CN109745336A - Codonopsis pilosula polysaccharide is as the application for adjusting human body intestinal canal flora drug - Google Patents

Abstract

Codonopsis pilosula polysaccharide is as the application for adjusting human body intestinal canal flora drug, drug of the Codonopsis pilosula polysaccharide as prevention colitis, the application of food or health care product.After preventing 21 days in healthy mice model respectively using oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e, induce establish acute colitis mouse model within 7 days using 3% dextran sulfate sodium.By the indexs variation such as assessment experiment mice changes of weight, colon lengths, disease pathology index, tissue pathological slice and activity of myeloperoxidase, compare three kinds of drugs to the alleviation situation of colitis macroscopic view pathological symptom.Using 250 microarray dataset of Illumina MiSeq, it is based on the variable region bacterial 16 S V3-V4, adjusting of the drug to colitis mice intestinal flora has been probed into, has specified the superiority bacteria spp in each pharmaceutical intervention group.Absorbing state of the highest monomer saponin of exposed amount in serum and caecum in Radix Codonopsis saponin(e；Illustrate the mechanism that Codonopsis pilosula polysaccharide acts on saponin(e metabolism, influences its performance disease treatment effect by changing intestinal flora.

Description

Codonopsis pilosula polysaccharide is as the application for adjusting human body intestinal canal flora drug

Technical field

The present invention relates to Codonopsis pilosula polysaccharides to the adjusting field of intestinal flora, in particular to Codonopsis pilosula polysaccharide is as adjusting human body The application of intestinal flora drug.

Background technique

Intestinal flora is by 100,000,000,000,000 archeobacterias and bacterium group as the essential a part of human gastrointestinal tract is constituted At complicated community, represent more than 1000 kinds of species, wherein be more than 90% species belong to Bacteroidetes, Firmicutes and Actinomyces door.A large amount of human body and rodent assays prove: intestinal flora is to maintain the indispensable " device of host health Official ".

Inflammatory bowel disease (Inflammatory Bowel Disease, IBD) is a kind of by diet, infection, metabolism, easily The autoimmune disease that many factors such as sense gene, immune, intestinal flora mediate jointly, principal pathogenetic are characterized in stomach and intestine Continue in mucous membrane, repeatedly and uncontrollable inflammatory reaction.According to the difference of its position of disease and feature: ulcer can be subdivided into Property colitis (UC) and Crohn disease (CD).

The therapeutic advance of 1 IBD of table

Table 1Treatment progress of IBD

Supplement alternative medicine (complementary and alternative medicine, CAM) refers to mainstream health care In addition, a series of general names that can be helped and make up mainstream health care insufficient diagnosis and control measure are broadly divided into replacement medicine and examine Treatment, idea therapy, biology feed several major class such as therapy and energy therapy.Different from traditional western medicine, CAM is because of its work With mild, effect, good and side effect is far below the advantages that conventional medicament, gradually by the welcome of patient and receiving.CAM method is normal Profitable probliotics, prebiotics and Chinese herbal medicine (HM) etc..

Radix Codonopsis sweet in flavor and neutral in nature, be strengthening spleen and nourishing blood, beneficial lung fluid classical Chinese medicine, be mainly used for preventing and treating spleen-lung Qi deficiency, food Few burnout, insufficiency of vital energy and blood, Heat Diabetes etc..Clinical treatment spleen deficiency is widely used in Radix Codonopsis Sijunzi Tang as main component Caused disease.Codonopsis pilosula polysaccharide tool enhancing anti-stress ability adjusts immunity of organism and anti-oxidant isoreactivity.Research shows that: Radix Codonopsis Decocting liquid can increase mouse macrophage quantity, enhancing gulps down by stomach-filling and muscle, vein or intraperitoneal injection in various degree Bite ability.Meanwhile the activity of intracellular nucleic thuja acid, carbohydrate and part enzyme can be significantly increased.Radix Codonopsis saponin(e is in intestinal flora Antitumor, antibacterial can be played under metabolism and inhibits the pharmacological actions such as the expression of relevant inflammatory factors.Supriya R. etc. is ground It is found when studying carefully effect of the Radix Codonopsis saponin(e in the mouse colitis that 2,4,6- trinitrobenzene sulfonic acid (TNBS) induces: codonopsis lanceolata Saponin A is mainly metabolized as secondary saponin class active small molecular substance through intestinal flora, and then is inhaled by enterocyte It receives, plays systemic effect into blood；It, which not only can inhibit, balances relevant inflammatory factors IL-1 β, TNF- with Th17/Treg The expression of α and IL-6 also can be relieved the damage of colitis mice mucosal tissue.

So far, Radix Codonopsis in the prevention and treatment of intestines problem and is underutilized.In addition, it is answered in long-term clinic In, Radix Codonopsis is mainly prepared and is administered orally in the form of water decoction；Because (Western medicine chemical component is simple, clear to Chinese medicine ) interaction between the complexity of material component (usually containing a variety of known or non-principal components) and each component is still unclear Chu；Its component for playing useful effect in treatment of diseases to finding, the mutual mechanism between active principle of tracking bring tired It is difficult.So furtheing investigate the interaction between Radix Codonopsis effective component and its playing drug effect in pipe intestinal digesting systemic disease When Coupling effects be of great significance.

Summary of the invention

To solve the above-mentioned problems of the prior art, the purpose of the present invention is to provide

Codonopsis pilosula polysaccharide is as the application for adjusting human body intestinal canal flora drug, and in the therapeutic process of IBD, Codonopsis pilosula polysaccharide can The advantageous variation of IBD intestinal flora is directly or indirectly induced as prebiotics, and the intestinal flora changed will promote Radix Codonopsis soap Glycosides is converted into active pharmacological molecules aglycon, and then plays disease treatment effect (Fig. 1).This project establishes the acute knot of DSS induction Enteritis mouse model, by high throughput sequencing technologies, probe into Codonopsis pilosula polysaccharide to the adjusting of colitis mice intestinal microflora and Flora influences the mechanism of Radix Codonopsis saponin(e metabolism.Using the content of SCFAs in GC measurement excrement；ELISA detect IBD in TH17/ The secretion of Treg balance relevant cell factor；Blood is analyzed using ultra high efficiency liquid phase-mass spectrometric hyphenated technique combination metabolism group method The metabolic alterations of Radix Codonopsis saponin(e in liquid and caecum；It illustrates Codonopsis pilosula polysaccharide and acts on the metabolism of Radix Codonopsis saponin(e by changing intestinal flora, And then its mechanism for playing disease treatment effect is influenced, it lays the foundation for clinical prevention, treatment IBD, new approaches is provided.

In order to achieve the above objectives, the technical solution of the present invention is as follows:

Codonopsis pilosula polysaccharide is used to adjust the application of human body intestinal canal flora, drug of the Codonopsis pilosula polysaccharide as prevention colitis, food Or the application of health care product.

Codonopsis pilosula polysaccharide is used to adjust the application of human body intestinal canal flora, drug of the Codonopsis pilosula polysaccharide as treatment colitis, food Or the application of health care product.

Codonopsis pilosula polysaccharide is used to adjust the application of human body intestinal canal flora, and Codonopsis pilosula polysaccharide is by acting on saponin(e Metabolism regulation people Body intestinal flora.

Further, Codonopsis pilosula polysaccharide is by lowering proinflammatory cytokine related to Th17/Treg balance: interleukins The secretion of [IL] -17A, IL-17F, IL-6, IL-22 and tumor necrosis factor [TNF]-α are raised anti-inflammatory factors IL-10 and are turned Change the expression of growth factor [TGF]-β, adjusts human body intestinal canal flora.

Further, Codonopsis pilosula polysaccharide passes through three kinds of selective stimulating important prebiotic Pseudomonas Bifidobacterium spp., Harmful Pseudomonas Desulfovibio spp. is lowered in the growth of Akkermansia spp and Lactobacillus spp, The abundance of Alistipes spp and Helicobacter spp, adjust the disorder of intestinal flora.

Further, Codonopsis pilosula polysaccharide increases the content of SCFAs in colitis mice enteron aisle, and rich by selectivity Collect to have and promotees the Blautia that SCFAs is generated, Prevotellaceae UCG-001, Oscillibacter and Quinella bacterium The growth of category improves the system and local energy supply situation of SCFAs.

Further, the Codonopsis pilosula polysaccharide is drug, food or health care product.

Further, the Codonopsis pilosula polysaccharide is for oral administration or exterior-applied formulation.

Further, the Codonopsis pilosula polysaccharide be tablet, pill, dispersing agent, granule, the prepared slices of Chinese crude drugs, spray, or Ointment plaster.

Compared with the existing technology, the invention has the benefit that

The result shows that: oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e intervention prevention and treatment can improve DSS induction in various degree Colitis mice macroscopical pathological condition；Lower proinflammatory cytokine related to Th17/Treg balance: interleukins The secretion of [IL] -17A, IL-17F, IL-6, IL-22 and tumor necrosis factor [TNF]-α are raised anti-inflammatory factors IL-10 and are turned Change the expression of growth factor [TGF]-β.16SrRNA high-flux sequence is as the result is shown: three kinds of medical treatments can improve in various degree The structure and diversity of colitis mice intestinal flora.Wherein, oligofructose effect is optimal, but has no between Codonopsis pilosula polysaccharide Significant difference.Meanwhile Codonopsis pilosula polysaccharide passes through three kinds of selective stimulating important prebiotic Pseudomonas Bifidobacterium spp., Harmful Pseudomonas Desulfovibio spp. is lowered in the growth of Akkermansia spp and Lactobacillus spp, The abundance of Alistipes spp and Helicobacter spp, improve the disorder of colitis mice intestinal flora.GC result Display: oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e not only increase the content of SCFAs in colitis mice enteron aisle, and There is the Blautia, Prevotellaceae UCG-001, Oscillibacter for promoting SCFAs and generating by selective enrichment With the growth of Quinella Pseudomonas, the system and local energy supply situation of SCFAs are improved.In addition, research discovery: three kinds After medical treatment, by alleviating the imbalance of colitis mice intestinal flora, the highest monomer of exposed amount in Radix Codonopsis saponin(e is improved Compound Cs-415.2 is in the intracorporal absorptivity of colitis mice, wherein oligofructose effect is optimal, and Codonopsis pilosula polysaccharide takes second place.It is comprehensive Upper described: Codonopsis pilosula polysaccharide has good prebiotics characteristic, shows good application during the prevention and treatment of acute colitis Prospect.

One aspect of the present invention confirms the potential prebiotic effect of Codonopsis pilosula polysaccharide, on the other hand also reveals in Radix Codonopsis altogether The polysaccharide deposited influences the mechanism of saponin(e metabolism by restoring the disorder of colitis mice intestinal flora.For with Codonopsis pilosula polysaccharide Novel probiotics are developed as prebiotics to lay the foundation, while also providing theoretical foundation for discovery Chinese drugs.

Detailed description of the invention

Fig. 1 is experimental design process figure of the present invention.

Fig. 2 is the colitis model Establishing process figure of DSS induction

Macroscopical situation of Fig. 3 acute colitis mouse model.(A) weight of experiment mice changes situation；(B) colon is long Degree variation；(C) model group and normal group mouse Colon situation；(D) disease activity index.*P<0.05, **P<0.01,***P< 0.001

Fig. 4 acute colitis mouse Colon changes in microstructure.(A) mouse Colon tissue HE colored graph is normally organized；(B) Model group mouse Colon tissue HE colored graph；(C) Histological injury's assessment result.**P<0.01

Fig. 5 is that different pharmaceutical intervenes chmice acute colitis schematic diagram experimental period.

Change of Fig. 6 different pharmaceutical to acute mouse colitis physiological status.(A) model phase each experimental mice weight Knots modification；(B) disease pathology index；(C) colon lengths；(D) myeloperoxidase enzyme activity；(E) pathology index；(F) colonopathy Reason slice (under 100 times of mirrors).* P < 0.001 P < 0.05, * * P < 0.01, * * *.

Under Fig. 7 pharmaceutical intervention, the expression of relevant cell factor is balanced in colitis mice enteron aisle with Th17/Treg. (A) IL-22；(B)IL-6；(C)IL-17A；(D)IL-17F；(E)TNF-α；(F)TGF-β；(G)IL-10.* P < 0.05, * * P < 0.01, * P < 0.001 * *.

Fig. 8 drug is to acute colitis mouse intestinal flora richness and multifarious adjusting.(A-C) each experimental group exists 1st day, the 21st day and the 30th day sample dilutes linearity curve figure；(D-F) each experimental group was at the 1st day, the 21st day and the 30th day Shannon index map.

Fig. 9 is the species composition of colitis mice intestinal flora under three kinds of pharmaceutical interventions.(A-B) prevention phase and knot are tested The Venn of colitis model phase schemes；(C) the Hierarchical clustering analysis histogram that group forms between sample.

Adjusting of the tri- kinds of pharmaceutical interventions of Figure 10 to acute colitis mouse intestinal flora structure.(A-B) prevent phase and colon The principal component analysis figure of scorching model phase.

The composition analysis of acute colitis mouse intestinal flora under Figure 11 pharmaceutical intervention.(A) Men Shuiping histogram (B) is thick The ratio of wall bacterium door and Bacteroidetes；(C) the model phase belongs to horizontal Heatmap figure；(D) belong to horizontal histogram.* P < 0.05, * * P < 0.01, * P < 0.001 * *.

The specific Pseudomonas that Figure 12 acute colitis mouse plays response to three kinds of pharmaceutical interventions is analyzed.(A) prevent phase and model Phase sample clustering chadogram；(B) prevent the LDA histogram distribution of phase and model phase based on microorganism significant difference between group.

Under Figure 13 different pharmaceutical is intervened, the variation of mouse models of colitis excrement SCFAs content.(A) acetic acid；(B) third Acid；(C) isobutyric acid；(D) n-butyric acie；(E) isovaleric acid；(F) positive valeric acid.*P<0.05；**P<0.01；***P< 0.001.

Under tri- kinds of pharmaceutical interventions of Figure 14 in acute colitis mouse intestinal to the variation that produces the related Pseudomonas abundance of SCFAs. (A) SCFAs is promoted to generate the abundance of related Pseudomonas；(B) SCFAs is inhibited to generate related Pseudomonas abundance.

The full scanning of the mass spectrum figure of Figure 15 Radix Codonopsis saponin(e.

Specific embodiment

Technical solution of the present invention is described in further detail with reference to the accompanying drawings and detailed description:

Experimental example 1:

As shown in Figure 1, establishing suitable animal model is to probe into disease incidence mechanism and the clinical Drugs in Therapy effect of assessment Key point.For IBD, establishing mouse colitis model is to probe into the most common method of its pathomechanism.Common knot Colitis model has: spontaneous, gene modification and induction Colitis Model etc..Wherein, chemical substance glucan sulphur is taken The acute mouse colitis model that sour sodium (DSS) stimulation generates is most widely used at present.So this chapter has probed into DSS modeling Experimental period, prepare for later experiments.Material

Experimental animal: the cleaning grade C57BL/6 female mice purchased from Hunan SJA Laboratory Animal Co. , Ltd (18±2g；4 week old)；

Instrument: NC-MS electronic balance, HMS-350 turbula shaker, DH-500 constant incubator, HS-202 paraffin section Machine；

Reagent: DSS (molecular weight: 36-50kDa) is purchased from Sigma-Aldrich；Before modeling, according to document The method of report is configured to 3% DSS solution using sterile purified water.Dimethylbenzene, dehydrated alcohol, formaldehyde, hematoxylin, Phenol red dye liquor reaches section's biology Co., Ltd purchased from Shenzhen.

Experimental method

The construction method of the acute colitis model of DSS induction

Cleaning grade female C57BL/6 mouse 16, assess the health status of every animal；At 20 ± 4 DEG C, 12h is provided Illumination/dark cycle alternating, the environment that relative humidity is 35-55%, then free adaptive feeding 7 days is randomly divided into pair According to group and model group, every group each 8；All experiment mices give conventional feed, the mouse of model group by 5g/ pcs/day of amount It allows mouse freely to drink by 6ml/ pcs/day of amount to move back within 3%DSS solution continuous 7 days except (molten every the DSS that 2 days more renew Liquid), it is changed to distilled water and continues to put to death after drinking 2 days in the 10th day.The mouse of control group is freely drunk distilled water 9 days daily It is put to death afterwards with model group mouse together cervical dislocation；Lower distal colon is taken to prepare tissue pathological slice.In addition, whole experiment process In, observe two groups of experiment mice physiological properties and metamorphosis.Weight, daily diet and the model group of record test mice daily The hematochezia situation of mouse.Detailed process is shown in Fig. 2.

The evaluation and observation of colitis model

The assessment of disease activity index (DAI): the daily weight of record mouse observes its feature of defecating, living in conjunction with disease Dynamic degree index score standard obtains the DAI of every mouse, evaluates its disease event.

The assessment of Histological injury: it is carried out according to the colon general form of experiment mice after dissection and its ulcer level Assessment.

Colonic tissue pathological section is made, under an electron microscope, is commented using double-blind study according to institutional framework damage index Minute mark standard assesses colon sample slice.

Data statistics and analysis

Statistical analysis is carried out using 22 software of SPSS, and the data in figure are analyzed to be shown in the form of mean value ± standard error Show.Difference between each group is compared using one-way analysis of variance (ANOVA), is assessed using the test of double tails.

Experimental result

The overall evaluation of acute colitis model

Find during the acute colitis model for establishing DSS induction using C57BL/6 female mice: model group is small Mouse (the 11st day) when continuously drinking DSS aqueous solution 4 days of 3%, weight does not occur significantly to change, and part is small There are also the trend risen for the weight of mouse, but the mouse criminal of having begun is lazy, does not like；Rough coat, the symptoms such as do not feel like eating.With Control group is compared, experimental mice weight loss since the 5th day (the 12nd day), starts within the 6th day (the 13rd day) can see that tight Weight bloody stool, and mouse weight drastically reduces, and drinks DSS aqueous solution and food-intake is greatly reduced.It will at (the 15th day) the 8th day After DSS aqueous solution removes, the weight loss of experimental mice is to minimum.And compared with the control group, difference is extremely significant (Fig. 3 A). Do not occur the case where dead mouse in whole experiment process.

Experiment terminates, and cervical dislocation is put to death mouse and dissected, when obtaining colonic tissue and measuring colon lengths using ruler It was found that: the colonic pathological change of model group mouse is concentrated mainly on lower distal colon, while entire enteric cavity hyperemia expansion, no shaped granule Shape excrement exists, intestinal wall crisp fritter and shrinkage-void (Fig. 3 C).In addition, comparing with normal group, model mice colon lengths obviously contract Short (P < 0.05) (Fig. 3 B).According to mouse weight variation, stool and situation of occulting blood using the assessment discovery of DAI standards of grading: Otherness between the DAI disease index of model group experiment mice and normal group is extremely significant (P < 0.001).

The variation of colon histopathologic slide structure

After choosing lower distal colon 0.6cm production experiment mice colon histopathologic slide, it is observed that DSS induction It is that colon epithelial cell largely lacks that acute colitis pathological characteristics, which mainly show, and colon crypt and goblet cell are damaged tight Weight, and can not identify.Meanwhile occurring a large amount of inflammatory cytokine in entire colonic mucosa, mucous membrane is arrived in part even infiltration Lower layer (Fig. 4 B).It is assessed, is further demonstrated using damaged degree of the histological damage score standard to colonic tissue State result (Fig. 4 C).

It tests brief summary and discusses

This laboratory has used C57BL/6 female mice to be successfully established the acute colitis model that DSS is induced；Therefore this hair Bright selection C57BL/6 female mice.

Exploring suitable DSS modeling concentration and period is successfully to construct the pass of chordapsus model and curative effect of medication research Key.Research is found: when the total intake of DSS is higher than its lowest threshold 30mg/g dosage, will generate inflammatory symptom, also It is to say that small-scale concentration difference will not influence the final result of modeling.The present invention uses 3% DSS dosage modeling, C57BL/6 female mice was begun to decline again in the 5th day body, and the 6th day starts dilute bloody stool occur, and weight sharply declines, food-intake and DSS aqueous solution intake is greatly decreased；Even stop edible.So the administration time and concentration of increase DSS solution have no too greatly Meaning.In addition, with the extension of DSS intake time, colitis disease course can be aggravated, and then may cause dead mouse；Institute In the 7th day removal DSS solution of modeling.Then, it gives distilled water and continues feeding 2 days, observe modeling mouse disease pathology shape The variation of state.We have found that the weight and hematochezia situation of modeling mouse are not substantially change because of the removal of DSS solution, And mouse death rate is zero.Cervical dislocation puts to death mouse within 10th day, observes its colon lengths, and enteric cavity congestive state, weight become Change, assessed jointly in conjunction with colon histopathologic slide, DAI standards of grading and histological damage score standard, finally confirms modeling Obvious lesion occurs for mouse Colon institutional framework；Meet the standard of acute colitis model.Meanwhile further illustrating C57BL/6 Female mice, the DSS induction 3% can establish stable acute colitis model after a week；It is mentioned to carry out pharmaceutical intervention experiment Good research model is supplied.

Effect study of 2 drug of experimental example to colitis mice intestinal flora and Th17/Treg

In recent years, with the proposition of " Chinese medicine multicomponent acts on multiple target spots simultaneously " viewpoint, restore the enterobacteriaceae of disorder The novel research target spot of group, remodeling Th17/Treg balance as treatment IBD, has become research hotspot.Herbal polysaccharide because Its pharmacological action is extensive, and immunological regulation advantage is significant, and intestinal flora can be degraded by number of mechanisms by body benefit With, be in Chinese medicine play drug effect key component.Classical Chinese medicine of the Radix Codonopsis as tonifying middle-Jiao and Qi, representative activity pharmacology Molecule Codonopsis pilosula polysaccharide is not yet clear to IBD intestinal flora and with the influence of Th17/Treg balance relevant cell factor expression.Base In this, the present invention is goed deep into using the mouse colitis of Codonopsis pilosula polysaccharide prevention and treatment DSS induction using 16SrRNA high throughput sequencing technologies Adjusting of the Codonopsis pilosula polysaccharide to colitis mice intestinal flora is explored, detects in colon and balances relevant cell factor with Th17/Treg Expression；Assess the potential prebiotic effect of Codonopsis pilosula polysaccharide and the prospect of the prebiotic product of exploitation.

Experimental material

Experimental animal: SPF grades of C57BL/6 female mice (18 ± 2g；4 week old), it is dynamic up to experiment purchased from Hunan Si Laike scape Object Co., Ltd；Medicinal material: Codonopsis pilosula polysaccharide (56%) and Radix Codonopsis saponin(e (95%) are purchased from Lanzhou Waters biology Co., Ltd；

Instrument: NC-MS electronic balance, HMS-350 turbula shaker, high speed freezing centrifuge (U.S. Thermo Scientific company), UV-6100BS ultraviolet-uisible spectrophotometer, PCR instrument: ABI9700 types；

Reagent: DSS (molecular weight: 36-50kDa)；Phenol red, haematoxylin；Oligofructose is purchased from Mannheim, Germany company；MPO Enzymatic determination kit；Mouse enzyme-linked immunosorbent assay kit.

Experimental method

The assay of Codonopsis pilosula polysaccharide

Content identification is carried out to the Codonopsis pilosula polysaccharide bought using Anthrone-sulfuricacid method.It is specific as follows:

The embodiment of different pharmaceutical group intervention colitis mice

Cleaning grade female C57BL/6 mouse 60 (weight is about 18 ± 2g), at 20 ± 4 DEG C, provide the illumination of 12h/ Dark cycle is alternately and the relative humidity of 35-55% formally starts to test after adaptive feeding 7 days.According to processing method With the difference of administration kind, it is specifically divided into following 5 groups:

A: oligofructose group (Fro), n=12, every morning in the same period, stomach-filling 300mg/kg dosage it is oligomeric Fructose aqueous solution；

B: Codonopsis pilosula polysaccharide group (Cp), n=12, every morning, the Radix Codonopsis of stomach-filling 300mg/kg dosage was more in the same period Sugar aqueous solution (according to the common dose range of the codonopsis pilosula of 2010 editions States Pharmacopoeia specifications: 9-30g, in conjunction with specific experiment and correlation Document takes the maximum dosage 30g of States Pharmacopoeia specifications, referring to the body surface area conversion method of human body and animal, finally determines party The dosage 300mg/kg of gracilis polysaccharide)；

C: Radix Codonopsis saponin(e group (Cs), n=12, every morning is in same period, the Radix Codonopsis soap of stomach-filling 300mg/kg dosage Glycosides aqueous solution (dosing method is same as above)；

D: control group (Con), n=12 do not give DSS solution during experiment, every morning is in same period, stomach-filling Isodose PBS buffer solution；

E: colitis model group (Mod): n=12, every morning, the isodose PBS buffering of stomach-filling was molten in the same period Liquid；

As Fig. 5 shows the experimentation with 3%DSS inducing mouse acute colitis；Entire experiment is divided into the prevention phase (Day1-21) and treatment phase (Day22-30), totally 30 days.In addition to control group, after the prevention phase, since the 22nd day, continuously Give 3% 7 days induction acute colitis of DSS solution；During this period, fresh DSS solution is prepared daily.It removes within 29th day DSS solution, after fasting 12h, it is primary (the 30th day) that all experimental groups give Radix Codonopsis saponin(e solution (300mg/kg)；And 1,21,30 day collection excrement.The morphological feature and activity situation of each group mouse are observed, while recording the daily weight of mouse.

At the end of experiment, mouse is anesthetized with ether and cervical dislocation is used to put to death.Method by plucking eyeball blood sampling Quick Acquisition blood sample, and colon is taken out rapidly, longitudinally slit, PBS is gently cleaned.Meanwhile according to the literature second Standards of grading in the 2.2.3 of chapter carry out visual assessment to disease grade, and colonic tissue is used for MPO enzyme activity and elisa assay. During entire animal experiment study, all experimental animals are all to strictly observe experimental animal in " international animal protection criterion " Love and using standard carry out.In addition, all methods that this experiment uses are entrusted according to Lanzhou University's experimental animal ethics The scheme that member can ratify carries out.

The preparation of colon MPO enzyme activity determination and histotomy

The measurement of colon MPO enzyme activity: the colonic tissue weighing frozen is taken out from -80 DEG C of refrigerators, by tissue weight than being added The PBS solution of 10 times of volumes is fully ground.Under the conditions of 4 DEG C, 1000g/min is centrifuged 5min, aspirates tissue supernatant, and presses Subsequent operation is carried out according to MPO enzyme reagent kit specification.

The measurement of relevant cell factor is balanced in colon with Th17/Treg

The colonic tissue weighing frozen, the PBS solution by tissue weight than 15 times of volumes are added are taken out from -80 DEG C of refrigerators It is fully ground.Under the conditions of 4 DEG C, 8000g/min is centrifuged 15min, collects supernatant.According to commercially available mouse ELISA kit (Dakewe) specification is to pro-inflammatory cytokine: IL-17A, IL-17F, IL-6, IL-22, TNF-α and anti-inflammatory cell The factor: the expression of TGF-β and IL-10 is analyzed.

3.2.5Illumina the sequencing of MiSeq 250 and bioinformatic analysis

Product is sequenced in 250 microarray dataset of Illumina MiSeq according to " sequencing approach " handbook.This class Extraction, PCR amplification, the building in the library corresponding Illumina PE250 and the high pass for the fecal specimens DNA being related in topic measure Program process is completed by Shanghai Ling En Biotechnology Co., Ltd.

Data statistics processing and analysis

Origin 8.0 and R 3.2.4 language mapping software dissect intestinal flora；SPSS 22 is for statistical analysis, data It is drawn in figure in the form of mean value ± standard error (SE).Difference between each group is compared using ANOVA analysis, utilizes double tails Test is assessed.P value is can determine whether less than 0.05 as with otherness.

Experimental result

The assay result of Codonopsis pilosula polysaccharide

Anthrone-sulfuricacid method is used to measure the content of Codonopsis pilosula polysaccharide for 55.82%, with 56% base marked on purchase label This is consistent.

3.3.2 drug improves colitis mice macroscopic view disease condition

Fig. 6 shows effect of the different pharmaceutical intervention to the DSS C57BL/6 mouse colitis physiological status induced.Research It was found that: DSS induction start within the 4th day, the weight knots modification of model group mouse drastically reduces, and it is significant lower than control group (P < 0.01)；And last 2 days and model group that each pharmaceutical intervention group (oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e) induces in DSS It compares, significantly suppresses mitigation (P < 0.001 of mouse weight；Fig. 6 A).Meanwhile being induced through DSS, although assessment colon is scorching The DAI of disease degree scores in the extremely significant increase (P < 0.01) of model group；But compared with model group, each pharmaceutical intervention is significantly improved The degree of inflammation of colitis mice, reduces DAI index (P < 0.05) (Fig. 6 B).In addition, the model group handled through DSS is small Mouse colon lengths significantly shorten (P < 0.01), and each pharmaceutical intervention group (oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e) and model Group is compared, then obviously inhibits shortening (P < 0.05 of experiment mice colon；Fig. 6 C).

MPO enzyme activity is the marker for evaluating inflammatory cell infiltration in tissue.Research is found: after DSS processing, and being compareed Group is compared, and the MPO enzymatic activity in model group sharply increases；But the MPO enzyme activity value of each administration group significantly reduces compared with model group (Fig. 6 D).Section of colon (Fig. 6 F) display of H&E dyeing: control group mice colonic epithelia tissue structural integrity can be observed bright Aobvious goblet cell and crypts structure.After DSS processing, the integrality of model group mouse Colon epithelial structure is caused seriously to be destroyed, Crypts, goblet cell are a large amount of damaged and lack, and inflammatory cell and submucosa show a large amount of inflammatory cell infiltration.Each medicine Object group shows different degrees of protective effect to the integrality of colonic tissue inflammation and crypts structure.In addition, histology is commented Dividing, which will also recognize that, shows the difference (6E) of each experimental group；The histological score of control group is substantially less than model group (P < 0.01)； And three kinds of pharmaceutical interventions have been significantly reduced the histological damage score (P < 0.05) of colon.

The expression of relevant cell factor is balanced in experiment mice enteron aisle with Th17/Treg

According to the specification of mouse ELISA cytokine assay kit, according to returning for the standard solution of each cell factor Return equation, acquires the content of each sample cell factor.As shown in Fig. 7 A-E, compared with the control group, the colitis of DSS induction is small The level of IL-17A in mouse, IL-17F, IL-6, IL-22 and TNF-α dramatically increases (P < 0.01).It is different compared with model group Pharmaceutical intervention prevention and treatment, then significantly suppress IL-17A, IL-17F, IL-6, the expression of IL-22 and TNF-α proinflammatory factor (P < 0.05).As shown in Fig. 7 F-G, TGF-β and IL-10 are as the main anti-inflammatory cytokines for participating in Treg immune function, in DSS Expression in the colitis mice colonic mucosa of induction is significantly lower than control group (P < 0.05).In contrast, each drug is dry The expression of TGF-β and IL-10 are significantly higher than colitis model group (P < 0.05) in the mouse intestinal mucosa organized in advance.

Experiment mice intestinal flora richness and multifarious variation

16SrRNA sequencing is carried out using Illumina PE250, finally obtaining 2716932 altogether from 60 samples can Gene order and 787 OTU.Linearity curve figure (Fig. 8 A-C) comparison discovery is diluted according to the sample of different time nodes: being surveyed The increase of sequence data volume promotes sample OTU quantity to increase, and dilution linearity curve figure is in rising trend, but increased amplitude is more next Smaller, final curves tend to flat；If increasing continuously sequencing depth, also only has a small amount of new OTU and occur.Therefore, explanation Sequencing data amount is reasonable, and dilution linearity curve can reflect the sequencing depth profile of sample.Meanwhile being treated through each pharmaceutical intervention The sequencing depth of experimental mice increased.Wherein, Codonopsis pilosula polysaccharide and oligofructose group are significantly greater than control group and model Group.In terms of the shannon index results of different time points each group mouse (Fig. 8 D-F): either in the prevention phase of pharmaceutical intervention, Or the treatment phase of the colitis mice in DSS induction, the shannon index of each pharmaceutical intervention group is on the rise, wherein Oligofructose and Codonopsis pilosula polysaccharide group obviously rise.

The species composition of experiment mice intestinal flora is analyzed

Have studied what 3 kinds of drugs formed acute colitis mouse intestinal flora using Wei Entu (Venn diagram) It adjusts, OTU number (Fig. 9 A-B) exclusive between more each experimental group sample and shared.In terms of result, after the prevention phase, Exclusive 12 OTUs of oligofructose group, peculiar 23 OTUs of Codonopsis pilosula polysaccharide group, peculiar 13 OTUs of Radix Codonopsis saponin(e；Control group is special There are 32 OTUs.It is each although OTU number common to experimental group increases to 286 after DSS processing causes inflammation to occur OTU quantity specific to pharmaceutical intervention group is reduced, and each medical treatment group and OTU quantity common to model group are bright It is aobvious to increase (oligofructose group 431-444OTUs；Codonopsis pilosula polysaccharide group: 307-333OTUs；Radix Codonopsis saponin(e group: 428-502OTUs). That is, DSS processing changes the species composition in sample.

Hierarchical clustering analysis (bray-curtis algorithm) histogram (Fig. 9 C) that group forms between sample is also shown: no matter It is the model phase of prevention phase or DSS induction, three kinds of pharmaceutical interventions cause influence to the structure of community of sample.It is specific next It says, compared with control group and model group, after the prevention phase, the enteron aisle of oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e group mouse Flora is slightly changed, but itself and control group are still in same branch.And the colitis model phase handled in DSS, with Control group is compared, and apparent change has occurred in the mouse Bacterial community of either colitis model group or each pharmaceutical intervention group Change, and itself and control component are in different branches.In addition, being compared with regard to model group with each pharmaceutical intervention group, it has been found that low Farther out, intestinal flora composition difference is larger, but Radix Codonopsis for the distance of Fructooligosaccharides group and Codonopsis pilosula polysaccharide group apart from colitis model group Saponin(e group is nearest at a distance from model group, so intestinal flora composition is the most similar.

The variation of experiment mice intestinal microflora

Principal component analysis (PCA) based on Euclidean distance, determines prevention phase and colitis model using multivariate statistical method Phase, the similitude of five experimental mice sample groups composition.From the point of view of analysis result in conjunction with PCA (Figure 10 A-B): testing The prevention phase, there is no notable differences between each administration group and control group sample group composition.But after DSS induction, with control group phase Than significant change has occurred in the overall structure of colitis mice intestinal flora.Meanwhile model group and each pharmaceutical intervention treatment group Mouse intestinal flora structural difference is larger；And the holistic community's structure and control group of each pharmaceutical intervention treatment group mouse are more It is close；And in all pharmaceutical intervention groups, oligofructose group and the structure of community of control group are the most similar.

Experiment mice intestinal flora composition analysis

Heatmap figure is used as in intuitive reflection different classifications level, flora composition identity and specificity between sample Important indicator.Hierarchical clustering analysis histogram is usually formed with sample group and structure of community histogram connects common point It analyses in different classifications level, the flora difference between each experimental group sample.

In door level, histogram (Figure 11 A) is formed in conjunction with structure of community, it has been found that each experimental group contains Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobia, Actinobacteria etc..In reality After testing the prevention phase, compared with the control group, three kinds of pharmaceutical interventions significantly increase Bacteroidetes and The abundance (P < 0.05) of Actinobacteria has lowered the abundance (P < 0.05) of Firmicutes.Meanwhile oligomeric fruit is administered Sugar and Codonopsis pilosula polysaccharide significantly increase the ratio (P < 0.05) of Verrucomicrobia in enteron aisle.In the colitis of DSS induction After the model phase, compared with the control group, Bacteroidetes, Verrucomicrobia in colitis model group mouse intestinal, The growth of Proteobacteria and Actinobacteria is suppressed significantly (P < 0.01), and Firmicutes, The abundance of Tenericutes, Deferribacteres and Spirochaetae dramatically increase (P < 0.01).In addition, Radix Codonopsis is more The sugared growth (P < 0.01) that Spirochaetae is inhibited with oligofructose, and Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e selective depression The growth of Tenericutes (P < 0.05).By comparing the abundance ratio of Firmicutes and Bacteroidetes in each experimental group Firmicutes/Bacteroidetes (F/B) (Figure 11 B), it has been found that the ratio of F/B is significantly higher than control group in model group (P < 0.01), hence it is evident that be higher than oligofructose group and Codonopsis pilosula polysaccharide group (P < 0.05).

It is horizontal belonging to, as shown in Figure 11 C-D, after the prevention phase, compared with the control group, in all pharmaceutical intervention groups The abundance of Odoribacter, Lachnospiraceae NK4A136group and Rikenellaceae RC9 gut group are equal It is obviously suppressed (p < 0.05), and oligofructose and Codonopsis pilosula polysaccharide have selectively promoted Akkermansia, Lactobacillus, Allobaculum, PrevotellaceaeNK3B31 group, Bifidobacterium and The growth (P < 0.01) of Ruminococcaceae UCG-014 strains Pseudomonas.In addition, compared with the control group, Codonopsis pilosula polysaccharide The selective depression growth (P < 0.05) of Erysipelotrichaceae_uncultured Pseudomonas.In the model of DSS induction Phase, Alistipes, Lachnospiraceae NK4A136 group in colitis model group mouse intestinal, Bacteroides, Desulfovibrio, Helicobacter, Parasutterella, Paraprevotella, Parabacteroides and Odoribacter Pseudomonas abundance dramatically increases (P < 0.01), becomes in model group mouse intestinal Predominant bacteria.However, different pharmaceutical interventions is treated, the Desulfovibrio in each administration group mouse intestinal, The abundance of Alistipes, Helicobacter, Parasutterella and Odoribacter Pseudomonas is significantly lower than model group (P <0.05).In addition, Radix Codonopsis saponin(e has selectively promoted Alloprevotella in enteron aisle compared with model group, The growth (P < 0.05) of 2 Pseudomonas of Bacteroides, Roseburia and Treponema；And oligofructose and Codonopsis pilosula polysaccharide choosing Selecting property increases Bifidobacterium, Lactobacillus, the abundance of Turicibacter and Quinella Pseudomonas (P < 0.05).It is worth noting that: the Bacteroides abundance in Codonopsis pilosula polysaccharide group and oligofructose group is but significantly lower than model group (P<0.05)。

Each experimental mice plays the specific Pseudomonas of response to pharmaceutical intervention

LEfSe analysis takes different grouping mode to be clustered according to taxology group paired samples, and is differentiated using line style It analyzes (LDA), finds out the Pseudomonas between sample with significant difference.The sample clustering tree drawn according to biostatistics And LDA score histogram can intuitively show between the Pseudomonas and group and group that play main function in each group sample exist The Pseudomonas (Figure 12 A and B) of significant difference.At the end of testing the prevention phase, it has been found that: main make is played in oligofructose group Having discrepant with and with other experimental groups is to be under the jurisdiction of Saccharibacteria, Proteobactreia respectively, 18 plants of bacterium of 4 doors of Firmicutes and Actinobacteria, play in Codonopsis pilosula polysaccharide group main function and with other groups That with significant difference is the Anaeroplasma for belonging to two doors of Tenericutes and Firmicutes, Anaeroplasmatales, Anaeroplasmataceae, Acetitomaculum and ClostridialesvadinBB60 5 plants of bacterium of group；Radix Codonopsis saponin(e group is the Olsenella bacterium for belonging to Actinobacteria；Control group is to belong to The Eisenbergiella of Firmicutes, Lachnospiraceae NK4A136 group and Roseburia；Model group is then It is 9 plants of bacterium for belonging to Firmicutes and Proteobactreia.We are it is also seen that administration oligofructose as a result, The species diversity that key effect is played in mouse intestinal is increased with Codonopsis pilosula polysaccharide.In the colitis model phase knot of DSS induction Shu Hou, it has been found that play key effect in oligofructose group is Saccharibacteria, Tenericutes and Firmicutes 13 plants of bacterium, wherein there is 7 plants of bacterium not play a crucial role in the prevention phase, and play key effect originally 12 plants of dominant bacterias also disappear.Belong to four plants of bacterium of Proteobacteria and Firmicutes in Codonopsis pilosula polysaccharide group: Bilophila, LachnospiraceaeNK4A136group, Allobaculum and FamilyXIIIUCG_002 play crucial make With；And it is entirely different with the dominant bacteria of prevention phase.Dominant bacteria in Radix Codonopsis saponin(e group is to belong to Firmicutes, 18 plants of bacterium of Deferribacteres and Bacteroidetes；Compared with the prevention phase, newly there are 18 plants of dominant bacterias, and former The superiority bacteria spp come has not been its Pseudomonas for playing key effect.There are 14 plants to belong to Actinobacteria in control group, Firmicutes and Bacteroidetes, and only had found in Mod group and belong to Firmicutes's Ruminococcaceae UCG_005.In general, the acute colitis mouse intestinal flora diversity of DSS induction obviously drops It is low.

Trifle and discussion

Mammalian gastrointestinal tract is the home of fine and close microbiologic population, this group is referred to as intestinal flora.Intestinal flora It is coexisted in a manner of mutual reciprocity and mutual benefit with host, and provides micronutrient for host, increase the digestion and nutrient absorption of food Deng, and secrete the metabolin of aid nutrition absorption and systemic physiological.In healthy individuals, microorganism has diversity, goblet cell By generating thick and solid colonic mucus layer, to form a physical protection barrier, invasion, the enrichment of pathogenic microorganism are prevented Specific mucus is resident microorganism.When IBD occurs, intestinal bacilli illness causes microbial diversity to reduce, and is mainly due to The reduction of prevotella Prevotella species and Actinobacteria in Bacteroidetes, A large amount of growths of Adhesion, Invasion Escherichia coli Escherichia coli and Fusobacteria in Proteobacteria. In addition, lipid-metabolism is also affected after Function of intestinal mucosa changes.Paneth's cell and the reduction of goblet cell quantity and its function Missing causes the secretion of antibacterial material and rete malpighii thickness to reduce, finally causes mucosal integrity and barrier function damage.This Process not only increases bacterial translocation, and stimulates DC and M Φ cell activation to induce and change T cell spectrum, it is made to generate secretion IFN-γ/TNF-α Th1, the Th2 for secreting IL-6/TNF- α and the Th17 lymphocyte for promoting IL-17 to generate, lead to inflammation Reaction and tissue damage, are further degrading imbalance and the chronic inflammatory reaction of intestinal flora.So extensive in several ways Multiple IBD intestinal bacilli illness, increasing microbial diversity is to alleviate the key point for even curing IBD.In addition, it is ground by flora " multicomponent acts on multiple target spots " for studying carefully initiation has become for research hotspot, this is also prebiotics, probiotics and conjunction at this stage The welcome reason of raw member.

Radix Codonopsis pharmacological activity is extensive, as the classical Chinese medicine of clinical tonifying middle-Jiao and Qi, even can be used as replacing for ginseng sometimes Dai Pin, main active include polysaccharide, triterpene compound, Phenylpropanoid Glycosides class, alkaloids and polyacetylene class etc..According to Document is recorded, and so far, has isolated the water-soluble polysaccharide and divide that molecular weight is more than 10kDa from Radix Codonopsis root respectively The pectin polysaccharide that son amount is 14.5kDa.These discovery illustrate in codonopsis pilosula bioactive polysaccharide molecular weight distribution covering compared with It is wide.Codonopsis pilosula polysaccharide can not only inhibit the activity of human gastric adenocarcinoma and liver cancer cells, and effectively be dropped in a manner of dose-dependent Blood glucose and insulin level in low empty stomach mice serum reduce malonaldehyde (MDA) content.Meanwhile research is found: water-soluble Codonopsis pilosula polysaccharide can improve the gastric mucosa damage of gastric ulcer model mouse；And in vitro in immunological investigation, with dose-dependent The lymphopoiesis that mode stimulates concanavalin A or lipopolysaccharides (LPS) to induce.In addition, Polysaccharide from Portulaca oleracea, Radix Astragali, dragon Eye polysaccharide etc. has had proven to flora adjustment effect, and Sijunzi Tang is being probed into colitis intestinal flora by laboratory seminar Effect when have been surprisingly found that: primary water-soluble polysaccharide-Codonopsis pilosula polysaccharide (35%) in Sijunzi Tang can increase colitis mice Beneficial to the ratio of Pseudomonas in enteron aisle, inhibits the growth of harmful Pseudomonas, alleviate colitis to a certain extent.But it is most main due to testing The purpose wanted not is the interactively probed between Codonopsis pilosula polysaccharide and intestinal flora, and tests the Codonopsis pilosula polysaccharide purity selected It is relatively low, so and not yet explicitly specific influence of the Codonopsis pilosula polysaccharide on intestinal flora.Therefore, during this investigation it turned out, we establish The acute colitis model of DSS induction simulates enteric microorganism ecological disturbance, using the Codonopsis pilosula polysaccharide of high-purity probes into it To colitis mice intestinal microflora and multifarious adjustment effect, the prebiotic mechanism of Codonopsis pilosula polysaccharide is further clarified.

The study found that although different pharmaceutical intervention prevention and treatment can improve the colitis model mice of DSS induction in various degree The damage of colonic mucosa reduces disease activity index, inhibits to balance associated inflammatory cytokine with Th17/Treg in colon The expression of TNF-α, IL-17A, IL-17F, IL-6, IL-22 increases the quantity of anti-inflammatory cytokines IL-10 and TGF-β, finally Alleviate colitis reaction；But the function and effect of Codonopsis pilosula polysaccharide are substantially better than Radix Codonopsis saponin(e, and consistent with oligofructose group. This may be attributed to the fact that the extensive immunopharmacological activity of Codonopsis pilosula polysaccharide and anti-inflammatory effect.This phenomenon also further demonstrates reality early period Test the conclusion of seminar: Codonopsis pilosula polysaccharide has the function of alleviating colitis.

It is analyzed using the flora of different classifications level, searches acute colitis mouse intestinal bacterium after three kinds of pharmaceutical interventions Group structure and multifarious specific difference.In door level, research is found: after the prevention phase, three kinds of pharmaceutical interventions are increased The abundance of Bacteroidetes and Actinobacteria；And oligofructose and Codonopsis pilosula polysaccharide significantly increase The ratio of Verrucomicrobia.Document report: Bacteroidetes encodes 3976 carbohydrate activity enzymes It (CAZymes), is that macromolecular substances metabolism is essential.Although many in Actinobacteria will lead to the mankind Disease, but part Pseudomonas especially Streptomyces spp can be metabolized some drugs and generate many biologically active metabolites productions Object, and then antibacterial is played, it is antiviral, the effects of immunological regulation.Certain Pseudomonas in Verrucomicrobia can be metabolized sulphur And mucoprotein of degrading.The abundance of Proteobacteria significantly rises after the model phase of DSS induction, three kinds of pharmaceutical intervention treatments It is high；Meanwhile Codonopsis pilosula polysaccharide and oligofructose inhibit the growth of Spirochaetae.Document is recorded: in Proteobacteria Many Pseudomonas can induce IgA secretion so that adjust enteron aisle homeostasis, and in Spirochaetae most of Pseudomonas tool Have pathogenic, perhaps can aggravate the pathogenesis of colitis.The ratio of F/B is not only closely related with obesity, but also is assessment The key index of IBD inflammatory reaction.We have found that the ratio of F/B is significant compared with the control group in acute colitis mouse intestinal Increase, this is consistent with document report.In addition, the ratio of Codonopsis pilosula polysaccharide and oligofructose group F/B are relatively small, show Radix Codonopsis Polysaccharide and oligofructose can alleviate colitis by adjusting the ratio of Firmicutes and Bacteroidetes in enteron aisle.

It is horizontal belonging to, Codonopsis pilosula polysaccharide have stimulated simultaneously two kinds of important prebiotic Pseudomonas Bifidobacterium spp with The growth of Lactobacillus spp, it is suppressed that harmful Pseudomonas Desulfovibio spp., Alistipes spp and The abundance of Helicobacter spp has restored the disorder of colitis mouse intestinal flora；Its flora tune to colitis mice Section effect is substantially better than Radix Codonopsis saponin(e group, and simultaneously there was no significant difference between putative oligofructose, so having good Good prebiotic effect.In addition, Codonopsis pilosula polysaccharide and oligofructose can promote Akkermansia spp, Quinella, The growth of Allobaculum, Turicibacter inhibit Parasutterella and Paraprevotella abundance.Research report Road Akkermansia spp can induce Treg (Foxp3+T cell) cell and mitigate type II diabetes symptom, adjust enteron aisle mucus Thickness maintains intestinal barrier integrity；And daily intake oligofructose, short-chain carbohydrates etc. can increase in enteron aisle The quantity of Akkermansia spp；Parasutterella and Paraprevotella suffers from IBD patient and colon cancer respectively It is significantly increased in person's body；And Quinella, Turicibacter and Allobaculum are then related with fermentation generation SCFAs.

It is excellent in the colitis mice of DSS induction that LEfSe analysis further illustrates that three kinds of pharmaceutical interventions increase in various degree The diversity of gesture bacterium.In addition, we can see that certain Pseudomonas in Firmicutes belong to crucial bacterium in each experimental group Belong to, it may be related with the colitis that DSS is induced.Deferribacteres and Bacteroidetes in Radix Codonopsis saponin(e group The reason of door category dominant bacteria door may be that some of which Pseudomonas is caused by participating in the important Pseudomonas of metabolism.In short, no matter It is in the prevention phase or in colitis treatment phase, Codonopsis pilosula polysaccharide can be divided into two to the relaxation effect of chmice acute colitis Aspect: on the one hand, Codonopsis pilosula polysaccharide is after oral administration reaches enteron aisle, by thousands of kinds of carbon hydrates of enteric microorganism group coding The degradation of object organized enzyme is broken down into small-molecule substance by the fermentation of enteric microorganism, and then is absorbed by host and play its medicine Manage active function；On the other hand, Codonopsis pilosula polysaccharide provides nutriment for intestinal flora, while in auxiliary intestinal flora and disease When opportunistic pathogen competes limited space, by enhancing the immune response of body, promotes the growth of prebiotic Pseudomonas in enteron aisle, inhibit disease The field planting of opportunistic pathogen, and then restored enteron aisle homeostasis.

Influence of 3 drug of experimental example to colitis mice SCFAs content and related Pseudomonas

Source of the SCFAs colon cell energy, can be by improving intestinal permeability and interior environment, adjusting body Energy homeostasis and immunocyte chemotaxis play anti-inflammatory and antibacterial action.Acute colitis is acted in order to probe into Codonopsis pilosula polysaccharide Afterwards, if will affect the variation that SCFAs content in SCFAs Pseudomonas and excrement is produced in colitis mice enteron aisle.The present invention passes through gas Phase chromatography (GC) has detected the SCFAs content in the acute colitis stool in mice of DSS induction, and 16SrRNA is combined to be sequenced The variation of Pseudomonas related to SCFAs is produced in interpretation of result colitis mouse intestinal.

Experimental material

Instrument: Agilent 7890B gas chromatograph (including fid detector)；NC-MS electronic balance, the whirlpool HMS-350 Revolve oscillator, high speed freezing centrifuge (Thermo Scientific company, the U.S.)；

Drug and reagent: acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, the 2 Ethylbutanoic acid reference substance (U.S. Sigma company), metaphosphoric acid, PBS solution.

Experimental method

Chromatographic condition

Efficient capillary chromatographic column: DB-FFAP (30m*0.25mmid, 0.25um), Max Temperature:250 DEG C, Carrier gas be high-purity nitrogen (99.999%), flow velocity 0.8ml/min, fid detector temperature: 230 DEG C, injector temperature 250 DEG C, split ratio 30:1,60 DEG C of initial injector temperature keeps 1min, is warming up to 220 DEG C with 20 DEG C/min rate, keeps 3min.

The preparation of reference substance solution

It compares the preparation of mixed solution: accurately weighing acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid and appropriate valeric acid, add Enter in 10ml volumetric flask, is settled to graduation mark with the metaphosphoric acid solution containing internal standard 2 Ethylbutanoic acid, is configured to each constituent concentration Be the stock solution of 200mM, be stored in -4 DEG C it is spare.

The preparation of internal standard stock solution

Precision weighs 25g metaphosphoric acid and the 2 Ethylbutanoic acid of 0.217ml is added in the volumetric flask of 100ml, fixed with distilled water Hold to graduation mark, be configured to the metaphosphoric acid deproteinized solution of 25% containing internal standard 2 Ethylbutanoic acid, be stored in -4 DEG C it is spare.

The drafting of standard curve

Mixed reference substance solution 1.5ml is taken to be placed in Agilent GC sample injection bottle, respectively with 0.1ul, 0.2ul, 0.4ul, The sample volume (controlled concentration) of 0.6ul, 0.8ul, 1ul difference gradient carries out sample analysis, with the SCFAs and internal standard detected Peak area ratio (Y) carries out linear regression to concentration (X) and obtains regression equation.The processing method of stool in mice sample

The different times being collected into, the different other stool in mice samples of group are taken out from -80 DEG C of refrigerators, every 0.1g is suspended in In the PBS solution of 0.5ml, whirlpool is mixed.Under the conditions of 4 DEG C, 3000g/min is centrifuged 10min, and supernatant 1ml is taken to be added to In 1.5ml centrifuge tube, the deproteinized solution of 0.2ml is added, is mixed；After ice-water bath stands 30min, in 4 DEG C in 10000g/ Min is centrifuged 12min, takes supernatant to be placed in new centrifuge tube, 4 DEG C stored refrigerated spare.

The analysis of SCFAs correlation Pseudomonas and mathematical statistics

According to chapter 3 16S rRNA sequencing data, in conjunction with the measurement result of SCFAs content in fecal sample；Using Origin 8.0 and the analysis DSS induction of R 3.2.4 language mapping software and pharmaceutical intervention is anti-controls the inferior, in enteron aisle with produce SCFAs phase The Pseudomonas of pass changes.For statistical analysis using SPSS 22, data are drawn in the form of mean value ± standard error in figure. ANOVA analysis carries out group difference comparison, and is assessed using double tail tests.P value is can determine whether less than 0.05 as with difference Property.

Experimental result

The variation of SCFAs content in stool in mice sample

Using GC chromatograph analyzed and researched DSS induction colitis mice excrement in SCFAs content variation.Such as figure Shown in 13, compared with the control group, acetic acid in the colitis model group stool in mice of DSS induction, propionic acid, butyric acid, isobutyric acid and different The content of valeric acid significantly reduces (p < 0.05).Compared with model group, three kinds of pharmaceutical interventions significantly increase acetic acid, propionic acid, positive fourth The content (p < 0.05) of acid, isobutyric acid and isovaleric acid, the wherein acetic acid in Radix Codonopsis saponin(e group stool in mice, propionic acid and butyric acid contain It measures extremely significant higher than model group (p < 0.01).It should be noted, however, that either model group or each pharmaceutical intervention are treated Group, there were significant differences between the positive valeric acid content and control group in fecal sample.

The variation of SCFAs correlation Pseudomonas in enteron aisle

This trifle combines the measurement result and chapter 3 16S rRNA sequencing data of SCFAs content in excrement, compares intestines The variation (Figure 14 A-B) of Pseudomonas content related to SCFAs generation in road.As shown in Figure 14 A, in experiment prevention phase, Codonopsis pilosula polysaccharide The total abundance of related Pseudomonas for promoting SCFAs to generate in group is higher than other each experimental groups (p < 0.01).After DSS induction, each drug The total abundance of Pseudomonas for promoting SCFAs to generate in intervention group and specific Pseudomonas Blautia, Oscillibacter and Quinella Abundance be all remarkably higher than model group (p < 0.05).In addition, it is worth noting that, it is generated with SCFAs in just in Radix Codonopsis saponin(e group Relevant Pseudomonas totality abundance is not influenced by DSS processing, is always maintained at the trend of sustainable growth, and alternative rush Into the growth (p < 0.01) of Prevotellaceae UCG-001 Pseudomonas；And in oligofructose and Codonopsis pilosula polysaccharide intervention group The abundance of Prevotellaceae NK3B31 and Prevotellaceae UCG-001 Pseudomonas is influenced significant by DSS processing It reduces (p < 0.05).As shown in Figure 14B, press down in each pharmaceutical intervention group mouse intestinal compared with the control group in the experiment prevention phase Total abundance that SCFAs processed generates related Pseudomonas significantly reduces (p < 0.05).After DSS is induced, specific Pseudomonas Ruminococcaceae_uncultured, Coprococcus 1, Odoribacter and Clostridiales The abundance of vadinBB60group_norank is all remarkably higher than control group (P < 0.01).Fortunately, in oligofructose, The total abundance of negatively correlated Pseudomonas and specified germ are generated with SCFAs in Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e different pharmaceutical intervention group Belong to Coprococcus 1 and ClostridialesvadinBB60 group_norank abundance be substantially less than model group (p < 0.05).In addition, 1 Pseudomonas abundance of Clostridium sensu stricto in Radix Codonopsis saponin(e group lower than model group (p < 0.01)。

It tests brief summary and discusses

SCFAs be host from the non-digest carbohydrate of external world's intake in colon with commensal gut bacterium fermentation The product of generation mainly includes the acetic acid for not waiting carbon atoms to form by 1-6, propionic acid, butyric acid, isobutyric acid, isovaleric acid and penta Acid.SCFAs, not only can be with as the signal of interest molecule of monitoring enteron aisle homeostasis variation and the energy source of colon cell Body circulation is absorbed by enteric epithelium and plays systemic effect, and the energy balance of adjustable host, improvement enteron aisle are penetrating Property and homeostasis, adjust energy homeostasis, metabolism and immunocyte chemotaxis, while also having anti-inflammatory, anticancer and antibacterial living Property.

So the content of SCFAs plays therapeutic effect to drug to further investigation flora imbalance in measurement stool in mice It influences, the interactively inquired between intestinal flora and SCFAs has certain directive significance.Research report, 1 Pseudomonas of Coprococcus can inhibit the generation of propionic acid, and the generation of Odoribacter and acetic acid, propionic acid are negatively correlated, and Clostridiales vadinBB60group_norank Pseudomonas inhibits the content of propionic acid and butyric acid.In the present invention, I Find that administration Codonopsis pilosula polysaccharide not only increases the generation of colitis mice enteron aisle SCFAs, and pass through selective enrichment document It is reported have promote SCFAs generate Blautia, Prevotellaceae UCG-001, Oscillibacter and Quinella Pseudomonas inhibits Coprococcus 1, Odoribacter and Clostridiales vadinBB60 group_ The growth of certain commensal gut bacterium such as norank improves the system and local energy supply situation of SCFAs, Quinella hair Ferment carbohydrate generates acetic acid and lactic acid；This experimental result is consistent with document report.In addition to this, research report, enterobacteriaceae The generation that group adjusts SCFAs also relates to the main mechanism of SCFAs signal transduction: the inhibition of HDAC and the signal of GPCR pass It leads.In the present invention, it has been found that the expression that Codonopsis pilosula polysaccharide passes through and inhibits proinflammatory cytokine, promotes anti-inflammatory cytokines, Connecing reduces colonic epithelium mucosa injury, and has restored the balance of Th17/Treg in colitis Mice Body, this may be due to Polysaccharide affects result caused by the intestinal flora for participating in SCFAs metabolism.

4 Codonopsis pilosula polysaccharide of embodiment, which adjusts intestinal flora, influences the pharmacokinetic of Radix Codonopsis saponin(e

Complexity between Chinese medicine and intestinal flora, which contacts, allows enteric bacteria to mediate the interaction between traditional Chinese medicine ingredients. In order to probe into active pharmacological molecules Codonopsis pilosula polysaccharide and the Coupling effects of Radix Codonopsis saponin(e in the course of disease treatment in codonopsis pilosula. The present invention has probed into the effect that Codonopsis pilosula polysaccharide adjusts intestinal flora using ultra high efficiency liquid phase-mass spectrometer (UPLC-TOF-MS) Under, absorbing state of the Radix Codonopsis saponin(e in acute colitis mouse model serum.

Experimental material

Instrument: 1260 UPLC-6460 Triple Quad LC-MS combined instrument of Agilent, NC-MS electronic balance, HMS-350 vortex concussion instrument, (U.S. Thermo Scientific is public for HMS-350 turbula shaker, high speed freezing centrifuge Department)；

Reagent: hplc grade methanol, Watson ultrapure water, chromatographic grade acetonitrile, chromatographic grade formic acid is purchased from Dakewe biology has Limit company (China Shenzhen), PBS

Experimental method

Chromatographic condition

Liquid-phase condition: chromatographic column is Agilentporoshell 120EC-C₁₈(2.1mm*100mm, 2.7um), flowing Phase-stationary phase: 0.1% formic acid solution (A)-acetonitrile (B)；Sample volume is 2ul, 30 DEG C of column temperature.

Mass Spectrometry Conditions: ion source is electron spray (ESI) ion source, and negative ions ionize mode；Dry gas (N₂), temperature It is 350 DEG C, atomization gas (N₂) pressure: 15psi, flow 12L/min；Electron spray voltage 3000V, scanning mode are more reaction prisons (MRM) mode of survey；Quota ion m/z:415.2.

2 ultra high efficiency liquid phase of table-mass spectrometry gradient elution

The processing of blood sample

After the mouse fasting 12h prevented and treated through different pharmaceutical, it is primary that Radix Codonopsis saponin(e (300mg/kg) is given in stomach-filling.It is giving Medicine 1h, 3h, 6h, 9h, 12h, for 24 hours after, pluck eyeball and collect each group blood sample, and under the conditions of 4 DEG C, 10000g/min centrifugation 15min collects serum and is stored in -20 DEG C until analysis.

Methanol 300ul is added in accurate measuring mice serum 100ul, is vortexed and mixes.Under the conditions of 4 DEG C, 8000 g/min centrifugation 12min takes in sample solution injection UPLC-TOF-MS and is analyzed, calculates the highest saponin(e of exposed amount in sample with external standard method The relative mass concentration of monomeric compound.

The preparation of serum standards curve

It takes mouse blank serum to be homogenized 100ul, the 1ng/ml Radix Codonopsis saponin(e aqueous solution through methanol serial dilution is added 300ul is vortexed and mixes.Under the conditions of 4 DEG C, 8000g/min is centrifuged 12min, takes supernatant in sample injection bottle, respectively with 0.01, The sample volume injection UPLC-TOF-MS of 0.03,0.05,0.1,0.2,0.4,0.8ul (concentration control) difference gradient is divided Analysis records sample peak area.Make linear regression using sample quality concentration (X) and peak area (Y), obtains the maximum soap of exposed amount The serum standards curvilinear equation of glycosides monomeric compound.Data Analysis Services

It uses 2.0 software of PKsolver (China Medicine University) with the analysis of non-compartment model, calculates Radix Codonopsis saponin(e in urgency Pharmacokinetic parameter in property mouse models of colitis blood and caecum.

Experimental result

The full scanning of the mass spectrum result of Radix Codonopsis saponin(e

Scanned respectively under negative ions ionization mode using MRM mode with ESI ion source: research finds Radix Codonopsis soap Glycosides is under negative ESI mode almost without mass-basis response；And there is great mass-basis response under positive ESI mode, and in [M- H] the saponin monomer molecule of highest light exposure, hereinafter referred to as Cs-415.2 are obtained at-m/z 415.2.Figure 15 shows Radix Codonopsis soap The full scanning of the mass spectrum figure of glycosides.

The full scanning of the mass spectrum of 3 Radix Codonopsis saponin(e of table analyzes parameter

Content distribution of the Radix Codonopsis saponin(e in serum

Make linear regression according to blood serum sample mass concentration (X) and sample peak area (Y), obtains the maximum Radix Codonopsis of exposed amount The calibration curve equation that saponin monomer molecule Cs-415.2 is metabolized in serum is as follows: Y=1948721.689038*X+ 157855.395005；R²=0.993938.

Using UPLC-TQ-MS technology determine colitis mice oral administration Radix Codonopsis saponin(e it is primary after, exposed amount is most Changes of contents of the big saponin monomer molecule Cs-415.2 in inherent serum for 24 hours.We have found that after oral Radix Codonopsis saponin(e, The overall absorption amount of Cs-415.2 saponin monomer molecule is successively for 24 hours in range, in each experimental group serum are as follows: oligofructose group > right According to group > Codonopsis pilosula polysaccharide group > Radix Codonopsis saponin(e group > model group.In addition, Cs-415.2 saponin monomer molecule is in oligofructose group and right According to the maximum absorption in group serum in 6h or so；Maximum absorption in Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e group about in 3h, and It is then 1h or so in model group.PKsolver software is used successfully to have studied Cs-415.2 in serum with non-compartment model Pharmacokinetics.After Radix Codonopsis saponin(e 300mg/kg is administered orally, Cs- in each experimental mice serum 415.2 main pharmacokinetic parameter.Compared with the control group, in model group mice serum Cs-415.2 pharmacokinetics Parameter is substantially change.Specifically, the AUC of the Cs-415.2 in model group mice serum_0-infAnd C_maxSubstantially less than pair According to group (P < 0.05).However, after intervening 1 month through oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e, it has been found that with mould Type group is compared, the AUC of Cs-415.2 in Codonopsis pilosula polysaccharide group and Radix Codonopsis saponin(e group serum_0-infAnd C_maxSignificantly improve (p < 0.05).In addition, in oligofructose group serum Cs-415.2 AUC_0-infAnd C_maxIt is extremely significant to be higher than model group (p < 0.01).

After Radix Codonopsis saponin(e is administered in table 4, pharmacokinetic parameter of the maximum monomer molecule Cs-415.2 in serum

There is conspicuousness (P < 0.05) between a-c subscript expression group and group

It tests brief summary and discusses

At this stage, in natural drug active pharmacological molecules research due to a lack of standard items and its in the biological sample Low biological concentration and high variation features and be limited.Therefore, during this investigation it turned out, we select exposure in Radix Codonopsis saponin extract It measures highest monomeric compound to be studied, it is desirable to thus probe into Radix Codonopsis saponin(e in the intracorporal quantitative change rule of colitis mice. Research is found: the molecular formula of the highest monomeric compound of exposed amount is C in Radix Codonopsis saponin extract₂₉H₅₀O, with beans steroid -7- Alkene -3- alcohol (Stigmast-7-en-3-ol) relative molecular weight having the same.

Using " exposing method relatively ", in the case where no standard items, to exposure in experiment mice blood and caeca samples Highest exogenous Radix Codonopsis saponin monomer compound Cs-415.2 is measured, carries out relative quantification using " thinner ratio calibration curve method "； Obtain " the opposite blood concentration " of Radix Codonopsis saponin monomer compound Cs-415.2, not practical blood concentration.Research report, is removed The pharmacokinetic parameter that absolute blood concentration, the method and standard items detection methods obtain has no significant difference.Research is found Colitis mice is after the Radix Codonopsis saponin extract of 300mg/kg dosage is given in stomach-filling, the highest monomeric compound of exposed amount It is best in the absorption of oligofructose group, it is worst in the absorptivity of model group, in Radix Codonopsis saponin(e group, control group and Codonopsis pilosula polysaccharide group Absorptivity it is unanimous on the whole.That is, enhancing Cs- for a period of time through oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e intervention 415.2 absorption in the colitis mice serum of DSS induction has delayed Radix Codonopsis saponin(e to play the time of disease treatment effect. On the one hand, this may be to alleviate colitis mice intestinal flora to a certain extent by oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e Imbalance, promote participate in saponin(e metabolism Related Bacteria Lactobacillus spp, Bacteroides spp and The growth of Bifidobacterium spp, and then improve Radix Codonopsis saponin(e and be degraded to small-molecule substance and be easier to be absorbed by organisms and drawn It rises.It on the other hand is also likely to be oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e by promoting to produce SCFAs's The growth of Akkermansia, Bifidobacterium and Bacteroides Pseudomonas, and then it is small so that SCFAs is improved colitis The breakage of mouse gut barrier finally improves caused by the intestines metabolism of Radix Codonopsis saponin(e.But either which kind of reason, ours grinds It is consistent with document report to study carefully result.Future is by the interaction mechanism being primarily upon between natural drug bioactive molecule and its in machine The structure feature research of interior metabolism product is modernization of Chinese medicine application based theoretical.

Conclusion

1) that gives C57BL/6 female mice 3% can be successfully established stability, repeatability for DSS aqueous solution 7 days well, and Without the acute mouse colitis model that death occurs, it is suitble to the assessment of later period curative effect of medication.

2) oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e can be sick by improving the macroscopic view of the colitis mice of DSS induction Reason situation；The expression for raising anti-inflammatory factors related to Th17/Treg balance, lowers the secretion of proinflammatory factor；Adjust its enterobacteriaceae Group structure and diversity；And then alleviate colitis in various degree.Wherein, the function and effect of Codonopsis pilosula polysaccharide and oligofructose group without Significant difference, but it is better than Radix Codonopsis saponin(e group.

3) content that oligofructose, Codonopsis pilosula polysaccharide and Radix Codonopsis saponin(e increase SCFAs in colitis mice enteron aisle is administered, There is the Blautia, the Prevotellaceae UCG-001 that promote SCFAs to generate by the way that selective enrichment document is reported, Oscillibacter and Quinella Pseudomonas inhibits Coprococcus 1, Odoribacter and Clostridiales The growth of certain commensal gut bacterium such as vadinBB60group_norank improves the system and local energy supply of SCFAs Situation；Wherein Codonopsis pilosula polysaccharide and oligofructose function and effect are optimal.

4) after oligofructose, Codonopsis pilosula polysaccharide and the prevention and treatment of Radix Codonopsis saponin(e, by the mistake for adjusting colitis mice intestinal flora It adjusts, enhances absorption of the highest monomeric compound Cs-415.2 of exposed amount in Radix Codonopsis saponin(e in colitis mice, delay Radix Codonopsis saponin(e plays the time of disease treatment effect.Wherein, oligofructose effect is optimal, and Codonopsis pilosula polysaccharide takes second place.

5) Codonopsis pilosula polysaccharide passes through three kinds of selective stimulating important prebiotic Pseudomonas Bifidobacterium spp, The growth of Akkermansia spp and Lactobacillus spp inhibit harmful Pseudomonas Desulfovibio spp, The abundance of Alistipes spp and Helicobacter spp, have restored the disorder of colitis mouse intestinal flora；It increases SCFAs is promoted to generate the growth of Pseudomonas in enteron aisle；Improve the highest monomeric compound Cs-415.2 of exposed amount in Radix Codonopsis saponin(e Absorptivity, extend its play drug effect action time；With good prebiotics characteristic, in the prevention and treatment of acute colitis Good application prospect is shown in the process.

The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, appoints The change or replacement why not expected by creative work, should be covered by the protection scope of the present invention.Therefore, this hair Bright protection scope should be determined by the scope of protection defined in the claims.

Claims (9)

1. the application that Codonopsis pilosula polysaccharide is used to adjust human body intestinal canal flora, which is characterized in that Codonopsis pilosula polysaccharide is as prevention colitis The application of drug, food or health care product.

2. the application that Codonopsis pilosula polysaccharide is used to adjust human body intestinal canal flora, which is characterized in that Codonopsis pilosula polysaccharide is as treatment colitis The application of drug, food or health care product.

3. the application that Codonopsis pilosula polysaccharide is used to adjust human body intestinal canal flora, which is characterized in that Codonopsis pilosula polysaccharide is by acting on saponin(e generation Thank to adjusting human body intestinal canal flora.

4. application according to claim 3, which is characterized in that Codonopsis pilosula polysaccharide is related to Th17/Treg balance by lowering Proinflammatory cytokine: the secretion of interleukins [IL] -17A, IL-17F, IL-6, IL-22 and tumor necrosis factor [TNF]-α, The expression of anti-inflammatory factors IL-10 and transforming growth factor [TGF]-β are raised, human body intestinal canal flora is adjusted.

5. application according to claim 3, which is characterized in that Codonopsis pilosula polysaccharide passes through three kinds of important probiotics of selective stimulating Belong to Bifidobacterium spp., harmful Pseudomonas is lowered in the growth of Akkermansia spp and Lactobacillus spp The abundance of Desulfovibio spp., Alistipes spp and Helicobacter spp, adjust the disorder of intestinal flora.

6. application according to claim 3, which is characterized in that Codonopsis pilosula polysaccharide increases SCFAs in colitis mice enteron aisle Content, and by selective enrichment have promote SCFAs generate Blautia, Prevotellaceae UCG-001, The growth of Oscillibacter and Quinella Pseudomonas improves the system and local energy supply situation of SCFAs.

7. application according to claim 3, which is characterized in that the Codonopsis pilosula polysaccharide is drug, food or health care product.

8. application according to claim 3, which is characterized in that the Codonopsis pilosula polysaccharide is for oral administration or exterior-applied formulation.

9. application according to claim 8, which is characterized in that the Codonopsis pilosula polysaccharide is tablet, pill, dispersing agent, particle Agent, the prepared slices of Chinese crude drugs, spray or ointment plaster.