CN100493498C - Application of piperlonguminine and dihydropiperlonguminine in the preparation of the medicine for curing senile dementia - Google Patents
Application of piperlonguminine and dihydropiperlonguminine in the preparation of the medicine for curing senile dementia Download PDFInfo
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- CN100493498C CN100493498C CNB2007100143363A CN200710014336A CN100493498C CN 100493498 C CN100493498 C CN 100493498C CN B2007100143363 A CNB2007100143363 A CN B2007100143363A CN 200710014336 A CN200710014336 A CN 200710014336A CN 100493498 C CN100493498 C CN 100493498C
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Abstract
An application of a piperlonguminine and a dihydropiperlonguminine for preparing a drug for treating senile dementia is disclosed in the invention, wherein, the piperlonguminine and the dihydropiperlonguminine having mass ratio of 1:1.08 are separated and extracted from pepper stem of FuJian. It is approved that the piperlonguminine and the dihydropiperlonguminine are active components for suppressing APP expression by the pepper stem via RT-PCR and Western Blot in the invention, with preferred drug concentration of 3.28 mu g/ml-6.56 mu g/ml. Accordingly, the piperlonguminine and the dihydropiperlonguminine has inviting developing prospect for preparing novel drug for treating the senile dementia.
Description
Technical field
The present invention relates to the application of piperlonguminine and dihydropiperlonguminine, relate in particular to piperlonguminine and dihydropiperlonguminine in preparation treatment alzheimer disease, the application in the medicine of minimizing APPmRNA and APP protein expression.
Background technology
Alzheimer (Alzheimer ' s disease, AD) be senile dementia main diseases because of.In the pathogenesis of AD, the deposition of amyloid (A β) is the co-channel of the various causes of disease.Experiment confirm: A β derives from amyloid precursor protein (APP), if suppress the APP overexpression, can reduce A β and generate in brain and deposit, and therefore becomes the important target spot of treatment AD.
Caulis Piperis Kadsurae (caulis piperis kadsurae) is the dry rattan of Piperaceae Piper plant Radix seu Caulis fici Martinii [Piperkadsura (choisy) Ohwi].Tcm clinical practice is mainly used in treatment of diseases such as asthma, rheumatism and arthralgia, but rarely is useful on the report of senile dementia treatment.
In recent years, the applicant adopts Northern blot method to confirm that Caulis Piperis Kadsurae can suppress the app gene expression by selectivity, adopts the method for RT-PCR and SABC to find that first piperlonguminine (piperlonguminine) and dihydropiperlonguminine (dihydropiperlonguminine) can suppress SK-N-SH cell amyloid precursor protein (APP) mRNA and the proteic expression of APP simultaneously.This research is looked into newly by retrieval, does not appear in the newspapers as yet both at home and abroad at present.
Summary of the invention
To deficiency, the problem to be solved in the present invention provides a kind of piperlonguminine and dihydropiperlonguminine is treated alzheimer disease in preparation at prior art, the application in the medicine of minimizing APPmRNA and APP protein expression.
Main technological route of the present invention is:
1. extract by the Fujian Caulis Piperis Kadsurae and separate piperlonguminine and dihydropiperlonguminine.
2. from the cell purchaser of institute of Shanghai Chinese Academy of Sciences neuroblastoma cell system (SK-N-SH), set up stable continuous cell line.
3. Caulis Piperis Kadsurae suppresses amyloid precursor protein (APP) gene expression experiment (APP mRNA): set up cell RNA and extract and the RT-PCR reaction system.
4. Caulis Piperis Kadsurae suppresses amyloid precursor protein (APP) experiment: set up total protein of cell and extract and Western blot reaction system.
The application in preparation treatment alzheimer disease medicine of piperlonguminine of the present invention and dihydropiperlonguminine.
Wherein: described piperlonguminine and dihydropiperlonguminine can suppress APPmRNA and the proteic expression of APP from cellular level.
Wherein: described piperlonguminine is separated, is extracted by the Fujian Caulis Piperis Kadsurae with dihydropiperlonguminine, and the mass ratio of piperlonguminine and dihydropiperlonguminine is 1:0.8.
Above-mentioned piperlonguminine and dihydropiperlonguminine are specifically made by following method:
1. the extraction of Caulis Piperis Kadsurae effective ingredient
1) Caulis Piperis Kadsurae is produced available from Bozhou, Anhui Province Chinese Medicinal Materials Markets, through the teaching and research Wa Shi Fructus Piperis that is accredited as (Piper wallichii) of pharmaceutical college of the Shandong University pharmacognosy in Fujian.
2) Caulis Piperis Kadsurae medical material (18kg) is with behind the water extraction, and extracting solution concentrates and obtains extractum 1788.06g.After extractum is dissolved in water, use petroleum ether successively, ethyl acetate, n-butanol extraction obtains petroleum ether extraction phase 4.577g respectively, and ethyl acetate extraction phase 37.95g and n-butanol extraction be 198.25g mutually.
2. extraction phase cytotoxic assay:
The activity of extraction phase all is converted into suitable crude drug concentration, so that have comparability between each extraction phase and crude drug.
The solution effects concentration that makes each extraction phase is carried out mtt assay with human neuroblastoma cell line (SK-N-SH) and is detected when 50g/l (being equivalent to crude drug) is following.
The result shows: do not have obvious cytotoxicity.
3. the extraction phase active site is definite:
1) utilizes RNA to extract and the RT-PCR reaction system, use the suitable concentration (being lower than 50g/l) of three extraction phases of Caulis Piperis Kadsurae to act on cell respectively, observe its inhibitory action.
By experiment, the active site of Caulis Piperis Kadsurae inhibition app gene expression is determined in the ethyl acetate extraction phase the most at last.
2) reaction system of application Protein Extraction and Western blot has reconfirmed that active site is the ethyl acetate extraction phase.
4. the determination of active ingredients of Caulis Piperis Kadsurae:
(1) separation of monomer component and evaluation:
1) ethyl acetate extraction phase (37.95g) with 95% dissolve with ethanol after, add silica gel mixed sample, through silica gel (the 100-200 order, 1.52kg, the column chromatography of 10cm * 1m), chloroform-acetone gradient elution is collected eluting stream part, 500ml/ flow part, collects 151 parts of stream parts altogether.Wherein chloroform-acetone (9:1) eluting stream part Fr.31 obtains chemical compound 1 (called after HFT-1) 20mg with behind petroleum ether-acetone repeated crystallization.
2) HFT-1 analyzes
Data according to hydrogen spectrum, carbon spectrum, and contrast with document, determine that the HFT-1 crystallization is by compd A piperlonguminine (piperlonguminine, A) and B dihydropiperlonguminine (dihydropiperlonguminine, B) form, determine that by its hydrogen spectral integral the mass ratio of A and B is 1:0.8 in this crystallization.
Compd A, B molecular formula are as follows:
A: piperlonguminine (piperlonguminine, A)
B: dihydropiperlonguminine (dihydropiperlonguminine, B)
In order to understand the action effect of essence of the present invention and chemical compound of the present invention better,, further set forth its application in preparation treatment alzheimer disease medicine below in conjunction with the pharmacological evaluation and the result of piperlonguminine and dihydropiperlonguminine.
1. the dissolving of monomer component and spraying medicine concentration:
It is 105mg/ml that Caulis Piperis Kadsurae monomer component HFT-1 is made its final concentration with the DMSO dissolving.
Become 1:16 (6.56mg/ml) with the DMSO doubling dilution, 1:32 (3.28mg/ml) before the dosing.
In the ratio dosing of 1ulDMSO medicinal liquid adding 1ml culture medium, making DMSO concentration is 1 ‰, and the ultimate density of HFT-1 is respectively 6.56ug/ml and 3.28ug/ml.
2. monomer component cytotoxic assay:
Carry out mtt assay with human neuroblastoma cell line (SK-N-SH) and detect, the result shows: two experimental concentration of HFT-1 all do not have obvious cytotoxicity (mtt assay).
3. monomer component determination of activity:
1) utilize the human neuroblastoma cell line of having set up (SK-N-SH), RNA extracts and the reaction system of RT-PCR is tested, and the result shows: HFT-1 can optionally suppress app gene and express (mRNA).(see figure 1)
2) use of the expression of the method for Western blot from protein level confirmation HFT-1 energy obvious suppression amyloid precursor protein (APP).(see figure 2)
By above-mentioned experiment and result thereof, can draw as drawing a conclusion:
The active site that Caulis Piperis Kadsurae suppresses the APP expression is that the Caulis Piperis Kadsurae composition is through chemically separated ethyl acetate extraction phase, the active ingredient of Caulis Piperis Kadsurae is piperlonguminine and dihydropiperlonguminine, confirm that by RT-PCR and Western Blot piperlonguminine and dihydropiperlonguminine are that Caulis Piperis Kadsurae suppresses the effective ingredient that APP expresses, its preferable drug level is 3.28ug/ml～6.56ug/ml, indication piperlonguminine and dihydropiperlonguminine have comparatively tempting development prospect in the new drug development and application of preparation treatment alzheimer disease.
Description of drawings
Fig. 1 HFT-1 optionally suppresses app gene to express
Wherein: left side figure is APP; Right figure is 18s (reference gene).
1. normal control.2.DMSO contrast (1 ‰).3.HFT-1(3.28ug/ml)。
Shown in the figure, HFT-1 (being piperlonguminine and dihydropiperlonguminine) is the expression that 3.28ug/ml can suppress SK-N-SH cell APP mRNA in concentration.
Fig. 2 HFT-1 suppresses the expression of amyloid precursor protein (APP)
Wherein: last figure is APP; Figure below is β-Actin (with reference to an albumen).
1. normal control.2. positive control (crude drug concentration 35g/l).3.DMSO contrast (1 ‰).4.HFT-1(3.28ug/ml)。5.HFT-1(6.56ug/ml)。
Shown in the figure, concentration is that the HFT-1 (being piperlonguminine and dihydropiperlonguminine) of 6.56ug/ml can suppress the proteic expression of SK-N-SH cell APP.
The specific embodiment
The Chemical Decomposition of the effective ingredient of embodiment 1 Caulis Piperis Kadsurae is extracted.
Caulis Piperis Kadsurae medical material (18kg) is with behind the water extraction, and extracting solution concentrates and obtains extractum 1788.06g.After extractum is dissolved in water, use petroleum ether successively, ethyl acetate, n-butanol extraction obtains petroleum ether extraction phase 4.577g respectively, and ethyl acetate extraction phase 37.95g and n-butanol extraction be 198.25g mutually.Ethyl acetate extraction phase (37.95g) with 95% dissolve with ethanol after, add silica gel mixed sample, through silica gel (the 100-200 order, 1.52kg, the column chromatography of 10cm * 1m), chloroform-acetone gradient elution is collected eluting stream part, 500ml/ flow part, collects 151 parts of stream parts altogether.Wherein chloroform-acetone (9:1) eluting stream part Fr.31 obtains chemical compound 1 (called after HFT-1) 20mg with behind petroleum ether-acetone repeated crystallization.
HFT-1 is according to the data of hydrogen spectrum, carbon spectrum, determine this crystallization by the chemical compound piperlonguminine (piperlonguminine, A) and dihydropiperlonguminine (mass ratio of A and B is 1:0.8 in this crystallization for dihydropiperlonguminine, B) composition.
Caulis Piperis Kadsurae monomer component HFT-1 (piperlonguminine and dihydropiperlonguminine) is dissolved (final concentration is 105mg/ml) with a small amount of DMSO.
Become 1:16 (6.56mg/ml) with the DMSO doubling dilution before the dosing.
In the ratio dosing of 1ulDMSO medicinal liquid adding 1mlMEM culture medium, making DMSO concentration is 1 ‰, and the ultimate density of HFT-1 is respectively 6.56ug/ml.
Added the SK-N-SH cell culture 24-48 hour, routine is extracted RNA and is carried out the RT-PCR reaction, finds that HFT-1 can suppress the expression of APP mRNA.(see figure 1)
Extract the SK-N-SH cell protein, found that with Western blot method HFT-1 can suppress the expression of amyloid precursor protein.(see figure 2)
Claims (2)
1. piperlonguminine and dihydropiperlonguminine are as the application of unique active component in preparation treatment alzheimer disease medicine.
2. application as claimed in claim 1 is characterized in that: described piperlonguminine is separated, is extracted by Caulis Piperis Kadsurae with dihydropiperlonguminine, and the mass ratio of piperlonguminine and dihydropiperlonguminine is 1:0.8.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US11485725B2 (en) | 2017-12-15 | 2022-11-01 | Auransa Inc. | Derivatives of piperlongumine and uses thereof |
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| CN114652712B (en) * | 2022-03-24 | 2024-01-26 | 河南中医药大学 | Application of kadsura pepper quinuclidine in preparation of medicine for preventing and treating senile dementia |
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Non-Patent Citations (10)
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| Selective inhibition of haifengteng in gene expression ofbeta-amyloid precursor protein. En-ji，Han等.中国临床康复，第8卷第13期. 2004 |
| Selective inhibition of haifengteng in gene expression ofbeta-amyloid precursor protein. En-ji，Han等.中国临床康复，第8卷第13期. 2004 * |
| 广东海风藤提取物对痴呆鼠脑内β淀粉样前体蛋白基因表达的影响. 罗焕敏等.中国老年学杂志，第23卷第6期. 2003 |
| 广东海风藤提取物对痴呆鼠脑内β淀粉样前体蛋白基因表达的影响. 罗焕敏等.中国老年学杂志，第23卷第6期. 2003 * |
| 海风藤对β-APP基因表达的影响. 韩恩吉等.山东大学学报(医学版)，第41卷第1期. 2003 |
| 海风藤对β-APP基因表达的影响. 韩恩吉等.山东大学学报(医学版)，第41卷第1期. 2003 * |
| 海风藤抑制淀粉样前体蛋白基因表达的研究. 韩恩吉等.中国中药杂志，第23卷第11期. 1998 |
| 海风藤抑制淀粉样前体蛋白基因表达的研究. 韩恩吉等.中国中药杂志，第23卷第11期. 1998 * |
| 海风藤的研究概况. 罗永明.江西中医学院学报，第6卷第1期. 1994 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11485725B2 (en) | 2017-12-15 | 2022-11-01 | Auransa Inc. | Derivatives of piperlongumine and uses thereof |
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