WO2021063075A1 - Albiziae cortex lignan compound and application thereof - Google Patents

Abstract

The present invention relates to the technical field of biomedicine, and disclosed are an albiziae cortex lignan compound and an application thereof. The compound prepared by the present invention can effectively treat FFAs-induced lipid metabolism disorders and fatty degeneration at a concentration, and can enable the lipid droplet area to reduce by about 3 times at an administration concentration of 20 μM 24 h after administration; the compound can significantly enhance cell activity of HUVEC and significantly promote self-replication of cells, so as to promote proliferation of endothelial cell HUVEC. The cell viability can reach 242.233% in control at an administration concentration of 40 μM 24 h after administration. The compound prepared by the present invention can significantly inhibit the production of HG-induced reactive oxygen species (ROS) of the HUVEC cells, and can reduce the average fluorescence intensity of an HG group from 818.37% to 176.36%.

Description

Albizia bark lignan compounds and their applications Technical field

The invention relates to albizia bark lignan compounds and applications, 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 a new lignan compound of Albizia Julibrissin, the structural formula of which is shown in formula 1;

In one embodiment, the compound is a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate of the compound represented by formula 1.

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

In one embodiment, the composition is a pharmaceutical composition.

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 three objectives of the present invention are to provide a method for preparing the compound represented by formula 1, which comprises the following steps: (1) The albizia bark is pulverized with a pulverizer, and the ethanol concentration is 70-80% according to the ratio of material to liquid. Mix in a ratio of 1:4-10, 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 extracts obtained in step (1), freeze-dry, Collect the crude extract, pulverize, suspend, and extract with ethyl acetate and n-butanol in sequence; (3) Recover the n-butanol extract and dry to obtain 254g. D101 macroporous adsorption resin is used, and the volume fraction is 30% and 50%. , 70% and 95% ethanol for elution separation, respectively enriched 30% ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, 95% ethanol elution section; 30% ethanol elution section The segment was subjected to silica gel column chromatography, using 40:1, 20:1, 16:1, 10:1, 8:1 and 6:1 dichloromethane-methanol mixture gradient elution, and the same components were combined by TLC detection , Collect the 10:1 elution section.

In one embodiment, the recovery in step (3) is to recover the n-butanol by rotary evaporation under reduced pressure at 80° C., and dry the n-butanol extract in a vacuum drying oven.

In one embodiment, the recovery in step (3) is to recover the n-butanol by rotary evaporation under reduced pressure at 80° C., and dry the n-butanol extract in a vacuum drying oven.

In one embodiment, collect the dichloromethane-methanol mixture 10:1 elution section, pass the 10:1 elution section through a reverse silica gel column, and finally 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:

In one embodiment, the elution peak when phase A is 42% CH ₃ OH is eluted and separated according to the following semi-preparative separation conditions:

Time/min	CH 3 OH volume percentage	H 2 O volume percentage
0	10%	90%
4	10%	90%
5	26%	74%
45	28%	72%
60	32%	68%
70	50%	50%
80	100%	0%

Collect the compound with the retention time of 26.50min, that is,

The fourth 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 fifth object of the present invention is to provide the application of the compound in the preparation of drugs that promote the proliferation and migration of endothelial cells.

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 effectively treat the lipid metabolism disorder and steatosis induced by FFAs. At a dosage of 20 μM, the area of lipid droplets can be reduced by about 3 times after 24 hours of administration;

The compound prepared by the present invention can significantly enhance the cell activity of HUVEC, and can significantly promote cell self-replication, thereby promoting the proliferation of HUVEC of endothelial cells. After the administration of 40μM, 24h after administration, the cell viability can reach 242.233% of the control.

The compound prepared by the invention can significantly inhibit the production of reactive oxygen species (ROS) induced by HG in HUVEC cells, and can reduce the average fluorescence intensity of the HG group from 88.37% to 176.35%.

Description of the drawings

Figure 1 is an analytical high performance liquid chromatogram with 10:1 elution segment.

Figure 2 is a 10:1 component Davisil C18 reverse column 42% CH ₃ OH elution section semi-prepared high performance liquid chromatogram.

Figure 3 is a ¹ H-NMR chromatogram of compound Aj4.

Figure 4 is a ¹³ C-NMR chromatogram of compound Aj4.

Figure 5 shows the HR-ESI-MS chromatogram of albioside A.

Figure 6A shows that compound albioside A inhibits FFAs-induced lipid droplet formation (×400); B is the accumulation area value of lipid droplets; where ^* P<0.05 compared with the control group, ^# P<0.05 compared with the FFAs group, n= 3/group.

Figure 7 shows that the compound albioside A promotes cell proliferation activity, ^** P<0.01 compared with the control group.

Figure 8 shows that the compound albioside A promotes HUVEC cell migration.

Figure 9 shows that the compound albioside A enhances the cloning or self-replication ability of HUVEC cells.

Figure 10 shows the detection of intracellular ROS levels of compounds by DCFH-DA fluorescence (×200); B. Intracellular ROS fluorescence values. *P compared with control P<0.05, compared with HG ^# P<0.05, n=3/group.

Figure 11 is an analytical high performance liquid chromatogram with 8:1 elution segment.

Figure 12 is a semi-prepared high performance liquid chromatogram of an 8:1 component Davisil C18 reverse column with 32% CH _{3 OH elution section.}

Figure 13 is a HR-ESI-MS chromatogram of compound Aj7.

Figure 14 is a ¹ H-NMR chromatogram of compound Aj7.

Figure 15 is a ¹³ C-NMR chromatogram of compound Aj7.

Detailed ways

Example 1 Preparation of Compound

(1) Extraction: Take 20kg of dried Albizia julibrissin bark, pulverize, and extract with 5 times 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. The crude extract was crushed and suspended 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 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. Desorption, enrichment of 30% ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, and 95% ethanol elution section. The 30% ethanol elution section of the macroporous adsorption resin was chromatographed on a silica gel column with a dichloromethane-methanol mixture as the mobile phase, and a gradient solvent of dichloromethane (CH ₂ Cl ₂ ): methanol (CH ₃ OH) = ( 40:1, 20:1, 16:1, 10:1, 8:1 and 6:1) for gradient elution (mobile phase volume is 3 times the column volume, real-time TLC detection of whether the target product is obtained), respectively The mobile phase was eluted with about 4 times the column volume, and the eluate was collected separately in a 250ml Erlenmeyer flask, and the same components were combined by TLC detection to obtain each elution section.

Analytical high performance liquid (HPLC) detection was performed on the elution segment of dichloromethane (CH ₂ Cl ₂ ): methanol (CH ₃ OH) solvent 10:1 (chromatographic column is X-Bridge C18, 5μm, 4.6×250mm, flow rate 1ml/min, column temperature 30℃), UV detection wavelength is 254nm, elution conditions:

The result is shown in Figure 1. According to the retention time of the compound under the liquid phase detection conditions in Figure 1, it is determined that the main peak of the silica gel 10:1 elution section corresponds to the retention time of the elution condition on the C18 reverse column as 23min, 30min, 36min, 60min, and The methanol concentration corresponding to each retention time is 41% CH ₃ OH, 52% CH ₃ OH, 62% CH ₃ OH, and 100% CH ₃ OH.

The 10:1 elution segment was passed through a reverse silica gel column (Davisil C18, 50μm), and methanol and water were used as elution phases for elution. Because Figure 1 is an analytical liquid 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%. According to Figure 1, determine the elution phase combination is 31% CH ₃ OH, 42% CH ₃ OH, 52% CH ₃ OH, 100% CH ₃ OH. The elution components were detected by HPLC, according to the analytical liquid phase groping 42 The separation conditions of %CH ₃ OH elution section are determined and the best separation conditions are determined, as shown in Table 1. Finally, the 42% CH ₃ OH elution section was subjected to semi-preparative liquid phase separation (the chromatographic column was X-Bridge C18, 5μm, 10×250mm, the flow rate was 4ml/min, and the column temperature was 30°C). The results are shown in Figure 2. , The compound with retention time t=26.50min is named albiosideA(Aj4).

Table 1 Different separation conditions of semi-preparative liquid chromatography

Among the separation conditions shown in Table 1, condition 1, condition 2, and condition 3 cannot effectively separate Aj4, and there is noise interference near the retention time. The separation condition 4 can effectively separate compound Aj4. The separation conditions used in this experiment are The best separation method further optimized on the basis of four, its separation effect is shown in Figure 2. Collect the compound at the retention time t _R =26.50 min, and evaporate under reduced pressure at 75° C. to recover the solvent and dry in a vacuum drying oven to obtain the Aj4 monomer compound.

(3) Identification of the structure (FIGS. 3 to 5): Aj4 as a pale yellow powder by UPLC-ESI-MS detection, ESI-MS m / z 633.1932 [M + Cl -], by Monoisotopic Mass Even Electron Ions determined, Its molecular formula is C ₂₈ H ₃₈ O ₁₄ .

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 Aj4 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 Determination of the optical rotation of compound Aj4

The result shows negative optical activity

(c 0.002, CH ₃ OH) is a levorotatory compound.

The sample Aj4 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)δ6.76(2H,s,H-2',6'), 6.74(2H,s,H-2,6), 6.56(1H,d,J=16.0Hz ,H-7'), 6.34(1H,dt,J=15.8,5.5Hz,H-8'), 4.81(1H,d,J=7.2Hz,H-7), 4.65(4H,s), 4.29 (1H,dd,J=9.0,4.8Hz,H-1”), 4.24(2H,d,J=5.4Hz,H-9'), 3.96–3.88(2H,m), 3.85(6H,s, 3,5-OMe), 3.83(6H,s,3',5'-OMe), 3.77(1H,s), 3.73–3.63(4H,m), 3.60(1H,dd,J=8.4,3.9Hz ),3.56–3.39(4H,m),3.22(s,1H). ¹³ C NMR(101MHz,CD ₃ OD)δ153.08(C-3',5'),152.39(C-3,5), 138.12(C-4'),134.99(C-4),134.13(C-1'),133.30(C-1),129.95(C-7'),128.51(C-8'),104.62(C- 2,6),104.26(C-1”),103.51(C-2',6'),85.66(C-8),76.94(C-3”),76.37(C-5”),74.32(C -2”),72.64(C-7),69.92(C-4”),62.16(C-9),61.17(C-9'),60.23(C-6”),55.61(3',5' -OMe), 55.28 (3,5-OMe).

According to the mass spectrum, ¹ HNMR, ¹³ CNMR, the final structure is as follows:

Chemical name 2-[4-(3-Hydroxy-1-propenyl)-2,6-dimethoxy-phenoxy]-3-hydroxy-3-(4-hydroxy-3,5-dimethoxy-phenyl)propyl-β-D -glucopyranoside. It is named albioside A (Aj4).

Example 2 Drugs Containing Albizia Neolignoside A

Combining the Albizia xyloside A prepared in Example 1 with pharmaceutically acceptable salts, or combining with various solid or liquid pharmaceutical excipients and/or adjuvants, into a suitable administration form that can be used for humans Or dosage form.

The pharmaceutically acceptable salts include, but are not limited to, the salts formed by albizia lignoside A with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, phosphorous acid, hydrobromic acid and nitric acid, and with various organic acids such as horses. It is a salt of acetic acid, malic acid, fumaric acid, succinic acid, tartaric acid, citric acid, acetic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, palmitic acid, etc.

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, lyophilized 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.

Example 3 Compounds used to improve lipid metabolism disorders 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. HepG2 was cultured 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 culture dish and then passaged at a confluence ratio of 1:3. Use the cells between the 2nd and 5th passages to verify the effect of Albizia Julibrissin A in 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, the normal DMEM medium is discarded, and 0.3mM FFAs (oleic acid: palmitic acid = 2:1) is added, in which oleic acid is dissolved in DMSO, and palmitic acid is dissolved in ultrapure water at 75°C in a water bath. Dissolve in 40mM, quickly dissolve and mix with 40% BSA in PBS, cool through a 0.22μm sterile filter, and dilute with DMEM medium to 0.3mM FFAs high-fat medium) DMEM high-fat medium for 24h, that is After HepG2 cells were modeled as a lipid metabolism disorder model, each hole was added with the compound prepared in Example 1 with different concentration gradients, and the administration concentration was set at 5 concentration gradients (HepG2 cells cultured in normal medium were used as the control, and the control group was added The same volume of PBS is used as a blank control group), and the rest are at final concentrations of 5μM, 10μM, 20μM, 40μM, 80μM (albioside A is dissolved in DMSO to 100mM, and then diluted with sterile PBS, and finally 20μL of high-concentration monomer is added to each well Drugs, make the final concentration of the culture medium the set concentration gradient) continue to cultivate for 24h.

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 staining solution (oil red storage solution: deionized water = 3:2) at room temperature for 20 to 30 minutes, observe the staining of the cells under a microscope, discard the oil red staining solution, and differentiate to interstitium with 60% isopropanol Clear (differentiation is about 5-10 seconds, excessive differentiation will cause the oil red O to fade), and 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. At this time, the average lipid droplet area compared with the blank control group is 988.23% ( *P<0.05, n=3/group), when the administration concentration is 10μM 24h after administration, albioside A can effectively reduce the formation of lipid droplets, when the administration concentration is 20μM, compared with the blank control group The average droplet area is 240.165% (compared with FFAs induction group #P<0.05, n=3/group), indicating that albioside A can effectively treat FFAs-induced lipid metabolism disorders and steatosis. In Figure 6B, the lipid accumulation area value was processed using Image-ProPlus 6.0 software under the same parameters.

Hepatic steatosis is a significant feature of type 2 diabetes (T2DM), which may lead to non-alcoholic fatty liver disease and cardiovascular disease. This example shows that albioside A can effectively treat FFAs-induced lipid metabolism disorders and steatosis, can be used as a potential natural drug for the treatment of non-alcoholic fatty liver and related hepatocellular diseases, and can also be used as a drug to prevent type 2 diabetes and cardiovascular disease .

Example 4 Compound used to promote endothelial cell proliferation activity

Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium 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 culture dish and then passaged at a confluence ratio of 1:3. Cells between the 2nd and 5th passages were used for the verification of the activity of albizia cortex lignoside A to promote endothelial cell proliferation. Take the HUVEC cells in the logarithmic growth phase, use a 96-well plate as a culture container, add 100 μL of medium to each well, and inoculate 3000 cells. After culturing for 12 hours to adhere to the wall, apply the compound prepared in Example 1, and continue to incubate for 24 hours. Then add 10 μL CCK-8 to each well. After incubating for 1 hour at 37°C in a cell incubator, use a microplate reader to detect the absorbance (OD) at 450 nm wavelength. ) Value to calculate cell viability.

The results are shown in Figure 7. Compared with the blank control group, HUVEC cell viability increased significantly after 24 hours of administration. In this experiment, when 40μM was administered, the cell viability was 242.233% compared with 100% of the blank control group. It shows that albioside A can significantly enhance the cell proliferation activity of HUVEC.

Therapeutic angiogenesis refers to the transfer of exogenous angiogenesis-inducing factors into tissues to promote collateral capillary angiogenesis in the ischemic area. It is an important method for the treatment of ischemic diseases in recent years. The angiogenesis process is closely related to the migration and proliferation of vascular endothelial cells. The results show that albioside A can effectively promote the migration of endothelial cells, enhance cell viability and self-replication ability, and can effectively promote angiogenesis.

Example 5 Compound used to promote HUVEC cell migration

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% CO2. 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, the cells between the 2nd and 5th passages were used for the verification of the activity of albizia cortex lignoside A to promote the migration of HUVEC cells. Take the HUVEC cells in the logarithmic growth phase and culture them in a 6-well plate. Inoculate 200,000 cells per well. After culturing for 24 hours to allow the cells to grow adherently, the cells in the dish are scratched, and then washed twice with PBS, and added Culture medium containing 40μM albioside A monomer compound, continue to culture for 24 hours, observe the cell migration state. The experimental results show that albioside A can significantly promote cell migration. The results are shown in Figure 8. After scratching, 40μM is administered and cultured for 24 hours, and taken with an inverted microscope. Albioside A can significantly promote cell migration. This is also true in in vivo experiments. Promote wound healing.

Example 6 Compound used to promote angiogenesis

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.} Change the medium every 1-2 days, and pass 85-90% confluent cells at a ratio of 1:3 confluence. In all experiments, cells between passage 2 and passage 5 were used to promote HUVEC cell proliferation. Take the HUVEC cells in the logarithmic growth phase, digest them with trypsin and pipette them into single cells, culture them in a 6-well plate, add 2ml medium to each well, inoculate 100 cells per well, and place them at 37°C with 5% CO _2. Culture in the cell incubator. When there are macroscopically visible clones in the petri dish, stop the culture. The supernatant was discarded and carefully immersed twice with PBS. Add 4% paraformaldehyde to fix for 15 minutes. Then remove the fixative solution, add appropriate amount of GIMSA and apply the dye solution to dye for 10-30 minutes, then slowly wash off the dye solution with running water, and air dry. Finally, the number of clones was observed. The results are shown in Figure 9. The number of clones in the administration group was significantly more than that in the control group. The results showed that the monomer compound albioside A can significantly promote cell self-replication, thereby promoting the proliferation of HUVEC endothelial cells, that is, angiogenesis.

Example 7 Compound 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 culture dish and then passaged at a confluence ratio of 1:3. In all experiments, the cells between the 2nd and 5th passages were used to verify the anti-oxidative stress effect of Albizia Julibrissin A. In order to test the protective effect of Albioside A (Albioside A) in the oxidative stress of HUVEC cells induced by high concentration glucose (HG), HUVEC cells in the logarithmic growth phase were taken and cultured in a 12-well plate for 12 hours until the cells After adherent growth, add 80μM albioside A and continue to incubate for 12h (5 repeat holes are set), and then add pre-prepared sterile glucose solution (after glucose is dissolved in PBS, pass through 0.22μm sterile filter membrane, and then add to each hole 50μL high-concentration glucose solution, blank control group plus 50μL PBS), make the final concentration of glucose 35mM, continue to incubate for 24h. The production of ROS in cells was detected by the 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 10. 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 818.37 ( ^* P<0.05 vs. Control, n=3/group). When treated with 80μM albioside A for 24h, its fluorescence intensity was 176.335 (( ^# P<0.05vs.HG, n=3/group)) The experiment showed that exposure of HUVEC cells to HG increased the accumulation of reactive oxygen species, DCFH- DA staining results showed that albioside A eliminated the production of reactive oxygen species induced by HG, and when treated with 80μM, the accumulation of ROS was significantly reduced. In Figure 10B, the average fluorescence intensity value was processed using Image-Pro Plus 6.0 software under the same parameters.

Because the accumulated reactive oxygen species (ROS) is a response to high glucose (High glucose or HG), 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 accumulate ROS, and albioside A can significantly eliminate accumulated ROS and protect endothelial cell damage induced by high glucose. It also shows that it can be used as a potential drug for the treatment of complications such as diabetes.

Comparative example 1

The specific implementation mode is the same as in Example 1, the difference is that the analytical high performance liquid (HPLC) detection is performed on the 8:1 elution section of silica gel (the chromatographic column is X-Bridge C18, 5μm, 4.6×250mm, and the flow rate is 1ml/min. Column temperature 30℃), UV detection wavelength is 254nm,

The detection results are shown in Figure 11. According to the retention time of each component in the analytical high performance liquid chromatogram in Figure 11, 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 11 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 11, determine 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. For 32 The eluted fraction of %CH ₃ OH was detected 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 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 12 (UV detection (Wavelength 290nm), the compound at the retention time t _R =41.0min is Dendrobium c (Picraquassioside C) (Aj7),

Aj7 is a white powder, detected by UPLC-ESI-MS, and its mass spectrum is shown in Figure 13. HR-ESI-MS m/z 633.1925[M+Cl] ^- _{, its molecular formula is C 28} H ₃₈ O ₁₄ determined by Monoisotopic Mass, Even Electron Ions. The sample Aj7 was dissolved in a nuclear magnetic tube with deuterated methanol, and the ¹ H-NMR and ¹³ C-NMR were measured by a fully digital nuclear magnetic resonance spectrometer. The results are as follows:

¹ H-NMR (400MHz, CD ₃ OD) δ 6.76 (2H, s, H-2, 6), 6.74 (2H, s, H-2', 6'), 6.57 (1H, d, J = 15.8 Hz,H-7'), 6.33(1H,dt,J=15.8,5.5Hz,H-8'), 4.94(1H,d,J=5.6Hz,H-7), 4.81(1H,d,J ＝7.3Hz,H-1”), 4.29(1H,dd,J＝9.0,4.8Hz,H-8), 4.24(1H,d,J＝5.4Hz,H-9'),3.98–3.89(2H ,m), 3.87(1H,s), 3.85(6H,s,3,5-OMe), 3.83(6H,s,3',5'-OMe), 3.77(1H,s), 3.73--3.58( 4H,m),3.52-3.41(4H,m),3.37(1H,s),3.22(s,2H). ¹³ C-NMR(101MHz,CD ₃ OD)δ153.09(C-3',5'),152.40(C-3,5),138.12(C-1),135.02(C-4'),134.17(C-4),133.31(C-1'),129.96(C-8'),128.53(C-7'),104.66(C-2,6),104.29(C-1”),103.55(C-2',6'),85.68(C-8), 76.94(C-5”), 76.39(C-3”), 74.34(C-2”), 72.67(C-7), 69.95(C-4”), 62.17(C-9'), 61.20(C-6”), 60.25(C -9), 55.63(3,5-OMe), 55.31(3',5'-OMe).

The optical rotation was tested and calculated according to the method of Example 1, and the results are shown in Table 3.

Table 3 Determination of the optical rotation of compound Aj7

The result shows negative optical activity

(c 0.002, CH ₃ OH) is a levorotatory compound.

According to mass spectrometry, ¹ HNMR and ¹³ CNMR, its structure is finally determined as follows. The Chinese name is Dendrobium C, and the English name is Picraquassioside C. It is isomers with albioside A (albioside A).

Comparative example 2

The specific embodiments are the same as those in Examples 3-7. The difference is that the compound albioside A is replaced with Dendrobium sylvestris C. Although the two are isomers of each other and have similar structures, Dendrobium sylvestris C does not promote endothelial blood vessels. Regenerate, improve steatosis, and pharmacological activity against oxidative stress.

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 (14)

1. Compound

   

   Or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein the pharmaceutically acceptable salt comprises a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate of the compound.
3. A composition containing the compound of claim 1.
4. The composition according to claim 3, wherein the composition is a pharmaceutical composition.
5. The composition according to claim 4, characterized in that it contains a pharmaceutically acceptable carrier.
6. The composition according to claim 5, wherein the pharmaceutically acceptable carrier comprises solvents, propellants, solubilizers, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, Lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavors, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrating agents, penetration enhancers, pH Regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids or release Blocker.
7. The composition according to any one of claims 4 to 6, wherein the dosage form is a tablet, capsule, granule, powder, syrup, oral liquid or injection.
8. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of medicines.
9. The application according to claim 8, wherein the medicine comprises: a medicine for preventing or treating a lipid metabolism disorder, a medicine for promoting the proliferation and migration of endothelial cells, a medicine for anti-oxidative stress, or a medicine for protecting endothelial damage.
10. The use of the compound of claim 1 in the preparation of food or health food.
11. A preparation compound

    

    The method is characterized in that it comprises the following steps:

    (1) Crush the albizia bark with a pulverizer, mix it with ethanol with a concentration of 70 to 80% in a ratio of 1: (4 to 10), reflux and extract at 70 to 90°C for 1 to 3 times, 1 time each time ~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) Recover the n-butanol extract, dry to obtain a solid substance, reconstitute it, and then use D101 macroporous adsorption resin, eluting and separating with 30%, 50%, 70%, and 95% ethanol, respectively, enriched Set 30% ethanol elution section, 50% ethanol elution section, 70% ethanol elution section, 95% ethanol elution section; perform silica gel column chromatography on the 30% ethanol elution section, using 40:1, 20:1 , 16:1, 10:1, 8:1 and 6:1 dichloromethane-methanol mixture gradient elution, the same components were combined by TLC detection, and the 10:1 elution segment was collected.
12. The method according to claim 11, characterized in that the step (3) recovers the n-butanol by rotary evaporation under reduced pressure at 80° C., and the n-butanol extract is dried in a vacuum drying oven.
13. The method according to claim 12, characterized in that the 10:1 elution section of the dichloromethane-methanol mixture is collected, the 10:1 elution section is passed through a reverse silica gel column, and finally 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:

    
14. The method according to claim 13, wherein the _{elution peak when phase A is 42% CH 3} OH is eluted and separated according to the following semi-preparative separation conditions:

    Time/min CH ₃ OH volume percentage H ₂ O volume percentage 0 10% 90% 4 10% 90% 5 26% 74% 45 28% 72% 60 32% 68% 70 50% 50% 80 100% 0%

    Collect the compound with the retention time of 26.50min, that is,

    

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