CN103599162A

CN103599162A - Application of Tibet rhodiola crenulata extract in preparing medicament for resisting intestinal inflammation

Info

Publication number: CN103599162A
Application number: CN201310594361.9A
Authority: CN
Inventors: 金丽华; 朱彩霞; 关法春; 兰小中
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2013-11-22
Filing date: 2013-11-22
Publication date: 2014-02-26
Anticipated expiration: 2033-11-22
Also published as: CN103599162B

Abstract

The invention provides an application of Tibet rhodiola crenulata extract in preparing a medicament for resisting intestinal inflammation. The rhodiola crenulata extract is obtained by adopting a water extraction method, and is subjected to a drosophila intestinal inflammatory injury repairing experiment. Results prove that the rhodiola crenulata extract has excellent anti-inflammation and anti-oxidation activities, and can be used for preparing medicaments for resisting intestinal inflammation.

Description

The application of Tibet Rhodiola crenulata extract in the anti-enteritis medicine of preparation

Technical field

The present invention relates to the application of Tibet Rhodiola crenulata extract in the anti-enteritis medicine of preparation, relate to the field of Chinese medicines and fruit bat natural immunity technical field.

Background technology

Intestinal mucosa contacts the different Antigens materials such as a large amount of food, antibacterial and virus every day, in intestinal, host produces immunoreation to pathogenic microorganism, and food antigens and other normal enteric cavity content are produced to immunologic tolerance etc., all needs intestinal mucosal barrier to play a role.The intestinal mucosa immunity of enantiopathy pathogenic microorganism can be divided into innate immunity and the day after tomorrow acquired immunity, wherein innate immunity forms the basis of intestinal mucosal barrier.Intestinal innate immunity is the important defense mechanism of intestinal, is the first line of defence that host resists pathogenic microorganism and harmful substance.

Fruit bat lacks the acquired immune system that mammal has, and in body, there is no T cell and B cell, also there is no antibody and complement system, can only infecting of pathogenic microorganism be made and being replied by natural immune system.In addition, fruit bat has similar 26S Proteasome Structure and Function with mammiferous intestinal; The differentiation mechanism of intestinal stem cell and natural immunity mechanism are also quite similar.Therefore, can utilize fruit bat as the model of research mammalian immune reaction, the more important thing is the generation that can utilize its announcement and mankind's relevant disease.When fruit bat intestinal is subject to external environment stimulation, its intestinal epithelial cell can be made and replying Internal and external factors, as produce active oxygen (reactive oxygen species, ROS) and various antibacterial peptide (antibacterial peptides) etc. remove the pathogenic microorganism of invasion, thereby maintain intestinal environment stable state.

Radix Rhodiolae [Rhodiola crenulata (Hook, f.et thoms.) H.Ohba] be perennial herb or the semishrub plant of Crassulaceae (Crassulaceae) rhodiola (Rhodiola), the west areas such as Tibet, Sichuan and Yunnan mainly distribute.The chemical composition of Radix Rhodiolae mainly contains rhodioside, Radix Rhodiolae element, butyl alcohol, Radix Rhodiolae polysaccharide, flavone compound, phenolic compound etc., and wherein rhodioside is its main effective ingredient.Research shows that Radix Rhodiolae has the immunity of enhancing, resisting fatigue, anoxia enduring, antitumor, radioprotective, antiviral and armour and avoids many-sided pharmacological actions such as radical damage.But up to now, the research of the anti-enteritis of Tibet Rhodiola crenulata extract aspect is had no to report.

Summary of the invention

The object of the invention is to disclose the application of Tibet Rhodiola crenulata extract in the anti-enteritis medicine of preparation.

Above-mentioned purpose of the present invention is achieved by following scheme:

Utilize spore, Micrococcus luteus and the surfactant SDS feeding fruit bat of pathogenic microorganism Beauveria bassiana to set up fruit bat intestinal immunity damage model first respectively, bring out fruit bat enteritis, by relatively having or not interpolation Rhodiola crenulata extract to probe into the repair of Rhodiola crenulata extract to enteritis damage to the impact of the indexs such as expression, intestinal epithelial cell ROS level and Intestinal Tract Morphology variation of antibacterial peptide in drosophila survival rate, intestinal epithelial cell death condition, intestinal.Found that this extract has good antiinflammatory, antioxidation, removing free radical isoreactivity, can be used for preparing the medicine of anti-enteritis.

Accompanying drawing explanation

Fig. 1 is that Tibet Rhodiola crenulata extract is processed the impact of rear fruit bat survival rate on fungus M.luteus, B.bassiana and pro-inflammatory cytokine SDS;

Fig. 2 is the impact of fruit bat intestinal epithelial cell after Tibet Rhodiola crenulata extract is processed SDS;

Fig. 3 is that Tibet Rhodiola crenulata extract infects the impact of peptide expression amount in rear intestinal on B.bassiana;

Fig. 4 is the impact of ROS level in fruit bat intestinal epithelial cell after Tibet Rhodiola crenulata extract is processed SDS;

Fig. 5 is that Tibet Rhodiola crenulata extract is processed the impact of rear intestinal form and intestinal walls thickness on SDS.

The specific embodiment

The preparation of embodiment 1 Tibet Radix Rhodiolae extracting solution

Take Tibet Radix Rhodiolae dry root 20g, after pulverizing, in 200mL deionized water, soak 12h, little fiery heated and boiled 3h, filter, adding 200mL deionized water to use the same method extracts 2 times again, merge 3 extracting solution and be concentrated into 200mL, obtain 0.1g/ml(10%) Radix Rhodiolae extracting solution, 4 ℃ of sealed storage are standby.High effective liquid chromatography for measuring Radix Rhodiolae extracting solution is not less than 0.5% containing rhodioside, meets the quality standard of < < Chinese Pharmacopoeia > > 2010 version ().

Embodiment 2 Tibet Rhodiola crenulata extracts are processed the impact of rear fruit bat survival rate on fungus B.bassiana, M.luteus and pro-inflammatory cytokine SDS

The preparation of B.bassiana spore liquid: B.bassiana is applied on fresh LB solid medium, 25 ℃ of standing cultivation 7d, the spore in culture medium filters after collecting with aseptic PBS, measures spore concentration.After spore is centrifugal, abandon supernatant, in precipitation, add 5% sucrose solution (matched group) or containing the Radix Rhodiolae extracting solution (experimental group) of 5% sucrose, be mixed with A ₆₀₀=40 spore solution, 4 ℃ store for future use.

The preparation of M.luteus bacterium liquid: M.luteus (LB culture medium, 37 ℃) be inoculated into the amplification culture of spending the night in 200mL fluid medium, by bacterium liquid 4000r/min, 25 ℃, centrifugal 10min, abandon supernatant, add 5% sucrose solution (matched group) or containing the Radix Rhodiolae extracting solution (experimental group) of 5% sucrose, be mixed with the bacterium liquid of A600=180,4 ℃ store for future use.

With the fruit bat cultivated in ordinary culture medium as a control group, containing the fruit bat of cultivating in the culture medium of Rhodiola crenulata extract as experimental group.The sprouted wings fruit bat of 3～4d of random collecting, female, male each 15.First fruit bat hunger is processed to 2h, then move on in the fruit bat pipe that is placed with 5 metafiltration paper, in filter paper, add 450 μ L reagent or bacterium liquid, filter paper is fully infiltrated, and absorb unnecessary liquid.Every 24h changes 1 filter paper, records fruit bat death toll simultaneously, calculates survival rate, records altogether 6～7d, and every group of experiment repeats 3 times.

Result shows: fruit bat is carried out to M.luteus and infect after 6d, matched group drosophila survival rate is 5.6%, the survival rate of Radix Rhodiolae group is 47.6%, and the survival rate of Radix Rhodiolae group has been compared significant difference (P<0.01) (Figure 1A) with matched group.While being infected by B.bassiana spore, the experimental group survival rate of feeding Rhodiola crenulata extract is apparently higher than matched group.As shown in Figure 1B, matched group drosophila survival rate is 15.6%, and the survival rate of Radix Rhodiolae group is 80.0%, and the survival rate of Radix Rhodiolae group is compared difference extremely significantly (P<0.001) with matched group.Adult fruit bat SDS(0.6%) process after 6d, the survival rate of matched group is only 3.3%, and the survival rate of Radix Rhodiolae group is 59.2%, and Radix Rhodiolae group matched group has been compared utmost point significant difference (P<0.001) (Fig. 1 C).These results show, Rhodiola crenulata extract can resist pathogenic microorganism and pro-inflammatory cytokine SDS stimulates the damage of fruit bat, improves significantly the survival rate of fruit bat.

Embodiment 3 Tibet Rhodiola crenulata extracts are processed the impact of rear fruit bat intestinal epithelial cell on SDS

In order to explore SDS, process dark one deck mechanism that rear Rhodiola crenulata extract improves drosophila survival rate, this experiment utilizes the death condition of amino D actinomycin D (7-Aminoactionomycin D, 7-AAD) the staining analysis intestinal epithelial cell of 7-.7-AAD can only enter in dead cell and be combined with its DNA as a kind of nucleic acid dye, after exciting, can produce red fluorescence.

Sprouted wings 30～40 of the female fruit bats of 3～4d of random collecting, feeding is containing the SDS(0.6% of 5% sucrose) solution 96h.Isolate under the microscope complete intestinal, utilize 7-AAD dyeing 30min, paraformaldehyde is 10min fixedly, the DAPI 10min that dyes, fluorescence microscopy Microscopic observation taking pictures after glycerol mounting.Every component from approximately 10 of fruit bat intestinals, experiment repeats 3 times.

Result shows: after adult fruit bat SDS(0.6%) processing, mortality appears in the midgut epithelial cell of matched group fruit bat, and in the experimental group of feeding Rhodiola crenulata extract, dead cell quantity obviously reduces (Fig. 2).The above results explanation Tibet Rhodiola crenulata extract can obviously be alleviated the damage that SDS causes intestinal epithelial cell.

Embodiment 4 Tibet Rhodiola crenulata extracts infect the impact of peptide expression amount in rear intestinal on B.bassiana

Sprouted wings 30～40 of the female fruit bats of 3～4d of random collecting, the spore (A of feeding B.bassiana ₆₀₀=40) intestinal of separated fruit bat under microscope after 4h, extracts total RNA of intestinal, and reverse transcription utilizes sxemiquantitative PCR method to detect the expression of multiple antibacterial peptide after becoming cDNA.

Result shows: the drosophila melanogaster antimicrobial peptide Dfn(Defencin of Radix Rhodiolae group), Drs(Drosomycin), AttA(Attacin A), CecA1(Cecropin A2) and CecA2(Cecropin A2) gene expression amount compared with matched group, all raise, this shows that Tibet Rhodiola crenulata extract can improve the peptide expression amount of fruit bat intestinal, strengthens the ability (Fig. 3) that intestinal is resisted pathogenic microorganism.

Embodiment 5 Tibet Rhodiola crenulata extracts are processed the impact of ROS level in rear fruit bat intestinal epithelial cell on SDS

ROS is significant at aspects such as antibacterial, antiinflammatory and inhibition tumors.But when body is subject to disease or some exogenous medicine and poisonous substance invasion, the antioxidant system in body may get muddled, Radical Metabolism dysequilibrium.Wherein, the excessive generation of ROS can cause cell injury, and to a certain extent, the cell of these excessive damage will be eliminated by apoptosis mechanism, and still, surpassing a certain amount of ROS will cause organ dysfunction extremely or lesion tissue by lipid peroxidation.The ability of removing ROS in order to detect Radix Rhodiolae extract, this experiment is carried out SDS(1% to fruit bat) process 48h, utilize the content of ROS in dihydro second pyridine (Dihydroethidium, DHE) colouring method analysis of cells.

In fruit bat intestinal epithelial cell, the detecting step of ROS level dyes with 7-AAD.

Result shows: the most cells of matched group promesenteron and rear middle intestinal cell over half have produced a large amount of ROS, and in the experimental group fruit bat of feeding Rhodiola crenulata extract, in midgut epithelial cell, ROS content obviously reduces (Fig. 4).This explanation Tibet Rhodiola crenulata extract can suppress intestinal epithelial cell and produce excessive ROS, and protection body is avoided oxidative damage.

Embodiment 6 Tibet Rhodiola crenulata extracts are processed the impact of rear intestinal form and intestinal walls thickness on SDS

Sprouted wings 30～40 of the female fruit bats of 3～4d of random collecting, feeding is containing the SDS(0.6% of 5% sucrose) solution 96h.Isolate under the microscope complete intestinal in PBS, micro-Microscopic observation is also taken pictures.Every component from approximately 10 of fruit bat intestinals, experiment repeats 3 times.

Result shows: with SDS(0.6%) to process after 4d, matched group fruit bat is compared with the normal fruit bat of a feeding sucrose, and fruit bat intestinal length obviously shortens, and has occurred obvious melanoma at middle intestinal and hindgut junction.And the experimental group fruit bat intestinal length of feeding Rhodiola crenulata extract is 2 times of matched group, its intestinal length is slightly shorter than the normal fruit bat of a feeding sucrose, and does not find melanoma (Fig. 5 A, B).In addition, to SDS(0.6%) different disposal fruit bat intestinal walls varied in thickness observation discovery after the time, while processing 4d, the intestinal walls thickness of matched group and experimental group is close; While processing 6d, the obvious attenuation of intestinal walls thickness of matched group fruit bat, and the experimental group fruit bat intestinal walls thickness of feeding Rhodiola crenulata extract does not have significant change (Fig. 5 C).This result has further proved that Tibet Rhodiola crenulata extract can protect intestinal epithelial cell to avoid the inflammation damnification of SDS induction, maintains the Intestinal Morphology of fruit bat.

Claims

1. a Tibet Rhodiola crenulata extract is to obtain by the following method:

Take dry Tibet Radix Rhodiolae root, after pulverizing, in 200mL deionized water, soak 12h, little fiery heated and boiled 3h, filters, then adds 200mL deionized water to use the same method to extract 2 times, merge 3 extracting solution and be concentrated into 200mL.

2. Tibet claimed in claim 1 Rhodiola crenulata extract is for the preparation of the medicine of anti-enteritis.

3. Tibet according to claim 2 Rhodiola crenulata extract purposes, is characterized in that: described medicine is any one in tablet, capsule, granule, oral liquid.