CN117084936A - Application of polyterpene compounds as kallikrein inhibitor in cosmetics - Google Patents

Abstract

The invention discloses application of a polyterpene compound as a kallikrein inhibitor in cosmetics, wherein the polyterpene compound is selected from vitamin K2, coenzyme Q or carotenoid. The polyterpene compound has an inhibitory effect on kallikrein KLKs. The polyterpene compound is added into cosmetics as a small molecular kallikrein inhibitor, can effectively inhibit the activity of kallikrein, promote the balance of protease and inhibitor thereof, and repair damaged skin barrier, thereby maintaining the dynamic balance of the skin barrier.

Description

Application of polyterpenoids as kallikrein inhibitors in cosmetics

Technical field

The invention belongs to the technical field of cosmetics, and particularly relates to the application of polyterpenoids in cosmetics as a kallikrein targeted small molecule inhibitor that can effectively repair skin barriers.

Background technique

The skin is the outermost layer of the human body and has barrier, absorption, secretion, excretion, metabolism, temperature regulation and sensory functions. Among them, the skin barrier function is the basis. On the one hand, the skin barrier can protect the skin from being invaded by adverse external factors; on the other hand, it can prevent the loss of water, nutrients, electrolytes and other substances in the skin and the body, helping to maintain a relatively stable environment within the body.

Impaired skin barrier function will cause dry skin and weakened antioxidant and anti-radiation functions, thereby aggravating skin aging and causing atopic dermatitis, eczema, solar dermatitis, sensitivity, acne, etc.

The physical barrier of the skin is mainly composed of stratum corneum-related structures and tight junctions. The stratum corneum is located in the outermost layer of the skin, forming a "brick wall structure" and is the main component of the skin barrier. Tight junctions are also important in maintaining the integrity of the skin barrier. Crucial. Keratinocytes have a physical barrier function that can reduce mechanical damage and invasion of external substances. At the same time, their components and structures are important components of the skin barrier, including filaggrin, keratinized envelope, corneal desmosomes, proteases, and cells. Lipids, etc.

Keratinocytes 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 keratinocytes 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 stratumcorneum[J ].J Invest Dermatol,2005,124(1):198-203.). KLKs belong to the serine protease family and are expressed in the granule layer. KLKs can cleave desmosomes, KLK5 and KLK14 have trypsin activity and can cleave desmoglein, and KLK7 has chymotrypsin activity and can cleave desmoglein and cutodesmoglein. In particular, KLK5 and KLK7 are highly related to desquamation and can induce inflammatory factors. Among them, KLK5 can degrade cuticular 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.

The stability of the skin barrier depends on the balance between the differentiation of keratinocytes in the granular layer into keratinocytes and the shedding (i.e., desquamation) of keratinocytes in the surface layer of the skin. The desquamation process of the skin includes desmosomes participated by (kallikrein) KLKs. Because desmosomes are the adhesion proteins of keratinocytes, the degradation of desmosomes will cause keratinocytes to fall off and cause skin desquamation. Evidence shows that local or temporary imbalance in the control of KLKs activity is one of the main reasons for the loss of skin barrier stability in patients with atopic dermatitis.

In the field of cosmetics, Han Dan et al. (Methods and Applications for Repairing Skin Barrier Function, Chemical Science, Jul. 2020.VOL.43NO.7) discussed the composition and damage factors of the skin barrier, and discussed lipids, natural Moisturizing factors, humectants, active ingredients, pH value and other aspects have proposed solutions for skin barrier repair. This literature does not involve or take into account the damage caused by the dysregulation or abnormal expression of kallikrein KLKs, which may therefore affect its barrier repair effect. Currently, inhibitors of KLKs are relatively rare in the field of skin care cosmetics.

Kallikrein is involved in regulating the physiological functions of cardiovascular, kidney, nervous system, etc., and is closely related to the occurrence of heart disease, kidney disease, inflammatory reaction, cancer and other diseases. In the medical field, research on kallikrein inhibitors in the cardiovascular system is progressing rapidly. 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. This inhibitor mainly targets KLK7, and the metabolism of keratinocytes is mainly regulated by the hydrolysis cascade of KLK5, KLK7, KLK14 and other related proteases. For the prevention and treatment of skin-specific dermatitis, KLKs regulation is becoming a direction of development.

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

Contents of the invention

The object of the present invention is to provide a polyterpenoid compound used as a kallikrein inhibitor in cosmetics.

The present invention finds that some compounds in polyterpenoids have an inhibitory effect on kallikrein KLKs, and therefore provides a polyterpenoid compound for use in cosmetics as a kallikrein inhibitor, wherein the polyterpene The compound is selected from vitamin K2, coenzyme Q or carotenoids.

Further, the vitamin K2 is selected from vitamin K2 (MK-4), vitamin K2 (MK-7), vitamin K2 (MK-8), DMK-7 or DMK-8.

Further, the coenzyme Q includes coenzyme Q6, coenzyme Q7, coenzyme Q8, coenzyme Q9, and coenzyme Q10.

Further, the carotenoid is selected from the group consisting of lycopene, β-carotene, canthaxanthin, zeaxanthin, astaxanthin, lutein, capsanthin, streptozolin, antheraxanthin, cryptoxanthin, Xanthin.

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

The polyterpenoid compound has been proven to have a high targeted inhibition rate on the activity of kallikrein KLK5, KLK7, and KLK14, especially KLK5. As a small molecule kallikrein inhibitor, it has a high target inhibition rate on KLK5, KLK7 , KLK14 can inhibit and regulate its activity at the same time.

The polyterpenoids 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, and help cuticles Cells normally shed from the surface of the stratum corneum to repair the damaged skin barrier, thereby maintaining the dynamic balance of the skin barrier.

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

Beneficial effects: Based on the inhibitory effect of kallikrein KLKs discovered by polyterpenoids selected from vitamin K2, coenzyme Q or carotenoids, the present invention provides the compound as a small molecule inhibitor of kallikrein. The agent is used in cosmetics to simultaneously inhibit and regulate the activity of kallikrein KLKs, especially KLK5, which has significant activity. When used in cosmetics, it can quickly and effectively inhibit the overexpression of kallikrein and promote the balance of proteases and their inhibitors. Long-term use can dilute dark circles, reduce redness, increase skin elasticity, eliminate fine wrinkles, slow down the deepening of nasolabial folds, and is effective for It has the effects of firming, anti-aging, anti-inflammatory and repairing barrier function of the skin.

Description of the drawings

Figure 1 Enzyme activity curve of kallikrein KLK5 added to MK-7;

Figure 2 Enzyme activity curve of kallikrein KLK5 added with astaxanthin;

Figure 3 Enzyme activity curve of kallikrein KLK5 added with ectoine;

Figure 4. Enzyme activity curve of kallikrein KLK5 added with ginsenoside.

Detailed ways

The technical solutions described in the present invention will be 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.

Some of the polyterpenoid compounds with the inhibitory effect of kallikrein KLKs in the present invention are selected from vitamin K2, coenzyme Q or carotenoids. The specific compounds are shown in Table 1. These polyterpenoids have been confirmed to have an inhibitory effect on kallikrein KLKs, while most of the active substances in cosmetics, such as ectoine, ginseng, α-hederin, sodium aescin, astragalus, ginsenoside, etc. Kallikrein KLKs have no inhibitory effect.

Table 1 Polyterpenoids with kallikrein inhibitory effects

In the following examples, the targeted inhibition rates of different substances selected from polyterpenoids on the activities of kallikrein KLK5, KLK7 and KLK14 are measured in order to screen out safe and efficient kallikrein small molecules that can be used in cosmetics. Inhibitors.

The results show that the polyterpenoids in Table 1 can inhibit and regulate KLK5, KLK7, and KLK14 at the same time as small molecule inhibitors. Among them, vitamin K2 (MK-7) and astaxanthin are mild and safe in nature, and their inhibitory effect on kallikrein KLKs is more significant.

In the following examples, vitamin K2 (MK-7), astaxanthin and kallikrein KLK5 are taken as examples to illustrate the inhibitory effect of polyterpenoids in the present invention on their activities.

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

(1)Materials:

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

Boc-VPR-AMC Fluorogenic Peptide Substrate, purchased from R&D system, Catalog#:ES011, stock solution 10mM stock in DMSO (protect from light)

Analytical solution: 0.1M NaH ₂ PO ₄ , pH 8.0

96-well plate: Nunc, Catalog, 475515

(2)Experimental method:

1. The aliquoted substrate is 10mM, 10ul per tube;

2. Dilute 10ul of the packed substrate to 1mL, 100uM;

3. KLK5 is divided into 0.1ug/each tube, 10ul per tube, each tube is configured to 200ul, add 10ul to each hole, that is, 0.05ug per hole;

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

5. Read from 380nm to 460nm on the microplate reader;

6. Analysis of experimental results.

Example 1

The inhibitory effect of vitamin K2 (MK-7) (hereinafter abbreviated 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. The samples are divided into 6 groups from group A to group F. Group A only contains KLK5 and is a blank control group. Group B to group F except that KLK5 is the same as group A, add 50uM, 40uM, and 25.6uM respectively. , 12.8uM, 6.4uM MK-7. Set up 2 samples in each group. Add the test sample and substrate to a 96-well plate to react with KLK5. Samples are taken every 45 seconds. The enzyme activity is measured with a microplate reader. The results of the two parallel samples are averaged.

The activity curves are shown in Figure 1. From top to bottom in the figure are the activity curves of Group A, Group F, Group E, Group D, Group C and Group B respectively. Part of the data obtained by sampling every 3 minutes is shown in Table 2.

Table 2

Time(min) Group A Group B Group C Group D Group E Group F 0 4300 790 1095 1273 2108 2989 3 8946 1186 1944 2420 4232 6346 6 13992 1710 2774 3392 6767 10049 9 19353 2211 3697 4674 9757 14230 12 24704 2752 4672 5953 12573 19539 15 30091 3211 5438 6962 14594 23005 18 34110 3556 6179 8015 16311 25895 twenty one 37971 3786 6638 8675 17675 28442 twenty four 41397 3911 7024 9332 18811 30086 27 43681 4079 7585 9790 20115 31683 30 46136 4210 7769 10222 20893 33191 33 48292 4273 8014 10459 21445 34096 36 49911 4378 8146 10825 22072 34839 40 52084 4504 8437 11081 22709 36750

As can be seen from the figure, the KLK5 enzyme activity in group A is the highest (the curve is at the top), and adding 6.4uM MK-7 has a significant inhibitory effect on the enzyme activity. As the concentration of added MK-7 increases, a stronger inhibitory effect on KLK5 enzyme activity occurs, and the KLK5 enzyme activity in groups C and B is strongly inhibited. After calculation, its IC50=11.78uM.

Example 2

The inhibitory effect of astaxanthin on KLK5 was determined according to the same method as Example 1. The samples are divided into 7 groups from group A to group G. Group A only contains KLK5 and is a blank control group. Group B to group G except that KLK5 is the same as group A, add 50uM, 25uM, 12.5uM, 6.25uM, respectively. 3.125uM, 1.562uM astaxanthin. Set up 2 samples in each group. Add the test sample and substrate to a 96-well plate to react with KLK5. Samples are taken every 45 seconds. The enzyme activity is measured with a microplate reader. The results of the two parallel samples are averaged.

The activity curves are shown in Figure 2. From top to bottom in the figure are the activity curves of Group A, Group G, Group F, Group E, Group D, Group C and Group B respectively. Part of the data obtained by sampling every 3 minutes is shown in Table 3.

table 3

As can be seen from the figure, the KLK5 enzyme activity in group A is the highest (the curve is at the top), and adding 1.562uM astaxanthin has a significant inhibitory effect on the enzyme activity. As the concentration of astaxanthin increases, a stronger inhibitory effect on KLK5 enzyme activity occurs, and the KLK5 enzyme activity in groups C and B is strongly inhibited. After calculation, its IC50=28.78uM.

Comparative example 1

The inhibitory effect of ectoine (ectoine) on KLK5 was measured according to the same method as Example 1. The samples are divided into 6 groups from group A to group F. Group A only contains KLK5 and is a blank control group. Group B to group F except that KLK5 is the same as group A, add 5mM, 500uM, 50uM, 5uM, 0.5uM respectively. of ectoine. Samples were taken every 45 seconds to measure enzyme activity. The activity curves are shown in Figure 3. From top to bottom in the figure are the activity curves of Group B, Group E, Group A, Group D, Group C and Group F respectively.

It can be seen from Figure 3 of the changes in KLK5 protease activity that ectoine (ectoine) has no inhibitory effect on KLK5 at all.

Comparative example 2

The inhibitory effect of ginsenoside on KLK5 was measured in the same manner as in Example 1. The samples were divided into four groups from group A to group D. Group A only contained KLK5 and was a blank control group. Group B to group D were the same as group A except for KLK5, but added 10uM, 100nM, and 1nM ginsenosides respectively. Samples were taken every 45 seconds to measure enzyme activity. The activity curves are shown in Figure 4. From top to bottom in the figure are the activity curves of Group B, Group C, Group D and Group A respectively.

From Figure 4 of the changes in KLK5 protease activity, it can be seen that ginsenoside has no inhibitory effect on KLK5 at all.

Example 3

The MK-7 is added to the emulsion as a small molecule kallikrein inhibitor. The formula is as shown in Table 4. The emulsion can quickly and effectively inhibit the overexpression of kallikrein, promote the balance of proteases and their inhibitors, and repair damage. skin barrier.

Table 4 Emulsion 1

Example 4

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

Table 5 Emulsion 2

Claims (5)

1. The application of polyterpenoids as kallikrein inhibitors in cosmetics, wherein the polyterpenoids are selected from vitamin K2, coenzyme Q or carotenoids. 2. Application according to claim 1, characterized in that the vitamin K2 is selected from the group consisting of vitamin K2 (MK-4), vitamin K2 (MK-7), vitamin K2 (MK-8), DMK-7 or DMK-8. 3. The application according to claim 1, characterized in that the coenzyme Q is coenzyme Q6, coenzyme Q7, coenzyme Q8, coenzyme Q9 or coenzyme Q10. 4. Application according to claim 1, characterized in that the carotenoid is selected from the group consisting of lycopene, β-carotene, canthaxanthin, zeaxanthin, astaxanthin, lutein, and capsanthin. pigment, neurosporin, antheraxanthin or cryptoxanthin. 5. Application according to claim 1, characterized in that the polyterpenoid compound is selected from vitamin K2 (MK-7) or astaxanthin.