CN111728969B - Application of anti-aging and anti-oxidation compound in preparation of medicines or anti-aging cosmetics - Google Patents

Abstract

The invention relates to application of an anti-aging and anti-oxidation compound in preparation of a medicament or an anti-aging cosmetic. The invention proves that the compound shown in the formula (I) has better antioxidant effect and the function of preventing cell aging. The compound has good commercial value by being prepared into a corresponding anti-aging medicament or anti-aging cosmetic.

Description

Application of anti-aging and anti-oxidation compound in preparation of medicines or anti-aging cosmetics

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of an anti-aging and anti-oxidation compound in preparation of medicines or anti-aging cosmetics.

Background

The anti-aging mechanism is one of the hot topics studied at present, and with the continuous development of biochemistry and molecular biology technologies, researches related to cell aging such as oxygen radicals, antioxidants, stem cell technologies, telomeres, genes and the like are more and more emphasized, and become hot spots of anti-aging researches. Studies have shown that with age, the production of intracellular free radicals increases, which have a damaging effect on the cells, and the next generation of damaged cells is also damaged, increasing cell damage and death, and thus causing aging. In the experimental research of anti-aging, peroxisome exists in the organism, and the main function of peroxisome is to generate and eliminate H ₂0₂Has the important function of preventing active oxygen damage in vivo. How to reduce the generation of free radicals is also a hot issue in the present research.

Research shows that in the aging process, harmful factors such as external physical damage, biochemical poison, DNA replication error in vivo, spontaneous hydrolysis reaction, Reactive Oxygen Species (ROS) and the like continuously damage DNA, so that gene mutation, translocation, telomere shortening, chromosome number abnormality and the like are caused, and the integrity and stability of a genome are damaged. Accumulation of somatic mutations can be observed in cells of an aging body. In order to maintain the stability of genome, the body develops a complex DNA repair mechanism, if the repair mechanism is destroyed, the aging of the body and the occurrence of related diseases are accelerated, and a plurality of diseases which are shown as premature senility, such as xeroderma pigmentosum, Werner syndrome, Bloom syndrome, Cockayne syndrome and the like are all involved in the defect of the DNA damage repair mechanism. Along with the progress of aging, the expression level of the mitotic checkpoint gene BubR1 in tissues is reduced, and studies show that over-expression of BubR1 can ensure accurate separation of chromosomes in vivo and prolong the life of mice. In addition to nuclear genes, mitochondrial genes (mtDNA) play an important role in the aging process of the body. The DNA polymerase gamma is responsible for the replication of mtDNA, and the mutation or deletion of the mtDNA of the mouse knocked out by the DNA polymerase gamma gene is shown as premature senility and shortened life span. Unlike nuclear DNA, mtDNA lacks an integral repair mechanism, and early mutated mtDNA can clonally amplify, causing mitochondrial respiratory chain damage, thereby promoting the onset of senescence. The nuclear fiber layer provides a scaffold for nuclear membrane and chromatin, and also plays an important role in maintaining genome stability.

Cellular senescence refers to the arrest of the cell cycle, permanently disabling division. Aging is a process of decreased cell adaptation, cessation of proliferation and death, and the properties of tumor cells are opposite, but both are caused by progressive accumulation of lesions and are merely different in performance. Unlike quiescent and terminally differentiated cells, senescent cells are mainly characterized by the following characteristics: high expression of p16INK4a and p19ARF, cell cycle arrest at G1 phase; chromatin remodeling to form senescence-associated heterochromatin aggregates (SAHF); expressing senescence-associated β -galactosyl glycinase (SABG); DNA Damage Response (DDR) persists with increased expression of P53. However, increasing the expression of P16. sup. INK4a, P19. sup. ARF, and P53 in mice can not only inhibit tumor but also prolong life span, and decrease the expression of P53 can accelerate aging, because cellular aging is also a compensatory mechanism of the body. The immune system can recognize and eliminate senescent cells, progenitor cells in tissues are differentiated to form young cells to replace the senescent cells, decay or diffusion of potential harmful cells such as tumor cells is prevented, but as the body ages, cell aging is accelerated, the elimination of the senescent cells by the immune system is slowed, and the number of the senescent cells in the body is increased. The phenomenon that senescent cells act on surrounding cells by secreting pro-inflammatory factors and matrix metalloproteinases is called the senescence-associated secretory phenotype (SASP). SASP and NF-kB are over activated, the immune system is disordered, autophagy function is reduced and other factors act on the body together to cause inflammatory aging. Many inflammatory factors such as IL-1, Tumor Necrosis Factor (TNF), interferon, etc. are present in increasing amounts in the aging body. Inflammatory aging is associated with the development of many ARD's, such as atherosclerosis, type 2 diabetes, alzheimer's disease, tumors, and the like. The aged mice tend to be younger by inhibiting NF- κ B through drug or gene manipulation. The expression of NF-. kappa.B in the hypothalamus results in a decrease in the secretion of gonadotropin-releasing hormone (GnRH), thereby promoting aging of the body. The long-term aspirin administration can prolong the life of mice and promote the healthy aging of human bodies.

Many anti-aging drugs are researched at present, and many drugs play an anti-aging role by acting on signal paths related to aging, and representative drugs comprise rapamycin, metformin, resveratrol and the like. Rapamycin is a novel immunosuppressant, and acts on mTOR to inhibit the mTOR signaling pathway, so that the downstream production of mTORC1 and S6K is reduced, and the anti-aging effect is achieved. The medicine can prolong the life of mouse by 60%, and the rapamycin analog RAD001 can improve the immunosenescence of the elderly. Rapamycin causes side effects including cataract, hematopoietic dysfunction, metabolic disorders (e.g., hyperglycemia, hyperinsulinemia, insulin resistance), etc., some of which are due to the fact that rapamycin inhibits mTORC1 as well as mTORC 2. However, whether rapamycin or its analogs, there is currently a lack of sufficient clinical evidence to demonstrate its anti-aging effects in humans. Metformin is a common drug for treating type 2 diabetes in clinic, and the incidence of cardiovascular diseases and cancers can be reduced by taking the drug for a long time. Metformin can prolong the life of nematodes and mice, and the anti-aging effect is considered to be related to activation of AMPK pathway, so that the anti-aging effect of metformin in human body is to be proved by further clinical tests. Resveratrol is a polyphenol compound, can improve the activity of SIRT1, and maintains the normal function of mitochondrial respiratory chain. Resveratrol can prolong the life of mice on high-fat diet but can not prolong the life of mice on normal diet, and the metabolic disorder can be improved by administering resveratrol to obese people, while the metabolic function of the body can not be enhanced by adding resveratrol to non-obese women with normal sugar tolerance. Spermidine is a polyamine, can prolong the life of yeast, Drosophila and mouse, has autophagy inducing effect, and can improve memory of aged Drosophila and improve cardiovascular health of mouse.

CN103936608B discloses a novel chalcone compound with anti-aging effect, which can obviously inhibit H₂O₂The induced telomerase activity is reduced, and the preparation method can be used as a raw material medicine for preparing various dosage forms of medicines related to delaying senility or regulating immunity and promoting brain function, and can be used as a raw material medicine for preparing various dosage forms of medicines related to delaying senility or regulating immunity and promoting brain function.

Although some anti-aging drugs exist at present, the types and types of drugs are not abundant enough, and the selection opportunities are not many, so that continuous research and development are needed to meet the social needs.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a new application of a new compound capable of delaying senility.

When the applicant researches skin anti-aging, the existing compound library is utilized to carry out specific screening on skin cells, and the compound capable of delaying skin cell aging is obtained.

The compound is of formula (I):

(formula I).

The compounds of formula I are typically used in the form of pharmaceutical compositions comprising one or more compounds of formula I and a pharmaceutically acceptable carrier. Preferably, these compositions are in unit dosage forms, such as tablets, pills, capsules, powders, granules, suspensions, metered aerosol or liquid sprays, drops, ampoules, transdermal patches; for painting or coating or otherwise administering the drug.

The principal active ingredients are typically mixed in a pharmaceutically acceptable carrier such as conventional tableting ingredients, for example corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate and dicalcium phosphate, or gums, dispersants, suspending agents or surfactants such as sorbitan monooleate and polyethylene glycol, and other pharmaceutically acceptable diluents such as water, to form a homogeneous preformulation composition containing the compound of the invention or a pharmaceutically acceptable salt thereof.

When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

In a further aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a compound of formula I. Preferably, the pharmaceutical composition is in a unit dosage form suitable for topical administration, such as an ointment or spray.

Further, the application of the compound shown in the formula I in preparing a pharmaceutical composition for treating human epidermal keratinocyte senescence is also provided.

Further, the human epidermal keratinocytes are human epidermal keratinocytes commercialized in the art.

The invention also provides a cosmetic for preventing skin aging, which contains the compound shown in the formula I.

Further, the application of the compound in the formula I in preparing cosmetics for preventing skin aging is also provided.

Advantageous effects

According to the invention, researches show that the compound shown in the formula (I) has a good antioxidant effect and has a function of preventing cell aging. The compound has good commercial value by being prepared into a corresponding anti-aging medicament or anti-aging cosmetic.

Drawings

FIG. 1 is a graph showing the results of the effect of compounds on ROS expression in human epidermal keratinocytes.

FIG. 2H at different concentrations₂O₂Graph of results for human epidermal keratinocytes under injury.

FIG. 3 Compound at H₂O₂Functional outcome plots in injury.

FIG. 4 Compound at H₂O₂Functional verification result chart in the damage.

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure.

EXAMPLE 1 preparation of the Compound of formula (I)

0.73ml of 30% H was added at 0 deg.C₂O₂The solution was added to a solution of 1g 4-bromothioanisole in 5ml acetic acid. The mixture was stirred at RT for 12h and worked up with 10% NaOH solution. The aqueous phase was extracted with ethyl acetate. The organic phase was washed with saturated NaCl solution and Na₂SO₄And (5) drying. The solvent was removed to give 0.99g of crude product A. 223mg of sodium azide are added dropwise at 0 ℃ to a solution of 900mg of product A in 18ml of chloroform. The mixture was stirred at 0 ℃ for 12h and ice water was added. Conventional work-up gave 592.6mg of product B. 253mg of 3-hydroxy-2-oxo-pyrrolidine-3-carboxylic acid-3-chloro-5-fluoro-benzylamide, 359mg of potassium carbonate, 382mg of N ', N' -dimethyl-ethane-1, 2-diamine and 330mg of copper iodide were added successively to a 12ml degassed dioxane solution of 200mg of product B from the above procedure. The mixture was reacted in a microwave oven at 140 ℃ for 2 h. The mixture was filtered through celite, and the solvent was removed in vacuo. Preparative HPLC gave 53.4mg of the compound of formula (I); the identification result is as follows:¹H NMR 400 MHz , DMSO-d₆:δ[ppm] 8.79 (t，J = 6.40 Hz , 1H) , 7.90-7.96(m，4H) , 7.26-7.29 (m，1H) , 7.20 (s，1H) , 7.10 (d，J = 9.60 Hz , 1H) , 6.87 (s ,1H) , 4.35-4.41 (m，1H) , 4.20-4.27 (m，2H) , 3.90-3.93 (m，2H) , 3.04 (d , J =0.80 Hz , 3H) 2.58-2.62 (m, 1H), 2.12-2.19 (m, 1H), the formula is shown in the following formula (I). The preparation procedure was repeated to obtain sufficient amount of the compound for subsequent experiments.

(formula I)

EXAMPLE 2 Effect of Compounds on ROS expression in human epidermal keratinocytes

(1) Human epidermal keratinocytes (Cat. 2110, Sciencell) in logarithmic growth phase were taken and adjusted to a density of 5X 10⁵Perml, add to 6 well plates (100. mu.L/well), 37 ℃ 5% CO₂And (5) adherent culture.

(2) The compound prepared in example 1 was diluted to a range of concentrations: 1. 10 and 50 mu g/mL, and the concentration of quercetin as a positive control is 50 mu g/mL. Serum-free medium was used as a negative control.

(3) After 24h of attachment, compound was added. After 12h incubation, the original medium was aspirated off and serum-free medium containing DCFH-DA (20 mM) was added to each well, except for the blank.

(4) The fluorescence value, excitation wavelength (485 nm), emission wavelength (538 nm) and detection time are measured immediately by using a multifunctional microplate reader at 37 ℃ every 5 min, wherein the detection time is 120 min.

(5) And (4) calculating a result: and calculating the relative fluorescence value of the compound according to the fluorescence values of the compound treatment group and the blank control group, calculating the time point with the maximum relative fluorescence value, and obtaining the relative expression amount of the ROS.

All samples were tested in 3 replicates. The results are shown in FIG. 1 below. The result of detecting the change of the ROS content in human epidermal keratinocytes shows that the ROS content of the negative control group is set to be 1, and the relative expression quantity of the ROS of the quercetin positive control group is as follows: 0.452 ± 0.070. While the relative expression of ROS in cells treated with different concentrations of compound was between 0.127 and 0.355. Has certain cell concentration dependence, wherein the compound with the concentration of 50 mug/mL has the best effect of inhibiting the ROS in cells, and the content is only 0.127 +/-0.031.

EXAMPLE 3 inhibition of the MAPK pathway by Compounds

Culturing human epidermal keratinocyte, collecting cells, and making into cosmetic⁵one/mL. Human epidermal keratinocytes were first seeded into Transwell cell chambers (2 mL/well). PBS (2 mL/well) was added and the dose was selected to be 100. mu.j/cm²The irradiation is carried out for 2 min. After irradiation, the expression level of p38MAPK is partially detected. The compound was added in part, and the cells in the Transwell plate were placed in a 6-well plate plated with HFF cells and after incubation for 24h, the cells were taken to determine the amount of p38MAPK expression. The results are shown in FIG. 2, with normal non-irradiated epidermal keratinocytes as a negative control. As can be seen from FIG. 2, 50. mu.g/mL of the compound significantly reduced the MAPK associated factor p38MAPK in human epidermal keratinocytes. Compared with the irradiation group, the compound addition group after irradiation can reduce the expression amount of p38MAPK to 54.22 +/-1.27%. The result shows that the irradiation can cause high expression of key factors of the MAPK signal pathway, and the compound can remarkably inhibit the over-expression of the key factors of the MAPK signal pathway after being added. Indicating that the compound can protect epidermal keratinocytes from radiation damage by inhibiting the p38MAPK signaling pathway.

Example 4H₂O₂Establishment of human epidermal keratinocyte model under injury

Taking the cells in good growth state, discarding the original culture medium, washing with PBS buffer solution for three times, digesting with pancreatin, adding the culture medium to terminate digestion, gently blowing and beating the cell suspension, counting the cells with a cell counting plate, taking out a 96-well plate, wherein the number of cells in each well is about 1 × 10⁴After 24h of incubation, the original medium was discarded. Separately adding different concentrations of H to the 96-well plate plated with cells₂O₂The culture medium of (4) is also provided with a blank control group. After 24h of culture, the survival rate of each cell is detected by a CCK-8 method, the optimal molding concentration is explored, the experiment is repeated for 3 times, and the average value is taken. As shown in FIG. 3, different concentrations of H were used₂O₂After 24H of action on the cells, the experimental results obtained show that with H₂O₂The survival rate of the cells is reduced along with the increase of the concentration, andat H₂O₂The viability of the cells was significantly reduced at a concentration of 500. mu. mol/l (P)<0.05), indicating that 500. mu. mol/l is the optimum H for the cell₂O₂And (5) successfully constructing a model concentration and oxidation damage model.

Example 5 Compounds in H₂O₂Functional verification in injury

500umol/l of H₂O₂Adding into human epidermal keratinocyte, adding keratinocyte culture medium containing 50 μ g/mL compound, and arranging blank control group, positive control group (containing quercetin 50 μ g/mL), and H₂O₂ Modeling of the model set (no compound treatment). After 24h of culture, the survival rate of the cells in each well is detected by a CCK-8 method, the experiment is repeated for 3 times, and the average value is taken. The results are shown in fig. 4, where the survival rate of human epidermal keratinocytes induced by hydrogen peroxide is reduced, as can be seen from the model group. The survival rate of the experimental group cells added with the compound is remarkably increased compared with that of the positive control group, and the result shows that the compound can improve the cell damage caused by hydrogen peroxide induction and prevent cell aging and apoptosis better than quercetin by improving the oxidation resistance of the cells.

Example 6 cell safety assay

Respectively culturing human erythrocyte, human embryo lung fibroblast and human epidermal keratinocyte by conventional culture method, and collecting cell with concentration of 1XlO⁵And (4) respectively. 5 XlO³Density of individual cells/well cells were added to 96-well plates separately and compounds were added and incubated with the cells for 4 hours, with no drug added in PBS solution as a control. The cell density of the drug-added group is not less than that of the control group by detecting the cell density, which shows that the composition provided by the invention has no obvious killing effect on normal cells and has better application prospect.

Example 7 Compound safety assay

Test animals: common-grade experimental animal white rabbit. The animals were examined for abnormalities. Environmental conditions: room temperature: 22-25 ℃, relative humidity: 60% -70%; the test method comprises the following steps:

according to the test of acute skin irritation in the cosmetic hygiene code (2007 edition), the hair on both sides of the spine of the tested animal is cut off in a range of 3 cm by 3 cm 24 hours before the test. The test compound was applied directly to the right skin, covered with clean gauze and fixed with a non-irritating adhesive tape for 2 hours. The other side was a negative control. Skin reaction at the contact site of the test animal was observed and recorded at 1 hour, 24 hours, 48 hours and 72 hours after the removal of the test substance. Under the test condition, the compound is classified as nonirritant to acute skin irritation test of white rabbits after 72 hours according to skin irritation strength, and has better safety.

Claims (6)

1. The use of a compound of formula (I) in the manufacture of a medicament for the treatment of cellular oxidation and cellular senescence; the compound has a structural formula as follows:

(formula I), wherein the cell is a human epidermal keratinocyte.

2. Use according to claim 1, characterized in that: the medicament further comprises a pharmaceutically acceptable carrier.

3. Use according to claim 1 or 2, characterized in that the medicament is a tablet, pill, capsule, powder, granule, suspension, metered aerosol or liquid spray, drop, ampoule, transdermal patch.

4. The use of a compound of formula I for the preparation of a cosmetic for preventing skin aging; the compound has a structural formula as follows:

(formula I).

5. Use according to claim 4, characterized in that the cosmetic product also contains a whitening component.

6. Use according to claim 5, characterized in that the cosmetic product is in the form of an emulsion.

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