Application of euphorbia fischeriana extract in preparation of medicine for preventing and treating ulcerative colitis

Abstract

The invention relates to the technical field of medicine, in particular to application of euphorbia fischeriana extract in preparation of medicine for preventing and treating ulcerative colitis. The preparation method of the euphorbia fischeriana extract comprises the following steps: cleaning collected euphorbia fischeriana, removing surface soil and other impurities, then putting the euphorbia fischeriana in an electrothermal blowing drying box at 40 DEG C for drying, crushing the euphorbia fischeriana by using a traditional Chinese medicine crusher for 15 minutes after drying, and removing uncrushed parts by using a 50-mesh screen to obtain the euphorbia fischeriana extract. Placing the treated sample in a sample bag and storing the sample in a refrigerator at-20 DEG C; mixing 100g of dried euphorbia fischeriana powder with 1L of 95% ethanol, extracting for 1h by using a condensation reflux method under the water bath condition of 40 DEG C, and filtering an alcohol extracting solution by using filter paper and a negative pressure filtering device to remove residual substances in the alcohol extracting solution;

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of a Euphorbia chamaejasme extract in the preparation of a medicine for preventing and treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) belongs to the category of inflammatory bowel disease. It is a nonspecific intestinal disease characterized by chronic inflammatory changes and ulcer formation in the colorectal mucosa. The clinical manifestations are recurrent diarrhea, bloody stools with mucus and pus, and abdominal pain, often accompanied by varying degrees of systemic symptoms and extraintestinal manifestations. It is currently believed that its pathogenesis is related to immune response. Traditional Chinese medicine believes that UC is mostly caused by damp-heat evil toxins accumulating in the colon and viscera, stagnation of qi and blood, and damage to the intestinal network, and belongs to the category of "evil poison" in traditional Chinese medicine. With the changes in living environment and dietary structure in recent years, the incidence and prevalence are showing a rapid growth trend. UC can accumulate in joints, skin, liver and gallbladder, mucosa and other organs, and can increase the risk of colon cancer.

[0003] Ulcerative colitis will lead to the destruction of the intestinal barrier, causing dysfunction of epithelial cells, macrophages, neutrophils and lymphocytes in the lamina propria, and causing a series of pathological changes. Macrophages are important innate immune cells of the body's immune system. Under UC, monocytes are continuously recruited from the microcirculatory system to the intestinal lamina propria, and come into contact with excessive pathogenic microorganisms. Then, monocytes highly express CD14 and become pro-inflammatory M1 macrophages, causing the destruction of colon immune homeostasis. Studies have shown that the activation of macrophages in the course of UC involves many important signaling pathways, such as Toll-like receptor 4 (TLR4)-mediated multifunctional nuclear transcription factor (NF-K B), PI3K-AKT-mTOR, ROS, NLRP3 inflammasome signaling pathways, etc.

[0004] Since the pathogenesis of UC is not yet clear, current drug treatments cannot completely cure it, and treatments mainly focus on controlling symptoms and improving the quality of life of patients. Traditional Chinese medicine has advantages in treating UC, such as multi-level, multi-pathway, multi-target, and few side effects. It can maintain long-term treatment and is increasingly attracting attention. Therefore, it is of far-reaching significance to explore effective traditional Chinese medicine for the treatment of UC and further analyze and explain its mechanism of action.

[0005] Euphorbia fischeriana Steud. is a plant of the Euphorbia genus of the Euphorbiaceae family. It is a perennial herb, mainly distributed in Mongolia, Russia (Eastern Siberia) and China; in China, it is distributed in Heilongjiang, Jilin, Liaoning, Inner Mongolia (Eastern) and Shandong (Yantai, Laoshan). Wolfsbane contains a variety of compounds such as esters, terpenes, volatile oils, sterols, anthraquinones, etc. It is one of the important traditional Chinese herbal medicines in my country. According to medical books such as "Shennong's Herbal Classic", "Ben Jing Jizhu" and "Ming Yi Bie Lu", wolfsbane tastes pungent, is neutral in nature, and is highly toxic. It is mainly used to treat edema and abdominal distension, phlegm and food worm accumulation, heart and abdominal pain, chronic tracheitis, asthma, lymph nodes, bone tuberculosis, etc.; as a morning medicine, wolfsbane is often used to treat psoriasis, eczema, neurodermatitis and other diseases, but no research on wolfsbane has been found in the prevention and treatment of ulcerative colitis. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a Euphorbia chamaejasme extract in the preparation of a drug for preventing and treating ulcerative colitis, so as to solve the problems raised in the above background technology.

[0007] To achieve the purpose of the invention, the present invention provides the following technical solution: The preparation method of the Euphorbia chamaejasme extract comprises the following steps:

[0008] Step 1: Clean the collected Euphorbia chamaejasme, remove the surface soil and other impurities, and then place it in a 40℃ electric blast drying oven for drying. After drying, use a Chinese medicine grinder to crush it for 15 minutes, and use a 50-mesh sieve to remove the uncrushed part. The processed samples are placed in sample bags and stored in a -20℃ refrigerator.

[0009] Step 2: Take 100 g of the dried Euphorbia chamaejasme powder and mix it with 1 liter of 95% ethanol, and extract it using the condensation reflux method in a 40°C water bath for 1 hour, and filter the alcohol extract using filter paper and a negative pressure filtration device to remove residual substances.

[0010] Step 3: Place the alcohol extract at 40°C for reduced pressure drying to obtain a jelly, then dissolve the jelly with a small amount of water, place it in a -80°C refrigerator overnight, freeze-dry it, and grind it to obtain the Euphorbia chamaejasme extract.

[0011] The invention also provides a use of the Euphorbia chamaejasme extract in inhibiting LPS-induced macrophage inflammation.

[0012] The present invention further provides an application of the Euphorbia chamaejasme extract in preparing a medicine for preventing and treating ulcerative colitis.

[0013] Effects of the Invention

[0014] 1. Discover a method for preparing a Euphorbia chamaejasme extract

[0015] 2. Euphorbia chamaejasme extract has preventive and therapeutic effects on ulcerative colitis BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown is the effect of Euphorbia chamaejasme extract on the NLRP3 signaling pathway in macrophages.

[0017] Figure 2 Shown is the effect of Euphorbia chamaejasme extract on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) disease model in mice.

[0018] Figure 3 The figure shows the secretion of inflammatory factors in the colon tissue of UC mice. The secretion of inflammatory factors in the group treated with wolfsbane extract was significantly lower than that in the UC disease model group. DETAILED DESCRIPTION

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] In the present invention, Figure 1 The results show the effect of Euphorbia chamaejasme extract on the NLRP3 signaling pathway of macrophages. In the LPS-induced macrophage model, Euphorbia chamaejasme extract can significantly inhibit the expression of inflammatory factors IL-1β, Caspase-1, NLRP3, ASC and LDH. Figure 2 The effect of Euphorbia chamaejasme extract on the mouse ulcerative colitis (UC) disease model induced by dextran sodium sulfate (DSS) is shown. The colon inflammation in the group treated with Euphorbia chamaejasme extract was significantly improved compared with the UC model group. Figure 3 The results show that the secretion of inflammatory factors in the serum of experimental mice was significantly lower in the group treated with wolfsbane extract than in the UC disease model group.

[0021] In the present invention, Euphorbia chamaejasme is collected in Changbai Mountain area of ​​Jilin Province in mid-May every year; the collected Euphorbia chamaejasme is cleaned to remove surface soil and other impurities, and then placed in a 40°C electric blast drying oven for drying, and then crushed for 15 minutes using a traditional Chinese medicine crusher, and a 50-mesh sieve is used to remove the uncrushed part, and the processed sample is placed in a sample bag and stored in a -20°C refrigerator; the whole plant is directly extracted and processed without the need for more pretreatment steps in the early stage.

[0022] In the present invention, 100 g of dried Euphorbia chamaejasme powder is mixed with 1 liter of 95% ethanol, and extracted for 1 hour using a condensation reflux method under a 40° C. water bath condition, and the alcohol extract is filtered using filter paper and a negative pressure filtration device to remove residual substances therein. Compared with the prior art, the processing steps are simplified and the processing time is short.

[0023] In the present invention, the alcohol extract is placed at 40°C for reduced pressure drying to obtain a jelly, then the jelly is dissolved with a small amount of water and placed in a refrigerator at -80°C overnight, then freeze-dried and ground.

[0024] The present invention explores the effect of Euphorbia chamaejasme extract on iNOS and COX-2 inflammatory mediators secreted by macrophages.

[0025] The present invention also explores the effect of the Euphorbia chamaejasme extract in a mouse ulcerative colitis disease model induced by dextran sodium sulfate (DSS).

[0026] Example 1

[0027] This embodiment provides a method for preparing a Euphorbia chamaejasme extract, which comprises the following steps:

[0028] (1) Euphorbia chamaejasme was collected in the Changbai Mountain area of ​​Jilin Province in mid-May every year.

[0029] (2) After collection, the Euphorbia chamaejasma was cleaned to remove surface dirt and other impurities, and then placed in a 40°C electric blast drying oven for drying. After drying, it was crushed using a traditional Chinese medicine grinder for 15 minutes, and a 50-mesh sieve was used to remove the uncrushed part. The processed samples were placed in a sample bag and stored in a -20°C refrigerator.

[0030] (3) 100 g of the dried Euphorbia chamaejasme solid powder was mixed with 1 liter of 95% ethanol, and extracted by condensation reflux method in a 40° C. water bath for 1 hour. The alcohol extract was filtered using filter paper and a negative pressure filtration device to remove residual substances.

[0031] (4) The alcohol extract is placed at 40°C for vacuum drying to obtain a jelly, and then the jelly is dissolved with a small amount of water and placed in a refrigerator at -80°C overnight, followed by freeze drying and grinding to obtain the Euphorbia chamaejasme extract.

[0032] Example 2

[0033] Effects of Euphorbia chamaejasme extract on NLRP3 signaling pathway in macrophages.

[0034] MH7A (from RIKEN cell bank); LPS (L8274), ATP (A7699), dimethyl sulfoxide (DMSO), etc. were purchased from Sigma; DMEM, antibiotics, fetal bovine serum (FBS), etc. were purchased from Gibco; IL-1β, Caspase-1, NLRP3, ASC, LDH, ATP, β-actin (SC-1616), BCAprotein assay kit, etc. were purchased from Thermo.

[0035] (1) Macrophage culture

[0036] Macrophage Raw264.7 cells (derived from RIKEN Cell Bank) were cultured in DMEM high-glucose medium containing 10% (v / v) FBS and 1% (v / v) antibiotics (penicillin and streptomycin, 100 U / mL) at 37°C, relative humidity of 95%, and CO. 2 The content is 5%. Digestion is performed using a digestion solution containing 0.25% trypsin and 0.02% EDTA, and the digestion is terminated with a serum-containing DMEM high-glucose medium for subculture.

[0037] (2) Euphorbia chamaejasma stimulates macrophages

[0038] (3) Sample pretreatment:

[0039] Macrophages were cultured at 2.6 × 10 6 cells / well / 2mL were plated on a 6-well plate and cultured overnight for 12h. After the macrophages were treated with LPS (100ng / ml) medium for 3h, different doses of Euphorbia chamaejasma extract were added to treat the cells for 30min, and then ATP was added to stimulate the cells for 30min to collect the cell supernatant and cell lysate. After the WB experiment was terminated, the supernatant was discarded. 100μL RIPA lysis buffer was added to each well and fully scraped with a cell spatula. The obtained cell lysate was ice-bathed for 30min and then centrifuged (13000rpm, 15min) to obtain the supernatant. In order to ensure the accuracy of the sample loading in subsequent experiments, the protein concentration was determined by the BCA method. A standard curve was prepared based on the BCA standard to calculate the protein content of each well. The obtained cell lysate and loading buffer were completely mixed at a volume ratio of 4:1, placed in boiling water at 100℃ for 5min, and cooled for use.

[0040] The expression level of each protein was detected by immunoblotting. Prepare 10% separation gel and pour it between the glass plates. Immediately add 0.5-1mL of methanol. After 15 minutes, the liquid surface is layered and the upper layer of methanol is discarded. Make 5% concentrated gel and pour it between the glass plates and immediately insert the sample comb. After 15 minutes, pull out the sample comb. Assemble the electrophoresis system, add electrophoresis buffer, add 5μL Marker to the first well, and add sample solution to the remaining wells in sequence according to the quantitative results of BCA. The electrophoresis conditions are constant voltage 120V, 70min. After the electrophoresis is completed, the gel is assembled with the PVDF membrane that has been activated by soaking in methanol. The assembly order is: sponge-filter paper-PVDF membrane-gel-filter paper-sponge, and the transfer conditions are constant voltage 70V, 210min. After the transfer is completed, the PVDF membrane is cut to the appropriate size according to the molecular weight of the relevant protein. The membrane was washed 3 times with PBS-T (5 min / time), blocked with 10% skim milk at room temperature for 1 hour, and then incubated with primary antibody (dilution ratio 1:1000) at 4°C overnight. According to the properties of the primary antibody, a suitable secondary antibody (dilution ratio 1:5000) was selected and incubated at room temperature for 1 hour. ECL chemical developer was prepared and exposed to the image using a gel imager. The images were processed using ImageJ-2 and the relative expression of each protein was calculated.

[0041] (4) Summary

[0042] According to the results of the gel electrophoresis experiment, the expression level of inflammatory factors secreted by the NLRP3 signaling pathway of macrophages in the Euphorbia chamaejasme extract group was significantly higher than that in the normal group, indicating that LPS can induce inflammatory response in macrophages, while the expression level of inflammatory factors secreted by the NLRP3 signaling pathway of macrophages in the Euphorbia chamaejasme extract-treated group was significantly lower than that in the LPS-treated group, indicating that Euphorbia chamaejasme extract can inhibit the inflammatory response of macrophages induced by LPS.

[0043] Example 3

[0044] Effects of Euphorbia chamaejasma extract on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) disease model in mice.

[0045] (1) Preparation of ulcerative colitis (UC) model and treatment with Euphorbia chamaejasme extract

[0046] According to the experimental requirements, male C57 / BL6 mice (6-8 weeks old, weighing 15-18g) were divided into 3 groups, with 6 mice in each group: 1) normal group, 2) UC model group, and 3) Euphorbia chamaejasme extract group. Starting from the first day of the experiment, the mice were given oral gavage every day according to the grouping to pre-protect the mice. Starting from the fourth day of the experiment, DSS (3g) was dissolved in 100mL drinking water, and the mice were allowed to drink freely, and 3% DSS drinking water was replaced every 1d. Note that during the experiment, the drug was administered at the same time every day as much as possible, and the mice were recorded every day to observe the daily diarrhea, bloody stools, and weight changes of the rats for DAI scores. The mice were euthanized on the 10th day, and the serum, intestinal tissue, and feces of the mice were collected, and the relevant data were recorded and analyzed.

[0047] (2) IL-1β, IL-6, and TNF-α in mouse tissue sections and intestinal tissues

[0048] After the mouse intestinal tissue was removed and soaked in formalin for 24 hours, the tissue was cut into pieces, and then placed in an embedding box, washed with water for 24 hours, and placed in an automatic tissue dehydrator for dehydration → transparent → wax dipping. The tissue was then placed in an embedding machine for embedding. After the paraffin block solidified, it was trimmed, and the slicer was adjusted to make the slice thickness 4μm. The sliced ​​slices were placed in a 37℃ water bath for spreading, fixed on a slide, and dried using a 37℃ slide oven. H&E staining was used for staining, and the stained slices were sealed with neutral resin and observed under a microscope to observe the pathological changes of the colon.

[0049] Collect the intestinal tissue after grinding and centrifugation, and measure it according to the instructions of the ELISA kit. Add 100μL / well of the prepared coating solution to the ELISA plate and incubate it overnight at 4℃ for 12 hours. Discard the coating solution, add 200μL PBS-T to each well and wash 4 times, add 3% skim milk to block for 1 hour, continue to wash the plate 4 times, add 50μL of the sample or standard to be tested to each well and incubate for 2 hours, wash the plate 4 times, add 50μL of detection antibody to each well, wash the plate 6 times, add TMB to the reaction in the dark, decide whether to terminate the reaction according to the color, and add 100μL 2N H 2 SO 4 The reaction was stopped. The OD value of each well was measured at a wavelength of 450 nm using an ELISA reader, and a standard curve was prepared based on the OD value of the standard to calculate the secretion of inflammatory factors in different groups.

[0050] (3) Summary

[0051] In the in vivo mouse test, it was clearly observed that the degree of colon damage in the UC model group was significantly higher than that in the normal group, indicating that the mouse UC model was successfully established. The degree of colon damage in the foot of mice eliminated by the Euphorbia chamaejasme extract was significantly lower than that in the UC model group, indicating that the Euphorbia chamaejasme extract can effectively prevent and treat ulcerative colitis in mice.

[0052] In the HE staining of mouse colon tissue, the UC model group had obvious colon tissue damage, increased tissue gaps and tissue infiltration compared with the normal group, while the colon tissue in the Euphorbia chamaejasme extract group was not much different from that of the normal group and was significantly better than that of the UC model group, indicating that the Euphorbia chamaejasme extract can effectively prevent and treat the occurrence of ulcerative colitis in mice.

[0053] In the detection of inflammatory factors in colon tissue, it can be found that the contents of IL-1β, IL-6, and TNF-α in the Euphorbia chamaejasme extract group were significantly lower than those in the UC model group, and there was a significant difference. This shows that the Euphorbia chamaejasme extract can effectively prevent and treat the production of inflammatory factors in rheumatoid arthritis in mice.

[0054] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. Use of a Euphorbia chamaejasme extract in preparing a drug for preventing and treating ulcerative colitis.

2. Use of a Euphorbia chamaejasme extract in the preparation of a drug for inhibiting LPS-induced macrophage inflammation.

3. A method for preparing a Euphorbia chamaejasme extract, the method comprising the following steps: Step 1: Clean the collected Euphorbia chamaejasme, remove the soil and other impurities on the surface, and then place it in a 40℃ electric blast drying oven for drying. After drying, use a Chinese medicine grinder to crush it for 15 minutes, and use a 50-mesh screen to remove the uncrushed part. The processed samples are placed in sample bags and stored in a -20℃ refrigerator: Step 2: Mix 100 g of the dried Euphorbia chamaejasme powder with 1 liter of 95% ethanol, and extract the mixture using a condensation reflux method in a 40° C. water bath for 1 hour, and filter the alcohol extract using filter paper and a negative pressure filter to remove residual substances; Step 3: Place the alcohol extract at 40°C for vacuum drying to obtain a jelly, then dissolve the jelly with a small amount of water, place it in a -80°C refrigerator overnight, freeze-dry it, and then grind it.

4. The preparation method according to claim 3, wherein The extract is selected from the whole plant of Euphorbia chamaejasme collected in the Changbai Mountain area of ​​Jilin Province in mid-May every year.

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