Saussurea involucrata sesquiterpene lactone compound for promoting expression of antioxidant enzyme cat in cells and application thereof

Abstract

The application discloses a saussurea involucrata sesquiterpene lactone compound for promoting expression of antioxidant enzyme CAT in cells and application thereof, and is compounds 1-7. The compound is obtained by extraction and separation from aerial parts of saussurea involucrata, is safe and reliable in source, meets the safety requirement for biological use, and has the effects of promoting expression of antioxidant enzyme CAT in cells and significantly reversing up-regulation of MDA caused by H2O2, and has the effects of anti-aging and anti-oxidation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Saussurea, and in particular to a sesquiterpene lactone compound in Saussurea involucrata for promoting expression of antioxidant enzyme CAT in cells and application thereof. BACKGROUND

[0002] Sesquiterpene lactones are a class of important natural products with various biological activities and structural diversity. The unique structure of sesquiterpene lactones enables them to have various biological activities, including anti-inflammatory, antimicrobial, antioxidant, melanin production inhibition, anti-malaria, and immune regulation activities. Sesquiterpene lactones exert their biological activity by inhibiting key signaling pathways such as NF-κB, MAPK, JAK-STAT, PI3K / Akt / m-TOR, and NLRP3, which play an important role in the regulation of inflammation and immune response. The regulation of inflammation and immune response is closely related to many 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 defense mechanism of cells against oxidative damage.

[0003] Sesquiterpene lactones are mainly derived from organisms such as plants, insects, and microorganisms. In plants, sesquiterpene lactones are widely distributed, especially in Magnoliaceae, Rutaceae, and Compositae. They often exist in the form of alcohols, ketones, and lactones in volatile oils, and are the main components of high-boiling fractions in volatile oils.

[0004] Saussurea involucrata grows in high-altitude areas of Central Asia and is a perennial herbaceous plant belonging to the genus Saussurea of the Asteraceae family. Existing studies have shown that Saussurea involucrata is rich in sesquiterpene lactones, and about 30 sesquiterpene lactones have been isolated from its aboveground parts, including most guaiane-type and some eudesmane-type sesquiterpenes and sesquiterpene lactones.

[0005] CN202311765420.4 discloses that Saussurea involucrata extract has the effects of reducing acne marks, whitening, and inhibiting acne regeneration, but there is no related report on the antioxidant activity and whitening activity of sesquiterpene components contained in Saussurea involucrata.

[0006] The information disclosed in the background section is only intended to increase the understanding of the general background of the application and should not be considered as admitting or in any form suggesting that the information constitutes prior art known to those of ordinary skill in the art. SUMMARY

[0007] The present application provides a saussurea involucrata sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells and an application thereof.

[0008] The present application provides a saussurea involucrata sesquiterpene lactone compound for promoting the expression of antioxidant enzyme CAT in cells, which is compound 1-7.

[0009]

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

[0011] 1) After obtaining the extract from the aboveground parts of saussurea involucrata as raw material, gradient elution is performed after extraction and silica gel column chromatography, and the obtained fractions are combined by TLC detection, and 8 components Fr.E1-Fr.E8 are obtained;

[0012] 2) Selecting component Fr.E5 to obtain compounds 1-4 by separation;

[0013] 3) Selecting component Fr.E6 to obtain compounds 5-7 by separation.

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

[0015] Fr.E5 is taken and separated by reversed-phase ODS column chromatography to obtain Fr.E51-Fr.E53; specifically, the elution system is methanol-water, and methanol-water with a volume ratio of 20:80, 70:30 and 90:10 is used for elution in turn.

[0016] Fr.E52 is taken and separated by silica gel column chromatography, and the obtained fractions are combined by TLC detection, and 5 components Fr.E52A-Fr.E52E are obtained; specifically, the elution solvent is petroleum ether-acetone, and gradient elution is performed using petroleum ether-acetone with a volume ratio of 30:1, 10:1, 1:1 and 0:1 in turn.

[0017] Fr.E52B is taken and separated by column chromatography to obtain 5 components Fr.E52B1-Fr.E52B6; specifically, the column used for elution is Sephadex LH-20 column, and the elution solvent is dichloromethane-methanol (V:V=1:1).

[0018] Compound 1 and compound 2 are obtained by separating Fr.E52B4 by semi-preparative high performance liquid chromatography (HPLC); specifically, the parameters of semi-preparative high performance liquid chromatography are as follows: the chromatographic column is Waters X-bridge with a specification of 5 μm, 10*150 mm, the volume ratio of acetonitrile to water is 35:75, the flow rate of the mobile phase is 3 mL / min, and the column temperature is 30°C.

[0019] The fractions obtained from the silica gel column chromatography of Fr.E52B3 were combined according to the TLC detection, and 7 fractions, Fr.E52B31-Fr.E52B37, were obtained. Specifically, the elution solvents were dichloromethane-methanol, and the gradient elution was performed using dichloromethane-methanol with volume ratios of 30:1, 10:1 and 0:1, respectively.

[0020] Compound 3 was obtained by semi-preparative HPLC separation of Fr.E52B34. Specifically, the parameters of the semi-preparative HPLC were as follows: the column was Waters X-bridge with a size of 5 μm and a length of 10 x 150 mm, the mobile phase was acetonitrile:water with a volume ratio of 40:60, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30 °C.

[0021] Compound 4 was obtained by semi-preparative HPLC separation of Fr.E52B37. Specifically, the parameters of the semi-preparative HPLC were as follows: the column was Waters X-bridge with a size of 5 μm and a length of 10 x 150 mm, the mobile phase was acetonitrile:water with a volume ratio of 30:70, the flow rate of the mobile phase was 3 mL / min, and the column temperature was 30 °C.

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

[0023] The fractions obtained from the ODS column chromatography of Fr.E6 were combined according to the TLC detection, and 5 fractions, Fr.E61-Fr.E65, were obtained. Specifically, the elution solvents used in the ODS column chromatography were methanol-water, and the elution was performed using methanol-water with volume ratios of 40:60, 70:30, 90:10 and 100:0, respectively.

[0024] The fractions obtained from the silica gel column chromatography of Fr.E61 were combined according to the TLC detection, and 6 fractions, Fr.E61A-Fr.E61F, were obtained. Specifically, the elution solvents used in the silica gel column chromatography were dichloromethane-methanol, and the gradient elution was performed using dichloromethane-methanol with volume ratios of 40:1, 20:1, 10:1 and 0:1, respectively.

[0025] The fractions obtained from the column chromatography of Fr.E61D were combined, and 6 fractions, Fr.E61D1-Fr.E61D6, were obtained. Specifically, the elution solvents were dichloromethane-methanol (V / V = 1:1).

[0026] Compound 5 and compound 6 were obtained by semi-preparative high performance liquid chromatography (HPLC) separation from Fr.E61D2; specifically, the parameters of semi-preparative high performance liquid chromatography were as follows: Waters X-bridge column with a size of 5 μm, 10 x 150 mm, acetonitrile: water volume ratio of 25:75, flow rate of mobile phase of 3 mL / min, and column temperature of 30 °C.

[0027] Compound 7 was obtained by semi-preparative high performance liquid chromatography (HPLC) separation from Fr.E61D3; specifically, the parameters of semi-preparative high performance liquid chromatography were as follows: Waters X-bridge column with a size of 5 μm, 10 x 150 mm, acetonitrile: water volume ratio of 30:70, flow rate of mobile phase of 3 mL / min, and column temperature of 30 °C.

[0028] Preferably, the preparation method of the extract of the aerial part of Saussurea involucrata Kar. et Kir. comprises: filtering the combined extract after alcohol extraction of the powdered aerial part of Saussurea involucrata Kar. et Kir. for at least 3 times to obtain an extract; and concentrating the extract to obtain a crude extract; specifically, the aerial part of Saussurea involucrata Kar. et Kir. used is a mixture of stems, leaves and flowers of Saussurea involucrata Kar. et Kir. in a mass ratio of 3:1:2.

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

[0030] Preferably, the extraction operation is as follows: the crude extract is dissolved in water and extracted with ethyl acetate for at least 3 times, 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 as follows: gradient elution is performed once with petroleum ether: acetone mixed solution in a volume ratio of 50:1, 25:1, 10:1 and 1:1, respectively, and then gradient elution is performed with dichloromethane-methanol in a volume ratio of 10:1, 1:1 and 0:1, respectively, to obtain the flow fraction for TLC detection.

[0032] Preferably, compounds 2-5 have the effect of significantly increasing CAT enzyme activity and reversing the down-regulation of CAT enzyme activity caused by H2O2.

[0033] Compounds 1-4, 6-7 have the effect of significantly reversing the up-regulation of MDA caused by H2O2.

[0034] Preferably, the concentration of compounds 2-5 for exerting the effect of significantly increasing CAT enzyme activity is greater than or equal to 12.5 μM; specifically, the concentrations of each compound for exerting the effect of significantly increasing CAT enzyme activity are as follows: the concentration of compound 2 is 12.5-25 μM, the concentration of compound 3 is 12.5 μM, the concentration of compound 4 is 25 μM, and the concentration of compound 5 is 12.5-50 μM.

[0035] The compounds 1-4, 6-7 exert a significant reverse effect on the up-regulation of MDA caused by H2O2 at a concentration of 12.5 μM or more. Specifically, the concentration of each compound exerting a significant reverse effect on the up-regulation of MDA caused by H2O2 is: 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 compounds 2-5 exert a significant effect on increasing the activity of CAT enzyme at a concentration of 12.5-50 μM.

[0037] The compounds 1-4, 6-7 exert a significant reverse effect on the up-regulation of MDA caused by H2O2 at a concentration of 12.5-50 μM.

[0038] Another aspect of the present application also provides the use of the saussurea involucrate sesquiterpene lactone compound for promoting the expression of the antioxidant enzyme CAT in cells for the preparation of a medicine or cosmetic having antioxidant and anti-aging purposes.

[0039] The composition can be used for the preparation of various substances having antioxidant and anti-aging purposes.

[0040] The composition can be prepared into a medicine or cosmetic as needed, and the corresponding pharmaceutical or cosmetic adjuvants can be selected for configuration according to the purpose, for example, adding the necessary amount of adjuvants in the composition, including but not limited to: fillers, diluents, binders, lubricants, disintegrants, and other commonly used pharmaceutical adjuvants. Cosmetic adjuvants include but are not limited to: solvents, excipients, preservatives, antibacterial agents, thickening agents, humectants, and other commonly used cosmetic adjuvants. Various commonly used cosmetic adjuvants. Those skilled in the art can select from commonly used adjuvants according to the function and type of the product to be prepared.

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

[0042] Preferably, the composition is a cosmetic composition; the dosage form is at least one of a cream, an emulsion, an aqueous agent, a gel, an oil agent, and a powder.

[0043] The beneficial effects that can be produced by the present application include:

[0044] 1) The sesquiterpene lactone compound provided by the application for promoting the expression of antioxidant enzyme CAT in cells and its application, the compound is obtained by extraction and separation from the aerial part of Saussurea involucrata, which is safe and reliable in source and meets the biological use safety requirements. Meanwhile, the obtained compounds 1-7 have the effects of promoting the expression of antioxidant enzyme CAT in cells and significantly reversing the up-regulation of MDA caused by H2O2, and have the effects of anti-aging and anti-oxidation.

[0045] 2) The sesquiterpene lactone compound provided by the application for promoting the expression of antioxidant enzyme CAT in cells and its application, the compound can be used as a raw material in whitening, freckle-removing cosmetics or anti-oxidation drugs. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The structural formula of the compounds 1-7 obtained in the examples provided by the application is shown in the figure.

[0047] Figure 2 The figure of the detection results of the effects of the compounds 1-7 on the activity of antioxidant enzyme CAT in HepG2 cells is shown in the figure, wherein A-G are the experimental result figures of the compounds 1-7, respectively; in each figure, "H2O2-, compound-" represents the blank group; "H2O2+, compound-" represents the H2O2 is the model group induced by hydrogen peroxide; the horizontal coordinates 50, 25, 12.5 (μM) in each figure represent the concentration of the corresponding compound in the administration group; the data is represented as mean ± SD, compared with the blank group, P ### <0.001; compared with the model group, P*** <0.001, P** <0.01.

[0048] Figure 3 The figure of the influence of the compounds 1-7 on the MDA content in HepG2 cells is shown in the figure, wherein A-G are the experimental result figures of the compounds 1-7, respectively; in each figure, "H2O2-, compound-" represents the blank group; "H2O2+, compound-" represents the H2O2 is the model group induced by hydrogen peroxide, the horizontal coordinates 50, 25, 12.5 (μM) in each figure represent the addition concentration of each compound in the treatment group; the data is represented as mean ± SD, 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 application will be further described in detail below in combination with the drawings and examples, but in no way limits the application, any transformation or improvement based on the teaching of the application falls within the protection scope of the application.

[0050] Examples

[0051] The materials and instruments used in the following examples were obtained from commercial sources unless otherwise specified; the detection methods used were conventional unless otherwise specified.

[0052] Preparation of compounds 1-7 in Example 1:

[0053] 1) The aerial parts (a mixture of stems, leaves and flowers at a mass ratio of 3:1:2, 9.9 kg) of Saussurea involucrata Kar. et Kir. were ground into powder, and then extracted with 95% ethanol solution (30 L, at a solid-liquid ratio of 1:3) by hot reflux extraction for 3 times. The extract was combined and filtered to obtain the ethanol extract of Saussurea involucrata Kar. et Kir.

[0054] The ethanol extract of Saussurea involucrata Kar. et Kir. was concentrated to dryness, and then dissolved in water (at a solid-liquid ratio of 1:8). The solution was extracted with ethyl acetate for 3 times (at a solid-liquid ratio of 1:1). The ethyl acetate extract was mixed with silica gel (60-80 mesh), and then subjected to column chromatography on silica gel (200-300 mesh) with a gradient elution system of petroleum ether-acetone-dichloromethane-methanol. The gradient elution was performed with petroleum ether-acetone at a volume ratio of 50:1, 25:1, 10:1 and 1:1, respectively, and then with dichloromethane-methanol at a volume ratio of 10:1, 1:1 and 0:1, respectively. The fractions were combined according to TLC detection, and 8 fractions were obtained, namely Fr.E1-Fr.E8.

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

[0056] Fr.E5 was subjected to column chromatography on ODS with a gradient elution system of methanol-water. The gradient elution was performed with methanol-water at a volume ratio of 20:80, 70:30 and 90:10, respectively, to obtain 3 fractions, namely Fr.E51-Fr.E53.

[0057] Fr.E52 was subjected to column chromatography on silica gel with a gradient elution system of petroleum ether-acetone. The gradient elution was performed with petroleum ether-acetone at a volume ratio of 30:1, 10:1, 1:1 and 0:1, respectively. The fractions were combined according to TLC detection, and 5 fractions were obtained, namely Fr.E52A-Fr.E52E.

[0058] Fr.E52B was subjected to column chromatography on Sephadex LH-20 with a gradient elution system of dichloromethane-methanol (V:V=1:1), to obtain 5 fractions, namely Fr.E52B1-Fr.E52B6.

[0059] Fr.E52B4 was separated by semi-preparative HPLC (Waters X~bridge, 5 μm, 10 x 150 mm, acetonitrile: water (35:75), 3 mL / min, 30 °C) to give compound 1 (2.0 mg, t R = 13.5 min) and compound 2 (2.2 mg, t R = 20.5 min).

[0060] Fr.E52B3 was separated by column chromatography on silica gel with dichloromethane-methanol as elution solvent, gradient elution was performed with dichloromethane-methanol (30:1, 10:1, 0:1, by volume) and the fractions of the same component were combined according to TLC detection, to give 7 fractions, Fr.E52B31~Fr.E52B37.

[0061] Fr.E52B34 was separated by semi-preparative HPLC (Waters X~bridge, 5 μm, 10 x 150 mm, acetonitrile: water (40:60), 3 mL / min, 30 °C) to give compound 3 (6.4 mg, t R = 10.2 min).

[0062] Fr.E52B37 was separated by semi-preparative HPLC (Waters X~bridge, 5 μm, 10 x 150 mm, acetonitrile: water (30:70), 3 mL / min, 30 °C) to give compound 4 (4.0 mg, t R = 12.5 min).

[0063] Fr.E6 was separated by column chromatography on ODS with methanol-water as elution solvent, gradient elution was performed with methanol-water (40:60, 70:30, 90:10, 100:0, by volume) and the fractions of the same component were combined according to TLC detection, to give 5 fractions, Fr.E61~Fr.E65.

[0064] Fr.E61 was separated by column chromatography on silica gel with dichloromethane-methanol as elution solvent, gradient elution was performed with dichloromethane-methanol (40:1, 20:1, 10:1, 0:1, by volume) and the fractions of the same component were combined according to TLC detection, to give 6 fractions, Fr.E61A~Fr.E61F.

[0065] Fr.E61D was separated by Sephadex LH-20 column chromatography with dichloromethane-methanol (V:V = 1:1) as eluent, and six fractions were obtained: Fr.E61D1~Fr.E61D6.

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

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

[0068] Compound 1, white amorphous powder; (c 0.20, MeOH); UV (MeOH) λ max (log ε): 195 (4.3); IR (KBr) v max : 3428, 2934, 1765, 1639, 1448, 1384, 1259, 1082, 1021 cm -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-like crystal; (c 0.05, MeOH); UV (MeOH) λ max (log ε): 195 (4.3); IR (KBr) v max : 3428, 2934, 1765, 1639, 1448, 1384, 1259, 1082, 1021 cm -1HR-ESI-MS (positive) at m / z 249.1483 ([M+H] + 1), calcd for C + ECD (MeOH, nm) λ (Δε) 216 (+1.64). max ECD (MeOH, nm) λ (Δε) 216 (+1.64). 1 H and 13 C-NMR data are listed in Table 1.

[0070] Compound 3, white amorphous powder; (c 0.15, MeOH); UV (MeOH) λ (log ε): 195 (3.5); IR (KBr) v max (log ε): 195 (3.5); IR (KBr) v max : 3435, 3071, 2969, 1641, 1409, 1363, 1296, 1255, 1085, 776 cm -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 (Δε) 245 (+3.27), 298 (-1.04). max ECD (MeOH, nm) λ (Δε) 216 (+1.64). 1 H and 13 C-NMR data are listed in Table 1.

[0071] Compound 4, white amorphous powder; (c 0.15, MeOH); UV (MeOH) λ (log ε): 195 (3.5); IR (KBr) v max (log ε): 195 (3.5); IR (KBr) v max : 3435, 3071, 2969, 1641, 1409, 1363, 1296, 1255, 1085, 776 cm -1 ; HR-ESI-MS (positive) at m / z 255.0987 [M+Na] + , calcd for 255.0992); ECD (MeOH, nm) λ (Δε) 211 (-7.24), 242 (+6.42). max ECD (MeOH, nm) λ (Δε) 216 (+1.64). 1 H and 13 C-NMR data are listed in Table 1.

[0072] Compound 5, colorless needle-like crystal; (c 0.15, MeOH); UV (MeOH) λ (log ε): 195 (3.5); IR (KBr) v max(log e): 195 (3.9); IR (KBr) v max : 3438, 2917, 1743, 1642, 1418, 1369, 1325, 901, 772 cm -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 listed in Table 2.

[0073] Compound 6, colorless oil; (c 0.12, MeOH); UV (MeOH) λ max (log e): 195 (4.7); IR (KBr) v max : 3427, 2930, 1755, 1723, 1639, 1438, 1383, 1344, 1305, 1008, 970, 636 cm -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 listed in Table 2.

[0074] Compound 7, colorless oil; (c 0.18, MeOH); UV (MeOH) λ max (log e): 195 (4.1); IR (KBr) v max : 3431, 3077, 2981, 2947, 2909, 1637, 1442, 1374, 1307, 1199, 1105, 969, 754 cm -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 given in Table 2.

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

[0076]

[0077]

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

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

[0080] c Indicated NMR signals are overlapping.

[0081] Table 2 C-NMR data of compounds 5-7 in deuterated chloroform 1 H and 13 C NMR data

[0082]

[0083]

[0084]

[0085] a Indicated 1 H (500 MHz) and 13 C (125 MHz) NMR data b Indicated 1 H (600 MHz) and 13 C (150 MHz) NMR data c Indicated NMR signals are overlapping.

[0086] Evaluation of antioxidant activity of compounds 1-7 of Example 2

[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 medium were purchased from Wuhan Ponsay Biotech Co., Ltd; Penicillin-streptomycin mixture (Beijing Solabio Technology Co., Ltd); Trypsin digestion solution (Shanghai Biyun Tian Biotechnology Co., Ltd); CAT hydrogen peroxide test kit and MDA malondialdehyde test kit (Nanjing Jiancheng Bioengineering Institute).

[0090] 1.2 Instruments

[0091] Full-wavelength microplate reader (Thermo Fisher); carbon dioxide incubator (Thermo Fisher); centrifugal precipitator (Shanghai Surgical Instrument Factory); micro tabletop refrigerated centrifuge (D-37520 Thermo); analytical balance (AG135, Metler Toledo, 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, and when the cell confluence reached about 80%, they were carefully and uniformly inoculated in a 6-well plate at a density of 2.5 x 10 5 After about 24 h, the cells adhered, the blank control group and the model group discarded the old culture medium, and new DMEM complete medium was added; the compound administration group discarded the old culture medium, and DMEM complete medium containing 12.5, 25, and 50 μmol / L of compounds 1-7 was added, and incubated for 24 h.

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

[0095] After 4 h of modeling induced by the above method, the cells were collected and centrifuged to obtain cell pellets. 200 μL of cell lysis solution (prepared by mixing RIPA strong lysis solution, phosphatase inhibitor, and PMSF in a volume ratio of 100:1:1, and used immediately) was added to the cell pellets, and lysed on ice for 30 min. The broken cell sample was obtained, and the broken cell sample was used to determine the protein concentration and enzyme activity. The specific determination steps are described in the CAT and MDA kit instructions.

[0096] 2) Results analysis

[0097] The results of the effect of compounds 1-7 on the antioxidant capacity of H2O2-induced HepG2 cells were detected by CAT and MDA kits, as shown in Table 1.Figure 2 As shown in A-G, compared with the blank control group (H2O2, drug =-), the expression of CAT in each compound model group was significantly down-regulated compared with the blank group (P<0.001). Compound 2 at a concentration of 12.5-25 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2 (P<0.001); similarly, compound 3 at 12.5 μM could significantly increase the CAT enzyme activity. Compound 4 at 25 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2; similarly, compound 5 at a concentration of 12.5-50 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2.

[0098] As shown in A-G, compared with the blank control group (H2O2, drug =-), the expression of CAT in each compound model group was significantly down-regulated compared with the blank group (P<0.001). Compound 2 at a concentration of 12.5-25 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2 (P<0.001); similarly, compound 3 at 12.5 μM could significantly increase the CAT enzyme activity. Compound 4 at 25 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2; similarly, compound 5 at a concentration of 12.5-50 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2. Figure 3 As shown in A-G, compared with the blank control group (H2O2, drug =-), the expression of CAT in each compound model group was significantly down-regulated compared with the blank group (P<0.001). Compound 2 at a concentration of 12.5-25 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2 (P<0.001); similarly, compound 3 at 12.5 μM could significantly increase the CAT enzyme activity. Compound 4 at 25 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2; similarly, compound 5 at a concentration of 12.5-50 μM could significantly reverse the down-regulation of CAT enzyme activity caused by H2O2.

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

[0100] Example 3

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

[0102] 2. At least one of the compounds 1-7 prepared in Example 1 is dissolved in DMSO, and then dissolved in sterile water for injection, stirred to dissolve, filtered through a sterile suction filter funnel, and then sterilely filtered, divided into ampoules, and then sterilely sealed after low-temperature freeze-drying to obtain a powder injection.

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

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

[0105] 5. At least one of the compounds 1-7 prepared in Example 1 is prepared into an oral liquid according to a conventional method for preparing an oral liquid.

[0106] 6. At least one of the compounds 1-7 prepared in Example 1 is added to excipients in a mass ratio of 5:1, and granulated into capsules.

[0107] 7. At least one of the compounds 1-7 prepared in Example 1 is added to excipients in a mass ratio of 5:1, and granulated into granules.

[0108] Example 4 Toner containing the compounds 1-7

[0109] The toner is prepared by mixing the compounds 1-7 in sterile water to a concentration of 25 μM.

[0110] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that modifications can be made to the foregoing embodiments, or other embodiments can be used, without departing from the spirit and scope of the application. Accordingly, the disclosure of the present application is intended to be illustrative, but not limiting, of the scope of the application, which is set forth in the following claims.

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, 3, 4, and 7:

2. The inoscine solanum 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 parts of Saussurea tianshanica as raw material, the extract was subjected to extraction, silica gel column chromatography, and gradient elution. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain a total of 8 components: Fr.E1 to Fr.E8; The preparation method of the extract of the aerial part of Saussurea involucrata comprises: extracting the aerial part of Saussurea involucrata powder at least three times with alcohol, combining the extracts and filtering to obtain an extract; concentrating the extract to obtain an extract extract; The solvent used for alcohol extraction is 95% ethanol solution, and the solvent is added at a material-liquid ratio of 1:3-4; Extraction procedure: The extract was dissolved in water and extracted at least three times with ethyl acetate as solvent. The obtained ethyl acetate phase was subjected to silica gel column chromatography; The elution system used for gradient elution was petroleum ether-acetone-dichloromethane-methanol; the gradient elution was performed by sequentially performing gradient elution with a petroleum ether:acetone mixture solution with a volume ratio of 50:1, 25:1, 10:1, and 1:1, followed by gradient elution with dichloromethane-methanol with a volume ratio of 10:1, 1:1, and 0:1, to obtain fractions for TLC detection; 2) Component Fr.E5 was selected and separated to obtain compounds 1, 3, and 4; 3) Component Fr.E6 was selected and separated to obtain compound 7; 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 obtained fractions were analyzed by TLC. The same fractions were combined to obtain 5 components: Fr.E52A to Fr.E52E; Fr.E52B was separated by column chromatography to obtain 6 components: Fr.E52B1 to Fr.E52B6; Fr.E52B4 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 1; The fractions obtained by silica gel column chromatography of Fr.E52B3 were combined and analyzed by TLC to obtain 7 components: Fr.E52B31 to Fr.E52B37; Fr.E52B34 was separated by semi-preparative HPLC to obtain compound 3; Fr.E52B37 was separated by semi-preparative HPLC to obtain compound 4; The separation of component Fr.E6 comprises the following steps: The fractions of Fr.E6 were separated by ODS column chromatography and analyzed by TLC, and the same fractions were combined to obtain 5 components: Fr.E61 ​​to Fr.E65; The fractions obtained by silica gel column chromatography of Fr.E61 ​​were combined and analyzed by TLC 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.E61D3 was separated by semi-preparative high performance liquid chromatography (HPLC) to obtain compound 7.

3. A use of the inoscine solanum sesquiterpene lactone compound for promoting the expression of the antioxidant enzyme CAT in cells as claimed in any one of claims 1 or 2, characterized in that: Used for the preparation of drugs or cosmetics with antioxidant uses; compounds 3 and 4 have the effect of significantly increasing CAT enzyme activity and reversing the downregulation of CAT enzyme activity caused by H2O2; Compounds 1, 3, 4, and 7 significantly reversed the upregulation of MDA induced by H2O2.

4. A composition for promoting the expression of antioxidant enzyme CAT in cells, characterized in that: The method at least comprises: the inoscine solanum sesquiterpene lactone compound for promoting the expression of the antioxidant enzyme CAT in cells as claimed in any one of claims 1 or 2.

5. The composition according to claim 4, 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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