CN113209154B - Application of chicken bone extract in preparation of medicine for preventing and/or treating ulcerative colitis - Google Patents
Application of chicken bone extract in preparation of medicine for preventing and/or treating ulcerative colitis Download PDFInfo
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention discloses application of a chicken bone extract in preparing a medicament for preventing and/or treating ulcerative colitis. The research of the invention finds that the chicken bone extract can obviously relieve the symptoms of colon shortening, colon damage, diarrhea, hematochezia and the like, relieve the infiltration of tissue inflammatory cells, and simultaneously reduce the expression of Inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) at the colon part, which indicates that the chicken bone extract can relieve ulcerative colitis by protecting the inherent structure of colon tissues and improving colon inflammation, thereby providing a novel natural medicine selection for preventing and/or treating ulcerative colitis.
Description
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to application of a chicken bone extract in preparing a medicament for preventing and/or treating ulcerative colitis.
Background
The incidence of Ulcerative Colitis (UC) increases year by year worldwide, and the incidence of the disease also tends to increase in China. The pathogenesis of UC is unknown, and is mainly related to genetic susceptibility, epithelial barrier defects, immune response disorders, environmental factors, and the like. The disease is chronic intestinal mucosa inflammation, most of pathological changes are in rectum and even can spread to near colon, and the main clinical manifestations are bloody diarrhea, abdominal pain, hematochezia, weight loss and the like. UC has a long course of disease and is easy to repeatedly attack, even can develop into colon cancer, and is confirmed by WHO as a modern refractory disease, and the pathogenesis of UC is very complex and is not clear at present.
The clinical conventional drug treatment method comprises an aminosalicylic acid preparation, steroids, immunosuppressive agents and the like, and the colon cancer or part of intractable ulcerative colitis needs colectomy treatment, which causes great trouble to the life quality of patients. Currently, amino salicylic acid preparations including mesalazine and sulfasalazine are commonly used in patients with mild and moderate UC, but the preparations have large administration dosage, obvious side effect and poor patient compliance. The immunosuppressive agent infliximab is commonly used for patients with moderate and severe UC, but the price is extremely high, and the reaction is often accompanied with drug withdrawal. Therefore, the development of more safe and effective therapeutic drugs is urgently needed.
The raw plant of chicken bone essence is Croton crassifolius Geisel (European bacteria family) of Croton genus of Euphorbiaceae family, and is used as root medicine. Widely distributed in Guangdong, Hainan, Guangxi, Yunnan, etc. The chicken bone is fragrant, bitter and pungent in taste and warm in nature, enters heart, lung, liver, stomach and kidney channels, and has the effects of regulating qi to alleviate pain, dispelling wind and removing dampness, and relieving swelling and pain. The chicken bone incense can be used for treating gastric and duodenal ulcer, gastrointestinal dysfunction, and flatulence. Experimental research shows that the chicken bone fragrant alcohol extract and the chicken bone fragrant water extract both have anti-inflammatory effect, in addition, patents CN110294766A and CN110540530A both disclose that the chicken bone fragrant root extract has anti-tumor activity, but at present, no relevant report about the effect of the chicken bone fragrant on ulcerative colitis exists, and development and research are needed.
Disclosure of Invention
The invention aims to provide application of a chicken bone extract in preparing a medicine for preventing and/or treating ulcerative colitis, develops new application of the chicken bone extract in preventing and/or treating ulcerative colitis, and provides a novel natural medicine choice for preventing or treating ulcerative colitis.
The above purpose of the invention is realized by the following technical scheme:
the invention provides application of a chicken bone extract in preparing a medicament for preventing and/or treating ulcerative colitis.
The inventor of the invention has conducted extensive and intensive studies, and adopts Dextran Sodium Sulfate (DSS) to induce an ulcerative colitis mouse animal model, wherein the mouse model group has the advantages of weight reduction, diarrhea and hematochezia, obvious colon shortening accompanied by blood stool and ulcer, mucosa lamina propria damage and ulcer of colon tissue, local disappearance or deformation of crypt structure and goblet cells, large infiltration of inflammatory cells, obvious thymus atrophy and degeneration, immune function reduction, and obvious increase of COX-2 protein and iNOS protein expression level in the colon tissue of the mouse. After the chicken bone aroma extract is given, the chicken bone aroma extract is unexpectedly found for the first time to obviously relieve the symptoms of colon shortening, colon damage, diarrhea, hematochezia and the like, relieve the infiltration of tissue inflammatory cells and simultaneously reduce the expression of Inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) at the colon part. The chicken bone extract has the effects of preventing and/or treating the ulcerative colitis, is beneficial to further development and utilization of chicken bone plant resources, and provides a novel natural medicine selection for preventing or treating the ulcerative colitis.
Preferably, the prevention and/or treatment of ulcerative colitis is the alleviation of colon shortening, colon damage and diarrheal hematochezia.
Preferably, the prevention and/or treatment of ulcerative colitis is a reduction in inflammatory cell infiltration of colonic tissue.
Preferably, the prevention and/or treatment of ulcerative colitis is inhibition of the expression of colonic inflammatory proteins COX-2 and iNOS.
In addition, the invention also requests a medicament for preventing and/or treating ulcerative colitis, which comprises the extract of the chicken bone and a pharmaceutically acceptable carrier. In the medicine, the chicken bone extract is used as the only active component, and the balance is substances such as a pharmaceutically acceptable carrier and other additives.
Preferably, the carrier can be selected from sugar powder, microcrystalline cellulose, starch, dextrin, sterilized single distilled water or physiological saline, etc.
Preferably, the medicament can be prepared into pills, granules, capsules, tablets, powder, paste, oral liquid, injection or syrup and the like. The dosage form of the drug of the present invention may be various, and any dosage form may be used as long as it allows the active ingredient to efficiently reach the body of a mammal.
As a preferable scheme, the preparation method of the chicken bone extract comprises the following steps: heating and refluxing the chicken bone essence serving as a raw material and low-carbon alcohol serving as a solvent, concentrating an extracting solution to obtain an extract, and freeze-drying to obtain the chicken bone essence extract.
Preferably, the lower alcohol is one or more of alcohols containing 1-6 carbon atoms. Most preferably, the lower alcohol is ethanol.
Preferably, the concentration of the lower alcohol is 70-95% (v/v).
Preferably, the feed-liquid ratio in the heating reflux extraction is 1:8 to 12.
Preferably, the heating reflux time is 0.5-3 h.
Preferably, the heating reflux temperature is 45-65 ℃.
Preferably, the heating reflux time is 1-3 times.
The subject to which the medicament for the prophylaxis and/or treatment of ulcerative colitis according to the present invention is applied is a human or an animal. By "animal" in the present invention is meant any animal that benefits from the improvement or reduction of the loss or other deterioration of the animal's age-related visual system, including humans, birds (avians), bovine (bovine), canine (Canine), equine (equine), feline (feline), caprine (hicrine), wolfine (lupine), murine (murine), ovine (ovine), and porcine (porcine), and preferably domestic animals. Animals domesticated therein include animals such as dogs, cats, birds, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, recreational horses, cows, goats, sheep, donkeys, pigs, and more exotic species fed by humans.
Preferably, when the application object is a mouse, the effective dose of the chicken bone essence extract is 150-600 mg/kg.
When the application object is a human, the dosage can be correspondingly converted into the dosage of the human body according to the relevant administration standard in the pharmaceutical field. In the case of a large single dose, administration may be carried out several times a day.
Compared with the prior art, the invention has the following beneficial effects:
the research of the invention finds that the chicken bone extract can obviously relieve the symptoms of colon shortening, colon damage, diarrhea, hematochezia and the like, relieve the infiltration of tissue inflammatory cells, and simultaneously reduce the expression of Inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) at the colon part. This indicates that the extract of chicken bone can improve colonic inflammation by protecting the inherent structure of colonic tissue, thereby alleviating ulcerative colitis. The invention develops a new application of the chicken bone extract in the aspect of preventing and/or treating ulcerative colitis, and provides a novel natural medicine selection for preventing and/or treating ulcerative colitis.
Drawings
FIG. 1 shows the results of the weight changes of the mice in each group in the experimental examples of the present invention;
FIG. 2 shows the disease activity index results of various groups of mice in the experimental examples of the present invention;
FIG. 3 shows representative colon lengths (A) and statistical results (B) of various groups of mice in the experimental example of the present invention;
FIG. 4 shows colon pathological sections (A) and scoring results (B) of various groups of mice in the experimental example of the present invention;
FIG. 5 shows the results of analyzing thymus index of each group of mice in the experimental example of the present invention;
FIG. 6 shows the expression results (A) of inflammatory protein COX-2 and iNOS protein in colon tissues of various groups of mice in the experimental examples of the present invention;
whereinIn each figure*P<0.05，**P<0.01 indicates that the model group is statistically different from the normal group;#P<0.05，##P<0.01 indicates that the difference between the administered group and the model group is statistically significant.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
In the present invention, a "pharmaceutically acceptable" component is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
Laboratory animal
C57BL/6 mouse, male, 20 ± 2g, SPF grade, sbeft (beijing) biotechnology limited, license number: SCXK (Jing) 2014-. The animal experiment was conducted at the laboratory animal center of Guangzhou university of traditional Chinese medicine (university City), and all animals were approved by the laboratory animal use Committee of Guangzhou university of traditional Chinese medicine, with a license number: no. 00215301.
Reagent and apparatus
The chicken bone-fragrant medicinal material is purchased from Guangzhou Qingping market and is identified as a certified product;
dextran Sulfate Sodium (DSS) was purchased from Millipore, usa;
sodium carboxymethylcellulose (CMC-Na) and tissue fixative, available from gangrenum biotechnology limited;
95% ethanol and sodium chloride, available from Tianjin Damao chemical reagent factory;
a human urine-feces occult blood test kit (benzidine method) purchased from Nanjing, built institute of bioengineering;
h & E staining kit and neutral gum, purchased from Beijing Solebao scientific Co., Ltd;
mesalazine enteric coated tablets (huidi) were purchased from the sunflower pharmaceutical group jiausi luling pharmaceutical limited.
Example 1 preparation of extract of chicken bone
1250g of chicken bone fragrant medicinal material is taken, respectively added with 95 percent (volume) ethanol with the weight of 10 times of that of the medicinal material, heated and refluxed at 60 ℃ for 2 hours, 1 hour and 45 minutes for 3 times of extraction, filtered, the reflux liquid is combined and condensed into thick paste, and the chicken bone fragrant extract is obtained after decompression, freeze drying.
Example 2
First, experiment method
1. Establishment and administration of ulcerative colitis experimental animal model
All C57BL/6 mice were SPF grade and were acclimatized for one week. Feeding at 25 + -1 deg.C, 55 + -5% humidity and 12 hr day and night circulation. The experimental animals were randomly divided into 5 groups of 6 animals each, and were fed freely.
(1) Control group (Control group): all mice were freely drunk sterile distilled water and gavaged with CMC-Na solution of the corresponding volume for 10 days;
(2) model group (DSS group): mice were given 3% DSS water solution for free drinking for 7 days and gavaged with a corresponding volume of CMC-Na solution for 10 consecutive days, once a day;
(3) mesalazine group (Mesalazine group): the mice were given 3% DSS aqueous solution for free drinking for 7 days, and were gavaged for 10 days with the corresponding volume of mesalamine (100mg/kg, mesalamine enteric coated tablets were ground into powder, mesalamine powder was dissolved in 0.05% CMC-Na solution to make 10mg/mL), ready to use, once a day;
(4) chicken bone-fragrant low dose group (CCGL group): mice were given 3% DSS aqueous solution for free drinking for 7 days, and were gavaged continuously for 10 days with a corresponding volume of low dose of chicken bone essence (150mg/kg, dissolved in 0.05% CMC-Na solution to make 15mg/mL suspension) once a day;
(5) chicken bone-flavored high dose group (CCGH group): mice were given 3% DSS aqueous solution for free drinking for 7 days, and were gavaged for 10 consecutive days with a corresponding volume of high dose of chicken bone essence (600mg/kg, dissolved in 0.05% CMC-Na solution to make a 60mg/mL suspension) once daily.
On the 11 th day of the experiment, blood is taken from the orbit and placed in a 1.5mL sterile EP tube, after standing for 1h at room temperature, the tube is centrifuged at 4000r/min for 10min, and upper serum is sucked; thymus and colon were collected simultaneously. Measuring the length of colon, washing with pre-cooled physiological saline, fixing 0.5cm colon of each mouse in 4% paraformaldehyde for hematoxylin-eosin (H & E) staining; all remaining samples were stored at-80 ℃.
2. Mouse sign observation and disease activity index scoring
The body weight of each mouse was recorded at the same time every day, and the effect of the drug on the body weight of the mice was observed. Observing the physiological state of the experimental mice in the molding administration process, recording the weight change, the stool character change and the fecal occult blood condition of each group of mice in time, and counting the daily scoring result according to the scoring standard in the table 1 to be used as a Disease Activity Index (DAI). Grading standard: a weight loss rate score of 0 ═ weight unchanged; 1, reducing the weight by 1-5%; 2, the weight is reduced by 6-10%; reducing the weight by 11-15% when the weight is 3; weight loss was greater than 15%. Stool trait score DAI ═ (weight loss score + stool trait score + hematochezia score)/3.
TABLE 1 disease Activity index Scoring criteria
Note: the occult blood of the feces of each mouse was observed with occult blood kit every day and relevant records were made.
After the experiment is finished, the thymus is taken and weighed, and the thymus index of each mouse is calculated. Thymus index is thymus (mg)/mouse weight (g).
3. Colon pathological tissue section and pathological scoring
The colons of each group of mice were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and dewaxed, then stained with H & E, and the changes in the pathological tissues of the colons of the mice were observed under a microscope. The pathology scoring criteria were as follows: no obvious damage is seen when 0 is equal to zero; 1-damage to the surface epithelium; 2, ulcer of mucosa, infiltration of inflammatory cells of lamina propria and edema of submucosa; transmural inflammation and ulceration, epithelial deformity, necrosis, desquamation; transmural inflammation and ulceration, with normal mucosa spared; 5-transmural inflammation and ulceration, without normal mucosa.
4. Expression of inflammatory proteins COX-2 and iNOS in colonic tissue
Approximately 60mg of colon tissue was taken, the mass accurately recorded, and placed in a 1.5mL centrifuge tube. The colon tissue in the centrifuge tube was minced with sterilized scissors, 1000. mu.L of RIPA lysate and two sterile steel beads were added, the mixture was trimmed and placed in a multi-sample cryomill precooled in advance, and homogenized for 1min at a shaking frequency of 60 Hz. Taking out the centrifuge tube, placing in a centrifuge, centrifuging at 15000g rotation speed at 4 deg.C for 10min, collecting supernatant, and storing at-80 deg.C for use to avoid repeated freeze thawing.
5. Data analysis
And (3) statistically analyzing the data of the measured data by adopting SPSS 20.0 software, and then drawing a statistical chart by adopting GraphPad Prism 6.0 software. Firstly, performing a normality test on each group of data, and if the data do not accord with normal distribution, performing pairwise comparison by adopting an independent sample Mann-Whitney U test; if two groups of data meet the conditions of normal distribution and homogeneous comparison of variance, two independent samples are adopted for t test; one-way ANOVA was used if multiple sets of data met the normal distribution. In the process of single-factor analysis of variance, if data meet homogeneity of variance, Dunnett test is adopted when two-by-two comparison is carried out; if the data do not meet the homogeneity of the variances, Dunnett T3 test is adopted when two data are compared; if the statistical result is that P is less than 0.05, the difference has statistical significance; if the statistical result is P < 0.01, the difference is very significant statistically.
Second, experimental results
1. Body weight change in acute UC mouse model
As can be seen from fig. 1, the body weight of the normal group mice showed a slow rising trend during the experiment, whereas the body weight of the UC model mice did not differ much from the normal group four days before the 3% DSS was freely drunk, and the model group started to significantly decrease on the fifth day, and the body weight of the model group mice decreased significantly from the normal group by the tenth day (P < 0.01). After the high-dose administration of the chicken bone essence, the weight of the mice is obviously increased compared with that of a model group and a positive medicine group, but the difference in the groups is large, and the statistical significance is avoided.
2. Diarrhea and hematochezia condition of acute UC mouse model
The feces of the normal group mice appeared in a dark wheat grain during the experiment. Compared with the normal group of mice, the anus of the model group of mice is adhered with blood sample mucus-shaped excrement, the excrement is loose and has blood color, the hair color is not glossy, the spirit is listened, and the daily activities are reduced. This is very similar to the symptoms of patients with UC clinically, characterized experimentally by an index of disease activity. As can be seen from FIG. 2, the feces of the model group mice began to show hematochezia, and the diarrheal hematochezia worsened sharply with time, and the diarrheal hematochezia of the model group mice was still not improved after the stop of the administration of DSS on day 7 to day 10. The daily activities of the chicken bone fragrance high-dose administration group mice four days before the experiment are not much different from those of the normal group, and the excrement is black and formed soft excrement without occult blood and hematochezia. The disease activity indexes of the mice in each experimental group are analyzed, the disease activity indexes of the mice are reduced to a certain extent after administration treatment, and the drug effect of the chicken bone-flavored high-dose group is particularly obvious.
3. Colon appearance morphology observation and scoring of acute UC mouse model
The graph A in FIG. 3 is an appearance graph of colon, and the graph B is a statistical result of the length of colon, and it can be known from the graph B in FIG. 3 that the colon of the model group mouse is significantly shortened (P < 0.01) and is accompanied by obvious bloody stool and ulcer, which indicates that DSS successfully induces UC model mouse in the experiment. As can be seen from the appearance of the colon, after the chicken bone incense is administrated for treatment, the hematochezia and ulcer conditions of the colon are obviously reduced compared with a model group, and the length of the colon is obviously increased (P < 0.01).
4. Colon pathological section and score of acute UC mouse model
As can be seen from the A diagram of FIG. 4, the mucosal lamina propria, crypt glands and goblet cells of the colon tissue of the normal group of mice are intact, and inflammatory cells have no or little infiltration; the mucosa lamina propria of the model group mice is broken and ulcerated, the crypt structure and goblet cells are locally disappeared or deformed, and inflammatory cells are largely infiltrated. After the prognosis of mesalazine and chicken bone dried meat, symptoms such as crypt disappearance, epithelial cell injury, inflammatory cell infiltration and the like in colon tissues of UC mice are effectively relieved, and particularly, ulcer and edema are relieved after the chicken bone dried meat is administrated, and neutrophilic granulocytes in the colon tissues are reduced. As can be seen from the graph B in fig. 4, the pathological section score shows that both the mesalazine and the chicken bone aroma high and low dose groups can remarkably alleviate the pathological changes of the UC mouse colon tissue (P < 0.01), and protect the inherent structure of the colon tissue. This suggests that the pathological changes of the UC mouse colon tissue can be significantly alleviated by the high and low doses of the chicken bone essence.
5. Thymus degeneration condition of acute UC mouse model
As can be seen from fig. 5, the thymus of the model group mice was markedly atrophied and degenerated (P < 0.01) and the immune function was decreased as compared to the normal group. Compared with other administration groups, the breast gland degeneration condition is obviously reduced after the high-dose intervention of the chicken bone essence and is close to the level of a normal group.
Expression of colon inflammatory proteins COX-2 and iNOS in acute UC mouse model
As can be seen from the graph A of FIG. 6, the expression level of COX-2 protein in colon tissue was increased in the model group of mice as compared to the normal group; the expression quantity of COX-2 protein in the UC mouse colon tissue is obviously reduced after the chicken bone essence is administrated in high dose (P < 0.01). As is clear from the B diagram in fig. 6, the expression level of iNOS protein in the model group was significantly increased (P < 0.01) as compared with the normal group. And iNOS was significantly reduced compared to the model group after high dose treatment of chicken bone aroma (P < 0.01). The expression levels of inflammatory proteins COX-2 and iNOS are reduced after the chicken bone incense is administrated, which indicates that the chicken bone incense can relieve intestinal inflammation by inhibiting the expression of the inflammatory proteins COX-2 and iNOS.
In conclusion, the intervention of the positive drug and the tested drug has different degrees of improvement effect on the mouse acute ulcerative colitis induced by the DSS. The chicken bone essence with high dose can remarkably reduce the weight loss, colon shortening, diarrhea and rectal bleeding of UC mice, inhibit the expression of inflammatory proteins COX-2 and iNOS in the colon tissues of the UC mice, and improve the inflammatory injury of the colon tissues of the UC mice. This suggests that the extract of chicken bone may ameliorate colonic inflammation by protecting the inherent structure of colonic tissue, thereby alleviating ulcerative colitis.
The applicant declares that the above detailed description is a preferred embodiment described for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, i.e. it does not mean that the present invention must be implemented by means of the above embodiment. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (3)
1. The application of the chicken bone extract in preparing the medicine for preventing and/or treating ulcerative colitis;
the chicken bone incense is prepared from chicken bone incense of Croton of Euphorbiaceae;
the preparation method of the chicken bone extract comprises the following steps: heating and refluxing the chicken bone essence serving as a raw material and v/v 95% ethanol serving as a solvent, concentrating an extracting solution to obtain an extract, and freeze-drying to obtain the chicken bone essence extract;
the material-liquid ratio in the heating reflux extraction is 1: 8-12; the heating reflux time is 0.5-3 h; the heating reflux temperature is 45-65 ℃.
2. The use according to claim 1, wherein the prevention and/or treatment of ulcerative colitis is the alleviation of colon shortening, colon damage and diarrheal hematochezia.
3. The use according to claim 1, wherein the prevention and/or treatment of ulcerative colitis is a reduction in inflammatory cell infiltration of colonic tissue.
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