CN105796678A - Application of raspberry freeze-dried powder or extract thereof to preparation of medicine for treating acute/chronic inflammatory bowel diseases - Google Patents

Abstract

The invention relates to application of raspberry freeze-dried powder or extract thereof to preparation of medicine for treating acute/chronic inflammatory bowel diseases. According to the application, it is found through analysis and research on the inflammation relieving function of an in-vivo inflammatory bowel disease mouse model and mouse feces flora that the raspberry freeze-dried powder or the extract thereof has a remarkable reducing function on abnormal increase of inflammatory factors, has an obvious inhibiting function on intestinal pathogenic bacteria in the inflammatory bowel disease mouse model and has a promoting function on intestinal beneficial bacteria such as butyrate producing bacteria. The result shows that the raspberry freeze-dried powder or the extract thereof can regulate and control the intestinal flora and then has a remarkable treatment function on the inflammatory bowel diseases.

Description

Fructus Rubi lyophilized powder or the application in preparation treatment urgency/chronic inflammatory bowel disease medicament of its extract

Technical field

The invention belongs to pharmaceutical technology field, relate generally to the application in preparation treatment urgency/chronic inflammatory bowel disease medicament preparation of fruit of raspberry lyophilized powder or its extract (including Fructus Rubi mature fruit, Leaves From Raspberry extract).

Background technology

Inflammatory bowel, is called for short IBD, is a kind of special chronic gut inflammation disease, mainly includes Crohn disease (CD) and ulcerative colitis (UC).Clinically, patient can show as stomachache repeatedly, diarrhoea, mucus, hemafecia, various systemic complications even occurs, such as blurred vision, arthralgia, erythra etc..In the U.S., nearly 1,400,000 Americans suffer from inflammatory bowel disease (IBD), and have 30,000 new cases every year；Ulcerative colitis can promote checking and ulcer at large intestine epithelium layer, and ulcerative colitis can increase the risk of patient's colorectal carcinoma.Over nearly 30 years, China's IBD sickness rate has constantly soaring trend.Reporting according to domestic literature, the case load of nearly 5 years is 8 times of the same period nineties in last century, and IBD has been increasingly becoming the commonly encountered diseases of Gastroenterology dept. of China.

IBD Intestinal Mucosal Injury in Patients Undergoing can not the carbohydrate of normal absorption feed, protein, fat, vitamin and various trace elements, it is likely to result in plus enteritis or the medicine taken and is off one's feed, therefore IBD is often along with malnutrition in various degree, even affects the normal growth promoter of child.Nutritional support is to IBD remission and promotes that healing has important effect, and therefore keeping self good nutritional status is treatment mono-part and parcel of IBD.There is not special special diet in IBD, the tolerant food of different people has different, and therefore total for IBD diet principle is to ensure that oneself diet balance avoids some to make the food of disease progression simultaneously.

Therefore the medicine being developed for inflammatory bowel disease is the direction that this area is constantly researched and developed.Expert thinks, inflammatory bowel is not fearful, as long as forming healthy lifestyles, ensure feed, rest, medicine law, the regular follow-up monitoring state of an illness, inflammatory bowel passes through immunization therapy, Biotherapeutics and nutritional support treatment, and the application of some micromolecular compounds and use in conjunction can control.

Fructus Rubi is the perennial fallen leaves half filling property fruit tree of Rosaceae rubus, because its Mature fruit color has black, redness and yellow, and it is called red raspberry, black raspberry and yellow Fructus Rubi, as third generation fruit, it is nutritious and complete, is easily absorbed by the body and the indispensable nutrient of human body containing multiple.If ascorbic content is 5 times of Fructus Mali pumilae, aminoacid and ferrum, zinc phosphorus equal size higher than Fructus Mali pumilae and Fructus Vitis viniferae, especially with bioactive chemical classes various.

In prior art, there is not Fructus Rubi or its extract relevant report in treatment urgency/chronic inflammatory bowel disease.

Summary of the invention

It is an object of the invention to provide Fructus Rubi lyophilized powder or its extract disorderly with intestinal flora metabolism in preventing and treating, intestinal wall ulcerative lesions and mastocyte excessive activation are the application in the urgency/chronic inflammatory bowel disease of principal character.

The present invention, with Fructus Rubi lyophilized powder or its extract for effective ingredient, its total effective dose is: Fructus Rubi lyophilized powder 20-40g/ days, Fructus Rubi lyophilized powder extract 1-100mg/kg/ days.

The present invention, Fructus Rubi lyophilized powder or its extract can individually be applied, it is also possible to the Elental use in conjunction general with treatment of inflammatory bowel clinically.

The present invention, is mixed and made into pharmaceutical dosage form by Fructus Rubi lyophilized powder or its extract with pharmaceutically acceptable excipient.Described pharmaceutical dosage form is peroral dosage form.Described peroral dosage form is tablet, powder, suspension, emulsion, capsule, granule, coated tablet, pill, liquid, syrup or limonada.

The invention has the beneficial effects as follows: the present invention, by the inflammation improvement result of internal inflammatory bowel disease mouse model and the analysis and research of stool in mice flora are found, Fructus Rubi lyophilized powder or its extract raise for the exception of inflammatory factor and significantly reduce effect, obvious inhibitory action is served for enteropathic antibacterial in inflammatory bowel disease model mouse, and intestinal beneficial bacterium such as butyrate producing strains is served facilitation.It is shown that inflammatory bowel disease by regulating and controlling intestinal microbial population and then can be had significant therapeutic effect by Fructus Rubi lyophilized powder or its extract.

Accompanying drawing explanation

Fig. 1 be raspberry extract for inflammatory bowel stool in mice DNA based on T-rflp collection of illustrative plates cluster analysis dendrogram.

Fig. 2 is the raspberry extract action diagram to probiotics eisseria；

* p < 0.05vs model group, * * p < 0.01vs model group.

Fig. 3 is the raspberry extract action diagram to probiotics butyrate producing strains；

* p < 0.05vs model group, * * p < 0.01vs model group.

Fig. 4 is the raspberry extract action diagram to pathogenic bacterium Campylobacter；

* p < 0.05vs model group, * * p < 0.01vs model group.

Fig. 5 is the raspberry extract action diagram to pathogenic bacterium Bacteroides；

* p < 0.05vs model group, * * p < 0.01vs model group.

Fig. 6 is that antibacterial T-rflp analyzes significance fragment.

Fig. 7 is that raspberry extract is to the abnormal inhibitory action figure raised of inflammatory bowel mice enteral inflammatory cytokine.

Fig. 8 is the variable concentrations raspberry extract inhibitory action figure for the external bacteria group that spreads cultivation of inflammatory bowel stool in mice.

Detailed description of the invention

The application in preparation treatment urgency/chronic inflammatory bowel disease medicament of embodiment 1 raspberry extract

(1) black raspberry mature fruit extract (BRBFE)

Black raspberry mature fruit being smashed to pieces, cross 0.02 inch aperture screen cloth, cross elimination seed, make puree, puree is poured in the lyophilization dish of 1 inch of surface painting polyethylene thin film, preserves in the refrigerating chamber ventilated.Carry out lyophilization by sublimating apparatus further, obtain black raspberry fruit lyophilized powder.Black raspberry fruit lyophilized powder is added ethanol: water (80:20) solvent, soak extraction, temperature 25 DEG C；Time 10h；Extraction into ethyl acetate removes fats composition in fruit, obtains the deduction of black raspberry mature fruit and take thing (BRBFE) after decompression and solvent recovery.

(2) model

The foundation of ulcerative colitis mouse model

1.4-6 week old C57BL/J mice, random packet, if blank group, model group, experimental group (two dosage: 5% and 10%) totally four groups.Blank group and the edible normal diet of model group mice, experimental mice eats BRBFE.

2. giving lumbar injection azoxymethane (Azoxymethane, AOM) according to Mouse Weight, the 1st, 8 days, model group and experimental mice lumbar injection AOM, injection dosage was 10mg/kg.

3. model group and experimental mice 2% dextran sulfate sodium (DextranSodiumnsulfate, DSS) take the photograph water, continuous 7 days, the 8th day, change normal drinking-water and continue 14 days.Blank group is normal drinking water.

4.2%DSS takes the photograph water totally 3 circulations, namely 1,4,7 weeks respectively 2%DSS take the photograph water, other times are normal water.

(3) BRBFE studies based on T-rflp cluster analysis for inflammatory bowel stool in mice DNA

Blank group and the edible normal diet of model group mice, experimental mice eats BRBFE.Model group and experimental mice 2%DSS take the photograph water, continuous 7 days, the 8th day, change normal drinking-water and continue 14 days.Blank group is normal drinking water.2%DSS takes the photograph water totally 3 circulations, namely 1,4,7 weeks respectively 2%DSS take the photograph water, other times are normal water.After sacrifice, pick fresh excreta in intestinal, extract DNA in stool in mice according to test kit description.T-rflp method analyzes Flora dynamics situation with three kinds of enzymes (HaeIII, HhaI and MSPI).Testing result is done T-rflp collection of illustrative plates cluster analysis, result as it is shown in figure 1, in figure B represent BRBFE group, C represents blank group, M representative model group.

Result: as seen from Figure 1, dendrogram modal data shows: compared with inflammatory bowel disease models Mus, larger through mouse intestinal flora and the blank group similarity of the BRBFE feeding process group of 9 weeks, and has comparatively significant difference compared with inflammatory bowel disease models Mus.Show that Fructus Rubi lyophilized powder or its extract mouse intestinal flora to causing inflammatory of the present invention are inhibited qualitatively.

(4) BRBFE is to the effect of probiotics and pathogenic bacterium in inflammatory bowel stool in mice

After sacrifice, pick fresh excreta in intestinal, extract DNA in stool in mice according to test kit description.T-rflp method analyzes Flora dynamics situation with three kinds of enzymes (HaeIII, HhaI and MSPI).Such as Fig. 2-Fig. 5: antibacterial T-rflp analyzes significance fragment, such as Fig. 6: T-rflp atlas analysis.

1, butyrate is the oxidation substrates that colonic epithelium is best, account for the 80% of colon cell oxygen consumption, butanoic acid needs not move through liver and gall and absorbs and complicated tricarboxylic cycle system, directly can provide energy for enterocyte, it it is the fast energy source of enterocyte, especially the energy that enterocyte (caecum and colon cell) is first-selected, and very easily absorb in enteric cavity, the prevention of intestinal mucosa reparation and colitis and colon cancer is worked.In the BRBFE stool in mice processed, butyrate producing strains content increases, and improves 6%, and model group content is 1% compared with blank group.In the BRBFE stool in mice processed, eisseria content increases, and improves 5%, and model group content is 0% compared with blank group.Result shows, BRBFE has obvious positivity regulating and controlling effect for beneficial bacteria of intestinal tract.

2, Campylobacter and bacteroid are pathogenic bacterium main in intestinal, and in the BRBFE stool in mice processed, Campylobacter, helicobacter pylori, product succinic acid irrigate honest and clean bacterium, and content reduces, and reduces 15% compared with model group.Bacteroides and general Bordetella in the BRBFE stool in mice processed, content reduces, and reduces 72% compared with model group, and result shows, BRBFE has significant inhibitory action for pathogenic entero becteria.

(5) BRBFE is for the abnormal inhibitory action raised of inflammatory bowel mice enteral inflammatory cytokine

The extraction of mice enterocyte

Jejunum, ileum, colon longitudinally being cut open, PBS cleans, and respectively takes one section and inserts 20ml ice PBS, then will move into 37 DEG C of 15mMEDTA30ml, 37 DEG C of vibrations, 220r, 30min by each tissue above.First vibrate with hands, then after agitator fully vibrates, remove myenteron meat tissue.4 DEG C are centrifuged, 5000r, 5min, first supernatant discarded, and the cold PBS re-suspended cell of 5ml is sub-packed in 1.5mlEP pipe.4 DEG C are centrifuged, 3000r, 5min.Supernatant discarded, EP pipe is uncapped and is placed in liquid nitrogen.After taking-up, uncap and be placed in-80 DEG C of liquid excessively.Next day is by EP lid pipe.

The extraction of mice enterocyte RNA and the synthesis of cDNA

Extract mice enterocyte RNA by Trizol method, according to cDNA the first chain synthetic agent box description, total serum IgE is carried out reverse transcription synthesis cDNA, deposits for-20 DEG C.

Design of primers and Real-TimePCR

IL-1:R:5’-GCAACT-GTTCCTGAACTCAACT-3’,

F:5’-ATCTTTTGGGGT-CCGTCAACT-3’；

IL-6:R:5’-TAGTCCTTCCTACCCCAAT-TTCC-3’,

F:5’-TTGGTCCTTAGCCACTCCTTC-3’；

IL-10:R:5’-GCTCTTACTGACTGGCATGAG-3’,

F:5’-CGCAGCTCTAGGAGCATGTG-3’；

COX2:R:5’-TGAGCAACTA-TTCCAAACCAGC-3’,

F:5’-GCACGTAGTCTTCGATCA-CTATC-3’；

TNF-a:R:5’-CCCTCACACTCAGATCATCTTCT-3’,

F:5’-GCTACGA-CGTGGGCTACAG-3’；

β-actin:R:5’-AGGCAAACCGTGAAAAGATG-3’,

F:5’-AGGCAAACCGTGAAAAGATG-3。

With cDNA for template, carry out Real-TimePCR by UItraSYBRMixture reagent description, process experimental data, obtain Fig. 7.

Such as Fig. 7, in 5%BRBFE and the 10%BRBFE mice enterocyte processed, the expression of IL-1 reduces, and reduces 30% and 55% compared with model group respectively.In 5%BRBFE and the 10%BRBFE mice enterocyte processed, the expression of IL-6 reduces, and reduces 58% and 19% compared with model group respectively.In 5%BRBFE and the 10%BRBFE mice enterocyte processed, the expression of IL-10 reduces, and reduces 23% and 58% compared with model group respectively.In 5%BRBFE and the 10%BRBFE mice enterocyte processed, the expression of COX2 reduces, and reduces 40% and 55% compared with model group respectively.In 5%BRBFE and the 10%BRBFE mice enterocyte processed, the expression of TNF α reduces, and reduces 67% and 21% compared with model group respectively.

(6) the variable concentrations BRBFE impact on the faecal microbiota of In vitro culture

With 96 orifice plates, BRBFE Mlc being screened, each group of stool in mice flora supernatant 1 milliliter adds amplification culture in 200 milliliters of liquid culture medium, 37 DEG C, 180 turns, detects its light absorption value after 14 hours, each group light absorption value is at 600nm place OD=0.4.Adding in 96 orifice plates after bacteria suspension is diluted 100 times, every hole adds 200 microlitres, and the fluid medium taking same volume does blank group.The BRBFE2 microlitre taking each concentration adds in bacteria suspension, takes ammonia benzyl mycin 2 microlitre simultaneously and does positive controls, do respectively three groups parallel, put into cultivation 24 hours in bacteriological incubator after labelling is good.Detecting every hole light absorption value by microplate reader after 24 hours and be calculated mapping, result is as shown in Figure 8.

As seen from Figure 8, when 0-250 μ g/mlBRBFE concentration, its fungistatic effect also strengthens gradually, suppression ratio respectively 0.96%, 4.8%, 40.3%, 59.4%, 59.7%, 70.2%, when 500 μ g/ml and 2500 μ g/mlBRBFE concentration, its fungistatic effect reduces, it is suppressed that rate respectively 68.4% and 47.5%.Positive controls suppression ratio is 38.2%.

Claims (9)

1. Fructus Rubi lyophilized powder or the application in preparation treatment urgency/chronic inflammatory bowel disease medicament of its extract.

2. apply as claimed in claim 1, it is characterised in that: Fructus Rubi lyophilized powder or its extract and the Elental use in conjunction that treatment of inflammatory bowel is general clinically.

3. apply as claimed in claim 1, it is characterised in that: Fructus Rubi lyophilized powder or its extract are mixed and made into pharmaceutical dosage form with pharmaceutically acceptable excipient.

4. apply as claimed in claim 3, it is characterised in that: described pharmaceutical dosage form is peroral dosage form.

5. apply as claimed in claim 4, it is characterised in that: described peroral dosage form is tablet, powder, suspension, emulsion, capsule, granule, coated tablet, pill, liquid, syrup or limonada.

6. the application as described in as arbitrary in claim 1-5, it is characterised in that: total effective dose of Fructus Rubi lyophilized powder or its extract respectively 20-40g/ day and 1-100mg/kg/ day.

7. the application as described in as arbitrary in claim 1-5, it is characterised in that: described Fructus Rubi lyophilized powder or its extract improve the profitable strain eisseria in intestinal, butyrate producing strains and addicted to the application in the medicine of ammonia clostridium in preparation.

8. the application as described in as arbitrary in claim 1-5, it is characterised in that: described Fructus Rubi lyophilized powder or its extract suppress the application in the medicine of the pathogenic bacterium Campylobacter in intestinal, helicobacter pylori, Bacteroides and general Bordetella in preparation.

9. the application as described in as arbitrary in claim 1-5, it is characterised in that: described Fructus Rubi lyophilized powder or its extract suppress enteritis factor IL-1, IL-6, IL-10, COX2 and TNF.