cell viability of sebaceous gland cells.

[0018] Figure 2 Schematic diagram of the effect of the Camellia chrysantha extract prepared in Example 1 of the present application on the secretion of oil droplets by sebaceous gland cells (wherein, Figure 2 a is a schematic diagram of the cell status of the control group; Figure 2 b is a schematic diagram of the cell status of the sample group with 20 ppm of Camellia chrysantha extract added; Figure 2 c is a schematic diagram of the cell status of the model group; Figure 2d is a schematic diagram of the cell status of the sample group with 100 ppm of Camellia chrysantha extract added). DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Unless otherwise specified, 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 specified, the test temperature of each parameter mentioned in this application is 25°C and the test pressure is standard atmospheric pressure.

[0023] 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.

[0024] Camellia nitidissima is a precious plant of the genus Camellia in the family Theaceae. Known for its unique golden-yellow flowers, it primarily grows in Guangxi and Yunnan, China. Its rich active ingredients (such as saponins and flavonoids) possess antioxidant, anti-inflammatory, and immunomodulatory properties, offering broad application prospects in the pharmaceutical, health care, and cosmetics industries. Current research on Camellia nitidissima focuses primarily on component analysis; the extraction and processing of active ingredients typically involve the preparation of single ingredients and extraction using organic solvents, resulting in environmentally unfriendly results. There is a lack of research on technologies for the simultaneous enrichment of flavonoids and saponins, and the removal of interfering components such as starch, protein, and phenolic acids has not been effectively addressed. Furthermore, the resulting Camellia nitidissima extract exhibits poor antioxidant, soothing, and oil-control effects.

[0025] In view of the above problems, the inventors have improved the extraction method of Camellia chrysantha medicinal material.

[0026] The first aspect of the embodiment of the present application provides a preparation method of a camellia chrysantha extract, comprising the steps of mixing camellia chrysantha pollen with water and performing an extraction treatment, filtering to obtain a first crude extract of camellia chrysantha; treating the first crude extract of camellia chrysantha with an enzyme or an alcohol agent to obtain a second crude extract of camellia chrysantha; centrifuging and membrane filtering the second crude extract of camellia chrysantha to obtain a camellia chrysantha extract; concentrating the camellia chrysantha extract to obtain a camellia chrysantha extract; mixing the camellia chrysantha extract with a resolvent to obtain a camellia chrysantha concentrate; mixing the camellia chrysantha concentrate with an auxiliary material to obtain a camellia chrysantha suspension; spray-drying the camellia chrysantha suspension to obtain a camellia chrysantha extract; wherein the enzyme treatment comprises using a first complex enzyme and a second complex enzyme to The first crude extract of Camellia chrysantha is treated with an enzyme agent, wherein the first complex enzyme includes at least tannase, pectinase and acid protease, and the second complex enzyme includes at least cellulase and α-amylase, the mass ratio of tannase, pectinase and acid protease is (0.1-0.3):(0.01-0.4):(0.01-1), and the mass ratio of cellulase and α-amylase is (0.1-1):(0.25-1); the alcohol agent treatment includes using a complex alcohol agent to treat the first crude extract of Camellia chrysantha, the complex alcohol agent includes a combination of ethanol and isopropanol, or a combination of ethanol and n-propanol; the mass ratio of ethanol to isopropanol is 5:(2-5); and the mass ratio of ethanol to n-propanol is 5:(2-5).

[0027] Preferably, mixing the Camellia chrysantha pollen with water comprises crushing Camellia chrysantha flowers and mixing the crushed flowers with water.

[0028] By using the above-mentioned specific formula of complex enzyme to treat the first crude extract of Camellia chrysantha, unstable particles in the crude extract of Camellia chrysantha can be degraded, and the influence of other impurities such as polyphenols, polysaccharides, alkaloids, and proteins on the targeted components can be reduced. At the same time, the enzyme reaction is relatively mild, and the side effects of the targeted components are reduced, which is beneficial to improving the extraction rate of flavonoids and saponins in the Camellia chrysantha extract; at the same time, it can also improve the efficacy of Camellia chrysantha extract in anti-oxidation, soothing, oil control and other aspects.

[0029] By treating the first crude extract of Camellia chrysantha with the composite alcohol agent of the specific formula, unstable particles such as proteins, starches, and polysaccharides can be removed from the extract, increasing the solubility of the targeted components and thereby improving their content. Furthermore, the antioxidant, soothing, and oil-control effects of the Camellia chrysantha extract can be enhanced. Preferably, the mass ratio of tannase, pectinase, and acid protease is (0.1-0.2):0.01:(0.05-0.5).

[0030] As an example, the mass ratio of tannase, pectinase and acid protease can be 0.1:0.01:0.1, 0.1:0.02:0.1, 0.1:0.03:0.1, 0.1:0.04:0.1, 0.1:0.05:0.1, 0.2:0.01:0.1, 0.3:0.01:0.1, 0.1:0.01:0.2, 0.1:0.01:0.3, 0.1:0.01:0.4, 0.1:0.01:0.5.

[0031] As an example, the mass ratio of cellulase to α-amylase can be 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1:1.

[0032] As an example, the mass ratio of ethanol to isopropanol may be 5:1, 5:2, 5:3, 5:4, or 5:5.

[0033] As an example, the mass ratios of ethanol and n-propanol are 5:1, 5:2, 5:3, 5:4, and 5:5.

[0034] In some embodiments, the extraction treatment includes more than two extraction treatments; the extraction treatment includes two extraction treatments, wherein the solid-liquid ratio in the first extraction treatment is 1:(10-20) (g / g), and the solid-liquid ratio in the second extraction treatment is 1:(5-15) (g / g); or, the preliminary treatment includes three extraction treatments, wherein the solid-liquid ratio in the first extraction treatment is 1:(10-20) (g / g), the solid-liquid ratio in the second extraction treatment is 1:(5-15) (g / g), and the solid-liquid ratio in the third extraction treatment is 1:(1-10) (g / g).

[0035] As an example, the extraction process includes two extraction processes, wherein the solid-liquid ratio in the first extraction process is 1:15 (g / g), and the solid-liquid ratio in the second extraction process is 1:10 (g / g).

[0036] In some embodiments, the extraction process includes three extraction processes, wherein the solid-liquid ratio in the first extraction process is 1:15 (g / g), the solid-liquid ratio in the second extraction process is 1:10 (g / g), and the solid-liquid ratio in the third extraction process is 1:5 (g / g).

[0037] Increasing the amount of extraction solvent and the number of extractions can reduce the concentration of the extracted components in the solvent, making it easier for the extraction solvent water to enter the Camellia chrysantha cells, which is beneficial for the active ingredients to flow from high concentration to low concentration of the extraction solvent, thereby improving the extraction efficiency of the active ingredients.

[0038] Optionally, the extraction process further includes centrifugation and filtration.

[0039] In some embodiments, the membrane filtration is microfiltration membrane filtration and / or reverse osmosis membrane filtration.

[0040] The enzyme-treated camellia chrysantha extract is filtered through a microfiltration membrane and a reverse osmosis membrane, or the alcohol-treated camellia chrysantha extract is filtered through a microfiltration membrane to obtain a clear and transparent camellia chrysantha extract, which removes impurities and suspended particulate matter, improves the purity of the active ingredients and their subsequent applicability, and reduces the effect of long-term heating temperature on the content of the active ingredient in the reduced pressure concentration step.

[0041] In some embodiments, the volume fraction of the complex alcohol agent in the second crude extract of Camellia chrysantha is 20% to 70%. Preferably, the volume fraction of the complex alcohol agent in the second crude extract of Camellia chrysantha is 50% to 70%.

[0042] As an example, the volume fraction of the complex alcohol agent in the second crude extract of Camellia chrysantha can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%.

[0043] In some embodiments, the resolvent comprises at least one of water, ethanol, isopropanol, glycerol, propylene glycol, butylene glycol, 1,3-propanediol, 1,3-butylene glycol and dipropylene glycol; and / or, the excipient comprises maltodextrin, resistant dextrin, cyclodextrin and its derivatives, trehalose, starch and its derivatives.

[0044] As an example, the excipients include one or more of maltodextrin, β-cyclodextrin, methylcyclodextrin, hydroxypropyl-β-cyclodextrin, and the like.

[0045] Preferably, the auxiliary material comprises maltodextrin.

[0046] In some embodiments, during the enzymatic extraction of the Camellia chrysantha crude extract using the first and second complex enzymes, the temperature of the first complex enzyme is 40°C to 50°C; and / or the temperature of the second complex enzyme is 65°C to 75°C.

[0047] As an example, during the enzymatic extraction of the crude extract of Camellia chrysantha using the first complex enzyme and the second complex enzyme, the temperature of the first complex enzyme can be 40°C, 42°C, 45°C, 48°C or 50°C.

[0048] As an example, the temperature of the second complex enzyme can be 65°C, 68°C, 70°C, 72°C or 75°C.

[0049] Controlling the temperature of the first complex enzyme to 40°C to 50°C and the temperature of the second complex enzyme to 65°C to 75°C can ensure normal enzyme activity. Too low or too high a temperature will affect the activity of the enzyme, preventing normal operation and affecting the processing of the Camellia chrysantha extract, resulting in a low content or purity of active substances.

[0050] A second aspect of the embodiments of the present application provides a Camellia chrysantha extract, which is prepared according to the preparation method described in the first aspect. The Camellia chrysantha extract includes saponins and flavonoids.

[0051] In some embodiments, the mass proportion of saponins in the Camellia chrysantha extract is 25% to 40%; and / or the mass proportion of flavonoids in the Camellia chrysantha extract is 10% to 20%.

[0052] As an example, the mass proportion of saponins in the Camellia chrysantha extract can be 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.

[0053] As an example, the mass proportion of flavonoids in the Camellia chrysantha extract can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0054] The third aspect of the embodiment of the present application provides a camellia chrysantha extract prepared by the preparation method described in the first aspect or the use of the camellia chrysantha extract described in the second aspect in the preparation of an external care composition, wherein the mass proportion of the camellia chrysantha extract in the external care composition is 0.001% to 5%.

[0055] As an example, the mass proportion of Camellia chrysantha extract in the topical care composition can be 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.05%, 0.1%, 0.5%, 1% or 5%.

[0056] In some embodiments, the dosage form of the topical care composition includes at least one of liquid, powder, semisolid, or solid.

[0057] Camellia chrysantha extract can be used as a functional raw material in external care compositions such as personal care products.

[0058] Example

[0059] 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.

[0060] Example 1

[0061] Example 1 of the present application provides a method for preparing a camellia chrysantha extract, comprising:

[0062] S1, grinding freeze-dried Camellia chrysantha flowers into about 40 meshes to obtain Camellia chrysantha pollen;

[0063] S2, crushing the golden camellia flowers and mixing them with water and performing two extraction treatments, wherein, in the first extraction treatment step, the solid-liquid ratio of the golden camellia flower powder to water is 1:15 (g / g), the system pH is about 5.5, and extraction is performed at 85° C. for 2 hours, followed by centrifugation and filtration to obtain a first filtrate and a first residue; in the second extraction treatment step, water is added to the first residue, the solid-liquid ratio of the first residue to water is 1:10 (g / g), the extraction is repeated at 85° C. once, and then centrifugation and filtration are performed to obtain a second filtrate, and the first filtrate and the second filtrate are combined to obtain a first crude extract of the golden camellia;

[0064] S3, treating the first crude extract of Camellia chrysantha with an enzyme to obtain a second crude extract of Camellia chrysantha;

[0065] The specific steps of enzyme treatment include:

[0066] First, tannase, pectinase, and acid protease were added to the crude extract of Camellia chrysantha, and enzymatic hydrolysis was carried out at 45°C for 2 hours. The mass ratio of tannase, pectinase, and acid protease was 1:0.1:1. Based on the mass of the crude extract of Camellia chrysantha, the added amount of tannase was 1g / kg, the added amount of pectinase was 0.1g / kg, and the added amount of acid protease was 1g / kg.

[0067] The liquid temperature was then raised, and cellulase and α-amylase were added. Enzymatic hydrolysis was carried out at 70°C for 2 hours, and finally the liquid temperature was raised to 100°C to inactivate the enzymes. The mass ratio of cellulase to α-amylase was 1:0.4, and the amount of cellulase added was 1g / kg and 0.4g / kg, respectively, based on the mass of the crude Camellia chrysantha extract.

[0068] S4, centrifuging the second crude extract of Camellia chrysantha and separating the supernatant, and filtering the supernatant with a microfiltration membrane and an RO membrane to obtain a Camellia chrysantha extract;

[0069] S5, concentrating the camellia chrysantha extract under reduced pressure and vacuum to obtain a dry extract to obtain a camellia chrysantha extract;

[0070] S6, mixing the camellia chrysantha extract with water to obtain a camellia chrysantha concentrate;

[0071] The specific steps include:

[0072] Mixing the camellia chrysantha extract with water and soaking overnight to obtain a first solution, wherein the amount of water added is 80% of the mass of the camellia chrysantha pollen; then mixing the first solution with water and ultrasonically treating until the dry extract is completely dissolved to obtain a second solution, which is the camellia chrysantha concentrate;

[0073] S5, mixing the camellia chrysantha concentrate with maltodextrin as an auxiliary material, and simultaneously performing ultrasonic and stirring treatment to obtain a camellia chrysantha suspension;

[0074] S6. Using a spray dryer, the camellia chrysantha suspension is prepared into a powder, namely, a camellia chrysantha extract, wherein the mass percentage of maltodextrin in the camellia chrysantha extract is 20% (w / w).

[0075] Example 2

[0076] Example 2 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 1 only in step S2:

[0077] S2, crushing the golden camellia and mixing it with water and performing three extraction treatments, wherein, in the first extraction treatment step, the solid-liquid ratio of the golden camellia powder to water is 1:15 (g / g), the system pH is about 5.5, and the extraction is carried out at 85°C for 2h, followed by centrifugation and filtration to obtain a first filtrate and a first residue; in the second extraction treatment step, water is added to the first residue, the solid-liquid ratio of the first residue to water is 1:10 (g / g), the extraction is repeated at 85°C once, and then centrifugation and filtration are performed to obtain a second filtrate and a second residue, water is added to the second residue, the solid-liquid ratio of the second residue to water is 1:5 (g / g), the extraction is repeated at 85°C once, and then centrifugation and filtration are performed to obtain a third filtrate, and the first filtrate, the second filtrate and the third filtrate are combined.

[0078] Example 3

[0079] Example 3 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 1 only in steps S3 and S4:

[0080] S3, treating the first crude extract of Camellia chrysantha with an alcohol agent to obtain a second crude extract of Camellia chrysantha;

[0081] The specific steps of alcohol treatment include:

[0082] The ethanol and isopropanol complex was mixed with the first crude extract of Camellia chrysantha, and the mixture was slowly stirred until uniformly mixed. The mixture was stirred at room temperature for 1 hour, and allowed to stand overnight. The upper layer was separated and the second crude extract of Camellia chrysantha was obtained. The mass ratio of ethanol to isopropanol was 5:2, and the volume fraction of the complex in the mixture was 50% (v / v).

[0083] S4, centrifuging the second crude extract of Camellia chrysantha and separating the supernatant, filtering the supernatant with a microfiltration membrane to obtain a Camellia chrysantha refined extract.

[0084] Example 4

[0085] Example 4 of the present application provides a method for preparing a Camellia chrysantha extract solution, comprising:

[0086] Weigh 0.5 g of the Camellia chrysantha extract obtained in Example 1, add water to 100 g, stir and mix until completely dissolved, filter, and take the filtrate to which 1,2-hexanediol and p-hydroxyacetophenone are added as preservatives to prepare a Camellia chrysantha extract solution. The mass percentage of 1,2-hexanediol in the Camellia chrysantha extract solution is 2%, and the mass percentage of p-hydroxyacetophenone in the Camellia chrysantha extract solution is 0.5%.

[0087] The obtained camellia chrysantha extract solution can be directly added into cosmetic formulas as a raw material for application.

[0088] Example 5

[0089] Example 5 of the present application provides a method for preparing a Camellia chrysantha extract solution, comprising:

[0090] Weigh 1 g of the Camellia chrysantha extract obtained in Example 2, add 50% by mass aqueous glycerol solution and dilute to a total volume of 100 g. Stir and mix until completely dissolved, filter, and take the filtrate to which 1,2-pentanediol and ethylhexylglycerin are added as preservatives to obtain a Camellia chrysantha extract solution. The mass percentage of 1,2-pentanediol in the Camellia chrysantha extract solution is 2%, and the mass percentage of ethylhexylglycerin in the Camellia chrysantha extract solution is 0.1%.

[0091] The obtained camellia chrysantha extract solution can be directly added into cosmetic formulas as a raw material for application.

[0092] Comparative Example 1

[0093] Comparative Example 1 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 1 only in that an equal amount of pectinase in step S3 is replaced by tannase.

[0094] Comparative Example 2

[0095] Comparative Example 2 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 1 only in that the mass ratio of tannase, pectinase and acid protease in step S3 is adjusted to 0.5:0.1:1.

[0096] Comparative Example 3

[0097] Comparative Example 3 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 1 only in that an equal amount of cellulase in step S3 is replaced by xylanase.

[0098] Comparative Example 4

[0099] Comparative Example 4 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 1 only in that the mass ratio of cellulase to α-amylase in step S3 is adjusted to 2:3.

[0100] Comparative Example 5

[0101] Comparative Example 5 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 3 only in that an equal amount of isopropyl alcohol in step S3 is replaced by glycerol.

[0102] Comparative Example 6

[0103] Comparative Example 6 of the present application provides a method for preparing a Camellia chrysantha extract, which differs from Example 3 only in that the mass ratio of ethanol and isopropanol in step S3 is adjusted to 5:1, and the volume fraction of the complex alcohol agent in the mixed system is 15% (v / v).

[0104] Comparative Example 7

[0105] Without the addition of enzymes and alcohols, the content of active ingredients is reduced.

[0106] Performance Testing

[0107] The performance test of the Camellia chrysantha extracts prepared in Examples 1-3 and Comparative Examples 1-7 was performed using the following test methods:

[0108] 1. Test Method

[0109] 1. Detection of flavonoid content

[0110] Taking rutin as a reference, the flavonoid content in the camellia chrysantha extracts prepared in Examples 1-3 and Comparative Examples 1-7 was detected with reference to Huang Yonglin, Wen Yongxin, et al. Determination of total flavonoid content in five types of camellia chrysantha [J]. Chinese Journal of Traditional Chinese Medicine and Pharmacy, 2009, 16(1): 38-39.

[0111] 2. Detection of saponin content

[0112] Taking ginsenoside Rb1 as a benchmark and referring to the group standard T / AHFIA 004-2018 "Spectrophotometric method for determination of total saponin content in food", the saponin content in the Camellia chrysantha extracts prepared in Examples 1-3 and Comparative Examples 1-7 was detected.

[0113] 3. Effect on DPPH free radicals

[0114] Taking ascorbic acid as a reference substance, referring to Hu Jinrui, Cao Rongan, et al. Effect of ultrasound-assisted enzymatic hydrolysis on the structure and antioxidant activity of mung bean protein hydrolysate [J]. Journal of Chinese Institute of Food Science and Technology, 2024, 1-14, the effects of the golden camellia extracts prepared in Examples 1-3 and Comparative Examples 1-7 on DPPH free radical scavenging at different concentrations were investigated. The specific preparation method is as follows:

[0115] (1) 0.1% sample group: The Camellia chrysantha extracts prepared in Examples 1-3 and Comparative Examples 1-7 were dissolved in water to prepare a test solution with a mass fraction of 0.1%;

[0116] (2) 1% sample group: The Camellia chrysantha extracts prepared in Examples 1-3 and Comparative Examples 1-7 were dissolved in water to prepare a test solution with a mass fraction of 1%.

[0117] 4. Oil control test

[0118] The test method refers to the group standard T / ZHCA 002-2018 "Test Method for Oil Control Efficacy of Cosmetics". Ten volunteers were tested on the forehead. The forehead was cleaned and equilibrated in the environment for 30 minutes. Four 1.5*1.5cm test areas were selected on the tester's forehead. The CK Sebumeter SM 815 test probe was used to test the oil content after equilibration for 30 minutes and after applying the test sample for 2 hours. The application amount of the test sample was 0.1uL. The oil change results after 2 hours are shown in Table 2.

[0119] The test sample preparation method includes:

[0120] (1) Take the Camellia chrysantha extracts prepared in Example 3, Comparative Example 1, Comparative Example 3, Comparative Example 5, and Comparative Example 7;

[0121] (2) adding 0.1% by mass of each of the above extracts to a 0.5% by mass xanthan gum solution;

[0122] (3) stirring and mixing to obtain sample groups S-1 to S-5;

[0123] (4) A 0.5% xanthan gum solution without extract was set as the control group S-0.

[0124] Grease reduction rate = (grease content of reference substance - grease content of test sample) / grease content of reference substance * 100%; the average grease reduction rate is the sum of the grease reduction rates of 10 people / 10.

[0125] 5. Cytotoxicity test

[0126] Sebaceous gland cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 The cells were cultured in DMEM medium supplemented with 100 mg / L penicillin and 100 mg / L streptomycin at 37°C in a 5% CO2 incubator until the cell confluence reached 85% to 95%. The cells in the logarithmic growth phase were digested with 0.05% trypsin, and the digestion reaction was terminated with DMEM medium supplemented with 10% serum.

[0127] Count the cells using a cell counting plate and adjust the cell suspension to 2 × 10 5 / mL, inoculated into 96-well plates at 100μL / well, cultured in a 37°C, 5% CO2 incubator until the cell confluence is 45% to 60%, the old culture medium was removed, and 100μL of serum-free culture medium containing different concentrations (31.25ppm, 62.5ppm, 125ppm, 250ppm, 500ppm, 1000ppm) of the Camellia chrysantha extract of Example 1 was added respectively. Three replicates were set for each dose and used as the sample group. The blank group was 100μL serum-free culture medium without HaCaT cells; the control group was sebaceous gland cells, and 100μL serum-free culture medium was added; the culture was continued for 24h, 10μL CCK-8 was added to each well, and the culture was continued for 1h. The absorbance values ​​of the above sample group, blank group and control group were measured at 450nm, and the cell viability of each group was calculated. The formula:

[0128] Cell viability (%) = (sample group - blank group) / (control group - blank group) × 100%.

[0129] 6. Cell lipid content detection

[0130] 6.1 Cell culture: Take logarithmic phase sebaceous gland cells, wash them with PBS, add 0.25% trypsin, let it stand for 4 minutes, stop digestion with culture medium, centrifuge, resuspend the cells with complete DMEM culture medium, and adjust the cell concentration to 1×10 5 / mL, 0.5mL was taken from each well and added to a 24-well plate. After 24 hours, the cells were attached to the wall, and 5α-DHT and different concentrations of the Camellia chrysantha extract samples prepared in Example 1 were added.

[0131] 6.2 Cell Model and Testing: A lipid model was constructed using 5α-DHT (0.5 μM). For the model group, 450 μL of complete culture medium and 50 μL of 5α-DHT were added to each well. For the sample group, 50 μL of different concentrations of the Camellia chrysantha extract prepared in Example 1 and 50 μL of 5α-DHT were added to each well. For the control group, the same concentrations of culture medium were added to each well. Three parallel experiments were performed for each group. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0132] 6.3 Oil content detection: Stain the constructed and cultured cells with Oil Red O staining solution, remove the staining solution, wash three times with 40% isopropanol, add 50 μL sterile water, observe under an inverted microscope, and take pictures for comparison.

[0133] 2. Test Results

[0134] 1. The test results of flavonoid content and saponin content are shown in Table 1 below.

[0135] Table 1

[0136] name Saponin content% Flavonoid content% Example 1 32.07％ 13.28％ Example 2 29.12％ 10.32％ Example 3 35.64％ 11.90％ Comparative Example 1 24.78％ 9.85％ Comparative Example 2 33.29％ 7.76％ Comparative Example 3 22.45％ 9.18％ Comparative Example 4 22.81％ 9.23％ Comparative Example 5 7.69％ 3.82％ Comparative Example 6 20.39％ 8.73％ Comparative Example 7 15.72％ 6.21％

[0137] As can be seen from Table 1, compared with Comparative Example 1 in which pectinase is not used, Comparative Example 3 in which cellulase is not used, and Comparative Example 2 and Comparative Example 4 in which the dosage ratio of the composite enzyme is changed, the composite enzyme formula with the specific components and dosage ratio of the present application is used to treat the Camellia chrysantha medicinal material with enzymes (Example 1, Example 2), and its saponin content and flavonoid content can be greatly improved, and the saponin content can reach up to 32.07%, and the flavonoid content can reach up to 13.28%; compared with Comparative Example 5 in which isopropanol is not used, the composite alcohol with the specific components and dosage ratio of the present application is used to treat the Camellia chrysantha medicinal material with alcohol (Example 3), and its saponin content and flavonoid content can be significantly improved, and the saponin content can reach up to 35.64%, and the flavonoid content can reach up to 11.90%.

[0138] In Comparative Example 1, no pectinase was added, and only tannase and acid protease were added, resulting in the crude extract of Camellia chrysantha containing large molecular micelles, which can adsorb some active ingredients - flavonoids and saponins, resulting in a decrease in the content of the two. In addition, the clarity of the liquid was reduced, the refining and filtration processing time was increased, and the cost increased.

[0139] In Comparative Example 2, the amount of tannase was increased, resulting in a decrease in the flavonoid content. The color of the obtained Camellia chrysantha extract powder prepared into a 0.5% concentration liquid tended to deepen.

[0140] In Comparative Example 3, cellulase was replaced by xylanase, resulting in incomplete degradation of macromolecular particles in the Camellia chrysantha extract, reduced liquid transparency, and reduced saponin and flavonoid contents.

[0141] In Comparative Example 4, the mass ratio of cellulase and α-amylase was adjusted to 2:3. On the one hand, this resulted in a lower molecular weight of sugar in the crude extract of Camellia chrysantha, and the obtained Camellia chrysantha extract contained less lower molecular weight sugar components; on the other hand, it adsorbed saponins or flavonoids in the Camellia chrysantha extract, resulting in a loss of content.

[0142] In Comparative Example 5, an equal amount of isopropyl alcohol was replaced with glycerol. Glycerol has a high viscosity, has a small solubility and extraction effect on active ingredients such as saponins and flavonoids, is not easily volatile, causes waste and loss, and cannot be prepared into powder.

[0143] In Comparative Example 6, the mass ratio of ethanol to n-propanol is 5:1, and the proportion of the composite alcohol agent in the mixed system is 15%, resulting in incomplete alcohol precipitation, causing loss of active ingredients and reduced saponin and flavonoid contents.

[0144] In summary, it is shown that the use of the composite enzyme or composite alcohol formula with the specific components and dosage ratios of the present application to extract the medicinal material Camellia chrysantha can effectively increase the saponin content and flavonoid content in the extract.

[0145] 2. Effect on DPPH free radical scavenging

[0146] The scavenging effects of different groups of Camellia chrysantha extracts on DPPH free radicals are shown in Table 2.

[0147] Table 2

[0148]

[0149]

[0150] It can be seen from Table 2 that the scavenging effect of 0.1% mass fraction of Camellia chrysantha extract on DPPH free radicals is weaker than that of 0.1% ascorbic acid, and the scavenging effect of 1% Camellia chrysantha extract on DPPH free radicals is stronger than that of 0.1% ascorbic acid. Compared with the Camellia chrysantha extract samples of the comparative example, the Camellia chrysantha extract samples of Examples 1-3 have better scavenging effects on DPPH free radicals, that is, they have significant antioxidant capacity.

[0151] 3. The test results of the oil control effect of different Camellia chrysantha extracts are shown in Table 3.

[0152] Table 3

[0153] Sample name Average oil reduction rate (%) S-0 / S-1 33.91 S-2 26.57 S-3 23.54 S-4 10.73 S-5 14.29

[0154] As can be seen from the above table, the oil-control effect of the Camellia chrysantha extract prepared in Example 3 is better than that of the Camellia chrysantha extracts in other groups.

[0155] 4. Cytotoxicity test results

[0156] Figure 1 This is the cytotoxicity test result. It can be seen that when the addition amount of Camellia chrysantha extract is within 500ppm, it will not have a significant effect on the cell activity of sebaceous gland cells.

[0157] 5. Cellular lipid content test results

[0158] Test results are shown in Figure 2 The red spots in the picture are oil droplets. Figure 2 a is a schematic diagram of the cell status of the control group. Figure 2 c is a schematic diagram of the cell state of the model group, which shows that the cells in the model group secreted a large number of oil droplets; Figure 2 b and Figure 2 d are photos of the sample groups with 20ppm and 100ppm of Camellia chrysantha extract added, respectively. Compared with the model group, the sample group has significantly fewer oil droplets than the model group 2c, and with the increase of the concentration of Camellia chrysantha extract, the amount of oil droplets secreted by the cells decreases; this indicates that the Camellia chrysantha extract prepared by the extraction process of the present invention can significantly inhibit the secretion of oil droplets by cells, and within a certain range, the effect of inhibiting cell oil secretion is proportional to the concentration of Camellia chrysantha extract.

[0159] 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 Camellia chrysantha extract, characterized in that: include, Mixing camellia chrysantha pollen with water, performing extraction treatment, and filtering to obtain a first crude camellia chrysantha extract; treating the first crude extract of Camellia chrysantha with an enzyme or alcohol to obtain a second crude extract of Camellia chrysantha; The second crude extract of Camellia chrysantha is centrifuged and subjected to membrane filtration to obtain a refined extract of Camellia chrysantha; concentrating the camellia chrysantha extract to obtain a camellia chrysantha extract; mixing the camellia chrysantha extract with a resolvent to obtain a camellia chrysantha concentrate; Mixing the camellia chrysantha concentrate with auxiliary materials to obtain a camellia chrysantha suspension; spray-drying the Camellia chrysantha suspension to obtain the Camellia chrysantha extract; The enzyme treatment comprises treating the first crude extract of Camellia chrysantha with a first complex enzyme and a second complex enzyme, wherein the first complex enzyme comprises at least tannase, pectinase and acid protease, and the second complex enzyme comprises at least cellulase and α-amylase, and the mass ratio of the tannase, pectinase and acid protease is (0.1-0.3): (0.01-0.4): (0.01-1), and the mass ratio of the cellulase and α-amylase is (0.1-1): (0.25-1); The alcohol treatment comprises treating the first crude extract of Camellia chrysantha with an alcohol treatment agent using a composite alcohol agent, wherein the composite alcohol agent comprises a combination of ethanol and isopropanol, or a combination of ethanol and n-propanol; The mass ratio of ethanol to isopropanol is 5:(2-5); The mass ratio of the ethanol to the n-propanol is 5:(2-5).

2. The preparation method according to claim 1, characterized in that The extraction process includes two or more extraction processes; The extraction process includes two extraction processes, wherein the material-liquid ratio in the first extraction process is 1:(10-20) (g / g), and the material-liquid ratio in the second extraction process is 1:(5-15) (g / g); or, The extraction process includes three extraction processes, wherein the solid-liquid ratio in the first extraction process is 1:(10-20) (g / g), the solid-liquid ratio in the second extraction process is 1:(5-15) (g / g), and the solid-liquid ratio in the third extraction process is 1:(1-10) (g / g).

3. The preparation method according to claim 1, characterized in that The volume fraction of the compound alcohol agent in the second crude extract of Camellia chrysantha is 20% to 70%. Preferably, the volume fraction of the compound alcohol agent in the second crude extract of Camellia chrysantha is 50% to 70%.

4. The preparation method according to claim 1, characterized in that During the enzymatic treatment of the first crude extract of Camellia chrysantha using the first complex enzyme and the second complex enzyme, the temperature of the first complex enzyme is 40°C to 50°C; and / or the temperature of the second complex enzyme is 65°C to 75°C.

5. The preparation method according to claim 1, characterized in that The membrane filtration is microfiltration membrane filtration and / or reverse osmosis membrane filtration.

6. The preparation method according to claim 1, characterized in that The complex solvent comprises at least one of water, ethanol, isopropanol, glycerol, propylene glycol, butylene glycol, 1,3-propylene glycol, 1,3-butylene glycol and dipropylene glycol; and / or, The auxiliary materials include maltodextrin, resistant dextrin, cyclodextrin and its derivatives, trehalose, starch and its derivatives.

7. A Camellia chrysantha extract, characterized in that The camellia chrysantha extract is prepared according to the preparation method according to any one of claims 1 to 6, wherein the camellia chrysantha extract comprises saponins and flavonoids.

8. The Camellia chrysantha extract according to claim 7, characterized in that The mass proportion of the saponin in the Camellia chrysantha extract is 25% to 40%; and / or, The mass proportion of the flavonoids in the camellia chrysantha extract is 10% to 20%.

9. Use of the Camellia chrysantha extract prepared by the preparation method according to any one of claims 1 to 6 or the Camellia chrysantha extract according to any one of claims 7 to 8 in preparing a topical care composition, characterized in that: The mass proportion of the Camellia chrysantha extract in the external care composition is 0.001% to 5%.

10. The use according to claim 9, characterized in that The dosage form of the external care composition includes at least one of liquid, powder, semisolid or solid.

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