TWI787527B - USE OF EMBLICA EXTRACTION IN PREPARINGPHARMACEUTICAL COMPOSITION FOR REDUCING PRODUCTION OF AMYLOID β-40 ASSOCIATED WITH NEURODEGENERATIVE DISEASE - Google Patents

Abstract

A use of an emblica extraction in preparing a pharmaceutical composition for treating or preventing a neurodegenerative disease comprises providing the pharmaceutical composition that includes the emblica extraction to the cell, so that the generation of one or more biological factors in this cell is lower than that in other cell without receiving the pharmaceutical composition that includes the emblica extraction.

Description

Emlical extracts used in preparations to reduce neurodegenerative diseases Use of a pharmaceutical composition for generating β-amyloid 40

The present invention relates to the preparation of a pharmaceutical composition for treating or preventing neurodegenerative diseases, in particular to the application of a medicinal composition for treating or preventing neurodegenerative diseases using the extract of Amla emblica.

Nerve cells, also known as neurons, are one of the structural and functional units of the nervous system. They can sense changes in the environment and then transmit information to other neurons for a collective response. There are tens of billions of nerve cells in the human brain, and the nerve cells are connected to each other by synapses to jointly perform the functions of the brain.

Alzheimer's disease, commonly known as Alzheimer's disease, is a neurodegenerative disease. Recent studies have pointed out that amyloid plaques (Amyloid Plaques) produced by the formation and aggregation of β-amyloid tend to reduce the connection of nerve cells in the early brain development period, and easily lead to the degeneration or apoptosis of nerve cells in old age. Death is considered the leading cause of Alzheimer's disease.

In view of the above, an embodiment of the present invention provides the use of an extract of Amla emblica for preparing a pharmaceutical composition for treating or preventing neurodegenerative diseases.

According to the use of the emblica extract disclosed in the present invention for preparing a pharmaceutical composition for treating or preventing neurodegenerative diseases, the extract of emblica is provided to cells, so that the generation of biological factors related to neurodegenerative diseases in the cells The amount was lower than the production amount of biological factors associated with neurodegenerative diseases in another cell from which the extract of Amla emblica was not obtained. In this way, after the subject's nerve cells are subjected to the action of the emblica extract aqueous solution or the pharmaceutical composition containing the emblica extract, the production of biological factors associated with neurodegenerative diseases in the cells is reduced.

The above description of the disclosure and the following description of the implementation are used to demonstrate and explain the spirit and principle of the present invention, and provide a further explanation of the patent application scope of the present invention.

The detailed features and advantages of the present invention are described in detail below in the implementation mode, and its content is enough to make any person familiar with the related art understand the technical content of the present invention and implement it accordingly, and according to the content disclosed in this specification, the scope of the patent application and the drawings , anyone skilled in the art can easily understand the purpose and advantages of the present invention. The following examples are to further describe the concept of the present invention in detail, but not to limit the scope of the present invention in any way.

Emblica (such as Phyllanthus Emblica or Emblica Officinale), also known as yuzu, tangerine, Amalaka, Pokok Melaka, Indian Gooseberry, belongs to the Euphorbiaceae genus Emblica ) is a deciduous subtree distributed from India to Malaysia and southern China, and India is generally considered to be its origin.

The method of obtaining the emblica extract used in the present invention is, for example, using carbon dioxide as a supercritical fluid to extract the emblica fruit, or using methanol, ethanol, acetone, ethyl acetate, and an aqueous sodium chloride solution with a concentration of 0.1 to 5% by weight ( salt water), potassium chloride aqueous solution with a concentration of 0.1 to 5% by weight, an aqueous solution of calcium chloride with a concentration of 0.1 to 5% by weight, an aqueous solution of magnesium chloride with a concentration of 0.1 to 5% by weight, and an aqueous solution with a concentration of 0.1 to 5% by weight 5% sodium chloride ethanol solution, potassium chloride ethanol solution with a concentration of 0.1 to 5% by weight, calcium chloride ethanol solution with a concentration of 0.1 to 5% by weight or magnesium chloride ethanol solution with a concentration of 0.1 to 5% by weight The solution is used as a solvent to extract the emblica fruit to obtain a primary extract. Next, the primary extract is filtered and purified to obtain the extract of Amla emblica used in the present invention. The emblica extract can be dried by spray drying (Spray dry) or vacuum drying to obtain an easy-to-preserve emblical extract powder.

In one or more embodiments of the present invention, the emblica extract contains 0.45-14.52 mg/g gallic acid (Gallic acid) and 1.6-4.2 mg/g ellagic acid (Ellagic acid).

The structure of gallic acid is shown in Formula 1. formula one

The structure of ellagic acid is shown in formula 2. formula two

Please refer to FIGS. 1A-1C and FIGS. 2A-2C . Fig. 1A is the tandem mass spectrometer collection of the gallic acid standard product in one or more embodiments of the present invention; Fig. 1B is the concentration gradient figure of the gallic acid in one or more embodiments of the present invention; Fig. 1C It is the tandem mass spectrometer spectrum contained in the emblica extract in one or more embodiments of the present invention; Fig. 2A is the tandem mass spectrometer spectrum of the ellagic acid standard in one or more embodiments of the present invention; Fig. 2B is a concentration gradient diagram of ellagic acid in one or more embodiments of the present invention; and FIG. 2C is a tandem mass spectrometer spectrum contained in the extract of Amla emblica in one or more embodiments of the present invention.

As shown in Figure 1A, the Agilent 1100 LC/ API 4000 tandem mass spectrometer collection of gallic acid standards has a characteristic peak signal at 5.61 ± 0.5 minutes, and as shown in Figure 1C, one or more implementations of the present invention The spectrum of the Agilent 1100 LC/ API 4000 tandem mass spectrometer of the extract of Amla emblica used in the example also has a characteristic peak signal at 5.61±0.5 minutes, indicating that it contains gallic acid. Further, the area of the characteristic peak at 5.61 ± 0.5 minutes of the emblical extract was calculated, and compared to the concentration gradient diagram shown in Figure 1B, the concentration of gallic acid contained in the emblical extract could be deduced. Taking the example in FIG. 1C as an example, the extract of Amla emblica contained gallic acid at a concentration of 0.91 mg/g.

As shown in Figure 2A, the Agilent 1100 LC/API 4000 tandem mass spectrometer spectrum of the ellagic acid standard has a characteristic peak signal at 7.29 ± 0.5 minutes, and as shown in Figure 2C, one or more embodiments of the present invention The Agilent 1100 LC/ API 4000 tandem mass spectrometer spectrum of the used emblica extract also has a characteristic peak signal at 7.29±0.5 minutes, indicating that it contains ellagic acid. Further, the area of the characteristic peak at 7.29 ± 0.5 minutes of the extract of Amla emblica was calculated, and compared to the concentration gradient diagram shown in Figure 2B, the concentration of ellagic acid contained in the extract of Amla emblica could be deduced. Taking the example in FIG. 2C as an example, the emblica extract shown therein contains ellagic acid at a concentration of 1.93 mg/g.

Further, the above-mentioned Figures 1C and 2C are tandem mass spectrometer spectra generated by using the same emblica extract as an input source and performing mass analysis on the chromatography products of different time periods. That is, the emblica extract containing the tandem mass spectrometer patterns shown in FIGS. 1C and 2C contained gallic acid at a concentration of 0.91 mg/g and ellagic acid at a concentration of 1.93 mg/g.

When the concentration of 50 to 100 micrograms per milliliter (μg/ml) of emblica extract aqueous solution or the pharmaceutical composition containing the concentration of emblica extract is provided to the cells, the biological factors associated with neurodegenerative diseases in the cells The production of biofactors associated with neurodegenerative diseases was lower in another cell that did not receive the emblica extract. Wherein, the emblica extract described here is, for example, the extract of emblica described in the previous embodiment with the tandem mass spectrometer spectrum in Figure 1C and 2C; the neurodegenerative disease is, for example, Alzheimer's disease; the The biological factors associated with neurodegenerative diseases include at least one β-amyloid protein; and the cells can be neural cells, such as neural cells of Down's syndrome patients (DS-iPSCs).

The method of providing the emblica extract or the pharmaceutical composition containing the emblica extract to the cells, for example, is to ingest the emblica extract or the pharmaceutical composition containing the emblica extract orally in the form of food. When the emblica extract is provided to the cells in the form of food, the effective dosage of the emblica extract is 540 mg to 1080 mg. The effective dose here is calculated according to the conversion formula between the effective dose of the cell experiment and the kilograms of the human body and the general conversion rules commonly used in the field. The conversion formula is as follows: human effective dose (mg) = effective dose of cell experiment (μg/ml) × mouse weight (kg) × conversion factor × human body weight (kg). The conversion factor is obtained by looking up the conversion factor table of the dose per kilogram of body weight for animals and humans. When the weight of the mouse is 20g and the body weight in kilograms is 60kg, the conversion coefficient is 9.01.

For the convenience of ingesting the emblica extract or the pharmaceutical composition containing the emblica extract by mouth, the emblica extract or the pharmaceutical composition containing the emblica extract can be made into liquid, solid, Processed products of emblica extract in the form of granules, powders, pastes or gels. The processed products of emblica extract can be mixed with excipients or flavoring agents as additives to enhance the flavor and facilitate consumption.

Examples of excipients are wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin and other starches; crystalline cellulose; lactose, glucose, granulated sugar, reduced maltose, maltose, fructooligosaccharides, emulsified oligosaccharides Sugars such as sorbitol, erythritol, xylitol, lactitol, mannitol and other sugar alcohols.

Taste agents are, for example, various fruit juice extracts such as longan extract, lychee extract, and grapefruit extract; various fruit juices such as apple juice, orange juice, and lemon juice; various spices such as peach flavors, plum flavors, and yoghurt flavors; Various sweeteners such as potassium phosphate, sucralose, erythritol, oligosaccharides, mannose, xylitol, and isomerized sugars; various sour agents such as citric acid, malic acid, tartaric acid, and gluconic acid; green tea, oolong tea, Various tea ingredients such as Banaba Tea, Eucommia tea, Tieguanyin tea, Coix tea, Aescin tea, wild rice stem tea, and kelp tea; Coffee Arabica, Coffee Robusta, Various coffee ingredients such as Liberia (Coffee Liberica).

Furthermore, the emblica extract or the pharmaceutical composition containing the emblica extract can also be coated in capsules to facilitate oral intake of the emblica extract. The emblica extract or the pharmaceutical composition containing the emblica extract can be coated in a hard capsule in the form of dry powder. The emblica extract or the pharmaceutical composition containing the emblica extract can also be coated in soft capsules in the form of solution, suspension, paste, powder or granules.

Oils used to dissolve or disperse Amla emblica extract in soft capsules are calyx pear oil, almond oil, linseed oil, cumin oil, white sage oil, olive oil, olive squalene, sweet orange oil, pectoralis Orange roughy oil, sesame oil, garlic oil, cocoa butter, pumpkin seed oil, chamomile oil, carrot oil, cucumber oil, tallow fatty acid, macadamia nut oil, lingonberry oil, brown rice germ oil, rice oil, wheat germ oil, safflower oil, shea butter, liquid shea butter, perilla oil, soybean oil, evening primrose oil, camellia oil, corn oil, rapeseed oil, saw palmetto extract oil, coix seed oil, Peach Kernel Oil, Celery Seed Oil, Castor Oil, Sunflower Oil, Grapeseed Oil, Borage Oil, Macadamia Oil, Meadowfoam Oil, Cottonseed Oil, Peanut Oil, Turtle Oil, Mink Oil, Egg Butter, Fish oil, palm oil, palm kernel oil, wood wax, coconut oil, long-chain/medium-chain/short-chain fatty acid triglycerides, diglycerides, tallow, lard, squalene, squalane, pristine Alkanes, and hydrogenated products of such oils and fats. Among them, borage oil and evening primrose oil contain a large amount of gamma-linolenic acid (Gamma-linolenic acid, GLA), gamma-linolenic acid is an essential fatty acid in the human body, which has the functions of moisturizing, promoting cell regeneration and enhancing the activity of brown fat. Degree to promote fat burning function.

In addition, colorants, preservatives, tackifiers, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, pH value regulators, etc. are added in accordance with government regulations It can also be added to processed products of Amla emblica extract according to the dosage standards stipulated by government units and the needs of processing and production.

Please refer to Figures 3 to 5 to illustrate the use of the emblica extract proposed in the present invention to reduce the number of biological substances associated with neurodegenerative diseases in cells through the cytotoxicity test and the comparison between Examples 1 to 3 and the control example. Use of a factor (eg at least one β-amyloid protein) in a pharmaceutical composition for treating or preventing neurodegenerative diseases. Among them, Fig. 3 is a schematic diagram of the results of the cytotoxicity test of various emblica extract concentrations in an embodiment of the present invention; Fig. 4 is the β- Schematic diagram of the measurement results of the production of amyloid 40; FIG. 5 is a schematic diagram of the measurement results of the production of β-amyloid 42 in the cells of Example 1, Example 2, Example 3 and the control example of the present invention .

The emblica extract used in Examples 1 to 3 of the present invention and the cytotoxicity test is to immerse the fruit of Emblica Officinalis in an aqueous solution of 1% by weight of sodium chloride, and then heat it at 65°C to 75°C. Heated in a steam bath (Steam bath) for 1 hour, then filtered, then refrigerated for 3 days, and then passed through a spray drying (Spray dry) procedure to obtain the powder of Amla emblica extract. The dried emblica extract is brown powder. The emblica extract contains 0.45~14.52 mg/g gallic acid and 2.6~4.2 mg/g ellagic acid. The emblica extract of the present invention is not limited to the extract of Emblica Officinalis , other extracts of emblica with different scientific names but similar components also have the same effect.

The cells used in the experiments corresponding to the schematic diagrams of the results in Figures 3 to 5 can be nerve cells, especially nerve cells from Down syndrome patients (DS-iPSCs) as mentioned above. Specifically, the purpose proposed by the present invention is to provide a pharmaceutical composition containing emblica extract to cells, so that the production of at least one β-amyloid protein in the cells is lower than that of cells that do not contain emblica extract. The production amount of at least one β-amyloid protein in another cell of the pharmaceutical composition, wherein the pre-amyloid protein gene (Amyloid precursor protein, APP) expressing this protein is located on chromosome 21, and Down's syndrome patients There is an extra copy of chromosome 21, so the experiments described below use nerve cells from patients with Down's syndrome as experimental samples, but this case does not limit the application to nerve cells from patients with Down's syndrome.

The cell culture system adopts the method of cell subculture, in which the medium condition used in the first generation is Neurobasal medium containing 2% B27 cell culture supplement (B27 supplement), and the medium condition used in the subculture is containing 2% B27 Neurobasal medium with cell culture supplement (B27 supplement) and 10 μM ROCK inhibitor (ROCK inhibitor Y-27632).

Figure 3 is a schematic diagram of the results corresponding to the cytotoxicity test of the extract of Amla emblica. In the cytotoxicity test, the aqueous solution of extracts of Amla emblica with various predetermined concentrations was administered to the above-mentioned nerve cells. In detail, the cytotoxicity test includes the following steps.

Day 1: Implant 200,000 cells (approximately 1ml) into each well of a plate (for example, 24 wells), and add 250 μL of DMEM/F12 containing 1% Matrigal (Dulbecco's Modified Eagle's Medium/Nutrient Mixture F -12), and carry out the coating (Coating) process at room temperature for more than 1 hour. DMEM/F12 was carefully aspirated from the wells prior to the seeding procedure.

The next day: Add 200 μL of various predetermined concentrations of emblica extract aqueous solutions (including 20, 25, 50, 100, 150, 250, 500 and 1000 μg/ml in the experiment shown in Figure 3) to each of the cells where the cells are located. hole and soak for 24 hours at 37°C.

Day 3: Add 500 μL of 10% biological indicator (such as Alamar Blue) to each well, and incubate for 4 hours in the dark at 37 degrees Celsius, and then detect OD530/595nm by spectrophotometer Count the viable cell rate.

As shown in Figure 3, when the concentration of the aqueous solution of the emblica extract is below 100 micrograms (μg/ml) per milliliter (inclusive), the cell survival rate does not decrease significantly compared with the control example without providing the aqueous solution of the emblica extract, It means that the aqueous solution of emblica extract with a concentration below 100μg/ml (inclusive) will not produce harmful toxicity to cells.

Therefore, in the experiment corresponding to FIG. 4 and FIG. 5 , the aqueous solution of the emblica extract at a concentration that would not cause harm to the cells was provided to the cells, and the concentration of β-amyloid protein in the cells was measured. Among them, the cells treated with the aqueous solution of emblica extract at a concentration of 25 μg/ml, 50 μg/ml and 100 μg/ml were respectively used as the first embodiment, the second embodiment and the third embodiment, and the cells not treated with the aqueous solution of the emblica extract were used as Control example.

As mentioned above, the use of the emblica extract proposed in the present invention for the preparation of a pharmaceutical composition for treating or preventing neurodegenerative diseases includes providing the pharmaceutical composition containing the emblica extract to cells, so that at least The production of one beta-amyloid is lower than the production of at least one beta-amyloid in another cell that has not received the pharmaceutical composition comprising the emblica extract. Wherein, the at least one β-amyloid protein is particularly associated with Alzheimer's disease.

Furthermore, on the cell membrane of nerve cells, amyloid precursor protein (Amyloid Precursor Protein, APP) for cell expression is inlaid. This protein will be hydrolyzed by three enzymes (Proteolysis), namely α-secretase, β-secretase, and γ-secretase (alpha, beta, and gamma-Secretase). Among them, α-secretase and γ-secretase can hydrolyze APP to release a peptide containing 40 amino acids, called β-amyloid 40 (Aβ40); and α-secretase and β- Secretory enzymes hydrolyze APP to release a peptide containing 42 amino acids, called β-amyloid 42 (Aβ42). The released β-amyloid proteins 40 and 42 tend to tangle with each other and aggregate to form amyloid plaques. The accumulation of plaques will lead to damage or even death of nerve cells, and then lead to Alzheimer's disease. From the above, it can be seen that the production of β-amyloid 40 and β-amyloid 42 in cells is related to the probability of suffering from Alzheimer's disease. Therefore, the experiments corresponding to FIG. 4 and FIG. 5 take the β-amyloid protein 40 and the β-amyloid protein 42 as measurement targets respectively.

In each embodiment, the flow of giving the aqueous solution of the emblica extract to the cells and measuring the concentration of β-amyloid in the cells includes the following steps.

Day 1: Implant 200,000 cells (approximately 1ml) into each well of a plate (for example, 24 wells), and add 250 μL of DMEM/F12 containing 1% Matrigal (Dulbecco's Modified Eagle's Medium/Nutrient Mixture F -12), and carry out the coating (Coating) process at room temperature for more than 1 hour. DMEM/F12 was carefully aspirated from the wells prior to the seeding procedure.

The next day: add 1ml of a predetermined concentration of emblica extract aqueous solution (25 μg/ml in Example 1; 50 μg/ml in Example 2; 100 μg/ml in Example 3) into the wells where the cells are located.

Day 5: The experimental sample was centrifuged at 1000 rpm and 4°C for 5 minutes, and the supernatant was stored at -80°C.

Day 6: Measure the concentrations of β-amyloid 40 and β-amyloid 42 in the experimental samples with ligand screening reagents (kit) for β-amyloid 40 and β-amyloid 42, respectively.

The aforementioned medium for cell culture and the cell environment for the measurement of β-amyloid protein do not contain raw materials for the production of high-density lipoprotein (High-Density Lipoprotein, HDL), that is, in the above-mentioned experimental mechanism, there will be no There is formation of high-density lipoprotein.

The measurement results of β-amyloid protein are shown in Figure 4 and Figure 5, the concentration of β-amyloid protein 40 in the cells of Example 3 (100 μg/ml) was significantly lower than that of the control example (p>0.05); Example The concentrations of β-amyloid protein 42 in the nerve cells of the second (50 μg/ml) and the third (100 μg/ml) were significantly lower than those of the control example (p>0.05). It can be seen from Figure 4 and Figure 5 that compared with the nerve cells not treated with the aqueous solution of the emblica extract, the nerve cells treated with the aqueous solution of the emblical extract of 50 μg/ml and the nerve cells treated with the aqueous solution of the emblical extract of 100 μg/ml The production of β-amyloid 40 and 42 in nerve cells is low. It can be inferred that the aqueous solution of emblica extract can reduce the condition of nerve cell death triggered by the accumulation of amyloid plates, thereby reducing the risk of Alzheimer's disease in the subjects Chances of mutism.

According to the use of the emblica extract disclosed in the present invention for preparing a pharmaceutical composition for treating or preventing neurodegenerative diseases, the extract of emblica is provided to cells to increase the production of biological factors related to neurodegenerative diseases in the cells The production of biological factors associated with neurodegenerative diseases was lower than that in another cell that did not receive the emblica extract. In this way, after the subject's nerve cells are subjected to the action of the emblica extract aqueous solution or the pharmaceutical composition containing the emblica extract, the production of biological factors associated with neurodegenerative diseases in the cells is reduced.

Although the present invention is disclosed by the aforementioned embodiments, they are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, all changes and modifications are within the scope of patent protection of the present invention. For the scope of protection defined by the present invention, please refer to the appended scope of patent application.

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FIG. 1A is a tandem mass spectrometer spectrum of a gallic acid standard in one or more embodiments of the present invention. Figure 1B is a concentration gradient diagram of gallic acid in one or more embodiments of the present invention. Fig. 1C is a tandem mass spectrometer spectrum contained in the extract of Amla emblica in one or more embodiments of the present invention. Fig. 2A is a tandem mass spectrometer spectrum of an ellagic acid standard in one or more embodiments of the present invention. Figure 2B is a concentration gradient diagram of ellagic acid in one or more embodiments of the present invention. Fig. 2C is a tandem mass spectrometer spectrum contained in the extract of Amla emblica in one or more embodiments of the present invention. Fig. 3 is a schematic diagram of the results of cytotoxicity tests of various concentrations of Amla emblica extracts in an embodiment of the present invention. 4 is a schematic diagram of the measurement results of the production of β-amyloid protein 40 in the cells of Example 1, Example 2, Example 3 and the control example of the present invention. 5 is a schematic diagram of the measurement results of the production of β-amyloid protein 42 in the cells of Example 1, Example 2, Example 3 and the control example of the present invention.

Claims (7)

Use of an emblica extract for preparing a pharmaceutical composition for reducing the production of β-amyloid 40 associated with neurodegenerative diseases, wherein the emblica extract is provided to a cell in the form of an aqueous solution of emblica extract , and the concentration of the emblica extract aqueous solution is 50 to 100 micrograms per milliliter (μg/ml), wherein the emblica extract contains 0.45~14.52mg/g of gallic acid (Gallic acid) and 2.6~4.2mg /g of ellagic acid (Ellagic acid). The use as described in claim 1, comprising: providing the pharmaceutical composition comprising the extract of Emlical emblica to a cell, so that the production amount of the β-amyloid protein 40 in the cell is lower than that without the extract containing the emblical seed The production amount of the β-amyloid protein 40 in another cell of the pharmaceutical composition of the extract. The use as described in claim 1, wherein the neurodegenerative disease is Alzheimer's disease. The use as described in claim 1, wherein the neurodegenerative disease is associated with neurodegeneration of Down's syndrome. The use as described in claim 1, wherein the emblica extract is obtained by extracting the fruit of emblica with saline as a solvent. The use as described in claim 1 comprises eating the pharmaceutical composition comprising the extract of Amla emblica. The use as described in claim 6, wherein the effective dosage of the emblica extract is 540 mg to 1080 mg.

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