阅读说明：本技术 吐叶醇作为有效成分在免疫抑制剂中的应用 (Application of the vomifoliol as effective component in immunosuppressor ) 是由 徐静 李钢 岑举人 邓勤 于 2019-09-19 设计创作，主要内容包括：本发明公开了吐叶醇作为有效成分在免疫抑制剂中的应用,本发明通过从角果木中筛选出了具有显著的钙调蛋白磷酸酶(CN)抑制活性的吐叶醇,其免疫抑制作用与临床广泛使用的免疫抑制剂环孢菌素A相当,作用机制研究显示吐叶醇为靶向CN/NFAT信号通路发挥免疫抑制活性,且对脾细胞毒性很弱,是一种高效、低毒的免疫抑制剂。(Application the invention discloses vomifoliol as effective component in immunosuppressor, the present invention from ceriops tagal by having filtered out the vomifoliol with significant Calcineurin (CN) inhibitory activity, its immunosuppressive action is suitable with clinical widely used immunosuppressor cyclosporin A, study on mechanism shows that vomifoliol is that targeting CN/NFAT signal path plays immunosuppressive activity, and it is very weak to splenocyte toxicity, it is a kind of immunosuppressor efficiently, less toxic.)

1. application of the vomifoliol as effective component in immunosuppressor, the inhibitor is immunosuppressive drug.

2. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: spit Leaf-alcohol to Jurkat cell missing cytotoxic activity, without promotion cell Proliferation or inhibits cell to increase in 1~40 μm of concentration range Effect.

3. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: spit The vigor for the Jurkat cell that leaf-alcohol is used to that PMA/Io to be inhibited to stimulate proliferation differentiation.

4. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: spit Leaf-alcohol is used to inhibit the vigor to Calcineurin (CN).

5. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: spit Leaf-alcohol is as the dephosphorylized immunosuppressor of NFAT albumen for inhibiting Intracellular phosphorylation.

6. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: spit Leaf-alcohol is used to inhibit the generation of the interleukin -2 (IL-2) of Jurkat cell.

7. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: spit Leaf-alcohol is by the resulting compound of separating-purifying in ceriops tagal.

8. application of the vomifoliol according to claim 1 as effective component in immunosuppressor, it is characterised in that: institute The molecular formula for stating vomifoliol is C₁₃H₂₀O₃: structural formula are as follows:

Technical field

The present invention relates to the purposes of vomifoliol, in particular to vomifoliol as effective component answering in immunosuppressor With.

Background technique

Calcineurin (calcineurin, CN) is a kind of dependence Ca²⁺With the serine/threonine of calmodulin Specificity phosphatase, this enzyme are extracted from yeast fermentation broth, since content is high in nerve fiber for it, so again Often it is known as Calcineurin (Klee et al., 1979).When biochemist and neurobiologist study this When kind of new albumen, it is not intended to the immunosuppressor cyclosporin A (CsA) of new discovery, it be by with intracellular close ring egg White (cytosolic protein) combines into compound, and then has significant immunosuppressive effect, is widely used in In organ transfer operation (Borel et al., 1976；Handschumacher et al.,1984).

Phosphorylation and the dephosphorylation effect of protein are a kind of extremely important adjustment mechanism, adjusting in vital movement Mechanism almost penetrates into during each vital movement, this process is protein kinase (protein kinase, PKase) and egg The result that white phosphatase (protein phosphatase, PPase) dynamic action is adjusted jointly.For protein kinase, egg White phosphatase is currently known that type is less, and the research of the adjustment mechanism in cell activities also know it is few.Calcium tune The a member of phosphoprotein phosphatase (Calcineurin, CN) as protein phosphatase enzyme family, activity is directly by intracellular second messenger Ca²⁺The adjusting of/CaM (calmodulin), immunological regulation, learning and memory and in terms of play it is of crucial importance Effect the great interest and height weight of vast researcher are caused due to its property, the particularity of structure and function Depending on, the discovery of its substrate, the research of physiological function and enzyme activity inhibitor in terms of carried out relatively broad grind Study carefully, wherein the research achievement of most breakthrough is nineteen ninety Calcineurin to be determined as immunosuppressive drug ring spore The specificity target enzyme of rhzomorph A (cyclosporin A, CsA), FK506.But existing Calcineurin inhibitor such as ring spore Plain A and tacrolimus are current clinically common immunosuppressor, but have renal toxicity, hyperglycemia, to the non-specific of cell A variety of adverse reactions such as property.

The present invention is that have external inhibition calmodulin by filtering out in ceriops tagal using Calcineurin as target enzyme Phosphatase (CN) active compound, and in-depth study its mechanism mechanism, exist for studying and disclosing Calcineurin Function and mechanism of action in vital movement are extremely important, and the high-efficiency low-toxicity immunosuppressor to develop new provides New way.

Summary of the invention

In consideration of it, the present invention proposes application of the vomifoliol as effective component in immunosuppressor.

The technical scheme of the present invention is realized as follows:

Application of the vomifoliol as effective component in immunosuppressor, the inhibitor are immunosuppressive drug.

The present invention is target enzyme by immune Calcineurin (CN), using pNPP as at the beginning of the immunosuppressive activity of substrate Sieve carries out immunosuppressive activity screening, discovery vomifoliol during primary dcreening operation to the monomeric compound separated in ceriops tagal Show preferable immunosuppressive activity.Continue further to study vomifoliol, establishes PMA/Io induction Jurkat cell Model is activated, the expression of CN vigour changes in Jurkat cell, the variation of IL-2 content and NFAT albumen is detected, illustrates The mechanism of action of vomifoliol.

Vomifoliol is in 1~40 μm of concentration range to Jurkat cell missing cytotoxic activity, without promotion cell Proliferation or suppression The effect that cell processed increases.

The present invention handles Jurkat in the toxicity assessment to Jurkat cell of vomifoliol, by the vomifoliol of various concentration Cell 48h has found that under different processing time conditions, in used concentration range (1~40 μM), Jurkat cell is deposited Significant difference is not present in motility rate, and cell survival rate is all 90% or more.Continue to rise with the compound concentration, high agent The vomifoliol of amount group (75,100 μM) shows slight inhibiting effect to Jurkat cell, and the survival rate of cell is reduced to 90% Below.Illustrate that vomifoliol does not have cytotoxic activity to Jurkat cell in 1~40 μM of concentration range and without promoting cell to increase The effect grown or cell is inhibited to increase.

The vigor for the Jurkat cell that vomifoliol is used to that PMA/Io to be inhibited to stimulate proliferation differentiation.

The present invention has used Calcineurin activity test method, using pNPP as substrate, to activity in ceriops tagal at Divide and carry out screening active ingredients, discovery vomifoliol has strong inhibiting effect to CN.And further it is made by Jurkat cell It is further analyzed with mechanism.Jurkat cell Proliferation, Differentiation is stimulated by PMA/Io, T cell is simulated and receives external antigen thorn Physiological environment after swashing.Jurkat cell first stimulates proliferation differentiation through PMA/Io, then the vomifoliol through various concentration co-cultures. The results show that vomifoliol to PMA/Io stimulate proliferation differentiation Jurkat cell have certain inhibiting effect.Illustrate vomifoliol The immunocompetence of cell can be inhibited to a certain extent.

Vomifoliol is used to inhibit the vigor to Calcineurin.

Vomifoliol is as the dephosphorylized immunosuppressor of NFAT albumen for inhibiting Intracellular phosphorylation.

Vomifoliol is used to inhibit the generation of the interleukin -2 (IL-2) of Jurkat cell.

Calcineurin (CN) is a kind of and NFAT activates closely bound up enzyme, generally under normal circumstances, into the cell NFAT albumen exists only in cytoplasm, and when cell is activated by environmental stimuli, intracellular CN can be such that it goes in conjunction with NFAT albumen Phosphorylation generates corresponding immune molecule (such as interleukin -2) in conjunction with the transcription factor in downstream hence into nucleus.This Invention not only screens compound from extracellular Calcineurin (CN) Inhibition test, tentatively confirmed to spit leaf The immunosuppressive activity of alcohol has also set up PMA/Io induction Jurakat cell proliferation and differentiation model, to activated cell through spitting Leaf-alcohol processing has detected its intracellular Calcineurin (CN) activity, intracellular IL-2 content and to passing through respectively Cell pathway level is studied in western blot experiment.As a result, it has been found that the cell without vomifoliol processing, hence it is evident that living Change, intracellular proleulzin (IL-2) content increases, and intracellular Calcineurin (CN) vigor rises.It can by WB result Know, is substantially reduced through NFAT degree in the processed Jurkat cell of vomifoliol.Illustrate vomifoliol by inhibiting intracellular Ca2+ The activity of heregulin phosphatase (CN) reduces NFAT and enters in nucleus and tie to reduce the dephosphorylation degree of NFAT Downstream albumen is closed, and then IL-2 level reduces.Therefore, the present invention successfully discloses the mechanism of action of vomifoliol, opens for vomifoliol The immunosuppressor that hair becomes novel high-efficiency low-toxicity provides theoretical foundation.

Vomifoliol is by the resulting compound of separating-purifying in ceriops tagal.

The molecular formula of the vomifoliol is C₁₃H₂₀O₃: structural formula are as follows:

Compared with prior art, the beneficial effects of the present invention are: the present invention is shown by having filtered out to have from ceriops tagal The vomifoliol of Calcineurin (CN) inhibitory activity of work, and in-depth study its mechanism mechanism, immunosuppressive action Suitable with clinical widely used immunosuppressor cyclosporin A, study on mechanism shows that vomifoliol is targeting CN/NFAT letter Number access plays immunosuppressive activity, and very weak to splenocyte toxicity, using vomifoliol as effective component in immunosuppressor Application, provide new way to develop novel efficient, less toxic immunosuppressor.

Detailed description of the invention

Fig. 1 is the active primary dcreening operation schematic diagram of Calcineurin (CN) of the invention；

Fig. 2 is the schematic diagram of the embodiment of the present invention enzyme activity testing tube

Fig. 3 is column analysis chart of the embodiment of the present invention vomifoliol to the toxicity of Jurkat cell；

Fig. 4 is that the column that the embodiment of the present invention vomifoliol inhibits PMA/Io to stimulate Jurkat cell proliferation function is analyzed Figure；

Fig. 5 is that the embodiment of the present invention vomifoliol analyzes the column of the inhibiting effect of Calcineurin (CN) vigor Figure；

Fig. 6 is column analysis chart of the embodiment of the present invention vomifoliol to the inhibiting effect of IL-2 in Jurkat cell；

Fig. 7 is the electrophoretic band figure of influence of the embodiment of the present invention vomifoliol to NFAT；

Fig. 8 is the column analysis chart of influence of the embodiment of the present invention vomifoliol to NFAT；

Fig. 9 is the hydrogen spectrogram of the embodiment of the present invention compound HZ-2 obtained by separating-purifying in ceriops tagal；

Figure 10 is the carbon spectrogram of the embodiment of the present invention compound HZ-2 obtained by separating-purifying in ceriops tagal；

Specific embodiment

In order to be best understood from the technology of the present invention content, specific embodiment is provided below, the present invention is described further.

Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.

Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.

One, experimental material and instrument

1. cell origin

Jurkat cell strain, is purchased from Chinese Academy of Sciences's stem cell bank, is passed on, frozen in this laboratory liquid used in this experiment In nitrogen tank.

2. material and reagent vomifoliol (vomifoliol, abbreviation Vom), purity >=90%.

1 main agents of table

Table1 Main reagents

3. key instrument equipment

2 major experimental instrument of table

Table 2Main experimental instruments

Two, experimental method and interpretation of result