Euphrasia line compound as well as preparation method and application thereof

Abstract

The invention provides an eyebright compound as well as a preparation method and application thereof, the compound is cetyl-4-lactone-13-0-beta-D-glucopyranose (1-> 2)-beta-D-glucopyranoside, and the preparation method comprises the following steps: extracting a water extract of dry eyebright herb with ethyl acetate, and recovering a solvent to obtain an ethyl acetate extraction part; separating the water layer by using macroporous adsorption resin, eluting by using pure water and ethanol as eluents, and recovering the solvent to obtain an ethanol elution part; separating the obtained ethanol elution part by using normal phase silica gel column chromatography, carrying out gradient elution by using dichloromethane: methanol as an eluent, separating an obtained fraction Fr.8 by using MCI column chromatography, carrying out gradient elution by using methanol-water as an eluent to obtain a fraction Fr.8.2, and carrying out preparative HPLC (High Performance Liquid Chromatography), so that the HaCaT cell activity can be remarkably improved; the compound has a protection effect on HaCaT cell oxidative damage caused by UVB, and can be used for resisting skin photoaging.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a Euphrasia 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, sagging, and a rough appearance. 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 the action of UVB 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 has the effects of clearing heat and detoxifying, and diuretic, and is used for treating feverish thirst, cough due to lung heat, sore throat, dysuria, etc. Further development and research on Euphrasia pectinata Tenore using modern traditional Chinese medicine technology are 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 Euphrasia compound.

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

[0006] Another technical problem to be solved by the present invention is to provide an application of the above Euphrasia compound.

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

[0008] A Euphrasia compound, which is hexadecane-4-olide-13-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside, has the structure shown in (I):

[0009]

[0010] The preparation method of the above Euphrasia compound is as follows: The water extract of dry Euphrasia whole herb is extracted with ethyl acetate and the solvent is recovered to obtain the ethyl acetate extraction part. The water layer is separated by macroporous adsorption resin and eluted with pure water and ethanol as eluents, and the ethanol elution part is obtained after recovering the solvent; The obtained ethanol elution part is separated by normal-phase silica gel column chromatography and gradient eluted with dichloromethane:methanol as eluents to obtain 4 fractions, Fr.6-Fr.9. Fr.8 is separated by MCI column chromatography and gradient eluted with methanol-water as eluents to obtain fractions Fr.8.1 and Fr.8.2. Fr.8.2 is obtained by preparative HPLC (acetonitrile-water as the mobile phase) to obtain this compound.

[0011] Preferably, the preparation method of the above Euphrasia compound is specifically as follows:

[0012] (1) The dry Euphrasia whole herb 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. It is extracted with ethyl acetate 3 times, and the solvent is recovered to obtain the ethyl acetate extraction part. The water layer is separated by D101 macroporous adsorption resin and eluted with pure water and 95% ethanol as eluents, and the 95% ethanol elution part is obtained after recovering the solvent;

[0013] (2) The obtained 95% ethanol elution part is separated by normal-phase silica gel column chromatography and gradient eluted with dichloromethane:methanol as eluents (100∶0-0∶100) to obtain 4 fractions, Fr.6-Fr.9. Fr.8 is separated by MCl column chromatography and gradient eluted with methanol-water as eluents (40∶60-100∶0) to obtain fractions Fr.8.1 and Fr.8.2; Fr.8.2 is obtained by preparative HPLC (acetonitrile-water 32∶68 as the mobile phase) to obtain this compound.

[0014] The application of the above Euphrasia compound in the preparation of HaCaT cell protection products or HaCaT cell oxidative damage repair products.

[0015] The application of the above Euphrasia compound in the preparation of anti-skin photoaging products.

[0016] Preferably, for the application of the above Euphrasia officinalis compound, the product is an anti-skin photoaging drug or cosmetic.

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

[0018] The above Euphrasia officinalis compound can significantly improve the activity of HaCaT cells, has a protective effect on the oxidative damage of HaCaT cells caused by UVB, can be used for anti-skin photoaging, and provides a research basis for the further development and utilization of its resources. Description of the Drawings

[0019] Figure 1 1H-1H COSY and HMBC correlation diagrams crucial for LEP-2.

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

[0021] Figure 3 1H-NMR (500 MHz, pyridine-d5) spectrum of LEP-2.

[0022] Figure 4 13C-NMR (125 MHz, pyridine-d5) spectrum of LEP-2.

[0023] Figure 5 DEPT 135 (500 MHz, pyridine-d5) spectrum of LEP-2.

[0024] Figure 6 1H-1H COSY (500 MHz, pyridine-d5) spectrum of LEP-2.

[0025] Figure 7 1H-13C HMQC (500 MHz, pyridine-d5) spectrum of LEP-2.

[0026] Figure 8 1H-13C HMBC (500 MHz, pyridine-d5) spectrum of LEP-2.

[0027] Figure 9 TCOSY (500 MHz, pyridine-d5) spectrum of LEP-2.

[0028] Figure 10 Effect of different concentrations of LEP-2 on the activity of HaCaT cells (n = 12), where *** P < 0.001 vs Control ** P < 0.01 vs Control.

[0029] Figure 11 Effect of different concentrations of LEP-2 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.

[0030] Figure 12 Effect of different concentrations of LEP-2 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

[0031] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners.

[0032] The experimental instruments, materials and reagents used in the following examples are as follows:

[0033] 1. Experimental instruments

[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 the plant Euphrasia pectinata Ten of the Orobanchaceae family.

[0037]

[0038] 3. Experimental reagents

[0039]

[0040] Example 1

[0041] Separate and extract eyebright, and the separation process is as follows:

[0042] 4.5 kg of dried whole Euphrasia herb was soaked in 8-fold volume of water and refluxed for extraction three times, each time for 2 h. After combining the filtrates, they were dried under reduced pressure to obtain a water extract. The water extract was extracted three times with ethyl acetate. After recovering the solvent, 7.6 g of the ethyl acetate extraction fraction was obtained. The aqueous layer was separated using D101 macroporous adsorption resin and eluted with pure water and 95% ethanol as eluents. After recovering the solvent, 97.2 g of the 95% ethanol elution fraction was obtained. 80 g of the obtained 95% ethanol elution fraction was separated by normal-phase silica gel column chromatography and gradient eluted with dichloromethane:methanol as the eluent (100:0 - 0:100) to obtain four fractions (Fr.6 - Fr.9). Fr.8 (1.25 g) was separated by MCI column chromatography and gradient eluted with methanol-water as the eluent (40:60 - 100:0) to obtain fractions Fr.8.1 and Fr.8.2; 381 mg of Fr.8.2 was separated by preparative HPLC (acetonitrile-water 32:68) to obtain 25 mg of this compound, labeled as LEP-2.

[0043] As Figures 1-9 shown, the structure of the obtained compound was analyzed:

[0044] White amorphous powder, soluble in methanol. It showed a purple-red color when stained with 10% sulfuric acid ethanol. The specific rotation was -8 (c 0.20, MeOH), and Q-TOF-MS gave its quasi-molecular ion peak m / z 593.3145 [M-H] - , (Calcd for C 28 H 50 O 13 , 593.3179) and the fragment ion 431.2616 [M-H-162] - produced by the loss of one molecule of glucose determined its molecular formula to be C 28 H 50 O 13 . The calculated degree of unsaturation of the compound was 4.

[0045] In 1 1H NMR (pyridine-d5, 500 MHz), δ H 5.33 (1H, d, J = 7.8 Hz, H-1″), 4.97 (1H, d, J = 7.7 Hz, H-1′) suggested the presence of two glycosides, and based on the coupling constant, the sugar was speculated to be in the β configuration. In the high-field region, 4.36 (1H, m, H-4), 2.48 (2H, dd, J = 9.4, 7.0 Hz, H-2), 2.10 (1H, m, H-3a), 1.66 (1H, m, H-3b) were characteristic hydrogen signals of the γ-lactone ring. 1.82 - 1.23 (21H, m) suggested the presence of an aliphatic side chain in the structure, and 0.86 (3H, t, J = 7.5 Hz, H-16) was the terminal methyl hydrogen signal.

[0046] In 13 C NMR (pyridine-d5, 125 MHz) and the Dept 135 spectrum, the anomeric carbons δ of the two sugars C 106.5 (C-1″), 102.3 (C-1′), further verified that the sugars were in the β configuration based on the chemical shift values. The carbon signals of the methine C-2′ to C-5″ on the sugars were 84.2 - 72.0, and the carbon signals of the methylene C-6″ and C-6′ on the sugars were 63.3 and 63.1. The above were the signals of β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside. 177.6, 81.2, 29.3, 28.5 were the characteristic carbon signals of the γ-lactone ring. In the high field region, 36.1 - 23.5 were the carbon signals of -[CH2] 12 -. The carbon signal of the terminal methyl was 14.6 (C-16).

[0047] 1 H- 1 In the H- H HCOSY spectrum, the correlations of H-4 (δ H 4.36) and H-3 (δ H 2.10), H-3 (δ H 2.10) and H-2 (δ Figure 6 ) ([[]] H suggested the presence of a γ-lactone ring (fragment A). The correlations of H-13 (δ H 3.96) with H-12a (δ H 1.79), H-12b (δ H 1.72), H-14 (δ H 1.50), the correlations of each hydrogen at 1.82 - 1.23 and the correlation of H-15 (δ H 1.30) with H-16 (δ Figure 1 ) [[[]] Figure 1 suggested the presence of the structure of dodecane (fragment B), and the correlations of each hydrogen at 5.33 - 3.89 [[[]]

[0048] Combining 1 H, 13 C, 1 H- 1 H COSY, HSQC for carbon-hydrogen assignment, see Table 1.

[0049] In the HMBC spectrum, the long-range correlations of H-4 (δ H 4.36) with C-1 (δ C 177.6), O-2 (δ C 29.3) and O-3 (δ C 28.5), H-2 (δH The long-range correlations of H-3 (δ C 2.48) with C-1 (δ C 177.6), H-4 (δ H 84.2) with C-1 (δ C 177.6) determined the structure of the five-membered lactone. The long-range correlations of H-3 (δ H 2.10, 1.66) with C-5 (δ C 25.9), H-4 (δ H 4.36) with C-5 (δ C 25.9), H-5 (δ H 1.34, 1.26) with C-3 (δ C 28.5) determined that the 12-alkyl group was substituted at the 4-position of the five-membered lactone. The correlation of H-1′ (δ H 4.97) with C-13 (δ C 80.0), and the correlation of H-13 (δ H 3.96) with O-1′ (δ C 102.3) determined that glucose was substituted at the 13-position of the carbon chain. The correlation of H-1″ (δ H 5.33) with C-2′ (δ C 84.2), and the correlation of H-2′ (δ H 4.16) with C-1″ (δ C 106.5) determined that the two glucoses were in a 1→2 substitution.

[0050] Absolute configuration of the sugar: The compound was identified as D-glucose after acid hydrolysis, derivatization experiments with L-cysteine methyl ester hydrochloride and o-tolyl isothiocyanate.

[0051] Table 1 1H-NMR (pyridine-d5, 500 MHz) and 13 C-NMR (pyridine-d5, 125 MHz) data assignment of LEP-2

[0052]

[0053]

[0054] In summary, the structure of the compound was identified as 16-alkane-4-olide-13-O-β-D-glucopyranosy(1→2)-β-D-glucopyranoside. The confirmed structure of the obtained compound is:

[0055]

[0056] Example 2

[0057] Protective effect of compound LEP-2 in Example 1 on oxidative damage of HaCaT cells induced by UVB

[0058] 1 Experimental materials

[0059] LEP-2, an extract from Euphrasia officinalis, 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.

[0060] 2 Experimental methods

[0061] 2.1 Preparation of solutions

[0062] (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;

[0063] (2) Preparation of Euphrasia extract: Weigh 1.18 mg of LEP-2 precisely and add 20 μL of DMSO to dissolve it completely to obtain a stock solution of 100 mM, and store at 4°C for later use; prepare its 1, 10, and 100 μM sample solutions by serial dilution with DMEM complete culture medium;

[0064] (3) Preparation of 1×CCK-8 working solution: Dilute the 10×CCK-8 solution 10-fold with PBS solution according to the detection requirements and mix well.

[0065] 2.2 HaCaT cell culture

[0066] 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 and wash twice, then 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 along the wall of the flask 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. 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 a culture flask, observe the degree of cell dispersion under the microscope, place it in the incubator for culture, regularly observe the cell status, and subculture and seed plates when it grows to about 80%.

[0067] Take a clean hemocytometer, wipe the hemocytometer and coverslip clean with an alcohol cotton ball. Place the coverslip on the hemocytometer, blow the cell suspension evenly, aspirate the cell suspension with a 10 μL pipette and inject it from the middle of the hemocytometer, taking care not to generate bubbles. Let it stand for a while to allow the cells to settle onto the hemocytometer. Place the hemocytometer on the stage of the microscope, first find the counting area under the low-power microscope, and then switch to the high-power microscope 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.

[0068] Counting formula = number of cells / mL = number of cells in four large grids / 4 × 10000.

[0069] Seed HaCaT cells into a 96-well plate at a density of 1×10 5 cells / mL and culture them in an incubator at 37°C and 5% CO2.

[0070] 2.3 Effect of extracts from Euphrasia officinalis on the cytotoxicity of HaCaT cells

[0071] Take HaCaT cells in the logarithmic growth phase, seed them into a 96-well cell culture plate at a density of 1×10 5 cells / mL. After they adhere and grow to 70 - 80%, add different concentrations of LEP-2 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 and discard 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 on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The experiment is repeated 2 times (n = 12).

[0072] 2.4 Effect of extracts from Euphrasia officinalis on the viability of UVB-damaged HaCaT cells

[0073] Take logarithmic growth phase HaCaT cells and inoculate them into 96-well plates at a density of 1×10 5 cells / mL. Wait until the cells in the 96-well plates grow to about 60-70%, then perform pre-drug administration. Add different concentrations of LEP-2 and irradiate with UVB after culturing for 24 h. The UVB radiation dose is 60 mJ / cm 2 . Set up Control group, UVB group and sample solution + UVB group. The Control group is not irradiated with UVB. After UVB irradiation, take out the cells after continuing to culture them in the incubator for 24 h, aspirate the supernatant and place it in another new 96-well plate, and detect the LDH release amount according to the operation of the LDH kit. After rinsing the original 96-well plate once with PBS, add 100 μL of 1×CCK-8 working solution to each well and incubate at 37 °C for 1 h. Measure the OD value at 450 nm in an enzyme-linked immunosorbent assay reader. Set 6 replicates for each group and repeat the experiment 2 times (n = 12).

[0074] 3 Statistical analysis

[0075] All data were processed using SPSS 26.0 statistical analysis software and expressed as mean ± standard deviation . One-way ANOVA was used between groups, and P < 0.05 indicated that the difference was statistically significant.

[0076] 4 Results

[0077] 4.1 Effects of extracts from Euphrasia officinalis on the cytotoxicity of HaCaT cells

[0078] As shown in Table 2 Figure 10 , there was no significant difference in the viability of HaCaT cells in the LEP-2 groups of 1, 10, and 100 μM compared with the normal control group, which were safe concentrations for HaCaT cells. 1, 10, and 100 μM of LEP-2 were selected for subsequent experiments.

[0079] Table 2 Effects of different concentrations of LEP-2 on the activity of HaCaT cells (n = 12)

[0080]

[0081] * P < 0.05 vs Control

[0082] 4.2 Effects of extracts from Euphrasia officinalis on the viability of UVB-damaged HaCaT cells

[0083] As shown in Table 3 Figure 11As shown, 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 treatment with 1, 10, and 100 μM of LEP-2, compared with the UVB group, the cell viability in the 1, 10, and 100 μM LEP-2 groups was significantly increased.

[0084] Table 3 Effects of different concentrations of LEP-2 on the viability of UVB-damaged HaCaT cells (n = 12)

[0085]

[0086] ### P < 0.01 vs Control *** P < 0.001 vs Model ** P < 0.01 vs Model * P < 0.05 vs Model

[0087] 4.3 Effects of extracts from Euphrasia officinalis and Centella asiatica on the LDH release of UVB-damaged HaCaT cells

[0088] As shown in Table 4, Figure 12 compared with the Control group, the LDH release of cells increased significantly after UVB irradiation. After treatment with 1, 10, and 100 μM of LEP-2, compared with the UVB group, the LDH release of cells in the 1, 10, and 100 μM LEP-2 groups decreased significantly.

[0089] Table 4 Effects of different concentrations of LEP-2 on the LDH release of UVB-damaged HaCaT cells (n = 12)

[0090]

[0091] ### P < 0.01 vs Control *** P < 0.001 vs Model ** P < 0.01 vs Model * P < 0.05 vs Model

[0092] The above-described embodiments are merely 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 of the present invention.

Claims

1. Euphrasia compound, which is hexadecane-4-lactone-13-O-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside, and is characterized in that: It has the structure shown in (I):

2. The preparation method of the eyebright compound according to claim 1, characterized in that: The steps are as follows: The ethyl acetate extract of the water extract of the dried whole herb of Euphrasia officinalis is obtained by extraction with ethyl acetate and recovery of the solvent. The aqueous layer is separated by macroporous adsorption resin and eluted with pure water and ethanol as eluents. After recovery of the solvent, the ethanol elution fraction is obtained; the obtained ethanol elution fraction is separated by normal-phase silica gel column chromatography and gradient eluted with dichloromethane:methanol as the eluent. The fraction Fr.8 is obtained and separated by MCI column chromatography and gradient eluted with methanol-water as the eluent to obtain the fraction Fr.8.

2. The compound is obtained by preparative HPLC.

3. The preparation method of the Euphrasia compound according to claim 2, characterized in that: The specific steps are as follows: (1) The dried whole herb of Euphrasia officinalis is soaked in 8 times the amount of water and refluxed and extracted 3 times, 2 hours each time. After combining the filtrates, it is dried under reduced pressure to obtain a water extract. It is extracted with ethyl acetate 3 times, and after recovery of the solvent, the ethyl acetate extraction fraction is obtained. The aqueous layer is separated by D101 macroporous adsorption resin and eluted with pure water and 95% ethanol as eluents. After recovery of the solvent, the 95% ethanol elution fraction is obtained; (2) The obtained 95% ethanol elution fraction is separated by normal-phase silica gel column chromatography and gradient eluted with dichloromethane:methanol as the eluent. The fraction Fr.8 is obtained and separated by MCI column chromatography and gradient eluted with methanol-water as the eluent to obtain the fraction Fr.8.

2. The compound is obtained by preparative HPLC.

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

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

6. Use of the eyebright compound according to claim 5, characterized in that: The product is an anti-skin photoaging drug or cosmetic.

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