Flavonoid carbonyl glycoside Ligqianside A and its extraction and separation method from Ligustrum lucidum leaves

Abstract

The present invention discloses flavonoid carbon glycoside Ligqianside A and a method for extracting and separating it from Ligustrum lucidum leaves. Flavonoid carbon glycoside Ligqianside A is extracted for the first time. Its preparation method comprises four steps: extraction, coarse fractionation by microporous resin column, separation by dextran gel LH‑20 medium-pressure chromatography column, and purification by reverse-phase high-pressure preparative column. The technical means adopted in the present invention can be used for large-scale production: the raw materials are widely planted, low in cost, and easy to prepare in batches; the extraction is performed by cold soaking with methanol at room temperature, which is easy to operate; the separation adopts coarse fractionation by microporous resin, and the microporous resin separation material can be installed in a medium-pressure column chromatography system, which is easy to scale up; the reverse-phase high-pressure preparative liquid chromatography used in separation and purification is a rapid isocratic method and can ensure that the product purity is greater than 98%.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicinal chemistry, and in particular to a flavonoid carbon glycoside Ligqianside A and a method for extracting and separating the flavonoid carbon glycoside Ligqianside A from Ligustrum lucidum leaves. Background Art

[0002] Ligustrum qianluoenenst (Ligustrum qianluoenenst TZXU) is a perennial shrub of the genus Ligustrum (Ligustrum L.) in the family Oleaceae, growing 0.5 to 2 meters tall. It is primarily found along the coastal areas of the Shandong Peninsula. Its branches, leaves, fruit, and roots are all used medicinally. Its nutritious leaves can be used as a tea substitute. Qianluoenenst leaf tea has the benefits of clearing heat and detoxifying, killing bacteria and reducing inflammation, strengthening the stomach and eliminating stagnation, relieving cough and phlegm, promoting salivation and quenching thirst, refreshing the mind, lowering blood pressure and weight, inhibiting and preventing cancer, and providing anti-aging benefits. To date, no literature or patents have been found regarding flavonoid glycosides in Ligustrum qianluoenenst. To further accelerate the quality evaluation, production and sales of Ligustrum qianluoenenst, and the development of related new drugs, it is necessary to identify more structurally novel active ingredients. Summary of the Invention

[0003] Based on the above problems, the present invention separates and extracts a new flavonoid carbon glycoside Ligqianside A. The flavonoid carbon glycoside Ligqianside A of the present invention is separated and extracted for the first time.

[0004] A new flavonoid carbon glycoside Ligqianside A was found in the leaves of Ligustrum lucidum. The flavonoid carbon glycoside compound is in the form of yellow powder and is named Ligqianside A. Its molecular formula is C 26 H 28 O 14 , the chemical structure is:

[0005]

[0006] A method for extracting and isolating flavonoid carbon glycoside Ligqianside A from Ligustrum lucidum leaves is proposed for the first time. The method for isolating and preparing flavonoid carbon glycoside Ligqianside A comprises the following steps:

[0007] Step 1, extraction: dry the leaves of Ligustrum lucidum and add a methanol solution 10 times its weight, cold-immerse and extract three times at room temperature, each time for 6 to 8 hours, combine the filtrates to obtain filtrate A, and mix the filtrate A with a sample in a ratio of silica gel mass to Ligustrum lucidum leaf mass of 1:4 and dry under reduced pressure to obtain the crude Ligustrum lucidum leaf extract sample;

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

[0009] Step 3, separation using a Sephadex LH-20 medium-pressure chromatography column: the primary target component Fr4 is dissolved in a 30% to 70% methanol-water solution to prepare a sample concentration of 0.5 to 1.0 g / mL, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, i.e., filtrate B. The filtrate B is separated by a Sephadex LH-20 medium-pressure chromatography column and detected by an ultraviolet detector with a detection wavelength of 210 nm. The corresponding chromatographic peak fraction Fr45 in the reverse-phase chromatogram of the filtrate B is collected and dried under reduced pressure to obtain the secondary target component Fr45.

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

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

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

[0013] Furthermore, in step 3, the operating parameters of the Sephadex LH-20 column liquid chromatography separation refer to a chromatographic column size of 1000×15 mm, a stationary phase of 80-160 μm Sephadex column filler, a mobile phase of methanol, an analysis time of 3500 minutes, an injection volume of 1-8 mL, and a flow rate of 0.5 mL / min.

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

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] (1) The separation and extraction method of the present invention is proposed for the first time, and the flavonoid carbon glycoside Ligqianside A separated and extracted from the leaves of Ligustrum lucidum is discovered for the first time. Moreover, the flavonoid carbon glycoside Ligqianside A of the present invention has the activity of resisting myocardial damage.

[0017] (2) The present invention has low cost and high product purity.

[0018] The extraction solvent used in the present invention, the solvent used for microporous resin coarse fractionation, the solvent used for dextran gel LH-20 column liquid chromatography separation, and the solvent used for reversed-phase C18 column liquid chromatography purification can all be recycled; the chromatographic separation material (microporous resin) used can be reused, and the recycled solvent and reused separation material ensure that the average cost in the separation process is relatively low. The two-step medium-pressure separation (microporous resin column coarse fractionation and dextran gel separation) and reversed-phase C18 liquid phase purification can ensure that the purity of the product is greater than 95%.

[0019] (3) The technical means adopted by the present invention can be used for large-scale production.

[0020] The raw material requirements of the present invention are low, the cost is low, it is widely planted in the Jiaodong area, and it is easy to prepare in batches; the methanol cold soaking extraction at room temperature is easy to operate; the microporous resin column is used for enrichment, and the separation material can be installed in a medium-pressure column system and connected to a preparative liquid chromatography, which is easy to scale up; the reversed-phase C18 column liquid chromatography used in the purification is a rapid isocratic method and is very suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a chromatogram of the microporous resin column crude fraction of the sample of the present invention;

[0022] Figure 2 This is a chromatogram of the separation of the Fr4 component of the present invention using a Sephadex LH-20 medium-pressure chromatography column;

[0023] Figure 3 This is a reverse phase high pressure preparative column purification chromatogram of the Fr45 component of the present invention;

[0024] Figure 4This is a purity verification diagram of the new flavonoid carbon glycoside compound Ligqianside A isolated by the present invention;

[0025] Figure 5 This is the high-resolution mass spectrum of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0026] Figure 6 The present invention is to separate and obtain the new flavonoid carbon glycoside compound Ligqianside A 1 H NMR spectrum;

[0027] Figure 7 The present invention is to separate and obtain the new flavonoid carbon glycoside compound Ligqianside A 13 C NMR spectra;

[0028] Figure 8 This is the HMBC diagram of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0029] Figure 9 This is the HSQC chart of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0030] Figure 10 The COSY diagram of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0031] Figure 11 This is the ROESY diagram of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0032] Figure 12 This is the DEPT diagram of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0033] Figure 13 This is the infrared spectrum of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0034] Figure 14 This is the UV spectrum of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0035] Figure 15 The optical rotation diagram of the new flavonoid carbon glycoside compound Ligqianside A isolated and obtained in the present invention;

[0036] Figure 16 This is the planar structure diagram of the new flavonoid carbon glycoside compound Ligqianside A separated and obtained in the present invention.

[0037] Figure 17 The effect of Ligqianside A on the hatching rate of zebrafish embryos.

[0038] Figure 18 The effect of Ligqianside A on the heart rate of zebrafish.

[0039] Figure 19(a) shows the effect of Ligqianside A on the SV-BA spacing in zebrafish;

[0040] Figure 19(b) shows the effect of Ligqianside A on zebrafish heart circularization. DETAILED DESCRIPTION

[0041] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] Example 1

[0043] A method for separating and preparing a new flavonoid carbon glycoside Ligqianside A from Ligustrum lucidum leaves comprises the following steps:

[0044] Step 1, extraction: take 1000g of dried Ligustrum lucidum leaves, add methanol solution 10 times its mass, cold soak extraction at room temperature 3 times, each time for 6 hours, combine the filtrates to obtain filtrate A, and mix the filtrate A according to the mass of silica gel: mass of Ligustrum lucidum leaves = 1:4 and dry under reduced pressure, that is, 410.2g of the crude extract of Ligustrum lucidum leaves is mixed; the conditions for reduced pressure drying are vacuum degree 100mbar and temperature 40℃.

[0045] Step 2, microporous resin column crude fractionation: 43 g of the crude extract of Ligustrum lucidum leaf was taken, and the sample was separated by a medium pressure chromatographic column equipped with microporous resin, and detected by an ultraviolet detector with a detection wavelength of 210 nm, and the fourth chromatographic peak fraction (Fr4) in the preparation chromatogram was collected (see Appendix Figure 1 The fraction was dried under reduced pressure to obtain 6.9 g of the target component; wherein the conditions for drying under reduced pressure were a vacuum degree of 100 mbar and a temperature of 40° C.; the medium-pressure chromatography separation operating parameters of the microporous resin column were as follows: a chromatographic column length of 460 mm and a diameter of 49 mm, the stationary phase was CHP20P, the mobile phase A was water, B was methanol, and C was dichloromethane, the chromatographic conditions were 0-200 min, 0-100% B; 200-260 min, 0-100% C, the injection volume was 50 g, and the flow rate was 50 mL / min.

[0046] Step 3, Sephadex LH-20 medium pressure column chromatography separation: 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, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, i.e., filtrate B. The filtrate B was separated by a Sephadex LH-20 medium pressure column chromatography, and detected by an ultraviolet detector with a detection wavelength of 210 nm. The corresponding chromatographic peak fraction Fr45 (attached) in the reverse phase chromatogram of the filtrate B was collected. Figure 2 The target component (shown in Figure 2) was dried under reduced pressure to obtain 398 mg of the target component. The reduced pressure drying conditions were a vacuum of 100 mbar and a temperature of 40°C. The operating parameters for the Sephadex LH-20 column liquid chromatography separation were a column size of 1000 × 15 mm, a Sephadex column packing material of 80-160 μm in diameter, methanol as the mobile phase, an analysis time of 3500 minutes, an injection volume of 5 mL, and a flow rate of 0.5 mL / min.

[0047] Step 4, reverse phase high pressure preparative column purification: 398 mg of the target component Fr45 was dissolved in a 30% methanol-water solution to prepare a sample concentration of 100.0 mg / mL, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, i.e., filtrate C. The filtrate C was purified by reverse phase high pressure C18 chromatographic column liquid chromatography, and detected by an ultraviolet detector with a detection wavelength of 210 nm. The corresponding chromatographic peak fraction Fr453 (attached) in the reverse phase chromatogram of the filtrate C was collected. Figure 3 The purified product (shown as shown) was dried under reduced pressure to obtain 50 mg of a flavonoid carbonyl glycoside compound with a purity greater than 95%, which was named Ligqianside A. The reduced pressure drying conditions were a vacuum of 100 mbar and a temperature of 40°C. The operating parameters for the reversed-phase C18 column liquid chromatography purification were a 250×20 mm column, a 5 μm stationary phase using pure water-resistant ReproSil-Pur 120C18 AQ column packing, a 16% acetonitrile-water mobile phase, an injection volume of 0.5 mL, and a flow rate of 19 mL / min.

[0048] The purity verification diagram of flavonoid carbon glycoside compound Ligqianside A is shown in the attached figure. Figure 4 As shown, its chemical properties and NMR signal assignments are as follows:

[0049] Flavonoid carbon glycosides 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] + ,1 H-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 The above NMR data were combined with mass spectrometry, ultraviolet and infrared data, and the literature data were searched for comparison, and it was found that compound Fr453 was a new compound, named Ligqianside A.

[0050] Its structural characterization and structure are shown in Figure 5-16 .

[0051] Example 2

[0052] A method for separating and preparing a new flavonoid carbon glycoside Ligqianside A from Ligustrum lucidum leaves comprises the following steps:

[0053] Step 1, extraction: take 100g of dried Ligustrum lucidum leaves, add 10 times its mass of methanol solution, cold soak extraction at room temperature 3 times, each time for 8 hours, combine the filtrates to obtain filtrate A, and reduce the pressure to dry the filtrate A, that is, 41g of crude extract of Ligustrum lucidum leaves; the conditions for reduced pressure drying are vacuum degree 200mbar and temperature 50℃.

[0054] Step 2, microporous resin column crude fractionation: 4.3 g of the crude extract sample of Ligustrum lucidum leaf is taken, and the sample is separated by a medium-pressure chromatography tower equipped with microporous resin. The sample is detected by an ultraviolet detector with a detection wavelength of 210 nm, and the fourth main chromatographic peak fraction (Fr4) in the preparative chromatogram is collected. The fraction is dried under reduced pressure to obtain 0.7 g of a component containing the target component; wherein, the conditions for reduced pressure drying are a vacuum degree of 200 mbar and a temperature of 50°C; the medium-pressure chromatography separation working parameters of the microporous resin column are: the chromatographic column is 460 mm long and 49 mm in diameter, the stationary phase is CHP20P, the mobile phase A is water, B is methanol, and C is dichloromethane, the chromatographic conditions are 0-200 min, 0-100% B; 200-260 min, 0-100% C, the injection volume is 30 g, and the flow rate is 40 mL / min.

[0055] Step 3, Sephadex LH-20 medium-pressure column separation: 240 mg of the target component, fraction Fr4, was dissolved in a 60% methanol-water solution to a sample concentration of 0.8 g / mL. The sample was filtered through a 0.45 μm microporous membrane to obtain a filtrate, filtrate B. This filtrate B was separated on a Sephadex LH-20 medium-pressure column and detected with a UV detector at a wavelength of 210 nm. The corresponding peak fraction Fr45 in the reverse-phase chromatogram of filtrate B was collected and dried under reduced pressure to obtain 40 mg of the target component. The reduced pressure drying conditions were a vacuum of 200 mbar and a temperature of 50°C. The Sephadex LH-20 column liquid chromatography separation operating parameters were: column dimensions of 1000 × 15 mm, a Sephadex column packing material of 80-160 μm in diameter, methanol as the mobile phase, an analysis time of 3500 min, an injection volume of 7 mL, and a flow rate of 0.5 mL / min.

[0056] Step 4, reverse-phase high-pressure preparative column purification: 40 mg of the component Fr45 containing the target component was dissolved in a 60% methanol-water solution to prepare a sample concentration of 120.0 mg / mL, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, i.e., filtrate C. The filtrate C was purified by reverse-phase high-pressure C18 chromatographic column liquid chromatography, and detected by an ultraviolet detector with a detection wavelength of 210 nm. The corresponding chromatographic peak fraction Fr453 in the reverse-phase chromatogram of the filtrate C was collected and dried under reduced pressure to obtain 5 mg of a flavonoid carbon glycoside compound with a purity greater than 95%, which is Ligqianside A. The conditions for reduced pressure drying were a vacuum degree of 200 mbar and a temperature of 50°C. The operating parameters for reversed-phase C18 column liquid chromatography purification were a column size of 250×20 mm, a stationary phase of 5 μm pure water-resistant ReproSil-Pur 120C18 AQ column packing, a mobile phase of 16% acetonitrile-water solution, an injection volume of 0.8 mL, and a flow rate of 19 mL / min.

[0057] Pharmacological experiments: Flavonoid glycosides Ligqianside A isolated from the leaves of Ligustrum lucidum

[0058] Zebrafish share 87% genetic similarity with humans, 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. Male and female zebrafish in good condition were selected for mating and spawning. After spawning, the eggs were washed with deionized water and the normally developing embryos were selected for use in the experiment. A zebrafish heart injury model was established using 35 μM Dox. Embryos that developed normally 6 hours after fertilization (6 hpf) were selected as the starting point, and the experiment ended at 96 hours.

[0059] (1) Effects of Ligqianside A on zebrafish embryo hatching

[0060] Normally developed zebrafish embryos were selected under a stereomicroscope, and 6hpf zebrafish embryos were transferred to a 6-well plate at 30 per well, with three parallel wells in each group. Ligqianside A was treated for different times with four concentration gradients of 15μg / mL, 30μg / mL, 60μg / mL, and 120μg / mL. The solution was changed every 24h, and the experimental endpoint was reached after 96h. After reaching the end point of the experimental effect, the solution was removed and then washed three times with PBS. Eight zebrafish embryos were taken from each group and placed under a microscope to observe the effects on the hatching and morphology of the zebrafish embryos and take photos for record. The experimental results are shown in the figure. Figure 17 shown.

[0061] (2) Effects of Dox on the heart rate of zebrafish embryos

[0062] A zebrafish heart injury model was established using 35μM Dox. The solution was removed after 96 hours when the experimental endpoint was reached, and then the cells were washed three times with PBS. A drop of 0.016% tricaine solution was added to the glass slide, and the zebrafish embryo was gently aspirated using a rubber-tipped dropper. When the zebrafish embryo automatically settled to the tip of the rubber-tipped dropper, the rubber-tipped dropper was gently squeezed to allow the zebrafish embryo to slowly slide onto the glass slide with the tricaine solution added. Let it stand at room temperature for 2 minutes. Place the glass slide on the microscope stage, and observe and record the number of heartbeats of the zebrafish embryo in 30 seconds. The experimental results are as follows. Figure 18 shown.

[0063] (3) Effects on cardiac cyclization:

[0064] Normally developed 6hpf zebrafish embryos were selected under a stereomicroscope, and the solution was changed every 24 hours for up to 96 hours. After reaching the end point of the experimental effect, the solution was removed and then washed three times with PBS. Pictures were taken, and then the distance between the point where blood flows into the heart (i.e., the arterial bulb position Bulbus Arteriosus, BA) and the point where blood flows out of the heart (i.e., the venous sinus position Sinus Venosus, SV) was measured using a DMi1 optical inverted microscope as a key indicator for quantifying the degree of cardiac circularization. The experimental results are shown in Figures 19(a)(b).

[0065] The above experimental results were derived from three independent repeated experiments. GraphPad Prism8 software and IBM SPSS Statistics 20 were used for graphing and statistical analysis. * or # indicates P < 0.05, indicating statistical significance.

[0066] The above research results show that the concentration of flavonoid carbon glycoside Ligqianside A below 120μg / mL has no significant effect on the hatching rate and morphology of zebrafish embryos ( Figure 17 ), indicating that the compound is basically non-toxic. Using 35μM Dox can significantly cause zebrafish heart damage, specifically manifested as the heartbeat of zebrafish embryos slowing down ( Figure 18 ), the SV-BA distance was significantly increased compared with the normal control group (P < 0.01), indicating incomplete cardiac cyclization. Ligqianside A can significantly increase the heart rate of zebrafish embryos, reduce the SV-BA distance, and promote cardiac cyclization; and its protective effect is concentration-dependent. The above shows that Ligqianside A, a new flavonoid carbon glycoside in the leaves of Ligustrum lucidum of the present invention, can effectively resist zebrafish myocardial damage caused by doxorubicin and has anti-myocardial damage activity.

[0067] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A flavonoid carbon glycoside Ligqianside A, characterized by: The flavonoid carbon glycoside compound is in the form of yellow powder, and its molecular formula is C 26 H 28 O 14 , the chemical structure is: 。 2. The method for separating and preparing the flavonoid carbon glycoside Ligqianside A according to claim 1, characterized in that The steps include: Step 1, extraction: dry the Ligustrum lucidum leaves and add a methanol solution 10 times its weight, perform cold soak extraction at room temperature for 3 times, each time for 6-8 hours, combine the filtrates to obtain filtrate A, mix the filtrate A with a sample at a ratio of silica gel mass:Ligustrum lucidum leaves mass = 1:4, and dry under reduced pressure to obtain a sample of the crude Ligustrum lucidum leaf extract; Step 2, microporous resin column crude fractionation: the crude extract of Ligustrum lucidum leaf is mixed with a sample and separated by a medium-pressure chromatographic column equipped with a microporous resin. The sample is detected by an ultraviolet detector with a detection wavelength of 210 nm, and the fourth chromatographic peak fraction Fr4 in the preparative chromatogram is collected. The fraction is dried under reduced pressure to obtain the primary target component Fr4; The medium-pressure chromatography separation parameters of the microporous resin column are as follows: the column length is 460 mm and the diameter is 49 mm. The stationary phase of the microporous resin column is CHP20P. The mobile phases A are water, B is methanol, and C is dichloromethane. The chromatographic conditions are 0-200 min, 0-100% B; 200-260 min, 0-100% C, the injection volume is 15-50 g, and the flow rate is 40-50 mL / min. Step 3, Sephadex LH-20 medium-pressure column separation: the primary target component Fr4 was dissolved in a 30% to 70% methanol-water solution to prepare a sample concentration of 0.5 to 1.0 g / mL, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, i.e., filtrate B. The filtrate B was separated on a Sephadex LH-20 medium-pressure column and detected by an ultraviolet detector at a detection wavelength of 210 nm. The chromatographic peak fraction Fr45 corresponding to the reverse-phase chromatogram of the filtrate B was collected and dried under reduced pressure to obtain the secondary target component Fr45; The operating parameters of the Sephadex LH-20 column liquid chromatography separation are as follows: column size 1000×15 mm, stationary phase 80-160 μm Sephadex column filler, mobile phase methanol, analysis time 3500 min, injection volume 1-8 mL, flow rate 0.5 mL / min; Step 4, reverse phase high pressure preparative column purification: the secondary target component Fr45 was dissolved in a 30% to 70% methanol-water solution to prepare a sample concentration of 100.0 to 150.0 mg / mL, and filtered through a 0.45 μm microporous filter membrane to obtain a filtrate, i.e., filtrate C. The filtrate C was purified by reverse phase high pressure C18 chromatographic column liquid chromatography, and detected by an ultraviolet detector with a detection wavelength of 210 nm. The chromatographic peak fraction Fr453 corresponding to the reverse phase chromatogram of the filtrate C was collected and dried under reduced pressure to obtain a flavonoid carbon glycoside compound with a purity greater than 95%, which was named Ligqianside A; The working parameters of the reversed-phase C18 column liquid chromatography purification are as follows: the column size is 250×20 mm, the stationary phase is 5 μm pure water-resistant ReproSil-Pur 120 C18 AQ column filler, the mobile phase is 16% acetonitrile-water solution, the injection volume is 0.1~1 mL, and the flow rate is 19 mL / min.

3. The separation and preparation method according to claim 2, wherein: In step 1, step 2, step 3 and step 4, the conditions for reduced pressure drying are: vacuum degree 100-250 mbar, temperature 40-50°C.

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