Application of flavonoid carbon glycosides in preparation of anti-myocardial injury drugs

Abstract

The application discloses application of flavone carbon glycosides in preparation of anti-myocardial injury drugs. Flavone carbon glycosides Ligqianside A is obtained through four steps of extraction, coarse separation of a micropore resin column, separation of a dextran gel LH-20 medium pressure chromatographic column and purification of a reversed-phase high-pressure preparation column. The flavone carbon glycosides Ligqianside A separated and extracted from the leaves of Ligustrum robustum Blume is found for the first time. It is found through pharmacological tests that the flavone carbon glycosides Ligqianside A has the activity of resisting myocardial injury, and can be used for the purposes of preparation of a heart protection drug and application in preparation of an anti-myocardial injury drug.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, specifically to the application of flavonoid C-glycosides in the preparation of drugs for treating myocardial injury. Background Technology

[0002] *Ligustrum qianluoenenst* TZXU is a perennial shrub belonging to the genus *Ligustrum* in the family Oleaceae, growing to a height of 0.5–2 meters. It is mainly distributed along the coastal areas of the Shandong Peninsula. The branches, leaves, fruits, and roots of *Ligustrum qianluoenenst* are all used medicinally. Its leaves are rich in nutrients and can be used as a tea substitute. Drinking *Ligustrum qianluoenenst* leaf tea is believed to have effects such as clearing heat and detoxifying, sterilizing and reducing inflammation, strengthening the stomach and aiding digestion, relieving cough and phlegm, promoting saliva production and quenching thirst, refreshing the mind, lowering blood pressure and aiding weight loss, inhibiting and preventing cancer, and anti-aging. To date, no literature or patent reports have been found concerning flavonoid glycosides in *Ligustrum qianluoenenst*. To further accelerate the quality evaluation, production, sales, and related new drug development of *Ligustrum qianluoenenst*, it is necessary to explore more structurally novel active ingredients from it. Summary of the Invention

[0003] Based on the above problems, this invention proposes the application of flavonoid C-glycosides in the preparation of drugs for treating myocardial injury. The flavonoid C-glycoside of this invention is a novel flavonoid C-glycoside extracted from the leaves of *Ligustrum lucidum*, and this flavonoid C-glycoside is a first-time isolated and extracted compound. The novel flavonoid C-glycoside extracted from *Ligustrum lucidum* leaves is a yellow powder, named Ligqianside A, with the molecular formula C0. 26 H 28 O 14 .

[0004] The application of flavonoid C-glycosides in the preparation of drugs for treating myocardial injury, wherein the chemical structural formula of the flavonoid C-glycosides is as follows:

[0005]

[0006] The method for isolating and preparing the flavonoid C-glycoside Ligqianside A includes the following steps:

[0007] Step 1, Extraction: After drying the leaves of *Ligustrum lucidum*, add 10 times its weight of methanol solution and extract by cold soaking 3 times at room temperature, each time for 6-8 hours. Combine the filtrates to obtain filtrate A. Mix filtrate A with silica gel at a ratio of 1:4 to *Ligustrum lucidum* leaves and dry under reduced pressure to obtain the crude extract of *Ligustrum lucidum* leaves.

[0008] Step 2, coarse separation with microporous resin column: The crude extract of Ligustrum lucidum leaves is mixed with a sample and separated in a medium-pressure chromatographic column packed with microporous resin. The sample is detected by an ultraviolet detector with a detection wavelength of 210 nm. The fraction of the fourth chromatographic peak (Fr4) in the preparation chromatogram is collected and dried under reduced pressure to obtain the primary target component Fr4.

[0009] Step 3, separation using a dextran gel LH-20 medium-pressure chromatographic column: The primary target component Fr4 is dissolved in a 30%–70% methanol-water solution to prepare a sample concentration of 0.5–1.0 g / mL. The solution is then filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B is then separated using a dextran gel LH-20 medium-pressure chromatographic column and detected by a 210 nm UV detector. The corresponding peak fraction Fr45 in the reversed-phase chromatogram of filtrate B is collected and dried under reduced pressure to obtain the secondary target component Fr45.

[0010] Step 4, reversed-phase high-pressure preparative column purification: The secondary target component Fr45 is dissolved in 30%–70% methanol-water solution to prepare a sample concentration of 100.0–150.0 mg / mL. The solution is filtered through a 0.45 μm microporous membrane to obtain filtrate, i.e., filtrate C. Filtrate C is purified by reversed-phase high-pressure C18 column liquid chromatography and detected by a UV detector with a detection wavelength of 210 nm. The chromatographic peak fraction Fr453 corresponding to the reversed-phase chromatogram of filtrate C is collected and dried under reduced pressure to obtain a flavonoid C-glycoside compound with a purity greater than 95%, named Ligqianside A.

[0011] Furthermore, in steps 1, 2, 3, and 4, the conditions for vacuum drying are: vacuum degree 100–250 mbar and temperature 40–50 °C.

[0012] Furthermore, in step 2, the medium-pressure chromatographic separation parameters of the microporous resin column are as follows: column length 460 mm, diameter 49 mm, stationary phase of microporous resin column is CHP20P, mobile phase A is water, B is methanol, C is dichloromethane, chromatographic conditions are 0–200 min, 0–100% B; 200–260 min, 0–100% C, injection volume is 15–50 g, and flow rate is 40–50 mL / min.

[0013] Furthermore, in step 3, the operating parameters for the dextran gel LH-20 column liquid chromatography separation are as follows: the column size is 1000×15mm, the stationary phase is 80-160μm dextran gel column packing material, the mobile phase is methanol, the analysis time is 3500 minutes, the injection volume is 1-8mL, and the flow rate is 0.5mL / min.

[0014] Furthermore, in step 4, the working parameters for reversed-phase C18 column liquid chromatography purification are as follows: column size 250×20mm, stationary phase 5μm ReproSil-Pur 120 C18 AQ column packing material resistant to pure water, mobile phase 16% acetonitrile-water solution, injection volume 0.1-1mL, and flow rate 19mL / min.

[0015] Beneficial effects:

[0016] The separation and extraction method of this invention is proposed for the first time, and the flavonoid C-glycoside Ligqianside A isolated and extracted from Ligustrum lucidum leaves is discovered for the first time. Pharmacological tests have shown that the flavonoid C-glycoside Ligqianside A of this invention has activity against myocardial injury and can be used in the preparation of cardioprotective drugs and anti-myocardial injury drugs. Attached Figure Description

[0017] Figure 1 This is a crude chromatogram of the microporous resin column of the sample of the present invention;

[0018] Figure 2 This is the chromatogram of the dextran gel LH-20 medium-pressure column separation of the Fr4 component of this invention;

[0019] Figure 3 This is a reverse-phase high-pressure preparative column chromatogram of the Fr45 component of this invention.

[0020] Figure 4 This is a purity verification diagram of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0021] Figure 5 The high-resolution mass spectrum of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0022] Figure 6 To obtain a novel flavonoid C-glycoside compound, Ligqianside A, in this invention. 1 H NMR MRI;

[0023] Figure 7 To obtain a novel flavonoid C-glycoside compound, Ligqianside A, in this invention. 13 C10 NMR MRI;

[0024] Figure 8 HMBC diagram of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0025] Figure 9 The HSQC diagram of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0026] Figure 10 COSY diagram of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0027] Figure 11 The ROESY diagram of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0028] Figure 12 DEPT image of Ligqianside A, a novel flavonoid C-glycoside compound isolated in this invention;

[0029] Figure 13 The infrared spectrum of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0030] Figure 14 The ultraviolet spectrum of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0031] Figure 15 The optical rotation diagram of the novel flavonoid C-glycoside compound Ligqianside A isolated in this invention;

[0032] Figure 16 The diagram shows the planar structure of Ligqianside A, a novel flavonoid C-glycoside compound isolated in this invention.

[0033] Figure 17 The effect of Ligqianside A on zebrafish embryo hatching rate.

[0034] Figure 18 The effect of Ligqianside A on the heart rate of zebrafish.

[0035] Figure 19(a) shows the effect of Ligqianside A on the SV-BA spacing in zebrafish;

[0036] Figure 19(b) Effect of Ligqianside A on cardiac circumduction in zebrafish. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] A novel method for isolating and preparing Ligqianside A, a flavonoid C-glycoside, from the leaves of Ligustrum lucidum, comprising the following steps:

[0040] Step 1, Extraction: Take 1000g of dried Ligustrum lucidum leaves, add 10 times its weight of methanol solution, and extract by cold soaking three times at room temperature for 6 hours each time. Combine the filtrates to obtain filtrate A. Mix filtrate A with silica gel at a ratio of 1:4 to Ligustrum lucidum leaves and dry under reduced pressure to obtain 410.2g of crude Ligustrum lucidum leaf extract sample. The conditions for reduced pressure drying are a vacuum of 100mbar and a temperature of 40℃.

[0041] Step 2, coarse separation using microporous resin column: 43g of the crude extract of *Ligustrum lucidum* leaves was taken and separated using a medium-pressure chromatographic column packed with microporous resin. The sample was detected by a UV detector with a wavelength of 210nm, and the fraction representing the fourth chromatographic peak (Fr4) in the preparative chromatogram was collected (see attached image). Figure 1 As shown), the fraction was dried under reduced pressure to obtain 6.9 g of the component containing the target component; wherein, the conditions for reduced pressure drying were a vacuum degree of 100 mbar and a temperature of 40 °C; the working parameters for medium-pressure chromatography separation of the microporous resin column were: column length 460 mm, diameter 49 mm, stationary phase CHP20P, mobile phase A being water, B being methanol, and C being dichloromethane, chromatographic conditions being 0–200 min, 0–100% B; 200–260 min, 0–100% C, injection volume being 50 g, and flow rate being 50 mL / min.

[0042] Step 3, separation using a dextran gel LH-20 medium-pressure chromatographic column: 2.4 g of the target component Fr4 was dissolved in a 30% methanol-water solution to prepare a sample concentration of 1.0 g / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B was then separated using a dextran gel LH-20 medium-pressure chromatographic column and detected using a 210 nm UV detector. The corresponding peak fraction Fr45 (see attached image) in the reversed-phase chromatogram of filtrate B was collected. Figure 2 (As shown) The target component was obtained in 398 mg after vacuum drying. The vacuum drying conditions were 100 mbar vacuum and 40 °C temperature. The working parameters for the LH-20 dextran gel column liquid chromatography separation were as follows: column size 1000 × 15 mm, stationary phase 80–160 μm dextran gel column packing, mobile phase methanol, analysis time 3500 min, injection volume 5 mL, and flow rate 0.5 mL / min.

[0043] Step 4, reversed-phase high-pressure preparative column purification: 398 mg of the target component Fr45 was dissolved in 30% methanol-water solution to prepare a sample concentration of 100.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate C. Filtrate C was purified by reversed-phase high-pressure C18 column liquid chromatography and detected by a 210 nm UV detector. The corresponding peak fraction Fr453 (see attached image) in the reversed-phase chromatogram of filtrate C was collected. Figure 3 (As shown) After vacuum drying, 50 mg of a flavonoid C-glycoside compound with a purity greater than 95% was obtained and named Ligqianside A. The vacuum drying conditions were a vacuum degree of 100 mbar and a temperature of 40 °C. The working parameters for reversed-phase C18 column liquid chromatography purification were: a column size of 250 × 20 mm, a stationary phase of 5 μm water-resistant ReproSil-Pur 120 C18 AQ column packing material, a mobile phase of 16% acetonitrile-water solution, an injection volume of 0.5 mL, and a flow rate of 19 mL / min.

[0044] The purity verification diagram of the flavonoid C-glycoside compound Ligqianside A is attached. Figure 4 As shown, its chemical properties and NMR signal assignments are as follows:

[0045] The flavonoid glycoside Ligqianside A is a yellow powder with the chemical formula C. 26 H 28 O 14 Molecular weight 564.15, MS m / z: 565.1579 [M+H] + 563.1803[M+H] + , 1H-NMR (600MHz, DMSO-d6): δ6.88(1H,s,H-3),7.65(1H,brs,H-2'),6.93(1H,d,J=8.4Hz,H-5'),7.59(1H,brs,H-6'), 3.91(3H,s,H-OCH3),13.75(1H,brs,H-5-OH),9.29(1H,brs,H-7-OH),9.95(1H,brs,H-7'-OH),4.57(1H,d,J=9.4Hz,H -1”),4.08(1H,brs,H-2”),3.15(1H,t-like,H-3”),3.40(1H,m,H-4”),3.08(1H,t-like,H-5”),3.78(1H,m,H-5”),4. 94(1H,brs,H-1"'),3.80(1H,m,H-2"'),3.48(1H,dd,J=8.9Hz,2.3Hz,H-3"'),3.85(1H,brs,H-4"'),3.76,3.90(each 1H,m,H-5”'); 13 C-NMR (151MHz, DMSO-d6): δ163.6(C-2),102.9(C-3),182.4(C-4),159.9(C-5),109.2(C-6),16 1.7(C-7),103.5(C-8),153.4(C-9),103.2(C-10),121.7(C-1'),110.4(C-2'),147.9(C-3'),15 0.6 (C-4'), 115.8 (C-5'), 120.6 (C-6'), 55.8 (C-OCH3), 73.8 (C-1"), 70.0 (C-2"), 79.3 (C-3"), 70.0 (C-4"), 70.3 (C-5"), 75.5 (C-1"'), 70.0 (C-2"'), 74.0 (C-3"'), 68.4 (C-4"'), 70.3 (C-5"'). Combining these NMR data with mass spectrometry, UV, and IR data, and comparing with literature data, compound Fr453 was identified as a new compound and named Ligqianside A.

[0046] Its structural characterization and structure are shown in Figure 5-16 .

[0047] Example 2 Pharmacological test: Ligqianside A, a flavonoid C-glycoside isolated and extracted from Ligustrum lucidum leaves

[0048] Zebrafish and humans share 87% genetic homology, and their embryonic development is transparent, making it easier to observe and analyze the effects of drugs on the heart and blood circulation. Therefore, zebrafish are widely used as a model organism in medical research. Healthy male and female zebrafish were selected for mating and spawning. After spawning, the embryos were washed with deionized water, and normally developing embryos were selected for the experiment. A zebrafish heart injury model was established using 35 μM Dox. Embryos that had developed normally for 6 hours (6 hpf) after fertilization were selected as the starting point, and 96 hours was the experimental endpoint.

[0049] (1) Effects of Ligqianside A on zebrafish embryo hatching

[0050] Normally developing zebrafish embryos were selected under a stereomicroscope. 6 hpf zebrafish embryos were transferred at a rate of 30 embryos / well to 6-well plates, with three parallel wells per group. Four concentration gradients of Ligqianside A (15 μg / mL, 30 μg / mL, 60 μg / mL, and 120 μg / mL) were used for treatment at different time points. The solution was changed every 24 hours, and the experiment reached its endpoint after 96 hours. After reaching the endpoint, the solution was removed, and the embryos were washed three times with PBS. Eight zebrafish embryos from each group were placed under a microscope to observe the effects on embryo hatching and morphology, and photographs were taken for recording. The experimental results are as follows: Figure 17 As shown.

[0051] (2) Effects on Dox-induced heart rate in zebrafish embryos

[0052] A zebrafish heart injury model was established using 35 μM Dox. After 96 hours, the experimental endpoint was reached, and the solution was removed. The embryos were then washed three times with PBS. One drop of 0.016% tricaine solution was added to a glass slide. Zebrafish embryos were gently aspirated using a dropper. Once the embryos had settled to the tip of the dropper, the dropper was gently squeezed to allow them to slowly slide onto the slide containing the tricaine solution. The slide was allowed to stand at room temperature for 2 minutes. The slide was then placed on the microscope stage, and the number of heartbeats in the zebrafish embryos over 30 seconds was observed and recorded. The experimental results are as follows: Figure 18 As shown.

[0053] (3) Effects on cardiac circumduction:

[0054] Normally developing 6hpf zebrafish embryos were selected under a stereomicroscope. The solution was changed every 24 hours, and the drug was administered for up to 96 hours. After reaching the experimental endpoint, the solution was removed, and the embryos were washed three times with PBS. Images were taken, and the distance between the points where blood flowed into the heart (Bulbus Arteriosus, BA) and where blood flowed out of the heart (Sinus Venosus, SV) was measured using a DMi1 optical inverted microscope. This distance served as a key indicator for quantifying the degree of cardiac circumduction. The experimental results are shown in Figures 19(a) and (b).

[0055] All experimental results above are derived from three independent replicate experiments. GraphPad Prism8 software and IBM SPSS Statistics 20 were used for plotting and statistical analysis. * or # indicates P < 0.05, which means it is statistically significant.

[0056] The above experimental results show that the concentration of the flavonoid C-glycoside Ligqianside A below 120 μg / mL has no significant effect on the hatching rate and morphology of zebrafish embryos. Figure 17 This indicates that the compound is essentially non-toxic. The use of 35 μM Dox significantly caused cardiac damage in zebrafish, specifically manifested as a slowing of the heartbeat in zebrafish embryos. Figure 18 The SV-BA distance was significantly increased compared to the normal control group (P < 0.01), indicating incomplete cardiac circumduction. Ligqianside A significantly increased the heart rate in zebrafish embryos, decreased the SV-BA distance, and promoted cardiac circumduction; its protective effect was concentration-dependent. These findings indicate that Ligqianside A, a novel flavonoid glycoside from *Ligustrum lucidum* leaves, can effectively resist doxorubicin-induced myocardial damage in zebrafish, and that Ligqianside A has potential applications in the preparation of drugs to combat myocardial injury.

[0057] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of flavonoid C-glycosides in the preparation of drugs for treating myocardial injury, characterized in that... The chemical structural formula of the flavonoid C-glycoside compound is as follows:

Images