CN105920137A - Application of sanguisorba total saponin to treatment of inflammatory bowel diseases - Google Patents
Application of sanguisorba total saponin to treatment of inflammatory bowel diseases Download PDFInfo
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- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/739—Sanguisorba (burnet)
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- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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Abstract
The invention relates to application of sanguisorba total saponin to treatment of inflammatory bowel diseases, relates to a treatment effect of sanguisorba total saponin on inflammatory bowel diseases, belongs to the pharmaceutical field and provides application of sanguisorba total saponin to significant reversal of colon injury caused by TNBS. Experiments prove that sanguisorba saponin can obviously reduce the phenomenon that the weight of rats induced by TNBS and suffered from colitis is reduced and also can obviously reduce the degree of pathological changes of the colitis of the rats induced by TNBS; inflammatory cell infiltration is reduced; the mucosal epithelial degeneration is reduced; TNBS causes severe inflammation response in the rats and also causes increase of expression of proinflammatory factors including TNF-alpha, IL-1beta and IL-6 in serum of the rats; sanguisorba saponin can reverse the phenomenon that the inflammatory factors in the serum of the rats are increased; TNBS simultaneously causes increase of expression of MPO and MDA and reduction of expression of SOD in colon oxygen free radical systems of the rats; therefore, sanguisorba saponin can reverse the phenomenon that the colon oxygen free radical systems of the rats are disordered. The invention further provides influence of sanguisorba saponin on inflammatory factors in LPS-excited RAW 264.7 macrophages; results show that sanguisorba saponin can significantly reduce expression of TNF-alpha, IL-6 and IL-1beta, so that the anti-inflammatory effect is achieved; sanguisorba total saponin can be applied to treatment of inflammatory bowel diseases.
Description
Technical field
The present invention relates to the radix sanguisorbae total saponin therapeutic action to IBD, increase the indication of radix sanguisorbae total saponin, promote ground
The exploitation of elm total saposins, belongs to drug world.
Background technology
Garden burnet is abundant in china natural resources, of a great variety, and has had the most skillful artificial cultivation technique can carry out big face
Long-pending breeding.Containing the multiple chemical composition such as tannin and Polyphenols, triterpene and glycoside thereof, flavones and glycoside thereof in garden burnet, have
Hemostasis, anti-inflammation detumescence, antitumor, the pharmacological action such as anti-oxidant and antiallergy[17-20].Sanguisorbin and aglycon composition thereof with
Triterpene saponin componds is main, and its structure type mainly has Ursane, oleanane type, dammarane type and lupinane
Type, wherein ursane (ursane) type and oleanane (oleanane) type are most commonly seen.Sanguisorbin bioavilability is low,
Permeability is poor, be extremely difficult to valid density in vivo；But, sanguisorbin has clear and definite hemostasis, anti-inflammation detumescence, anti-swollen
Knurl, the pharmacological action such as anti-oxidant and antiallergy.The incoherent phenomenon of this PK Yu PD points out us, and sanguisorbin may
It is to be played a role by a kind of indirect mechanism.
IBD (inflammatory bowel disease, IBD) is a kind of agnogenic chronic nonspecific inflammation
Property enteropathy, IBD at developed country's incidence of disease higher than developing country, but recently as the raising of China's economic level, people
Group's eating habit and the change of habits and customs, IBD has the trend raised year by year at China's incidence of disease.Due to pathogenesis the most not
Clearly, the most often recurrent exerbation is shown as and to cure difficulty big.Diet and other factors can affect the easy of hypoimmunity
Sense person so that it is resistivity immunoreactive to enteric bacteria reduces, thus lose the tolerance to normal flora, finally lead
Cause the generation of disease.Having a large amount of flora in human body intestinal canal, broad-spectrum antibiotic can prevent IBD to occur, and IBD morbidity and bacterium are described
Group is relevant.In the pathogenesis of IBD, immune factor has highly important effect.In the colonic mucosa of IBD patient
All can detect that substantial amounts of thick liquid cell, lymphocyte, neutrophil leucocyte and macrophage, these immunocytes are the most multiple
Cell factor, such as IL-1 β, IL-6 and TNFs etc. belong to pro-inflammatory cytokine, can cause generation and the development of inflammation.
The phenomenons such as IBD pathological process has enteric cavity internal pressure to increase, SA enhancing, endogenous vaso-excitor material activity mediator,
Make intestines CBF reduce, or re perfusion phenomenon occurs after transient ischemic, can cause for hydrogen reduction incomplete, cause a large amount of oxygen
Free radical is formed, and damages intestinal mucosa.
Therefore, from pharmacokinetics-pharmacodynamics angle system investigate sanguisorbin to the therapeutic action of IBD can
Row and possibility, promote the secondary development of sanguisorbin.
Summary of the invention
In view of this, it is an object of the invention to investigate sanguisorbin to inflammation from pharmacokinetics-pharmacodynamics angle system
The feasibility of the therapeutic action of disease property enteropathy and possibility, promote the secondary development of sanguisorbin.
At medicine in material base research process, it is primarily based on high-resolution, high sensitivity, the LC-Q-TOF/MS of high analyte speed
Saponin component in Radix Sangusorbae extract is identified by technology, builds IBD rat model, investigates sanguisorbin and exists
Normal and IBD rat model In vitro metabolism difference, finds and affect sanguisorbin normally and rat model inside and outside generation
Thank to the principal element of difference.Build IBD rat model, investigate sanguisorbin normal and inflammatory bowel disease model is big
Mouse In vitro metabolism difference, in the process, utilizes TNB to build inflammatory bowel disease model.Investigate garden burnet soap respectively
Glycosides drug-induced constituents spectrum in normal rat and IBD rat model blood plasma, urine, excrement, gut flora equal samples
Difference, finds the medicine generation-effective substance affecting sanguisorbin treatment colitis.
During sanguisorbin is to the pharmacology activity research of the therapeutic action of IBD, set up the mouse of LPS induction
The external inflammatory model of RAW264.7 macrophage, by ELISA detection variable concentrations sanguisorbin to neoplasm necrosis
The factor-α (TNF-α), interleukin-6 (IL-6) and the impact of interleukin-1 ' beta ' (IL-1 β).Experiment in vivo part
Use TNB to build Ulcerative Colitis Model in rat body, be divided into control group, model group, low dose group
(10mg/kg), middle dosage group (25mg/kg), high dose group (50mg/kg) and positive drug group (250mg/kg).Modeling
Rear drug treatment, after 7 days, compares different groups of changes of body mass situations, measures IL-1 β, IL-6 and the table of TNF-α in serum
Reach；Taking the obvious place of colonic ulcer and carry out pathological analysis, conventional H E dyes, and evaluates each group of sample degree of inflammation；Take knot simultaneously
Its myeloperoxidase (MPO), MDA (MDA), superoxide dismutase (SOD) is measured after intestinal tissue homogenate
Expression in colon.It was found that sanguisorbin dose-dependently reverses the TNF-α (P ＜ 0.01) caused by TNBS
The rise expressed with IL-1 β (P ＜ 0.01), IL-6 (P ＜ 0.01) has significant difference with model group under 50 μ g/mL concentration.
Experiment in vivo shows, sanguisorbin substantially alleviates the rat colonitis pathological change degree of TNBS induction；Model group rats body
Heavily being substantially less than control group (P ＜ 0.001), low dose group, middle dosage group, high dose group can significantly raise with positive drug group and make
Rat body weight after mould；After modeling, in rat blood serum, IL-6 expresses and raises (P ＜ 0.05), and compared with model group, sanguisorbin is low
Dosage, high dose and positive drug group can significantly reduce the colitis reaction induced by TNBS；After modeling in rat blood serum
IL-1 β express raise (P ＜ 0.05), compared with model group, sanguisorbin low dosage, middle dosage and positive drug group can significantly under
Adjust by the expression of IL-1 β in serum；After modeling, in rat blood serum, TNF-α expresses rising, has conspicuousness poor compared with control group
Different (P ＜ 0.01), the TNF-α of sanguisorbin treatment group expresses the trend having certain downward.
By above research, investigate sanguisorbin from pharmacokinetics-pharmacodynamics angle system IBD is controlled
The feasibility for the treatment of effect and possibility, promote the secondary development of sanguisorbin.
Accompanying drawing illustrates:
The impact on RAW264.7 cytoactive of Fig. 1: the radix sanguisorbae total saponin.
Impact (A) TNF-α (B) that the RAW264.7 cell cytokine that LPS is induced by Fig. 2: sanguisorbin is expressed
IL-1β(C)IL-6。
Modeling rat weight is affected by Fig. 3: sanguisorbin.
Therapeutic action (A) control group (B) of rat colon lesion tissue result and radix sanguisorbae total saponin thereof after Fig. 4: TNBS modeling
Model group (C) treatment group (D) positive drug group.
Impact (A) MPO (B) SOD (C) MDA that each index in rat colon tissue is expressed by Fig. 5: sanguisorbin.
Fig. 6: sanguisorbin is on impact (A) IL-6 (B) IL-1 β (C) TNF-α of cell factor in rat blood serum.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment, the present invention is further detailed.
The qualitative analysis of saponin component in embodiment 1 Radix Sangusorbae extract
The first step: the fragmentation pattern analysis of sanguisorbin: saponin component can occur desugar base to react, 162Da represents and sloughs
Hexose (glucose, Glc-), 132Da represents and sloughs pentose (arabinose, Ara-).Analytical fragments INFORMATION DISCOVERY,
In sanguisorbin I and II secondary fragment mass spectrogram, 453.33,471.34 and 585.38 have higher intensity.Therefore,
We select fragments characteristic ion m/z 453.33,471.34, the 585.38 and neutral m/z 132,162 that loses as soap
Methods of glycosides characteristic information, sets up and is applicable to the fragments characteristic ion diagnosis technology of sanguisorbin Rapid identification and neutral loss skill
Art.
Second step: by characteristic ion diagnostic techniques and neutral loss technology, the most comprehensively and rapidly identify saponins
Dividing 24 kinds, correlated results (retention time, molecular formula, accurate molecular masses, mass deviation, fragment ion) is listed in table 1.
The saponins compound configuration identified is as Figure 1-4.Wherein Ursane saponin component has 18 kinds, oleanane
Type saponin component has a kind, and other type saponin component has 5 kinds.
Saponin component relevant information in table 1 Radix Sangusorbae extract
Embodiment 2 sanguisorbin is identified at normal and IBD rat model In vitro metabolism thing
Step one: the preparation of colitis model made by TNBS bowel lavage: be dissolved in by TNBS in ethanol solution (1: 1),
Being configured to concentration is 100mg/kg solution.After lumbar injection 7% chloral hydrate anesthesia rat,.Draw with syringe and configure
TNBS solution, with bowel lavage after vaseline sufficient lubrication catheter tip, the catheter tip about 5cm of the anus away from rat, will note
After TNBS in emitter is slowly pushed into colon, it is inverted rat 1min.Control group mice gives the physiological saline of Isodose.
Step 2: prepared by gut flora: rat femoral sacrificed by exsanguination, takes out ileocaecal sphineter, exposes its content, weighs intestines
Road content quality.1g content adds 3mL Sample storage liquid (20% glycerine adds 1.8% sodium chloride), after piping and druming uniformly
Be dispensed in 1.5mL aseptic EP pipe, be saved in-80 DEG C standby.
Step 3: medicine altogether temperature external with gut flora is incubated: with PY culture medium, bacterium solution is diluted 5 times in aseptic operating platform,
Bacterium solution after dilution in shaking table 37 DEG C, after 200rpm temperature incubates 12h, takes 90 μ L bacterium solution and 10 μ L in aseptic operating platform
Sanguisorbin solution mixes, and is placed in 2.5L temperature and incubates in box, puts into rapidly anaerobism bag, in shaking table 37 DEG C, and 200rpm is respectively
Temperature incubates 1h.Temperature terminates reacting and carrying out extracting (n=5) with 1mL ice n-butanol at the end of incubating.10,000g are centrifuged 10min,
Taking supernatant 0.8mL, put in 45 DEG C of traditional vacuum enrichment facilities and volatilize, 200 μ L acetonitrile dissolved residues, 40,000g are centrifuged
10min, 5 μ L sample introductions are analyzed.Result of study is shown in Table 2 and table 3.
Sanguisorbin metabolin composition in table 2 normal group gut flora
Sanguisorbin metabolin composition in table 3 model group gut flora
Embodiment 3 sanguisorbin is metabolism research in normal and IBD rat model body
Step one: animal used as test and packet: healthy male SD rat 24, is divided into normal group and model group.Model group
Anus gave TNBS (100mg/kg) after 7 days, and two groups of gavages give sanguisorbin (50mg/kg).
Step 2: biological specimen collection: adopt blank plasma samples before administration, 1h femoral artery sacrificed by exsanguination after gastric infusion,
Take model group and control group blood plasma respectively in the flexible pipe adding liquaemin.Gastric infusion, is respectively put in metabolic cage, receives
Before collection is administered and after being administered 0～24h, 24～the urine of 48h, 48～72h, 72～96h and ight soil.
Step 3: biological sample processes: take in control group and model group rats blood plasma 200 μ l respectively, add the positive fourth of 1mL
Alcohol vibration 2min, 10,000g are centrifuged 10min, take supernatant 0.8mL, put in 45 DEG C of traditional vacuum enrichment facilities and volatilize,
200 μ L acetonitrile dissolved residues, 40,000g are centrifuged 10min, 5 μ L sample introductions.Separately take 200 μ L urines, add 1mL
N-butanol vibration 2min, 10,000g are centrifuged 10min, take supernatant 0.8mL, put in 45 DEG C of traditional vacuum enrichment facilities and wave
Dry, 200 μ L acetonitrile dissolved residues, 40,000g are centrifuged 10min, 5 μ L sample introductions.After fecal specimens is weighed, by 5mL/g
Ratio add ultra-pure water grind to form suspension.Take 200 μ L excrement suspensions, add 1mL n-butanol vibration 2min, 10,000
G is centrifuged 10min, takes supernatant 0.8mL, puts in 45 DEG C of traditional vacuum enrichment facilities and volatilizes, 200 μ L acetonitrile dissolved residues,
40,000g are centrifuged 10min, 5 μ L sample introductions.Analysis result is shown in Table 4～6.
Table 4 sanguisorbin metabolin composition in rat model blood plasma
Table 5 sanguisorbin metabolin information in model group rats urine
Table 6 sanguisorbin metabolin information in model group rats ight soil
The treatment IBD drug action research of embodiment 4 sanguisorbin
Step one: using mtt assay detection cytoactive, (concentration is respectively 10,25,50 and 100 with radix sanguisorbae total saponin
μ g/mL) process after RAW 264.7 mononuclear macrophage, under 10,25,50 μ g/mL concentration, sanguisorbin cell is not
There will be obvious cell-cytotoxic reaction (Fig. 1), under 100 μ g/mL concentration, sanguisorbin has cytotoxicity (P ＜ 0.01).
TNF-α (P ＜ 0.001) in RAW 264.7 cell, IL-6 (P ＜ 0.001) have significantly been raised in step 2: LPS stimulation
Expression with IL-1 β (P ＜ 0.001).Compared with LPS group, sanguisorbin has dose-dependently been lowered is stimulated institute by LPS
The TNF-α caused, the expression of IL-1 β and IL-6 raises.Elm saponin(e low concentration (P ＜ 0.01), middle concentration (P ＜ 0.01) and
High concentration (P ＜ 0.01) can significantly lower the TNF-α secretion amount of the RAW264.7 macrophage of LPS induction.Garden burnet soap
Glycosides low concentration (P ＜ 0.01), middle concentration (P ＜ 0.01) and high concentration (P ＜ 0.01) can significantly lower the RAW of LPS induction
The IL-1 β of 264.7 macrophages expresses.Sanguisorbin high concentration (P ＜ 0.01) can significantly reduce the RAW264.7 of LPS induction
The IL-6 of macrophage expresses, and sees Fig. 2.
Step 3: control rats is active, body weight increases, and stool is normal, in dry graininess.Model group rats is independently lived
Dynamic minimizing, body weight increases significant difference (P ＜ 0.001) compared with control group.Various dose sanguisorbin treatment group is different
Degree alleviates rat body weight downward trend, low dose group (P ＜ 0.05), middle dosage group (P ＜ 0.05), high dose group (P ＜ 0.05)
And positive drug group (P ＜ 0.01) is variant compared with model group.In garden burnet, dosage group and positive drug group and control group are without statistics
Difference.Show that sanguisorbin can be alleviated the rat body weight that TNBS causes and alleviate phenomenon (see Fig. 3).
Step 4: the impact of colon's pathomorphism is shown in Fig. 4 by sanguisorbin.
Normal group: Colonic Struture Changes understands, it is seen that mucous layer, submucosa, muscle layer and adventitia, mucosal epithelial cells without
Sex change, necrosis and come off, lamina propria is shown in more glands of large intestine, and interstitial has no congested, oedema, without cell infiltration.
Model group: all tissue pathologies change is seen by colon.Each example mucosal epithelial cells is substantially complete, have no sex change,
Necrosis and disappearance.Mucous layer and submucosa severe cell infiltration, a side region intestinal wall seriously narrows, and enteraden lacks in a large number.
Treatment group: each example mucosal epithelial cells is substantially complete, has no sex change, necrosis and disappearance.In mucous layer and submucosa
Degree cell infiltration, a large amount of enteraden of lamina propria is cystic dilatation, and the squeezed buckling of cell is flat, and subregion submucosa fiber is tied
Form the slight hyperplasia of tissue
Positive drug group: all example intestinal tissue Cha Jian tissue pathologies changes.Mucous layer and the slight cell infiltration of submucosa,
Submucosa loosens oedema, and subregion enteraden lacks.
Step 5: the impact of rat colon tissue middle finger target is included by sanguisorbin: to MPO, SOD, MDA content
Impact, result is as shown in Figure 5.In ulcerative colitis in rats colon, MPO expresses significantly raised (P ＜ 0.05),
In each dosage treatment group, rat colon tissue MPO expresses and reduces in various degree, low dose group (P ＜ 0.05), middle dosage group
(P ＜ 0.01), high dose group (P ＜ 0.05) and positive drug group (P ＜ 0.01) have statistical significant difference compared with model group.
After modeling, in rat colon tissue, SOD expresses reduction (P ＜ 0.05), shows that body Scavenging ability declines.Give ground
After the treatment of elm saponin(e, SOD expresses the trend returning to normal level.Rat MDA expression showed increased after modeling
(P ＜ 0.05).MDA is the product of peroxidization, has cytotoxicity.It is with lipid effect and peroxidization occurs,
The cross-linked polymeric of the life macromolecule such as protein, nucleic acid can be caused.After sanguisorbin treatment, MDA returns to become normally
Gesture.
Claims (3)
1. containing complicated saponin component, predominantly Ursane and oleanane glycoside, Qi Zhonghan in sanguisorbin extract
Measuring higher composition is sanguisorbin I and sanguisorbin II.
2. sanguisorbin extract as claimed in claim 1, it is characterised in that: I phase, II phase and desugar is there is in rat inside and outside
Base metabolism.
3. sanguisorbin extract as claimed in claim 1, it is characterised in that: the therapeutic action to IBD.
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| CN111972354A (en) * | 2019-05-24 | 2020-11-24 | 凯斯艾生物科技（苏州）有限公司 | Method for constructing interstitial pneumonia model with autoimmune characteristics |
| CN112274522A (en) * | 2020-11-12 | 2021-01-29 | 成都中医药大学 | New anti-inflammatory application of phenol glycoside compounds in garden burnet and extraction and separation method thereof |
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| CN116832109A (en) * | 2023-03-27 | 2023-10-03 | 南京鼓楼医院 | Method for regulating TNBS immune response by low-dose paris polyphylla saponin |
| CN118203627A (en) * | 2024-03-19 | 2024-06-18 | 鲁南厚普制药有限公司 | Application of elm-cape jasmine hemostatic granule in preparing medicine for preventing or treating inflammatory bowel disease |
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