8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone from Tianshan Saussurea involucrata for whitening and / or anti-aging, and its application

Abstract

This application discloses an 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone from Tianshan Saussurea involucrata for whitening and / or anti-aging, and its application. The structural formula is as follows: This compound can reduce ROS levels in HepG2 cells, increase the activities of the antioxidant enzymes CAT, SOD, and GSH, reduce MDA content, and improve oxidative stress in HepG2 cells. This compound can be used to prepare antioxidant and anti-aging compositions, and this substance can be used to prepare compositions that improve oxidative stress in HepG2 cells.

Description

Technical Field

[0001] The present application relates to the technical field of Saussurea involucrata, in particular to an 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone for whitening and / or anti-aging in Saussurea involucrata and its application. Background Art

[0002] Saussurea involucrata is the dried aerial part of the Asteraceae plant. It is warm in nature and slightly bitter in flavor, known for warming the kidneys and promoting yang, as well as promoting menstruation and blood circulation. Modern research indicates that it has anti-inflammatory, analgesic, lipid-regulating, anti-obesity, antioxidant, and anti-tumor properties.

[0003] Tianshan Snow Lotus has multiple effects on the skin, such as the Tianshan Snow Lotus extract and the skin care composition containing it disclosed in CN202311765420.4. The snow lotus extract improves the extraction rate through the composite biological enzyme-ultrasonic extraction technology to ensure the activity of the effective ingredients. The skin care composition containing the above-mentioned Tianshan Snow Lotus extract maintains the microecological balance of the skin by compounding lactobacilli, thereby achieving more effective resistance to the invasion of free radicals, reducing the speed of skin aging, and maintaining the youthful state of the skin. At the same time, it also has the effects of strengthening the skin barrier, renewing skin cells, inhibiting melanin deposition, smoothing acne pits, reducing acne marks, improving skin texture and inhibiting acne regeneration, making the skin smooth as before.

[0004] However, the extraction method of the above composition is simple, and the effective components of the composition are unclear, which cannot provide a basis for further in-depth research on the efficacy of snow lotus extract on the skin.

[0005] For example, CN202111406538.9 discloses a composition containing Tianshan Saussurea cell culture, its preparation method, and application. The raw materials of the composition include: Tianshan Saussurea cell culture and Schisandra chinensis, with a mass ratio of 1:3 to 4:1; the preparation method of the composition includes: S1, mixing Tianshan Saussurea cell culture, Schisandra chinensis, and 1,3-butanediol, and extracting them under hot reflux; S2, fine filtration, and compounding with resveratrol to obtain the composition; the above composition has application in the preparation of anti-inflammatory and / or whitening products. The process of obtaining Tianshan Saussurea cell culture is cumbersome, the production process is complex, and the production requirements are high, making it unsuitable for large-scale industrial production.

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

[0007] In response to the above technical problems, the present application provides a sesquiterpene lactone 8-hydroxy-11,13-dihydrodehydrocostus lactone from Tianshan Snow Lotus for whitening and / or anti-aging, and its application. The substance is safe and non-toxic, can effectively improve the oxidative stress of HepG2 cells and exert an antioxidant effect.

[0008] The present application provides an 8-hydroxy-11-hydroxy-2-nitropropionic acid obtained from Tianshan Snow Lotus for whitening and / or anti-aging.

[0009] ,13-Dihydrodehydrocostus lactone sesquiterpene lactone, the structural formula is as follows:

[0010]

[0011] Preferably, the preparation method comprises the following steps: extracting the raw materials and separating them by silica gel column chromatography;

[0012] The raw material is Tianshan Snow Lotus or Centaurea canariensis of the Asteraceae family.

[0013] Preferably, when using Saussurea involucrata as raw material, the Saussurea involucrata is subjected to alcohol extraction before extraction, and the alcohol extract is suspended in water; the extraction agent is a mixed solution of petroleum ether and ethyl acetate;

[0014] Preferably, when the Asteraceae plant Centaurea canariensis is used as the raw material, the extraction solvent is diethyl ether and petroleum ether in a volume ratio of 1:2.

[0015] The natural compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone can be extracted and separated from the plant Saussurea involucrata by various existing methods. For example, Saussurea involucrata is used as a raw material and subjected to 95% ethanol hot reflux extraction, the ethanol extract is suspended in water, and extracted with petroleum ether and ethyl acetate respectively, and the ethyl acetate fraction is separated by silica gel column chromatography to obtain the compound.

[0016] The compound can also be isolated from Centaurea canariensis, a plant of the Asteraceae family found at high altitudes in Chile. This is achieved through extraction with a mixed solvent of ether and petroleum ether (1:2 by volume), followed by repeated silica gel column chromatography and preparative thin-layer chromatography. This substance belongs to the sesquiterpene lactone class.

[0017] This compound reduces the ROS level in HepG2 cells by activating the Nrf2 / Keap1 signaling pathway, enhances the activity of antioxidant enzymes CAT, SOD and GSH, reduces MDA content, and improves oxidative stress. It can be used as an antioxidant drug or cosmetic.

[0018] Preferably, the 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone has no cytotoxicity to HepG2 cells when the concentration is less than or equal to 50 μM.

[0019] Preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is greater than or equal to 12.5 μM, it can reduce the ROS content in cells after H2O2 stimulation. More preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is between 12.5 and 50 μM, it can reduce the ROS content in cells after H2O2 stimulation. This also includes when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is 25 μM.

[0020] Preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is greater than or equal to 12.5 μM, the ROS content in HepG2 cells induced by H2O2 is reduced;

[0021] Preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is 12.5 to 50 μM, the ROS content in HepG2 cells induced by H2O2 is reduced.

[0022] Preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is greater than 6.25 μM, it can reverse the down-regulation of CAT and GSH in HepG2 cells induced by H2O2; more preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is 6.25-25 μM, it can reverse the down-regulation of CAT and GSH in HepG2 cells induced by H2O2;

[0023] Preferably, 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can reverse the downregulation of SOD in HepG2 cells induced by H2O2 when the concentration is greater than 12.5 μM;

[0024] Preferably, 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can reverse the downregulation of SOD in HepG2 cells induced by H2O2 when the concentration is 12.5 to 25 μM;

[0025] Preferably, 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can reverse the upregulation of MDA in HepG2 cells induced by H2O2 when the concentration is greater than 6.25 μM;

[0026] Preferably, when the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is 6.25-25 μM, it can reverse the upregulation of MDA in HepG2 cells induced by H 2 O 2 .

[0027] Preferably, 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can reverse the downregulation of Nrf2 and Keap1 mRNA and improve cellular oxidative damage;

[0028] Preferably, 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can activate the Nrf2 / Keap1 signaling pathway for antioxidant protection.

[0029] Another aspect of the present application provides an anti-aging composition, comprising at least: the above-mentioned 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone.

[0030] Preferably, the composition includes: cosmetic excipients or pharmaceutical excipients.

[0031] The added excipients can be selected from the common excipients in the field according to the intended use of the composition. For example, the composition can be prepared as a skin care cream, essence, isolation cream, isolation milk, BB cream, foundation or toner; when preparing corresponding types of cosmetics, corresponding excipients can be selected.

[0032] The added excipients can be selected from the common excipients in the art according to the pharmaceutical preparation to be prepared, for example, the combination can be made into tablets, granules, pills, capsules or injections.

[0033] Another aspect of the present application further provides an antioxidant composition, comprising at least: the above-mentioned 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone.

[0034] Preferably, the composition includes: cosmetic excipients or pharmaceutical excipients.

[0035] The added excipients can be selected from the common excipients in the field according to the intended use of the composition. For example, the composition can be prepared as a skin care cream, essence, isolation cream, isolation milk, BB cream, foundation or toner; when preparing corresponding types of cosmetics, corresponding excipients can be selected.

[0036] For example, the combination can be made into tablets, granules, pills, capsules or injections.

[0037] The beneficial effects of this application include:

[0038] 1) 8-Hydroxy-11-hydroxy-11-hydroxy-2-nitropropionic acid for whitening and / or anti-aging in the Tianshan Saussurea involucrata provided in the present application

[0039] ,13-Dihydrodehydrocostus lactone sesquiterpene lactone, application, separation and purification of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone from Tianshan Snow Lotus and study of its pharmacological effects are of great significance for the development and utilization of Tianshan Snow Lotus resources in the fields of anti-aging and whitening cosmetics or antioxidant drugs.

[0040] 2) 8-Hydroxy-11-hydroxy-11-hydroxy-2-nitropropionic acid for whitening and / or anti-aging in the Saussurea involucrata provided in this application

[0041] 1,3-Dihydrodehydrocostus lactone is a sesquiterpene lactone. This compound can reduce ROS levels in HepG2 cells, increase the activities of the antioxidant enzymes CAT, SOD, and GSH, reduce MDA content, and improve oxidative stress in HepG2 cells. This compound can be used to prepare antioxidant and anti-aging compositions, and this substance can be used to prepare compositions that improve oxidative stress in HepG2 cells. This substance has anti-aging properties and can effectively inhibit the formation of pigmentation, thereby achieving whitening and antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a graph showing the results of the CCK-8 assay for the toxic effects of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on HepG2 cells at concentrations of 10, 20, 30, 40, and 50 μM in the examples of this application;

[0043] Figure 2 This is a graph showing the results of flow cytometry detection of the effects of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 12.5, 25, and 50 μM on the ROS content of HepG2 cells damaged by oxidative damage; Figure 2 (A), the horizontal axis represents the fluorescence intensity, and the vertical axis represents the number of collected cells. Figure 2 A shows the results of the blank group, the hydrogen peroxide-induced model group (H2O2), the positive control group (Vc), and the treatment groups with 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 50, 25, and 12.5 μM. Figure 2 (B) Yes Figure 2 (A) Quantification results. Compared with the blank control group, P ### <0.001; compared with the model, P***<0.001, P**<0.01.

[0044] Figure 3 This is a graph showing the results of antioxidant enzyme activity detection in the examples of this application, wherein Figure 3 (A) Effect of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on CAT activity; Figure 3 (B) Effect of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on SOD activity; Figure 3 (C) shows the effect of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on GSH activity; Figure 3(D) Effects of the compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on MDA activity; the blank group is the normal culture group of HepG2 cells, H2O2 is the hydrogen peroxide-induced model group, and the horizontal axes 50, 25, and 12.5 represent the treatment groups with different concentrations of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone; Figure 3 The "-" in the horizontal axis indicates that the corresponding substance of the end head is not added, and "+" indicates that the substance of the end head is added; C584 is 8α-hydroxy-11β,13-dihydrodehydrocostus lactone; the data are expressed as average values

[0045] ±SD, compared with blank, P ### <0.001; compared with the model, P***<0.001, P**<0.01.

[0046] Figure 4 This is a graph showing the effect of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the mRNA expression of Nrf2 and Keap1 at the transcriptional level using PCR in the examples of this application. Figure 4 (A) Effects of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the mRNA expression of Nrf2; Figure 4 (B) shows the effect of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the mRNA expression of Keap1; the groups and references corresponding to the horizontal axis are as follows: Figure 3 ; Data are expressed as mean ± SD, compared with blank, P ### <0.001; compared with the model, P***<0.001, P**<0.01.

[0047] Figure 5 This is the result of Western blotting to detect the activation of Nrf2 / Keap1 signaling pathway by compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone to improve oxidative damage in HepG2 cells in the examples of this application. Figure 5 (A) Effects of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the expression of key proteins in the Nrf2 / Keap1 signaling pathway; Figure 5 (B) Effects of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on Nrf2 protein expression; Figure 5 (C) Effects of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on Keap1 protein expression; grouping and designation are as follows: Figure 3 ; Data are expressed as mean ± SD, compared with blank, P ### <0.001; compared with the model, P***<0.001, P**<0.01. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below with reference to 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 scope of protection of the present invention.

[0049] Example

[0050] 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.

[0051] Example 1 Preparation of Sesquiterpene Lactone Compound (8α-Hydroxy-11β,13-Dihydrodehydrocostus lactone)

[0052] The substance is prepared by referring to the method disclosed in the existing literature (Sesquiterpene lactones from Saussureainvolucrata, Fitoterapia 82 (2011) 983–987), using Saussurea involucrata as a raw material. The Saussurea involucrata is crushed and then immersed in 95% ethanol. The mixture is extracted three times with 95% ethanol under hot reflux. The combined extracts are concentrated, and the extracts are suspended in water. The mixture is extracted with petroleum ether and ethyl acetate, respectively. The ethyl acetate fraction obtained by the extraction is subjected to silica gel column chromatography and eluted with dichloromethane:methanol (50:1) to obtain 8α-hydroxy-11β,13-dihydrodehydrocostus lactone. The yield of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone is 0.0016%.

[0053] Example 2 Sesquiterpene lactone compound (8α-hydroxy-11β,13-dihydrodehydrocostus lactone) prevents oxidative stress in HepG2 cells

[0054] (1) Experimental cells

[0055] HepG2 cells were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China).

[0056] (2) Instruments and reagents

[0057] Full wavelength microplate reader (Thermo Fisher);

[0058] CO2 incubator (Thermo Fisher);

[0059] Centrifugal sedimentation device (Shanghai Surgical Instrument Factory);

[0060] Micro desktop refrigerated centrifuge (D-37520TherMo);

[0061] flow cytometer (Becton Dickinson and Company);

[0062] Nanodrop 20000 ultramicro-volume spectrophotometer (Thermo Fisher);

[0063] QuantStudio 3and 5Real Time PCR (Thermo Fisher);

[0064] Electrophoresis apparatus (EPS600 Tanon);

[0065] Transfer tank (Mini-PROTEAN Tetra SysteM, BIO-RAD);

[0066] Multicolor fluorescence, chemiluminescence, and visible light imager (ProteinsiMple FluorcheM Q);

[0067] Fetal bovine serum (Pnocet);

[0068] DMEM medium (Pnosel);

[0069] Penicillin-streptomycin mixture (Beijing Solebow Technology Co., Ltd.);

[0070] Trypsin digestion solution (Shanghai Biyuntian Biotechnology Co., Ltd.);

[0071] CAT Hydrogen Peroxide Test Kit (Nanjing Jiancheng Biotechnology Co., Ltd.)

[0072] SOD superoxide dismutase test kit (Nanjing Jiancheng Biotechnology Co., Ltd.)

[0073] GSH reduced glutathione test kit (Nanjing Jiancheng Biotechnology Co., Ltd.)

[0074] MDA malondialdehyde test kit (Nanjing Jiancheng Biotechnology Co., Ltd.)

[0075] DCFH-DA (Shanghai Biyuntian Biotechnology Co., Ltd.)

[0076] Ascorbic acid (Aladdin)

[0077] Cell lysis buffer RIPA (strong) (Shanghai Biotech Co., Ltd.);

[0078] Trizol reagent (Thermo Fisher);

[0079] PMSF (protease inhibitor, Beijing Solebow Technology Co., Ltd.);

[0080] protein phosphatase inhibitors (Beijing Solaibao Technology Co., Ltd.);

[0081] BCA protein concentration determination kit (Beijing Solebow Technology Co., Ltd.);

[0082] 5× protein loading buffer (Beijing Solebow Technology Co., Ltd.);

[0083] Rainbow 180 spectral protein marker (Beijing Solebow Technology Co., Ltd.);

[0084] PVDF membrane (Immobilon-P SQ Transfer Membrane.Merck Millpore Ltd.,pore size:0.2μM);

[0085] ECL Plus ultrasensitive luminescent liquid (Beijing Solebow Technology Co., Ltd.);

[0086] Tris-base, tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), glycine (Beijing Solaibao Technology Co., Ltd.)

[0087] TBST (Wuhan Saiweier Biotechnology Co., Ltd.);

[0088] 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone: prepared from Saussurea involucrata. For the specific preparation method, see Example 1.

[0089] The primers used are as follows:

[0090] Table 1 Primer sequences

[0091]

[0092] The sources of the antibodies used in Table 1 are as follows:

[0093] primary antibody

[0094] β-Actin Mouse MAb Antibody (Proteintech, 66009);

[0095] NRF2,NFE2L2 Polyclonal antibody(Proteintech, 16396-1-AP)

[0096] KEAP1 Polyclonal antibody (Proteintech, 10503-2-AP);

[0097] Secondary Antibodies

[0098] HRP (horseradish peroxidase)-labeled rabbit anti-goat IgG (Wuhan Saiweier Biotechnology Co., Ltd.);

[0099] HRP-labeled goat anti-mouse IgG (Wuhan Saiweier Biotechnology Co., Ltd.)

[0100] (3) Experimental methods

[0101] 1) CCK-8 assay for cell viability

[0102] HepG2 cells were cultured in complete DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 79-83% confluency. The supernatant was carefully discarded and the cells were washed two to three times with 2 mL of PBS buffer.

[0103] Subsequently, 1 mL of trypsin digestion solution was added for 2 minutes, and then 2 mL of DMEM complete medium was added to stop the digestion. The cells were completely pipetted off, collected into a centrifuge tube, and placed in a centrifuge at 1050 rpm for 5 minutes.

[0104] The supernatant was discarded, 1 mL of DMEM complete medium was added, and the cell suspension was formed by gently pipetting.

[0105] Finally, 300 μL of cell suspension was pipetted and added into sterilized clean culture flasks, and 3 mL of DMEM complete medium was added to each culture flask.

[0106] HepG2 cells in logarithmic growth phase were cultured at a density of 2×10 5 Cells were inoculated with 100 μg / mL of culture medium in a 96-well plate and incubated in a constant temperature incubator for 24 hours. After 24 hours of incubation, the original culture medium was filtered and replaced with fresh DMEM medium for the blank group and with pre-prepared DMEM medium containing 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 10, 20, 30, 40, and 50 μmol / L for the drug-treated group. After 24 hours of incubation in the incubator, 10 μL of CCK-8 solution (protected from light) was added to each well. The cells were incubated in a 37°C oven for 40 minutes. After mixing using a microplate reader, the OD values ​​of the solutions obtained from the blank and drug-treated groups were measured at a wavelength of 450 nm. The blank and drug-treated experiments were repeated three times for each well, and the results were analyzed.

[0107] 2) Flow cytometry detection of ROS content in oxidatively stressed HepG2 cells

[0108] HepG2 cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. When the cell confluence reached 79-83%, 2.5×10 5The cells were carefully and evenly seeded in a 6-well plate at a density of 100 cells / mL and placed in a 37°C, 5% CO2 incubator for culture. After about 24 hours, the cells adhered to the wall. The blank control group ( Figure 2 B is control), model group ( Figure 2 The horizontal axis in B is H2O2). The old culture medium was discarded and new DMEM complete culture medium was added to the positive drug-treated group; the DMEM complete culture medium containing 50 μM ascorbic acid (Vc) was replaced in the positive drug-treated group; the old culture medium was discarded in the compound-treated group, and DMEM complete culture medium with 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 12.5, 25, and 50 μM was added and incubated for 24 hours.

[0109] After 24 h, the blank control group discarded the original culture medium and added new DMEM culture medium, while the model group, positive drug group and compound treatment group replaced it with DMEM culture medium containing 500 μM H2O2 (protected from light).

[0110] After 4 hours of induction using the above method, the supernatant was discarded and 2 mL of cell culture medium containing the DCFH-DA probe (final concentration of 10 μM) was added for incubation in the dark for 20-30 minutes. After incubation, the supernatant was discarded, and the cells were rinsed twice with PBS buffer. 0.5 mL of trypsin was added to each well for 2 minutes, and the cells were collected by pipetting with PBS.

[0111] The cell suspension was collected into a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 min to obtain a precipitate. The cells were then resuspended in 0.5 mL of PBS buffer and passed through a 300-mesh nylon sieve. The ROS (reactive oxygen species) content in the cells was detected by flow cytometry.

[0112] 3) The effects of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the antioxidant capacity of HepG2 cells induced by H2O2 were detected by CAT, SOD, GSH and MAD kits.

[0113] Cell culture, group setting, and treatment methods were the same as those in Experiment 2). Four hours after modeling, cells were harvested and centrifuged to obtain a cell pellet. 200 μL of cell lysis buffer (prepared at a 100:1:1 volume ratio of RIPA strong lysis buffer: phosphatase inhibitor: PMSF, prepared immediately before use) was added to the cell pellet and lysed on ice for 30 min. Disrupted cell samples were obtained and used to measure protein concentration and enzyme activity. Specific assay procedures are described in the instructions for the CAT, SOD, GSH, and MAD kits.

[0114] 4) PCR detection of the effect of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone-related oxidative factor mRNA expression

[0115] 4.1 RNA extraction

[0116] Cell culture, group assignment, and processing methods were the same as above. Cells were harvested 4 h after modeling. Cell culture medium was discarded, and the cells were rinsed twice with PBS buffer and discarded. 1 mL of Trizol reagent was added to thoroughly cover the cell surface. Cells were then repeatedly pipetted to dislodge. The lysate was transferred to a 1.5 mL enzyme-free centrifuge tube and pipetted repeatedly until fully lysed. The tube was allowed to stand at room temperature for 5 min. 0.2 mL of chloroform was added, the tube was tightly capped, shaken vigorously for 15 s, and incubated at room temperature for 2-3 min. The mixture was centrifuged at 12,000 rpm for 15 min at 4°C. The mixture separated into three phases. 0.5 mL of the upper aqueous phase was carefully transferred to a new 1.5 mL enzyme-free centrifuge tube. 0.5 mL of isopropanol was added to precipitate RNA. The mixture was mixed by inversion and incubated on ice for 10 min. The RNA precipitate was collected by centrifugation at 12,000 rpm for 10 min. The supernatant was discarded, and any remaining liquid was completely removed with a pipette. Wash the RNA pellet once with 75% ethanol. Add 1 mL of 75% ethanol, vortex to mix the sample, and then centrifuge at 7500 rpm for 5 minutes at 4°C. Repeat the wash. Completely remove the ethanol and air-dry the RNA pellet for 10 minutes.

[0117] 4.2 Reverse transcription reaction

[0118] ① RNA volume: The required RNA mass is 1000 ng, X (RNA volume) = 1000 ng / RNA concentration (ng / μL), calculate the RNA volume.

[0119] ② Synthesize cDNA: Prepare cDNA using gDNA-depleted RNA as a template. The gDNA-depleted reaction system is shown in Table 2. After thorough mixing, reverse transcription is performed according to the reaction conditions. The reverse transcription conditions are shown in Table 3. Note: X (RNA volume) = 1000 ng / RNA concentration (ng / μL).

[0120] Table 2 gDNA removal reaction

[0121]

[0122]

[0123] Table 3 Reverse transcription reaction system

[0124]

[0125] 4.3 Polymerase chain reaction

[0126] Table 4 PCR amplification system

[0127]

[0128] ③Except for the cDNA solution, all other reagents were prepared and mixed in advance, then transferred to an 8-tube strip, and finally the cDNA solution was added. Amplification was performed using the QuantStudio 3 Real Time PCR System. -△△Ct The relative expression levels were calculated using the β-actin method, with β-actin as the internal reference.

[0129] 5) Western blot analysis of the effect of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the expression levels of related proteins in cells

[0130] 5.1 Extraction of total protein from cells

[0131] Cells were seeded in 6-well plates according to the method described in "1) Determination of Cell Viability by CCK-8 Assay." Blank control, model, and compound-treated groups were set up and incubated for another 48 hours. The supernatant was discarded and the cells were gently washed twice with PBS. 1 mL of PBS was added to each well. The cells were completely dislodged using a pipette and transferred to a 1.5 mL EP tube. The cells were collected by centrifugation to obtain a cell pellet. Cell lysis mixture (prepared at a volume ratio of RIPA strong lysis buffer: phosphatase inhibitor: PMSF of 100:1:1, prepared immediately before use) was added to the cell pellet and lysed on ice for 30 minutes.

[0132] Centrifuge the lysate at 3500 rpm for 10 minutes at 4°C. Aspirate the supernatant and record the volume. Add protein loading buffer to the supernatant at a volume ratio of 1:4. Heat in a metal bath at 100°C for 10 minutes to denature the protein. Cool to room temperature and store in a -20°C refrigerator.

[0133] 5.2 Determination of protein concentration

[0134] The protein concentration in the sample solution was determined according to the BCA protein concentration assay kit instructions. The absorbance of the sample solution at a wavelength of 562 nm was measured using a microplate reader. A standard curve was constructed according to the instructions and the protein concentration in the sample was calculated based on the obtained curve.

[0135] 5.3 Glue making

[0136] Prepare 10% separation gel and 5% stacking gel according to Table 1

[0137] Table 1 Separation gel and concentration gel ratio system

[0138]

[0139] 5.4 Electrophoresis

[0140] Add marker and protein sample, transfer the gel to the electrophoresis tank after loading, adjust the voltage to 80V, run for 30 minutes, then adjust the voltage to 120V, continue running until the gap between the marker bands near the target protein is separated, and stop electrophoresis.

[0141] 5.5 Transfer

[0142] Cut the gel containing the target protein, compare the size of the gel, cut the PVDF membrane and let it stand in methanol for activation for 2-3 minutes. Transfer the gel to the PVDF membrane and place it in the transfer tank for transfer. Set the voltage to 72V for 60 minutes. During the operation, use 1× transfer buffer (first prepare 10× transfer buffer: weigh 30.3g Tris-base, 142.6g glycine, dissolve it in ultrapure water and dilute to 1L. 1× transfer buffer preparation: add 350mL ultrapure water and 100mL methanol to 50mL 10× transfer buffer in sequence, mix thoroughly) to keep it moist, avoid dehydration of the gel, and avoid the formation of bubbles.

[0143] 5.6 Closure

[0144] After the transfer is completed, the PVDF membrane is removed, marked, placed in 5% skim milk powder, and gently shaken on a shaker at room temperature for 2 hours to block.

[0145] 5.7 Primary Antibody Incubation

[0146] After blocking, rinse the PVDF membrane with skim milk powder in TBST. Cut the target bands according to the Maker's molecular weight instructions and mark them. Incubate the bands in the corresponding primary antibody on a shaker at 4°C overnight. After incubation, wash the membrane five times with TBST, 5 minutes each time.

[0147] 5.8 Secondary Antibody Incubation

[0148] Remove the washed strips, place in the corresponding secondary antibody, and incubate at room temperature for 2 hours. Wash the membrane 5 times, 5 minutes each time.

[0149] 5.9 Development

[0150] ECL Plus ultrasensitive luminescent solution was evenly applied to the strips and developed using a developer. β-actin was used as an internal reference, and the grayscale values ​​of the strips were quantitatively analyzed using Image J software.

[0151] 5.10 Data Analysis

[0152] One-way ANOVA was performed using SPSS 19.0 software, and the experimental results are expressed as mean ± standard deviation. All bar graphs were analyzed using GraphPad Prism 7.0 software, and P < 0.05 was considered statistically significant.

[0153] (6) Analysis and discussion of experimental results

[0154] 6.1 The CCK-8 assay was used to detect the cytotoxic effects of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 10, 20, 30, 40, and 50 μmol / L on HepG2 cells (see “1) Determination of cell viability by CCK-8 assay” for the experimental procedure). The results were as follows: Figure 1 As shown in the figure: compared with the blank group, 8 α 8α-hydroxy-11β,13-dihydrodehydrocostus lactone had no cytotoxicity to HepG2 cells in the concentration range of 10 to 50 μM, so a concentration less than 50 μM was selected for activity screening of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone.

[0155] 6.2 Experiment 2) Flow cytometry detection of ROS content in HepG2 cells under oxidative stress A cell oxidative stress model was established using H2O2, and flow cytometry was used to detect the ROS content in HepG2 cells. The results are as follows: Figure 2 As shown in the results of the treatment groups in (A), it can be seen that compared with the blank group, after H2O2 stimulation, flow cytometry detection found that the peak of the model group after H2O2 stimulation was significantly shifted to the right, and the intracellular fluorescence intensity was enhanced, indicating that the intracellular ROS content increased at this time. After the administration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 12.5, 25, and 50 μM, the peak shape was significantly shifted to the left, indicating that the fluorescence intensity was weakened, indicating that the intracellular ROS content decreased at this time.

[0156] Figure 2 As shown in (B), the blank control group did not contain H2O2, so the ROS content was low. 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 50, 25, and 12.5 μM all reduced the ROS content in HepG2 cells to varying degrees, and significantly improved the oxidative stress in HepG2 cells at concentrations of 25 and 12.5 μM (P < 0.001). The inhibitory effect of 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone on H2O2-induced ROS production in HepG2 cells at concentrations of 25 and 12.5 μM was lower than that of VC, indicating that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone can inhibit H2O2-induced ROS production in HepG2 cells, reduce intracellular oxidative stress, and prevent cell damage.

[0157] 6.3 Figure 3 The results of experiment "3) Detecting the effect of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the antioxidant capacity of HepG2 cells induced by H2O2 using CAT, SOD, GSH and MAD kits" are as follows: Figure 3 As shown in A to D, compared with the blank control group (yellow column, i.e., neither H2O2 nor C584 was added), the model group (blue data column, i.e., the treatment group with only H2O2 added) showed a significant downregulation of CAT, SOD, and GSH activity (P<0.001), while a significant upregulation of MDA activity (P<0.001). 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone at concentrations of 25, 12.5, and 6.25 μM significantly reversed the downregulation of CAT and GSH induced by H2O2 (P<0.001).

[0158] At 25 and 12.5 μM, it could significantly reverse the downregulation of SOD caused by H2O2 (P<0.001);

[0159] At 25, 12.5 and 6.25 μM, it was able to significantly reverse the upregulation of MDA caused by H2O2 (P<0.001).

[0160] This indicates that the addition of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone can enhance the activity of antioxidant enzymes CAT, SOD and GSH, reduce MDA activity, reduce intracellular oxidative stress response and avoid cell damage.

[0161] The antioxidant mechanism of this compound is expected to be further explored.

[0162] These studies demonstrate that 8α-hydroxy-11β,13-dihydrodehydrocostus lactone has the potential to reduce ROS levels in oxidatively damaged HepG2 cells, increase CAT, SOD, and GSH activities, and reduce MDA activity. Therefore, to further clarify the mechanism by which 8α-hydroxy-11β,13-dihydrodehydrocostus lactone improves oxidative damage, PCR and Western blot were used to examine its effects on signaling pathways in HepG2 cells.

[0163] The results of experiment 6.4 “PCR detection of the mRNA expression of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone-related oxidative factors” are shown in Figure 4 , Figure 4 The PCR experiment was used to detect the effect of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the mRNA expression of Nrf2 and Keap1 at the transcriptional level. The results are shown in Figure 2. Figure 4As shown in A and B, compared with the blank control group (yellow column, i.e., neither H2O2 nor C584 was added), the model group (blue data column, i.e., the group treated with only H2O2) showed significantly downregulated mRNA expression of Nrf2 and Keap1 (P<0.001), while the 8α-hydroxy-11β,13-dihydrodehydrocostus lactone treatment group was able to significantly reverse the downregulation of Nrf2 and Keap1 mRNA induced by H2O2 (P<0.001). This suggests that 8α-hydroxy-11β,13-dihydrodehydrocostus lactone may improve oxidative damage in HepG2 cells through the Nrf2 / Keap1 signaling pathway.

[0164] The results of experiment 6.5 "Western blot analysis of the effect of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the expression level of related proteins in cells" are as follows Figure 5 As shown in A to C, the experiment used H2O2 to treat cells, causing oxidative stress to the cells, blocking the Nrf2 / Keap1 signaling pathway, and the protein levels of related signaling pathways were as follows: Figure 5 See Figure 5 A shows that 8α-hydroxy-11β,13-dihydrodehydrocostus lactone has no effect on the expression of β-actin, but has an effect on the expression of Keap1 and a significant effect on the expression of Nrf2; Figure 5 (B) and (C) show the effects of compound 8α-hydroxy-11β,13-dihydrodehydrocostus lactone on the expression of Nrf2 and Keap1 proteins, respectively. Figure 5 (B) Treatment of HepG2 cells with 8α-hydroxy-11β,13-dihydrodehydrocostus lactone at concentrations of 25 and 12.5 μM increased the expression level of Keap1 protein in the cells.

[0165] The results of this study suggest that 8α-hydroxy-11β,13-dihydrodehydrocostus lactone may exert its antioxidant effects by activating the Nrf2 / Keap1 signaling pathway, suggesting that 8α-hydroxy-11β,13-dihydrodehydrocostus lactone may be used in the development of antioxidant cosmetics or drugs.

[0166] Example 2 Toner containing 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone

[0167] 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone was added to sterile water to a concentration of 6.25 μM and mixed thoroughly to obtain a toner. The solubility of 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone in water is 1 mg in 200 μL of water, and the toner was prepared according to this ratio.

[0168] Example 3 Injection containing 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone

[0169] 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone was added to sterile water to a concentration of 6.25 μM and mixed thoroughly to obtain an injection. The solubility of 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone in water is 1 mg per 200 μL of water, and this ratio is used for preparation.

[0170] 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 scope of protection of the present invention.

Claims

1. The use of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone in Tianshan Saussurea involucrata in the preparation of antioxidant, whitening and / or anti-aging preparations, characterized in that: The structural formula is as follows:

2. The use according to claim 1, characterized in that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone had no cytotoxicity to HepG2 cells when the concentration was less than or equal to 50 μM.

3. The use according to claim 1, characterized in that When the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is greater than or equal to 12.5μM, it can reduce the ROS content in cells after H2O2 stimulation.

4. The use according to claim 1, characterized in that When the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone is 12.5-50 μmol / L, it can reduce the ROS content in cells after H2O2 stimulation.

5. The use according to claim 1, characterized in that When the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone was greater than or equal to 12.5 μM, it reduced the ROS content in HepG2 cells induced by H2O2.

6. The use according to claim 1, characterized in that 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone reduced the ROS content in H2O2-induced HepG2 cells at a concentration of 12.5-50 μM.

7. The use according to claim 1, characterized in that When the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone is greater than 6.25μM, it can reverse the downregulation of CAT and GSH in HepG2 cells induced by H2O2.

8. The use according to claim 1, characterized in that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone concentrations of 6.25 to 25 μM can reverse the downregulation of CAT and GSH in HepG2 cells induced by H2O2.

9. The use according to claim 1, characterized in that When the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone is greater than 12.5μM, it can reverse the downregulation of SOD in HepG2 cells induced by H2O2.

10. The use according to claim 1, characterized in that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone concentrations of 12.5 to 25 μM can reverse the downregulation of SOD in HepG2 cells induced by H2O2.

11. The use according to claim 1, characterized in that When the concentration of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone is greater than 6.25 μM, it can reverse the upregulation of MDA in HepG2 cells induced by H2O2.

12. The use according to claim 1, characterized in that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone concentrations of 6.25-25 μM can reverse the upregulation of MDA in HepG2 cells induced by H2O2.

13. The use according to claim 1, characterized in that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can reverse the downregulation of Nrf2 and Keap1 mRNA and improve cellular oxidative damage.

14. The use according to claim 1, characterized in that 8α-Hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone can activate the Nrf2 / Keap1 signaling pathway for antioxidant protection.

15. The use according to claim 1, characterized in that The preparation method of 8α-hydroxy-11β,13-dihydrodehydrocostus lactone sesquiterpene lactone comprises the following steps: The raw materials are obtained by extraction and silica gel column chromatography; The raw material is Tianshan Snow Lotus or Centaurea canariensis of the Asteraceae family.

16. The use according to claim 15, characterized in that When the Tianshan Saussurea involucrata is used as the raw material, the Tianshan Saussurea involucrata is subjected to alcohol extraction before extraction, and the alcohol extract is suspended in water; the extraction solvent is a mixed solution of petroleum ether and ethyl acetate.

17. The use according to claim 15, characterized in that When the Asteraceae plant Centaurea canariensis is used as the raw material, the extraction solvent is ether and petroleum ether in a volume ratio of 1:2.

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