CN110950917B - Separation method and application of apiose isoliquiritin - Google Patents
Separation method and application of apiose isoliquiritin Download PDFInfo
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- CN110950917B CN110950917B CN201911123148.3A CN201911123148A CN110950917B CN 110950917 B CN110950917 B CN 110950917B CN 201911123148 A CN201911123148 A CN 201911123148A CN 110950917 B CN110950917 B CN 110950917B
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- Prior art keywords
- isoliquiritin
- apiose
- converting enzyme
- angiotensin converting
- application
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- YNWXJFQOCHMPCK-UHFFFAOYSA-N Isoliquiritin Natural products OC1C(O)C(O)C(CO)OC1OC(C=C1)=CC=C1C=CC(=O)C1=CC=C(O)C=C1O YNWXJFQOCHMPCK-UHFFFAOYSA-N 0.000 title claims abstract description 38
- AVGPOAXYRRIZMM-UHFFFAOYSA-N D-Apiose Natural products OCC(O)(CO)C(O)C=O AVGPOAXYRRIZMM-UHFFFAOYSA-N 0.000 title claims abstract description 31
- ASNHGEVAWNWCRQ-LJJLCWGRSA-N D-apiofuranose Chemical compound OC[C@@]1(O)COC(O)[C@@H]1O ASNHGEVAWNWCRQ-LJJLCWGRSA-N 0.000 title claims abstract description 31
- ASNHGEVAWNWCRQ-UHFFFAOYSA-N D-apiofuranose Natural products OCC1(O)COC(O)C1O ASNHGEVAWNWCRQ-UHFFFAOYSA-N 0.000 title claims abstract description 31
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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Abstract
The invention provides an application of apigenin as an angiotensin converting enzyme inhibitor, wherein the apigenin has stronger angiotensin converting enzyme inhibition activity and can be used for preventing and/or treating various diseases requiring angiotensin converting enzyme inhibition, such as cardiovascular diseases, type 2 diabetes, diabetic nephropathy and the like. The invention also provides a separation method of the apiose isoliquiritin, the separation method is very simple and efficient, the operation is simple and convenient, the controllability and the repeatability are good, and the purity of the apiose isoliquiritin obtained by separation is high.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a separation method of apiose isoliquiritin and application of the apiose isoliquiritin in preparation of an angiotensin converting enzyme inhibitor.
Background
The licorice is a perennial herb of leguminous, is an indispensable traditional Chinese medicinal material in clinic, and is called as the Chinese aged because of being capable of harmonizing various medicines and relieving various toxins. Licorice root, originally recorded in Shen nong Ben Cao Jing (Shen nong's herbal), is called Mei Cao, Mi Gao and listed as the top grade, and is thought to be mainly responsible for pathogenic factors of cold and heat in five zang-organs and six fu-organs, strengthening tendons and bones, growing muscles, strengthening strength, swelling of incised wound and removing toxicity. The licorice is mainly used for treating inflammation, cardiovascular and cerebrovascular diseases, oxidative aging, tumor and the like, and has inseparable pharmacological action with active ingredients of the licorice extract.
The main active components of Glycyrrhrizae radix include glycyrrhizin, flavonoids, and polysaccharides, and the specific structure of apiose Isoliquiritin (CAS number: 120926-46-7) is shown as following formula:
angiotensin Converting Enzyme (ACE) is obtained by converting the C-terminal amino acid of peptide into two segments, and can hydrolyze dipeptide residue at C-terminal of peptide chain. ACE is widely distributed in various tissues of human body, and has rich contents of epididymis, testis and lung, wherein the ACE activity of lung capillary endothelial cells is the highest. It adheres to the surface of endothelial cells and can be broken down and released into the blood circulation. Angiotensin II directly contracts vascular smooth muscle, stimulates aldosterone production, activates the sympathetic nervous system and increases sodium reabsorption, leading to elevated blood pressure. Angiotensin converting enzyme is an ideal target for treating hypertension, heart failure, type 2 diabetes, diabetic nephropathy and the like.
Angiotensin Converting Enzyme Inhibitor (ACEI) has blood pressure lowering effect, can delay and reverse ventricular remodeling, prevent further development of myocardial hypertrophy, improve vascular endothelial function and cardiac function, and reduce arrhythmia, and can reduce systemic blood pressure and intrarenal blood pressure, improve permeability of glomerular filtration membrane, reduce proteinuria discharge, inhibit renal tissue cell hardening process including proliferation of mesangial cells, endothelial cells, tubular epithelial cells and interstitial fibroblasts, and secrete chemotactic factor, inflammatory factor and profibrotic factor, thereby inhibiting renal tissue inflammatory reaction and hardening. Currently, clinically used ACEI includes captopril, enalapril, benazepril, fosinopril, ramipril and the like. In the development of ACEI drugs and drug targets, natural plants are one of the most important sources, and traditional Chinese medicines are important parts in the plant medicine treasury, so that the search for new angiotensin converting enzyme inhibitory drugs in traditional Chinese medicines is particularly important.
In the prior art, the research on the total flavonoids of licorice is more, and Guo et al (Guo A, He D, Xu H B, et al, promotion of regulation T cell induction by immunoloregulation of lipid metabolism and its two constraints [ J ]. Scientific reports,2015,5:14046.) find that the total flavonoids of licorice can improve the body weight, intestinal bleeding and stool of animals of ulcerative colitis model, and has the effect of treating ulcerative colitis. Jiang et al (Jiang Y X, Dai Y, Pan Y F, et al. Total flavone from Radix Glycyrrhiza exitart anti-inflammation and anti-tumor effects by activating iNOS signaling pathway [ J ]. Evaporation-Based comparative and Alternative Medicine, Volume 2018.) found that licorice total flavonoids can effectively block NO generation and iNOS expression induced by LPS/interferon-gamma (IFN-gamma), have NO cytotoxicity and can achieve the anti-tumor effect by establishing a breast tumor in-situ transplantation model. The research on the antidepressant action of the liquorice general flavone shows that after the liquorice general flavone (30, 100 and 300mg/kg) is used for drying, the standing times of rats in open field tests are increased, the number of the penetrated lattices is increased, the number of the excrement grains is obviously reduced, the immobility time of forced swimming is obviously reduced, and the immobility time of tail suspension tests is obviously reduced; the serum corticosterone of the rats after the dry prognosis by using the dose of 300mg/kg of licoflavone is obviously lower than that of the model group.
However, pharmacological action research on apioside liquiritin is relatively few, and no relevant research report on the aspect of treating cardiovascular diseases is found.
Disclosure of Invention
The invention aims to provide application of apiose isoliquiritin in inhibiting angiotensin converting enzyme, and the apiose isoliquiritin has a strong inhibiting effect on the angiotensin converting enzyme.
The invention also aims to provide a method for separating the apiose isoliquiritin, which is simple and efficient, and the purity of the separated apiose isoliquiritin is higher.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an application of apiose isoliquiritin in preparing an angiotensin converting enzyme inhibitor.
The invention also provides application of the apiose isoliquiritin in preparing a medicine for treating and/or preventing related diseases needing to inhibit the activity of angiotensin converting enzyme.
According to a large number of experiments, the invention discovers that the specific active component apigenin in liquorice has extremely strong angiotensin converting enzyme inhibition activity, so that the active component apigenin can control blood pressure, reduce load before and after the heart, inhibit hypertrophy and hyperplasia of cardiac muscle and blood vessels, delay or reverse ventricular and blood vessel reconstruction, improve the contractile function of the heart and the blood vessels, and improve the compliance of the cardiac muscle and the blood vessels, thereby being capable of being used for treating and/or preventing diseases related to angiotensin converting enzyme, such as hypertension, heart failure, type 2 diabetes, diabetic nephropathy and the like.
Based on the mechanism, the apioside isoliquiritin has the application of inhibiting the activity of angiotensin converting enzyme, so the invention also provides the application of the apioside isoliquiritin in preparing medicaments for treating and/or preventing cardiovascular diseases, type 2 diabetes or diabetic vascular complications.
The present invention is not particularly limited to the diabetic vascular complications, but those skilled in the art are familiar with the diabetic vascular complications, such as diabetic macroangiopathy, diabetic microangiopathy including diabetic ocular disease, diabetic nephropathy, etc.
In the application of the invention, the prepared medicine comprises apioside and at least one pharmaceutic adjuvant. The choice of the pharmaceutical excipients depends on the administration route and the action characteristics, and generally refers to the pharmaceutical excipients which are conventional in the pharmaceutical field, and comprises a filling agent, an adhesive, a wetting agent, an absorption enhancer, a surfactant, a flavoring agent, a sweetening agent and the like.
The filler can adopt starch, sucrose or microcrystalline cellulose; the adhesive can adopt starch slurry, hydroxypropyl cellulose, gelatin or polyethylene glycol; the humectant can be magnesium stearate, silica gel micropowder or polyethylene glycol; the absorption enhancer can be polysorbate or lecithin; the surfactant may be poloxamer, sorbitan fatty acid, or polysorbate.
Experiments show that when the concentration of the apiose isoliquiritin is below 50 mu M, the apiose isoliquiritin has no influence on the proliferation of cells. The concentration of the apiose isoliquiritin in the medicine is 0.01-50 mu M, and preferably 12.5-25 mu M.
The medicaments may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.
The medicine can be in any dosage form, including tablets, pills, powder, dispersible tablets, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft capsules, hard capsules, sterile injection, liniments or suppositories and the like, and can also be prepared into conventional, quick-release, sustained-release or delayed-release preparations. The medicament can be administered in any form, including oral (solid or liquid preparation), nasal, intramuscular, subcutaneous, intravenous, etc.
In a second aspect, the invention also provides a method for separating apioside isoliquiritin, which comprises the following steps:
(1) reflux-extracting pulverized Glycyrrhrizae radix with solvent, filtering, mixing filtrates, and recovering solvent to obtain concentrated extract;
(2) suspending the extract concentrated solution in water, removing insoluble substances to obtain macroporous adsorption resin sample liquid, passing through a macroporous adsorption resin column, and standing for 0-8 h; and eluting with water, 15-25% ethanol and 35-60% ethanol in sequence, collecting 35-60% ethanol eluate, concentrating and drying, and separating by preparative liquid chromatography to obtain apiose isoliquiritin.
In the step (1), apiose isoliquiritin is extracted from liquorice firstly, the liquorice is crushed and then is extracted by refluxing with 0-100% ethanol, dregs of a decoction are filtered, the filtrate is combined, and a solvent is recovered to obtain a concentrated solution. The source of licorice is not particularly limited in the present invention, and commercially available. In the extraction process, in order to fully extract the apiose isoliquiritin, the reflux extraction frequency is 1-3 times, and each time lasts for 1-2 hours; the number of reflux extractions is preferably 2, each for 1 h.
The solvent for reflux extraction is preferably water or ethanol with any concentration, and experiments in the invention find that the apioside isoliquiritin in liquorice can be effectively enriched only by using water and ethanol which are easy to remove. The reflux extraction, filtration of the combined filtrate and recovery of the solvent are all techniques well known to those skilled in the art and are not particularly limited herein.
In the step (2), the concentrate obtained by the extraction is continuously subjected to subsequent separation, the extract concentrate is firstly suspended in water, insoluble substances are removed, a macroporous adsorption resin sample liquid is obtained, the macroporous adsorption resin sample liquid passes through a macroporous adsorption resin column, standing is carried out for 0-8 h, then water, 15-25% ethanol and 35-60% ethanol are sequentially used for elution, and 35-60% ethanol eluent is collected.
The type of the macroporous adsorption resin column is not particularly limited in the invention, and the type of the macroporous adsorption resin column known to those skilled in the art can be used.
In order to ensure the content and purity of the finally collected apigenin and isoliquiritin, the method is preferably to elute with water, 18-22% ethanol and 35-45% ethanol and collect 35-45% ethanol eluent. Further preferably, the flow rate of the sample liquid and the eluent of the macroporous adsorption resin is preferably 0.5-3 BV/h (column volume/hour), and the dosage of the eluent is preferably 2-6 BV (column volume).
The concentration of ethanol in the present invention is volume fraction, and the loading, elution and collection of the macroporous adsorbent resin are well known to those skilled in the art, and are not particularly limited herein.
The method comprises the steps of concentrating and drying eluent collected by macroporous adsorption resin, separating and collecting chromatographic peak for 35min by using preparative liquid chromatography, and recovering solvent under reduced pressure to obtain apiose isoliquiritin.
In order to further ensure the content and purity of the final collected apigenin and isoliquiritin, the conditions for preparing the liquid chromatographic separation are preferably as follows: a chromatographic column: agilent Zorbax SB-C18Columns (250X 21.2mm,7 μm); mobile phase: the phase A is water, and the phase B is acetonitrile;
linear elution gradient: 0min, 15% B; 10min, 22% B; 30min, 27% B; 40min, 30% B; 60min, 100% B; flow rate: 8 mL/min.
The preparative liquid chromatography separation method is a technical means well known to those skilled in the art, and the present invention gives only the main parameter conditions, and is not particularly limited herein.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a separation method of apiose isoliquiritin, which is very simple and efficient, simple and convenient to operate, controllable and good in repeatability, and the purity of the apiose isoliquiritin obtained by separation is higher.
(2) The invention also provides application of the apiose isoliquiritin in preparing an angiotensin converting enzyme inhibitor, wherein the apiose isoliquiritin has stronger inhibiting effect on angiotensin converting enzyme, controls blood pressure by inhibiting the activity of the angiotensin converting enzyme, reduces the load of the heart, inhibits the hypertrophy and hyperplasia of cardiac muscle and blood vessel, delays or reverses the reconstruction of ventricle and blood vessel, improves the contraction function of heart and blood vessel, and improves the compliance of cardiac muscle and blood vessel, thereby defining a new action way and a treatment mechanism for treating cardiovascular diseases, type 2 diabetes and diabetic vascular complications by the apiose isoliquiritin; provides a basis for clinical medication of the apiose isoliquiritin.
Drawings
FIG. 1 is a line graph showing the inhibition rate of apigenin to angiotensin converting enzyme at different concentrations.
Detailed Description
The following examples are given for the purpose of illustration and are not intended to limit the scope of the invention.
Example 1: separation of apiose isoliquiritin
Extracting pulverized Glycyrrhrizae radix with water under reflux for 2 times, each for 1 hr, filtering to remove residue, mixing filtrates, and concentrating to obtain concentrated extract; suspending the extract concentrated solution in water, and passing through a macroporous adsorption resin column at the flow rate of 2 BV/h; flushing with 4BV of water at a flow rate of 2 BV/h; rinsing with 5BV of 20% (volume fraction) ethanol at a flow rate of 2 BV/h; rinsing with 4BV of 40% ethanol at a flow rate of 2 BV/h; rinsing with 4BV of 95% ethanol at a flow rate of 2 BV/h; collecting 40% ethanol eluate, concentrating, drying, separating by preparative liquid chromatography for 52min, and recovering solvent under reduced pressure to obtain apiose isoliquiritigenin with purity of more than 95%.
Wherein, the separation conditions of the preparative liquid chromatography are as follows:
the instrument comprises the following steps: agilent 1200 preparation of liquid chromatograph fitted with DAD detector.
A chromatographic column: agilent Zorbax SB-C18Column (250X 21.2mm,7 μm).
Mobile phase: the phase A is water; and the phase B is acetonitrile.
Linear elution gradient: 0min, 15% B; 10min, 22% B; 30min, 27% B; 40min, 30% B; 60min, 100% B. Flow rate: 8 mL/min.
Application example: measurement of angiotensin converting enzyme inhibitory Activity
The inhibitory activity of angiotensin converting enzyme is detected by using a TPE-SDKP probe, and the specific detection method is shown in the literature: wang H, Huang Y, ZHao X, et al. A novel aggregation-induced emission protocol for an Antibiotic Conversion Enzyme (ACE) assay and inhibition screening [ J ]. Chemical Communications,2014,50(95): 15075-.
The specific detection method comprises the following steps: mu.L of Tris buffer at a final concentration of 50mM, 10. mu.L of TPE-SDKP probe at a final concentration of 50. mu.M, and 10. mu.L of angiotensin-converting enzyme (12.5mU/mL) were added to a black 96-well cell culture plate (purchased from Corning), and apiose isoliquiritigenin at final concentrations of 50. mu.M, 25. mu.M, and 12.5. mu.M, respectively, while blank control wells were made, and each well was repeated three times. After incubation at 37 ℃ for 2h, ZnCl was added to a final concentration of 3mM2After incubating the aqueous solution for 1h again, absorbance values were measured at an excitation wavelength of 320mm and an absorption wavelength of 470mm and compared with blank control wells, and the inhibition was calculated, and the results are shown in FIG. 1.
As can be seen from fig. 1, different concentrations of apigenin have significant inhibitory effects on angiotensin converting enzyme, and the inhibitory rate increases with increasing concentration, and reaches over 40% when the concentration of apigenin is 50 μ M.
Comparative example: determination of angiotensin converting enzyme inhibitory Activity of other chalcone Components in Glycyrrhiza
Angiotensin converting enzyme inhibitory activity of other chalcone components extracted from Glycyrrhrizae radix, Isoliquiritigenin (Isooliquitinin, CAS number: 961-29-5) and Isoliquiritin (Isooliquitinin, CAS number: 5041-81-6).
The determination method of the two licorice components is basically consistent with the application example, and specifically comprises the following steps: 70 μ L of Tris buffer at 50mM final concentration, 10 μ L of TPE-SDKP probe at 50 μ M final concentration, and 10 μ L of angiotensin-converting enzyme (12.5mU/mL) were added to a black 96-well cell culture plate (purchased from Corning Corp.), and isoliquiritigenin and isoliquiritin at 50 μ M final concentration were added, respectively, while making blank control wells, and repeated three times. After incubation at 37 ℃ for 2h, ZnCl was added to a final concentration of 3mM2After incubating the aqueous solution for 1h again, absorbance values were measured at an excitation wavelength of 320mm and an absorption wavelength of 470mm and compared with blank control wells to calculate the inhibition.
The experimental result shows that isoliquiritigenin and isoliquiritin with the concentration of 50 mu M have no inhibiting effect on angiotensin converting enzyme.
Claims (5)
1. An application of apiose isoliquiritin in preparing angiotensin converting enzyme inhibitor is provided.
2. An application of apiose isoliquiritin in preparing the medicines for preventing and/or treating the diseases associated with the inhibition of angiotensinamide convertase activity is disclosed.
3. The use of claim 1, wherein the inhibitor is for the treatment and/or prevention of cardiovascular disease.
4. The use according to claim 1, wherein the inhibitor is for use in a medicament for the treatment and/or prevention of type 2 diabetes or diabetic vascular complications.
5. The use according to any one of claims 1 to 4, wherein the concentration of apioglycoside in the medicament is 12.5 to 25 μ M.
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| CN1651384A (en) * | 2004-11-27 | 2005-08-10 | 江南大学 | Synthesis method of glycyrrhizin |
| CN102247339A (en) * | 2011-05-26 | 2011-11-23 | 首都医科大学 | Application of isoliquiritigenin to preparation of drugs for preventing and/or treating cardiovascular and cerebrovascular diseases |
| CN102297917A (en) * | 2011-01-26 | 2011-12-28 | 辽宁中医药大学 | Method for measuring contents of seven components in liquorice and processed product thereof |
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| CN102247339A (en) * | 2011-05-26 | 2011-11-23 | 首都医科大学 | Application of isoliquiritigenin to preparation of drugs for preventing and/or treating cardiovascular and cerebrovascular diseases |
| CN107510710A (en) * | 2017-10-12 | 2017-12-26 | 沈阳药科大学 | A kind of method and medical usage that diabetes B target spot inhibitor is enriched with from Glycyrrhiza uralensisFisch residue |
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