CN110638822A - Albizzia julibrissin glycoside compound for promoting endothelial cell proliferation and application thereof - Google Patents
Albizzia julibrissin glycoside compound for promoting endothelial cell proliferation and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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Abstract
The invention discloses an albizzia bark lignan glycoside compound for promoting endothelial cell proliferation and an application thereof, belonging to the technical field of biological medicines. The invention provides icariside E5The new application in promoting endothelial cell proliferation and migration and promoting angiogenesis. Experiments prove that the HUVEC cell activity after 24 hours of administration at 40 mu M can be improved to 133.65% of that of a control, cell migration can be obviously promoted, and wound healing is promoted in-vivo experimentsAnd (4) acting. Monomer compound icariside E prepared by the invention5Can obviously promote the proliferation activity of cells, promote the migration and self-replication of HUVEC cells, and can be used for transferring exogenous angiogenesis inducing factors into tissues, promoting angiogenesis and enhancing the proliferation capacity of cells.
Description
Technical Field
The invention relates to an albizzia julibrissin glycoside compound for promoting endothelial cell proliferation and application thereof, belonging to the technical field of biological medicines.
Background
Cortex Albizziae (Albiziae Cortex) is the bark of Albizzia julibrissin Durazz (Albzia julibrissin Durazz) belonging to Leguminosae, is a common traditional Chinese medicine, has sweet and mild properties and taste, and has the effects of resolving stagnation, regulating blood, calming heart and relieving swelling. In recent years, with the intensive research on albizzia julibrissin durazzini by scholars, chemical components and pharmacological and medicinal effects of albizzia julibrissin durazzini are continuously discovered.
To date, a variety of compounds have been isolated from albizia plants, including triterpenes, flavones, lignans, and the like. The literature indicates that the content of lignans in the albizzia julibrissin durazzini is low, and certain difficulty is caused to the comprehensive evaluation of the extraction and purification process of water-soluble lignans in the albizzia julibrissin durazzini. Meanwhile, the cortex albiziae is often used as a component of a compound medicine and rarely applied independently, so that the pharmacological activity of various chemical components contained in the cortex albiziae is not clear.
Disclosure of Invention
The invention aims to provide the application of lignan glycoside compounds in preparing medicaments for promoting endothelial cell proliferation and angiogenesis.
In one embodiment, the lignan glycoside compound is represented by formula 1;
in one embodiment, the effective amount of the lignan glycoside compound is 20 μ M or more.
The second object of the present invention is to provide a pharmaceutical composition comprising the compound represented by formula 1.
In one embodiment, the composition is a pharmaceutical composition.
In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.
In one embodiment, the pharmaceutically acceptable carrier comprises a diluent, excipient, or solvate.
In one embodiment, the pharmaceutical composition is in the form of tablet, capsule, granule, powder, syrup, oral liquid or injection
The use of a compound of formula 1 in the manufacture of a medicament.
It is three objects of the present invention to provide a method for preparing a compound represented by formula 1, comprising the steps of: (1) crushing cortex albiziae by using a crusher, mixing the crushed cortex albiziae with 70-80% ethanol according to the material-liquid ratio of 1: 4-10, and performing reflux extraction at 70-90 ℃ for 1-3 times for 1-3 hours each time; (2) filtering to remove cortex Albizziae residues, mixing the extractive solutions obtained in step (1), freeze drying, collecting crude extract, pulverizing, suspending, and sequentially extracting with ethyl acetate and n-butanol; (3) and (3) taking the n-butyl alcohol extract, carrying out reduced pressure rotary evaporation at 80 ℃ to recover n-butyl alcohol, and drying the n-butyl alcohol extract in a vacuum drying oven. Taking 254g of n-butanol, separating by using D101 macroporous adsorption resin, eluting by using 2-3 times column volume of ethanol-water mixed solution as a mobile phase, and enriching a 30% ethanol elution section, a 50% ethanol elution section, a 70% ethanol elution section and a 95% ethanol elution section; performing silica gel column chromatography on the 30% ethanol elution section, eluting by using a gradient solvent, taking dichloromethane-methanol mixed solution with the solvent of 40:1,20:1,16:1,10:1,8:1 and 6:1 as a mobile phase, eluting by using the mobile phase with about 4 times of column volume, collecting eluent, and combining the same components through TLC detection to obtain each elution section.
The fourth purpose of the invention is to provide the application of the compound in preparing medicaments for promoting the proliferation and migration of endothelial cells.
Has the advantages that: the invention provides icariside E5The new application in promoting endothelial cell proliferation and migration and promoting angiogenesis. Experiments prove that the HUVEC cell activity can be improved to 133.65% of the control after 24h after 40 mu M administration. The scratch test result shows that after the administration of 40 mu M and the continuous culture for 24 hours, icariside E5Can obviously promote cell migration, namely the function of promoting wound healing in vivo experiments. The number of cell clones cultured after 40. mu.M administration of the compound was significantly greater than that culturedThe results of the control group show that the monomeric compound icariside E5Can obviously promote the proliferation activity of cells, promote the migration and self-replication (cloning) of HUVEC cells, and can be used for transferring exogenous angiogenesis inducing factors into tissues, promoting angiogenesis and enhancing the cell proliferation capacity.
Drawings
FIG. 1 is a diagram: 8:1 elution fraction analysis type high performance liquid chromatogram.
FIG. 2 is a diagram of: davisil C18 reverse column 40% CH3And (3) performing a semi-preparative high performance liquid chromatogram of the OH elution section.
FIG. 3 is a diagram of: icariside E5ESI-MS chromatogram of (1).
FIG. 4 shows icariside E5Is/are as follows1H-NMR chromatogram.
FIG. 5 shows icariside E5Is/are as follows13C-NMR chromatogram.
FIG. 6 shows icariside E5Promote HUVEC cell proliferation.
FIG. 7 shows icariside E5Promote HUVEC cell migration.
FIG. 8 shows icariside E5The cloning capacity of the HUVEC cells is enhanced, namely the self-replication capacity.
Detailed Description
Preparation of the Compound of example 1
(1) Extracting 20kg of dried cortex Albizziae, pulverizing, extracting with 5 times of 75% ethanol (water) 100L each time under reflux at 80 deg.C for 2 times each time for 2 hr. Filtering to remove cortex Albizziae residue, mixing 75% ethanol extractive solutions of cortex Albizziae, and freeze drying to obtain 1.6kg ethanol crude extract of cortex Albizziae. The crude extract was ground and suspended in 2L of deionized water to dissolve it as far as possible. Suspending, sequentially extracting with ethyl acetate and saturated n-butanol, extracting for several times by adding ethyl acetate and saturated n-butanol, stopping extraction until the color of the extractive solution becomes light to colorless, and respectively mixing the extractive solutions to obtain ethyl acetate extract and n-butanol extract.
(2) Separating and taking 254g of n-butanol, dissolving and suspending with deionized water, taking 254g of n-butanol, separating by adopting D101 macroporous adsorption resin, and performing gradient elution by respectively using 2-3 times of column volume of ethanol-water mixed solution as a mobile phase to enrich a 30% ethanol elution section, a 50% ethanol elution section, a 70% ethanol elution section and a 95% ethanol elution section; performing silica gel column chromatography on the 30% ethanol elution section, eluting by using a gradient solvent, taking dichloromethane-methanol mixed solution with the solvent of 40:1,20:1,16:1,10:1,8:1 and 6:1 as a mobile phase, eluting by using the mobile phase with about 4 times of column volume, collecting eluent by using a 250ml conical flask respectively, and merging the same components through TLC detection to obtain each elution section. Carrying out analytical High Performance Liquid Chromatography (HPLC) detection on the silica gel 8:1 elution section (the chromatographic column is X-Bridge C18,5 mu m, 4.6X 250mm, the flow rate is 1ml/min, the column temperature is 30 ℃), the ultraviolet detection wavelength is 254nm, and the elution conditions are as follows:
time CH3OH H2O
The results are shown in FIG. 1. Retention time of each component in analytical high performance liquid chromatogram according to FIG. 1 for CH2Cl2:CH3The OH 8:1 elution fraction was subjected to a reverse phase silica gel column (Davisil C18,50 μm) under the conditions of tRCH corresponding to 22min, 24min, 26min, 60min3OH concentrations 39.8%, 42.9%, 46.2%, 100% (since FIG. 1 is an analytical liquid chromatogram, the column is X-Bridge C18(5 μm, 4.6X 250 mm.) and the Davisil C18 reverse column packing is 50 μm, the mobile phase methanol concentrations are all reduced by 10%, i.e., 29.8%, 32.9%, 36.2%, 100%). Eluting with methanol and water as eluting phase. The elution phase composition was determined to be 29% CH according to FIG. 13OH(H2O),32％CH3OH(H2O),36％CH3OH(H2O),100％CH3And (5) OH. Eluting with 2-3 times of column volume, collecting eluate in 250ml conical flask, and eluting with 36% CH3The OH eluate fraction was subjected to analytical HPLC assay and the separation conditions (as shown in table 1) were investigated to determine the optimum separation method. For 36% CH3The OH elution fraction was subjected to semipreparative liquid phase separation (column chromatography X-Bridge C18,5 μm, 10X 250mm, flow rate 4ml/min, column temperature 3%0 ℃ C.), as shown in FIG. 2 (UV detection wavelength 254nm), retention time tRThe compound at 37.25min is icariside E5(Aj8)。
TABLE 1 conditions of liquid chromatography separation
In the separation conditions shown in table 1, condition one, condition two and condition three can not effectively separate Aj8, and the retention time is near to have strong interference of miscellaneous peak5(Aj8) whose semi-preparative chromatogram is shown in FIG. 2. Collecting the retention time tRThe compound was rotary evaporated at 75 ℃ under reduced pressure for 37.25min, and the solvent was recovered and dried in a vacuum oven to obtain Aj8 monomeric compound.
(3) Structural identification
Aj8 is white powder, and its mass spectrum is shown in FIG. 3 by UPLC-ESI-MS detection. ESI-MS M/z 557[ M + Cl ]-]Is combined with1H-NMR and13C-NMR (FIGS. 4 to 5) determined that the molecular formula is C26H34O11。
Dissolving the sample Aj8 in a nuclear magnetic tube by using deuterated methanol, and determining by using a full-digital nuclear magnetic resonance spectrometer1HNMR、13CNMR, the results are as follows:1H NMR(400MHz,CD3OD)δ6.94(2H,d,J＝4.8Hz,H-2,6),6.59(3H,t,J＝7.0Hz,H-2’,5’,6’),6.50(1H,d,J＝8.0Hz,H-7),6.32(1H,dt,J＝15.6,5.6Hz,H-8),4.69(2H,d,J＝7.2Hz),4.24(2H,d,J＝5.5Hz,H-9),4.03–3.94(1H,m),3.92–3.85(1H,m),3.84(3H,s,3-OMe),3.82–3.74(3H,m),3.72(1H,s),3.71(3H,s,3’-OMe),3.67(1H,d,J＝7.2Hz),3.62–3.35(4H,m),3.19–3.11(1H,m),2.99(1H,dd,J＝13.8,5.5Hz),2.74(1H,dd,J＝13.7,9.4Hz).13CNMR(101MHz,CD3OD)δ152.05(C-3),147.02(C-3’),143.94(C-4’),143.61(C-4),137.53(C-5),134.01(C-1),131.82(C-1’),130.10(C-8),128.29(C-7),121.22(C-6’),117.76(C-6),114.29(C-5’),112.41(C-2’),107.75(C-2),103.97(C-1”),76.67(C-5”),76.46(C-3”),74.54(C-2”),69.85(C-4”),65.45(C-9’),62.27(C-9),61.06(C-6”),55.02(3’-OCH3),54.89(5’-OCH3) 41.39(C-8 '), 37.76 (C-7'). according to1HNMR、13CNMR and mass spectrum finally determined that Aj8 is Icariside E5(Icariside E)5). The results are as follows:
example 2 icariside E5Promoting endothelial cell proliferation activity
(1) Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin in a 37 ℃ incubator containing 5% CO 2. Every 1-2 days the medium was changed and 85-90% confluent cells were plated at a rate of 1: 3 confluent ratio passage. In all experiments, cells were used between passage 2 and passage 5. HUVEC cells in logarithmic growth phase were plated in 96-well plates with 100. mu.L of medium per well and 3000 cells. After plating for 12 hours, after the cells are applied to the wall, the cells are cultured for 24 hours, 10 mu LCCK-8 is added into each hole, and after the cells are incubated for 1 hour in a cell culture box at 37 ℃, the absorbance (OD) value is detected at the wavelength of 450nm by using an enzyme labeling instrument. The results are shown in FIG. 6, the HUVEC cell viability was significantly increased after 24h administration compared to the blank control group, and the cell viability was 133.65% when compared to 100% of the blank control group in this experiment when 40. mu.M administration was performed, indicating that icariside E is present5Can obviously enhance the cell proliferation activity of HUVEC.
Example 3 icariside E5Effect on migration of HUVEC cells.
Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin in a 37 ℃ incubator containing 5% CO 2. Every 1-2 days the medium was changed and 85-90% confluent cells were plated at a rate of 1: 3 confluent ratio passage. In all experiments, cells were used between passage 2 and passage 5.Taking HUVEC cells in logarithmic growth phase, laying in 6-well plates, laying 20 thousands of cells in each well, culturing for 24 hours, namely after the cells adhere to the wall, scratching the cells in the dish, washing for 2 times by PBS (phosphate buffer solution), and adding a culture medium (containing 40 mu M Icariside E)5Monomeric compounds) were cultured for 24 hours, and the cell migration state was observed. The experimental result shows that icariside E5Can significantly promote cell migration, the result is shown in FIG. 7, after scratching, 40 μ M is administered to continue culturing for 24h, the photograph is taken by an inverted microscope, icariside E is added5Can obviously promote cell migration, namely the function of promoting wound healing in vivo experiments.
Example 4
Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in a 37 ℃ incubator with 5% CO 2. Every 1-2 days the medium was changed and 85-90% confluent cells were plated at a rate of 1: 3 confluent ratio passage. In all experiments, cells were used between passage 2 and passage 5. Taking HUVEC cells in logarithmic growth phase, digesting with pancreatin, beating into single cells, spreading on 6-well plate, adding 2ml culture medium into each well, inoculating 100 cells into each well, and standing at 37 deg.C containing 5% CO2The cell culture box of (3). When macroscopic colonies appeared in the culture dish, the culture was terminated. The supernatant was discarded and carefully rinsed 2 times with PBS. Fixed with 4% paraformaldehyde for 15 minutes. Then removing the fixing solution, adding a proper amount of GIMSA to dye by using the dyeing solution for 10-30 minutes, then slowly washing off the dyeing solution by using running water, and drying in air. Finally, the clone numbers were observed, and the results are shown in FIG. 8, in which the clone numbers of the administration group were significantly higher than those of the control group, and the results showed that the monomeric compound icariside E5Can obviously promote the self-replication of cells, thereby promoting the proliferation of endothelial cells HUVEC, namely angiogenesis.
Comparative example 1:
the present embodiment is the same as example 1, except that the separation conditions were adjusted to conditions 1,2 and 3, which indicated icariside E5Cannot be effectively separated, and the optimal separation method in the research can effectively separate and obtain pure icariside E5A monomeric compound. Compared with the traditional normal phase silica gel separation method, the method for separating the monomerThe ODS column, the gel column and other chromatographic columns are properly adopted in the process of the compound, so that the target product can be effectively purified, the effect of removing pigments is also achieved, and a shortcut is provided for further separation to obtain the target product. If the solvent condition, extraction temperature and extraction time are adjusted, the result will effectively increase icariside E5The yield of (a).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The application of lignan glycoside compounds in preparing medicines for promoting endothelial cell proliferation and/or angiogenesis is characterized in that the lignan glycoside compounds have the structural formula
2. The use of claim 1, wherein the effective amount of the compound is 20 μ M or more.
3. The use according to claim 1 or 2, wherein the compound is replaced by a pharmaceutically acceptable salt thereof.
4. Use according to any one of claims 1 to 3 for the preparation of an agent for promoting endothelial cell or angiogenesis.
5. Use according to claim 1, characterized in that the preparation of said compound comprises the following steps: (1) crushing cortex albiziae by using a crusher, mixing the crushed cortex albiziae with 70-80% ethanol according to the material-liquid ratio of 1: 4-10, and performing reflux extraction at 70-90 ℃ for 1-3 times for 1-3 hours each time; (2) filtering to remove cortex Albizziae residues, mixing the extractive solutions obtained in step (1), freeze drying, collecting crude extract, pulverizing, suspending, and sequentially extracting with ethyl acetate and n-butanol; (3) taking the n-butanol extract, carrying out reduced pressure rotary evaporation at 80 ℃ to recover n-butanol, and drying the n-butanol extract in a vacuum drying oven; taking 254g of n-butanol, separating by adopting D101 macroporous adsorption resin, eluting by using 2-3 times of column volume of ethanol-water mixed solution as a mobile phase, and enriching a 30% ethanol elution section; performing silica gel column chromatography on the 30% ethanol elution section, eluting with gradient solvent, taking dichloromethane-methanol mixed solution with solvent of 40:1,20:1,16:1,10:1,8:1 and 6:1 as mobile phase, eluting with mobile phase with 4 times column volume, combining the same components through TLC detection, and collecting the 8:1 elution section.
6. A pharmaceutical composition comprising a compound
7. The pharmaceutical composition of claim 6, wherein the composition further comprises a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 6 or 7, wherein the pharmaceutically acceptable carrier comprises a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive, an integrating agent, an osmotic enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a coating agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, or a release retardant.
9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the dosage form is tablet, capsule, granule, powder, syrup, oral liquid or injection.
10. Compound (I)
The application of the extract in preparing health products for improving endothelial cell proliferation and/or angiogenesis functions.
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| CN201910945579.1A CN110638822A (en) | 2019-09-30 | 2019-09-30 | Albizzia julibrissin glycoside compound for promoting endothelial cell proliferation and application thereof |
| PCT/CN2020/102569 WO2021063078A1 (en) | 2019-09-30 | 2020-07-17 | Lignan glycoside compound from bark of albizia julibrissin for promoting endothelial cell proliferation and use thereof |
| ZA2021/10483A ZA202110483B (en) | 2019-09-30 | 2021-12-15 | Lignan glycoside compound from bark of albizia julibrissin for promoting endothelial cell proliferation and use thereof |
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| CN101167787B (en) * | 2007-10-17 | 2011-06-22 | 江南大学 | Albizzia plant extraction composition for inhibiting angiogenesis and preparation and application thereof |
| US20130053438A1 (en) * | 2010-01-19 | 2013-02-28 | Universitat Innsbruck | Pharmaceutical compositions comprising lignans and their derivatives for the medical management of angiogenesis and hypovascularity |
| CN110563781B (en) * | 2019-09-30 | 2021-06-25 | 江南大学 | Preparation method of albizzia bark neolignan monomer compound |
| CN110638822A (en) * | 2019-09-30 | 2020-01-03 | 江南大学 | Albizzia julibrissin glycoside compound for promoting endothelial cell proliferation and application thereof |
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| YUNG-HUSAN CHEN 等: "Identification of antioxidants from rhizome of Davallia solida.", 《FOOD CHEMISTRY》 * |
| 任刚等: "半边风苯丙素类成分及抑制中性粒细胞呼吸爆发活性研究", 《中国实验方剂学杂志》 * |
| 蔚冬红: "合欢皮活性成分研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
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