Seawater pearl oyster extract and application thereof

Abstract

The invention relates to the technical field of marine resource development and application, in particular to a seawater pearl oyster extract and application thereof. The seawater pearl oyster extract comprises at least one of a mantle extract, a shell meat extract and a mucus extract of seawater pearl oysters, is rich in active ingredients, and has excellent anti-oxidation, anti-inflammatory and anti-aging effects.

Description

Technical Field

[0001] The present application relates to the field of marine resource development and application technology, and in particular to a seawater pearl oyster extract and its application. Background Art

[0002] Pearl oysters are a general term for marine or freshwater shellfish that can secrete nacre. Due to their rich bioactive ingredients, they are widely used in cosmetics, food and health products.

[0003] The pearls, shells, and meat of pearl oysters have been developed into a variety of products, such as pearl powder, pearl hydrolyzate, pearl enzymatic hydrolyzate, etc. These products have whitening, anti-oxidation, anti-aging, skin barrier repair and other effects, and are widely used in the cosmetics field; in addition, pearls are also used to develop new food additives, such as pearl wine, pearl yogurt, etc. These products not only meet consumers' needs for healthy diet, but also enhance the added value and market competitiveness of the products; pearls and their derivatives contain a variety of trace elements and active ingredients, and have also been developed into health products with specific functions. For example, pearl powder, pearl oral liquid, etc. are believed to have the effects of enhancing immunity, delaying aging, improving memory, etc., and have always been popular with consumers.

[0004] During the research on pearl oysters, it was found that the current applications of pearl oysters mainly focus on pearls, shells and shellfish meat. Pearls and shells are usually ground directly for use, and shellfish meat is usually enzymatically hydrolyzed and prepared into enzymatic hydrolysate for use, or separated and extracted to obtain specific polypeptides as a polypeptide resource library. However, other components of pearl oysters, such as the mantle, mucus and other components of the shellfish meat have not yet been fully developed and applied.

[0005] Among the cosmetics and health products commonly used on the market, pearl extracts mostly use freshwater pearl oysters as the main raw material, while seawater pearl oysters are rarely used. Seawater pearl oysters are mainly used to produce expensive seawater pearls, and the shells themselves are rarely used, resulting in serious waste of marine resources. Summary of the Invention

[0006] Based on this, one or more embodiments of the present application provide a seawater pearl oyster extract, which includes at least one of the mantle extract, shellfish meat extract and mucus extract of the seawater pearl oyster, is rich in active ingredients and has better antioxidant, anti-inflammatory and anti-aging effects than traditional pearl oyster extracts such as hydrolyzed pearl powder and pearl powder extract.

[0007] The technical solution of this application is as follows:

[0008] A seawater pearl oyster extract comprises at least one of a seawater pearl oyster mantle extract, a pearl oyster meat extract and a mucus extract.

[0009] Furthermore, the preparation method of the seawater pearl oyster extract described above comprises the following steps:

[0010] Isolation and collection of mantle, flesh and mucus from marine pearl oysters;

[0011] The mantle and the shellfish meat are extracted respectively by water extraction method;

[0012] Centrifuging the liquid extracted from the mantle and the shellfish meat and the mucus to separate the supernatant and the precipitate;

[0013] The supernatants obtained by centrifugation were subjected to ultrafiltration to obtain feed solutions with a concentration of <10 kd;

[0014] The feed solutions obtained by ultrafiltration are freeze-dried respectively to obtain the mantle extract, the shellfish meat extract and the mucus extract.

[0015] Furthermore, the centrifugal speed is 9000 rcf~11000 rcf, and the time is 10min~20min.

[0016] In some embodiments, the step of extracting the mantle by water extraction comprises:

[0017] Shearing and separating the mantle to obtain mantle fragments;

[0018] A first water is added to the mantle fragments and the mixture is stirred at a first temperature for a first time.

[0019] Furthermore, the weight ratio of the mantle fragments to the first water is 1:(4-6);

[0020] The first temperature is -20°C to -10°C, and the first time is 5 minutes to 15 minutes.

[0021] In some embodiments, the step of extracting the shellfish meat using a water extraction method comprises:

[0022] Shearing and separating the shellfish meat to obtain shellfish meat fragments;

[0023] A second amount of water is added to the shellfish meat fragments, and the mixture is stirred at a second temperature for a second time.

[0024] Furthermore, the weight ratio of the shellfish meat fragments to the second water is 1:(4-6);

[0025] The second temperature is -20°C to -10°C, and the second time is 5 minutes to 15 minutes.

[0026] One or more embodiments of the present application further provide a use of the seawater pearl oyster extract described above in the preparation of an anti-aging product. In this embodiment, the anti-aging product includes at least one of cosmetics, food, and health products having anti-aging effects.

[0027] One or more embodiments of the present application further provide a use of the seawater pearl oyster extract described above in the preparation of an antioxidant product. In this embodiment, the anti-aging product comprises at least one of a cosmetic, a food, and a health product having an antioxidant effect.

[0028] One or more embodiments of the present application further provide a use of the seawater pearl oyster extract described above in the preparation of an anti-inflammatory product. In this embodiment, the anti-aging product comprises at least one of cosmetics, foods, health products, and medicines having anti-inflammatory effects.

[0029] The seawater pearl oyster extract of the present application extracts components other than the pearl body of the seawater pearl oyster, especially the mantle, shellfish meat and mucus. The extract has excellent anti-aging, anti-inflammatory and antioxidant effects, thereby making more full use of marine resources and avoiding resource waste. The extract is taken from seawater pearls of natural origin, which solves the human safety issues caused by organic solvent residues in anti-aging, anti-inflammatory and antioxidant products on the market, and has better efficacy than common anti-aging, anti-inflammatory and antioxidant active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0031] Figure 1 The regulatory effects of the seawater pearl oyster extracts of Examples 1 to 3 of the present application on aging-related genes;

[0032] Figure 2 The inhibitory effect of the seawater pearl oyster extracts of Examples 1 to 3 of the present application on the activity of galactosidase;

[0033] Figure 3 The regulatory effects of the seawater pearl oyster extract of Example 3 of the present application on aging-related genes at different concentrations;

[0034] Figure 4 The results are a comparison of the regulatory effects of the seawater pearl oyster extract of Example 3 of the present application and other commonly used cosmetic additives on aging-related genes;

[0035] Figure 5 This is a comparison of the regulatory effects of the seawater pearl oyster extract of Example 3 of the present application and the freshwater pearl oyster extract of Comparative Example 1 on aging-related genes;

[0036] Figure 6 This is a comparison of the regulatory effects of the seawater pearl oyster extract of Example 3 of the present application and other commonly used pearl extracts on aging-related genes;

[0037] Figure 7 The regulatory effects of the seawater pearl oyster extracts of Examples 1 to 3 of the present application on inflammation-related genes;

[0038] Figure 8 The regulatory effect of the seawater pearl oyster extract of Example 3 of the present application on inflammation-related genes at different concentrations;

[0039] Figure 9 This is a comparison of the regulatory effects of the seawater pearl oyster extract of Example 3 of the present application and other commonly used cosmetic additives on inflammation-related genes;

[0040] Figure 10 This is a comparison of the regulatory effects of the seawater pearl oyster extract of Example 3 of the present application and the freshwater pearl oyster extract of Comparative Example 1 on inflammation-related genes;

[0041] Figure 11 This is a comparison of the regulatory effects of the seawater pearl oyster extract of Example 3 of the present application and other commonly used pearl extracts on inflammation-related genes;

[0042] Figure 12 The inhibitory effect of the seawater pearl oyster extracts of Examples 1 to 3 of the present application on cellular oxidation levels;

[0043] Figure 13 The inhibitory effect of the seawater pearl oyster extract of Example 3 of the present application on the cellular oxidation level at different concentrations;

[0044] Figure 14 This is a comparison of the inhibitory effects of the seawater pearl oyster extract of Example 3 of the present application and other commonly used cosmetic additives on cellular oxidation levels;

[0045] Figure 15 This is a comparison of the inhibitory effects of the seawater pearl oyster extract of Example 3 of the present application and the freshwater pearl oyster extract of Comparative Example 1 on cellular oxidation levels;

[0046] Figure 16 This is a comparison of the inhibitory effects of the seawater pearl oyster extract of Example 3 of the present application and other commonly used pearl extracts on cellular oxidation levels;

[0047] Figure 17 This is the comparative result of the effects of the extracts obtained using different ultrafiltration membranes during the experimental condition screening stage of this application. DETAILED DESCRIPTION

[0048] Below in conjunction with embodiment and example, further elaborate the application.It should be understood that these examples are only used to illustrate the application and are not used to limit the scope of the application.In addition, it should be understood that after reading the content taught in this application, those skilled in the art can make various changes or modifications to the application, and these equivalent forms also fall within the protection scope of the claims appended hereto.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0050] the term

[0051] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0052] The term "and / or" as used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0053] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0054] In this application, terms such as "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, terms such as "first," "second," and "third" are used only for non-exhaustive enumeration and description purposes and should not constitute closed-ended limitations on quantity.

[0055] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0056] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0057] Unless otherwise specified, the temperature parameters in this application may be either constant temperature or fluctuating within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the precision range of the instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0058] In this application, weight can be μg, mg, g, kg and other mass units commonly known in the fields of cosmetics, food (health products), medicines, etc.

[0059] In one aspect of the present application, a seawater pearl oyster extract is provided, comprising at least one of a seawater pearl oyster mantle extract, a shellfish meat extract, and a mucus extract.

[0060] The seawater pearl oyster extract of the present application extracts components other than the pearl body of the seawater pearl oyster, especially the mantle, shellfish meat and mucus. The extract has excellent anti-aging, anti-inflammatory and antioxidant effects, thereby making more full use of marine resources and avoiding resource waste. The extract is taken from seawater pearls of natural origin, which solves the human safety issues caused by organic solvent residues in anti-aging, anti-inflammatory and antioxidant products on the market, and has better efficacy than common anti-aging, anti-inflammatory and antioxidant active ingredients.

[0061] The preparation method of the seawater pearl oyster extract of the present application comprises the following steps:

[0062] Isolation and collection of mantle, flesh and mucus from marine pearl oysters;

[0063] The mantle and shellfish flesh were extracted separately by water extraction method;

[0064] The liquid and mucus extracted from the mantle and shellfish meat are centrifuged separately to separate the supernatant and the precipitate; the centrifugation speed is preferably 9000 rcf ~ 11000 rcf, and the time is 10 min ~ 20 min;

[0065] The supernatants obtained by centrifugation were subjected to ultrafiltration to obtain feed solutions with a concentration of <10 kd.

[0066] During the experiment, the present applicant unexpectedly discovered that after centrifugation at a speed of approximately 10,000 rcf and separation using a 10kd ultrafiltration membrane, the mantle and shellfish meat can be effectively extracted at the same time, so that the mantle extract, shellfish meat extract and mucus extract of seawater pearl oysters can be obtained using the same set of equipment and extraction procedures; in addition, for mucus, after omitting the water extraction step, direct centrifugation can also achieve effective extraction, which can reduce the freeze-drying time and reduce processing costs.

[0067] In some embodiments, the centrifugation speed is 10,000 rcf and the time is 15 min.

[0068] In some embodiments, the step of extracting the mantle by water extraction comprises:

[0069] The mantle is sheared and separated to obtain mantle fragments;

[0070] A first water is added to the mantle fragments and the mixture is stirred at a first temperature for a first time.

[0071] Furthermore, the weight ratio of the mantle fragments to the first water is 1:(4-6);

[0072] The first temperature is -20°C to -10°C, and the first time is 5 minutes to 15 minutes.

[0073] In some embodiments, the step of extracting shellfish meat using water extraction comprises:

[0074] Shearing and separating the shellfish meat to obtain shellfish meat fragments;

[0075] A second amount of water is added to the shellfish meat fragments, and the mixture is stirred at a second temperature for a second time.

[0076] Furthermore, the weight ratio of the shellfish meat fragments to the second water is 1:(4-6);

[0077] The second temperature is -20°C to -10°C, and the second time is 5 minutes to 15 minutes.

[0078] Another aspect of the present application provides a use of the seawater pearl oyster extract described above in the preparation of an anti-aging product. In this embodiment, the anti-aging product comprises at least one of cosmetics, food, and health products having anti-aging effects.

[0079] Another aspect of the present application provides a use of the seawater pearl oyster extract described above in the preparation of an antioxidant product. In this embodiment, the anti-aging product comprises at least one of a cosmetic, a food, and a health product having an antioxidant effect.

[0080] Another aspect of the present application provides a use of the seawater pearl oyster extract described above in the preparation of an anti-inflammatory product. In this embodiment, the anti-aging product comprises at least one of cosmetics, foods, health products, and medicines having anti-inflammatory effects.

[0081] The following are some specific examples.

[0082] For experimental parameters not specified in the following specific examples, reference is made to the guidance given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturer.

[0083] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known methods.

[0084] The mantle, shellfish meat and mucus used in the following examples (or comparative examples) were obtained by the following method:

[0085] 1. Collect seawater (or freshwater) pearl oysters and remove impurities from the shells;

[0086] 2. Open the shells and collect the mantle attached to the shell and flesh;

[0087] 3. Sieve the remaining shellfish meat and mucus through a perforated plastic sieve to separate the mucus and shellfish meat.

[0088] 1. Obtaining Pearl Shell Extract

[0089] Example 1

[0090] This embodiment provides a seawater pearl oyster extract of the present application, the raw material source is seawater pearl oyster, and the preparation method comprises the following steps:

[0091] 1. Shear and separate the shellfish meat to obtain shellfish meat fragments;

[0092] 2. Add 500 g of water to 100 g of shellfish meat fragments. Stir the mixture at -15°C for 10 minutes and then centrifuge at 10,000 rcf to obtain the supernatant.

[0093] 3. Ultrafiltration of the supernatant using a separation tube (10 kd membrane) to obtain a feed solution <10 kd;

[0094] 4. Freeze-dry the liquid to obtain shellfish meat extract.

[0095] Example 2

[0096] This embodiment provides a seawater pearl oyster extract of the present application, the raw material source is seawater pearl oyster, and the preparation method comprises the following steps:

[0097] 1. Shear and separate the mantle to obtain mantle fragments;

[0098] 2. Add 500 g of water to 100 g of mantle fragments. Stir the mixture at -15°C for 10 minutes and then centrifuge at 10,000 rcf to obtain the supernatant.

[0099] 3. Ultrafiltration of the supernatant using a separation tube (10 kd membrane) to obtain a feed solution <10 kd;

[0100] 4. Freeze-dry the liquid to obtain the mantle extract.

[0101] Example 3

[0102] This embodiment provides a seawater pearl oyster extract of the present application, the raw material source is seawater pearl oyster, and the preparation method comprises the following steps:

[0103] 1. Centrifuge the mucus at 10,000 rcf to obtain the supernatant;

[0104] 2. Ultrafiltration of the supernatant using a separation tube (10 kd membrane) to obtain a feed solution <10 kd;

[0105] 3. Freeze-dry the liquid to obtain a mucus extract.

[0106] Comparative Example 1

[0107] This comparative example provides a freshwater pearl oyster extract, the raw material of which is freshwater pearl oyster, and is prepared using a method basically the same as that in Example 3.

[0108] Hereinafter, BN represents the seawater pearl oyster extract of Example 1; MN represents the seawater pearl oyster extract of Example 2; AKN represents the seawater pearl oyster extract of Example 3; CTRL represents pure water; and DSN represents the freshwater pearl oyster extract of Comparative Example 1.

[0109] In the following text, hydrolyzed pearl powder comes from Huakang Raw Materials; pearl extract comes from Xiya Biological;

[0110] The UVA source is a UVA irradiation device, the brand is Xuzhou Aijia Electronic Technology;

[0111] Hexapeptide was sourced from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0112] The source of bosine was Shanghai MacLean Biochemical Technology Co., Ltd.;

[0113] Carnosine was sourced from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0114] NR was sourced from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0115] The source of EGT was Shanghai Ergothioneine Biotechnology Group Co., Ltd.

[0116] 2. Anti-aging effect test

[0117] 1. Regulatory effects of BN, MN, and AKN on aging-related genes

[0118] Human skin fibroblasts (HSF) were seeded in 6-well plates at a rate of 6 × 10 5 The CTRL group, UVA group, BN group, MN group, and AKN group were treated with equal volumes of pure water, pure water, BN aqueous solution (600 μg / ml), MN aqueous solution (600 μg / ml), and AKN aqueous solution (600 μg / ml) for 12 h. The CTRL group was not treated further. The other groups were treated with 25 J / cm 2 HSF were stimulated with UVA and stabilized for 24 hours, and cell samples were collected to detect gene expression.

[0119] The results are as follows Figure 1 As shown in the results, the aging-related genes p16, p21, and p53 in skin fibroblasts were significantly upregulated after ultraviolet irradiation, while the expression of aging-related genes was reduced after pre-treatment with mother-of-pearl extract, among which the decrease was most significant in the AKN-treated group.

[0120] 2. Inhibitory effects of BN, MN, and AKN on galactosidase activity

[0121] Human skin fibroblasts (HSF) were seeded in 6-well plates at a rate of 6 × 10 5The CTRL group, UVA group, BN (200) group, BN (600) group, MN (200) group, MN (600) group, AKN (200) group, and AKN (600) group were treated with equal volumes of pure water, pure water, BN aqueous solution (200 μg / ml), BN aqueous solution (600 μg / ml), MN aqueous solution (200 μg / ml), MN aqueous solution (600 μg / ml), AKN aqueous solution (200 μg / ml), and AKN aqueous solution (600 μg / ml), respectively, for 12 h. The CTRL group was not further treated, and the other groups were treated with 25 J / cm 2 HSF was stimulated with UVA and stabilized for 24 hours, and the β-galactosidase activity in the samples was detected.

[0122] The results are as follows Figure 2 As shown in the figure, the activity of β-galactosidase in skin fibroblasts was significantly upregulated after ultraviolet irradiation, which was manifested by a significant increase in the blue staining area. After pre-treatment with BN, MN or AKN, the intracellular β-galactosidase activity decreased, indicating that the aging phenotype of the cells was restored.

[0123] 3. Regulatory effects of different concentrations of AKN on aging-related genes

[0124] Human skin fibroblasts (HSF) were seeded in 6-well plates at a rate of 6 × 10 5 The CTRL group, UVA group, AKN (100) group, AKN (200) group, AKN (400) group, AKN (600) group, and AKN (800) group were treated with equal volumes of pure water, pure water, AKN aqueous solution (100 μg / ml), AKN aqueous solution (200 μg / ml), AKN aqueous solution (400 μg / ml), AKN aqueous solution (600 μg / ml), and AKN aqueous solution (800 μg / ml), respectively, for 12 h. The CTRL group was not further treated, and the other groups were treated with 25 J / cm 2 HSF were stimulated with UVA and stabilized for 24 hours, and cell samples were collected to detect gene expression.

[0125] The results are as follows Figure 3 As shown in the results, AKN can significantly reduce the expression of aging-related genes in cells, among which the p53 gene has an obvious trend of increasing inhibition effect with increasing concentration.

[0126] 4. Comparison of the regulatory effects of AKN and other commonly used cosmetic additives on aging-related genes

[0127] Human skin fibroblasts were seeded in 6-well plates at 6 × 10 5 / mL, the CTRL group, UVA group, hexapeptide group, bosin group, carnosine group, NR group, EGT group, and AKN group were treated with equal volumes of pure water, pure water, an aqueous solution of hexapeptide (final concentration 400μg / ml), an aqueous solution of bosin (400μg / ml), an aqueous solution of carnosine (400μg / ml), an aqueous solution of NR (400μg / ml), an aqueous solution of EGT (400μg / ml), and an aqueous solution of AKN (400μg / ml) for 12h. The CTRL group was not further treated, and the other groups were treated with 25J / cm 2 HSF were stimulated with UVA and stabilized for 24 hours, and cell samples were collected to detect gene expression.

[0128] The results are as follows Figure 4 As shown in the figure, the expression of senescence-related genes in HSF cells after AKN treatment was significantly lower than that in the UVA and other additive groups.

[0129] 5. Comparison of the regulatory effects of AKN and DSN on aging-related genes

[0130] Human skin fibroblasts were seeded in 6-well plates at 6 × 10 5 The CTRL group, UVA group, DSN group, and AKN group were treated with equal volumes of pure water, pure water, DSN aqueous solution (final concentration 400 μg / ml), and AKN aqueous solution (400 μg / ml) for 12 h. The CTRL group was not treated further. The other groups were treated with 25 J / cm 2 HSF were stimulated with UVA and stabilized for 24 hours, and cell samples were collected to detect gene expression.

[0131] The results are as follows Figure 5 As shown in the figure, the expression of senescence-related genes in HSF cells after AKN treatment was significantly lower than that in UVA and DSN.

[0132] 6. Comparison of the regulatory effects of AKN and other commonly used pearl extracts on aging-related genes

[0133] Human skin fibroblasts (HSF) were seeded in 6-well plates at a rate of 6 × 10 5 The CTRL group, UVA group, hydrolyzed pearl powder group, pearl extract group, and AKN group were treated with equal volumes of pure water, pure water, suspension of hydrolyzed pearl powder (400 μg / ml), diluted pearl extract (400 μg / ml), and aqueous solution of AKN (400 μg / ml) for 12 h. The CTRL group was not further treated. The other groups were treated with 25 J / cm 2 HSF were stimulated with UVA and stabilized for 24 hours, and cell samples were collected to detect gene expression.

[0134] The results are as follows Figure 6As shown in the results, the expression of aging-related genes in HSF cells after AKN treatment was significantly lower than that in the UVA and hydrolyzed pearl powder or pearl extract groups.

[0135] 3. Anti-inflammatory effect test

[0136] 1. Regulatory effects of BN, MN, and AKN on inflammation-related genes

[0137] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was left untreated, and the other groups were stimulated with 1 μg / ml LPS. Subsequently, equal volumes of pure water, pure water, BN aqueous solution (400 μg / ml), MN aqueous solution (400 μg / ml), and AKN aqueous solution (400 μg / ml) were added to the CTRL group, LPS group, BN group, MN group, and AKN group, respectively, for treatment. After each group stabilized for 10-12 hours, cell samples were collected to detect gene expression.

[0138] The results are as follows Figure 7 As shown in the results, the inflammation-related genes IL6, TNF-A, and IL6 were significantly upregulated in mouse glioma cells BV2 after lipopolysaccharide (LPS) induction, while the expression of inflammation-related genes was reduced after treatment with mother-of-pearl extract, among which the decrease in the mother-of-pearl mucus (AKN) treatment group was the most significant.

[0139] 2. Regulatory effects of different concentrations of AKN on inflammation-related genes

[0140] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was not treated, and the other groups were stimulated with 1μg / ml LPS. Subsequently, the CTRL group, LPS group, AKN (100) group, AKN (200) group, AKN (400) group, AKN (600) group, and AKN (800) group were treated with equal volumes of pure water, pure water, AKN aqueous solution (100μg / ml), AKN aqueous solution (200μg / ml), AKN aqueous solution (400μg / ml), AKN aqueous solution (600μg / ml), and AKN aqueous solution (800μg / ml), respectively. After each group stabilized for 10-12 hours, cell samples were collected to detect gene expression.

[0141] The results are as follows Figure 8 As shown, AKN concentrations below 600 μg / ml can significantly reduce the expression of inflammation-related genes in cells, among which 400 μg / ml and 200 μg / ml have the most obvious inhibitory effects.

[0142] 3. Comparison of the anti-inflammatory effects of AKN and other commonly used cosmetic additives

[0143] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was not treated, and the other groups were stimulated with 1μg / ml LPS. Subsequently, the CTRL group, LPS group, hexapeptide group, bosine group, carnosine group, NR group, EGT group, and AKN group were treated with equal volumes of pure water, pure water, an aqueous solution of hexapeptide (400μg / ml), an aqueous solution of bosine (400μg / ml), an aqueous solution of carnosine (400μg / ml), an aqueous solution of NR (400μg / ml), an aqueous solution of EGT (400μg / ml), and an aqueous solution of AKN (400μg / ml), respectively. After each group stabilized for 10-12 hours, cell samples were collected to detect gene expression.

[0144] The results are as follows Figure 9 As shown in the figure, the expression of inflammation-related genes in BV2 cells after AKN treatment was significantly lower than that in the LPS and other additive groups.

[0145] 4. Comparison of anti-inflammatory effects of AKN and DSN

[0146] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was left untreated, and the other groups were stimulated with 1 μg / ml LPS. Subsequently, equal volumes of pure water, pure water, DSN aqueous solution (400 μg / ml), and AKN aqueous solution (400 μg / ml) were added to the CTRL group, LPS group, DSN group, and AKN group, respectively, for treatment. After each group stabilized for 10-12 hours, cell samples were collected to detect gene expression.

[0147] The results are as follows Figure 10 As shown in the figure, the expression of inflammation-related genes in BV2 cells after AKN treatment was significantly lower than that in the LPS and freshwater pearl mucus DSN groups.

[0148] 5. Comparison of the anti-inflammatory effects of AKN and other commonly used pearl-related cosmetic ingredients

[0149] Mouse glioma cells BV2 were used to study the inhibitory effects of different additives on the lipopolysaccharide (LPS)-induced cellular inflammation model at the cellular level. BV2 cells were seeded in 12-well plates, 3×10 5 After 12 hours of cell attachment, BV2 cells were stimulated with 1 μg / ml LPS and treated with different concentrations of AKN or solvent (pure water). After stabilization for 10-12 hours, cell samples were collected to detect gene expression. Figure 9As shown in the figure, the expression of inflammation-related genes in BV2 cells after AKN treatment was significantly lower than that in the LPS and other raw material groups.

[0150] The results are as follows Figure 11 As shown, the expression of inflammation-related genes in BV2 cells after AKN treatment was significantly lower than that in the LPS and other pearl raw material groups.

[0151] 3. Antioxidant Effect Test

[0152] 1. Inhibitory effects of BN, MN, and AKN on cellular oxidation levels

[0153] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was not treated, and the other groups were stimulated with 1 μg / ml LPS. Subsequently, equal volumes of pure water, pure water, BN aqueous solution (400 μg / ml), MN aqueous solution (400 μg / ml), and AKN aqueous solution (400 μg / ml) were added to the CTRL group, LPS group, BN group, MN group, and AKN group for treatment, respectively. After each group was stable for 10-12 hours, DCFH-DA was incubated for 40 minutes, and cell samples were collected. The ROS levels were detected by flow cytometry.

[0154] The results are as follows Figure 12 As shown in the results, the oxidation level in mouse glioma cells BV2 was significantly upregulated after lipopolysaccharide (LPS) induction, while the oxidation level was reduced after treatment with mother-of-pearl extract, among which the decrease in the mother-of-pearl mucus (AKN) treatment group was the most significant.

[0155] 2. Comparison of the inhibitory effects of different concentrations of AKN on cellular oxidation levels

[0156] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was not treated, and the other groups were stimulated with 1μg / ml LPS. Subsequently, the CTRL group, LPS group, AKN (100) group, AKN (200) group, AKN (400) group, AKN (600) group, and AKN (800) group were treated with equal volumes of pure water, pure water, AKN aqueous solution (100μg / ml), AKN aqueous solution (200μg / ml), AKN aqueous solution (400μg / ml), AKN aqueous solution (600μg / ml), and AKN aqueous solution (800μg / ml), respectively. After each group was stable for 10-12 hours, DCFH-DA was incubated for 40 minutes, and cell samples were collected. The ROS level was detected by flow cytometry.

[0157] The results are as follows Figure 13As shown in the results, AKN can significantly reduce the expression of oxidative stress in cells, and the inhibitory effect tends to increase with increasing concentration.

[0158] 3. Comparison of the antioxidant effects of AKN and other commonly used cosmetic additives

[0159] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was not treated, and the other groups were stimulated with 1 μg / ml LPS. Subsequently, the CTRL group, LPS group, hexapeptide group, bosine group, carnosine group, NR group, EGT group, and AKN group were treated with equal volumes of pure water, pure water, an aqueous solution of hexapeptide (400 μg / ml), an aqueous solution of bosine (400 μg / ml), an aqueous solution of carnosine (400 μg / ml), an aqueous solution of NR (400 μg / ml), an aqueous solution of EGT (400 μg / ml), and an aqueous solution of AKN (400 μg / ml), respectively. After each group was stable for 10-12 hours, DCFH-DA was incubated for 40 minutes, and cell samples were collected. The ROS levels were detected by flow cytometry.

[0160] The results are as follows Figure 14 As shown, the oxidation level in BV2 cells after AKN treatment was significantly lower than that in the LPS and other raw material groups.

[0161] 4. Comparison of the antioxidant effects of AKN and DSN

[0162] Mouse glioma BV2 cells were seeded in 12-well plates at 3 × 10 5 / mL. After 12 hours of cell attachment, the CTRL group was left untreated, and the other groups were stimulated with 1 μg / ml LPS. Subsequently, equal volumes of pure water, pure water, DSN aqueous solution (400 μg / ml), and AKN aqueous solution (400 μg / ml) were added to the CTRL group, LPS group, DSN group, and AKN group, respectively, for treatment. After each group was stable for 10-12 hours, DCFH-DA was incubated for 40 minutes, and cell samples were collected. The ROS levels were detected by flow cytometry.

[0163] The results are as follows Figure 15 As shown, the oxidation level in BV2 cells after AKN treatment was significantly lower than that in the LPS and freshwater pearl mucus DSN groups.

[0164] 5. Comparison of the anti-inflammatory effects of AKN and other commonly used pearl-related cosmetic ingredients

[0165] Mouse glioma cells BV2 were used to study the inhibitory effect of pearl-related cosmetic raw materials on the lipopolysaccharide (LPS)-induced cell oxidation model at the cellular level. BV2 cells were seeded in 12-well plates, and 3×10 5After 12 hours of cell attachment, BV2 was stimulated with 1 μg / ml LPS and treated with different pearl materials or solvents (pure water). After stabilization for 10-12 hours, DCFH-DA was incubated for 40 minutes, and cell samples were collected. ROS levels were detected by flow cytometry. Figure 16 As shown in the figure, the oxidation level in BV2 cells after AKN treatment was significantly lower than that in the LPS and other pearl raw material groups.

[0166] The results are as follows Figure 16 As shown in the figure, the oxidation level in BV2 cells after AKN treatment was significantly lower than that in the LPS and other pearl raw material groups.

[0167] 3. Condition Optimization

[0168] 1. Membrane pore size screening

[0169] The ultrafiltration membranes were screened and the pore sizes of 1kd, 3kd and 10kd were selected to obtain mucus extracts with pore sizes of <1, <3, <10, 1-3 and 1-10kd, respectively. The bioactivity of the extracts was determined as shown in the following table. Figure 17 As shown in the figure, the anti-aging effect of the component <10kd is significantly better than that of other size components, so a 10kd ultrafiltration membrane is selected for separation in the examples of the present application.

[0170] 2. Extraction condition screening

[0171] The centrifugal speeds of 5000 rcf, 8000 rcf, and 13000 rcf were used respectively. The results showed that the solid matter could not be separated well using the centrifugal speeds of 5000 rcf and 8000 rcf, and the time cost was increased exponentially. The centrifugal speed of 13000 rcf would cause damage to the centrifuge tube. Therefore, the centrifugal speed of 10000 rcf was selected in the embodiment of the present application.

[0172] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0173] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0174] The embodiments described above only express several implementation methods of the present application, but they should not be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A seawater pearl oyster extract, characterized in that The invention comprises at least one of a mantle extract, a shellfish meat extract and a mucus extract of a seawater pearl oyster.

2. The seawater pearl oyster extract according to claim 1, characterized in that The preparation method comprises the following steps: Isolation and collection of mantle, flesh and mucus from marine pearl oysters; The mantle and the shellfish meat are extracted respectively by water extraction method; Centrifuging the liquid extracted from the mantle and the shellfish meat and the mucus to separate the supernatant and the precipitate; The supernatants obtained by centrifugation were subjected to ultrafiltration to obtain feed solutions with a concentration of <10 kd; The feed solutions obtained by ultrafiltration are freeze-dried respectively to obtain the mantle extract, the shellfish meat extract and the mucus extract.

3. The seawater pearl oyster extract according to claim 2, characterized in that The centrifugal speed is 9000 rcf-11000 rcf, and the time is 10 min-20 min.

4. The seawater pearl oyster extract according to claim 2, characterized in that The steps of extracting the mantle by water extraction include: Shearing and separating the mantle to obtain mantle fragments; A first water is added to the mantle fragments and the mixture is stirred at a first temperature for a first time.

5. The seawater pearl oyster extract according to claim 4, characterized in that The weight ratio of the mantle fragments to the first water is 1:(4-6); The first temperature is -20°C to -10°C, and the first time is 5 minutes to 15 minutes.

6. The seawater pearl oyster extract according to claim 2, characterized in that The step of extracting the shellfish meat by water extraction comprises: Shearing and separating the shellfish meat to obtain shellfish meat fragments; A second amount of water is added to the shellfish meat fragments, and the mixture is stirred at a second temperature for a second time.

7. The seawater pearl oyster extract according to claim 6, characterized in that The weight ratio of the shellfish meat fragments to the second water is 1:(4-6); The second temperature is -20°C to -10°C, and the second time is 5 minutes to 15 minutes.

8. Use of the seawater pearl oyster extract according to any one of claims 1 to 7 in the preparation of anti-aging products.

9. Use of the seawater pearl oyster extract according to any one of claims 1 to 7 in the preparation of an antioxidant product.

10. Use of the seawater pearl oyster extract according to any one of claims 1 to 7 in the preparation of anti-inflammatory products.

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