CN114931519A

CN114931519A - Synthesis method of hydroxytyrosol and derivatives thereof and application of hydroxytyrosol and derivatives thereof in cosmetics

Info

Publication number: CN114931519A
Application number: CN202210754577.6A
Authority: CN
Inventors: 蒋昌盛; 何皓; 董海平; 刘阳
Original assignee: Shanghai Xianding Biological Technology Co ltd
Current assignee: Shanghai Xianding Biological Technology Co ltd
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-08-23

Abstract

The invention provides a synthetic method of hydroxytyrosol and derivatives thereof and application of the hydroxytyrosol and derivatives thereof in preparing cosmetics, and application of a mixture of one or more of hydroxytyrosol and derivatives thereof as an antioxidant in preparing cosmetics with effects of resisting wrinkles, improving firmness, whitening, preventing sunburn and the like, wherein the mixture of one or more of hydroxytyrosol and derivatives thereof in the cosmeticsThe content of the compound is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

in the formula I, R is selected from one of hydrogen, hydroxyl, halogen, methyl, and linear chain, branched chain, cyclic saturated or unsaturated alkyl with 2-6 carbon atoms; and occupies any position on the aromatic ring other than the phenolic hydroxyl group; n is 1 to 10. The hydroxytyrosol and the derivatives thereof synthesized by the application can efficiently remove free radicals such as ROS (reactive oxygen species), have good antioxidant activity, and can be applied to anti-wrinkle, tightening, whitening, sun-screening and other functional cosmetics as an antioxidant.

Description

Synthesis method of hydroxytyrosol and derivatives thereof and application of hydroxytyrosol and derivatives thereof in cosmetics

Technical Field

The invention relates to the field of skin care cosmetics, in particular to a synthesis method of an antioxidant active compound hydroxytyrosol and derivatives thereof and application of the antioxidant active compound hydroxytyrosol and derivatives thereof in preparing cosmetics with the effects of resisting wrinkles, improving firmness, whitening, preventing sunburn and the like.

Background

With the improvement of living standard, people have higher and higher demands on cosmetics, particularly cosmetics with real efficacy. The cosmetics with the functions of resisting aging and whitening occupy an important position in the cosmetic market. According to the statistics of union lihua company, the anti-aging effect product only occupies 38 percent of the cosmetic market, the anti-aging market in 2021 of China is nearly 1000 hundred million RMB, and the global market is more as high as 2160 hundred million dollars and rapidly increases. Excessive Reactive Oxygen Species (ROS) in human skin tissue can accelerate skin aging, increase skin wrinkles, and become loose, rough and dull; free radicals also participate in the formation of melanin and glycation reactions in the skin, causing skin darkening and stain production. The antioxidant active compound can assist a human body to eliminate redundant free radicals, resist the generation of skin wrinkles or enable loose skin to become compact, reduce skin color spots, and is a core functional raw material of cosmetics with the effects of resisting aging, whitening and the like.

At present, antioxidants such as ergothioneine, acetyl hexapeptide-8, vitamin E and the like commonly used in the cosmetic industry are either expensive or have a common effect, and development of a novel antioxidant active compound which is efficient, safe, controllable in cost and capable of being used in cosmetics is urgently needed.

The cosmetics are applied on the skin surface of human body, and besides the effect, the cosmetics are also safe and harmless, so that a new trend is to search and develop new cosmetic raw materials from medicinal active molecules and foods, and N-acetylneuraminic acid (cubilose acid) which is the first new raw material approved by the national drug administration after the cosmetics are reformed in 2021 year is the effective component in the cubilose extract. Olive oil is one of the main food materials in the Mediterranean region, belongs to a super food with rich nutrition, and because of low incidence rate of cancer, cardiovascular diseases, diabetes and other modern common diseases, Mediterranean drinkers are always subjected to the key research of modern medicine. It is considered that the antioxidant activity of phenolic compounds such as hydroxytyrosol in olive oil is a major factor for reducing the incidence of these diseases. The hydroxytyrosol has strong antioxidant activity, can promote regeneration of cartilage tissue, can resist and prevent cancer, has anti-inflammatory and antibacterial effects, and can be used for preventing cardiovascular and cerebrovascular diseases, and sugar and lipid metabolism diseases. However, the use of hydroxytyrosol as a single pure product in cosmetics has not been systematically studied.

Hydroxytyrosol exists in various parts of plant olives in the form of different esters or oleuropein (oleuropein), which are gradually degraded into hydroxytyrosol and other phenolic or polyphenolic compounds only when the olives are mature or extracted into olive oil, so that the content of hydroxytyrosol in olive oil extracts is very small, and it is difficult to obtain high purity hydroxytyrosol in large quantities by purification methods.

So far, several documents have reported about the synthesis method of hydroxytyrosol, and international patent W02007009590 discloses a synthesis route using catechol and glyoxylic acid as raw materials, and obtains the target compound through four-step reaction, the total yield is not higher than 52.7%, and lithium aluminum hydride which is expensive and inconvenient to operate is needed. Chinese patent CN110128246A discloses a synthetic route using 3, 4-dimethoxyphenylacetic acid as raw material, the total yield of four-step reaction is 41%, HBr and metallic sodium are needed, the operation is inconvenient and harmful to the environment. Patent CN103664536A discloses another route using catechol as starting material, which requires five steps with total yield of only 24%, bromine and boron tribromide in the reaction, and is environmentally hazardous and inconvenient to operate. Bovicelli et al (Synthetic Communications, 37(23), 4245-4252; 2007) report that tyrosol is used as a raw material, the ortho-position of phenolic hydroxyl group of the tyrosol is brominated under the action of sodium bromide and Oxone, and then the tyrosol is obtained through five reactions of methoxylation, protective reduction and the like. The above reports all have the problems of low total yield due to long route, expensive reaction raw materials, inconvenient operation, harm to the environment and the like. Hydroxytyrosol has wide application prospect in the fields of medicines, foods and cosmetics, and a synthetic route which is simple in route, low in cost and harmless to the environment is needed.

Disclosure of Invention

In view of the above disadvantages of the prior art, the present invention aims to provide a method for synthesizing hydroxytyrosol and its derivatives, and to systematically study the possible uses of the synthesized high-purity hydroxytyrosol and its derivatives in cosmetics, so as to solve the problem of the prior art that the application of hydroxytyrosol and its derivatives as single pure products in cosmetics is blank.

The structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

in the formula I, R is selected from one of hydrogen, hydroxyl, halogen, methyl, and linear, branched, cyclic saturated or unsaturated alkyl with 2-6 carbon atoms; and occupies any position on the aromatic ring other than the phenolic hydroxyl group; n is 1 to 10.

When R is hydrogen and n is 2, the compound is hydroxytyrosol.

The invention firstly provides a method for synthesizing hydroxytyrosol and derivatives thereof, which takes a compound with a structural formula shown as a formula II as a raw material to carry out hydroxylation reaction to prepare the hydroxytyrosol and the derivatives thereof;

in the formula II, R is selected from one of hydrogen, hydroxyl, halogen, methyl, and linear, branched, cyclic saturated or unsaturated hydrocarbon groups with 2-6 carbon atoms; and occupies any position on the aromatic ring other than the phenolic hydroxyl group; n is 1 to 10.

Preferably, a catalyst and an inorganic strong base are also added in the hydroxylation reaction.

The synthesis method only needs two steps of reaction, and the reaction route is as follows:

the invention mainly considers the direct hydroxylation reaction of bromine, and finds that the reaction can not be well carried out under the condition that copper salts such as copper chloride, cuprous chloride, oxine copper and the like are selected as catalysts and singly or under the combined action of ligand 8-hydroxyquinaldine, and firstly finds that the bromo intermediate shown in the formula II reacts with excessive inorganic strong base under the catalysis of copper powder or copper sulfate, takes water as a solvent, under the condition of heating and refluxing, the hydroxyl can smoothly replace bromine, and the target compound hydroxytyrosol and the derivative thereof shown in the formula I are directly generated after acidification.

Further, the catalyst for hydroxylation reaction of the bromo-intermediate shown in the formula II is one of copper powder or copper sulfate, and the copper sulfate can be anhydrous copper sulfate or copper sulfate hydrate. The molar ratio of the intermediate 3 to the copper powder is 1: 0.05-0.3, and the molar ratio of the intermediate 3 to the copper sulfate is 1: 0.1-0.3. Both catalysts have a good catalytic effect within the range of the amount used, the reaction efficiency is reduced below the range, the yield cannot be improved above the range, unnecessary waste is brought, and excessive waste is formed.

Further, the inorganic strong base is one of sodium hydroxide, potassium hydroxide or lithium hydroxide. The reaction efficiency of the three is almost the same, and sodium hydroxide is used preferably because it is the lowest cost and easy to scale up.

By utilizing the optimal reaction conditions of the invention, the yield of hydroxytyrosol synthesized by hydroxylation of the bromo-intermediate shown in the formula II can reach 87.9%, and the product purity can reach 99%. According to the reaction conditions reported by Bovicelli and the like, the yield of the bromo-intermediate shown in the formula II generated by brominating the tyrosol derivative shown in the formula III can reach 93.6%, and the total yield of the two-step reaction exceeds 80%. The raw materials used in the two-step reaction are cheap and easy to obtain, and the operation is simple and easy to industrialize.

The invention further utilizes the high-purity hydroxytyrosol system prepared by the synthesis method to research the application of the hydroxytyrosol in cosmetics so as to prove the effectiveness of the hydroxytyrosol in the aspects of anti-aging, whitening, sun protection and the like.

Skin aging mainly takes two forms of natural aging and photoaging, and the natural aging is mainly caused by inherent factors such as heredity and the like; photoaging refers to the accumulation of ultraviolet injury during skin aging, and is the result of combined action of natural aging and ultraviolet radiation, and is manifested by increased skin wrinkles at exposed parts, coarse epidermis with poor keratosis, irregular pigmentation, and dermal elastic fiber deformation with loose skin. One of the important causes of photoaging is that human skin generates excessive Reactive Oxygen Species (ROS) and other free radicals after being irradiated by ultraviolet rays, and the influence on human skin is reflected in three aspects: 1) the redundant free radicals of the human body can react with unsaturated fatty acid in the body, so that the unsaturated fatty acid in the cell membrane is reduced, the saturated fatty acid is relatively increased, the softness of the cell membrane is reduced, the cell membrane is abnormal in function, the skin is dry when the cell membrane is expressed on the skin, and wrinkles become more and deeper; 2) mitochondria are a cell power plant, 85% of energy of a human body generates the energy, and under the action of excessive free radicals, mitochondrial DNA is subjected to point mutation or massive deletion, so that the function of fibroblasts is reduced, the structure and the function of the mitochondria are changed, adenine nucleotide triphosphate (ATP) generation is influenced, the energy supply is insufficient, the metabolic capability of the body is reduced, and a series of aging changes occur; 3) the free radicals form glycosylation end products (AGEs) on the surface of collagen to over-crosslink the collagen, the skin half-bridge structure loses elasticity, the skin becomes flaccid, loses elasticity, and is pigmented. Natural aging is irreversible, but photoaging can be alleviated by scavenging excess ROS and free radicals from the body, so that the antioxidant active compounds that scavenge free radicals and ROS can reduce skin wrinkles, restore skin elasticity and serve as anti-aging effects.

Because the free radicals play an important promoting role in the conversion process of the dopaquinone to the melanin, play a key role in the saccharification process of AGEs formed by metabolizing redundant sugar and collagen on the surface of human skin to generate senile plaques, and the removal of the free radicals can prevent the formation of the melanin and the senile plaques, the antioxidant active compound capable of removing the free radicals and ROS can play a whitening role in cosmetics.

Ultraviolet radiation damage to human skin is an oxidative stress process that causes a range of tissue damage by generating oxygen free Radicals (ROS), and scavenging or reducing ROS can block or slow tissue damage or promote post-sunburn repair, which is an indirect sunscreen effect. From the end biological effects of sun protection, antioxidants with the ability to scavenge free radicals may be used as biological sunscreens.

The invention researches the capacity of hydroxytyrosol as an antioxidant for eliminating free radicals and ROS and the possibility of the hydroxytyrosol applied to cosmetics from three aspects of in vitro, animal models and human bodies.

1) DPPH radical scavenging method

The free radical scavenging ability of hydroxytyrosol as an antioxidant in vitro was tested by using 1, 1-Diphenyl-2-trinitrophenylhydrazine (1, 1-Diphenyl-2-picryl hydrazyl, DPPH for short) free radical scavenging method. DPPH is a stable long-lived radical, with a dark purple color in ethanol and a strong absorption near 517 nm. In the presence of the free radical scavenger, the DPPH ethanol solution absorbs less light due to its one-electron pairing. The degree of decolorization of DPPH ethanol solution is in linear relation with the number of electrons received by DPPH ethanol solution, so that the capability of eliminating free radicals and the magnitude of antioxidant activity of experimental samples can be evaluated.

The DPPH clearance in the presence of various concentrations of hydroxytyrosol is shown in Table 1:

TABLE 1 Hydroxytyrosol DPPH clearance

TABLE 2 vitamin E DPPH clearance

As can be seen from Table 1, the clearance has reached 77.52% with hydroxytyrosol addition at a concentration of 0.02mg/mL, IC ₅₀ But only 0.01 mg/mL. As can be seen from Table 2, the clearance of vitamin E widely used in the cosmetics industry is only 39.85% under the same concentration, when the concentration is added to 0.04mg/mL, the clearance reaches 74.45%, and the IC of the vitamin E is ₅₀ It was 0.026 mg/mL. The above experimental results clearly demonstrate that hydroxytyrosol has a good free radical scavenging ability in vitro and can be used as an antioxidant.

2) Zebrafish embryo ROS clearance test method

Hydroxytyrosol was tested for its antioxidant capacity using the ROS scavenging assay of zebrafish embryos. The gene similarity of zebra fish and human is up to 87%, and the national institutes of health of the United states of America in 2003 is the third model organism following rat and mouse, which is called as the 'mouse' in water. The physiological and developmental metabolism of the zebra fish skin is highly similar to that of mammals, the skin structure and the human body similarity of the zebra fish skin are high, and the zebra fish skin also has a basal layer, a spinous layer, a granular layer, a transparent layer, an epidermal keratinocyte layer, an inherent layer, a desmosome, a melanocyte, a blood vessel, a subcutaneous fat cell and the like, wherein the skin structure of the human body is the same. Therefore, the zebra fish can be applied to acute toxic substance testing and efficacy testing and has been widely accepted in the cosmetic industry.

The ROS regulation mechanism in the zebra fish is the same as that of a human body, and ROS in the zebra fish embryo can be monitored by vital staining of a fluorescent probe 2 ', 7' -dichlorofluorescein diacetate (H2DCFDA) and is marked as green fluorescence. The zebra fish embryos are small in size, photographing is convenient to conduct under a microscope, green fluorescence signals which are enhanced along with increase of ROS in the zebra fish embryos are quantitatively read through software analysis, ROS level changes of the fish embryos of a treatment group and a blank control group are tested and compared, and the ROS removing capacity of hydroxytyrosol is calculated.

The test result is shown in figure 1, when the formula is added with 2.5 percent of hydroxytyrosol (the test concentration is 0.125g/L), the ROS clearance rate of the zebra fish embryo can reach 35 percent, and the effect is obvious; further increases in concentration to 7.5% (test concentration 0.375g/L), 12.5% (test concentration 0.625g/L), further increases in ROS clearance to 40% and 47%, clearly showing that hydroxytyrosol as an antioxidant is highly effective in scavenging ROS in zebrafish embryos. The survival rate of the zebra fish is over 90 percent under all the detection concentrations, which indicates that the hydroxytyrosol is safe and harmless.

The oxidation resistance effect of the ergothioneine which is an expensive active raw material widely used in the market is detected by using a zebra fish embryo ROS clearance test method, under the condition of the same formula addition concentration of 7.5%, the ergothioneine ROS clearance rate is only 24%, and hydroxytyrosol can reach 40%. The ROS clearance rate of another expensive antioxidant raw material acetyl hexapeptide-8 is only 14% when the addition concentration of the formula is 1% and 1.4%, and the ROS clearance rate is reduced to 6% when the addition concentration is increased to 20%. The vitamin C derivative ascorbyl glucoside has an ROS clearance of 13% at an added concentration of 0.4%, but is not tolerated by zebrafish at the further added formula concentration, and the specific results are shown in fig. 2.

The above experimental results clearly demonstrate that hydroxytyrosol has a strong ROS-scavenging ability in zebrafish and a much higher scavenging efficiency than expensive ergothioneine and acetyl hexapeptide-8 at the same concentration. This is probably because hydroxytyrosol not only has a strong radical scavenging ability, but also is an amphiphilic small molecule soluble in both water and oil, which passes more easily through the skin barrier of zebrafish. Due to the strong similarity between the skin of zebra fish and the skin of human body, hydroxytyrosol as an antioxidant can be expected to play a good role in cosmetics such as anti-aging, whitening and sunscreen.

3) Human body efficacy experiment

In order to further prove the real efficacy of hydroxytyrosol as an antioxidant in human bodies, the invention carries out human body tests to verify the actual efficacy of hydroxytyrosol in resisting wrinkles and aging on human skin. The safety of hydroxytyrosol on human body is firstly verified by using a patch test before an actual efficacy test.

3.1) test of blocking Patch on human skin

The invention adopts a skin-closed patch test to evaluate the safety of the hydroxytyrosol in the use of human skin. According to the requirements of technical Specification for safety of cosmetics (2015), 30 qualified subjects were selected and subjected to skin-enclosed patch test. First, a test area on the arm of a subject was examined, and an aqueous solution containing 2% hydroxytyrosol was injected into a patch tester, and the patch tester was attached to the test area of the subject for 24 hours. Skin reactions were observed at 30 minutes (after disappearance of the impression), 24 hours, and 48 hours after patch removal according to the criteria of table 3, and the observations were recorded.

TABLE 3 skin reaction grading Standard of skin Enclosed Patch test and test results

The human occlusion patch test was performed on a 2% hydroxytyrosol aqueous solution according to the above specified method, and the test results of table 3 show that: all of 29 subjects showed negative reactions (score: grade 0), and only one of the subjects showed suspicious reactions, indicating that the test subjects did not have adverse reactions to human skin.

3.2) human body anti-wrinkle and tightening effect test

After the safety and effectiveness of the hydroxytyrosol to a human body are confirmed, efficacy evaluation methods such as skin parameter measurement, skin rapid three-dimensional imaging, skin ultrasonic diagnosis and facial image analysis are adopted to evaluate the anti-aging effects such as anti-wrinkle, firmness improvement and the like of the hydroxytyrosol in cosmetics.

The method comprises the following specific steps: 30 female volunteers with age of 35-50 years, identical wrinkle grade of left and right canthus and period of non-preparation pregnancy, lactation and pregnancy more than 1 are selected, the left and right sides are randomly divided into an experimental side and a control side, the night cream prepared in the example 11 with 2% hydroxytyrosol added is used on the experimental side, and the blank night cream without hydroxytyrosol is used on the control side. The experiment requires volunteers to use 2 times a day in the morning and evening, with a dose not less than 3g each time, and ensures testingIndoor activities are taken as the main part during the period, and the long-time unprotected exposure under the sunlight is avoided. Before each test, the subject needs to clean the face, dry the skin, sit still in a room at a temperature of 21 + -1 deg.C and a humidity of 50 + -5% for more than 30 minutes, and wait for the skin to reach a stable state. The changes of skin wrinkle parameters including the number of wrinkles, average wrinkle depth (. mu.m), and wrinkle volume (mm) were measured at D0, D14, and D28 ³ ) And the like.

The test instrument used was:

facial image analyzer from Canfield corporation, usa, model: VISIA-CR;

multifunctional skin tester from CK company, germany, model: MPA 4;

model of skin ultrasonic diagnostic apparatus of Germany CK company: UC 22;

skin rapid three-dimensional imaging system model number of CK company, germany: PRIMOS lite;

the significance of the data difference is analyzed by adopting t test, and the data is marked as 'NS' when P is more than or equal to 0.05; p < 0.05 is marked as "S" and the results are shown in FIG. 3.

As can be seen from FIG. 3, the number of wrinkles of the skin of the tested part is obviously reduced by 46.52%, which is 1.58 times that of the control group, compared with that before the use, when the skin is continuously used for 28 days; the average wrinkle depth was 62.89% less than 1.52 times that of the control group; the volume of wrinkles is reduced by 9.42 percent, which is 1.33 times that of the control group; the values were all significantly different (P < 0.05).

The depth and density variation of the dermis and epidermis layers, respectively, of the skin area tested is shown in fig. 4 and 5. As can be seen from the test results of fig. 4 and 5, after 28 consecutive days of use, the dermal layer depth of the skin of the subject was increased by 23.48% compared to that before use, which was 2.3 times that of the control group; the density is improved by 87.50 percent, which is 1.75 times of that of the control group. The depth of the epidermal layer is improved by 13.95 percent and is 1.09 times of that of the control group; the density of the epidermis layer is improved by 48.84 percent, which is about 1.5 times that of the control group. The depth of the epidermis layer removed was not significantly different (P > 0.05), and the other values were statistically significant (P < 0.05).

A subject aged 30 years was selected and observed for changes in the epidermal and dermal layers of the skin before and 28 days after continuous use of the test sample (fig. 6), and the changes were found to be significant.

The above human efficacy data clearly demonstrate that subjects using the hydroxytyrosol-containing night cream had a significant reduction in skin wrinkles and volume, and a significant increase in dermal depth and density, after 28 days. As can be seen from the facial images of the testees, most of the testees have fair and glossy skin after being used for 28 days, which shows that the hydroxytyrosol can help the skin to resist wrinkle and tighten, achieve the anti-aging effect and simultaneously have a certain whitening effect.

The three groups of experiments firstly prove that hydroxytyrosol as an antioxidant has very excellent capability of scavenging free radicals such as ROS and the like from two aspects of in vitro and animal models, and is superior to the antioxidant commonly used in the cosmetic industry at present, such as vitamin E, ergothioneine, acetyl hexapeptide-8 and the like. Further human body efficacy data show that hydroxytyrosol is not only safe and harmless to human bodies, but also shows good anti-wrinkle and anti-aging effects, and is particularly suitable for being used as an antioxidant in cosmetics.

The synthesis method provided by the invention can be used for conveniently synthesizing the high-purity hydroxytyrosol and the derivatives thereof shown in the formula I. The hydroxytyrosol and its derivatives have certain solubility in water phase and oil phase, and can be conveniently added into various cosmetics such as solution, cream, facial mask, lotion, gel, spray, etc.

As shown in the foregoing, excessive free radicals such as ROS in human body can cause photoaging such as skin roughness and wrinkles, and hydroxytyrosol and derivatives 1 thereof as antioxidant can effectively eliminate free radicals such as ROS, so that the effects of resisting wrinkle and improving firmness can be achieved in cosmetics, the anti-aging effect is achieved, and the human body effect experiment is clearly and definitely verified. Hydroxytyrosol and its derivative 1 can play a key role in cosmetics with anti-wrinkle and firming effects as antioxidant, and the recommended addition concentration of the formula is in the range of 0.1-20% (w/w), more preferably in the range of 0.1-10% (w/w), and particularly preferably in the range of 0.5-5% (w/w).

Since the formation of melanin and senile plaque is closely related to free radicals, hydroxytyrosol and its derivative 1 as antioxidant can play a key role in whitening cosmetics, and the recommended addition concentration of the formula is in the range of 0.01-20% (w/w), more preferably 0.1-10% (w/w), and particularly preferably 0.5-5% (w/w).

The antioxidant with the ability of scavenging free radicals can be used as a biological sunscreen. Hydroxytyrosol and its derivatives 1 are recommended to be added as antioxidant in the range of 0.01-20% (w/w), more preferably 0.1-10% (w/w), and especially preferably 0.5-5% (w/w) in the formula of cosmetic with sunscreen effect.

Excessive free radicals such as ROS are closely related to inflammation, and a large number of documents report that hydroxytyrosol has an anti-inflammatory function. Since problems such as acne, pimple, and skin allergy on the skin surface are caused by inflammation, hydroxytyrosol and its derivative 1 as an antioxidant can be used as an auxiliary material for cosmetics having effects of removing acne, soothing and repairing, and caring scalp, and the recommended addition concentration of the formula is in the range of 0.01% to 20% (w/w), more preferably 0.1% to 10% (w/w), and particularly preferably 0.5% to 5% (w/w).

As described above, the method for synthesizing hydroxytyrosol and its derivatives and the use thereof for preparing cosmetics according to the present invention have the following beneficial effects: the synthetic route of hydroxytyrosol and derivatives thereof provided by the application is simple, the raw materials used in the two-step reaction are cheap and easy to obtain, the operation is simple, the yield can reach 87.9%, the product purity can reach 99%, and the industrialization is easy to realize; the synthesized hydroxytyrosol and derivatives thereof can efficiently scavenge free radicals such as ROS (reactive oxygen species), have good antioxidant activity, can be used as an antioxidant to be applied to cosmetics with potential effects of resisting wrinkles, improving firmness, whitening, preventing sunburn and the like, and human body effect tests further prove that the hydroxytyrosol can play the effects in the cosmetics.

Drawings

FIG. 1 is a graph showing the results of the ROS scavenging test of hydroxytyrosol on zebrafish embryos.

FIG. 2 is a graph showing the results of ROS scavenging test of zebrafish embryos with antioxidants commonly used in cosmetics.

Figure 3 shows a graph of hydroxytyrosol against the change of effective wrinkles in humans.

Fig. 4 shows a plot of the change in cortical depth of the dermis and epidermis layers, respectively, of the tested skin area for hydroxytyrosol.

FIG. 5 is a graph showing the cortical density variation of hydroxytyrosol on the dermal and epidermal layers, respectively, of an area of skin tested.

Figure 6 shows a plot of hydroxytyrosol versus cortical changes in individual subjects.

FIG. 7 shows the NMR spectrum of hydroxytyrosol obtained in example 1.

FIG. 8 shows a flow chart of a zebrafish embryo ROS clearance test method.

Detailed Description

The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.

Furthermore, it is to be understood that one or more method steps recited in the present disclosure are not exclusive of other method steps that may also be present before or after the recited combination of steps or that other method steps may also be inserted between the explicitly recited steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Example 1

The embodiment provides a synthesis method of 3-bromo-4-hydroxyphenylethanol, which comprises the following steps:

dissolving a raw material 4-hydroxyphenylethanol (100g, 0.72mol) in a mixed solvent of 30mL of acetone and 72mL of water, adding potassium hydrogen peroxysulfate composite salt (356g, 0.58mol) in batches under the cooling of an ice water bath, then dropwise adding a 50% sodium bromide (82g, 0.8mol) aqueous solution, continuing to react for 1 hour in the ice water bath after finishing dripping, then dropwise adding a sodium thiosulfate aqueous solution to quench the reaction, concentrating under reduced pressure to remove the acetone, filtering, washing and drying the precipitated solid to obtain 147g of 3-bromo-4-hydroxyphenylethanol, wherein the yield is 93.6%.

¹ H NMR(400MHz，CDCl ₃ )：δ7.34(s，1H)，7.08(d，J＝8.0Hz，1H)，6.95(d，J＝8.4Hz，1H)，5.52(br，1H)，3.82(t，J＝6.6Hz，2H)，2.78(t，J＝6.4Hz，2H)。

Example 2

The embodiment provides a method for synthesizing hydroxytyrosol, which comprises the following steps:

dissolving sodium hydroxide (138g, 3.45mol) in 900mL of water, adding copper sulfate pentahydrate (5.74g, 0.023mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating to 110 ℃, refluxing for 7 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting for 4 times by using ethyl acetate, then concentrating the combined organic phase under reduced pressure to remove the solvent, and distilling the residual substance under reduced pressure to obtain 26.4g of hydroxytyrosol, wherein the yield is 74.3%. The NMR spectrum of the hydroxytyrosol product obtained in this example is shown in FIG. 7, and the characteristic peaks are as follows: ¹ H NMR(400MHz，D2O)：66.84(d，J＝8.0Hz，1H)，6.79(d，J＝2.0Hz，1H)，6.69(dd，J＝8.2，2.2Hz，1H)，3.74(t，J＝6.8Hz，2H)，2.70(t，J＝6.8Hz，2H)。

example 3

dissolving sodium hydroxide (138g, 3.45mol) in 900mL of water, adding copper sulfate pentahydrate (17.23g, 0.069mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating to 110 ℃, refluxing for 7 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting the combined organic phase for 4 times by using ethyl acetate, concentrating under reduced pressure to remove the solvent, and distilling the residual under reduced pressure to obtain 31g of hydroxytyrosol product, wherein the yield is 87.3%.

Example 4

The embodiment provides a synthetic method of hydroxytyrosol, which comprises the following steps:

dissolving sodium hydroxide (138g, 3.45mol) in 900mL of water, adding anhydrous copper sulfate (11g, 0.069mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating and refluxing for 7 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting for 4 times by using ethyl acetate, carrying out pressure-reducing concentration on the combined organic phase to remove the solvent, and carrying out reduced-pressure distillation on the residual substance to obtain 31.2g of hydroxytyrosol product, wherein the yield is 87.9%.

Example 5

dissolving lithium hydroxide (82.6g, 3.45mol) in 900mL of water, adding anhydrous copper sulfate (11g, 0.069mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating to 110 ℃, refluxing for 7 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting for 4 times by using ethyl acetate, then concentrating the combined organic phase under reduced pressure to remove the solvent, and distilling the residual substance under reduced pressure to obtain 30.4g of hydroxytyrosol product, wherein the yield is 85.6%.

Example 6

dissolving sodium hydroxide (138g, 3.45mol) in 900mL of water, adding copper powder (1.46g, 0.023mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating to 110 ℃, refluxing for 20 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting for 4 times by using ethyl acetate, concentrating the combined organic phase under reduced pressure to remove the solvent, and distilling the residual substance under reduced pressure to obtain 28.3g of hydroxytyrosol product with the yield of 79.7%.

Example 7

dissolving sodium hydroxide (138g, 3.45mol) in 900mL of water, adding copper powder (4.38g, 0.069mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating to 110 ℃, refluxing for 20 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting for 4 times by using ethyl acetate, concentrating the combined organic phase under reduced pressure to remove the solvent, and distilling the residual substance under reduced pressure to obtain 28.5g of hydroxytyrosol product with the yield of 80.2%.

Example 8

dissolving sodium hydroxide (138g, 3.45mol) in 900mL of water, adding copper powder (0.73g, 0.0115mol), then adding 3-bromo-4-hydroxyphenylethanol (50g, 0.23mol), heating to 110 ℃, refluxing for 20 hours, cooling the reaction solution, adjusting the pH value to 2-3 by using concentrated hydrochloric acid, extracting for 4 times by using ethyl acetate, concentrating the combined organic phase under reduced pressure to remove the solvent, and distilling the residual substance under reduced pressure to obtain the hydroxytyrosol product 24.3g with the yield of 68.5%.

Example 9

This example provides a DPPH radical scavenging experiment for hydroxytyrosol:

the test method is that DPPH and hydroxytyrosol samples are respectively or jointly dissolved in water or 95% ethanol, and the absorbance is measured at 517 nm. The detection mode is shown in the following table 4:

TABLE 4 DPPH radical scavenging Experimental test conditions

DPPH free radical clearance calculation formula: clearance (%) - (1- (T-T0)/(C-C0)). 100%

T-sample tube light absorption value, namely the light absorption value of the solution after the sample reacts with DPPH;

t0- -sample background absorbance;

C-DPPH tube light absorption value, namely the light absorption value of DPPH solution when no sample is added;

c0-background absorbance of solvent.

Example 10

This example provides a tyrosol zebrafish embryo ROS clearance test:

the detection experiment method comprises the steps of respectively exposing 24-tailed zebra fish embryos in 48 hours to hydroxytyrosol solutions of 0.625g/L, 0.375g/L and 0.125g/L, setting a blank control group, carrying out H2DCFDA staining on the fish embryos after 24 hours of exposure, measuring ROS signal intensity by fluorescence photography, and carrying out statistical analysis. The detection flow is shown in fig. 8.

The final Reactive Oxygen Species (ROS) clearance is then calculated as follows:

the clearance rate is [ (C-T)/C ] 100%

In the formula, T is the average value of ROS (reactive oxygen species) average signal intensity of a test object treated fish embryo; c is the average value of ROS 'average signal intensity' of fish embryos in the blank control group.

Example 11

This example provides a stability test of hydroxytyrosol as an antioxidant for preparing cosmetics, specifically using the formula of the anti-wrinkle efficacy test cosmetics for human body as shown in table 5:

TABLE 5 cosmetic formula for human body wrinkle resistance test

The method comprises the following operation steps: respectively heating the phase A and the phase B to 80-85 ℃, completely dissolving the phases uniformly, adding the phase B into the phase A under the stirring condition, and homogenizing at the speed of 3000r/min for 3-5 min; continuously stirring and cooling to 70 ℃, and adding the phase C; and cooling to below 45 ℃, sequentially adding the raw materials of the phase D, and uniformly stirring to obtain a cream sample.

Test sample formulation stability:

the test sample was divided into A, B, C, D, E five portions, where sample a was placed in a conventional room temperature.

1. And (3) centrifugal test: and putting the sample B into a centrifuge tube, putting the centrifuge tube into a centrifuge, centrifuging the sample B at the speed of 3000r/min for 30min, and taking the sample out to observe whether the sample has the phenomena of layering, precipitation, demulsification and the like.

2. Heat resistance test: placing the sample C in a constant temperature box, storing for 7 days at 40 +/-1 ℃, taking out, comparing with the sample A, and observing whether the sample C has the phenomena of layering, discoloration, taste change, coarsening and the like so as to evaluate the heat resistance of the product;

3. cold resistance test: placing a sample D in a refrigerator, storing for 7 days at the temperature of minus 5 +/-1 ℃, comparing the sample D with the sample A after being taken out, and observing whether the sample D has the phenomena of layering, color change, taste change, thickness reversion and the like so as to evaluate the cold resistance of the product;

4. cold-hot cycling test: and (3) sampling a sample E, testing for 7 days under the circulating conditions of keeping at 45 ℃ for 12h, keeping at 25 ℃ for 12h, keeping at-10 ℃ for 12h and keeping at 25 ℃ for 12h, and observing whether the sample E is layered, discolored, smelled, coarsened and the like so as to evaluate the cold and hot circulating stability of the product.

And (3) stability test: through a centrifugal test, a heat resistance test, a cold stability test and a cold and heat cycle test for 7 days, the sample containing the 2% hydroxytyrosol cream has no phenomena of layering, discoloration, taste change, coarse reversion and the like, and the stability of the sample in cosmetics is good.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. Use of one or more mixtures of hydroxytyrosol and its derivatives as antioxidant for preparing cosmetic with anti-wrinkle and tightening effects, characterized in that the content of one or more mixtures of hydroxytyrosol and its derivatives in the cosmetic is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

in the formula I, R is selected from one of hydrogen, hydroxyl, halogen, methyl, and linear chain, branched chain, cyclic saturated or unsaturated alkyl with 2-6 carbon atoms; and occupies any position on the aromatic ring other than the phenolic hydroxyl group; n is 1 to 10.

2. The application of the hydroxytyrosol as an antioxidant in preparing cosmetics with anti-wrinkle and firming effects is characterized in that the content of the hydroxytyrosol in the cosmetics is 0.01-20 wt%.

3. Use of hydroxytyrosol and one or more mixtures of its derivatives as antioxidant for the preparation of a cosmetic with whitening effect, characterized in that: the content of one or more mixtures of hydroxytyrosol and derivatives thereof in the cosmetic is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

4. Use of hydroxytyrosol and one or more mixtures of its derivatives as antioxidants for the preparation of a cosmetic product with sunscreen effect, characterized in that: the content of one or more mixtures of hydroxytyrosol and derivatives thereof in the cosmetic is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

5. Use of hydroxytyrosol and one or more mixtures of its derivatives as antioxidant for the preparation of a cosmetic with soothing and healing effects, characterized in that: the content of one or more mixtures of hydroxytyrosol and derivatives thereof in the cosmetic is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

6. Use of hydroxytyrosol and one or more mixtures of its derivatives as antioxidant for the preparation of a cosmetic with anti-acne effect, characterized in that: the content of one or more mixtures of hydroxytyrosol and derivatives thereof in the cosmetic is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

7. Use of hydroxytyrosol and one or more mixtures of its derivatives as antioxidant for the preparation of a cosmetic product with scalp care efficacy, characterized in that: the content of one or more mixtures of hydroxytyrosol and derivatives thereof in the cosmetic is 0.1-20% (w/w); the structural formula of the hydroxytyrosol and the derivatives thereof is shown as the formula I:

8. The synthetic method of hydroxytyrosol and derivatives thereof is characterized in that: taking a compound with a structural formula shown as a formula II as a raw material, and carrying out hydroxylation reaction to prepare hydroxytyrosol and derivatives thereof;

in the formula II, R is selected from one of hydrogen, hydroxyl, halogen, methyl, and linear chain, branched chain, cyclic saturated or unsaturated alkyl with 2-6 carbon atoms; and occupies any position on the aromatic ring other than the phenolic hydroxyl group; n is 1-10;

9. The method of synthesis according to claim 8, characterized in that: a catalyst and inorganic strong base are also added in the hydroxylation reaction; the inorganic strong base is selected from one of sodium hydroxide, potassium hydroxide and lithium hydroxide, and the catalyst is selected from one of copper powder and copper sulfate.

10. The method of synthesis according to claim 9, characterized in that: the catalyst is copper powder, and the molar ratio of the compound with the structural formula shown in the formula II to the copper powder is 1: 0.05-0.3; or the catalyst is copper sulfate, and the mol ratio of the compound shown in the structural formula II to the copper sulfate is 1: 0.1-0.3.