Use of smilagenin in the preparation of a medicament for preventing or treating atherosclerosis

Abstract

The application provides application of sarsasapogenin in preparation of a medicine for preventing or treating atherosclerosis. The sarsasapogenin can significantly improve multiple indexes of atherosclerosis mice. Pharmacokinetics and in-vivo distribution test results show that the sarsasapogenin can effectively enter peripheral blood circulation to play a vascular protection role, and is not easy to accumulate in the liver, thereby avoiding liver toxicity caused by a first pass effect; in the safety test, the sarsasapogenin shows extremely high in-vivo safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to new uses of active ingredients of natural products, particularly the application of Sarsasapogenin in the preparation of drugs for the prevention or treatment of atherosclerosis (AS) and cardiovascular-related diseases. Background Technology

[0002] Atherosclerosis (AS) is the main pathological basis for acute and chronic cardiovascular diseases such as coronary heart disease, cerebral infarction, and peripheral vascular disease. Its pathogenesis is complex, mainly manifested as lipid metabolism disorders, vascular endothelial damage, smooth muscle cell proliferation and migration, macrophage-derived foam cell formation, and chronic vascular wall inflammation. Currently, statins (such as simvastatin) are commonly used clinically for lipid-lowering and plaque-stabilizing treatment; however, their therapeutic effect is limited, reducing plaque reversal by only 0.3% to 1.2%, and long-term use carries adverse reactions such as liver and kidney damage and rhabdomyolysis. Furthermore, its efficacy is limited in some patients with a "residual inflammation risk."

[0003] Sarsasapogenin (molecular formula: C) 27 H 44 O3 is a natural steroidal saponin widely found in plants of the Liliaceae family (such as Anemarrhena asphodeloides and Smilax china). Previous studies have shown that it has certain antitumor, antidepressant, and anti-inflammatory effects. However, there are currently no clear reports on the use of smilax saponin alone for the prevention or treatment of ankylosing spondylitis (AS), nor are there any systemic pharmacological patents disclosed regarding its molecular regulatory mechanisms on aortic plaque lesions, lipid deposition, and vascular endothelial adhesion. Therefore, in-depth research on the role of smilax saponin in anti-AS is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a drug that is highly effective, safe, and effective in preventing or treating AS in both male and female individuals, namely, a new use of Sarsasapogenin in AS.

[0005] This invention provides the use of smilax saponin in the preparation of drugs for the prevention or treatment of atherosclerosis.

[0006] Furthermore, the prevention or treatment of atherosclerosis includes one or more of the following: reducing plaque area, reducing lipid deposition, increasing collagen content in plaques, and inhibiting vascular endothelial inflammatory response.

[0007] Furthermore, the sarsaponin plays a role in preventing or treating atherosclerosis by at least one of reducing aortic lipid deposition, reducing aortic plaque area, and increasing aortic collagen fiber content to increase plaque stability.

[0008] Furthermore, the sarsaponin can inhibit vascular endothelial inflammatory response and exert an anti-atherosclerotic effect by downregulating the expression of inflammatory factors (TNF-α, IL-1β) and vascular endothelial adhesion molecules (VCAM-1, ICAM-1).

[0009] Furthermore, the drug is an oral preparation (such as tablets, capsules) or injection prepared with smilax saponin as the sole active ingredient, or with the addition of pharmaceutically acceptable excipients.

[0010] This invention also provides the use of smilax saponin in the preparation of medicaments for the prevention or treatment of cardiovascular-related diseases.

[0011] Furthermore, the cardiovascular-related diseases include atherosclerotic cardiovascular diseases.

[0012] Furthermore, the cardiovascular-related diseases include coronary heart disease or stroke.

[0013] This invention also provides the application of smilax saponin in the preparation of pharmaceuticals; The drug is used for at least one of the following effects: downregulating the expression of inflammatory factors (TNF-α, IL-1β) and vascular endothelial adhesion molecules (VCAM-1, ICAM-1), thereby inhibiting vascular endothelial inflammatory responses.

[0014] Beneficial effects This invention provides a drug that targets blood vessels and has no hepatotoxicity or nephrotoxicity. Pharmacokinetic studies show that smilax saponin is detectable in plasma after administration, indicating its ability to enter peripheral blood circulation; however, no accumulation was detected in liver tissue, suggesting a low risk of first-pass effect and hepatic accumulation. Blood biochemistry (ALT, AST, BUN, CREA-S, AMS, CK, LDH) and liver and kidney histopathological examinations showed no significant differences between the administered group and the control group, indicating that the compound does not exhibit significant toxicity to the liver, kidneys, pancreas, and myocardium, providing a new approach for the development of anti-atherosclerotic drugs.

[0015] This invention also utilizes sarsaki saponin to treat atherosclerotic female and male mice, and evaluates aortic lipid deposition, plaque area, and collagen fiber content in both mice. The results show that sarsaki saponin significantly reduces high-fat diet-induced apolipoprotein E gene knockout (ApoE). - / - It promotes aortic plaque formation and lipid accumulation in both female and male mice, increases collagen deposition, and enhances the stability of vulnerable plaques.

[0016] This invention also detected the release levels of vascular endothelial inflammatory factors. The test results showed that sarsaponin could significantly downregulate the mRNA and protein expression of vascular adhesion molecules (ICAM-1, VCAM-1) and pro-inflammatory cytokines (TNF-α, IL-1β) in a dose-dependent manner, indirectly suggesting that it may inhibit adhesion and improve the vascular endothelial microenvironment.

[0017] In summary, the sarsaponin of the present invention is a potential drug for treating atherosclerosis or cardiovascular-related diseases. Attached Figure Description

[0018] Figure 1 The pharmacokinetic characteristics and in vivo distribution of sarsaki saponin are presented. Among them, Figure 1 In diagram A, the experimental design flowchart is shown. Figure 1 Image B is a mass spectrometry result obtained by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), showing the content of sarsaki saponin in blood and liver tissue.

[0019] Figure 2 These are the results of a safety study on sarsaki saponin. Among them, Figure 2 In diagram A, the experimental design flowchart is shown. Figure 2 The BE values ​​represent the liver, kidney, pancreas, and myocardial damage indicators, as well as the toxicity biochemical indicators of endotoxins in mice treated with smilax saponin.

[0020] Figure 3 These are the results of a safety study on sarsaki saponin. Among them, Figure 3 In diagram A, the experimental design flowchart is shown. Figure 3 The results of HE staining in the middle section show the morphology of liver and kidney tissues in the drug-treated group.

[0021] Figure 4 Smilax saponin to ApoE - / - Morphological evaluation of the effects of aortic plaque and lipid deposition on female and male mice. Among them, Figure 4 In diagram A, the experimental design flowchart is shown. Figure 4 B in the middle is ApoE - / - Gross diagram of lipid deposition in the aorta of female and male mice.

[0022] Figure 5 Smilax saponin to ApoE - / Morphological evaluation of the effects of aortic plaque and lipid deposition in male mice. Figure 5 In the middle, AB represents the HE staining diagram. Figure 5 The middle CD is an image stained with Oil Red O and Figure 5 E in the middle is the Masson staining diagram ( Figure 5 In the text, "*" indicates a comparison with the normal group; "#" indicates a comparison with the model group.

[0023] Figure 6 Smilax saponin to ApoE - / - Morphological evaluation of the effects of aortic plaque and lipid deposition on female mice. Figure 6 In the middle, AB represents the HE staining diagram. Figure 6 The middle CD is an image stained with Oil Red O and Figure 6 E in the middle is the Masson staining diagram ( Figure 6 In the text, "*" indicates a comparison with the normal group; "#" indicates a comparison with the model group.

[0024] Figure 7 This diagram illustrates the mechanism by which sarsaki saponin regulates the expression of atherosclerosis-related inflammation and adhesion molecules. It includes relative mRNA expression levels and Western blotting analysis. Figure 7 In the middle, A represents the result at the gene transcription level (mRNA); Figure 7 BC represents the protein translation level (Western Blot) results. Figure 7 In the text, "*" indicates a comparison with the normal group; "#" indicates a comparison with the model group. Detailed Implementation

[0025] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0026] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0027] This invention provides the application of smilax saponin in the preparation of anti-atherosclerotic drugs.

[0028] This invention does not specifically limit the source and preparation method of sarsaki saponin; it can utilize common commercially available products in the field, or be prepared according to known extraction or preparation methods. The sarsaki saponin used in the embodiments of this invention was purchased from Hubei Cuiyuan Biotechnology Co., Ltd.

[0029] Example 1 1. Drug source Smilax saponin was purchased from Hubei Cuiyuan Biotechnology Co., Ltd.

[0030] 2. Establishment and grouping of animal models SPF-grade healthy 8-week-old female and male ApoE - / - Mice, including SPF-grade male and female C57BL / 6J mice. After one week of acclimatization, ApoE...- / - An ankylosing spondylitis (AS) mouse model was established by feeding mice a high-fat diet (40% fat, 20% protein, 40% carbohydrates, Cat#D12108C) for 12 weeks. Successfully established AS mice were randomly divided into a high-fat diet (HFD), a low-dose smilax glabra aglycone group (Sar-L), a medium-dose smilax glabra aglycone group (Sar-M), a high-dose smilax glabra aglycone group (Sar-H), and a positive control simvastatin group (SIM). A Ctrl group was also included. From week 12, the Sar-L, Sar-M, and Sar-H groups received smilax glabra aglycone via gavage at low (40 mg / kg), medium (60 mg / kg), and high (80 mg / kg) doses, respectively. The Ctrl and HFD groups received an equal volume of saline via gavage, and the SIM group received simvastatin (4 mg / kg) via gavage. All interventions lasted for 4 weeks. Mice were sacrificed in week 16 to obtain aortic tissue.

[0031] 3. In vivo pharmacokinetic analysis and tissue distribution Male C57BL / 6J mice were randomly divided into a normal control group and a smilax saponin administration group (n=6 in each group). The administration group received smilax saponin (80 mg / kg) daily by gavage, while the normal control group received an equal volume of physiological saline. This treatment continued for 4 weeks. After the last administration, blood was collected via the orbital sinus, and serum was separated. Mice were sacrificed, and liver tissue was harvested. Serum samples were directly processed, and liver tissue was homogenized. Proteins were precipitated by acetonitrile, and the distribution concentrations of smilax saponin in plasma and liver were determined using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Figure 1 (A)

[0032] Mass spectrometry results show ( Figure 1 In the plasma sample (B), sarsaki saponin (retention time approximately 8.5 min, calculated theoretical molecular weight m / z 417.33685) was significantly detected, but not in the liver sample. This indicates that sarsaki saponin can effectively enter the peripheral blood circulation to exert a vascular protective effect, and is not prone to accumulation in the liver, thus avoiding the liver toxicity caused by the first-pass effect.

[0033] 4. Security Testing Mice in the Ctrl and Sar-H groups were administered smilax saponin via gavage daily for four consecutive weeks. Twenty-four hours after the last administration, blood was collected from the orbital cavity for sacrifice. Serum was collected, and biochemical indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (CREA-S), lipase (LPS), amylase (AMS), creatine kinase (CK), and lactate dehydrogenase (LDH) were measured. Simultaneously, the liver and kidneys were harvested, fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a light microscope to assess pathological changes in the liver and kidney tissues.

[0034] Biochemical results showed ( Figure 2 In the AD group (represented by Sar in the figure), there were no significant differences (ns) in the serum levels of liver function indicators (ALT, AST), kidney function indicators (BUN, CREA-S), pancreatic indicators (AMS), myocardial injury indicators (CK, LDH), and endotoxin (LPS) in the Sar-H (represented by Sar in the figure) group compared with the Ctrl group.

[0035] Pathological results showed: liver and kidneys ( Figure 3 HE staining (10× and 40×) of the Sar-H group showed that, compared with the Ctrl group, the Sar-H group had normal cell morphology, intact structure, and no necrosis or inflammatory infiltration. This indicates that sarsaponin has extremely high in vivo safety.

[0036] 5. ApoE - / - Atherosclerotic plaque area and lipid deposition in mice The aorta was completely removed, peripheral adipose tissue was removed, and lipid deposition in the aorta was observed under a gross microscope. Mouse aorta sections were immersed in Oil Red O working solution (0.5% Oil Red O dissolved in isopropanol:water = 3:2) for 15 min, then separated with 60% isopropanol until the background was clear. After rinsing with running water, images were acquired using a digital pathological slide scanner. Paraffin sections of mouse aorta were routinely dewaxed to water, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol, stained with eosin for 2 min, dehydrated and cleared, and mounted with neutral resin. The morphology of aortic sinus plaques, necrotic cores, and inflammatory cell infiltration were observed. Paraffin sections of mouse aorta were stained with Weigert iron hematoxylin for nuclei, Biebrich scarlet-acid fuchsin for cytoplasm and collagen, differentiated with phosphomolybdic acid, stained with aniline blue for collagen fibers, treated with acetic acid, dehydrated, and mounted. Collagen fibers (blue), muscle fibers (red), and cellular components within the plaque were distinguished, and plaque stability was assessed. Figure 4 (A)

[0037] Gross microscopy results show ( Figure 4In the Ctrl group (B group), the aortic wall was smooth with no lipid accumulation; compared with the Ctrl group, the aortic lipid accumulation in the HFD group was significantly increased; after intervention with different doses of Sar (L / M / H) and the positive control drug SIM, aortic lipid accumulation was reduced, and the effect of sarsaki saponin showed a clear dose-dependent effect. Sex difference analysis showed that there was no significant difference in protective effect between male and female mice, and the efficacy was highly consistent.

[0038] HE staining results showed that ( Figure 5 and 6 In the AB group and the Ctrl group, the aortic intima was smooth and no plaques formed. Compared with the Ctrl group, the aortic intima in the HFD group was significantly thickened, forming huge atherosclerotic plaques with large areas of necrotic cores. After intervention with smilax saponin (40 mg / kg, 60 mg / kg, 80 mg / kg), the aortic plaque area in both male and female mice was significantly reduced, showing a typical dose-dependent decrease, with effects comparable to simvastatin (SIM).

[0039] Oil Red O staining results showed ( Figure 5 and 6 In the CD group, compared with the Ctrl group, the HFD model group showed a large number of red-stained lipid deposition areas on the tube wall. After intervention with sarsaparilla saponin (40 mg / kg, 60 mg / kg, 80 mg / kg), compared with the HFD model group, the red-stained lipid areas on the tube wall of male and female mice in each dose group of sarsaparilla saponin were significantly reduced, and the effect of the high-dose group of sarsaparilla saponin was comparable to that of simvastatin (SIM).

[0040] Masson trichrome staining results showed that ( Figure 5 and 6 (E): In the HFD group, the blue collagen fibers in the plaques were sparse, indicating that the plaques were vulnerable. After treatment with smilax saponin, the area indicated by the red arrow in the figure clearly showed a significant increase in the blue collagen fibers on the surface and inside of the plaques, and a significant thickening of the fibrous cap, indicating that it can effectively increase the stability of the plaques and prevent plaque rupture and secondary thrombosis.

[0041] 6. Detection of vascular endothelial inflammation and adhesion molecules The following groups were set up: Ctrl group, HFD group, low-, medium-, and high-dose sarsakinin groups (Sar-L, Sar-M, Sar-H), and SIM group. Total RNA and total protein were extracted from aortic tissue, and the expression levels of target genes and proteins were detected by qRT-PCR and Western Blot, respectively.

[0042] Gene transcription level (mRNA) results showed ( Figure 7(A) Compared with the Ctrl group, the mRNA expression levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and pro-inflammatory cytokines (TNF-α, IL-1β) were significantly upregulated in the HFD group. P <0.001). After intervention with different doses of sarsaki saponin (L / M / H) and the positive control drug SIM, the expression of the above-mentioned inflammation-related genes in each treatment group was downregulated, and the sarsaki saponin treatment group showed a dose-dependent trend, indicating that sarsaki saponin can effectively inhibit inflammatory signals in the process of atherosclerosis.

[0043] Protein translation level (Western Blot) results showed ( Figure 7 (BC): Protein band and quantitative statistical results were consistent with the mRNA trend. Compared with the Ctrl group, the expression of ICAM-1, VCAM-1, TNF-α and IL-1β proteins increased in the HFD group; compared with the HFD group, the expression levels of these four pathogenic proteins decreased significantly with increasing dose in the Sar-L, Sar-M and Sar-H treatment groups.

[0044] The test results above show that sarsaponin plays a core role in anti-atherosclerosis by inhibiting the infiltration of inflammatory cells into the blood vessel wall through dose-dependent blocking of the release of inflammatory factors and the expression of endothelial adhesion molecules.

[0045] In summary, sarsaki saponin significantly improved multiple indicators in mice with atherosclerosis. Pharmacokinetic and in vivo distribution tests showed that sarsaki saponin effectively entered the peripheral blood circulation to exert vascular protective effects and did not easily accumulate in the liver, avoiding the hepatotoxicity caused by the first-pass effect. In safety tests, sarsaki saponin demonstrated extremely high in vivo safety. At the plaque lesion level, sarsaki saponin reduced aortic plaque area and lipid deposition, increased collagen fiber content, and improved plaque stability. In inflammatory response tests, sarsaki saponin reduced the mRNA and protein expression levels of ICAM-1, VCAM-1, TNF-α, and IL-1β in the aorta, alleviating endothelial cell inflammation, comprehensively demonstrating the therapeutic potential of sarsaki saponin for atherosclerosis. This patent systematically elucidates the molecular mechanism by which sarsaponin exerts its anti-atherosclerotic effect by regulating the inflammatory response, providing a solid experimental basis and theoretical support for its clinical translation and application, while also opening up new candidate directions for the development of innovative anti-atherosclerotic drugs.

[0046] The exemplary embodiments of the present invention have been described above. However, the scope of protection of the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of smilax saponin in the preparation of drugs for the prevention or treatment of atherosclerosis.

2. The application according to claim 1, wherein, The anti-atherosclerotic effect includes one or more of the following: reducing plaque area, reducing lipid deposition, increasing collagen content in plaques, and inhibiting vascular endothelial inflammatory response.

3. The application according to claim 1 or 2, wherein, The smilax saponin plays a role in preventing or treating atherosclerosis by at least one of reducing aortic lipid deposition, reducing aortic plaque area, and increasing aortic collagen fiber content to increase plaque stability.

4. The application according to any one of claims 1-3, wherein, The sarsaponin aglycone exerts its anti-atherosclerotic effect by downregulating the expression of inflammatory factors (TNF-α, IL-1β) and vascular endothelial adhesion molecules (VCAM-1, ICAM-1).

5. The application according to any one of claims 1-4, wherein, The drug is an oral or injectable preparation made with smilax saponin as the sole active ingredient, or with pharmaceutically acceptable excipients.

6. Application of sarsaponin in the preparation of drugs for the prevention or treatment of cardiovascular-related diseases.

7. The application according to claim 6, wherein, The cardiovascular-related diseases mentioned include atherosclerotic cardiovascular diseases.

8. The application according to claim 6 or 7, wherein the cardiovascular-related disease includes coronary heart disease or stroke.

9. Application of sarsaki saponin in drug preparation; The drug is used for at least one of the following effects: downregulating the expression of inflammatory factors (TNF-α, IL-1β) and vascular endothelial adhesion molecules (VCAM-1, ICAM-1), thereby inhibiting vascular endothelial inflammatory response.

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