CN110711207B - Application of Wenwang Yizhen extract in preparation of medicine for treating ulcerative colitis of digestive tract - Google Patents
Application of Wenwang Yizhen extract in preparation of medicine for treating ulcerative colitis of digestive tract Download PDFInfo
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Classifications
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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)
- A61K36/185—Magnoliopsida (dicotyledons)
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- 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
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- 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
- 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
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- 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
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Abstract
The invention relates to application of a Wen king one-pen extract in preparing a medicine for treating ulcerative colitis of a digestive tract. The extract is supernatant and precipitate obtained by water extraction and alcohol precipitation of a Wen king pen, the supernatant is decompressed to recover ethanol, ethyl acetate and n-butanol are sequentially used for extraction to obtain an ethyl acetate extract and an n-butanol extract respectively, and the precipitate is washed by alcohol and dried to obtain the water extraction and alcohol precipitation extract. The total extract (Extzw) of Wen king one pen and the extract of ethyl acetate part and water extraction and alcohol precipitation part prepared from the total extract have obvious improvement effect on ulcerative colitis of mice induced by Dextran Sodium Sulfate (DSS), can be applied to preparing medicines for treating ulcerative colitis of digestive tracts, and can even be applied to preparing medicines for treating other intestinal inflammatory diseases.
Description
Technical Field
The invention relates to the technical field of application of a Wen king one-pen extract, in particular to application of the Wen king one-pen extract in preparation of a digestive tract anti-ulcerative colitis medicament.
Background
Inflammation is a biological response involving multiple types of immune and non-immune cells that protects the body from damage caused by harmful stimuli. Typically, inflammatory responses are triggered by exogenous substances and tissue injury products, accompanied by the production of pro-inflammatory cytokines, recruitment and activation of immune cells, and the production of free radicals. Normally, these inflammatory processes clear foreign bodies and pathogens and promote tissue regeneration, however, in certain disease processes, the inflammatory response becomes excessive, causing acute or chronic inflammatory responses and associated tissue/organ damage. Thus, controlling excessive inflammatory responses in the body has an important role in maintaining the health of the body.
Ulcerative colitis (ulcerative colitis, UC) is a multifactorial, multi-layered, non-specific inflammatory disease of the digestive tract with an ambiguous etiology, the lesion being mainly located in the colorectal, presenting diffuse, continuous and superficial inflammation of the mucous membrane and submucosa, with a corresponding histological change; clinically, the symptoms of abdominal pain, diarrhea, mucopurulent bloody stool and tenesmus are mainly clinical manifestations; the disease course has the characteristics of being chronic, lifelong and easy to recur.
In recent years, with the development of science, basic research and clinical treatment of ulcerative colitis achieve a certain achievement, and various treatment modes such as Western medicine treatment, cell treatment, microecological preparation treatment, traditional Chinese medicine compound treatment, acupuncture treatment and the like are formed. The traditional drugs for Western medicine treatment include aminosalicylic acid, glucocorticoid, immunosuppressant, etc. Wherein, the aminosalicylic acid is mainly used as a main medicine for maintenance treatment in the remission stage of ulcerative colitis, is not suitable for treatment in the acute stage of diseases, and can produce a series of adverse reactions such as nausea, vomiting, dizziness and the like; the glucocorticoid is a medicine for effectively controlling acute active phase inflammation of ulcerative colitis, and is particularly suitable for severe patients or light and moderate patients with poor treatment effect of aminosalicylic acid; however, adverse reactions such as cushing's syndrome, osteoporosis and the like can occur after long-term administration; immunosuppressants exert an immunosuppressive effect mainly by blocking proliferation and activation of T lymphocytes in patients with ulcerative colitis, and their main side effects are bone marrow suppression manifestations such as leukopenia and the like and increase the incidence of infection. The Chinese herbal compound, such as paeonia lactiflora decoction, ginseng, poria cocos and bighead atractylodes rhizome powder, dark plum pills, pain relieving and diarrhea treating prescription, chinese pulsatilla root decoction and the like, has a certain curative effect on ulcerative colitis, but often has slow effect, and is not suitable for treating ulcerative colitis in an acute active phase. In view of the above, there is currently no very desirable drug for treating ulcerative colitis; in addition, in recent years, the incidence rate of ulcerative colitis in China is in an increasing trend, so that the research and development of corresponding novel therapeutic drugs for ulcerative colitis are further carried out, and the method has important theoretical and practical significance.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of the extract of the Wenyuang Yizhen in preparing medicines for treating ulcerative colitis of digestive tracts, exact anti-inflammatory activity and application of the extract in preparing anti-inflammatory medicines, in particular to preparing medicines for treating ulcerative colitis.
The technical scheme of the invention is as follows:
application of Wen Wang Yizhen extract in preparing medicine for treating colonitis, wherein colonitis is ulcerative colitis. The ulcerative colitis of the digestive tract is a chronic nonspecific inflammatory disease of colon and rectum with unclear etiology, and the lesions are limited to the mucous membrane and submucosa of the large intestine. Lesions are mostly located in the sigmoid colon and rectum, but can also extend to the descending colon, even the entire colon.
Preferably, the ulcerative colitis is Dextran Sodium Sulfate (DSS) -induced ulcerative colitis in mice.
Preferably, the medicine is medicinal liquor, tincture, suspension, capsule, tablet, pill, dripping pill, powder or injection.
Preferably, the extract of the Wen king one pen comprises an extract of the Wen king one pen, which is extracted by water, and an extract of an alcohol-soluble part obtained by alcohol precipitation treatment after the extraction of the Wen king one pen by water.
Further preferably, the extract of the Wen king one pen is an ethyl acetate part extract and an n-butanol part extract which are respectively obtained by extracting the Wen king one pen with water, then performing alcohol precipitation treatment on the supernatant, recovering ethanol under reduced pressure, and sequentially extracting with ethyl acetate and n-butanol.
Further preferably, the extract of the Wen king one pen is a precipitate obtained by extracting the Wen king one pen with water and then carrying out alcohol precipitation treatment, and the extract of the water extracted and alcohol precipitated part is obtained after alcohol washing and drying.
Further preferably, the alcohol is ethanol or industrial alcohol or methanol extraction, and the volume fraction of the ethanol is 80% -95%.
Still more preferably, the dosage of the Wen king one pen extract is 9-18 g, the dosage is reduced into the dosage of each extract, the dosage of the Wen king one pen water extract (Extzw) is 3-9 g, and the dosage of the ethyl acetate part extract is 0.1-1.5 g; the usage amount of the water extraction and alcohol precipitation part extract is 0.1-3 g.
Still more preferably, the composition of the ethyl acetate part extract and the water extraction and alcohol precipitation part extract of the King's pen is used in an amount of 0.1-6 g, and the weight ratio of the ethyl acetate part extract to the water extraction and alcohol precipitation part extract is (1-10): (1-10).
The extraction method of the Wen king one pen extract comprises the following steps:
s1: digging a raw plant of Wenwang, removing soil and impurities, slicing, drying in the shade, cutting or crushing whole plant, extracting with water as solvent under reflux for 0.5-3 hr for 1-3 times, filtering with solvent 6-25 times of the weight of the medicinal materials; collecting filtrate, concentrating, filtering again, and fixing volume to obtain water extract;
s2: taking the water extract obtained in the step S1, and obtaining a Wen king one pen water extract after freeze drying;
s3: stirring the water extract obtained in the step S1 at 40-70 ℃, and slowly adding 80-95% ethanol or industrial alcohol or methanol to ensure that the final alcohol concentration of a liquid medicine system is 30-80%; then standing for 12-36 hours at normal temperature or low temperature (4-10 ℃), and separating supernatant and precipitate;
s4: taking the supernatant obtained in the step S3, recovering the solvent under reduced pressure, concentrating and drying to obtain an alcohol solution (Frs);
s5: collecting the supernatant obtained in the step S3, recovering the solvent under reduced pressure, adding water to disperse the supernatant, sequentially extracting with ethyl acetate and n-butanol for 1-4 times, combining the chemical solvent extraction layers of each time, recovering the solvent under reduced pressure, concentrating and drying to obtain an extract of an ethyl acetate part and an extract of an n-butanol part respectively; taking the precipitate obtained in the step S3, washing the precipitate with 80% -95% ethanol or industrial alcohol or methanol for several times, removing the solvent, and drying to obtain an extract of the water extraction and alcohol precipitation part;
the extraction of the extract of the King of Chinese pencils is completed.
The invention has the following beneficial effects:
1. extracts of the Wen Wang Yi, especially water extract (Extzw), and water extract and ethanol precipitation part extract (Frp) and ethyl acetate part extract (Fre) prepared from the extracts can obviously inhibit NO release stimulated by LPS and secretion of IL-1 beta inflammatory factors in vitro RAW264.7 cells, and can inhibit expression of inflammatory factor mRNA such as TNF-alpha, IL-1 beta and the like; can obviously inhibit the expression of inflammatory factor mRNA such as TNF-alpha, IL-1 beta and the like in mouse liver tissues stimulated by LPS in vivo; meanwhile, the water extract (Extzw), the water extract and alcohol precipitation part extract (Frp) and the ethyl acetate part extract (Fre) have good protection and improvement effects on the ulcerative colitis induced by the DSS.
2. The extracts of the Wen king one pen, especially the water extraction and alcohol precipitation part (Frp) extract and the ethyl acetate part extract (Fre) prepared from the total extract of the Wen king one pen, show good anti-inflammatory effect on the in vitro cell level. At the same time, it also shows good anti-inflammatory effect at animal level and good protection and improvement effect on ulcerative colitis induced by Dextran Sodium Sulfate (DSS). During the animal experiment, no death of the mice was found, nor was there any significant liver and kidney dysfunction observed, indicating a better safety.
3. The total extract (Extzw) of Wen Wang Yi Zhi and the water extraction and alcohol precipitation part extract (Frp) and the ethyl acetate part extract (Fre) prepared from the total extract can be used for preparing medicines for treating ulcerative colitis of the digestive tract whether the total extract is used alone or in a form of combined use of the water extraction and alcohol precipitation part and the ethyl acetate part.
4. The ulcerative colitis is a non-specific inflammatory disease of the digestive tract, and lesions are mostly located in the sigmoid colon and the rectum, and can also extend to the descending colon and even the whole colon, and the total extract (Extzw) of the Wenyang pen and the extracts (Fre) of water extraction and alcohol precipitation parts (Frp), ethyl acetate parts and the like prepared from the total extract (Extzw) show good anti-inflammatory effect and safety in the in-vitro and in-vivo level, so that the ulcerative colitis-resistant extract can be used for preparing medicines of the ulcerative colitis of the digestive tract and can also be used for preparing medicines for treating other intestinal inflammatory diseases.
Drawings
The characteristic spectrum of each extracted part of Wang Yi Zhi (A, lambda=254 nm; B, lambda=210 nm) in the text of FIG. 1;
fig. 2 effect of each extract of wang pen on RAW264.7 cell growth (< 0.05 p, n=3);
FIG. 3 influence of extracts of Wang Yi Zhen extract on NO release amount # p<0.05,vs Mock group；*p<0.05,vs LPS group，n＝3)；
FIG. 4 influence of extracts of Wang Yi Zhi Ding on IL-1 beta secretion # p<0.05,vs Mock group；*p<0.05,vs LPS group，n＝3)；
FIG. 5 influence of Wang's extract on the expression of inflammatory factor mRNA in RAW264.7 cells ((1) mock (2) LPS (3) LPS+Frs (4) LPS+Fre (5) LPS+Extzw (6) LPS+Frp (7) LPS+Frb);
fig. 6: influence of extracts of Wenwang on mRNA expression of inflammatory factor in mouse liver tissue induced by LPS ((1) mock (2) LPS (4) LPS+Fre-High (5) LPS+Extzw (6) LPS+Frp-Low (7) LPS+Frp-High (8) LPS+Frb);
fig. 7 effect of the venturi-king water extraction and alcohol precipitation site (Frp), total extract (Extzw) and ethyl acetate site (Fre) on DSS-induced ulcerative colitis colon length (< 0.05 p, n=5);
FIG. 8 effect of Wang Yi water extract and alcohol precipitation (Frp), total extract (Extzw) and ethyl acetate (Fre) on score of DSS-induced ulcerative colitis inflammation (< 0.05 p, n=5, 200X) (A: mock; B:3.5%DSS;C:3.5%DSS+Extzw;D:3.5%DSS+Frp I;E:3.5%DSS+Fre II);
fig. 9 effect of Wang Ying water extraction and alcohol precipitation site (Frp), ethyl acetate site (Fre) and total extract (Extzw) on DSS-induced expression of mRNA of colonic tissue inflammation-related factor of ulcerative colitis (n=5).
Detailed Description
The present invention will be further illustrated by the following examples, but the scope of the invention is not limited to the examples.
Example 1
1. Medicine source, medicine extraction and preparation
The Wang Yi branch pen is collected in temple county of Shiwei city of Hubei province, and is identified as whole grass of Balanophora serrulata Balanophora involucrata, which is one of the medicinal plants of Balanophora. We have also collected a variety of Balanophora medicinal plants in the Western areas of Enshi, yichang, etc., as well as in the areas of Yunnan, sichuan, guangdong, jiangxi, hunan, hubei, guizhou, etc.
Digging a raw plant of Wenwang, removing soil and impurities, slicing, and drying in the shade. Cutting or grinding whole plant, reflux-extracting with water as solvent under heating for 3 times (each for 2 hr) with solvent 15 times of the weight of the medicinal materials, and filtering. The filtrate was collected, concentrated, filtered again and fixed in volume. At this time, the relative density of the liquid medicine at room temperature is 1.05-1.15.
And (3) taking part of the liquid medicine, and freeze-drying to obtain the Wen Wang Yizhi water extract (Extzw). Taking the rest liquid medicine, and slowly adding 95% ethanol or industrial alcohol under the conditions of heat preservation and stirring at 40-70 ℃ to ensure that the final alcohol concentration of a liquid medicine system is 70%. Then, the mixture was allowed to stand at room temperature for 12 hours, and the supernatant and the precipitate were separated. Taking out part of the supernatant, recovering ethanol under reduced pressure, concentrating, and drying to obtain alcohol solution (Frs). Taking the rest supernatant, recovering ethanol under reduced pressure, adding water to disperse, extracting with ethyl acetate and n-butanol sequentially for 3 times, mixing the chemical solvent extraction layers, recovering solvent under reduced pressure, concentrating, and drying to obtain ethyl acetate (Fre) and n-butanol (Frb) respectively. Taking out precipitate in water extraction and alcohol precipitation, washing with 95% ethanol or industrial alcohol for several times, removing ethanol as much as possible, and drying to obtain water extraction and alcohol precipitation part (Frp).
2. Feature spectrogram of each extraction part of Wenwang Yizhen
HPLC characteristic spectrum researches are carried out on the Wenwang water extract (Extzw), and alcohol solution (Frs), ethyl acetate part (Fre), n-butanol part (Frb), water extraction and alcohol precipitation part (Frp) and the like prepared from the Wenwang water extract. The column was packed with octadecylsilane chemically bonded silica (250X 4.6mm, particle size: 5 μm). The mobile phase system uses acetonitrile as a mobile phase A and water as a mobile phase B, and gradient elution is carried out, wherein the elution procedure is 0min and 5% A;10min,15% A;35min,25% A;50min,30% A;70min,65% A. The column temperature was 30℃and the flow rate was 1.0ml/min, and the detection wavelengths were 254nm and 210nm.
3. Anti-inflammatory effect research of each extraction part of Wenwang Yizhen
3.1 materials and methods
3.1.1 cell culture and Experimental animals, reagents
RAW264.7 cells were purchased from Shanghai life sciences institute of China academy of sciences cell resource center, cultured in DMEM medium containing 10% fetal bovine serum, and placed at 37deg.C, CO 2 Culturing in an incubator with a volume fraction of 5%. DMEM broth and fetal bovine serum were purchased from Gibco company, usa; kunming mice were purchased from the university of three gorges laboratory animal center.
3.1.2 cell proliferation assay
RAW264.7 cells were seeded into 96-well plates (1X 10) 4 After 24 hours of cell adhesion, adding a Wanwang water extract (Extzw) with different mass fractions, and alcohol soluble substances (Frs), ethyl acetate part (Fre), n-butanol part (Frb) and water extraction and alcohol precipitation part (Frp) with mass fractions ranging from 25 mug/mL to 150 mug/mL. Then, the absorbance at 570nm was measured (A570) by MTT method (5 g/L MTT) on days 1, 2, 3 and 4 for each extracted site, and the experiment was repeated 3 times.
3.1.3 Nitric Oxide (NO) detection
RAW264.7 cells were seeded into 96-well plates (1X 10) 4 Individual/hole)After 24 hours of cell adhesion, 50 mug/mL of Wanwang water extract (Extzw), alcohol soluble substances (Frs), ethyl acetate part (Fre), n-butanol part (Frb) and water extraction and alcohol precipitation part (Frp) are respectively added, 1 mug/mL of Lipopolysaccharide (LPS) is added into each hole, and zeroing holes, reagent control groups (Mock), LPS groups and LPS+Wanwang extract groups are arranged, wherein each group comprises 4 compound holes. After 24 hours of drug action, the supernatant NO release was measured using the NO kit.
3.1.4 cytokine detection
RAW264.7 cells were seeded into 96-well plates (1X 10) 4 After 24 hours of cell attachment, 50 μg/mL of Wanwang water extract (Extzw), alcohol soluble (Frs), ethyl acetate (Fre), n-butanol (Frb) and water-extracted alcohol precipitation (Frp) were added to each well, 1 μg/mL Lipopolysaccharide (LPS) was added to each well, and zeroing wells, reagent control (Mock), LPS group, lps+Wanwang extract group were set, 4 multiplex wells each. After 24 hours of drug action, the supernatant was assayed for IL-1. Beta. Secretion using ELISA kits.
3.1.5 RT-PCR detection of expression of inflammatory related factor mRNA at cellular level
RAW264.7 cell suspensions were seeded into 6-well plates (2X 10) 5 After the cells are attached, 50 mug/mL of Wanwang water extract (Extzw), alcohol soluble substances (Frs), ethyl acetate part (Fre), n-butyl alcohol part (Frb) and water extraction and alcohol precipitation part (Frp) are respectively added to act on the cells for 12 hours, the cells are set as reagent control (Mock) groups, LPS groups and LPS+Wanwang extract groups, 1 mug/mL of LPS are added to jointly stimulate for 12 hours, total RNA is extracted according to a Trizol kit, cDNA is reversely transcribed, and expression of inflammatory related factors mRNA such as TNF-alpha, IL-1 beta and the like is amplified by PCR.
3.1.6RT-PCR detection of expression of inflammatory-related factor mRNA in liver tissue of mice
Male Kunming mice (20-23 g) of 6 weeks of age were supplied by the university of three gorges animal center; the experimental animal procedure was approved by the experimental animal ethics committee of the university of three gorges. After 1 week of adaptation to SPF-grade sterile animal room, the Kunming mice were divided into reagent control (Mock), LPS, total extract (Extzw), ethyl acetate fraction (Fre), water-extracted alcohol-precipitated fraction (Frp), etc., each group of 6 mice. Each group of mice was free to eat and drink water. Fre and Frp were divided into low dose group (L) and high dose group (H) according to the results of the pre-cell level experiments. The stomach is infused with the extract of the Wen king one pen of each group, once a day, and the dosages of each group are as follows: extzw is 1000mg/kg; fre-L is 200mg/kg, fre-H is 400mg/kg; frp-L is 200mg/kg, and Frp-H is 400mg/kg; the same volume of solvent was infused into each of Mock and LPS. After 6 days, LPS was injected into each group of mice except for Mock at a dose of 20mg/kg (Mock group was injected with an equal volume of physiological saline), and after 8 hours, the mice were sacrificed, liver tissues were removed, total RNA was extracted according to Trizol kit, reverse transcribed into cDNA, and expression of inflammatory-related factor mRNA such as TNF- α, IL-1β, etc., was amplified by PCR.
3.1.7 study of protective action against ulcerative colitis in mice
Male Kunming mice (20-23 g) of 6 weeks of age were supplied by the university of three gorges animal center; the experimental animal procedure was approved by the experimental animal ethics committee of the university of three gorges. After 1 week of adaptation of the SPF grade sterile animal room, the Kunming mice were divided into reagent control (Mock), 3.5% DSS, 3.5% DSS+Extzw, 3.5% DSS+Fre, 3.5% DSS+Frp, etc., each group of 5 mice, each group of mice being free to eat and drink water. In each administration group of the Wen Wang Yi extract, the stomach is continuously irrigated with 1000mg/kg of Extzw, 400mg/kg of Fre and 400mg/kg of Frp for 22 days; gastric lavage of the corresponding doses of solvent in Mock and 3.5% dss groups, respectively; at the last 7 days of gastric lavage, each group except for Mock group changed the free drinking water to 3.5% dss (double distilled water formulation). When the experiment is carried out for 22 days, treating the mice, dissecting the abdominal cavity to take the colon and the small intestine, washing the mice cleanly by PBS, measuring the length and photographing; taking 0.5 cm colon and small intestine, fixing 4% paraformaldehyde and embedding in paraffin, and observing under HE (high-performance organic) dyeing and microscope for pathological grading; extracting total RNA from the rest colon tissue according to Trizol kit, reversely transcribing into cDNA, and then amplifying the expression of the inflammatory related factors mRNA such as TNF-alpha, IL-1 beta and the like by PCR.
3.1.8 statistical treatments
The experimental results are expressed as mean ± standard deviation (x ± s), analyzed using statistical software SPSS 11.5, and the differences between the groups were significant using One-Way ANOVA (One Way ANOVA) with p < 0.05.
4. Results of the study
4.1 characteristic patterns of a Wen king one pen water extract and various chemical parts: the results are shown in FIG. 1.
4.2 Effect of Wenwang Yizhen Water extract and chemical parts on RAW264.7 cell growth
As shown in FIG. 2, in RAW264.7 cells cultured in vitro, after treating the cells with 100. Mu.g/mL of each extract for 4 days, the cell growth was significantly slowed down (p < 0.05) compared to the control group after treating the cells with 150. Mu.g/mL of the extract for 3 days. The results suggest that the extract of Wen king pen has no obvious effect on RAW267.4 cell growth in the concentration range of less than 100 mug/mL.
4.3 research on anti-inflammatory action of Wenwang Yizhan extract and chemical parts in vitro RAW264.7 cells
After RAW264.7 cells were treated with water extract (Extzw), water extract and alcohol precipitation site (Frp) and ethyl acetate site (Fre) respectively, LPS-induced NO release was significantly inhibited (FIG. 3); inhibiting secretion of the inflammatory factor IL-1. Beta. Induced by LPS (FIG. 4); also can significantly inhibit the expression of RAW264.7 intracellular inflammatory related factors IL-1 beta and other mRNA (figure 5). The results suggest that the Wen Wang one pen water extract (Extzw), the water extraction and alcohol precipitation part (Frp) and the ethyl acetate part (Fre) have anti-inflammatory effect in vitro RAW264.7 cells.
4.4 research on anti-inflammatory action of each extracted part of Wenwang Yizhen in vivo
After mice are fed with the total extract of Wen king and each extraction part, the mice are treated by LPS, as shown in figure 6, the total extract (Extzw), the water extraction and alcohol precipitation part (Frp) and the ethyl acetate part (Fre) can inhibit the expression of partial related inflammatory factors to a certain extent, and the Frp and Fre high dose effects are more obvious, which indicates that the Wen king total extract (Extzw) and the water extraction and alcohol precipitation part (Frp) and the ethyl acetate part (Fre) have anti-inflammatory effect in vivo.
4.5 study of protective effect of Wen Wang one Stroke extracts on ulcerative colitis the total extract of Wen Wang one Stroke and the extracts were fed to mice for 22 days and treated with 3.5% dss for 7 days as shown in figures 7-9: the colon length in 3.5% DSS model group is obviously shortened compared with normal group, and obvious bleeding point is present; colon length increased, bleeding improved, colon mass to length ratio decreased after Extzw and Frp, fre administration (fig. 7A-C, <0.05, n=5). Microscopic observation after colon HE staining, in 3.5% dss model group, colonic mucosa was severely damaged, broken, goblet cells decreased, crypt number decreased, inflammatory cell infiltration (compared to Mock group); after Extzw, frp, and Fre, the number of crypt, goblet cells increased, colonic mucosa integrity increased, inflammatory cell infiltration decreased (compared to 3.5% dss model group); after calculation of inflammation scores, extzw and Frp, fre showed a significant decrease in colonic inflammation scores after administration (fig. 8, <0.05 for p, n=5). Reverse transcription PCR results for colon tissue showed: after the Extzw, the Frp and the Fre are used, the expression of inflammatory factors mRNA such as TNF-alpha, IL-1 beta and the like of colon tissues is obviously reduced, and the Frp I group effect is more obvious. These results suggest that Extzw, frp, and Fre in each extraction site of the Wenwang pen have protective effects on ulcerative colitis.
Claims (2)
1. The application of the extract of the Wen king Yinchen in preparing the anti-ulcerative colitis medicament is characterized in that the extract of the Wen king Yinchen is an ethyl acetate part extract or an aqueous extraction and alcohol precipitation part extract; the extraction method of the Wen king one pen extract comprises the following steps: s1: digging a raw plant of Wenwang, removing soil and impurities, slicing, drying in the shade, cutting or crushing whole plant, extracting with water as solvent under reflux for 0.5-3 hr for 1-3 times, filtering with solvent 6-25 times of the weight of the medicinal materials; collecting filtrate, concentrating, filtering again, and fixing volume to obtain water extract;
s2: taking the water extract obtained in the step S1, and obtaining a Wen king one pen water extract after freeze drying;
s3: stirring the water extract obtained in the step S1 at 40-70 ℃, and slowly adding 80-95% ethanol to ensure that the final alcohol concentration of a liquid medicine system is 70%; then standing for 12-36 hours at normal temperature or at a low temperature of 4-10 ℃ to separate supernatant and precipitate;
s4: collecting the supernatant obtained in the step S3, recovering the solvent under reduced pressure, adding water to disperse the supernatant, sequentially extracting with ethyl acetate and n-butanol for 1-4 times, combining the chemical solvent extraction layers of each time, recovering the solvent under reduced pressure, concentrating and drying to obtain ethyl acetate part extract and n-butanol part extract respectively; and (3) washing the precipitate obtained in the step (S3) with 80% -95% ethanol or industrial alcohol or methanol for several times, removing the solvent, and drying to obtain the water extraction and alcohol precipitation part extract.
2. Use according to claim 1, characterized in that the medicament is a medicinal liquor, a tincture, a suspension, a capsule, a tablet, a pill, a drop pill, a powder or an injection.
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