A kind of Herba Erodii extract
Technical field
The present invention relates to a kind of new Herba Erodii extract and preparation method thereof, relate to Herba Erodii extract that contains corilagin and preparation method thereof particularly, the purposes that also relates to described Herba Erodii extract gastric acid inhibitory secretion, reduction gastric acidity, and the purposes for the treatment of or preventing diseases such as hyperchlorhydria, peptic ulcer, chronic gastritis.
Background technology
Herba Erodii is China's medical material commonly used, it is of a great variety, comprise yak Seedling section yak Seedling platymiscium yak Seedling (Erodium stephanianum Willd) and equal Geranium plant Herba Erodii (Geraniumwilfordii Maxim), and nepal cranesbill (Geranium nepalense Sweet.), thick root Herba Erodii (G.dahuricum DC), Pelargonium (G.sibiricum L.), Carolina Cranesbill Herb (G.caroliniznum L.), Herba Geranii eriostemonis (G.eriostemon Fisch.ex.DC.), G.vlassouianum Fisch. (Geranium vlassovianum), Geranium tsingtauense Yabe (Geranium tsingtauense Yabe), Geranium macrorrhizum Zdravets (Geranium macrorrhizum) and geranium thunbergii (G.thunbergii Sieb.et Zucc.) etc.Chemical constituent in the Herba Erodii is numerous, mainly comprises tannin, flavonoid, organic acid and volatilization wet goods.Along with people to the deepening continuously of Herba Erodii research, its increasing pharmacological action constantly is found, as antiinflammatory, analgesic activity, antibacterial action, antivirus action, antioxidation, immunization, antitussive effect, anti-diarrhea effect, hepatoprotective effect, antitumor action, blood sugar reducing function, anastalsis etc.Yet yet there are no the active relevant reports in aspect such as the secretion of Herba Erodii gastric acid inhibitory, reduction gastric acidity.The inventor is engaged in the experimentation of Herba Erodii for a long time, finds that Herba Erodii has the gastric acid inhibitory secretion, reduces the activity of gastric acidity, aspect the diseases such as treatment or prevention hyperchlorhydria, peptic ulcer, chronic gastritis good effect is being arranged.
Hyperchlorhydria is meant gastric juice (comprising compositions such as hydrochloric acid and pepsin) supersecretion, and makes the patient feel stomach discomfort, acid regurgitation water, heartburn (stomach burn feeling).Slight hyperchlorhydria mainly shows as and occurs stomach burn feeling, acid regurgitation repeatedly, if hyperchlorhydria infringement gastric mucosa, also can cause stomachache, feel sick, symptom such as vomiting, hyperchlorhydria also may be further developed into peptic ulcer, even perforated ulcer occurs.
The medicine of hyperchlorhydria can be divided three classes: the first kind be in and the gastric acid medicine, as sodium bicarbonate, aluminium hydroxide etc.; Second class is for pressing down sour medicine, as cimetidine, ranitidine, famotidine and proton pump inhibitor etc.; The 3rd class is for increasing the medicine of gastric mucosal defense ability, as bismuth preparation etc.
Peptic ulcer is a kind of global commonly encountered diseases and frequently-occurring disease, estimates that about 10% crowd was once suffering from this disease in life.Peptic ulcer mainly means gastric ulcer and duodenal ulcer, it is a kind of multi-pathogenesis disease, its morbidity is relevant with multiple factor, as gastric acid, pepsin, helicobacter pylori (Hp) infection, heredity, body constitution, environment, diet, living habit, neural Nervous and Mental Factors etc., by different approaches or mechanism, cause above-mentioned invasion and attack effect to strengthen and or preventing mechanism weaken, all can inspire ulcer and take place.Along with people's deepening continuously to gastric acid generting machanism and gastric mucosa defense function understanding, Therapeutic Method to peptic ulcer is also being updated, at present main Therapeutic Method have (referring to: the time space, the treatment of gastric ulcer and gastric acid secretion, circulate a notice of of biology, 1994,29 (12), 14-15):
Antiacid pharmacotherapy:
At present, generally acknowledge that it is satisfied ulcer healing environment and indispensable treatment condition that stomach inner pH value maintains more than 3.5-4.0.Therefore gastric acid inhibitory secretion, improving gastric pH is the basis of treatment of peptic ulcer.Antacid generally is an alkalescent medicine, as sodium bicarbonate etc., by reducing gastric acidity with hydrochloric acid generation neutralization reaction, but this neutral effectiveness can only temporarily be lived in peace with each other, and gastric acid again can supersecretion soon, if reuse alkalescence medicine neutralizes repeatedly, can cause that more gastric acid secretes in a large number, be as good as and add fuel to the flames.This is because this class medicine can only neutralize and be secreted into the gastric acid of gastral cavity, result's gastral cavity pH value at short notice raises rapidly, this has weakened the effect of the negative feedback inhibition gastric acid secretion of gastric acid on the contrary, make parietal cell generate more gastric acid, cause " knock-on ", so antacid only is used as the adjuvant drug of ulcer treatment at present.
The receptor blocking agent therapy:
In vivo, have three kinds of materials can directly act on parietal cell, promote the generation and the secretion of gastric acid, they are respectively: acetylcholine, gastrin, histamine.When it is found that these three kinds of physiological stimulating factors all are to act on the receptor on the parietal cell film and behind the gastric acid secretion, just attempt to seek corresponding receptor blocking agent, the receptor with on the blocking-up parietal cell film reduces the generation of gastric acid.Being used for clinical the earliest is cholinoceptor blocker atropine (atropine).Atropine can only reduce the generation of a part of gastric acid, and the patient is effective to part.But, thereby side effect such as xerostomia, the dimness of vision are arranged because of atropine also acts on the m receptor of body other parts simultaneously.In recent years, the discovery of specific M receptor blocker pirenzepine (pirenzepine) has substituted atropinic use, and effect is better than atropine.H receptor blocker mainly contains medicines such as cimetidine, ranitidine, famotidine, nizatidine, roxatidine, and they are by blocking-up parietal cell H
2The secretion of receptor and gastric acid inhibitory has characteristics such as determined curative effect, untoward reaction are few, economical and practical.Though gastrin-receptor is still at the experimental stage at present through seeking for many years, is not used in clinical treatment.Because three kinds of stimulating factors independently play a role by each autoreceptor, thereby the use receptor blocking agent is treated, can only be to a certain extent or only effective to a part of patient.
Proton pump inhibitor (PPI) therapy:
Because proton pump is the final step that produces gastric acid, thereby reducing the effective method of gastric acid is the activity that suppresses proton pump.Yan Zhi proton pump inhibitor such as omeprazole (Omeprazole) can directly act on proton pump in recent years, suppress the effect of its secretion gastric acid.Omeprazole has been widely used in clinical treatment abroad, and effect is fine.But because gastric ulcer is a kind of chronic disease, and sour pump metabolism is very fast, must adhere to taking for a long time omeprazole, and this causes treating the cost costliness, has exceeded many patients' ability to bear, and therefore, this medicine also is not suitable for extensive at home clinical use.
Eradicate the Drug therapy of Hp:
Should accept the eradication therapy of Hp to the male ulcer of Hp, main therapy is to unite to use antibiotic and antacid.Antibiotic such as clarithromycin, amoxicillin, tinidazole etc. play antibacterial or bactericidal action, but these antibiotic are all not acidproof, and its drug effect depends on pH value; Acid inhibitor such as PPI can provide a suitable pH environment, and many acid nonfast antibiotic will be brought into play maximum effectiveness under this environment.
Chronic gastritis also is a kind of commonly encountered diseases, frequently-occurring disease, and it is the first that its sickness rate occupy in various gastropathy, accounts for outpatient service gastroscopy patient's 90%, and the age is big more, and sickness rate is high more, and particularly middle-older patient more more sees.The general treatment method of chronic gastritis normally adopts the proton pump inhibitor treatment, reduces the stimulation of gastric acid to gastric mucosa, mitigation symptoms; Or colloidal bismuth class Drug therapy, this type of medicine is owing to its colloid property, and the protein binding that can ooze out with mucosa under sour environment still forms firm protective at gastric mucosa together; Or antibiotics or other non-medicine auxiliary treatment etc.(referring to: 1, Zhang Yan, the treatment present situation of chronic gastritis, the 24th the 4th phase of volume of Li Ganzhi's journal; 2, Guo Shoujun, the diagnosis and treatment of chronic gastritis, Chinese clinic study magazine, the 13rd the 2nd phase of volume of January in 2007).
In sum, diseases such as hyperchlorhydria, peptic ulcer, chronic gastritis are common frequently-occurring disease, and their treatment need reduce gastric acidity mostly, and continuing to seek safely and effectively, the broad-spectrum curing medicine is still the problem that needs solve.The inventor is through experimentation for many years, find that unexpectedly a kind of Herba Erodii extract has the activity of good restraining gastric acid secretion, reduction gastric acidity, aspect the diseases such as treatment or prevention hyperchlorhydria, peptic ulcer, chronic gastritis good effect is being arranged, and finishing the present invention based on above discovery.
Summary of the invention
One aspect of the present invention provides a kind of new Herba Erodii extract, and the percentage ratio that the corilagin of described Herba Erodii extract Chinese style I accounts for the extract gross mass is 5%-25%, and is preferred 15%-25%, and most preferably 20%.
Formula I
The present invention provides the preparation method of described Herba Erodii extract on the other hand, it is characterized in that, Herba Erodii (Geranium dahuricum) is pulverized the alcohol reflux of 5-8 times of amounts 2-4 times, extracting solution merges concentrated, after being dissolved in water, organic solvent extraction, extracting solution is concentrated into dried, column chromatography for separation, the chloroform methanol of different proportion is an eluant, and the eluent of collecting chloroform: methanol=5:1 merges, and is concentrated into and does promptly.Wherein said organic solvent is selected from petroleum ether, ethyl acetate.
Concrete preparation method is: Herba Erodii (Geranium dahuricum) is pulverized, the ethanol that adds 5-8 times of amounts 40%, reflux, extract, 2-4 times, elimination medicinal residues merge extractive liquid,, concentrate extractum, after being dissolved in water, use petroleum ether successively, ethyl acetate extracts, with the acetic acid ethyl acetate extract concentrating under reduced pressure as for, divide and pull on silicagel column, use chloroform successively, chloroform: methanol=10:1, chloroform: methanol=8:1, chloroform: methanol=5:1 carries out eluting, the eluent of chloroform: methanol=5:1 is merged, be concentrated into driedly, obtain containing the Herba Erodii extract of corilagin.
Preparation method is more specifically: get Herba Erodii (Geranium dahuricum) and pulverize, add the ethanol of 8 times of amounts 40%, reflux, extract, three times, each 2 hours.The elimination medicinal residues merge 3 extracting solution, and concentrating under reduced pressure gets extractum, add the suitable quantity of water dissolving after, extract with petroleum ether, ethyl acetate successively.Acetic acid ethyl acetate extract is evaporated to dried, divides and to pull on silicagel column (100-200 order silica gel G), use chloroform successively, chloroform: methanol=10:1, chloroform: methanol=8:1, chloroform: methanol=5:1 carries out eluting, detects with thin layer chromatography.The eluent of chloroform: methanol=5:1 is merged, be concentrated into driedly, obtain the described Herba Erodii extract that contains corilagin, wherein the content of corilagin is 5%-25%.
Further aspect of the present invention provides described Herba Erodii extract gastric acid inhibitory secretion, reduces the purposes of gastric acidity.
Further aspect of the present invention provides described Herba Erodii extract treatment or prevents the purposes of diseases such as hyperchlorhydria, peptic ulcer, chronic gastritis.
Further aspect of the present invention provides a kind of gastric acid inhibitory secretion that is used for, and reduces the pharmaceutical composition of gastric acidity, and it comprises described Herba Erodii extract and one or more acceptable accessories.
Further aspect of the present invention provides a kind of pharmaceutical composition that is used for the treatment of or prevents diseases such as hyperchlorhydria, peptic ulcer, chronic gastritis, and it comprises described Herba Erodii extract and one or more acceptable accessories.
Herba Erodii described in the present invention mainly is selected from one or more the plant in thick root Herba Erodii (Geranium dahuricum) in the Mang cattle Seedling section plant, Carolina Cranesbill Herb (Geranium caroliniznum), Herba Geranii eriostemonis (Geranium eriostemon), G.vlassouianum Fisch. (Geranium vlassorianum), Geranium tsingtauense Yabe (Geranium tsingtauense), the Geranium macrorrhizum Zdravets (Geranium macrorrhizum).
The content of corilagin adopts high performance liquid chromatography to detect in the Herba Erodii extract provided by the invention,
Actual conditions and method are as follows:
Instrument: high performance liquid chromatograph
Pump: Tianjin, island LC-10AT vp
Detector: Tianjin, island SPD-10A vp
Work station: the NC2000 of Zhejiang University
Mobile phase: acetonitrile-1% phosphoric acid solution (13:87)
Wavelength X: 268nm
Chromatographic column: Thermo Hypersil BDS C18 (4.6x250mm, 5 μ m)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Reference substance: corilagin (making according to the method among the Chinese patent ZL97104154.7), area normalization content is 95%
Computational methods: the about 20 μ g/ml of reference substance solution concentration (C to) prepare the about 100 μ g/ml of solution concentration to be measured (C sample), sample size 20 μ l, record reference substance solution peak area (A to), solution peak area to be measured (A sample)
Pharmaceutical composition provided by the invention can be Herba Erodii extract and the mixing acceptable accessories that contains corilagin, pharmaceutical preparation technology according to routine, the any pharmaceutical preparation of use, for example capsule, tablet, pill, granule, the oral liquid etc. clinically of being fit to that are prepared into.
Acceptable accessories comprises pharmaceutically acceptable filler, disintegrating agent, binding agent and lubricant, correctives etc.
Suitable filler is selected from lactose, microcrystalline Cellulose, pregelatinized Starch, starch, dextrin, mannitol, cellulose, sucrose, glucose, sorbitol, xylitol etc.
Suitable disintegrants is selected from polyvinylpolypyrrolidone, carboxymethyl starch sodium, low-substituted hydroxypropyl cellulose, crosslinked hydroxypropyl emthylcellulose etc.
Suitable bonding is selected from polyvidone, hydroxypropyl cellulose, methylcellulose, sodium carboxymethyl cellulose, hydroxypropyl emthylcellulose, ethyl cellulose etc.
Examples of suitable lubricants is selected from micropowder silica gel, magnesium stearate, zinc stearate, calcium stearate, stearic acid, refining hydrogenated vegetable oil, Palmic acid, Pulvis Talci, Polyethylene Glycol, sodium lauryl sulphate, Stepanol MG etc.
Suitable correctives is selected from sucrose, simple syrup, glycerol, sorbitol, mannitol, stevioside, saccharin sodium etc.
Peptic ulcer of the present invention comprises gastric ulcer and duodenal ulcer.
The present invention is by ligation rat pylorus, collect 6 hours excretory gastric juice and estimate the described influence that contains the Herba Erodii extract of corilagin to gastric acid secretion, result of the test shows that described Herba Erodii extract can effectively suppress the gastric acid secretion of rat, reduces gastric acidity.When the therapeutic effect of estimating peptic ulcer, adopt following three kinds of experimental animal models: pyloric ligation ulcers rat gastric ulcer model, water logging irritability rat gastric ulcer model, the acetic acid type rat gastric ulcer model of burning.Wherein, pylorus ligature law is a Shay ' s traditional classical rat gastric ulcer modelling, and behind the ligation pylorus, gastric juice is detained in the stomach, and the coat of the stomach defence capability weakens, and causes gastric ulcer to form.Microscopically is observed and is seen leukocyte infiltration and tissue necrosis, and ulcer reaches the flesh layer deeply.This model is similar to the gastric ulcer that human hyperchlorhydria causes.The modeling mechanism of water logging irritability rat gastric ulcer model is, after animal was subjected to stress stimulation, the sympathetic nervous system irritability raise, and vasoconstriction causes the mucosa hypoxic-ischemic, and resistance descends.Parasympathetic nervous-hypophysis-adrenal system is excited to raise, and causes that gastric acid, pepsin and gastrin secretion increase, thereby causes stress ulcer.The acetic acid method is directly burnt the stomach serous coat with acetic acid and is formed ulcer, and this method is reliable, good reproducibility, and ulcer is dark and big, and is very similar to the mankind's chronic gastric ulcer.Its normal healing needs about 60 days, individual animal is also arranged in still disunion more than 200 days, thereby is applicable to the effect of observing medicine promotion ulcer healing, can be used as the experimental model of screening treatment chronic gastric ulcer medicine.
Animal test results shows: the Herba Erodii extract that contains corilagin provided by the invention can reduce gastric acid secretion, reduces ulcer incidence rate and the ulcer index of rat in pyloric ligation ulcers and the water logging stress gastric ulcer model; And the healing of rat gastric ulcer in the acetic acid burn type gastric ulcer model had significant facilitation, and can increase the ulcer healing percentage rate, reduce ulcer index.
The Herba Erodii extract preparation technology who contains corilagin provided by the invention is simple, and can reduce gastric acid secretion, reduce gastric acidity, reduce the incidence rate and the ulcer index of ulcer, have good curing or the prevention peptic ulcer effect, and can be prepared into easily according to the conventional method of this area the treatment peptic ulcer pharmaceutical composition for clinical use.
The specific embodiment
Describe content of the present invention in detail with specific embodiment below, following examples also do not mean that any limitation of the invention.
Embodiment 1 preparation contains the Herba Erodii extract of corilagin
Get thick root Herba Erodii (Geranium dahuricum) 2000g, pulverize, add the ethanol of 8 times of amounts 40%, reflux, extract, three times, each 2 hours.The elimination medicinal residues merge 3 extracting solution, and concentrating under reduced pressure gets extractum 280g, add the suitable quantity of water dissolving after, use petroleum ether (500ml * 4), ethyl acetate (500ml * 4) to extract successively.Acetic acid ethyl acetate extract is merged, be evaporated to driedly, divide and pull on silicagel column (silica gel with 100~200 purpose silica gel G), use chloroform successively, chloroform: methanol=10:1, chloroform: methanol=8:1, chloroform: methanol=5:1 carries out eluting, adopts thin layer chromatography to follow the tracks of and detects.The eluent of chloroform: methanol=5:1 is merged, be concentrated into driedly, obtain containing the Herba Erodii extract 25g of corilagin, detecting wherein with high performance liquid chromatography, the content of corilagin is 20%.
Embodiment 2 preparations contain the Herba Erodii extract of corilagin
Get Geranium tsingtauense Yabe (Geranium tsingtauense) 2000g, pulverize, add the ethanol of 8 times of amounts 40%, reflux, extract, twice, each 2 hours.The elimination medicinal residues merge 2 extracting solution, and concentrating under reduced pressure gets extractum 220g, add the suitable quantity of water dissolving after, use petroleum ether (500ml * 4), ethyl acetate (500ml * 4) to extract successively.Acetic acid ethyl acetate extract is merged, be evaporated to driedly, divide and pull on silicagel column (silica gel with 100~200 purpose silica gel G), use chloroform successively, chloroform: methanol=10:1, chloroform: methanol=8:1, chloroform: methanol=5:1 carries out eluting, adopts thin layer chromatography to follow the tracks of and detects.The eluent of chloroform: methanol=5:1 is merged, be concentrated into driedly, obtain containing the Herba Erodii extract 18g of corilagin, detecting wherein with high performance liquid chromatography, the content of corilagin is 25%.
Embodiment 3 preparations contain the Herba Erodii extract of corilagin
Get G.vlassouianum Fisch. (Geranium vlassovianum) 2000g, pulverize, add the ethanol of 5 times of amounts 40%, reflux, extract, four times, each 2 hours.The elimination medicinal residues merge 4 extracting solution, and concentrating under reduced pressure gets extractum 250g, add the suitable quantity of water dissolving after, use petroleum ether (500ml * 4), ethyl acetate (500ml * 4) to extract successively.Acetic acid ethyl acetate extract is merged, be evaporated to driedly, divide and pull on silicagel column (silica gel with 100~200 purpose silica gel G), use chloroform successively, chloroform: methanol=10:1, chloroform: methanol=8:1, chloroform: methanol=5:1 carries out eluting, adopts thin layer chromatography to follow the tracks of and detects.The eluent of chloroform: methanol=5:1 is merged, be concentrated into driedly, obtain containing the Herba Erodii extract 16g of corilagin, detecting wherein with high performance liquid chromatography, the content of corilagin is 6%.
Embodiment 4
Contain of the influence of the Herba Erodii extract of corilagin to rat tolerance secretion
1 material
1.1 reagent
Herba Erodii extract makes according to the method for embodiment 1
Ranitidine hydrochloride is provided by Jiangxi Huiren Pharmaceutical Co., Ltd
Omeprazole is provided by AstraZeneca pharmaceutical Co. Ltd
Sodium carboxymethyl cellulose (CMC-Na) is provided by Chemical Reagent Co., Ltd., Sinopharm Group
Benzylpenicillin sodium for injection is provided by Huabei Pharmaceutic Co., Ltd
Chloral hydrate is provided by Chemical Reagent Co., Ltd., Sinopharm Group
Sodium hydroxide is provided by Nanjing chemical reagent one factory
Phenolphthalein cause Nereid analyses Chemical Industry Science Co., Ltd to be provided
1.2 animal
Cleaning level SD rat, male, body weight 180-200g, Shanghai Slac Experimental Animal Co., Ltd.
1.3 instrument
Sartorius BS110S electronic balance Beijing Sai Duolisi balance company limited
LDZ5-2 centrifuge Beijing Medical Centrifugal Machine Factory
1.4 group and drug dose setting
Model group CMC-Na (0.5%)
Ranitidine group 100mg/kg
Omeprazole group 13.6mg/kg
Herba Erodii extract 200mg/kg
Herba Erodii extract 100mg/kg
1.5 medicine preparation and route of administration: medicine is with CMC-Na (0.5%) suspendible, gastric infusion, 10ml/kg.
2 methods
2.1 modeling method
Each organizes the rat oral gavage administration, and 1 time/d, continuous 5d.Fasting 48h before the experiment.During experiment with rat with chloral hydrate (0.30g/kg) anesthesia, carry out pyloric ligation, and duodenal administration 1 time, water is prohibited in the postoperative fasting, puts to death animal behind the 6h, collects gastric juice in graduated centrifuge tube.With 3000r/min, centrifugal 15min gets supernatant.
2.2 observation index
2.2.1 gastric juice amount
Behind the modeling 6h, put to death rat, collect gastric juice in graduated centrifuge tube.With 3000r/min, centrifugal 15min gets supernatant, accurately measures the gastric juice volume.
2.2.2 total acidity
Behind the modeling 6h, put to death rat, collect gastric juice in graduated centrifuge tube.With 3000r/min, centrifugal 15min gets supernatant, measures total acidity with 0.01mol/L NaOH by acid base neutralization titration (phenolphthalein is indicator), and calculates total acid output according to gastric secretion.
2.3 statistical procedures
Data are represented with X ± SD, carry out statistical procedures with SPSS11.0, relatively use the least significant difference method in twos between each group.
3. experimental result
Table 1. Herba Erodii extract is to the excretory influence of pylorus ligation rat gastric juice (X ± SD)
Annotate: compare with the blank group,
*P<0.05,
*P<0.01.
4 conclusions
Herba Erodii extract can reduce rat total acidity gastric juice and total acid output, and the prompting Herba Erodii extract has the excretory effect of gastric acid inhibitory, can be used for the treatment of hyperchlorhydria, chronic gastritis.
Embodiment 5 contains the anti-gastric-ulcer zoopery of the Herba Erodii extract of corilagin
1 experiment material:
1.1 animal:
The SD rat, male, 180-220g is provided by the Yangzhou University experimental center
1.2 medicine and reagent:
Herba Erodii extract: the method according to embodiment 1 makes
Positive drug: ranitidine hydrochloride capsules is provided by middle promise pharmaceutcal corporation, Ltd
Acetic acid: Shanghai is insulted wind chemical reagent company limited and is provided
Pentobarbital sodium: Chinese Medicine Shanghai chemical reagents corporation provides
1.3 equipment:
The rat fixing head, operating scissors, scalpel, tweezers, needle holder, microsyringe, balance
1.4 experiment condition:
The experimental rat sub-cage rearing, the feed full-valence pellet feed is freely drunk water, 20 ± 2 ℃ of room temperatures, humidity 50-70%
2. pyloric ligation ulcers rat gastric ulcer model
2.1 experimental technique
Rat is divided into 4 groups at random by body weight, 10 every group: blank group, ranitidine group (135mg/kg), Herba Erodii extract high dose group (150mg/kg), Herba Erodii extract low dose group (75mg/kg).According to the continuous gastric infusion of 10ml/kg 5 days, the blank group gave the equivalent distilled water to administration group rat respectively.Rat begins fasting and can't help water after the 1st administration, the last administration after 2 hours with rat with 0.7ml/ 1% shallow fiber crops of pentobarbital sodium only, from ensiform process of sternum lower edge ventrimeson open abdomen, the about 2-3cm of otch.At costal margin position, left side, up one push away gently with referring to, make stomach be exposed to otch.Wear a line (not injuring blood vessel) with pylorus ligation (other vascular ligations that will not be close to) under the stomach pylorus, sew up incision of abdominal wall, clean with normal saline, iodine disinfection is with bundle cutting mouth.Fasting is put to death animal after prohibiting water 18h, opens the abdominal cavity, behind the ligation cardia stomach is taken out.Collect gastric juice, volume calculated.Cut off stomach along greater gastric curvature,, be tiled on the blank sheet of paper, observe the situation that glandular stomach portion ulcer takes place, write down every group of ulcerogenic Mus number, ulcer number, ulcer level, ulcer area and pathological changes situation, calculate ulcer index with normal saline flush away gastric content.
The ulcer index assessment method: the rat that gastric perforation do not occur is calculated ulcer index with aphtha number and ulcer area.Calculate the area summation that all maximum gauges surpass the ulcer of 1mm, the area of each ulcer is according to S=π * r
1* r
2Calculate r
1And r
2Be respectively diameter and wide footpath; All count according to aphtha less than the ulcer of 1mm in diameter and wide footpath, with aphtha number and the ulcer gross area as the Comprehensive Assessment index.Evaluation criteria sees the following form:
Ulcer index |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
The aphtha number |
0 |
1-5 |
6-30 |
31-60 |
>60 |
|
|
Ulcer area mm
2 |
0 |
1-10 |
11-20 |
21-30 |
31-40 |
41-50 |
50 or the perforation |
Ulcer suppresses percentage rate %=(blank group ulcer index-medication group ulcer index)/blank group ulcer index * 100%
The ulcer incidence rate: the number of animals that ulcer takes place accounts for the percentage ratio of laboratory animal number.
2.2 experimental result
To the influence of pyloric ligation ulcers gastric ulcer (X ± s)
Group |
Dosage mg/kg |
Number of animals |
Ulcer index |
Ulcer suppresses percentage rate % |
Ulcer incidence rate % |
Gastric juice volume ml |
Blank |
- |
10 |
3.9±1.7 |
|
100 |
18.8±3.0 |
Ranitidine |
135 |
10 |
1.5±1.0
** |
61.5 |
80 |
14.6±4.5
* |
Herba Erodii extract |
150 |
10 |
0.9±1.1
** |
76.9 |
50 |
9.7±3.5
** |
Herba Erodii extract |
75 |
10 |
2.2±0.9
* |
43.6 |
100 |
14.3±4.2
* |
Compare with the blank group
*Represent P<0.05,
*Represent P<0.01;
Above result shows: form tangible ulcer behind the rat pylorus ligation in this experiment, show the modeling success.Positive control medicine ranitidine can reduce gastric acid secretion, reduces the incidence rate and the ulcer index of ulcer.The high and low dose of Herba Erodii extract can both reduce gastric acid secretion, reduces the incidence rate and the ulcer index of ulcer.Herba Erodii extract reduces gastric acid secretion when high dose, effects such as the incidence rate of reduction ulcer and ulcer index are better than the ranitidine group.
3 water logging irritability rat gastric ulcer models
3.1 experimental technique
Rat is divided into 4 groups at random by body weight, 10 every group: blank group, ranitidine group (135mg/kg), Herba Erodii extract high dose group (150mg/kg), Herba Erodii extract low dose group (75mg/kg).Rat was pressed the continuous gastric infusion of 10ml/kg 5 days, the blank group gives the equivalent distilled water, and last administration rat fasting in preceding 24 hours was fixed in the extremity colligation of rat on the plank after the administration in 1 hour, be dipped in the tank about 20 ℃, liquid level remains on the ensiform process of sternum level.Put to death rat after 20 hours, open the abdominal cavity, behind ligation cardia and the pylorus stomach is taken out, inject 1% formalin 10ml to gastric, and stomach was immersed in 1% formalin 10 minutes, fixedly ectonexine by pylorus.Cut off stomach along greater gastric curvature,, be tiled on the blank sheet of paper, observe the situation that glandular stomach portion ulcer takes place with normal saline flush away gastric content.The degree of ulcer is represented with ulcer index, and the incidence rate of calculating ulcer inhibition percentage rate and ulcer is estimated the effect of medicine.
Ulcer index: the major diameter of ulcer is directly measured length greater than 1mm person, and every 1mm is 1 minute; If then score greater than 1mm person and double in wide footpath, for major diameter and wide footpath all less than 1mm person, by the score of ulcer number, each ulcer meter 0.5 minute.The accumulative total total points of every animal is the ulcer index of this animal.
Ulcer suppresses percentage rate %=(blank group ulcer index-medication group ulcer index)/blank group ulcer index * 100%
The ulcer incidence rate: the number of animals that ulcer takes place accounts for the percentage ratio of experimental animal number.
3.2 experimental result
To the influence of water logging stress gastric ulcer model (X ± s)
Group |
Dosage (mg/kg) |
Number of animals |
Ulcer index |
Ulcer suppresses percentage rate % |
Ulcer incidence rate % |
Blank |
- |
10 |
11.9±5.4 |
0 |
100 |
Ranitidine |
135 |
10 |
2.6±1.9
** |
78.2 |
80 |
Herba Erodii extract |
150 |
10 |
2.3±2.3
** |
80.7 |
80 |
Herba Erodii extract |
75 |
10 |
6.6±2.8
* |
44.5 |
100 |
Compare with the blank group
*Represent P<0.05,
*Represent P<0.01;
Experimental result shows: formed tangible gastric ulcer after the rat water logging in this experiment, shown the modeling success.Positive control medicine ranitidine has the obvious suppression effect to ulcer, and can significantly reduce ulcer index.Close during the Herba Erodii extract high dose with the action effect of ranitidine, the ulcer of irritability rat gastric ulcer model is had inhibitory action, and dose-effect relationship is obvious, the high dose group effect is better than low dose group.
The 4 acetic acid type rat gastric ulcer model of burning
4.1 experimental technique
Rat is divided into 4 groups at random by body weight, 10 every group: blank group, ranitidine group (135mg/kg), Herba Erodii extract high dose group (150mg/kg), Herba Erodii extract low dose group (75mg/kg).The rat fasting after 24 hours, is carried out intraperitoneal injection of anesthesia with 5mg/100g body weight pentobarbital sodium, then its dorsal position is fixed on the operating-table.Cut the stomach wall hair, use the ethanol routine disinfection, under xiphoid-process to the about 2cm of lower edge ventrimeson clip, stomach is shifted out the abdominal cavity, at the facies ventralis of stomach, body of stomach and pyloric antrum intersection, thrust 0.4-0.5mm under the serous coat with microsyringe is flat, inject the acetic acid of 50 μ l 10%, form pimple.Stomach is sent back to gently, sew up abdominal muscle, skin suture then.With iodine tincture, alcohol disinfecting, the conventional raising.From performing the operation next day, each organizes every day by the 10ml/kg gastric infusion, successive administration 7 days, and the blank group is given the equivalent distilled water.Animal was all put to death in the 7th day.Open fu jie and prick pylorus and cardia, get stomach and be dipped in 1% formalin, fix 10 minutes.Cut off along greater gastric curvature, stomach is turned up, outwell content, reuse nipple suction pipe washes out food debris gently.Ulcer is rounded or oval, and central concave is swelled all around slightly, and blood capillary gathers.The ulcer that has healed has protuberance on every side slightly, and the surface is ruddy, and as seen radial microgroove is arranged.With the equal value representation ulcer index of the major diameter of ulcer and minor axis, carry out statistical procedures to calculate the ulcer healing percentage rate, the evaluation medicine is to the facilitation of ulcer healing.
Ulcer healing percentage rate (%)=(blank group ulcer diameter average summation-medication group ulcer diameter average summation)/blank group ulcer diameter average summation * 100%
4.2 experimental result
To the burn influence (X ± s) of type gastric ulcer of rats acetic acid
Group |
Dosage mg/kg |
Number of animals |
Ulcer index mm |
Ulcer healing percentage rate % |
The blank group |
- |
10 |
5.5±1.6 |
|
Ranitidine |
135 |
10 |
3.2±1.5
** |
41.8 |
Herba Erodii extract |
150 |
10 |
2.8±1.3
** |
49.1 |
Herba Erodii extract |
75 |
10 |
4.3±1.8 |
21.8 |
Compare with the blank group
*Represent P<0.05,
*Represent P<0.01;
Healing to rat gastric ulcer during the Herba Erodii extract high dose has significant facilitation, can increase the ulcer healing percentage rate, reduces ulcer index, and effects such as the incidence rate of reduction ulcer and ulcer index are all near with the ranitidine winding.Little during low dosage to the influence of ulcer healing and ulcer index.