Use of polyterpenoids as kallikrein inhibitors in cosmetics

Abstract

The use of polyterpenoids as kallikrein inhibitors in cosmetics. The polyterpenoids are selected from vitamin K2, coenzyme Q or a carotenoid. The polyterpenoids have an inhibitory effect on kallikreins, i.e., KLKs. The polyterpenoids are added to cosmetics as small molecule kallikrein inhibitors, so that the activity of kallikreins can be effectively inhibited, the balance of proteases and inhibitors thereof can be promoted, the damaged skin barrier can be repaired, and the dynamic balance of the skin barrier can be maintained.

Description

Application of polyterpenoids as kallikrein inhibitors in cosmetics Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to the application of polyterpenoid compounds in cosmetics as a small molecule inhibitor targeting kallikrein that effectively repairs the skin barrier. Background Art

[0002] The skin, the outermost layer of the human body, performs barrier, absorption, secretion, excretion, metabolism, temperature regulation, and sensory functions. The skin barrier is fundamental to this function. It protects the skin from adverse external factors while also preventing the loss of moisture, nutrients, and electrolytes from the skin and the body, helping to maintain a relatively stable internal environment.

[0003] Damage to the skin barrier function can cause dry skin, weaken antioxidant and anti-radiation functions, thereby aggravating skin aging and causing atopic dermatitis, eczema, photodermatitis, sensitivity, acne, etc.

[0004] The skin's physical barrier is primarily composed of structures associated with the stratum corneum and tight junctions. The stratum corneum, located at the outermost layer of the skin, forms a "brick wall" structure and is the primary component of the skin barrier. Tight junctions are also crucial for maintaining the integrity of the skin barrier. Keratinocytes act as a physical barrier, reducing mechanical damage and the intrusion of foreign substances. Their components and structures are also crucial components of the skin barrier, including filaggrin, cornified envelope, corneodesmosomes, proteases, and intercellular lipids.

[0005] Corneocytes are constantly shed from the surface of the stratum corneum, which is of great significance for maintaining the homeostasis of the stratum corneum. The shedding of corneocytes is mainly regulated by kallikreins (KLKs)-related peptidases, such as KLK5, KLK7, and KLK14 (Brattsand M, Stefansson K, Lundh C, et al. A proteolytic cascade of kallikreins in the stratum corneum [J]. J Invest Dermatol, 2005, 124 (1): 198-203.). KLKs belong to the serine protease family and are expressed in the granular layer. KLKs can cleave corneous desmosomes. KLK5 and KLK14 have trypsin activity and can cleave desmoglein, while KLK7 has chymotrypsin activity and can cleave desmocollin and corneous desmosome protein. In particular, KLK5 and KLK7 are highly correlated with desquamation and can induce inflammatory factors. Among them, KLK5 can degrade corneal desmosomes, destroy the integrity of the stratum corneum, inhibit the release of lamellar bodies, hinder the repair of permeability barrier function, and induce the expression of inflammatory factors, playing an important role in the inflammatory response of skin diseases.

[0006] The stability of the skin barrier depends on the balance between the differentiation of keratinocytes in the stratum granulosum into keratinocytes and the shedding of keratinocytes in the surface layers of the skin (i.e., desquamation). The desquamation process involves the degradation of desmosomes, which are involved in the degradation of desmosomes by KLKs (kallikreins). Since desmosomes are the adhesion proteins of keratinocytes, the degradation of desmosomes leads to the shedding of keratinocytes and skin desquamation. Evidence suggests that local or temporary dysregulation of KLK activity is one of the main reasons for the loss of skin barrier stability in patients with atopic dermatitis.

[0007] In the cosmetics field, Han Dan et al. (Methods and Applications for Repairing Skin Barrier Function, Journal of Chemical Science, July 2020, Vol. 43, No. 7) discussed the composition and damage factors of the skin barrier, and proposed solutions for skin barrier repair from several aspects, including lipids, natural moisturizing factors, moisturizers, active ingredients, and pH. This literature does not address or consider damage caused by dysregulation or abnormal expression of kallikreins (KLKs), which may affect the effectiveness of barrier repair. Currently, KLK inhibitors are relatively rare in the field of skin care cosmetics.

[0008] Kallikreins are involved in regulating the physiological functions of the cardiovascular, renal, and nervous systems, and are closely related to the occurrence of diseases such as heart disease, kidney disease, inflammatory response, and cancer. In the medical field, research on kallikrein inhibitors in the cardiovascular system has made rapid progress. For the prevention and treatment of skin diseases, CN201580005402.3 discloses a new kallikrein 7 inhibitor and the use of KLK7 inhibitors for the prevention and treatment of skin diseases. The inhibitor mainly targets KLK7, and the metabolism of keratinocytes is mainly regulated by the hydrolysis cascade of related proteases such as KLK5, KLK7, and KLK14. For the prevention and treatment of skin atopic dermatitis, KLKs regulation is becoming a development direction.

[0009] Therefore, in the field of cosmetic applications, the development of small molecule inhibitors of kallikreins that are active against KLKs (KLK5, KLK7, and KLK14) is very important for skin barrier repair.

[0010] Summary of the Invention

[0011] The present invention aims to provide a polyterpenoid compound for use as a kallikrein inhibitor in cosmetics.

[0012] The present invention finds that some polyterpenoid compounds have an inhibitory effect on kallikreins (KLKs). Therefore, a polyterpenoid compound is provided for use as a kallikrein inhibitor in cosmetics, wherein the polyterpenoid compound is selected from vitamin K2, coenzyme Q or carotenoids.

[0013] Furthermore, the vitamin K2 is selected from vitamin K2 (MK-4), vitamin K2 (MK-7), vitamin K2 (MK-8), DMK-7 or DMK-8.

[0014] Furthermore, the coenzyme Q includes coenzyme Q6, coenzyme Q7, coenzyme Q8, coenzyme Q9, and coenzyme Q10.

[0015] Furthermore, the carotenoids are selected from lycopene, β-carotene, canthaxanthin, zeaxanthin, astaxanthin, lutein, capsanthin, streptozocin, antheraxanthin, and cryptoxanthin.

[0016] Preferably, the polyterpenoid compound is selected from vitamin K2 (MK-7) or astaxanthin.

[0017] The polyterpenoid compound has been shown to have a high targeted inhibition rate on the activity of kallikreins KLK5, KLK7, and KLK14, especially KLK5. As a small molecule kallikrein inhibitor, it can simultaneously inhibit and regulate the activities of KLK5, KLK7, and KLK14.

[0018] The polyterpenoid compounds are added to cosmetics (lotions, creams, etc.) as small molecule kallikrein inhibitors, which can quickly and effectively inhibit the activity of kallikrein, promote the balance of proteases and their inhibitors, help keratinocytes to fall off normally from the surface of the stratum corneum, repair damaged skin barriers, and thus maintain the dynamic balance of the skin barrier.

[0019] The polyterpenoids are green and safe, and are mostly derived from or used in food and medicine. They can be used as a highly safe skin barrier repair agent. A typical example is vitamin K2 (MK-7), which is a mild and safe inhibitor.

[0020] Beneficial Effects: Based on the discovery that polyterpenoid compounds selected from vitamin K2, coenzyme Q, or carotenoids inhibit kallikreins (KLKs), the present invention provides the use of these compounds as small molecule kallikrein inhibitors in cosmetics. These compounds simultaneously inhibit and modulate the activity of kallikreins (KLKs), particularly exhibiting significant activity against KLK5. When used in cosmetics, these compounds can quickly and effectively inhibit overexpression of kallikreins and promote the balance of proteases and their inhibitors. Long-term use can reduce dark circles, redness, increase skin elasticity, eliminate fine wrinkles, and slow the deepening of nasolabial folds, providing skin firming, anti-aging, anti-inflammatory, and barrier function repair benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is an enzyme activity curve of kallikrein KLK5 with the addition of MK-7;

[0022] FIG2 is an enzyme activity curve of kallikrein KLK5 with the addition of astaxanthin;

[0023] FIG3 is an enzyme activity curve of kallikrein KLK5 with the addition of ectoine;

[0024] FIG4 shows the enzyme activity curve of kallikrein KLK5 with the addition of ginsenosides. DETAILED DESCRIPTION

[0025] The technical solutions of the present invention are further described in detail below through specific examples. However, it is necessary to point out that the following examples are only used to describe the content of the invention and do not constitute a limitation on the scope of protection of the present invention.

[0026] The polyterpenoid compounds of the present invention having a kallikrein KLKs inhibitory effect are selected from vitamin K2, coenzyme Q, or carotenoids, and the specific compounds are shown in Table 1. These polyterpenoid compounds have been shown to have an inhibitory effect on kallikrein KLKs, while most active substances in cosmetics, such as ectoine, ginseng, α-hedera saponin, sodium aescin, astragalus, and ginsenosides, have no inhibitory effect on kallikrein KLKs.

[0027] Table 1 Polyterpenoid compounds with kallikrein inhibitory activity

[0028] In the following examples, the targeted inhibition rates of different substances selected from polyterpenoid compounds on the activities of kallikreins KLK5, KLK7, and KLK14 were determined in order to screen out safe and effective small molecule kallikrein inhibitors that can be used in cosmetics.

[0029] The results showed that the polyterpenoid compounds listed in Table 1 can simultaneously inhibit and regulate KLK5, KLK7, and KLK14 as small molecule inhibitors. Among them, vitamin K2 (MK-7) and astaxanthin are mild and safe, and have more significant inhibitory effects on kallikrein KLKs.

[0030] In the following examples, vitamin K2 (MK-7), astaxanthin and kallikrein KLK5 are used as examples to illustrate the inhibitory effect of the polyterpenoid compounds of the present invention on their activities.

[0031] In the embodiment, the method for detecting KLK5 protease activity is as follows:

[0032] (1) Materials:

[0033] Recombinant Human Kallikrein 5 Protein, purchased from R&D system, Catalog #: 1108-SE;

[0034] Boc-VPR-AMC Fluorogenic Peptide Substrate, purchased from R&D system, Catalog #: ES011, 10 mM stock in DMSO (store in the dark)

[0035] Assay solution: 0.1M NaH2PO4, pH 8.0

[0036] 96-well plate: Nunc, Catalog, 475515

[0037] (2) Experimental methods:

[0038] 1. The substrate is packaged at 10mM, 10ul per tube;

[0039] 2. Dilute 10ul of the aliquoted substrate to 1mL, 100uM;

[0040] 3. KLK5 was dispensed into 0.1ug / tube, 10ul per tube, each tube was configured to 200ul, 10ul was added to each well, i.e. 0.05ug per well;

[0041] 4. 96-well plate preparation system: add a certain concentration volume of test solution, enzyme, and substrate;

[0042] 5. Read the readings on a microplate reader at 380 nm to 460 nm.

[0043] 6. Analysis of experimental results.

[0044] Example 1

[0045] The inhibitory effect of vitamin K2 (MK-7) (hereinafter referred to as MK-7) on kallikrein KLK5 (abbreviated as KLK5) is expressed by the change in KLK5 protease activity after adding MK-7 to KLK5. The experimental method is as above, and the samples are divided into 6 groups from group A to group F. Group A contains only KLK5 and serves as the blank control group. Groups B to group F are the same as group A, except that KLK5 is added to 50uM, 40uM, 25.6uM, 12.8uM, and 6.4uM of MK-7, respectively. Two samples are set for each group. The test sample and substrate are added to a 96-well plate to react with KLK5. Samples are taken every 45 seconds, and the enzyme activity is measured using a microplate reader. The results of the two parallel samples are averaged.

[0046] The activity curves are shown in Figure 1 , which, from top to bottom, are the activity curves of Group A, Group F, Group E, Group D, Group C, and Group B. Some of the data obtained by sampling every 3 minutes are shown in Table 2 .

[0047] Table 2

[0048] As shown in the figure, KLK5 enzyme activity was highest in Group A (the curve is at the top), and the addition of 6.4 μM MK-7 significantly inhibited the enzyme activity. Increasing the concentration of MK-7 resulted in a stronger inhibitory effect on KLK5 activity, with strong inhibition observed in Groups C and B. The calculated IC50 was 11.78 μM.

[0049] Example 2

[0050] The inhibitory effect of astaxanthin on KLK5 was determined using the same method as in Example 1. The samples were divided into seven groups, Group A to Group G. Group A contained only KLK5 and served as the blank control group. Groups B to Group G contained the same KLK5 as Group A, but astaxanthin was added at concentrations of 50 uM, 25 uM, 12.5 uM, 6.25 uM, 3.125 uM, and 1.562 uM, respectively. Two samples were set up in each group. The test sample and substrate were added to a 96-well plate to react with KLK5. Samples were taken every 45 seconds, and the enzyme activity was measured using a microplate reader. The results of the two replicate samples were averaged.

[0051] The activity curves are shown in Figure 2 , which show, from top to bottom, the activity curves of Group A, Group G, Group F, Group E, Group D, Group C, and Group B. Some of the data obtained by sampling every 3 minutes are shown in Table 3 .

[0052] Table 3

[0053] As shown in the figure, KLK5 enzyme activity was highest in Group A (the curve is at the top), and the addition of 1.562 μM astaxanthin significantly inhibited the enzyme activity. Increasing the concentration of astaxanthin produced a stronger inhibitory effect on KLK5 enzyme activity, with KLK5 activity in Groups C and B being strongly inhibited. The calculated IC50 was 28.78 μM.

[0054] Comparative Example 1

[0055] The inhibitory effect of ectoine on KLK5 was determined using the same method as in Example 1. The samples were divided into six groups, Groups A to F. Group A contained only KLK5 and served as the blank control. Groups B to F were the same as Group A, except that KLK5 was added at 5mM, 500uM, 50uM, 5uM, and 0.5uM ectoine, respectively. Samples were collected every 45 seconds, and enzyme activity was measured. The activity curves are shown in Figure 3, which show the activity curves for Groups B, E, A, D, C, and F, from top to bottom.

[0056] As shown in FIG3 , which shows the changes in KLK5 protease activity, ectoine has no inhibitory effect on KLK5.

[0057] Comparative Example 2

[0058] The inhibitory effect of ginsenosides on KLK5 was determined using the same method as in Example 1. Samples were divided into four groups, Groups A, D, and C. Group A contained only KLK5 and served as a blank control. Groups B, B, and D contained the same KLK5 as in Group A, but also added 10 μM, 100 nM, and 1 nM ginsenosides, respectively. Samples were collected every 45 seconds for enzyme activity measurement. Activity curves are shown in Figure 4, with the activity curves for Groups B, C, D, and A, from top to bottom.

[0059] As shown in Figure 4 , which shows the changes in KLK5 protease activity, ginsenosides have no inhibitory effect on KLK5.

[0060] Example 3

[0061] The MK-7 is added to the emulsion as a small molecule kallikrein inhibitor, and the formula is shown in Table 4. The emulsion can quickly and effectively inhibit the overexpression of kallikrein, promote the balance of protease and its inhibitors, and repair the damaged skin barrier.

[0062] Table 4 Emulsion 1

[0063] Example 4

[0064] Astaxanthin is added to the emulsion as a small molecule kallikrein inhibitor, the formula of which is shown in Table 5. The emulsion has the function of repairing damaged skin barrier.

[0065] Table 5 Emulsion 2

Claims

1. Application of polyterpenoids as kallikrein inhibitors in cosmetics, wherein the polyterpenoids are selected from vitamin K2, coenzyme Q or carotenoids.

2. The use according to claim 1, characterized in that: The vitamin K2 is selected from vitamin K2 (MK-4), vitamin K2 (MK-7), vitamin K2 (MK-8), DMK-7 or DMK-8.

3. The use according to claim 1, characterized in that: The coenzyme Q is coenzyme Q6, coenzyme Q7, coenzyme Q8, coenzyme Q9 or coenzyme Q10.

4. The use according to claim 1, characterized in that: The carotenoid is selected from lycopene, β-carotene, canthaxanthin, zeaxanthin, astaxanthin, lutein, capsanthin, streptozocin, antheraxanthin or cryptoxanthin.

5. The use according to claim 1, characterized in that: The polyterpenoid compound is selected from vitamin K2 (MK-7) or astaxanthin.

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