CN113368100A - Application of icaritin in preparation of medicine for preventing and treating gastrointestinal hemorrhage - Google Patents

Abstract

The invention belongs to the field of medicines, relates to medical application of icariin, and particularly relates to application of icariin in preparation of a medicine for preventing and treating gastrointestinal hemorrhage. The invention respectively researches the digestive tract hemorrhage caused by peptic ulcer, the digestive tract ulcer hemorrhage caused by medicaments and the digestive tract hemorrhage caused by tumor radiotherapy and chemotherapy. The research result shows that: the icariin has obvious effect of reducing the incidence rate of the gastrointestinal hemorrhage caused by various reasons, has definite curative effect and obvious effect on the gastrointestinal hemorrhage, and is expected to become an efficient and rapid medicament for preventing and treating the gastrointestinal hemorrhage.

Description

Application of icaritin in preparation of medicine for preventing and treating gastrointestinal hemorrhage

Technical Field

The invention belongs to the field of medicines, relates to medical application of icariin, and particularly relates to application of icariin in preparation of a medicine for preventing and treating gastrointestinal hemorrhage.

Background

The digestive tract comprises an upper digestive tract, a middle digestive tract and a lower digestive tract, wherein the upper digestive tract hemorrhage is an internal emergency and can be caused by factors such as gastric mucosa injury, ulcer of the upper digestive tract and the like. Peptic Ulcer (PU) is one of the common diseases in the digestive system, and refers to chronic ulcers occurring in the stomach and duodenum. PU has the characteristic of rapid disease progression and acute onset of disease with acute upper gastrointestinal hemorrhage (UGIH), and clinical symptoms mainly include hematemesis, dark stool, bloody stool and the like, and severe cases may cause shock or death. In addition, many drugs such as non-steroidal anti-inflammatory drugs, thrombolytics, anti-thrombolytics, and various acute stress conditions can cause upper gastrointestinal bleeding. Therefore, how to rapidly and effectively stop bleeding of the gastrointestinal hemorrhage patients is of great significance in the disease treatment process.

The herba Epimedii is dried stem and leaf of Epimedium brevicornum Maxim belonging to berberidaceae, Epimedium sagittatum Maxim, Epimedium pubescens Maxim, or Epimedium koreanum Nakai. The traditional Chinese medicine composition is mainly used for treating kidney-yang deficiency, impotence, frequent micturition and infertility clinically; rheumatism pain, numbness and contracture of limbs, flaccidity of bones and muscles, and difficulty in walking; kidney yang deficiency, cough and dyspnea with shortness of breath. Icariin has effects of increasing blood flow of cardiovascular and cerebrovascular vessels, promoting hematopoiesis, immunity and bone metabolism, invigorating kidney, supporting yang, resisting aging and tumor.

Icaritin (IT) is a polyhydroxy flavonoid monomer component in herba Epimedii of Epimedium of berberidaceae. Pharmacological research shows that: the IT has stronger osteoporosis resisting effect than other flavonoid glycoside compounds in epimedium, and has the effects of promoting osteoblast activity and inhibiting osteoclast activity in vitro. Icariin and icariin, which are important active ingredients in epimedium, have been receiving more and more attention from medical workers in recent years. For example, patent application CN101637467A discloses the application of icariin in preparing medicine for treating osteoporosis. Patent US6399579 discloses the use of anhydroicaritin for the treatment of sexual dysfunction.

At present, no report on the application of icariin in the aspect of treatment of gastrointestinal hemorrhage exists in the literature.

Disclosure of Invention

In order to find a medicine for quickly and efficiently reducing the incidence rate of the gastrointestinal hemorrhage, the invention provides the following technical scheme:

the results of the study on the effect of icaritin on gastrointestinal bleeding caused by peptic ulcer of the present invention in specific examples 1 to 3 show that icaritin not only can lower the bleeding index of gastric bleeding caused by acute peptic ulcer due to ethanol, but also can significantly reduce the incidence of gastric bleeding caused by chronic peptic ulcer due to helicobacter pylori and aspirin, and that the effect is dose-dependent.

In the specific embodiment 4, the icaritin has the effect of preventing gastric bleeding caused by thrombolytic drugs, and the test results show that: the icaritin can reduce the incidence rate of gastrointestinal hemorrhage caused by thrombolytic drugs, and the high-dose icaritin also has remarkable prevention effect on gastrointestinal hemorrhage caused by thrombotic diseases, and the effects show a dose-dependent relationship.

Specific example 5 intervention effect of icaritin on acute duodenal radiation injury hemorrhage, test results show that: the icaritin can remarkably reduce the rate of duodenal mucosal hemorrhage caused by radiotherapy and chemotherapy, reduce the rate of hematochezia in mice, and has remarkable prevention and treatment effects on gastrointestinal hemorrhage caused by tumor radiotherapy and chemotherapy.

Application of icaritin in preparing medicine for preventing and treating gastrointestinal hemorrhage is provided.

The gastrointestinal hemorrhage is stress ulcer which is caused by thrombus diseases and is represented by peripheral organ damage.

The gastrointestinal hemorrhage is the gastrointestinal hemorrhage caused by tumor radiotherapy and chemotherapy.

Further, the medicine is one or more of non-steroidal anti-inflammatory drugs or thrombolytic and anti-thrombotic drugs. The non-steroidal anti-inflammatory drug is aspirin. The thrombolytic drug is urokinase; the antithrombotic drug is one or more of anticoagulant drugs or antiplatelet drugs.

Icaritin has obvious effect of reducing the bleeding rate of peptic ulcer caused by various reasons.

The effect of reducing the rate of bleeding from the digestive tract caused by drugs includes, but is not limited to, the drugs listed in the above examples.

The icaritin administration routes include gastrointestinal routes and parenteral routes, including but not limited to subcutaneous, intradermal, arterial, intravenous, intramuscular, articular, intrathecal, intracranial, thoracic, intraperitoneal injection or instillation, nasal, buccal, sublingual, tracheal, urethral, rectal or focal topical administration, and the like.

The invention not only provides the application of the icariin in the preparation of the medicine for preventing and treating the gastrointestinal hemorrhage, but also provides a medicinal preparation used for treating the diseases, which comprises the icariin and pharmaceutically acceptable pharmaceutic adjuvant.

The pharmaceutical preparation includes but is not limited to injection, powder injection, capsule, tablet, microemulsion, dripping pill, and enteric soft capsule.

The pharmaceutically acceptable pharmaceutical excipients used in the invention refer to any substance which does not interfere with the physiological action of icariin and has no toxicity to subjects including human beings.

The pharmaceutical excipients used in the formulations of the invention are the usual excipients known to those skilled in the art. For example, common pharmaceutical excipients for preparing microemulsion formulations include, but are not limited to, soybean oil, polyoxyethylene-23-lauryl ether, 1, 2-propylene glycol, hydrogenated coconut oil glyceride, lauroyl macrogol-32-glyceride, polyethylene glycol 3350, safflower oil, cottonseed oil, decaglycerol monostearate; the common pharmaceutic adjuvants for preparing the dripping pill preparation comprise but are not limited to polyethylene glycol 6000 and polyethylene glycol 1000; common pharmaceutical excipients used in the preparation of capsule formulations include, but are not limited to, lactose and corn starch. The pharmaceutically acceptable carriers commonly used for preparing soft capsule preparations include, but are not limited to, medium-chain fatty glyceride, polyoxyethylene castor oil, 1, 2-propylene glycol, and the like.

Those skilled in the art can select suitable pharmaceutical excipients according to actual needs and formulate the preparation of the present invention by methods known in the art. Such formulations include, but are not limited to, solids, liquids, oils, emulsions, gels, aerosols, inhalants, sprays, capsules, pills, patches, suppositories, and the like.

In the treatment of the above diseases, icaritin is administered in an amount of 0.01mg/kg to 500mg/kg, preferably 0.1mg/kg to 50 mg/kg.

Compared with the prior art, the icariin has the following advantages when reducing the bleeding rate of the digestive tract:

(1) has obvious curative effect

The icaritin has obvious effect of reducing the incidence rate of various gastrointestinal hemorrhage and has the characteristic of high-efficiency hemostasis.

(2) Safe, nontoxic and convenient for administration

The icaritin is extracted from traditional Chinese medicine, has the advantages of less toxic and side effects, and has the characteristics of convenient administration and good patient compliance compared with the existing treatment method.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention is further described below by way of specific embodiments, but the present invention is not limited to only the following examples.

Formulation example 1 icaritin tablet

The preparation process comprises mixing icariin and adjuvants including microcrystalline cellulose and sodium carboxymethyl starch, adding appropriate amount of starch slurry to make soft mass, sieving with 16 mesh sieve, and granulating. Drying wet granules at 60 deg.C, sieving dry granules with 20 mesh sieve, grading, sieving to obtain fine powder, mixing with magnesium stearate, mixing with dry granules, and tabletting to obtain tablet of about 200 mg.

Formulation example 2 icaritin capsule formulation

The preparation process comprises the following steps: mixing icariin 100g, lactose 120g and corn starch 130g in a mixer for 10-15 minutes, adding magnesium stearate 5g, mixing for 1-3 minutes, and filling into capsule shell of 1000 granules.

Formulation example 3 icaritin microemulsion formulation

The preparation process comprises the following steps: weighing soybean oil, polyoxyethylene-23-lauryl ether and 1, 2-propylene glycol according to the prescription amount, mixing, uniformly stirring, adding icariin for dissolving, or performing ultrasonic treatment to accelerate dissolving to obtain a clear solution, namely the icariin microemulsion preparation. The particle size was measured by a laser particle size analyzer, and the average particle size was 15 nm.

Formulation example 4 icaritin microemulsion formulation

The preparation process comprises the following steps: weighing the formula amounts of hydrogenated cocoglyceride, lauroyl polyethylene glycol-32-glyceride, 1, 2-propylene glycol and polyethylene glycol 3350, mixing, stirring uniformly, adding icaritin for dissolving, or performing ultrasonic treatment to accelerate dissolving to obtain a clear solution, namely the icaritin microemulsion preparation. The particle size was measured by a laser particle size analyzer, and the average particle size was 40 nm.

Formulation example 5 enteric-coated icaritin Soft Capsule preparation

The rubber formula comprises:

gelatin 10g

Glycerol 5g

Purified Water 10g

Prescription of enteric coating liquid:

the preparation process comprises the following steps: weighing medium-chain fatty glyceride, polyoxyethylene castor oil, 1, 2-propylene glycol and absolute ethyl alcohol according to the prescription amount, mixing, uniformly stirring, adding icariin for dissolving, and performing ultrasonic treatment to accelerate dissolving to obtain a clear concentrated solution, namely the icariin microemulsion concentrate. Adding water to the obtained microemulsion concentrate according to the proportion of 1: diluting to clear solution at weight ratio of 10-20 to obtain soft capsule microemulsion content. Weighing gelatin, glycerol and purified water according to the prescription amount, uniformly mixing, pressing into a rubber, weighing Eudragit L30D-55, triethyl citrate, talcum powder and purified water according to the prescription amount, and uniformly mixing to obtain the enteric coating solution. Wrapping the microemulsion content of the soft capsule containing icariin with a rubber to obtain soft capsule, and wrapping the soft capsule with enteric coating to obtain enteric soft capsule.

Preparation example 6 icaritin drop pill preparation

Icaritin 5.0g

Polyethylene glycol-600014.5 g

Polyethylene glycol-10005.0 g

Making into 1000 pieces

The preparation process comprises the following steps: weighing icariin with the prescription amount and passing through a 100-mesh sieve, adding the icariin into the mixed solution which is heated and melted on a water bath and contains the polyethylene glycol 6000 and the polyethylene glycol 1000 with the prescription amount, fully stirring the mixed solution to be uniform, filling the mixed solution into a dropping bottle, and dropping the mixed solution at the temperature of 95 +/-2 ℃; dripping into glass condensation column containing 4-6mL of methyl silicone oil, molding, taking out, and absorbing with absorbent paper to remove the adhered methyl silicone oil.

Formulation example 7 icaritin injection

The preparation process comprises the following steps: mixing ethanol and tween-80, adding icariin, stirring for dissolving, adding water for injection to 10L, stirring, adding 0.5% injectable active carbon, stirring, and removing carbon.

Formulation example 8 icaritin injection

The preparation process comprises the following steps: adding icariin into PEG-400, stirring to dissolve, adding 0.9% sodium chloride solution to 10L, stirring, adding 0.5% active carbon for injection, stirring, and removing carbon.

Formulation example 9 icaritin injection

Icaritin 1g

Ethanol 3.3L

Adding water for injection to 10L

The preparation process comprises the following steps: adding icariin into ethanol according to the prescription amount, stirring for dissolving, adding water for injection to 10L, stirring uniformly, adding 0.5% of active carbon for injection, stirring, and removing carbon to obtain the injection.

Formulation example 10 icariin powder injection

The preparation process comprises the following steps: weighing icariin raw materials for injection in a prescription amount, and adding a proper amount of water for injection to dissolve. Then, adding a specified amount of uniformly mixed materials which are subjected to sterilization and pyrogen removal treatment in advance, and then adding water for injection to the specified amount of 1000 ml; adding 5g of activated carbon for injection into the liquid medicine, heating for 30 minutes at 60-80 ℃, filtering by using a filter membrane, and collecting the filtrate. And (3) performing positive pressure sterilization filtration on the filtrate by using a sterilization filter according to an aseptic operation method, filtering by using a 0.22 mu M microporous filter membrane, performing pyrogen examination and semi-finished product content examination on the filtrate, and subpackaging by using penicillin bottles. Pre-freezing at-40 deg.C for 1.5-3.5 hr, sublimating at vacuum degree until the free water content is 90%, heating, drying (the highest temperature should not exceed 35 deg.C), and freeze-drying to obtain icariin aglycone powder for injection.

Pharmacodynamic example 1 preventive action of icaritin on ethanol-induced gastrointestinal bleeding

1. Test method

1.1 animal pretreatment

30min before model making, 60 SD rats are randomly divided into A, B, C, D, E five groups, wherein A, B two groups are injected into the abdominal cavity with physiological saline according to 2mL/kg, and C, D, E groups are respectively administered with icariin through intragastric administration at the dosage of 2mg/kg, 6mg/kg and 18 mg/kg.

1.2 preparation of upper gastrointestinal hemorrhage model of rat

After fasting for 24h (but with free water), except for group A, the rats were gavaged with 4mL/kg of physiological saline, and the other groups were gavaged with 4mL/kg of absolute ethanol. 30min after the gavage, 20% urethane solution (1mI/150g) is used for intraperitoneal injection anesthesia.

1.3 mucosal Bleeding Index (BI) determination

The stomach was removed from the abdominal cavity, cut along the greater curvature, flushed with saline at first glance, flattened, and the lesion was measured with a ruler under an optical microscope. The length of the spots, linear lesions on the gastric mucosa were observed and recorded and scored according to literature standards: and (3) scoring the lesion length of less than 1mm by 1 point, scoring the lesion length of 1-2 mm by 2 points, scoring the lesion length of 2-3 mm by 3 points, scoring the lesion length of 3-4 mm by 4 points, if the lesion length is greater than 4mm, scoring the lesion length of more than 2mm by multiplying the score by 2, and finally, taking the sum of the scores of all the lesions of the whole stomach as BI.

1.4 statistical methods

SPSSll.5 statistical analysis software is adopted for data processing, the measured data are all expressed by mean value standard deviation (z +/-5), the comparison of multiple groups of questions is tested by Oneway ANOVA,

pairwise comparisons between groups were checked by q.

2 results of the experiment

The measurement results of the gastric mucosal hemorrhage indexes of rats in each group are shown in table 1, and it can be seen from the table that the gastric mucosal hemorrhage indexes of the dose groups of the icaritin are remarkably reduced compared with the model group, and have statistical significance (P <0.01 or P <0.05), and the gastric mucosal hemorrhage indexes are more remarkably reduced along with the increase of the dose, which indicates that the icaritin can remarkably reduce the hemorrhage rate caused by acute gastric mucosal injury, and presents a dose-dependent relationship.

TABLE 1 comparison of index of bleeding of gastric mucosa in rats of each group

Group of	Number of animals (n)	BI
			A	12	0
B	12	35.33±4.12
			C	12	22.12±3.34#
D	12	16.66±2.34##
			E	12	11.53±5.09##

In comparison to the group B (model group),^##P<0.01，^#P<0.05。

pharmacodynamic example 2 preventive action of icaritin against gastrorrhagia caused by helicobacter pylori

1. Grouping and gavage infection of animals

60 Mongolian gerbils were divided into a control group, an H.pyri-infected group, and a drug intervention group. Each group had 20 animals that were fasted 24h before gavage and water deprived 4h before gavage. H.pyrori-infected group and drug-dried group were each administered with 0.5ml of intragastric lavage solution, and control group was administered with 0.5ml of intragastric buchner broth. And after the treatment is finished, the water feeding is resumed for 4 hours. Gavage 1 time every other day for 5 times. The icariin is administrated by intragastric administration according to the dose of 18mg/kg while the first intragastric lavage liquid is used, and the intragastric administration is carried out once a day later until the test at the 24 th week is finished.

2. Animal material drawing and observation indexes

2.1 blood sampling of the eyeball after the last administration to determine the platelet number.

2.2 the animals are killed under anesthesia, the stomach tissue of the animals is separated and longitudinally cut along the greater curvature of the stomach, the residues in the stomach are washed by normal saline, and the ulcer and bleeding of the stomach tissue are observed.

3. Test results

3.1 platelet number determination

The platelet measurement results show that: the platelet number of the normal control group, the infection group and the drug intervention group has no significant difference.

3.2 Observation of gastric tissue ulceration and bleeding

Visible bleeding and ulcer lesion of gastric mucosa in an infected group, wherein ulcer mostly occurs in antrum of the stomach, and is single-shot or multiple (2 or more), the ulcer is round or oval, the diameter is 0.2-0.5cm, and the edge is slightly convex; the bleeding incidence rate of the drug control group is obviously reduced, and the ulcer lesion is obviously slight; no changes such as mucosal erosion, bleeding and ulcer were observed in the normal control group during the whole experiment.

The bleeding of the stomach of each group of animals is shown in table 2, and it can be seen from the table that: the bleeding rate of the drug intervention group is obviously lower than that of the infection group, and the statistical significance is achieved (P is less than 0.01), which indicates that the icaritin can obviously reduce the incidence rate of the gastric bleeding caused by helicobacter pylori and improve the gastric ulcer state.

TABLE 2 comparison of bleeding in gastric mucosa of animals in each group

Group of	Animal number (n)	Bleeding number (n)	Bleeding Rate (%)
				Normal control group	20	0	-
Infectious group	20	11	55
				Pharmaceutical intervention group	20	2	10##

Compared with the group with the infection,^##P<0.01。

pharmacodynamic example 3 preventive action of icaritin on Aspirin-induced gastrointestinal hemorrhage

1. Moulding and grouping

24 SD rats are infused with aspirin at a dose of 500mg/kg to prepare a gastrointestinal bleeding model, another 12 SD rats are infused with normal saline with the same volume as the former to serve as a normal control, the bleeding model rats are randomly divided into a model group and an icariin group, the icariin group is infused with icariin at a dose of 18mg/kg during model preparation, and the model group is infused with normal saline with the same volume as the former.

2. Dissecting and observing gastric mucosa ulcer bleeding

After 24 hours of gastric lavage, the rats were anesthetized and sacrificed, dissected and observed for bleeding, erosion, ulceration on the gastric mucosal surface of the rats.

3. Test results and discussion

The occurrence of bleeding in the gastric mucosa of rats in each group is shown in Table 3, from which it can be seen that: the normal control group has no bleeding, the bleeding rate of the model group is 66.7%, compared with the model group, the icariin group rat has a remarkable slight gastric mucosa bleeding, the bleeding rate is remarkably reduced compared with the model group, the statistical significance is realized (P is less than 0.01), and the icariin is shown to be capable of remarkably reducing the incidence rate of the gastrointestinal bleeding caused by aspirin.

TABLE 3 comparison of bleeding in gastric mucosa of rats in each group

Group of	Animal number (n)	Bleeding number (n)	Bleeding Rate (%)
				Normal control group	12	0	-
Model set	12	8	66.7
				Icariin group	12	2	16.7##

In comparison to the set of models,^##P<0.01。

pharmacodynamic example 4 preventive action of icaritin on gastric bleeding caused by thrombolytic drug

1. Moulding and grouping

120 SD rats are divided into a pseudo-surgery group, a model group, a urokinase group, an icariin high dose + urokinase group, an icariin medium dose + urokinase group and an icariin low dose + urokinase group by a random digital method, wherein each group comprises 20 rats; wherein, the icariin dosage groups are respectively administrated by intragastric administration 4 days before operation, the administration dosage is respectively 2mg/kg, 6mg/kg and 18mg/kg, and the dummy operation group, the model group and the urokinase group are administrated by intragastric administration with equal volume of normal saline; except for the sham operation group, the other groups were administered via catheter via regional artery, urokinase was used at a dose of 5000u/kg, concentration was 200u/μ L, and the volume of administration was 20 μ L, and the model group and the regional artery of icariin group were administered with the same volume of physiological saline. The cerebral artery is blocked by autothrombus and thrombus to prepare the cerebral ischemia animal model of thromboembolism and keep the catheter.

2. Observation indicator and measurement method

2.1 bleeding Rate calculation

At 24h after arterial administration, rats in each group were anesthetized and dissected, and statistical cases of bleeding observed with naked eyes and under optical lens were counted to calculate the ratio of bleeding to total number of cases.

2.2 pathological observation of gastric tissue:

cutting left lung and antrum, fixing with 100g/L neutral formaldehyde solution, dehydrating, transparentizing, soaking in wax, embedding, slicing, staining, and observing pathological morphology change under light microscope. The degree of damage is graded from mild to severe according to the degree of tissue congestion, edema and cell degeneration, wherein the grade I is 0-25%, the grade II is 26-50%, the grade III is 51-75%, and the grade IV is more than 75%

(bleeding under the mirror).

3. Experimental results and discussion

3.1 comparison of bleeding rates

The comparison of the gastric bleeding rates of rats in each group is shown in table 4, and the gastric bleeding of the model group compared with the gastric bleeding of the sham operation group can be seen from the table, which indicates that cerebral ischemia can cause gastric bleeding complications to a certain extent; compared with the model group, the urokinase group has obviously increased bleeding rate and has obvious difference (P <0.01), which indicates that the thrombolytic drug can cause digestive tract bleeding complications when carrying out thrombolytic treatment on thrombotic diseases; compared with the urokinase group, the bleeding rate of each dose group of urokinase + icaritin is obviously reduced, and has obvious difference (P <0.01), which indicates that the icaritin has the function of reducing the gastrointestinal bleeding rate caused by thrombolytic drugs, and the function presents a dose-dependent relationship.

Compared with the model group, the icariin high-dose group has significant difference (P <0.01), which shows that the icariin also has a certain prevention effect on gastrointestinal hemorrhage caused by thrombotic diseases.

TABLE 4 comparison of gastrorrhagia rates of rats in each group

Compared with the false operation group, the operation table has the advantages that,^﹩﹩P<0.01；

compared with the urokinase group, the compound has the advantages that,^##P<0.01；

in comparison to the set of models,^&P<0.05。

3.2 pathological examination of stomach tissue

The pathological observation results of the gastric tissues of rats in each group are shown in table 5, and the results show that compared with a model group, the urokinase group has obviously higher gastric injury rate and obvious difference (P <0.01), which indicates that the thrombolytic drug can cause complications such as gastric tissue congestion, edema, cell degeneration and even hemorrhage when being used for thrombolytic treatment of thrombotic diseases; compared with the urokinase group, the incidence rate of the complications is remarkably reduced in each dose group of urokinase + icaritin, and has a remarkable difference (P <0.01 or P <0.05), which shows that the icaritin has the function of reducing the complications such as congestion, edema and cell degeneration even bleeding of the digestive tract tissue caused by the thrombolytic drug, and compared with the model group, the incidence rate of the complications is remarkably reduced in each dose group of urokinase + icaritin, and the results show that: the icaritin can reduce incidence rate of gastrointestinal hemorrhage caused by thrombolytic drug, relieve gastrointestinal tissue congestion, edema and cell degeneration, and recover digestive tract injury.

In addition, the results compared to the model set show that: icaritin also has certain prevention and treatment effects on the gastrointestinal injury caused by ischemic diseases.

TABLE 5 comparison of results of pathological grading of rat stomach tissue in each group

Compared with the false operation group, the operation table has the advantages that,^﹩﹩P<0.01；

compared with the urokinase group, the compound has the advantages that,^##P<0.01；

in comparison to the set of models,^&P<0.05。

pharmacodynamic example 5 intervention action of icaritin on acute duodenal radiation injury hemorrhage

1. Experimental animal grouping and establishment of acute radiation enteritis icariin intervention model

60 clean-grade Balb/c female mice, 6-8 weeks old, 18-20g, randomly divided into 3 groups according to body weight: group a blank control group (20): no irradiation and drug intervention, group B, single irradiation group (20): 12Gy60CO ray single abdominal irradiation + physiological saline lavage, C group irradiation drug intervention group (20): 12Gy60CO ray single belly irradiation plus medicine intervention, grouping, putting into SPF animal room for adaptive feeding for 3 days, controlling the temperature (21 +/-2) DEG C and the relative humidity of 60 percent in day and night 12 hours, feeding with free drinking water and laboratory standard feed, and measuring the weight of the mouse regularly every day.

Modeling of acute radiation enteritis: the simple irradiation group and the irradiation drug intervention group are administered with no food water for 6 hours before irradiation, and are administered with intraperitoneal injection for anesthesia 30 minutes before irradiation, and the anesthetic is fast dormancy-inducing: ketamine is 1.5:2, 0.04 ml/mouse, the ketamine is fixed on a self-made special mouse fixing frame after anesthesia, 60Co rays are given for abdominal one-time irradiation, the irradiation range is from xiphoid process to pubic symphysis, the irradiation area is about 2.0 x 2.0cm, the total irradiation dose is 12Gy, the irradiation dose rate is 137.31cGy/min, the irradiation time is 8min27s, the irradiation drug intervention group is started to administrate icariin at 18mg/kg in intragastric manner 2 days before irradiation until 3.5 days after irradiation, and the irradiation group is started to give physiological saline equal to that of the irradiation drug intervention group 2 days before irradiation until 4 days after irradiation.

2. Taking materials

2.1 collecting the feces of each group of mice and observing the hematochezia condition.

2.2 day 4 after irradiation, all mice were sacrificed by decapitation, fasted for 12 hours before sacrifice without water, and a vertical incision was made in the center of the abdomen, and the duodenum was placed in a glass dish containing physiological saline.

3. Observation index

3.1 general conditions of mice such as mental, activity, fur, stool, water intake, etc. and weight changes.

3.2 taking the duodenal tissues for fixation, embedding, HE staining and pathological section and observing the structural change of the duodenal mucosa under a microscope: including villus length, mucosal thickness, crypt depth, etc., and intestinal mucosal bleeding was recorded for each group of mice.

4. Statistical treatment: statistical analysis was performed using SPSS13.0, the measurements were expressed as mean ± SD, one-way anova was used for the comparisons between groups, t-test was used for the comparisons between two groups, the test level α was 0.05, and P <0.05 was statistically significant.

5. Results

5.1 general comparison of groups of mice

The blank control group has no phenomena of loose stool and hematochezia, and all indexes of spirit, appetite, water intake and weight are normal; the phenomena of loose stool and hematochezia with different degrees appear in the simple irradiation group at day 2 after irradiation, and the phenomena of mental retardation, appetite reduction, water intake reduction, weight reduction and the like with different degrees appear, 3 people die before death at day 4 after irradiation, and the death rate is 15%; the phenomenon of loose stool appears 3 days after the irradiation of the drug intervention group, but the phenomenon of hematochezia is obviously reduced compared with the single irradiation group, and the phenomena of poor spirit, reduced appetite, reduced water intake, reduced weight and the like are also obviously lighter than the single control group, and no death occurs before death at 4 days after the irradiation.

The hematochezia status of each group of mice is shown in the following table, from which it can be seen that: compared with the single irradiation group, the drug intervention group has significantly reduced hematochezia rate and statistical significance (P is less than 0.01), which indicates that icariin has significant prevention effect on hemorrhage caused by radiation injury of intestinal mucosa.

TABLE 6 hematochezia status of mice in each group

Group of	Animal number (n)	Number of hematochezia (n)	Percentage of hematochezia (%)
				Blank control group	20	0	-
Simple irradiation group	17	6	35.3
				Pharmaceutical intervention group	20	1	5##

Compared with the single-shot group,^##P<0.01。

5.2 pathological Observation of radiation injury to the duodenum in acute radiation enteritis model

5.2.1 visual inspection

The intestinal canal of the blank control group mouse is pink, and congestion, edema and intestinal cavity expansion are avoided; the intestinal canal of the single irradiation group is dark red, the intestinal cavity is dilated and congested, the brittleness of the intestinal wall is found to be increased during dissection, the intestinal wall can be cracked by slight touch, and the intestinal wall is thinned and the mucosa is bleeding to different degrees; the phenomena of intestinal cavity expansion and congestion of the drug intervention group are obviously slight compared with those of the simple irradiation group, and the phenomenon of mucosal hemorrhage rarely occurs.

5.2.2 Observation under the mirror

The blank control group had long and regular intestinal villi, complete structure, clear and visible central chylomicron, and normal glandular structure. The intestinal villi of the single-irradiation group becomes short and sparse, the epithelium is incomplete, the gland is exposed to form erosion, and the crypt cavity is reduced; the intestinal villus structure of the drug intervention group is complete, the villus is slightly edematous, the central chylomicron duct is visible, and the gland erosion degree is obviously slight compared with that of the simple irradiation group.

Intestinal mucosal bleeding in each group of mice is shown in table 7. As can be seen from the table: compared with the single irradiation group, the medicine intervention group has the advantages that the intestinal mucosa bleeding rate is remarkably reduced, the statistical significance is realized (P is less than 0.01), and the icariin can remarkably reduce the intestinal mucosa bleeding rate caused by radiotherapy and chemotherapy and has a remarkable repairing effect on the intestinal mucosa injury caused by the radiotherapy and chemotherapy.

TABLE 7 comparison of intestinal mucosal hemorrhage in mice

Group of	Animal number (n)	Bleeding number (n)	Bleeding Rate (%)
				Blank control group	20	0	-
Simple irradiation group	17	8	47.1
				Pharmaceutical intervention group	20	2	10##

Compared with the single-shot group,^##P<0.01。

Claims (10)

1. application of icaritin in preparing medicine for preventing and treating gastrointestinal hemorrhage is provided.

2. The use according to claim 1, wherein the gastrointestinal bleeding is gastrointestinal bleeding caused by peptic ulcer, radiation damage bleeding or drug.

3. The use according to claim 2, wherein the drug is a non-steroidal anti-inflammatory drug or one or more of a thrombolytic drug and an anti-thrombotic drug.

4. The use according to claim 3, wherein the NSAID is aspirin.

5. The use according to claim 3, wherein the thrombolytic agent is urokinase; the antithrombotic agent is one or more of anticoagulant drugs or antiplatelet drugs.

6. The use according to claim 1, wherein the gastrointestinal bleeding is a stress ulcer manifested by peripheral organ damage caused by a thrombotic disease.

7. The use according to claim 1, wherein the gastrointestinal bleeding is gastrointestinal bleeding caused by tumor radiotherapy and chemotherapy.

8. The use of claim 1, wherein the pharmaceutical formulation comprising icariin is solid, liquid, oil, emulsion, gel, aerosol, inhalant, spray, capsule, pill, patch and suppository thereof.

9. The use according to claim 1, wherein the medicament is administered in a dose of 0.01mg/kg to 500mg/kg for the prevention and treatment of gastrointestinal bleeding.

10. The use according to claim 9, wherein the medicament is administered in a dose of 0.1mg/kg to 50mg/kg for the prevention and treatment of gastrointestinal bleeding.