Rose general flavone as well as preparation method and application thereof

Abstract

The invention relates to the technical field of natural product extraction, in particular to rose general flavone as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting roses by using C1-C3 alcohol solvents to form a crude extract; mixing the crude extract with water, adjusting the pH value to 9-10, and filtering to form a first supernatant; and adjusting the pH value of the first supernatant to 4-5, filtering to form a second supernatant, and carrying out chromatography separation on the second supernatant by using macroporous adsorption resin. The rose general flavone provided by the embodiment of the invention has a good treatment effect on hypercholesterolemia, improves abnormal blood lipid level and visceral organ lesion caused by high-fat and high-cholesterol feed, relieves liver injury and lipid deposition, and also can regulate intestinal flora imbalance.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, in particular to total flavonoids from roses, a preparation method thereof and applications thereof. Background Art

[0002] Hypercholesterolemia is an abnormal lipid metabolism caused by elevated levels of cholesterol (TC) and low-density lipoprotein (LDL-C) and decreased levels of high-density lipoprotein (HDL-C). It is a major risk factor for atherosclerosis, myocardial infarction, coronary heart disease, and other diseases. It also increases the risk of chronic kidney disease and diabetes. Currently, clinical treatments for hypercholesterolemia are primarily divided into pharmacological and non-pharmaceutical approaches, with the latter primarily focusing on diet, lifestyle, and surgical intervention. Drug therapy remains the mainstay of treatment, with commonly used drugs including statins, fibrates, ezetimibe, and probucol. Statins are the most widely used drugs. While they are rapidly effective and well-tolerated, prolonged use can often lead to adverse reactions such as liver damage, type 2 diabetes, diarrhea, and insomnia. Therefore, the search for highly effective and low-toxic plant-based lipid-lowering drugs has become both a hot topic and a challenge. Double-petaled red rose (Rose rugosa cv.plena), a Chinese herbal medicine with both medicinal and edible properties, is rich in volatile oils, flavonoids, fatty acids and other chemical components, which has aroused great interest among researchers. However, there is no research on the effect of total flavonoids from roses on hypercholesterolemia in the existing technology.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The present invention aims to provide rose total flavonoids, a preparation method thereof, and applications thereof. The present invention provides rose total flavonoids that have a good therapeutic effect on hypercholesterolemia, improve abnormal blood lipid levels and organ lesions caused by a high-fat, high-cholesterol diet, alleviate liver damage and lipid deposition, and regulate intestinal flora imbalance.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a method for preparing total flavonoids from roses, comprising: extracting roses using a C1-C3 alcohol solvent to form a crude extract;

[0007] The crude extract is mixed with water, and the pH is adjusted to 9-10, and then filtered to form a first supernatant;

[0008] Adjusting the pH of the first supernatant to 4-5, filtering to form a second supernatant,

[0009] The second supernatant is separated by macroporous adsorption resin chromatography.

[0010] In an optional embodiment, the macroporous adsorption resin chromatography separation step includes: eluting with water and then eluting with 3-5% ethanol and 55-65% ethanol in sequence, and collecting the eluate formed by eluting with 60% ethanol.

[0011] In an optional embodiment, the step of macroporous adsorption resin chromatography separation includes: eluting with 8-12BV volume of water, then eluting with 2-3BV volume of 5% ethanol, and then eluting with 3-5BV volume of 60% ethanol, and collecting the eluate formed by 60% ethanol elution.

[0012] In an optional embodiment, the method comprises: extracting rose flowers by reflux using 8-12 BV of 85-95% ethanol to form a crude extract;

[0013] The crude extract is mixed with water, and the pH is adjusted to 9-10, and then allowed to stand for 10-14 hours and filtered to form a first supernatant;

[0014] The pH of the first supernatant is adjusted to 4-5, and the mixture is allowed to stand for 10-14 hours and then filtered to form a second supernatant;

[0015] The second supernatant was separated by chromatography using D-101 macroporous adsorption resin.

[0016] In a second aspect, the present invention provides a rose total flavonoids, which is prepared by the preparation method of rose total flavonoids according to any one of the aforementioned embodiments.

[0017] In an optional embodiment, the total flavonoids of rose include the following active ingredients: quercetin, isoquercetin, guajava glycoside, astilbin, luteolin, myricetin, apigenin, isorhamnetin, dioscorea alkaloids, eriodictyol, hydroxygenkwain, kaempferol, naringenin, chamaecyparis glycosides, luteolin and hyperoside.

[0018] In a third aspect, the present invention provides a use of the total flavonoids from roses described in the aforementioned embodiment in the preparation of a medicament for treating hypercholesterolemia.

[0019] In a fourth aspect, the present invention provides a use of the total flavonoids from roses described in the aforementioned embodiment in the preparation of a medicament for treating atherosclerosis.

[0020] In a fifth aspect, the present invention provides a use of the total flavonoids from roses described in the aforementioned embodiment in the preparation of a medicament for treating liver damage;

[0021] Preferably, the liver damage includes hepatitis and liver fat accumulation.

[0022] In a sixth aspect, the present invention provides a use of the total flavonoids from roses described in the aforementioned embodiment in the preparation of a medicament for improving the imbalance of intestinal microbiota.

[0023] The present application has the following beneficial effects: the rose total flavonoids provided by the embodiments of the present application contain various active ingredients and can improve hypercholesterolemia. Specifically, the rose total flavonoids can reduce the levels of TC, LDL-C, AST, ALT, AKP in serum and TC in liver, and can also increase the levels of HDL-C in serum and TC in feces, and can relieve liver inflammatory lesions and fat vacuoles. The rose total flavonoids can inhibit the activity of endogenous cholesterol synthesis enzyme HMG-CoAR and promote the activity of cholesterol decomposition enzyme CYP7A1. Meanwhile, the rose total flavonoids can improve the intestinal flora structure disorder, increase the relative abundance of bacteria such as Firmicutes and its subordinate classification Oxyfarcimlales NK4A136, Bacteroidetes and its subordinate classification Muribaculaceae, Rikenellaceae and Prevotellaceae, and reduce the relative abundance of bacteria such as Verrucomicrobia and Proteobacteria. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0025] Figure 1 The total ion chromatogram of rose total flavonoids in Example 1 provided for characterization of the present application, wherein A represents negative ion mode and B represents positive ion mode;

[0026] Figure 2 The traditional Chinese medicine component-common target-pathway network diagram provided for Analysis Example 1 of the present application;

[0027] Figure 3 The result diagram of blood lipid level of mice provided for animal experiments of the present application, wherein A is TC, B is TG; C is LDL-C; and D is HDL-C;

[0028] Figure 4 The influence diagram of atherosclerosis index and blood lipid comprehensive index of mice provided for animal experiments of the present application, wherein A is AI and B is LCI;

[0029] Figure 5 The influence diagram of liver function of mice provided for animal experiments of the present application, wherein A is AST, B is ALT, and C is AKP;

[0030] Figure 6 The influence diagram of liver macro and micro of mice provided for animal experiments of the present application, wherein A is liver macroscopic morphology diagram, B is liver histopathology section diagram, 100x;

[0031] Figure 7 This is an effect diagram of the lipid content in mouse feces provided by the animal experiment of the present invention; wherein A is TC and B is TG;

[0032] Figure 8 This is an effect diagram of the activities of lipid metabolism-related enzymes in mouse liver provided by the animal experiment of the present invention, wherein A is HMG-CoAR activity; B: CYP7A1 activity;

[0033] Figure 9 The effect diagram of the short-chain fatty acid content in the mouse cecal contents provided for the animal experiment of the present invention, wherein A is acetic acid, B is propionic acid, C is isobutyric acid, D is n-butyric acid, E is isovaleric acid, and F is valeric acid;

[0034] Figure 10 This is an influence diagram of the mouse intestinal microbial alpha diversity provided by the animal experiment of the present invention, where A is the Ace index, B is the Chao1 index, C is the observed_features, D is the Shannon index, E is the Simpson index, and F is faith_pd;

[0035] Figure 11 This is the influence diagram of mouse intestinal microbial Beta diversity provided by the animal experiment of the present invention, where A is the PCoA score diagram; B is the NMDS score diagram;

[0036] Figure 12-13 The figure shows the influence diagram of the intestinal microbial community composition at the phylum level of mice, where A is the species composition diagram at the phylum level; B to E are the relative abundances of the bacterial groups with significant differential abundance at the phylum level;

[0037] Figure 14-15 Figure 1 is an influence diagram of the intestinal microbial community composition at the family level in mice, where A is the species composition diagram at the family level; B to D are the relative abundances of the bacterial groups with significantly different abundances at the family level;

[0038] Figure 16-17 The figure shows the influence diagram of the intestinal microbial community composition at the genus level in mice, where A is the species composition diagram at the genus level; B to D are the relative abundances of the bacterial groups with significantly different abundances at the genus level;

[0039] Figure 18-19 The figure shows the species difference analysis between mouse intestinal microbial community groups, where A is the LEfSe evolutionary branch diagram; B is the LDA value distribution bar graph. DETAILED DESCRIPTION

[0040] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0041] In a first aspect, the present invention provides a method for preparing total flavonoids from roses, comprising:

[0042] The roses are pretreated, specifically, the roses are crushed and then sieved.

[0043] After sieving, extraction is performed. Specifically, a C1-C3 alcohol solvent is used to extract to form a crude extract, wherein the C1-C3 alcohol solvent is selected from solvents such as methanol, ethanol, and propanol. For example, the solvent can be 85% ethanol, 90% ethanol, 95% ethanol, or any value between 85-95%. The amount of solvent used is 8-12 BV, for example, 8 BV, 9 BV, 10 BV, 11 BV, 12 BV, or any value between 8-12 BV. The extraction method is reflux extraction. The extraction number can be multiple, and the extracts formed by multiple extractions are combined and concentrated.

[0044] Next, the crude extract is mixed with water, the pH is adjusted to 9-10, and then the mixture is allowed to stand for 10-14 hours before filtering to form a first supernatant; the pH of the first supernatant is then adjusted to 4-5, the mixture is allowed to stand for 10-14 hours, and then filtered to form a second supernatant; then, the second supernatant is separated by macroporous adsorption resin chromatography. Specifically, the second supernatant is separated by chromatography using D-101 macroporous adsorption resin. The macroporous adsorption resin chromatography separation step includes: eluting with 8-12 BV of water, then eluting with 2-3 BV of 5% ethanol, and then eluting with 3-5 BV of 60% ethanol, collecting the eluate formed by eluting with 60% ethanol, and then drying the 60% ethanol eluate, and freeze-drying to obtain total flavonoids from roses.

[0045] It should be noted that the above BV refers to column volume.

[0046] In a second aspect, an embodiment of the present invention provides a total flavonoids from roses, which is prepared by the preparation method of the total flavonoids from roses described in any one of the aforementioned embodiments.

[0047] Specifically, the total flavones of rose flowers include 37 compounds, wherein 36 chemical components and 1 isomer. Specifically, the total flavones of rose flowers include (+)-catechin, astilbin, kaempferol-3,4'-di-O-glucoside, (+)-catechin-7-O-beta-D-xyloside or isomer, hyperoside, quercetin-3-O-glucuronide, rutin, isoquercitrin, quercetin-3-O-xyloside, (+)-catechin-7-O-beta-D-xyloside or isomer, echinatoside, galuteolin, swertiamarin, kaempferol-3-O-rutinoside, quercitrin, zephyranthine, isorhamnetin-3-O-glucoside, 2'-O-galloylquercetin, kaempferol-3-O-arabinoside, afzelin, eriodictyol, 3,4',5,7-tetrahydroxy-8-methoxy-flavone, quercetin, myricetin, luteolin, tiliroside, pinocembrin, pinocembrin-7-O-beta-D-glucoside, naringenin, apigenin, kaempferol, hydroxyaurone, isorhamnetin, cyanidin, dihydroguaiaretic acid and prunin. Further analysis shows that the total flavones of rose flowers include the following active components, which have good therapeutic effect on hypercholesterolemia: quercetin, isoquercitrin, swertiamarin, astilbin, luteolin, myricetin, apigenin, isorhamnetin, dihydroguaiaretic acid, eriodictyol, kaempferol, naringenin, pinocembrin, tiliroside, hyperoside and hyperin.

[0048] The embodiment of the present application also provides the application of the total flavones of rose flowers. Specifically, the total flavones of rose flowers can improve the intestinal flora structure disorder, increase the relative abundance of the bacterial flora of Firmicutes and the subordinate classification of Lachnospiraceae NK4A136, Bacteroidetes and the subordinate classification of Muribaculaceae, Rikenellaceae and Prevotellaceae, and reduce the relative abundance of the bacterial flora of Verrucomicrobia and Proteobacteria. The total flavones of rose flowers can reduce the levels of TC, LDL-C, AST, ALT, AKP in serum and TC in liver, increase the levels of HDL-C in serum and TC in feces, relieve liver inflammation and fat vacuoles, inhibit the activity of endogenous cholesterol synthesis enzyme HMG-CoAR, promote the activity of cholesterol decomposition enzyme CYP7A1, and treat hypercholesterolemia.

[0049] The features and performances of the present application are further described in detail in combination with the following embodiments.

[0050] Embodiment 1

[0051] The embodiment of the present application provides a preparation method of total flavones of rose flowers, which comprises the following steps:

[0052] About 5 kg of dried rose flower buds are crushed in a pulverizer and passed through a 60-mesh sieve. After sieving, 10 BV of 95% ethanol is added, and reflux extraction is performed for 1 h. While hot, filtration is performed, and the extraction is performed for a total of 3 times. The extraction liquid is combined, and concentrated at 50 DEG C under reduced pressure to obtain TFR crude extract.

[0053] Take the crude extract of TFR, dissolve and dilute it with an appropriate amount of distilled water, adjust the pH value to 9.5, let it stand overnight, and filter the next day. Obtain the supernatant by filtration, adjust the pH value to 4.5 with HCl, let it stand overnight, and filter the next day. Pre-treat the D-101 macroporous adsorption resin and wet-pack it into the column. Slowly pour the supernatant into the resin and adsorb the sample. After the sample is loaded, rinse the macroporous resin with 10BV of water, then rinse 2BV with 5% ethanol, and then rinse with 4BV of 60% ethanol. Collect the 60% ethanol segment, recover the solvent under reduced pressure, and freeze-dry to obtain the TFR sample, which is the total flavonoids of rose.

[0054] Characterization

[0055] The TFR sample of Example 1 was detected using UPLC-Q-Exactive Orbitrap MS, where the detection conditions were as follows:

[0056] Chromatographic column: InfinityLab Poroshell 120EC-C18 (2.1×100 mm, 2.7 μm); mobile phase: 0.1% formic acid aqueous solution (A)-acetonitrile solution (B), gradient elution (0-3 min, 5% B; 3-10 min, 5%-9% B; 10-32 min, 9%-21% B; 32-40 min, 21%-30% B; 40-48 min, 30%-80% B; 48-60 min, 80%-100% B); column temperature 30°C, injection volume 3 μL, flow rate 0.3 mL min -1 .

[0057] Electrospray ionization (ESI) source was used for the acquisition in positive and negative ion modes. Orbitrap was used as the primary mass spectrometer with a resolution of 70,000 and a scanning range of m / z 80 to 1500. Ion trap was used as the secondary mass spectrometer with a resolution of 17,500.

[0058] Test results see Figure 1Peak 1 is (+)-catechin, with the main fragments in negative ion mode being 245.082 6, 203.071 7, 151.040 3, 125.024 6, and 109.029 8; and the main fragments in positive ion mode being 245.080 7, 207.0651, 167.033 9, 151.038 9, and 139.038 9. Peak 2 is astilbin, with the main fragments in negative ion mode being 269.0460, 152.012 3, 125.024 6, and 83.013 8. Peak 3 is dactylin, with the main fragments in negative ion mode being 314.0437, 299.020 2, 271.025 2, and 255.030 5; and the main fragments in positive ion mode being 317.065 4, 302.041 9, and 85.028 3. Peak 4 is kaempferol-3,4'-di-O-glucoside, with the main fragments in negative ion mode being 284.033 2, 255.030 3, and 227.0353; and the main fragments in positive ion mode being 287.054 9, 127.038 8, 97.028 5, and 85.0284. Peak 54 is (+)-catechin-7-O-β-D-xyloside or its isomers. The main fragments in negative ion mode are: 169.0144, 151.003 8, 125.024 6, 123.008 9, 59.013 9. Peak 6 is hyperoside. The main fragments in negative ion mode are: 301.035 5, 300.028 2, 271.025 5, 255.030 7; the main fragments in positive ion mode are: 303.049 9, 229.049 4, 153.017 5, 85.028 4. Peak 7 is quercetin-3-O-glucuronide. The main fragments in negative ion mode are: 301.035 7, 178.998 8, 151.003 8; the main fragments in positive ion mode are: 303.049 9, 229.049 7, 153.018 4, 85.028 4. Peak 8 is rutin. The main fragments in negative ion mode are: 301.035 4, 300.028 0, 271.025 1, 255.030 1; the main fragments in positive ion mode are: 303.049 8, 85.028 3, 71.049 1. Peak number 9 is isoquercetin. The main fragments in negative ion mode are: 301.035 4, 300.028 0, 271.025 3, 255.030 1, 151.0039; the main fragments in positive ion mode are: 303.049 9, 285.040 5, 257.044 6, 229.049 7, 127.038 8.Peak 10 is quercetin-3-O-xyloside. The main fragments in negative ion mode are: 301.035 3, 300.028 0, 271.025 4, 255.030 3; the main fragments in positive ion mode are: 303.049 9, 257.044 3, 229.049 9, 153.018 4. Peak 11 is (+)-catechin-7-O-β-D-xyloside or its isomers. The main fragments in negative ion mode are: 169.014 5, 151.003 9, 125.024 7, 123.008 9. The main fragments in positive ion mode are: 153.018 1, 127.038 8, 109.028 2, 91.054 4. Peak 12 is echinopsin, with the main fragments in negative ion mode being 285.040 5, 255.030 3, and 227.035 4; and the main fragments in positive ion mode being 287.055 0, 153.017 4, and 85.028 3. Peak 13 is luteolin, with the main fragments in negative ion mode being 284.033 2, 255.030 4, and 227.035 3; and the main fragments in positive ion mode being 287.054 9, 97.028 4, and 85.028 3. Peak 14 is guajavarin, with the main fragments in negative ion mode being 301.035 5, 300.027 9, 271.025 1, and 255.030 1; and in positive ion mode being 303.049 8, 257.044 0, 229.049 8, and 73.028 1. Peak 15 is kaempferol-3-O-rutinoside, with the main fragments in negative ion mode being 284.0331, 255.030 2, and 227.035 4. Peak 16 is quercetin. The main fragments in negative ion mode are: 301.035 6, 300.0280, 271.025 4, 255.030 3, 151.003 8; the main fragments in positive ion mode are: 303.049 9, 287.054 9, 85.028 3. Peak 17 is narcissin. The main fragments in negative ion mode are: 315.051 5, 285.041 3, 151.004 1. Peak 18 is isorhamnetin-3-O-glucoside. The main fragments in negative ion mode are: 314.043 8, 299.020 2, 271.0251, 243.030 3. Peak number 19 is 2'-O-quercetin gallate. The main fragments in negative ion mode are: 313.056 8, 285.041 0, 257.046 1, 229.050 9, 151.003 8; the main fragments in positive ion mode are: 287.055 0, 237.039 9, 191.033 6, 153.018 1, 127.039 0.Peak 20 is kaempferol-3-O-arabinoside. The main fragments in negative ion mode are: 285.040 8, 284.033 2, 255.030 3, 227.035 5; the main fragments in positive ion mode are: 299.053 1, 287.054 8, 153.018 1, 121.028 7. Peak 21 is afudamidin. The main fragments in negative ion mode are: 285.040 7, 255.030 3, 227.035 3; the main fragments in positive ion mode are: 287.054 9, 129.054 6, 85.028 4, 71.049 1. Peak No. 22 is eriodictyol. The main fragments in negative ion mode are: 151.003 8, 135.045 3, 107.014 0; the main fragments in positive ion mode are: 271.06 00, 163.038 9, 153.018 1, 145.028 5.

[0059] Peak No. 23 is 3,4',5,7-tetrahydroxy-8-methoxy-flavone, the main fragments in negative ion mode are: 271.061 7, 256.038 1, 227.035 6, 80.965 2. Peak No. 24 is quercetin, the main fragments in negative ion mode are: 178.998 9, 151.003 9, 121.029 7, 107.014 1; the main fragments in positive ion mode are: 257.043 9, 229.049 6, 165.017 5, 153.018 0, 137.024 0. Peak No. 25 is myricetin, the main fragments in negative ion mode are: 271.025 3, 178.998 9, 151.003 9, 137.024 5, 109.029 7. Peak No. 26 is luteolin, the main fragments in negative ion mode are: 199.040 5, 151.004 1, 133.029 7, 107.014 3. Peak No. 27 is tiglioside, the main fragments in negative ion mode are: 447.093 2, 285.040 9, 255.030 5, 227.035 5, 145.029 9; the main fragments in positive ion mode are: 287.055 0, 147.044 0, 119.049 1. Peak No. 28 is pinocembrin, the main fragments in negative ion mode are: 213.056 7, 151.004 4, 107.013 9, 83.013 8; the main fragments in positive ion mode are: 215.070 5, 173.059 9, 153.018 2, 131.049 1. Peak No. 29 is pinocembrin-7-O-β-D-glucoside, the main fragments in positive ion mode are: 257.080 9, 153.018 2, 131.049 2, 103.054 1. Peak No. 30 is naringenin, the main fragments in negative ion mode are: 177.019 7, 151.003 9, 119.050 5, 107.014 0, 93.034 7; the main fragments in positive ion mode are: 179.033 6, 171.029 3, 153.018 2, 147.044 0, 119.049 2. Peak No. 31 is apigenin, the main fragments in negative ion mode are: 225.055 8, 159.045 5, 151.003 8, 117.034 8; Peak No. 32 is kaempferol, the main fragments in negative ion mode are: 257.046 0, 239.034 7, 185.061 4, 169.065 5, 151.003 6; the main fragments in positive ion mode are: 213.055 0, 165.018 6, 153.018 4, 121.028 3. Peak No. 33 is hydroxyacacetin, the main fragments in negative ion mode are: 284.033 2, 256.037 9, 227.035 7.Peak No. 34 is isorhamnetin, the main fragments in negative ion mode are: 300.028 0, 271.025 4, 164.011 7, 151.003 9, 107.014 0; the main fragments in positive ion mode are: 317.065 5, 285.039 4. Peak No. 35 is cyanidin, the main fragments in negative ion mode are: 344.054 5, 329.031 0, 314.007 5, 286.012 6; the main fragments in positive ion mode are: 346.068 3, 331.044 7, 303.049 8. Peak No. 36 is diosmetin, the main fragments in positive ion mode are: 286.047 2, 258.052 5, 153.018 4. Peak No. 37 is prunetin, the main fragments in negative ion mode are: 268.038 2, 221.155 0, 193.160 2; the main fragments in positive ion mode are: 270.052 5, 242.057 2, 167.033 8.

[0060] Analysis Example 1 - “Traditional Chinese Medicine Ingredients - Common Targets - Pathways”

[0061] The interactions between TFR, active ingredients, common targets, diseases and KEGG enriched pathways were constructed and visualized by Cytoscape v3.7.2 software, and the built-in network function was used for topological analysis to screen out the core active ingredients, targets and pathways.

[0062] The results are shown in Figure 2 By visual analysis through the “Analyze Network” tool, 483 nodes and 4536 edges were obtained, and the rose flavonoid active ingredients were sorted according to the Degree value. It was speculated that quercetin, isoquercitrin, guanxinkang, astilbin, luteolin and other chemical components might be the key active ingredients of the total flavonoids of rose flowers TFR provided in Example 1 for the treatment of hypercholesterolemia.

[0063] Animal experiments

[0064] Experimental plan: 72 SPF male C57BL / 6J mice, 6 weeks old, weighing 20±2g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were housed in an SPF barrier environment at the Animal Experimental Center of Shandong University of Traditional Chinese Medicine. After one week of adaptive feeding, they were weighed and randomly divided into 6 groups according to their body weight, namely the normal control group (Con), the high-fat and high-cholesterol model group (Mod), the positive treatment group (SV), the low-dose treatment group of rose total flavonoids (TFR-25), the medium-dose treatment group of rose total flavonoids (TFR-50), and the high-dose treatment group of rose total flavonoids (TFR-100), with 12 mice in each group. The normal control group was fed with a blank control feed (LAD0011), and the other groups were fed with a high-fat and high-cholesterol feed (TP28900). The low-, medium-, and high-dose groups of rose total flavonoids were given 25, 50, and 100 mg·kg-1 BW rose total flavonoids aqueous solution, respectively, and the positive treatment group was given 10 mg·kg -1 Simvastatin aqueous solution was administered orally once every afternoon at a dose of 10 mL kg -1 ·d -1 The normal control group and the high-fat and high-cholesterol model group were given equal volumes of distilled water for 6 consecutive weeks.

[0065] (1) Serum biochemical index detection

[0066] Kits were used to determine the levels of cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AKP) in mouse serum, and the arteriosclerosis index (AI) and lipid comprehensive index (LCI) were calculated according to the following formulas.

[0067]

[0068] Results see Figure 3 ,according to Figure 3Compared with the Con group, the Mod group significantly increased TC and LDL-C levels (P < 0.01), while significantly decreased TG and HDL-C levels (P < 0.05) due to the high-fat, high-cholesterol diet. Both SV and TFR interventions improved dyslipidemia induced by the high-fat, high-cholesterol diet. Compared with the Mod group, the SV group had significantly lower serum TC and LDL-C levels (P < 0.01), while the HDL-C level increased but did not differ significantly (P > 0.05). There were no significant differences in lipid profiles between the TFR-25 group and the Mod group (P > 0.05). Only the serum LDL-C level was significantly decreased in the TFR-50 group compared with the Mod group (P < 0.05). However, the TFR-100 group had significantly lower serum TC and LDL-C levels (P < 0.05), while the HDL-C level was significantly increased (P < 0.05).

[0069] Results see Figure 4 ,according to Figure 4 Compared with the Con group, the AI ​​and LCI levels of mice in the Mod group were significantly increased (P < 0.01). After supplementation with SV and TFR, we found that only the AI ​​level in the SV group was significantly reduced (P < 0.05), while the AI ​​and LCI levels in the TFR-100 group were significantly decreased compared with the Mod group (P < 0.05).

[0070] Results see Figure 5 ,according to Figure 5 After 6 weeks of feeding, the Mod group had significantly higher serum AST, ALT, and AKP levels than the Con group (P < 0.01), indicating that consuming a high-fat, high-cholesterol diet can damage the liver. However, after SV and TFR treatment, these conditions improved to varying degrees. Compared with the Mod group, the serum AST level of the SV group decreased significantly (P < 0.01), while the ALT and AKP levels decreased but showed no significant difference (P > 0.05). The serum AST and AKP levels of the TFR-25 and TFR-50 groups decreased significantly (P < 0.05), and the serum AST, ALT, and AKP levels of the TFR-100 group decreased significantly (P < 0.05).

[0071] (2) Histopathological examination of mouse liver

[0072] The mouse liver tissue fixed in 4% paraformaldehyde tissue fixative was removed, dehydrated, paraffin-embedded, sectioned, and counterstained with hematoxylin and eosin. Finally, the pathological changes of the liver tissue were observed under a 100x ordinary optical microscope.

[0073] Results see Figure 6 ,according to Figure 6It can be seen that the livers of mice in the Con group were dark red, smaller in size, and had a clear and smooth surface. Compared with the Con group, the livers of mice in the Mod group were yellow-red, enlarged, and had a rough surface due to the stimulation of a high-fat and high-cholesterol diet. The morphology of the liver was significantly improved after SV and TFR treatment, especially the color and size of the livers of mice in the SV and TFR-100 groups were similar to those of mice in the Con group. In order to further elucidate the pathological changes in the liver of mice, we performed histopathological examinations on the livers of mice in different drug-treated groups by H&E staining and observed them under an optical microscope (100 times). The results showed that ( Figure 6 Middle B), the liver tissue of mice in the Con group was clear, the hepatocyte morphology was normal and regularly arranged, no obvious fat vacuoles were observed, and there was no lipid degeneration or inflammatory infiltration. In contrast, after 6 weeks of high-cholesterol diet induction, a large number of fat vacuoles appeared in the Mod group, the hepatocytes were significantly swollen, the cell volume increased, and obvious fatty degeneration and inflammatory lesions of the liver were observed. However, under the intervention of SV and TFR, the degeneration of hepatocytes and inflammatory lesions were significantly alleviated, and the fat vacuoles were also significantly reduced. Among them, only a small amount of inflammatory infiltration and fat vacuoles were observed in the TFR-100 group, and the hepatocyte morphology was close to that of the normal group. Based on the above results, we found that a high-cholesterol diet can induce fatty lesions and inflammatory cell infiltration in the liver of mice, damage the liver tissue of mice, but the intervention of TFR can effectively alleviate these adverse effects.

[0074] (3) Lipid content in mouse feces

[0075] Accurately weigh about 160 mg of liver tissue, add 9 times the amount of normal saline according to the ratio of body weight (g): volume (mL) = 1:9, and mechanically homogenize under ice water bath, centrifuge (3000r·min -1 The collected feces were dried at 37°C for 6 h (force drying), weighed and ground evenly, and 9 times the volume of anhydrous ethanol was added at a ratio of weight (g): volume (mL) = 1:9. After ultrasonication for 30 min, the supernatant was taken for testing. -1 Centrifuge for 10 minutes and collect the supernatant for testing. TC and TG in mouse liver and feces were detected and analyzed using relevant kits.

[0076] Results see Figure 7 ,according to Figure 7 It can be seen that the fecal TC and TG levels of the Mod group mice were significantly higher than those of the Con group (P < 0.01), and there were no significant differences in the fecal TC and TG levels of the SV group, TFR-25 group and TFR-50 group compared with the Mod group mice (P > 0.05), while the fecal TC and TG levels of the TFR-100 group mice were significantly higher than those of the Mod group (P < 0.01), indicating that the TFR-100 group can promote the excretion of cholesterol and triglycerides in mice.

[0077] (4) Activity of enzymes related to lipid metabolism in mouse liver

[0078] The activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) and cholesterol 7α-hydroxylase (CYP7A1) in the liver of mice were detected by enzyme-linked immunosorbent assay (ELISA).

[0079] Results see Figure 8 ,according to Figure 8 It can be seen that compared with the Con group, the HMG-CoAR activity of the mice in the Mod group was significantly increased (P < 0.05), and the CYP7A1 activity was significantly decreased (P < 0.05). Compared with the Mod group, there was no significant difference in the HMG-CoAR and CYP7A1 activities in the liver of the mice in the TFR-25 group (P > 0.05). However, after intervention with the SV, TFR-50, and TFR-100 groups, the abnormal enzyme activity caused by hypercholesterolemia was reversed to varying degrees. It is worth noting that the HMG-CoAR activity in the liver of the mice in the TFR-100 group reached 60.81 ± 13.41 pg mL -1 , which was significantly lower than that in the Mod group (77.88±10.31pg·mL -1 , P < 0.05). Similarly, the liver CYP7A1 activity of mice in the TFR-100 group (51.23 ± 9.21 pg mL -1 ) was significantly higher than that in the Mod group (32.74±7.80 pg·mL -1 , P<0.01), and were close to those of the Con group.

[0080] (5) Short-chain fatty acids in mice

[0081] Before the end of the experiment, mice were placed in clean metabolic cages and allowed to move normally for 3-5 hours, with normal diet and water intake. Feces were collected and transferred with forceps into sterile, enzyme-free cryotubes, labeled, frozen in liquid nitrogen, and stored at -80°C. During the autopsy, cecal contents were removed and placed in sterile cryotubes, stored at -80°C until use.

[0082] Gas chromatography was used to analyze the effects of TFR on the SCFAs content in the cecum of hypercholesterolemia mice.

[0083] Results see Figure 8 ,according to Figure 8The results showed that the levels of acetic acid, propionic acid, isobutyric acid, isovaleric acid, and valeric acid in the cecal contents of the Mod group mice were significantly lower than those in the Con group (P < 0.05), indicating that a high-fat, high-cholesterol diet inhibits the production of SCFAs in the mouse intestine. After SV and TFR intervention, the levels of SCFAs in each group of mice were upregulated to varying degrees. Specifically, the levels of butyric acid and isovaleric acid in the cecal contents of the SV group mice were significantly higher than those in the Mod group (P < 0.05). In the cecal contents of the TFR-50 group mice, only the level of isobutyric acid was significantly increased compared with the Mod group (P < 0.05). In contrast, the levels of acetic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid in the TFR-100 group increased by 0.8, 1.2, 1, 2.9, and 2.5 times, respectively, after intervention (P < 0.05), indicating the best therapeutic effect, which was closest to that of the Con group.

[0084] (6) Alpha diversity of mouse intestinal microorganisms

[0085] In order to comprehensively evaluate the diversity of mouse intestinal microbial communities, the QIIME2 2021.11 software was used to evaluate the sample Alpha diversity index, mainly including Ace, Chao1, Observed_features, Shannon, Simpson and faith_pd indices.

[0086] Results see Figure 10 ,according to Figure 10 It can be seen that compared with the Con group, the Ace, Chao1, Observed_features, Shannon, Simpson, and faith_pd indices of the mice in the Mod group were significantly reduced (P < 0.01), indicating that a high-fat, high-cholesterol diet reduces the richness and diversity of intestinal microorganisms in mice, which is similar to the study of Liu et al.

[20] . However, the intake of TFR can reverse this trend to varying degrees, among which the TFR-100 group has the best and significant effect (P < 0.05).

[0087] (7) Beta diversity of mouse intestinal microorganisms

[0088] Beta diversity analysis was performed using QIIME software to compare the similarity of species diversity among different samples. PCoA and NMDS analyses were performed using the Python (V2.7.18) matplotlib library and the R (V3.6.2) vegan package, respectively, based on the weighted Unifrac distance matrix.

[0089] The results are shown in 11. Figure 11PCoA analysis revealed that the contribution rates of the first principal coordinate (PCoA1) and the second principal coordinate (PCoA2) to the intestinal microbiota were 39.02% and 19.19%, respectively, with a cumulative contribution rate of 58.21%, demonstrating the validity and reliability of the model, with high predictive power. The blank group was primarily distributed in the left quadrant, while the model group was primarily distributed in the right quadrant, indicating significant differences in the intestinal microbiota between normal mice and hypercholesterolemia model mice. NMDS analysis also showed similar results. Following SV and TFR intervention, changes in the intestinal microbiota of mice occurred. Compared with the TFR-25 and TFR-50 groups, the SV and TFR-100 groups were more similar to the blank group. In particular, there was some overlap between the TFR-100 and Con groups, indicating that the TFR-100 group can partially restore the changes in the intestinal microbiota induced by a high-fat, high-cholesterol diet.

[0090] It should be noted that: Figure 11 Each color represents a group, and each dot represents an individual sample. The horizontal axis represents the first principal coordinate, and the percentage represents the contribution of the first principal coordinate to the sample differences. The vertical axis represents the second principal coordinate, and the percentage represents the contribution of the second principal coordinate to the sample differences.

[0091] (8) Analysis of species community structure

[0092] Based on the abundance and annotation information of ASV, the proportion of sequences at different taxonomic levels (phylum, family, and genus) of each sample was statistically analyzed to evaluate the species annotation resolution and species complexity of the samples. R (V3.6.2) was used to draw stacked bar charts of species abundance at different taxonomic levels.

[0093] LEfSe (Line Discriminant Analysis (LDA) Effect Size) analysis is an analytical method that combines the nonparametric Kruskal-Wallis and Wilcoxon rank sum tests with the effect size of linear discriminant analysis (LDA). It can identify statistically significant biomarkers between different groups. Therefore, based on the relative abundance of ASVs, the software LefSe (http: / / huttenhower.sph.harvard.edu / lefse / ) was used to identify differentially expressed species at each taxonomic level (kingdom, phylum, class, order, family, genus, and species). LEfSe cladograms and LDA value distribution histograms were also plotted. The screening criteria were LDA score > 3 and P-value < 0.05.

[0094] (A) Door level

[0095] Results see Figure 12 and Figure 13 ,according to Figure 12 and 13 It can be seen that the most abundant microbial groups in the intestines of mice in each experimental group are mainly Bacteroidota, Firmicutes, Verrucomicrobiota, and Proteobacteria, accounting for more than 85% of the total intestinal microbial population. Figure 13 Compared with the Con group, the intestinal microbiota of mice fed a high-fat, high-cholesterol diet underwent significant changes. The relative abundance of the characteristic bacteria phyla Bacteroidetes and Firmicutes decreased significantly (P < 0.05), while the levels of Verrucomicrobia and Proteobacteria increased significantly (P < 0.01). The relative abundance of Verrucomicrobia and Proteobacteria in the intestines of mice in the SV and TFR-50 groups decreased significantly compared with the Mod group (P < 0.01), while there were no significant differences in Bacteroidetes and Firmicutes (P > 0.05). In contrast, the relative abundance of Bacteroidetes and Firmicutes in the intestines of mice in the TFR-100 group increased significantly compared with the Mod group (P < 0.05), while the levels of Verrucomicrobia and Proteobacteria decreased significantly (P < 0.01). Notably, the TFR-administered group showed a dose-dependent effect and gradually returned to Con levels.

[0096] (B) Subject level

[0097] Results see Figure 14-15 ,according to Figure 14-15 Compared with the Con group, the relative abundance of Akkermansiaceae in the intestines of Mod mice was significantly increased (P < 0.01), while the levels of Rikenellaceae and Prevotellaceae were significantly decreased (P < 0.05). Compared with the Mod group, the relative abundance of Akkermansiaceae in the SV group was significantly decreased (P < 0.01), while the relative abundance of Rikenellaceae was significantly increased (P < 0.01). In the TFR-25 group, only the level of Prevotellaceae was significantly increased (P < 0.05). The relative abundance of Akkermansiaceae in the TFR-50 and TFR-100 groups was significantly decreased (P < 0.01), while there was no significant difference in the Rikenellaceae and Prevotellaceae (P > 0.05).

[0098] (C) Level

[0099] Results see Figure 16-17 ,according to Figure 16-17It can be seen that at the genus classification level, the relative abundance of Akkermansia in the intestine of mice in the Mod group was significantly higher than that in the Con group (P < 0.01), while the relative abundances of Alistipes and Lachnospiraceae_NK4A136_group were significantly decreased (P < 0.01). After SV and TFR intervention, the relative abundances of Akkermansia, Alistipes and Lachnospiraceae_NK4A136 were all restored. The levels of Akkermansia in the SV group, TFR-50 group and TFR-100 group were significantly downregulated compared with the Mod group (P < 0.01), the levels of Alistipes in the SV group were significantly higher than that in the Mod group (P < 0.05), and the relative abundance of Lachnospiraceae_NK4A136 in the TFR-100 group was significantly increased compared with the Mod group (P < 0.05).

[0100] (D) Analysis of species differences among groups

[0101] The results are shown in 18-19. Figure 18 This is a LEfSe evolutionary branch diagram, which shows the different microbial communities between groups at each level. From the inside to the outside, they represent the classification levels of kingdom, phylum, class, order, family, genus, and species. Nodes of different colors represent microbial communities that play an important role in the group represented by the color, and the diameter size is proportional to the relative abundance. Species with no significant differences are uniformly colored yellow. Figure 19 This is a histogram of the LDA value distribution, showing species with significant differences and LAD score > 3.0. The vertical axis represents the microbial communities with significant differences between groups, and the horizontal axis represents the scores corresponding to the LDA analysis. The larger the score, the more significant the species difference.

[0102] According to data from 18-19, the characteristic bacteria in the Con group are Firmicutes, Clostridia, Lachnospirales, Lachnospiraceae, Muribaculaceae, Muribaculaceae, Clostridia_UCG_014, etc. In sharp contrast, the Mod group is mainly composed of Ekmansia, Ekmansia, Verrucomicrobiales and its subordinate Verrucomicrobiales, Sutterellaceae, etc. as the dominant bacteria. Therefore, the pathogenesis of hypercholesterolemia may be related to the changes in these bacteria. Compared with the Mod group, the Tannerellaceae family, Parabacteroides genus, Bilophila genus, Ruminococcus_torques_group genus, Butyricimonas genus, etc. were considered to be the key microorganisms in SV-intervention mice. We also observed that the Bacteroidetes family and its subordinate classification Bacteroides genus, Proteobacteria phylum, Gammaproteobacteria class, Alloprevotella genus were clearly differentiated in the TFR-25 group. In the TFR-50 group, Bacteroides species_vulgatus, Desulfobacterota phylum, Desulfovibrionaceae family, Roseburia genus, Turicibacter genus, etc. dominated. Mucispirillum genus, Ferrobacter phylum and its subordinate classification Ferrobacter class and Ferrobacteriaceae genus were significantly enriched in the TFR-100 group.

[0103] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing total flavonoids from rose, characterized in that: include: extracting rose flowers using a C1-C3 alcohol solvent to form a crude extract; The crude extract is mixed with water, and the pH is adjusted to 9-10, and then filtered to form a first supernatant; Adjusting the pH of the first supernatant to 4-5, filtering to form a second supernatant, The second supernatant is separated by macroporous adsorption resin chromatography.

2. The preparation method according to claim 1, characterized in that The macroporous adsorption resin chromatography separation step includes: eluting with water, then eluting with 3-5% ethanol and 55-65% ethanol in sequence, and collecting the eluate formed by eluting with 60% ethanol.

3. The preparation method according to claim 1, characterized in that The macroporous adsorption resin chromatography separation step includes: eluting with 8-12BV of water, then eluting with 2-3BV of 5% ethanol, and then eluting with 3-5BV of 60% ethanol, and collecting the eluate formed by 60% ethanol elution.

4. The preparation method according to any one of claims 1 to 3, characterized in that include: Reflux extraction of rose flowers using 8-12 BV of 85-95% ethanol to form a crude extract; The crude extract is mixed with water and the pH is adjusted to 9-10, and then allowed to stand for 10-14 hours, followed by filtration to form a first supernatant; The pH of the first supernatant is adjusted to 4-5, and the mixture is allowed to stand for 10-14 hours and then filtered to form a second supernatant; The second supernatant was separated by chromatography using D-101 macroporous adsorption resin.

5. A total flavonoids from rose, characterized in that: The total flavonoids from roses are prepared by the preparation method of any one of claims 1 to 4.

6. The rose total flavonoids according to claim 5, characterized in that The total flavonoids of rose flower include the following active ingredients: quercetin, isoquercetin, guajava glycoside, astilbin, luteolin, myricetin, apigenin, isorhamnetin, diosgenin, eriodictyol, hydroxygenkwain, kaempferol, naringenin, chamaecyparisin, luteolin and hyperoside.

7. Use of the total flavonoids from roses according to claim 5 in the preparation of a medicament for treating hypercholesterolemia.

8. Use of the total flavonoids from roses according to claim 5 in preparing a medicine for treating atherosclerosis.

9. Use of the total flavonoids from rose flowers according to claim 5 in the preparation of a medicament for treating liver damage; Preferably, the liver damage includes hepatitis and liver fat accumulation.

10. Use of the total flavonoids from roses according to claim 5 in the preparation of a medicament for improving intestinal microbial imbalance.

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