Saussurea involucrata sesquiterpene lactone compound for promoting expression of antioxidant enzyme CAT in cells and application of saussurea involucrata sesquiterpene lactone compound

Abstract

The invention discloses a saussurea involucrata sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells and application of the saussurea involucrata sesquiterpene lactone compound, the saussurea involucrata sesquiterpene lactone compound is a compound 1-7: # imgabs0 #, the compound is obtained by taking the overground part of saussurea involucrata as a raw material through extraction and separation, the source is safe and reliable, the safety requirement of biological use is met, and the compound has a good application prospect. Meanwhile, the obtained compounds 1-7 have the effects of promoting the expression of antioxidant enzyme CAT in cells and remarkably reversing MDA up-regulation caused by H2O2, and have anti-aging and anti-oxidation effects.

Description

Technical Field

[0001] The present application relates to the technical field of snow lotus application, and in particular to a snow lotus sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells and its application. Background Art

[0002] Sesquiterpene lactones are an important class of natural products with multiple biological activities and structural diversity. The unique structure of sesquiterpene lactones gives them multiple biological activities, including anti-inflammatory, antimicrobial, antioxidant, inhibition of melanin production, anti-malarial and immunomodulatory activities. Sesquiterpene lactones exert their biological activities by inhibiting key signaling pathways such as NF-κB, MAPK, JAK-STAT, PI3K / Akt / m-TOR, and NLRP3. These pathways play an important role in the regulation of inflammation and immune response, and the regulation of inflammation and immune response is closely related to multiple functions of the human body and the occurrence of diseases. Existing studies have shown that sesquiterpene lactones can be used to regulate the Nrf-2 / Keap-1 pathway, which is a key factor in the cell defense mechanism against oxidative damage.

[0003] Sesquiterpene lactones are mainly derived from plants, insects, microorganisms and other organisms. In plants, sesquiterpene lactone compounds are widely distributed, especially in a variety of plants in the Magnoliaceae, Rutaceae, and Compositae families. In plants, they often exist in volatile oils in the form of alcohols, ketones, lactones, etc., and are the main components of the high-boiling point part of volatile oils.

[0004] Saussurea involucrata grows in high altitude areas of Central Asia and is a perennial herb of the genus Saussurea in the Asteraceae family. Existing studies have shown that Saussurea involucrata is rich in sesquiterpenoid lactones, and about 30 sesquiterpenoids have been isolated from its aerial parts, including most guaiacane-type and some eucalyptane-type sesquiterpenoids and sesquiterpenoid lactones.

[0005] For example, CN202311765420.4 discloses that the extract of Saussurea involucrata has the effects of reducing acne scars, whitening and inhibiting acne regeneration, but there is currently no report on the antioxidant and whitening activities of the sesquiterpenoid components contained in Saussurea involucrata.

[0006] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In response to the above technical problems, the present application provides a saussurea involucrata sesquiterpene lactone compound and its application for promoting the expression of antioxidant enzyme CAT in cells.

[0008] The present application provides a sesquiterpene lactone compound of Saussurea involucrata for promoting the expression of antioxidant enzyme CAT in cells, which are compounds 1 to 7:

[0009]

[0010] Preferably, the preparation method comprises the following steps:

[0011] 1) After obtaining an extract from the aerial part of Saussurea tianshanica as a raw material, the extract was subjected to extraction, silica gel column chromatography and gradient elution, and the obtained fractions were combined by TLC detection to obtain 8 components in total: Fr.E1 to Fr.E8;

[0012] 2) Select component Fr.E5 and separate to obtain compounds 1 to 4;

[0013] 3) Component Fr.E6 was selected and separated to obtain compounds 5 to 7.

[0014] Preferably, the separation of component Fr.E5 comprises the following steps:

[0015] The Fr.E5 segment was separated by reverse phase ODS column chromatography to obtain Fr.E51 to Fr.E53; during the specific elution, the elution system was methanol-water, and the elution was carried out with methanol-water in volume ratios of 20:80, 70:30, and 90:10, respectively.

[0016] Fr.E52 was separated by silica gel column chromatography and gradient elution. The obtained fractions were combined by TLC detection to obtain 5 components in total: Fr.E52A to Fr.E52E. Specifically, the elution solvent was petroleum ether-acetone, and gradient elution was performed with petroleum ether-acetone in volume ratios of 30:1, 10:1, 1:1, and 0:1, respectively.

[0017] Fr.E52B was separated by column chromatography to obtain five components: Fr.E52B1-Fr.E52B6; specifically, the column used for elution was Sephadex LH-20 column, and the elution solvent was dichloromethane-methanol (V:V=1:1).

[0018] Fr.E52B4 was separated by semi-preparative HPLC to obtain compound 1 and compound 2; specifically, the parameters of the semi-preparative HPLC were as follows: the chromatographic column was Waters X-bridge, with a specification of 5 μm, 10×150 mm, the volume ratio of acetonitrile: water was 35:75, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30°C.

[0019] The fractions obtained by separating Fr.E52B3 by silica gel column chromatography were combined after TLC detection to obtain 7 components in total: Fr.E52B31 to Fr.E52B37; specifically, the elution solvent was dichloromethane-methanol, and gradient elution was performed with dichloromethane-methanol in volume ratios of 30:1, 10:1, and 0:1, respectively.

[0020] Fr.E52B34 was separated by semi-preparative HPLC to obtain compound 3; specifically, the parameters of the semi-preparative HPLC were as follows: the chromatographic column was Waters X-bridge, with a specification of 5 μm, 10×150 mm, the volume ratio of acetonitrile: water was 40:60, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30°C.

[0021] Fr.E52B37 was separated by semi-preparative HPLC to obtain compound 4. Specifically, the parameters of the semi-preparative HPLC were as follows: the chromatographic column was Waters X-bridge, with a specification of 5 μm, 10×150 mm, a volume ratio of acetonitrile: water of 30:70, a flow rate of the mobile phase of 3 mL / min, and a column temperature of 30°C.

[0022] Preferably, the separation of component Fr.E6 comprises the following steps:

[0023] The Fr.E6 segment was separated by ODS column chromatography and the obtained fractions were combined after TLC detection to obtain 5 components in total: Fr.E61 ​​to Fr.E65; specifically, the elution solvent used in the ODS column chromatography separation was methanol-water, and the elution was carried out with methanol-water in volume ratios of 40:60, 70:30, 90:10, and 100:0, respectively.

[0024] The fractions obtained by silica gel column chromatography separation of Fr.E61 ​​were combined after TLC detection to obtain 6 components in total: Fr.E61A to Fr.E61F; specifically, the elution solvent for silica gel column chromatography separation was dichloromethane-methanol, and gradient elution was performed with dichloromethane-methanol in volume ratios of 40:1, 20:1, 10:1, and 0:1, respectively.

[0025] Fr.E61D was separated by column chromatography to obtain 6 components: Fr.E61D1 to Fr.E61D6; specifically, the elution solvent was dichloromethane-methanol (V / V=1:1).

[0026] Fr.E61D2 was separated by semi-preparative HPLC to obtain compound 5 and compound 6; specifically, the parameters of the semi-preparative HPLC were as follows: the chromatographic column was Waters X-bridge, with a specification of 5 μm, 10×150 mm, the volume ratio of acetonitrile: water was 25:75, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30°C.

[0027] Fr.E61D3 was separated by semi-preparative HPLC to obtain compound 7. Specifically, the parameters of the semi-preparative HPLC were as follows: the chromatographic column was Waters X-bridge, with a specification of 5 μm, 10×150 mm, a volume ratio of acetonitrile: water of 30:70, a flow rate of the mobile phase of 3 mL / min, and a column temperature of 30°C.

[0028] Preferably, the method for preparing the extract of the aerial part of Saussurea tianshanica comprises: extracting the powdered aerial part of Saussurea tianshanica at least 3 times with alcohol, combining the extracts and filtering them to obtain an extract; concentrating the extract to obtain an extractum extract; specifically, the aerial part of Saussurea tianshanica used is a mixture of the stems, leaves and flowers of Saussurea tianshanica in a mass ratio of 3:1:2.

[0029] Preferably, the solvent used for alcohol extraction is a 95% ethanol solution, and the solvent is added at a solid-liquid ratio of 1:3-4;

[0030] Preferably, the extraction operation is as follows: the extract is dissolved in water and then extracted at least three times with ethyl acetate as solvent, and the obtained ethyl acetate phase is subjected to silica gel column chromatography;

[0031] Preferably, the elution system used for gradient elution is petroleum ether-acetone-dichloromethane-methanol; the gradient elution is: gradient elution is performed once with a petroleum ether:acetone mixed solution with a volume ratio of 50:1, 25:1, 10:1, and 1:1, and then gradient elution is performed with dichloromethane-methanol with a volume ratio of 10:1, 1:1, and 0:1 to obtain a fraction for TLC detection.

[0032] Preferably, compounds 2 to 5 have the effect of significantly increasing CAT enzyme activity and reversing the downregulation of CAT enzyme activity caused by H2O2;

[0033] Compounds 1-4, 6-7 had a significant effect of reversing the upregulation of MDA caused by H2O2.

[0034] Preferably, the concentrations of compounds 2 to 5 that significantly increase CAT enzyme activity are greater than or equal to 12.5 μM; specifically, the concentrations of each compound that significantly increase CAT enzyme activity are: compound 2 at a concentration of 12.5 to 25 μM, compound 3 at a concentration of 12.5 μM, compound 4 at a concentration of 25 μM, and compound 5 at a concentration of 12.5 to 50 μM.

[0035] The concentrations of compounds 1-4, 6-7 that significantly reverse the up-regulation of MDA caused by H2O2 are greater than or equal to 12.5 μM. Specifically, the concentrations of each compound that significantly reverse the up-regulation of MDA caused by H2O2 are: 25 μM for compounds 1 and 7, 25-50 μM for compounds 2 and 6, 12.5-25 μM for compound 3, and 12.5-25 μM for compound 4.

[0036] Preferably, the concentration of compounds 2 to 5 that significantly increase CAT enzyme activity is 12.5 to 50 μM;

[0037] The concentrations of compounds 1-4, 6-7 that significantly reversed the up-regulation of MDA caused by H2O2 were 12.5-50 μM.

[0038] Another aspect of the present application also provides an application of the involucrata sesquiterpene lactone compound as described above for promoting the expression of the antioxidant enzyme CAT in cells, for preparing drugs or cosmetics with antioxidant and anti-aging uses.

[0039] The composition can be used to prepare various substances with anti-oxidation and anti-aging uses.

[0040] The composition can be made into medicine or cosmetics for use as needed, and can be configured with corresponding common excipients of medicine or cosmetics according to the purpose. For example, necessary amount of excipients can be added to the combination, including but not limited to: fillers, diluents, adhesives, lubricants, disintegrants and other common excipients of medicines. Cosmetic excipients include but are not limited to: solvents, excipients, preservatives, antibacterial agents, thickeners, moisturizers, etc. Common excipients for various cosmetics. Those skilled in the art can choose from common excipients according to the function and type of the product to be prepared.

[0041] Another aspect of the present application provides a composition for promoting the expression of antioxidant enzyme CAT in cells, comprising at least an effective amount of: the Saussurea involucrata sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells as described in any one of claims 1 to 8.

[0042] Preferably, the composition is a cosmetic composition; the dosage form is at least one of creams, emulsions, aqueous solutions, gels, oils, and powders.

[0043] The beneficial effects of this application include:

[0044] 1) The present application provides a sesquiterpene lactone compound of Saussurea schizandrica for promoting the expression of the antioxidant enzyme CAT in cells and its application. The compound is obtained by extraction and separation from the aerial part of Saussurea schizandrica, and the source is safe and reliable, meeting the safety requirements for biological use. At the same time, the obtained compounds 1 to 7 have the effect of promoting the expression of the antioxidant enzyme CAT in cells and significantly reversing the upregulation of MDA caused by H2O2, and have anti-aging and antioxidant effects.

[0045] 2) The involucrata sesquiterpene lactone compound provided in the present application for promoting the expression of the antioxidant enzyme CAT in cells and its application. The compound can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition, and can also be used as a raw material in whitening, freckle removal cosmetics or antioxidant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the structural formula of compounds 1 to 7 obtained in the examples provided in this application.

[0047] Figure 2 The results of the detection of the antioxidant enzyme CAT activity in HepG2 cells by compounds 1-7 in the examples provided in the present application, wherein A to G are the experimental results of compounds 1 to 7, respectively; in each figure, "H2O2-, compound-" represents a blank group; "H2O2+, compound-" represents a model group induced by hydrogen peroxide; the abscissas 50, 25, and 12.5 (μM) of each figure represent the concentrations of the corresponding compounds as the drug-treated group; the data are expressed as mean ± SD, and compared with the blank group, P ### <0.001; compared with the model group, P***<0.001, P**<0.01.

[0048] Figure 3 The results of the effects of compounds 1 to 7 on the MDA content in HepG2 cells in the examples provided in the present application are shown in Figures A to G, respectively; "H2O2-, compound-" in each figure represents a blank group; "H2O2+, compound-" represents a model group induced by hydrogen peroxide; the abscissas 50, 25, and 12.5 (μM) of each figure represent the added concentrations of each compound in the treatment group; the data are expressed as mean ± SD, and compared with the blank group, P ### <0.001; compared with the model group, P***<0.001, P**<0.01, P*<0.1. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0050] Example

[0051] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0052] Example 1 Preparation of compounds 1 to 7:

[0053] 1) The aerial part of Saussurea tianshanica (a mixture of stems, leaves and flowers in a mass ratio of 3:1:2, 9.9 kg) was crushed into powder, and then the aerial part was extracted three times with a 95% ethanol solution (30 L, a solid-liquid ratio of 1:3) by hot reflux, and the extracts were combined and filtered to obtain an alcohol extract of Saussurea tianshanica.

[0054] The Tianshan Saussurea ethanol extract was concentrated to dryness, and then the extract was dissolved in water (solid-liquid ratio 1:8), extracted with ethyl acetate three times (solid-liquid ratio 1:1), and the ethyl acetate extract obtained was mixed with 60-80 mesh silica gel, and chromatographed on a 200-300 mesh silica gel column. The elution system was petroleum ether-acetone-dichloromethane-methanol, and gradient elution was performed with a petroleum ether:acetone mixed solution with a volume ratio of 50:1, 25:1, 10:1, and 1:1, and then gradient elution was performed with dichloromethane-methanol with a volume ratio of 10:1, 1:1, and 0:1. The obtained fractions were detected by TLC and the same fractions were combined to obtain a total of 8 components: Fr.E1 to Fr.E8.

[0055] LC-QTOF-MS / MS analysis revealed that components Fr.E5 and Fr.E6 might contain the target sesquiterpenes.

[0056] Fr.E5 was separated by reverse phase ODS column chromatography with a methanol-water elution system, and eluted with methanol-water in volume ratios of 20:80, 70:30, and 90:10, respectively, to obtain three components: Fr.E51 to Fr.E53.

[0057] Fr.E52 was separated by silica gel column chromatography with petroleum ether-acetone as the elution solvent, and gradient elution was performed with petroleum ether-acetone in volume ratios of 30:1, 10:1, 1:1, and 0:1, respectively. The obtained fractions were detected by TLC and the same fractions were combined to obtain a total of 5 components: Fr.E52A~Fr.E52E.

[0058] Fr.E52B was separated by Sephadex LH-20 column chromatography with dichloromethane-methanol (V:V=1:1) as the eluting solvent, and a total of 5 components were obtained: Fr.E52B1-Fr.E52B6.

[0059] Fr.E52B4 was separated by semi-preparative HPLC (chromatographic column: Waters X-bridge, specification: 5 μm, 10×150 mm, acetonitrile: water volume ratio: 35:75, mobile phase flow rate: 3 mL / min, column temperature: 30°C) to obtain compound 1 (2.0 mg, t R =13.5min) and compound 2 (2.2mg, t R =20.5min).

[0060] Fr.E52B3 was separated by silica gel column chromatography with dichloromethane-methanol as the elution solvent. Gradient elution was performed with dichloromethane-methanol in volume ratios of 30:1, 10:1, and 0:1, respectively. The obtained fractions were detected by TLC and the same fractions were combined to obtain a total of 7 components: Fr.E52B31~Fr.E52B37.

[0061] Fr.E52B34 was separated by semi-preparative HPLC (chromatographic column: Waters X-bridge, specification: 5 μm, 10×150 mm, acetonitrile: water volume ratio: 40:60, mobile phase flow rate: 3 mL / min, column temperature: 30°C) to obtain compound 3 (6.4 mg, t R =10.2min).

[0062] Fr.E52B37 was separated by semi-preparative HPLC (chromatographic column: Waters X-bridge, specification: 5 μm, 10×150 mm, acetonitrile: water volume ratio: 30:70, mobile phase flow rate: 3 mL / min, column temperature: 30°C) to obtain compound 4 (4.0 mg, t R =12.5min).

[0063] The Fr.E6 segment was separated by ODS column chromatography with methanol-water as the elution solvent. The elution was carried out with methanol-water in volume ratios of 40:60, 70:30, 90:10, and 100:0, respectively. The obtained fractions were detected by TLC and the same fractions were combined to obtain a total of 5 components: Fr.E61 ​​to Fr.E65.

[0064] Fr.E61 ​​was separated by silica gel column chromatography with dichloromethane-methanol as the elution solvent. Gradient elution was performed with dichloromethane-methanol in volume ratios of 40:1, 20:1, 10:1, and 0:1, respectively. The obtained fractions were detected by TLC and the same fractions were combined to obtain a total of 6 components: Fr.E61A to Fr.E61F.

[0065] Fr.E61D was separated by Sephadex LH-20 column chromatography with dichloromethane-methanol (V:V=1:1) as the eluting solvent, and a total of 6 components were obtained: Fr.E61D1-Fr.E61D6.

[0066] Fr.E61D2 was separated by semi-preparative HPLC (chromatographic column: Waters X-bridge, specification: 5 μm, 10×150 mm, acetonitrile: water volume ratio: 25:75, mobile phase flow rate: 3 mL / min, column temperature: 30°C) to obtain compound 5 (2.7 mg, t R =15.2min) and compound 6 (2.2mg, t R =18.5min).

[0067] Fr.E61D3 was separated by semi-preparative HPLC (chromatographic column: Waters X-bridge, specification: 5 μm, 10×150 mm, acetonitrile: water volume ratio: 30:70, mobile phase flow rate: 3 mL / min, column temperature: 30°C) to obtain compound 7 (3.4 mg, t R =16.7min).

[0068] Compound 1, white amorphous powder; (c 0.20,MeOH); UV(MeOH)λ max (logε):195(3.9); IR(KBr)v max :3451,3084,2968,2913,1744,1642,1453,1375,1291,1026cm -1 ;HR-ESI-MS(positive)at m / z 247.1326[M+H] + ,calcd.for C 15 H 18 O3,247.1329);ECD(MeOH,nm)λ max (Δε)215(-2.15). 1 H and 13 C-NMR data are shown in Table 1.

[0069] Compound 2, colorless needle-shaped crystals; (c 0.05, MeOH); UV(MeOH)λ max (logε):195(4.3); IR(KBr)v max :3428,2934,1765,1639,1448,1384,1259,1082,1021cm -1;HR-ESI-MS(positive)at m / z 249.1483([M+H] + ,calcd.for249.1485); ECD(MeOH,nm)λ max (Δε)216(+1.64). 1 H and 13 C-NMR data are shown in Table 1.

[0070] Compound 3, white amorphous powder; (c 0.22,MeOH); UV(MeOH)λ max (logε):195(3.6); IR(KBr)v max :3434,2931,2859,1767,1639,1453,1382,1223,1147,1080,1015cm -1 ;HR-ESI-MS(positive)at m / z 257.1146([M+Na] + ,calcd.for C 14 H 18 O3,257.1154);ECD(MeOH,nm)λ max (Δε)245(+3.27),298(-1.04). 1 H and 13 C-NMR data are shown in Table 1.

[0071] Compound 4, white amorphous powder; (c 0.15, MeOH); UV(MeOH)λ max (logε):195(3.5); IR(KBr)v max :3435,3071,2969,1641,1409,1363,1296,1255,1085,776cm -1 ;HR-ESI-MS(positive)at m / z 255.0987[M+Na] + ,calcd for 255.0992); ECD(MeOH,nm)λ max (Δε)211(-7.24),242(+6.42). 1 H and 13 C-NMR data are shown in Table 1.

[0072] Compound 5, colorless needle-shaped crystals; (c 0.15, MeOH); UV(MeOH)λ max(logε):195(3.9); IR(KBr)v max :3438,2917,1743,1642,1418,1369,1325,901,772cm -1 ;HR-ESI-MS(positive)at m / z 465.2114([M+H] + ,calcd for 465.2119); ECD(MeOH,nm)λ max (Δε)196(-6.42),247(+0.57). 1 H and 13 C-NMR data are shown in Table 2.

[0073] Compound 6, colorless oily substance; (c 0.12, MeOH); UV(MeOH)λ max (logε):195(4.7); IR(KBr)v max :3427,2930,1755,1723,1639,1438,1383,1344,1305,1008,970,636cm -1 ;HR-ESI-MS(positive)at m / z 465.2114([M+H] + ,calcd for 465.2119); ECD(MeOH,nm)λ max (Δε)197(-2.94),218(+1.30). 1 H and 13 C-NMR data are shown in Table 2.

[0074] Compound 7, colorless oily substance; (c 0.18,MeOH); UV(MeOH)λ max (logε):195(4.1); IR(KBr)v max :3431,3077,2981,2947,2909,1637,1442,1374,1307,1199,1105,969,754cm -1 ;HR-ESI-MS(positive)at m / z 419.2042([M+Na] + ,calcd for 419.204); ECD(MeOH,nm)λ max (Δε)λ max (Δε)195(+4.24),208(-3.45). 1 H and 13C-NMR data are shown in Table 2.

[0075] Table 1 Compounds 1-4 in deuterated chloroform 1 H and 13 C-NMR data

[0076]

[0077]

[0078] a express 1 H(600MHz) and 13 C (150 MHz) NMR data

[0079] b express 1 H(500MHz) and 13 C (125 MHz) NMR data

[0080] c Indicates overlapping NMR signals.

[0081] Table 2 Compounds 5-7 1 H and 13 C NMR data

[0082]

[0083]

[0084]

[0085] a Indicates that the compound is in deuterated methanol 1 H(500MHz) and 13 C (125 MHz) NMR data b Indicates that the compound is in deuterated pyridine 1 H(600MHz) and 13 C (150 MHz) NMR data c Indicates overlapping of NMR signals.

[0086] Example 2 Evaluation of Antioxidant Activity of Compounds 1-7

[0087] 1) Materials and methods

[0088] 1.1 Materials

[0089] HepG2 cells were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). Fetal bovine serum and DMEM culture medium were purchased from Wuhan Procell Life Science Co., Ltd.; penicillin-streptomycin mixture (Beijing Solebold Technology Co., Ltd.); trypsin digestion solution (Shanghai Bio-Technology Co., Ltd.); CAT hydrogen peroxide test kit and MDA malondialdehyde test kit (Nanjing Jiancheng Biotechnology Co., Ltd.).

[0090] 1.2 Instruments

[0091] Full wavelength microplate reader (Thermo Fisher); CO2 incubator (Thermo Fisher); centrifugal precipitator (Shanghai Surgical Instrument Factory); micro desktop refrigerated centrifuge (D-37520TherMo). Analytical balance (AG135, MetlerToledo, China).

[0092] 1.3 Experimental process

[0093] HepG2 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. When the cell confluence reached about 80%, 2.5×10 5 The cells were carefully and evenly inoculated in a 6-well plate at a density of 100 μmol / mL and placed in a 37°C, 5% CO2 incubator for culture. After about 24 hours, the cells adhered to the wall, and the old culture medium was discarded in the blank control group and the model group, and new DMEM complete culture medium was added; the old culture medium was discarded in the compound administration group, and DMEM complete culture medium with compound 1-7 at 12.5, 25, and 50 μmol / L was added and incubated for 24 hours.

[0094] After 24 h, the original culture medium was discarded from the blank control group and new DMEM culture medium was added, while the DMEM culture medium containing 500 μmol / L H2O2 (protected from light) was replaced in the model group and compound treatment group.

[0095] After 4 hours of induction of the model by the above method, the cells were collected and centrifuged to obtain a cell pellet. 200 μL of cell lysis solution (prepared and mixed at a volume ratio of RIPA strong lysis solution: phosphatase inhibitor: PMSF of 100:1:1, prepared before use) was added to the cell pellet and lysed on ice for 30 minutes. The broken cell samples were obtained, and the broken cell samples were used to determine the protein concentration and enzyme activity. The specific determination operation steps are shown in the instructions for use of the CAT and MDA kits.

[0096] 2) Result analysis

[0097] The results of the effects of compounds 1 to 7 on the antioxidant capacity of HepG2 cells induced by H2O2 were tested by CAT and MDA kits. Figure 2 As shown in A to G, compared with the blank control group (hydrogen peroxide and drug are both -), the expression of CAT in the model group of each compound was significantly downregulated relative to the blank group (P<0.001). Compound 2 at a concentration of 12.5-25μM can significantly reverse the downregulation of CAT enzyme activity caused by H2O2 (P<0.001); similarly, compound 3 at 12.5μM can significantly increase CAT enzyme activity. Compound 4 at 25μM can significantly reverse the downregulation of CAT enzyme activity caused by H2O2; similarly, compound 5 at a concentration of 12.5-50μM can significantly reverse the downregulation of CAT enzyme activity caused by H2O2.

[0098] like Figure 3 A to G show the experimental results of compounds 1 to 7, respectively. Compared with the blank control group, the expression of MDA in the model group of each compound was significantly increased (P<0.001). Compounds 1 and 7 at a concentration of 25 μM can significantly reduce the increase in MDA content caused by H2O2. Similarly, compounds 2 and 6 at a concentration of 25-50 μM can significantly reverse the upregulation of MDA caused by H2O2; compound 3 at a concentration of 12.5-25 μM can significantly reverse the upregulation of MDA caused by H2O2; compound 4 at a concentration of 12.5-50 μM can significantly reverse the upregulation of MDA caused by H2O2.

[0099] The above results show that the addition of compounds 2-5 can increase the activity of antioxidant enzyme CAT in cells, and the addition of compounds 1-4 and 6-7 can reduce the level of MDA, reduce the oxidative stress response in cells, and avoid cell damage. Sesquiterpene lactone compounds 1-7 in Tianshan Saussurea involucrata have significant antioxidant activity and can be used as drugs for related diseases caused by excessive cell oxidation and as raw materials in whitening and anti-freckle cosmetics.

[0100] Example 3

[0101] At least one of the compounds 1-7 prepared in Example 1 was dissolved in DMSO, and then added with water for injection according to a conventional method, finely filtered, and sterilized by filling to prepare an injection solution, wherein the concentration of the injection solution was 0.5-5 mg / mL.

[0102] 2. Dissolve at least one of the compounds 1-7 prepared in Example 1 in DMSO, dissolve it in sterile water for injection, stir to dissolve it, filter it with a sterile suction funnel, filter it with sterile fine filtration, pack it into ampoules, freeze-dry it at low temperature, and seal it with sterile sealing to obtain a powder injection.

[0103] 3. At least one of the compounds 1-7 prepared in Example 1 was added to the excipient at a mass ratio of 9:1 to the excipient to prepare a powder.

[0104] 4. At least one of the compounds 1-7 prepared in Example 1 was added to the excipient at a mass ratio of 5:1, and granulated and tableted.

[0105] 5. Prepare at least one of the compounds 1-7 prepared in Example 1 into an oral liquid according to a conventional oral liquid preparation method.

[0106] 6. At least one of the compounds 1-7 prepared in Example 1 was added to the excipient at a mass ratio of 5:1 to the excipient to prepare capsules.

[0107] 7. At least one of the compounds 1-7 prepared in Example 1 was added to the excipient at a mass ratio of 5:1 to the excipient to prepare granules.

[0108] Example 4 Toner containing compounds 1-7

[0109] Compounds 1-7 were mixed in sterile water to a concentration of 25 μM to obtain toner.

[0110] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A sesquiterpene lactone compound of Saussurea involucrata for promoting the expression of antioxidant enzyme CAT in cells, characterized in that: For compounds 1 to 7:

2. The inositol sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells according to claim 1, characterized in that: The preparation method comprises the following steps: 1) After obtaining an extract from the aerial part of Saussurea tianshanica as a raw material, the extract was subjected to extraction, silica gel column chromatography and gradient elution, and the obtained fractions were combined by TLC detection to obtain 8 components in total: Fr.E1 to Fr.E8; 2) Select component Fr.E5 and separate to obtain compounds 1 to 4; 3) Component Fr.E6 was selected and separated to obtain compounds 5 to 7.

3. The inositol sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells according to claim 2, characterized in that: The separation of component Fr.E5 comprises the following steps: The Fr.E5 segment was separated by reverse phase ODS column chromatography to obtain Fr.E51 to Fr.E53; Fr.E52 was separated by silica gel column chromatography, and the fractions obtained by gradient elution were detected by TLC and combined to obtain 5 components: Fr.E52A to Fr.E52E; Fr.E52B was separated by column chromatography to obtain five components: Fr.E52B1 to Fr.E52B6; Fr.E52B4 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 1 and compound 2; The fractions obtained by separating Fr.E52B3 by silica gel column chromatography were combined by TLC detection to obtain 7 components: Fr.E52B31 to Fr.E52B37; Fr.E52B34 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 3; Fr.E52B37 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 4.

4. The inositol sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells according to claim 2, characterized in that: The separation of component Fr.E6 comprises the following steps: The fractions obtained by ODS column chromatography separation of Fr.E6 were combined by TLC detection to obtain 5 components: Fr.E61 ​​to Fr.E65; The fractions obtained by separating Fr.E61 ​​by silica gel column chromatography were combined by TLC detection to obtain 6 components: Fr.E61A to Fr.E61F; Fr.E61D was separated by column chromatography to obtain 6 components: Fr.E61D1 to Fr.E61D6; Fr.E61D2 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 5 and compound 6; Fr.E61D3 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 7.

5. The inositol sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells according to claim 2, characterized in that: The preparation method of the extract of the aerial part of Saussurea tianshanica comprises: extracting the aerial part of Saussurea tianshanica in powder form with alcohol for at least 3 times, combining the extracts and filtering to obtain an extract; concentrating the extract to obtain an extract extract; Preferably, the solvent used for alcohol extraction is a 95% ethanol solution, and the solvent is added at a solid-liquid ratio of 1:3-4; Preferably, the extraction operation is as follows: the extract is dissolved in water and then extracted at least three times with ethyl acetate as solvent, and the obtained ethyl acetate phase is subjected to silica gel column chromatography; Preferably, the elution system used for gradient elution is petroleum ether-acetone-dichloromethane-methanol; the gradient elution is: gradient elution is performed once with a petroleum ether:acetone mixed solution with a volume ratio of 50:1, 25:1, 10:1, and 1:1, and then gradient elution is performed with dichloromethane-methanol with a volume ratio of 10:1, 1:1, and 0:1 to obtain a fraction for TLC detection.

6. The inositol sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells according to claim 3, characterized in that: Compounds 2 to 5 significantly increased CAT enzyme activity and reversed the downregulation of CAT enzyme activity caused by H2O2; Compounds 1-4, 6-7 had a significant effect of reversing the upregulation of MDA caused by H2O2.

7. The inositol sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells according to claim 6, characterized in that: The concentrations of compounds 2 to 5 that significantly increased CAT enzyme activity were greater than or equal to 12.5 μM; The concentrations of compounds 1-4, 6-7 that significantly reversed the up-regulation of MDA caused by H2O2 were greater than or equal to 12.5 μM; Preferably, the concentration of compounds 2 to 5 that significantly increase CAT enzyme activity is 12.5 to 50 μM; Preferably, the concentration of compounds 1-4, 6-7 that significantly reverses the up-regulation of MDA caused by H2O2 is 12.5-50 μM.

8. A use of the inositol sesquiterpene lactone compound of any one of claims 1 to 7 for promoting the expression of the antioxidant enzyme CAT in cells, characterized in that: Used for preparing medicines or cosmetics with antioxidant and anti-aging effects.

9. A composition for promoting the expression of antioxidant enzyme CAT in cells, characterized in that: The method comprises at least an effective amount of the inositol sesquiterpene lactone compound of any one of claims 1 to 8 for promoting the expression of the antioxidant enzyme CAT in cells.

10. The composition according to claim 9, characterized in that The composition is a cosmetic composition; the dosage form is at least one of creams, emulsions, aqueous solutions, gels, oils, and powders.

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