CN101716262B - Extract from Iris tenuifolia, preparation method and application thereof - Google Patents

Abstract

The invention discloses an extract from Iris tenuifolia, a preparation method and application thereof. The method comprises the following steps of: 1) taking an underground part of the Iris tenuifolia, soaking the underground part in absolute methanol, collecting extracting solution, and then removing the absolute methanol of the extracting solution to obtain a total extract of the underground part of the Iris tenuifolia; and 2) dissolving the total extract of the underground part of the Iris tenuifolia obtained by the step 1) in water, then adding chloroform into the water for chloroform extraction, collecting a chloroform phase, and then removing the chloroform in the chloroform phase to obtain a chloroform extract. The chloroform extract has the effect of inhibiting the aggregation of amyloid beta, which indicates that the chloroform extract has certain effect of treating senile dementia.

Description

A kind of extract from Iris tenifolia and its preparation method and application

technical field

The invention relates to an extract from Iris tenifolia and its preparation method and application.

Background technique

Alzheimer's disease (AD) is a progressive neurodegenerative disease that causes dementia in the elderly population. Different pathological signatures and progressive deposition of senile plaques and neurofibrillary tangles leading to neuronal degeneration accompany cognitive and memory decline. A typical feature of AD is the accumulation of amyloid-β peptide (Aβ) (amyloid-β peptide chain (Aβ)) derived from the hydrolysis of the transmembrane glycoprotein β-amyloid precursor protein (βAPP). The two main Aβ (Alzheimer's β-amyloid) variants consist of 40 and 42 amino acids, respectively, and they promote pro-inflammatory responses and activate neurotoxic pathways that lead to abnormal function and even death of brain cells. Relevant studies have shown that when Aβ aggregates in the form of fibrils, it has neurotoxic effects both in cells and in vivo. Therefore, finding small molecular compounds that can prevent Aβ aggregation has become very valuable in AD treatment (Ce'line Rivie`re etc, Inhibitory activity of stilbenes on Alzheimer's β-amyloid fibrils in vitro, Bioorganic & Medicinal Chemistry 15(2007) 1160- 1167).

At present, there are four main types of drugs for the treatment of Alzheimer's disease, namely cholinesterase inhibitors, antioxidant drugs, hormone replacement therapy drugs and non-steroidal anti-inflammatory drugs, but these drugs have risks or risks to a certain extent. limit. At present, the screening and research of small molecular lead compounds that can inhibit Aβ aggregation has become one of the hotspots in the development of treatment for Alzheimer's disease.

Plants of the genus Iridaceae. In my country, it is mainly distributed in the southwest, northwest and northeast regions. There are about 300 species in this genus, which are widely distributed all over the world. There are about 60 species and 13 varieties in China, (Mahmood U, KaulVK, Jirovetz L. Alkylated benzoquinones from Iris kumaonensis. Phytochemistry, 2002, 2(61): 923-926. Qin MJ, Xu LS, Tanaka T, et al. A preliminary study on the distribution pattern of isoflavones in rhizomes of Iris from China and its systematic significance. Acta Phyto Sinica, 2000, 38: 343-349.) This genus has been It is used as a traditional folk medicine to treat a variety of diseases such as cancer, inflammation, bacterial and viral infections. At present, compounds isolated from Iris have anti-tumor, anti-oxidation, anti-plasmodium and anti-tuberculosis effects (1.Atta-ur-RahmnM, et al.Anti-inflammatory isoflavonoids from the rhizomes of Iris germanica.Journal of Ethnopharmacology, 2003, 86: 177-180.2. Bonfils JP et al. Cytotoxicity of iridals, triterpenoids from Iris. on human tumor cell lines A2780 and K562. Planta Med, 2001, 67: 79-82).

Iris tenuifolia (Iris tenuifolia), is Iridaceae Iris plant, its chemical composition has certain reports, (1.Kojima, K., Gombosurengyin, P., Ondogyni, P., Begzsurengyin, D., Zevgeegyin, O ., Hatano, K., Ogihara, Y. Flavanones from Iris tenuifolia. Phytochemistry, 1997, 44(4): 711-714; 2.M.I.Choudhary, S.Hareem and H.Siddiqui, et al.A benzil andisoflavone from Iris tenuifolia, Phytochemistry, 2008, 69, 1880-1885). We continued to study the chemical components of this plant, and isolated and identified a new compound; biological activity experiments showed that this compound can prevent Aβ aggregation, which indicates that it has certain curative effect on Alzheimer's disease.

Contents of the invention

The object of the present invention is to provide a preparation method of the extract from Iris tenifolia.

The preparation method provided by the invention comprises the following steps:

1) taking the underground part of Iris tenifolia, extracting it with anhydrous methanol, collecting the extract, and then removing the anhydrous methanol in the extract to obtain the total extract of the underground part of Iris tenifolia;

2) Dissolving the total extract of the underground part of Iris tenifolia obtained in step 1) in water, adding chloroform for chloroform extraction, collecting the chloroform phase, and removing the chloroform in the chloroform phase to obtain the protected extract of the present invention.

Further, in the above step 1), the extraction is repeated 3 times.

In the above step 2), the chloroform extraction can be repeated 5 times.

The extract prepared by the above method also falls within the protection scope of the present invention.

The application of the above-mentioned extracts in the preparation of Aβ aggregation-preventing products also falls within the protection scope of the present invention.

Further, the above-mentioned product may be a drug.

The application of the above-mentioned extracts in the preparation of medicaments for treating Alzheimer's disease also falls within the protection scope of the present invention.

Another object of the present invention is to provide a medicine for treating senile dementia.

The medicament for treating senile dementia provided by the present invention has an active ingredient of the above-mentioned extract.

Experiments have proved that the extract provided by the invention can prevent Aβ aggregation (entanglement). Because Aβ aggregation is a typical feature of senile dementia, the extract provided by the invention can be used to treat senile dementia.

Description of drawings

Fig. 1 is the flow chart of compound preparation in the present invention.

Fig. 2 is the result figure that the extract of the present invention makes Aβ entanglement degree decline, and wherein aggregates represents the control group that does not add sample; 9 is the extract of the present invention, and 10 is the aqueous phase extract, and the ordinate of Fig. 2 is the The fluorescence intensity of the control group is recorded as 1, and the ratio of the fluorescence intensity of the other groups to the fluorescence intensity of the control group is the corresponding ordinate.

Detailed ways

Example 1. Preparation of a compound 1 capable of inhibiting amyloid beta aggregation:

1. Preparation of Compound 1

The preparation process is shown in Figure 1.

1) Soak in anhydrous methanol

Get 1661g of fresh underground part of Iris tenifolia, soak it with anhydrous methanol at room temperature for three times (each time 5L anhydrous methanol/1kg material), combine the three extracts, reclaim methanol with a rotary evaporator, and obtain the total extraction of the underground part of Iris tenifolia Compound 241.8g.

2) Chloroform extraction

The extract obtained in step 1) was dissolved in 300mL distilled water, moved into a 1000mL separatory funnel, added an equal volume of chloroform for extraction, the extraction was carried out 5 times, combined the chloroform extracts, and used a rotary evaporator to remove the chloroform in the chloroform extracts. Recover, obtain the 198.6g of the chloroform extract of the underground part of Iris tenifolia, and collect 23.2g of the aqueous phase extract at the same time. The chloroform extract is also the extract provided by the present invention.

Experimental example 2, activity evaluation of the extract provided by the present invention in inhibiting amyloid β aggregation

The chloroform extract and aqueous phase extract obtained in Example 1 were subjected to the following activity analysis.

Reagent source: Aβ protein (1-40, M=4329.84) was purchased from Bachem Company of Switzerland, Thioflavin T (Tht) was purchased from Sigma Company

The activity evaluation experiment method refers to the literature Ce'line Rivie`re etc. Inhibitory activity of stilbenes on Alzheimer's-amyloid fibrils in vitro, Bioorganic & Medicinal Chemistry15: 1160-1167 (2007), the specific steps are as follows:

1. Aβ tangle

1mg Aβ protein + 2ml PBS (phosphate buffer, PH = 7.4) to obtain a 0.5mg/ml Aβ solution, add EDTA (1mM) to promote entanglement, incubate at 37°C, 300rmp for 72h, and store in a -80°C refrigerator.

2. Preparation of Thioflavin T (Tht)

Make up 300uM Tht, store it in a refrigerator at 4°C for later use, and dilute it to 3uM when measuring fluorescence.

3. Sample + Aβ

Add 20ul of Aβ solution with a concentration of 20uM (the Aβ solution saved in step 1 was diluted to 20uM) and 2ul of sample 1 or sample 2 into the tube, mix well, and incubate at 37°C for 30 minutes. Sample 1 is that the chloroform extract that embodiment 1 obtains is dissolved in dimethyl sulfoxide (DMSO), forms the solution of 10ug/ml; Sample 2 is that the aqueous phase extract that embodiment 1 obtains is dissolved in dimethyl sulfoxide (DMSO), forming a 10ug/ml solution.

4. Fluorescence measurement

Take 1500ul of 3uM Tht in a new tube, add 15ul of the mixture incubated in step 3 for fluorescence measurement, excitation wavelength: 442nm, measurement wavelength: 480nm.

Statistical method: SPSS statistical software analysis of variance, **P<0.001.

The experiment was repeated 3 times, and the experimental results are shown in Figure 5. The degree of entanglement of Aβ in the experimental group of the chloroform extract decreased significantly, while the effect of the aqueous phase extract was weak, indicating that the chloroform extract provided by the present invention can prevent the aggregation of Aβ. Because Aβ aggregation is a typical feature of senile dementia, the extract provided by the invention can be used to treat senile dementia.

Claims (8)

1. a preparation method from the extract of Iris tenifolia, comprising the steps of: 1) taking the underground part of Iris tenifolia, soaking it in anhydrous methanol, collecting the extract, and then removing the anhydrous methanol in the extract to obtain the total extract of the underground part of Iris tenifolia; 2) Dissolving the total extract of the underground part of Iris tenifolia obtained in step 1) in water, then adding chloroform for chloroform extraction, collecting the chloroform phase, and then removing the chloroform in the chloroform phase to obtain the extract. 2. The method according to claim 1, characterized in that: in the step 1), soaking in anhydrous methanol is repeated 3 times. 3. The method according to claim 1 or 2, characterized in that: in the step 2), the chloroform extraction is repeated 5 times. 4. the extract prepared by the arbitrary described method of claim 1-3. 5. The application of the extract according to claim 4 in the preparation of products preventing the aggregation of amyloid-beta peptide chains. 6. The use according to claim 5, characterized in that: the product is a medicine. 7. the application of the extract described in claim 4 in the preparation of the medicine for the treatment of senile dementia. 8. A medicine for treating senile dementia, whose active ingredient is the extract according to claim 4.

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