CN112608306B

CN112608306B - Preparation method and application of flavonoid saponin new ketone A in spina gleditsiae

Info

Publication number: CN112608306B
Application number: CN202011524948.9A
Authority: CN
Inventors: 曹彦刚; 曾梦楠; 冯卫生; 郑晓珂; 齐曼; 阚玉璇
Original assignee: Henan University of Traditional Chinese Medicine HUTCM
Current assignee: Henan University of Traditional Chinese Medicine HUTCM
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-03
Anticipated expiration: 2040-12-22
Also published as: CN112608306A

Abstract

The invention relates to a preparation method and application of flavonoid compound sapogenone A in spina gleditsiae, which can effectively solve the application problem of quickly extracting flavonoid compound vitexin from spina gleditsiae and realizing preparation of an anti-sepsis acute kidney injury medicament, the spina gleditsiae is extracted by acetone, concentrated under reduced pressure, suspended by distilled water, sequentially treated by petroleum ether and ethyl acetate, the solvent is recovered under reduced pressure, the extract of the ethyl acetate extraction part is primarily separated by silica gel column chromatography, further separated by the silica gel column chromatography, purified by semi-preparative liquid phase, the eluent is collected, concentrated under reduced pressure and dried to obtain 4'' - (6-hydroxy-2, 6-dimethyl-2, 7-dieneooctanoyl) -vitexin, the invention has the advantages of high separation efficiency, short time, small solvent consumption, high sample recovery rate and high purity, and the obtained extract has better protection effect on lipopolysaccharide-induced kidney injury, is effectively used for preparing the medicine for treating the kidney injury caused by the sepsis or other infection factors.

Description

Preparation method and application of flavonoid saponin new ketone A in spina gleditsiae

Technical Field

The invention relates to medicine, in particular to a preparation method and application of a flavonoid saponin new ketone A in spina gleditsiae.

Background

Acute Kidney Injury (AKI) is a common complication of sepsis and is an independent risk factor for increased mortality in patients with sepsis, and its severity significantly correlates with high mortality from sepsis. With the aggravation of the aging of the population in China, invasive medical means and the application of immunosuppressants, the incidence rate of acute kidney injury caused by sepsis is on the trend of increasing year by year. Research shows that the incidence rate of acute kidney injury in patients with hospitalized sepsis is 68.4%, and the in-hospital mortality rate is up to 30.2%. Patients with sepsis are susceptible to acute kidney injury, which aggravates the condition of patients with sepsis. The sepsis combined with acute kidney injury patients has worse prognosis, and the death rate is about 2 times of that of patients without acute kidney injury. Survivors are also at risk of chronic kidney disease and even end stage renal disease, placing a heavy burden on the family and society. Acute kidney injury caused by sepsis becomes one of more and more important diseases which endanger the health of residents in China, so that economic burden and adverse effects on social development are increasingly highlighted. How to prevent and treat acute kidney injury which is a complication of sepsis becomes a very serious medical problem.

The application history of Chinese herbal medicines in the aspect of cell protection is long, active substances with kidney cell protection are searched from the Chinese herbal medicines, and important social benefits and economic benefits are achieved by developing novel medicines with strong specificity and small toxic and side effects.

Spina Gleditsiae is dry spina Gleditsiae (Gleditsia sinensis Lam.) of Gleditsia, and has repercussive, pus expelling, and parasite killing effects. Spina Gleditsiae is widely distributed in Henan, Hebei and Jiangsu provinces of China, is widely distributed in Japan and Korea, and can be harvested all the year round. Chemical component research shows that the spina gleditsiae mainly contains flavonoid, phenolic acid, terpenes and other components, and the flavonoid compound is the main active component in the spina gleditsiae and has the activities of resisting inflammation, resisting tumors, resisting oxidation and the like. However, no published report of extracting flavonoid compounds from spina gleditsiae for preparing the medicament for resisting sepsis acute kidney injury exists so far.

Disclosure of Invention

Aiming at the situation and overcoming the defects of the prior art, the invention aims to provide a preparation method and application of flavonoid saponin new ketone A in spina gleditsiae, which can effectively solve the application problem of rapidly extracting the flavonoid saponin new ketone A from the spina gleditsiae and realizing preparation of the anti-sepsis acute kidney injury medicament.

The technical scheme for solving the problem is that the flavonoid saponin new ketone A in the spina gleditsiae has a molecular structural formula as follows:

the preparation method comprises the following steps:

(1) preparing a crude extract by crushing spina gleditsiae, sieving with a 40-mesh sieve, extracting 3 times with acetone with volume concentration of 50% 8-10 times the weight of spina gleditsiae each time and 2-3min each time, combining the extracting solutions for 3 times, concentrating under reduced pressure to a concentrate with crude drug content of 1.2g/mL, adding distilled water with the weight and volume of 3-5 times that of the concentrate to suspend, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part (spina gleditsiae crude extract); the weight volume refers to that the solid is counted by g, the liquid is counted by mL, the same is shown below, if the concentrate is 1g, the distilled water is 3-5 mL;

(2) performing primary separation by silica gel column chromatography, performing gradient elution by using a dichloromethane-methanol system with volume ratio of 25:1 and 12:1 at flow rate of 20mL/min and flow rate of 1 fraction per 200mL for 250min, and collecting elution fractions with volume ratio of 12:1 from dichloromethane-methanol;

(3) separating again, namely further separating the eluted fractions of the primarily separated dichloromethane-methanol with the volume ratio of 12:1 by silica gel column chromatography, performing gradient elution by adopting a dichloromethane-methanol system with the volume ratio of 20:1 and 10:1 at the flow rate of 10mL/min and 1 fraction per 50mL for 120min, and collecting the eluted fractions of the secondarily separated dichloromethane-methanol with the volume ratio of 10: 1;

(4) and (3) purifying, namely purifying the eluted part of the separated dichloromethane-methanol with the volume ratio of 10:1 by using a semi-preparative liquid phase, eluting by using methanol-water with the volume ratio of 39:61 at the flow rate of 3mL/min for 30min, collecting eluent, concentrating under reduced pressure at 45 ℃, and drying to obtain the sapogenone A (compound 1).

The invention adopts silica gel column chromatography to enrich target components, and then combines the silica gel column chromatography with preparative liquid phase separation and purification, thereby establishing a rapid preparation method of trace flavonoid saponin new ketone A in saponin thorn. The method has the advantages of high separation efficiency, short time, small solvent consumption, high sample recovery rate, high purity of the obtained target component, good protective effect of the obtained extract on renal injury induced by lipopolysaccharide, effective application in preparing medicines for treating renal injury caused by sepsis or other infection factors, development of medicinal value and commercial value of spina gleditsiae, and remarkable economic and social benefits.

Drawings

FIG. 1 is a molecular structural diagram of Saponin A, a compound of the present invention.

FIG. 2 is a graph showing the effect of saponarin A, a compound of the present invention, on the mouse kidney plasma inflammatory factor, wherein A, B, C, D is a graph showing the expression of the inflammatory factor in plasma measured by ELISA method according to TNF- α, IL-6, IFN- γ and IL-17 assay kits, respectively.

Detailed Description

The following detailed description of the embodiments of the present invention will be given with reference to the accompanying specific cases and examples.

In particular, the invention may be embodied as set forth in the following examples.

Example 1

In the specific implementation of the invention, the preparation method of the flavonoid saponin new ketone A in the spina gleditsiae comprises the following steps:

(1) preparing a crude extract, namely crushing 5.12kg of spina gleditsiae, sieving the crushed spina gleditsiae with a 40-mesh sieve, extracting the spina gleditsiae for 3 times and 2min for each time by using acetone with the volume concentration of 50% and the weight of 8 times of the weight of the spina gleditsiae, combining extracting solutions for 3 times, concentrating the extracting solutions under reduced pressure to obtain a concentrate with the crude drug content of 1.2g/mL, adding distilled water with the weight and the volume of 4 times of the concentrate to suspend the concentrate, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering a solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part (92.7g of the spina gleditsiae crude extract);

(2) performing primary separation, namely mixing 100g of silica gel which is sieved by a 100-plus 200-mesh sieve with the extract at the ethyl acetate extraction part, loading the mixture to a silica gel column containing 1000g of silica gel which is sieved by the 100-plus 200-mesh sieve, performing primary separation by silica gel column chromatography, performing gradient elution by adopting a dichloromethane-methanol system with the volume ratio of 25:1 and 12:1, wherein the flow rate is 20mL/min, 1 fraction is obtained per 200mL, each gradient elution is 250min, and collecting the elution fraction with the volume ratio of 12:1 of dichloromethane-methanol which is subjected to primary separation;

(4) and (3) purifying, namely purifying the eluted part of the dichloromethane-methanol with the volume ratio of 10:1 of the re-separated dichloromethane-methanol by adopting a semi-preparative liquid phase, eluting by adopting methanol-water with the volume ratio of 39:61 at the flow rate of 3mL/min for 30min, collecting eluent, concentrating under reduced pressure at 45 ℃, and drying to obtain the saponin new ketone A (compound 1).

Example 2

(1) preparing a crude extract, namely crushing 4.57kg of spina gleditsiae, sieving the crushed spina gleditsiae with a 40-mesh sieve, extracting the spina gleditsiae for 3 times and 3min each time by using acetone with the volume concentration of 50 percent, the weight of which is 10 times that of the spina gleditsiae, combining extracting solutions for 3 times, concentrating the extracting solutions under reduced pressure to a concentrate with the crude drug content of 1.2g/mL, adding distilled water with the weight and the volume of which are 3 times that of the concentrate to suspend the concentrate, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering a solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part (83.5g of the spina gleditsiae crude extract);

(2) performing primary separation, namely mixing 90g of silica gel which is sieved by a 100-plus 200-mesh sieve with the extract at the ethyl acetate extraction part, loading the mixture to a silica gel column containing 900g of silica gel which is sieved by the 100-plus 200-mesh sieve, performing primary separation by silica gel column chromatography, performing gradient elution by adopting a dichloromethane-methanol system with the volume ratio of 25:1 and 12:1, wherein the flow rate is 20mL/min, 1 fraction is obtained per 200mL, each gradient elution is 250min, and collecting the elution fraction with the volume ratio of 12:1 of dichloromethane-methanol which is subjected to primary separation;

Example 3

(1) preparing a crude extract, namely crushing 5.76kg of spina gleditsiae, sieving the crushed spina gleditsiae by a 40-mesh sieve, extracting the spina gleditsiae for 3 times and 3min each time by acetone with the volume concentration of 50 percent, the weight of the spina gleditsiae is 9 times that of the spina gleditsiae, combining the extracting solutions for 3 times, concentrating the extracting solutions under reduced pressure to a concentrate with the crude drug content of 1.2g/mL, adding distilled water with the weight and volume of 5 times that of the concentrate to suspend the concentrate, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extracting part (95.8g of the spina gleditsiae crude extract);

Example 4

In the specific implementation of the invention, the preparation method of the sapogenone A which is a flavonoid compound in the spina gleditsiae comprises the following steps:

(1) preparing a crude extract, namely crushing 4.82kg of spina gleditsiae, sieving the crushed spina gleditsiae with a 40-mesh sieve, extracting the spina gleditsiae for 3 times and 2min for each time by using acetone with the volume concentration of 50% and the weight of 8 times of the weight of the spina gleditsiae, combining extracting solutions for 3 times, concentrating the extracting solutions under reduced pressure to obtain a concentrate with the crude drug content of 1.2g/mL, adding distilled water with the weight and the volume of 5 times of the concentrate to suspend the concentrate, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering a solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part (87.6g of the crude extract of the spina gleditsiae);

Firstly, compound structure identification (taking example 1 as an example):

the above compounds are subjected to nuclear magnetic resonance spectroscopy (¹H-NMR、¹³C-NMR、HSQC、HMBC、¹H-¹H COSY, NOESY) and high resolution mass spectrum (HR-ESI-MS) spectral technology identify the chemical structure:

compound 1: yellow powder, readily soluble in methanol.

–12.1(c 0.02,CH₃OH), obtaining the quasi-molecular ion peak [ M + H ] by HR ESI MS measurement]⁺m/z:599.2120(C₃₁H₃₅O₁₂Calculated 599.2123).

¹H NMR(500MHz,CD₃OD), 2 single hydrogen broad singlet peaks at δ 4.81 and 4.74, suggesting that these 2 hydrogens are two hydrogen signals on the terminal double bond; δ 8.07(2H, d, J ═ 8.8Hz, H-2',6') and 6.98(2H, d, J ═ 8.8Hz, H-3',5') are hydrogen signals on a 1, 4-disubstituted benzene ring; six hydrogen signal peaks are arranged between delta 3-4 of the high field region, a sugar structure is guessed to exist according to delta 4.79(1H, d, J is 9.9Hz, H-1 "), and delta 4.79 is an end group hydrogen signal of the sugar; δ 1.82(3H, s, 9' -CH)₃)，1.18(3H，s，10”'-CH₃) Is the hydrogen signal of two methyl groups.

¹³C NMR(125MHz,CD₃OD) and delta 182.3 are carbonyl carbon signals, and the HSQC and HMBC are integrated to obtainLanes delta 182.3(C-4), 164.0(C-2), 162.8(C-7), 161.5(C-4'), 160.8(C-5), 156.1(C-9), 129.0(C-2',6'), 121.7(C-1'), 116.2(C-3',5'), 104.4(C-10), 104.3(C-8), 102.6(C-3), 98.4(C-6), are flavone structural fragments C-3₆-C₃-C₆The 6 carbon signals on the binding sugars, delta 79.7(C-5 "), 75.8 (C-3"), 73.8(C-1 "), 71.9 (C-4"), 71.4(C-2 "), 61.4 (C-6"), are essentially identical to vitexin. In addition, there are 10 carbon signals in the carbon spectrum, and the observation of chemical shifts revealed that these signals were those of 6-hydroxy-2, 6-dimethyl-2, 7-dienoctanoyl.

In HMBC spectra, H-4 ' (delta 5.03) is remotely related to C-1' suggesting that 6-hydroxy-2, 6-dimethyl-2, 7-dienoctanoyl is attached to C-4 '. By combining the above analyses, the structure of the compound was found to be 4' - (6-hydroxy-2, 6-dimethyl-2, 7-dienoctanoyl) -vitexin, and the compound was found to be a new compound by literature search and named sapogenone A.

TABLE 1 Nuclear magnetic data of Saponin A

a NMR spectroscopic data were recorded in CD₃OD at 500MHz(¹H NMR)and 125MHz(¹³CNMR)

The compound belongs to a flavonoid sapogenone A with renal cell protection activity in spina gleditsiae.

The compound powders of examples 2 to 4 were subjected to nuclear magnetic resonance (1H-NMR, 13C-NMR, HSQC, HMBC,¹H-¹H COSY, NOESY) and high resolution mass spectrometry (HR-ESI-MS) (the same as the compound in example 1), and the molecular structures are the same and are flavonoid compounds, namely saponarin A with renal cell protection activity in saponaria molitor.

Second, Activity test

Experiments prove that the saponin neoketone A (compound 1) extracted and separated by the method can effectively inhibit apoptosis of rat renal tubular epithelial cells (NRK 52e) induced by Lipopolysaccharide (LPS), and in-vivo experimental results show that the saponin neoketone A can effectively relieve damage of the LPS to the mouse kidney, can be used for preparing medicines for treating renal damage caused by various reasons, and is fully proved by the experiments, and the related experimental data are as follows:

1. experiment on the protective effect of sapogenone a (compound 1) on lipopolysaccharide-induced rat renal tubular epithelial cells (NRK 52e cells):

recovering and passaging the cells by using a DMEM culture medium, taking the cells in a logarithmic phase, and dispersing the cells by using the culture medium to obtain the cells with the density of 2 multiplied by 10⁴cell·mL^-1Inoculating 0.2mL of the single cell suspension into a 96-well plate, culturing for 24h, dividing the cells into a control group, a model group and different medicine groups, adding an equal amount of culture medium into a normal group, and adding 1 mg/mL of culture medium into the model group^-1LPS Medium solution (final concentration: 1. mu.g. mL)^-1) The test compound with the concentration of 0.1,1,10,50 and 100 mu M and the concentration of 1 mu g/mL are added into different drug groups^-1The lipopolysaccharide of (4). After 24 hours of incubation, MTT solution (5 mg. multidot.mL) was added to each well^-1) After further incubation at 37 ℃ for 4 hours in 20. mu.l, the medium was carefully aspirated, 150. mu.l DMSO was added to each well and the mixture was shaken for 10 minutes to completely dissolve the crystals. Adjusting to zero with DMSO, measuring absorbance value (A) of each well with enzyme-labeling instrument at 490nm, calculating inhibition rate of drug on cells with the following formula, and processing with SPSS 18.0 software according to the calculated inhibition rate of each concentration to obtain half Effective Concentration (EC)₅₀) The test was repeated 3 times, and the average was taken as the final result.

By examining the effect of saporin A on LPS-induced apoptosis of NRK 52e cells, EC₅₀The values were 3.0. mu.M, respectively, and identical or similar results were obtained by repeated experiments.

2. Experiment of saponin neone a (compound 1) against LPS-induced acute renal function injury in mice:

(1) experimental grouping and administration

32 Balb/c mice were selected, and after adaptive feeding, they were randomly divided into a control group, a model group, a positive group, and a treatment group. LPS (5mg/kg) is injected into the abdominal cavity of the model group, the positive group and the treatment group for modeling, the control group is injected with physiological saline with the same volume into the abdominal cavity, after 2 hours of modeling, Trolox (25mg/kg) is administered into the positive group by intragastric administration, compound 1(20mg/kg) is administered into the treatment group by intragastric administration, and physiological saline of an isosomal machine is administered into the control group and the model group for 3 days and twice a day. Animals were sacrificed 3 days after the last dose and kidney tissue and blood were collected for further analysis.

(2) Hematoxylin-eosin (HE) staining of kidney tissue

Mouse kidney tissue was soaked with 4% paraformaldehyde for 24h, then embedded with paraffin, sliced, and approximately 5 μm thick. After sectioning, HE staining was performed, and morphological changes of kidney tissues were observed under an optical microscope. Compared with the normal group, the mouse in the model group has the symptoms of swelling and vacuolar degeneration of renal tubular epithelial cells, and the symptoms of the positive group and the treatment group are obviously reduced compared with the model group.

(3) Expression experiment for detecting inflammatory factors in blood plasma by ELISA method

The following steps are carried out according to the specification of the TNF-alpha, IL-6, IFN-gamma and IL-17 detection kit: firstly, diluting and sampling a standard substance; sample adding; thirdly, incubation; fourthly, preparing the liquid; washing; sixthly, adding enzyme; seventhly, incubation and washing; eighthly, developing color; ninthly, stopping; and (c) measuring and calculating. The experimental results are shown in figure 2, the levels of TNF-alpha, IL-6, IFN-gamma and IL-17 in the plasma of the model group are obviously higher than those of the control group, and the levels of TNF-alpha, IL-6, IFN-gamma and IL-17 in the plasma of the positive group and the control group are obviously lower than those of the model group.

The method adopts silica gel column chromatography to enrich target components, and combines the silica gel column chromatography with the preparation of liquid phase separation and purification, so as to establish a rapid preparation method of the flavonoid saponin new ketone A in the saponin thorn. The saponin neoketone A prepared by the invention can effectively inhibit the apoptosis of kidney cells induced by LPS, can be effectively used for preparing medicines for treating kidney injury caused by sepsis or other infection factors, opens up a new application of spina gleditsiae in medicines with the effect of protecting the kidney cells, is an innovation in medicines for treating the kidney injury, opens up a new medicinal way and a new commercial value of the spina gleditsiae, and has remarkable economic and social benefits.

Claims

1. A flavonoid saponin new ketone A in spina Gleditsiae is 4' - (6-hydroxy-2, 6-dimethyl-2, 7-diene octanoyl) -vitexin, and has a molecular structural formula as follows:

2. the method for preparing sapogenone a, a flavonoid in spina gleditsiae, according to claim 1, comprising the steps of:

(1) preparing a crude extract by crushing spina gleditsiae, sieving with a 40-mesh sieve, extracting 3 times with acetone with volume concentration of 50% 8-10 times the weight of spina gleditsiae each time and 2-3min each time, combining the extracting solutions for 3 times, concentrating under reduced pressure to a concentrate with crude drug content of 1.2g/mL, adding distilled water with weight and volume of 3-5 times the weight and volume of the concentrate to suspend, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part; the weight volume refers to that the solid is counted by g, the liquid is counted by mL, the same is shown below, if the concentrate is 1g, the distilled water is 3-5 mL;

(4) purifying by using a semi-preparative liquid phase, eluting by using methanol-water with a volume ratio of 39:61 at a flow rate of 3mL/min for 30min, collecting the eluent, concentrating under reduced pressure at 45 ℃, and drying to obtain the saponin new ketone A, wherein the volume ratio of dichloromethane to methanol is 10: 1.

3. The method for preparing sapogenone A, a flavonoid in spina gleditsiae, according to claim 2, characterized in that it comprises the following steps:

(1) preparing a crude extract by crushing 5.12kg of spina gleditsiae, sieving with a 40-mesh sieve, extracting 3 times and 2min each time with acetone with volume concentration of 50% and weight of 8 times of the spina gleditsiae, combining the extracting solutions for 3 times, concentrating under reduced pressure to obtain a concentrate with crude drug content of 1.2g/mL, adding distilled water with weight and volume of 4 times of that of the concentrate for suspension, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspension, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part;

4. The method for preparing sapogenone A, a flavonoid in spina gleditsiae, according to claim 2, characterized in that it comprises the following steps:

(1) preparing a crude extract by crushing 4.57kg of spina gleditsiae, sieving with a 40-mesh sieve, extracting 3 times each time with acetone with the volume concentration of 50% and the weight of 10 times of the spina gleditsiae for 3min, combining the extracting solutions for 3 times, concentrating under reduced pressure to a concentrate with the crude drug content of 1.2g/mL, adding distilled water with the weight and the volume of 3 times of that of the concentrate to suspend, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extraction part;

(2) the initial separation is carried out, the extract of the ethyl acetate extraction part is mixed with 90g of silica gel which passes through a 100-sand 200-mesh sieve, the mixture is loaded on a silica gel column which contains 900g of silica gel which passes through the 100-sand 200-mesh sieve, the initial separation is carried out by silica gel column chromatography, a dichloromethane-methanol system with the volume ratio of 25:1 and 12:1 is adopted for gradient elution, the flow rate is 20mL/min, 1 fraction is used for every 200mL, each gradient elution is carried out for 250min, and the dichloromethane-methanol volume ratio which is obtained by the initial separation is 12:1 is collected;

(3) separating again, namely further separating the dichloromethane-methanol elution flow with the volume ratio of 12:1 subjected to the primary separation by silica gel column chromatography, performing gradient elution by adopting a dichloromethane-methanol system with the volume ratio of 20:1 and 10:1 at the flow rate of 10mL/min and 1 flow per 50mL for 120min, and collecting the dichloromethane-methanol elution flow with the volume ratio of 10:1 subjected to the secondary separation;

(4) and (3) purifying the eluted part of the dichloromethane-methanol with the volume ratio of 10:1 by using a semi-preparative liquid phase, eluting by using methanol-water with the volume ratio of 39:61 at the flow rate of 3mL/min for 30min, collecting the eluent, concentrating under reduced pressure at 45 ℃, and drying to obtain the saponin new ketone A.

5. The method for preparing sapogenone A, a flavonoid in spina gleditsiae, according to claim 2, characterized in that it comprises the following steps:

(1) preparing a crude extract, namely crushing 5.76kg of spina gleditsiae, sieving the crushed spina gleditsiae by a 40-mesh sieve, extracting the spina gleditsiae for 3 times and 3min each time by acetone with the volume concentration of 50 percent, the weight of the spina gleditsiae is 9 times that of the spina gleditsiae, combining the extracting solutions for 3 times, concentrating the extracting solutions under reduced pressure to a concentrate with the crude drug content of 1.2g/mL, adding distilled water with the weight and volume of 5 times that of the concentrate to suspend the concentrate, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extracting part;

6. The method for preparing sapogenone A, a flavonoid in spina gleditsiae, according to claim 2, characterized in that it comprises the following steps:

(1) preparing a crude extract, namely crushing 4.82kg of spina gleditsiae, sieving the crushed spina gleditsiae by a 40-mesh sieve, extracting the spina gleditsiae for 3 times and 2min each time by acetone with the volume concentration of 50 percent and the weight of 8 times of the weight of the spina gleditsiae, combining the extracting solutions for 3 times, concentrating the extracting solutions under reduced pressure until the extracting solution is equivalent to 1.2g/mL of crude drug, adding distilled water with the weight and the volume of 5 times of the weight and the volume of the extracting solution into the extracting solution for suspension, sequentially adding petroleum ether and ethyl acetate with the same volume as the suspending solution into the extracting solution, and recovering the solvent under reduced pressure to obtain an extract of an ethyl acetate extracting part;

7. The use of a flavonoid of spina Gleditsiae sapogenone A of claim 1 in preparing a medicament for treating acute kidney injury caused by sepsis.