Application of sulforaphane in preparation of product for preventing or treating oxidative stress or inflammatory response induced diseases
Sulforaphane-based pharmaceuticals and health foods target oxidative stress and inflammatory diseases by inhibiting relevant biomarkers, offering a therapeutic solution for conditions like allergy and autoimmune diseases.
Patent Information
- Application Number
- CN202411276147.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-12
- Publication Date
- 2024-12-13
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing technologies have not effectively addressed the preventive or therapeutic effects of sulforaphane on diseases induced by oxidative stress or inflammatory response.
Development of pharmaceutical compositions and health foods containing sulforaphane as an active ingredient to target specific biomolecules associated with oxidative stress and inflammatory diseases, including conditions such as allergy, Alzheimer's disease, and autoimmune diseases.
Sulforaphane effectively inhibits the expression of inflammatory markers and oxidative stress-related proteins, providing a potential therapeutic benefit for a range of inflammatory and oxidative stress-induced diseases.
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Figure CN119112861A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medicine, and in particular to the use of sulforaphane in the preparation of medicines or health foods for preventing or treating diseases induced by oxidative stress or inflammatory response. Background Art
[0002] Sulforaphane (SFN) is a natural plant compound found in many cruciferous vegetables, such as broccoli, cabbage, cauliflower and kale. More than 120 glucosinolates have been found in plants, with the highest content in cruciferous crops, which are distributed in all parts of the plant, especially in seeds. The content of glucosinolates in plants is affected by the external growth environment, so the content of sulforaphane is also affected by external environmental conditions. The content of glucosinolates in different plant species is also different. Generally, the content of glucosinolates in Brassica vegetables is 500-2000 μg·g-1, among which broccoli has the highest content, especially in seeds and seedlings, where the content of glucosinolates is 10-100 times that of mature plants. Therefore, broccoli is an ideal raw material for extracting sulforaphane.
[0003] The edible parts of 9 cruciferous vegetables were dried at 50℃, 70℃ and 90℃ under normal pressure, and the sulforaphane content was determined by HPLC. The results showed that the sulforaphane content of the 9 cruciferous vegetables was Brassica>Raphanus; the sulforaphane content of broccoli was the highest in Brassica, which was 480μg.g-1, and the lowest in cauliflower, which was 35.2μg.g-1; the sulforaphane content of carrots was the highest in Raphanus, which was 80.9μg.g-1, and the lowest in white radish, which was 19.8μg.g-1; and in the treatment of the same sample at these three different drying temperatures, the sulforaphane content in the sample dried at 90℃ was the highest. Sulforaphane not only has strong anti-cancer activity, but also has strong antioxidant capacity. It is recognized as one of the natural products with anti-cancer, anti-cancer and cosmetic effects. As early as the beginning of the 20th century, researchers studied the anti-cancer and anti-cancer effects of sulforaphane and found that sulforaphane has an inhibitory effect on liver cancer, colon cancer, breast cancer, prostate cancer, etc., induces Phase II enzymes, activates the production of anti-cancer substances, and kills white blood cells; in addition, sulforaphane can adjust the outermost layer of the human immune system - the skin, and enhance human immunity. Experiments on humans and mice by American researchers have shown that sulforaphane helps the lungs clear harmful bacteria.
[0004] The process of removing free radicals in the body is a process of "redox reaction". Oxidative stress refers to the imbalance between oxidation and antioxidant effects in the body, which tends to oxidize. At this time, the oxidative tension in the cells increases (free radicals increase). The degree of oxidation caused by free radicals to the cells will exceed the cells' ability to remove "free radicals". The redox balance system is broken (imbalanced), resulting in cell damage (oxidative damage). This process is oxidative stress. Oxidative stress destroys the balance between oxidants and antioxidants. Excessive oxidative stress is the main cause of cell damage.
[0005] Whether sulforaphane has a preventive or therapeutic effect on diseases induced by oxidative stress or inflammatory response has not been reported in the literature. Summary of the invention
[0006] In order to address the deficiencies of the prior art, one of the objectives of the present invention is to provide a use of sulforaphane in the preparation of a product for preventing or treating diseases induced by oxidative stress or inflammatory response.
[0007] As a specific embodiment, the oxidative stress or inflammatory response-induced disease exhibits an altered level of one or more biomolecules selected from cluster of differentiation 40 ligand (CD-40L), eotaxin, fibrinogen, growth hormone (GH), keratinocyte-derived cytokine or GRO1 oncogene (KC / GRO), interleukin-1β (IL-1β), IL-6, IL-18, lymphocyte chemotactic factor, myeloperoxidase (MPO), tissue inhibitor of metalloproteinase 1 (TIMP-1), vascular endothelial growth factor A (VEGF-A), C-reactive protein (CRP), macrophage-derived chemokine (MDC), macrophage inflammatory protein-1α (MIP-1α), vWF or oncostatin.
[0008] As a specific embodiment, the inflammatory disease is allergy, Alzheimer's disease, ankylosing spondylitis, asthma, autoimmune disease, arthritis, atherosclerosis, carpal tunnel syndrome, celiac, Crohn's disease, diverticulitis, eczema, fibrosis, Guillain-Barre disease, lupus, multiple sclerosis, nephritis, neuropathy, pancreatitis, Parkinson's disease, psoriasis, polymyalgia rheumatica, rheumatoid arthritis, scleroderma or vasculitis.
[0009] A second object of the present invention is to provide a pharmaceutical composition for achieving the above application, wherein the active ingredient includes sulforaphane.
[0010] A third object of the present invention is to provide a medicament for achieving the above application, characterized in that it comprises sulforaphane and pharmaceutically acceptable excipients.
[0011] As a specific embodiment, the medicament is a capsule, tablet, powder, granule, injection, oral liquid, wine, pill, mixture or tincture.
[0012] A fourth object of the present invention is to provide a health food comprising sulforaphane for realizing the above application. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 The production of ROS in RAW264.7 macrophages infected with Mycobacterium tuberculosis was quantified using a reactive oxygen species assay kit under a fluorescence microscope; green indicates ROS, and red indicates Mycobacterium tuberculosis;
[0014] Figure 2 The production of ROS in primary peritoneal macrophages infected with Mtb in WT mice was measured using a reactive oxygen species assay kit; green represents ROS and red represents Mtb;
[0015] Figure 3 Hoechst33342 / PI double staining analysis of NRF2- / - mice infected with Mycobacterium tuberculosis for 24 h;
[0016] Figure 4 Hoechst33342 / PI double staining analysis of NRF2- / - mice infected with Mycobacterium tuberculosis for 72 h. DETAILED DESCRIPTION
[0017] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.
[0018] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.
[0019] The following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting.
[0020] Mycobacterium tuberculosis (H37Ra) was purchased from GeneOptimal Biotechnology Co., Ltd. (Shanghai, China) and cultured in 7H9 complete medium containing 10% OADC (Middlebrook, NY, USA) at 30°C.
[0021] RAW264.7 macrophages were provided by Hycyte Biotechnology Co., Ltd., Quanzhou, China. Cells were cultured in DMEM (Gibco, Grand Island, NY, USA) and 10% FBS (Sorfa, Beijing, China) in a humidified chamber (37° C., 5% CO 2 ).
[0022] Example 1
[0023] WT and NRF2- / - mice were injected intraperitoneally with 3 mL of 3% thioglycolate broth for 3 days. Subsequently, all mice were killed. The peritoneal cavity was rinsed with 4 mL of cold PBS and the abdomen was gently massaged for 5 minutes. The peritoneal primary macrophage fluid was collected, centrifuged at 1000 rpm for 5 minutes, resuspended in RPMI1640 medium supplemented with 10% FBS, and the cells were implanted in a culture dish. After 2 hours, non-adherent cells were discarded, leaving primary macrophages as adherent cells.
[0024] Cells were lysed using RIPA buffer (Solarbio, Beijing), and supernatants were concentrated using the PierceBCA protein assay kit (Thermo, Rockford, IL, USA). Protein samples were electrophoresed on SDS-PAGE gels at 110 V for 1.5 h, and then the proteins were transferred to PVDF membranes by electroblotting at 110 V for 1 h. The PVDF membranes containing proteins were blocked with 5% skim milk in TBS-T at room temperature for 2 h. After washing with TBS-T, the PVDF membranes were incubated with the secondary antibody at 4 °C overnight or at room temperature for 1 h. The PVDF membranes were incubated with enzyme-labeled secondary antibodies in TBS-T solution for 1 h at room temperature. Blotting was performed using Super ECL (CWBIO, Beijing, China). -Antibodies were iNOS (CST, Danvers, MA, USA), COX-2, NRF2, HO-1, NQO-1, β-actin, enzyme-labeled secondary antibody (ab clone, Wuhan, China), NLRP3, ASC, Caspase-1, GSDMD, and IL-1β (Bioss, Beijing, China).
[0025] Example 2
[0026] After infection, the cells were washed three times with cold PBS (5 minutes each time). Subsequently, the cells were fixed with 4% paraformaldehyde for 10 minutes and then washed with cold PBS. Subsequently, the cells were treated with 0.1% TritonX-100 in PBS for 10 minutes and then washed with cold PBS. The cells were incubated in a solution of 5% BSA dissolved in PBS for 1 hour at room temperature. Next, the cells were incubated with anti-nrf2-antibody (diluted at a ratio of 1:300) in PBS containing 5% BSA at 4°C overnight and then washed again with cold PBS. A highly cross-adsorbed secondary antibody AlexaFluor was used using donkey anti-rabbit IgG (h+L). TM488 (diluted to a ratio of 1:2000) was incubated for another 1 h at room temperature and then washed again with cold PBS. Finally, cells were incubated with DAPI for 5 min, followed by another round of washing with cold PBS. Images were observed and recorded using a Nikon fluorescence microscope (Nikon, Tokyo, Japan).
[0027] Example 3
[0028] After infection, total RNA was extracted from cells using Trizol (TransGen Biotech, Beijing, China). RNA was then reverse transcribed into cDNA using the TransScript first-strand cDNA synthesis tool SuperMix (TransGen Biotech, Beijing). Gene expression was detected using MonAmpTM ChemoHSspecificPlusqPCRMix (Monad, Shanghai, China). The sample preparation premix was 2 μL sample + 0.5 μL forward primer + 0.5 μL reverse primer + 5 μL premix + 2 μL nuclease-free water, with a total volume of 10 μL. The RT-qPCR execution program included an initial denaturation at 95°C for 5 minutes, followed by 35 cycles, each cycle including denaturation at 95°C for 10 seconds, primer annealing at 58°C for 10 seconds, and extension at 72°C for 30s. The primer sequences are: NLRP3,
[0029] F: 5′-GAGCTGGACCTCAGTGACAATGC-3′,
[0030] R: 5′-ACCAATGCGAGATCCTGACAACAC-3′; ASC, f: 5′-
[0031] cagagagcagcagcaagag-3′,
[0032] r: 5'-caagagcgtccaggatggcatc-3';
[0033] Caspase1, F: 5′-ACAACCACTCGTACACGTCTTGC-3′,
[0034] R: 5′-CCAGATCCTCCAGCAGCAACTTC-3′;
[0035] β-actin,F:5'-GTCAGGTCATCACTATCGGCAAT-3′,
[0036] R: 5'-AGAGGTCTTTACGGATGTCAACGT-3'.
[0037] The relative gene expression was determined by the 2-ΔΔCT method, with β-actin as the internal parameter.
[0038] Example 4
[0039] The ROS level in macrophages was quantified using a reactive oxygen species assay kit (Solarbio, CA1410, Beijing, China). DCFH-DA was diluted to a concentration of 10 μM using DMEM or RPMI1640 medium without FBS. After Mtb infection, the medium was replaced with the DCFH-DA dilution and incubated at 37°C in a humidified environment for 20 min. Subsequently, the cells were observed and photographed using a fluorescence microscope.
[0040] Example 5
[0041] Malondialdehyde (MDA) content assay kit (Solarbio, BC0025, Beijing, China) was used to quantify MDA in macrophages. After infection with Mycobacterium tuberculosis, cells were harvested using extract at a ratio of 5 million cells per mL of extract. Cell lysis was achieved using an ultrasonic processor under the following parameters: power of 200 W; ultrasonication for 3 s; interval of 10 s; 30 repetitions. The resulting lysate was then centrifuged at 8000 × g for 10 min and the supernatant was collected. For sample preparation premix, 300 μL MDA detection solution + 100 μL cell lysis supernatant + 100 μL reagent were added to a 1.5 mL EP tube. The mixture in the tube was heated at 100 °C for 1 h, and then a volume of 200 μL of the mixture was transferred to a 96-well plate and the absorbance at 532 nm and 600 nm was measured.
[0042] Example 6
[0043] Reduced glutathione (GSH) levels in macrophages were quantified using a reduced glutathione (GSH) assay kit (Solarbio, BC1175, Beijing, China). After infection with Mycobacterium tuberculosis, 5 million cells per mL of extract were harvested using reagent 1. Cell lysis was performed using an ultrasonic processor with the following parameters: power 200 W; ultrasonication for 3 s; interval 10 s; 30 repetitions. The lysate was then centrifuged at 12,000 × g for 10 min, and the supernatant was collected as the sample. For sample preparation, a premix of 20 μL sample + 140 μL reagent 2 + 40 μL reagent 3 was added to a 96-well plate. After incubation at room temperature for 2 minutes, the absorbance at a wavelength of 412 nm was measured.
[0044] Example 7
[0045] Lactate dehydrogenase (LDH) activity assay kit (Solarbio, BC0685, Beijing, China) was used for the quantification of macrophage GSH. Cells were collected by centrifugation and resuspended in the extract at a ratio of 5 million cells per mL of extract. Cell lysis was performed using an ultrasonic processor under the following parameters: power 200 W; ultrasonification 3 s; interval 10 s; 30 repetitions.
[0046] After centrifugation at 8000×g for 10 minutes, collect the supernatant (sample). When preparing the sample premix, add 10μL sample + 50μL reagent 1 + 10μL reagent 2 to a 96-well plate. Incubate the plate containing the sample in a water bath at 37°C for 15 minutes. Subsequently, add reagent 3 (50μL) to the sample premix and incubate in a water bath at 37°C for another 15 minutes. Finally, add reagent 4 (150μL) to the sample premix, allow to stand at room temperature for 3 minutes, and then measure the absorbance at a wavelength of 450nm.
[0047] Example 8
[0048] Mouse IL-1β (MM-0040M1) and (MM-0169M1) ELISA kits were produced by Shanghai mlbio. After the cells were treated with SFN and Mycobacterium tuberculosis, the cell culture supernatant was collected and centrifuged at 4000rpm for 10min to remove particles and polymers. The resulting supernatant was diluted 5 times and added to the ELISA 96-well plate. Subsequently, 100μL of enzyme-labeled reagent was added to each well, covered with a sticky strip, and incubated at 37°C for 60min. The plate was washed 5 times with washing solution, each for 1min. Chromogen solution A (50μL) and chromogen solution B (50μL) were added to each well, mixed gently, and incubated at 37°C for 15min. Finally, stop solution (50μL) was added to each well, and the OD was read at a wavelength of 450nm using a microtiter plate reader.
[0049] Example 9
[0050] Macrophage staining was performed using the Hoechst33342 / PI double staining kit (Solarbio, CA1120, Beijing, China). Cells were seeded in 24-well plates containing coverslips. After infection with Mycobacterium tuberculosis, the cells were stained in an ice bath for 30 min with a staining solution consisting of 2.5 μL Hoechst staining solution and 2.5 μL PI staining solution in a total volume of 500 μL. The coverslips were then washed with cold PBS and sealed with gelatin before observation and photography under a fluorescence microscope.
[0051] Example 10
[0052] Approximately 50,000 primary peritoneal macrophages were seeded in each well of a 96-well plate and incubated overnight. Subsequently, SFN treatment was performed at a final concentration of 0 to 100 μM in each well, including a DMSO control group. Each experimental group consisted of 6 replicates. After 24 h of SFN treatment, CCK8 reagent was added and incubated at 37 °C for 30 m. Finally, the absorbance at 450 nm was measured using a microplate reader.
[0053] The data of the above examples are expressed as mean ± SEM. One-way ANOVA was used to analyze the significant effect, followed by Tukey's HSD test (*p<0.05, **p<0.01, ***p<0.001). Each experiment in this study was repeated at least 3 times. The experimental results showed that SFN effectively inhibited the expression of IL-1β induced by Mtb infection and inhibited the increase in IL-18 levels caused by Mtb infection. The results of the study of RAW264.7 macrophages and primary peritoneal macrophages showed that Mycobacterium tuberculosis infection upregulated the expression of iNOS and COX-2, while supplementation of SFN inhibited their expression. SFN inhibits the production of ROS induced by Mycobacterium tuberculosis infection, which can reduce the oxidative stress of Mycobacterium tuberculosis infected macrophages. That is, SFN can effectively inhibit oxidative stress and play an anti-pyroptosis effect in mtb-infected macrophages.
[0054] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and will inspire those skilled in the art to make various modifications or changes thereto, and will be included in the spirit and scope of the present application. In addition, any element or limitation of any invention disclosed herein or any implementation thereof may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are intended to be included within the scope of the present invention, but are not limited thereto.
Claims
1. Application of sulforaphane in the preparation of products for preventing or treating diseases induced by oxidative stress or inflammatory response.
2. The use according to claim 1, Features: The oxidative stress or inflammatory response-induced disease exhibits altered levels of one or more biomolecules selected from cluster of differentiation 40 ligand (CD-40L), eotaxin, fibrinogen, growth hormone (GH), keratinocyte-derived cytokine or GRO1 oncogene (KC / GRO), interleukin-1β (IL-1β), IL-6, IL-18, lymphocyte chemotactic factor, myeloperoxidase (MPO), tissue inhibitor of metalloproteinase 1 (TIMP-1), vascular endothelial growth factor A (VEGF-A), C-reactive protein (CRP), macrophage-derived chemokine (MDC), macrophage inflammatory protein-1α (MIP-1α), vWF or oncostatin.
3. The use according to claim 1, Features: The oxidative stress or inflammatory response-induced disease is selected from allergy, Alzheimer's disease, ankylosing spondylitis, asthma, autoimmune disease, arthritis, atherosclerosis, carpal tunnel syndrome, celiac, Crohn's disease, diverticulitis, eczema, fibrosis, Guillain-Barre disease, lupus, multiple sclerosis, nephritis, neuropathy, pancreatitis, Parkinson's disease, psoriasis, polymyalgia rheumatica, rheumatoid arthritis, scleroderma or vasculitis.
4. A pharmaceutical composition for realizing the use according to any one of claims 1 to 3, It is characterized in that The active ingredient comprises the sulforaphane used in any one of claims 1 to 3.
5. A medicament for realizing the use according to any one of claims 1 to 3, It is characterized in that The invention comprises the sulforaphane used in the application according to any one of claims 1 to 2 and a pharmaceutically acceptable excipient.
6. The medicament according to claim 5, It is characterized in that The medicine is in the form of capsules, tablets, powders, granules, injections, oral liquids, alcoholic preparations, pills, mixtures or tinctures.
7. A health food, It is characterized in that The invention comprises the sulforaphane used in any one of claims 1 to 3.