Chrysopa septempunctata extract and application thereof

Abstract

The invention discloses a chrysopa mossambica extract and application thereof, belongs to the field of traditional Chinese medicines, and particularly relates to preparation of the chrysopa mossambica extract and application of the chrysopa mossambica extract in preparation of renal injury medicines. According to the invention, the protective effect of the Chrysopa mossambica extract on a mouse model with acute kidney injury caused by cis-platinum is researched and analyzed, and the result shows that the Chrysopa mossambica extract can reduce the kidney coefficient of a mouse with cis-platinum exposed, reduce the expression level of serum creatinine and urea nitrogen, and improve the pathological change of kidney tissue caused by cis-platinum at the same time. Meanwhile, according to metabonomics, it is characterized that the Chrysopa septempunctata extract can adjust 38 differential metabolites in the kidney and mainly relates to glycerophosphatide metabolism, purine metabolism and metabolic pathways of nicotinic acid, nicotinamide and thiamine, the metabonomics and network pharmacology are further combined, and five key targets are screened out. The compound is expected to be used for preparing related medicines for treating renal injury, and has a wide application prospect and a relatively high medical value.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to an Acanthus macrocystis extract and application thereof in preparing a medicine for treating kidney injury. Background Art

[0002] Acute kidney injury (AKI) refers to a clinical syndrome caused by a rapid decline in renal function due to a variety of causes, which is manifested by a sharp increase in nitrogen products such as serum creatinine and urea nitrogen. The basic pathophysiological mechanism of AKI is not fully understood, and it mainly involves damage and apoptosis of renal tubular epithelial cells, especially the proximal tubules, leading to a sharp decline in renal function. At present, the treatment of AKI mainly relies on renal replacement therapy and supportive treatment, and there is still a lack of specific means. The prevention and treatment of AKI has become a public problem that needs to be solved urgently worldwide.

[0003] The larvae of the genus Megalodon, also known as the genus Megalodon ( hellgrammite, Hel ), belonging to the class Insecta, order Broadwinger, family Odontophoridae. According to the record of Xichang Chinese Herbal Medicine, Hel is warm in nature and sweet in taste, and has the effects of replenishing qi and kidney, suppressing deficiency and strengthening the foundation, strengthening and nourishing, treating injuries from falls, promoting blood circulation and removing blood stasis, warming yang and stopping drowning, etc., and has the reputation of "animal human kidney" and "insect ginseng". According to the record of Yunnan Chinese Herbal Medicine: It has a warm and tonic nature, and is a holy medicine for deficiency syndrome, deficiency stagnation, deficiency swelling, and deficiency pain, and its effect is better than that of nine fragrant insects. Hel is delicious, nutritious, rich in various proteins and essential amino acids for the human body. It is a traditional food and medicine tonic in southwest Sichuan, northeast Yunnan and other regions. It is also used by the Yi, Miao, Hani and Naxi peoples to treat enuresis in children and nocturia in the elderly due to physical weakness, suggesting that Hel may have the effect of protecting renal function and repairing renal damage. At present, the research on Hel by modern medicine mainly focuses on the research of nutritional components, and there is little research on pharmacological activity. So far, there has been no literature report on its application in drugs for the prevention and treatment of acute kidney injury. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned background technical difficulties and provide an Acarina macrophylla extract and its application.

[0005] In order to achieve the above purpose, the technical solution adopted is: A method for preparing an extract of Hel from Acanthida macrodon, comprising the following steps: drying the Hel and crushing it to obtain a powder; soaking the Hel powder in methanol for extraction for 24 hours and concentrating it to obtain a crude methanol extract; extracting the crude Hel methanol extract with n-butanol and concentrating it to obtain a Hel extract.

[0006] Furthermore, in the above steps, the concentration equipment is a rotary evaporator.

[0007] And the application of the above-mentioned Acanthacorydalis extract in the preparation of drugs for preventing and / or treating kidney injury.

[0008] Furthermore, the kidney injury is acute kidney injury caused by cisplatin.

[0009] And the analysis method for the mechanism of the above-mentioned Acanthacorydalis extract for treating kidney injury drugs is as follows: 1). Select 3 groups of kidney samples: blank control group, cisplatin model group, Acanthacorydalis extract administration group; add methanol, vortex and let stand, centrifuge at 4°C for 15 minutes, and take the supernatant for LC-MS detection; 2). Then, through kidney metabolomics profile analysis, compare the metabolites of the blank control group and the cisplatin model group, screen out the differential metabolites of the cisplatin acute kidney injury model, and compare with the Acanthacorydalis extract to screen out the differential metabolites with the mechanism of improving acute kidney injury for callback; 3). Perform pathway analysis on the specific differential metabolites obtained in the above steps through the MetaboAnalyst online analysis software; 4). Obtain the relevant targets of the Acanthacorydalis extract intervention callback differential metabolites through the MetScape plugin in Cytoscape; 5). In the Genecards and OMIM databases, retrieve the target genes related to cisplatin-induced kidney injury; 6). Match the relevant targets of the differential metabolites in step 4) with the targets in step 5), and confirm the selected repeated targets as the effective targets of the Acanthacorydalis extract in improving cisplatin-induced kidney injury, and screen out the changes in the relevant pathways through KEGG and GO enrichment analysis; 7). Verify the top 5 key target groups of the protective effect of the Acanthacorydalis extract screened in step 6) on cisplatin-induced acute kidney injury.

[0010] The achievements of the present invention: 1. The present invention first reveals that the Hel extract can significantly improve CP-induced AKI; 2. The present invention first uses metabolomics combined with network pharmacology technology to analyze the better molecular mechanism of the Hel extract on CP-induced AKI from aspects such as the component analysis, action targets and pathways of the Hel extract; 3. The present invention first verifies the action targets of the Hel extract on CP-induced AKI mice - NT5E, PNP, PLA2G6, PLD1 and PLD2.

[0011] In this invention, we used metabolomics combined with network pharmacology methods to further clarify the mechanism of action of Hel extract in preventing or / and treating drug-induced AKI represented by cisplatin (CP)-induced AKI. We established a CP-induced AKI mouse model, evaluated its therapeutic effect by conventional pharmacodynamics, and used metabolomics analysis to study the metabolic effects of Hel extract on AKI mice. Then, metabolite network pharmacology was applied to reveal the key targets and pathways of Hel extract in CP-induced AKI mice. This provides certain theoretical guidance for the development and utilization of Hel resources and the study of its active ingredient basis. Brief Description of the Drawings

[0012] Figure 1 Shows the effect of Hel extract on renal function indexes in the serum of AKI mice in this invention.

[0013] Figure 2 Shows the effect of Hel extract on renal histopathology of AKI mice in this invention.

[0014] Figure 3 Is the total ion current chromatogram of Hel extract in positive and negative ion modes.

[0015] Figure 4 Is the PCA plane score plot of the Control group, CP group, and CP+Hel-H administration group provided in the implementation of this invention.

[0016] Figure 5 Is the OPLS-DA plane score plot, 200 permutation test plot, and S score plot of the Control group, CP group, and CP+Hel-H administration group provided in the implementation of this invention.

[0017] Figure 6 Is the content analysis of biomarkers of the Control group, CP group, and CP+Hel-H administration group provided in the implementation of this invention.

[0018] Figure 7 Is the analysis of metabolic pathways of the Control group, CP group, and CP+Hel-H administration group provided in the implementation of this invention. Detailed Embodiments

[0019] The following will clearly and completely describe the concept and technical effects of this invention in combination with embodiments to fully understand the purpose, features, and effects of this invention. Based on the embodiments of this invention, other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of this invention. In addition, for better illustration of this invention, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that this invention can still be implemented without some specific details.

[0020] The present invention analyzes the effect and mechanism of Hel extract in improving CP-induced AKI based on conventional pharmacology, metabolomics and network pharmacology.

[0021] The following steps are involved: Step 1: Preparation of Hel Extract Hel was dried and crushed to obtain Hel powder, which was added to methanol and soaked for 24 hours, and concentrated by rotary evaporator to obtain methanol crude extract, and then extracted with n-butanol, and then concentrated to obtain n-butanol extract of Hel.

[0022] Step 2: Analysis of the amino acid composition of Hel Step 3: Serum creatinine (BUN) and urea nitrogen (SCr) were measured in mouse serum samples.

[0023] Step 4: Perform HE staining on mouse kidney tissue.

[0024] Step 5: Metabolomics analysis Mouse kidney tissue samples were analyzed using liquid chromatography-mass spectrometry, including sample preparation, chromatographic separation, and mass spectrometry detection; differential metabolite screening was performed after data processing and matching with the database, annotation, and data processing.

[0025] Step 6: Metabolite network pharmacology analysis Network pharmacology was used to identify disease targets, drug targets, and related targets of differential metabolites recalled by Hel extracts. This included: 1) Collection of related targets of Hel-recalled differential metabolites, AKI disease targets, and CP drug targets, and protein-protein interaction construction; 2) Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis pathway enrichment analysis; 3) Metabolite-target-pathway network construction.

[0026] The abbreviations of the terms used in the following embodiments and drawings are defined as follows: Abbreviations Full English Name Full Chinese Name Hel Hellgrammite Genus model dobsonfly CP Cisplatin Cisplatin AKI Acute kidney injury Acute kidney injury TCM Traditional Chinese medicine Traditional Chinese medicine BUN Blood urea nitrogen Serum creatinine SCr Serum creatinine Blood urea nitrogen QC Quality control Quality control sample PCA Principal component analysis Principal component analysis PLS-DA Partial least-squares discriminant analysis Partial least-squares discriminant analysis OPLS-DA Orthogonal projections to latent structures discriminate analysis Orthogonal partial least squares discriminant analysis Example 1 Preparation of Hel extract The Acanthida genus used in the experiment was collected in Yuxi, Yunnan, and was identified by experts in the field as the larvae of the Acanthida genus. The preparation steps of Hel extract are as follows: Hel was dried and crushed to obtain Hel powder, which was added to methanol and soaked for 24 hours, and concentrated by rotary evaporator to obtain methanol crude extract, and then extracted with n-butanol, and then concentrated by rotary evaporator to obtain n-butanol extract of Hel.

[0027] Example 2 Pharmacodynamic study of Hel extract on AKI mice In this chapter, the total butanol extract of A. megalodon was prepared using system solvents. Based on cisplatin-induced acute renal injury mice and guided by the renal protective effect, the key metabolites of A. megalodon that have a renal protective effect were screened, laying the foundation for the identification of its key targets.

[0028] 1. Experimental Materials 1.1 Main experimental materials CP, from Qilu Pharmaceutical Co., Ltd., catalog number; HPLC grade acetonitrile and methanol were purchased from Darmstadt, Germany. Acetic acid was purchased from Shanghai Yien Chemical Technology Co., Ltd. Ammonium formate, ammonia water and formic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Sodium carboxymethyl cellulose was purchased from Beijing Solaibao Technology Co., Ltd.; butanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0029] 1.2 Experimental animals SPF grade ICR mice, male, weight (20±2g), were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., production license: SCXK2019-0004. Housing conditions: temperature 23±2℃, humidity 50+10%, 12 h day and night cycle, adaptive feeding for one week before the experiment, free access to water and food during the feeding period. All experimental operations were in accordance with the regulations of the Laboratory Animal Medical Ethics Committee of Zunyi Medical University (ethics number: ZMU21-2304-009).

[0030] 2. Experimental Methods 2.1 Experimental groups and drug administration After 60 ICR mice were adaptively fed for 1 week, they were randomly divided into a blank control group (Control group), a CP group, a CP+Hel 2.55 mg / kg (CP+Hel-L) group, a CP+Hel 5.10 mg / kg (CP+Hel-H) group, and a Hel 5.10 mg / kg (Hel-H) group, with 12 mice in each group. Mice in the CP+Hel-L group, CP+Hel-H group, and Hel-H group were given Hel by gavage at doses of 2.55 mg / kg / day, 5.10 mg / kg / day, and 5.10 mg / kg / day, respectively, once a day for 7 consecutive days. The Control group and the CP group were given an equal amount of normal saline by gavage every day for 7 consecutive days. The CP group, CP+Hel-L group, and CP+Hel-H group were intraperitoneally injected with CP at a single dose of 20 mg / kg on the third day after drug administration.

[0031] 2.2 Serum biochemical indexes The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) in serum samples were measured using a Beckman Coulter automatic biochemical analyzer in the United States.

[0032] 2.3 Gross anatomical observation and kidney organ index On the 7th day after drug administration, the kidneys of the mice were dissected and removed, rinsed thoroughly with pre-cooled normal saline, and then the dissected tissues were observed macroscopically and photographed to record changes in their size, shape, color, or texture, weighed, and their organ indexes were calculated.

[0033] 2.4 Histopathological detection of mouse kidney tissues HE staining was used to observe the morphological structure of the mouse kidneys. The kidney tissues were placed in embedding cassettes and fixed in 4% paraformaldehyde for 24 h. They were successively dehydrated in ethanol at different concentrations, cleared with xylene, and the kidney tissue wax blocks were cut into sections about 4 μm thick after paraffin embedding, and stained with hematoxylin and eosin (HE). The conditions of renal tubules, glomeruli, protein casts, and inflammatory cell infiltration in mouse kidney tissues were observed under a microscope.

[0034] 2.5 Study on the improvement of Hel extract on the kidney metabolomics of CP-induced AKI mice 2.5.1 Sample preparation 1) Preparation of kidney samples All kidney samples were thawed at 4°C, then ground and added to 70% methanol aqueous internal standard extraction solution, and then centrifuged at 12000 rpm for 10 minutes to obtain the supernatant, which was made into the test sample.

[0035] 2) Preparation of QC samples For methodological verification, 10 μL of all kidney samples were taken and mixed to prepare QC samples, which were evenly divided into 3 QC samples. QC samples were added each time kidney samples were processed. The preparation method of QC samples was the same as that of kidney samples to control the quality of sample processing in this batch.

[0036] 2.5.2 Chromatography and Mass Spectrometry Acquisition Conditions 1) T3 Chromatographic Conditions 1. Chromatographic column: Waters ACQUITY Premier HSS T3 Column 1.8 μm, 2.1 mm*100mm 2. Mobile phase A: 0.1% formic acid / water; Mobile phase B: 0.1% formic acid / acetonitrile 3. Column temperature of the instrument: 40 °C; Flow rate: 0.4 mL / min; Injection volume: 4 μL Table 1: Mobile Phase Gradient Conditions of T3 Chromatographic Column Time (min) A（%） B（%） 0.0 95 5 2.0 80 20 5.0 40 60 6.0 1 99 7.5 1 99 7.6 95 5 10.0 95 5 Table 2: Mass Spectrometry Conditions of AB Triple TOF 6600 English name Chinese name ESI+ ESI- Duration (min) Collection time (min) 10 10 IonSpray Voltage (V) Ionization voltage (V) 5000 -4000 Temperature (℃) Ion source temperature (℃) 550 450 Ion Source Gas1 (psi) Spray gas (psi) 50 50 Ion Source Gas2 (psi) Auxiliary heating gas (psi) 60 60 Curtain Gas (psi) Curtain gas (psi) 35 35 Declustering Potential (V) Decustering voltage (V) 60 -60 MS1 Collision Energy (V) MS1 collision energy (V) 10 -10 MS2 Collision Energy (V) MS2 collision energy (V) 30 -30 Collision Energy Spread (V) Collision energy step (V) 15 15 MS1 TOF Masses (Da) MS1 mass range (Da) 50-1000 50-1000 MS2 TOF Masses (Da) MS2 mass range (Da) 25-1000 25-1000 MS1 Accumulation (s) MS1 collection time (s) 0.2s 0.2s MS2 Accumulation (s) MS2 collection time (s) 0.04s 0.04s Candidate ions Number of candidate ions 18 18 2.6 Network Pharmacology Study on the Mechanism of Hel in Protecting the Kidneys and Regulating Kidney Metabolites 2.6.1 Collection of Targets Related to Kidney Injury Diseases Using "Acute kidney injury" as the keyword, targets related to AKI diseases were searched from the OMIM (https: / / omim.org) and Genecards (https: / / genecards.org) databases. Using "Cisplatin" as the keyword, drug targets related to CP were searched from the CTD (https: / / ctdbase.com / ) database, and duplicate targets were deleted.

[0037] 2.6.2 Targets of Metabolites Recalled by Hel Extract Metabolites can act as "endogenous drug substances" in the body and play a role in biological processes by interacting with target proteins. For traditional Chinese medicines with unclear Hel components, in this study, the KEGG IDs of the callback differential metabolites obtained by intervening Hel extracts in CP-induced AKI mice were imported into the Metscape plugin of Cytoscape 3.9.1 to construct a "metabolite-target (M-T)" network. The protein-protein interaction (PPI) of metabolic targets was analyzed using the String database (https: / / string-db.org / Version 11.0), and hub genes were screened based on the average degree value. Important targets were identified in the KEGG pathway and GO-BP function analysis. According to the corrected P <0.05, a bubble chart of the top 20 pathways was drawn.

[0038] 2.7 Statistical analysis All data are expressed as mean ± standard error of the mean (mean ± SEM). SPSS 29.0 software was used for data statistical analysis. For data with homogeneous variance and conforming to a normal distribution, one-way ANOVA was used for comparison among multiple groups; for data with inhomogeneous variance and not conforming to a normal distribution, non-parametric tests were used. P <0.05 indicates that the difference is statistically significant, P <0.01 indicates that the difference is significantly statistically significant.

[0039] 3. Experimental results 3.1 Determination of the amino acid content in Hel A total of 21 amino acids were detected in Hel, including 8 essential amino acids and 13 non-essential amino acids (Table 1), and the results are as follows.

[0040] Table 1 Content of 21 amino acids in Hel

[0041] 3.2 Effects of Hel extracts on biochemical indexes of CP-induced AKI mice As Figure 1 shown in A, kidney index; B, BUN content; C, SCr content; compared with the normal group, ## p <0.01, compared with the cisplatin model group, * p <0.05, ** p <0.01; Control, normal group; CP, cisplatin model group; CP+Hel-L, low-dose administration group of Acanthacorydalis fruhstorferi; CP+Hel-H, high-dose administration group of Acanthacorydalis fruhstorferi; Hel-H, high-dose administration group alone. Figure 1It was shown that compared with the Control group, the expression levels of renal function indexes BUN and SCr in the CP group were significantly increased, and the kidney index was significantly increased. Compared with the CP group, the CP+Hel group could dose-dependently reduce the expression levels of BUN and SCr and the kidney index.

[0042] 3.3 Effects of Hel extract on the renal tissue morphology of CP-induced AKI mice Pathological histological examination of mouse kidneys was as Figure 2 shown in the figure: 1, renal tubular necrosis; 2, tissue vacuolization; 3, inflammatory cell infiltration; 4, protein casts; Control, normal group; CP, cisplatin model group; CP+Hel-L, low-dose administration group of Acanthacorydalis fruhstorferi; CP+Hel-H, high-dose administration group of Acanthacorydalis fruhstorferi; Hel-H, high-dose administration group alone. Figure 2 It was shown that the renal tissue structures of the Control group and the Hel group alone were normal, with regular-shaped renal tubules arranged neatly, normal glomerular spaces, and no obvious lesions. In the CP group, the renal tissues of mice showed renal tubular dilation, necrosis, cast formation, inflammatory cell infiltration, and tissue vacuolization.

[0043] In the CP+Hel-L group, the renal tubular dilation was improved and less cast formation was observed. In the CP+Hel-H group, the renal tissue damage was significantly reduced. The above results showed that cisplatin caused pathological damage to the renal tissues of mice, while the extract of Acanthacorydalis fruhstorferi could improve the renal pathological changes caused by cisplatin and had a renal protective effect.

[0044] 3.4 Metabolomics analysis of CP-induced AKI by Hel extract 3.4.1 Metabolomic profiling As Figure 3 shown: A, positive ion mode; B, negative ion mode; Control, normal group; CP, cisplatin model group; CP+Hel-H, high-dose administration group of Acanthacorydalis fruhstorferi.

[0045] The total ion current (TIC) chromatograms of QC samples in ESI+ and ESI- modes were used to evaluate the repeatability and stability of the system. The results showed that the retention times and peak intensities of the total ion current curves for detecting metabolites highly overlapped, indicating good signal stability of the mass spectrometry in analyzing the same sample at different times (Figure 3A, 3B). In addition, in the PCA plot, the score plots of QC samples were closely distributed ( Figure 3 Figure 3C, 3D), indicating good reproducibility of the experiment.

[0046] To investigate the potential protective effect of Hel on CP-induced AKI, we analyzed the metabolic profiles of different groups by PCA. We performed PCA metabolic profile analysis on samples from the Control group, CP group, and CP+Hel-H group. PCA score plot ( Figure 3 C, 3D) showed that under ESI+ and ESI- ion modes, the metabolic profiles of the Control group, CP group, and CP+Hel-H group were separated from each other, indicating that CP-induced AKI affected the endogenous metabolic profile of the body. The CP+Hel-H group had a significant trend toward the Control group, which indicated that Hel extract had a significant intervention effect on CP-induced AKI.

[0047] 3.4.2 Screening and identification of differential metabolites The OPLS-DA method was used to analyze the control group and CP group ( Figure 3 A-3F), CP group and CP+Hel-H ( Figure 3 The endogenous metabolites of mice in the G-3L group were analyzed to screen for potential differential metabolites. The predictive ability and reliability of the OPLS-DA model were verified by 200 permutation tests. The intercept of the Q2 regression line was less than 0, indicating that the OPLS-DA model was not overfitted, indicating that the model has high reliability in screening differential metabolites. We used VIP>1, P <0.05, FC>2.5 or FC<0.5 to screen differential metabolites, and a total of 83 differential metabolites were screened between the Control group and the CP group. After intervention with Hel extract, 38 of the differential metabolites partially returned to normal levels, and their relative abundance changes were as follows Figure 5 C. The above results indicate that Hel extract treatment can partially reverse the renal differential metabolite disorder caused by CP and improve CP-induced AKI.

[0048] like Figure 4 : A, positive ion mode; B, negative ion mode; Control, normal group; CP, cisplatin model group; CP+Hel-H, Acanthida macroglossum high-dose administration group.

[0049] 3.4.3 Metabolic pathway analysis and identification of potential biomarkers To further reveal the therapeutic mechanism of Hel, we used the Metaboanalyst 6.0 database to perform metabolic pathway enrichment analysis on the screened differential metabolites. Figure 5 :A, B, C, G, H, I, positive ion mode; D, E, F, J, K, L, negative ion mode; Control, normal group; CP, cisplatin model group; CP+Hel-H, Acanthida macrocypris high-dose administration group.Figure 5 As shown in A, according to pathway impact > 0.1, P and < 0.05, important pathways were screened out. CP mainly affected five metabolic pathways, including purine metabolism, pantothenate and CoA biosynthesis, glycerophospholipid metabolism, thiamine metabolism, and niacin and nicotinamide metabolism, as Figure 5 shown in B. After the intervention treatment with Hel extract, 4 of these pathways were involved in regulation to improve CP-induced AKI, including purine metabolism, glycerophospholipid metabolism, thiamine metabolism, and niacin and nicotinamide metabolism. Figure 5 D is the metabolic network of significantly different metabolites. The content changes of metabolites related to the pathways are as Figure 6 shown. Figure 6 In the figure: A, allantoin acid; B, adenine; C, adenosine monophosphate; D, deoxyadenosine; E, deoxyinosine monophosphate; F, uric acid; G, lysophosphatidylcholine; H, choline; I, lysophosphatidylcholine; J, choline; K, nicotinamide adenine dinucleotide; L, nicotinamide riboside; M, palmitoylethanolamide; N, phosphocholine; O, thiamine; P, thiamine pyrophosphate; Control, normal group; CP, cisplatin model group; CP+Hel-H, high-dose administration group of Acanthacorydalis fruhstorferi.

[0050] 3.4.4 Metabolite network pharmacology Input the KEGG IDs of the differential metabolites recalled by Hel extract into the Cytoscape software for metscape analysis, and a total of 133 metabolite targets associated with differential metabolites were obtained ( Figure 7 A). Figure 7 In the figure: A, metabolic pathway map of Control group and CP group; B, metabolic pathway map of CP group and CP+Hel-H administration group; C, integrated metabolic pathway map; Control, normal group; CP, cisplatin model group; CP+Hel-H, high-dose administration group of Acanthacorydalis fruhstorferi. Then we imported 133 metabolite targets into the Cytoscape software to construct a PPI network, and further screened 65 metabolite targets higher than the average value (25.23) as core targets ( Figure 7 B). In addition, we predicted 11,063 CP drug targets from the CTD database ( Figure 7C), and 9,019 kidney injury disease targets were predicted from the OMIM and Genecards databases. Combining the above results, we integrated the metabolite targets and predicted targets using a Venn diagram, and a total of 31 important potential targets related to Hel extract intervention in CP-induced AKI were obtained, including NT5E, PNP, PLA2G6, PLD1, PLD2, PLA2G4A, PLA2G2A, NAMPT, PARP1, PLA2G1B, CS, ADA, PLA2G5, ACLY, QPRT, BST1, TYMP, HPRT1, GOT2, PLA2G4C, NADSYN1, ITPA, CD38, PARP2, SIRT6, NNT, ENPP2, XDH, PARP4, LPL, and GOT1. Among them, 5 important targets including NT5E, PNP, PLA2G6, PLD1, and PLD2 had higher significance, and we verified them using immunoblotting experiments.

[0051] To clarify the biological functions of these targets, we performed KEGG pathway analysis ( Figure 7 D) and GO biological process analysis ( Figure 7 E) on the 31 important targets using the DAVID database. KEGG analysis found that Hel extract intervention in CP-induced AKI was mainly related to lipid metabolism, Ras signaling pathway, and GnRH signaling pathway, and metabolic pathways including nicotinic acid and nicotinamide metabolism, glycerophospholipid metabolism, linoleic acid metabolism, nucleotide metabolism, and purine metabolism. GO analysis found that Hel extract intervention in CP-induced AKI was mainly related to DNA repair, energy metabolism, inflammation, including response to NAD biosynthetic process, AMP catabolic process, and cholate secretion. The metabolite-target-pathway results are shown in Figure 7 Figure F.

[0052] The extract of Acanthacorydalis fruhstorferi prepared in this invention can relieve kidney injury by reducing kidney index, serum creatinine, and urea nitrogen levels, and alleviating kidney pathological tissue damage. It plays a kidney protection role by regulating purine metabolism, pantothenate and CoA biosynthesis, glycerophospholipid metabolism, thiamine metabolism, and nicotinic acid and nicotinamide metabolism. It plays a kidney protection role by regulating NT5E, PNP, PLA2G6, PLD1, and PLD2.

[0053] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing an extract of Acanthida macrodon Hel, characterized in that: The following steps are involved: The Hel was dried and crushed to obtain powder; the Hel powder was soaked and extracted with methanol for 24 hours, and concentrated to obtain a crude methanol extract; the crude Hel methanol extract was extracted with n-butanol, and concentrated to obtain a Hel extract.

2. The method for preparing the Acanthidae macrocystis extract according to claim 1, characterized in that: The concentration device in the step adopts a rotary evaporator.

3. Use of the Acarina macrophylla extract according to claim 1 in the preparation of a drug for preventing and / or treating kidney damage.

4. The use of an Acarina macrophylla extract according to claim 3, characterized in that: The renal injury is acute renal injury caused by cisplatin.

5. The method for analyzing the mechanism of the Acarina macrophylla extract for treating kidney injury according to claim 1, characterized in that the steps as follows: 1) Select three groups of kidney samples: blank control group, cisplatin model group, and Acanthus macrodon extract administration group; add methanol, vortex and let stand, centrifuge at 4°C for 15 minutes, and take the supernatant for LC-MS detection; 2) Then, by renal metabolomics profile analysis, the metabolites of the blank control group and the cisplatin model group were compared to screen out the differential metabolites of the cisplatin acute kidney injury model, and the differential metabolites with the mechanism of improving acute kidney injury were screened out by comparing with the drugs given to the Acanthida model; 3) Perform pathway analysis on the specific differential metabolites obtained in the above steps using MetaboAnalyst online analysis software; 4) Obtain the relevant targets of differential metabolites of Acanthus macrodon extract intervention through the MetScape plug-in in Cytoscape; 5) Search for target genes associated with cisplatin-induced renal injury in the Genecards and OMIM databases; 6) Match the related targets of the differential metabolites in step 4) with the targets in step 5), confirm that the selected duplicate targets are effective targets for the Acanthidae extract to improve cisplatin-induced renal injury, and screen out the changes in related pathways through KEGG and GO enrichment analysis; 7) Verify the top 5 key target groups screened in step 6) for the protective effect of Acarina macrodon extract on cisplatin-induced acute kidney injury.

Images