Methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, its preparation method and application

Abstract

The application discloses 2-(2,5-dihydroxyphenyl)-2-methoxy methyl acetate as well as a preparation method and application thereof, and relates to the technical field of marine fungus active ingredient production and application. The application discloses that 2-(2,5-dihydroxyphenyl)-2-methoxy methyl acetate has good anti-UVB-induced HaCaT cell oxidative damage activity, and the compound has low cytotoxicity, and has good development prospects in the preparation of cosmetics, medicines and health products for resisting skin damage.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of marine fungus active ingredient production and application, in particular to 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester and a preparation method and application thereof. BACKGROUND

[0002] Fungal natural products are a treasure trove of new cosmetic raw materials, and their development has important scientific significance and commercial value, and helps to solve the existing raw material bottleneck. Fungi are diverse in species and rich in metabolites, providing a broad space for the development of new raw materials. At the same time, the demand of consumers for natural, safe and efficient products can be met: fungal natural products have the characteristics of novel structure, diverse activity and high safety, which meet the expectations of modern consumers for cosmetic raw materials. Promote the development of green cosmetics: the culture conditions of marine microorganisms are mild and controllable, and the production process is environmentally friendly, which meets the green trend of the cosmetic industry. Consumers' demand for natural skincare products is rising, and bio-fermented skincare products are leading the way. After detailed literature research, no literature on the application of 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester in medicine and cosmetics is found. SUMMARY

[0003] The purpose of the present application is to provide 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester and a preparation method and application thereof. The present application finds that 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester has good anti-UVB-induced HaCaT cell oxidative damage activity, and the compound has low cytotoxicity, and has good development prospects in the preparation of anti-skin damage cosmetics, medicines and health products.

[0004] The technical scheme of the present application is as follows: a 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester has the following structural formula:

[0005] .

[0006] A preparation method of 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester comprises the following steps:

[0007] S1, using marine fungi After activation, inoculate in a liquid culture medium, and perform fermentation culture, Preserved in the China Center for Type Culture Collection, with a preservation number of ;

[0008] S2, after the fermentation culture is ended, the mycelium and the fermentation liquor are separated, the mycelium is added with an organic solvent for leaching, the leaching liquor is separated, the leaching liquor is concentrated and then suspended with distilled water to obtain a water suspension, and then the water suspension is extracted with ethyl acetate or n-butanol to obtain an extraction liquor A; the fermentation liquor is stirred with diatomite, and then extracted with ethyl acetate or n-butanol under reflux to obtain an extraction liquor B;

[0009] S3, the extraction liquor A or the extraction liquor B is concentrated and then subjected to normal phase silica gel column chromatography separation, gradient elution is carried out with dichloromethane / methanol mixed solution with a volume ratio of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1 and 10:1 in sequence, the fraction eluted by the dichloromethane / methanol mixed solution with a volume ratio of 10:1 is collected, and then reversed phase silica gel column chromatography and high performance liquid chromatography separation are carried out to obtain the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate.

[0010] In the aforementioned method for preparing the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, the fermentation culture is static culture at a temperature of 20-30℃ for 10-40 days.

[0011] In the aforementioned method for preparing the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, the specific steps for preparing the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate from the fraction eluted by the dichloromethane / methanol mixed solution with a volume ratio of 10:1 in S3 and then subjected to reversed phase silica gel column chromatography and high performance liquid chromatography separation are as follows: gradient elution is carried out with methanol / water mixed solution with a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 in sequence, each gradient elution is carried out for 5 times, fractions 18-25 are combined as sub-fraction 7, and fractions 26-28 are combined as sub-fraction 8; then high performance liquid chromatography separation is carried out, and the peaks with a retention time of 14 minutes in sub-fraction 7 under elution of methanol / water mixed solution with a volume ratio of 44% are the compounds.

[0012] Use of the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in the preparation of health care products for resisting skin damage.

[0013] In the aforementioned use of the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in the preparation of health care products for resisting skin damage, the skin damage includes skin damage and related complications caused by ultraviolet rays, and the health care product is prepared by adding acceptable food additives to the derivative of the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate.

[0014] Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in preparation of anti-skin damage cosmetics.

[0015] In the use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in preparation of anti-skin damage cosmetics, the skin damage includes UV-induced skin damage and related complications.

[0016] Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in preparation of anti-skin damage drugs.

[0017] In the use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in preparation of anti-skin damage drugs, the skin damage includes UV-induced skin damage and related complications, and the drugs are prepared by adding pharmaceutically acceptable adjuvants to the main active ingredient methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate according to the preparation method of the preparation recorded in pharmacy.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] Firstly, the application uses the polarity difference of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate to extract and separate methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate with a novel structure from the fermentation culture of marine fungi, and the method is simple in operation, high in extraction yield and high in product purity, and is suitable for large-scale production.

[0020] Secondly, the in vitro anti-UVB-induced HaCaT cell oxidative damage activity test shows that the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate provided by the application has good anti-UVB-induced HaCaT cell oxidative damage activity. Further cytotoxicity test shows that the compound provided by the application has low cytotoxicity, and has good development prospects in preparation of anti-skin damage cosmetics, drugs and health products. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the structural formula of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate of the application;

[0022] Figure 2 is a graph showing the effect of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate of the application on keratinocyte toxicity;

[0023] Figure 3 is a ROS detection result chart of the present application;

[0024] Figure 4 is a ROS removal rate chart of the present application. DETAILED DESCRIPTION

[0025] The present application is further described below in conjunction with the accompanying drawings and examples, but is not limited to the basis of the description.

[0026] Example 1. A methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, whose structural formula is:

[0027] .

[0028] A preparation method of a methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, comprising the following steps:

[0029] S1, using a marine fungus After activation, inoculate in a liquid medium, and perform fermentation culture, The microorganism is a gift from Zhejiang University. The microorganism has been disclosed in a patent with publication number CN103641791A, and was preserved in China General Microbiological Culture Collection Center on September 14, 2013, with a preservation number of ; the fermentation culture is static culture at a temperature of 20-30℃ for 10-40 days, and the preferred fermentation culture temperature is 22-26℃. More preferably, the fermentation culture is performed at a temperature of 25℃ for 20 days, and under this condition, the yield of the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate is the highest;

[0030] The fermentation liquid used is any one of the following fermentation liquids:

[0031] a1, in a volume of 1L, the liquid medium comprises the following raw materials: starch 1-5g, bran 10-20g, yeast extract 3-15g, KH2PO4 1-8g, MgSO4•7H2O 0.1-0.8g, and the rest is water;

[0032] a2, in a volume of 1L, the liquid medium comprises the following raw materials: potato 200-600g, peptone 2-10g, yeast extract 1-5g, glucose 5-20g, and the rest is water; the initial pH value of the culture solution is 6.0-7.0;

[0033] a3. The liquid medium comprises the following raw materials in a volume of 1 L: sucrose 10-40 g, corn powder 5-20 g, NaNO3 1-4 g, yeast extract 1-4 g, KH2PO4 0.2-0.8 g, MgSO4·7H2O 0.2-1 g, KCl 0.2-1 g, FeSO4 0.001-0.005 g, and the balance is water;

[0034] a4. The liquid medium comprises the following raw materials in a volume of 1 L: malt extract powder 20-30 g, glucose 15-20 g, casein peptone 1-2 g, and the balance is water;

[0035] S2. After the fermentation culture is completed, the mycelium and the fermentation liquor are separated, the mycelium is extracted by adding an organic solvent, the extract is separated, the extract is concentrated and then suspended in distilled water to obtain a water suspension, and the water suspension is extracted with ethyl acetate or n-butanol to obtain extract A; the fermentation liquor is stirred with diatomite, and then extracted by refluxing with ethyl acetate or n-butanol to obtain extract B;

[0036] S3. The extract A or extract B is concentrated and then subjected to normal phase silica gel column chromatography, gradient elution is performed with dichloromethane / methanol mixtures in a volume ratio of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1 and 10:1, respectively, the fraction eluted by the dichloromethane / methanol mixture in a volume ratio of 10:1 is collected, and then reversed phase silica gel column chromatography and high performance liquid chromatography are performed to obtain 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester; the fraction collected is subjected to reversed phase silica gel column chromatography, gradient elution is performed with methanol / water mixtures in a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, respectively, each gradient elution is performed 5 times, fractions 18-25 are combined as sub-fraction 7, and fractions 26-28 are combined as sub-fraction 8; then high performance liquid chromatography is performed, and the peaks with a retention time of 14 minutes in sub-fraction 7 are the compound eluted by the methanol / water mixture in a volume ratio of 44%.

[0037] I. Structural identification of the compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester in step S3.

[0038] The target compound prepared in step S3 is subjected to purity identification by HPLC, the sample with a purity greater than 98% is subjected to structural identification by mass spectrometry and nuclear magnetic resonance technology, the nuclear magnetic resonance is determined by TMS as an internal standard; high resolution mass spectrometry determined by electrospray mass spectrometry ​ Determination.

[0039] The chemical structure of compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester is shown in Figure 1 The NMR data are shown in Table 1 below.

[0040] Table 1. NMR data of compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester and

[0041]

[0042] II. Cytotoxicity analysis of compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester on keratinocytes

[0043] Keratinocytes (HaCaT) were evenly inoculated at 10000 cells per well in a 96-well plate, 100 μL per well. The 96-well plate was incubated in an incubator (37 ℃, 5% CO2) overnight. When the cell confluence rate in the 96-well plate reached 40%-60% under microscopic observation, the old culture medium was discarded and the cells were grouped and dosed. 100 μL of culture medium containing the corresponding concentration of the test substance was added to each well of the sample group, and 100 μL of culture medium was added to each well of the blank control group. The 96-well plate was incubated in the incubator for 24 h. After incubation, the old culture medium was discarded, and 100 μL of culture medium containing 10% CCK-8 was added to each well of the sample group and the blank control group, and the plate was incubated in the incubator for 0.5 h. After incubation, the plate was shaken to mix well, and the absorbance value (OD) was measured at 450 nm. All values are expressed as mean ± SD. Statistical comparisons were made using t-test or one-way ANOVA, with P<0.05 being considered significantly different, which was statistically significant.

[0044] The results show that at a concentration of 10 μM or less, compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester has no toxicity to keratinocytes.

[0045] In the process of obtaining 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester from mycelium, the mycelium is soaked in an organic solvent for 7-14 days, the mycelium is fully broken, and the intracellular substances are effectively dissolved. The organic solvent is one or two of methanol, ethanol, ethyl acetate, and acetone.

[0046] Table 2. Toxicity study of compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester on keratinocytes

[0047]

[0048] III. Analysis of the repair effect activity of compound 2-(2,5-dihydroxyphenyl)-2-methoxyethyl acetate on UVB-induced HaCaT cell damage.

[0049] 1. Experimental grouping: HaCaT cells were used for UVB test, and the experimental grouping is as follows: one group without UVB irradiation and without drug addition was recorded as blank control group (BC); one group with UVB irradiation and without drug addition was recorded as model control group (NC); one group with UVB irradiation and with Vc intervention before and after UVB irradiation was recorded as positive control group (PC); one group with UVB irradiation and with compound 2-(2,5-dihydroxyphenyl)-2-methoxyethyl acetate intervention before and after UVB irradiation was recorded as sample group (10 μM). Each group had at least 3 replicate wells.

[0050] 2. Cell culture and inoculation: HaCaT cells were cultured to 90% confluence, the cells were digested with 0.25% trypsin solution, centrifuged at 1200 rpm / min for 5 min, and the cells were resuspended in DMEM (containing 10% FBS) culture medium. The cells were inoculated into 12-well plates at a seeding amount of 1.5 x 104 cells / well, 1 mL per well. The 12-well plates were incubated in an incubator (37 ℃, 5% CO2) overnight.

[0051] 3. Liquid change and drug incubation before irradiation: when the confluence rate of cells in the 12-well plate reached 40-60% under a microscope, the old culture medium was discarded, and the liquid was changed and dosed according to the grouping. The blank control group was added with 1 mL of culture medium per well; the model control group was added with 1 mL of culture medium per well; the sample group was added with 1 mL of culture medium containing 10 μM of compound 2-(2,5-dihydroxyphenyl)-2-methoxyethyl acetate per well; the positive control group was added with 1 mL of culture medium containing 20 μg / mL of Vc per well. After the sample was added, the 12-well plates were incubated in the incubator for 4 h.

[0052] 4. UVB irradiation: the blank control group was not treated and not irradiated. The other groups (model control group, positive control group and sample group) were treated as follows: after the incubation for 4 h was completed, the original liquid in the wells was aspirated, 500 μL of HBSS solution was added to each well, and the cells were irradiated with 200 mJ / cm2 dose of UVB. After the irradiation, the culture medium was replaced, 1 mL of 10% FBS DMEM culture medium was added to each well of the model control group; 1 mL of culture medium containing 10 μM of compound 2-(2,5-dihydroxyphenyl)-2-methoxyethyl acetate was added to each well of the sample group; 1 mL of culture medium containing 20 μg / mL of Vc was added to each well of the positive control group. The blank control group was also replaced with new DMEM culture medium. After the sample was added, the 12-well plates were incubated in the incubator overnight.

[0053] ​5. DCFH-DA probe incubation: After overnight incubation, discard the old culture medium, and wash once with 500 μL HBSS per well. After washing, discard the HBSS in the well plate; add 500 μL of the diluted DCFH-DA probe to each well. Incubate in the incubator for 30 min.

[0054] 6. Flow cytometry detection:

[0055] a. After washing and probe incubation, wash twice with HBSS to remove residual probe solution.

[0056] b. Sample collection: add 500 μL of 0.25% trypsin solution to each well to digest the cells, centrifuge at 1200 rpm / min for 5 min, discard the supernatant; wash the cells with 500 μL of HBSS, centrifuge at 1200 rpm / min for 5 min, discard the supernatant, repeat this washing step twice; finally obtain the detection sample precipitate, and resuspend the cells with 300 μL of HBSS.

[0057] c. On-machine detection: set the target cell number to 10000, the detection speed to medium, and use the 488 nm wavelength channel (i.e. the FITC channel) for fluorescence detection.

[0058] 7. Data analysis

[0059] Perform data analysis on the experimental results to obtain the average fluorescence value (Geometric Mean, hereinafter referred to as GE) of each group. The GE values of each group can be used to draw a graph output, with the GE value as the vertical coordinate, the concentration of the sample to be tested and the sample category as the horizontal coordinate, for statistical analysis and graphing. All numerical values are expressed as mean values. Use t-test or one-way ANOVA for statistical comparison, with P<0.05 being significantly different, with statistical significance.

[0060] The average fluorescence value (GE value) of each group is shown in the accompanying drawings Figure 3 , wherein # indicates comparison with the BC group, "#" indicates P<0.05, "##" indicates P<0.01, "###" indicates P<0.001; "####" indicates P<0.0001; * indicates comparison with the NC group, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001; "****" indicates P<0.0001.

[0061] According to the average fluorescence value (GE value) of each group, calculate the ROS clearance rate, and the calculation formula is as follows:

[0062] .

[0063] The sample group GE is respectively brought into the positive control group and the sample group average fluorescence value data, the model group GE is brought into the model control group average fluorescence value data, and the blank control GE is brought into the blank control group average fluorescence value data.

[0064] Table 3 Activity oxygen (ROS) detection results of compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester

[0065]

[0066] The results show that, compared with the model control group and the blank control group, the average fluorescence intensity of the positive control group and the sample group has a significant difference, and the ROS clearance rates are 28.65% and 49.92% respectively, which indicates that the compound 2-(2,5-dihydroxyphenyl)-2-methoxyacetic acid methyl ester has a significant antioxidant effect, and the antioxidant effect is better than Vc.

Claims

1. Methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, characterized in that: The structural formula is: 。 2. A process for the preparation of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate, characterized in that, It comprises the following steps; S1, using marine fungi After activation, inoculate in liquid medium, fermentation culture, Preserved in China Center for Type Culture Collection, preservation number is ; S2, after the fermentation culture is finished, the mycelium and the fermentation liquor are separated, the mycelium is added with an organic solvent for leaching, the leaching liquor is separated, the leaching liquor is concentrated and then suspended with distilled water to obtain a water suspension, and then the water suspension is extracted with ethyl acetate or n-butanol to obtain an extraction liquor A; the fermentation liquor is stirred with diatomite, and then extracted with ethyl acetate or n-butanol under reflux to obtain an extraction liquor B; S3, the extraction liquor A or the extraction liquor B is concentrated and then subjected to normal phase silica gel column chromatography separation, gradient elution is carried out with dichloromethane / methanol mixed liquor with a volume ratio of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1 and 10:1 in sequence, the fraction eluted by the dichloromethane / methanol mixed liquor with a volume ratio of 10:1 is collected, and then reversed phase silica gel column chromatography and high performance liquid chromatography separation are carried out to obtain the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate.

3. A process for the preparation of methyl 2-(2,5-dihydroxyphenyl)-2- methoxyacetate according to claim 2, characterized in that, The fermentation culture is static culture at a temperature of 20-30 DEG C for 10-40 days.

4. The method for preparing methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate according to claim 2, characterized in that, The specific steps for obtaining the methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate by collecting the fraction eluted by the dichloromethane / methanol mixed liquor with a volume ratio of 10:1 in S3 and then carrying out reversed phase silica gel column chromatography and high performance liquid chromatography separation are as follows: gradient elution is carried out with methanol / water mixed liquor with a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 in sequence, each gradient elution is carried out 5 times, fractions 18-25 are combined as sub-fraction 7, and fractions 26-28 are combined as sub-fraction 8; then high performance liquid chromatography separation is carried out, and the peaks with a retention time of 14 minutes under elution of methanol / water mixed liquor with a volume ratio of 44% in sub-fraction 7 are the compounds.

5. Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in the preparation of a health care product for resisting skin damage.

6. Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate according to claim 5 for the preparation of a nutraceutical product against skin damage, characterized by the fact that it contains from 0.1 to 10% by weight of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate. The skin damage includes skin damage caused by ultraviolet rays and related complications.

7. Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in the preparation of a cosmetic product for resisting skin damage.

8. Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate according to claim 7 for the preparation of a cosmetic product against skin damage, characterized in that, The skin damage includes skin damage caused by ultraviolet rays and related complications.

9. Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate in the preparation of a pharmaceutical product for resisting skin damage.

10. Use of methyl 2-(2,5-dihydroxyphenyl)-2-methoxyacetate according to claim 9 for the preparation of a medicament for the treatment of skin lesions, characterized in that, The skin damage includes skin damage caused by ultraviolet rays and related complications.

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