Triterpene compound and application thereof in myocardial protection medicine
The triterpenoid compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol was extracted and isolated from ginseng or American ginseng by acid hydrolysis and high performance liquid chromatography, which solved the problem of insufficient novelty of compound structure in the prior art and realized the preparation of compound with cardioprotective effect and high purity.
Patent Information
- Application Number
- CN202511244337.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-18
AI Technical Summary
In the existing technology, the compounds isolated from ginseng and American ginseng have low structural novelty, lack novel compounds with cardioprotective effects, and the extraction and separation methods are complex and not environmentally friendly.
The triterpenoid compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol was extracted and separated from the processed products of ginseng or American ginseng by acid hydrolysis, ethanol extraction, macroporous adsorption resin enrichment, silica gel column chromatography, ODS column chromatography and high performance liquid chromatography. The process was simplified to five steps, using conventional reagents and improving purity.
25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol with a purity of over 90% was successfully extracted and separated, exhibiting significant cardioprotective effects. The extraction and separation process was simplified, making it environmentally friendly and efficient.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of traditional Chinese medicine chemistry and natural product separation, specifically to a triterpenoid compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol and its application in cardioprotective drugs. It is a new triterpenoid compound extracted and separated from the hydrolysis product of ginseng or black ginseng processed from ginseng, and its preparation method and application. Background Technology
[0002] Ginseng is the plant ginseng of the Araliaceae family. Panax ginseng The dried roots and rhizomes of *Panax quinquefolius*, a plant belonging to the Araliaceae family. Panax quinquefolium The dried roots and rhizomes of L. Both ginseng and its stems and leaves contain large amounts of ginsenosides. Black ginseng, a processed product of ginseng and American ginseng, contains high levels of rare saponins such as ginsenosides Rk1, Rk3, Rg3, Rg5, CK, F4, Rg6, Rh4, and Rs5, which are used for anti-cancer, anti-heart failure, hypoglycemic, and antioxidant purposes.
[0003] Currently, in addition to preparing rare saponins from ginseng and American ginseng through nine steaming and nine processing steps, the preparation of rare saponins and their aglycones through acid hydrolysis is also widely used.
[0004] Most of the chemical components isolated from ginseng and American ginseng are currently known, and their structural novelty is low. Therefore, it is urgent to develop novel compounds with better activity from ginseng and American ginseng processed products. Summary of the Invention
[0005] To address the problems existing in the prior art, this invention provides a novel compound, 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol, extracted from black ginseng, black American ginseng, or their stem and leaf processing products prepared by repeated steaming and drying of ginseng and American ginseng. Studies have found that the compound of this invention has a cardioprotective effect. At the same time, a simple, rapid, environmentally friendly, and highly pure extraction and separation method for the compound of this invention is provided.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.
[0007] This invention discloses a triterpenoid compound, characterized in that it is named 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol, with the molecular formula C 32 H 56 O4 has the following chemical structure: .
[0008] Furthermore, the above-mentioned compounds are used as active ingredients in the preparation of drugs for treating myocardial ischemia-reperfusion injury.
[0009] The present invention also discloses a pharmaceutical composition characterized in that it comprises the above-described 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
[0010] Furthermore, the use of the pharmaceutical composition described above in the preparation of a drug for treating myocardial ischemia-reperfusion injury.
[0011] The present invention also discloses a method for preparing the above-described compound, characterized by comprising the following steps: (a) Prepared by acid hydrolysis of ginseng plant raw materials or by black ginseng or black American ginseng; (b) Extract the hydrolysate obtained in step (a) or its residue or black ginseng powder with an alcohol solvent to obtain an extract; (c) The extract obtained in step (b) is enriched by macroporous adsorption resin, eluted with ethanol aqueous solution, and the eluent is collected. (d) The eluent obtained in step (c) is subjected to silica gel column chromatography and eluted with a dichloromethane-methanol gradient to collect the target eluent fraction; (e) The target elution fraction obtained in step (d) is subjected to ODS column chromatography and eluted with a methanol-water gradient to collect the target elution fraction; (f) The target elution fraction obtained in step (e) is separated and purified by high performance liquid chromatography to obtain the compound.
[0012] Furthermore, the acid used in step (a) for acid hydrolysis is hydrochloric acid with a concentration of 0.05-0.1 mol / L, a hydrolysis temperature of 60-80℃, and a hydrolysis time of 3-7 h.
[0013] Further, the alcohol solvent mentioned in step (b) is a 60-90% aqueous methanol solution or an aqueous ethanol solution.
[0014] Further, the dichloromethane-methanol gradient elution in step (d) includes elution stages with volume ratios of 100:0, 50:1, 20:1, 10:1, and 5:1, with the target elution fraction obtained from the 20:1 elution stage.
[0015] Further, the methanol-water gradient elution in step (e) includes elution stages with volume ratios of 10:90, 30:70, 70:30, and 100:0, and the target elution fraction is obtained from the 70:30 elution stage.
[0016] Furthermore, the high-performance liquid chromatography separation described in step (f) uses a methanol-water system as the mobile phase for isocratic or gradient elution.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.
[0018] The separation and pharmacological activity study of the novel compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol described in this invention have not been reported in the prior art. This invention provides an extraction and separation method for this novel compound, which involves sequentially using acid hydrolysis, ethanol reflux extraction, silica gel column chromatography, ODS medium-pressure column chromatography, and high performance liquid chromatography for separation, purification, and preparation. A compound was successfully extracted and separated. The method involves only five steps, is simple and fast, and mainly uses conventional reagents such as ethanol, dichloromethane, and methanol in the extraction and separation process. The process is simple and environmentally friendly, and the purity of the compound obtained by this method is high, all greater than 90%. In addition, studies have shown that this compound has cardioprotective effects. Therefore, the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention can be used as a lead compound for the synthesis of other compounds, as well as a raw material for new drug development and pharmacological activity studies. It can also be used to prepare drugs for cardioprotection. Attached Figure Description
[0019] Figure 1 This is the high-resolution mass spectrum of the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention. Where A represents [M+COOH]. - B is [M+H] + .
[0020] Figure 2 The compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention 13 C-NMR spectrum.
[0021] Figure 3 The compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention 1 H-NMR spectrum.
[0022] Figure 4 The HSQC spectrum of the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention is shown.
[0023] Figure 5The HMBC spectrum of the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention is shown.
[0024] Figure 6 The ROESY diagram of the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.
[0026] Unless otherwise specified, all reagents and materials used in this invention are commercially available.
[0027] Example 1.
[0028] This invention provides a novel triterpenoid compound, 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol, with the molecular formula C 32 H 56 O4, the chemical structural formula of which is shown below: .
[0029] The compound was named 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol based on its structure. Table 1 shows the NMR data of this compound: 1 H-NMR (600MHz) and 13 C-NMR (150MHz) in C5D5N.
[0030] Table 1: NMR data of the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of the present invention.
[0031] The structure of the novel compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of this invention was identified and deduced.
[0032] The compound is a white powder, readily soluble in methanol and pyridine, but sparingly soluble in water. HR-ESI-MS yielded a quasi-molecular ion peak at m / z 549.4273 [M+COOH]. - The calculated values are 549.4161 and 505.4276 [M+H].+ The calculated value is 505.4251, and the molecular formula is C. 32 H 56 O4. Compound 13 The C-NMR spectrum (C5D5N, 150MHz) yielded 32 carbon signals: the proton spectrum showed the presence of one double bond hydrogen signal at δ5.30 (1H,t,J=7.3), three oxymethyl hydrogens at δ4.46 (1H,m), 3.96 (1H,m), and 3.55 (dd,1H,J = 11.7, 4.8), two methylene hydrogens at 3.37 (2H,q,J=8.7), and 27 methyl hydrogens at δ2.02 (3H,s), 1.97 (3H,s), 1.48 (3H,s), 1.19 (3H,t,J=6.9), 1.19 (3H,s), 1.18 (6H,s), and 1.05 (6H,s). The carbon spectroscopy revealed that the compound contained two double-bonded carbons at δ 140.3 and 126.3, five oxygen-bonded carbons at δ 79.3, 75.1, 73.4, 68.6 and 57.3, and nine methyl carbons at δ 32.9, 26.8, 26.8, 20.9, 18.6, 18.4, 18.0, 17.5 and 17.4. Comprehensive analysis of the hydrocarbon data revealed that the compound is highly similar to the known compound Ginsenotransmetin B. The main difference lies in the shift of the chemical shift of the methyl carbon in the double bond, from δ13.2 to a lower field and then to δ20.9. Simultaneously, the chemical shift of the hydrogen in the double bond also shifts, from δ5.52 to a higher field and then to δ5.30. The chemical shift of the hydrogen at position 17, connected to the double bond, also shows a significant shift, from δ2.81 to a higher field and then to δ2.11. Furthermore, the ROESY spectrum shows NOE effects at 1.97 and 5.30, but not at 5.30 and 2.11. Therefore, the compound exhibits cis-trans isomerism with similar compounds, confirming its planar structure.
[0033] Based on the above information, this compound can be identified as having the structure described above.
[0034] The present invention also provides a method for extracting and separating the above-mentioned new compounds, the specific steps of which are as follows.
[0035] Step 1: Weigh 500g of dried ginseng powder, add 1000mL of 0.06mol / L hydrochloric acid aqueous solution, and extract with magnetic stirring in a water bath at 70℃ for 5h. After the extract cools, add sodium carbonate to adjust the pH to 7. After centrifugation, add 1000mL of 80% ethanol to the residue and extract with ultrasound for 30min. After evaporating to dryness under reduced pressure at 45℃, dissolve in boiling water and transfer to a beaker to obtain a concentrated solution for later use.
[0036] Alternatively, a concentrated solution can be prepared using black ginseng: Weigh 500g of dried black ginseng powder, add 1000mL of 80% ethanol, reflux and extract twice, 2 hours each time. After the extract is concentrated under reduced pressure, the concentrated solution is ready for use.
[0037] Step 2: Inject the concentrated solution from Step 1 into 1L of AB-8 macroporous adsorption resin, and elute sequentially with water (500mL) and 95% ethanol (3L), and dry the 95% ethanol eluent.
[0038] Step 3: Dissolve the 95% ethanol eluent from Step 2 in methanol, mix with 100-200 mesh silica gel, and separate using a 200-300 mesh silica gel column. Elute using a dichloromethane-methanol gradient (100:0, 50:1, 20:1, 10:1, 5:1, v / v). Develop the dichloromethane-methanol (20:1) eluent with vanillin-concentrated sulfuric acid. Combine identical components and concentrate under reduced pressure to dryness for later use.
[0039] Step 4: The product obtained in Step 3 is further separated by pretreated ODS column (Octadecylsilyl, octadecylsilane bonded silica gel packing material) chromatography, eluted with methanol-water gradient (10:90, 30:70, 70:30, 100:0, v / v) to obtain 4 elution fractions. The 70% methanol elution fraction is concentrated to dryness under reduced pressure to obtain the concentrate for later use.
[0040] Step 5: The concentrate obtained in Step 4 was separated by HPLC (high performance liquid chromatography) using 70% methanol-water as the mobile phase for isocratic elution at a flow rate of 2 mL / min and a detection wavelength of 210 nm. The novel compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol was finally obtained, with a normalized purity of 98%.
[0041] Example 2: Cardioprotective effect of the compounds of the present invention.
[0042] 1. Main materials.
[0043] 1.1 Drugs and Reagents: The new compounds used in the experiment were prepared by the above method, with a purity of 98%. Accurate weighing was performed, and the compounds were dissolved in DMSO and diluted to the required concentration with culture medium. DMEM culture medium, fetal bovine serum (Hyclone, USA); penicillin, streptomycin (Hangzhou Sijiqing Pharmaceutical Co., Ltd.), and CCK-8 reagent kit (Nanjing Jiancheng Biotechnology Co., Ltd.) were also used.
[0044] 1.2 Experimental animals: SD rats that are 2 days old (Liaoning Changsheng Biotechnology Co., Ltd., SCXK2020-0001).
[0045] 1.3 Grouping: The group was divided into a normal group, a model group, and low, medium, and high drug administration groups (culture medium containing DMSO solvent).
[0046] 2. Experimental methods.
[0047] 2.1 In vitro isolation and culture of neonatal cardiomyocytes from SD rats.
[0048] Take newborn SD mice within 3 days of birth, remove the apical portion of the heart, transfer it to a culture dish containing pre-chilled PBS, and cut the heart into 1mm pieces using ophthalmic forceps. 3 Small pieces on the left and right.
[0049] Tissue fragments were digested with trypsin in a 37°C water bath by shaking. The supernatant was carefully aspirated and transferred to DMEM containing 5 mL of 10% FBS (this step was repeated 5-8 times).
[0050] Combine all cell suspensions, pass through a 200-mesh sieve, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in DMEM containing 10% FBS, and then culture in a culture dish for differential culture.
[0051] After 1.5 hours, carefully aspirate the non-adherent cells from the upper layer into another culture dish and incubate at 37°C in a 5% CO2 incubator until the cells extend their pseudopodia and begin to beat synchronously, then proceed to the next experiment.
[0052] Cytotoxic activity assay.
[0053] Rat cardiomyocytes were treated with pancreatic enzymes to prepare a solution with a concentration of 1×10⁻⁶. 4 Cells / well single-cell suspensions were seeded into 96-well plates (200 μL), with 3 parallel wells per group.
[0054] Discard the original culture medium and add 200 μL of 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol culture medium at concentrations of 1 μM, 10 μM, and 30 μM to each well. After incubation for 24 h, aspirate the liquid from the wells and add 200 μL of fresh complete culture medium containing 5 μL of CCK-8 cell viability assay reagent to each well. Incubate the cells at 37°C for 4 h, and measure the absorbance of each well at 450 nm using an ELISA kit to determine the cytotoxic activity of the compound.
[0055] 2.2 Establishment of an in vitro hypoxia / reoxygenation model of cardiomyocytes.
[0056] Cardiac cells were cultured in a constant temperature incubator at 37°C, 95% N2, and 5% CO2. When the cell confluence was >90%, the cells were used for experiments, plated in 96-well plates, and allowed to adhere to the wells and extend pseudopodia.
[0057] Discard the DMEM medium containing 10% FBS, add sugar-free and serum-free medium, and pre-saturate with 95% N2 and 5% CO2 before starting the experiment.
[0058] Replace the culture medium in the 96-well plate with sugar-free and serum-free medium, place it in an anoxic device, replace the air in the device with 95% N2 and 5% CO2 for half an hour, seal the device, and incubate in an incubator at 37°C for 4 hours to establish an anoxic model.
[0059] After the hypoxia period ended, the medium was replaced with DMEM containing 10% FBS and cultured in a normal incubator for 4 hours to obtain a hypoxia / reoxygenation myocardial injury cell model.
[0060] 2.3 CCK-8 assay was used to detect the protective effect of the compound against hypoxia / reoxygenation-reperfusion injury of cardiomyocytes.
[0061] Rat cardiomyocytes were treated with pancreatic enzymes to prepare a solution with a concentration of 1×10⁻⁶. 4 Cells / well single-cell suspensions were seeded into 96-well plates (200 μL), with 3 parallel wells per group.
[0062] Discard the original culture medium of HRI model cardiomyocytes and add 200 μL of each concentration of 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol to each well. After incubation for 24 h, aspirate the liquid from the wells and add 200 μL of fresh complete culture medium containing 5 μL of CCK-8 cell viability assay reagent to each well. Incubate the cells at 37°C for 4 h, and measure the absorbance of each well at 450 nm using an ELISA kit to determine its protective effect on HRI model cardiomyocytes.
[0063] Relative cell viability = OD value of the drug-treated cell group / OD value of the normal cell group.
[0064] All data were processed using SPSS 20.0 software and expressed as mean ± SD. Independent group t-tests were used for comparisons, and a p-value < 0.05 was considered statistically significant.
[0065] 3. Experimental results.
[0066] At concentrations of 1 μM, 10 μM, and 30 μM, 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol did not exhibit cytotoxic effects.
[0067] Table 2 shows the effect of 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol on the viability of damaged myocardium at concentrations of 1μM, 10μM, and 30μM.
[0068] Table 2: Protective effect of 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol on hypoxia / reoxygenation cardiomyocyte injury model.
[0069] Note: ##P<0.01 compared with the normal group; *P<0.05 compared with the model group; **p<0.01 compared with the model group.
[0070] Experimental results show that 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of the present invention at various concentrations have a good protective effect on HRI-induced cardiomyocytes, and the protective ability against myocardial damage cells is significantly improved with increasing drug concentration.
[0071] In summary, this invention provides a novel method for the extraction and separation of compounds. The method involves sequential ethanol reflux extraction, silica gel column chromatography, ODS medium-pressure column chromatography, and liquid phase separation, successfully separating a novel compound. This method is simple, rapid, and environmentally friendly, and the compound obtained by this method has high purity. Due to the unique chemical structure of the obtained compound, which is extracted for the first time from the hydrolysis products of ginseng and related traditional Chinese medicines, it exhibits cardioprotective effects. Therefore, the compound and its composition of this invention can be used as natural products for the development of new traditional Chinese medicine drugs, showing broad prospects.
[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A triterpenoid compound, characterized in that, Named 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol, with the molecular formula C 32 H 56 O4 has the following chemical structure: 。 2. The use of the compound 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol of claim 1 in the preparation of a drug for treating myocardial ischemia-reperfusion injury.
3. A pharmaceutical composition, characterized in that, It comprises the 25-Ethoxy-dammar-20(22)Z-ene-3β,6α,12β-triol compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
4. The use of the pharmaceutical composition according to claim 3 in the preparation of a drug for treating myocardial ischemia-reperfusion injury.
5. A method for preparing the compound according to claim 1, characterized in that, Includes the following steps: (a) Black ginseng and black American ginseng are prepared by acid hydrolysis of ginseng plant raw materials or by steaming and sun-drying ginseng and American ginseng. (b) Extract the hydrolysate obtained in step (a) or its residue, black ginseng powder, or black American ginseng with an alcohol solvent to obtain an extract; (c) The extract obtained in step (b) is enriched by macroporous adsorption resin, eluted with ethanol aqueous solution, and the eluent is collected. (d) The eluent obtained in step (c) is subjected to silica gel column chromatography and eluted with a dichloromethane-methanol gradient to collect the target eluent fraction; (e) The target elution fraction obtained in step (d) is subjected to ODS column chromatography and eluted with a methanol-water gradient to collect the target elution fraction; (f) The target elution fraction obtained in step (e) is separated and purified by high performance liquid chromatography to obtain the compound.
6. The method according to claim 5, characterized in that, The acid used in step (a) for acid hydrolysis is hydrochloric acid with a concentration of 0.05-0.1 mol / L, a hydrolysis temperature of 60-80℃, and a hydrolysis time of 3-7 h.
7. The method according to claim 5, characterized in that, The alcohol solvent mentioned in step (b) is a 60-90% aqueous methanol solution or an aqueous ethanol solution.
8. The method according to claim 5, characterized in that, The dichloromethane-methanol gradient elution in step (d) includes elution stages with volume ratios of 100:0, 50:1, 20:1, 10:1, and 5:1, with the target elution fraction obtained from the 20:1 elution stage.
9. The method according to claim 5, characterized in that, The methanol-water gradient elution in step (e) includes elution stages with volume ratios of 10:90, 30:70, 70:30, and 100:0, with the target elution fraction obtained from the 70:30 elution stage.
10. The method according to claim 5, characterized in that, The high-performance liquid chromatography separation in step (f) uses a methanol-water system as the mobile phase for isocratic or gradient elution.