CN101491533B

CN101491533B - Use of baicalin in preparing medicine

Info

Publication number: CN101491533B
Application number: CN 200810207294
Authority: CN
Inventors: 杨骥; 李明
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2008-12-18
Filing date: 2008-12-18
Publication date: 2011-05-04
Anticipated expiration: 2028-12-18
Also published as: CN101491533A

Abstract

The invention relates to application of scutelloside in pharmacy. The scutelloside promotes a T cell to be transformed toward Treg with immunological suppression effect by up-regulating the expression of Foxp3; at the same time, the scutelloside inhibits Th17 with pro-inflammatory effect and inhibits vasculitis mediated by the Th17. A main mechanism of inhibting the Th17 of the scutelloside is to inhibit the amplification of the Th17 mediated by IL-6. At the same time, a further experiment proves that the scutelloside taken as a novel immunomodulator plays good therapeutic effect on treating lupus erythenlatosus nephritis and rheumatoid arthritis.

Description

The application of baicalin in pharmacy

Technical field

The present invention relates to the new purposes of baicalin, relate in particular to the application in preparation prevention and treatment lupus erythematosus nephritis, rheumatoid arthritis and vasculitic medicine.

Background technology

Autoimmune disease is a kind of disease of harm humans health of immunocyte mediation, and often with the damage of many organs and multisystem, weight person threatens life.Immunosuppressant such as hormone are as a line medicine for the treatment of at present, and the Chang Yinwei serious adverse aggravates disease, even treatment stops.And along with the change of environmental pollution and life and work pressure etc., the sickness rate of autoimmune disease is increasing year by year.Seeking the medicine for the treatment of autoimmune disease has safely and effectively become the problem of world wide further investigation.

Autoimmune disease is often with the destruction of immunologic tolerance.Immunologic tolerance is for keeping in the body immunologic balance and preventing that autoimmune disease is very important.Thymus at first can be removed autoreactive T cell, if thymus can not be removed autoreactive T cell fully, this moment, autoimmune disease did not take place healthy individual, because have the periphery immunologic tolerance.Regulatory T cells (Treg) has immunosuppressive action, plays a crucial role in the periphery immunologic tolerance, can prevent the generation of autoimmune disease.Increasing research support autoimmune disease as: lupus erythematosus, rheumatoid arthritis etc. are all with the attenuating of Treg cell quantity, and promote the T cell will play an important role to the treatment autoimmune disease to the medicine of Treg cell transformation.Forked head/wing shape spiral transcription factor (Forkhead/winged helix transcription factor (Foxp3)) is the core transcription factor in the Treg cell development process, and regulation and control naivety T cell is to the Treg cell transformation.Serious autoimmune disease can take place in the mice at Foxp3 clpp gene place, illustrates that Foxp3 plays an important role in the generation of autoimmune disease keeping immunologic tolerance and prevent.The medicine with rise Foxp3 of generally acknowledging so far is very few, and natural drug does not have discovery so far.

The CD4+T cell (Th17) of secretion IL-17 is a new T cell subsets of finding recently, it is mainly by secreting leukocytes mesonium-17A (IL-17A), Interleukin-17 F (IL-17F), tumor necrosis factor-alpha (TNF-α) and interleukin-6 cytokines such as (IL-6) stimulate vascular endothelial cell secretion adhesion molecule, and the mediation inflammatory cell is to the infiltration of tissue and the damage of tissue; The Th17 cell also can the upright damaged tissue that connects of secretory cell poisonous substance simultaneously.More and more studies show that the Th17 cell some autoimmune diseasees as: play a crucial role in the morbidity of rheumatic arthritis, lupus erythematosus, psoriasis and multiple sclerosis.Therefore seek the medicine that can suppress the Th17 cell subsets simultaneously and just might extenuate autoimmune disease.Radix Scutellariae is traditional heat-clearing and detoxifying herb, and bitter in the mouth is cold in nature, is used for the treatment of diseases such as damp and hot feeling of fullness, cough due to lung-heat, carbuncle sore tumefacting virus in the traditional medicine.(7-glucuronicacid-5 6-dihydroxyflavone) is the Main Ingredients and Appearance of Radix Scutellariae to baicalin, and molecular formula is C ₂₁H ₁₈O ₁₁, molecular weight is 446.35, structural formula:

In application and research in the past, known baicalin has hepatic cholagogic, blood pressure lowering, diuresis, antimicrobial effect, but baicalin does not appear in the newspapers in the effect of modulating T cell in Treg and Th17 conversion, does not appear in the newspapers in therapy system lupus erythematosus nephritis and rheumatic arthritis simultaneously yet.

Summary of the invention

The purpose of this invention is to provide the new purposes of baicalin in pharmacy.

In order to achieve the above object, technical scheme of the present invention provides the application of baicalin in the cell-mediated vasculitic medicine of preparation prevention and treatment lupus erythematosus nephritis, rheumatoid arthritis and Th17.

Pharmacological evaluation and result with baicalin illustrates its new purposes in pharmaceutical field below.

The external evoked T cell of baicalin transforms to Treg, suppresses simultaneously to transform to Th17: at first we use the inmature T lymphocyte (CD4 in the selected by flow cytometry apoptosis mouse spleen ⁺CD25 ^-), cultivate every hole 1 * 10 in 96 orifice plates ⁶Individual cell adds the anti-CD3 of 2 μ g/ml and the anti-CD28 of 2 μ g/ml simultaneously.Add transforming growth factor (TGF-β) 5ng/ml and induce cell transformation to Treg; Add transforming growth factor (TGF-β) 5ng/ml+ interleukin-6 (IL-6) 20ng/ml and induce cell transformation to Th17.The baicalin (being dissolved in dimethyl sulfoxide (DMSO)) that in culture fluid, adds 20 μ M simultaneously.Behind the cell culture 3 days, detect CD4 by flow cytometer ⁺Foxp3 ⁺And CD4 ⁺IL-17 ⁺The T cell at CD4 ⁺Percentage rate in the cell, the result as shown in Figure 1.This figure illustrated baicalin can external modulating T cell to Treg and Th17 transformation.

Baicalin raises the expression of Foxp3 mRNA: at first we cultivate every hole 1 * 10 with the inmature T lymphocyte in the selected by flow cytometry apoptosis mouse spleen in 96 orifice plates ⁶Individual cell is established three multiple holes.Add the anti-CD3 of 2 μ g/ml and the anti-CD28 of 2 μ g/ml simultaneously.The baicalin of independent adding transforming growth factor (TGF-β) 5ng/ml, 20 μ M or both add simultaneously.Behind the cell culture 3 days, by the expression of real-time quantitative polymerization integrated enzyme reaction (real-time quantitative PCR) detection Foxp3 mRNA, the result as shown in Figure 2.This figure has illustrated that baicalin can raise the expression of Foxp3mRNA.

Baicalin improves the kidney injury of spontaneous lupus mice: 12 spontaneous lupus mices in age in May (MRL/lpr) are available from Shanghai Inst. of Life Science, CAS Shanghai Slac Experimental Animal Co., Ltd..100mg/kg/ days baicalins (be dissolved in normal saline, be kept at-20 ℃ refrigerator) of treatment group lumbar injection treated for 9 weeks continuously.Collect mice twenty-four-hour urine liquid in the 0th week, 3 weeks, 6 weeks and 9 weeks respectively, detect urine protein content, calculate relative urine protein increase=time urine protein-Di 0 all urine protein to be measured.As shown in Figure 3, *＜0.001 illustrates that baicalin can improve the twenty-four-hour urine albumen of lupus mice.The 9th week was put to death animal, got kidney and did pathology haematoxylin-Yihong (HE) and Masson trichrome stain understanding kidney injury situation, as shown in Figure 4, illustrated that baicalin can improve the kidney injury of lupus mice.Whether extracting spleen cell detects baicalin and can promote the T cell to the Treg transformation, as shown in Figure 5, illustrates that baicalin can promote that the T cell transforms to Treg in the lupus mice body.

Baicalin is alleviated adjuvant-induced arthritis mice joint injury: 18 8 age in week male C57BL/6 mice available from Shanghai Inst. of Life Science, CAS Shanghai Slac Experimental Animal Co., Ltd..Modeling uses 100 μ l to contain the complete Buddhist formula adjuvant immunity mice metapedes of tubercule bacillus of the 10mg/ml of heat shock, and mice 2 all posterior joint inflammation shapes arrive peaks, and begin lumbar injection 100mg/kg/ days 1 week of baicalin this moment.Get the capable pathological section of joint tissue and estimate therapeutic effect, as shown in Figure 6, illustrate that baicalin can improve the joint injury of arthritis mice, get the mice joint tissue and detect the influence of baicalin Interleukin-17 (IL-17) mRNA by real-time quantitative PCR, as shown in Figure 7, illustrate that baicalin can suppress the expression of IL-17mRNA in the arthritis mice joint tissue.

Baicalin suppresses the expression of the inductive human umbilical vein endothelial cell of IL-17 (HUVEC) inflammatory factor: HUVEC cultivates in 6 orifice plates, when treating that cell to 80% merges, add 50ng/ml IL-17 and 20 μ M baicalins, and continue to cultivate after 24 hours, collecting cell, detect adhesion molecule-1 (ICAM-1) between the baicalin pair cell by real-time quantitative PCR, the influence of adhesion molecule between vascular endothelial cell-1 (VCAM-1) and IL-17 mRNA expression, and with 18S rRNA as confidential reference items.The result illustrates that baicalin can suppress the expression of the inductive adhesion molecule mRNA of IL-17 as shown in Figure 8.

Baicalin suppresses the adhesion of the inductive T cell of IL-17 to human umbilical vein endothelial cell: human umbilical vein endothelial cell is cultivated in 12 orifice plates, when treating that cell to 80% merges, add 50ng/ml IL-17 and 20 μ M baicalins, and continue to cultivate after 24 hours; With 5: 1 ratio adding human leukemia lymphocyte (Jurkat), continue to cultivate 24 hours then.Cultivate and finish the back with the not adherent lymphocyte of normal saline flush away, microscopically is observed adherent cell.As shown in Figure 9, illustrate that baicalin can suppress the adhesion of the inductive T cell of IL-17 endotheliocyte.

The present invention is converted into target spot with modulating T cell, finds that the baicalin modulating T cell transforms the application in the treatment autoimmune disease, and its concrete advantage is as follows:

1, the present invention has excavated new medical application to the known compound baicalin, has opened up a new application.

2, baicalin promotes the conversion that the T cell transforms to the Th17 of short scorching effect to the Treg of antiinflammatory action transformation and suppressor T cell;

3, astragaloside can further suppress the vasculitis by the Th17 cell induction;

4, the immunoregulation effect of baicalin can be applied in widely on the autoimmune disease.

Description of drawings

Fig. 1 detects figure for the mouse T cell flow cytometer;

Fig. 2 is the real-time quantitative polymerase connection reaction detection figure that Foxp3 mRNA expresses in the mouse T cell;

Fig. 3 is that mice twenty-four-hour urine protein quantification detects figure;

Fig. 4 is mouse kidney haematoxylin-Yihong and Masson trichrome stain figure;

Fig. 5 is mouse spleen CD4 ⁺Foxp3 ⁺The cell flow cytometer detects figure;

Fig. 6 is mice joint tissue haematoxylin-Yihong colored graph;

Fig. 7 is the polymerase connection reaction detection figure that mice joint tissue IL-17mRNA expresses;

Fig. 8 suppresses the polymerase connection reaction detection figure that vascular endothelial cell adhesion molecule mRNA expresses for baicalin;

Fig. 9 is the adhesion figure of baicalin suppressor T cell to vascular endothelial cell.

The specific embodiment

Specify the present invention below by embodiment.

Embodiment 1: the extraction of baicalin

Adopt Labiatae Scutellaria plant Radix Scutellariae 2.5kg, use the ethanol of 95vol% to extract twice, each two hours at 65 ℃, extracting solution is at 70 ℃ of concentrating under reduced pressure, and the concentrated solution cool to room temperature that obtains is centrifugal, filter, filtrate is at 70 ℃ of concentrating under reduced pressure, and the concentrated solution that obtains adds ethanol and carries out precipitate with ethanol to 85vol%, alcohol deposit fluid leaves standstill filtration, gets supernatant, cross and filter to remove impurity, in 70 ℃ of concentrating under reduced pressure, spray drying, the ultraviolet sterilization obtains baicalin.

Embodiment 2: capsular preparation:

The baicalin 300g that embodiment 1 is obtained, add 300g Polyethylene Glycol (PEG-400), the 30g triethanolamine, 20g HP-20g and glycerol 40g, mixed dissolution is made solution, adopts pressed film method to prepare soft capsule, and soft capsule is through solidifying by cooling in wind, drying, wash ball, do eventually, promptly with appropriate solvent.

The present invention can also prepare other dosage forms by conventional method, as soft capsule, enteric coated capsule, microcapsule, powder, granule, syrup, injection, suppository, pill, gel, patch, Emulsion, suspension, solution etc.Preparation among the present invention can be the coating or the form of coating not, depends on the needs.Pharmaceutic adjuvant among the present invention comprises the excipient that is used for solid preparation, lubricant, binding agent, disintegrating agent, stabilizing agent, foaming agent, coating materials etc., or be used for solution, solubilizing agent, suspending agent, isotonic agent, buffer agent, emollient, emulsifying agent of semi-solid preparation, liquid preparation etc., in addition, also can use other medical additive such as antiseptic, antioxidant, coloring agent, sweeting agent and flavoring agent etc. as required.

Claims

1. the application of baicalin in the medicine of preparation treatment or prevention lupus erythematosus nephritis.