TW201427676A - Neurotrophic agents - Google Patents

Abstract

The present invention relates to an herbal extracts and its preparation method which can effectively promote the growth of neural cells for the purpose of treatment or prevention of Alzheimer's disease. Especially the extracts of Angelica sinensis and Acorus tatrinowii.

Description

Neurotropic substance

The invention relates to a Chinese herbal medicine extract and a preparation method thereof, which can effectively promote the growth of nerve cells for the purpose of treating or preventing Alzheimer's disease. Especially the extract of Angelica and Shichangpu.

Alzheimer's disease (AD) is a neurodegenerative disease in which patients lose memory and multiple cognitive abilities, and even produce changes in personality traits. More than 35 million people worldwide suffer from Alzheimer's disease, one of the most common dementias in the elderly.

The most important pathological changes in Alzheimer's disease are amyloid-like plaques and neurofibrillary tangles containing highly phosphorylated tau. The choline hypothesis was proposed because of the degeneration of acetylcholine nerve in the basal forebrain of Alzheimer's disease patients and the significant reduction of choline acetyltransferase. Cholinergic Hypothesis). At present, four main treatments for Alzheimer's disease are developed according to this theory, including Cognex® tacrine, Aricept® donepezil, and Exelon® Galantamine. Reminyl® rivastigmine, these drugs are all acetycholinesterase inhibitors. In addition, based on the theory of excitatory neurotoxins, another therapeutic drug: memantine (Ebixa®, Witgen®, Manotin®) is a non-selective aspartic acid (N- methyl-D-aspartate (NMDA) receptor inhibitors prevent excitatory nerve damage caused by glutamate, thereby improving the symptoms of Alzheimer's patients. However, the above drug treatment can only achieve a moderate symptom improvement effect, and can not prevent the disease from continuing to deteriorate. So for the treatment of neurodegenerative diseases It is an ideal treatment strategy to try to save the damaged nerve cells and stimulate the regeneration of the nerve cells.

Most of the neurophilic substances are derived from amino acids, which can help the development and survival of neurons. Scientists believe that the introduction of these substances into the brain of Alzheimer's patients may help repair damaged neurons, nerve growth factors. (nerve growth factor; NGF) is one of them. NGF is the first discovered neurotrophin, which has the dual biological functions of neurotrophic nutrition and synaptic growth. It expresses the development, differentiation, growth, regeneration and functional properties of central and peripheral neurons. Has an important regulatory role.

According to previous research results, many neurophilic substances have been discovered by scientists, including Glial-derived Neurotrophic Factor (GDNF) and Brain-derived Neurotrophic (Brain derived Neurotrophic). Factor, BDNF), nerve growth factor (NGF), neurotrophin-3 (NT3), neurotrophin 4 (NT4), and platelet-derived growth factor (PDGF) )Wait. Although these neurotropic substances have been shown to have an activity of promoting nerve cell survival, they have many clinical limitations. Due to the administration of these neurotropic substances into living organisms, these neurotropic substances have a large molecular weight and do not easily cross the Blood Brain Barrier (BBB) to reach the brain. Therefore, it is not practical to directly develop protein drugs for oral administration, so small molecule active molecules such as nerve growth factors can be used as nerve growth factor analogs (mimics) to promote the proliferation of nerve cells and maintain their characteristics, even Promoting its differentiation into functional neuronal cells will help to prevent or treat it as a degenerative neurological disease.

CN 1742968 relates to a medicine for treating vascular dementia, which is composed of Chinese herbal medicines Polygonum multiflorum, astragalus root, salvia root, ligusticum root, acorus root, and albinia. Fruit), ginkgo leaf total flavone, earthworm, leech and gastrodia root. Treating 30 cases of senile dementia with Siqi Decoction (made in Pinellia, Zhu Xi, Shichangpu, Yushi, Yujin), compared with the control group, it has significant curative effect, and has good treatment for senile dementia, stroke and dementia. effect. The compound of Bupleurum, turmeric, perilla, and Shichangpu (called SYJN) has neuroprotective effects and can increase the exposure of rats to long-term unpredictable stress (chronic unpredictable). Stress; CUS) The expression of neurotrophin-3 and nerve growth factor (NGF).

According to the above results, the Chinese herbal compound has an effect in preventing or treating neurodegenerative diseases, but the performance of the compound is still difficult to evaluate separately. The present invention accurately confirms that the extract of Angelica sinensis and Acorus calamus promotes nerve growth, and obviously further provides an effective extract for preventing or treating neurodegenerative diseases.

It is an object of the present invention to provide an herbal extract which promotes the growth of nerve cells, which is an extract of Angelica sinensis and an extract of Acorus calamus.

Another object of the present invention is to provide a method of preparing an herbal extract which promotes the growth of nerve cells.

Another object of the present invention is to provide a use of the herbal extract for promoting nerve cell growth of the present invention for preventing or treating Alzheimer's disease.

The invention is described in detail in the following sections. Other features, objects, and advantages of the invention are readily apparent from the embodiments of the invention and the appended claims.

The first figure is a high performance liquid chromatogram of the water extract of Angelica sinensis.

The second figure is a high performance liquid chromatogram of the aqueous extract of Acorus calamus.

The third figure shows the effect of Angelica extract and Acorus calamus extract on the growth of PC-12 cells.

The fourth figure is the effect of the extract of A. glabra L. against PC-12 cells from apoptosis induced by Aβ _1-42 /apoE4.

The following examples are intended to further illustrate the technical scope of the present invention and are not intended to limit the scope of the application.

Manufacturing Example: Manufacture of Angelica Extract, as described in the general process, via Angelica 100 g of raw medicinal material was crushed, 2 liters of water was added, and the mixture was heated and boiled and refluxed for 1-4 hours. The filtered extract was concentrated and dried to obtain 30% of Angelica sinensis extract, and its high-performance liquid chromatogram is shown in FIG. The extract of the iris can be made as described in the general process, and 100 g of the rehmannia radix can be crushed, 2 liters of water is added, heated and boiled for 1-4 hours, and the filtered extract is concentrated and dried to obtain 30% of the stone. The HPLC chromatogram of the iris extract is shown in Figure 2. The column of high performance liquid chromatography is Thermo Hypersil-Keystone RPC 18, 4.6×250 mm; the mobile phase of high performance liquid chromatography is 20% acetonitrile (Acetonitrile, containing 0.1% formic acid). 0 minutes 20% acetonitrile (Acetonitrile, containing 0.1% formic acid) for 10 minutes, 100% acetonitrile (Acetonitrile, containing 0.1% formic acid) for 40 minutes, 100% acetonitrile (Acetonitrile, containing 0.1% formic acid (formic acid) )) 50 minutes, 20% acetonitrile (Acetonitrile, containing 0.1% formic acid) for 60 minutes; high-performance liquid chromatography flow rate of 1 ml per minute (1 mL / min); the detector is DAD, The wavelength is set at 230, 254, 280 nm.

Test Example 1: An example of an ex vivo model activity test of a herbal extract which promotes nerve cell proliferation. The rat pyelochamocytoma cell line PC-12 cells were used as an in vitro screening mode. PC-12 cells can be induced by nerve growth factor (NGF) to grow synapses and differentiate into cells resembling sympathetic neurons. Therefore, PC-12 cells are often used to study neuronal differentiation and neurodegenerative diseases. Mode. Many current theories of Alzheimer's disease, including Aβ, which leads to increased oxidative stress in nerve cells, and oxidative stress imbalance leading to apoptosis, have been confirmed in the PC-12 cell model. Studies have also been conducted to induce apoptosis in PC-12 cells using Aβ, and to establish a cell model that mimics Alzheimer's disease as a cell model for screening for anti-Alzheimer's disease drugs.

Rat adrenal medullary pheochromocytoma cell line PC-12 cells were cultured for 24 hours in medium containing fetal bovine serum and horse serum, and test samples were added to treat PC-12 cells for 48 hours to observe cell type and MTT. Color analysis was used to assess cell viability.

* Test Analysis The effect of the extract of the present invention on the growth and differentiation of PC-12 cells was observed by microscopic observation of cell type.

* Experimental analysis The growth and proliferation of the extract of the present invention on PC-12 cells was performed by calculating the cell viability in each well of a 96-well microplate, and the survival rate of the cancer cells was evaluated by MTT colorimetric assay.

※MTT color analysis: MTT is a tertrazolium salt, the full name is 3-[4,5-Dimenthylthialzol-2-yl]2,5-diphenyltetrazolium bromide, which is a yellow dye that can be absorbed by living cells and in the granules. The body is reduced to blue formazan by succinate-tetrazolium reductase, which is often used to screen the effects of substances on cell growth and proliferation.

According to the method of the above-mentioned ex vivo model activity test, more than twenty kinds of Chinese herbal medicine extracts were used to treat PC-12 cells for 48 hours, and it was found that the extract of Angelica sinensis and the extract of Acorus calamus L. can promote the proliferation of PC-12 cells, and the proliferation effect thereof. They were 278.9% and 171.3%, respectively, which was better than the control group. The proliferation effect of Ginkgo biloba extract EGb761 was 124.8%, among which Angelica sinensis extract promoted nerve cell growth (278.9%) and nerve growth factor NGF treatment (50 ng/mL). The group effect (310.8%) is equivalent (as shown in Figure 3).

Test Example 2: An example of an ex vivo model activity test for protecting herbal cells from apoptosis. The Aβ _1-42 powder was dissolved in Tris-HCl buffer (100 mM, pH 7.4) and purified and replaced with buffer apoE4 protein mixed in iDMEM culture medium, which was formulated to contain Aβ _1-42 5 μM and apoE4 20 The culture solution of μg/mL was then cultured in a cell culture incubator (37 ° C, 5% CO ₂ /95% air) for 48 hours to allow Aβ _1-42 to form a complex with apoE4. The culture medium containing Aβ _1-42 /apoE4 and the herbal extract to be tested were simultaneously treated with PC-12 cells that had been inoculated into a 96-well microplate and stimulated for differentiation for 48 hours, and then the Chinese herbal medicine was used to protect PC-12 cells by MTT method. The ability to cause apoptosis due to Aβ _1-42 /apoE. In this test, Ginkgo biloba extract EGb761 was used as a control group. Numerous studies have indicated that EGb761 has protective effects on nerve cells, including direct inhibition of necrosis and apoptosis of nerve cells. Studies have also indicated that EGb761 has a hippocampal gyrus for learning and memory of the main division. The protective effect, in addition, EGb761 can also increase the α-secretase activity in SD rats, and relatively reduce the production of Aβ. EGb761 also inhibits the aggregation of Aβ and reduces the activity of caspase-3, thus confirming that EGb761 has the potential to delay the progression of Alzheimer's disease.

The extract of the iris of the present invention was treated according to the method of the above-described ex vivo model activity test, and it was found that the extract of the iris of the present invention can increase the survival rate of PC-12 cells under the toxicity of Aβ _1-42 /apoE4 (188.5%). The effect is superior to the control of Ginkgo biloba extract EGb761 of 153.1% (as shown in Figure 4).

Claims (7)

A neurotropic substance comprising an herbal extract that is effective in promoting the growth of nerve cells. The herbal extract according to item 1 of the patent application scope is an extract of angelica. The herbal extract described in item 1 of the scope of the patent application is an extract of Acorus calamus. The extract of Angelica sinensis according to item 2 of the patent application refers to an aqueous extract. The extract of sarcophagus according to item 3 of the patent application refers to an aqueous extract. The extract of Acorus calamus according to item 3 of the patent application refers to an ethanol extract. Promoting nerve cell growth according to item 1 of the scope of the patent application means preventing and/or treating Alzheimer's disease.