CN102846597A

CN102846597A - Medicinal use of artemisinin and artesunate against alzheimer disease

Info

Publication number: CN102846597A
Application number: CN2012103808783A
Authority: CN
Inventors: 李萍; 陈君; 徐晓军; 王菊; 石子琪
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2012-10-10
Filing date: 2012-10-10
Publication date: 2013-01-02

Abstract

The invention relates to the field of natural medicaments, and in particular relates to a medicinal use of artemisinin and artesunate against alzheimer disease. Researches find that artemisinin and artesunate have the effects on inhibition of generation of Abeta 42, and Ic50 is in the range of 1.6-4.0 mu m. Molecular weights of artemisinin and artesunate are less than 500 Daltons, and mean that the compounds have the potential capability of permeating brain-blood barriers, more importantly, artemisinin and artesunate do not affect cytotoxicity and cell apoptosis in the range of inhibiting the concentration of Abeta 42, and do not have side effects on a Notch signal path.

Description

The medical application of arteannuin and artesunate anti-Alzheimer disease

Technical field

The present invention relates to natural medicine field, be specifically related to arteannuin and the artesunate medical application in anti-Alzheimer disease.

Background technology

Herba Artemisiae Annuae is the dry aerial parts of feverfew Herba Artemisiae annuae Artemisia annua L..Its main effect is clearing away summer-heat, except steaming preventing the attack (or recurrence) of malaria.The clinical application of Herba Artemisiae Annuae is quite extensive, and is also very high to its degree of concern both at home and abroad.Its effective ingredient arteannuin is a kind of efficient, and quick-acting antimalarials is used for tertian malaria, and subtertian malaria is particularly rescued cerebral malaria good effect.The derivant artesunate of developing on the basis of arteannuin also has Antimalarial.Arteannuin and artesunate molecular formula are respectively C ₁₅H ₂₂O ₅, C ₁₉H ₂₈O ₈Molecular weight is 282.33 and 384.43.Wherein arteannuin is colourless acicular crystal, and artesunate is white crystalline powder, and the two all is soluble in ethanol, is insoluble in water, and chemical structural formula is as follows:

Arteannuin

Artesunate

This compounds of bibliographical information is arranged except having Antimalarial, also have the effects such as anti-pregnant, anti-schistosomiasis, arrhythmia, antitumor and immunomodulating.(Guo Yan, Wang Jun, Chen Zhengtang. the pharmacological action new development of artemisinin-based drug. Chinese Clinical pharmacology and therapeutics, 2006,11 (6): 615-620)

Studies show that in a large number amyloid-beta (β-amyloidpeptide, A β) is the key factor of Alzheimer formation and development in corticocerebral abnormal stacking.One of them hypotype A β ₄₂Be more prone to autohemagglutination, if can not and advance to remove, then can the extremely insoluble A beta oligomers of very fast formation and amyloid fibril.Therefore be produced as purpose research and become focus in the treatment of alzheimer research field to remove A β or to suppress A β.（Richard?SS，Hendrie?HC.Diagnosis，management，and?treatment?of?Alzheimer’s?disease.Arch.Intern.Med.，1?999，1?59：789-798.）

Summary of the invention

The invention discloses the effect of the anti-Alzheimer disease of arteannuin and artesunate, namely arteannuin and artesunate can be used for preparing the medicine for the treatment of Alzheimer, and effect is excellent.

The inventor finds that in the pharmacology activity research to arteannuin and artesunate arteannuin and artesunate have the A of inhibition β ₄₂The effect that produces, its IC ₅₀Scope at 1.6-4.0 μ M.And the molecular weight of arteannuin and artesunate means that all less than 500 dalton (Dalton, D) these chemical compounds have the potential ability that sees through blood brain barrier.The more important thing is that arteannuin and artesunate are suppressing A β ₄₂Concentration range on cytotoxicity and apoptosis without impact, simultaneously the Notch signal path is had no side effect.

Below in conjunction with embodiment the anti-senile dementia activity of arteannuin and artesunate is further set forth.

Description of drawings

Fig. 1 is the A β that artemisinin action HEK293sw cell AlphaLISA method detects secretion ₄₂IC50 result;

Fig. 2 is the A β that artemisinin action SHSY5Ysw cell AlphaLISA method detects secretion ₄₂IC50 result;

Fig. 3 is that artemisinin action HEK293sw cell AlphaLISA method detects intracellular A β ₄₂IC50 result;

Fig. 4 is the A β that artesunate effect HEK293sw cell AlphaLISA method detects secretion ₄₂IC50 result;

Fig. 5 is the A β that artesunate effect SHSY5Ysw cell AlphaLISA method detects secretion ₄₂IC50 result;

Fig. 6 is that artesunate effect HEK293sw cell AlphaLISA method detects intracellular A β ₄₂IC50 result;

Fig. 7 is the cytoactive analysis result of artemisinin action SHSY5Ysw cell;

Fig. 8 is the cytoactive analysis result of artesunate effect SHSY5Ysw cell;

Fig. 9 is the apoptosis activity analysis result of artemisinin action SHSY5Ysw cell;

Figure 10 is the apoptosis activity analysis result of artesunate effect SHSY5Ysw cell;

Figure 11 is that arteannuin is suppressing A β ₄₂During the concentration that produces for the impact analysis result of Notch path;

Figure 12 is that artesunate is suppressing A β ₄₂During the concentration that produces for the impact analysis result of Notch path;

The specific embodiment

Embodiment 1

The AlphaLISA method estimates arteannuin and artesunate suppresses A β ₄₂The activity that produces

The HEK293sw cell (is stable cell strain, contains Swidish APP sudden change fragment, can produce in a large number secretion A β ₄₀With A β ₄₂) (be the stable cell strain of neuroglial cytoma, contain Swidish APP sudden change fragment, can secrete a large amount of A β with the SHSY5Ysw cell ₄₀With A β ₄₂) be seeded in respectively in 96 orifice plates, cell density is 5 * 104/100 μ l/well.To screen candidate compound (% suppression ratio〉50%) and do the Concentraton gradient analysis, the initial concentration of chemical compound is 10 μ M, 3 times of dilutions, totally 11 concentration point.Set up the known inhibitors of gamma-secretase of DAPT() positive medicine matched group, without the positive matched group of compound treatment cell, acellular is the background matched group without chemical compound, it is the test group that testing compound is processed cell.Positive compound to be measured and DAPT were hatched 24 hours with cell respectively.Then it is for subsequent use to collect the culture fluid supernatant.According to the AlphaLISA description, carry out A β ₄₂Detection.Arteannuin and artesunate detect respectively A β in extracellular and the cell in HEK293sw cell and SHSY5Ysw cell ₄₂Content, the result as shown in Figure 1 in the HEK293sw cell arteannuin and positive compound DAPT can suppress A β ₄₂Secretion, its IC50 is respectively: 1.6 μ M and 220 nM; Shown in Figure 2 in the HSY5Ysw cell arteannuin and positive compound DAPT can suppress A β ₄₂Secretion, its IC50 is respectively: 1.7 μ M and 730 nM; Can suppress A β in the cell such as Fig. 3 arteannuin and positive compound DAPT in the HEK293sw cell ₄₂Generation, its IC50 is respectively: 1.7 μ M and 510 nM.

Artesunate and positive compound DAPT can suppress A β in the HEK293sw cell as shown in Figure 4 ₄₂Secretion, its IC50 is respectively: 3.1 μ M and 220 nM; Shown in Figure 5 in the HSY5Ysw cell artesunate and positive compound DAPT can suppress A β ₄₂Secretion, its IC50 is respectively: 4.0 μ M and 730 nM; Artesunate and positive compound DAPT can suppress A β in the cell in the HEK293sw cell as shown in Figure 6 ₄₂Generation, its IC50 is respectively: 2.9 μ M and 510 nM.

Embodiment 2

Cytoactive and apoptosis activity analysis

Cytotoxic effect and apoptosis all can cause A β ₄₂The minimizing that produces, the A β that therefore causes in order to get rid of cell death that the toxicity of compound effect causes and apoptosis ₄₂Reduce, we use WST-1 model and casapse 3/7 activity analysis test kit to detect above positive compound to the impact of SHSY5Ysw cell.

With arteannuin and the artesunate compound treatment SHSY5Ysw cell of 100 μ M, overnight incubation, the WST-1 kit detection cell is active.Wherein 10 μ M staurosporines are established as the positive compound contrast, have cytotoxic effect, and 1% dimethyl sulfoxide (DMSO) is established as normal control.The result show the arteannuin of 100 μ M as shown in Figure 7 and Figure 8 and artesunate process the HSY5Ysw cell compare with normal control (1%DMSO processes cell) on the activity of cell without impact, show the acellular toxic action of arteannuin and artesunate, have cytotoxicity and positive compound 10 μ M staurosporines can significantly suppress the activity of HSY5Ysw cell.So arteannuin and artesunate for cytoactive without obviously with the impact.

Fig. 9 and Figure 10 show 10 μ M arteannuin and artesunate process the HSY5Ysw cell compare with normal control (1%DMSO processes cell) on the apoptosis activity of cell without impact, show the acellular apoptosis induction active function of arteannuin, positive compound 5 μ M staurosporines can significantly increase the apoptosis activity of HSY5Ysw cell.

Embodiment 3

The impact of Notch signal path

The HEK293 cell is seeded in the 3cm orifice plate with 3 * 105/300 μ l/ holes, plasmid pcDNA 3.2 these plasmids of sp Ntoch Δ E-GVP(are contained one section coding Ntoch Δ E-GVP(acid 1694-2205) fragment and have the Gal4/VP16 dna fragmentation of transcriptional activation activity) and plasmid pGL 4.3 1(luc2P/GAL4UAS/Hygro available from promega, having the fluorescence excitation activity) common transient transfection is in the HEK293 cell, the cell of transfection is processed respectively testing compound (test group), 100 μ M DAPT(positive drug matched groups), and set up without the positive signal matched group of compound treatment cotransfection cell, setting up and processing cell without transfection is the background group.37 ℃ of 5%CO ₂After hatching 24 hours, it is active to set chemiluminescence mode detection Notch signal path at the Envision instrument.Calculate the % suppression ratio.

Because the Notch signal path is the approach of a transfer cell interphase interaction signal that plays a crucial role in the specialization of various kinds of cell, and with some nervous system disease substantial connection is arranged.So must investigate candidate compound for the impact of Notch signal path.Such as Figure 11 and shown in Figure 12,10 μ M arteannuin and artesunate have been processed cotransfection respectively and have been contained the HEK293 cell of Notch Δ E fragment and reporter plasmid, compare (1%DMSO processes cell) with normal control active in impact on the Notch letter, show arteannuin and artesunate without the effect of Notch signal suppressing, positive compound 100 μ M DAPT can significantly suppress the activity of Notch signal path.Therefore, arteannuin and artesunate are suppressing A β ₄₂During the concentration that produces, for Notch without impact.

Claims

1. arteannuin or artesunate are for the preparation of the purposes of the medicine for the treatment of Alzheimer.