Application of sargassum fusiforme aqueous extract
By using the water extract of yam citrus, the Nrf2/ARE signaling pathway was activated, and the problem of adverse reactions in the prior art was solved, and the oxidative stress and insulin resistance of type 2 diabetes cells was effectively improved, and the drug characteristics were safe.
Patent Information
- Application Number
- CN202411732558.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-03-14
AI Technical Summary
In the prior art, although some oral hypoglycemic drugs can improve insulin resistance, most drugs have severe adverse reactions and fewer drugs from natural origin are used to treat type 2 diabetes.
The Nrf2/ARE signaling pathway was used to activate phase II detoxification enzymes and antioxidant enzymes to reduce ROS production and activate the insulin signaling pathway by performing oxidative stress and improvement of insulin resistance on HepG2 cells.
The water extract of yamba cabbage can effectively promote the consumption of extracellular glucose of IR-HepG2, improve the oxidative stress state of cells, significantly reverse insulin resistance, and have safe and non-toxic side effects.
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Figure CN119607032A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicines, and particularly relates to an application of a Sargassum fusiformis water extract. Background Art
[0002] Diabetes mellitus (DM) is a chronic disease caused by endocrine and metabolic disorders. Diabetes mellitus can be divided into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T2DM is caused by people's unhealthy diet and lifestyle habits, and its main symptoms include insulin resistance (IR), hyperglycemia and insulin deficiency. Insulin maintains blood glucose homeostasis by stimulating multiple physiological responses in peripheral tissues such as the liver, adipose tissue and skeletal muscle. When IR occurs, it can lead to fatal complications of multiple organs such as cardiovascular disease and diabetic nephropathy, and become a key link in the occurrence and development of T2DM. A large number of studies have shown that oxidative stress is the main upstream event in the development of IR, which initiates a series of physiological signaling pathways that lead to IR and T2DM. ROS increases the phosphorylation of the IRS-1Ser307 site by activating c-Jun amino-terminal kinase (JNK), while reducing the normal phosphorylation of IRS-1Tyr632, blocking the transduction of insulin signals, and ultimately inhibiting the transport of GLUT4 in cells, causing insulin resistance. However, Nrf2 binds to ARE, as the most important endogenous anti-oxidative stress pathway discovered so far, and has been proven to reduce oxidative stress damage and improve insulin resistance. As a nuclear transcription factor, Nrf2 mainly acts through the kelch-like epichlorohydrin-related protein (Keap) 1-Nrf2-ARE signaling pathway. Normally, Keap1 and Nrf2 maintain a stable binding state in the cytoplasm. When the body is stimulated by oxidative stress, Nrf2 is separated from Keap1 and released, and migrates to the nucleus to bind to the ARE region to activate the transcription of downstream genes. Once the Nrf2 / ARE pathway is activated, it will further activate phase II detoxification enzymes and antioxidant enzymes, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), reduce the production of ROS, and activate the transduction of the insulin signaling pathway (IRS1 / PI3K / AKT / GLUT4), thereby improving oxidative stress and insulin resistance.
[0003] Although some oral hypoglycemic drugs can effectively improve insulin resistance, most of them, including biguanides, thiazolidinediones and sulfonylureas, can cause serious adverse reactions. This has prompted people to be interested in natural drugs for the treatment of T2DM. Sargassum fusiforme is a plant of the genus Sargassum in the family Sargassum, distributed in Liaoning, Shandong, Zhejiang, Fujian, Guangdong and other provinces in China. It is used in traditional Chinese medicine to treat many diseases such as preventing balloon injuries, dehydrating soft phlegm and reducing swelling. Studies have shown that Sargassum fusiforme has multiple biological activities. Therefore, this application aims to explore the effects of Sargassum fusiforme aqueous extract (SFE) on oxidative stress and insulin resistance in HepG2 cells, in order to provide a basis for the research and development of drugs for type 2 diabetes. Summary of the invention
[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide an application of a water extract of Sargassum fusiformis, which is specifically achieved through the following technical solutions:
[0005] Application of Sargassum fusiformis water extract in preparing medicine for treating type 2 diabetes.
[0006] Application of Sargassum fusiformis water extract in the preparation of drugs for improving cellular oxidative stress and insulin resistance
[0007] Furthermore, the cells are IR-HepG2 cells.
[0008] Furthermore, the Sargassum fusiformis water extract is prepared by the following method:
[0009] The Sargassum fusiformis is soaked in water for desalination, washed and impurity-free, and then used for later use; the Sargassum fusiformis is added with deionized water and heated at 100° C. in a pressure cooker; after the heating, the tap water is cooled to room temperature, the solution is collected and centrifuged to obtain the supernatant, and the supernatant is concentrated and freeze-dried to obtain the Sargassum fusiformis water extract SFE.
[0010] Furthermore, the desalination time is 3 h, and the material-liquid ratio of Sargassum fusiformis to deionized water is 1 g:10 L.
[0011] Furthermore, the pressure cooker heating time is 15 minutes, the centrifugal condition is 4000r / min, and the centrifugation is 20 minutes.
[0012] The Sargassum fusiformis water extract prepared by the present invention can effectively promote the consumption of extracellular glucose in IR-HepG2 cells, effectively improve the oxidative stress state of IR-HepG2 cells and significantly reverse the down-regulation of key proteins (HO-1, Keap1) in insulin-resistant MOD group cells, and can be used for preparing drugs for treating type 2 diabetes, with safety and no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1Effects of different doses of SFE on HepG2 cell viability and glucose consumption of IR-HepG2 cells (A: Effects of different doses of SFE on HepG2 cell viability; B: Effects of SFE on glucose consumption of insulin-resistant HepG2 cells. Data are expressed as mean ± SD, n = 5. Compared with the CON group,
[0014] ****p<0.000);
[0015] Figure 2 Effects of SFE on oxidative stress in IR-HepG2 cells (A: MDA; B: ROS; C: GSH-Px; D: SOD. Data are expressed as mean ± standard deviation, n = 5. Compared with the CON group, **P < 0.01, ***P < 0.001, ****P < 0.000);
[0016] Figure 3 Western Blot was used to detect the effect of SFE on key proteins in the Nrf2 / ARE signaling pathway of insulin-resistant HepG2 cells (A: expression levels of Nrf2, Keap1 and HO-1 proteins; BD: protein quantitative bar graphs of Nrf2, Keap1 and HO-1; data are expressed as mean ± standard deviation, n = 3; compared with the MOD group, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with the CON group, #P < 0.05, ##P < 0.01, ###P < 0.001);
[0017] Figure 4 qPCR was used to detect the effect of SFE on key genes in the Nrf2 / ARE signaling pathway of insulin-resistant HepG2 cells (A: HO-1; B: IRS1; C: Nrf2; data are expressed as mean ± standard deviation, n = 3; compared with the MOD group, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with the CON group, #P < 0.05, ##P < 0.01, ###P < 0.001). DETAILED DESCRIPTION
[0018] The present invention is further described below in conjunction with the accompanying drawings in order to better understand the technical solution.
[0019] Reagents: Penicillin / streptomycin (P / S), fetal bovine serum (FBS), high-glucose DMEM medium, low-glucose DMEM medium, phosphate buffered saline (PBS), cell counting kit (CCK8), and RIPA lysis buffer were purchased from Biotech Co., Ltd. (Shanghai). Glucose kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing). SDS-PAGE gel preparation kit (4-20%) was purchased from Nanjing Aisiyi Biotechnology Co., Ltd. (Changzhou, China). ECL chemiluminescence kit and blocking buffer were purchased from Biotech Co., Ltd. (Changzhou, China).
[0020] Instruments: decolorization shaker, Beijing Liuyi Biotechnology Co., Ltd.; chromatography experiment refrigerator, Boyikang Instrument Co., Ltd.; eight-row tube centrifuge, Hangzhou Aosheng Instrument Co., Ltd.; electrophoresis instrument, gel imaging system, fluorescence quantitative PCR instrument, Bio-Rad Company.
[0021] Preparation of Sargassum fusiformis aqueous extract: Sargassum fusiformis was soaked in water for desalination for 3 hours, washed and cleaned for use. 500 g of Sargassum fusiformis (1:10, m / V) was added with deionized water and heated at 100°C for 15 minutes in a pressure cooker. After the tap water was cooled, it was centrifuged at 4000 r / min for 20 minutes to collect the supernatant, and the supernatant was concentrated and freeze-dried to obtain Sargassum fusiformis aqueous extract (SFE).
[0022] Establishment of HepG2 cell IR model and experimental grouping: HepG2 cells were derived from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The cells were cultured in a cell culture incubator containing 90% DMEM and 10% fetal bovine serum at 37°C and 5% CO2. After the passaged HepG2 cells grew stably, the CON group was incubated with 5.5mM low-glucose DMEM complete medium; the MOD group was incubated with 50mM high-glucose DMEM complete medium to induce insulin resistance in HepG2 cells; the ROSI group was incubated with 50mM high-glucose medium and 25μM rosiglitazone solution was added as a positive control; the 50mM high-glucose medium was prepared into SFE with a final concentration of 0.25, 0.5, 1.0 and 2.0mg / mL for cell incubation to obtain the drug-administered group. After the HepG2 cells were incubated in a CO2 incubator for 48h after administration, relevant experimental tests were performed.
[0023] Experimental Example 1: Cell Viability Assay
[0024] The cell viability was determined by CCK-8 method. HepG2 cells were cultured in 96-well plates, with 1×105 cells per well. After the cells adhered, different concentrations of SFE were dissolved in 50 mM high-glucose DMEM medium and incubated with the cells for 48 h. The cell viability of each group was then determined using the CCK-8 detection kit.
[0025] The CCK8 method was used to detect the effects of different concentrations of SFE (0.25, 0.5, 1.0, 2.0 mg / mL) on the viability of HepG2 cells. Figure 1 As shown in A, compared with the CON group, different drug concentrations of SFE treated for 24 h showed no cytotoxicity. There was no significant effect on the proliferation and cell viability of HepG2 cells. Therefore, we selected 0.25, 0.5, 1.0 and 2.0 mg / mL of SFE for subsequent experiments.
[0026] Experimental Example 2: Determination of glucose consumption of IR-HepG2 cells by SFE
[0027] After IR-HepG2 cells were cultured and stabilized according to the grouping requirements, the glucose oxidase kit was used to determine the residual glucose content in each well, and the absorbance value was measured using an enzyme reader. The glucose content in each well was calculated based on the standard curve measured by the standard concentration. Glucose consumption = glucose content (blank control group) - glucose content (groups to be tested).
[0028] The results are as follows Figure 1 As shown in B, compared with the CON group, the glucose consumption of the MOD group was significantly reduced, while the ROSI positive drug promoted the glucose consumption of IR-HepG2 cells. At the same time, the glucose consumption of IR-HepG2 cells treated with 0.25-2.0 mg / mL SFE increased significantly. Therefore, SFE can promote the consumption of extracellular glucose in IR-HepG2 cells.
[0029] Experimental Example 3: Effect of SFE on oxidative stress state of IR-HepG2 cells
[0030] The corresponding kits were used to detect the intracellular oxidative stress markers ROS, lipid oxidation MDA, and the activities of antioxidant enzymes GSH-Px and SOD to explore the effect of SFE on the oxidative stress state of IR-HepG2 cells.
[0031] Oxidative stress is one of the main causes of IR. In this experimental example, the corresponding assay kit was used to detect the regulatory effect of SFE on ROS, MDA, GSH-Px and SOD, the key factors of oxidative stress in IR-HepG2 cells. The results showed that compared with the CON group, the levels of ROS and MDA in the MOD group were significantly increased, and the levels of SOD and GSH-Px were significantly decreased. Figure 2 AD. However, after treatment with SFE at concentrations of 2 mg / ml, 1 mg / ml, and 0.5 mg / ml, the expression of ROS and MDA was significantly reduced, and the expression of SOD and GSH-Px antioxidant factors was significantly increased. These data confirm that SFE can improve the oxidative stress state of IR-HepG2 cells.
[0032] Experimental Example 4: Western Blot detection of the effect of SFE on the expression of key proteins in the Nrf2 / ARE pathway of insulin-resistant IR-HepG2 cells
[0033] The cells in each group were collected and lysed on ice with 1% PMSF-containing RIPA cell lysis buffer. The supernatant was centrifuged and the total protein concentration was determined by BCA method. The proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF. The membranes were blocked with 5% skim milk at room temperature for 1 h, incubated overnight at 4°C after adding the primary antibody, washed 3 times with TBST for 10 min each time, incubated with the corresponding secondary antibody at room temperature for 1 h, processed with ECL gel imaging system, and the bands of each group were counted and statistically analyzed.
[0034] In the Western Blot experiment to detect the effect of SFE on the expression of key proteins in the Nrf2 / ARE pathway of IR-HepG2 cells, the antibodies of nuclear Nrf2, keap1 and HO-1 were normalized to the internal reference GAPDH band. The results showed that compared with the CON group, the expression of cells in the high glucose-induced insulin resistance MOD group was significantly reduced, such as Figure 3 As shown in A and 3B, the 0.25-2.0 mg / mL SFE administration group significantly increased the expression of Nrf2 protein in a dose-dependent manner. At the same time, different concentrations of SFE also showed significant regulatory effects on key proteins in the Nrf2 / ARE signaling pathway. Figure 3 As shown in C and D, different concentrations of SFE significantly reversed the downregulation of key proteins (HO-1, Keap1) in cells of the insulin resistance MOD group, indicating that SFE improved oxidative stress and insulin resistance in IR-HepG2 cells by activating protein expression in the Nrf2 / ARE signaling pathway.
[0035] Experimental Example 5: qPCR detection of the effect of SFE on gene expression in the Nrf2 / ARE pathway of IR-HepG2 cells
[0036] Cells from each group were collected separately, RNA lysis buffer was added to each well for cell lysis, RNA dilution buffer was added to the lysate, total DNA was extracted, cDNA was obtained by reverse transcription, and the expression of related genes was detected by PCR amplification. GAPDH was used as the internal reference gene, and the relative expression of Nrf2, HO-1, and IRS-1 was calculated according to 2-ΔΔCt. The primer sequences (5′-3′) are shown in Table 1.
[0037] Table 1: Nucleotide sequences of primer sequences
[0038]
[0039] In the experiment of qPCR detection of the effect of SFE on the expression of key genes in the Nrf2 / ARE pathway of IR-HepG2 cells, GAPDH was used as the internal reference gene, and the mRNA expression of the CON group was normalized compared with that of the other groups. Figure 4 As shown in the figure, compared with the CON group, the mRNA expression levels of HO-1, Nrf2, and IRS1 in the MOD group were significantly downregulated (P<0.001). The levels of HO-1, Nrf2, and IRS1 in the ROSI and SFE groups were significantly increased (P<0.001), indicating that SFE can exert antioxidant activity by regulating the HO-1 gene. On the other hand, after the addition of ROSI and SFE, the IRS-1 gene level was significantly increased, indicating that SFE improved the insulin resistance of HepG2 cells. In summary, SFE improved the oxidative stress and insulin resistance of IR-HepG2 cells by regulating the expression of key genes in the Nrf2 / ARE pathway.
Claims
1. Application of Sargassum fusiformis aqueous extract in the preparation of drugs for the treatment of type 2 diabetes.
2. Application of Sargassum fusiformis aqueous extract in the preparation of drugs for improving cellular oxidative stress and insulin resistance.
3. The use according to claim 2, characterized in that The cells are IR-HepG2 cells.
4. The use according to any one of claims 1 to 3, characterized in that The Sargassum fusiformis water extract is prepared by the following method: The Sargassum fusiformis is soaked in water for desalination, washed and impurity-free, and then used for later use; the Sargassum fusiformis is added with deionized water and heated at 100° C. in a pressure cooker; after the heating, the tap water is cooled to room temperature, the solution is collected and centrifuged to obtain the supernatant, and the supernatant is concentrated and freeze-dried to obtain the Sargassum fusiformis water extract SFE.
5. The use according to claim 4, characterized in that The desalting time was 3 h, and the material-liquid ratio of Sargassum fusiformis to deionized water was 1 g:10 L.
6. The use according to claim 4, characterized in that The pressure cooker heating time was 15 min, and the centrifugal condition was 4000 r / min for 20 min.