Steroid saponin compound in longstalk allium macrostemon, preparation method of steroid saponin compound and application of steroid saponin compound in preparation of medicine for improving and / or treating myocardial infarction

Abstract

The invention discloses steroid saponin compounds in longstalk allium macrostemon, a preparation method of the steroid saponin compounds and application of the steroid saponin compounds in preparation of medicines for improving and / or treating myocardial infarction. According to the invention, two compounds, i.e., long-stalk allium macrostemon saponin A and degalactotigonin, are separated from long-stalk allium macrostemon. In an MI mouse model, the two compounds can significantly improve heart function indexes (EF and FS), reduce myocardial infarction area, inhibit inflammatory infiltration and collagen deposition, and improve pathological injury; the levels of cTnI, LDH and CK-MB are reduced; the expression of inflammation related factors and proteins (caspase-1, ASC, GSDMD, IL-1beta and IL-18) can be inhibited. In-vitro experiments prove that the two compounds can effectively protect H9c2 myocardial cells damaged by oxygen-glucose deprivation. The two compounds can be used for preparing medicines for improving and / or treating myocardial infarction, and can be used for manufacturing chemical raw material medicines and preparations.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a steroidal saponin compound from Allium macrostemon, its preparation method, and its application in the preparation of drugs for improving and / or treating myocardial infarction. Background Technology

[0002] Acute myocardial infarction (AMI), also known as myocardial infarction, is a life-threatening disease caused by acute obstruction of the coronary arteries, leading to insufficient blood supply to the corresponding myocardial region and resulting in myocardial necrosis. This disease is mainly caused by multiple factors, including insufficient blood supply, myocardial hypoxia, and excessive myocardial oxygen consumption due to various causes such as coronary atherosclerosis, arrhythmia, and respiratory failure. Although reperfusion therapy can reduce mortality in patients with acute myocardial infarction, some patients miss the treatment opportunity due to delayed medical attention, and there is a certain probability of no-reflow after reperfusion. After myocardial infarction, myocardial cells in the ischemic area die, subsequently initiating an inflammatory repair response; necrotic tissue is gradually replaced by fibrous scar tissue, losing its contractile function. Long-term effects can lead to ventricular remodeling, ultimately resulting in heart failure. Therefore, intervening in the progression of the infarcted area after myocardial infarction is a crucial step in reducing mortality.

[0003] Saponins are widely distributed in the plant kingdom, among which steroidal saponins, as an important class of bioactive substances, are commonly found in plants of the Liliaceae and Dioscoreaceae families. These components are widely used in the pharmaceutical field due to their various effects, including anti-myocardial ischemia, anti-inflammation, anti-cancer, and antifungal activity. The anti-myocardial ischemia activity of steroidal saponins has been extensively studied, and diosgenin has been developed into a drug for clinical use. Steroidal saponins are the main pharmacologically active components of Allium species. *Allium longipeense* is a plant of the Liliaceae family and the Allium genus. Allium neriniflorum The bulb of *Allium macrostemon* (Herb.) Baker is used in Mongolian medicine to treat chest and rib pain, angina pectoris, cough with excessive phlegm, dysentery, and to detoxify pufferfish poisoning. Chest and rib pain is similar to the symptoms of acute myocardial infarction in modern medicine, and it is also used in traditional Chinese medicine as a treatment for ischemic myocardial diseases. Currently, research on the extraction, separation, structural identification, and related pharmacological studies of *Allium macrostemon* is still in its initial stages both domestically and internationally. Steroidal saponins are one of its main chemical components, but there are currently no reports on the anti-acute myocardial infarction effects of *Allium macrostemon* or its monomeric compounds. Therefore, systematic and in-depth research on the steroidal saponins in *Allium macrostemon*, the discovery of new active steroidal saponin compounds and highly effective, low-toxicity drug lead compounds, is of great significance for their use in the manufacture of chemical pharmaceutical raw materials and formulations. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a steroidal saponin compound from Allium macrostemon.

[0005] A second objective of this invention is to provide a method for preparing steroidal saponin compounds from the aforementioned Allium tuberosum.

[0006] A third object of the present invention is to provide the use of the steroidal saponin compounds in the *Allium chinense* in the preparation of medicaments for improving and / or treating myocardial infarction.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: A steroidal saponin compound from Allium tuberosum, named (25R)-5α-spirostane-2-one 3-O- β -D-glucopyranosyl(1→2)-[ β -D-xylanose-1→3]- β -D-glucopyranosyl(1→4)- β -D-galactopyranoside ((25R)-5 α -spirostan-2-one 3-O- β -D-glucopyranosyl(1-2)-[ β -D-xylopyranosyl(1-3)- β -D-glucopyranosyl(1-4)- β -D-galactopyranoside), named Allium longiflorum saponin A, has the following structural formula (I): Formula (I).

[0008] The present invention also provides a method for preparing steroidal saponin compounds from *Allium chinense*, comprising the following steps: S1. Solvent extraction: Long-stemmed Allium macrostemon was extracted by reflux with 60-90% ethanol, and the solvent was removed from the extract under reduced pressure to obtain an extract. S2. Extraction: The extract is dissolved and then extracted with water-saturated n-butanol to obtain n-butanol extract; S3. Purification: The n-butanol extract was subjected to silica gel column chromatography with a dichloromethane:methanol solvent system of 50:1→20:1→10:1→5:1→3:1→1:1→0:1 to obtain the 3:1 elution fraction, which was then washed and purified to obtain the insoluble total saponins. S4. Separation: The sparingly soluble total saponins were separated by preparative high performance liquid chromatography using a C18 reverse-phase preparative column with acetonitrile-water as the mobile phase and a retention time of 15 min.

[0009] Preferably, the long-stemmed Allium tuberosum mentioned in step S1 is freshly harvested long-stemmed Allium tuberosum. The raw material is immediately soaked in 70-80% ethanol after harvesting, which can inhibit the degradation of saponin structure by enzymes.

[0010] Further, the ethanol mentioned in step S1 is 70-80% ethanol (preferably 75% ethanol).

[0011] Preferably, the ethanol reflux extraction in step S1 is performed by heating and refluxing with 70-80% ethanol (preferably 75% ethanol) 2-4 times (preferably 3 times), each time for 1-3 hours (preferably 2 hours), followed by filtration and combining the extracts.

[0012] Further, the extraction in step S2 involves sequential extraction with n-hexane, ethyl acetate, and water-saturated n-butanol. Hexane is primarily used to extract low-polarity components, such as oils and small-molecule terpenes; ethyl acetate is mainly used to extract medium-polarity components, such as flavonoids and coumarins; and water-saturated n-butanol is mainly used to extract medium-to-high-polarity components, such as saponins. This allows for better enrichment of saponins.

[0013] Furthermore, the washing and purification in step S3 involves washing with hexane, methanol, and water sequentially to remove free steroids, oligosaccharides, and methanol-soluble compounds mixed in the components, thereby enriching the target saponins.

[0014] Furthermore, the acetonitrile-water volume ratio in step S4 is 75:25.

[0015] Furthermore, the elution flow rate in step S4 is 4–6 mL / min (preferably 5 mL / min).

[0016] In a preferred embodiment, the method for preparing steroidal saponin compounds from *Allium chinense* is as follows: freshly harvested *Allium chinense* is promptly soaked in 75% ethanol to prevent saponin degradation, and an extract is obtained by extraction with 75% ethanol. The extract is dispersed in water and extracted sequentially with n-hexane, ethyl acetate, and water-saturated n-butanol to obtain the corresponding components. The water-saturated n-butanol fraction is crudely separated by open silica gel column chromatography, using dichloromethane:methanol (50:1 to 0:1) as the mobile phase for elution. The 3:1 eluted fraction is placed in a Buchner funnel lined with filter paper and washed sequentially with n-hexane, methanol, and water. Filtering removes the free steroids, oligosaccharides, and methanol-soluble compounds mixed in the fraction, thus obtaining the insoluble total saponins of the n-butanol fraction of *Allium chinense*. Subsequently, preparative HPLC was performed for chromatographic separation using a C18 reverse preparative column and elution with 75% acetonitrile (75:25 acetonitrile:water) as the mobile phase to obtain the steroidal saponin compounds.

[0017] This invention isolated two steroidal saponin compounds from *Allium longipes* using the above method: Compound 1: *Allium longipes* saponin A (a new compound, structural formula shown in formula (I) above); Compound 2: degalactoside (a known compound), named (25R)-5α-spirostane 3-O- β -D-glucopyranosyl(1→2)-[β-D-xylopyranosyl(1→3)]-β-D-glucopyranosyl(1→4)- β -D-galactopyranoside ((25R)-5 α -spirostan 3-O- β -D-glucopyranosyl(1-2)-[ β -D-xylopyranosyl(1-3)- β- D-glucopyranosyl(1-4)- β -D-galactopyranoside), the structure is shown in equation (II) below: Equation (II).

[0018] In the n-butanol fraction of *Allium longipes*, compound 1 accounted for 4.74% and compound 2 accounted for 14.78%. The high content of both compounds in *Allium longipes* indicates that this plant is suitable as a source of raw materials for the preparation of compounds 1 and 2.

[0019] In existing technologies, degalactostatin possesses broad-spectrum antitumor activity, anti-influenza virus activity (inhibiting both type A and type B influenza viruses), and potential for treating acute and chronic bronchitis, with significant antitussive effects. However, its role in acute myocardial infarction has not been reported. This invention further investigates the application of Allium macrostemon saponin A and degalactostatin in the treatment of acute myocardial infarction.

[0020] In this invention, both Allium longiflorum saponin A and degalactoside can reduce the mortality rate of mice with acute myocardial infarction, with Allium longiflorum saponin A more significantly reducing the mortality rate of the mouse model of acute myocardial infarction.

[0021] In this invention, *Allium longiflorum* saponin A and degalactoside tagarotin can significantly improve cardiac dysfunction caused by acute myocardial infarction. The results of the embodiments of this invention demonstrate that *Allium longiflorum* saponin A and degalactoside tagarotin can significantly improve cardiac contractile function in a mouse model of acute myocardial infarction, reverse changes in echocardiographic parameters in the mouse model of acute myocardial infarction, and achieve the effect of improving cardiac dysfunction.

[0022] In this invention, *Allium longiflorum* saponin A and degalactotigan saponin can significantly inhibit the inflammatory response induced by acute myocardial infarction. The results of the embodiments of this invention demonstrate that *Allium longiflorum* saponin A and degalactotigan saponin can efficiently regulate the levels of inflammatory factors in serum and myocardial tissue in a mouse model of acute myocardial infarction, and have an inhibitory effect on the inflammatory response.

[0023] In this invention, *Allium longiflorum* saponin A and degalactoside A can significantly inhibit changes in serum LDH and CK-MB levels. The results of the examples provided by this invention demonstrate that these two compounds can significantly reduce serum LDH and CK-MB levels in a mouse model of acute myocardial infarction.

[0024] In this invention, *Allium longipes* saponin A and degalactoside can significantly reduce the infarct area, improve cardiac function, and inhibit inflammatory responses, thereby achieving highly effective improvement and / or treatment of acute myocardial infarction and reducing the mortality rate of acute myocardial infarction. The results of the embodiments of this invention show that both compounds achieve good effects in improving and / or treating acute myocardial infarction, and *Allium longipes* saponin A is more effective than degalactoside in improving and / or treating acute myocardial infarction.

[0025] This invention proposes a possible structure-activity relationship between Allium longiflorum saponin A and degalactoside: 1. Key fragments of anti-myocardial infarction activity: (1) Substituents: ketone carbonyl group (C=O) substitution at C-2 position; (2) Specific tetrasaccharide chain: The C-3 position is composed of the core fragment Glu(1→2)-[Xyl(1→3)]-Glu(1→4)-Gal.

[0026] (3) Interaction between C-2 and C-3 positions: The sugar chain at C-3 position is eventually exposed by the metabolic breakdown in vivo; the exposed C-3 position -OH and the ketone carbonyl group (C=O) at the adjacent C-2 position are very likely to form an intramolecular hydrogen bond; such hydrogen bond is crucial for the key conformation of the active metabolite and may be related to its biological activity.

[0027] 2. Speculated mechanism of activity: Among spirosterosteroids, allium longipesin A exhibited the strongest cardioprotective effect. Structure-activity relationship analysis showed that allium longipesin A has a ketone carbonyl substitution at the C-2 position. The C-2 ketone carbonyl group may coordinate with free iron ions (Fe2+) through coordination. 2+ / Fe 3+The binding of the ketone carbonyl group inhibits the iron-mediated Fenton reaction, thereby reducing the generation of reactive oxygen species (ROS), blocking the activation of the ferroptosis pathway, and thus exerting an antioxidant effect. In addition, the strong electron-withdrawing property of the ketone carbonyl group can regulate the redox activity of the adjacent hydroxyl group, enhance the free radical scavenging ability, and alleviate oxidative stress damage in the OGD / R model.

[0028] The C-3 position glycan substitution may enhance molecular polarity and water solubility, promoting binding to cell membrane surface receptors. It also protects the steroid nucleus from enzymatic degradation, thus prolonging drug efficacy.

[0029] Therefore, the present invention also provides the use of Allium longiflorum saponin A or its derivative desgalactotigonin in the preparation of drugs for improving and / or treating myocardial infarction.

[0030] Furthermore, the improvement and / or treatment of myocardial infarction includes any one or more of the following (1) to (8): (1) Reduce the area of ​​myocardial infarction; (2) Reduce the mortality rate of myocardial infarction; (3) Reduce collagen deposition after myocardial infarction; (4) Improves cardiac dysfunction; (5) Inhibits inflammatory response; (6) Reduce serum levels of cTnI, LDH, and MDA, and increase SOD activity; (7) Inhibits the expression of inflammatory factors and proteins caspase-1, ASC, GSDMD, IL-1β, and IL-18; (8) Protects H9c2 cardiomyocytes damaged by oxygen and glucose deprivation.

[0031] Furthermore, the myocardial infarction is an acute myocardial infarction.

[0032] The present invention also provides a drug for treating myocardial infarction, the drug containing steroidal saponin compounds from Allium macrostemon as shown in formula (I) above.

[0033] Furthermore, the dosage form of the drug includes, but is not limited to, powder, oral liquid, injection, honey pill, or granules.

[0034] This invention, through the isolation and activity experiments of the main component compounds with high content in *Allium chinense*, obtained new monomeric compounds with large content, good activity, and easy preparation. This provides a scientific basis and experimental research foundation for in-depth research on *Allium chinense* and its monomeric compounds in the field of myocardial ischemia, as well as the development and utilization of *Allium chinense* natural resources.

[0035] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a novel steroidal saponin compound extracted from *Allium macrostemon* and its preparation method. The preparation method includes solvent extraction, extraction, silica gel column chromatography, and preparative HPLC separation. It also provides the application of this novel compound and its derivative, degalactostatin, in the treatment of myocardial infarction (MI). Pharmacological studies have shown that in a mouse model of MI, the two compounds significantly improve cardiac function indicators (EF, FS), reduce infarct size, inhibit inflammatory infiltration and collagen deposition, and improve pathological damage; reduce the levels of cTnI, LDH, and CK-MB; and inhibit the expression of inflammatory factors and proteins (caspase-1, ASC, GSDMD, IL-1β, IL-18). In vitro experiments have confirmed that the two compounds effectively protect H9c2 cardiomyocytes damaged by oxygen and glucose deprivation. This indicates that the two compounds can be used to prepare drugs for improving and / or treating myocardial infarction. Furthermore, the two compounds are high in content, easy to prepare, and have significant therapeutic effects, making them suitable for use as raw materials and in the manufacture of chemical pharmaceuticals, demonstrating great development potential. Attached Figure Description

[0036] Figure 1 This is the hydrogen spectrum of steroidal saponin compound 1.

[0037] Figure 2 This is the carbon spectrum of steroidal saponin compound 1.

[0038] Figure 3 This is the dept 135 spectrum of steroidal saponin compound 1.

[0039] Figure 4 This is the dept 90 spectrum of steroidal saponin compound 1.

[0040] Figure 5 H is a steroidal saponin compound 1 1 -H 1 COSY spectrum.

[0041] Figure 6 This is the HSQC spectrum of steroidal saponin compound 1.

[0042] Figure 7 The image shows the HMBC spectrum of steroidal saponin compound 1.

[0043] Figure 8 This is the NOESY spectrum of steroidal saponin compound 1.

[0044] Figure 9 This is the hydrogen spectrum of steroidal saponin compound 2.

[0045] Figure 10 This is the carbon spectrum of steroidal saponin compound 2.

[0046] Figure 11 The survival rate of mice in each group is shown in Example 1.

[0047] Figure 12 Figure 1 shows the results of typical left ventricular M-mode echocardiography of the short axis of the sternum in each group of mice in Example 1 and the analysis of related parameters.

[0048] Figure 13 The graph shows the results of detecting cardiac troponin I (cTnI), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) in each group of mice in Example 1.

[0049] Figure 14 The graph shows the results of IL-1β and IL-18 detection in each group of mice in Example 1.

[0050] Figure 15 The results of TTC staining and the percentage of myocardial infarction area in each group of mice in Example 1 are presented.

[0051] Figure 16 The image shows the HE staining results of mice in each group of Example 1.

[0052] Figure 17 The image shows the results of Masson staining in each group of mice in Example 1.

[0053] Figure 18 The image shows the GSDMD immunofluorescence results of mice in each group of Example 1.

[0054] Figure 19 The image shows the immunofluorescence results of ASC and caspase-1 in each group of mice in Example 1.

[0055] Figure 20 The graph shows the survival rate of H9C2 cardiomyocytes in each group of the oxygen-glucose deprivation model applied in Example 2. Detailed Implementation

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0057] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0058] Example 1: Preparation of steroidal saponins from Allium macrostemon longiflorum 150 kg (equivalent to 35 kg dry weight) of freshly harvested long-stalked Allium macrostemon was immediately soaked in 75% ethanol to prevent saponin enzymatic hydrolysis. It was then extracted three times by reflux with 10 times the volume of 75% ethanol, 2 hours each time. The extracts were filtered through gauze, and all extracts were combined. The solvent was removed under reduced pressure to obtain an extract (1300 g). The extract was dissolved in 10 L of deionized water and extracted sequentially with n-hexane, ethyl acetate, and water-saturated n-butanol. The solvent was removed under reduced pressure after each extraction to obtain a hexane layer (200 g), an ethyl acetate layer (120 g), and a water-saturated n-butanol layer (475 g).

[0059] The water-saturated n-butanol fraction was crudely separated by open silica gel column chromatography using a gradient elution system of dichloromethane:methanol (50:1-20:1-10:1-5:1-3:1-1:1-0:1). A large amount of white, poorly soluble components were observed in the 3:1 elution fraction. This fraction was placed in a Buchner funnel lined with filter paper and washed sequentially with n-hexane, methanol, and water. Filtering removed the free steroids, oligosaccharides, and methanol-soluble compounds, yielding the poorly soluble total saponins from the n-butanol fraction of *Allium longipes*. Subsequently, the poorly soluble total saponins were separated by preparative HPLC using a C18 reverse-phase column with 75% acetonitrile (75:25 acetonitrile:water) as the mobile phase at a flow rate of 5 mL / min. Compound 2 eluted at a retention time of 11.2 min, and compound 1 eluted at a retention time of 15 min. Repeated preparative HPLC column chromatography with acetonitrile-water (75:25) yielded degalactoside (70.2 g) and Allium longipes saponin A (22.5 g), which are the steroidal saponin compounds of this invention. In the n-butanol fraction of Allium longipes, compound 1 accounted for 4.74%, and compound 2 accounted for 14.78%. The high content of both compounds in Allium longipes indicates that this plant is suitable as a source of raw materials for the preparation of compounds 1 and 2.

[0060] Example 2: Structural identification of compound 1 The structure of compound 1 obtained in Example 1 was identified, and the spectral data are as follows: Table 1. Saponins A from Allium longiflorum 1 H-NMR and 13 C-NMR data ( δ Unit: ppm J (unit: Hz)

[0061] Compound 1 is a white powder, and its high-resolution mass spectrometry result is [M+Na] at m / z 1071.4990. + The ion peak (calculated value 1071.4983) indicates that the molecular weight of compound 1 is 1048, suggesting its molecular formula is C. 50H 80 O 23 Compound 1 was hydrolyzed with 2 mol / L hydrochloric acid. The hydrolysate was derivatized with sugars and analyzed by GC-MS, which identified it as D-glucose, D-galactose and D-xylose.

[0062] The proton spectrum of compound 1 is shown below. Figure 1 As shown, in 1 H-NMR (400 MHz, Pyridine- d 5) Middle: δ 5.62 (1H, d, J = 6.4 Hz, H-1'''), 5.28 (1H, d, J = 7.1 Hz, H-1''); 5.16 (1H, d, J = 7.0 Hz, H-1''''); 4.97 (1H, d, J = 7.7 Hz, H-1'); 4.87 (1H, dd, J = 11.7, 7.1 Hz, H-3); 4.72(2H, t, J = 9.7 Hz, H-6'), 4.60 (2H, s, H-4', 6'''), 4.54 (2H, m, H-2'', 6''), 4.48 (1H, m, H-2'), 4.42 (2H, m, H-6'''), 4.31 (t, J = 9.0 Hz, H-4'''), 4.24(m, H-6'), 4.14 (m, H-3', 5', 3'', 3''', 2'''', 4'''', 5''''), 4.99 (m, H-2'''), 3.89 (m, H-4'', 5''), 3.83 (m, H-5''', 3''''), 3.69 (t, J = 10.3 Hz, H-5''''), 3.60 (1H, d, J = 10.7 Hz, H-26a), 3.51 (1H, t, J = 10.7 Hz, H-26b); 2.47(2H, d, J= 12.4 Hz, H-1), 2.25 (2H, m, H-4), 2.01 (2H, m, H-1, 15), 1.63 (4H,m, H-12, 4, 23, 24), 1.58 (1H, m, H-7), 1.41 (1H, m, H-15), 1.26 (2H, m, H-6,11), 1.13 (1H, m, H-11), 1.00 (2H, m, H-6, 12); 1.94 (1H, m, H-20), 1.78 (1H,t, J = 7.4 Hz, H-17), 1.54 (3H, m, H-8, 16, 25), 1.43 (1H, m, H-5), 0.71 (1H,m, H-9); 0.70 (3H, d, J = 3.9 Hz, H-27), 0.59 (3H, s, H-19). 0.78 (3H, s, H-18), 1.15 (3H, d, J = 6.3 Hz, H-21).

[0063] The carbon spectrum of compound 1 is shown below. Figure 2 As shown, in 13 C-NMR (100 MHz, Pyridine- d 5) Middle: δ208.2 (C-2),109.6 (C-22), 105.8 (C-1''''), 105.3 (C-1''), 105.2 (C-1'''); 102.3 (C-1'),87.4 (C-3''), 81.6 (C-2''), 81.4 (C-16), 80.9 (C-4'), 79.5 (C-3), 79.1 (C-3''''), 79.0 (C-5'''), 78.3 (C-3'''), 78.0 (C-5''), 76.5 (C-5'), 76.2 (C-2'''', d), 75.5 (C-2'''), 75.4 (C-3'), 72.8 (C-2'), 71.0 (C-4''''), 70.7 (C-4'''), 67.7 (C-5''''), 67.2 (C-26), 63.2 (C-17), 63.2 (C-6''), 62.5 (C-6'''), 60.8 (C6'), 56.5 (C-14), 53.98 (C-9), 53.4 (C-1), 36.8 (C-4), 27.9 (C-6), 32.2(C-7), 21.6 (C-11), 40.1 (C-12), 32.4 (C-15); 44.1 (C-5), 42.3 (C-20), 41.5(C-13), 41.0(C-10), 34.8(C-8), 30.9 (C-25); 32.1 (C-23), 29.6 (C-24), 17.6 (C-27), 16.8 (C-18), 15.3 (C-21), 12.8 (C-19).

[0064] The data for this compound are similar to those for the known compound tigogenin 3-O- β -D-glucopyranosyl(1-2)-[ β -D-xylopyranosyl(1-3)- β -D-glucopyranosyl(1-4)- β Compared to -D-galactopyranoside, the data for the 3-position sugar chain are highly consistent; the difference lies in the carbon at the C-2 position of compound 1. δ 32.7 Displaced towards the lower field region δ208 indicates a carbonyl substitution at the C-2 position. This is further supported by the increased chemical shifts at the adjacent C-1 and C-3 positions, which enhance the deshielding effect of the carbonyl group at C-2. Combined with DEPT spectroscopy... Figure 3 , Figure 4 ), through HSQC spectrum ( Figure 6 The hydrogen atom was assigned to the carbon atom it was directly bonded to, through... 1 H- 1 H COSY ( Figure 5 ) spectrum and HMBC spectrum ( Figure 7 This confirmed the correctness of the compound's structural hypothesis. The NOESY spectrum (…) Figure 8 H can be seen in ) β -19 ( δ H 0.59) and H-1 ( δ H 2.47) is related, so H-1 ( δ H 2.01) is α Configuration, and H-1 can also be seen ( δ H 2.01) and H-3 ( δ H 4.87) is related, thus determining that 3-OH is β Configuration, and H-3 is visible at the same time. δ H 4.87) and H-5 ( δ H 1.43) is related, thus determining that 5-H is α Configuration.

[0065] Based on the above information, the structure of compound 1 is determined to be (25R)-5. α -spirostan-2-one 3-O- β -D-glucopyranosyl(1-2)-[ β -D-xylopyranosyl(1-3)- β -D-glucopyranosyl(1-4)- β -D-galactopyranoside((25R)-5α-spirostane-2-one3-O- β -D-glucopyranosyl(1→2)-[ β -D-xylanose-1→3]- β -D-glucopyranosyl(1→4)- β -D-galactopyranoside), with the structural formula shown in formula (I): Formula (I). A search on SciFinder confirmed it as a new compound, named neriniflorumside A. Because its structure is that of degalactotigonin, a derivative with a carbonyl substitution at the C-2 position, it is also called 2-carbonyl-degalactotigoninone (2-CDGT).

[0066] Example 3: Structural identification of compound 2 The structure of compound 2 obtained in Example 1 was identified, and the spectral data are as follows: Table 2. Galactosidase saponins 1 H-NMR and 13 C-NMR data ( δ Unit: ppm J (unit: Hz)

[0067] Compound 2 is a white powder, and its high-resolution mass spectrometry shows [M+Na] at m / z 1057.5195. + The ion peak (calculated value 1057.5190) indicates that the molecular weight of compound 2 is 1034, suggesting its molecular formula is C. 50 H 82 O 22 .

[0068] The proton spectrum of compound 2 is shown below. Figure 9 As shown, in 1 H-NMR (400 MHz, Pyridine- d 5) In: 5.58 (1H, d, J = 7.4 Hz, H-1'''), 5.21 (1H, d, J = 8.0 Hz, H-1''), 5.25 (1H, d, J = 7.9 Hz, H-1''''), 4.90 (1H, d, J= 7.8 Hz, H-1'), 4.71 (1H, m, H-6'), 4.61 (1H, m, H-4'), 4.55 (3H, m, H-16, 6'', 6'''), 4.42 (4H, m, H-2', 2'', 6'', 6'''), 4.13 (9H,m, H-3', 5'', 3'', 2''', 3''', 4''', 2'''', 3'''', 5''''), 4.03 (1H, m, H-4''''), 3.92 (3H, m, H-3, 5'', 5'''), 3.83 (1H, t, J = 9.1 Hz, H-4''), 3.69(1H, t, J = 10.3 Hz, H-5''''), 3.61 (m, H-26a), 3.52 (t, J = 10.2 Hz, H-26b),2.04 (m, H-6), 0.80 (1H, m, H-1), 1.82 (2H, m, H-4), 1.67 (2H, m, H-12, 23), 1.41 (2H, m, H-15, 23), 1.37 (2H, m, H-24), 1.23 (1H, m, H-11), 1.57 (m, H-1,2, 4, 6, 7, 11, 12), 1.96 (1H, q, J = 6.9 Hz, H-20), 0.50 (1H, m, H-9), 1.80 (2H, m, H-17), 1.02 (2H, m, H-5, 14), 1.61 (m, H-8, 25), 0.83 (3H, s, H-18), 0.71 (3H, d, J = 5.2 Hz, H-27), 0.63 (3H, s, H-19), 1.15 (3H, d, J = 7.1 Hz, H-21).

[0069] The carbon spectrum of compound 2 is as follows Figure 10 As shown, in 13 C-NMR (100 MHz, Pyridine- d 5) Middle: δ109.5 (C-22), 105.2 (C-1''), 105.1 (C-1'''), 102.7 (C-1'), 105.4 (C-1''''), 87.0 (C-3''), 81.6 (C-2''), 80.2 (C-4'), 79.0 (C-3''''), 78.9 (C-5'''), 78.0 (C-3'''), 75.9 (C-2'''), 75.6 (C-2'''), 75.3 (C-3'), 73.4 (C-2'), 71.3 (C-4'''), 71.0 (C-4'''), 70.7 (C-4'''), 67.6 (C-5''''), 77.7 (C-5''), 76.5 (C-5'), 63.3 (C-6''), 62.7 (C-6'''), 60.9 (C-6'), 81.4 (C-16), 77.9 (C-3), 67.19(C-26), 56.7 (C-14), 54.7 (C-9), 44.9 (C-5), 42.3 (C-20), 35.6 (C-8), 30.9(C-25), 41.1 (C-13), 36.1 (C-10), 40.4 (C-12), 37.5 (C-1), 35.1 (C-4), 32.7(C-2), 32.4 (C-15), 32.1 (C-7), 30.2 (C-23), 29.5 (C-24), 29.2 (C-6), 21.5(C-11), 17.6 (C-27), 16.9 (C-18), 15.3 (C-21), 12.6 (C-19). Based on the above information, and comparing it with existing literature... 1 H-NMR and 13 C-NMR data comparison confirmed that compound 2 is degalactotigonin (DGT), a known compound, and all C- and H-NMR data for compound 2 were assigned. Compound 2 was named (25R)-5α-spirostane 3-O- β -D-glucopyranosyl(1→2)-[β-D-xylopyranosyl(1→3)]-β-D-glucopyranosyl(1→4)- β -D-galactopyranoside ((25R)-5 α -spirostan 3-O- β -D-glucopyranosyl(1-2)-[ β-D-xylopyranosyl(1-3)- β- D-glucopyranosyl(1-4)- β -D-galactopyranoside), the structure is shown in equation (II) below: Equation (II).

[0070] Application Example 1: In vivo pharmacodynamic activity Establishment of an Acute Myocardial Infarction Model in Mice: Six- to eight-week-old C57BL / 6 mice, weighing 20-25 grams, were used to establish an acute myocardial infarction model by ligating the left anterior descending coronary artery (LAD). All surgical instruments were sterilized at high temperature and disinfected with povidone-iodine. Mice were fasted for 12 hours preoperatively and anesthetized with 1.25% tribromoethanol at a dose of 0.2 mL / 10 g via intraperitoneal injection. After anesthesia, the hair in the left thoracic region was shaved and the mice were fixed to the operating table. The surgical area was disinfected with povidone-iodine. A longitudinal incision was made in the left third intercostal space. The intercostal muscles were bluntly dissected, and the thoracic cavity was opened with a retractor. The origin of the LAD was located (between the lower edge of the left atrial appendage and the pulmonary artery conus). A sterile 8-0 suture was used to ligate the LAD 2 mm below the origin. Immediate observation after ligation was indicated by pallor of the left ventricular anterior wall. The chest was closed layer by layer, and the intercostal muscles, outer muscle layer, and outer skin were sutured sequentially using sterile 4-0 sutures. After suturing, the wound was disinfected with povidone-iodine and penicillin sodium powder was applied. The rat was placed on a 37°C warming pad and returned to its cage after waking up. In the sham surgery group, only the LAD site was sutured without ligation, and the rest of the procedure was the same as above.

[0071] Experimental grouping and drug administration: The compounds from Example 1 were dissolved in physiological saline to obtain a suspension. The model group was designated as the MI group, the sham-operated group as the Sham group, the model group plus Allium macrostemon saponin A as the MI+2-CDGT group, and the model group plus degalactoside as the MI+DGT group. The MI+2-CDGT and MI+DGT groups were administered the drug by gavage daily on the day following surgery, with a dosage of 40 mg / kg per mouse. The MI and Sham groups were administered the same volume of physiological saline by gavage daily on the day following surgery.

[0072] Mice were administered the above-described drug via gavage for 28 consecutive days, and mortality was observed and recorded during this period. Cardiac function was assessed in surviving mice, and serum levels of lactate dehydrogenase and creatine isoenzyme were measured. On day 14 of gavage administration, echocardiography was performed under tribromoethanol anesthesia to assess cardiac function. On day 28 of administration, mice were anesthetized with sodium pentobarbital, and serum and heart tissue were collected.

[0073] Survival rates of mice in each group were recorded. The mortality rate of mice in each group was observed and recorded within 28 days after gavage administration.

[0074] The results are as follows Figure 11 As shown, within 28 days of gavage administration, the survival rate of the Sham group was 92.5%; the survival rate of the MI group was 27.4%; the survival rate of the MI+DGT group was 45%; and the survival rate of the MI+2-CDGT group was 55%, all of which were statistically significant compared with the model group. The results indicate that *Allium longipes* saponin A and degalactoside can effectively reduce the mortality rate of mice after myocardial infarction.

[0075] Cardiac ultrasound was performed on surviving mice in each group 14 days after oral administration of the drug. Rats were anesthetized with 2.5% tribromoethanol. After anesthesia, the rats were placed on the operating table. A high-frequency ultrasound probe was used, and conductive adhesive was applied to the rat's chest to ensure good contact between the probe and the skin and to reduce air interference. The systolic and diastolic diameters of the left ventricle were recorded in the short-axis section, and the fractional shortening (FS) and ejection fraction (EF) were calculated.

[0076] Figure 1 shows the results of typical left ventricular M-mode echocardiography in the parasternal short-axis view of mice and the analysis of related parameters in each group. Figure 12 As shown, the Sham group mice had normal cardiac function with an LVEF of 94.55±1.70%. Compared with the Sham group, the MI group had a decreased cardiac ejection fraction with an LVEF of 37.56±2.92%. Compared with the MI group, the MI+DGT group and the MI+2-CDGT group had increased cardiac ejection fractions with LVEF values ​​of 61.38±3.28% and 72.52±4.11%, respectively. The LVFS value of the Sham group was 46.42±6.10%. Compared with the Sham group, the MI group had a decreased cardiac shortening fraction with an LVFS value of 19.54±1.88%. Compared with the MI group, the MI+DGT group and the MI+2-CDGT group had enhanced cardiac contractile function with LVFS values ​​of 28.24±1.92% and 30.85±2.58%, respectively. These results indicate that *Allium longipes* saponin A and degalactotiocin can improve cardiac function in mice after myocardial infarction.

[0077] After 28 days of oral administration of ELSA to detect serum and cardiac infarction markers in mice, serum cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and creatine kinase isoenzyme (CK-MB) in cardiac tissue were measured strictly according to the ELSA kit instructions. All data were analyzed using ANOVA and statistical analysis with Graphad Prism 9.4.1. P < 0.05 was considered statistically significant. Data are expressed as mean ± standard deviation (x±s). Intergroup comparisons were performed using SPSS 18.0 software with a one-way ANOVA test; P < 0.05 was considered statistically significant.

[0078] The results are as follows Figure 13As shown, compared with the Sham group rats, the MI group had significantly higher levels of cTnI, CK-MB, and LDH. P <0.01); Compared with the MI group, the DGT group and 2-CDGT group significantly reduced the levels of LDH and cTnI in mouse serum ( P <0.01), and can also significantly reduce the CK-MB content in the heart tissue of MI mice ( P <0.01), among which the group treated with saponin A from Allium tuberosum had the most significant efficacy.

[0079] The levels of inflammatory factors in the serum of mice in each group were detected by ELSA assay strictly according to the instructions of the ELSA kit. The results are as follows: Figure 14 As shown, serum IL-1β and IL-18 levels in the MI group mice were significantly higher than those in the Sham group, and the differences were statistically significant. P <0.01). Compared with the MI group, the serum levels of L-1β and IL-18 in mice in the DGT and 2-CDGT groups were significantly reduced ( P <0.01). Based on the above results, it can be concluded that Allium longiflorum saponin A and degalactoside can reduce the content of inflammatory factors in the heart tissue of MI rats.

[0080] The TTC staining method was used to detect the myocardial infarction area in each group of mice. The rat heart slice mold and blade were pre-cooled at -80℃ for 30 minutes. Heart tissue collected that day was removed and placed on the pre-cooled mold. The heart tissue was quickly sliced ​​into thin sections and placed in 5mL centrifuge tubes containing TTC staining solution, wrapped in aluminum foil to protect from light. The tubes were heated in a water bath at 37℃ for 15-30 minutes. Heating was stopped when the infarcted area turned white. The heart tissue was removed with forceps and fixed in centrifuge tubes containing 4% paraformaldehyde, wrapped in aluminum foil to protect from light. The tubes were then stored at 4℃. The next day, the tissue was photographed, and the infarct area was calculated using ImageJ software. The TTC staining method was used to detect the proportion of myocardial infarction area in each group of mice. The staining results and the percentage of myocardial infarction area in mice are shown below. Figure 15 As shown. Twenty-eight days after the left anterior descending coronary artery in mice, the ligated area of ​​the heart turned white. TTC staining revealed that the myocardium in mice after surgery was divided into red and white, with red representing normal myocardium and white representing necrotic myocardium. Compared with the Sham group, the MI group showed a large infarct area in the myocardium, with an infarct area of ​​31.31±2.84%. Compared with the MI group, the infarct area in the DGT and 2-CDGT groups was reduced, with infarct areas of 20.12±0.76% and 18.53±0.67%, respectively, showing a significant difference compared with the MI group. p<0.01). The results showed that allium longiflorum saponin A and degalactoside effectively reduced the infarct size after acute myocardial infarction in mice, with the allium longiflorum saponin A group being superior to the degalactoside group.

[0081] For HE staining, tissue fixed with paraformaldehyde was used. The paraffin in the sections was removed with xylene, and the xylene was washed away with ethanol to fully hydrate the tissue. The sections were then immersed in hematoxylin for 4 min, rinsed with tap water for 10 min, differentiated with hydrochloric acid-ethanol for 3 s, rinsed with tap water for 10 min, eosin for 25 s–1 min, dehydrated with anhydrous ethanol, permeabilized with xylene, mounted with neutral resin, and allowed to air dry before being photographed and observed under a microscope.

[0082] The results are as follows Figure 16 As shown, HE staining results indicated that no obvious inflammatory infiltration was observed in the heart tissue of the Sham group. In the Model group, small-scale epicardial connective tissue hyperplasia was observed in the heart tissue of the blood vessels, accompanied by a small amount of lymphocyte infiltration. The number of subcapsular cardiomyocytes was reduced, and a small amount of connective tissue replaced the original necrotic cardiomyocytes. The cytoplasm of small-scale cardiomyocytes was loose, and a small number of cardiomyocytes showed tiny vacuoles in their cytoplasm. Compared with the MI group, long-stemmed Allium saponin A and degalactoside reduced the necrotic myocardial tissue.

[0083] Masson staining: Stain with prepared Weigert iron hematoxylin staining solution for 8 minutes, differentiate with acidic ethanol differentiation solution, restore blue with Masson blue solution, wash with distilled water, stain with Ponceau S and fuchsin staining solution, wash with weak acid working solution for 1 minute; wash with phosphomolybdic acid solution for 1 minute, wash with weak acid working solution for 1 minute; stain with aniline blue staining solution for 2 minutes, wash with weak acid for 1 minute; dehydration and clearing: rapid dehydration with 95% ethanol for 2-3 seconds, dehydration with anhydrous ethanol 3 times, 5-10 seconds each time, clearing with xylene 3 times, 1-2 minutes each time, and mounting with neutral resin; staining results: collagen fibers appear blue, and muscle appears red.

[0084] The results are as follows Figure 17 As shown, Masson staining revealed that collagen fiber deposition was not obvious in the Sham group, while collagen fibers increased in the Model group. Compared with the MI group, the administration of Allium longiflorum saponin A and degalactoside A to the MI group could alleviate the collagen fiber deposition phenomenon in rats after MI.

[0085] 10. Fluorescence Analysis Paraffin sections require dewaxing and rehydration, followed by microwave thermal retrieval using citrate buffer (pH 6.0). Incubation with 0.3% Triton X-100 at room temperature for 10 minutes, followed by blocking with 5% goat serum or BSA at room temperature for 30 minutes to 2 hours. Diluted primary antibody is incubated overnight at 4°C or for 2 hours at 37°C to specifically bind the target antigen. Washing three times with TBST buffer, 5 minutes each time, removes unbound antibodies. Incubation with species-matched fluorescently labeled secondary antibody in the dark for 1 hour, followed by washing, nucleus staining with DAPI, and nucleus labeling with 1 μg / mLDAPI in the dark for 10 minutes. Mounting with antifluorescent quenching mounting medium, avoiding air bubbles, selecting the excitation wavelength according to the fluorescent group, and timely image acquisition to prevent quenching.

[0086] The results are as follows Figure 18 and Figure 19 As shown, compared with the Sham group, the expression levels of ASC, caspase-1, and GSDMD in the heart tissue of MI group mice were significantly increased, suggesting that inflammasome activation and pyroptosis are involved in the pathological process of MI. Meanwhile, *Allium tuberosum* saponin A and degalactoside significantly inhibited the expression of ASC, caspase-1, and GSDMD, suggesting that their role in reducing and alleviating myocardial damage may be related to blocking inflammasome assembly or downstream signaling.

[0087] Application Example 2: In vitro activity study of DGT and 2-CDGT 1. Effects of DGT and 2-CDGT on cardiomyocyte viability 3×10 4 Cells were seeded at 100 μL / well in 96-well plates. After adhesion, different concentrations (25, 50, 100, 200, 400, 800, 1000 nM) of DGT and 2-CDGT were administered to observe the effects of the two monomers on cell viability. The results showed that allium longiflorum saponin A and degalactoside A had no effect on cardiomyocyte viability at concentrations of 25–1000 nM.

[0088] 2. Effects of DGT and 2-CDGT on cell survival in different groups of the oxygen-glucose deprivation / reoxygenation (OGD / R) model of cardiomyocytes. Rat H9C2 cardiomyocytes were cultured in a high-glucose medium containing 10% serum and 1% penicillin and streptomycin, and modeling and drug administration experiments were conducted at passage 3-4. Cells were seeded at a density of 3000 cells / well in 96-well plates. After 24 hours, the culture medium was replaced with serum-free and glucose-free medium. The cells were subjected to oxygen-glucose deprivation (OGD) for 9 hours in an anaerobic environment of 5% CO2 / 95% N2. After reoxygenation, the medium was replaced with normal medium. The cells were then treated in the following groups: (1) Blank group (normal culture); (2) Blank group + VX765 (5 μM treatment for 2 hours followed by normal medium for 24 hours); (3) OGD / R model group (OGD / R 9h / 24h); (4) Model group + VX765 (OGD 9h + 5 μM VX765 added at the beginning of reoxygenation and reglucose treatment for 2 hours, followed by normal medium for 24 hours); (5) DGT group (200 nM, OGD 9h, continuous administration for 24 hours during the reoxygenation period); (6) DGT + VX765 group (OGD (7) 2-CDGT group (200 nM, administered for 24 hours during the reoxygenation period); (8) 2-CDGT+VX765 group (OGD 9h+reoxygenation and reglucose were started with 5 μM VX765 added for 2 hours, then replaced with normal culture medium for 24 hours). Cell viability was detected by CCK8 assay.

[0089] The results are as follows Figure 20 As shown, after cellular oxygen-glucose deprivation and reoxygenation injury, the cell nuclei shrank, exhibiting a pyroptosis morphology. Treatment with 200 nM of *Allium longiflorum* saponin A and degalactoside increased the viability of cardiomyocytes. The effect of adding the caspase-1 inhibitor VX-765 on improving cell viability was reduced.

[0090] The above results indicate that Allium longiflorum saponin A and degalactoside can be used to improve and / or treat myocardial infarction.

Claims

1. A steroidal saponin compound from Allium macrostemon, characterized in that, The compound was named (25R)-5α-spirostane-2-one 3-O- β -D-glucopyranosyl(1→2)-[ β -D-xylanose-1→3]- β -D-glucopyranosyl(1→4)- β -D-galactopyranoside, with the structural formula shown in formula (I): Formula (I).

2. The method for preparing steroidal saponin compounds from *Allium tuberosum* according to claim 1, characterized in that, Includes the following steps: S1. Solvent extraction: Long-stemmed Allium macrostemon was extracted by reflux with 60-90% ethanol. The solvent was removed from the extract under reduced pressure to obtain an extract. S2. Extraction: The extract is dissolved and then extracted with water-saturated n-butanol to obtain n-butanol extract; S3. Purification: The n-butanol extract was subjected to silica gel column chromatography with a dichloromethane:methanol solvent system of 50:1→20:1→10:1→5:1→3:1→1:1→0:1 to obtain the 3:1 elution fraction, which was then washed and purified to obtain the insoluble total saponins. S4. Separation: The sparingly soluble total saponins were separated by preparative high performance liquid chromatography using a C18 reverse preparative column and elution with acetonitrile-water as the mobile phase.

3. The preparation method according to claim 2, characterized in that, The ethanol mentioned in step S1 is 70-80% ethanol.

4. The preparation method according to claim 2, characterized in that, The acetonitrile-water volume ratio in step S4 is 75:

25.

5. The preparation method according to claim 2, characterized in that, The elution flow rate in step S4 is 4–6 mL / min.

6. The use of the steroidal saponin compounds or their derivatives, degalactoside, from *Allium tuberosum* as described in claim 1 in the preparation of drugs for improving and / or treating myocardial infarction, characterized in that... The structural formula of the galactosidase saponin is shown in formula (II) below: Equation (II).

7. The application according to claim 6, characterized in that, The improvement and / or treatment of myocardial infarction includes any one or more of the following (1) to (8): (1) Reduce the area of ​​myocardial infarction; (2) Reduce the mortality rate of myocardial infarction; (3) Reduce collagen deposition after myocardial infarction; (4) Improves cardiac dysfunction; (5) Inhibits inflammatory response; (6) Reduce serum levels of cTnI, LDH, and MDA, and increase SOD activity; (7) Inhibits the expression of inflammatory factors and proteins caspase-1, ASC, GSDMD, IL-1β, and IL-18; (8) Protects H9c2 cardiomyocytes damaged by oxygen and glucose deprivation.

8. The application according to claim 6, characterized in that, The myocardial infarction mentioned refers to acute myocardial infarction.

9. A drug for treating myocardial infarction, characterized in that, It contains steroidal saponin compounds from the long-stemmed Allium macrostemon as described in claim 1.

10. The myocardial infarction treatment drug according to claim 9, characterized in that, The dosage form of the drug is powder, oral liquid, injection, honey pill, or granules.

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