CN110559307A

CN110559307A - albizzia julibrissin new lignan compound and new application thereof

Info

Publication number: CN110559307A
Application number: CN201910945560.7A
Authority: CN
Inventors: 邱丽颖; 史学林; 刘艺筱; 李双双
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-09-30
Filing date: 2019-09-30
Publication date: 2019-12-13

Abstract

The invention discloses a cortex albiziae neolignanoid compound and a new application thereof, belonging to the technical field of biological medicines. The compound of the invention can effectively treat FFAs-induced lipid metabolism disorder and steatosis after being administrated for 24h, wherein the administration concentration is 5 mu M. And can eliminate active oxygen generation induced by HG, and the average fluorescence intensity in cells is 300.86 after 24h of treatment at the dose of 80 mu M, and is remarkably reduced compared with the fluorescence intensity of a control group 813.87.

Description

Albizzia julibrissin new lignan compound and new application thereof

Technical Field

The invention relates to a cortex albiziae neolignanoid compound and a new 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 first object of the present invention is to provide a lignan compound for use in the preparation of a medicament for ameliorating or preventing steatosis.

In one embodiment, the lignan compound is represented by formula 1;。

In one embodiment, the effective dose of the lignan compound is 5 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 a tablet, capsule, granule, powder, syrup, oral liquid or injection.

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 with D101 macroporous adsorbent resin, and enriching 30% ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, and 95% ethanol elution section; performing silica gel column chromatography on the 30% ethanol elution section, eluting with gradient solvent of dichloromethane-methanol mixed solution of 40:1,20:1,16:1,10:1,8:1 and 6:1, and mixing the same components by TLC detection to obtain each elution section.

The fourth purpose of the invention is to provide the application of the compound in preparing a medicament for preventing or treating lipid metabolism disorder.

The fifth purpose of the invention is to provide the application of the compound in preparing the medicines for resisting oxidative stress and protecting endothelial injury.

Has the advantages that: the compound prepared by the invention is administrated for 24h, the administration concentration is 5 mu M, and the average area of lipid droplets is 222.365 percent compared with a blank control group (compared with an FFAs induction group)^#P<0.05, n-3/group), is effective in treating FFAs-induced lipid metabolism disorders and steatosis.

The average intracellular fluorescence intensity of the (+) -Lyoniresinol-9' -O-glucoside prepared by the invention is 300.86 (after being treated for 24 hours at the dose of 80 mu M^#P<Hg at 0.05vs. n 3/group) had a significant decrease in fluorescence intensity compared to control 813.87. DCFH-DA staining results showed that (+) -Lyoniresinol-9' -O-glucoside abolished HG-induced reactive oxygen species production, with a significant reduction in ROS accumulation when treated with 80. mu.M.

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 32% CH₃Half-preparation of OH elution sectionHigh performance liquid chromatogram.

FIG. 3 is a diagram of: ESI-MS chromatogram of (+) -Lyoniresinol-9' -O-glucoside.

FIG. 4 is a diagram of: process for preparation of (+) -Lyoniresinol-9' -O-glucoside¹HNMR map.

FIG. 5 is a diagram: process for preparation of (+) -Lyoniresinol-9' -O-glucoside¹³A CNMR map.

FIG. 6A is (+) -Lyoniresinol9' -O-glucoside inhibits FFAs-induced lipid droplet generation (X400); b is the lipid droplet accumulation area value. P <0.05vs. control, # P <0.05vs FFAs, n 3/group.

FIG. 7A is the intracellular ROS levels detected by DCFH-DA fluorescence (x 200); and B is the intracellular ROS fluorescence value. P <0.05 compared to control, # P <0.05 compared to HG, n 3/group.

Detailed Description

(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, and mixing ethyl acetate phase and saturated n-butanol phase extractive solutions to obtain ethyl acetate extract and n-butanol extract.

(2) And (3) separating and taking 254g of n-butanol, dissolving and suspending with deionized water, and separating and purifying by adopting D101 macroporous adsorption resin, namely a 30% ethanol elution section, a 50% ethanol elution section, a 70% ethanol elution section and a 95% ethanol elution section. Subjecting the macroporous resin-absorbed 30% ethanol elution segment to silica gel column chromatography, and eluting dichloromethane (CH) with gradient solvent₂Cl₂): methanol (CH)₃OH) (40:1,20:1,16:1,10:1,8:1 and 6:1) was subjected to gradient elution (3 column volumes for mobile phase, and whether the desired product was obtained was detected by TLC in real time), and the same fractions were combined by TLC detection to obtain each elution fraction. Performing High Performance Liquid Chromatography (HPLC) detection on the 8:1 elution section, wherein the ultraviolet detection wavelength is 254nm, and the result is shown in figure 1; detection conditions：

Time CH₃OH H₂O

0min 5％ 95％

60min 100％ 0％

determining CH according to retention time of each component in analytical high performance liquid chromatogram of figure 1₂Cl₂:CH₃The peak positions in the OH 8:1 elution fraction were eluted through a reverse silica gel column (Davisil C18,50 μm) under the condition of t_RCH corresponding to 22min, 24min, 26min, 60min₃OH concentrations 39.8%, 42.9%, 46.2%, 100%, CH since the C18 filler used was 50 μm in diameter₃The OH concentrations are 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％CH₃OH, analytical HPLC assay was performed on each eluted fraction, and the separation conditions were investigated to determine the optimal separation method (as shown in table 1). For 32% CH₃The OH elution phase was subjected to semi-preparative liquid phase separation as shown in FIG. 2 (UV detection wavelength 290nm), retention time t_RThe compound at 31.75min was (+) -Lyoniresinol-9' -O-glucoside (Aj 5).

TABLE 1

Although the target monomeric compound can be separated under the conditions of one to four in the liquid phase separation method described in the present study, the purity of the obtained product Aj5 is poor due to the interference of impurity peaks, and the optimal separation conditions optimized on the basis can eliminate the interference of the impurity peaks on the target product, so that the high-purity monomeric compound is separated, and the semi-preparative separation chromatogram is shown in fig. 2.

(3) Structural identification

Aj5 is white powder, and mass spectra thereof are shown in FIGS. 3-5 by UPLC-ESI-MS detection. ESI-MS M/z617[ M + Cl ]^-]By Monosotopic Mass, Even Electron Ions determining its molecular formula as C₂₈H₃₈O₁₃.

Dissolving the sample Aj5 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:

¹HNMR(400MHz,MeOD)δ6.60(1H,s,H-8),6.45(2H,s,H-2’,6’),4.44(1H,d,J＝6.2Hz,H-4),4.30(1H,d,J＝7.7Hz,anomeric-H),3.95–3.81(6H,m,5,7-OMe),3.76(6H,s,3’,5’-OMe),3.67(2H,dd,J＝11.8,4.9Hz,H-3a),3.56(1H,dd,J＝10.9,6.6Hz),3.47(1H,dd,J＝9.8,3.9Hz),3.39(1H,t,J＝7.7Hz),3.36(3H,s),3.28–3.23(2H,m,H-2a),2.77–2.59(2H,m,H-1),2.09(1H,d,J＝5.7Hz,H-3),1.73(1H,s,H-2).¹³CNMR(101MHz,MeOD)δ147.59(C-3’,5’),147.24(C-5),146.19(C-7),137.95(C-1’),137.51(C-6),133.10(C-4’),128.81(C-9),125.03(C-10),106.47(C-8),105.55(C-2’,6’),103.43(C-1”),76.85(C-5”),76.54(C-3”),73.79(C-2”),70.27(C-4”),70.11(C-3α),64.84(C-2α),61.44(C-6”),58.80(5-OMe),55.48(3’,5’-OMe),55.23(7-OMe),45.30(C-3),41.39(C-4),39.22(C-2),32.43(C-1).

Based on mass spectrum and¹HNMR、¹³CNMR finally determined the structure as follows, chemical name (+) -Lyoniresinol9' -O-glucoside.

Example 2 amelioration of lipid metabolism disorder and steatosis

HepG2 cells (American Type Culture collection, USA) were cultured in DMEM containing 25% glucose, 10% FBS (fetal bovine serum, Gibco), 100U/ml penicillin and 100/ml streptomycin. The culture was carried out 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. HepG2 cells were harvested in logarithmic growth phase and plated in 12-well plates, 10 ten thousand cells per well. After 12h of adherent growth of the cells, the normal DMEM high-sugar medium was discarded, DMEM high-sugar medium containing 0.3mM FFAs (oleic acid: palmitic acid 2:1) was added and the cells were cultured for 24h, that is, after HepG2 cells were modeled as a model of lipid metabolism disorder, lignan compound Aj5 prepared in example 1 was administered to each well, and 5 concentration gradients were set, that is, final concentrations of 5 μ M, 10 μ M, 20 μ M, 40 μ M, and 80 μ M were further cultured for 24h, and HepG2 cells cultured in normal DMEM medium were used as a control.

The culture medium of the 12-well plate is discarded, the culture medium is washed for three times by PBS, 4% paraformaldehyde is fixed for 30min, the culture medium is fully washed by PBS after the fixation is finished, then the culture medium is soaked and washed for 10-15 seconds by 60% isopropanol, and then the culture medium is washed for three times by PBS. Dyeing with oil red O (oil red storage solution: deionized water: 3: 2) at room temperature for 20-30 min, observing the cell staining condition under a microscope, discarding the oil red staining solution, differentiating with 60% isopropanol until the stroma is clear (differentiation is about 5-10 seconds, and fading of the oil red O can be caused by over-differentiation), finally washing with PBS for three times, adding 1ml of PBS into each hole, sealing and taking a picture.

The results of oil red O staining are shown in FIG. 4, and FFAs can significantly induce accumulation and steatosis of HepG2 cells after being cultured in high-fat medium for 24h compared with the blank control group with 100% lipid droplet area, wherein the average lipid droplet area is 970.01% ((C) ((B) ((C))^*P<0.05, 3/group), and the mean area of lipid droplets was 222.365% at a concentration of 5 μ M after 24h administration (compared with FFAs-induced group) compared with that of blank control group (compared with FFAs-induced group)^#P<0.05, n-3/group), indicating that (+) -Lyoniresinol-9' -O-glucoside is effective in the treatment of FFAs-induced lipid metabolism disorders and steatosis. Hepatic steatosis is a prominent feature of type 2 diabetes (T2DM) and may lead to nonalcoholic fatty liver disease and cardiovascular disease. Therefore, the (+) -Lyoniresinol-9'-O-glucoside can effectively treat lipid metabolism disorder and steatosis induced by FFAs, and the result shows that the (+) -Lyoniresinol-9' -O-glucoside can be used as a potential natural medicine for treating nonalcoholic fatty liver and related liver cell diseases and can also be used as a medicine for preventing type 2 diabetes and cardiovascular diseases.

Example 3 resistance to oxidative stress

Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in DMEM 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. To test the protective effect of (+) -Lyoniresinol-9'-O-glucoside in the oxidative stress induced by HUVEC cells with high concentration glucose (HG), HUVEC cells in logarithmic growth phase were taken, and spread on 12-well plates, after 12h of cell adhesion, 80. mu.M of (+) -Lyoniresinol-9' -O-glucoside was added for further culture for 12h (5 multiple wells were set), followed by addition of pre-prepared sterile glucose solution (after glucose was dissolved in PBS, 0.22. mu.m sterile filter, 50. mu.L of high concentration glucose solution was added per well, 50. mu.LPBS was added for blank control group) to make the final concentration 35mM, and culture was continued for 24h. Intracellular ROS production was detected by fluorescent probe DCFH-DA. HUVEC were washed 3 times with PBS and then incubated with 10. mu.M DCFH-DA (DCFH-DA fixed in PBS) for 30 minutes at 37 ℃ in the dark. After washing the cells with PBS, pictures were taken on a fluorescence microscope (80i, Nikon, Japan).

As shown in FIG. 5, 35mM HG significantly increased the accumulation of reactive oxygen species in HUVEC cells, i.e., promoted endothelial injury, compared to Control group ROS fluorescence intensity of 100%, at which ROS fluorescence intensity was 813.87: (^*P<Control, n 3/group) at 0.05vs. (300.86) (average intensity of fluorescence obtained after 24h when treated with 80. mu.M (+) -Lyoniresinol-9' -O-glucoside)^#P<HG, n-3/group) experiments showed that exposure of HUVEC cells to HG increased reactive oxygen species accumulation, and DCFH-DA staining showed that (+) -Lyoniresinol-9' -O-glucoside abolished HG-induced reactive oxygen species production, with a significant reduction in ROS accumulation when treated with 80 μ M.

It has been shown that the accumulated Reactive Oxygen Species (ROS) are responsible for High glucose (High glucose or HG) and are also the main cause of cell damage and apoptosis. Existing studies indicate that reactive oxygen species play a major role in HG-induced endothelial apoptosis. Hyperglycemia is one of the most important features in diabetes, which leads to various cardiovascular complications. The adverse reaction mechanism of hyperglycemia to the cardiovascular system is complex, wherein active oxygen participates in the pathogenesis of cardiovascular injury caused by hyperglycemia, and the generation of active oxygen and the dysfunction of endothelial cells can be caused by continuous hyperglycemia. The results show that HG can remarkably induce endothelial cell injury and ROS accumulation, and that (+) -Lyoniresinol-9'-O-glucoside can remarkably eliminate accumulated ROS and protect high-sugar-induced endothelial cell injury, and also indicate that the (+) -Lyoniresinol-9' -O-glucoside can be used as a potential medicament for treating complications such as diabetes mellitus.

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

1. The use of a compound of formula 1 for the manufacture of a medicament for the amelioration or prevention of steatosis and diseases associated therewith;

2. The use according to claim 1, wherein the effective amount of lignan compound is 5 μ M or more.

3. The use of claim 1, wherein the steatosis-associated disease comprises hepatic steatosis, or non-alcoholic fatty liver disease.

4. A pharmaceutical composition comprising a compound represented by formula 1;

5. The pharmaceutical composition of claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier comprises a diluent, excipient, or solvate.

7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the dosage form is tablet, capsule, granule, powder, syrup, oral liquid or injection.

8. The use of a compound of formula 1 for the preparation of a medicament for combating oxidative stress and/or protecting against endothelial damage;

9. The use according to claim 8, wherein the medicament is for combating elevated levels of ROS in the body.

10. the use according to claim 8, wherein the medicament is for the prevention or treatment of type II diabetes.