WO2021063076A1

WO2021063076A1 - Application of albiziae cortex lignan compound in improving steatosis

Info

Publication number: WO2021063076A1
Application number: PCT/CN2020/102547
Authority: WO
Inventors: 邱丽颖; 史学林; 侯豹; 蔡维维; 李忠杰; 刘艺筱; 李双双
Original assignee: 江南大学
Priority date: 2019-09-30
Filing date: 2020-07-17
Publication date: 2021-04-08

Abstract

Both of the two Albiziae Cortex lignan compounds provided are chiral compounds, and have the effect of improving lipid metabolism disorders and steatosis. 24 h after administration, when the administration concentration is 5 μM or more, it can effectively treat FFAs-induced lipid metabolism disorders and steatosis. R-type compounds can eliminate the production of reactive oxygen species induced by HG, and after 24 hours of treatment at a dose of 80 μM, the average intensity of intracellular fluorescence is 300.86, which is a significant decrease compared to the fluorescence intensity of the control group of 813.87.

Description

Application of Albizia Julibrissin Lignosome Compounds in Improving Fatty Degeneration

Technical field

The present invention relates to the application of Albizia Julibrissin lignan compound in improving steatosis, and belongs to the technical field of biomedicine.

Background technique

Albiziae Cortex is the bark of the legume Albzia julibrissin Durazz. It is a commonly used traditional Chinese medicine. It has a sweet, calming nature and has the effects of relieving depression, healing blood, calming the heart and reducing swelling. In recent years, with the in-depth study of Albizia bark by scholars, its chemical components and pharmacological activities have been continuously discovered.

So far, many compounds have been isolated from Albizia plants, including triterpenes, flavonoids, lignans and so on. Documents have shown that the content of lignans in Albizia Julibrissin is relatively low, which has caused certain difficulties for the comprehensive evaluation of the extraction and purification process of water-soluble lignans in Albizia Julibrissin. At the same time, Albizia Julibrissin is often used as a component of compound drugs and is rarely used alone. Therefore, the pharmacological activities of the various chemical components contained in it are not clear.

Summary of the invention

The first object of the present invention is to provide the application of the compound in the preparation of medicines for improving or preventing steatosis and related diseases; the compound is

In one embodiment, the effective dose of the lignan compound is ≥5 μM.

In one embodiment, the lignan compound is

The effective dose is 5μM.

In one embodiment, the effective dose of the compound is ≥10 μM.

In one embodiment, the lignan compound is

The effective dose is 10μM.

In one embodiment, the steatosis-related disease includes liver steatosis, or non-alcoholic fatty liver disease.

In one embodiment, the steatosis-related disease is a disease caused by a lipid metabolism disorder, including but not limited to diabetes, hyperlipidemia, or fatty liver.

The second object of the present invention is to provide a pharmaceutical composition containing

In one embodiment, the composition further contains a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutically acceptable carrier includes a diluent, excipient or solvate.

In one embodiment, the dosage form of the pharmaceutical composition is a tablet, capsule, granule, powder, syrup, oral liquid or injection.

The present invention also claims the application of the compound represented by Formula 1 in the preparation of drugs for anti-oxidative stress and/or protection of endothelial damage;

In one embodiment, the drug is used to counteract the increase in the level of ROS in the body.

The three objectives of the present invention are to provide a preparation

The method of the compound shown, the method comprises the following steps:

(1) Crush the albizia bark with a pulverizer and mix with ethanol with a concentration of 70-80% in a ratio of 1:4-10 to the material-to-liquid ratio, reflux and extract at 70-90°C for 1 to 3 times, each for 1 to 3 hours (2) Filter to remove the residue of Albizia julibrissin, combine the extract obtained in step (1), freeze-dry, collect the crude extract, pulverize, suspend and extract with ethyl acetate and n-butanol in sequence; (3) take n-butanol The extract was subjected to vacuum rotary evaporation at 80°C to recover the n-butanol, and the n-butanol extract was dried in a vacuum drying oven. Take 254g of n-butanol fraction and use D101 macroporous adsorption resin to separate, enrich 30% ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, 95% ethanol elution section; for 30% ethanol The elution section is subjected to silica gel column chromatography, using a gradient solvent elution, the solvent is 40:1, 20:1, 16:1, 10:1, 8:1 and 6:1 dichloromethane-methanol mixture. TLC detection combines the same components to obtain each elution section.

In one embodiment, collect the 8:1 elution section of the dichloromethane-methanol mixture, pass the 8:1 elution section through a reverse silica gel column, and then use CH ₃ OH as phase A and water as phase B. Perform liquid phase detection under the following elution conditions: C18 chromatographic column, flow rate 1～1.2mL/min, column temperature 29.5～30.5, elution phase gradient elution according to the following procedure:

The elution peak of 32% CH ₃ OH was separated by semi-preparative elution according to the following elution conditions:

Time/min	CH ₃ OH volume percentage	H ₂ O volume percentage
0	10%	90%
4	10%	90%
5	twenty three%	77%
50	twenty three%	77%
55	100%	0%
60	100%	0%

, Collect the compound whose retention time is 31.75min.

The fourth object of the present invention is to provide a preparation

The method includes the following steps:

(1) The albizia bark is pulverized with a pulverizer and mixed with ethanol with a concentration of 70 to 80% in a ratio of 1:4 to 10, and refluxed at 70 to 90°C for 1 to 3 times, each time for 1 to 3 hours (2) Filter to remove the residue of Albizia julibrissin, combine the extract obtained in step (1), freeze-dry, collect the crude extract, pulverize, suspend and extract with ethyl acetate and n-butanol in sequence; (3) take n-butanol The extract was subjected to vacuum rotary evaporation at 80°C to recover the n-butanol, and the n-butanol extract was dried in a vacuum drying oven; 254g of n-butanol was taken and separated by D101 macroporous adsorption resin to enrich the 30% ethanol elution section, 50 % Ethanol elution section, 70% ethanol elution section, 95% ethanol elution section; the 30% ethanol elution section is subjected to silica gel column chromatography, using gradient solvent elution, the solvent is 40:1,20:1,16 :1, 10:1, 8:1 and 6:1 dichloromethane-methanol mixtures, the same components were combined by TLC detection to obtain each elution segment.

, Collect the compound at the retention time of 35.2min, that is,

The fifth object of the present invention is to provide the application of the compound in the preparation of drugs for preventing or treating lipid metabolism disorders.

The sixth objective of the present invention is to provide the application of the compound in the preparation of drugs for anti-oxidative stress and protection of endothelial damage.

Beneficial effects: The compound prepared by the present invention can improve the accumulation of lipid droplets and steatosis of HepG2 cells induced by FFAs.

24h, under the condition of 20μM administration concentration, the lipid droplet area can be reduced from 954.15% (*P<0.05, n=3/group) to an average lipid droplet area of 134.19% (compared with FFAs induction group #P <0.05, n=3/group),

During administration

24h, the administration concentration was 5μM, and the average area of lipid droplets was 222.365% compared with the blank control group (compared with FFAs induction group ^# P<0.05, n=3/group), after 24h treatment at a dose of 80μM, The average intracellular fluorescence intensity is 300.86 ( ^# P<0.05 vs. HG, n=3/group), which has a significant decrease compared with the fluorescence intensity of the control group 813.87. The compound also has the effect of anti-oxidative stress. DCFH-DA staining results show that (+)-Lyoniresinol-9'-O-glucoside eliminates the production of reactive oxygen species induced by HG. When treated with 80μM, the accumulation of ROS is significantly reduced.

Description of the drawings

Figure 1 is an analytical high performance liquid chromatogram of 8:1 elution segment.

Figure 2 is a semi-prepared high performance liquid chromatogram of the _{32% CH 3} OH elution section of the Davisil C18 reverse column.

Figure 3 shows the ESI-MS chromatogram of (+)-Lyoniresinol-9'-O-glucoside.

^{Figure 4 is a 1} HNMR chart of (+)-Lyoniresinol-9'-O-glucoside.

^{Figure 5 is a 13} CNMR chart of (+)-Lyoniresinol-9'-O-glucoside.

Figure 6A shows that (+)-Lyoniresinol 9'-O-glucoside inhibits FFAs-induced lipid droplet formation (×400); B is the accumulation area value of lipid droplets. *P<0.05vs.control, #P<0.05vs FFAs, n=3/group.

Figure 7A is the intracellular ROS level detected by DCFH-DA fluorescence (×200); B is the intracellular ROS fluorescence value. *P compared with control P<0.05, compared with HG #P<0.05, n=3/group.

Figure 8 is a chromatogram of the components of the 8:1 elution segment detected by analytical high performance liquid chromatography.

Figure 9 is a _{chromatogram of the components of the 32% CH 3} OH elution section of the Davisil C18 reverse column under the detection of semi-prepared high performance liquid chromatography.

Figure 10 is an ESI-MS chromatogram of (-)-Lyoniresinol-9'-O-glucoside.

^{Figure 11 is a 1} HNMR spectrum of (-)-Lyoniresinol-9'-O-glucoside.

^{Figure 12 shows the 13} CNMR spectrum of (-)-Lyoniresinol-9'-O-glucoside.

Figure 13A shows that (-)-Lyoniresinol-9'-O-glucoside inhibits FFAs-induced lipid droplet formation (×400); B is the accumulation area of lipid droplets; ^* P<0.05 compared with the blank control group, ^# P<0.05 Compared with the FFAs model group, n=3/group.

Detailed ways

Example 1 Preparation of compound (+)-Lyoniresinol-9'-O-glucoside

(1) Extraction: Take 20 kg of dried Albizia julibrissin bark, pulverize, and extract with 5 times of 75% ethanol (water), namely 100L each time, reflux at 80°C for 2 times, 2 hours each time. The residue of Albizia julibrissin was removed by filtration, and the 75% ethanol extract of Albizia julibrissin was combined and freeze-dried to obtain 1.6 kg of crude ethanol extract of Albizia julibrissin. Grind the crude extract and suspend it in 2L of deionized water to make it as soluble as possible. After suspension, it was extracted with ethyl acetate and saturated n-butanol successively, and the extraction was performed by adding ethyl acetate and saturated n-butanol a few times each time. The extracts of ethyl acetate phase and saturated n-butanol phase were combined separately. The ethyl acetate part and the n-butanol part extract were obtained.

(2) Separation: Take 254g of n-butanol, dissolve and suspend in deionized water, and use D101 macroporous adsorption resin to separate and purify 30% ethanol elution section, 50% ethanol elution section, and 70% ethanol elution section , 95% ethanol elution section. The 30% ethanol elution section of macroporous liposuction was chromatographed on a silica gel column, and a gradient solvent was used to elute dichloromethane (CH ₂ Cl ₂ ): methanol (CH ₃ OH) = (40:1,20:1,16: 1,10:1, 8:1 and 6:1) perform gradient elution (the mobile phase volume is 3 times the column volume, and the target product is detected by TLC in real time). The same components are combined by TLC detection to obtain each elution segment. High performance liquid phase (HPLC) detection was performed on the 8:1 elution segment, and the UV detection wavelength was 254nm. The results are shown in Figure 1; detection conditions:

According to the retention time of each component in the analytical high performance liquid chromatogram in Figure 1, it is determined _{that each peak position in the elution section of CH 2} Cl ₂ :CH ₃ OH = 8:1 has been subjected to reverse silica gel column (Davisil C18 , 50 m) elution condition _{t R = 22min, 24min, 26min} , CH 3 OH concentration 60min corresponding to 39.8%, 42.9%, 46.2%, 100%, since the C ₁₈ packing had a diameter of 50 m, so CH ₃ The OH concentration needs to be subtracted by 10%, namely 29.8%, 32.9%, 36.2%, 100%. The elution was carried out with methanol and water as the elution phases. According to Figure 1, determine the elution phase combination is 29% CH ₃ OH (H ₂ O), 32% CH ₃ OH (H ₂ O), 36% CH ₃ OH (H ₂ O), 100% CH ₃ OH, for each The eluted components were tested by analytical HPLC, and the separation conditions were explored to determine the best separation method (as shown in Table 1). Semi-preparative liquid phase separation was performed on the 32% CH ₃ OH elution section, as shown in Figure 2 (UV detection wavelength _{290nm), the compound at the retention time t R} =31.75min is (+)-Lyoniresinol-9'-O- glucoside (Aj5).

Table 1 Liquid Chromatography Conditions

In the liquid phase separation method described in this study, although conditions 1 to 4 can also separate the target monomer compound, the purity of the product Aj5 is poor due to the interference of the impurity peak. The optimal separation conditions optimized on this basis can be Eliminate the interference of impurity peaks on the target product, and separate high purity monomer compounds. The semi-preparative separation chromatogram is shown in Figure 2.

(3) Structural identification

Aj5 is a white powder, detected by UPLC-ESI-MS, and its mass spectrum is shown in Figures 3 to 5. ESI-MS m / z 617.1992 [ M + Cl -], by Monoisotopic Mass, Even Electron Ions determine the molecular formula C ₂₈ H ₃₈ O _13.

Use Autopol IV automatic polarimeter to measure the optical rotation of monomer compounds. At a specific concentration, the vibration direction and propagation direction of a beam of light are affected by the optical rotation of the solution, and its deflection angle is related to the concentration of the solution. Ethanol, methanol and acetone are not optically active by themselves as solvents. When the measured value is negative, the measured substance is left-handed, and when the measured value is positive, the measured substance is right-handed. In this experiment, set the polarimeter to measure the polarized light wavelength at 589nm, the measuring temperature t=25°C, and the sample tube length L=1dm. The concentration of the compound Aj5 solution is c=0.001 g/mL. The specific rotation ratio [α] is calculated as follows.

The results of the optical rotation measurement are shown in the table below.

Table 2 Optical rotation measurement results of compound Aj5

The result shows negative optical activity

(c 0.001, CH ₃ OH) is a levorotatory compound.

The sample Aj5 was dissolved in a nuclear magnet tube with deuterated methanol, and ¹ HNMR and ¹³ CNMR were measured by a fully digital nuclear magnetic resonance spectrometer. The results are as follows:

¹ HNMR(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). ¹³ CNMR(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).

According to the mass spectrum, ¹ HNMR and ¹³ CNMR, the final structure is as follows, the chemical name is (+)-Lyoniresinol 9'-O-glucoside, and the chemical formula is

Example 2 Compound (+)-Lyoniresinol 9'-O-glucoside is used to improve 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), 100 U/ml penicillin and 100/ml streptomycin. Incubate in a 37°C constant temperature incubator containing 5% CO _2. The medium is changed every 1-2 days, and the cells are cultured to account for 85-90% of the volume of the petri dish and then passaged at a confluence ratio of 1:3. Use cells between the 2nd and 5th passages to verify the effect of (+)-Lyoniresinol 9'-O-glucoside on improving lipid metabolism disorders and steatosis. Take HepG2 cells in logarithmic growth phase, use a 12-well plate as a culture container, and inoculate 100,000 cells per well. After culturing for 12 hours until the cells adhere to the wall, discard the normal DMEM high glucose medium, add 0.3mM FFAs (oleic acid: palmitic acid = 2:1) in DMEM high glucose medium and continue culturing for 24 hours to make HepG2 cells modeled After becoming a lipid metabolism disorder model, add the lignan compound Aj5 prepared in Example 1 to each well, and set 5 concentration gradients, namely, at the final concentration of 5μM, 10μM, 20μM, 40μM, 80μM, and continue to incubate for 24h. Culture with normal DMEM Basal cultured HepG2 cells were used as controls.

Discard the medium of the 12-well plate, wash three times with PBS, fix with 4% paraformaldehyde for 30 minutes, wash thoroughly with PBS after the fixation, then soak with 60% isopropanol for 10-15 seconds, then wash with PBS three times all over. Stain with oil red O (oil red storage solution: deionized water=3:2) at room temperature for 20-30 minutes, observe the staining of the cells under a microscope, discard the oil red dye solution, and differentiate with 60% isopropanol until the interstitium is clear ( Differentiation takes about 5-10 seconds. Excessive differentiation will cause the oil red O to fade). Finally, wash with PBS three times, add 1ml PBS to each well to mount and take pictures.

The oil red O staining results are shown in Figure 6. Compared with the blank control group with 100% lipid droplet area, FFAs can significantly induce lipid droplet accumulation and steatosis in HepG2 cells after 24h culture in high-fat medium. Compared with the blank control group, the average is 970.01% ( ^* P<0.05, n=3/group). After 24 hours of administration, when the administration concentration is 5μM, the average area of lipid droplets is 222.36% compared with the blank control group (compared with FFAs). treated group compared to ^{# P <0.05, n = 3} / group), indicating (+) - Lyoniresinol-9'- O-glucoside effective in treating FFAs induced steatosis and dyslipidemia. Hepatic steatosis is a significant feature of type 2 diabetes (T2DM), which may lead to non-alcoholic fatty liver disease and cardiovascular disease. Therefore, this application (+)-Lyoniresinol-9'-O-glucoside can effectively treat FFAs-induced lipid metabolism disorders and steatosis, and the results show that (+)-Lyoniresinol-9'-O-glucoside can be used as a treatment for non-alcoholic Potential natural medicines for fatty liver and related hepatocellular diseases, and can also be used as medicines for the prevention of type 2 diabetes and cardiovascular diseases.

Example 3 Compound (+)-Lyoniresinol 9'-O-glucoside is used for anti-oxidative stress

Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in a 37°C constant temperature incubator _{containing 5% CO 2.} The medium is changed every 1-2 days, and the cells are cultured to account for 85-90% of the volume of the petri dish and then passaged at a confluence ratio of 1:3. Cells between the 2nd and 5th passages were used to verify the activity of (+)-Lyoniresinol9'-O-glucoside against oxidative stress. In order to test the protective effect of (+)-Lyoniresinol-9'-O-glucoside in the oxidative stress of HUVEC cells induced by high concentration of glucose (HG), HUVEC cells in the logarithmic growth phase were taken and cultured in a 12-well plate as a culture vessel. After 12h to cell adhesion, add 80μM (+)-Lyoniresinol-9'-O-glucoside and continue to incubate for 12h (set 5 groups in parallel), and then add pre-prepared sterile glucose solution (after glucose is dissolved in PBS, after 0.22μm sterile filter membrane, and then add 50μL high concentration glucose solution to each hole, and add 50μL PBS to the blank control group to make the final concentration 35mM, and continue to incubate for 24h. The production of ROS in cells was detected by fluorescent probe DCFH-DA. The HUVEC was washed 3 times with PBS, and then incubated with 10 μM DCFH-DA (DCFH-DA dissolved in PBS) at 37°C for 30 minutes in the dark. After washing the cells with PBS, they were photographed on a fluorescence microscope (80i, Nikon, Japan).

The results are shown in Figure 7. Compared with the ROS fluorescence intensity of 100% in the Control group, 35mM HG significantly increased the accumulation of reactive oxygen species in HUVEC cells and promoted endothelial damage. At this time, the ROS fluorescence intensity was 813.87 ( ^* P<0.05 vs. Control, n=3/group). When treated with 80μM (+)-Lyoniresinol-9'-O-glucoside for 24h, the average fluorescence intensity was 300.86 ( ^# P<0.05vs.HG, n=3/group) The experiment showed that HUVEC cells were exposed to HG Increased the accumulation of reactive oxygen species. DCFH-DA staining results showed that (+)-Lyoniresinol-9'-O-glucoside eliminated the production of reactive oxygen species induced by HG. When treated with 80μM, the accumulation of ROS was significantly reduced.

Studies have shown that the accumulated reactive oxygen species (ROS) is the response to high glucose or HG, and it is also the main cause of cell damage and apoptosis. Existing studies have shown that reactive oxygen species play a major role in endothelial cell apoptosis induced by HG. 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 complicated. Among them, active oxygen participates in the pathogenesis of cardiovascular damage caused by hyperglycemia, and continuous hyperglycemia can cause reactive oxygen generation and endothelial cell dysfunction. The results show that HG can significantly induce endothelial cell damage and ROS accumulation. (+)-Lyoniresinol-9'-O-glucoside can significantly eliminate accumulated ROS and protect endothelial cell damage induced by high glucose. It also shows that the (+)- Lyoniresinol-9'-O-glucoside can be used as a potential drug for the treatment of complications such as diabetes.

Example 4 Preparation of compound (-)-Lyoniresinol-9'-O-glucoside

(2) Separation: Take 254g of n-butanol, dissolve and suspend it in deionized water, and use D101 macroporous adsorption resin for separation and purification. Use 2 to 3 column volumes of ethanol-water mixed solution as the mobile phase for gradient washing. Remove, enriched 30% ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, 95% ethanol elution section. The 30% ethanol elution section of macroporous liposuction was chromatographed on a silica gel column with a gradient solvent eluted with dichloromethane (CH ₂ Cl ₂ ): methanol (CH ₃ OH) = (40:1, 20:1, 16:1, 10:1, 8:1 and 6:1) as the mobile phase. Gradient elution was performed with a mobile phase of about 4 times the column volume. The eluent was collected in a 250ml Erlenmeyer flask. TLC detection combines the same components to obtain each elution section. Analytical high performance liquid (HPLC) detection on the 8:1 elution segment of silica gel (the chromatographic column is X-Bridge C18, 5μm, 4.6×250mm, the flow rate is 1ml/min, the column temperature is 30°C), and the UV detection wavelength is 254nm , Elution conditions:

The detection result is shown in Figure 8. According to the retention time of each component in the analytical high performance liquid chromatogram in Figure 8, it is determined that the CH ₂ Cl ₂ :CH ₃ OH = 8:1 elution section has been reversed silica gel The elution conditions of the column (Davisil C18, 50μm) are t _R = 22min, 24min, 26min, and the corresponding CH ₃ OH concentration at 60min is 39.8%, 42.9%, 46.2%, 100%, because Figure 1 shows the analytical liquid phase In the chromatogram, the chromatographic column is X-Bridge C18 (5μm, 4.6×250mm), and the Davisil C18 reverse column packing is 50μm, so the mobile phase methanol concentration is reduced by 10%, namely 29.8%, 32.9%, 36.2%, 100 %. The elution was carried out with methanol and water as the elution phases. According to Figure 8, it is determined that the elution phase combination is 29% CH ₃ OH (H ₂ O), 32% CH ₃ OH (H ₂ O), 36% CH ₃ OH (H ₂ O), 100% CH ₃ OH. Analytical HPLC detection was performed on the eluted fractions of 32% CH ₃ OH, and the separation conditions were explored to determine the best separation method (as shown in Table 1). Semi-preparative liquid phase separation for the 32% CH ₃ OH elution section (chromatographic column is X-Bridge C18, 5μm, 10×250mm, flow rate is 4ml/min, column temperature is 30℃), as shown in Figure 9 (UV detection (Wavelength 290nm), the compound at the retention time t _R =35.20min is (-)-Lyoniresinol-9'-O-glucoside(Aj6).

In the process of obtaining the liquid phase separation method described in the foregoing embodiments, the separation conditions from Condition 1 to Condition 4 have also been tried. Although these separation conditions can also separate the target monomer compound, they are interfered by the impurity peak, and the product Aj6 is obtained. The purity is poor, and it is difficult to carry out the next step of separation and identification. The optimal separation conditions optimized on this basis can eliminate the interference of impurity peaks on the target product, and separate high-purity monomer compounds. The semi-preparative separation chromatogram is shown in Figure 9. Collect the compound at the retention time t _R =35.20 min, evaporate under reduced pressure at 75° C., recover the solvent, and dry in a vacuum drying oven to obtain the Aj6 monomer compound.

(3) Structural identification

Aj6 is a light yellow powder, detected by UPLC-ESI-MS, and its mass spectrum is shown in Figures 10-12. ESI-MS m / z 617.2001 [ M + Cl -], by Monoisotopic Mass, Even Electron Ions determine the molecular formula C ₂₈ H ₃₈ O _13.

Use Autopol IV automatic polarimeter to measure the optical rotation of monomer compounds. At a specific concentration, the vibration direction and propagation direction of a beam of light are affected by the optical rotation of the solution, and its deflection angle is related to the concentration of the solution. Ethanol, methanol and acetone are not optically active by themselves as solvents. When the measured value is negative, the measured substance is left-handed, and when the measured value is positive, the measured substance is right-handed. In this experiment, set the polarimeter to measure the polarized light wavelength at 589nm, the measuring temperature t=25°C, and the sample tube length L=1dm. The concentration of the compound Aj6 solution is c=0.001 g/mL. The specific rotation ratio [α] is calculated as follows.

The results of the optical rotation measurement are shown in the table below.

Table 3 Optical rotation measurement results of compound Aj6

The result shows negative optical activity

(c 0.001, CH ₃ OH) is a left-handed compound.

The sample Aj6 was dissolved in a nuclear magnet tube with deuterated methanol, and ¹ HNMR and ¹³ CNMR were measured by a fully digital nuclear magnetic resonance spectrometer. The results are as follows:

¹ H NMR(400MHz,CD ₃ OD)δ6.59(1H,s,H-8), 6.43(2H,s,H-2',6'), 4.24(1H,d,J=5.3Hz,H -4), 4.15(1H,d,J=7.7Hz,H-1”), 3.87(3H,s,-OCH ₃ ), 3.84(3H,d,J=3.6Hz,-OCH ₃ ), 3.77( 6H,s,3',5'-OCH ₃ ), 3.72 (1H, d, J = 5.2 Hz), 3.69 (1H, d, J = 5.1 Hz), 3.62 (5H, dt, J = 22.8, 6.5 Hz ), 3.49(1H,dd,J=12.5,7.2Hz), 3.44–3.35(2H,m), 3.30–3.13(3H,m), 2.70(2H,t,J=8.0Hz,H), 2.13( 1H,dd,J=13.1,6.9Hz,H-3),1.70(1H,d,J=6.3Hz,H-1). ¹³ C NMR(101MHz,CD ₃ OD)δ147.61(C-3',5'),147.29(C-5),146.15(C-7),138.06(C-1'),137.49(C-6),133.21(C-4'),128.83(C-9),124.84(C-10),106.41(C-8),105.72(C-2',6'),102.85(C-1”),76.79(C-5”),76.58(C-3”),73.67( C-2”), 70.61(C-3α), 70.16(C-4”), 64.83(C-2α), 61.31(C-6”), 58.72(5-OMe), 55.53(7-OMe), 55.23(3',5'-OMe), 45.19(C-3), 41.83(C-4), 39.85(C-2), 32.43(C-1).

According to the mass spectrum, ¹ HNMR and ¹³ CNMR, the final structure is as follows, and the chemical name is (-)-Lyoniresinol-9'-O-glucoside.

Example 5 Compound (-)-Lyoniresinol-9'-O-glucoside is used to improve lipid metabolism disorder and steatosis

(1) HepG2 cells (American Type Culture collection, USA) were cultured in DMEM containing 25% glucose, 10% FBS (fetal bovine serum, Gibco), 100 U/ml penicillin and 100 U/ml streptomycin. Incubate in a 37°C constant temperature incubator containing 5% CO _2. The medium is changed every 1-2 days, and the cells are cultured to account for 85-90% of the volume of the petri dish and then passaged at a confluence ratio of 1:3. In all experiments, cells between the 2nd and 5th passages were used to verify the effect of (-)-Lyoniresinol-9'-O-glucoside on improving lipid metabolism disorders and steatosis. Take HepG2 cells in the logarithmic growth phase and spread a 12-well plate with 100,000 per well. After 12 hours of cell growth, the normal DMEM high glucose medium was discarded, and DMEM high glucose medium containing 0.3 mM FFAs (oleic acid: palmitic acid = 2:1) was added and cultured for 24 hours, that is, HepG2 cells were modeled into After the lipid metabolism disorder model, 5 concentration gradients were set for each well (the Control group was HepG2 cells cultured in normal DMEM medium), that is, the final concentration of 5μM, 10μM, 20μM, 40μM, 80μM was continued to be cultured for 24h.

(2) Discard the medium of the 12-well plate, wash with PBS three times, fix with 4% paraformaldehyde for 30 minutes, wash thoroughly with PBS after the fixation, and then soak with 60% isopropanol for 10-15 seconds, and then with Wash three times with PBS. Stain with oil red O (oil red storage solution: deionized water=3:2) at room temperature for 20-30 minutes, observe the staining of the cells under a microscope, discard the oil red dye solution, and differentiate with 60% isopropanol until the interstitium is clear ( Differentiation takes about 5-10 seconds. Excessive differentiation will cause the oil red O to fade). Finally, wash with PBS three times, add 1ml PBS to each well to mount and take pictures.

(3) The oil red O staining result is shown in Figure 13 (Figure 13B, the lipid accumulation value area was processed using Image-Pro Plus 6.0 software under the same parameters). Compared with 100% of the lipid droplet area of the blank control group, FFAs can significantly induce lipid droplet accumulation and steatosis in HepG2 cells after 24 hours of culture in the high-fat medium. At this time, the lipid droplet area averaged 954.15% compared with the blank control group (* P<0.05, n=3/group), 24h after administration, when the administration concentration is 10μM, it can significantly improve the lipid metabolism disorder of HepG2 cells induced by FFAs and improve steatosis. When the administration concentration is 20μM, the average lipid droplet area is 134.19% compared with the blank control group (compared with the FFAs induction group #P<0.05, n=3/group), indicating (-)-Lyoniresinol-9'-O -glucoside can effectively treat lipid metabolism disorders and steatosis induced by FFAs.

Example 6 Drugs containing (+)-Lyoniresinol-9'-O-glucoside or (-)-Lyoniresinol-9'-O-glucoside

(+)-Lyoniresinol-9'-O-glucoside prepared in Example 1 or (-)-Lyoniresinol-9'-O-glucoside prepared in Example 2 is compatible with pharmaceutically acceptable salts, or with various solids Or liquid pharmaceutical excipients and/or adjuvants are combined to prepare an appropriate administration form or dosage form for human use.

The pharmaceutically acceptable salt includes but is not limited to (+)-Lyoniresinol-9'-O-glucoside or (-)-Lyoniresinol-9'-O-glucoside and inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid , Hydrobromic acid and nitric acid, and with various organic acids, such as maleic acid, malic acid, fumaric acid, succinic acid, tartaric acid, citric acid, acetic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, palmitic acid Wait for the resulting salt.

The drug can be administered to a host in need, such as humans, through enteral or parenteral routes. The pharmaceutical dosage form administered through the intestinal tract is an oral preparation, such as: tablets, capsules, granules, suspensions, sustained-release agents, etc. The product of the present invention administered parenterally may be in the form of injections, topical preparations such as skin patches, or sprays.

The drug can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum, or rectum. Dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, lozenges, suppositories, freeze-dried powder injections Wait. It can be ordinary preparations, sustained-release preparations, controlled-release preparations, and various particulate drug delivery systems. In order to prepare a unit dosage form into a tablet, various carriers known in the art can be widely used. Carriers can be diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting Agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, glucose solution, gum arabic, gelatin syrup, sodium carboxymethyl cellulose, shellac, Methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dried starch, alginate, agar powder, alginate, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fat Acid esters, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary Ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the administration unit into a pill, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , Ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dried starch, alginate, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose, etc. In order to make the administration unit into a suppository, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like. In order to make the administration unit into a capsule, the active ingredient is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard clear capsule or a soft capsule. The active ingredients can also be made into microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules or made into injections for application. In order to prepare the administration unit into injection preparations, such as solutions, emulsions, freeze-dried powder injections and suspensions, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3 -Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation, and in addition, conventional solubilizers, buffers, pH adjusters, etc. can also be added.

In addition, if necessary, coloring agents, preservatives, flavors, flavors, sweeteners or other materials can also be added to the pharmaceutical preparations.

Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims

Application of the compound in the preparation of medicines for improving or preventing steatosis and related diseases; the compound is
The application according to claim 1, wherein the effective dose of the lignan compound is ≥5 μM.
The use according to claim 1, wherein the effective dose of the compound is ≥10 μM.
The application according to claim 1, wherein the steatosis-related diseases include hepatic steatosis or non-alcoholic fatty liver disease.
The application according to claim 1, wherein the steatosis-related diseases are diseases caused by lipid metabolism disorders, including but not limited to diabetes, hyperlipidemia or fatty liver.
A pharmaceutical composition characterized in that it contains
The pharmaceutical composition according to claim 6, wherein the composition further contains a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier comprises a diluent, excipient or solvate.
The pharmaceutical composition according to any one of claims 6 to 8, wherein the dosage form is a tablet, capsule, granule, powder, syrup, oral liquid or injection.
Application of the compound represented by formula 1 in the preparation of a medicine for preventing oxidative stress and/or protecting endothelial damage;
The application according to claim 10, wherein the medicine is used to resist the increase of ROS level in the body.
A method for preparing a compound, characterized in that it comprises the following steps:

(1) The albizia bark is pulverized with a pulverizer and mixed with ethanol with a concentration of 70 to 80% in a ratio of 1:4 to 10, and refluxed at 70 to 90°C for 1 to 3 times, each time for 1 to 3 hours ；

(2) Filtering to remove the residue of Albizia julibrissin, combining the extracts obtained in step (1), freeze-drying, collecting the crude extract, pulverizing, suspending and then extracting with ethyl acetate and n-butanol;

(3) Take the n-butanol extract, recover the n-butanol by rotary evaporation under reduced pressure at 80°C, and dry the n-butanol extract in a vacuum drying oven; take 254g of the n-butanol fraction and use D101 macroporous adsorption resin for separation and enrichment for 30 % Ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, 95% ethanol elution section; silica gel column chromatography was performed on the 30% ethanol elution section, eluted with a gradient solvent, and the solvent was 40 :1,20:1,16:1,10:1,8:1 and 6:1 dichloromethane-methanol mixtures, the same components are combined by TLC detection, and each elution segment is obtained.
The method according to claim 12, characterized in that the 8:1 elution section of the dichloromethane-methanol mixture is collected, the 8:1 elution section is passed through a reverse silica gel column, and then CH 3 OH is used as phase A, Water is phase B, and liquid phase detection is carried out according to the following elution conditions: C18 chromatographic column, flow rate 1～1.2mL/min, column temperature 29.5～30.5, elution phase gradient elution according to the following procedure:

The elution peak of 32% CH 3 OH was separated by semi-preparative elution according to the following elution conditions:

Time/min CH 3 OH volume percentage H 2 O volume percentage 0 10% 90% 4 10% 90% 5 twenty three% 77% 50 twenty three% 77% 55 100% 0% 60 100% 0%

, The compound whose retention time is 31.75min is the compound
The method according to claim 12, characterized in that the 8:1 elution section of the dichloromethane-methanol mixture is collected, the 8:1 elution section is passed through a reverse silica gel column, and then CH 3 OH is used as phase A, Water is phase B, and liquid phase detection is carried out according to the following elution conditions: C18 chromatographic column, flow rate 1～1.2mL/min, column temperature 29.5～30.5, elution phase gradient elution according to the following procedure:

The elution peak of 32% CH 3 OH was separated by semi-preparative elution according to the following elution conditions:

Time/min CH 3 OH volume percentage H 2 O volume percentage 0 10% 90% 4 10% 90% 5 twenty three% 77% 50 twenty three% 77% 55 100% 0% 60 100% 0%

, Collect the compound with a retention time of 35.2min, and obtain