New application of s-mu-conopeptide CnIIIC

Abstract

The invention discloses a novel application of s-mu-conopeptide CnIIIC, relates to the technical field of active polypeptides, and particularly relates to an application of the s-mu-conopeptide CnIIIC in inhibiting ACE activity and reducing or removing under-eye puffiness.

Description

Technical Field

[0001] The present disclosure relates to the technical field of active polypeptides, and in particular to a new use of s-μ-conopeptide CnIIIC. Background Art

[0002] People have always admired personal aesthetics. With the development of the times and the advancement of science and technology, personal aesthetics has also developed from light makeup or heavy makeup in ancient times to modern popular medical cosmetology. In contemporary society, in addition to the most common makeup, people also pay great attention to the state of the skin itself, and hope to improve the skin state through external intervention to pursue individual beauty. In the process of paying attention to skin beauty, the problem of eye bags has become one of the troubles that bothers many people. Many factors can cause eye bags, such as age factors, lack of sleep, kidney disease, etc., which can cause fluid accumulation in the eyelids and form eye bags. Promoting blood circulation in the eyes and eliminating edema are conducive to improving the problem of eye bags.

[0003] Angiotensin converting enzyme (ACE), also known as kininase II or peptidyl-carboxypeptidase, is a membrane-bound glycoprotein that can hydrolyze the histidine and leucine residues at the C-terminus of the angiotensin I peptide chain to form angiotensin II, which can cause vasoconstriction and increase blood pressure. ACE can also directly act on the adrenal cortex to promote aldosterone secretion. In addition, ACE also catalyzes the hydrolysis of bradykinin. Therefore, ACE participates in the regulation of bradykinin and the renin-angiotensin-aldosterone system, affecting a variety of physiological functions of the human body. When ACE activity is inhibited, the conversion of angiotensin I to angiotensin II is inhibited, the content of angiotensin II in the blood or tissue is reduced, and the release of aldosterone is also reduced, which can reduce the retention of water and sodium in local tissues, thereby achieving the effect of relieving edema. At present, the active substances commonly used in the field of skin care products to eliminate edema and remove eye bags are acetyl tetrapeptide-5 (eye serine peptide), which can effectively inhibit ACE activity, eliminate eyelid edema, and achieve the effect of removing eye bags.

[0004] There are still not many active substances that can inhibit ACE activity and eliminate eye puffiness. In order to meet the needs of consumers, it is very necessary to continue to develop active substances that have the effect of removing eye bags. Summary of the invention

[0005] 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.

[0006] In the present disclosure, the term "s-μ-conopeptide CnIIIC" refers to a polypeptide composed of 22 amino acid residues with a sequence of pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2, which are connected into a ring by three pairs of disulfide bonds, and the three pairs of disulfide bonds are located at the positions of Cys3-Cys15, Cys4-Cys21 and Cys10-Cys22, respectively.

[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.

[0008] In the present disclosure, the term "periocular skin" should be understood as the skin tissue covering the orbital area, including the upper eyelid, lower eyelid, canthus and tail of the eye.

[0009] In one aspect, the present disclosure provides use of s-μ-conopeptide CnIIIC or a salt thereof in preparing a composition for reducing or removing eye bags, and / or for inhibiting the activity of angiotensin converting enzyme.

[0010] In some embodiments, the composition is used to reduce or remove eye bags caused by edema of the lower eyelid.

[0011] In some embodiments, the mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-5%.

[0012] In some embodiments, the mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-1%.

[0013] In some embodiments, the mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-0.1%.

[0014] In some embodiments, the mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-0.05%.

[0015] In some embodiments, the mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-0.01%.

[0016] In some embodiments, the mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-0.0025%.

[0017] The s-μ-conopeptide CnIIIC or its salt disclosed in the present invention can inhibit ACE activity, improve microcirculation, and thus alleviate local tissue edema. Therefore, when s-μ-conopeptide CnIIIC or its salt acts on the skin around the eyes, especially the lower eyelid, it can alleviate edema, thereby achieving the effect of reducing or removing eye bags.

[0018] Another aspect of the present disclosure provides a method for reducing or removing eye bags, comprising the following steps: topically applying an effective amount of s-μ-conopeptide CnIIIC or a salt thereof, or a composition containing an effective amount of s-μ-conopeptide CnIIIC or a salt thereof, to the skin area around the eyes; the method is non-therapeutic.

[0019] The effective amount of the disclosed s-μ-conopeptide CnIIIC 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 the s-μ-conopeptide CnIIIC.

[0020] "Effective amount" means an amount of the s-μ-conopeptide CnIIIC of the present disclosure that is non-toxic but sufficient to provide the desired effect. The s-μ-conopeptide CnIIIC 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.

[0021] In some embodiments, the periocular skin area to which the s-μ-conopeptide CnIIIC or a salt thereof, or a composition containing s-μ-conopeptide CnIIIC or a salt thereof is applied includes the upper eyelid, the lower eyelid, the corner of the eye, and the tail of the eye.

[0022] In some embodiments, the composition is a periocular skin care preparation.

[0023] In some embodiments, the formulation of the composition includes eye cream, eye mask, serum or facial mask.

[0024] In another aspect of the present disclosure, an eye cream is provided, comprising s-μ-conopeptide CnIIIC or a salt thereof, wherein the mass percentage concentration of the s-μ-conopeptide CnIIIC or a salt thereof in the eye cream is 0.0001%-0.0025%.

[0025] In another aspect of the present disclosure, an eye mask is provided, comprising s-μ-conopeptide CnIIIC or a salt thereof, wherein the mass percentage concentration of the s-μ-conopeptide CnIIIC or a salt thereof in the eye mask is 0.0001%-0.0025%.

[0026] Another aspect of the present disclosure provides an eye essence, comprising s-μ-conopeptide CnIIIC or a salt thereof, wherein the mass percentage concentration of the s-μ-conopeptide CnIIIC or a salt thereof in the eye essence is 0.0001%-0.0025%.

[0027] The present disclosure has the following advantages and effects:

[0028] 1. In vitro experiments have shown that the s-μ-conopeptide CnIIIC disclosed in the present invention can effectively inhibit ACE activity; clinical trials have shown that the s-μ-conopeptide CnIIIC disclosed in the present invention can significantly reduce or remove eye bags. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solution of the present disclosure, the drawings required for use in the description of the present disclosure will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is a result diagram of the eye bag volume value in the human eye bag removal efficacy test of Example 3 in the present disclosure.

[0031] Figure 2 This is a result diagram of the rate of change of eye bag volume in the human eye bag removal efficacy test of Example 3 of the present disclosure. DETAILED DESCRIPTION

[0032] 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 the accompanying drawings and 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 this field without creative work are within the scope of protection of the claims attached to the present disclosure.

[0033] Unless otherwise specified, the experimental reagents and materials used in the present disclosure can be obtained commercially.

[0034] Example 1 Preparation of Cono Peptide Emulsion

[0035] A cono peptide emulsion, the preparation method is as follows: Components Mass percentage s-μ-conopeptide CnIIIC 0.0025% Carbomer 0.1% Hexanediol 1% Pentylene glycol 2% water Top up to 100% Arginine Moderate

[0036] According to the formula, take the formulated amount of carbomer, hexylene glycol, pentanediol, add appropriate amount of water, and heat to swell; after the carbomer is completely swollen, it is homogenized and crushed, and then s-μ-conopeptide CnIIIC and the remaining amount of water are added, stirred evenly, and finally arginine is used to adjust the pH value of the emulsion to 5.5-6.5 to obtain a conopeptide emulsion.

[0037] Example 2 ACE inhibition test

[0038] 2.1 Reagents and Materials

[0039] PBS buffer (pH 8.3), N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) (purchased from McLean), and angiotensin converting enzyme (ACE) (purchased from Sigma).

[0040] 2.2 Instruments

[0041] Microplate reader, gas bath constant temperature oscillator.

[0042] 2.3 Samples to be tested

[0043] s-μ-conopeptide CnIIIC, tested at 1ppm, 100ppm, 500ppm and 1000ppm; acetyl tetrapeptide-5, tested at 100ppm. The samples were dissolved in PBS buffer (pH 8.3).

[0044] 2.4 Experimental Methods

[0045] Divide into four groups according to the sample group, sample zero adjustment group, blank control group and blank zero adjustment group. Take a 96-well plate, add 50μL sample and 50μL FAPGG to the sample group and sample zero adjustment group respectively; add 50μL PBS buffer and 50μL FAPGG to the blank control group and blank zero adjustment group respectively. After incubation at 37℃ for 5min, add 10μL ACE to each well of the blank control group and sample group, and add 10μL PBS to each well of the blank zero adjustment group and sample zero adjustment group, and incubate at 37℃ for 30min. Measure the absorbance A after enzyme catalysis reaction at 340nm.

[0046] Enzyme activity inhibition rate (%) =

[0047] Where: A1 is the OD of the blank control group 340value, A2 is the OD of the blank zero adjustment group 340 value, A3 is the OD of the sample group 340 A4 is the OD of the sample zeroing group. 340 value.

[0048] 2.5 Experimental Results

[0049] Angiotensin converting enzyme (ACE) is a key enzyme in the renin-angiotensin system (RAS), which can hydrolyze angiotensin I to produce angiotensin II with vasoconstriction. In addition, angiotensin converting enzyme can degrade bradykinin. Bradykinin is a circulating nonapeptide that stimulates lymphatic drainage by activating muscles around the vascular network. Therefore, by inhibiting angiotensin converting enzyme, the level of angiotensin II can be reduced, the microcirculation of the eye skin can be improved, and the level of bradykinin can be increased, the lymphatic circulation can be improved, thereby promoting the excretion of harmful fluids in the skin, eliminating edema, and playing a role in removing eye bags. Angiotensin converting enzyme can specifically degrade the substrate FAPGG, and the change in the OD value at 340 nm is proportional to the disappearance of FAPGG, which can quantify the activity of ACE. Therefore, this experiment quantitatively analyzes the ACE activity and inhibition rate by detecting the OD value of each sample at 340 nm.

[0050] The results showed that the inhibition rate of acetyl tetrapeptide-5 on ACE activity was 13.98% at 100ppm, while the inhibition rate of s-μ-conopeptide CnIIIC on ACE activity was 23.30% at 1ppm. It can be seen that s-μ-conopeptide CnIIIC showed a significant inhibitory effect on ACE activity at 1ppm, and its ACE activity inhibitory effect was stronger than that of acetyl tetrapeptide-5 at 100ppm. The inhibition rate of s-μ-conopeptide CnIIIC on ACE activity was 48.21% at a concentration of 100ppm, which was significantly higher than that of acetyl tetrapeptide-5. The inhibition rate of s-μ-conopeptide CnIIIC on ACE activity was 56.09% at a concentration of 500ppm; the inhibition rate of s-μ-conopeptide CnIIIC on ACE activity was 67.20% at a concentration of 1000ppm. The above results prove that s-μ-conopeptide CnIIIC can significantly inhibit the activity of ACE, improve the microcirculation and lymphatic circulation of the eye skin, eliminate edema, and reduce or remove eye bags.

[0051] Example 3 Test on the efficacy of removing eye bags on human body

[0052] 3.1 Subjects

[0053] There were 15 subjects in each group, for a total of 45 subjects.

[0054] Inclusion criteria: Volunteers aged 30-55 years, male or female, with eye bags around the eyes.

[0055] Exclusion criteria: those who have applied any topical medication to the test site within the past two months; those who have used antihistamines in the past week or immunosuppressants in the past month; those who have clinically unhealed inflammatory skin diseases; insulin-dependent diabetes patients; patients with asthma or other chronic respiratory diseases being treated; those who have received anti-cancer chemotherapy in the past six months; patients with immunodeficiency or autoimmune diseases; breastfeeding or pregnant women; those who have undergone bilateral mastectomy and bilateral axillary lymph node resection; those whose skin areas to be measured have scars, pigments, atrophy, port wine stains or other blemishes that affect the determination of test results; those who participate in other clinical test researchers; those with highly sensitive constitutions; those who are involuntary participants or cannot complete the specified content according to the test requirements. Those who fail to use the test product as required and cannot determine the efficacy; those who withdraw on their own or fail to complete the specified observation period, which affects the determination of efficacy.

[0056] 3.2 Samples to be tested

[0057] s-μ-conopeptide CnIIIC (25 ppm), the conopeptide emulsion of Example 1;

[0058] Acetyl tetrapeptide-5 (25 ppm), prepared according to the preparation method of Example 1, except that the conopeptide in Example 1 was replaced by acetyl tetrapeptide-5;

[0059] The placebo was prepared according to the preparation method of Example 1, except that the cono peptide in Example 1 was replaced by an equal weight of water.

[0060] 3.3 Test Instruments

[0061] Skin rapid three-dimensional imaging system PRIMOS-CR; intelligent skin detector.

[0062] 3.4 Purpose

[0063] By recruiting subjects to try the test products s-μ-conopeptide CnIIIC, acetyl tetrapeptide-5 and placebo, and combining the skin rapid three-dimensional imaging system PRIMOS-CR and the intelligent skin detector to jointly detect the volume of facial eye bags, a comprehensive evaluation of the eye bag removal effect of s-μ-conopeptide CnIIIC and acetyl tetrapeptide-5 was conducted.

[0064] 3.5 Methods

[0065] Each time the subject visits, he / she washes his / her face with the designated cleansing liquid and dries it with a dandruff-free absorbent paper towel. He / she sits quietly in a constant temperature and humidity room for 30 minutes. After the meditation, the data of the instrument test is collected for the subjects who participated in the test.

[0066] After the test product is distributed, the researchers will explain the use of the test product to the subjects and guide them to try the test product on site, fully covering the area to be measured with the test product. From the second visit onwards, the subjects need to bring the test product back and hand it over to the researchers for inspection, and record the subjects' use.

[0067] The eye bag volume data of each subject were collected before the subject used the product (Day 0) and on the 14th day after using the product.

[0068] Change rate = (data after using the product - data before using the product) / data before using the product * 100%

[0069] 3.6 Results

[0070] The test data of each group of 15 people in this experiment are all valid data, and no one is missing from the group. Figure 1 , Figure 2 Compared with the 0th day before use, the volume of eye bags of the subjects decreased significantly after using s-μ-conopeptide CnIIIC for 14 days ( P <0.01), the rate of change of eye bag volume was -8.08%, indicating that s-μ-conopeptide CnIIIC can reduce eye bags at low concentrations; after using acetyl tetrapeptide-5 for 14 days, the eye bag volume value of the subjects did not change significantly, and there was no statistical difference in the eye bag volume value before and after use, and the rate of change of eye bag volume was 1.23%, indicating that acetyl tetrapeptide-5 failed to effectively improve the eye bag volume at low concentrations; after using the placebo for 14 days, the eye bag volume value of the subjects increased and was not significant, and the rate of change of eye bag volume was 0.60%.

[0071] The above results prove that s-μ-conopeptide CnIIIC has a significant clinical effect of reducing the volume of eye bags, and can be used to reduce or remove eye bags, and is particularly effective in reducing or removing eye bags caused by lower eyelid edema.

[0072] 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.

[0073] 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 s-μ-conopeptide CnIIIC or a salt thereof in the preparation of a composition for reducing or removing eye bags, and / or for inhibiting the activity of angiotensin converting enzyme.

2. The use according to claim 1, characterized in that The composition is used to reduce or remove eye bags caused by lower eyelid puffiness.

3. The use according to claim 1 or 2, characterized in that The mass percentage concentration of s-μ-conopeptide CnIIIC or its salt in the composition is 0.0001%-5%.

4. A method for reducing or removing eye bags, characterized in that: The following steps are involved: An effective amount of s-μ-conopeptide CnIIIC or a salt thereof, or a composition containing an effective amount of s-μ-conopeptide CnIIIC or a salt thereof is topically applied to the periocular skin area; the method is non-therapeutic.

5. The method according to claim 4, characterized in that The periocular skin area includes the upper eyelid, lower eyelid, corner of the eye and tail of the eye.

6. The method according to claim 4 or 5, characterized in that: The composition is a skin care preparation around the eyes.

7. The method according to claim 4 or 5, characterized in that: The preparation of the composition includes eye cream, eye mask, essence or facial mask.

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