CN103705502A - Application of flavonoid compound in treating inflammatory diseases - Google Patents
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Abstract
The invention discloses application of flavonoid compound 5,7,3',4',5'-pentamethyl flavanone (PMFA) as an active ingredient for treating inflammatory diseases. The 5,7,3',4',5'-pentamethyl flavanone (PMFA) taken as an anti-inflammatory active ingredient has the characteristics of small toxicity and good treatment effect, and has an obvious inhibitory effect on inflammatory factors and especially macrophage cytokines. Thus, the 5,7,3',4',5'-pentamethyl flavanone (PMFA) can be taken as an anti-inflammatory preparation used for the inflammatory diseases in which other various macrophage cytokines are participated such as sepsis, arthritis, immune-type enteritis, diabetes and the like, and especially has a good treatment effect on the sepsis which is difficult to cure.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of flavone compound 5,7,3 ', 4 ', 5 '-pentamethyl flavanone, as the application in active component treatment diseases associated with inflammation, is mainly used in the diseases associated with inflammation that multiple macrophage participates in, as arthritis, immunologic pattern enteritis, diabetes etc., especially the sepsis that is difficult to cure is had to good therapeutical effect.
Background technology
Macrophage, as the sincere advice constituent of natural immune system, participates in the pathological process of multiple solemn and just property disease, comprises sepsis, arthritis, immunologic pattern enteritis, diabetes etc.Wherein, sepsis remains a huge medical difficult problem at world wide, is attended by serious systemic inflammatory syndrome, causes multiple organ failure, MOF, and causes high mortality rate.In north america, infer that pathogenesis of sepsis rate surpasses 600000 examples every year, mortality rate is up to 30%-50%.Clinically pyemic treatment is still lacked to effective healing means at present.The inflammatory reaction that macrophage participates in is considered to the very important factor of pathogenesis of sepsis process, suppress macrophage inflammatory reaction and can effectively alleviate sepsis symptom, so the macrophage-mediated inflammation of targeting can be used as the screening index for the treatment of of sepsis medicine.In addition, there is macrophage in patient with rheumatoid arthritis synovial tissue place, secretion proinflammatory factor, somatomedin, chemotactic factors etc., bring out synovial membrane inflammation, the inflammatory reaction of regulation and control macrophage causes joint injury, so also can be used as the evaluation index of arthritis medicine.
Flavone compound is studied widely as the effective ingredient of plurality of Chinese preparation, and its effect comprises antiinflammatory, defying age, antioxidation etc.5,7,3 ', 4 ', 5 '-pentamethyl flavanone, is dissolved in the organic solvents such as methanol, ethanol, acetone and chloroform, but the research that improves diseases associated with inflammation by regulation and control macrophage function about flavone compound also seldom.
5,7,3 ', 4 ', 5 '-pentamethyl flavanone, chemical name: 5,7,3 ', 4 ', 5 '-pentamethoxyflavanone, No. CAS: 479672-30-5, molecular structure:
Summary of the invention
The object of the invention is to explore 5,7,3 ', 4 ', 5 '-pentamethyl flavanone (PMFA) is the application in sepsis and related inflammation disease as anti-inflammatory drug.
The present invention is by studies have shown that, and 5,7,3 ', 4 ', the main anti-inflammatory mechanisms of 5 '-pentamethyl flavanone (PMFA), for adjusting macrophage polarization, can be used for treating the diseases associated with inflammation such as sepsis.
5,7,3 ', 4 ', 5 '-pentamethyl flavanone (PMFA) is that separation and purification obtains or synthetic can obtaining from natural plants.PMFA does not have toxicity to normal macrophage, and this shows that PMFA may be able to avoid the side effect of some common immunoregulation compounds.Meanwhile, PMFA, in the situation that not affecting macrophage survival rate, suppresses the expression of its inflammatory factor, shows that PMFA has good antiinflammatory action.We carry out testing and detecting its anti-inflammatory efficacy in body subsequently.Compare with matched group, PMFA can significantly improve the survival rate of mice when the dosed administration of 30mg/kg, improves lung injury.Simultaneously to blood inflammatory cytokines levels in non-diabetic IL-1 β, IL-6, TNF-α has remarkable inhibitory action.STAT1 is important transcription factor, inflammatory factor IL-1 β, and IL-6, transcribing of TNF-α affected by it all.PMFA suppresses the activation of STAT1, thereby suppresses the expression of downstream inflammatory factor.
Beneficial effect of the present invention compared with the prior art: 5,7,3 ', 4 ', 5 '-pentamethyl flavanone (PMFA) derives from the extract of natural plants, as anti-inflammatory activity composition, there is little, the eutherapeutic feature of toxicity, inflammatory factor especially macrophage cytokines is had to remarkable inhibitory action, therefore can be used as the diseases associated with inflammation that anti-inflammatory preparation participates in for other multiple macrophage, as sepsis, arthritis, immunologic pattern enteritis, diabetes etc., especially the sepsis that is difficult to cure is had to good therapeutical effect.
The specific embodiment
By the specific embodiment, effect of the present invention is described further below:
Embodiment 1.PMFA toxicity detects
Raw264.7 cell is with 5*10
4individual cells/well kind, in 96 orifice plates, adds 100ng/ml LPS or isopyknic PBS, adds PMFA37 ℃ to hatch after 24 hours simultaneously, the impact of mtt assay detection compound on cell strain survival.
The normal mouse macrophage strain Raw264.7 cultivating and the PMFA of variable concentrations are hatched after 24 hours jointly, mtt assay detects its survival rate, dosage reaches 60 μ M, and cell survival rate is compared with normal group without obviously reducing, and shows that strain does not have toxicity to PMFA to macrophage.Further experiment shows, PMFA does not have inhibitory action (Figure 1B and C) to the survival rate of the macrophage of LPS activation yet.
Embodiment 2.PMFA anti-inflammatory activity detects
Raw264.7 cell is with 1*10
6individual cells/well kind, in 6 orifice plates, adds 37 ℃ of PMFA and 100ng/ml lipopolysaccharide (LPS) to hatch after 6 hours simultaneously, and QPCR method detects IL-1 β, the variation of IL-6mRNA level.
Experimental result shows: after LPS stimulates, macrophage is activated, cytokine IL-1 β, IL-6mRNA level rising (Fig. 1 D and E).And under PMFA effect, IL-1 β, IL-6mRNA level significantly reduces (Fig. 1 D and E)).The result of comprehensive Figure 1B and C, show, PMFA suppresses macrophage activation under the prerequisite that does not affect macrophage survival, disclose PMFA inflammatory factor especially macrophage cytokines is had to remarkable inhibitory action, therefore can be used as the diseases associated with inflammation that anti-inflammatory preparation participates in for other multiple macrophage, as sepsis, arthritis, immunologic pattern enteritis, diabetes etc.
Embodiment 3.PMFA is to pyemic effect
1) the pyemic induction of mice
Get 6-8 C57/BL6 mice in age in week, lumbar injection 100 microlitre 10mg/kg lipopolysaccharide (LPS), normal group injected in mice 100 microlitre PBS are in contrast.
2) observation of mouse survival rate
First 2 hours of injection LPS, the 100 microlitre PMFA that mouse peritoneal injection olive oil is dissolved, normal group and model group lumbar injection 100 microlitre olive oil are as contrast.Every 4 hours, observe and record mouse survival rate, until mouse survival rate is stable, no longer change.
3) serum levels of inflammatory cytokines IL-1 β, IL-6, the detection of TNF-α
Separately get a collection of injected in mice LPS first 2 hours, the 100 microlitre PMFA that mouse peritoneal injection olive oil is dissolved, normal group and model group lumbar injection 100 microlitre olive oil are as contrast.After 4 hours, mice is put to death, pluck eyeball and get blood, after 37 ℃ of water-bath 30min, 3500rpm is centrifugal 15 minutes, draws serum ELISA method and detects inflammatory factor IL-1 β, IL-6, TNF-alpha levels.
4) observation that lungs histopathology changes
The mice of putting to death is got lungs tissue simultaneously.A part 10% formalin solution is fixed, and paraffin embedding, does the crown section of 4-5 micron thickness, and dye with hematoxylin-eosin.Tissues observed pathological changes and inflammatory cell infiltration.
5) detection of lungs histiocytokine mRNA level
The mice of putting to death, gets after a part of lungs tissue adds Trizol homogenate and extracts total RNA, gets 1g RNA reverse transcription after quantitative and becomes cDNA, and reaction system is as follows: the total RNA of 1 μ g, and 2000pM Oligo dT, 1.0mM dNTP, 200U reverse transcriptase, 5 * transcribe buffer.Full genome cDNA is used Auele Specific Primer to increase, and reaction system is as follows: 1 μ l cDNA, 0.5 μ M upstream and downstream primer, 5 μ l iQ
tMsYBR
green permix.PCR primer and Amplification are in Table 1.Amplification is used BioRad CFX manager software to analyze.
The parameter of each cytokine of table 1
6) lungs are organized the detection of p-STAT1 level
The mice of putting to death, get after a part of lungs tissue adds cell pyrolysis liquid homogenate and extract total protein, quantitatively, get 25g albumen and carry out SDS-PAGE electrophoretic separation, trace is transferred to pvdf membrane, and 5% skim milk room temperature sealing 1 hour, with p-STAT1 or 4 ℃ of overnight incubation of STAT1 antibody, wash film and remove the antibody into combination, with corresponding two anti-hatching 2 hours, wash film, develop.
Result shows:
2 hours pneumoretroperitoneum injection LPS of PMFA (15mg/kg, 30mg/kg) of mouse peritoneal injection various dose.LPS brings out mice and produces serious acute inflammatory reaction, and serum levels of inflammatory cytokines raises, and a plurality of organ generation pathological changes, follow higher mortality rate.Compare with model group, PMFA high dose group (30mg/kg) mouse survival rate significantly raises.Meanwhile, short-term experiment result shows, PMFA treatment group mice serum inflammatory factor IL-1 β, and IL-6, TNF-alpha levels is significantly suppressed (Fig. 2).Illustrate that PMFA can significantly improve the sepsis mice state of an illness.
Under LPS effect, the many organs of mice are impaired, and wherein lungs sustain damage at first.After LPS effect 4 hours, mice lungs tissue morphology is damaged, and non-viable non-apoptotic cell increases, and follows obvious inflammatory cell infiltration.And mice lungs have effectively been protected in PMFA treatment.Wherein, lesion tissue is eased, and the inflammatory cell of non-viable non-apoptotic cell, infiltration reduces.Meanwhile, lungs histiocyte inflammatory factor IL-1 β, IL-6, TNF-α mRNA level is suppressed, has organized the destruction of inflammatory factor to internal organs.Illustrate that PMFA can significantly improve sepsis mice lung lesion (Fig. 3).
STAT1 is important transcription factor, inflammatory factor IL-1 β, and IL-6, transcribing of TNF-α affected by it all.Under LPS stimulates, STAT1 is phosphorylated (its activated form) and is indexed into nucleus from endochylema, is combined with specific DNA fragmentation, starts transcribing of corresponding gene.The STAT1 of the dirty activated form of mouse lung that LPS stimulates increases.Immunohistochemical assay result shows, after LPS effect, STAT1 more appears at nucleus, exercises its functional transcription.In PMFA treatment group mice lungs core, STAT1 obviously reduces (Fig. 4 A).Meanwhile, western blot experimental result shows, PMFA treatment group mice lungs homogenate Protein S TAT1 phosphorylation level reduction (Fig. 4 B).These results show, the activation of PMFA inhibition STAT1, thereby the expression of inhibition downstream inflammatory factor.
Accompanying drawing explanation
Fig. 1: PMFA does not affect the survival rate of macrophage, suppresses the activation of macrophage simultaneously.
(A) HPLC method detects PMFA purity.
(B) Raw264.7 cell is with 5*10
4individual cells/well kind, in 96 orifice plates, adds PBS to add PMFA37 ℃ to hatch after 24 hours simultaneously, the impact of mtt assay detection compound on cell strain survival.
(C) Raw264.7 cell is with 5*10
4individual cells/well kind, in 96 orifice plates, adds 100ng/ml lipopolysaccharide (LPS) to add PMFA37 ℃ to hatch after 24 hours simultaneously, the impact of mtt assay detection compound on cell strain survival.
(D, E) Raw264.7 cell is with 1*10
6individual cells/well kind, in 6 orifice plates, adds PMFA and 100ng/ml LPS37 ℃ to hatch after 6 hours simultaneously, and QPCR method detects IL-1 β, the variation of IL-6mRNA level.Result represents with mean ± s.e.m.
*p<0.05,
*p<0.01, vs LPS group.
Fig. 2: PMFA improves mice sepsis.
(A) 2 hours pneumoretroperitoneum injection LPS of PMFA (15mg/kg, 30mg/kg) of mouse peritoneal injection various dose.Observe and record mouse survival rate.N=12,
*p<0.01, vs LPS group.
(B) 2 hours pneumoretroperitoneum injection LPS of PMFA (15mg/kg, 30mg/kg) of mouse peritoneal injection various dose.After four hours, mice is put to death, pluck eyeball and get blood, ELISA method detects serum levels of inflammatory cytokines IL-1 β, IL-6, TNF-alpha levels.N=6, result represents with mean ± s.e.m.
*p<0.05,
*p<0.01, vs LPS group.
Fig. 3: PMFA improves sepsis mice lung lesion.
(A) 2 hours pneumoretroperitoneum injection LPS of PMFA (15mg/kg, 30mg/kg) of mouse peritoneal injection various dose.After four hours, mice is put to death, a part of lungs organize 10% formalin solution to fix, and paraffin embedding is done the crown section of 4-5 micron thickness, and dyeed with hematoxylin-eosin.Tissues observed pathological changes and inflammatory cell infiltration.
(B) a part of lungs tissue extracts total RNA after adding Trizol homogenate, and QPCR method detects IL-1 β, the change of IL-6TNF-α mRNA level.N=6, result represents with mean ± s.e.m.
*p<0.05,
*p<0.01, vs LPS group.
Fig. 4: PMFA suppresses lungs and organizes STAT1 activation.
2 hours pneumoretroperitoneum injection LPS. of PMFA (15mg/kg, 30mg/kg) of mouse peritoneal injection various dose
(A) the section warp that is unstained of making is de-cured, antigen retrieval, and sealing waits processes rear and p-STAT1 antibody hybridization, and uses Real Envision Detection kit to detect its expression.
(B) separately get a part of lungs tissue and add after cell pyrolysis liquid homogenate, western blot method detects the expression of p-STAT1.
Claims (3)
1.5,7,3 ', 4 ', 5 '-pentamethyl flavanone is applied to treat diseases associated with inflammation as active component.
2. purposes according to claim 1, is characterized in that 5,7,3 ', 4 ', and 5 '-pentamethyl flavanone can treat as active component the diseases associated with inflammation that macrophage participates in.
3. purposes according to claim 2, is characterized in that described diseases associated with inflammation is sepsis, arthritis, immunologic pattern enteritis, diabetes etc.
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| CN110776517A (en) * | 2019-10-14 | 2020-02-11 | 广东药科大学 | Flavanone compound and application thereof |
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| CN102038699A (en) * | 2009-10-19 | 2011-05-04 | 周亚伟 | Novel application of paeoniflorin to inflammation resistance |
| CN102302483A (en) * | 2011-07-08 | 2012-01-04 | 中国科学院生物物理研究所 | Application of flavonoid small molecule medicine in anti-inflammation and associated diseases |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038699A (en) * | 2009-10-19 | 2011-05-04 | 周亚伟 | Novel application of paeoniflorin to inflammation resistance |
| CN102302483A (en) * | 2011-07-08 | 2012-01-04 | 中国科学院生物物理研究所 | Application of flavonoid small molecule medicine in anti-inflammation and associated diseases |
Non-Patent Citations (2)
| Title |
|---|
| JIAYU ZHANG ET AL.: "Simultaneous Screening and Identifying Four Categories of Particular Flavonoids in the Leaves of Murraya exotica L. by HPLC–DAD–ESI-MS-MS", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 * |
| 吴龙火等: "九里香指纹图谱与其抗炎活性的灰关联度分析", 《中国实验方剂学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110776517A (en) * | 2019-10-14 | 2020-02-11 | 广东药科大学 | Flavanone compound and application thereof |
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