CN107582574B

CN107582574B - Application of nostoc commune alcohol extract in preparation of medicine for treating inflammatory bowel disease

Info

Publication number: CN107582574B
Application number: CN201710968603.4A
Authority: CN
Inventors: 段小群; 王宇晖; 徐笑天; 苑博; 朱梦玲; 曹后康
Original assignee: Guilin Medical University
Current assignee: Guilin Medical University
Priority date: 2017-10-17
Filing date: 2017-10-17
Publication date: 2019-12-24
Anticipated expiration: 2037-10-17
Also published as: CN107582574A

Abstract

The invention discloses an application of nostoc commune alcohol extract in preparing a medicament for treating inflammatory bowel disease, wherein the preparation method of the nostoc commune alcohol extract comprises the following steps: extracting herba Gelidii Piloselloidis with low carbon alcohol as solvent under heating condition, collecting extractive solution, and recovering solvent. The applicant finds that the nostoc commune alcohol extract obviously improves the weight reduction of a mouse model UC induced by DSS, inhibits the increase of DAI score, reduces the reduction of the length of the colon of the mouse caused by UC, and simultaneously reduces inflammatory cell infiltration and tissue damage. In addition, the nostoc commune alcohol extract obviously reduces the content of colon tissue cell factors TNF-alpha and IL-6, and the effect is closely related to the activation of a key transcription factor NF-kappa B for inhibiting an inflammatory signal channel. The experimental results of the applicant show that the nostoc commune ethanol extract can be used for treating inflammatory bowel diseases, relieving the inflammatory reaction of a DSS-induced UC model and improving the severity of UC.

Description

Application of nostoc commune alcohol extract in preparation of medicine for treating inflammatory bowel disease

Technical Field

The invention relates to application of a plant extract, in particular to application of a nostoc commune alcohol extract in preparing a medicament for treating inflammatory bowel diseases.

Background

Inflammatory Bowel Disease (IBD) is an idiopathic inflammatory bowel disease mainly affecting the ileum, rectum and colon, and clinically manifested as diarrhea, abdominal pain and even bloody stool. The disease includes Ulcerative Colitis (UC) and Crohn's Disease (CD). Ulcerative colitis is a continuous inflammation of the mucosal layer and submucosa of the colon, and the disease usually affects the rectum first and gradually spreads to the whole colon; crohn's disease affects the whole digestive tract, is a non-continuous, full-thickness inflammation, and is most commonly affected in the terminal ileum, colon and perianal region. UC, a typical inflammatory bowel disease, is better in young and strong years of 30-40 years old, has no sex difference, is manifested by clinical symptoms such as diarrhea, abdominal pain, mucopurulent bloody stool and the like, and is mostly complicated by intestinal perforation, polyp, ulcer and the like, and seriously affects the life quality and survival of patients. Epidemiological studies have shown that UC incidence in european regions is 505/100,000 high, in canadian regions 248/100,000 and in the united states 214/100,000. With the change of life style and diet rules, the incidence of UC in Asia and middle east is increasing year by year.

Currently, the current practice is. Drug therapy and surgical resection are the primary methods of managing UC, and the following 5 classes of commonly used therapeutic drugs: (1) aminosalicylates, such as sulfasalazine and mesalamine; (2) glucocorticoids, such as dexamethasone and beclomethasone dipropionate; (3) immunosuppressants such as 6-mercaptopurine and cyclosporine; (4) biologicals, such as the TNF-alpha inhibitor infliximab; (5) anti-infective drugs, such as the antibiotics metronidazole and ciprofloxacin. The medicines mainly reduce local inflammation of colon and prevent disease progression by inhibiting abnormal immune response, but have the defects of unstable curative effect, more adverse reactions, poor tolerance, unsuitability for long-term administration, high price and the like. Therefore, the method for searching the UC-resistant medicine with definite curative effect, less adverse reaction and controllable quality from the traditional Chinese medicine has important value.

An increasing number of studies have shown that an excess of cytokines, chemokines and growth factors, as well as reactive oxygen metabolites (such as ROS and NO), among others, can induce an inflammatory cascade leading to colon damage. Of these mediators, IL-1 β, TNF- α and IL-6 are thought to play important roles in the inflammatory response of UC. In UC patients with colonic mucosa, blood and feces, the expression of IL-1 beta, TNF-alpha and IL-6 is obviously increased. Targeted therapies against TNF-a and IL-6 have become an important tool for the treatment of UC, and various anti-TNF-a agents (such as infliximab and adalimumab) have been used to treat patients with UC, and monoclonal antibodies against IL-6 hold promise for the treatment of UC. However, the drugs are expensive, the medication route is single, and off-target effects occur in part of patients, so that the drugs are difficult to be widely used clinically.

NF-kB is an important transcription factor involved in inflammatory response genes and is considered as a target of various inflammatory diseases. Early studies have shown that NF-. kappa.B activation is significantly increased in colon tissue from IBD patients and colitis model animals. NF-. kappa.B is usually a heterodimer composed of two subunits, p50 and p65, and exists in the cytoplasm in the form of a complex with the inhibitory protein Iκ B at rest. Various stimuli can activate the NF- κ B-I κ B complex, leading to phosphorylation, ubiquitination and degradation of I κ B proteins. The NF-. kappa.B heterodimer then translocates rapidly into the nucleus, binds to the DNA binding site of the target gene, and regulates transcription of various target genes such as TNF-. alpha.and IL-6. Although treatment of UC against a single inflammatory factor may be an alternative treatment, the therapeutic effect is often poor. Indeed, the genes for a single factor, such as a cytokine, chemokine or adhesion molecule, represent only one of the downstream target genes, while NF-. kappa.B is the ultimate common pathway or rate-limiting step of the inflammatory cascade.

Nostoc commune, a academic name of common Nostoc commune Vauch, namely Nostoc commune, pachira hirta, Nostoc commune and the like, is sheet algae of Nostoc of cyanobacteriaceae, and is favored to grow on the surface of moist soil or be mixed among stems and leaves of the root of weeds, the base of big trees and moss flora and is distributed almost all over the country. The nostoc commune is cool in nature and sweet in taste and enters liver meridian; the cortex schizophragmatis integrifolii radicis has the effects of clearing heat, improving eyesight, astringing and benefiting qi, is rich in protein, various vitamins and mineral substances such as phosphorus, zinc, calcium and the like, and scientific researches of researches on Wenzmann Israel find that one component contained in the cortex schizophragmatis integrifolii radicis can inhibit the activity of acetylcholinesterase in human brains, so that the effect of treating senile dementia can be achieved. In addition, patent publication No. CN103059112A discloses that a protein having anti-intestinal cancer activity and a molecular weight of 35-40kDa can be obtained by extracting Gelidium japonicum with PBS buffer at low temperature and subjecting the extract to SDS-PAGE gel electrophoresis. However, no report on the application of the nostoc commune ethanol extract in treating inflammatory bowel diseases is found at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the nostoc commune ethanol extract in preparing the medicine for treating inflammatory bowel diseases.

The technical scheme of the invention is as follows: application of herba Geranii alcohol extract in preparing medicine or health product for treating inflammatory bowel disease is provided.

In the technical scheme of the invention, the preparation method of the nostoc commune alcohol extract preferably comprises the following steps: taking nostoc commune, taking low-carbon alcohol as a solvent, extracting under a heating condition, collecting an extracting solution, and recovering the solvent to obtain the nostoc commune alcohol extract. The nostoc commune alcohol extract obtained by the method can be directly applied to preparation of medicines or health-care products for treating inflammatory bowel diseases. In order to improve the purity of the nostoc commune alcohol extract, the preparation method of the nostoc commune alcohol extract further comprises a purification step, and specifically, the nostoc commune alcohol extract is recrystallized by using low-carbon alcohol to obtain the purified nostoc commune alcohol extract.

In the preparation method of the nostoc commune alcohol extract, the nostoc commune can be fresh, can be dried, and is preferably dried.

In the above preparation method of the nostoc commune alcohol extract, the lower alcohol may be one or a combination of two or more of alcohols containing 1-6 carbon atoms, preferably methanol, ethanol or propanol, and most preferably ethanol. The concentration of the lower alcohol may be 10 to 100 v/v%, preferably 50 to 100 v/v%, 70 to 100 v/v%.

In the preparation method of the nostoc commune ethanol extract, the extraction is carried out under the heating condition, so that protein components contained in the extract are cracked and denatured, and various effective components which can act on inflammatory bowel disease medicines are better obtained. The extraction may be carried out at a temperature ranging from 50 ℃ to the boiling point of the solvent, preferably at a temperature ranging from 60 ℃ to the boiling point of the solvent, more preferably at a temperature ranging from 70 ℃ to the boiling point of the solvent. The extraction mode is preferably reflux extraction, and the extraction times can be carried out according to requirements, and are usually 1-3 times; the amount of solvent used in each extraction is the same as that of the conventional extraction operation, and specifically can be 3-12 times of the weight of the raw materials; the time of each extraction can be 0.5-3h, or longer.

The invention also comprises a medicament or health-care product for treating inflammatory bowel diseases, which contains the nostoc commune alcohol extract with a therapeutically effective dose. The nostoc commune alcohol extract is prepared by the method. The dosage form of the medicine or the health care product can be pharmaceutically acceptable dosage forms, such as conventional dosage forms of capsules, tablets or granules.

The applicant finds that the nostoc commune ethanol extract can obviously reduce the weight reduction of enteritis model mice induced by Dextran Sodium Sulfate (DSS), inhibit the increase of Disease Activity Index (DAI) scores of colitis model mice, improve the reduction of colon length of colitis model mice and reduce the infiltration of colon tissue inflammatory cells of colitis model mice through a large number of experiments. In addition, the nostoc commune alcohol extract can obviously reduce the content of proinflammatory factors TNF-alpha and IL-6 in colon tissues of a model mouse, and the effect is closely related to the activation of a key transcription factor NF-kappa B for inhibiting an inflammatory signal channel. Therefore, the nostoc commune alcohol extract can relieve the inflammatory reaction of a DSS-induced inflammatory bowel disease model mouse, improve the severity of inflammatory bowel disease and be used for preparing a medicament or a health-care product for treating inflammatory bowel disease.

Drawings

FIG. 1 is a graph showing the effect of alcohol extract of Gelidium japonicum on weight loss of mice with UC induced by DSS, wherein normal indicates normal group, DSS indicates model group, AENC (20mg/kg) indicates low dose (20mg/kg) group, AENC (100mg/kg) indicates high dose (100mg/kg) group, and Mesaiazine (200mg/kg) indicates mesalazine (200mg/kg) group;

FIG. 2 is a graph showing the effect of alcohol extract of Gelidium japonicum on DAI score of mice with DSS-induced UC, wherein normal indicates normal group, DSS indicates model group, AENC (20mg/kg) indicates low dose (20mg/kg) of alcohol extract of Gelidium japonicum, AENC (100mg/kg) indicates high dose (100mg/kg) of alcohol extract of Gelidium japonicum, and Mesaiazine (200mg/kg) indicates mesalazine (200 mg/kg);

FIG. 3 is a relationship between the influence of alcohol extract of Gelidium japonicum on the length of colon of UC mouse induced by DSS, wherein DSS represents a model group, AENC (mg/kg) represents an alcohol extract group of Gelidium japonicum, and Mesaiazine (mg/kg) represents a mesalazine group;

FIG. 4 is a bar graph of the effect of alcohol extract of Gelidium japonicum on MPO activity of mice UC induced by DSS, where DSS represents the model group, AENC (mg/kg) represents the alcohol extract of Gelidium japonicum group, and Mesaiazine (mg/kg) represents the mesalazine group;

FIG. 5 is a graph showing the effect of HE staining on colon tissues, wherein (a) is a normal group, (b) is a model group, (c) is a low-dose (20mg/kg) group, (d) is a high-dose (100mg/kg) group, and (e) is mesalamine (200mg/kg) group;

fig. 6 is a bar graph of the effect of alcohol extract of nostoc commune on DSS-induced mouse pathomorphology of UC, wherein DSS represents the model group, aecc (mg/kg) represents the alcohol extract of nostoc commune, and mesalazine (mg/kg) represents the mesalazine group;

FIG. 7 is a bar graph of the effect of alcohol extract of Gelidium japonicum on the content of TNF- α in UC mouse pro-inflammatory factor induced by DSS, where DSS represents the model group, AENC (mg/kg) represents the alcohol extract group of Gelidium japonicum, and Mesaiazine (mg/kg) represents the mesalamine group;

FIG. 8 is a bar graph of the effect of alcohol extract of Gelidium japonicum on the content of IL-6 in UC mouse proinflammatory factor induced by DSS, where DSS represents the model group, AENC (mg/kg) represents the alcohol extract of Gelidium japonicum group, and Mesaiazine (mg/kg) represents the mesalamine group;

FIG. 9 is a bar graph of the effect of alcohol extract of Gelidium japonicum on the content of UC mouse proinflammatory factor IL-1 β induced by DSS, where DSS represents the model group, AENC (mg/kg) represents the alcohol extract of Gelidium japonicum group, and Mesaiazine (mg/kg) represents the mesalamine group;

FIG. 10 is a Western blot graph of the activation of mouse pro-inflammatory factor NF-. kappa.B induced by DSS by the alcohol extract of Geranium, wherein DSS represents a model group, AENC (mg/kg) represents an alcohol extract group of Geranium, Mesaiazine (mg/kg) represents a mesalamine group, p-p65 represents the phosphorylation of p65, i.e., the activation of NF-. kappa.B, and GAPDH represents an internal reference;

fig. 11 is a bar graph of the effect of alcohol extracts of nostoc commune on DSS-induced UC mouse pro-inflammatory factor NF- κ B activation, where DSS represents the model group, aecc (mg/kg) represents the alcohol extract group of nostoc commune, and mesalazine (mg/kg) represents the mesalazine group.

Detailed Description

Example 1

Drying nostoc commune in an electric heating constant temperature blast drying oven at 80 ℃ for 8 hours to obtain dried nostoc commune; placing the dried nostoc commune in an extraction tank, adding 80 v/v% ethanol, and extracting under reflux for 3 times (the addition amount of solvent in each extraction is respectively 10 times, 8 times and 5 times of the mass of the dried nostoc commune, and the extraction time for 3 times is respectively 2 hours, 1.5 hours and 1 hour); mixing extractive solutions, filtering, and concentrating the filtrate until there is no alcohol to obtain herba Geranii ethanol extract.

Example 2

Drying nostoc commune in an electric heating constant temperature blast drying oven at 60 ℃ for 5 hours to obtain dried nostoc commune; placing the dried nostoc commune in an extraction tank, adding 100 v/v% ethanol, and extracting under reflux for 1 time (the addition of solvent is 12 times of the mass of the dried nostoc commune, and the extraction time is 3 hours); filtering, and concentrating the filtrate until no alcohol exists to obtain the nostoc commune alcohol extract.

Example 3

Drying nostoc commune in an electric heating constant temperature blast drying oven at 40 ℃ for 12 hours to obtain dried nostoc commune; placing the dried nostoc commune in an extraction tank, adding 20 v/v% methanol, and extracting under reflux for 2 times (the addition amount of solvent in each extraction is respectively 10 times and 10 times of the mass of the dried nostoc commune, and the extraction time for 2 times is respectively 3 hours and 2 hours); mixing extractive solutions, filtering, and concentrating the filtrate until there is no alcohol to obtain herba Geranii ethanol extract.

Example 4

Drying nostoc commune in an electric heating constant temperature blast drying oven at 60 ℃ for 8 hours to obtain dried nostoc commune; placing the dried nostoc commune in an extraction tank, adding 100 v/v% propanol, and extracting under reflux for 3 times (the addition amount of solvent in each extraction is 6 times, 5 times and 5 times of the mass of the dried nostoc commune, and the extraction time for 3 times is 2 hours, 1 hour and 1 hour respectively); mixing extractive solutions, filtering, and concentrating the filtrate until there is no alcohol to obtain herba Geranii ethanol extract.

Example 5

Drying nostoc commune in an electric heating constant temperature blast drying oven at 80 ℃ for 8 hours to obtain dried nostoc commune; placing the dried nostoc commune in an extraction tank, adding 60 v/v% butanol, and extracting under reflux for 3 times (the addition amount of solvent in each extraction is respectively 10 times, 8 times and 5 times of the mass of the dried nostoc commune, and the extraction time for 3 times is respectively 2 hours, 1 hour and 1 hour); mixing extractive solutions, filtering, and concentrating the filtrate until there is no alcohol to obtain herba Geranii ethanol extract.

The application of the nostoc commune alcohol extract to the treatment of inflammatory bowel diseases is described in the following by combining specific experiments.

In animal experiments, a classical 2.5% DSS solution is adopted to induce a C57/BL6 mouse to construct a UC model, meanwhile, a low dose (20mg/kg), a high dose (100mg/kg) and a positive drug mesalazine (200mg/kg) are administered to the model mouse by stomach irrigation, the improvement effect of the alcohol extract of the nostoc commune on UC is comparatively analyzed, the influence of the alcohol extract of the nostoc commune on the contents of proinflammatory factors TNF-alpha, IL-6 and IL-beta and the activation of NF-kB is further examined, and the mechanism of the alcohol extract of the nostoc commune for resisting UC is discussed.

1. Materials and methods

1.1 Experimental animals

SPF grade C57/BL6 mice, female, 6-8 weeks old, having a body weight of 20 + -2 g, purchased from Schleickford laboratory animals, Inc. of Hunan, license number: SCXK (xiang) 2016-: SYXK 2013-. Feeding in an environment with temperature of 25 + -2 deg.C and humidity of 55 + -10%, and freely taking food and drinking water. Is suitable for being used after being fed for one week.

1.2 Experimental reagents

The nostoc commune ethanol extract is prepared by the method of the embodiment 1 of the invention; mesalazine, shanghai love pharmaceutical co ltd, cat #: 151007; dextran Sulfate Sodium (DSS), MP Biomedicals corporation, usa, cat #: 160110; myeloperoxidase (MPO) kit, tokyo institute of bioengineering, lot number: 20170717, respectively; NF-. kappa. B p65 polyclonal antibody, Proteitech corporation, cat #: 10745-1-AP; p-p65 polyclonal antibody, Bioworld, manufacturing lot number: CA 36131; a mouse IL-1 beta enzyme-linked immunosorbent assay (ELISA) detection kit is provided, wherein the kit is manufactured by Elapscience, and the product number is AK0016MAR 09007; a mouse IL-6 enzyme-linked immunosorbent (ELISA) detection kit, CUSABIO company, the cat number is Z01018255; mouse TNF-alpha enzyme-linked immunosorbent (ELISA) detection kit, Elabscience, cat #: AK0016AUG 30005; polyvinylidene fluoride (PVDF) membrane, Millipore corporation, usa, cat #: k5NA 8022H; bicinchoninic acid (BCA) protein quantification kit, pecan biotechnology institute, cat No.: P0010S; TRIzol reagent, Invitrogen, usa, cat #: 152104, respectively; bone Servicebumin (BSA), solarbio, lot number: 316S052, cat no: a8020; glycine (glycine), solarbio, lot number: 1203P 0633; tris (Tris-base), solarbio, lot number: 1217S 074; tween-20 (tween-20), national drug group chemical limited, lot number: f20100722; sodium Dodecyl Sulfate (SDS), solarbio corporation, lot number: 1207G 034; phenylmethylsulfonyl fluoride (PMSF), solarbio, cat # n: p0100, lot number: 20160826, respectively; tetramethylethylenediamine (TEMED), inc: biyuntian biotechnological research institute, product number: ST 728; ECL chemiluminescence liquid, Gutai biotechnology Limited liability company, Wuhan City, Cat number: g2020-2; other reagents are all commercially available analytical reagents, national drug group chemical reagents, Inc.

1.3 Experimental instruments

2. Experimental methods

2.1 establishment of mouse colitis model

Female C57BL/6 mice, 6-8 weeks old, weight 20 + -2 g. The other groups, except the normal group, were freely drunk 2.5% DSS for 7 days, followed by 3 days with tap water. The day of the start of molding was d1, and the body weight of the mouse at this time was recorded as the initial body weight.

In order to investigate the inhibitory effect of the nostoc commune alcohol extract on the DSS-induced colitis of mice, the mice were randomly divided into the following 5 groups: normal group (normal), model group (DSS), low dose (20mg/kg) group of the alcohol extract of nostoc commune (aecc (20mg/kg)), high dose (100mg/kg) group (aecc (100mg/kg)) and mesalazine (200mg/kg) (Mesaiazine (200mg/kg)), 8 animals per group. Gavage (0.1mL/10g) was started on the day of molding, once a day for 10 consecutive days. The normal group and the model group were gavaged with the corresponding volumes of mixed solution (composed of ethanol and PBS buffer at a volume ratio of 5: 95).

2.2 disease Activity index assessment

Observing the mental state, hair color, stool character, activity state, hematochezia and other conditions of the mice every day, recording the weight, diarrhea index and fecal occult blood condition of the mice, and calculating Disease Activity Index (DAI), wherein the DAI is (weight reduction + diarrhea index + fecal occult blood)/3. The DAI scoring criteria are detailed in table 1.

Table 1: DAI scoring criteria

And (3) occult blood detection: by adopting an o-toluidine method, a small amount of excrement is picked by a cotton swab, 0.3mL of o-toluidine glacial acetic acid solution is firstly dripped, then 0.3mL of 3% hydrogen peroxide solution is quickly dripped, and blue brown is positive within 2 min.

2.3 specimen Collection

Collecting blood from fundus venous plexus 1h after the last administration, standing at 4 deg.C for 2h, centrifuging (3500rpm,4 deg.C) for 15min, sucking upper layer serum, and subpackaging at-80 deg.C for freezing and storing. The colon was removed at a distance of 1cm from the anus, the length of the colon was measured with a ruler and photographed, the colon was washed 2 times with PBS, 0.4cm of the colon was taken from the end close to the rectum, fixed in 4% paraformaldehyde solution, and the remaining colon was frozen at-80 ℃.

2.4 colonic MPO content determination

Weighing colon tissue 40mg, adding phosphate buffer solution to prepare 10% homogenate, measuring absorbance (OD) at 460nm according to MPO kit instruction, and calculating according to formula to obtain MPO content. The calculation formula is as follows:

MPO activity unit/g tissue (determination tube OD value-control tube OD value). times.2/22.6 Xsample size (g)2.5 Colon histopathological examination

Taking colon tissues fixed in 4% paraformaldehyde solution (not less than 24H), neutralizing with 5% sodium sulfate solution for 36H, washing with running water overnight, dehydrating with gradient concentration absolute ethyl alcohol, embedding in paraffin, dewaxing conventionally, staining with hematoxylin and eosin (H & E), sealing with neutral resin, and observing pathological and histological changes of intestinal wall under an optical microscope, wherein the specific scores are as follows: (1) the severity of the intestinal wall inflammation, scored as 1, 2 and 3, represents mild, moderate and severe inflammation, respectively; (2) the severity of the lesions, scored as 1, 2 and 3, i.e., lesions located in the mucosal layer, mucosa and submucosa and transmural lesions; (3) the extent of crypt damage was scored as 1, 2, 3 and 4. Namely 1/3 crypt lesions, 2/3 crypt lesions, crypt loss but both surface epithelium intact and crypt surface epithelium are missing.

2.6 cytokine detection

40mg of colon tissue was weighed, 10% homogenate was prepared by adding physiological saline, and supernatant of the colon tissue was prepared by centrifugation (3,000rpm, 4 ℃) for 10min, and the protein concentration was measured by Coomassie Brilliant blue method. The levels of the cytokines TNF-. alpha.and IL-6 were determined and were performed according to the kit instructions. And calculating the content of the cell factor according to a standard curve obtained by the standard substance, ensuring that the OD value of the sample falls within the range of the standard curve, and if the OD value exceeds the range of the standard curve, diluting again and detecting. The concentration of colon cytokines is expressed as: pg/ml colon tissue protein.

2.7Western blot analysis

2.7.1 extraction and quantification of Total protein from animal tissues

The tissue to be tested was weighed to 40mg, washed twice with pre-cooled PBS and minced. Adding protein lysate (1% RIPA, 50mM Tris-HCl pH 8.0, 0.02% NaN)₃150mM NaCl, 10. mu.L of 100mM PMSF per 1mL of lysate), 400. mu.L, ground for 10min, lysed in an ice bath for 20min, and centrifuged at 12000rpm for 10 min. The supernatant was transferred to a 1.5mL eppendorf tube and protein quantification was performed as described in the BCA kit instructions.

2.7.2 gel electrophoresis and transfer of membranes

According to the difference of the molecular weight of the protein to be detected, 8% or 10% of separation gel and 4% of compression gel are prepared and filled into SDS-PAGE gel. After adding a proper amount of 1 × electrophoresis buffer, a pre-stained protein marker (1 μ L) and a sample to be detected are sequentially added. And (4) performing 80V stabilized electrophoresis for about 30min, and after the sample enters the separation gel, adjusting the voltage to 120V to continue electrophoresis. And (5) referring to the pre-stained protein marker, and ending electrophoresis when the target band reaches a proper position. The gel at the corresponding position is cut according to the size of the target protein and put into the transmembrane buffer for balancing. And (3) putting the sheared PVDF membrane into methanol for activation for 1min, then putting the PVDF membrane into a membrane conversion buffer solution for soaking for 30min, and putting the filter paper into the membrane conversion buffer solution for soaking simultaneously. "sandwich" transmembrane was made in the order "(+) splint-filter paper-PVDF membrane-gel-filter paper-splint (-)" to ensure no air bubbles and then transmembrane was performed. And (5) setting the membrane conversion voltage and time according to the molecular weight of the protein to be detected. After the membrane transfer is completed, the PVDF membrane is dyed with ponceau S dye solution for about 1min, the protein on the membrane is observed, and then the membrane is washed with deionized water for 3 min.

2.7.3 development

And (3) putting the PVDF membrane into a prepared confining liquid, and incubating for 2h at room temperature. PBST was washed 4 times for 5min each. Subsequently, it was placed in a hybridization bag and coated overnight at 4 ℃ with a primary antibody. The next day, PBST was washed 4 times for 5min each, placed in a new hybridization bag and coated with room temperature secondary antibody for 2 h. PBST was washed 4 times for 5min each. The films were photographed using a Bio-Rad gel imaging system and the optical density was analyzed using IPP software.

2.8 data analysis

All data are expressed as means ± s.e.m. and statistical differences between groups were tested using one-way ANONA and Dunnett's in the SPSS software. A p-value less than 0.05 is considered to be significantly different.

3. Results of the experiment

3.1 Effect of alcohol extract of Gelidium japonicum on weight loss of mice with UC induced by DSS

Mice drinking DSS causes symptoms of colitis, mainly characterized by weight loss, diarrhea or loose stools, and macroscopic hematochezia. Fig. 1 shows the effect of alcohol extract of nostoc commune on the reduction of body weight in mice with UC induced by DSS.^#p<0.05,^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The results of this study show that compared to the normal groupThe weight of mice in the DSS model group is obviously reduced (p is less than 0.01). Gavage of the alcohol extract of nostoc commune at low dose (20mg/kg), high dose (100mg/kg) and positive drug (200mg/kg) attenuated the weight loss in the model mice compared to the DSS model group. Wherein, the improvement effect of the high dose (100mg/kg, p < 0.01) of the nostoc commune alcohol extract on the weight reduction of the model mice is equivalent to that of a positive drug (200mg/kg, p < 0.01).

3.2 Effect of Alcoholic extract of Gelidium japonicum on DAI Scoring in mice with UC induced by DSS

DSS-induced colitis mice have a gradual increase in the disease activity index DAI, manifested primarily as weight loss, diarrhea and hematochezia. Fig. 2 shows the effect of alcohol extract of nostoc commune on the reduction of body weight in mice with UC induced by DSS.^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The results of the study show that the DAI score of mice in the DSS model group is obviously increased (p is less than 0.01) compared with that in the normal group. Low dose (20mg/kg), high dose (100mg/kg) and positive drug (200mg/kg) of the nostoc commune alcohol extract are administrated by gastric lavage to obviously reduce DAI (disease activity index) scores of model mice. Wherein, the inhibition effect of the nostoc commune alcohol extract on the DAI score is equivalent to that of a positive drug (200mg/kg, p is less than 0.01) in a low dose (20mg/kg, p is less than 0.05), and the inhibition effect of the nostoc commune alcohol extract on the DAI score of a model mouse is stronger than that of the positive drug (200mg/kg, p is less than 0.01) in a high dose (100 mg/kg).

3.3 Effect of alcohol extract of Geranium strictipes on Colon Length of UC mice induced by DSS

The colon length of DSS-induced colitis mice is obviously shortened. Fig. 3 is a graph of the effect of alcohol extract of nostoc commune on colon length of DSS-induced UC mice.^##p<0.01 verses normal group; p<0.01 versis DSS model group. The results of this study show that the colon length of mice in the DSS model group is significantly shortened (p < 0.01) compared to the normal group. The stomach-perfused nostoc commune ethanol extract obviously protects the colon of a model mouse from shortening at low dose (20mg/kg, p is less than 0.01) and high dose (100mg/kg, p is less than 0.01), and the colon shortening inhibition capability of the stomach-perfused nostoc commune ethanol extract is equivalent to that of a positive drug (200mg/kg, p is less than 0.01).

3.4 Effect of Dictamnus dasycarpus alcohol extract on MPO activity of DSS-induced UC mice

DSS-induced colitis mouse colon tissue neutrophil infiltration is obvious, MPO activity is clearIt was significantly elevated. FIG. 4 shows the effect of alcohol extract of nostoc commune on the activity of UC mouse MPO induced by DSS.^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The results of the study show that compared with the normal group, the activity of MPO (p is less than 0.01) of the colon tissue of the mice in the DSS model group is obviously improved. The low dose (20mg/kg, p < 0.05) and the high dose (100mg/kg, p < 0.01) of the nostoc commune ethanol extract are administrated by gastric lavage to obviously inhibit the MPO activity of a model mouse, and the inhibition capability of the nostoc commune ethanol extract on the MPO activity is equivalent to that of a positive drug (200mg/kg, p < 0.01).

3.5 Effect of the alcohol extract of Gelidium japonicum on the pathologic morphology of mice UC induced by DSS

The colon tissue of the mice in the normal group has no obvious pathological change, the lesion of the colon tissue of the mice in the DSS model group mainly affects a mucous layer and a submucosa layer, and inflammatory cell types are mainly mononuclear macrophage and neutrophil. The local mucosa in the severe inflammation area is necrosed in a whole layer to form ulcer.

Fig. 5 is a staining graph of HE staining of colon tissues, and fig. 6 is an effect of swiss chard alcohol extract on DSS-induced UC mouse pathomorphology.^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The results of the study show that compared with the normal group, the DSS model group mice have colon tissue inflammatory cell infiltration and crypt damage (p is less than 0.01). The administration of low (20mg/kg, p < 0.05) and high (100mg/kg, p < 0.01) doses of the extract of nostoc commune by gavage significantly reduced the above histopathological changes, with an effect on reducing the score of histopathology comparable to that of the positive (200mg/kg, p < 0.01).

3.6 Effect of the alcohol extract of Gelidium japonicum on the content of TNF-alpha in UC mouse proinflammatory factor induced by DSS

A plurality of proinflammatory factors are involved in the generation and development of UC, and TNF-alpha and IL-6 are particularly important. FIG. 7 shows the effect of the alcohol extract of Gelidium japonicum on the content of UC mouse proinflammatory factor TNF-alpha induced by DSS.^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The results of the study show that compared with the normal group, the content of TNF-alpha in colon tissues of mice in the DSS model group is obviously increased (p is less than 0.01). The injection of high dose (100mg/kg, p is less than 0.05) and positive drug (200mg/kg, p is less than 0.01) of nostoc commune alcohol extract for gastric lavage can obviously reduce the knot of model miceTNF-alpha content in intestinal tissue.

3.7 Effect of the alcohol extract of Gelidium japonicum on the content of IL-6 in UC mouse proinflammatory factor induced by DSS

DSS-induced colitis mouse colon tissue IL-6 expression is obviously increased. FIG. 8 shows the effect of the alcohol extract of Geranium strictipes on the content of UC mouse proinflammatory factor IL-6 induced by DSS.^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The results of the study show that compared with the normal group, the content of IL-6 in colon tissues of mice in the DSS model group is obviously increased (p is less than 0.01). The content of IL-6 in colon tissues is obviously reduced by intragastrically administering the nostoc commune ethanol extract with low dose (20mg/kg, p is less than 0.05) and high dose (100mg/kg, p is less than 0.01), and the reduction degree of the IL-6 content is equivalent to that of a positive drug (200mg/kg, p is less than 0.01).

3.8 Effect of the alcohol extract of Gelidium japonicum on the content of IL-1 beta of UC mouse proinflammatory factor induced by DSS

The DSS-induced colitis mouse model mouse has obviously raised colon tissue IL-1 beta content. FIG. 9 shows the effect of the alcohol extract of Geranium strictipes on the content of UC mouse proinflammatory factor IL-1 beta induced by DSS.^##p<0.01 verses normal group; p<0.05 versiss DSS model group. The results of the study show that compared with the normal group, the content of IL-1 beta in colon tissues of mice in the DSS model group is obviously increased (p is less than 0.01). The content of IL-1 beta is slightly reduced by intragastrically administering low dose (20mg/kg) and high dose (100mg/kg) of the nostoc commune alcohol extract, and the reduction of the content of IL-1 beta is weaker than that of a positive drug (200mg/kg, p is less than 0.05).

3.9 Effect of the alcohol extract of Geranium strictipes on the activation of NF-kB of UC mouse proinflammatory factor induced by DSS

NF-kB is a key transcription factor and is involved in regulating the expression of inflammation target genes such as pro-inflammatory factors in the UC pathogenesis process. FIG. 10 is a Western blot diagram of activation of UC mouse pro-inflammatory factor NF-kB induced by DSS by the nostoc commune alcohol extract; FIG. 11 is a bar graph of the effect of alcohol extract of Gelidium japonicum on the activation of UC mouse pro-inflammatory factor NF- κ B induced by DSS.^##p<0.01 verses normal group; p<0.05,**p<0.01 versis DSS model group. The research result shows that the DSS model group mice colon tissue NF-kB activation is obviously enhanced compared with the normal group. (p < 0.01). Stomach-irrigation for feeding nostoc commune alcoholThe low dose (20mg/kg, p < 0.05) and the high dose (100mg/kg, p < 0.01) of the extract obviously inhibit the activation of NF-kB of colon tissues, and the degree of the inhibition of the activation of the NF-kB is equivalent to that of a positive drug (200mg/kg, p < 0.01).

4. Conclusion and discussion

The research finds that the nostoc commune alcohol extract obviously improves the weight reduction of a mouse model UC induced by DSS, inhibits the increase of DAI score, reduces the shortening of the colon length of the mouse caused by UC, and simultaneously reduces inflammatory cell infiltration and tissue damage. In addition, the nostoc commune alcohol extract obviously reduces the content of colon tissue cell factors TNF-alpha and IL-6, and the effect is closely related to the activation of a key transcription factor NF-kappa B for inhibiting an inflammatory signal channel. These conclusions suggest that the extract of nostoc commune can be used for treating inflammatory bowel diseases, relieving inflammatory reaction of UC model induced by DSS, and improving severity of UC, and the mechanism of the extract is probably related to inhibiting expressions of cytokines TNF-alpha, IL-6 and the like regulated by NF-kB signal channel.

Claims

1. The application of the nostoc commune alcohol extract in preparing the medicine for treating ulcerative colitis is as follows: taking nostoc commune, extracting the nostoc commune by using low-carbon alcohol as a solvent under a heating condition, collecting an extracting solution, and recovering the solvent to obtain a nostoc commune alcohol extract; recrystallizing the nostoc commune alcohol extract with low-carbon alcohol to obtain a purified nostoc commune alcohol extract; wherein the lower alcohol is one or the combination of more than two of alcohols containing 1-6 carbon atoms.

2. Use according to claim 1, characterized in that: the extraction is carried out at a temperature ranging from 50 ℃ to the boiling point of the solvent.