Application of methyl 3, 4-dihydroxybenzoate in whitening

Abstract

The invention relates to the technical field of traditional Chinese medicine extracts, and discloses application of methyl 3, 4-dihydroxybenzoate in whitening. The 3, 4-dihydroxy methyl benzoate is extracted from balanophora involucrata, it is found that the 3, 4-dihydroxy methyl benzoate can achieve whitening by inhibiting the activity of tyrosinase, the inhibition rate of the 3, 4-dihydroxy methyl benzoate to the tyrosinase is 59.10% when the concentration is 0.125 mg / ml, the IC50 value is 0.2107 mg / ml, the inhibition rate is about one half of that of positive drug beta-arbutin (the IC50 value is 0.3763 mg / ml), and the inhibition activity is higher. The method has a wide application prospect when being applied to whitening products.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extracts, and in particular to application of 3,4-dihydroxybenzoic acid methyl ester in whitening. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Deer fairy grass is the whole herb of Balanophora involucrata Hook.f. and Balanophora harlandii Hook.f., plants of the genus Balanophora in the family Banophoraceae. It is a dicotyledonous perennial parasitic fleshy herb, also known as Wenwang's pen and Jixinqi. Among them, Balanophora involucrata often parasitizes on the roots of plants of the genus Rhododendron, while Balanophora harlandii mainly parasitizes on the roots of plants such as Ironwood, Hainan Sparrow Bean Vine, Indian Blood Vine, Rock Bean Vine, Density Flower Bean, Palm Leaf Ficus, Pilea, Red Mold Water Kudzu, Water Hemp, and Feathered Whitehead Tree. It is distributed in Fujian, Hubei, Guangdong, Guangxi, Sichuan, Yunnan and other places, and is also distributed in Japan and India. It is produced in Sichuan, Yunnan, Guizhou and Hunan.

[0004] The whole plant of Deer Herb is used as medicine, with a bitter, sweet and flat taste; cool in nature; and enters the heart, spleen, stomach and kidney meridians. It is beneficial to the kidney and nourishes yin; it has the effects of clearing away heat and stopping bleeding, strengthening yang and tonifying the kidney, regulating qi and strengthening the stomach, clearing away heat and detoxifying, stopping bleeding and promoting tissue regeneration. It is mainly used to treat kidney deficiency and low back pain; consumptive bleeding; hemorrhoid bleeding. It is used for impotence, spermatorrhea, edema, menorrhagia, leucorrhea, neurosis, chronic hepatitis, stomach pain, gastrointestinal bleeding, wind-heat macules, lung heat cough, and traumatic bleeding. It is mostly used in folk medicine, as Miao medicine, Zhuang medicine, Yi medicine, and is also used clinically. It plays an important role in the treatment of chronic hepatitis, traumatic bleeding, drunkenness, anti-tumor, sobering up, protecting the liver and lowering blood sugar.

[0005] Uneven skin tone, dullness and the formation of spots are generally caused by excessive melanin deposition. Tyrosine in melanocytes in the basal layer of the epidermis is oxidized to DOPA (DOPA) under the action of tyrosinase (TYR), and then further oxidized to dopaquinone, which is related to the formation of melanin. At present, there are few reports on the whitening active ingredients of Luxiancao. Therefore, in this study, the chemical components of Luxiancao were extracted and separated, and the isolated monomer components were studied for their in vitro anti-tyrosinase activity, in order to provide literature references for the development of the whitening active ingredients of Yi medicine Luxiancao.

[0006] The prior art states that a herbaceous deer stalk extract, preparation method and application thereof have the effect of inhibiting tyrosinase activity to achieve whitening, and the extracted ingredients include 3-methoxy-quercetin 1.92-10.38 mg / g, 3,4-dihydroxybenzoic acid 1.50-6.47 mg / g, apigenin 1.32-3.79 mg / g, kaempferol 1.97-8.59 mg / g, and herbaceous deer stalk sterone 25-500 μg / g. However, the prior art points out that the above multiple substances have synergistic whitening, but the types of extracts developed are limited.

[0007] In the prior art, studies on the synthesis of 3,4-dihydroxybenzoic acid alkyl esters and their biological effects pointed out that 3,4-dihydroxybenzoic acid methyl ester, ethyl ester, propyl ester, butyl ester and pentyl ester have no significant inhibitory effect on tyrosinase, and pointed out that the inhibitory ability of 3,4-dihydroxybenzoic acid alkyl esters on tyrosinase increases with the increase of the carbon chain. Summary of the invention

[0008] The purpose of the present invention is to solve the problem of limited development and research of the current deer jelly grass extract, provide the application of 3,4-dihydroxybenzoic acid methyl ester in whitening, provide a new deer jelly grass extract with whitening effect, and enhance the application value of the deer jelly grass.

[0009] The technical solution of the present invention is as follows:

[0010] In one aspect, the present invention provides use of methyl 3,4-dihydroxybenzoate in preparing a whitening composition.

[0011] Preferably, the whitening composition includes whitening cosmetics, skin care products, medicines and the like.

[0012] According to a preferred embodiment, the structural formula of methyl 3,4-dihydroxybenzoate is as follows:

[0013]

[0014] According to a preferred embodiment, the methyl 3,4-dihydroxybenzoate is white needle crystals with a molecular formula of C8H8O4.

[0015] According to a preferred embodiment, the methyl 3,4-dihydroxybenzoate is extracted from Herba Lycopodii.

[0016] Another aspect of the present invention provides a method for preparing methyl 3,4-dihydroxybenzoate, which is prepared by extracting from Herba Lycopodii, and specifically comprises the following steps:

[0017] Step S1: taking the whole herb of Luxiancao, crushing and sieving it, and extracting it with 85% ethanol by heating and refluxing for 5 times, combining and concentrating the extracts under reduced pressure to obtain an extract;

[0018] Step S2: the ethanol extract is suspended in water, filtered, extracted 5 times with 1:1 volume of petroleum ether and ethyl acetate, and the fractions are combined and concentrated under reduced pressure to obtain a petroleum ether fraction and an ethyl acetate fraction;

[0019] Step S3: Take the ethyl acetate fraction and mix it with 100-200 mesh silica gel in equal amounts of 1:1, and apply it to a silica gel column. Degrease it with 2 column volumes of petroleum ether, and then mix the petroleum ether:ethyl acetate in the ratio of 30:1, 10:1, 5:1, and 1:1 in sequence to obtain 4 components.

[0020] Step S4: Take a 1:1 segment of petroleum ether: ethyl acetate, and subject it to normal silica gel chromatography with a gradient elution of petroleum ether-ethyl acetate in the ratio of 30:1, 20:1, 15:1, 10:1, 5:1, 3:1, and 1:1 to obtain 7 segments. Take a 20:1 segment of petroleum ether-ethyl acetate, Fr.4.2, and separate it by Sephadex LH-20 chromatography (methanol). Then, pass through a secondary gel to obtain Fr.4-2-1-2. Fr.4-2-1-2 is purified by semi-preparative liquid phase (acetonitrile-water 27:73; injection volume 20uL, volume flow rate 3mL / min; detection wavelength: 210, 280, 254, 365nm) to obtain compound 3,4-dihydroxybenzoic acid methyl ester (5mg, tR=12.787min).

[0021] Another aspect of the present invention provides a whitening composition comprising methyl 3,4-dihydroxybenzoate.

[0022] Preferably, the concentration of methyl 3,4-dihydroxybenzoate is 0.1 mg / ml-0.25 mg / ml.

[0023] Preferably, the concentration of methyl 3,4-dihydroxybenzoate is 0.125 mg / ml-0.21 mg / ml.

[0024] Preferably, the whitening composition includes whitening cosmetics, skin care products, medicines and the like.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention has found that 3,4-dihydroxybenzoic acid methyl ester can be extracted from Deer Herb;

[0027] 2. Provided is a method for treating skin whitening with methyl 3,4-dihydroxybenzoate, which has the effect of inhibiting tyrosinase activity and achieving skin whitening. It can be used in skin whitening products to exert skin whitening effects. The inhibition rate of tyrosinase is 59.10% at a concentration of 0.125 mg / ml, and the IC 50 The value is 0.2107mg / ml, which is about the positive drug β-arbutin (IC 50The value is 0.3763mg / ml), which is half of the inhibitory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Methyl 3,4-dihydroxybenzoate 1 H-NMR spectrum;

[0029] Figure 2 Methyl 3,4-dihydroxybenzoate 13 C-NMR (DEPT) spectrum;

[0030] Figure 3 This is the concentration inhibition curve of the positive drug β-arbutin on tyrosinase;

[0031] Figure 4 This is the concentration inhibition curve of 3,4-dihydroxybenzoic acid methyl ester on tyrosinase. DETAILED DESCRIPTION

[0032] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0034] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0035] Example 1 Separation of 3,4-dihydroxybenzoic acid methyl ester from Deer Herba

[0036] Step S1: 70 kg of dried whole herb of Herba Luxiancao was crushed and sieved (10 meshes), and extracted with 3 times the amount of 85% ethanol by heating and refluxing for 5 times, each time for 2 hours, and the extracts were combined and concentrated under reduced pressure to obtain 38 kg of extract.

[0037] Step S2: the ethanol extract was suspended in water, filtered through a 400-mesh nylon cloth, and extracted five times with 1:1 volume of petroleum ether and ethyl acetate, respectively. After combining the fractions, they were concentrated under reduced pressure to obtain a petroleum ether fraction (100 g) and an ethyl acetate fraction (3.4 kg).

[0038] Step S3: Take the ethyl acetate fraction (3.4 kg) and mix it with 100-200 mesh silica gel in an equal amount of 1:1 and load it onto a silica gel column. Degrease it with 2 column volumes of petroleum ether, and then flush and segment the column with petroleum ether: ethyl acetate in the ratio of 30:1, 10:1, 5:1, and 1:1 in sequence to obtain 4 components.

[0039] Step S4: Take a 1:1 segment of petroleum ether: ethyl acetate (137.5 g) and subject it to normal silica gel chromatography (200-300 mesh), and use petroleum ether-ethyl acetate (30:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1) gradient elution to obtain 7 segments (Fr.4.1-Fr.4.7), take a 20:1 segment of petroleum ether-ethyl acetate, Fr.4.2, and separate it by Sephadex LH-20 chromatography (methanol), and then pass through a secondary gel to obtain Fr.4-2-1-2, Fr.4-2-1-2 is purified by semi-preparative liquid phase (acetonitrile-water 27:73; injection volume 20uL, volume flow rate 3mL / min; detection wavelength: 210, 280, 254, 365nm) to obtain compound 3,4-dihydroxybenzoic acid methyl ester (5mg, tR=12.787min).

[0040] The separated 3,4-dihydroxybenzoic acid methyl ester was tested by nuclear magnetic resonance hydrogen spectrum and carbon spectrum. The hydrogen spectrum results are as follows Figure 1 As shown, 1 H-NMR (600MHz, CD3OD)δ H :7.42 (1H, d, J=7.8, 2.0Hz, H-6), 7.42 (1H, d, J=2.0Hz, H-2), 6.78 (1H, d, J=7.8Hz, H-5), 3.82 (3H, s, OCH3); 13 C-NMR (150MHz, CD3OD)δ C :122.5(C-1), 115.8(C-2), 146.2(C-3), 151.8(C-4), 117.4(C-5), 123.6(C-6), 168.9(COOCH3), 52.2(-OCH3); The carbon spectrum results are as follows Figure 2As shown, ESI-MS m / z: 169.1[M+H] + The molecular formula of methyl 3,4-dihydroxybenzoate is C8H8O4.

[0041] Example 2 Study on the effect of 3,4-dihydroxybenzoic acid methyl ester in Herba Luxiancao on anti-tyrosinase activity

[0042] 1. Tyrosinase Inhibition Activity Test

[0043] 1. Solution Preparation

[0044] (1) Preparation of 250U tyrosinase solution: Accurately pipette 25mL of PBS phosphate buffer (0.01mol / L, pH 6.5) into 25KU tyrosinase and mix well. Dispense into 25 tubes of 1000units / mL tyrosinase solution. Accurately take 0.25mL of PBS buffer and dilute to 1mL with 0.75mL of PBS buffer to obtain 250units / mL tyrosinase test solution. Place the test solution in a -20℃ refrigerator for later use.

[0045] (2) Preparation of L-tyrosine solution: Accurately weigh 2 mg of L-tyrosine powder into a 50 mL centrifuge tube, add 20 mL of PBS buffer, mix well to obtain a 0.1 mg / mL L-tyrosine solution, set aside for use, and prepare freshly each time.

[0046] (3) Preparation of sample solution: Accurately weigh 1 mg of methyl 3,4-dihydroxybenzoate, add 30 uL DMSO to completely dissolve it, then accurately add 970 uL PBS and mix well to make the concentration 1 mg / mL. After diluting each sample with PBS, the concentrations are as follows: 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL; set aside.

[0047] 2. Determination of tyrosinase activity inhibition rate

[0048] The experiment was divided into a sample reaction group (6 concentrations of methyl 3,4-dihydroxybenzoate + 250U tyrosinase reagent), a sample control group (6 concentrations of methyl 3,4-dihydroxybenzoate + PBS buffer), a reagent reaction group (250U tyrosinase reagent + PBS buffer), and a reagent control group (PBS buffer).

[0049] Each sample solution of each concentration was pipetted into a 96-well plate with a pipette, 40uL per well, 40uL of prepared tyrosinase solution was added to each well of the sample reaction group, and 40uL of PBS buffer was added to each well of the sample control group; 40uL of prepared tyrosinase solution and an equal amount of PBS buffer were added to each well of the reagent reaction group, and 80uL of PBS buffer was added to each well of the reagent control group. After the addition of liquid, incubate at 37°C for 10min in a constant temperature incubator, then add L-tyrosine solution with a pipette, 40uL per well, and each group was added. After the addition of liquid, the 96-well plate was placed in a constant temperature incubator at 37°C for 20min. After the incubation was completed, it was immediately placed in a multifunctional microplate reader to measure its absorbance at 492nm and record the data. Three parallel groups were performed for each concentration experiment. The average value of the three groups of data was used to calculate the inhibition rate of 3,4-dihydroxybenzoic acid methyl ester on tyrosinase according to the following formula:

[0050] Inhibition rate = [1 - (sample reaction - sample control) / (reagent reaction - reagent control)] × 100%.

[0051] 3. Data Processing and Analysis

[0052] IC 50 The calculation results were calculated using IBM SPSS Statistics 26. The experiment was repeated 3 times for each sample to calculate the IC value of each compound for tyrosinase inhibition. 50 The mean and standard deviation were compared with the positive drug β-arbutin using a one-way ANOVA test. LSD and Duncan were used for post hoc comparison.

[0053] 4. Experimental Results and Analysis

[0054] 1. Tyrosinase inhibition test results

[0055] The results of the positive drug β-arbutin's inhibition of tyrosinase activity are as follows Figure 3 As shown in the figure, the inhibition rates from high to low are 71.62%, 69.18%, 60.42%, 48.81%, 31.74% and 14.29%, respectively. The inhibition rates of positive drugs on tyrosinase increase with the increase of test solution concentration. When the concentration is less than 1 mg / ml, the inhibition rate of positive drugs increases faster with the increase of concentration. When the concentration is greater than 1 mg / ml, the increase is slower, showing concentration-dependent inhibition. Its IC 50 The value is 0.3763mg / ml.

[0056] The results of the concentration inhibition of the compound on tyrosinase activity are as follows Figure 4As shown in the figure, the inhibition rates are 61.96%; 59.28%; 59.10%; 47.41%; 42.66%; 34.61% from high to low. The inhibition rate of tyrosinase decreases with the increase of the test solution concentration. When the compound concentration is below 0.2 mg / ml, the inhibition rate decreases rapidly with the increase of concentration. When the concentration is greater than 0.2 mg / ml, the rate of decrease becomes slow, showing low concentration inhibition. Its IC 50 The value is 0.2107mg / ml.

[0057] 2. Results Analysis

[0058] The results of the inhibition rate experiment showed that the inhibition rate of the compound to tyrosinase was 59.10% when the concentration was 0.125mg / ml, while the inhibition rate of the positive drug β-arbutin was only 31.74%. 50 The value is 0.2107mg / ml, which is about the positive drug β-arbutin (IC 50 The value is 0.3763mg / ml), which is half of that of β-arbutin, indicating that the compound has a greater inhibitory activity on tyrosinase than β-arbutin and has a better whitening effect. The above results all show that the compound 3,4-dihydroxybenzoic acid methyl ester has better whitening activity than positive drugs. Moreover, this compound is isolated from Deer Herb for the first time and has good research prospects.

[0059] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. Application of methyl 3,4-dihydroxybenzoate in the preparation of whitening compositions.

2. The use according to claim 1, characterized in that: The whitening composition includes whitening cosmetics, skin care products or medicines.

3. The use according to claim 1, characterized in that: The 3,4-dihydroxybenzoic acid methyl ester achieves whitening by inhibiting the activity of tyrosinase.

4. The use according to claim 1, characterized in that: The structural formula of the 3,4-dihydroxybenzoic acid methyl ester is as follows:

5. The use according to claim 1, characterized in that: The 3,4-dihydroxybenzoic acid methyl ester is extracted from the herb of gypsophila.

6. A method for preparing methyl 3,4-dihydroxybenzoate, characterized in that: It is prepared by extracting from deer lily grass, and specifically comprises the following steps: Step S1: taking the whole herb of Luxiancao, crushing and sieving it, and extracting it with 85% ethanol by heating and refluxing for 5 times, combining and concentrating the extracts under reduced pressure to obtain an extract; Step S2: the ethanol extract is suspended in water, filtered, extracted 5 times with 1:1 volume of petroleum ether and ethyl acetate, and the fractions are combined and concentrated under reduced pressure to obtain a petroleum ether fraction and an ethyl acetate fraction; Step S3: Take the ethyl acetate fraction and mix it with silica gel in an equal amount of 1:1, load it onto a silica gel column, first defat it with petroleum ether, then flush and segment it with petroleum ether:ethyl acetate in a ratio of 30:1, 10:1, 5:1, and 1:1 in sequence to obtain four components; Step S4: Take a 1:1 segment of petroleum ether: ethyl acetate, and subject it to normal silica gel chromatography with a gradient elution of petroleum ether-ethyl acetate in the ratio of 30:1, 20:1, 15:1, 10:1, 5:1, 3:1, and 1:1 to obtain 7 segments. Take a 20:1 segment of petroleum ether-ethyl acetate, Fr.4.2, and separate it by Sephadex LH-20 chromatography. Then, pass it through a secondary gel to obtain Fr.4-2-1-2. Fr.4-2-1-2 is purified by semi-preparative liquid phase to obtain compound 3,4-dihydroxybenzoic acid methyl ester.

7. A whitening composition, characterized in that: Including methyl 3,4-dihydroxybenzoate.

8. A whitening composition according to claim 7, characterized in that: The concentration of the 3,4-dihydroxybenzoic acid methyl ester is 0.1 mg / ml-0.25 mg / ml.

9. A whitening composition according to claim 8, characterized in that: The concentration of methyl 3,4-dihydroxybenzoate is 0.125 mg / ml-0.21 mg / ml.

10. A whitening composition according to claim 7, characterized in that: The whitening composition includes whitening cosmetics, skin care products or medicines.

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