Pueraria flower-derived extracellular vesicle-like particles as well as preparation method and application thereof

Abstract

The invention provides pueraria flower-derived extracellular vesicle-like particles as well as a preparation method and application thereof, and particularly belongs to the technical field of biological medicines. The preparation method comprises the following steps: mixing flos puerariae with a PBS (Phosphate Buffer Solution), juicing and filtering to obtain a first filtrate; standing the first filtrate to obtain a first supernatant; performing first centrifugation on the first supernatant to obtain a second supernatant; carrying out tangential flow filtration and concentration on the second supernatant to obtain concentrated liquid; carrying out second centrifugation on the concentrated liquid to obtain a third supernatant; filtering and sterilizing the third supernatant to obtain a second filtrate; carrying out ultra-high-speed centrifugation on the second filtrate to obtain a precipitate; and mixing the precipitate with a PBS buffer solution, resuspending, filtering and sterilizing to obtain the pueraria flower-derived extracellular vesicle-like particles. The pueraria flower-derived extracellular vesicle-like particles obtained by the preparation method can protect the liver from alcohol injury, and have the potential of becoming an alcoholism antidote.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to Flos Pueraria-derived EV like particles (FP-EVLP) and a preparation method and application thereof. Background Art

[0002] Alcohol consumption is the most important risk factor for poor health among working-age populations worldwide [1] Excessive drinking can cause damage to multiple organs and tissues, mainly causing cerebral hemorrhage [2] , neuronal loss [3] , myocardial ischemia [4] , alcoholic liver disease [5] , upper gastrointestinal bleeding and enteritis [6] Etc., and even sudden death, which poses a great threat to human health. Therefore, it is necessary to take drugs that promote alcohol metabolism to prevent alcohol poisoning or accelerate the metabolism of ethanol in the early stage of alcohol poisoning to reduce the damage of ethanol to tissues and organs.

[0003] At present, the international research on alcohol detoxification drugs mainly starts from the following aspects: enhancing the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase (ALDH) [7] , inhibit hepatocyte apoptosis and necrosis [8] , Antioxidant [5] , and maintain the balance of intestinal flora [9] In the field of traditional Chinese medicine, Huangdi Neijing first proposed the name of "wine paradox", believing that the pathogenesis lies in the invasion of alcohol poison, phlegm covering the clear orifices, and dampness generating phlegm, etc. The treatment should focus on sobering up the spleen, clearing away heat and drying dampness, etc.

[0004] Kudzu flower is a traditional Chinese medicine for alcohol detoxification. The Southern Yunnan Materia Medica records that kudzu flower can "detoxify the spleen and stomach, and can cause alcohol poisoning, vomiting blood, and relieve heat and detoxify alcohol." Clinically, the use of kudzu flower decoction for enema treatment of severe acute alcohol poisoning can effectively shorten the sobering time and promote the recovery of patients' body temperature, blood pressure and breathing.

[10] After being treated with kudzu flower or its extract as the basic medicine, the sobering time can be significantly shortened, and it also has a certain therapeutic effect on alcoholic liver disease. [11-13] Current research has confirmed that the main active ingredient of kudzu flower is isoflavones, which are metabolized into irisin in the body, which can significantly improve mitochondrial function, inhibit oxidative stress, and shorten the half-life of ethanol.

[14] However, the high cost of extracting kudzu flower isoflavones, low biocompatibility, and poor bioavailability in vivo have restricted further research and application of kudzu flower.

[15] Therefore, it is necessary to explore the new material basis for the effect of kudzu flower in sobering up and protecting the liver, so as to provide a reliable basis for the research on the pharmacological effects and clinical application of kudzu flower.

[0005] References

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[0007] [2]Daviet R,G Aydogan,K Jagannathan,N Spilka,PD Koellinger,H RKranzler,G Nave,RR Wetherill.Associations between alcohol consumption and gray and white matter volumes in the UK Biobank[J].NATURE COMMUNICATIONS,2022,13(1):1175.

[0008] [3]Choi M,M Schneeberger,W Fan,A Bugde,L Gautron,K Vale,RE Hammer,YZhang,JM Friedman,DJ Mangelsdorf,S AKliewer.FGF21 counteracts alcoholintoxication by activating the noradrenergic nervous system[J].CELLMETABOLISM,2023,35(3):429-437.

[0009] [4]LiangY,X WXu,Q Li,Y Deng,M D Xie,YYZheng,W Ou,Q Q He,X Y Xu,W Wu,TLi.Chronic Alcohol Intake Exacerbates Cardiac Dysfunction After MyocardialInfarction[J].ALCOHOLANDALCOHOLISM,2020,55(5):524-530.

[0010] [5]Tsermpini E E,AP Iljes,V Dolzan.Alcohol-Induced Oxidative Stressand the Role of Antioxidants in Alcohol Use Disorder:A Systematic Review[J].ANTIOXIDANTS,2022,11(7):1374.

[0011] [6]Kim D H,Y Sim,J H Hwang,I S Kwun,J H Lim,J Kim,J I Kim,M C Baek,MAkbar,W Seo,D Kim,B J Song,Y E Cho.Ellagic Acid Prevents Binge Alcohol-Induced Leaky Gut and Liver Injury through Inhibiting Gut Dysbiosis andOxidative Stress[J].ANTIOXIDANTS,2021,10(9):1386.

[0012] [7]Srinivasan S,K K Dubey,R S Singhal.Influence offood commodities onhangoverbased on alcohol dehydrogenase and aldehyde dehydrogenase activities[J].CURRENT RESEARCH IN FOOD SCIENCE,2019,1:8-16.

[0013] [8]LouvetA,P Mathurin.Alcoholic liver disease:mechanisms of injury and targeted treatment[J].NATURE REVIEWS GASTROENTEROLOGY&HEPATOLOGY,2015,12(4):231-242.

[0014] [9] Song gutmicrobiota[J].FOOD&FUNCTION,2022,13(5):2441-2455.

[0015]

[10] Wang Ke. Clinical observation on the treatment of severe acute alcohol poisoning with Gehua Jiedun decoction enema[J]. Modern Distance Education of Traditional Chinese Medicine, 2023, 21(08): 106-108.

[0016]

[11] Li Xiaoling, Wu Yuhong, Yin Yinxia. Three cases of Professor Wu Yuhong using Gehua Jiedun Decoction to treat alcohol-related diseases[J]. Journal of Clinical Research in Traditional Chinese Medicine, 2022, 14(25): 123-125.

[0017]

[12] Zhang Jianfu. Study on the effect of kudzu root and kudzu flower on hangover[J]. Journal of Zhoukou Normal University, 2022, 39(05): 57-59.

[0018]

[13] Li Dongmei, Yang Jie, Li Baoyu, Liu Xuguang, Hao Zhiming, Lin Chunhua. Preparation and in vitro alcohol detoxification effect of Pueraria lobata compound alcohol detoxification solid beverage[J]. Food Industry, 2022, 43(04): 47-53.

[0019]

[14] Tan Zhenyuan, Liang Qiuyun, Huang Xingzhen. Research progress on the chemical composition of Pueraria lobata flower and its hangover-relief function[J]. Journal of Guangxi University of Traditional Chinese Medicine, 2017, 20(01): 72-75.

[0020]

[15] Wu Tong, Huang Yu, Hou Wanchao, Xia Jianli, Liu Chunming, Li Sainan. Isolation and identification of isoflavones from Pueraria lobata flowers[J]. Journal of Analytical Science, 2020, 36(03): 353-358. Summary of the invention

[0021] The purpose of the present invention is to provide a kudzu flower-derived extracellular vesicle-like particle and a preparation method and application thereof. The method of the present invention extracts kudzu flower-derived extracellular vesicle-like particles from kudzu flower dry products, studies the alcohol-detoxifying and liver-protecting effects of the particles, and explores the alcohol-detoxifying and liver-protecting potential of kudzu flower.

[0022] The present invention provides a method for preparing kudzu flower-derived extracellular vesicle-like particles, comprising the following steps:

[0023] Mixing kudzu flower with PBS buffer, squeezing juice, filtering, and obtaining a first filtrate;

[0024] After the first filtrate is allowed to stand, a first supernatant is obtained;

[0025] Performing a first centrifugation on the first supernatant to obtain a second supernatant;

[0026] Concentrating the second supernatant by tangential flow filtration to obtain a concentrated liquid;

[0027] The concentrated liquid is subjected to a second centrifugation to obtain a third supernatant;

[0028] The third supernatant is filtered and sterilized to obtain a second filtrate;

[0029] The second filtrate is subjected to ultra-high speed centrifugation to obtain a precipitate;

[0030] The precipitate is mixed with PBS buffer and resuspended, and then filtered for sterilization to obtain kudzu flower-derived extracellular vesicle-like particles.

[0031] Preferably, the kudzu flower is a dried kudzu flower, and the juicing is allowed to stand for 1 to 4 hours.

[0032] Preferably, the first centrifugation condition is 3000-4000g centrifugation for 30 min; the second centrifugation condition is 10000g centrifugation for 30 min; and the ultra-high-speed centrifugation condition is 100000-135000g centrifugation for 60-90 min.

[0033] Preferably, the filtration concentration is performed at a multiple of 20 times.

[0034] The present invention also provides kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme.

[0035] Preferably, it mainly includes lipids and lipid molecules, phenylpropanoids and polyketides, benzoic acids, alkaloids and their derivatives, organic oxygen compounds, organic heterocyclic compounds, fatty acyl groups, flavonoids and isoflavonoids, and steroids and steroid derivatives;

[0036] The alkaloids include berberine; the flavonoids include naringenin, kaempferol and luteolin; and the isoflavones include tectorius.

[0037] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of alcohol sobering products.

[0038] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of liver protection products.

[0039] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of a drug for treating acute alcoholic liver injury.

[0040] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of any one or more drugs for regulating inflammation, cell apoptosis, lipid metabolism and immune response.

[0041] The present invention provides a kudzu flower-derived extracellular vesicle-like particle and a preparation method and application thereof. The present invention creatively extracts extracellular vesicle-like particles FP-EVLP from the dried product of kudzu flower, a traditional Chinese medicine, and detects its characterization, targeting and biological activity. It is found that FP-EVLP has morphological characteristics similar to those of animal-derived extracellular vesicles, can stably exist in gastrointestinal fluid and alcohol, and can reduce the acetaldehyde concentration in the blood of model mice by increasing ALDH activity, prolong the drunkenness time, and shorten the sobering time, proving that it has the potential to sober up, and can be used to prepare sober up drugs. The experimental results show that the present invention, based on the study of the mouse model of acute alcoholic liver injury, found that FP-EVLP can reduce inflammatory infiltration in liver tissue, significantly reduce the levels of ALT and AST in mouse serum, and protect the liver mitochondrial structure, and inhibit cell apoptosis by reducing liver inflammatory response through multiple pathways. FP-EVLP can play a liver-protective role by regulating the overall gene expression level of the body, including the expression of genes related to inflammation, oxidative stress, immune regulation genes, and lipid metabolism, involving multiple signaling pathways such as cytokine-cytokine receptor interactions, NF-κB, TNF, PPAR, FoXO, PI3K / AKT, and MAPK. Therefore, the present invention discovered for the first time that FP-EVLP can protect the liver from alcohol damage from multiple pathways and angles. FP-EVLP has the potential to become an antidote for alcohol poisoning, and can be used in the treatment of chemical liver damage, acute hepatitis, and other fields. It can also be used as a drug carrier or vaccine adjuvant for anti-liver cancer and anti-viral hepatitis, and can be used to prepare alcohol-detoxifying and liver-protecting foods, health products, or medicines, and can be used in the production of traditional Chinese medicine alcohol-detoxifying and liver-protecting products. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0043] Figure 1 The results of extraction, characterization and stability of FP-EVLP provided by the present invention are shown in Figure 1; wherein A is a schematic diagram of FP-EVLP extraction; B is a transmission electron microscopy image of FP-EVLP; C is a graph showing the stability of FP-EVLP in gastrointestinal fluid; and D is a graph showing the stability of FP-EVLP in different concentrations of alcohol.

[0044] Figure 2 The metabolomics detection result diagram of FP-EVLP provided by the present invention; wherein A is the Superclass cluster analysis diagram; B is the Class cluster analysis diagram; C is the relative intensity diagram of the main components;

[0045] Figure 3 It is a result diagram of the alcohol sobering effect of FP-EVLP provided by the present invention; wherein A is an animal treatment mode diagram; B is a drunkenness score diagram; C is a sobering score diagram; D is an ALDH activity result diagram; E is a blood ethanol content result diagram; F is a blood acetaldehyde content result diagram; "**" indicates that the difference is extremely significant compared with the blank group, p<0.01; "***" indicates that the difference is extremely significant compared with the blank group, p<0.001; "****" indicates that the difference is extremely significant compared with the blank group, p<0.0001; "###" indicates that the difference is extremely significant compared with the model group, p<0.001; "####" indicates that the difference is extremely significant compared with the model group, p<0.0001;

[0046] Figure 4It is a result diagram of the liver protection effect of FP-EVLP provided by the present invention; wherein A is a result diagram of ALT content in serum; B is a result diagram of AST content in serum; C is a result diagram of CAT activity in liver tissue; D is a result diagram of MDA content in liver tissue; E is a HE staining image of liver tissue, with a scale of 200 μm; F is a transmission electron microscope image of liver mitochondria, with a scale of 1 μm; "**" indicates an extremely significant difference compared with the blank group, p<0.01; "***" indicates an extremely significant difference compared with the blank group, p<0.001; "****" indicates an extremely significant difference compared with the blank group, p<0.0001; "###" indicates an extremely significant difference compared with the model group, p<0.001; "####" indicates an extremely significant difference compared with the model group, p<0.0001; "&" indicates a significant difference compared with the Metadoxine group, p<0.05; "&&" indicates an extremely significant difference compared with the Metadoxine group, p<0.01, the same below;

[0047] Figure 5 The result graph of transcriptome sequencing data provided by the present invention; wherein A is a GO function enrichment analysis graph of differentially expressed genes between the FP-EVLP group and the MOD group; B is a KEGG pathway enrichment analysis graph of differentially expressed genes between the FP-EVLP group and the MOD group; C is a heat map of differentially expressed genes between the groups; D is a volcano map of differentially expressed genes between the FP-EVLP group and the MOD group; E is an immune infiltration analysis graph of differentially expressed genes between the FP-EVLP group and the MOD group;

[0048] Figure 6 The present invention provides a GSEA analysis and RT-qPCR verification of transcriptome sequencing data result graph; wherein A is a GSEA-KEGG analysis result graph of differentially expressed genes in the cytokine-cytokine receptor interaction pathway; B is a GSEA-KEGG analysis result graph of differentially expressed genes in the TNF signaling pathway; C is a GSEA-KEGG analysis result graph of differentially expressed genes in the NF-κB signaling pathway; D is a GSEA-KEGG analysis result graph of differentially expressed genes in the MAPK signaling pathway; E is a result graph of RNA-seq data verified by RT-qPCR; RT-qPCR data are expressed as relative gene expression levels. DETAILED DESCRIPTION

[0049] The present invention provides a method for preparing kudzu flower-derived extracellular vesicle-like particles, comprising the following steps:

[0050] Mixing kudzu flower with PBS buffer, squeezing juice, filtering, and obtaining a first filtrate;

[0051] After the first filtrate is allowed to stand, a first supernatant is obtained;

[0052] Performing a first centrifugation on the first supernatant to obtain a second supernatant;

[0053] Concentrating the second supernatant by tangential flow filtration to obtain a concentrated liquid;

[0054] The concentrated liquid is subjected to a second centrifugation to obtain a third supernatant;

[0055] The third supernatant is filtered and sterilized to obtain a second filtrate;

[0056] The second filtrate is subjected to ultra-high speed centrifugation to obtain a precipitate;

[0057] The precipitate is mixed with PBS buffer and resuspended, and then filtered for sterilization to obtain kudzu flower-derived extracellular vesicle-like particles.

[0058] The kudzu flower is mixed with PBS buffer, juiced, and filtered to obtain a first filtrate. The kudzu flower of the present invention is a dry kudzu flower. In a specific embodiment, before the mixing, the kudzu flower may be washed. Sterile water may be used for washing. The filtration of the present invention may be sterile gauze extrusion filtration.

[0059] After obtaining the first filtrate, the present invention allows the first filtrate to stand to obtain a first supernatant. In the present invention, the time for standing after the juice is squeezed and filtered is 1 to 4 hours, specifically 1 hour.

[0060] After obtaining the first supernatant, the present invention performs a first centrifugation on the first supernatant to obtain a second supernatant. In a specific embodiment, the first centrifugation is performed at 3000-4000 g for 30 min, specifically at 3000 g for 30 min or at 4000 g for 30 min.

[0061] After obtaining the second supernatant, the present invention performs tangential flow filtration and concentration on the second supernatant to obtain a concentrated liquid. In a specific embodiment, the filtration and concentration multiple is 20 times.

[0062] After obtaining the concentrated liquid, the present invention performs a second centrifugation on the concentrated liquid to obtain a third supernatant. In a specific embodiment, the condition of the second centrifugation is 10000g centrifugation for 30 minutes. In a specific embodiment, the temperature of the centrifugation is 4°C. The second centrifugation of the present invention can remove dead cells, shedding vesicles and apoptotic bodies.

[0063] After obtaining the third supernatant, the present invention performs filtration sterilization on the third supernatant to obtain a second filtrate. In a specific embodiment, a 0.22 μm filter membrane can be used for vacuum filtration sterilization.

[0064] After obtaining the second filtrate, the present invention performs ultra-high speed centrifugation on the second filtrate to obtain a precipitate. In a specific embodiment, the ultra-high speed centrifugation condition is 100000-135000g centrifugation for 60-90min, specifically 135000g centrifugation for 70min. In a specific embodiment, the centrifugation temperature is 4°C.

[0065] After obtaining the precipitate, the present invention mixes the precipitate with a PBS buffer solution and resuspends it, and filters and sterilizes it to obtain kudzu flower-derived extracellular vesicle-like particles. In a specific embodiment, the PBS buffer solution is precooled in advance. In a specific embodiment, it can be sterilized by filtering with a 0.22 μm filter membrane.

[0066] The present invention also provides kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme. In a specific embodiment, it mainly includes lipids and lipid molecules, phenylpropanoids and polyketide compounds, benzoic acids, alkaloids and their derivatives, organic oxygen compounds, organic heterocyclic compounds, fatty acyl groups, flavonoids and isoflavonoids, and steroids and steroid derivatives; the alkaloids include berberine; the flavonoids include naringenin, kaempferol and luteolin; the isoflavonoids include irisin. After obtaining kudzu flower-derived extracellular vesicle-like particles using the above extraction method, the present invention detects the microscopic morphology, particle size distribution, particle concentration and composition of the components in the vesicles, which provides a reliable basis for subsequent in-depth research on pharmacological effects.

[0067] The present invention also provides the use of kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical solution in the preparation of alcohol-detoxifying products. The present invention established an acute alcoholic liver injury mouse model by intragastric administration of 6 g / kg alcohol to 8-week-old male C57BL / 6J mice, and investigated the alcohol-detoxifying effect of FP-EVLP by evaluating the effects of FP-EVLP on the drunkenness time and sober-up time, serum ethanol and acetaldehyde concentrations, and acetaldehyde dehydrogenase activity.

[0068] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of liver protection products.

[0069] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of a drug for treating acute alcoholic liver injury.

[0070] The present invention also provides the use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method described in the above technical scheme in the preparation of any one or more drugs for regulating inflammation, cell apoptosis, lipid metabolism and immune response.

[0071] The present invention is based on an acute alcoholic liver injury mouse model. The serum biochemical analyzer is used to detect liver function-related indicators such as ALT, AST and ALP in mouse serum. The MDA, SOD and CAT detection kits are used to detect the liver oxidative stress level and HE staining is used to observe the liver pathological damage, and the protective effect of FP-EVLP on the liver is investigated. The results show that FP-EVLP can reduce inflammatory infiltration in liver tissue, reduce serum ALT levels, and protect liver mitochondrial structure. The present invention detects the overall transcription level of genes in the liver by liver transcriptomics, and then verifies the differential genes by RT-qPCR technology. The results show that FP-EVLP can play a liver-protecting role by regulating the overall gene expression level of the body, including the expression of genes related to inflammation, oxidative stress, immune regulation genes and lipid metabolism, involving multiple signaling pathways such as cytokine cytokine receptor interaction, NF-κB, TNF, PPAR, FoXO, PI3K / AKT and MAPK.

[0072] To further illustrate the present invention, the kudzu flower-derived extracellular vesicle-like particles provided by the present invention, as well as their preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] Extraction, characterization and stability of FP-EVLP

[0075] 1. The extraction method of FP-EVLP is as follows ( Figure 1 A) in:

[0076] 1) Sample pretreatment: Take a certain amount of dried kudzu flower, wash it with sterile water, and then put it in precooled PBS to extract the juice. After squeezing and filtering with sterile gauze, collect the juice in a sterile beaker, let it stand for 1 hour, collect the supernatant, centrifuge at 4000g for 30 minutes, discard the precipitate, and filter the collected supernatant by tangential flow filtration system to concentrate it 20 times.

[0077] 2) Removal of dead cells, shed vesicles and apoptotic bodies: The concentrated liquid was collected in a sterile 50 mL centrifuge tube, centrifuged at 10,000 g for 30 min at 4°C, and the supernatant was collected.

[0078] 3) Sterilization: Sterilize the supernatant by vacuum filtration using a 0.22 μm filter membrane.

[0079] 4) Collect vesicles: The filtered liquid was collected in a special centrifuge tube of an ultra-high speed centrifuge, centrifuged at 100,000 g for 60 min at 4°C, the supernatant was discarded, the precipitate was resuspended with pre-cooled 1× PBS buffer, and then filtered and sterilized again using a 0.22 μm filter membrane to obtain the FP-EVLP solution, which was stored in a -80°C refrigerator for later use.

[0080] 2. Characterization

[0081] FP-EVLP solution (1*10 11 The mixture was dropped onto a copper grid and then dried at room temperature. The morphology of the sample was monitored by transmission electron microscopy at 110 KV. The observation results showed that the FP-EVLP particles had a clear membrane structure and exhibited a typical cup-shaped and disc-shaped vesicle structure ( Figure 1 B).

[0082] 3. Stability

[0083] The particle number and particle size distribution of FP-EVLP in gastrointestinal fluid and alcohol solution were analyzed by nanoflow cytometry: the initial average particle size of FP-EVLP was 87nm, which is consistent with the extracellular vesicle particle size range reported in the literature. There was no significant change in the particle number and particle size distribution of FP-EVLP after incubation in artificial gastric fluid (SIF), artificial small intestinal fluid (SGF) and SIF+SGF for 30 minutes ( Figure 1 C); the number of FP-EVLP particles in the alcohol solution decreased, but the particle size distribution remained unchanged ( Figure 1 D), indicating that FP-EVLP has good stability.

[0084] Example 2

[0085] FP-EVLP contains characteristic ingredients of Pueraria lobata

[0086] The metabolites contained in FP-EVLP were qualitatively analyzed by non-targeted metabolomics. First, the metabolites were extracted by organic reagent precipitation metabolite method, and the extracted samples were randomly sorted and tested. QC samples were inserted before, during and after the samples as repeated evaluations of experimental techniques. The samples were scanned by mass spectrometry in positive and negative ion modes, respectively. Metabolite identification was performed using XCMS and metaX software. The identification results showed that the metabolites in FP-EVLP were mainly lipids and lipid molecules (40.42%), and glycerophospholipids (5.49%) were the main categories of lipids and lipid molecules. This component may be related to the phospholipid structure of the vesicles ( Figure 2 A in ). Followed by phenylpropanoids and polyketides, benzoic acids, alkaloids and derivatives, organic oxygen compounds, organic heterocyclic compounds, etc. ( Figure 2In addition, FP-EVLP contains a large number of fatty acyl groups, flavonoids and isoflavones, steroids and steroid derivatives. Among them, irisin, the hallmark ingredient of kudzu flower, accounts for the highest proportion, and the strength of traditional Chinese medicine ingredients such as naringenin, berberine, kaempferol, and luteolin in FP-EVLP is also high. These ingredients have been proven to have pharmacological activity, indicating that FP-EVLP has great pharmacological potential ( Figure 2 C).

[0087] Example 3

[0088] Evaluation of FP-EVLP alcohol detoxification function

[0089] The experimental animals were 8-week-old male C57BL / 6J mice. The model was established by intragastric administration of 53% alcohol solution, so that the alcohol dosage for each mouse reached 6 g / kg (w / w). 30 minutes before and 30 minutes after intragastric administration of alcohol, the FP-EVLP high-dose group (FP-EVLPH, 1×10 9 particles / piece) and FP-EVLP low-dose group (FP-EVLPL, 1×10 8 The mice in the model group were gavaged with the corresponding dose of FP-EVLP solution, and the mice in the model group were gavaged with the same amount of normal saline. Metadoxine was used as a positive drug control, with 24 replicates in each group. Six mice were randomly selected from each group at 1h, 4h, 8h and 24h after modeling, and their eyeballs were removed for blood collection after anesthesia. After separating the serum, the mice were killed by cervical dislocation, and the livers were removed ( Figure 3 A).

[0090] 1. Drunk and sober scores

[0091] The drunkenness time and soberness time of each group of mice were recorded by observing the righting reflex of mice. The time from the start of intragastric alcohol administration to the disappearance of the righting reflex of mice was the drunkenness time; the time from the disappearance of the righting reflex to the first appearance of the righting reflex was the soberness time. The drunkenness time and soberness time of each group of mice were scored according to the scoring criteria in Table 1, and the Mann-Whitney test was used for difference analysis. The results showed that compared with the model group, both FP-EVLPH and FP-EVLPL significantly prolonged the drunkenness time of mice and significantly shortened the soberness time of mice (p<0.05), indicating that FP-EVLP has a significant sobering effect, and its sobering effect is better than that of the positive control drug ( Figure 3 B and Figure 3 C).

[0092] Table 1 Rating criteria for drunkenness time and soberness time

[0093] Fraction Intoxication time scoring criteria Sobering time scoring criteria 0 0 min < Drunkenness time ≤ 5 min die 1 5 min < Drunkenness time ≤ 10 min 8h≤sobering time 2 10 min < Drunkenness time ≤ 30 min 3h≤Decline time<8h 3 30 min < Drunkenness time ≤ 60 min 1h≤Decline time<3h 4 60min < Drunkenness time 0h≤Decline time<1h 5 Not drunk Not drunk

[0094] 2. Detection of blood ethanol and blood acetaldehyde concentrations

[0095] First, the aldehyde dehydrogenase (ALDH) activity detection kit was used to detect the ALDH activity in liver tissue. Then, according to the operating instructions of the blood ethanol concentration detection kit and the blood acetaldehyde concentration detection kit, the ethanol and acetaldehyde concentrations in the serum were detected using a fully automatic serum biochemical analyzer. The test results showed that FP-EVLP can enhance ALDH activity in the early stage of drinking, thereby promoting the conversion of acetaldehyde into acetic acid ( Figure 3 In addition, compared with the model group, both high and low concentrations of FP-EVLP could significantly reduce the concentrations of ethanol and acetaldehyde in the blood of mice (p<0.05), and the effect was better than that of the positive control drug ( Figure 3 E and Figure 3 F).

[0096] Example 4

[0097] FP-EVLP protects the liver from alcohol damage

[0098] The animals were treated according to the method described in Example 3, and the results of the investigation are as follows:

[0099] 1. Detection of biochemical indicators related to liver damage

[0100] The liver damage was reflected by measuring the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum using a fully automatic serum biochemical analyzer. Figure 4 A and Figure 4 As shown in B, at 4h, 8h and 24h after alcohol modeling, FP-EVLP treatment can significantly reduce the levels of ALT and AST in the serum of drinking mice (p<0.01), and the effect is significantly better than Metadoxine (p<0.05).

[0101] At the same time, the present invention also used catalase (CAT) activity detection kit and malondialdehyde (MDA) detection kit to detect the oxidative stress level in the liver at 1h, 4h and 24h after alcohol modeling. The results showed that at 1h and 4h, FP-EVLP treatment significantly increased the CAT activity of the mouse liver compared with the MOD group, resulting in a significant decrease in the MDA level at 4h and 24h ( Figure 4 C and Figure 4 D in (p<0.05). This preliminarily indicates that FP-EVLP alleviates the oxidative stress caused by alcohol.

[0102] 2. Detection of liver pathology and mitochondrial ultrastructural changes

[0103] The liver pathological structure was observed by hematoxylin & eosin staining. Figure 4 As shown in Figure E, 24 hours after intragastric administration of alcohol, obvious inflammatory infiltration was observed in the liver of mice in the model group, while no obvious pathological changes were observed in the liver of mice in the FP-EVLP group. This result further demonstrates that FP-EVLP can protect the liver from alcohol damage.

[0104] Fresh liver tissue with a thickness of 1 mm was cut and fixed in 0.25% glutaraldehyde electron microscope fixative overnight, and then fixed with 1% osmium acid for 2 hours, dehydrated with gradient ethanol and acetone, resin impregnated, embedded, and polymerized (37°C oven for 12 hours, 60°C oven for 48 hours), and ultrathin sections were made, double-stained with uranyl acetate and lead citrate, and photographed with a transmission electron microscope. The results showed that the mitochondrial cristae in the model group were disordered, the mitochondrial inner membrane was dissolved, the mitochondria were swollen, and vacuolation occurred; the mitochondrial structure in the FP-EVLP group was relatively complete, and no obvious swelling and vacuolation were observed ( Figure 4 F). This indicates that FP-EVLP can maintain the integrity of mitochondrial structure in acute alcoholic liver injury.

[0105] Example 5

[0106] Transcriptome analysis of key genes in the hepatoprotective effect of FP-EVLP

[0107] Transcriptome sequencing analysis was performed on mice 24 hours after acute alcohol gavage. The Illumin NovaSeq6000 sequencing platform was used to detect gene expression levels in liver tissues. After conversion to sequence data (reads) by CASAVA base recognition, HISAT2v2.0.5 was used to build the index of the reference genome and the paired-end cleanreads were aligned with the reference genome. Feature Counts (1.5.0-p3) was used to calculate the gene expression of the samples, and DESeq2 (1.20.0) was used to perform differential expression between two comparison combinations (screening conditions P < 0.05, |log2FC| ≥ 1). The differentially co-expressed genes were aligned to the Gene Ontology (GO) and Kyoto Gene and Genome (KEGG) databases for annotation analysis and pathway enrichment analysis using clusterProfiler (3.8.1).

[0108] The results of differential gene statistics and cluster analysis showed that there were certain differences in gene expression between the model group and the blank group, and between the FP-EVLP group and the model group, and the cluster analysis showed significant differences in genes. Figure 5 C and Figure 5As shown in D, compared with the model group, the number of differentially expressed genes in the FP-EVLP group was 2894, of which 1970 were upregulated and 924 were downregulated. It is worth noting that compared with the model group, genes such as Bcl3, Nfkb2, Nfkbia, Il1b, Il6, Tnfrsf1b, Tnfaip3, Cxcl10, Map3k8 and Fos were significantly downregulated, and Ppara gene was significantly upregulated in the FP-EVLP group. This indicates that FP-EVLP can regulate inflammation and apoptosis and promote lipid metabolism.

[0109] GO functional enrichment and KEGG pathway enrichment were used to analyze the differentially expressed genes. The GO enrichment results showed that ( Figure 5 A in the figure, differentially expressed genes were mainly enriched in biological functions such as transmembrane transport, biological processes, inflammatory response, peroxisomes, metabolic processes, fatty acid synthesis and metabolism, mitochondria, mitochondrial depolarization, and mitochondrial DNA replication. KEGG( Figure 5 The B) pathways are mainly enriched in MAPK, TNF, PI3K-AKT, JAK-STAT, FoxO, PPAR, NF-κB, NOD-like receptor, AMPK and Toll-like receptor signaling pathways.

[0110] The immune infiltration results showed ( Figure 5 E), after FP-EVLP treatment, the number of initial B cells, plasma cells, CD8+T cells, M2 macrophages, Th17 cells and T cells increased significantly, while CD8+T cells, CD4 memory T cells and Th2 cells decreased significantly. The above results indicate that FP-EVLP may protect the liver by regulating immune response, reducing liver inflammatory damage and inhibiting cell apoptosis.

[0111] Example 6

[0112] GSEA analysis and RT-qPCR validation of transcriptome sequencing data

[0113] The results of GSEA-KEGG enrichment analysis showed that the differential genes between the FP-EVLP group and the model group were significantly enriched in the cytokine-cytokine receptor interaction pathway ( Figure 6 A in), TNF( Figure 6 B), NF-κB ( Figure 6 C) and MAPK ( Figure 6 D) and other signaling pathways. Figure 6As shown in Figure E, the expression of genes related to inflammation, apoptosis, lipid metabolism and immune response was verified by RT-qPCR, and it was found that FP-EVLP could significantly inhibit the expression levels of Rasd1, Nfkbia, Map3k8, Jun, Il1b, Tnfaip3, Tnfrsf1b, Fos, Nfkb2, Cd14, Fasn, Tgfbr2, Bcl3, Il1a, Il1r1 and Acod1 genes, and significantly up-regulate the expression levels of Ppara, Cxcl12, Ripk4 and Acp2 genes, further proving that FP-EVLP can reduce alcohol damage to the liver by regulating inflammation, apoptosis, lipid metabolism and immune response.

[0114] Example 7

[0115] The alcoholic fatty liver (ASH) model was established in male 8-week-old C57BL / 6J mice by chronic alcohol feeding, and the biological activity mechanism of FP-EVLP and the efficacy of treating alcoholic fatty liver were deeply explored from multiple angles. The ALT, TG and TC levels in the serum of mice were detected by serum biochemical analyzer, the pathological changes of liver tissue and intestinal tissue were detected by HE staining, and the changes in fat content in the liver were detected by Oil Red O staining. The evaluation results showed that after FP-EVLP treatment, the fat content in the liver of mice with alcoholic fatty liver decreased significantly, the inflammatory infiltration in the liver decreased, the vacuolation area of ​​hepatocytes decreased, the hepatic cords were neatly arranged and clear, and the ALT, TG and TC levels in the serum returned to normal levels. In addition, it was found that FP-EVLP can protect the integrity of the intestinal structure, reduce the damage of alcohol to the intestinal barrier, and further prevent harmful substances such as bacteria or endotoxins in the intestine from damaging the liver through the portal vein.

[0116] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing kudzu flower-derived extracellular vesicle-like particles, characterized in that: The following steps are involved: Mixing kudzu flower with PBS buffer, squeezing juice, filtering, and obtaining a first filtrate; After the first filtrate is allowed to stand, a first supernatant is obtained; Performing a first centrifugation on the first supernatant to obtain a second supernatant; Concentrating the second supernatant by tangential flow filtration to obtain a concentrated liquid; The concentrated liquid is subjected to a second centrifugation to obtain a third supernatant; The third supernatant is filtered and sterilized to obtain a second filtrate; The second filtrate is subjected to ultra-high speed centrifugation to obtain a precipitate; The precipitate is mixed with PBS buffer and resuspended, and then filtered and sterilized to obtain kudzu flower-derived extracellular vesicle-like particles.

2. The preparation method according to claim 1, characterized in that: The kudzu flower is a dried product of kudzu flower, and the juice is squeezed; the juicing is performed and the standing time after filtering is 1 to 4 hours.

3. The preparation method according to claim 1, characterized in that: The conditions for the first centrifugation are 3000-4000g centrifugation for 30 min; the conditions for the second centrifugation are 10000g centrifugation for 30 min; and the conditions for the ultra-high-speed centrifugation are 100000-135000g centrifugation for 60-90 min.

4. The preparation method according to claim 1, characterized in that: The filtration concentration was performed 20 times.

5. The kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method according to any one of claims 1 to 4.

6. The kudzu flower-derived extracellular vesicle-like particles according to claim 5, characterized in that They mainly include lipids and lipid molecules, phenylpropanoids and polyketides, benzoic acids, alkaloids and their derivatives, organic oxygen compounds, organic heterocyclic compounds, fatty acyl groups, flavonoids and isoflavonoids, and steroids and steroid derivatives; The alkaloids include berberine; the flavonoids include naringenin, kaempferol and luteolin; and the isoflavones include tectorius.

7. Use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of alcohol sobering products.

8. Use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of liver protection products.

9. Use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of a drug for treating acute alcoholic liver injury.

10. Use of the kudzu flower-derived extracellular vesicle-like particles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of any one or more drugs for regulating inflammation, apoptosis, lipid metabolism and immune response.

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