Use of extract of fructus aurantii in preparation of products for relieving and / or treating vascular calcification

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of extract of fructus aurantii in preparation of products for relieving and / or treating vascular calcification. The application discloses that the extract of fructus aurantii rich in flavones can significantly relieve vascular calcification, reduce serum calcium ion level, inhibit hydroxyapatite deposition in the vascular wall, improve vascular compliance, and maintain the thickness of the arterial wall and the inner diameter of the lumen. In addition, the application also proves that the extract can improve the intestinal microbial community structure, promote the colonization of beneficial bacteria, reduce the abundance of harmful bacteria, and enhance the phagocytosis by regulating metabolites, thereby stabilizing the calcium homeostasis in vascular smooth muscle cells, and can provide a new scheme for clinical treatment of vascular calcification.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Citrus reticulata extract in the preparation of products for relieving and / or treating vascular calcification. Background Technology

[0002] Vascular calcification is a pathological process characterized by the abnormal deposition of hydroxyapatite in the blood vessel walls. It is a common pathological change in many diseases, including atherosclerosis, diabetes, chronic kidney disease, and hypertension. This pathological process increases the workload of the left ventricle and causes insufficient organ perfusion. It is a key risk factor for heart failure, cardiovascular death, and chronic limb ischemia, and has become an increasingly serious global health burden.

[0003] The prevalence of vascular calcification increases with age, and is particularly pronounced in certain high-risk populations such as those with diabetes and chronic kidney disease. In patients with type 2 diabetes, the prevalence of lower extremity arterial calcification can reach 17%-42%; in patients with chronic kidney disease, especially those with end-stage renal disease, the burden of vascular calcification is extremely severe, with a prevalence as high as 80%-90%. In patients who undergo amputation due to severe limb ischemia, the detection rate of foot vascular calcification can reach 72%, an indicator directly correlated with extremely poor clinical prognosis.

[0004] The occurrence of vascular calcification is closely related to a series of traditional and metabolic risk factors, including advanced age, male sex, hypertension, dyslipidemia, diabetes, and chronic kidney disease. Among these, diabetes and chronic kidney disease are considered key "accelerators" of its progression, as the calcium and phosphorus metabolism disorders they induce jointly promote the deposition of calcium salts in the vascular media. From a health hazard perspective, vascular calcification impairs vascular elasticity, leading to increased cardiac afterload, reduced coronary artery and peripheral tissue perfusion, and is an independent predictor of heart failure. In the lower extremities, it is the core pathological basis for chronic limb-threatening ischemia, foot ulcers, and even amputation, and also significantly increases the difficulty and risk of peripheral vascular interventional therapy, reducing the effectiveness of revascularization.

[0005] Despite the serious health challenges posed by vascular calcification, current clinical practice lacks effective treatments to reverse or cure this pathological process. Existing treatment options primarily focus on controlling risk factors and directly intervening in the calcification process; however, both approaches have significant limitations. Regarding risk factor control, clinical practice mainly involves using phosphate binders (such as sevelamer and lanthanum carbonate) to lower serum phosphorus levels, or using calcimenimetrists (such as cinacalcet) to regulate calcium and phosphorus metabolism and parathyroid hormone levels. Meta-analyses have shown that cinacalcet may have a relative advantage in slowing the progression of vascular calcification in hemodialysis patients compared to other drugs, including sodium thiosulfate and bisphosphonates. However, the efficacy of these drugs is mainly reflected in improvements in biochemical indicators, and large-scale clinical trials are lacking to confirm their ability to significantly improve cardiovascular hard endpoint outcomes. Sodium thiosulfate acts as both a calcium chelator and an antioxidant. Although small studies have shown its potential to slow calcification progression, high-level clinical evidence remains scarce, and its long-term safety and impact on patient prognosis are still unclear.

[0006] In recent years, the academic community has recognized that vascular calcification is not simply a local vascular lesion; its occurrence and development are closely related to systemic metabolic disorders, and the structural and functional imbalance of the gut microbiota plays a crucial role in this process. In metabolic diseases such as chronic kidney disease, the gut microbiota often exhibits significant dysbiosis, manifested as decreased microbiota diversity and altered abundance of specific microbiota. This dysbiosis can participate in the occurrence and development of vascular calcification through multiple mechanisms. On the one hand, the excessive proliferation of harmful bacteria can directly produce or promote the production of pro-inflammatory and pro-calcification toxins. For example, studies have found a significant increase in the abundance of *Prevotella copri* in the feces of rats and patients with chronic kidney disease, and a positive correlation with aortic calcification scores. The lipopolysaccharide (LPS) produced by this bacterium can activate Toll-like receptor 4 (TLR4) on vascular smooth muscle cells (VSMCs), thereby initiating the nuclear factor κB (NF-κB) signaling pathway, driving VSMCs to transform into an osteoblastic phenotype, and thus accelerating vascular calcification. On the other hand, gut microbiota dysbiosis can lead to a decrease in the synthesis of protective metabolites. Short-chain fatty acids (SCFAs) are important metabolites produced by the fermentation of dietary fiber by gut microbiota, playing a crucial role in maintaining intestinal barrier integrity and regulating immune and metabolic homeostasis. Clinical observational studies have shown that SCFA levels (especially propionate) in blood and feces are independently negatively correlated with vascular calcification scores. Animal experiments have further confirmed that propionate or acetate supplementation can remodel gut microbiota (e.g., enrich Akkermansia), increase SCFA levels, improve intestinal barrier function, reduce systemic inflammation, and ultimately significantly reduce vascular calcification. However, the effects of SCFAs are complex, and not all SCFAs exert a protective effect. Studies have found that butyrate, also an SCFA, can promote high-phosphorus-induced osteogenic differentiation of VSMCs and vascular calcification in vitro and in vivo by inhibiting histone deacetylase (HDAC) and activating the NF-κB signaling pathway. Furthermore, antibiotics disrupting gut microbiota lead to a decrease in Bacteroidetes abundance and serum SCFA levels (such as acetate), thereby exacerbating vascular calcification; acetate supplementation can reverse this process. From a signaling pathway perspective, gut microbiota and its metabolites indirectly or directly influence vascular calcification by affecting key pathways related to bone homeostasis. For example, SCFAs can promote bone morphogenetic protein (BMP) / SMADs and Wnt / β-catenin signaling pathways by inhibiting HDACs; these pathways also play a crucial role in driving osteogenic differentiation of VSMCs. SCFAs can also indirectly affect bone formation by activating G protein-coupled receptors (such as GPR41) and peroxisome proliferator-activated receptor γ (PPARγ), and may have a similar effect on vascular calcification.In addition, direct translocation of gut microbiota (e.g., bacteria or their components entering the bloodstream) has been shown to exacerbate vascular calcification by activating Toll-like receptor 9 (TLR9) to mediate BMP-2 expression. Gut microbiota can also participate in the regulation of bone and blood vessel metabolic balance through various mechanisms, including modulating immune responses (e.g., regulating Treg cell function), influencing hormone secretion (e.g., glucagon-like peptide-1, GLP-1), and regulating calcium absorption.

[0007] In summary, the gut microbiota and its metabolites can create a dual imbalance of "increased harmful products and decreased beneficial products," directly regulating inflammatory pathways and interfering with osteogenic signaling pathway networks, thereby participating in the development and progression of vascular calcification. Therefore, if dietary or pharmacological methods can be used to reshape gut microbiota homeostasis, increase the production of beneficial metabolites such as propionate and acetate, and inhibit the release of harmful bacteria and their toxins, it may provide a novel approach to the prevention and treatment of vascular calcification. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention has confirmed through research that the extract of Citrus reticulata, which is rich in flavonoids, can significantly downregulate osteogenic transformation-related indicators of vascular smooth muscle cells in a vascular calcification model. At the same time, it can improve the intestinal microecological environment under vascular calcification and has significant regulatory activity on intestinal flora and metabolites. It is expected to be used to prepare products that alleviate and / or treat vascular calcification.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the use of Citrus reticulata extract in the preparation of drugs for relieving and / or treating vascular calcification.

[0010] This invention successfully established a mouse model of vascular calcification using vitamin D3 overload. It was found that a 200 mg / kg dose of Citrus reticulata extract exhibited excellent vascular repair effects in these mice, significantly alleviating vascular calcification damage (Alizarin Red and Von Kassa staining showed a reduction in staining area after Citrus reticulata extract intervention). Simultaneously, this invention found that a 200 mg / kg dose of Citrus reticulata extract could improve the intestinal microbiota composition of mice with vitamin D3 overload-induced vascular calcification, promoting the colonization of beneficial bacteria, reducing the abundance of harmful bacteria, and regulating cell burial function. Therefore, the use of Citrus reticulata extract can be an effective strategy for the prevention and treatment of vascular calcification.

[0011] Preferably, the flavonoid content in the Citrus reticulata extract is ≥70%.

[0012] More preferably, the flavonoid content in the Citrus reticulata extract is ≥75%. For example, it can be 76%, 78%, 80%, 85%, 90%, or 95%.

[0013] Citrus flavonoids are bioactive natural flavonoids extracted from Citrus reticulata peel. Studies have shown they possess anti-cancer properties, inhibiting tumor cell proliferation and inducing apoptosis. However, their application in treating vascular calcification-related diseases is rarely reported. Some studies have found that citrus flavonoids have a protective effect against diabetes, but in practical applications, higher dosages are required to achieve good protective effects, significantly limiting their application. This invention, however, demonstrates that a dose of only 200 mg / kg of Citrus reticulata peel extract can achieve excellent therapeutic effects on vascular calcification.

[0014] Preferably, the vascular calcification is a more severe type of vascular calcification that is prevalent in people with metabolic disorders, electrolyte imbalances, or acid-base imbalances; the metabolic disorders, electrolyte imbalances, or acid-base imbalances are caused by hyperlipidemia, hyperphosphatemia, vitamin D overdose, chronic kidney disease (CKD), or diabetes. The chronic kidney disease (CKD) includes end-stage renal disease.

[0015] More preferably, the vascular calcification includes vascular calcification caused by chronic kidney disease (CKD).

[0016] Vascular calcification poses serious risks, easily leading to myocardial ischemia, left ventricular hypertrophy, and heart failure, as well as thrombosis and plaque rupture. This invention has found that using 200 mg / kg of Citrus reticulata extract can significantly alleviate these symptoms and improve the gut microbiota composition in animal models of vascular calcification, promoting the colonization of beneficial bacteria, reducing the abundance of harmful bacteria, and regulating metabolites to enhance cell burial function, thus providing a therapeutic strategy for the treatment of vascular calcification.

[0017] Preferably, the relief and / or treatment of vascular calcification comprises at least one of the following effects: A) Improves decreased vascular compliance, thickened arterial walls, narrowed vascular lumens, atherosclerotic plaque instability, and plaque rupture caused by vascular calcification; B) Down-regulate the ankle-brachial index (ABI) and pulse wave velocity (PWV) values ​​in subjects with vascular calcification; C) Improves clinical symptoms in subjects with vascular calcification, such as dizziness, headache, chest tightness, chest pain, abdominal pain, refractory hypertension, and lower extremity gangrene; D) Reduces inflammation levels in subjects with vascular calcification and improves decreased arterial wall compliance; E) Regulate the gut microbiota structure of subjects with vascular calcification and increase the abundance of beneficial bacteria colonization in the gut; F) Upregulates cell burial, thereby improving vascular calcification in subjects.

[0018] Preferably, the preparation method of the Citrus reticulata extract includes the following steps: S1. Fresh tangerine peel is dried and then pulverized to obtain tangerine peel powder; S2. Mix the Citrus reticulata powder from S1 with water, extract with hot water, collect the filtrate, and then concentrate it to obtain the hot water extract. S3. Mix the hot water extract from S2 with the protein removal reagent, centrifuge after reaction to remove the supernatant solution, repeat the protein removal step 6 to 20 times, and concentrate under reduced pressure to obtain the crude extract. S4. Add ethanol to the crude extract of S3, let it stand, centrifuge and collect the precipitate. After redissolving the precipitate in water, dialyze and dry it to obtain the Citrus reticulata extract.

[0019] The method for preparing Citrus reticulata extract of the present invention can largely retain the active ingredients in Citrus reticulata, such as polysaccharides, flavonoids, and organic acids. Among them, polysaccharides are one of the main bioactive components. The monosaccharide composition is mainly composed of three monosaccharides: mannitol (7.74%), glucose (70.41%), and galactose (16.38%). It has multiple functions such as antioxidation, immune regulation, and anti-tumor activity. It can enhance the body's immunity, promote intestinal health, and help fight free radicals and delay aging. Flavonoids can help eliminate free radicals in the body, reduce oxidative stress, and protect cells from damage. Organic acids (such as malic acid and citric acid) help promote metabolism and improve the health of the digestive system.

[0020] More preferably, the Huajuhong includes Gutian Huajuhong and Huazhou Huajuhong.

[0021] More preferably, in S2, the volume ratio of the citrus powder to water is 1:20-40; the hot water extraction temperature is 90-110℃, the time is 1-4 h, and the hot water extraction is performed 1-4 times.

[0022] More preferably, in S3, the protein removal reagent comprises chloroform and / or n-butanol, and the volume ratio of the hot water extract to the protein removal reagent is 1:1 to 3.

[0023] Furthermore, the volume ratio of chloroform to n-butanol is 3 to 5:1.

[0024] More preferably, in step S4, the volume ratio of the crude extract to ethanol is 1:3 to 5, and the ethanol is an aqueous solution of ethanol with an ethanol content greater than 95%. For example, the volume ratio can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc. The ethanol solution can be 96%, 97%, 98%, 99%, 99.5%, or 99.9%, etc.

[0025] More preferably, in step S4, the settling is performed at 0–4°C for 8–12 h, the dialysis molecular weight cutoff is 2.5–3.5 kDa, and the time is 24–72 h. More preferably, it is 3 kDa.

[0026] More preferably, in S2, the volume of the concentrated solution is 1 / 4 to 1 / 15 of the volume of the filtrate.

[0027] More preferably, in S3, the centrifugation speed is 3000-5000 r / min and the time is 8-20 min.

[0028] More preferably, in S4, the centrifugation speed is 3000-8000 r / min and the time is 10-20 min.

[0029] More preferably, in S1, the drying method includes oven drying or vacuum freeze drying, wherein the temperature of the oven drying is 40–70°C.

[0030] More preferably, in S4, the mass-to-volume ratio of the precipitate to water is 1g:8-12mL.

[0031] More preferably, the drug further comprises pharmaceutically acceptable excipients and / or carriers.

[0032] Furthermore, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, lubricants, sweeteners, and flavorings.

[0033] Furthermore, the excipient includes water.

[0034] Furthermore, the filler includes at least one of starch and sucrose.

[0035] Furthermore, the adhesive includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

[0036] Furthermore, the wetting agent includes glycerin.

[0037] Furthermore, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0038] Furthermore, the absorption enhancer includes a quaternary ammonium compound.

[0039] Furthermore, the surfactant includes hexadecyl alcohol.

[0040] Furthermore, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0041] Furthermore, the vascular calcification treatment also includes a pharmaceutically acceptable carrier. Such a pharmaceutically acceptable carrier is generally recognized for this purpose and serves as an inactive ingredient in the drug. Compilations of pharmaceutically acceptable carriers can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994). For example, adsorbent carriers include at least one of kaolin and soap clay.

[0042] Furthermore, the dosage of the vascular calcification treatment drug can be adjusted according to the actual situation.

[0043] Furthermore, the vascular calcification treatment drug can be administered via the gastrointestinal tract or / and non-gastrointestinal routes.

[0044] Furthermore, the non-gastrointestinal route of administration is selected from injection, respiratory administration, skin administration, mucosal administration, or cavity administration.

[0045] Furthermore, the non-gastrointestinal drug dosage form is selected from injections, sprays, aerosols, patches, etc.

[0046] Furthermore, the dosage form of the drug administered via the gastrointestinal tract is selected from tablets, capsules, powders, granules, pills, solutions, emulsions, or syrups, etc.

[0047] More preferably, the drug is in the form of an oral formulation, an injection, or a topical formulation.

[0048] Furthermore, the oral preparations include tablets, capsules, pills, powders, granules, syrups, or solutions; the injectable preparations include injection solutions or lyophilized powder for injection; and the topical preparations include creams, ointments, sprays, aerosols, gels, poultices, or patches.

[0049] Furthermore, the carriers used in the preparation of oral formulations can be conventional pharmaceutical excipients such as starch, dextrin, cyclodextrin, various chemically modified cyclodextrins, sucrose, and stearates. The lyophilized powder injections can be prepared using methods such as aseptic spray drying, low-temperature vacuum drying, and freeze-drying. The subsequent preparation processes and equipment for each formulation are all conventional technologies in the pharmaceutical field, and this invention does not limit them. Other features and advantages of this invention will be described in the following description.

[0050] Compared with the prior art, the beneficial effects of the present invention are: This invention, through the construction of a vascular calcification model, found that the use of flavonoid-rich Citrus reticulata extract can significantly alleviate vascular calcification (such as vascular calcification induced by high-dose vitamin D3), reduce serum calcium ion levels, inhibit hydroxyapatite deposition in the vascular wall, improve vascular compliance, and maintain arterial wall thickness and luminal diameter. Furthermore, this invention also demonstrates that the extract can improve the intestinal microbiota structure, promote beneficial bacteria colonization, reduce the abundance of harmful bacteria, and enhance cell burial by regulating metabolites, thereby stabilizing intracellular calcium homeostasis in vascular smooth muscle cells, providing a novel approach for the clinical treatment of vascular calcification. Attached Figure Description

[0051] Figure 1 The effect of Citrus reticulata extract on mouse body weight; Figure 2 To investigate the effect of Citrus reticulata extract on vascular calcification in mice, A represents alizarin red staining of fresh aortic tissue, B represents calcium salt staining of paraffin sections of aortic rings, and C represents hematoxylin-eosin staining of paraffin sections of aortic rings. Figure 3 The dilution curves of mouse gut microbiota samples at the OTU level are shown for chao1, observed-feature, Shannon, and Simpson, where A is the chao1 dilution curve, B is the observed-feature dilution curve, C is the Shannon dilution curve, and D is the Simpson dilution curve. Figure 4 The results show the effect of Citrus reticulata extract on the β diversity of intestinal flora in mice. A is a visualization of PLS ​​analysis, B is the PCoA analysis result, and C is the NMDS analysis result. Figure 5 The results show the effects of Citrus reticulata extract on the species composition of mouse gut microbiota at the phylum and genus levels. In this figure, A represents the statistical results of species differences at the phylum level among the mouse groups, and B represents the statistical results of species differences at the genus level among the mouse groups. Figure 6 The results of differential analysis of dominant gut microbiota in mice are shown, where A is a taxonomic clade diagram of species and B is a bar chart of the distribution of LDA values ​​of significantly different species. Figure 7 The graphs show the quality control data of mouse metabolomics; A is the Pearson correlation heatmap of QC samples in negative ion mode, and B is the Pearson correlation heatmap of QC samples in positive ion mode. Figure 8The results of PCA and OPLS-DA analyses of vascular metabolites in mice are shown below. A represents the PCA analysis between the blank control group and the model group; B represents the OPLS-DA analysis between the blank control group and the model group; C represents the PCA analysis between the model group and the 50 mg / kg Citrus reticulata extract group; D represents the OPLS-DA analysis between the model group and the 50 mg / kg Citrus reticulata extract group; E represents the PCA analysis between the model group and the 100 mg / kg Citrus reticulata extract group; and F represents the OPLS-DA analysis between the model group and the 100 mg / kg Citrus reticulata extract group. Figure 9 The results show the effect of Citrus reticulata extract on the composition of fecal metabolites in mice. In this figure, A represents the Venn diagram analysis results, and B represents the statistical results of differential metabolites in each group. Figure 10 The enrichment pathway in metabolism after subcutaneous injection of excessive VD3-induced vascular calcification in mice; Figure 11 The enrichment pathway in fecal metabolism of mice after intervention with 50 mg / kg Citrus reticulata extract; Figure 12 The enrichment pathway in fecal metabolism of mice after intervention with 200 mg / kg Citrus reticulata extract. Detailed Implementation

[0052] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all available through conventional commercial channels. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0054] In the following examples, the fresh Huazhou tangerine peel used was extracted from Huazhou pomelo, and the process included the following steps: (1) Harvesting: Select unripe Huazhou pomelos that are about 30-45 days old (purchased from Huazhou, Guangdong).

[0055] (2) Blanching: Place the fresh fruit at 8-15℃, RH 55%-70%, and slight forced ventilation until the weight loss is about 12% (4 days), and then blanch it in hot water at 80-90℃ for 5 minutes.

[0056] (3) Drying: A segmented low-temperature baking process is adopted: in the first 24 hours, the temperature is set at 60℃ for slow drying; in the middle (24-72 hours), the temperature is raised to 70℃ to accelerate dehydration; in the later stage (after 72 hours), the temperature is lowered back to 60℃ until fully dry. The whole process takes about 4 days (72-96 hours) to ensure that the moisture content is reduced to below 12% and to avoid the decomposition of heat-sensitive components caused by high temperature.

[0057] (4) Flavor enhancement: Set the final high-temperature treatment (80±2℃) for about 2 hours.

[0058] Example 1: This embodiment provides a method for preparing Citrus reticulata extract, specifically including the following steps: (1) Place 100 g of fresh tangerine peel in a vacuum freeze dryer. After freeze drying and dehydration, use a pulverizer to pulverize the dried tangerine peel to 20-60 mesh to obtain tangerine peel powder.

[0059] The freeze-drying dehydration conditions are as follows: (1) Pre-freezing stage: set the pre-freezing temperature to -50 to -35 ℃ and pre-freeze at a constant temperature for 2 to 4 hours; (2) Sublimation drying stage: maintain the vacuum degree of the system at 10 to 30 Pa, the temperature of the heating plate at 35 to 45 ℃, and dry for 12 to 24 hours until the moisture content of the material drops to below 3% to 5%.

[0060] (2) Mix the above-mentioned citrus powder with distilled water at a mass-volume ratio of 1:30, and extract by reflux of boiling water for 2 hours. The extraction is performed once. Then, filter out the citrus powder residue with a filter screen, collect the filtrate, and concentrate it at 60°C using a rotary evaporator to obtain a concentrated extract with a volume of 1 / 4 to 1 / 5 of the original filtrate, i.e., hot water extract.

[0061] (3) The above hot water extract was subjected to deproteinization using the Sevag method, wherein the Sevag reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The specific method for deproteinization using the Sevag method is as follows: The above hot water extract was mixed with Sevag reagent at a volume ratio of 1:1 and placed on a shaker at 270 rpm for 2 h at 25 °C. Then, it was centrifuged at 3500 rpm for 10 min at 4 °C, and the supernatant was collected. This shaking and centrifugation process was repeated 10 times. The supernatant was collected and then concentrated under reduced pressure (60 °C) to 1 / 10 of its original volume to obtain the crude extract solution after protein removal. (4) Add 3 times the volume of anhydrous ethanol to the crude extract solution after protein removal and let it stand overnight at 4 °C; then centrifuge at 3500 r / min for 10 min, collect the precipitate, dissolve the precipitate in an appropriate amount of ultrapure water at a ratio of 1 g: 10 mL, dialyze at 4 °C for 48 h, the molecular weight cutoff of the dialyzed treatment is 3 kDa, and finally freeze dry under vacuum to obtain the Citrus reticulata extract for later use.

[0062] Example 2: This embodiment provides a method for preparing Citrus reticulata extract, specifically including the following steps: (1) After drying 100 g of fresh tangerine peel at 70℃ for 0.5 h, the dried tangerine peel was pulverized using a pulverizer to obtain tangerine peel powder.

[0063] (2) Mix the above-mentioned citrus powder with distilled water at a mass-volume ratio of 1:20, and extract with hot water at 90°C for 2 h, and extract three times. Collect the extract and centrifuge at 4000 r / min for 15 min. Collect the supernatant and concentrate it under reduced pressure at 60°C using a rotary evaporator to obtain a concentrated extract with 1 / 10 of the original filtrate volume, i.e., hot water extract.

[0064] (3) The above hot water extract was subjected to deproteinization using the Sevag method, wherein the Sevag reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The specific method for deproteinization using the Sevag method is as follows: The above hot water extract was mixed with Sevag reagent at a volume ratio of 1:1 and placed on a shaker at 270 rpm for 20 min at 25 °C. Then it was centrifuged at 3000 r / min for 10 min at 4 °C. The supernatant was collected, and the shaking and centrifugation steps were repeated 10 times. The supernatant was collected and then concentrated under reduced pressure at 60 °C to 1 / 10 of its volume to obtain the crude extract solution after protein removal.

[0065] (4) Add 3 times the volume of anhydrous ethanol to the crude extract solution after protein removal and let it stand overnight at 4 °C; then centrifuge at 8000 r / min for 10 min, collect the precipitate, dissolve the precipitate in ultrapure water at a ratio of 1 g: 10 mL, dialyze at 4 °C for 48 h, the molecular weight cutoff of the dialyzed treatment is 3 kDa, and finally freeze dry under vacuum to obtain the Citrus reticulata extract for later use.

[0066] Example 3: This embodiment provides a method for preparing Citrus reticulata extract, specifically including the following steps: (1) After drying 100 g of fresh tangerine peel at 60℃ for 0.5 h, the dried tangerine peel was pulverized using a pulverizer to obtain tangerine peel powder.

[0067] (2) Mix the above-mentioned citrus powder with distilled water at a mass-volume ratio of 1:30, and extract with hot water at 100℃ for 4 h, and extract 3 times; collect the extract and centrifuge at 8000 r / min for 10 min, collect the supernatant and concentrate it by rotary evaporation at 60℃ under reduced pressure to obtain a concentrated extract with 1 / 10 of the original filtrate volume, i.e. hot water extract.

[0068] (3) The above hot water extract was subjected to deproteinization using the Sevag method, wherein the Sevag reagent was prepared by mixing chloroform and n-butanol in a volume ratio of 4:1. The specific method for deproteinization using the Sevag method is as follows: The above hot water extract was mixed with Sevag reagent at a volume ratio of 1:1 and placed on a shaker at 270 rpm for 20 min at 25 °C. The mixture was then centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatant was collected. This shaking and centrifugation process was repeated 10 times, and the supernatant was collected and then concentrated under reduced pressure at 60 °C to 1 / 10 of its original volume to obtain the crude extract solution after protein removal.

[0069] (4) Add 3 times the volume of anhydrous ethanol to the crude extract solution after protein removal and let it stand overnight at 4 °C; then centrifuge at 4000 r / min for 15 min, collect the precipitate, dissolve the precipitate in ultrapure water at a ratio of 1 g: 10 mL, dialyze at 4 °C for 48 h, the molecular weight cutoff of the dialyzed treatment is 3 kDa, and finally freeze dry under vacuum to obtain the Citrus reticulata extract for later use.

[0070] Experimental Example 1: Determination of Sugar and Flavonoid Content in Citrus reticulata Extract 1. The sugar content of the Citrus reticulata extracts prepared in Examples 1-3 above was determined using the following methods: The sugar content in Citrus reticulata extract was determined using the phenol-sulfuric acid method. A standard curve was plotted with the concentration of glucose in the standard solution on the x-axis and the absorbance of the glucose solution at 490 nm on the y-axis.

[0071] The fitted standard curve formula is: y = 3.52598x + 0.0261005, r 2 = 0.99956 (r) 2≥0.99). The absorbance of the citrus velutipes extract solution prepared in Example 1 was measured to be 1.373 using the phenol-sulfuric acid method. Substituting this into the above standard curve formula, the sugar concentration in the citrus velutipes extract was found to be 0.382 mg / L. Further calculation showed that the sugar content of the citrus velutipes extract could reach 76.4%, indicating that the citrus velutipes extract prepared using the process method in Example 1 has high purity of citrus velutipes polysaccharide (Citrus velutipes extract) and can be used for subsequent research.

[0072] In addition, the average absorbance of the Citrus reticulata extract solution prepared in Example 2 was 1.956, and the sugar content was 67.9%. The average absorbance of the Citrus reticulata extract solution prepared in Example 3 was 1.989, and the sugar content was 69.8%.

[0073] 2. The flavonoid content of the Citrus reticulata extracts prepared in Examples 1-3 above was determined using the following methods: The total flavonoid content was determined using a Plant Flavonoid Content Detection Kit (Catalog No. BC1330) from Beijing Solarbio Technology Co., Ltd. The determination method employed was the aluminum ion colorimetric method (NaNO2-Al(NO3)3-NaOH system), and the procedure was performed according to the kit instructions. The specific method is as follows: (1) After accurately weighing the extract of Citrus reticulata, add 60% ethanol to dissolve it, extract by ultrasonication and centrifuge, and take the supernatant as the sample solution to be tested.

[0074] (2) Take a certain volume of sample solution and add the reagents provided in the kit in sequence (reagent 1: sodium nitrite solution; reagent 2: aluminum nitrate solution; reagent 3: sodium hydroxide solution). The reaction is carried out at room temperature to form a stable flavonoid-aluminum ion complex.

[0075] (3) After the reaction is complete, the absorbance is measured at a wavelength of 470 nm using a UV-Vis spectrophotometer.

[0076] (4) Preparation of standard curve: Using rutin as a reference, standard solutions of different concentrations were prepared with 60% ethanol. The colorimetric reaction was performed according to the instructions of the Beijing Solarbio Science & Technology Co., Ltd. Plant Flavonoid Content Detection Kit (BC1330), and the absorbance was measured at 470 nm. The standard curve equation was obtained through linear regression: A = 0.0048129 C + 0.010857, R 2 = 0.99997; Where A is the absorbance at 470 nm and C is the total flavonoid concentration in the sample, in μg / mL.

[0077] (5) Taking Example 1 as an example, 20.00 mg of Citrus reticulata extract was weighed, dissolved in 60% ethanol, and brought to a final volume of 10.00 mL. After ultrasonic extraction and centrifugation, the supernatant was collected. Since the total flavonoid content of the sample was high, the supernatant was diluted 20 times and subjected to a colorimetric reaction according to the kit instructions. The absorbance was measured at 470 nm. The total flavonoid content was calculated using the following formula: Total flavonoid content (mg RE / g) = C×V×D / m; Where C is the sample concentration (μg / mL) calculated from the standard curve, V is the total volume of the sample extract (mL), D is the dilution factor, and m is the sample weight (mg). In this test, V = 10.00 mL, D = 20, and m = 20.00 mg.

[0078] The total flavonoid content in the Citrus reticulata extract was determined to be 704.20 ± 6.60 mg RE / g, which is 70.42 ± 0.66% by mass percentage. The RSD of three parallel determinations was 0.94% (Table 1).

[0079] Table 1: Determination of total flavonoid content in Citrus reticulata extract Experiment Example 2: Effect of Citrus reticulata extract on vitamin D3-induced vascular calcification Taking the Citrus reticulata extract prepared in Example 1 as an example, the effect of Citrus reticulata extract on the vascular calcification model was detected by constructing a mouse vascular calcification model, specifically including the following contents.

[0080] 1. Establishment of a mouse model of vascular calcification Sixty male C57 BL / 6J mice (approximately 25g in weight, purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd.) aged 6-8 weeks were obtained and housed in an SPF-grade animal facility. The ambient temperature was controlled at 22-25℃, and the relative humidity at 55%-70%. A 12-hour / 12-hour light-dark cycle was used, and the mice had free access to water and food. All animal experiments conducted in this study strictly followed the relevant guidelines in the national standard "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T35892-2018).

[0081] After one week of acclimatization, mice were randomly divided into a blank control group, a model group, a 50 mg / kg Citrus reticulata extract group, a 100 mg / kg Citrus reticulata extract group, and a 200 mg / kg Citrus reticulata extract group. The mice were administered the drug via gavage once daily from the second day of modeling until the end of the modeling process (i.e., the seventh day of modeling).

[0082] 2. Aortic tissues from each group of mice were collected for vascular histopathological examination. The tissues were fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned and prepared, and then stained with alizarin red, calcium salt and hematoxylin-eosin (HE). The pathological morphological changes of vascular tissues were observed under an optical microscope.

[0083] 3. Effects of Citrus reticulata extract on mouse body weight and vascular calcification Figure 1 This section describes the changes in body weight of the experimental animals in each group during the experiment. At the beginning of the experiment, the initial body weight levels of the animals in each group were basically the same, indicating that the groups were comparable after randomization. As the experiment progressed, the body weight of the animals in each group showed different trends. The body weight of the animals in the normal control group continued to increase over time, showing a stable growth state; the body weight gain of the animals in the model group was significantly inhibited, and a downward trend was observed at some time points, suggesting that the modeling process affected the overall nutritional status and metabolic level of the animals. After intervention with Citrus reticulata extract, the trend of body weight change in the experimental animals was improved compared with that in the model group, and it could alleviate the model-induced weight loss to a certain extent. The above results indicate that the Citrus reticulata extract of the present invention has the effect of improving the overall metabolic state of model animals and maintaining physiological homeostasis.

[0084] The effects of Citrus reticulata extract on vascular morphology and pathology in mice are as follows: Figure 2 As shown in the figure. Calcium staining revealed significant calcium deposition on the vessel walls of the model group, which gradually decreased with increasing dosage of Citrus reticulata extract. HE staining showed disordered arrangement of smooth muscle cells in the vascular media and calcifications in the model group, while the vascular media structure in the Citrus reticulata extract intervention group was more regular and calcifications were reduced, further confirming that Citrus reticulata extract can improve VD3-induced vascular tissue pathological changes.

[0085] Experiment Example 3: Effects of Citrus reticulata extract on gut microbiota in a mouse model of vascular calcification This experiment investigated the effect of Citrus reticulata extract on the gut microbiota of a mouse model of vascular calcification. The construction and intervention methods of the vitamin D3-induced vascular calcification model were the same as those in Experiment 2, specifically including the following:

[0086] 1. Experimental Methods Microbial diversity analysis was conducted using the Illumina NovaSeq 6000 high-throughput sequencing platform. The experimental procedure was as follows: PCR amplification was performed using specific primers targeting the V4 region (515F / 806R), V4 region (528F / 706R), and ITS1 region (ITS5-1737F / ITS2-2043R) of the 16S rRNA gene. The reaction system included Plusion high-fidelity Master Mix. After 30 cycles of amplification, the products were purified using magnetic beads and mixed in equal volumes. For bioinformatics analysis, the PCR data were first split and paired-end spliced ​​(FLASH v1.2.11). Low-quality sequences were filtered using fastp v0.23.1, and chimeras (UCHIME) were removed by comparison with the Silva or Unie databases to obtain valid sequences. Subsequently, OTU clustering was performed using Uparse v7.0.1001 with 97% similarity to select representative sequences. Species annotation was performed using the Mothur (16S / Silva 138.1), RDP (18S / Silva 138.1), and BLAST (ITS / Unite v9.0) algorithms, with the Micro_NT database used as a supplement for regular regions; some annotation levels were completed using the NCBI database. Based on the OTU table, a phylogenetic tree was constructed using MUSCLE v3.8.31, and data were normalized using the minimum number of sample sequences. Alpha diversity was calculated using Qiime 1.9.1, including Observed OTUs, Chao1, Shannon, Simpson, and Faith PD indices, and dilution curves were plotted. Beta diversity was based on weighted / unweighted Unifrac distance, and differences in community structure among samples were analyzed using PCoA, NMDS, and UPGMA clustering. Differences between groups were tested using ANOSIM, Adonis, LEfSe, and Metagenomeseq methods. Furthermore, functional pathways and phenotypic predictions were performed using tools such as PICRUSt2, Tax4Fun, and BugBase.

[0087] 2. Experimental Results (1) Results of detection on the effect on the species composition of mouse gut microbiota The dilution curves of chao1, observed-features, shannon, and simpson at OTU levels in the gut microbiota samples of each group of mice are shown below. Figure 3 As shown in the figure, the dilution curves corresponding to the chao1 index, observed-features index, shannon index, and simpson index of each group all tend to flatten out at the end, indicating that the sequencing depth met the requirements and could cover the vast majority of bacterial diversity information in the sample.

[0088] Further β-diversity analysis was performed on the gut microbiota of each group of mice. Partial least squares discriminant analysis (PLS-DA) based on Bray-curtis distance was used for visualization to examine the distribution of gut microbiota samples in each group of mice. Results are as follows: Figure 4 As shown in the figure, there are clear boundaries between the blank control group, the model group, the 50 mg / kg Citrus reticulata extract group, and the 100 mg / kg Citrus reticulata extract group, indicating differences in the gut microbiota composition of the four groups of mice. Specifically, the sample distribution of the model group deviates significantly from that of the blank control group, while the sample distribution of the Citrus reticulata extract groups (especially the 200 mg / kg Citrus reticulata extract group) converges towards that of the blank control group.

[0089] The above results indicate that Citrus reticulata extract can alter the gut microbiota in mice with a vascular calcification model.

[0090] (2) Results of detection on the effect on the species composition of mouse gut microbiota The effects of Citrus reticulata extract on the species composition of mouse gut microbiota at the phylum level are as follows: Figure 5 As shown in Figure A, at the phylum level, the gut microbiota with relatively high abundance include Pseudomonadota, Bacillota, Bacteroidetes, Campylobacterota, Actinomycetota, Thermodesulfobacteriota, Deferribacterota, etc. (e.g., Figure 5 (As shown in A in the figure). Compared with the blank control group, the relative abundance of Pseudomonadota and Bacteroidota in the gut microbiota of the model group mice was significantly increased (P < 0.01), while the intervention of Citrus reticulata extract reduced the relative abundance of Pseudomonadota and Bacteroidota (P < 0.01).

[0091] Further analysis was conducted on the species composition of the mouse gut microbiota at the genus level, and the results were as follows: Figure 5Figure B shows the top ten most abundant gut microbiota at the genus level, including *Escherichia-Shigella*, *Desulfovibrio*, *Helicobacter*, *Lactobacillus*, *Ligilactobacillus*, *Staphylococcus*, *Adlercreutzia*, *Mammaliicoccus*, and *Bacteroides*. Compared with the control group, the relative abundance of *Escherichia-Shigella* in the gut microbiota of the model group mice was significantly increased (P < 0.001), while intervention with *Citrus reticulata* extract could reduce the relative abundance of *Escherichia-Shigella*.

[0092] The above results indicate that the extract of Citrus reticulata peel helps restore the gut microbiota of the model mice to a normal relative abundance.

[0093] (3) Results of detection on the influence of the dominant flora of mouse gut microbiota The LEfSe method was used to screen bacterial communities with a linear discriminant analysis value (LDA) greater than 3 (LDA > 3), and specific dominant bacterial communities in each group of mice were detected. The higher the LDA value, the more significant the difference between microbial colonies. The results of the differential analysis of dominant gut microbiota in mice are as follows: Figure 6 As shown in the figure, 23, 4, 2, and 6 dominant bacterial groups were screened in the blank control group, model group, 50 mg / kg Citrus reticulata extract group, and 100 mg / kg Citrus reticulata extract group, respectively. Among them, Firmicutes (p_Bacillota) and Lactobacillus (g_Lactobacillus) were the main dominant bacterial groups in the blank control group, while Pseudomonas (p_Pseudomonadota), Gammaproteobacteria (c_Gammaproteobacteria), Enterobacterales (o_Enterobacterales), and Enterobacteriaceae (f_Enterobacteriaceae) were the main dominant bacterial groups in the model group.

[0094] The above results indicate that the flavonoid-rich Citrus reticulata extract of the present invention can improve the composition of the intestinal microbiota, promote the colonization of beneficial bacteria, and reduce the abundance of harmful bacteria.

[0095] Experimental Example 4: Results of the detection of effects on mouse intestinal metabolism This embodiment tested the effect of Citrus reticulata extract on intestinal metabolism in mice with vascular calcification. The construction and intervention methods of the vascular calcification model were the same as in Example 2, and specifically included the following contents.

[0096] 1. Experimental Methods Fecal samples (80 mg each) from mice in each treatment group were collected, mixed with 200 μL of water and 5 grinding beads, and homogenized using a tissue homogenizer. Then, 800 μL of a methanol / acetonitrile mixture (1:1, v / v) was added to the tissue homogenate, and the mixture was centrifuged at 14000 g and 4℃ for 15 min. The supernatant was collected and concentrated to dryness. Before analysis, the dried sample was redissolved in 100 μL of an acetonitrile / water solution (1:1, v / v). Metabolomics analysis was performed using an Agilent 1290 Infinity liquid chromatography (LC) system and an AB SCIEX Triple TOF6600 triple quadrupole high-resolution time-of-flight mass spectrometer (MS) (non-targeted metabolomics sequencing of mouse tissues was performed by Novogene). Samples were analyzed using an ACQUIY upregulated LC BEH column (2.1 mm × 100 mm, 1.7 μm). The raw LC / MS data was converted into an mzXML file using the Proteo Wizard MSconventer software (version 3.0.8789), and then the data was processed using the XCMS online software.

[0097] 2. Experimental Results (1) Quality control of metabolomics data First, quality control (QC) samples were used to assess data quality to ensure the stability and reproducibility of the metabolomics method. The quality control results of mouse fecal metabolomics data are as follows: Figure 7 As shown in the figure, the Pearson correlation coefficient between QC samples is close to 1, indicating that the instrument is in good condition, the detection method is highly stable, and the obtained data is reliable. Next, principal component analysis (PCA) was performed on the obtained QC sample data matrix to further monitor system stability. The results show that the QC samples are well-clustered and densely distributed, confirming that the sample analysis process is effective.

[0098] (2) Results of the effects on metabolites and metabolic pathways in mouse vascular tissue PCA and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to determine metabolite differences between and within groups. The results of PCA and OPLS-DA analyses of mouse vascular metabolites are shown below. Figure 8 As shown in the figure, the three groups of samples can be clearly separated, indicating differences in metabolites among the groups.

[0099] Differential metabolites were further screened based on the thresholds VIP > 1.0 and P < 0.05, and the results are as follows: Figure 9 As shown in the figure, 835, 60, and 272 differentially expressed metabolites were identified between the blank control group and the model group, between the model group and the 50 mg / kg Citrus reticulata extract group, and between the model group and the 200 mg / kg Citrus reticulata extract group, respectively. Subsequently, 49 potential biomarkers were screened using a Venn diagram (Table 2), including aspergillamide D, N-(4-amino-5-methoxy-2-methylphenyl)benzamide, azoxystrobin, malicoline B, glyceryl monopalmitate, acetochlor ethanesulfonate, geranyl-β-D-glucopyranoside, N-arachidonicylmethionine, melphalan H, 7,14-dihydroxy-4,8,10,12-octadecathatetraenidylcarnitine, and glycyrrhizin chalcone. C,3'-Acetylininium oxoaminooxide, cadaverine, berberine, acetyl tributyl citrate, tongine D, (6S,8Z)-6-hydroxy-3-oxotetradecenoic acid, laria aglycone A, (2S)-2-[(1S)-1-carboxyundecyl]-5-oxotetrahydrofuran-2-carboxylic acid, lysophosphatidylcholine (18:2), taurine, glutamic acid-leucine-phenylalanine-alanine (tetrapeptide), methyl pentadecanoate, (3R,5R)-3 -(((3R,5R)-3,5-dihydroxydecanoyl)oxy)-5-hydroxydecanoic acid, 4-[5-(3,4-dimethoxyphenyl)-3,4-dimethyloxacyclopentan-2-yl]phenol, terazine A, methylnaphthoquinone-4 (vitamin K2), cis-7,10,13,16-docosahexaenoic acid, apo-8'-capsaicinaldehyde, 3-methoxy-4-[2-(4-methylpiperazin-1-yl)ethoxy]benzaldehyde, ganoderic acid B, crocin, glutamic acid -Alanine-Tryptophan (tripeptide), Sargenin B, sangfuran W, clovatin, undecyl glucoside, carbamate, icosperidin A, di[2-(2-butoxyethoxy)ethyl] adipic acid, lysophosphatidylcholine (20:3), duracil, bromoflavones A, 1-tetratel-2-hydroxy-sn-glycerol-3-phosphocholine, succinyl thiopicrol A, histidine-proline (dipeptide), dityrosine, ornithine, lysophosphatidylethanolamine (18:3).

[0100] Table 2: Detailed information on 49 potential biomarkers Continued from the previous table: Continued from the previous table: Continued from the previous table: Continued from the previous table: Further analysis compared the screened differentially expressed metabolites with the KEGG database and performed enrichment pathway analysis. The results showed that vitamin D3-induced vascular calcification significantly altered 20 metabolic pathways (e.g., Figure 10 As shown, P < 0.05, mainly including pyrimidine metabolism, nucleotide metabolism, biosynthesis of unsaturated fatty acids, and tryptophan metabolism. Intervention with Citrus reticulata extract significantly altered six metabolic pathways in the intestinal metabolites of the model group mice (e.g., ...). Figure 11 , 12 As shown (P < 0.05), this includes cell closure, taurine and taurine metabolism, primary bile acid biosynthesis, glycerophospholipid metabolism, choline metabolism in cancer, and bile secretion.

[0101] The above results indicate that Citrus reticulata extract can improve vitamin D3-induced vascular calcification by regulating the levels and metabolic pathways of the aforementioned differential metabolites.

[0102] In summary, this invention successfully established a mouse model of vascular calcification by subcutaneous injection of excessive vitamin D3, and simultaneously administered Citrus reticulata extract. The results confirmed that this intervention could reduce the degree of vascular calcification. Therefore, Citrus reticulata extract shows promise as an effective strategy for preventing and treating vascular calcification and can be used in the preparation of products related to alleviating and / or treating vascular calcification.

[0103] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of Citrus reticulata extract in the preparation of drugs for relieving and / or treating vascular calcification.

2. The application according to claim 1, characterized in that, The vascular calcification mentioned is a type of vascular calcification that is more common and more severe in people with metabolic disorders, electrolyte imbalances, or acid-base imbalances; the metabolic disorders, electrolyte imbalances, or acid-base imbalances are caused by hyperlipidemia, hyperphosphatemia, vitamin D excess, chronic kidney disease, or diabetes.

3. The application according to claim 1, characterized in that, The preparation method of the Citrus reticulata extract includes the following steps: S1. Fresh tangerine peel is dried and then pulverized to obtain tangerine peel powder; S2. Mix the Citrus reticulata powder from S1 with water, extract with hot water, collect the filtrate, and then concentrate it to obtain the hot water extract. S3. Mix the hot water extract from S2 with the protein removal reagent, centrifuge after reaction to remove the supernatant solution, repeat the protein removal step 6 to 20 times, and concentrate under reduced pressure to obtain the crude extract. S4. Add ethanol to the crude extract of S3, let it stand, centrifuge and collect the precipitate. After redissolving the precipitate in water, dialyze and dry it to obtain the Citrus reticulata extract.

4. The application according to claim 3, characterized in that, The Huajuhong mentioned includes Gutian Huajuhong and Huazhou Huajuhong.

5. The application according to claim 3, characterized in that, In S2, the volume ratio of the citrus powder to water is 1:20-40; the hot water extraction temperature is 90-110℃, the time is 1-4 hours, and the hot water extraction is performed 1-4 times.

6. The application according to claim 3, characterized in that, In S3, the protein removal reagent contains chloroform and / or n-butanol, and the volume ratio of the hot water extract to the protein removal reagent is 1:1 to 3.

7. The application according to claim 3, characterized in that, In S4, the volume ratio of the crude extract to ethanol is 1:3 to 5, the ethanol is an aqueous solution of ethanol, and the ethanol content in the ethanol solution is greater than 95%.

8. The application according to claim 3, characterized in that, In S4, the standing is carried out at 0-4°C for 8-12 hours, and the molecular weight cutoff for dialysis is 2.5-3.5 kDa for 24-72 hours.

9. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients and / or carriers.

10. The application according to claim 1, characterized in that, The drug is available in the form of an oral formulation, an injection, or a topical formulation.

Images