New application of radix pseudostellariae cyclic peptide B

Abstract

The invention discloses a novel application of radix pseudostellariae cyclic peptide B, relates to the technical field of active polypeptides, and particularly relates to an application of the radix pseudostellariae cyclic peptide B in oil control, acne removal, moisturizing, soothing or repairing.

Description

Technical Field

[0001] The present disclosure relates to the technical field of active polypeptides, and in particular to a new use of Pseudostigma tuberculin B. Background Art

[0002] Heterophyllin B is a cyclic peptide compound extracted from the traditional Chinese medicine Pseudostellaria heterophylla. Chinese patent application CN108402988A discloses that Heterophyllin B has the effects of promoting collagen production, scavenging free radicals, inhibiting MMPs, etc., and has the effect of firming and anti-wrinkle; Chinese patent application CN108498784A discloses that Heterophyllin B has the effect of resisting Candida albicans; Chinese patent application CN105106280A discloses that Heterophyllin B can be used to inhibit the production of tyrosinase and melanin, and has the effect of whitening.

[0003] In the field of cosmetics, the diversity of the efficacy of active ingredients is an important direction for product development. At present, the main application of Pseudostellariae Cyclic Peptide B in the field of cosmetics is to stimulate collagen production and firm and anti-wrinkle. However, as a natural compound with complex biological activity, Pseudostellariae Cyclic Peptide B still has the significance of continued exploration for its potential other functional uses. In-depth research on the other functional uses of Pseudostellariae Cyclic Peptide B can not only expand its scope of application in the field of cosmetics, but also provide new ideas and technical support for the development of multifunctional and efficient skin care products. The present disclosure aims to reveal the new functional uses of Pseudostellariae Cyclic Peptide B in cosmetics, fill the gaps in the prior art, and provide a scientific basis for its further development and application. Summary of the invention

[0004] In order to help those skilled in the art to accurately understand the technical solutions of the present disclosure, the present disclosure explains and defines the following terms.

[0005] In the present disclosure, the term "Pseudostellariae cyclotide B" refers to a cyclic polypeptide consisting of 8 amino acid residues with the following structure:

[0006]

[0007] In the present disclosure, the term "skin" is understood to refer to the multiple layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends are included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes, etc. In the present disclosure, the term "skin" includes the scalp.

[0008] In one aspect, the present disclosure provides use of Pseudostellariae Cyclotide B or a salt thereof in preparing a composition for oil control or acne removal.

[0009] In some embodiments, the composition is used to reduce skin oil synthesis or secretion.

[0010] In some embodiments, the composition is used to inhibit 5α-reductase expression.

[0011] In some embodiments, the composition is used to reduce the occurrence of acne and / or pimples due to oil clogging pores.

[0012] The Pseudostellariae Cyclic Peptide B or its salt disclosed in the present invention can inhibit the expression of 5α-reductase, and at the same time can reduce the synthesis or secretion of skin oil, thereby reducing the occurrence of acne and / or blackheads caused by oil blocking pores. Therefore, when Pseudostellariae Cyclic Peptide B or its salt acts on the skin, it can not only reduce the synthesis or secretion of skin oil to achieve the effect of oil control, but also help reduce the occurrence of acne and blackheads, and achieve the effect of acne removal.

[0013] On the other hand, the present disclosure provides use of Pseudostellariae Cyclotide B or a salt thereof in preparing a composition for moisturizing, soothing or repairing.

[0014] In some embodiments, the composition is used to inhibit the activity of hyaluronidase, reduce the neurological response of the skin to irritants, or enhance the skin's tolerance.

[0015] In some embodiments, the composition is used to increase the content of hyaluronic acid.

[0016] In some embodiments, the composition is used to alleviate skin inflammatory responses mediated by low molecular weight hyaluronic acid oligosaccharide fragments.

[0017] Low molecular weight hyaluronic acid oligosaccharide fragments (hyaluronan oligosaccharides, o-HA) are oligosaccharide fragments composed of 2-25 disaccharide units produced by endogenous hyaluronidase digestion of hyaluronic acid polymers, which have the activity of activating CD44 or TLR receptors, and can also induce macrophages to release IL-1β, thereby mediating the occurrence of skin inflammatory reactions. When the hyaluronidase activity in the body is abnormally increased, the hyaluronic acid polymer will be degraded into o-HA, resulting in an inflammatory reaction in the skin. The Pseudostellariae Radix Cyclotide B or its salt described in the present disclosure can inhibit the activity of hyaluronidase, thereby reducing the production of o-HA, and then reducing the occurrence of skin inflammatory reactions. Therefore, when Pseudostellariae Radix Cyclotide B or its salt acts on the skin, it can alleviate the skin inflammatory reaction and achieve the effect of soothing and de-redness.

[0018] Another aspect of the present disclosure provides a method for reducing skin oil secretion, comprising the following steps: topically applying an effective amount of Pseudostellariae Cyclic Peptide B or a salt thereof, or a composition containing an effective amount of Pseudostellariae Cyclic Peptide B or a salt thereof to the skin area to be applied; the method is for non-therapeutic purposes.

[0019] Another aspect of the present disclosure provides a method for improving skin sensitivity, comprising the following steps: topically applying an effective amount of Pseudostellariae Cyclic Peptide B or its salt, or a composition containing an effective amount of Pseudostellariae Cyclic Peptide B or its salt to the skin area to be applied; the method is non-therapeutic.

[0020] The effective amount of Pseudostellariae Cyclic Peptide B of the present disclosure to be administered and its dosage will depend on many factors, including the age, status of the user, severity of the condition, route and frequency of administration, and the specific properties of Pseudostellariae Cyclic Peptide B.

[0021] "Effective amount" means an amount of the Pseudostellariae Cyclic Peptide B of the present disclosure that is non-toxic but sufficient to provide the desired effect. The Pseudostellariae Cyclic Peptide B of the present disclosure is used in the composition of the present disclosure at an effective concentration to obtain the desired effect. In some embodiments, the concentration is between 0.00000001% (by weight) and 20% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.000001% (by weight) and 15% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 10% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 5% (by weight) relative to the total weight of the composition.

[0022] The present disclosure has the following advantages and effects:

[0023] 1. The Pseudostellariae Cyclic Peptide B disclosed in the present invention can effectively inhibit the activity of hyaluronidase and the expression of 5α-reductase, and can also effectively inhibit the synthesis and secretion of oil by human sebaceous gland cells. By applying an effective amount of Pseudostellariae Cyclic Peptide B on human skin, the oil secretion of the skin can be reduced, the skin sensitivity can be improved, and the effects of oil control, acne removal, moisturizing, soothing and repairing can be achieved. DETAILED DESCRIPTION

[0024] In order to make the objects, features and advantages of the present disclosure more obvious and understandable, the present disclosure is further described in detail below in conjunction with embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the claims attached to the present disclosure.

[0025] Unless otherwise specified, the experimental reagents and materials used in the present disclosure can be obtained commercially.

[0026] Example 1 Hyaluronidase inhibition test

[0027] 1.1 Reagents and Materials

[0028] Sodium acetate buffer (pH=5.6), hyaluronidase, calcium chloride, sodium hyaluronate, acetylacetone, anhydrous ethanol, P-DAB (obtained by uniformly mixing p-dimethylaminobenzaldehyde (0.8 g) and concentrated hydrochloric acid (15 mL) and an equal amount of glacial acetic acid).

[0029] 1.2 Instruments

[0030] ELISA reader, electronic balance, gas bath constant temperature oscillator.

[0031] 1.3 Samples to be tested and grouping

[0032] 1.3.1 Samples to be tested

[0033] Pseudostellariae cyclopeptide B and dipotassium glycyrrhizinate, the test concentrations are 1ppm, 10ppm, 20ppm, 50ppm, and 100ppm.

[0034] 1.3.2 Grouping

[0035] Sample group: test sample, hyaluronidase, sodium hyaluronate;

[0036] Sample zeroing group: test sample, sodium acetate buffer;

[0037] Blank control group: distilled water, hyaluronidase, sodium hyaluronate;

[0038] Blank zero adjustment group: distilled water, sodium acetate buffer.

[0039] 1.4 Experimental methods

[0040] Hyaluronidase and sodium hyaluronate were dissolved in sodium acetate buffer.

[0041] Take a 96-well plate, add 25μL of the sample to be tested and 25μL of hyaluronidase (500U / mL) to the sample group; add 25μL of the sample to be tested and 25μL of sodium acetate buffer to the sample zeroing group; add 25μL of distilled water and 25μL of hyaluronidase (500U / mL) to the blank control group; add 25μL of distilled water and 25μL of sodium acetate buffer to the blank zeroing group. After shaking in a 37℃ constant temperature air bath for 20min, add 5μL of calcium chloride solution (2.5mol / L) to each well, and shake in a 37℃ constant temperature air bath for 20min. Add 25μL of sodium hyaluronate (1mg / mL) to the sample group and the blank control group, add 25μL of sodium acetate buffer to the sample zeroing group and the blank zeroing group, shake in a 37℃ constant temperature air bath for 40min, and then place at room temperature for 10min. Add 25 μL of distilled water, 5 μL of sodium hydroxide solution (5 mol / L), and 25 μL of acetylacetone solution to each well, boil in water for 15 min, ice bath for 10 min, and finally place at room temperature for 10 min. Add 50 μL of P-DAB to each well, shake thoroughly, add 100 μL of anhydrous ethanol, place at room temperature for 30 min, and measure OD at 570 nm. 570 value.

[0042]

[0043] Where: A1 is the OD of the sample zeroing group 570 value, A2 is the sample group OD 570 A3 is the OD of the blank zero adjustment group. 570 A4 is the OD of the blank control group. 570 value.

[0044] 1.5 Experimental Results

[0045] Hyaluronidase activity is closely related to type I allergic reactions. Inhibiting hyaluronidase activity can play a soothing and anti-allergic role. Therefore, inhibiting hyaluronidase activity is often used as an evaluation indicator for studying soothing and anti-allergic effects. In addition, hyaluronidase is a hydrolase that degrades hyaluronic acid. Inhibiting hyaluronidase activity can reduce the degradation of hyaluronic acid in cells and increase the content of hyaluronic acid in cells, which is beneficial to increase the hydration of the skin or mucous membranes, repair the skin barrier, and play a moisturizing, soothing, and repairing role.

[0046] Sodium hyaluronate is a substrate of hyaluronidase. Under the catalytic action of hyaluronidase, sodium hyaluronate is degraded to generate glucuronic acid and N-acetylglucosamine, which develop color and absorb visible light of 570nm wavelength under the action of P-DAB. In this experiment, the test sample is used to treat hyaluronidase, and the reaction amount of sodium hyaluronate is detected to determine whether the test sample disclosed in the present invention can inhibit the activity of hyaluronidase.

[0047] The results of the effects of the test samples on the inhibition of hyaluronidase activity are shown in Table 1.

[0048] Table 1 Effect of the tested samples on the inhibition of hyaluronidase activity

[0049]

[0050] Note: Compared with the Control group, **P<0.01, ***P<0.001.

[0051] The results showed that dipotassium glycyrrhizinate at low concentrations did not show any effect on the activity of hyaluronidase, while Pseudostellaria baicalensis cyclic peptide B at low concentrations had a significant inhibitory effect on hyaluronidase. It can be used to improve skin sensitivity, inhibit type I allergic reactions, reduce the degradation of hyaluronic acid in cells, and repair the skin barrier, thereby reducing the skin's neurological response to irritants, enhancing skin tolerance, and has moisturizing, soothing and repairing effects.

[0052] Example 2 Oil content test

[0053] 2.1 Reagents and Materials

[0054] 0.25% trypsin digestion solution (prepared with 0.25 g trypsin in 100 mL water), PBS, FFA (prepared with linoleic acid and palmitic acid mixed in a molar ratio of 1:1), and Oil Red O staining kit.

[0055] 2.2 Instruments

[0056] Constant temperature CO2 incubator, clean bench, gas bath constant temperature oscillator.

[0057] 2.3 Cell lines

[0058] Human sebaceous gland cells SZ-95.

[0059] 2.4 Samples to be tested and grouping

[0060] 2.4.1 Samples to be tested

[0061] Pseudostellariae cyclotide B was dissolved in PBS and tested at concentrations of 20ppm, 50ppm, and 100ppm;

[0062] Simvastatin, tested at a concentration of 0.05 μM.

[0063] 2.4.2 Grouping

[0064] Experimental group: samples to be tested, FFA;

[0065] Normal group: PBS;

[0066] Model group: PBS, FFA.

[0067] 2.5 Experimental methods

[0068] Take a bottle of SZ-95 cells in good condition in the exponential growth phase, add 0.25% trypsin digestion solution, digest to make the attached cells fall off, and count (1-4)×10 5 / mL, and make a cell suspension. Take an appropriate amount of cell suspension and inoculate it on a 12-well plate containing complete culture medium, and culture it in a constant temperature CO2 incubator for 24h. Except for the normal group, PBS was added to each well of the remaining wells to make the final concentration of 225μmol / mL. While inducing modeling, the corresponding samples to be tested were added to the experimental groups respectively, and cultured in a constant temperature CO2 incubator for 48h. Aspirate the culture medium and stain according to the instructions of the Oil Red O staining kit.

[0069] The solution in the well plate was discarded, isopropanol was added to dissolve the Oil Red O dye solution, and the absorbance value was detected at 492 nm using an ELISA reader. The relative content of cellular lipid secretion was calculated based on the absorbance value.

[0070] 2.6 Experimental Results

[0071] Oil Red O is a fat-soluble dye that is highly soluble in fat. Its staining principle is that Oil Red O can specifically adsorb neutral triglycerides, lipids, and lipoproteins in tissues and cells to stain fat. FFA is an inducer. Under the stimulation and induction of FFA, SZ-95 cells will secrete a large amount of oil that can be stained with Oil Red O. This experiment used test samples to treat SZ-95 cells stimulated by FFA, and detected the amount of oil produced by SZ-95 cells to determine whether Pseudostellariae Cyclotide B can inhibit the secretion of oil by human sebaceous gland cells.

[0072] The results of the effects of the test samples on the lipid secretion of SZ-95 cells are shown in Table 2.

[0073] Table 2 Relative secretion of lipids in SZ-95 cells

[0074]

[0075]

[0076] Note: Compared with the normal group, ### P<0.001; ***P<0.001 compared with the model group.

[0077] Experimental results show that Pseudostellaria baicalensis cyclic peptide B can significantly inhibit the synthesis and secretion of oil by sebaceous gland cells and slow down oil deposition. It can be used to improve problems such as excessive skin oiliness and skin water-oil imbalance. It can reduce the occurrence of blackheads or acne, and also help the skin recover after blackheads or acne occur. It has the effect of controlling oil and removing acne.

[0078] Example 3 5α-Reductase Test

[0079] 3.1 Reagents and Materials

[0080] 0.25% trypsin digestion solution (prepared with 0.25 g trypsin in 100 mL water), PBS, FFA (prepared with linoleic acid and palmitic acid mixed in a molar ratio of 1:1), RIPA lysis buffer, 5α-reductase ELISA kit, and BCA protein quantification kit.

[0081] 3.2 Instruments

[0082] Constant temperature CO2 incubator, clean workbench.

[0083] 3.3 Cell lines

[0084] Human sebaceous gland cells SZ-95.

[0085] 3.4 Samples to be tested and grouping

[0086] 3.4.1 Samples to be tested

[0087] Pseudostellariae cyclotide B was dissolved in PBS and tested at concentrations of 20ppm, 50ppm, and 100ppm;

[0088] Simvastatin, tested at a concentration of 0.05 μM.

[0089] 3.4.2 Grouping

[0090] Experimental group: samples to be tested, FFA;

[0091] Normal group: PBS;

[0092] Model group: PBS, FFA.

[0093] 3.5 Experimental methods

[0094] Take a bottle of SZ-95 cells in good condition in the exponential growth phase, add 0.25% trypsin digestion solution, digest to make the attached cells fall off, and count (1-4)×10 5 / mL, and make a cell suspension. Take an appropriate amount of cell suspension and inoculate it on a 12-well plate containing complete culture medium, and culture it in a constant temperature CO2 incubator for 24h. Except for the normal group, which added PBS, FFA was added to each well of the remaining wells to make the final concentration of 225μmol / mL. While inducing modeling, the experimental groups added the corresponding samples to be tested and cultured in a constant temperature CO2 incubator for 48h. Collect the cells, centrifuge to obtain the cell pellet, add RIPA lysis buffer, shake it evenly with a vortex instrument 3 times (30s / time, 3min interval), and centrifuge at 12000rpm*10min. Aspirate the lysis supernatant and test it with an ELISA kit, and use a BCA protein quantification kit to detect the total protein concentration of the supernatant.

[0095] 3.6 Experimental Results

[0096] 5α-reductase is an important metabolic enzyme in the skin, which can irreversibly convert testosterone into dihydrotestosterone (DHT), which can induce excessive secretion of oil by sebaceous glands. Therefore, the expression of 5α-reductase can be tested to determine whether the test substance has the ability to relieve excessive secretion of oil by sebaceous glands. In this experiment, the test sample was used to treat SZ-95 cells induced by FFA stimulation, and the content of 5α-reductase in SZ-95 cells was detected to determine whether Pseudostellariae Cyclotide B can inhibit the expression of 5α-reductase in human sebaceous gland cells.

[0097] The results of the effects of the test samples on the 5α-reductase content in SZ-95 cells are shown in Table 3.

[0098] Table 3 Relative content of 5α-reductase

[0099]

[0100] Note: Compared with the normal group, ### P<0.001; compared with the model group, **P<0.01, ***P<0.001.

[0101] Experimental results show that Pseudostellariae Cyclic Peptide B can significantly inhibit the expression of 5α-reductase in sebaceous gland cells, thereby alleviating excessive oil secretion by sebaceous glands. It can be used to improve the problem of excessive oil secretion of the skin, reduce the occurrence of blackheads or acne, and also help the skin recover after blackheads or acne occur. It has the effect of controlling oil and removing acne.

[0102] In the present disclosure, relational terms such as first and second, etc. are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the statement "comprise one..." do not exclude the presence of other identical elements in the process, method, article or terminal device including the elements.

[0103] Although the specific embodiments of the present disclosure are described for the purpose of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. These modifications or improvements should all fall within the scope of the appended claims of the present disclosure.

Claims

1. Use of Pseudostellariae Cyclotide B or its salt in preparing a composition for oil control or acne removal.

2. The use according to claim 1, characterized in that The composition is used for reducing skin oil synthesis or secretion.

3. The use according to claim 1, characterized in that The composition is used for inhibiting the expression of 5α-reductase.

4. The use according to claim 1, characterized in that The composition is used to reduce the occurrence of acne and / or pimples due to oil clogging pores.

5. Use of Pseudostellariae Cyclic Peptide B or its salt in the preparation of a composition for moisturizing, soothing or repairing.

6. The use according to claim 5, characterized in that The composition is used for inhibiting the activity of hyaluronidase, reducing the neurological response of the skin to irritants or enhancing the tolerance of the skin.

7. The use according to claim 5, characterized in that The composition is used to increase the content of hyaluronic acid.

8. The use according to claim 5, characterized in that The composition is used for alleviating skin inflammatory response mediated by low molecular weight hyaluronic acid oligosaccharide fragments.

9. A method for reducing skin oil secretion, characterized in that: The following steps are involved: An effective amount of Pseudostellariae Cyclic Peptide B or its salt, or a composition containing an effective amount of Pseudostellariae Cyclic Peptide B or its salt is topically applied to the skin area to be applied; the method is non-therapeutic.

10. A method for improving skin sensitivity, characterized in that: The following steps are involved: An effective amount of Pseudostellariae Cyclic Peptide B or its salt, or a composition containing an effective amount of Pseudostellariae Cyclic Peptide B or its salt is topically applied to the skin area to be applied; the method is non-therapeutic.

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