CN111904955A - Hydroxytyrosol and its derivatives for relieving xeroderma and application thereof - Google Patents

Abstract

The present invention relates to the relieving effect of hydroxytyrosol and its derivatives on dry skin and its application. The hydroxytyrosol derivative is selected from at least one of hydroxytyrosol ester and hydroxytyrosol ether, and can be derived from chemical synthesis, microbial fermentation or animal and plant extraction. The hydroxytyrosol and its derivatives involved in the present invention can relieve dry skin, have a repairing effect on skin structural proteins, and then repair the skin barrier. The hydroxytyrosol and its derivatives of the present invention can be applied to external skin cosmetics or skin medicines in various dosage forms such as gels, facial cleansers, facial creams, and lotions.

Description

Relief effect of hydroxytyrosol and its derivatives on dry skin and its application

technical field

The invention belongs to the field of cosmetics, and in particular relates to the relieving effect of hydroxytyrosol and its derivatives on skin dryness and its application. The expression of keratin 1 (KRT1), filaggrin (FLG), loricrin (LOR), and involucrin (IVL) can effectively repair the damaged skin barrier caused by skin dryness. Used in cosmetics for skin care.

Background technique

The skin covers the surface of the whole body and is one of the largest organs of the human body. It is composed of polysaccharides, etc., which can resist external harmful substances, irritants, and sunlight, and at the same time have moisturizing and regulating anti-inflammatory effects.

The natural moisturizing system in human skin is mainly composed of water, lipids, and natural moisturizing factor (NMF). Lipids are filled between the stratum corneum cells in layers, and their main function is to form a water barrier to stop water loss. Most lipids are non-polar substances that limit the flow of water in and out of cells and between cells. Lipids associated with skin barrier function include sphingolipids, free cholesterol, and free fatty acids. The active epidermis is rich in phospholipids. However, in the process of differentiation from the basal layer to the stratum corneum, the contents of ceramides, cholesterol and fatty acids gradually increase, and the lipid distribution also changes. The lipids in the granular layer accumulate in the lamellar layer. In the granule, when the cells of the stratum granulosa are transformed into stratum corneum cells, these lipids are expelled and filled in the intercellular spaces, forming a barrier against water loss. When the skin barrier is damaged, its water barrier function is weakened, and transepidermal water loss (TEWL) will increase, resulting in dry skin and scaling, allergic dermatitis, psoriasis and other skin diseases. There are many reasons for the damage of the skin barrier. In addition to the influence of external physical factors and age, the damage of the skin barrier caused by skin inflammation is also a factor worthy of attention.

The sebum film can moisturize the skin and is the protective barrier of the skin. When the sebum secretion is insufficient or the moisture is insufficient, the skin will be dry, the outermost barrier of the skin will be damaged, and the health of the skin will be affected. It will stimulate sebum secretion and relieve dry skin. Ginseng polysaccharide contains water-absorbing groups, which can be used to increase the water content of the skin and relieve dryness.

Keratin is the main structural protein of the epidermis. KRT10 and KRT1 are characteristic markers of epithelial cell growth, differentiation and maturation and play important roles in epithelial cells. They allow the final differentiation of intermediate filaments in keratinocytes, thereby maintaining smooth physicochemical properties and metabolic processes of cells. If KRT10 and KRT1 proteins are disrupted, it will lead to filament aggregation, cytoskeleton instability and cell lysis in epidermal keratinocytes.

Filaggrin is the basic protein component of transparent keratinocytes in mammalian skin basal cells, which can gradually differentiate into the epidermal cell layer. FLG is a large, highly phosphorylated polypeptide that is an important component of granular keratin granules. After dephosphorylation and proteolysis, filaggrin is cleaved into functional filaggrin peptides during terminal differentiation of granulosa cells. The peptide binds and breaks down the keratin cytoskeleton, and each peptide is gradually degraded by enzymes into hydrophilic amino acids, including pyrrolidone, carboxylic acid, alanine, and transuric acid (imidazole acrylic acid). These amino acids and various ions are called natural moisturizing factors (NMF). Hydration in the stratum corneum. It plays a vital role in usage. The FLG then begins to accumulate keratin filaments, which disintegrate the non-nucleated granulosa cells into scales. The cytoskeleton is then cross-linked by transglutaminase to form the keratinized envelope of the stratum corneum and the outermost skin barrier. Therefore, FLG can play an important role in the formation of the skin barrier.

The keratinized envelope (CE) is the envelope of the stratum corneum cells that provides the correct binding sites for intercellular lipids. If the CE is immature, the wall-like structure of the pure stratum corneum cannot be formed even if the amount of intercellular lipids is sufficient. LOR protein is mainly expressed in the stratum granulosa and stratum corneum, which are the major components of terminally differentiated CE, accounting for about 80% of its protein. IVL protein is mainly expressed in the upper part of the spinal cell layer and granular layer, which is a marker protein of keratinocyte differentiation and is located in the outer layer of CE. The IVL is covalently bound to a ceramide containing a hydroxyl functional group, and then the lipid substrate and keratinocytes are linked together to play a supporting function.

The chemical name of hydroxytyrosol is 3,4-dihydroxyphenethyl alcohol, which is a natural polyphenolic compound. It mainly exists in various parts of olives in the form of esterified oleuropein. Free hydroxytyrosol can be obtained after hydrolysis. Studies have found that hydroxytyrosol has many biopharmacological activities that are beneficial to human health, and has received much attention from the biological and medical fields in recent years. Studies have shown that hydroxytyrosol has anticancer, antibacterial, anti-inflammatory and other biological and pharmacological activities. Through further research, it is found that hydroxytyrosol has a good relieving effect on dry skin, so hydroxytyrosol has broad application prospects in the field of cosmetics.

CN102657601B, an external medicine formulation and process for treating ichthyosis and dry skin, mainly used for external medicine for treating ichthyosis and dry skin. The formula is mainly composed of the following components: urea, light liquid paraffin, alpha-hydroxypropionic acid, glycerol mono and distearate, concentrated ammonia solution, polyoxyl stearate (40), cholesterol, glycerin, White petrolatum, stearyl alcohol, purified water. However, the preparation process of the invention uses concentrated ammonia solution, which has a heavy odor and affects product quality.

CN100490773C, insoluble powder, powder for restoring skin barrier function, powder for preventing or improving skin roughness and skin external preparation containing the same, insoluble powder is composed of one or more of metal ions lithium ion, sodium ion and zinc ion It has the function of restoring skin barrier function and improving skin roughness. The invention is an insoluble powder, which lacks convenience in the application of specific cosmetic formulations.

CN105434212B, an external skin care composition, emulsion and preparation method thereof with radiation protection and skin barrier repairing effects, the external skin care composition combines honeysuckle, salvia, dangshen, astragalus, Radix Ophiopogon and cactus together to form a compound, and As an active component, it is prepared into a skin care preparation, which has the functions of protecting and inhibiting the proliferation of fibroblasts caused by radiation, inhibiting skin pathogenic microorganisms and repairing skin barrier. The invention protects the skin barrier from the aspect of bacteriostasis, and does not fundamentally solve the problem of skin barrier repair.

At present, the main method for the treatment of dry skin is moisturizing treatment. At present, skin moisturizing agents mainly include oil-sealing moisturizing agents, hydrophilic matrix hygroscopic moisturizing agents, hydrating moisturizing agents, etc., but simple moisturizing agent treatment is only a temporary treatment. Hydration allows moisture to remain in the skin, which cannot fundamentally solve the problem of dry skin and achieve the effect of skin barrier repair.

The invention relieves skin dryness through hydroxytyrosol and its derivatives, in addition to the basic improvement of the transdermal water loss rate, the expression of skin structural proteins can be increased, and the skin barrier can be further repaired.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide the application of hydroxytyrosol and its derivatives in cosmetics for relieving dry skin.

In the present invention, the hydroxytyrosol derivative has the structure of general formula (I):

The structural formula of the hydroxytyrosol derivative is part or all of -OH and ester or ether in the compound represented by the structural formula (I). R, R ₁ , and R ₂ are H.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the experimental group that hydroxytyrosol and its derivatives have a certain alleviation effect on acetone/ether-induced xerosis in mice and improve the skin transdermal water loss rate.

Figure 2 is the analysis of mouse skin stratum corneum thickness, wherein, *P<0.05, **P<0.01.

Figures 3, 4, 5, 6, and 7 show the mRNA expression levels of KRT1, KRT10, FLG, IVL, and LOR proteins in the skin of mice in the control group, wherein **P<0.01.

Detailed ways

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. . Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, which can be purchased through commercial channels. The water may be deionized water, purified water or distilled water.

Example 1: Synthesis of Hydroxytyrosol Derivatives

1.1 Synthesis of hydroxytyrosol alkyl ether

Three-step synthesis of hydroxytyrosol alkyl ether, the reaction formula is as follows (n=1~4):

A round bottom flask was charged with 0.8 g of hydroxytyrosol, 25 mL of anhydrous acetone, 1.4 mL of benzyl bromide, and 2.9 g of potassium carbonate, and the resulting mixture was heated to reflux for 24 hours. The obtained suspension was filtered and

Concentration gave a crude residue which was further purified by column chromatography using a 1:2 mixture of ether/hexanes as eluent to give the intermediate product. A mixture of 334 mg of the above intermediate, 335 mg of KOH, 12 mL of dimethyl sulfoxide and the corresponding alkyl iodide was stirred at room temperature until the reaction was complete, then 25 mL of 3M HCl was added and the resulting mixture was extracted with _CHCl3 . The organic phase was washed with 25 mL of 2% NaHSO3 and 25 mL of water, then dried _{over Na2SO4} , filtered and evaporated, and the desired product was purified by flash column chromatography on silica gel. The above product and Pd-C were added to 20 mL of tetrahydrofuran, and the mixture was hydrogenated under a pressure of 4 bar for 24 hours using magnetic stirring, then the catalyst was removed, and the organic solvent was evaporated in vacuo to obtain hydroxytyrosol alkyl ether. Four kinds of hydroxytyrosol alkyl ether structural formulas are prepared according to the above method as follows:

1.2 Synthesis of hydroxytyrosol fatty acid ester derivatives

The reaction formula of hydroxytyrosol fatty acid ester derivatives is as follows:

在无水四氢呋喃中的以1：1的质量比加入羟基酪醇和相应脂肪酸氯化物搅拌下加入Er（OTf）_3，将混合物在氮气下室温下反应12小时。完成后将混合物倒入饱和NaHCO₃溶液中，并用CHCl₃萃取。合并有机层并用Na₂SO₄干燥并过滤，在减压下蒸发溶剂获得粗产物，随后用CHCl ₃ /MeOH=9.5/0.5作为洗脱剂，通过快速色谱纯化。根据上述方法制备四种羟基酪醇脂肪酸酯结构式如下：

Hydroxytyrosol and corresponding fatty acid chlorides were added in anhydrous tetrahydrofuran at a mass ratio of 1:1 and Er(OTf) _{3 was added with stirring, and} the mixture was reacted under nitrogen at room temperature for 12 hours. Upon completion, the mixture was poured into saturated _NaHCO3 solution and extracted with _CHCl3 . The organic layers were combined and dried over Na ₂ SO ₄ and filtered, and the solvent was evaporated under reduced pressure to obtain the crude product, which was then purified by flash chromatography using CHCl ₃ /MeOH=9.5/0.5 as eluent. Four kinds of hydroxytyrosol fatty acid ester structural formulas are prepared according to the above method as follows:

Example 2

This example is an observation on the relieving effect of hydroxytyrosol and its derivatives on dry skin.

2.1 Drug formulation and animal grouping

Weigh 50 mg of hydroxytyrosol (HT) and its derivatives A~H each (purity ≥98%), add 1 mL of butanediol, 3.5 mL of deionized water, and Tween 80 and mix well to prepare a drug that is formulated to 10 mg/mL solution, ready for use. 60 BABL/C mice (7-8 weeks), a group of 5, were divided into normal group (CON), model group (AEW), positive group (HA), hydroxytyrosol group (HT), hydroxytyrosol derived group There were 12 groups in total (A~H).

2.2 Mice modeling and administration method

2 days before the modeling, the mice were shaved and depilated with 6% NaS. On the day of modeling, acetone/ether-water (AEW) model: 2cm×2cm absorbent cotton was dipped in acetone:ether=1:1 mixture to cover the depilation area For 15s, another 2cm×2cm absorbent cotton was dipped in distilled water to cover the depilation area for 30s, and the administration group applied 100 μL of hydroxytyrosol solution 1h after modeling; twice a day for 7d.

2.3 Mouse skin percutaneous water loss rate test

The percutaneous water loss rate of the blank group (CON), model group (AEW), positive group (HA), hydroxytyrosol group (HT), and hydroxytyrosol derivative groups (A~H) was monitored by MPA instrument. The skin appearance changes, and the results are shown in Figure 1. Compared with the model group, both hydroxytyrosol and its derivatives could significantly improve the degree of water loss of the skin, and the improvement effect of hydroxytyrosol was the best.

2.4 Pathological observation of mouse skin stratum corneum

After the experiment, the mice were killed by dislocation, and the removed skin tissue was fixed with a 4% paraformaldehyde block. After the fixation was successful, it needed to be trimmed to 25mm*25mm*5mm, placed in an embedding box, and rinsed with running water (to remove the fixation in the tissue). solution) for 30 min, then place the tissue block in xylene, a clearing agent dissolved in both alcohol and paraffin, to make it transparent, replace the alcohol in the tissue block with xylene, and then embed the tissue block in wax. In the melted paraffin, put it into a wax melting box to keep warm, fix the embedded paraffin block on the microtome, cut it into 5μm thin slices, put it in heated water and iron it, and then paste it on a glass slide, and keep it at 45 ℃ constant temperature Drying in a box, dewaxing with xylene, graded with ethanol to 30% alcohol, stained with hematoxylin-eosinstaining (H&E), the hematoxylin staining solution is alkaline, It mainly makes the chromatin in the nucleus and the nucleic acid in the cytoplasm to be purple-blue; eosin is an acid dye, which mainly makes the components in the cytoplasm and the extracellular matrix red. The results of H&E staining epidermal thickness analysis showed that compared with the blank group, the model group had obvious stratum corneum hyperplasia, and hydroxytyrosol and its derivatives could inhibit the stratum corneum hyperplasia to a certain extent and reduce the thickness of the stratum corneum, as shown in Figure 2.

2.5 Mouse skin barrier repair index test

After the experiment, the mice were sacrificed by cervical dislocation, and RNA was extracted from the removed skin tissue. RT-PCR quantitatively analyzed the mRNA expression of FLG, LOR, KRT10, KRT1, and IVL in the skin tissue, as shown in Figure 3.

The keratinous intermediate filament protein will be converted into NMF in the stratum corneum under the catalytic degradation of bleomycin hydrolase. Significant effect in barrier repair.

(1) Extraction of total RNA (pipette tip and centrifuge tube are sterilized by moist heat, no RNase)

1) Take a homogenization tube, add 1 ml of Trizol Reagent, and place it on ice to pre-cool.

2) Take 100 mg of tissue and add it to the homogenization tube.

3) The homogenizer is fully ground until there are no visible tissue lumps.

4) Centrifuge at 12000 rpm for 10 min to take the supernatant.

5) Add 250 μl chloroform, invert the centrifuge tube for 15 s, mix well, and let stand for 3 min.

6) Centrifuge at 12000rpm for 10min at 4°C.

7) Transfer the supernatant to a new centrifuge tube, add 0.8 times the volume of isopropanol, and mix by inversion.

8) -20℃ for 15min.

9) Centrifuge at 12000rpm for 10min at 4℃, the white precipitate at the bottom of the tube is RNA.

10) Suction off the liquid, add 1.5 ml of 75% ethanol to wash the precipitate.

11) Centrifuge at 12000rpm for 5min at 4°C.

12) Remove the liquid by suction, place the centrifuge tube on the ultra-clean bench and blow for 3 minutes.

13) Add 15 μl of RNase-free water to dissolve the RNA.

14) Incubate at 55°C for 5 minutes.

15) Use Nanodrop 2000 to detect RNA concentration and purity: After the instrument is blanked to zero, take 2.5 μl of the RNA solution to be tested on the detection base, put down the sample arm, and use the software on the computer to start absorbance detection.

16) Dilute the RNA that is too high in concentration to a final concentration of 200ng/μl.

(2) Reverse transcription (pipette tip and PCR are sterilized by moist heat, no RNase)

1) Take a PCR tube and add a solution containing 2 μg RNA.

2) Add 1 μl oligo(dT)18.

3) Make up to 12 μl with RNase-free deionized water.

4) Incubate at 65°C for 5 min on the PCR machine, and then quickly cool on ice.

5) Add 4μl 5× Reaction Buffer, 2μl 10mM dNTP Mix, 1μl RiboLock Instruction Manual 4/5 Page 6 CN 110354154 A 6 RNAase Inhibitor (20U/μl) and 1μl RevertAi M-MuLV Reverse Transcriptase (200u/μl) in sequence. Aspirate to mix.

6) Incubate at 42°C for 60 minutes on a PCR machine, and inactivate reverse transcriptase by incubating at 70°C for 5 minutes after the end.

(3) Quantitative PCR

1) Take a 0.2ml PCR tube, prepare the following reaction system, prepare 3 tubes for each reverse transcription product.

2 × qPCR Mix 12.5μl

7.5μM Gene Primer 2.0μl

Reverse transcription product 2.5μl

ddH2O 8.0μl

2) PCR amplification

Pre-denaturation 95℃, 10min

Cycle (40 times) 95℃, 15s→60℃, 60s

Melting curve 60℃→95℃, heating 0.3℃ every 15s

(4) Result processing ΔΔCT method:

A=CT (target gene, sample to be tested) - CT (internal standard gene, sample to be tested)

B=CT (target gene, control sample) - CT (internal standard gene, control sample)

K=A-B

Expression fold=2 ^-K

The results are shown in Figures 3, 4, 5, 6, and 7. The mRNA expression levels of KRT1, KRT10, FLG, LOR, and IVL in the hydroxytyrosol and its derivatives group were higher than those in the AEW group, indicating that hydroxytyrosol and its derivatives It can effectively promote the expression of KRT1, KRT10, FLG, LOR, and IVL, relieve dry skin, and promote skin barrier repair.

Claims (10)

1. Hydroxytyrosol and its derivatives have effects of relieving xerosis cutis and its application; the hydroxytyrosol derivative has a general formula (I):

general formula (I).

2. The compound of formula (I) according to claim 1, wherein R is C₁-C₂₀Saturated or unsaturated alkyl, C₁-C₂₀Any of the saturated or unsaturated alkoxy group and the hydroxyl group.

3. The compound of formula (I) according to claim 1, wherein R₁、R₂Are respectively selected from H, C₁-C₆A saturated alkyl group.

4. Hydroxytyrosol and its derivatives as claimed in claim 1, which can be obtained from chemical synthesis, microbial fermentation or animal and plant extraction; the plant may be olive fruit, olive leaf or other plant containing hydroxytyrosol or its derivatives.

5. The use of claim 1, wherein the hydroxytyrosol and derivatives thereof are present in an amount of 0.5mg/cm²-5.0mg/cm²。

6. The use of claim 1, wherein the hydroxytyrosol and derivatives thereof are present in an amount of 2.0mg/cm²。

7. The use according to claim 1, wherein hydroxytyrosol and its derivatives are used for reducing the transdermal water loss rate of the skin, reducing the thickness of the stratum corneum of the skin and relieving the dryness of the skin.

8. Use according to claim 1, wherein hydroxytyrosol and its derivatives are used for treating impaired barrier caused by xeroderma and promoting the expression of skin structural proteins, such as: keratin 10, keratin 1, filaggrin, loricrin, and integrins.

9. Hydroxytyrosol and its derivatives according to claims 1-4, characterized in that the dosage form is a cosmetically or dermatologically acceptable diluent, carrier, excipient, adjuvant or vehicle.

10. The formulation according to claim 9, wherein the cosmetic or dermatological formulation is a gel, a face wash, a cream, a lotion.

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