CN115772204A

CN115772204A - Method for extracting marigold saponin E from radix Achyranthis bidentatae and application of marigold saponin E in preparation of medicine for treating acute liver injury

Info

Publication number: CN115772204A
Application number: CN202211402802.6A
Authority: CN
Inventors: 冯敖梓; 郭彭莉; 李婉; 李莉; 何宁霞; 刘萌; 张贝贝; 张钦钦
Original assignee: First Affiliated Hospital of Jinan University
Current assignee: First Affiliated Hospital of Jinan University
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2023-03-10

Abstract

The invention relates to a method for extracting calendula officinalis saponin E from radix achyranthis bidentatae and application of calendula officinalis saponin E in preparation of drugs for treating acute liver injury, which can effectively solve the problems of extracting calendula officinalis saponin E from radix achyranthis bidentatae and preparing drugs for treating acute liver injury ₇₀ ‑1～NX ₇₀ -6, merging NX ₇₀ -1 and NX ₇₀ -2, performing silica gel column chromatography and dichloromethane-methanol gradient elution to obtain a component NX ₇₀ ‑1‑1～NX ₇₀ -1-4; component NX ₇₀ Loading-1-2 on Sephadex LH-20 column, isocratically eluting with aqueous methanol to obtain component NX ₇₀ ‑1‑2‑1～NX ₇₀ -1-2-5; component NX ₇₀ Separating and purifying (1-2-3) by semi-preparative HPLC, and collecting t _R Fraction of which is 5.37 to 6.0min, concentrating and drying to obtain the marigold saponin E. The invention has rich raw materials, easy operation of the extraction method and development of a new approach of the liver-protecting medicine.

Description

Method for extracting calendula saponin E from radix Achyranthis bidentatae and application of calendula saponin E in preparation of medicine for treating acute liver injury

Technical Field

The invention relates to a medicine, in particular to a method for extracting calendula saponin E from radix achyranthis bidentatae and application of calendula saponin E in preparation of a medicine for treating acute liver injury.

Background

Acute liver injury is a short-term incidence of hepatocyte injury due to a variety of factors, usually manifested as elevated transaminase in serum. The Lipopolysaccharide (LPS) and D-galactosamine (GalN) induced liver injury animal model is similar to the pathological change of human liver injury, and the model is stable, thereby being an ideal animal model for liver injury pathogenesis and drug screening. LPS can induce endotoxin damage and stimulate immune cells to release inflammatory factors, so that liver cells are apoptotic and necrotic. D-GalN depletes uridine phosphate by competitive inhibition, thereby inhibiting nucleic acid and protein synthesis, causing hepatocyte damage. Under the action of LPS/D-GalN, liver cells undergo massive apoptosis and necrosis in a short time, so that liver function is seriously damaged, and the liver cell apoptosis and necrosis are widely used for researching pathogenesis of liver damage and screening of potential therapeutic drugs. The pathogenesis of acute liver injury is not completely clear at present, and researches show that the pathogenesis of the acute liver injury is that pathogenic microorganisms or poisons directly damage liver cells to induce downstream inflammatory reaction and activate the immune response of an organism, a large number of inflammatory cells are gathered in the liver, and the acute liver injury is aggravated. In addition, most liver diseases are characterized by severe mitochondrial dysfunction, which can lead to massive hepatocyte death, and studies have now shown that liver is closely associated with mitochondrial damage mediated by the AMPK/SIRT3 signaling pathway.

Achyranthes bidentata, which is a dried root of Achyranthes bidentata Bl (Achyranthaceae) and is one of the four major wye drugs, is produced in Henan county, wu\38495, boai, qinyang, etc.. At present, research on traditional Chinese medicinal materials as liver-protecting medicines is a hotspot, and a plurality of natural medicines with liver-protecting and anti-inflammatory activities are found clinically. Calendula saponin E is also called glucose-free panax japonicus saponin, is an active ingredient in achyranthes bidentata, is extracted from achyranthes bidentata and is used for treating liver injury, but is not reported in a public way till now.

Disclosure of Invention

In view of the above situation, in order to overcome the defects of the prior art, the invention aims to provide a method for extracting calendula saponin E from radix achyranthis bidentatae and application of calendula saponin E in preparation of a drug for treating acute liver injury, which can effectively solve the problem of extracting calendula saponin E from radix achyranthis bidentatae and preparing a drug for treating acute liver injury.

The technical scheme for solving the problem is that the method for extracting the marigold saponin E from the achyranthes bidentata is characterized in that the molecular structural formula of the marigold saponin E is as follows:

the extraction method comprises the following steps: chopping Achyranthis radix, extracting with ethanol under reflux for 3 times, filtering, mixing extractive solutions, concentrating under reduced pressure to recover ethanol, adsorbing with macroporous adsorbent resin column, eluting with ethanol-water gradient to obtain 6 components, and collecting 70% ethanol part (NX) ₇₀ ) Dissolving with 70% methanol, loading onto MCI chromatographic column, and gradient eluting with methanol-water system to obtain NX ₇₀ -1～NX ₇₀ -6, identifying by thin layer chromatography, pooling NX ₇₀ -1 and NX ₇₀ -2 two components denoted NX ₇₀ -1, silica gel column chromatography, gradient elution with dichloromethane-methanol system, thin layer chromatography, and pooling of the same fractions to obtain NX fraction ₇₀ -1-1～NX ₇₀ -1-4; wherein the component NX ₇₀ Loading on Sephadex LH-20 column at-1-2, isocratically eluting with aqueous methanol,thin layer chromatography, and mixing the same fractions to obtain NX fraction ₇₀ -1-2-1～NX ₇₀ -1-2-5; component NX ₇₀ Separating and purifying-1-2-3 by semi-preparative HPLC, and collecting with retention time t _R Fraction of which the time is 5.37-6.0 min, and concentrating and drying to obtain the compound marigold saponin E.

The marigold saponin E extracted by the method can be effectively used for treating acute liver injury, and the application of the marigold saponin E in preparing a medicine for treating acute liver injury is realized.

The calendula saponin E serving as the active ingredient for protecting the liver is extracted from radix achyranthis bidentatae, the raw materials are rich, the extraction method is easy to operate, and the calendula saponin E is stable and reliable, so that the medicinal value of the radix achyranthis bidentatae is exploited, the new application of the radix achyranthis bidentatae is widened, the new approach of a liver-protecting medicine is exploited, and remarkable economic and social benefits are achieved.

Drawings

FIG. 1 is a molecular structural diagram of the marigold saponin E of the present invention.

FIG. 2 is a mass spectrum of marigold saponin E of the present invention.

FIG. 3 shows the calendula saponin E compounds of the present invention ¹ H-NMR Spectroscopy (CD) ₃ OD,500 MHz) plot.

FIG. 4 shows the calendula saponin E compounds of the present invention ¹³ C-NMR Spectroscopy (CD) ₃ OD,125 MHz).

FIG. 5 is a graph of the effect of the compound calendula saponin E of the present invention on the pathological changes of the liver tissue and liver function index enzyme of a liver-injured mouse, wherein A. The liver morphology graph; B. liver tissue HE (20 μm); c, d. in turn represent the levels of ALT, AST in serum of liver-injured mice (n = 12); con represents the normal group; m represents a model group; SC represents silibinin group; E-Low represents calendula saponin E Low dose group; E-High represents the marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^## P<0.01, compared to M, ^** P<0.01。

FIG. 6 is a graph showing the effect of calendula saponin E of the present invention on oxidative stress and inflammation of the liver of liver-injured mice, wherein A: ROS flow chart and quantification of primary hepatocytes (n = 3); b: sequentially represents the levels of MDA, GSH-Px and SOD in the serum of the liver injury mouse (n = 12);c: fluorescence intensity and quantification of 2-DG probe in mice at 6h (n = 3); d: fluorescence intensity and quantification of PEG probes in mice at 6h (n = 3); e: sequentially representing the contents of TNF-alpha, IL-1 beta, IL-6, IFN-gamma and IL-2 in the liver of the liver injury mouse (n = 12); f: primary hepatocyte apoptosis flow chart and quantification results (n = 3). Con represents the normal group; m represents a model group; SC represents silibinin group; E-Low represents calendula saponin E Low dose group; E-High represents marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^## P<0.01, compared to M, ^* P<0.05, ^** P<0.01。

FIG. 7 is a graph showing the effect of calendula saponin E, a compound of the present invention, on liver immune cells of liver-injured mice (n = 3), wherein Con represents the normal group; m represents a model group; SC represents silibinin group; E-Low represents calendula saponin E Low dose group; E-High represents the marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^## P<0.01, compared to M, ^* P<0.05, ^** P<0.01。

FIG. 8 is a graph of the effect of calendula saponin E, a compound of the invention, on immune cells in peripheral blood of liver injured mice (n = 3), wherein Con represents the normal group; m represents a model group; SC represents silibinin group; E-Low represents calendula saponin E Low dose group; E-High represents the marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^# P<0.05, ^## P<0.01, compared to M, ^* P<0.05, ^** P<0.01。

FIG. 9 is a graph showing the effect of calendula saponin E of the compound of the present invention on the intestinal flora of liver-injured mice, wherein A: shannon index representing Alpha diversity analysis; b: PCoA analysis representing beta diversity analysis; c: relative abundance at the level of the mouse gut flora; D-E: quantification of the relative abundance of firmicutes and bacteroidetes in mouse feces (n = 5); f: the picrus 2 was used to predict KEGG signaling pathways. Con represents the normal group; m represents a model group; E-High represents marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^## P<0.01, compared to M, ^** P<0.01。

FIG. 10 shows the energy metabolism of liver-damaged mouse mitochondria by the compound calendula saponin E of the inventionWherein, a: primary hepatocyte mitochondrial membrane potential flow diagram; b: quantification results of mitochondrial membrane potential of primary hepatocytes (n = 3); c: mtDNA levels in mouse liver (n = 3); d: ATP content in mouse liver (n = 12); e: mitochondrial structure (1 μm) was observed by transmission electron microscopy. Con represents the normal group; m represents a model group; SC represents silibinin group; E-Low represents calendula saponin E Low dose group; E-High represents the marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^# P<0.05, ^## P<0.01, compared to M, ^* P<0.05, ^** P<0.01。

FIG. 11 is a graph of the effect of marigold saponin E of the compounds of the present invention on the AMPK-SIRT3 signaling pathway in mice with liver injury, wherein A, C: an AMPK-SIRT3 signal pathway western blot; b, D: relative expression of protein in mouse liver tissue (n = 3); e, F: AMPK-SIRT3 signaling pathway mRNA expression levels (n = 3); g: AMPK α 1/α 2, SIRT3, PGC-1 α immunofluorescence mapping (20 μm) and quantification (n = 3). Con represents the normal group; m represents a model group; SC represents silibinin group; E-Low represents marigold saponin E Low dose group; E-High represents marigold saponin E High dose group. Compared with the Con, the method has the advantages that, ^# P<0.05, ^## P<0.01, compared to M, ^* P<0.05, ^** P<0.01。

Detailed Description

The following detailed description is of specific embodiments of the invention.

The invention relates to a method for extracting calendula saponin E from radix achyranthis bidentatae, wherein the molecular structural formula of the calendula saponin E is as follows:

the extraction method comprises the following steps: taking 25.0kg of radix achyranthis bidentatae, cutting, carrying out reflux extraction for 3 times at 70 ℃ by using ethanol with the weight volume of 8 times and the volume concentration of 70%, wherein the weight volume is 1h each time in terms of g of solid and mL of liquid, combining extracting solutions, and recovering ethanol under reduced pressure to obtain an extract; adsorbing the extract with macroporous adsorbent resin column of 14.5 × 100.0cm for 5 hr, and adding ethanol at volume ratioWater =0, 10, 30; concentrating the 70% ethanol fraction under reduced pressure to obtain extract (1.8 kg), dissolving the extract with 70% methanol by volume concentration, loading on MCI chromatographic column, and purifying with methanol: water =40, 60, 50, 60: 30 90, 100, gradient elution with a flow rate of 3mL/min, once every 300mL, judging the elution end point by using anisaldehyde-concentrated sulfuric acid thin layer detection for each gradient mobile phase, and obtaining a component NX after 4d of elution is finished ₇₀ -1、NX ₇₀ -2、NX ₇₀ -3、NX ₇₀ -4、NX ₇₀ -5、NX ₇₀ -6; identifying by thin layer chromatography, and combining NX ₇₀ -1 and NX ₇₀ -2 two components denoted NX ₇₀ -1, and subjecting to silica gel column chromatography with a 100-200 mesh sieve, gradient elution with dichloromethane: methanol =100, 1, 90, 1, 70 ₇₀ -1-1、NX ₇₀ -1-2、NX ₇₀ -1-3、NX ₇₀ -1-4; wherein the component NX ₇₀ Loading on Sephadex LH-20 column (1-2), isocratic eluting with 2 times of 70% aqueous methanol at flow rate of 1mL/min, detecting with thin layer, and mixing the same fractions to obtain NX ₇₀ -1-2-1、NX ₇₀ -1-2-2、NX ₇₀ -1-2-3、NX ₇₀ -1-2-4、NX ₇₀ -1-2-5; component NX ₇₀ Separating and purifying-1-2-3 by semi-preparative HPLC with methanol-water volume ratio of 80: 20, flow rate of 2.5mL/min, and collection retention time t _R Fraction of 5.37-6.0 min, concentrating and drying to obtain the compound marigold saponin E (69.3 mg).

The above embodiments are only examples, which are used to illustrate specific embodiments of the present invention, and are not intended to limit the scope of the present invention, and all technical solutions substantially the same as the technical solutions of the present invention, which are made by equivalent and equivalent substitution means, belong to the scope of the present invention.

The calendula saponin E extracted by the method has the liver protection activity, can be applied to the preparation of the medicine for treating acute liver injury, and obtains very good beneficial technical effects through experiments, and the related data are as follows:

1. sample identification

1.1 Experimental instruments

Bruker AVANCE III 500 nuclear magnetic resonance Spectrometer (TMS internal standard) (Bruker), nicolet is 10 Microsphere Spectrometer (Thermo Scientific, USA) infrared Spectrometer, bruker maxis HD mass Spectrometer, shimadzu UV-2401PC appaatus ultraviolet Spectrometer, waters Alliance series 2695 high performance liquid system equipped with 2998 diode array detector, empoer 3 chromatographic datse:Sup>A workstation, LC50 high pressure preparative liquid chromatograph, UV200 ultraviolet detector [ Seiko Seiki (Beijing) science Co., ltd ], YMC-Pack ODS-A chromatographic column (250X 10mm. D.S-5mm, 12mm) (YMC Co., ltd.), the rest are N-1100 rotary evaporator (Shanghai Airy Instrument Co., ltd.), A-1000S type air extractor (Shanghai Langhai Airy Instrument Co., ltd.), N-1111 Shanghai Cooling Water circulation apparatus (Shanghai Liang Water circulation apparatus) (DFK, shanghai Liang apparatus Co., ltd.), and DFZ-Souchi Freeze drying apparatus (DFZ-Hailan Liang instruments Co., ltd.), and DFZ dry Freeze instruments, FDZ-A-1000S instruments, SUPERY instruments, SUPERLANG, ghai instruments, USA, and the like.

The normal phase chromatography filler is 200-300 mesh silica gel produced by Qingdao ocean factory; thin-layer chromatography plate silica gel, silica gel G and GF254 are produced by Qingdao ocean factory; the reversed-phase column chromatography material RP-18 (SMB 100-20/45) is produced by Fuji corporation; sephadex LH-20 is produced by Parmachia Biotech; the macroporous adsorption resin Diaion HP-20 and MCI gel CHP 20P are produced by Mitsubishi chemical company of Japan; toyopearl HW-40 is manufactured by TOSOH corporation of Japan; the preparative column was YMC-PACK ODS-A (250X 20mml.D S-5um, 12nm), and the semi-preparative column was Cosmosil 5C 18-MS-II 10ID X250 mm.

Color developing agent: (1) concentrated sulfuric acid anisaldehyde solution; ferric chloride potassium ferricyanide solution; a modified bismuth potassium iodide solution. (2) observing fluorescence under 254nm by using an ultraviolet instrument; other reagents used in the experiment are analytically pure and chromatographically pure, and are produced by Tianjin Si you reagent Co.

Plant: is collected from the Radix achyranthis bidentatae production base of Zhongnan province, zhongzhao county, the collection period is the Radix achyranthis bidentatae collection period and is identified as the root of Radix achyranthis bidentatae (Radix Achyranthus bidentata).

1.2 structural identification

The calendula saponin E extracted by the invention is colorless acicular crystal, the concentrated sulfuric acid-anisaldehyde reagent is mauve, and ESI-MS m/z:631.3[ 2 ] M-H] ^- Presumably, its molecular formula is C ₃₆ H ₅₆ O ₉ . From ¹ In an H-NMR spectrum, delta 5.24 (1H, brs) is a hydrogen proton on a double bond (see figure 3), delta 3.14 (1H, m) is hydrogen of an oxygen-linked carbon, 7 methyl hydrogen protons appear between delta 0.7 and 1.2 of a high field region, and a plurality of CH and CH2 hydrogen signal peaks appear between delta 1.0 and 2.1; ¹³ in the C-NMR spectrum, the delta 181.8 is carbonyl carbon (see FIG. 4), and the delta 145.1 and delta 123.7 are signals of a pair of double-bond carbons, which indicates that the compound has the structure of oleanolic acid. In the 13C-NMR spectrum, delta 172.6 is carbonyl carbon, and 4 methine oxygen connecting carbons appear between delta 71 and delta 78, which indicates that the structure of the compound contains glucuronic acid. From the 1H-NMR spectrum, a terminal hydrogen proton of one sugar was observed to be δ 4.37 (1H, d, J =7.5 Hz), and δ 107.0 corresponding thereto on the 13C-NMR spectrum was the terminal carbon of the sugar. When the hydroxyl hydrogen of the 3-position carbon is replaced by the sugar, the chemical shift value of the 3-position carbon is increased from delta 77-79 to delta 88-90, delta 91.0 can be observed on a 13C-NMR spectrum, the sugar is connected to the 3-position, and the compound is identified as the marigold saponin E, and the molecular structural formula is shown in figure 1.

TABLE 1 NMR data attribution (CD) of the Compound calendula saponin E ₃ OD)

2. In vivo experimental animals and reagents

2.1 Experimental animals

SPF-grade BALB/c mice 60, male, having a body weight of 18-22g were purchased from Experimental animals technology, inc. of Wei Tong Hua, beijing (license number: SCXK (Jing) 2021-0006). The experimental mice are placed in a standardized animal feeding room of Chinese medicine university in Henan, the relative humidity is 40-60%, the temperature is 23 +/-2 ℃, and the experimental mice can freely eat and drink water in a12 h light/12 h dark period. The animal experiments were approved by the institutional animal ethics committee of the university of traditional Chinese medicine in Henan (approval No. DWLL 2018080003).

2.2 Experimental drugs and reagents

Lipopolysaccharide (LPS) (cat # L2880), D-galactosamine (D-GalN) (cat # G0500) from Sigma, USA; silibinin capsules, available from Tianjin Tianshili Sheng Teddy drug Co., ltd (positive control drug, batch No.: 850704104, national drug Standard H20040299); glutathione peroxidase (GSH-PX) test kit (batch number: A005-1-2), total superoxide dismutase (SOD) test kit (batch number: A001-3-2), malondialdehyde (MDA) test kit (batch number: A003-1-1), glutamic oxaloacetic transaminase (AST/GOT) test kit (batch number: C010-2-1), and glutamic pyruvic transaminase (ALT/GPT) test kit (batch number: C009-2-1) were purchased from Nanjing institute of bioengineering research, inc.; IRDye 800CW PEG Contrast Agent (lot number: 926-50401), IRDye 800CW 2-DG Optical Probe (lot number: 926-08946) were purchased from LI-COR, USA; the PE Annexin V Apoptosis Detection Kit I Apoptosis Detection Kit (batch number: 559763) was purchased from BD Biosciences, USA; tumor necrosis factor-alpha (TNF-alpha) (batch No. MM-0132M 1), interleukin-1 beta (IL-1 beta) (batch No. MM-0040M 1), interleukin-2 (IL-2) (batch No. MM0701M 1), interleukin-6 (IL-6) (batch No. MM-0163M 1), gamma-interferon (IFN-gamma) (batch No. MM-0182M 1) kits were purchased from Jiangsu enzyme-immune Biotechnology, inc.; a whole protein extraction kit (batch number: BC 3710), a BCA protein quantification kit (batch number: PC 0020), an active oxygen detection kit (batch number: CA 1410), a mitochondrial membrane potential detection kit (batch number: M8650), an ATP content detection kit (batch number: BC 0305) and a total RNA extraction kit (batch number: R1200) are purchased from Beijing Solibao science and technology GmbH; sweScript RT I First Strand cDNA Synthesis Kit (lot: G3330-100), 2 × Universal Blue SYBR Green qPCR Master Mix (lot: G3326-05) was purchased from Wuhanseville Biotechnology, inc.;

Blood/Cell/Tissue/bacterial genomic DNA Kit (DC 112-01) for rapid purification of Blood/Cell/Tissue/bacterial genomic DNA was purchased from Nanjing Novophilia Biotech GmbH; bax, caspase-3. Caspase-9 (batch: ab32503, ab13847, ab32539, ab 92701) was purchased from Abcam, inc. of UK; bcl-2, AMPK, p-AMPK, SIRT3, beta-actin, GAPDH (batch: A0208, A12718, AP0116, A17113, AC026, AC 033) was purchased from Abclonal, inc., wuhan; PGC-1 α (batch No. 66369-1-lg) was purchased from Wuhan Sanying Biotechnology Ltd.

2.3 Experimental instruments

BD FACS Aria iii flow cytometer (BD Biosciences, usa); sorval ST16R Centrifuge5810R high speed refrigerated Centrifuge (Eppendorf, germany); bioTek multifunctional microplate reader (Bio-Rad, USA); micropipettes (Eppendorf, germany); a membrane transfer apparatus (Bio-Rad, USA); electrophoresis apparatus (Bio-Rad, USA); odyssey two-color infrared fluorescence imager (LI-COR, usa);

trilogy small animal in vivo imaging (LI-COR, usa); nanodrop one ultramicro uv spectrophotometer (Thermo Fisher, usa); quant Studio 5 real-time fluorescent quantitative PCR gene amplification instrument (Thermo Fisher company, USA); applied Biosystems ^TM 2720 thermal cycler (Thermo Fisher, usa).

3. In vivo experiments

3.1 animal experiments

The male BALB/c mice are divided into a normal group, a model group, a silibinin (48 mg/kg) group (converted into the dosage of the mice according to the dosage of human), a marigold saponin E low-dosage (15 mg/kg) group and a marigold saponin E high-dosage (30 mg/kg) group, wherein each group comprises 12 mice, the normal group and the model group are both administrated with distilled water with the same volume for intragastric administration, the drug group is intragastric administered with corresponding drugs, and the intragastric administration volume is 0.01mL/g,1 time/d and 7 days continuously. After 7 days of administration, the other groups except the normal group were modeled with 700mg/kg D-GalN and 10. Mu.g/kg LPS, and after 6 hours of modeling, the eye ball was picked up and blood was collected, serum was centrifuged at 3000rpm for 20min, and the blood was stored in a refrigerator at-80 ℃ and frozen at-80 ℃ for use.

3.2 histopathological evaluation

Collecting blood, fixing, cutting intact liver lobules, soaking in 4% paraformaldehyde solution, fixing, dehydrating with ethanol step by step, embedding in paraffin, slicing to 3-5 μm thick, staining with H & E, and observing pathological condition of mouse liver tissue with optical microscope.

3.3 liver function serum enzyme index detection

Serum was collected from each group of mice and ALT, AST were detected separately with reference to kit instructions.

3.4 near-Infrared fluorescence in vivo imaging of mice

IRDye 800CW 2-DG Optical Probe and IRDye 800CW PEG Optical Probe lyophilized powder were dissolved with 1mL sterile PBS, respectively, 2-DG at 0.l nmol/. Mu.L, PEG at 0.015 nmol/. Mu.L. After being stimulated by 700mg/kg D-GalN and 10 mug/kg LPS for 2h by intraperitoneal injection, each group of mice to be used is taken. Tail vein injection of 2-DG probe 10nmo1, PEG fluorescent dye 1.5nmol. After the probe is injected for 4 hours, the mouse is anesthetized by isoflurane air and placed into a near-infrared fluorescent animal imaging system, and the metabolism and distribution of the fluorescent probe 2-DG and the PEG fluorescent dye in the mouse are dynamically monitored respectively. The resulting images were visualized under the same fluorescence (800 nm) and White light (White) channels to observe the fluorescence intensity in mice.

3.5 oxidative stress indicator detection

Serum of each group of mice was collected, and total superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in the serum of mice were detected according to the kit instructions.

3.6 detection of ROS levels in liver and apoptosis in Primary hepatocytes

3 mice were randomly selected per group, dissected rapidly to take out fresh liver tissue, placed in a sterile petri dish, washed 3 times with sterile PBS, soybean granule-sized liver tissue was taken, placed in a 1.5mL sterile centrifuge tube, 1mL PBS was added, cut into 1mm pieces with ophthalmic forceps, centrifuged twice, two minutes (4 ℃,1500 rpm) at a time, and the supernatant was discarded. Adding 1mL of 0.25% trypsin digestion solution into the tissue mass, digesting at normal temperature for 5min, and adding a little serum to stop digestion. Filtering with a 70-micron filter screen, washing the tissue mass with 5-10 mL of PBS, collecting the filtrate, centrifuging for 2 times, each time for 5min (4 ℃,1500 rpm), discarding the supernatant, and collecting primary hepatocytes. According to the operation of a reactive oxygen species Detection Kit and a PE Annexin V Apoptosis Detection Kit I Apoptosis Detection Kit, the ROS and Apoptosis level of the liver cells are analyzed by combining a flow cytometer.

3.7 detection of the levels of IL-1 β, TNF- α, IL-6, IFN- γ, IL-2 in the liver

Preparing liver tissue homogenate according to the kit instruction, and detecting the levels of IL-1 beta, TNF-alpha, IL-6, IFN-gamma and IL-2 in the liver tissue of the mouse.

3.8 flow cytometry detection of immune cell levels in mouse liver

Mouse primary hepatocytes were obtained in the same manner as in 3.6. Cells were resuspended in 500. Mu.L PBS and divided into 4 flow tubes labeled Ths & Tcs, DCs, NKs and Tregs. Each tube was incubated with the corresponding antibody in the dark for 30min, centrifuged at 1200rpm for 5min, the supernatant was discarded, and resuspended in 2mL of PBS and centrifuged, and repeated twice. 300 μ L of PBS was added to each tube to resuspend the cells and test on the machine, where Tregs were stained with Foxp3 antibody after membrane disruption.

3.9 flow cytometry detection of immune cell levels in peripheral blood of mice

Blood was drawn from the mouse eyeballs and plasma was divided into 4 flow tubes, 100 μ L each, labeled as Ths & Tcs, DCs, NKs and Tregs. The staining procedure was identical to 3.8, and finally the cells were resuspended and tested on the machine.

3.10 immunohistochemical staining

Paraffin sections of liver tissues were taken, deparaffinized to water, antigen-repaired, 5% BSA-blocked, then antibodies F4/80 (1) and LY-6G (1 600) were added, incubated overnight at 4 ℃ for the next day, secondary antibody was incubated for 1h at room temperature, DAB was developed, then hematoxylin running water was added to turn blue, dehydrated, cleared, and mounted, and F4/80 and LY-6G expression was observed under an optical microscope.

3.11 16S rDNA sequencing

Extracting genome DNA of each group of mouse caecum content samples by adopting a CTAB method, and detecting the purity and the concentration of the DNA. The selected V3-V4 variable regions were PCR amplified using specific primers with Barcode and high fidelity DNA polymerase, depending on the selection of the 16S rDNA sequencing region. Detecting the PCR product by 2% agarose gel electrophoresis, cutting and recovering the target fragment, and recovering the gelAxyPrepDNA gel recovery kit (Axygen Biosciences, USA) was used. Referring to the preliminary quantitative result of electrophoresis, quantiFluor is used for the PCR amplification recovery product ^TM The quantitative determination of ST blue fluorescence system (Promega, USA) is carried out, and the mixture is mixed according to the sequencing quantity requirement of each sample. The pooled amplification products were prepared and sequenced on the machine using a sequencing library using Pacific Biosciences SMRTbellTM Template Prep kit 1.0.

TABLE 2 primer sequences

3.12 flow cytometry detection of cellular mitochondrial Membrane potential (JC-1) levels

Mouse primary hepatocytes were obtained in the same manner as in 3.6, and 500. Mu.L of JC-1 staining solution was added to each sample tube, followed by incubation in an incubator at 37 ℃ for 30min. JC-1 staining working solution was removed, 2mL of PBS was added for washing twice, then 500. Mu.L of pancreatin (1X) was added for digesting the cells, after the cells were completely digested, 1mL of FBS and 2mL of DMEM were added immediately to stop the digestion, the cells were transferred to 15mL of EP tubes and centrifuged at 1500rpm for 5min. The supernatant was discarded, 1mL of JC-1 staining buffer (1X) was added thereto, the mixture was mixed well and centrifuged at 1500rpm for 5min twice. Discarding the supernatant for the last time, adding 500. Mu.L JC-1 staining buffer (1X), mixing, detecting on the machine, and placing the sample on ice in the dark.

3.13 mtDNA copy number detection

DNA in liver tissue is extracted according to the instruction of the rapid purification blood/cell/tissue/bacterial genome DNA extraction kit, the copy number of mtDNA in the liver tissue is determined by adopting a qPCR technology, a mitochondrial coding gene ND1 is used as the copy number of mtDNA, PECAM is used as an internal reference, a primer is designed and synthesized by Wuhanseville biotechnology Limited company, and the sequence of the primer is shown in Table 3.

TABLE 3 primer sequences

3.14 ATP content detection

And (3) taking mouse liver tissues, and detecting the ATP content in the mouse liver tissues by adopting a micro-method.

3.15 detection of mitochondrial ultrastructure in liver tissue

Taking fresh mouse liver tissues, pre-fixing the liver tissues by 3% of glutaraldehyde, re-fixing 1% of osmium tetroxide, dehydrating acetone step by step, embedding Ep812, dyeing a semi-thin section by toluidine blue for optical positioning, dyeing the semi-thin section by a diamond knife for ultra-thin section, and dyeing the semi-thin section by uranium acetate and lead citrate by a JEM-1400FLASH transmission electron microscope for observation and photographing.

3.16 Western blot detection of protein expression level

Weighing liver tissue about 0.1g,10 times volume of protein lysate, adding 5 μ L protease inhibitor and phosphorylation protease inhibitor, homogenizing for 3-5min, incubating on ice for 30min, centrifuging, and collecting supernatant to obtain total protein. The final total protein concentration was determined by careful handling according to the BCA protein quantification kit instructions, and the remaining tissue protein samples were denatured by adding 5 × Load Buffer at a ratio of 1. The loading concentration was adjusted according to the total protein concentration, 60. Mu.g of protein was loaded, electrophoretically separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% BSA for 2h, added with primary antibodies Bax, caspase-3, caspase-9, bcl-2, AMPK, p-AMPK, SIRT3, beta-actin and GAPDH, incubated overnight at 4 ℃ respectively, added with secondary antibodies goat anti-rabbit (1. The two-color infrared laser imaging system was developed and the protein bands were analyzed using Image Studio software.

3.17 PCR detection of mouse liver mRNA expression level

Total RNA of liver tissues of each group of mice is extracted and cDNA is synthesized according to the instruction of the kit, and RT-PCR analysis is carried out. Related protein genes AMPK alpha 1, AMPK alpha 2, SIRT3, PGC-1 alpha, caspase-3, caspase-9, bax, bcl-2 and GAPDH internal reference sequences used in the experiment are designed and synthesized by Wuhanseville Biotechnology Limited company, and the primer sequences are shown in a table 4.

TABLE 4 primer sequences

3.18 tissue immunofluorescence staining

Liver tissue samples were fixed with 4% formaldehyde and paraffin-embedded and then deparaffinized. Sections were antigen repaired and then blocked with BSA for 30min. Liver tissues were tested for levels of AMPK α 1/α 2, SIRT3 and PGC-1 α expression using AMPK α 1/α 2, SIRT3 and PGC-1 α antibodies. Sections were incubated with AMPK α 1/α 2, SIRT3 (1: 100) and PGC-1 α (1. After 5 washes with PBST, sections were incubated with Cy3 fluorochrome-labeled secondary antibodies for 1h. Next, sections were washed 3 times with PBST and incubated with DAPI. Then, sections were washed 3 times with PBST and imaged using a microscope.

3.19 statistical processing

Statistical processing was performed using SPSS 26.0 for One-Way ANOVA, all data as mean. + -. Standard deviation

And (4) showing. P <0.05 indicates significant differences, and P <0.01 indicates very significant differences.

4. Results of in vivo experiments

4.1 Effect of Marigold saponin E on liver histopathological changes and liver function index enzyme of liver injury mice

As shown in fig. 5A and B, the liver tissues of the normal group were red and glossy, the liver cells were arranged in order, the structure was complete, the cell nuclei were clear, the cytoplasmic staining was uniform, no pathological changes such as inflammatory cell infiltration were observed, and the liver tissues of the model group had severe bleeding, black red, liver cell necrosis, cell nucleus shrinkage, and inflammatory cell infiltration. After the intervention of the calendula saponin E, the conditions of liver bleeding, hepatocyte necrosis and shrinkage are obviously relieved, inflammatory infiltration and the like are obviously improved. The levels of the serum enzymes ALT and AST, which reflect the degree of damage and outcome of the hepatocytes, were measured. The results of fig. 5C and D show that the liver function of the model group mice is significantly reduced compared with the normal group, and the ALT and AST levels are significantly increased (P < 0.01). After the drug is taken, the activity of liver function serum enzyme is obviously reduced (P is less than 0.01), and the marigold saponin E is suggested to obviously improve the liver function of the liver injury mice.

4.2 Effect of Marigold Saponin E on hepatic oxidative stress and inflammation in liver injured mice

As shown in FIGS. 6A and B, the oxidation stress level in mice is reflected by detecting the ROS level of primary hepatocytes and the levels of lipid peroxidation and antioxidase activity indexes MDA, SOD and GSH-Px in serum, and compared with a model group, the marigold saponin E remarkably reduces the ROS level of hepatocytes and MDA in serum of liver-injured mice, and increases the levels of SOD and GSH-Px (P <0.01 or P < 0.05). The fluorescence probe 2-DG reflects the collective inflammation accumulation level of the mice, as shown in figure 6C, compared with the normal group, stronger 2-DG fluorescence signals are observed at the abdomen of the model group, which indicates that the in vivo inflammation level is increased, and compared with the model group, the fluorescence intensity of the in vivo and liver of the mice is obviously reduced and the inflammation level is improved after the intervention of the marigold saponin E low and high dose group and the silibinin group (P is less than 0.01). The fluorescence probe PEG mainly reflects the leakage condition of blood vessels in the body of the mouse, as shown in figure 6D, compared with the normal group, the abdomen of the mouse in the model group observes a stronger PEG fluorescence signal, which indicates that the blood vessels in the body are seriously leaked, compared with the model group, the fluorescence intensity of the liver and the body of the mouse is obviously reduced after the low and high dose group of the calendula saponin E and the intervention of the silibinin, and the leakage condition of the blood vessels in the body is better (P < 0.01). Then, the levels of inflammatory factors IL-1 beta, TNF-alpha, IL-6, IFN-gamma and IL-2 in liver tissues of the liver injury mice are detected, as shown in figure 6E, and the levels of IL-1 beta, TNF-alpha, IL-6, IFN-gamma and IL-2 in the livers of the model mice are obviously increased (P is less than 0.01) compared with that of the normal group. The low and high dose group of calendula saponin E and the silybin group obviously reduce the content of IL-1 beta, TNF-alpha, IL-6, IFN-gamma and IL-2 in liver tissues of mice, and reduce the inflammation level (P is less than 0.01 or P is less than 0.05) in the liver tissues of the mice with liver injury. The apoptosis level of the primary hepatocytes is detected, and as shown in fig. 6F, the rate of apoptosis of hepatocytes in the model group is significantly increased (P < 0.01) compared with that in the normal group. The low and high dose groups of calendula saponin E and the silibinin group obviously reduce the ROS level in mouse liver cells and inhibit the apoptosis rate of the mouse liver cells (P is less than 0.01).

4.3 Effect of Marigold Saponin E on liver immune cells of liver-injured mice

The influence of the marigold saponin E on liver immune cells of liver-injured mice is shown in figure 7, and flow cytometry is adopted to detect Ths (CD 4+ helper T cells), tcs (CD 8+ killer T cells), DCs (dendritic cells) and NKs (natural killer cells), so that compared with normal mice, the levels of Ths, tcs, DCs and NKs in the livers of the model mice are obviously reduced (P is less than 0.01), and the level of Tregs is obviously increased (P is less than 0.01). Compared with the mice in the model group, the levels of Ths, tcs, DCs and NKs in the livers of the mice are obviously increased (P is less than 0.01 or P is less than 0.05) and the level of Tregs is obviously reduced (P is less than 0.01) after the intervention of the low-dose and high-dose group and the silybin group of the marigold saponin E. Immunohistochemical detection of macrophage (F4/80) and neutrophil (LY-6G) levels in mouse livers, significantly increased macrophage and neutrophil levels in model mouse livers compared to normal mice (P < 0.01). The low and high dose groups of marigold saponin E and the silibinin group intervened in mice with significantly reduced levels of macrophages and neutrophils in the liver compared to the model group (P < 0.01).

4.4 Effect of Marigold Saponin E on immune cells in peripheral blood of liver injured mice

As shown in figure 8, the effect of marigold saponin E on immune cells in peripheral blood of liver-injured mice is that levels of Ths, tcs, DCs and NKs in peripheral blood of model mice are obviously reduced (P < 0.01) and levels of Tregs are obviously increased (P < 0.01) compared with those of normal mice. Compared with the mice in the model group, the levels of Ths, tcs, DCs and NKs in the peripheral blood of the mice are obviously increased (P is less than 0.01 or P is less than 0.05) and the level of Tregs is obviously reduced (P is less than 0.01) after the intervention of the low-dose and high-dose group and the silybin group of the marigold saponin E.

4.5 Effect of Marigold Saponin E on intestinal flora in liver-injured mice

As shown in fig. 9A, alpha diversity analysis was performed according to the 16S rDNA sequencing result, and a violin graph of the Shannon diversity index of the mouse intestinal flora was plotted, and the result showed that the Shannon index of the model group was significantly decreased (P < 0.01) and marigold saponin E had a tendency to be retrograded to the Shannon index of the liver injury mouse intestinal flora. The PCoA principal component analysis result is shown in FIG. 9B, the model group is far away from the marigold saponin E group of the normal group, and the flora composition is obviously different. The results of relative abundance of mouse gut flora at phylum level are shown in fig. 9C-E, where model group mice have increased relative abundance of firmicutes and decreased relative abundance of bacteroidetes, and the marigold saponin E high dose group can modulate relative abundance at phylum level. Prediction of gut microbiota gene function (picrast 2) KEGG signal pathways significant signal pathways including energy metabolism pathways, membrane transport, carbohydrate metabolism, amino acid metabolism, etc. are shown in fig. 9F. Studies have shown that energy metabolism plays an important role in acute liver injury.

4.6 Effect of Marigold Saponin E on mitochondrial energy metabolism in liver-injured mice

Mitochondria are energy factories of cell life activities and are also centers of energy metabolism, so the indexes related to energy metabolism of mitochondria are detected, and the experimental results are shown in figures 10A-D, compared with a normal group, the mitochondrial membrane potential of cells in a model group is obviously reduced (P is less than 0.01); calendula saponin E significantly improved the decrease in cell mitochondrial membrane potential (P < 0.01) compared to the model group. Compared with a normal group, the liver mtDNA level and ATP content of the mouse in the model group are obviously reduced (P <0.05 or P < 0.01); compared with the model group, the low and high dose calendula saponin E and silybin group can obviously show mtDNA level and ATP content (P <0.05 or P < 0.01) in the liver of the liver-injured mouse after the dry prognosis, and the effect of the high dose calendula saponin E group is the best. The transmission electron microscope results are shown in fig. 10E, the mitochondria morphology of the normal group of mice is intact, the arrangement of the mitochondrial cristae is normal, the mitochondria swelling outer membrane and cristae of the model group are dissolved and broken, only the residual cristae is left, and the improvement is achieved after the intervention of the calendoside E.

4.7 Effect of Marigold Saponin E on the AMPK-SIRT3 Signal pathway in liver-injured mice

Researches show that the AMPK-SIRT3 signal path plays an important role in maintaining cell energy balance and regulating energy metabolism, in order to further reveal the relation between the AMPK-SIRT3 signal path and the liver protection effect of marigold saponin E, western blot, PCR and immunofluorescence are adopted to detect the levels of AMPK-SIRT3 signal path related protein and mRNA, and the experimental results are shown in figure 11. The results show that compared with the normal group, the protein expression and mRNA level of AMPK alpha 1, AMPK alpha 2, SIRT3, PGC-1 alpha and Bcl-2 in liver tissues of liver-injured mice are remarkably reduced, the protein expression and mRNA level of Caspase-3, caspase-9 and Bax are remarkably increased (P <0.05 or P < 0.01), compared with the model group, the protein expression and mRNA level of AMPK alpha 1, AMPK alpha 2, SIRT3, PGC-1 alpha and Bcl-2 in the low and high dose group and the silybin group of calendula saponin E are remarkably increased, and the protein expression and mRNA level of Caspase-3, caspase-9 and Bax are remarkably reduced (P <0.05 or P < 0.01).

5. Conclusion

Acute liver injury is mainly a disease in which sudden abnormality of liver function is caused by various causes in a short period of time. In order to prevent the occurrence and the deterioration of acute liver injury, an LPS (lipid-soluble protein) and GalN (GalN) induced liver injury animal model is selected to investigate whether the calendula saponin E can be used as a potential medicament for treating liver diseases on the basis of understanding the pathogenesis of the acute liver injury. The research result shows that after 10 mu g/kg LPS is injected into the abdominal cavity and 700mg/kg D-GalN is combined, the bleeding of liver tissues is more serious and blackish red, liver pathology shows that hepatocyte necrosis, cell nucleus shrinkage and inflammatory cell infiltration are more serious, the AST and ALT levels in mouse serum are obviously increased, and the successful establishment of a liver injury model of the LPS/D-GalN induced mouse is prompted. The pre-administration of the marigold saponin E and the silybin obviously improves the conditions of liver bleeding, hepatocyte necrosis, shrinkage, inflammatory infiltration and the like, and also reduces the activity of AST and ALT in serum, which indicates that the marigold saponin E can relieve liver injury induced by LPS/D-GalN.

There are studies that indicate that oxidative stress is essential for the development of LPS and GalN induced liver damage. Active oxygen in the body can react with unsaturated fatty acid in the phospholipid of the biological membrane to generate lipid peroxide, and the lipid peroxide can damage the body, wherein Malondialdehyde (MDA) is the most representative substance. On the other hand, non-enzymatic antioxidants, represented by antioxidant enzymes represented by superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), can scavenge ROS and relieve body injury. The experiment shows that the marigold saponin E can obviously inhibit excessive generation of ROS and MDA level, and obviously increase SOD and GSH-PX level, and shows that the marigold saponin E plays a role in protecting liver injury mice induced by LPS and GalN by weakening oxidative damage.

Inflammation and oxidative stress are interacting, and different types of liver injury are accompanied by a strong inflammatory response. Vascular responses are a central link in the inflammatory process, where the injury factors are diluted, killed and surrounded by inflammatory bleeding and exudative reactions. According to the experiment, through a live body experiment of a small animal, the 2-DG probe reflecting the inflammation accumulation level of a mouse body and the PEG probe reflecting the blood vessel leakage and the blood stasis degree of the mouse body are injected into a tail vein, and the marigold saponin E is found to be capable of reducing the inflammation level and the blood vessel leakage condition of the induced liver injury mouse for the first time. LPS is the most important pathogenic substance of G-bacteria, the immunostimulation effect is strong, and D-GalN can cause the diffuse necrosis of liver cells, thereby enhancing the hepatotoxicity of LPS. Causing a large amount of inflammatory mediators to be released, resulting in the over-systemic inflammation and further causing the systemic immune response. TNF-alpha is a cytokine produced by activated macrophages and monocytes, can promote and induce cytokines such as IL-6, IL-1 beta, IL-2, INF-gamma and the like to participate in the immunoregulation action of the organism and mediate inflammatory response. The experiment shows that the marigold saponin E can obviously reduce the level of inflammatory factors in a liver injury mouse induced by LPS/GalN for the first time.

The liver is the largest metabolic organ of the body, the liver and the intestinal tract are closely related in anatomy and physiology, and meanwhile, the liver is the organ which is most closely contacted with the intestinal tract and is exposed to a large amount of bacterial components and metabolites, and researches prove that various liver diseases such as acute liver injury, alcoholic liver disease, nonalcoholic liver disease and the like are related to the change of microbiome. An imbalance in gut microbiota may act on the host immune system and thereby affect the degree of liver inflammation. The influence of the marigold saponin E on the levels of DC, NK, thc, tregs, neutrophils and macrophages in liver and peripheral blood is detected in the research, and the result proves that the marigold saponin E can regulate the disturbance of the host immune system of the liver injury mouse induced by LPS/D-GalN. In order to further explore whether the marigold saponin E influences intestinal flora to change the disturbance of a host immune system, a 16S rDNA technology is adopted to detect the influence of the marigold saponin E on the intestinal flora of the liver injury mice caused by LPS/D-GalN, and the result proves that the marigold saponin E can improve the unbalance of bacteroides and firmicutes in the intestinal flora of the liver injury mice caused by LPS/D-GalN, and the KEGG signal pathway is predicted through the prediction of the gene function of the intestinal microbiota (PICRUSt 2), wherein the regulation of energy metabolism is possibly the action mechanism of the marigold saponin E on the improvement of the liver injury of the LPS/D-GalN induced mice.

Mitochondria are highly dynamic double-membrane organelles, are main energy sources of eukaryotic cells, play an important role in energy metabolism of the cells, and dysfunction of mitochondria can cause energy metabolism disorder. Normal mitochondrial membrane potential is a prerequisite for maintaining mitochondrial oxidative phosphorylation and generating Adenosine Triphosphate (ATP), and in addition, mitochondrial function depends on mitochondrial DNA integrity, and mitochondrial copy number (mtDNA) mutations or deletions must affect mitochondrial oxidative phosphorylation levels, reduce ATP production, and exacerbate liver damage. In order to explore the action mechanism of the marigold saponin E in interference with LPS and D-GalN induced liver injury of mice, the research detects the influence of the marigold saponin E on mitochondrial membrane potential, mtDNA, ATP and mitochondrial morphology, and the result proves that the marigold saponin E can interfere with mitochondrial injury caused by LPS/D-GalN, and supposes that the marigold saponin E can regulate energy metabolism through mediated mitochondrial injury to further improve liver injury caused by LPS/D-GalN.

AMPK is an important cellular energy sensor, and can maintain cellular energy balance, playing an important role in regulating energy metabolism. It mainly realizes the regulation of energy metabolism by regulating the activity of key enzymes for oxidation, synthesis and transcription of energy substances. The AMPK signal pathway is a signal transduction pathway for regulating cell energy metabolism, and can control the production and consumption pathways of Adenosine Triphosphate (ATP) in tissue cells, thereby influencing the energy metabolism process of the tissue cells. There are studies that have demonstrated that SIRT3 is central to energy regulation and is a downstream target of the AMPK signaling pathway, promoting mitochondrial biosynthesis and deacetylation of mitochondrial antioxidant enzymes and affecting mitochondrial function. However, the research on whether the marigold saponin E can improve mitochondrial apoptosis through the activation of AMPK-SIRT3 signals so as to reduce acute liver injury has not been reported yet. Therefore, the research detects the expression level of the marigold saponin E on AMPK-SIRT3 signal path related protein and mRNA, and the result proves that the marigold saponin E can inhibit the protein expression and mRNA level of AMPK alpha 1, AMPK alpha 2, SIRT3, PGC-1 alpha and Bcl-2 in liver tissue of an LPS/D-GalN induced liver injury mouse, caspase-3, caspase-9 and Bax protein expression and mRNA level are obviously increased, so that the marigold saponin E can play a role in improving the mouse liver injury caused by LPS/D-GalN through the AMPK-SIRT3 signal path.

In conclusion, the calendula saponin E has a treatment effect on liver injury induced by LPS/D-GalN, and can effectively improve the liver injury, and the mechanism is that the compound can influence intestinal flora imbalance and mitochondrial injury mediated by an AMPK-SIRT3 signal path by regulating and controlling a host immune system so as to influence in-vivo energy metabolism, so that the injury of the LPS/D-GalN to the liver is improved, the liver injury can be effectively improved, the medicinal value of radix achyranthis bidentatae is exploited, the new application of the radix achyranthis bidentatae is broadened, the new way of liver-protecting medicines is exploited, and the obvious economic and social benefits are achieved.

Claims

1. The invention relates to a method for extracting calendula saponin E from radix achyranthis bidentatae, wherein the molecular structural formula of the calendula saponin E is as follows:

the extraction method comprises the following steps: cutting Achyranthis radix 25.0kg, extracting with 8 times by weight of 70% ethanol at 70 deg.C under reflux for 3 times, wherein the weight and volume are solid in g and liquid in mL, each time for 1 hr, and filteringFiltering, mixing extractive solutions, and recovering ethanol under reduced pressure to obtain extract; adsorbing the extract for 5h on a macroporous adsorption resin column of 14.5 × 100.0cm, performing gradient elution with ethanol: water =0, 10, 90, 30, 70, 50, 95; concentrating the 70% ethanol part under reduced pressure to obtain an extract, dissolving the extract by using 70% methanol with volume concentration, loading the extract on an MCI chromatographic column, and mixing the extract with a methanol-water ratio of =40, 50: 30 90, 100, gradient elution with a flow rate of 3mL/min, once every 300mL, judging the elution end point by using anisaldehyde-concentrated sulfuric acid thin layer detection for each gradient mobile phase, and obtaining a component NX after 4d of elution is finished ₇₀ -1、NX ₇₀ -2、NX ₇₀ -3、NX ₇₀ -4、NX ₇₀ -5、NX ₇₀ -6; identifying by thin layer chromatography, and combining NX ₇₀ -1 and NX ₇₀ -2 two components denoted NX ₇₀ -1, and subjecting to silica gel column chromatography with a 100-200 mesh sieve, gradient elution with dichloromethane: methanol =100, 1, 90, 1, 70 ₇₀ -1-1、NX ₇₀ -1-2、NX ₇₀ -1-3、NX ₇₀ -1-4; wherein the component NX ₇₀ Subjecting to Sephadex LH-20 column at-1-2, isocratically eluting with 2 times of 70% aqueous methanol at flow rate of 1mL/min, detecting with thin layer, and mixing the same fractions to obtain NX fraction ₇₀ -1-2-1、NX ₇₀ -1-2-2、NX ₇₀ -1-2-3、NX ₇₀ -1-2-4、NX ₇₀ -1-2-5; component NX ₇₀ Separating and purifying-1-2-3 by semi-preparative HPLC with methanol-water volume ratio of 80: 20, flow rate of 2.5mL/min, and collection retention time t _R Fraction of which is 5.37 to 6.0min, and concentrating and drying to obtain the compound marigold saponin E.

2. The use of calendula saponin E prepared by the method of claim 1 in the preparation of a medicament for the treatment of acute liver injury.