Composition for reducing protein carbonylation level and application thereof
By using an anti-carbonylation composition composed of yeast extract and epigallocate gallate, the carbonylation of protein is inhibited, and the problem of dark yellow skin tone is solved, achieving a significant effect of reducing dull yellow skin.
Patent Information
- Application Number
- CN202411766390.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-04
- Publication Date
- 2025-02-28
AI Technical Summary
Dark yellow skin tone is caused by protein carbonylation reaction, which is much higher than the skin saccharification reaction caused by protein glycosylation, and the prior art is difficult to effectively solve this problem.
An anti-carbonylation composition is provided, comprising a yeast extract and epigallocate gallate, which essentially reduces the formation of the carbonylated protein by inhibiting the carbonylation of the protein.
This composition can significantly reduce protein carbonylation levels and prevent dull yellowing of the skin, especially dull yellowing of the facial skin, providing an effective beauty method.
Smart Images
Figure HDA0005170196490000011
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of functional compositions, in particular to an anti-carbonylation composition and application thereof. Background Art
[0002] Yellowish skin is a skin problem caused by various reasons, which leads to aging of the skin surface, yellowish complexion and lack of luster. It is the most common physiological aging phenomenon in middle-aged and elderly women, and it becomes more serious with age. For oriental beauty-loving women who pursue fair and moist skin, how to solve the problem of yellowish skin has become an urgent problem to be solved.
[0003] The present invention provides a composition that solves the above problems. Summary of the invention
[0004] The present invention is based on the following discovery: the skin yellowing effect caused by protein carbonylation is much higher than the skin glycation reaction caused by protein glycation. The present invention provides an anti-carbonyl compound that can inhibit protein carbonylation, substantially reduce the formation of carbonylated proteins, and further remove carbonylated proteins without considering the removal of carbonylated proteins by degradation.
[0005] In one aspect, provided is a composition comprising or consisting of 0.5-8 parts by weight of yeast extract and 0.5-5 parts by weight of epigallocatechin gallate.
[0006] In one embodiment, the composition is an anti-carbonylation composition.
[0007] In one embodiment, the anti-carbonylation composition comprises 0.6-6 parts by weight, 0.7-5 parts by weight, or 1-4 parts by weight of yeast extract. In one embodiment, the anti-carbonylation composition comprises 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 parts by weight of yeast extract.
[0008] In one embodiment, the anti-carbonylation composition comprises 0.6-6 parts by weight, 0.7-5 parts by weight, or 1-4 parts by weight of epigallocatechin gallate. In one embodiment, the anti-carbonylation composition comprises 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or 6 parts by weight of epigallocatechin gallate.
[0009] In one embodiment, the glutathione content of the yeast extract is ≥ 5 wt%.
[0010] In one embodiment, the weight ratio of yeast extract to epigallocatechin gallate is 1: 1-4: 1. In one embodiment, the weight ratio of yeast extract to epigallocatechin gallate is 2: 1. In one embodiment, the yeast extract is obtained by the following method, which comprises yeast fermentation, enzymatic hydrolysis and separation of molasses and a fermentation nutrient source, wherein the fermentation nutrient source is selected from any N source and / or P source.
[0011] In one embodiment, the method further comprises one or more steps of sterilizing, concentrating, drying and sieving.
[0012] In another aspect, an oral composition is provided, comprising the anti-carbonylation composition described herein and an oral carrier, diluent or excipient.
[0013] In one embodiment, the oral composition is used to prevent sallowness of the skin, especially facial skin, in a subject.
[0014] In one embodiment, the oral composition may be a drink, jelly, solid beverage, candy, soft capsule, and hard capsule.
[0015] In yet another aspect, provided is a method for preparing the anti-carbonylation composition described herein, comprising the step of mixing yeast extract and epigallocatechin gallate.
[0016] In yet another aspect, a method of preparing the oral composition described herein is provided, comprising the step of mixing the anti-carbonylation composition and an oral carrier, diluent or excipient.
[0017] In another aspect, provided is the use of an anti-carbonylation composition or an oral composition in food or health food, wherein the food or health food is used to inhibit the carbonylation of proteins or to prevent skin sallowness, especially facial skin sallowness.
[0018] In yet another aspect, a method of inhibiting protein carbonylation in a cell in vitro is provided, comprising the step of contacting the anti-carbonylation composition described herein with the cell. In one embodiment, the cell is a skin cell, such as a keratinocyte or a fibroblast.
[0019] In yet another aspect, there is provided a use of the anti-carbonylation composition or oral composition described herein for the non-therapeutic purpose of inhibiting protein carbonylation in a cell. In one embodiment, the cell is a skin cell, such as a keratinocyte or a fibroblast.
[0020] Advantages of the present invention include:
[0021] 1. The anti-carbonylation composition of the present invention can synergistically resist carbonylation.
[0022] 2. The anti-carbonylation composition of the present invention can be used to inhibit the carbonylation of proteins or to prevent skin yellowing, especially facial skin yellowing. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The protein carbonylation content of different experimental groups is shown. The total concentration of each experimental group is consistent, mean ± SD, n = 3. One-way ANOVA was used for difference analysis. Different superscript letters indicate significant differences (P < 0.05). DETAILED DESCRIPTION
[0024] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.
[0025] Introduction
[0026] There are misunderstandings about skin darkening. On the one hand, it is believed that skin darkening is the front-end characteristic of skin blackening. In fact, the mechanisms of the two are very different: skin darkening is related to melanin in the skin. Melanin is produced by melanosomes in melanocytes. According to skin physiology, the main factors that determine the depth of skin color are the content of melanin in the skin and its distribution. Melanocytes are located in the basal layer of the skin. The secretion of melanin by melanocytes is an extremely complex biological process, involving the occurrence of melanocytes, dendrite formation, melanosome maturation, transport and other mechanisms, and is closely related to keratinocytes and extracellular matrix. On the other hand, it is believed that skin darkening is caused by protein glycosylation. Protein glycosylation is the combination of the carbonyl group of reducing sugars (such as glucose or fructose) with proteins in a non-enzymatic reaction to form glycosylated proteins. However, the skin darkening effect caused by protein carbonylation is much higher than that of skin glycation. Proteins react with active carbonyl substances (RCS) under non-enzymatic conditions, and irreversible carbonyl modifications or self-oxidation occur to form carbonyl proteins.
[0027] Reactive Carbonyl Species (RCS) are some active aldehyde compounds containing unsaturated chemical bonds. The most common is malondialdehyde, which is very common in the skin. It plays a very important role in the human body. In the process of oxidants and free radicals attacking the human body and causing various damage mechanisms, RCS is a direct factor and participates in and initiates the aging process. The main target of carbonylation is protein. Protein reacts with active carbonyl substances RCS under non-enzymatic conditions and is modified by irreversible carbonyl groups. Under oxidative stress, it is oxidized to form carbonylated proteins. After protein carbonylation, the shape and function of the protein itself are changed, and the original protein loses its effect on the human body. Carbonylated proteins play a very important role in the human aging mechanism. In the process of oxidants and free radicals attacking human tissues and causing cytotoxicity, proteins modified by carbonylation will cause structural changes, increased damage, and exceeding human metabolism will cause the complete loss of protein function. Carbonylated proteins cause denaturation of collagen and elastin, resulting in dull and yellow skin tone, aggravated oxidative damage, reduced skin hydration capacity, affected skin barrier function, and aggravated dark and coarse pores. Protein carbonylation is an irreversible modification, so it can only be degraded and accumulated. If not cleaned up in time, it will cause the accumulation and aggregation of damaged proteins, induce changes in physiological functions, and accelerate the aging process. However, not all carbonylated proteins can be removed, and only a part of carbonylated proteins can be degraded by proteasomes. Over-oxidized and cross-linked proteins will have the property of resisting proteolysis, and can promote proteins to form high molecular weight and potentially cytotoxic polymers. The further cross-linking and aggregation of these polymers in the body will have the characteristics of proteasome antibodies, hindering the degradation of abnormal proteins by the ubiquitin-protease system. In elderly individuals, proteins will undergo obvious carbonylation modification. With increasing age, the level of protein carbonylation increases year by year.
[0028] The purpose of the present invention is to find a reasonable composition and ratio, and the composition can achieve a more significant reduction in protein carbonylation level through synergistic action. The present invention optimizes the ratio of yeast extract and EGCG, and the HaCaT cell experiment verifies that the composition described herein can achieve a more significant reduction in protein carbonylation level through synergistic action, especially when the composition contains 1-4 parts by weight of yeast extract and 1 part by weight of EGCG.
[0029] definition
[0030] As used herein, the term "comprising" is an open-ended term. Comprising listed components, steps, or methods need not be limited to only those elements, but may include other components, steps, or methods not explicitly listed. The term "consisting of" is a closed term that excludes components, steps, or methods not explicitly listed.
[0031] As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and where appropriate, include fractions thereof (e.g., tenths and hundredths of an integer).
[0032] As used herein, protein "carbonylation" refers to the process of carbonyl modification in protein molecules. When lipid peroxidation occurs in cells, polyunsaturated fatty acids are degraded to generate lipid-derived electrophilic small molecules, which can react with nucleophilic residues of proteins to form carbonyl modifications, thereby affecting the structure and activity of proteins. Protein carbonylation is regarded as a biomarker of oxidative stress and aging, and is a non-enzymatic and irreversible carbonyl modification of proteins. Carbonyl modification is closely related to a variety of diseases.
[0033] As used herein, "anti-carbonylation" composition refers to a composition that prevents protein from being carbonylated. The anti-carbonylation composition can be administered by any suitable means. Preferably, the anti-carbonylation composition is administered orally. For example, the anti-carbonylation composition can be prepared into beverages, jellies, solid beverages, candies, soft capsules and hard capsules. The anti-carbonylation composition is preferably a food or a health food. The anti-carbonylation composition can also be used as part of a food or a health food.
[0034] As used herein, the terms "sallow complexion" and "yellowing complexion" are used interchangeably and refer to a type of skin problem that causes aging of the skin surface, yellowing of the complexion, and lack of luster due to various reasons (including protein carbonylation and oxidation of lipids on the skin surface). Sallow complexion can be caused by various factors, especially protein carbonylation.
[0035] As used herein, "yeast extract" is well known in the art and has corresponding national standards. For example, the yeast extract is obtained by the following method, which comprises yeast fermentation, enzymolysis and separation of molasses and a fermentation nutrient source, wherein the fermentation nutrient source is selected from any N source and / or P source. Preferably, the method comprises yeast fermentation, enzymolysis, separation, sterilization, concentration, drying and screening of molasses and a fermentation nutrient source. Preferably, the yeast is baker's yeast or brewer's yeast, etc. The glutathione content of the yeast extract may be ≥5wt%, 10wt%, ≥11wt%, ≥12wt%, ≥13wt%, ≥14wt% or ≥15wt%.
[0036] As used herein, "glutathione" can react with free radicals through the electron transfer pathway, and the free radicals lose their activity by combining with electrons, thereby reducing the content of ROS in the body and reducing oxidative stress damage. It is reported that glutathione can inhibit the production of melanin and decompose melanin.
[0037] As used herein, "epigallocatechin gallate", referred to as EGCG, is an ingredient extracted from tea leaves and is the component with the highest content in catechins. It is reported that epigallocatechin gallate can induce tumor cell apoptosis, antioxidant effects and other effects. The anti-carbonylation composition can contain 0.6-6 parts by weight, 0.7-5 parts by weight or 1-4 parts by weight of epigallocatechin gallate, such as 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 parts by weight (or any range therebetween) of epigallocatechin gallate.
[0038] As used herein, a "subject" may be any suitable mammal, but is preferably a human.
[0039] The present invention provides an anti-carbonylation composition. The anti-carbonylation composition may comprise 0.5-8 parts by weight of yeast extract and 0.5-5 parts by weight of epigallocatechin gallate. Preferably, the anti-carbonylation composition is composed of 0.5-8 parts by weight of yeast extract and 0.5-5 parts by weight of epigallocatechin gallate. Preferably, the glutathione content of the yeast extract is ≥5wt%. The preparation method of the yeast extract is also not particularly limited. For example, the yeast extract is obtained by the following method, which includes yeast fermentation, enzymolysis and separation of molasses and a fermentation nutrient source, wherein the fermentation nutrient source is selected from any N source and / or P source. Preferably, the method includes a process of yeast fermentation, enzymolysis, separation, sterilization, concentration, drying and screening of molasses and a fermentation nutrient source. Preferably, the yeast is baker's yeast or brewer's yeast, etc. The specification of epigallocatechin gallate is not particularly limited, and can be substantially pure, for example, 90% or more pure, 91% or more pure, 92% or more pure, 93% or more pure, 94% or more pure, 95% or more pure, 96% or more pure, 97% or more pure, 98% or more pure, 99% or more pure, 100% pure. There is no particular requirement for the preparation method of the anti-carbonylation composition. For example, the preparation method may include thoroughly mixing yeast extract and epigallocatechin gallate.
[0040] Oral Composition
[0041] The anti-carbonylation composition described herein can be prepared into an oral composition together with one or more of an oral carrier, a diluent and an excipient. The oral carrier, diluent and excipient can be common in the art. The method for preparing the oral composition is also conventional in the art. The oral composition can be used to prevent the skin of the subject from being dark yellow, especially the facial skin from being dark yellow (especially the facial skin of women from being dark yellow). The oral composition can include the anti-carbonylation composition described herein and another component to prevent the skin from being dark yellow. The oral composition can be any suitable oral form, including but not limited to one or more of a drink, a jelly, a solid beverage, a candy, a soft capsule and a hard capsule.
[0042] Purpose or method
[0043] Also provided is the use of the anti-carbonylation composition or oral composition described herein in food or health food, wherein the food or health food is used to inhibit the carbonylation of proteins or to prevent skin sallowness, especially facial skin sallowness.
[0044] A cosmetic method is also provided, which comprises applying the anti-carbonylation composition or oral composition described herein to a subject. The cosmetic method can be used to prevent skin sallowness, especially facial skin sallowness. A method for inhibiting protein carbonylation in cells in vitro is also provided, which comprises the step of contacting the anti-carbonylation composition described herein with cells. The type of cells is not particularly limited, including but not limited to skin cells, such as keratinocytes or fibroblasts.
[0045] Also provided is the use of the anti-carbonylation composition or oral composition described herein for non-therapeutic purposes of inhibiting protein carbonylation in cells. The type of cells is not particularly limited, including but not limited to skin cells, such as keratinocytes or fibroblasts.
[0046] Example
[0047] In order to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail through examples below. It should be understood that the examples and specific features are detailed descriptions of the technical solution of the present application, rather than limitations on the technical solution of the present application. In the absence of conflict, the technical features between the implementation plans can be combined with each other.
[0048] Example 1: Preparation of anti-carbonylation composition
[0049] The formula of the anti-carbonylation composition is shown in Table 1. EGCG is a commercial product (EGCG product content ≥ 94%). The preparation method of the yeast extract is that molasses and a fermentation nutrient source (N source, P source) are subjected to yeast fermentation, enzymolysis, separation, sterilization, concentration, drying, and sieving, wherein the content of glutathione in the obtained yeast extract is ≥ 5%.
[0050] The preparation method of the anti-carbonylation composition is to uniformly mix the yeast extract and EGCG according to the formula in Table 1 to obtain the anti-carbonylation composition.
[0051] Table 1 Components of the anti-carbonylation composition
[0052] Components Yeast Extract EGCG Example 1 1 1 Example 2 2 1 Example 3 4 1 Comparative Example 1 1 2 Comparative Example 2 8 1 Comparative Example 3 0 1 Comparative Example 4 1 0
[0053] Embodiment 2: Activity test of anti-carbonylation composition
[0054] Experiment construction method
[0055] The test composition used in this experiment is the composition prepared in Examples 1-3 and Comparative Examples 1-4. The inhibition rate of protein carbonylation in HaCaT cells was measured to evaluate the anti-carbonylation effect of the composition. The specific experimental steps are as follows:
[0056] (1) Cultivation of immortalized human keratinocytes
[0057] Human immortalized keratinocytes (HaCaT) spontaneously transform into immortalized human keratinocytes after in vitro culture. They are widely used in skin biology and differentiation research. HaCaT cells are cultured in complete medium at 37°C in a humidified incubator containing 5% CO2; the medium is replaced every 3 days, and when the cell density reaches 90%, the cells are passaged at a ratio of 1:2-1:3.
[0058] (2) Effect of samples on UVA-induced carbonylation
[0059] Adjust the logarithmic growth phase HaCaT cells to 7.5x10 4 / mL was inoculated into a 24-well plate, 1mL per well, and cultured in a 37°C, 5% CO2 incubator for 24h. The cells were randomly divided into a control group, a model group, and a sample treatment group. Before UVA irradiation, the sample treatment group was treated with 31.25μg / mL of the test object composition (each of the compositions prepared in Examples 1-3 and Comparative Examples 1-4) for 24h, and the control group and the model group were incubated with culture medium. After incubation for 24h, the culture medium was discarded, 200μL PBS was added, the model group and the sample treatment group were irradiated with 25J UVA, and the control group was not treated with UVA irradiation. After the irradiation was completed, 1mL of the test object composition with a total concentration of 31.25μg / mL (each of the compositions prepared in Examples 1-3 and Comparative Examples 1-4) was added to the sample group, and an equal amount of culture medium was added to the control group and the model group, and incubated overnight. The culture medium was discarded, and the cells were washed with PBS 1-2 times. 300 μL PBS was added to each well. The well plate was placed at -80°C, and the cells were lysed by freeze-thaw method (i.e., the well plate was placed at -80°C, frozen, and then thawed at 4°C for 2-3 times), and the solution was collected. The supernatant was collected by centrifugation at 4°C and 5000 rpm for 10 min. The protein concentration in the supernatant was detected according to the instructions using the BCA kit (brand: Biyuntian, item number: P0010) and the concentration was adjusted to 10 μg / mL with PBS. The protein carbonylation level in the sample was detected according to the instructions using the Cell Biolabs protein carbonylation kit (brand: Cell Biolabs, item number: STA-310).
[0060] result
[0061] Table 2 shows the protein carbonylation content of different groups.
[0062] Table 2
[0063] Group Protein carbonylation content (nmol / mg) Comparison 0.94 Model 1.69 Comparative Example 3 1.43 Comparative Example 4 1.45 Comparative Example 1 1.44 Example 1 1.26 Example 2 1.07 Example 3 1.24 Comparative Example 2 1.43
[0064] Analysis of results: According to Table 2, the protein carbonylation content was the lowest (1.07 nmol / mg) when the optimal ratio of the anti-carbonylation composition (yeast extract: EGCG = 2:1), which was lower than the protein carbonylation content when yeast extract: EGCG = 2-4:1 (1.07-1.24 nmol / mg), and also lower than the protein carbonylation content when yeast extract: EGCG = 1-4:1 (1.07-1.26 nmol / mg).
[0065] Figure 1 The protein carbonylation content of different experimental groups is shown. The total concentration of each experimental group is consistent, mean ± SD, n = 3. One-way ANOVA was used for difference analysis. Different superscript letters indicate significant differences (P < 0.05). Figure 1It can be seen that compared with the control group, the protein carbonylation content of the model group increased significantly, while the anti-carbonylation composition treatment group could significantly reduce the protein carbonylation content (the protein carbonylation results of Examples 1-3 and Comparative Examples 1-4 were significantly different from those of the model group), among which yeast extract: EGCG = 1-4: 1 can more significantly reduce the protein carbonylation content than other ratios (the protein carbonylation results of Examples 1-3 were significantly different from those of Comparative Examples 1-4), among which yeast extract: EGCG = 2: 1 can reduce the protein carbonylation content most significantly (the protein carbonylation results of Example 2 were significantly different from those of Examples 1 and 3). Cell experiments showed that by synergistically inhibiting protein carbonylation in HaCaT cells, the inhibitory effect was most obvious when yeast extract was 2 parts and EGCG was 1 part (protein carbonylation content was 1.07nmol / mg), which was the best ratio; 2-4 parts of yeast extract and 1 part of EGCG could significantly reduce the protein carbonylation content (about 1.07-1.24nmol / mg), and 1-4 parts of yeast extract and 1 part of EGCG could significantly reduce the protein carbonylation content (about 1.07-1.26nmol / mg).
[0066] The combination ratio of the two materials in the anti-carbonylation composition can synergistically inhibit the carbonylation of HaCaT cell proteins.
[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. An anti-carbonylation composition comprising 0.5-8 parts by weight of yeast extract and 0.5-5 parts by weight of epigallocatechin gallate.
2. The anti-carbonylation composition according to claim 1, wherein the anti-carbonylation composition comprises 0.6-6 parts by weight, 0.7-5 parts by weight, or 1-4 parts by weight of yeast extract, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 parts by weight of yeast extract; Preferably, the anti-carbonylation composition comprises 0.6-6 parts by weight, 0.7-5 parts by weight or 1-4 parts by weight of epigallocatechin gallate, for example 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 or 6 parts by weight of epigallocatechin gallate.
3. The anti-carbonylation composition according to claim 1 or 2, wherein the weight ratio of yeast extract to epigallocatechin gallate is 1:1-4:1, preferably 2:
1.
4. The anti-carbonylation composition according to any one of claims 1 to 3, wherein the glutathione content of the yeast extract is ≥5 wt%.
5. An oral composition comprising the anti-carbonylation composition according to any one of claims 1 to 4, optionally further comprising an oral carrier, diluent, excipient and / or other food acceptable materials; Preferably, the oral composition is used to prevent sallowness of the skin, especially facial skin, in a subject.
6. The oral composition according to claim 5, which is one or more of a drink, a jelly, a solid beverage, a candy, a soft capsule and a hard capsule.
7. A method for preparing the anti-carbonylation composition according to any one of claims 1 to 4, comprising the step of mixing yeast extract and epigallocatechin gallate.
8. Use of the anti-carbonylation composition according to any one of claims 1 to 4 or the oral composition according to claim 5 or 6 in food or health food, wherein the food or health food is used to inhibit protein carbonylation or to prevent skin sallowness, especially facial skin sallowness; preferably, skin sallowness is caused by protein carbonylation.
9. A method for inhibiting protein carbonylation in cells in vitro, comprising the step of contacting the anti-carbonylation composition according to any one of claims 1 to 4 with cells, preferably the cells are keratinocytes or fibroblasts.
10. Use of the anti-carbonylation composition according to any one of claims 1 to 4 or the oral composition according to claim 5 or 6 for non-therapeutic purposes of inhibiting protein carbonylation in cells, preferably cells are keratinocytes or fibroblasts.