Application of 3-hydroxy-5, 6-epoxy-beta-ionone in haze damage resistance

Abstract

The invention discloses an application of 3-hydroxy-5, 6-epoxy-beta-ionone in preparation of a medicine or a health care product with an anti-haze damage effect, and belongs to the technical field of biological activity of compounds. The haze damage resistance is at least one of the following expressions: inhibition of PM-induced intracellular ROS and NO generation, and inhibition of PM-induced intracellular ROS and NO generation; the cell survival rate is improved and the cell death rate is reduced; the synthesis of collagen is promoted; the high expression of MMP protease in cells is inhibited; the generation amount of proinflammatory cytokines in cells is inhibited; and the high expression of p50, p65, p-c-Jun and p-c-Fos proteins can be inhibited. The monomeric compound HEBI is separated and purified from sargassum horneri, experiments prove that the monomeric compound HEBI has a remarkable haze damage resisting effect and is dose-dependent within a certain range, and a theoretical basis is provided for application of HBEI in the technical field of biological medicine.

Description

Technical Field

[0001] The invention relates to application of 3-hydroxy-5,6-epoxy-β-ionone in resisting haze damage, and belongs to the technical field of biological activity of compounds. Background Art

[0002] With the rapid development of modern industry and the acceleration of urbanization, smog is becoming increasingly frequent and its impact is expanding. Smog contains a large number of harmful substances, including fine particulate matter (such as PM2.5 and PM10), sulfur dioxide, nitrogen oxides, and various volatile organic compounds. These substances severely deteriorate air quality and pose a significant threat to human health. Harmful particles in smog can enter the human body through the respiratory tract, causing a range of respiratory illnesses such as coughing, asthma, bronchitis, and pneumonia. Long-term exposure can even increase the risk of malignant diseases such as lung cancer. Smog can also damage the cardiovascular and immune systems, impacting overall health.

[0003] Currently, protective measures against the hazards of smog primarily focus on physical isolation, such as wearing masks and using air purifiers. These methods can reduce human exposure to smog pollutants to a certain extent, but they cannot fundamentally address the damage smog causes to the internal body. Existing research and products for repairing and treating smog-induced damage have many shortcomings, and there is a lack of effective countermeasures.

[0004] 3-Hydroxy-5,6-epoxy-β-ionone (HEBI), CAS number 38274-01-0. Currently, there are no reports that 3-hydroxy-5,6-epoxy-β-ionone has the ability to protect against haze damage. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention provides a new use of 3-hydroxy-5,6-epoxy-β-ionone - its application in resisting haze damage.

[0006] The present invention is achieved through the following technical solutions: Application of 3-hydroxy-5,6-epoxy-β-ionone in the preparation of medicines or health products with anti-smog damage efficacy.

[0007] Furthermore, the effective concentration of the 3-hydroxy-5,6-epoxy-β-ionone is 5 to 30 μg / mL, preferably 6.25 to 25 μg / mL, more preferably 6.25 μg / mL, 12.5 μg / mL, and 25 μg / mL.

[0008] Furthermore, the haze damage is specifically manifested as at least one of the following: increased levels of reactive oxygen species (ROS), cell apoptosis, collagen degradation, up-regulation of matrix metalloproteinases (MMPs) expression, and increased secretion of pro-inflammatory cytokines.

[0009] Furthermore, the mechanism of resistance to haze damage may be at least one of the following: (a) Inhibit PM-induced intracellular ROS and nitric oxide (NO) production; (b) Improve the survival rate of cells after PM induction and reduce cell death rate; (c) Promote collagen synthesis; (d) Inhibit the PM-induced high expression of multiple proteases such as MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 in cells; (e) Inhibit the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in cells; (f) Inhibits the PM-induced high expression of key proteins in the NF-κB (p50, p65) and AP-1 (pc-Jun, pc-Fos) signaling pathways.

[0010] This study, using a PM-HDF (human dermal fibroblast) cell injury model, demonstrated that HEBI can reduce PM-induced intracellular ROS levels, increase cell survival, promote collagen synthesis, inhibit PM-induced overexpression of multiple proteases, including MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, suppress the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, and inhibit PM-induced overexpression of key proteins in the NF-κB (p50, p65) and AP-1 (pc-Jun, pc-Fos) signaling pathways. This study demonstrated significant protection against smog-induced damage, with the effect becoming more pronounced with increasing HEBI concentration within a certain range. This study also demonstrated that HEBI can inhibit PM-induced ROS and NO production, reduce cell mortality, and exert significant protection against smog-induced damage, with the protective effect becoming more pronounced with increasing HEBI concentration within a certain range.

[0011] The present invention separates and purifies the monomer compound HEBI from copper algae, and experiments confirm that it has significant anti-haze damage efficacy and is dose-dependent within a certain range. The present invention provides a theoretical basis for the application of HBEI in the field of biomedicine technology.

[0012] Various terms and phrases used herein have the general meanings that are well understood by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : The structural formula of HEBI.

[0014] Figure 2 : Determination results of intracellular ROS levels, where control represents the blank control group, and “0, 6.25, 12.5, 25” represent experimental group 1, experimental group 2, experimental group 3, and experimental group 4, respectively; the same below.

[0015] Figure 3 : The results of cell viability determination.

[0016] Figure 4 : Determination results of collagen content.

[0017] Figure 5 : The results of determination of MMP-1 expression level.

[0018] Figure 6 : The results of determination of MMP-2 expression level.

[0019] Figure 7 : The results of determination of MMP-8 expression level.

[0020] Figure 8 : The results of determination of MMP-9 expression level.

[0021] Figure 9 : The results of determination of MMP-13 expression level.

[0022] Figure 10 : Results of measurement of TNF-α production.

[0023] Figure 11 : Results of measurement of IL-6 production.

[0024] Figure 12 : Results of measurement of IL-1β production.

[0025] Figure 13 : Protein bands of p50, p65, pc-Jun, and pc-Fos.

[0026] Figure 14 : The results of determination of p50 expression levels.

[0027] Figure 15 : The results of determination of p65 expression level.

[0028] Figure 16 : The results of pc-Jun expression level determination.

[0029] Figure 17 : The results of pc-Fos expression level determination.

[0030] Figure 18 : DCFH2-DA staining results of zebrafish embryos.

[0031] Figure 19 : Measurement results of ROS generation.

[0032] Figure 20 : DAF-FM-DA staining results of zebrafish embryos.

[0033] Figure 21 : Measurement results of NO generation amount.

[0034] Figure 22 : Acridine orange staining results of zebrafish embryos.

[0035] Figure 23 : Results of cell death rate determination. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0037] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0038] Example 1 Extraction of HEBI Copper algae ( Sargassum horneri Copper algae (SC) is a common large marine brown algae rich in various bioactive components, such as polysaccharides, polyphenols, and terpenoids, exhibiting antioxidant, anti-inflammatory, and immunomodulatory activities. This study isolated a natural compound from Copper algae and identified it as 3-hydroxy-5,6-epoxy-β-ionone. The specific extraction method is as follows: (1) Add 50 g of copper algae to 500 mL of 80% methanol solution (volume percentage), extract at 37°C for 2 hours, and filter. Repeat this extraction 10 times, each time for 2 hours. Combine the filtrates and concentrate to obtain the methanol crude extract (MSC).

[0039] (2) MSCs were homogenized in water and fractionated according to polarity using n-hexane, chloroform, and ethyl acetate solvents to obtain chloroform fractions (CMSCs).

[0040] (3) HEBI was isolated from CMSC using a high-performance centrifugal partition chromatography (HPCPC) system. The optimal two-phase immiscible solvent system for separation was n-hexane:ethyl acetate:methanol:water = 5:5:5:5 (volume ratio). The pump pressure was 3.7 MPa and the flow rate was maintained at 2 mL / min. HEBI was present in the second fraction obtained by fractionation and had significant absorbance at 230 nm. Further purification was performed using preparative high-performance liquid chromatography (prep-HPLC) equipped with a photodiode array detector.

[0041] (4) The second fraction obtained from the above separation was purified by preparative high performance liquid chromatography (prep-HPLC) equipped with a photodiode array detector to obtain two high-purity compounds, which were temporarily named CMSC-Ba and CMSC-Bb.

[0042] (5) The CMSC-Ba and CMSC-Bb obtained above were characterized by Fourier transform nuclear magnetic resonance (NMR) 1 H. 13 C, DEPT, COSY and HMBC NMR spectra confirmed that the molecular formula of CMSC-Ba is C 11 H 16 O3, molecular weight 196.2 Da, is the same as the lolidolide [(-)-loliolide] reported in the prior art; the molecular formula of CMSC-Bb is C 13 H 20 O3, molecular weight 224.2 Da, structural formula Figure 1 As shown, it is the same as the 3-hydroxy-5,6-epoxy-β-ionone reported in the prior art.

[0043] The data for CMSC-Bb are as follows; 1 H NMR (400 MHz, methanol-d4) δ: 0.86 (s, 3H, H-11), 1.12 (s, 3H, H-12), 1.10 (s, 3H, H-13), 1.14 (ddd, J = 13.7, 4.6 and 1.8 Hz, 1H, H-2), 1.45 (dd, J = 13.7 and 2.7 Hz, 1H, H-2), 1.55 (dd, J = 14.7 and 2.7 Hz, 1H, H-4), 1.12 (s, 3H, H-10), 2.18 (ddd, J = 14.7, 5.5 and 1.8 Hz, 1H, H-4), 3.57 (tdd, J = 5.5, 4.6 and 2.7 Hz, 1H, H-3), 6.01 (d, J = 15.6 Hz, 1H, H-8) and 7.07 (d, J = 15.6 Hz, 1H, H-7); and13 C NMR (100 MHz, methanol-d4) δ: 19.7 (C-13), 24.8 (C-11), 27.3 (C-10), 29.0 (C-12), 34.5 (C-1), 40.4 (C-4), 46.5 (C-2), 61.7 (C-5), 66.9 (C-3), 68.8 (C-6), 132.2 (C-8), 143.4 (C-7), and 197.4 (C-9).

[0044] Experiment 1: Study on the in vivo efficacy of HEBI in protecting against haze damage An in vivo PM damage model was constructed using HDF cells to evaluate the efficacy of HEBI in resisting haze damage.

[0045] (1) Experimental process HDF cells (ATCC® PCS 201012™, Manassas, VA, USA) were cultured in a mixed medium of F-12 and DMEM (volume ratio 1:3) supplemented with 10% FBS and 1% P / S. HDF cells were seeded in 96-well plates (5.0 × 10 4 cells / mL), 200 μL per well, and subsequent experiments were performed.

[0046] Experimental group 1, experimental group 2, experimental group 3, experimental group 4 and blank control group were set up, among which, Experimental Group 1 was treated as follows: 200 μL of serum-free medium (i.e., HEBI concentration was 0) was added to each well and pretreated at 37°C for 24 hours. The supernatant was removed, and 200 μL of 400 μg / mL PM solution was added to each well and incubated for 24 hours. The serum-free medium was a mixture of F-12 and DMEM (volume ratio 1:3).

[0047] Experimental Group 2 was treated as follows: 200 μL of HEBI solution (HEBI concentration: 6.25 μg / mL) was added to each well and pretreated at 37°C for 24 hours. The supernatant was removed, and 200 μL of PM solution (400 μg / mL) was added to each well and incubated for 24 hours. In this example, the HEBI solution consisted of HEBI and serum-free culture medium.

[0048] The treatment method of experimental group 3 was as follows: 200 μL of HEBI solution (HEBI concentration was 12.5 μg / mL) was added to each well and pretreated at 37°C for 24 h; the supernatant was removed, and 200 μL of PM solution with a concentration of 400 μg / mL was added to each well and incubated for 24 h.

[0049] The treatment method of experimental group 4 was as follows: 200 μL of HEBI solution (HEBI concentration was 25 μg / mL) was added to each well and pretreated at 37°C for 24 h; the supernatant was removed, and 200 μL of PM solution with a concentration of 400 μg / mL was added to each well and incubated for 24 h.

[0050] The blank control group was treated as follows: 200 μL of serum-free medium (i.e., the concentration of HEBI was 0) was added to each well and pretreated at 37°C for 24 h; the supernatant was removed, and 200 μL of serum-free medium (i.e., the concentration of PM was 0) was added to each well and incubated for 24 h.

[0051] The PM is provided and certified by the National Institute of Environmental Studies (NIES), and its components are shown in Tables 1 and 2.

[0052]

[0053] AAS: atomic absorption spectrometry; HG-AAS: hydride generation-atomic absorption spectrometry; HG-ICP-AES: hydride generation-inductively coupled plasma-atomic emission spectrometry; ICP-AES: inductively coupled plasma-atomic emission spectrometry; ICP-MS: inductively coupled plasma-mass spectrometry; INAA: instrumental neutron activation analysis; PIXE: proton-induced X-ray emission spectrometry; XRF: X-ray fluorescence spectrometry.

[0054]

[0055] HG-AAS: hydride generation-atomic absorption spectrometry; HG-ICP-AES: hydride generation-inductively coupled plasma-atomic emission spectrometry; HR-ICP-MS: high-resolution inductively coupled plasma-mass spectrometry; ICP-AES: inductively coupled plasma-atomic emission spectrometry; ICP-MS: inductively coupled plasma-mass spectrometry; INAA: instrumental neutron activation analysis; PIXE: proton-induced X-ray emission spectrometry; XRF: X-ray fluorescence spectrometry.

[0056] (2) Testing indicators The following indicators were measured for the cells cultured in each group: Intracellular ROS levels were measured using the DCF-DA method; Cell viability was determined using the MTT assay; Collagen content was determined using an ELISA kit (Sigma, St. Louis, MO, USA); The expression levels of MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 were determined using ELISA kits (Sigma, St. Louis, MO, USA); The production of inflammatory factors TNF-α, IL-6, and IL-1β was measured using ELISA kits (Sigma, St. Louis, MO, USA); The expression levels of p50, p65, pc-Jun, and pc-Fos were determined by Western Blot (WB).

[0057] (3) Experimental results (1) Intracellular ROS levels The results of the determination of intracellular ROS levels are shown in Table 3. Figure 2 As shown, p < 0.05 is considered to be significantly different, "#" indicates that the group has significant damage repair efficacy compared with the blank control group, "*" indicates that the group has significant damage repair efficacy compared with the PM damage without HEBI repair group (i.e., experimental group 1); p < 0.01 is considered to be extremely significantly different, "##" indicates that the group has extremely significant damage repair efficacy compared with the blank control group, and "**" indicates that the group has extremely significant damage repair efficacy compared with experimental group 1; the same below.

[0058]

[0059] (2) Cell survival rate The results of cell viability determination are shown in Table 4. Figure 3 shown.

[0060]

[0061] (3) Collagen content The results of the determination of collagen content are shown in Table 5. Figure 4 shown.

[0062]

[0063] (4) Expression levels of MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 The results of the determination of the expression levels of MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 are shown in Table 6. Figures 5 to 9 shown.

[0064]

[0065] (5) Production of inflammatory factors TNF-α, IL-6, and IL-1β The results of the determination of TNF-α, IL-6, and IL-1β production are shown in Table 7. Figures 10-12 shown.

[0066]

[0067] (6) Expression levels of p50, p65, pc-Jun, and pc-Fos The results of the determination of the expression levels of p50, p65, pc-Jun, and pc-Fos are shown in Table 8. The protein bands of p50, p65, pc-Jun, and pc-Fos are shown in Table 8. Figure 13 The results of the determination of p50, p65, pc-Jun, and pc-Fos expression levels are shown in Figures 14-17 shown.

[0068]

[0069] (IV) Conclusion These experimental results demonstrate that HEBI can reduce PM-induced intracellular ROS levels, increase cell survival, promote collagen synthesis, inhibit PM-induced overexpression of multiple proteases, including MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13, suppress the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β, and inhibit PM-induced overexpression of key proteins in the NF-κB (p50, p65) and AP-1 (pc-Jun, pc-Fos) signaling pathways. HEBI exhibits significant efficacy against the damage caused by smog, and within a certain range, the effect becomes more pronounced with increasing HEBI concentration.

[0070] Experiment 2: Study on the in vitro efficacy of HEBI in preventing haze damage A PM-zebrafish larvae damage model was constructed to evaluate the anti-haze damage efficacy in vitro.

[0071] (1) Experimental process Adult zebrafish were induced to spawn naturally using light, and then embryos were collected. Zebrafish embryos were transferred into 12-well plates 2 days after fertilization. Five experimental groups (experimental group 1, experimental group 2, experimental group 3, experimental group 4 and blank control group) were set up, with 15 embryos in each group. Experimental Group 1 was treated with 2 mL of embryo culture medium (i.e., 0 HEBI concentration) added to each well for 1 hour at 28°C. Then, 2 mL of a 400 μg / mL PM solution was added to each well and incubated for 6 hours. The embryo culture medium consisted of a sterile aqueous solution containing 10% NaCl, 0.3% KCl, 0.3% CaCl₂, 0.8% MgSO₄, and a small amount of methylene blue.

[0072] Experimental Group 2 was treated with 2 mL of Hebi solution (6.25 μg / mL) per well for pretreatment at 28°C for 1 hour. Then, 2 mL of 400 μg / mL PM solution was added to each well and incubated for 6 hours. In this example, the Hebi solution consisted of Hebi and embryo culture medium.

[0073] The treatment method of experimental group 3 was as follows: 2 mL of HEBI solution (HEBI concentration was 12.5 μg / mL) was added to each well and pretreated at 28°C for 1 hour; then, 2 mL of PM solution with a concentration of 400 μg / mL was added to each well and incubated for 6 hours.

[0074] The treatment method of experimental group 4 was as follows: 2 mL of HEBI solution (HEBI concentration was 25 μg / mL) was added to each well and pretreated at 28°C for 1 hour; then, 2 mL of PM solution with a concentration of 400 μg / mL was added to each well and incubated for 6 hours.

[0075] The blank control group was treated as follows: 2 mL of embryo culture medium (i.e., the concentration of HEBI was 0) was added to each well and pretreated at 28°C for 1 hour; then, 2 mL of embryo culture medium (i.e., the concentration of PM was 0) was added to each well and incubated for 6 hours.

[0076] (2) Testing indicators After incubation, the larvae were stained with DCFH2-DA (20 μg / mL, 1 hour), DAF-FM-DA (10 μM, 3 hours), and acridine orange (10 μg / mL, 30 minutes), respectively. The ROS production, NO production, and cell death rate of each experimental group were measured, and the relative levels of each experimental group were calculated with the levels of the blank control group as 100%.

[0077] (3) Experimental results (1) ROS generation The DCFH2-DA staining results of zebrafish embryos are shown in Figure 2. Figure 18 The results of the determination of ROS generation are shown in Table 9. Figure 19 shown.

[0078]

[0079] (2) Nitric oxide (NO) production The results of DAF-FM-DA staining of zebrafish embryos are as follows Figure 20 The results of the measurement of NO generation are shown in Table 10. Figure 21 shown.

[0080]

[0081] (3) Cell death rate Acridine orange staining results of zebrafish embryos Figure 22 The results of the cell death rate determination are shown in Table 11. Figure 23 shown.

[0082]

[0083] (IV) Conclusion The above results show that HEBI can inhibit the production of ROS and NO induced by PM and reduce cell mortality. HEBI has significant anti-haze damage effect, and within a certain range, the effect is more obvious with the increase of HEBI concentration.

[0084] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of 3-hydroxy-5,6-epoxy-β-ionone in the preparation of medicines or health products with anti-smog damage effects.

2. The use according to claim 1, characterized in that: The effective concentration of the 3-hydroxy-5,6-epoxy-β-ionone is 5 to 30 μg / mL.

3. The use according to claim 2, characterized in that: The effective concentration of the 3-hydroxy-5,6-epoxy-β-ionone is 6.25 to 25 μg / mL.

4. The use according to claim 3, characterized in that: The effective concentration of the 3-hydroxy-5,6-epoxy-β-ionone is 6.25 μg / mL, 12.5 μg / mL or 25 μg / mL.

5. The use according to claim 1, characterized in that The haze damage is specifically manifested as at least one of the following: increased levels of reactive oxygen species, cell apoptosis, collagen degradation, up-regulation of matrix metalloproteinase expression, and increased secretion of pro-inflammatory cytokines.

6. The use according to claim 1, characterized in that The performance of the anti-haze damage is at least one of the following: (a) Inhibit PM-induced intracellular ROS and nitric oxide production; (b) Improve the survival rate of cells after PM induction and reduce cell death rate; (c) Promote collagen synthesis; (d) Inhibit the PM-induced high expression of multiple proteases such as MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 in cells; (e) Inhibit the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in cells; (f) Inhibits the high expression of p50, p65, pc-Jun, and pc-Fos proteins induced by PM.

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