Cyperus rotundus extract composition and application thereof

Abstract

The invention provides a rhizoma cyperi extract composition and application thereof, belongs to the technical field of modernization of traditional Chinese medicines, and particularly provides a preparation method of the rhizoma cyperi extract composition, which mainly comprises the following steps: extracting by using an ethanol solution with a certain concentration, performing ultrafiltration treatment, adsorbing by using D101 type macroporous resin, and eluting by using ethanol solutions with different concentrations, thereby obtaining the rhizoma cyperi extract composition. Collecting 50%-70% of eluent, concentrating and drying to obtain a rhizoma cyperi extract composition; the invention also discloses application of the rhizoma cyperi extract composition in preparation of drugs for treating inflammatory bowel diseases, neuroinflammation and / or toxic hepatitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine modernization, and particularly provides a Cyperus rotundus extraction composition and its application. Background Art

[0002] Cyperus rotundus L., as a traditional Chinese medicine, has been recorded as early as in "Supplementary Records of Famous Physicians". It is widely used for soothing the liver and relieving depression, regulating menstruation and alleviating pain, and regulating qi and relieving distension. It is known as "the general controller of qi diseases and the main agent for women's diseases". After being processed with vinegar (i.e., Cyperus rotundus processed with vinegar), its efficacy of soothing the liver and relieving pain, promoting digestion and resolving stagnation can be enhanced, and it is commonly used to treat gastrointestinal disorders, dysmenorrhea and hernia pain related to liver qi stagnation. Modern research shows that the active ingredients of Cyperus rotundus mainly include terpenoids (such as α-cyperone), flavonoids (luteolin) and volatile oils, etc.

[0003] Inflammatory diseases refer to pathological inflammatory reactions caused by abnormal immune responses or pathogen infections, covering multiple organ fields such as the nervous system, digestive system, and cardiovascular system. Existing therapeutic drugs, such as corticosteroids, TNF-α inhibitors, etc., mostly target a single organ or a specific inflammatory pathway (such as IL-6 / JAK-STAT), and it is difficult to cover the complex regulatory network of multi-system inflammation. In addition, long-term use of immunosuppressants can lead to an increased risk of infection, and non-steroidal anti-inflammatory drugs (NSAIDs) are prone to cause gastrointestinal damage, etc. Inflammatory diseases threaten national health through multiple pathways such as direct organ damage, progression of chronic diseases, and collapse of the immune system. Colitis, also known as inflammatory bowel disease (IBD), has an increasing incidence rate year by year. May 19th of each year is the World IBD Day. Ulcerative colitis (UC for short) mostly occurs in people aged 20 to 50, showing a trend of getting younger. Abdominal pain, diarrhea, mucus stools, purulent stools, and bloody stools are its typical symptoms, and the canceration rate of UC over five years has been increasing year by year.

[0004] Existing pharmacological studies on Cyperus rotundus and its vinegar-processed products mostly focus on traditional indications such as antidepressant and menstrual regulation. The development of compositions for inflammatory diseases (such as inflammatory bowel disease) still relies on chemically synthesized ingredients, lacking research on the synergistic effects of multiple targets based on traditional Chinese medicine.

[0005] Defects of traditional preparation processes: In the traditional extraction process (water decoction method), active ingredients are severely lost, and impurities (polysaccharides, tannins) interfere with the exertion of drug efficacy.

[0006] Existing drugs for the treatment of enteritis have the defects of large side effects and easy recurrence: the cause of colitis is unknown, and hospitals often use drugs, enemas, surgery and other methods for symptomatic treatment. Common drugs include aminosalicylic acid preparations (sulfasalazine enteric-coated tablets, mesalazine, olsalazine), glucocorticoids (hydrocortisone tablets, prednisone acetate tablets), immune preparations (cyclosporine, thiopurine tablets or mercaptopurine tablets) and biological preparations (live Clostridium butyricum, Bacillus cereus). These prescription drugs are very powerful in anti-inflammatory and bactericidal effects and can quickly control symptoms, but they are prone to relapse after discontinuation of the drug, and most patients require long-term treatment and lifelong self-management. Drugs are resistant and dependent, and the dosage will increase as time goes by, and the effect will become worse as time goes by.

[0007] The above problems limit the modern development and application of Cyperus rotundus in the treatment of inflammation. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a Cyperus rotundus extraction composition and application thereof.

[0009] The technical solution of the present invention is as follows:

[0010] A method for preparing a Cyperus rotundus extract composition comprises the following steps:

[0011] (4) crushing the Cyperus rotundus, using a 30%-80% ethanol aqueous solution as an extraction solvent, with a material-liquid mass volume ratio of 1:(10-15) g / mL, an extraction temperature of 60-80° C., extracting 1-3 times, each extraction time of 2-3 hours, and collecting the extract as a crude extract;

[0012] (5) the crude extract was treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the ultrafiltrate was collected;

[0013] (6) The ultrafiltrate is adsorbed on a D101 macroporous resin column and eluted with 30%, 50%, 70%, and 90% ethanol in sequence. The 50%-70% eluate is combined, concentrated, and dried to obtain a Cyperus rotundus extract composition.

[0014] Preferably according to the present invention, in step (1), the Cyperus rotundus includes raw Cyperus rotundus and / or vinegar Cyperus rotundus.

[0015] Further preferably, in step (1), the Cyperus rotundus is vinegar-cooked Cyperus rotundus.

[0016] Preferably according to the present invention, in step (1), the Cyperus rotundus is crushed and passed through a 40-50 mesh sieve.

[0017] Preferably, according to the present invention, in step (1), an ethanol aqueous solution with a volume fraction of 50% is used as the extraction solvent; and the extraction temperature is 60°C.

[0018] Preferably according to the present invention, in step (2), the transmembrane pressure for ultrafiltration treatment is 0.5 - 1.5 MPa, and the temperature is 25 - 40 °C.

[0019] Preferably according to the present invention, in step (3), the elution flow rate is 1 - 3 BV / h.

[0020] More preferably, in step (3), the elution flow rate is 2 BV / h.

[0021] Preferably according to the present invention, in step (3), rotary evaporation concentration and drying are carried out.

[0022] More preferably, in step (3), the temperature for concentration and drying is 40 - 45 °C.

[0023] Application of the Cyperus rotundus extraction composition prepared by the above method in any of the following:

[0024] ① Application in the preparation of anti-inflammatory drugs;

[0025] ② Application in the preparation of drugs for treating inflammatory bowel disease, neuroinflammation, and / or toxic hepatitis.

[0026] Preferably according to the present invention, the inflammatory bowel disease includes: ulcerative colitis;

[0027] The toxic hepatitis includes: alcoholic liver disease, drug-induced hepatitis.

[0028] More preferably, the alcoholic liver disease includes alcoholic fatty liver and / or alcoholic hepatitis;

[0029] Drugs causing drug-induced hepatitis include: isoniazid, rifampicin, pyrazinamide, cyclophosphamide, Tripterygium wilfordii, Polygonum multiflorum, or more than one of them.

[0030] Application of the Cyperus rotundus extraction composition prepared by the above method in the preparation of anti-arrhythmia drugs, in the preparation method, the Cyperus rotundus is Rhizoma Cyperi.

[0031] A drug containing the Cyperus rotundus extraction composition prepared by the above method.

[0032] Preferably according to the present invention, the drug contains pharmaceutically acceptable excipients.

[0033] More preferably, the excipients include one or more of disintegrants, binders, or sustained-release materials.

[0034] Preferably according to the present invention, the dosage form of the drug includes tablets, capsules, or granules.

[0035] The beneficial effects of the present invention at least include the following:

[0036] 1. The present invention first discloses a Cyperus rotundus extraction composition, which has the effects of improving inflammatory bowel disease, neuroinflammation and toxic hepatitis. The inventors also found that the composition extracted from Cyperus rotundus processed with vinegar not only has the above effects, but also has the effect of improving arrhythmia.

[0037] 2. The composition prepared by the present invention relieves oxidative stress, improves Fe 2+ accumulation, inhibits ferroptosis and the expression of inflammatory factors in cells, and improves the intestinal inflammatory state by regulating the intracellular antioxidant system, iron metabolism and lipid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a graph for the safety investigation experiment of the active ingredient enrichments of raw Cyperus rotundus and Cyperus rotundus processed with vinegar.

[0039] Figure 2 It is a graph for the effect of the active ingredient enrichments of raw Cyperus rotundus and Cyperus rotundus processed with vinegar on the number of inflammatory cells in zebrafish;

[0040] In the figure: RCR1, RCR2, and RCR3 respectively represent the drug concentrations of raw Cyperus rotundus as 25 μg / mL, 50 μg / mL, and 100 μg / mL; VCR1, VCR2, and VCR3 respectively represent the drug concentrations of Cyperus rotundus processed with vinegar as 25 μg / mL, 50 μg / mL, and 100 μg / mL.

[0041] Figure 3 It is a graph for the statistical results of the number of inflammatory cells in zebrafish treated with the active ingredient enrichments of raw Cyperus rotundus and Cyperus rotundus processed with vinegar;

[0042] In the figure: #, ##, and # represent P < 0.05, 0.01, and 0.0001 compared with the blank control group, and ***, **** respectively represent P < 0.001 and 0.0001 compared with the model group.

[0043] Figure 4 It is a graph for the effect of the active ingredient enrichment of Cyperus rotundus on the number of intestinal inflammatory cells in zebrafish with inflammatory bowel disease;

[0044] In the figure: RCR1, RCR2, and RCR3 respectively represent the drug concentrations of raw Cyperus rotundus as 12.5 μg / mL, 25 μg / mL, and 50 μg / mL; VCR1, VCR2, and VCR3 respectively represent the drug concentrations of Cyperus rotundus processed with vinegar as 12.5 μg / mL, 25 μg / mL, and 50 μg / mL;

[0045] # represents P < 0.0001 compared with the blank control group, and *, ***, **** respectively represent P < 0.05, 0.001, and 0.0001 compared with the model group.

[0046] Figure 5Effect diagram of the active ingredient enrichment of Cyperus rotundus on the intestinal structure of zebrafish with inflammatory bowel disease;

[0047] In the figure: Ctl is the blank control group; TNBS is the model control group; RCR1, RCR2, and RCR3 respectively represent the crude Cyperus rotundus drug concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL; VCR1, VCR2, and VCR3 respectively represent the vinegar-processed Cyperus rotundus drug concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL; the legend is 50 μm.

[0048] Figure 6 Verification diagram of the expression levels of significantly different metabolites based on transcriptome analysis.

[0049] Figure 7 Effect diagram of the active ingredient enrichment of Cyperus rotundus on the expression levels of ferroptosis-related genes.

[0050] Figure 8 Effect diagram of the active ingredient enrichment of Cyperus rotundus on the intestinal peristalsis ability of zebrafish with inflammatory bowel disease;

[0051] In the figure: # represents P < 0.0001 compared with the blank control group, and ***, **** respectively represent P < 0.001 and 0.0001 compared with the model group.

[0052] Figure 9 Experimental result diagram of the improvement effect of the active ingredient enrichment of Cyperus rotundus on liver injury in zebrafish with alcoholic liver injury;

[0053] In the figure: ## represents P < 0.01 compared with the blank control group, and *, **, ***, **** respectively represent P < 0.05, P < 0.01, 0.001, and 0.0001 compared with the model group.

[0054] Figure 10 Experimental result diagram of the improvement effect of the active ingredient enrichment of Cyperus rotundus on liver injury in zebrafish with acute liver injury;

[0055] In the figure: ## represents P < 0.0001 compared with the blank control group, and *, **, *** respectively represent P < 0.05, 0.01, and 0.001 compared with the model group.

[0056] Figure 11 Heart rate statistical result diagram of zebrafish with heart injury treated with the active site enrichment of vinegar-processed Cyperus rotundus. Detailed implementation mode

[0057] The technical solutions of the present invention will be further elaborated below in combination with the embodiments and the accompanying drawings of the specification, but the scope of protection of the present invention is not limited thereto.

[0058] For the content without specific conditions in the examples, the conventional conditions are followed; for the reagents or instruments without specified manufacturers, they are all common commercially available products.

[0059] Source of biological materials

[0060] The raw Cyperus rotundus and its vinegar-processed products described in the examples were purchased from the Hebei Anguo Traditional Chinese Medicine Wholesale Market, which are common commercially available traditional Chinese medicines.

[0061] The vinegar-processed Cyperus rotundus is also called vinegar-processed Cyperus rotundus and vinegar Cyperus rotundus.

[0062] D101 macroporous resin column: Purchased from Jinan Boyi Biotechnology Co., Ltd.; Glass column specification: 10 cm * 100 cm.

[0063] In the present invention, "%" of the ethanol solution refers to volume fraction; for example, 60% ethanol refers to an ethanol solution with a volume fraction of 60%.

[0064] Detection method

[0065] Detection of total flavonoids and total phenolic acids

[0066] Detection of total flavonoids content: The aluminum nitrate-sodium nitrite colorimetric method is adopted. Using rutin as the standard product, gradient dilution (0 - 100 μg / mL), adding 5% sodium nitrite, 10% aluminum nitrate and 4% sodium hydroxide solutions for color development, and drawing the standard curve; the Cyperus rotundus extract sample is dissolved with 60% ethanol, filtered to obtain the supernatant, color-developed according to the standard curve method, and the absorbance is measured, and the total flavonoids content is calculated according to the standard curve.

[0067] Detection of total phenolic acids content: The Folin-Ciocalteu method is adopted. Using gallic acid as the standard product, gradient dilution (0 - 200 μg / mL), adding Folin-Ciocalteu reagent and saturated sodium carbonate solution for color development, reacting in the dark for 30 minutes after color development, measuring the absorbance at a wavelength of 760 nm, and drawing the standard curve; the Cyperus rotundus extract sample is dissolved with 60% ethanol, filtered to obtain the supernatant, color-developed according to the standard curve method, and the absorbance is measured, and the total phenolic acids content is calculated according to the standard curve.

[0068] Identification of component composition by HPLC-MS

[0069] Chromatographic conditions: Chromatographic column: Agilent ZORBAX RRHD SB-Aq (2.1×100 mm, 1.8 μm); Column temperature: 30 °C; Flow rate: 0.3 ml / min; Injection volume: 2 μl; Detection wavelength: 190 - 400 nm; Mobile phase ratio: Phase A is acetonitrile, Phase B is 0.1% formic acid aqueous solution, and the gradient is shown in Table 1.

[0070] Table 1 Mobile phase gradient

[0071]

[0072] Mass spectrometry detection mode: ESI - Negative / Positive ion mode, mass spectrometry parameters: see Table 2.

[0073] Table 2 Mass parameters(Sciex Triple TOF 4600 LC - MS)

[0074]

[0075] Example 1

[0076] A preparation method of the active ingredient enriched product of Cyperus rotundus, namely the Cyperus rotundus extraction composition, comprises the following steps:

[0077] (1) Select 500 g of dried rhizomes of raw Cyperus rotundus, crush them to a particle size passing through a 40 - mesh sieve (40 - mesh sieve) to increase the extraction efficiency. Use a 50% ethanol aqueous solution (volume ratio) as the extraction solvent, the solid - liquid ratio is 1:10 (w / v, g / mL), the extraction temperature is 60 °C, extract twice, with each extraction time being 2 hours, and obtain a crude extract after filtration;

[0078] (2) The crude extract is treated with an ultrafiltration membrane with a molecular weight cut - off of 1000 Da, the transmembrane pressure is 1.0 MPa, the temperature is 25 °C, collect the ultrafiltrate, remove macromolecular impurities such as polysaccharides and tannins (molecular weight > 1000 Da), and reduce subsequent pharmacodynamic interference;

[0079] (3) The ultrafiltrate is adsorbed by a D101 macroporous resin column, eluted successively with 30%, 50%, 70%, and 90% ethanol solutions at a flow rate of 2 BV / h, combine the 50 - 70% eluate, concentrate and dry it by rotary evaporation at 45 °C to obtain 25 g of an extract paste, an extract (RCR) with a total flavonoid content of 24.3% by mass fraction and a total phenolic acid content of 2.5% by mass fraction.

[0080] (4) Its chemical composition is shown in Table 3.

[0081] Table 3 Identification of main components in the Cyperus rotundus extract

[0082]

[0083]

[0084] Example 2

[0085] The preparation of the active ingredient enriched product of vinegar - processed Cyperus rotundus, namely the Cyperus rotundus extraction composition, comprises the following steps:

[0086] (1) Select 500 g of commercially available vinegar-fried Cyperus rhizome, dry it, and crush it to a particle size passing through a 40-mesh sieve (40-mesh sieve) to increase the extraction efficiency. Use 50% ethanol aqueous solution (volume ratio) as the extraction solvent, the solid-liquid ratio is 1:10 (w / v, g / mL), the extraction temperature is 60 °C, extract twice, with each extraction time of 2 hours, and obtain the crude extract after filtration;

[0087] (2) The crude extract is treated with an ultrafiltration membrane with a molecular weight cut-off of 1000 Da, the transmembrane pressure is 1.0 MPa, the temperature is 25 °C, collect the ultrafiltrate, remove macromolecular impurities such as polysaccharides and tannins (molecular weight > 1000 Da), and reduce the interference of subsequent drug effects;

[0088] (3) The ultrafiltrate is adsorbed by a D101 macroporous resin column, eluted successively with 30%, 50%, 70%, and 90% ethanol at a flow rate of 2 BV / h, combine the 50 - 70% eluate, concentrate and dry it by rotary evaporation at 45 °C to obtain 22 g of the extract paste, an extract (VCR) with a total flavonoid content of 22.6% and a total phenolic acid content of 2.0%.

[0089] (4) Its chemical composition is shown in Table 3.

[0090] Experimental Example 1

[0091] Anti-inflammatory bowel disease activity of the active ingredient enriched extract of Cyperus (i.e., the Cyperus extract composition prepared in Examples 1 - 2) based on the zebrafish model

[0092] (1) Safety screening of the active ingredient enriched extract of Cyperus

[0093] Select healthy wild-type AB zebrafish as the experimental subjects. When the embryos develop to 3 dpf, select normal zebrafish larvae under a stereomicroscope, transfer them into a 24-well culture plate, set up gradient concentration drug treatment groups (25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL respectively). Add the Cyperus extract composition solution to each drug group, with 10 zebrafish in each group, and set 2 replicates at the same time. Add culture water to 2.0 mL. After covering, place each experimental group of zebrafish in an illumination incubator (28 °C) to allow the embryos to continue developing for 24 h, then take pictures under a fluorescence microscope, observe the development of zebrafish, and count the number of dead zebrafish. The results are as Figure 1 shown. The active ingredient enriched extracts of raw and vinegar-fried Cyperus have good safety when the drug concentration ≤ 400 μg / mL.

[0094] (2) Anti-inflammatory activity of the active ingredient enriched extract of Cyperus

[0095] Using green fluorescently labeled macrophage Tg(lyz:EGFP) zebrafish (which can be purchased from the National Zebrafish Resource Center) as experimental animals, a zebrafish model of neuroinflammation was constructed using copper sulfate. Healthy Tg(lyz:EGFP) zebrafish larvae were selected as experimental subjects. When the embryos developed to 3 dpf, normal zebrafish larvae were selected under a stereomicroscope and transferred into a 24-well culture plate. A blank control group (Control), a model group (CuSO4·5H 2 O), and different concentration drug treatment groups (25 μg / mL, 50 μg / mL, and 100 μg / mL respectively) were set up. The blank control group and the model group were added with embryo culture water, and the drug group was added with the extract composition solution of Cyperus rotundus. There were 10 zebrafish in each group, and 2 replicate wells were set at the same time. Culture water was added to 2.0 mL. After covering, each experimental group of zebrafish was placed in an illumination incubator (28 °C) respectively to allow the embryos to continue to develop. After 5 h, 20 μM of CuSO 4 ·5H 2 O was used to treat the zebrafish in the model group and the drug treatment group respectively. After incubating the zebrafish in the dark for 1 h, the zebrafish were washed, and the zebrafish larvae were anesthetized with tricaine (0.2%, w / v). Zebrafish larvae were randomly selected and photographed under a fluorescence microscope, and the number of neutrophils in the zebrafish intestine was counted. The results are as Figure 2 and Figure 3 shown. The active ingredient enrichments of raw Cyperus rotundus (RCR) and vinegar-fried Cyperus rotundus (VCR) both have good anti-inflammatory activities.

[0096] (3) Effect of the active ingredient enrichment of Cyperus rotundus on the number of inflammatory cells in the intestine of zebrafish with inflammatory bowel disease

[0097] Using green fluorescently labeled macrophage Tg(lyz:EGFP) zebrafish as experimental animals, a zebrafish model of inflammatory bowel disease (ulcerative colitis) was constructed using trinitrobenzenesulfonic acid (TNBS). The zebrafish were randomly divided into a blank control group (Control), a TNBS model control group (TNBS), and different concentration drug treatment groups (12.5 μg / mL, 25 μg / mL, and 50 μg / mL respectively). Healthy Tg(zlyz:EGFP) zebrafish 72 h after fertilization were selected and randomly transferred into a 24-well plate, with 10 in each well and 2 replicate wells. The zebrafish in the blank control group were incubated in zebrafish culture water without TNBS, and the zebrafish in other groups were incubated in zebrafish culture water containing TNBS (60 μM) under the same conditions. After 48 h, the extract samples were added to the 24-well plate respectively. After incubating for 24 h, the zebrafish larvae were anesthetized with tricaine (0.2%, w / v). Zebrafish larvae were randomly selected and photographed under a fluorescence microscope, and the number of neutrophils in the zebrafish intestine was counted. The results showed that, see Figure 4Both the enriched active ingredient of raw Cyperus rotundus L. (RCR) and that of its vinegar-processed product (VCR) have good anti-enteritis activities.

[0098] (4) Effects of the enriched active ingredients of Cyperus rotundus L. on the intestinal structure of zebrafish with inflammatory bowel disease;

[0099] Using zebrafish Tg(lyz:EGFP) with green fluorescently labeled macrophages as experimental animals, an inflammatory bowel disease zebrafish model was constructed using trinitrobenzenesulfonic acid (TNBS). The zebrafish were randomly divided into a blank control group (Control), a TNBS model control group (TNBS), and extract sample groups (12.5 μg / mL, 25 μg / mL, 50 μg / mL). Healthy Tg(zlyz:EGFP) zebrafish at 72 h post-fertilization were randomly transferred into 24-well plates, with 10 fish per well and 2 replicate wells. The zebrafish in the blank control group were incubated in zebrafish culture water without TNBS, and the zebrafish in other groups were incubated in zebrafish culture water containing TNBS (60 μM) under the same conditions. After 48 h, the extract samples were added to the 24-well plates. After incubation for 24 h, the zebrafish larvae were anesthetized with tricaine (0.2%, w / v). Zebrafish larvae were randomly selected for HE staining. After fixation, dehydration, and embedding, sections were made. After dewaxing and hydration, they were stained with hematoxylin and eosin, and morphological features such as the intestinal mucosal structure, goblet cell distribution, and inflammatory cell infiltration of zebrafish were observed under a microscope. The results showed that Figure 5 in the intestinal tissue of zebrafish in the model group, a small amount of epithelial cell necrosis and exfoliation was visible in the mucosal layer, with nuclear fragmentation (red arrow). In the intestinal tissue of zebrafish in the groups of the enriched active ingredients of raw Cyperus rotundus L. (RCR) and its vinegar-processed product (VCR), the staining was uniform, the epithelial structure in the mucosal layer was intact, the morphological structure of epithelial cells was normal and arranged regularly, and there was no obvious abnormality in the lamina propria and no obvious inflammation. The experimental results further demonstrated that both enriched active ingredients have good anti-enteritis activities.

[0100] Experimental Example 2

[0101] Study on the mechanism of action of the enriched active ingredients of Cyperus rotundus L. (i.e., the Cyperus rotundus L. extraction composition prepared in Examples 1-2) against inflammatory bowel disease based on transcriptomics

[0102] (1) KEGG enrichment analysis of the improvement effect of the enriched active ingredients of Cyperus rotundus L. on inflammatory zebrafish based on transcriptomics

[0103] Using zebrafish Tg(lyz:EGFP) with green fluorescently labeled macrophages as experimental animals, when the embryos developed to 3 dpf, normal zebrafish larvae were selected under a stereomicroscope and transferred into 24-well culture plates. A blank control group (Control) and a model group (CuSO 4 ·5H 2(O) and drug treatment groups with different concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL respectively). Embryo culture water was added to the blank control group and the model group, and the extract composition solution of Cyperus rotundus was added to the drug groups. There were 10 zebrafish in each group, and 2 replicate wells were set at the same time. Culture water was added to 2.0 mL. After covering, each experimental group of zebrafish was placed in an illumination incubator (28 °C) to allow the embryos to continue developing. After 5 h, 20 μM CuSO 4 ·5H 2 O was used to treat the zebrafish in the model group and the drug treatment groups respectively. After incubating the zebrafish in the dark for 1 h, the zebrafish were washed, and samples of each experimental group were collected. The zebrafish larvae were anesthetized and fixed with tricaine (0.2%, w / v) and stored at -80 °C for later use in transcriptome analysis. By multivariate statistical analysis (PCA, PLS-DA), differential metabolites between groups were screened. With the threshold of VIP value > 1, p < 0.05, and FC > 2, significant metabolites were determined. The differential metabolites were imported into the KEGG database, mapped to relevant metabolic pathways, and the significance of pathway enrichment was statistically analyzed. The experimental results showed that the differential metabolites in the Control vs CuSO 4 , RCR vs CuSO 4 , VCR vs CuSO 4 groups were all enriched in the ferroptosis pathway, indicating that it might be the key signal for Cyperus rotundus to improve intestinal inflammation.

[0104] Subsequently, based on the transcriptome analysis samples, with significant differential metabolites as detection indicators, the accuracy of the transcriptome results was verified. The results showed that the gene expression levels of the differential metabolites were consistent with the results of the transcriptome analysis ( Figure 6 ), proving that the results of the transcriptome analysis were feasible, and ferroptosis was the signaling pathway for Cyperus rotundus to improve zebrafish intestinal inflammation.

[0105] (2) Effects of the enriched active components of Cyperus rotundus on the expression levels of key genes in the ferroptosis-related pathways;

[0106] Based on the results of the transcriptome study, zebrafish samples were collected for detecting the expression levels of key genes in relevant pathways. The results showed that the enriched active components of Cyperus rotundus could significantly improve the down-regulation of the expression of the ferroptosis key genes slc40a1 and fth1 caused by CuSO 4 . At the same time, the expression levels of the oxidative stress-related genes NQO1 and gpx4a were up-regulated, and the expression of the inflammation-related genes il8 and il10 was inhibited, as shown in Figure 7 .

[0107] Experimental Example 3

[0108] The enriched active components of Cyperus rotundus are the extract composition of Cyperus rotundus prepared in Examples 1 - 2

[0109] (1) Effects of the enriched active components of Cyperi Rhizoma on the intestinal peristalsis ability of zebrafish with inflammatory bowel disease

[0110] Using zebrafish Tg(lyz:EGFP) with green fluorescent-labeled macrophages as experimental animals, the zebrafish were randomly divided into a blank control group (Control), a TNBS model control group (TNBS), a positive group (5-ASA), and gradient concentration sample groups (12.5 μg / mL, 25 μg / mL, 50 μg / mL). Select healthy Tg(zlyz:EGFP) zebrafish at 72 h post-fertilization, randomly transfer them into 24-well plates, with 10 fish per well and 2 replicate wells. The zebrafish in the blank control group were incubated in zebrafish culture water without TNBS, and the zebrafish in other groups were incubated in zebrafish culture water containing TNBS (60 μM) under the same conditions. After 48 h, the enriched active components of Cyperi Rhizoma samples were added to the 24-well plates, and the drug solution was incubated for 24 h. Observe under a microscope and record the intestinal peristalsis of zebrafish within 1 min. Taking the number of intestinal peristalsis of zebrafish as the evaluation index, the intestinal peristalsis ability of the samples was measured. The experimental results showed that, see Figure 8 , the crude Cyperi Rhizoma extraction composition and the vinegar-processed Cyperi Rhizoma extraction composition had good improvement effects on the intestinal peristalsis function of zebrafish with inflammatory bowel disease, and the effect of the vinegar-processed Cyperi Rhizoma extraction composition was better than that of the crude Cyperi Rhizoma extraction composition.

[0111] (2) Protective effects of the enriched active components of Cyperi Rhizoma on zebrafish with alcoholic liver injury

[0112] Using transgenic zebrafish Tg(L-FABP:EGFP) (Tg(-1.7apoa2:GFP) or Tg(fabp10a:DsRed) zebrafish can also be used, which can be purchased from the National Zebrafish Resource Center) with green fluorescent protein labeled in the liver as experimental animals. When the zebrafish developed to 72 hpf, select normal-developed zebrafish larvae under a stereomicroscope and randomly assign them to a blank control group, a model group, and gradient concentration drug treatment groups (6.25, 12.5 μg / mL, 25 μg / mL). The zebrafish in the control group were fed with normal fish culture water, the fish culture water in the model group was treated with ethanol, and the treatment group was treated with the test substance while adding ethanol (volume fraction 1.1%) for modeling, and then placed in an incubator for culture. After 24 hours of treatment, the zebrafish were anesthetized with an anesthetic (tricaine), and then the zebrafish were fixed on a glass slide with methylcellulose and photographed. Observe the effects of the drug on the overall morphology of zebrafish under white light of a microscope, and observe the effects of the test substance on the liver morphology of zebrafish larvae under a fluorescence microscope, and calculate the liver area (Area) and liver fluorescence intensity (IOD). The experimental results showed that, see Figure 9, the crude Cyperus rotundus extract composition and the vinegar-processed Cyperus rotundus extract composition have good improvement effects on liver injury in zebrafish with alcoholic liver injury, and the effect of the vinegar-processed Cyperus rotundus extract composition is better than that of the crude Cyperus rotundus extract composition.

[0113] (3) Protective effect of the active ingredient enrichment of Cyperus rotundus on zebrafish with acute liver injury

[0114] Using transgenic zebrafish with Tg(L-FABP:EGFP) liver-labeled green fluorescent protein as experimental animals, when the zebrafish developed to 72 hpf, under a stereomicroscope, select zebrafish larvae of the Tg transgenic line with normal development and green fluorescent-labeled liver fluorescence, and randomly divide them into a control group, a thioacetamide model group (10 mM TAA), an S-adenosylmethionine positive drug control group (50 μM SAM-e), and a drug treatment group (10 mM TAA + Cyperus rotundus enrichment (6.25, 12.5 μg / mL, 25 μg / mL)). Zebrafish in the control group were fed with normal fish-raising water, zebrafish in the model group were treated with TAA added to the fish-raising water, and zebrafish in the treatment group were treated with the active ingredient enrichment of Cyperus rotundus while being modeled with TAA. Each group was set with 3 replicate wells, with 20 larvae in each well. 5 mL of the treatment solution was added to each well and cultured in an incubator. 24 hours after treatment, the zebrafish were anesthetized with tricaine at a mass concentration of 0.3‰, and then fixed on a glass slide with 3% methylcellulose. The zebrafish were fixed in a lateral position for photography. The changes in the liver morphology, liver area, and liver fluorescence intensity of zebrafish larvae were observed under a fluorescence microscope. The experimental results show that Figure 10 , the crude Cyperus rotundus extract composition and the vinegar-processed Cyperus rotundus extract composition have good improvement effects on liver injury in zebrafish with acute liver injury, and the effect of the vinegar-processed Cyperus rotundus extract composition is better than that of the crude Cyperus rotundus extract composition.

[0115] (4) Effect of the active ingredient enrichment of Cyperus rotundus on the heart rate of zebrafish with arrhythmia

[0116] Using wild-type AB strain zebrafish larvae at 48 hours post-fertilization (hpf) (which can be purchased from the National Zebrafish Resource Center) as experimental animals, randomly select healthy zebrafish and transfer them into a 24-well plate, with 10 in each well, and use 10 μM terfenadine to create an arrhythmia model. Set a blank control group (Ctl), a model group (Ter), and a gradient concentration drug group (5 μg / mL, 10 μg / mL, 20 μg / mL). The blank control group was added with zebrafish rearing water, the model group was added with terfenadine solution, and the drug group was co-treated with the active ingredient enrichment of Cyperus rotundus at gradient concentrations and terfenadine solution. After 24 hours of drug administration, record the video for 1 minute under a microscope, and according to the video, record the number of heartbeats of the zebrafish to characterize the effect of the drug on the heart rate of the zebrafish. The experimental results show that Figure 11The ethanol extract composition of Rhizoma Cyperi processed with vinegar has a good effect on improving the heart rhythm of zebrafish with arrhythmia. Under the experimental concentration conditions, the ethanol extract composition of raw Rhizoma Cyperi shows no obvious improvement effect.

[0117] (5) Comparison of the anti-enteritis activities of different parts eluted by ethanol from Rhizoma Cyperi

[0118] Collect the fractions of the raw Rhizoma Cyperi extract in Example 1 for the following experiments.

[0119] Using zebrafish Tg(zlyz:EGFP) with green fluorescently labeled macrophages as experimental animals, an inflammatory bowel disease zebrafish model was constructed using trinitrobenzenesulfonic acid (TNBS). The zebrafish were randomly divided into a blank control group (Control), a TNBS model control group (TNBS), and sample groups of different elution parts (water elution part, 30% ethanol elution part, 50% ethanol elution part, 70% ethanol elution part, 95% ethanol elution part). Select healthy Tg(zlyz:EGFP) zebrafish at 72 h after fertilization and randomly transfer them into 24-well plates, with 10 fish per well and 2 replicate wells. The zebrafish in the blank control group were incubated in zebrafish culture water without TNBS, and the zebrafish in other groups were incubated in zebrafish culture water containing TNBS (60 μM) under the same conditions. After 48 h, the extract samples were added to the 24-well plates respectively. After 24 h of incubation, the zebrafish larvae were anesthetized with tricaine (0.2%, w / v). Zebrafish larvae were randomly selected and photographed under a fluorescence microscope, and the number of neutrophils in the zebrafish intestine was counted. The experimental results are shown in Table 4 below. It can be seen from the experimental results that the 50% ethanol elution fraction and the 70% ethanol elution fraction have more significant activities, and the difference between the two is not large.

[0120] Table 4. Number of neutrophils in the intestine of zebrafish with inflammatory bowel disease treated with different elution parts

[0121] Group Neutrophil count Significance (compared with the model group) Blank control group 16.00±0.08 ****P＜0.0001 TNBS model group 29.25±0.13 / Water elution group 26.75±0.04 ns 30% ethanol 25.63±0.10 *P＜0.05 50% ethanol 19.50±0.09 ****P＜0.0001 70% ethanol 19.25±0.10 ****P＜0.0001 95% ethanol 25.88±0.07 *P＜0.05

[0122] The present invention first discloses an extract composition of Rhizoma Cyperi. The preparation method of the composition provided by the present invention is simple and is conducive to popularization and application. The composition has the effects of improving inflammatory bowel disease, neuroinflammation, and toxic hepatitis. The inventors also found that the composition extracted from Rhizoma Cyperi processed with vinegar, in addition to having the above effects, also has the effect of improving arrhythmia.

Claims

1. A method for preparing a Cyperus rotundus extract composition, characterized in that: The steps include: (1) crushing Cyperus rotundus, using 30%-80% ethanol aqueous solution as an extraction solvent, with a mass volume ratio of material to liquid of 1:(10-15) g / mL, an extraction temperature of 60-80° C., extracting 1-3 times, each extraction time of 2-3 hours, and collecting the extract as a crude extract; (2) the crude extract was treated with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the ultrafiltrate was collected; (3) The ultrafiltrate is adsorbed on a D101 macroporous resin column and eluted with 30%, 50%, 70% and 90% ethanol in sequence. The 50%-70% eluate is combined, concentrated and dried to obtain a Cyperus rotundus extract composition.

2. The method according to claim 1, characterized in that In step (1), the Cyperus rotundus includes raw Cyperus rotundus and / or vinegar Cyperus rotundus; Preferably, in step (1), the Cyperus rotundus is vinegar-cooked Cyperus rotundus.

3. The method according to claim 1, characterized in that In step (1), the cyperus rotundus is crushed and passed through a 40-50 mesh sieve; Preferably, in step (1), a 50% by volume ethanol aqueous solution is used as the extraction solvent; and the extraction temperature is 60°C.

4. The method according to claim 1, characterized in that In step (2), the transmembrane pressure of the ultrafiltration treatment is 0.5-1.5 MPa and the temperature is 25-40°C.

5. The method according to claim 1, characterized in that In step (3), the elution flow rate is 1-3 BV / h; Preferably, in step (3), the elution flow rate is 2 BV / h; Preferably, in step (3), rotary evaporation, concentration and drying are performed; Preferably, in step (3), the temperature for concentrating and drying is 40-45°C.

6. Use of the Cyperus rotundus extract composition prepared by the method according to any one of claims 1 to 5 in any one of the following: ① Application in the preparation of anti-inflammatory drugs; ② Application in the preparation of drugs for treating inflammatory bowel disease, neuroinflammation and / or toxic hepatitis.

7. The use according to claim 6, characterized in that The inflammatory bowel diseases include: ulcerative colitis; The toxic hepatitis includes: alcoholic liver disease, drug-induced hepatitis; Preferably, the alcoholic liver disease includes alcoholic fatty liver and / or alcoholic hepatitis; Preferably, the drugs causing drug-induced hepatitis include: one or more of isoniazid, rifampicin, pyrazinamide, cyclophosphamide, tripterygium wilfordii, and Polygonum multiflorum.

8. Use of the Cyperus rotundus extract composition prepared by the method according to any one of claims 1 to 5 in the preparation of a drug for treating arrhythmia, wherein in the preparation method according to any one of claims 1 to 5, the Cyperus rotundus is vinegar-cured Cyperus rotundus.

9. A medicine comprising the Cyperus rotundus extract composition prepared by the method according to any one of claims 1 to 5.

10. The drug according to claim 9, characterized in that Contains pharmaceutically acceptable excipients; Preferably, the excipients include one or more of a disintegrant, a binder or a sustained-release material; Preferably, the dosage form of the drug includes tablets, capsules or granules.

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