CN116392554A - Application of red rice seed coat active ingredient in preparation of medicine for preventing and treating ulcerative colitis - Google Patents

Application of red rice seed coat active ingredient in preparation of medicine for preventing and treating ulcerative colitis Download PDF

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CN116392554A
CN116392554A CN202310442829.6A CN202310442829A CN116392554A CN 116392554 A CN116392554 A CN 116392554A CN 202310442829 A CN202310442829 A CN 202310442829A CN 116392554 A CN116392554 A CN 116392554A
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red rice
active ingredient
seed coat
rice seed
ulcerative colitis
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李晔
田雷
孙瑾
陈昱旭
赵志业
郭善成
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Kunming University of Science and Technology
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Kunming University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a new application of red rice seed coat active ingredient in preparing a medicament for preventing and treating ulcerative colitis, belonging to the technical field of biological medicine. The medicine containing the red rice seed coat active ingredient is applied to preventing and/or treating ulcerative colitis and can inhibit related inflammatory factors and Ca in colon tissues 2+ Improving the expression of oxidative stress indexes in colon tissues and improving pathological damage caused by inflammatory reaction of colon tissues; and the red rice seed coat active ingredient can effectively inhibit related inflammation in colon tissue of ulcerative colitis model mice under lower dosageSex factor and Ca 2+ Effectively improving the expression of oxidative stress indexes in colon tissues of the ulcerative colitis model mice and effectively improving pathological damage caused by inflammatory reaction in colon tissues of the ulcerative colitis model mice; the raw materials of the invention are from natural products, which are convenient to collect and have high safety.

Description

Application of red rice seed coat active ingredient in preparation of medicine for preventing and treating ulcerative colitis
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of red rice seed coat active ingredients in preparation of medicines for preventing and treating ulcerative colitis.
Background
Inflammatory bowel disease (Inflammatory bowel disease, IBD) is a chronic and recurrent inflammatory disease of the gastrointestinal tract, a digestive system disease with diarrhea, abdominal pain, bloody mucosae, weight loss as the main symptoms, is difficult to treat completely, severely affects human health and increases the risk of colon cancer. The incidence of Ulcerative Colitis (UC) has increased significantly over the last 20 years, and the etiology and pathogenesis of UC, although complex and ambiguous, has been linked to genetic susceptibility of the host, changes in the host immune system, inflammation and colonic barrier disease. Clinical drugs for UC are mainly glucocorticoids, immunosuppressants and salicylic acids, but most of these drugs only temporarily relieve symptoms, and long-term use can cause side effects on human health, including nausea, vomiting, headache, diarrhea, blood stasis, nephrotoxicity, hypokalemia, increased infection risk (such as upper respiratory tract infection and urinary tract infection), etc. In order to prevent or treat the ulcerative colitis, a safe and effective medicament for preventing or treating the ulcerative colitis is required to be searched from natural products.
Red rice belongs to colored rice varieties and also belongs to rare grain and oil crop resources in China, and meanwhile, the red rice belongs to medicine and food dual-purpose resources and is rich in protein, amino acid, vitamins, trace elements and functional components. The red rice contains anthocyanin substances, and anthocyanin is flavonoid, namely a group of compounds which can be red in the substances based on flavone cores, and is the main component of the active ingredients of the red rice seed coats. The red rice seed coat active ingredient has the characteristics of nature, nutrition and multifunction, and also has pharmacological effects of treating and preventing various diseases of human body and health care functions. The red rice seed coat active ingredient is reported to have cholesterol-lowering, anticancer, blood sugar-lowering, weight-losing, etc. activities, and the red rice seed coat active ingredient is safe and free from mutagenic effects. However, at present, no patent literature report exists on the research on the anti-inflammatory active ingredients of red rice seed coats at home and abroad.
Disclosure of Invention
The invention mainly aims to provide a new application of red rice seed coat active ingredients in preparing a medicament for preventing and treating ulcerative colitis.
In order to achieve the above purpose, the present invention provides the following technical solutions:
application of red rice seed coat active ingredient in preparing medicine for preventing and treating ulcerative colitis is provided.
The concentration of the red rice seed coat active ingredient in the medicine for preventing and treating ulcerative colitis is preferably 200-600 mg/day/kg. The preparation formulation, the auxiliary materials and the preparation method of the medicine for preventing and treating ulcerative colitis have no special requirements, and are well known to those skilled in the art. The novel medicament for preventing and/or treating the ulcerative colitis takes red rice seed coat active ingredients as active ingredients, is used for preparing medicaments for preventing and treating the ulcerative colitis, and can be added with one or more pharmaceutically acceptable auxiliary materials or compounded with other active ingredients to play a role in synergistically improving the ulcerative colitis. The medicine can be granules, pills, tablets, capsules, powder and the like in oral preparations.
The invention carries out a series of researches on the active ingredients of the red rice seed coats for preventing and treating ulcerative colitis. It is proved that the red rice seed coat active ingredient can effectively inhibit related inflammatory factors and Ca in colon tissue of ulcerative colitis model mice 2+ Is expressed by (a); can effectively improve the expression of oxidative stress indexes in colon tissues of ulcerative colitis model mice; can effectively improve pathological damage caused by inflammatory reaction in colon tissues of the ulcerative colitis model mouse; overall effective in improving inflammatory response of colon tissue of mice with model of DSS-induced ulcerative colitis and maintaining normal structure and physiological function of colon thereof, for preventing or treating ulcerative colitisThe effect of enteritis is almost equivalent to that of a blank control group. The red rice seed coat active ingredient provided by the invention has remarkable effect on preventing and treating ulcerative colitis, and the red rice has wide distribution, high safety, low cost and easily available raw materials; the red rice seed coat active ingredient has good development and application prospects in the preparation of the medicines for preventing and treating ulcerative colitis.
Preferably, the preparation method of the red rice seed coat active ingredient comprises the following steps:
(1) Pulverizing red rice, and sieving to obtain red rice powder; adding an extracting agent into red rice powder for ultrasonic extraction to obtain a mixture; centrifuging the mixture to obtain red rice powder precipitate and supernatant;
(2) Continuously adding an extracting agent into the red rice powder precipitate obtained in the step (1) for ultrasonic extraction for 2-3 times, and then carrying out centrifugal separation to collect supernatant;
(3) Combining the supernatants of step (1) and step (2) to obtain a combined supernatant; filtering the combined supernatant, concentrating the filtrate by rotary evaporation, and recovering the extractant by rotary evaporation until a water phase is obtained; continuously concentrating the water phase by rotary evaporation to obtain concentrated solution;
(4) And (3) performing low-temperature pre-freezing on the concentrated solution in the step (3), and performing freeze-drying by using a freeze dryer to obtain freeze-dried powder of the red rice seed coat active ingredient extract.
Preferably, the sieving in the step (1) is a 80-100 mesh sieve.
Preferably, the extractant in the step (1) and the step (2) contains 0.4 to 0.8 percent of citric acid, 60 to 80 percent of ethanol and the balance of water by mass percent.
Preferably, the mass volume ratio of the red rice powder to the extractant in the step (1) is 1 (16-18) g/mL.
Preferably, the ultrasonic extraction in the step (2) is carried out by ultrasonic waves of 30-35 min under the power of 200W, and the extraction temperature is 50-55 ℃.
Preferably, the centrifugation in step (1) and step (2) is performed at 4300 to 4800r/min for 10 to 15min.
Preferably, the low temperature prefreezing temperature in step (4) is-20 to-50 ℃.
Preferably, the medicament comprises an extract of red rice seed coat active ingredient.
Preferably, the drug is a drug that inhibits related inflammatory factors and Ca in colon tissue 2+ The expression of oxidative stress index in colon tissue, and the pathological injury caused by inflammatory reaction of colon tissue.
In summary, compared with the prior art, the invention has the following advantages:
1. the invention discloses a new application of the red rice seed coat active ingredient extract.
2. The animal model experiment shows that the red rice seed coat active ingredient can effectively inhibit the related inflammatory factors and Ca in colon tissues of ulcerative colitis model mice under lower dose 2+ Effectively improving the expression of oxidative stress indexes in colon tissues of the ulcerative colitis model mice and effectively improving pathological damage caused by inflammatory reaction in colon tissues of the ulcerative colitis model mice.
3. The raw materials of the invention are derived from natural products, so that the invention is convenient to collect and has high safety; the extraction process is simple, the cost is low, and the method is suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is a graph showing the results of liquid phase detection of the red rice seed coat active ingredient extract prepared in example 1 of the present invention;
FIG. 2 shows the effect of red rice seed coat active ingredient on related inflammatory factors and Ca in colon tissue of mice 2+ A histogram of effects;
FIG. 3 is a bar graph showing the effect of red rice seed coat active ingredients of the present invention on related oxidative stress indicators in colon tissue of mice;
FIG. 4 is a HE staining pattern of lung tissue sections of mice according to the invention;
FIG. 5 is a graph of ab-pas staining of lung tissue sections of mice of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
The terms "first," "second," "third," and the like in this application are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "first," "second," and "third" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. All directional indications (such as up, down, left, right, front, back … …) in the embodiments of the present application are merely used to explain the relative positional relationship, movement, etc. between the components in a particular gesture (as shown in the drawings), and if the particular gesture changes, the directional indication changes accordingly. Furthermore, the terms "comprise" and "have," as well as any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those listed steps or elements but may include other steps or elements not listed or inherent to such process, method, article, or apparatus.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
The red rice used in the following examples of the present invention was purchased from Yunnan Pu' er; the C57BL/6J mice used were purchased from Beijing Bei Fu laboratory animals Co. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of purple Rice seed coat extract
(1) Extraction of red rice seed coat active ingredient
Crushing the purchased red rice, and sieving the crushed red rice with a 80-mesh sieve to obtain red rice powder. Weighing 150g of red rice flour, adding 2400mL of an extracting agent, wherein the extracting agent is a mixed solution of 0.5% citric acid, 70% ethanol and 29.5% water (the volume ratio is 3:16), the mass-volume ratio of the red rice flour to the extracting agent is 1g:16mL, and then carrying out ultrasonic treatment for 30min at 53 ℃ under the power condition of 200W; centrifuging at 4500 r/min for 10min after ultrasonic extraction, and collecting supernatant; this procedure was repeated 3 times. Then carrying out suction filtration on the supernatant, carrying out rotary evaporation for concentration, carrying out rotary evaporation on the extracting solution, and recovering the extracting agent until a water phase is obtained; continuously concentrating the water phase by rotary evaporation to obtain a concentrated solution; pre-freezing the concentrated solution at-20deg.C, and lyophilizing with a lyophilizing machine to obtain lyophilized powder of red rice seed coat active ingredient extract. The application of red rice seed coat active ingredient obtained by the preparation method in preparing ulcerative colitis medicine.
(2) Qualitative analysis of main chemical components of red rice seed coat active ingredient extract by UPLC-MS/MS technology
The liquid phase detection results are shown in FIG. 1. As shown in FIG. 1, the main chemical components of the red rice seed husk active ingredient extract include N (6), N (6) -dimethyl-L-lysine, L-arginine, guanosine, N-hydroxytryptamine, 11-octadecenoic acid, 9,10, 13-trihydroxy-11-octadecenoic acid, elaidic acid, petroselinic acid, N-acetylisatoic acid, 3 '-glucosyl-6, 7-dihydroxy-N-methyl-benzyltetrahydroisoquinoline, choline, diisobutyl phthalate, 5,6,7,5' -tetramethoxy-3 ',4' -methylenedioxyflavonoid, medicagin-7-O-glucoside, and the like. The red rice seed coat active ingredient is rich in amino acids and derivatives, alkaloids, lipids, phenolic acids and flavonoids thereof.
EXAMPLE 2 evaluation of Activity of red Rice seed coat active ingredient extract on prevention and treatment of ulcerative colitis
2.1 establishment and Experimental Process of ulcerative colitis mouse model
Male C57BL/6J mice (about 22g in weight at 8 weeks of age) were used as subjects, and 6 mice were used in each group. And (3) inducing ulcerative colitis to occur by using a free drink 2.5% DSS distilled water mode, constructing a mouse ulcerative colon inflammation model, and modeling 7d. The control effect of the red rice seed coat active ingredient (freeze-dried powder) obtained in the stomach-filling example 1 on mice with the model of DSS-induced ulcerative colon inflammation is evaluated; mice were acclimatized for 7d in standard environment after purchase and were fed with standard feed. The specific experimental procedure and the grouping are as follows:
blank control group: continuously pouring the stomach for 14 days, and taking the distilled water with equal volume and freely drinking the distilled water;
negative control group: continuously performing gastric lavage for 14 days, and taking equal volume of solvent (10% DMSO+40% PEG300+5% Tween-80+45% Saline) for dissolving red rice seed coat active ingredient, and freely drinking distilled water;
model group: filling the stomach for 14 days continuously, feeding equal volume of distilled water, freely drinking distilled water containing 2.5% DSS for 7 days continuously from 8 th day, and molding for 7d;
red rice seed coat active ingredient low dose group: 200mg/kg red rice seed coat active ingredient was administered by continuous 14d gavage and distilled water containing 2.5% dss was freely drunk 7 days continuously from day 8;
high dose group of red rice seed coat active ingredient: 600mg/kg red rice seed coat active ingredient was administered by continuous 14d gavage and distilled water containing 2.5% dss was freely drunk 7 days continuously from day 8;
positive control group: 600mg/kg of 5-aminosalicylic acid (SASP) was administered by continuous 14d gavage and distilled water containing 2.5% DSS was freely consumed for 7 consecutive days starting on day 8.
2.2 detection of inflammatory factors and Ca in colon tissue of model mice by active ingredients 2+ Expression effects:
2.2.1 taking groups of mice, aseptically taking colon tissues, washing with aseptically pre-cooled PBS, taking 100mg of colon tissues, adding 900 μl of aseptically pre-cooled PBS, homogenizing at 4deg.C, centrifuging to obtain supernatant, and detecting NO, MPO, ca in the colon tissues of the groups of mice with corresponding kit 2+ Expression status.
2.2.2 experimental results
Inflammatory factors refer to various cytokines involved in inflammatory responses, and when colon tissue is stimulated from outside, cells in the colon tissue generate immune responses, secrete a large amount of inflammatory factors, and even trigger a stronger inflammatory response. Typical inflammatory factors include Nitric Oxide (NO), myeloperoxidase (MPO), interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), and the like. In the book of magic nitric oxide, it is mentioned that "99.9% of diseases in the human body are related to NO". NO has pro-inflammatory effects, and at the site of inflammation, NO acts on vascular smooth muscle cells, increasing its cGMP level, causing vasodilation and increased permeability, facilitating the arrival of inflammatory mediators and pain-causing substances at the site of action, and increasing the penetration of monocytes into the site of inflammation during the inflammatory response. In the pathogenesis of immune inflammation, elevated levels of NO in the body can affect the pathological process from a number of pathways. NO can also cause the opening of calcium ion channels on the cell membrane and the increase of intracellular calcium ion concentration. MPO is a heme protein, which is enriched in neutrophils, synthesized in bone marrow and stored in azurin-phaga granules before they enter the circulation. MPO can kill microorganisms in phagocytes by catalyzing and oxidizing chloride ions to generate hypochlorous acid, destroy various target substances, and play roles in various aspects such as organism generation and regulation of inflammatory reaction.
The invention detects NO, MPO, ca in colon tissues of each group of mice by using the corresponding kit 2+ The effect of red rice seed coat active ingredients on ulcerative colon inflammation model mice was examined, and the effect is shown in FIG. 2. In FIG. 2, graph A shows the amount of NO expressed in colon tissue; panel B shows the amount of MPO expressed in colon tissue; panel C shows Ca in colon tissue 2+ Is a factor (B) of the expression level of (C). Contro l is a blank control group; NC is a negative control group; mode l is a DSS induction model group; RRPL is a low dose lavage group (200 mg/kg); RRPH is high dose lavage group (600 mg/kg); SASP is a positive control group (5-aminosalicylic acid); * Represents p<0.05 represents p<0.01 x represents p<0.001, compared to NC negative control; # denotes p<0.05, # denotes p<0.01, # # denotes p<0.001, and Mode l group.
FIG. 2 shows that the red rice seed coat active ingredient can significantly reduce NO expression (#, p) in colon tissue of ulcerative colon inflammation model mice<0.05 MPO expression is significantly reduced (#, p)<0.05)，Ca 2+ Expression is significantly reduced (# #, p<0.001). The red rice seed coat active ingredient can obviously inhibit inflammatory factors and Ca related to colon tissues of mice with inflammation models 2+ Is expressed by (a).
2.3 detection of the effect of active ingredient on oxidative stress index in colon tissue of model mice:
2.3.1 taking groups of mice, aseptically taking colon tissues, flushing the colon tissues by using aseptic precooled PBS, adding 900 mu l of aseptic precooled PBS into 100mg of colon tissues to homogenate at 4 ℃, centrifuging the mixture to obtain supernatant, and detecting the expression conditions of SOD and MDA in the colon tissues of the groups of mice by using corresponding kits.
2.3.2 experimental results
Superoxide dismutase (SOD) has strong antiinflammatory effect, and can be used for treating pleurisy to reduce accumulation of pleural effusion and leucocyte. SOD can also inhibit arthritis, reduce the precipitation of Ig in glomeruli, and interfere the accumulation of leukocyte in glomeruli inflammation site. SOD can inhibit organ permeability change caused by acute tracheitis caused by xanthine/xanthine oxidase, and reduce polymorphonuclear leukocyte accumulation in lung. SOD has the effects of treating and improving idiopathic proctitis, and injecting proper amounts of bovine Cu and Zn-SOD can improve the appearance of joints, improve the joint movement capacity and further effectively treat osteoarthritis. During pathological changes in inflammation, when oxygen radicals in the inflamed tissue attack cell membrane lipids, lipid peroxidation occurs, yielding lipid peroxidation products that eventually degrade into the stable product Malondialdehyde (MDA). MDA can crosslink with lipids and proteins on the cell membrane, thereby rendering its function dysfunctional. Thus, MDA is one of the important indicators of inflammatory conditions in the body.
The invention examines the influence of the active ingredients of red rice seed coats on ulcerative colitis model mice by detecting the expression conditions of SOD and MDA in colon tissues of each group of mice by using a corresponding kit, and the effect is shown in figure 3. Wherein graph A shows the SOD expression level in colon tissue; panel B shows the expression level of MDA in colon tissue. Contro l is a blank control group; NC is a negative control group; mode l is a DSS induction model group; RRPL is a low dose lavage group (200 mg/kg); RRPH is high dose lavage group (600 mg/kg); SASP is a positive control group (5-aminosalicylic acid); * Represents p <0.05, p <0.01, p <0.001, compared to NC group; # denotes p <0.05, # denotes p <0.01, # denotes p <0.001, and Mode l group comparison.
The results in FIG. 3 show that the active ingredients of red rice seed coats can significantly increase SOD expression (#, p < 0.01) and significantly decrease MDA expression (#, p < 0.01) in colon tissues of mice with inflammation models. The red rice seed coat active ingredient can obviously improve the expression of related oxidative stress indexes in colon tissues of mice with an inflammation model.
2.5 detection of the effect of active ingredient on colon histopathology in model mice:
2.5.1 taking each group of mice, taking colon tissues by aseptic technique, washing with aseptic precooled PBS, fixing to prepare paraffin sections, and observing the change condition of tissue structures under a microscope after HE staining and ab-pas staining.
The preparation method of paraffin sections comprises the following steps:
(1) Drawing materials: fresh colon tissue was fixed with 10% formalin solution for more than 24 h. Taking out colon tissue from the fixing solution, trimming the tissue of the target part in a fume hood by using a surgical knife, and placing the trimmed tissue and a corresponding label in a dehydration box;
(2) Dewatering and immersing wax: and (5) placing the dehydration box into a dehydrator to sequentially carry out gradient alcohol dehydration. 75% alcohol 4h,85% alcohol 2h,90% alcohol 2h,95% alcohol 1h, absolute alcohol I30 min, absolute alcohol II 30min, alcohol benzene 5-10min, xylene I5-10 min, xylene II 5-10min, paraffin I1h melted at 65 ℃, paraffin II 1h melted at 65 ℃, paraffin III 1h melted at 65 ℃;
(3) Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20deg.C, solidifying, removing the wax block from the embedding frame, and trimming;
(4) Slicing: placing the trimmed wax block into a paraffin slicer for slicing, wherein the thickness of the wax block is 4 mu m; the slices float on warm water at 40 ℃ of a slice spreading machine to flatten the tissues, the glass slide drags the tissues out, the slices are baked in a baking oven at 60 ℃, and the slices are taken out for preservation at normal temperature after being baked with water and wax.
The HE staining method is as follows:
(1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water;
(2) Hematoxylin staining: slicing, dyeing with hematoxylin dye solution for 3-5min, washing with tap water, differentiating with differentiation solution, washing with tap water, and washing with running water;
(3) Eosin staining: the slices are dehydrated in gradient alcohol of 85% and 95% for 5min respectively, and then are dyed in eosin dye solution for 5min.
(4) And (3) removing the water sealing piece: sequentially slicing, adding absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-absolute ethyl alcohol III 5 min-xylene I5 min-xylene II 5min, and sealing with neutral resin;
(5) Microscopic examination, image acquisition and analysis.
The ab-pas staining method is as follows:
(1) Dewaxing paraffin sections to water, and washing with distilled water for 2min;
(2) Adding an aliskiren blue staining solution, and staining for 10-20 min;
(3) Distilled water is washed for 3 times, each time for 1-2 min;
(4) Adding a periodic acid solution, and oxidizing for 5min;
(5) Entering a Schiff Reagent for dip dyeing for 10-20 min;
(6) Pouring out the Schiff Reagent, and flushing with running water for 10min;
(7) Adding hematoxylin staining solution, and staining the core for 1-2 min;
(8) Differentiating for 2-5 s by using an acidic differentiation liquid, and washing with water;
(9) Returning blue with Scott blue-forming solution, and washing with water for 3 min;
(10) Step-by-step conventional ethanol dehydration; xylene is transparent, and the neutral gum is sealed;
(11) Microscopic examination, image acquisition and analysis.
2.5.2 experimental results
The experiment adopts HE staining to observe colon tissue structure of the mice, and the result is shown in figure 4.Contro l is a blank control group; NC is a negative control group; mode l is a DSS induction model group; RRPL is a low dose lavage group (200 mg/kg); RRPH is high dose lavage group (600 mg/kg); SASP was the positive control group (5-aminosalicylic acid).
As can be seen from the results of FIG. 4, the red rice seed coat active ingredient can significantly reduce myotissue edema, significantly reduce inflammatory cell infiltration of tissues and maintain the integrity of goblet cells. The red rice seed coat active ingredient can obviously improve pathological damage caused by inflammatory reaction of colon tissues of mice.
The experiment uses ab-pas staining for detecting the mucosal status and the number of goblet cells in the colon tissue of the mice, and the results are shown in FIG. 5.Contro l is a blank control group; NC is a negative control group; mode l is a DSS induction model group; RRPL is a low dose lavage group (200 mg/kg); RRPH is high dose lavage group (600 mg/kg); SASP was the positive control group (5-aminosalicylic acid).
As can be seen from the results of fig. 5, the red rice seed coat active ingredient can significantly inhibit DSS-induced mucosal injury and attenuate goblet cell loss. The red rice seed coat active ingredient can obviously improve pathological damage caused by inflammatory reaction of colon tissues of mice.
Example 3 preparation of purple Rice seed coat extract
(1) Extraction of red rice seed coat active ingredient
Crushing the purchased red rice, and sieving the crushed red rice with a 100-mesh sieve to obtain red rice powder. Weighing 150g of red rice flour, adding 2700mL of extracting agent which is a mixed solution of 0.8% citric acid, 80% ethanol and 19.2% water, wherein the mass-volume ratio of the red rice flour to the extracting agent is 1g to 18mL, and then carrying out ultrasonic treatment for 35min at 55 ℃ under the power condition of 200W; centrifuging and centrifuging after ultrasonic extraction, centrifuging at a rotation speed of 4800r/min for 15min, and collecting supernatant; this step was repeated 2 times. Then carrying out suction filtration on the supernatant, carrying out rotary evaporation for concentration, carrying out rotary evaporation on the extracting solution, and recovering the extracting agent until a water phase is obtained; continuously concentrating the water phase by rotary evaporation to obtain a concentrated solution; pre-freezing the concentrated solution at-30deg.C, and lyophilizing with a lyophilizing machine to obtain lyophilized powder of red rice seed coat active ingredient extract. The application of red rice seed coat active ingredient obtained by the preparation method in preparing ulcerative colitis medicine.
Example 4
Preparation of purple rice seed coat extract
(1) Extraction of red rice seed coat active ingredient
Crushing the purchased red rice, and sieving the crushed red rice with a 90-mesh sieve to obtain red rice powder. Weighing 150g of red rice flour, adding 2600mL of extracting agent which is a mixed solution of 0.4% citric acid, 60% ethanol and 39.6% water (volume ratio is 3:17), wherein the mass-volume ratio of the red rice flour to the extracting agent is 1g:17mL, and then carrying out ultrasonic treatment at 50 ℃ under 200W power condition for 32 min; centrifuging after ultrasonic extraction at a rotation speed of 4300 r/min for 13 min, and collecting supernatant; this procedure was repeated 3 times. Then carrying out suction filtration on the supernatant, carrying out rotary evaporation for concentration, carrying out rotary evaporation on the extracting solution, and recovering the extracting agent until a water phase is obtained; continuously concentrating the water phase by rotary evaporation to obtain a concentrated solution; pre-freezing the concentrated solution at-25deg.C, and lyophilizing with a lyophilizing machine to obtain lyophilized powder of red rice seed coat active ingredient extract. The application of red rice seed coat active ingredient obtained by the preparation method in preparing ulcerative colitis medicine.
In conclusion, the red rice seed coat active ingredient can effectively inhibit related inflammatory factors and Ca in colon tissues of ulcerative colitis model mice 2+ Is expressed by (a); can effectively improve the expression of oxidative stress indexes in colon tissues of ulcerative colitis model mice; can effectively improve pathological damage caused by inflammatory reaction in colon tissues of ulcerative colitis model mice. The red rice seed coat active ingredient provided by the invention has remarkable effect on preventing and treating ulcerative colitis, and the raw materials are from natural products, so that the red rice seed coat active ingredient is high in safety and low in cost, and is suitable for industrial production and market popularization and application.
The embodiments of the invention have been described in detail above, but they are merely examples, and the invention is not limited to the above-described embodiments. It will be apparent to those skilled in the art that any equivalent modifications or substitutions to this invention are within the scope of the invention, and therefore, all equivalent changes and modifications, improvements, etc. that do not depart from the spirit and scope of the principles of the invention are intended to be covered by this invention.

Claims (10)

1. Application of red rice seed coat active ingredient in preparing medicine for preventing and treating ulcerative colitis is provided.
2. The use according to claim 1, wherein the red rice seed coat active ingredient is prepared by the following method:
(1) Pulverizing red rice, and sieving to obtain red rice powder; adding an extracting agent into red rice powder for ultrasonic extraction to obtain a mixture; centrifuging the mixture to obtain red rice powder precipitate and supernatant;
(2) Continuously adding an extracting agent into the red rice powder precipitate obtained in the step (1) for ultrasonic extraction for 2-3 times, and then carrying out centrifugal separation to collect supernatant;
(3) Combining the supernatants of step (1) and step (2) to obtain a combined supernatant; filtering the combined supernatant, concentrating the filtrate by rotary evaporation, and recovering the extractant by rotary evaporation until a water phase is obtained; continuously concentrating the water phase by rotary evaporation to obtain concentrated solution;
(4) And (3) performing low-temperature pre-freezing on the concentrated solution in the step (3), and performing freeze-drying by using a freeze dryer to obtain freeze-dried powder of the red rice seed coat active ingredient extract.
3. The use according to claim 2, wherein the sieving in step (1) is by a 80-100 mesh sieve.
4. The use according to claim 2, wherein the extractant in step (1) and step (2) comprises 0.4-0.8% citric acid, 60-80% ethanol, and water in balance.
5. The use according to claim 2, wherein the mass-to-volume ratio of red rice powder to extractant in step (1) is 1 (16-18) g/mL.
6. The use according to claim 2, wherein the ultrasonic extraction in step (2) is carried out at 200W power for 30-35 min at 50-55 ℃.
7. The use according to claim 2, wherein the centrifugation in step (1) and step (2) is performed at 4300 to 4800r/min for 10 to 15min.
8. The use according to claim 2, wherein the low temperature pre-freezing in step (4) is at a temperature of-20 to-30 ℃.
9. The use according to claim 2, wherein the medicament comprises an extract of red rice seed coat active ingredient.
10. The use according to claim 9, wherein the medicament is for inhibiting related inflammatory factors and Ca in colon tissue 2+ The expression of oxidative stress index in colon tissue, and the pathological injury caused by inflammatory reaction of colon tissue.
CN202310442829.6A 2023-04-23 2023-04-23 Application of red rice seed coat active ingredient in preparation of medicine for preventing and treating ulcerative colitis Pending CN116392554A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120132652A (en) * 2011-05-27 2012-12-07 한국식품연구원 Novel use of rice extract for improving, preventing or treating sleep disorders, anxiety, or depression

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20120132652A (en) * 2011-05-27 2012-12-07 한국식품연구원 Novel use of rice extract for improving, preventing or treating sleep disorders, anxiety, or depression

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAPAPAN KANGWAN等: "Protective Effect of Red Rice Extract Rich in Proanthocyanidins in a Murine Colitis Model", BIOMEDICINES, vol. 11, pages 1 *
马烨: "红米原花青素的提取纯化与抗氧化活性研究", 中国优秀硕士学位论文全文数据库 工程科技I辑, no. 3, pages 10 *

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