Ferulate compound as well as preparation method and application thereof

Abstract

The invention provides a ferulate compound as well as a preparation method and application thereof, the ferulate compound is (5-formyl-2-furan) methyl dihydroferulate, the ferulate compound is prepared by the following steps: extracting a dry whole herb water extract of eyebright by using ethyl acetate, recovering a solvent to obtain an ethyl acetate extraction part, and separating by using normal-phase silica gel column chromatography to obtain the (5-formyl-2-furan) methyl dihydroferulate. Petroleum ether-ethyl acetate is used as an eluent for gradient elution, a fraction Fr.2 is obtained and separated by normal-phase silica gel column chromatography to obtain a fraction Fr.2. 2, Sephadex LH-20 gel column chromatography is performed to obtain a fraction Fr.2. 2.1, MC I column chromatography is performed to separate the fraction Fr.2. 2.1, methanol-water is used as an eluent for gradient elution to obtain a fraction Fr.2. 1.1, and then preparative HPLC (High Performance Liquid Chromatography) is performed to obtain the HaCaT cell activity. The composition has a protection effect on HaCaT cell oxidative damage caused by UVB, and can effectively resist skin photoaging.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, in particular to a ferulic acid ester compound, a preparation method thereof and an application thereof. Background Art

[0002] Skin aging is a complex biological phenomenon, including endogenous aging and exogenous aging. Exogenous aging is caused by exposure to external stimuli (such as ultraviolet radiation, environmental toxins and infectious substances), resulting in skin wrinkles, reduced elasticity, flaccidity and rough appearance changes. Sunlight is the most threatening external factor to the skin, and ultraviolet rays are an important component of sunlight. Ultraviolet rays (UV) include long-wave ultraviolet rays (ultraviolet A, UVA, 320-400 nm), medium-wave ultraviolet rays (ultraviolet B, UVB, 290-320 nm) and short-wave ultraviolet rays (ultraviolet C, UVC, 270-290 nm). Among them, UVB is one of the main factors inducing skin damage. Keratinocytes (HaCaT cells) are the main cells constituting the human epidermis, and UVB acting on the skin epidermis will cause oxidative damage and apoptosis of keratinocytes. Lactate dehydrogenase (LDH) is a stable protein present in the cytoplasm of normal cells. Once the cell membrane is damaged, LDH is released into the extracellular space, and the degree of cell damage can be judged by detecting the LDH activity in the cell culture supernatant.

[0003] Euphrasia pectinata Tenore is the whole herb of Euphrasia pectinata Ten, Euphrasia Pectinata Ten Subsp Simplex (Freyn) Hong or Euphrasia rege / ii Wettst of the genus Euphrasia in the family Orobanchaceae, and is widely distributed in Inner Mongolia, Hebei, Shanxi, Ningxia and other places. It is bitter in taste, slightly cold in nature, and belongs to the bladder meridian, and has the effects of clearing heat and detoxifying, and promoting diuresis. At present, it is mainly used for treating fever and thirst, cough due to lung heat, sore throat, dysuria and the like. Further development and research on Euphrasia pectinata Tenore by using modern traditional Chinese medicine technology is expected to develop more medical and health care uses. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a ferulic acid ester compound.

[0005] Another technical problem to be solved by the present invention is to provide a preparation method of the above ferulic acid ester compound.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above ferulic acid esters compounds.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A ferulic acid ester compound, (5-formyl-2-furanyl)methyl dihydroferulic acid ester, has the structure shown in (I):

[0009]

[0010] The preparation method of the above ferulic acid ester compound is as follows: The ethyl acetate extract the water extract of the dried whole plant of Euphrasia officinalis and recover the solvent to obtain the ethyl acetate extraction part; The obtained ethyl acetate extraction part is separated by normal phase silica gel column chromatography, and gradient elution is carried out with petroleum ether - ethyl acetate as the eluent to obtain 8 fractions Fr.1 - Fr.8. Fr.2 is separated by normal phase silica gel column chromatography to obtain fractions Fr.2.1 and Fr.2.2. Fr.2.2 is subjected to Sephadex LH-20 gel column chromatography to obtain fractions Fr.2.2.1 and Fr.2.2.2. Fr.2.2.1 is separated by MC l column chromatography, and gradient elution is carried out with methanol - water as the eluent to obtain fraction Fr.2.2.1.1, and then the compound is obtained by preparative HPLC (acetonitrile - water as the mobile phase).

[0011] Preferably, the preparation method of the above ferulic acid ester compound is as follows:

[0012] (1) The dried whole plant of Euphrasia officinalis is soaked in 8 times the amount of water and refluxed for extraction 3 times, 2 hours each time. After combining the filtrates, it is dried under reduced pressure to obtain the water extract, and then extracted with ethyl acetate 3 times. After recovering the solvent, the ethyl acetate extraction part is obtained;

[0013] (2) The obtained ethyl acetate extraction part is separated by normal phase silica gel column chromatography, and gradient elution is carried out with petroleum ether - ethyl acetate as the eluent (100:0 - 0:100) to obtain 8 fractions Fr.1 - Fr.8. Fr.2 is separated by normal phase silica gel column chromatography to obtain fractions Fr.2.1 and Fr.2.2; Fr.2.2 is subjected to Sephadex LH-20 gel column chromatography to obtain fractions Fr.2.2.1 and Fr.2.2.2; Fr.2.2.1 is separated by MC l column chromatography, and gradient elution is carried out with methanol - water as the eluent (40:60 - 100:0) to obtain fraction Fr.2.2.1.1, and then the compound is obtained by preparative HPLC (acetonitrile - water 38:62 as the mobile phase).

[0014] Use of the above ferulic acid esters in the preparation of HaCaT cell protection products or HaCaT cell oxidative damage repair products.

[0015] Use of the above ferulic acid esters in the preparation of anti-skin photoaging products.

[0016] Preferably, in the use of the above ferulic acid esters, the product is an anti-skin photoaging drug or cosmetic.

[0017] The beneficial effects of the present invention are:

[0018] The above ferulic acid esters can significantly improve the activity of HaCaT cells, have a protective effect on the oxidative damage of HaCaT cells caused by UVB, can effectively combat skin photoaging, and provide a research basis for the further development and utilization of its resources. Description of the Drawings

[0019] Figure 1 Key for LEP-1 1 H- 1 H COSY and HMBC correlation diagrams.

[0020] Figure 2 Q-TOF-MS spectrum of LEP-1.

[0021] Figure 3 1H-NMR (600 MHz, DMSO-d6) spectrum of LEP-1.

[0022] Figure 4 13C-NMR (150 MHz, DMSO-d6) spectrum of LEP-1.

[0023] Figure 5 DEPT 135 (600 MHz, DMSO-d6) spectrum of LEP-1.

[0024] Figure 6 1H-1H COSY (600 MHz, DMSO-d6) spectrum of LEP-1.

[0025] Figure 7 1H-13C HMQC (600 MHz, DMSO-d6) spectrum of LEP-1.

[0026] Figure 8 1H-13C HMBC (600 MHz, DMSO-d6) spectrum of LEP-1.

[0027] Figure 9 Effect of different concentrations of LEP-1 on the activity of HaCaT cells (n = 12), wherein,*** P < 0.001 vs Control ** P < 0.01 vs Control。

[0028] Figure 10 Effect of different concentrations of LEP-1 on the viability of HaCaT cells damaged by UVB (n = 12), where ### P < 0.01 vs Control *** P < 0.001 vs Model ** P < 0.01 vs Model * P < 0.05 vs Model。

[0029] Figure 11 Effect of different concentrations of LEP-1 on the LDH release of HaCaT cells damaged by UVB (n = 12), where ### P < 0.01 vs Control *** P < 0.001 vs Model ** P < 0.01 vs Model * P < 0.05 vs Model。 Detailed implementation manners

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following further details the technical solution of the present invention in conjunction with the accompanying drawings and specific implementation manners.

[0031] The experimental instruments, materials and reagents used in the following examples are as follows:

[0032] 1. Experimental instruments

[0033]

[0034]

[0035] 2. Experimental materials

[0036] The used eyebright was purchased from Anguo City, Baoding, Hebei (batch number: XMC20200404), and it is the whole herb of Euphrasia pectinata Ten of the Orobanchaceae family.

[0037]

[0038] 3. Experimental reagents

[0039]

[0040] Example 1

[0041] Euphrasia officinalis was separated and extracted, and the separation process was as follows:

[0042] 4.5 kg of dry Euphrasia officinalis whole herb was soaked in 8 times the amount of water and refluxed for extraction 3 times, 2 h each time. After combining the filtrates, it was dried under reduced pressure to obtain a water extract. It was extracted with ethyl acetate 3 times, and after recovering the solvent, 7.6 g of the ethyl acetate extraction part was obtained. The water layer was separated by D101 macroporous adsorption resin, eluted with pure water and 95% ethanol as eluents, and after recovering the solvent, 97.2 g of the 95% ethanol elution part was obtained. 7 g of the ethyl acetate extraction part was separated by normal-phase silica gel column chromatography, and gradient elution was carried out with petroleum ether - ethyl acetate as the eluent (100:0 - 0:100), and a total of 8 fractions (Fr.1 - Fr.8) were obtained. Fr.2 (1.23 g) was separated by normal-phase silica gel column chromatography to obtain fractions Fr.2.1 and Fr.2.2; Fr.2.2 (834 mg) was separated by Sephadex LH-20 gel column chromatography to obtain fractions Fr.2.2.1 and Fr.2.2.2; Fr.2.2.1 (100 mg) was separated by MC l column chromatography, and gradient elution was carried out with methanol - water as the eluent (40:60 - 100:0) to obtain fraction Fr.2.2.1.1, and then 3.2 mg of this compound was obtained by preparative HPLC (acetonitrile - water 38:62), labeled as LEP-1.

[0043] As Figure 1-8 shown, the structure of the obtained compound was analyzed:

[0044] Pale yellow jelly, soluble in methanol. It showed black when stained with 10% sulfuric acid ethanol. UV λmax(MeOH) 235 nm, 270 nm. High-resolution Q-TOF-MS gave its quasi-molecular ion peak m / z 303.0882 [M-H] - , determining its molecular formula to be C 16 H 16 O6 (Calcd for C 16 H 16 O6, 303.0874). The degree of unsaturation was 9.

[0045] 1 In the 1H-NMR (600 MHz, DMSO-d6) spectrum, in the low-field region, δ H9.58 (1H, s) indicates the presence of an aldehyde hydrogen, 8.74 (1H, s, 7-OH) indicates the presence of a phenolic hydroxyl hydrogen, 6.77 (1H, d, J = 2.0 Hz, H-5), 6.63 (1H, d, J = 8.0 Hz, H-8) and 6.56 (1H, dd, J = 8.0, 2.0 Hz, H-9) indicate the presence of a 1,3,4-trisubstituted benzene ring in the structure. In addition, there is a pair of coupled double bond hydrogen signals 7.52 (1H, d, J = 3.5 Hz, H-3′) and 6.77 (1H, d, J = 3.5 Hz, H-4′), and 3.71 (3H, s) indicates the presence of a methoxy group in the structure.

[0046] 13 In the C-NMR (150 MHz, DMSO-d6) and Dept 135 spectra, there are two carbonyl carbons in the low field region, δ C 178.6 (C-6′), 171.9 (C-1). In the aromatic region of the benzene ring, 144.8 (C-7), 147.4 (C-6), 131.1 (C-4), 120.3 (C-9), 115.3 (C-8), 112.4 (C-5) are signals of the trisubstituted benzene ring, and 155.5 (C-2′), 152.4 (C-5′), 123.9 (C-4′), 113.0 (C-3′) are carbon signals on the furan ring of hydroxymethylfurfural. 55.5 (6-OCH3) in the high field region indicates the presence of a methoxy group.

[0047] Comprehensive 1 H, 13 C, 1 H- 1 HCOSY and HSQC were used for carbon-hydrogen assignment, as shown in Table 1.

[0048] In the HMBC spectrum, the long-range correlation between the methoxy group δ H 3.71 and C-6 (δ C 147.4) determined that the methoxy group is substituted at the 6-position. The long-range correlation between the phenolic hydroxyl δ H 8.74 and 115.3 (C-8), C-6 (δ C 147.4) determined that the phenolic hydroxyl is substituted at the 7-position. The long-range correlation between H-3 (δ H 2.74) and C-4 (δ C 131.1), C-9 (δ C 120.3), C-5 (δ C 112.4) determined that the 4-position of the benzene ring is substituted. The long-range correlation between H-3 (δ H 2.74) and C-1 (δ C 171.9), C-2 (δ C 35.3), and H-2 (δH 2.64) and the long-range correlation with C-1 (δ C 171.9), C-4 (δ C 131.1) determined the structure of dihydroferulic acid

[61] . In addition, the long-range correlation of H-6′ (δ H 9.58) with C-5′ (δ C 152.4), the long-range correlation of H-4′ (δ C 7.52) with C-2′ (δ C 155.5), the long-range correlation of H-3′ (δ H 6.77) with C-5′ (δ C 152.4), and the HMBC long-range correlation of H-1′ (δ H 5.15) with C-3′ (δ C 113.0) determined the structure of 5-hydroxymethylfurfural.

[0049] In the HMBC spectrum, the long-range correlation of H-1′ (δ H 5.15) with C-1 (δ C 171.9) determined that the carboxyl group of dihydroferulic acid was esterified with the hydroxyl group of 5-hydroxymethylfurfural. To sum up, the structure of this compound was determined and named as (5-formyl-2-furanyl)methyl dihydroferulic acid ester. According to the search in the Sci finder database, this compound was determined to be a new compound.

[0050] Table 1 1H-NMR (DMSO-d6 600 MHz) and 13C-NMR (DMSO-d6 150 MHz) data attribution of LEP-1

[0051]

[0052]

[0053] To sum up, the structure of this compound was identified as (5-formyl-2-furanyl)methyl dihydroferulic acid ester ((5-Formyl-2-furanyl)mathyl dihydroferulic acid ester). The obtained compound was confirmed as:

[0054]

[0055] Example 2

[0056] Protective effect of compound LEP-1 in Example 1 on UVB-induced oxidative damage in HaCaT cells

[0057] 1 Experimental materials

[0058] Components extracted from Euphrasia officinalis LEP-1, MEM medium (Gibco, USA), fetal bovine serum FBS (Biolnd, Israel), 0.25% trypsin + 0.02% EDTA (Gibco, USA), penicillin-streptomycin (PS) (Gibco, USA), PBS phosphate buffer (Gibco, USA), DMSO (Sigma, USA), CCK-8 kit (Dojindo Lab, Japan), LDH kit (Dojindo Lab, Japan), HaCaT cells.

[0059] 2 Experimental methods

[0060] 2.1 Preparation of solutions

[0061] (1) Preparation of MEM complete culture medium: Take an appropriate amount of MEM basal medium, add 10% FBS and 1% PS, mix well, and store at 4°C for later use.

[0062] (2) Preparation of Euphrasia officinalis extract: Weigh 0.61 mg of LEP-1 precisely and add 20 μL of DMSO to dissolve it completely to obtain a 100 mM stock solution, which is stored at 4°C for later use. Prepare its 1, 10, 100 μM sample solutions by serial dilution with DMEM complete culture medium.

[0063] (3) Preparation of 1×CCK-8 working solution: Dilute the 10×CCK-8 solution 10-fold with PBS solution according to the detection requirement and mix well.

[0064] 2.2 HaCaT cell culture

[0065] When the cell density reaches 70 - 80%, normal subculture is carried out. During subculture, pour out the culture medium in the culture flask, add 1 mL of PBS to wash twice, add 1.5 mL of 0.25% trypsin (containing 0.02% EDTA), place it in the incubator for 5 min for digestion. Observe under the microscope. When the cells are all digested into spherical shapes, add an appropriate amount of MEM complete culture medium to terminate digestion. Gently pipette the cell suspension on the bottle wall to make the cells detached, collect the cells, transfer the cell suspension to a centrifuge tube, and centrifuge at 4°C and 1000 rpm for 3 min at low speed. Discard the supernatant, add MEM complete culture medium, gently pipette the cell pellet to disperse it into a uniform cell suspension. Transfer the cell suspension to the culture flask, observe the dispersion degree of the cells under the microscope, place it in the incubator for culture, regularly observe the cell status, and subculture and seed the plate when it grows to about 80%.

[0066] Take a clean hemocytometer, wipe the counting plate and coverslip clean with an alcohol cotton ball. Place the coverslip on the counting plate, blow the cell suspension evenly, suck the cell suspension with a 10 μL pipette and inject it from the middle of the counting plate, making sure not to generate bubbles. Let it stand for a while to allow the cells to settle on the counting plate. Place the hemocytometer on the stage of the microscope. First, find the counting area under the low-power objective, then switch to the high-power objective for observation and counting. To ensure the accuracy of counting and avoid double counting and missed counting, when counting, count the upper lines but not the lower lines, and count the left lines but not the right lines to reduce errors.

[0067] Counting formula = number of cells / mL = number of cells in four large grids / 4 × 10000.

[0068] Seed HaCaT cells at a density of 1×10 5 cells / mL into a 96-well plate and culture them in an incubator at 37°C and 5% CO2.

[0069] 2.3 Effect of extracts from Euphrasia officinalis on the toxicity of HaCaT cells

[0070] Take HaCaT cells in the logarithmic growth phase and seed them at a density of 1×10 5 cells / mL into a 96-well cell culture plate. After the cells adhere and grow to 70 - 80%, add different concentrations of LEP-1 to different cell culture wells, 100 μL per well, with 6 replicates for each concentration. At the same time, set up a Control group without adding the drug. After culturing for 24 h, aspirate the supernatant in the cell plate, wash it 2 - 3 times with PBS, add 100 μL of 1×CCK-8 working solution to each well, incubate at 37°C for 1 h, then measure the OD value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell survival rate. The experiment is repeated 2 times (n = 12).

[0071] 2.4 Effect of extracts from Euphrasia officinalis on the viability of UVB-damaged HaCaT cells

[0072] Take HaCaT cells in the logarithmic growth phase and seed them at a density of 1×10 5 cells / mL into a 96-well plate. When the cells in the 96-well plate grow to about 60 - 70%, perform pre-drug administration by adding different concentrations of LEP-1. After culturing for 24 h, perform UVB irradiation. The UVB radiation dose is 60 mJ / cm 2. The Control group, UVB group, and sample solution + UVB group were set up. The Control group was not irradiated with UVB. After UVB irradiation, the cells were placed in an incubator and cultured for another 24 h and then taken out. The supernatant was aspirated and placed in another new 96-well plate, and the LDH release was detected according to the operation of the LDH kit. After the original 96-well plate was rinsed once with PBS, 100 μL of 1×CCK-8 working solution was added to each well and incubated at 37 °C for 1 h. The OD value was measured at 450 nm in an enzyme-linked immunosorbent assay reader. Six replicates were set for each group, and the experiment was repeated 2 times (n = 12).

[0073] 3 Statistical analysis

[0074] All data were processed using SPSS 26.0 statistical analysis software and expressed as mean ± standard deviation . One-way analysis of variance was used between groups, and P < 0.05 indicated that the difference was statistically significant.

[0075] 4 Results

[0076] 4.1 Effects of extracts from Euphrasia officinalis on the cytotoxicity of HaCaT cells

[0077] As shown in Table 2, Figure 9 the viability of HaCaT cells in the LEP-1 groups at 1, 10, and 100 μM showed no significant difference compared with the normal control group, which was the safe concentration for HaCaT cells. LEP-1 at 1, 10, and 100 μM was selected for subsequent experiments.

[0078] Table 2 Effects of different concentrations of LEP-1 on the activity of HaCaT cells (n = 12)

[0079]

[0080] *P < 0.05 vs Control

[0081] 4.2 Effects of extracts from Euphrasia officinalis on the viability of UVB-damaged HaCaT cells

[0082] As shown in Table 3, Figure 10 compared with the Control group, the cell viability decreased by about 40% after UVB irradiation, indicating that the cell photoaging model was successfully constructed. After administration of 1, 10, and 100 μM of LEP-1, compared with the UVB group, the cell viability in the LEP-1 groups at 10 and 100 μM was significantly increased.

[0083] Table 3 Effects of different concentrations of LEP-1 on the viability of UVB-damaged HaCaT cells (n = 12)

[0084]

[0085] ### P < 0.01 vs Control *** P < 0.001 vs Model ** P < 0.01 vs Model * P < 0.05 vs Model

[0086] 4.3 Effects of extracts of Euphrasia pectinata Ten. and Centella asiatica (L.) Urb. on LDH release in UVB-damaged HaCaT cells

[0087] As shown in Table 4, Figure 11 after UVB irradiation, the LDH release in cells was significantly increased compared with the Control group. After administration of 1, 10, 100 μM LEP-1, compared with the UVB group, the LDH release in cells of the 1, 10, 100 μM LEP-1 groups was significantly decreased.

[0088] Table 4 Effects of different concentrations of LEP-1 on LDH release in UVB-damaged HaCaT cells (n = 12)

[0089]

[0090] ### P < 0.01 vs Control *** P < 0.001 vs Model ** P < 0.01 vs Model * P < 0.05 vs Model

[0091] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An ferulic acid ester compound, characterized in that: It is (5-formyl-2-furyl)methyl dihydroferulate, with the structure shown in (Ⅰ):

2. The preparation method of the ferulic acid ester compound according to claim 1, characterized in that: The steps are as follows: The ethyl acetate extraction part is obtained by extracting the water extract of the dried whole plant of Euphrasia officinalis with ethyl acetate and recovering the solvent; the obtained ethyl acetate extraction part is separated by normal-phase silica gel column chromatography, and gradient elution is carried out with petroleum ether-ethyl acetate as the eluent to obtain fraction Fr.

2. Fraction Fr.2 is separated by normal-phase silica gel column chromatography to obtain fraction Fr.2.

2. Fraction Fr.2.2 is obtained by Sephadex LH-20 gel column chromatography. Fraction Fr.2.2.1 is separated by MCI column chromatography, and gradient elution is carried out with methanol-water as the eluent to obtain fraction Fr.2.2.1.

1. Then the compound is obtained by preparative HPLC.

3. The preparation method of the ferulic acid ester compound according to claim 2, characterized in that: The specific steps are as follows: (1) The dried whole plant of Euphrasia officinalis is soaked in 8 times the amount of water and reflux-extracted 3 times, 2 hours each time. After combining the filtrates, it is dried under reduced pressure to obtain a water extract, which is extracted with ethyl acetate 3 times, and the ethyl acetate extraction part is obtained after recovering the solvent; (2) The obtained ethyl acetate extraction part is separated by normal-phase silica gel column chromatography, and gradient elution is carried out with petroleum ether-ethyl acetate as the eluent to obtain fraction Fr.

2. Fraction Fr.2 is separated by normal-phase silica gel column chromatography to obtain fraction Fr.2.

2. Fraction Fr.2.2 is obtained by Sephadex LH-20 gel column chromatography. Fraction Fr.2.2.1 is separated by MCI column chromatography, and gradient elution is carried out with methanol-water as the eluent to obtain fraction Fr.2.2.1.

1. Then the compound is obtained by preparative HPLC.

4. Use of the ferulate compound according to claim 1 in the preparation of a product for protecting HaCaT cells or a product for repairing oxidative damage of HaCaT cells.

5. Use of the ferulate compound according to claim 1 in the preparation of an anti-skin photoaging product.

6. The application of the ferulic acid ester compound according to claim 5, wherein: The product is an anti-skin photoaging drug or cosmetic.

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