CN116589515B - Preparation method of p-propenylphenol glycoside with whitening effect - Google Patents

Abstract

The invention discloses a preparation method of p-propenyl phenol glycoside with whitening effect, which comprises the following steps: the p-propenyl phenol glycoside is obtained by taking fennel as a raw material and extracting, separating and refining the fennel with ethanol. The p-propenyl phenol glycoside prepared by the method has tyrosinase inhibition effect and is expected to be developed into a whitening product.

Description

Preparation method of p-propenylphenol glycoside with whitening effect

Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a preparation method of p-propenylphenol glycoside with whitening effect.

Background technique

Skin is the largest organ of the human body. People usually use skin care products to create a good local environment for the skin, which is conducive to the survival and growth of the skin, thereby achieving the effects of anti-aging, anti-wrinkles, moisturizing and acne removal. The whiteness of the skin is closely related to melanin. Melanin is produced by melanocytes and then transferred to keratinocytes, epidermal cells and stratum corneum. Finally, it is excreted with the shedding of the stratum corneum, thereby affecting the color of the skin or forming stains. Tyrosinase (TYR) is the rate-limiting enzyme in the human body that regulates melanin production. Tyrosinase is only produced by melanocytes, and the amount of melanin synthesis is related to the activity of tyrosinase. It can be regulated by regulating the activity of tyrosinase in cells. The expression level can effectively regulate the production of melanin, thereby achieving the effect of skin whitening. Therefore, melanin biosynthesis inhibitors based on inhibiting tyrosinase activity have become a hot spot in the research field. Compounds are developed by combining with the tyrosinase active center. Inhibitors are one of the more interesting categories.

Fennel is the dried and mature fruit of Foeniculum vulgare Mill., an Umbelliferae plant. Its original name is Xiangxiang. Its medicinal history is long. It was first recorded in "Tang Materia Medica" and has a medicinal history of more than a thousand years. In addition to its medicinal uses, cumin is also a commonly used food spice, so it is a good product for both medicinal and food purposes. Fennel is widely distributed, mainly in the Mediterranean region. In my country, it is mainly distributed in Shanxi, Gansu, Inner Mongolia in the northwest, and Liaoning in the northeast. It is rich in resources and is mainly cultivated with a wide cultivation area. Shanxi has the largest output. , high-quality products near Hetao, Inner Mongolia.

Fennel mainly contains volatile oil, as well as phenylpropanoids, flavonoids, glycosides, fatty oils, phenolic acids and other types of compounds. Current research and development mainly focuses on volatile oil, and is mainly based on component analysis. There are not many reports on the separation, purification and identification of compounds. Its rich non-volatile components, such as phenylpropanoid glycosides, lack in-depth research and development. The present invention found that some phenylpropanoids have a good effect on inhibiting tyrosinase activity, suggesting that the phenylpropanoids in fennel may have Better whitening effect.

Contents of the invention

The object of the present invention is to provide a preparation method of p-propenylphenol glycoside with whitening effect.

The purpose of the present invention is achieved as follows:

The structural formula of p-propenylphenol glycoside is , its preparation method is:

(1) Ethanol extraction: 20 kg of cumin sample was crushed through a 10-mesh sieve, and extracted in 10 L of 75% ethanol with reflux, 10 L each time, 2 hours each time, and extracted 5 times; the extracts were combined and passed The solvent was removed under reduced pressure on a rotary evaporator to obtain 2.387 kg of crude extract A;

(2) Extraction and separation: Suspend the crude extract A with water, extract five times with petroleum ether, ethyl acetate, and n-butanol in sequence, combine the extracts, and concentrate under reduced pressure to obtain the petroleum ether phase and the ethyl acetate phase B. Butanol phase and water phase;

(3) Separation and purification: ethyl acetate phase B is subjected to 200-300 mesh forward silica gel chromatography, and is eluted with a dichloromethane-methanol gradient. The volume ratio of dichloromethane-methanol is 100:1-1:1, and the fraction is The forward silica gel plate was developed with petroleum ether-ethyl acetate 1:1 as the developing agent. The fractions Fr.7 with R _f value between 0.3 and 0.8 were combined. Fr.7 was subjected to forward silica gel chromatography with 100-200 mesh. Methyl chloride-methanol gradient elution, the volume ratio of dichloromethane-methanol is 50:1-1:1, the fractions are developed through the forward silica gel plate with dichloromethane-methanol 10:1 as the developing agent, and the combined R _f value is at The fractions Fr.7.6 and Fr.7.6 between 0.1~0.6 were subjected to reversed-phase ODS column chromatography and eluted with a methanol-water gradient. The volume ratio of methanol-water was 20:80~100:0. The fractions were passed through a forward silica gel plate. Use dichloromethane-methanol 10:1 plus 2 drops of formic acid as a developing agent. Combine the fractions Fr.7.6.2 with R _f value between 0.3 and 0.4. Fr.7.6.2 is separated by Sephadex LH-20 chromatography. Chromatography The conditions are: methanol, height: 1.5 meters, flow rate: 4 seconds per drop; then purified by semi-preparative liquid phase, chromatographic conditions are acetonitrile-water 15:85; volume flow rate: 3 ml/min; detection wavelength: 365 nm, the purified compound is obtained 4-(1 E )-1-propen-1-ylphenyl β - D -glucopyranoside [trans-4-(1-propenyl)-phenol- β - D -glucopyranoside], p-propenylphenol glycoside .

The fennel is salt-roasted fennel.

Preparation of the salt-roasted fennel: take fennel, add 100ml of salt water per kilogram of fennel, seal it, suffocate it for 30 minutes, place it in a frying container, heat it over a slow fire to a temperature of 110-120°C, fry it until it is slightly yellow, and take it out , let cool and set aside.

The brine is saturated salt water.

Application of p-propenylphenol glycoside obtained by this preparation method in preparing tyrosinase inhibitor products.

A whitening product containing p-propenylphenol glycoside obtained by the above preparation method.

Advantages of the invention:

The advantage and positive effect of the present invention is that a new preparation method of p-propenylphenol glycoside is developed.

The p-propenylphenol glycoside obtained by the preparation method of the present invention has tyrosinase inhibitory effect and can be used in whitening products.

Description of drawings

Figure 1 shows the chemical structure of p-propenylphenol glycoside.

Figure 2 is the ¹ H-NMR spectrum of p-propenylphenol glycoside.

Figure 3 is the ¹³ C-NMR (DEPT) spectrum of p-propenylphenol glycoside.

Figure 4 is the concentration inhibition curve of β -arbutin on tyrosinase.

Figure 5 is the concentration inhibition curve of p-propenylphenol glycoside on tyrosinase.

Detailed ways

The invention provides a preparation method of p-propenylphenol glycoside with whitening effect.

The structural formula of p-propenylphenol glycoside is , its preparation method is:

(1) Ethanol extraction: 20 kg of salt-roasted cumin sample was crushed and passed through a 10-mesh sieve, and extracted in 10 L of 75% ethanol with reflux, 10 L each time, 2 hours each time, and extracted 5 times; the extracts were combined , the solvent was removed under reduced pressure using a rotary evaporator to obtain 2.387 kg of crude extract A;

(2) Extraction and separation: Suspend the crude extract A with water, extract five times with petroleum ether, ethyl acetate, and n-butanol in sequence, combine the extracts, and concentrate under reduced pressure to obtain the petroleum ether phase and the ethyl acetate phase B. Butanol phase and water phase;

(3) Separation and purification: ethyl acetate phase B is subjected to 200-300 mesh forward silica gel chromatography, eluting with dichloromethane-methanol (100:1-1:1) gradient, and the fraction is passed through a forward silica gel plate and eluted with petroleum ether- Develop with ethyl acetate 1:1 as the developing solvent, combine fraction Fr.7 with R _f value between 0.3 and 0.8, Fr.7 is subjected to 100-200 mesh forward silica gel chromatography, and dichloromethane-methanol (50:1 -1:1) gradient elution, the fractions were developed through the forward silica gel plate using dichloromethane-methanol 10:1 as the developing agent, and the fractions Fr.7.6 with R _f value between 0.1 and 0.6 were combined. Phase ODS column chromatography, using methanol-water (20:80~100:0) gradient elution, the fractions were developed on a forward silica gel plate using dichloromethane-methanol 10:1 plus 2 drops of formic acid as a developing agent, and the R _f values were combined Fractions Fr.7.6.2 and Fr.7.6.2 between 0.3~0.4 were separated by Sephadex LH-20 chromatography. The chromatographic conditions were: methanol, height: 1.5 meters, flow rate: 4 seconds per drop; then use half of the preparation solution Phase purification, chromatographic conditions are acetonitrile-water 15:85; volume flow 3 ml/min; detection wavelength: 365 nm, purified compound 4-(1 E )-1-propen-1-ylphenyl β - D -pyran Glucoside [trans-4-(1-propenyl)-phenol- β - D -glucopyranoside] is p-propenylphenol glycoside.

Cumin is salt-roasted fennel.

Preparation of salt-roasted fennel: Take fennel, add 100ml salt water per kilogram of fennel, seal it, simmer it for 30 minutes, place it in a frying container, heat it over a slow fire to a temperature of 110-120°C, stir-fry until it is slightly yellow, take it out and put it away Cool and set aside.

Salt water is saturated salt water.

Application of p-propenylphenol glycoside obtained by this preparation method in preparing tyrosinase inhibitor products.

A whitening product containing p-propenylphenol glycoside obtained by the above preparation method.

The present invention will be further described below, but the present invention will not be limited in any way. Any transformation made based on the present invention shall fall within the protection scope of the present invention.

Example 1

Preparation of salt-roasted cumin

Take salt and add water to make a saturated solution, which is salt water.

Take 20kg of cumin, add 2000ml of salt water, seal, simmer for 30 minutes, place in a frying container, heat over low heat (110-120℃), fry until slightly yellow, take out, let cool, and set aside.

Example 2

Preparation method of p-propenylphenol glycoside

1.1 Materials

Chromatography silica gel (100~200 mesh, 200~300 mesh, Qingdao Ocean Chemical Factory); GF254 thin layer chromatography silica gel (Qingdao Ocean Chemical Factory); LH-20 hydroxypropyl dextran gel (Pharmacia, USA); reversed phase C18 column chromatography material (ODS, Merck Company of Germany); thin layer chromatography developer (10% sulfuric acid ethanol solution); reagents such as methanol, ethanol, acetone, n-butanol, ethyl acetate, methylene chloride, petroleum ether and other reagents are heavy Steamed industrial or chemically pure solvent; chromatographic acetonitrile (Shanghai Xingke High Purity Solvent Co., Ltd.).

Fennel medicinal materials were purchased from the Xinluosi Bay medicinal materials market in September 2022. Associate Professor Li Hongzhe of Yunnan University of Traditional Chinese Medicine identified them as the dried and mature fruits of Foeniculum vulgare Mill., an Umbelliferae plant.

1.2 Instruments

Bruker Avance III 400 MHz and Bruker DRX 500 MHz superconducting nuclear magnetic resonance instrument, TMS as internal standard (Bruker Company, Germany); high performance liquid chromatograph Agilent 1200 type (Agilent Company, USA); ion well time-of-flight mass spectrometer Esquire HCT type ( Bruker Company, Germany); One-tenthousandth analytical balance FA2004 (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.); Circulating water multi-purpose vacuum pump SHB-Ⅲ (Gongyi Yuhua Instrument Co., Ltd.); Heidorf rotary evaporator Hei-VAP Core HL/G (German Heidolph).

2 Extraction and separation process

20 kg of salt-roasted cumin sample was crushed and passed through a 10-mesh sieve, and then refluxed and extracted in 10 L of 75% ethanol, 10 L each time, 2 hours each time, and extracted 5 times. The extracts were combined, and the solvent was removed under reduced pressure using a rotary evaporator to obtain 2.387 kg of crude extract. The crude extract was suspended in water and extracted five times with petroleum ether, ethyl acetate, and n-butanol in sequence. The extracts were combined and the pressure was reduced. Concentrate to obtain 203.6 g of petroleum ether phase, 73.9 g of ethyl acetate phase, 316.7 g of n-butanol phase and 759.3 g of water phase. The ethyl acetate fraction was subjected to forward silica gel chromatography (200-300 mesh) and eluted with a dichloromethane-methanol (100:1-1:1) gradient. The fraction was passed through a forward silica gel plate and eluted with petroleum ether-ethyl acetate 1: 1 is the developing agent, combine the fractions Fr.7 with R _f value between 0.3 and 0.8, Fr.7 is subjected to forward silica gel chromatography (100-200 mesh), and dichloromethane-methanol (50:1-1: 1) Gradient elution, the fractions are developed through the forward silica gel plate using dichloromethane-methanol 10:1 as the developing solvent, and the fractions Fr.7.6 with R _f value between 0.1 and 0.6 are combined, and the fractions Fr.7.6 are passed through the reversed-phase ODS column. Chromatography, use methanol-water (20:80~100:0) gradient elution, the fractions are developed on a forward silica gel plate using dichloromethane-methanol 10:1 plus 2 drops of formic acid as a developing agent, and the combined R _f value is between 0.3~ The fractions Fr.7.6.2 and Fr.7.6.2 between 0.4 and 0.4 were separated by Sephadex LH-20 chromatography (methanol, height: 1.5 meters, flow rate: 4 seconds per drop), and then purified by semi-preparative liquid phase (acetonitrile-water 15:85; volume flow 3 ml/min; detection wavelength: 365 nm), purified compound 4-(1 E )-1-propen-1-ylphenyl β - D -glucopyranoside [trans-4- (1-propenyl)-phenol- β - D -glucopyranoside (9.9 mg)] is p-propenylphenol glycoside. The chemical structure of this compound is shown in Figure 1. Figure 2 is the 1H-NMR spectrum of p-propenylphenol glycoside. Figure 3 is the 13C-NMR (DEPT) spectrum of p-propenylphenol glycoside.

Physicochemical constants and spectral data of p-propenylphenol glycosides

White powdery crystal (methanol); molecular formula: C ₁₅ H ₂₀ O ₆ HR-ESI-MS, m/z 319.1152 [M+Na] ⁺ (calculated value: 319.1152). ¹ H-NMR (400MHz, CD ₃ OD) δ _H : 6.36 (1H, d, J = 15.8Hz, H-7), 6.15 (1H, dq, J = 15.8, 6.5Hz, H-8), 3.80 ( 6H, m, H-2′, 3′, 4′, 5′, 6′), 1.85 (3H, d, J = 6.6Hz, H-9). ¹³ C-NMR (100MHz, CD ₃ OD) δ _C : 158.1 (s, C-4), 133.8 (s, C-1), 131.6 (d, C-7), 127.8 (d, C-2, 6), 124.8 (d, C-8), 117.8 (d, C-3, 5), 102.4 (d, C-1′), 78.1 (d, C-3′), 77.9 (d, C-5′), 74.9 (d, C-2′), 71.4 (d, C-4′), 62.5 (t, C-6′), 18.5 (q, C-9).

Example 3

Study on the role of anti-tyrosinase activity

This method is modified based on the reference. (Li ZY, Zhao P, Song SJ, Huang XX. Chiral resolution of racemic phenylpropanoids with tyrosinaseinhibitoryactivities from the fruits of Crataegus pinnatifida Bge. J FoodBiochem. 2022 Oct;46(10):e14304.).

1 Materials and instruments

1.1 Materials

Tyrosinase (Sigma-Aldrich Company); L-tyrosine (Shanghai McLean Biochemical Technology Co., Ltd.) β -arbutin (Shanghai Yuanye Biotechnology Co., Ltd.).

1.2 Instruments

Synergy2 multifunctional microplate reader (BIOTEK, USA).

Tyrosinase inhibitory activity test

2.1 Solution configuration

2.1.1 Configuration of phosphate buffer solution

Precisely measure 20 ml of 10×PBS buffer, dilute it with ultrapure water to 200 ml, and finally use 0.2 mol/L sodium hydroxide to adjust the pH to the specified range (6.5~7.5) to obtain the PBS buffer.

Preparation of tyrosinase test solution

Precisely pipette 9 ml of 100 units/ml tyrosinase (SLCN1560) that has been packed in advance, add PBS buffer and dilute to 30 ml to obtain a 30 units/ml tyrosinase test solution, put the test solution into -20℃ refrigerator until use.

Preparation of tyrosine solution

Precisely weigh 5 mg of L-tyrosine (batch number: C14856769) powder into a 100 ml EP tube, add 50 ml of PBS buffer, and shake well to obtain a 0.1 mg/ml L-tyrosine solution. Set aside for use.

Sample solution configuration

(1) Configuration of positive drug solution

Precisely weigh 4 mg of β -arbutin (batch number: T17S6B1) powder, add 30 ul DMSO to dissolve it completely, and then use a pipette to accurately add 970 ul PBS to obtain a 4 mg/ml positive drug test solution. The gradient dilution concentration of the drug is 2 mg/ml, 1 mg/ml, 0.5 mg/ml, and 0.25 mg/ml, and is ready for use.

(2) Configuration of sample solution to be tested

Accurately weigh an appropriate amount of p-propenylphenol glycoside prepared in Example 2, add 30 ul DMSO to completely dissolve it, and then add PBS accurately to make the concentration sequentially 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, and 0.125 mg/ml, 0.0625 mg/ml, set aside.

Determination of tyrosinase activity inhibition rate

Use a pipette to add PBS buffer, compound test solutions with different concentration gradients, arbutin control solutions and tyrosinase solutions with different concentration gradients to the 96-well plate in sequence according to the volumes in Table 1, and incubate at 37°C for 10 minutes. Afterwards, add L-tyrosine test solution and incubate for 30 minutes under the same conditions. After the incubation is completed, immediately put it into a multifunctional microplate reader to measure the absorbance at 492 nm and record the data. Do 3 parallel groups for each concentration experiment. Using the average of the three sets of data, calculate the inhibitory rate of the tested compound on tyrosinase according to the following formula:

Inhibition rate = [1-(A _T -A _B )/( _AC -A _N )]×100%

Among them, T: is the reaction solution group with sample and tyrosine added; B: is the reaction solution group with sample and no tyrosine added; C: is the reaction solution group without sample and tyrosine is added. Different; N: is the reaction solution group without adding sample and without adding tyrosine. A _T : is the absorbance value measured at 492nm for the reaction solution of group T; A _B : is the absorbance value measured at 492nm for the reaction solution of group B; A _C : is the absorbance value measured at 492nm for the reaction solution of group C ; A _N : is the absorbance value measured at 492nm of the N group reaction solution.

Table 1 Reaction solution composition

Sample(ul) PBS(ul) Tyrosinase (ul) Tyrosine (ul) Total amount(ul) T 40 40 40 120 B 40 40 40 120 C 40 40 40 120 N 80 40 120

2.3 Tyrosinase inhibition experimental results

The concentration inhibition results of the positive drug β -arbutin on tyrosinase activity are shown in Figure 4. The inhibition rates from high to low are 62.79%; 50.24%; 41.48%; 33.19%; 18.73%. Among them, the positive drug The inhibition rate of tyrosinase increases with the concentration of the test solution. When the concentration is less than 1 mg/ml, the inhibition rate of the positive drug increases faster with the increase of concentration. When the concentration is greater than 1 mg/ml, The growth rate is relatively slow, showing concentration-dependent inhibition, and its IC ₅₀ value is 1.784 mg/ml.

The concentration inhibition results of the compound on tyrosinase activity are shown in Figure 5. The inhibition rates from high to low are 54.58%; 48.83%; 42.05%; 35.24%; 28.24%. The inhibition rates for tyrosinase are also the same. It increases with the concentration of the test solution. When the compound concentration is below 0.5 mg/ml, the inhibition rate increases rapidly with concentration. When the concentration is greater than 0.5 mg/ml, the growth rate becomes slower, showing concentration-dependent inhibition. Its IC ₅₀ value is 0.575 mg/ml.

Result analysis

The results of the inhibition rate experiment showed that when the compound concentration was 1 mg/ml, the inhibition rate of tyrosinase was 54.58%, while the inhibition rate of the positive drug β -arbutin was only 41.48%, and the compound IC ₅₀ value was 0.575 mg/ml. ml, which is one-third of the positive drug β -arbutin (IC ₅₀ value is 1.784 mg/ml), indicating that the compound’s tyrosinase inhibitory activity is much greater than β -arbutin and has a better whitening effect. . The above results all show that the compound p-propenylphenol glycoside has better whitening activity than the positive drug. This phenylpropanoid compound is isolated from cumin for the first time and has good research prospects.

Claims (4)

1. A preparation method of p-propenylphenol glycoside, characterized in that the structural formula of p-propenylphenol glycoside is , its preparation method is: (1) Ethanol extraction: 20kg of cumin sample was crushed and passed through a 10-mesh sieve, and extracted in 10L of 75% ethanol with reflux, 10L each time, 2 hours each time, and extracted 5 times; the combined extracts were obtained by rotary evaporation The solvent was removed under reduced pressure to obtain 2.387kg of crude extract A; (2) Extraction and separation: Suspend crude extract A with water, extract five times with petroleum ether, ethyl acetate, and n-butanol in sequence, combine the extracts, and concentrate under reduced pressure to obtain petroleum ether phase, ethyl acetate phase B, and n-butanol phase and water phase; (3) Separation and purification: ethyl acetate phase B is subjected to 200~300 mesh forward silica gel chromatography, and is eluted with a dichloromethane-methanol gradient. The volume ratio of dichloromethane-methanol is 100:1~1:1, and the fraction is The forward silica gel plate was developed with petroleum ether-ethyl acetate 1:1 as the developing agent. The fraction Fr.7 with R _f value between 0.3 and 0.8 was combined. Fr.7 was subjected to forward silica gel chromatography with 100 to 200 mesh. Methyl chloride-methanol gradient elution, the volume ratio of dichloromethane-methanol is 50:1~1:1, the fractions are developed through the forward silica gel plate with dichloromethane-methanol 10:1 as the developing agent, and the combined R _f value is at The fractions Fr.7.6 and Fr.7.6 between 0.1~0.6 were subjected to reversed-phase ODS column chromatography and eluted with a methanol-water gradient. The volume ratio of methanol-water was 20:80~100:0. The fractions were passed through a forward silica gel plate. Use dichloromethane-methanol 10:1 plus 2 drops of formic acid as a developing agent. Combine the fractions Fr.7.6.2 with an R _f value between 0.3 and 0.4. Fr.7.6.2 is separated by SephadexLH-20 chromatography. Chromatographic conditions It is: methanol, height: 1.5 meters, flow rate: 4 seconds per drop; then purified by semi-preparative liquid phase, chromatographic conditions are acetonitrile-water 15:85, volume flow 3mL/min, detection wavelength: 365nm, and the purified compound is propylene Phenol glycoside. 2. The preparation method according to claim 1, characterized in that the fennel is salt-roasted fennel, and the preparation method is: take fennel, add 100 mL of salt water per kilogram of fennel, seal, suffocate for 30 minutes, and place In a frying container, heat over low heat to a temperature of 110~120°C. When stir-fried until slightly yellow, take it out, let it cool, and set aside. 3. The preparation method according to claim 2, characterized in that the brine is saturated saline. 4. The use of p-propenylphenol glycoside obtained by the preparation method of claim 1 in the preparation of whitening products that inhibit tyrosinase.