KR102151594B1 - Efficient extracting method of punicalin or ellagic acid from pomegranate - Google Patents

Abstract

The present invention relates to a method for efficiently extracting punicalin or ellagic acid from pomegranate. The present invention selects varieties with excellent content of punicalin and ellagic acid, and performs high-temperature pressurization treatment in a solid state using discarded pericarp, and then ultrasonic treatment, and further treatment of enzymes to extract water and high-temperature pressurization alone. In comparison, a high content of punicalin or ellagic acid was obtained. In addition, since the pomegranate extract with an enhanced ellagic acid content extracted by the method of the present invention has an anti-stress or degenerative brain disease improvement effect, it can be used as a health functional food composition for preventing or ameliorating stress and degenerative brain diseases. .

Description

Efficient extracting method of punicalin or ellagic acid from pomegranate}

The present invention relates to a method for efficiently extracting punicalin or ellagic acid from pomegranate peel.

Pomegranate ( Punica granatum Linne) is a deciduous broadleaf belonging to the pomegranate family (Punicaceae), and has been widely used in diseases such as an astringent, dysentery, anthelmintic or ulcer for medicinal purposes. In particular, pomegranate peel accounts for 30% of the fruit, and is known to be effective in respiratory diseases and skin diseases.

Pomegranate skins are particularly rich in tannins and have been widely used as astringent dry placebos. These are mainly ellagitannin, where hexahydroxydiphenic acid (HHDP) is linked to the structure of glucose or quinic acid and ester bonds. Has been. It is hydrolyzed when exposed to strong acids, and is divided into punicalagin, which is a water-soluble phenolic structure of a polymer size, and the funicalazine is again composed of punicalin (4,6-(SS)-gallagyl D-glucose) and four polyphenols. It is divided into ring-shaped ellagic acid or hexahydroxydiphenic acid. In particular, ellagic acid is contained in about 45 kinds of fruits and nuts such as strawberries, grapes, pomegranate, raspberries (raspberries), bilberry (blueberries), and walnuts. Pomegranate peel per gram is 1.78~12.8mg , Red raspberry contains 1.50mg, strawberry contains 0.64mg, walnut contains 0.59mg.

Punicalin and ellagic acid have various effects, but it is difficult to extract large quantities from pomegranate. Therefore, there is a need for research on a method of obtaining a large amount of punicalin or ellagic acid from the same amount of raw materials by more efficiently extracting punicalin or ellagic acid from pomegranate. In addition, pomegranate skin is a material discarded in the industrial process, and if it is used, it can be used as a new source of income and environmental protection.

On the other hand, Korean Patent Publication No. 2011-0037347 discloses a method for efficiently separating ellagic acid from pomegranate through acid hydrolysis and functional cosmetics containing the same, but punicalin or punicalin from pomegranate peel using high temperature pressure and ultrasound The present invention regarding a method for efficiently extracting ellagic acid has not been disclosed.

The present invention was derived from the above requirements, and selected pomegranates through high-temperature pressurization, ultrasonic treatment, and enzyme treatment by selecting varieties and sites with a high content of punicalin or ellagic acid through comparison of pomegranate varieties and parts. Providing a method for efficiently extracting punicalin or ellagic acid from a site of a variety, and confirming the anti-stress or degenerative brain disease improvement effect of the pomegranate extract with an enhanced ellagic acid content prepared by the above manufacturing method, completing the present invention I did.

In order to solve the above problems, the present invention

1) subjecting the pomegranate skin powder to a high temperature pressure treatment; And

2) dissolving the pomegranate peel powder subjected to the high-temperature pressurization of step 1) in a solvent and then ultrasonicating the powder; providing a method of efficiently extracting punicalin or ellagic acid from pomegranate.

In addition, the present invention provides a health functional food composition for preventing or improving stress-related diseases and degenerative brain diseases, comprising a pomegranate extract having an enhanced ellagic acid content extracted by the above method as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of stress disorders and degenerative brain diseases, comprising a pomegranate extract having an enhanced ellagic acid content extracted by the above method as an active ingredient.

The present invention relates to a method for efficiently extracting punicalin or ellagic acid from pomegranate. The present invention compares the content of funicalazine, funicalin, ellagic acid, and gallic acid in the pericarp, fruit seeds and fruit juice extracts of flower scent, red scent, Oedo 1 and Oedo 2, and the content of punicalin and ellagic acid is excellent, Red-flavored peel with a high total content was selected as a raw material for efficiently extracting punicalin or ellagic acid.

In addition, it was confirmed that the content of punicalin or ellagic acid in the hot-pressurized extract was high by water extraction, methanol extraction, ultrasonic extraction, or high-temperature pressurization extraction of the red-scented peel. It was confirmed that the content of kaline or ellagic acid was further enhanced.

In addition, by reacting with Aspergillus niger- derived glucanase and arabinase in addition to the ultrasonic treatment product, the effect of further enhancing the ellagic acid content was confirmed.

In addition, the pomegranate extract with enhanced ellagic acid content extracted by the method of the present invention has an antioxidant effect, improves the survival rate of SH-SY5Y cells, a neuroblastoma from glutamate stimulation, and cortisol increased by glutamate stimulation ( cortisol) and reducing the activity of acetylcholineesterase, which is increased by stimulation of glutamate, contains pomegranate extract with enhanced content of ellagic acid extracted by the above extraction method. The composition of the present invention is a health functional food composition for preventing or improving stress diseases and degenerative brain diseases; Or it can be used as a pharmaceutical composition for the prevention or treatment of stress diseases and degenerative brain diseases.

1 is a C18-reverse phase column analysis result according to the first extraction method of the present invention.
2 is a C18-reverse phase column analysis result according to the extraction method of water extraction, high-temperature pressurization, or ultrasonic treatment after high-temperature pressurization of the present invention.
3 is a result of TLC analysis of the degree of ellagic acid generation after treatment with eight commercial enzymes (SPL, Viscozyme, CL, CP, PR, UF, TH, BG2) in a pomegranate sample subjected to high temperature pressure treatment. EA is ellagic acid, GA is gallic acid, PC is funicalin, PCG is funicalazine, and EX is loaded with pomegranate samples.
4 is a C18-reverse phase column analysis result according to the extraction method of water extraction, high temperature pressurization, ultrasonic treatment after high temperature pressurization, or ultrasonic treatment after high temperature pressurization and then enzyme treatment according to the present invention.
5 is a result of confirming the molecular weights of funicalin (A) and ellagic acid (B) by ESI(-)-MS/MS analysis.
6 is a three-dimensional schematic diagram of a reaction surface analysis of a punicalin product reacted by the central synthesis planning method according to pomegranate skin content, high temperature pressing time, and ultrasonic treatment time.
7 is a 3D schematic diagram of a reaction surface analysis of an ellagic acid product reacted by the central synthesis planning method according to pomegranate skin content, ultrasonic treatment time, and enzyme treatment concentration.
Figure 8 is a result of confirming the DPPH radical scavenging ability of the pomegranate extract with improved ellagic acid content of the present invention. VitC is vitamin C, and EA is ellagic acid.
9 is a result of confirming the protective effect of cranial nerve cells of the pomegranate extract having an enhanced ellagic acid content of the present invention. Glutamate is a substance that induces cranial nerve cell death. Theanine is a positive control, and Buffer is a solvent-treated sample.
FIG. 10 is a view confirming the effect of inhibiting cortisol secretion of the pomegranate extract with enhanced ellagic acid content of the present invention, and glutamate is a cortisol secretion-inducing substance. Theanine is a positive control, and Buffer is a solvent-treated sample.
11 is a view confirming the acetylcholinesterase activity inhibitory effect of the pomegranate extract with enhanced ellagic acid content of the present invention, glutamate is an acetylcholinesterase activity-inducing substance. Tacrine is a positive control.

The present invention

1) subjecting the pomegranate skin powder to a high temperature pressure treatment; And

2) dissolving the pomegranate peel powder subjected to the high-temperature pressurization of step 1) in a solvent and then ultrasonicating the powder; it relates to a method of efficiently extracting punicalin or ellagic acid from pomegranate.

The pomegranate peel powder of the present invention may be any pomegranate cultivar, preferably a red scented cultivar, but is not limited thereto.

The high-temperature pressurization treatment of step 1) may be performed at 110 to 130°C for 10 to 200 minutes, preferably at 121°C for 20 to 170 minutes, and more preferably at 121°C for 165 minutes. It is to process, but is not limited thereto.

In the high-temperature pressurization treatment, pressure is applied to a pressure of 1.1 to 2.0 atm, preferably a pressure of 1.3 to 1.7 atm, and more preferably, a pressure of 1.5 atm is applied, but is not limited thereto.

The solvent of step 2) may be water, a lower alcohol of C ₁ ~ C ₄ or a mixture thereof, preferably water or ethanol as a solvent, more preferably water as a solvent to dissolve However, it is not limited thereto.

The ultrasonic treatment of step 2) may be performed by irradiating for 3 to 7 seconds at an intensity of 600 to 800 W, 15 to 25 kHz, and then stopping 0.5 to 1.5 seconds for a total of 10 to 30 minutes, preferably 700 W, After 5 seconds of irradiation at a 20 kHz intensity, the process of stopping 1 second is performed for a total of 20 minutes, but is not limited thereto.

The present invention may further include a step of treating the enzyme after step 2), and preferably, after step 2), glucanase and arabinase are treated. The step of; may further include, more preferably after step 2), treating glucanase and arabinase for 0.5 to 10 hours; It includes, but is not limited to this.

Glucanase (glucanase) and arabinase (arabinanase) of the present invention is an enzyme derived from Aspergillus niger ( Aspergillus niger ).

Glucanase and arabinase can be treated for 0.5 to 10 hours, preferably for 1 to 7 hours, more preferably for 6 hours, but limited thereto. It is not.

In one embodiment of the present invention, the method for efficiently extracting punicalin from pomegranate

1) High-temperature pressurization of pomegranate skin powder at 110-130° C. for 10 to 200 minutes; And

2) After the above step 1), after dissolving the pomegranate peel powder subjected to high-temperature pressurization in water, irradiation for 3 to 7 seconds at an intensity of 600 to 800 W and 15 to 25 kHz, the process of stopping 0.5 to 1.5 seconds is stopped for a total of 10 to 30 minutes. It may include, but is not limited to, the step of performing ultrasonic treatment during the process.

In one embodiment of the present invention, the method for efficiently extracting ellagic acid from pomegranate is

1) High-temperature pressurization of pomegranate skin powder at 110-130° C. for 10 to 200 minutes;

2) After the above step 1), after dissolving the pomegranate peel powder subjected to high-temperature pressurization in water, irradiation for 3 to 7 seconds at an intensity of 600 to 800 W and 15 to 25 kHz, the process of stopping 0.5 to 1.5 seconds is stopped for a total of 10 to 30 minutes. Performing ultrasonic treatment during the process; And

3) treating the high-temperature pressurized and sonicated product of step 2) with glucanase and arabinase derived from Aspergillus niger for 1 to 6 hours; including However, it is not limited thereto.

The punicalin or ellagic acid may be separated and purified by a conventional method after the high-temperature pressurization and ultrasonic treatment step or enzyme treatment step. For example, each compound can be isolated using High Performance Liquid Chromatography (HPLC). The column used may be a C18-reverse phase column or a Hypersil APS-2 NH2 column.

In addition, the optimum yield condition for punicalin through the central synthesis planning method and surface reaction analysis method is 650 to 750 W, 15 after high temperature pressurization of pomegranate peels of 5 to 15% (w/v) content at 110 to 130°C for 100 to 200 minutes. After irradiation for 4 to 6 seconds at an intensity of ~25 kHz, the process of stopping 0.5 to 1.5 seconds may be subjected to ultrasonic treatment for a total of 15 to 25 minutes, preferably a pomegranate peel having a content of 10% (w/v) at 121° C. After high-temperature pressurization at 165 minutes, irradiation at 700W, 20 kHz for 5 seconds, and then stopping for 1 second is ultrasonicated for a total of 20 minutes, but is not limited thereto.

The optimum yield condition for ellagic acid through the central synthesis planning method and surface reaction analysis for pomegranate skin content, ultrasonic treatment conditions and enzyme treatment conditions is 5-15% (w/v) for pomegranate skin content, 650-750W for ultrasonic treatment, After irradiation for 4~6 seconds at an intensity of 15~25kHz, the process of stopping 0.5~1.5 seconds is treated for a total of 15~25 minutes, and the UF enzyme (1.25U/ml) is 1~2% (v/v) concentration It may be treated with, and preferably, the pomegranate skin content is 10% (w/v), the ultrasonic treatment condition is 700W, irradiation for 5 seconds at an intensity of 20 kHz, and then the process of stopping for 1 second is treated for a total of 20 minutes. , UF enzyme (1.25U/ml) is to be treated at a concentration of 1.65% (v/v), but is not limited thereto.

In addition, the present invention relates to a health functional food composition for the prevention or improvement of stress-related diseases and degenerative brain diseases containing as an active ingredient a pomegranate extract having an enhanced ellagic acid content extracted by the above method.

The degenerative brain disease is caused by the degradation of acetylcholine, and may be any one selected from the group consisting of dementia, Alzheimer's disease and Parkinson disease, preferably dementia or Alzheimer's. It may be a disease, but is not limited thereto.

The stressful disease may be one or more selected from the group consisting of a depressive disorder, a sleep disturbance, and an anxiety disorder caused by an increase in cortisol secretion. It is not limited thereto.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient can be appropriately used depending on the purpose of use (prevention or improvement). In general, when preparing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the total raw material. However, in the case of long-term intake for the purpose of health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the kind of the health functional food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, and vitamin complexes, and all health foods in the usual sense are included.

In addition, the health functional food composition of the present invention can be prepared as a food, particularly a functional food. Functional foods of the present invention include ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols ( For example, xylitol, sorbitol, erythritol, etc.) are preferable. The flavoring agent may be a natural flavoring agent (eg, taumatin, stevia extract, etc.) and a synthetic flavoring agent (eg, saccharin, aspartame, etc.).

In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent used in beverages. Although the ratio of the ingredients to be added is not very important, it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of stress-related diseases and degenerative brain diseases, comprising a pomegranate extract having an enhanced content of ellagic acid extracted by the above method as an active ingredient.

Each of the pharmaceutical compositions according to the present invention can be formulated and used in the form of oral dosage forms such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. I can.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent.

Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

A suitable dosage of the pharmaceutical composition of the present invention can be variously prescribed depending on factors such as the method of formulation, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient. I can.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for describing the present invention in more detail, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example 1. Pomegranate varieties and parts Punicalin or Ellagic acid Content check

In order to select varieties and parts with a high content of punicalin or ellagic acid, flower scent, red scent, skin, fruit seeds and juices of Oedo No. 1 and Oedo No. 2 were lyophilized and pulverized. Thereafter, it was dissolved in 50% (v/v) ethanol so that the final content was 4% (w/v), and 0.05M HCl was added, followed by ultrasonic extraction for 30 minutes. Thereafter, the extracted sample was centrifuged at 10000 rpm for 15 minutes, and the supernatant was filtered with a 0.22 μM membrane filter and used as an HPLC analysis sample.

For the analysis, a sample was loaded on an HPLC on an PDA-MD2015 instrument (JASCO, Kyoto, Japan) for pure separation. The column was a C18-reverse phase column (300x19mm id, Waters), and solvent A (water containing 0.1% (v/v) formic acid) and solvent B (methanol containing 0.1% (v/v) formic acid) Using the solvent B compared to the solvent A 5% to 20% for 5 minutes, 20% to 50% 10 minutes, 50% to 10% was passed through 10 minutes. The flow rate was 0.9 ml/min, and the sample was analyzed at 254 nm using an MD 2015 model PDA detector (JASCO), and the temperature of the column oven was 40°C.

Standard solutions of ellagic acid, gallic acid, punicalin, and funicalazine used in the analysis were diluted according to the concentration ratio using methanol.

As a result, as disclosed in Table 1, the total content of the red-scented pericarp of the red-scented cultivar was excellent, and the total content including punicalin, ellagic acid, and gallic acid was excellent. The total content was excellent. Therefore, since the total content of the red scented peel contains the highest amount of punicalazine, punicalin, ellagic acid, and gallic acid, and the contents of punicalin and ellagic acid are also excellent, the red scented peel was selected as the raw material for the experiment.

kind part Punicalagin Punicalin Ellagic acid Gallic acid Total contents (mg/g dry weight) Flower scent pericarp 47.99 ^c ±0.53 1.17 ^c ±0.03 2.20 ^d ±0.15 0.26 ^c ±0.01 311.40 ^d ±0.09 Fruit 1.14 ^a ±0.09 0.79 ^a ±0.06 0.31 ^a ±0.12 0.09 ^a ±0.01 77.05 ^b ±0.74 crush 0.94 ^b ±0.03 1.90 ^b ±0.03 0.17 ^a ±0.02 0.08 ^b ±0.01 66.03 ^c ±0.08 Red scent pericarp 60.11 ^b ±0.48 2.77 ^a ±0.02 4.89 ^a ±0.11 0.47 ^a ±0.03 354.47 ^a ±0.31 Fruit 0.88 ^b ±0.04 0.11 ^b ±0.01 0.22 ^b ±0.03 0.07 ^b ±0.01 80.41 ^a ±0.24 crush 0.94 ^b ±0.05 5.31 ^a ±0.06 0.05 ^b ±0.05 0.17 ^a ±0.03 73.48 ^b ±0.07 Affair 1 pericarp 72.72 ^a ±0.07 2.10 ^b ±0.00 4.03 ^b ±0.53 0.55 ^a ±0.05 339.43 ^b ±0.63 Fruit 0.86 ^b ±0.00 0.04 ^c ±0.01 0.03 ^d ±0.01 0.07 ^b ±0.00 71.77 ^c ±0.32 crush 1.14 ^a ±0.09 0.74 ^c ±0.04 0.01 ^c ±0.01 0.10 ^c ±0.02 63.08 ^d ±0.04 Affair 2 pericarp 27.22 ^d ±0.10 0.65 ^d ±0.02 2.77 ^c ±0.17 0.38 ^b ±0.03 327.09 ^c ±0.03 Fruit 1.18 ^a ±0.14 0.73 ^a ±0.03 0.05 ^c ±0.05 0.10 ^a ±0.01 69.76 ^d ±0.04 crush 0.73 ^c ±0.03 0.04 ^d ±0.01 0.03 ^b ±0.02 0.07 ^c ±0.02 75.04 ^a ±0.21

The letters a to d in the column mean there is a significant difference between them, and p<0.05.

Example 2. Investigation of polyphenols content according to the primary extraction method

The red scented peel, the raw material selected in Example 1, was extracted with water, 50% (v/v) ethanol, ultrasonically extracted, or hot-pressurized to investigate the content of polyphenols according to the extraction method.

The water extract was prepared by mixing 50 ml of water with 1 g of freeze-dried pomegranate peel powder, followed by hot water extraction at 100° C. for 3 hours.

50% (v/v) ethanol extract was prepared by mixing 50 ml of 50% (v/v) ethanol with 1 g of lyophilized pomegranate peel powder, followed by extraction at 80° C. for 3 hours.

The ultrasonic treatment product was prepared by mixing 50 ml of water with 1 g of lyophilized pomegranate peel powder, setting the intensity to 50% (700W, 20 kHz), irradiation for 5 seconds, and stopping for 1 second for 10 minutes.

The high-temperature pressurized product was prepared by solidifying lyophilized pomegranate peel powder at 121°C for 30 minutes. For subsequent analysis, 50 ml of water was mixed with 1 g of the powder.

The extract or treated product according to the preparation method was analyzed by the method of Example 1, and as a result of the analysis, as disclosed in Fig. 1 and Table 2 below, as a result of comparing the four preparation methods, punicalin when subjected to high temperature pressure treatment And it was confirmed that the content of ellagic acid was significantly improved.

Punicalagin Punicalin Ellagic acid Gallic Acid (mg/g dry weight) water 2.93 ^d ±0.25 0.30 ^d ±0.1 0.73 ^d ±0.16 0.04 ^b ±0.03 50% (v/v) ethanol 54.62 ^b ±0.33 0.68 ^c ±0.03 2.34 ^c ±0.10 0.04 ^b ±0.02 ultrasonic wave 66.61 ^a ±0.92 3.18 ^b ±0.13 6.8 ^b ±0.20 0.04 ^b ±0.01 High temperature pressure 25.52 ^c ±0.7 31.52 ^a ±0.07 9.48 ^a ±0.25 0.14 ^a ±0.08

The letters a to d in the column mean there is a significant difference between them, and p<0.05.

Example 3. High temperature pressure And ultrasound Processed Investigation of polyphenols content

After the high-temperature pressurization method, which is a manufacturing method showing the highest content of punicalin and ellagic acid in Example 2, ultrasonic treatment was added, and then the contents of punicalin and ellagic acid were analyzed.

The water extract was prepared by mixing 50 ml of water with 5 g of lyophilized pomegranate peel powder, followed by hot water extraction at 100° C. for 3 hours.

The high-temperature pressurized product was prepared by solidifying lyophilized pomegranate peel powder at 121°C for 30 minutes. For subsequent analysis, 50 ml of water was mixed with 5 g of the powder.

For the high-temperature pressurized and sonicated product, 50 ml of water is mixed with 5 g of the high-temperature pressurized pomegranate peel powder, and the intensity is set to 50% (700W, 20 kHz), irradiated for 5 seconds, and then the process of stopping for 1 second is stopped for 20 minutes. It was prepared by proceeding.

The content of polyphenols in the extracts and treated products prepared by the above preparation method was analyzed by the method of Example 1, and as a result, as disclosed in Table 3 and FIG. 2, when ultrasonic treatment was added after high-temperature pressure treatment, punicalin and ella It was confirmed that the acid content increased.

Punicalagin Punicalin Ellagic acid Gallic Acid (mg/g dry weight) Water extraction 4.13 ^c ±0.25 2.00 ^c ±0.1 1.83 ^c ±0.16 0.08 ^b ±0.03 High temperature pressure 28.31 ^a ±0.44 30.18 ^b ±0.13 10.28 ^b ±0.20 0.14 ^a ±0.01 High temperature pressure + ultrasonic 12.52 ^b ±0.71 60.93 ^a ±0.07 21.18 ^a ±0.55 0.12 ^a ±0.08

The letters a to c in the column mean that there is a significant difference between them, and p<0.05.

Example 4. Enzyme Decomposition Investigation of polyphenols content

The punicalin polymer was digested using eight commercially available enzymes.

The enzyme names and derived bacteria of commercial enzymes are as disclosed in Table 4 below.

The high-temperature pressurized pomegranate sample was treated with the enzymes and then the degree of ellagic acid was confirmed through TLC analysis.

TLC analysis was performed by dropping the sample 1 µl each on a Whatman K5 TLC plate (silica gel 60 F254 TLC plate (Merck Co.)), and then using the developing solvent butanol/formic acid (3:1, v/v) once in a TLC chamber. Or ethyl acetate/acetic acid/water (3:1:1, v/v/v) was used twice in a solvent system, and the color development was a sulfuric acid color developing solvent (0.3% (v/v) N- 1-naphthyl-ethylenediamine and methanol containing 5% (v/v) sulfuric acid) were soaked for 5 seconds and then baked in an oven at 121° C. for 10 minutes to confirm, and non-polar substances were confirmed by irradiation with UV 245 nm instead of sulfuric acid color development. .

As a result, it was confirmed that the generation of ellagic acid was most effective in biscozyme, PR, and UF as disclosed in FIG. 3, and as a result of treatment of the three enzymes by time, ellagic acid was produced when UF was treated for 1 to 6 hours. The efficiency was the best. Therefore, in subsequent experiments, UF enzyme was used to produce punicalin or ellagic acid.

Example 5. High temperature pressure , Sonication and enzyme Decomposition Investigation of polyphenols content

After the step of ultrasonic treatment after high temperature pressure, which is the production method in which the contents of punicalin and ellagic acid were the best in Example 3, the enzyme treatment of Example 4 was added to analyze the content of punicalin or ellagic acid.

The water extract was prepared by mixing 50 ml of water with 5 g of lyophilized pomegranate peel powder, followed by hot water extraction at 100° C. for 3 hours.

The high-temperature pressurized product was prepared by solidifying lyophilized pomegranate peel powder at 121°C for 30 minutes. For subsequent analysis, 50 ml of water was mixed with 5 g of the powder.

For the high-temperature pressurized and sonicated product, 50 ml of water is mixed with 5 g of the high-temperature pressurized pomegranate peel powder, and the intensity is set to 50% (700W, 20 kHz), irradiated for 5 seconds, and then the process of stopping for 1 second is stopped for 20 minutes. It was prepared by proceeding.

High-temperature pressurization, ultrasonic treatment and enzyme treatment were prepared by adding 1% (v/v) UF enzyme (1.25U/ml) to the high-temperature pressurized and ultrasonic treatment product and then reacting at 50° C. for 1 hour or 6 hours. .

Thereafter, the content of polyphenols in the extract and the treated product prepared by the above preparation method was analyzed by the method of Example 1, and as a result, as disclosed in Table 5 and FIG. 4 below, as disclosed in Table 5 and FIG. In the case, the content was the best, and the content was decreased by the enzyme treatment, but it was confirmed that it was increased compared to the high temperature press alone or the water extract.

The ellagic acid content was further increased after high temperature pressurization, ultrasonication, and enzyme treatment, and when the enzyme was treated for 6 hours compared to the enzyme treatment for 1 hour, the ellagic acid content increased significantly.

Punicalagin Punicalin Ellagic acid Gallic Acid (mg/g dry weight) Water extraction 4.13 ^d ±0.25 2.00 ^d ±0.1 1.83 ^e ±0.16 0.08 ^c ±0.03 High temperature pressure 28.31 ^a ±0.44 30.18 ^c ±0.13 10.28 ^d ±0.20 0.14 ^a ±0.01 High temperature pressurization + ultrasound 12.52 ^b ±0.71 60.91 ^a ±0.25 21.12 ^c ±0.55 0.12 ^b ±0.08 High temperature pressure + ultrasound + enzyme treatment (1h) 9.22 ^bc ±0.71 45.93 ^b ±0.09 61.53 ^b ±0.55 0.11 ^b ±0.12 High temperature pressure + ultrasound + enzyme treatment (6h) 8.52 ^bc ±0.71 25.64 ^c ±0.21 109.98 ^a ±0.55 0.11 ^b ±0.09

The letters a to e in the column mean that there is a significant difference between them, and p<0.05.

Example 6. Identification of substances

In some cases, natural product changes in components during decomposition process, so the substance was identified through LC/MS/MS analysis to identify the target substance.

For LC/MS/MS analysis, ESI-MS/MS (electrospray ionization tandem mass spectrometry) was used, and for Positive ESI-MS analysis, Synapt HDMS system (Waters) was used (conditions: 2.8kV, 35V, source temperature. 100° C., desolvation temperature 300° C., and desolvation gas flow rate of 400 L/Hr) were analyzed with an electrospray ionization source with lock spray interface.

Punicalin was identified from the high-temperature pressurized and ultra-high pressure treatment products with the highest content, and ellagic acid was identified from the high-temperature pressurized, ultra-high pressure, and enzyme treatment for 6 hours.

As a result, as disclosed in FIG. 5A, funicalin was found to be 781.1 in which 1 was added through positive mode LC/MS/MS with 780 molecular weight, and as disclosed in FIG. 5B, ellagic acid was 300 in positive mode with molecular weight. LC/MS/MS showed 301.1 with 1 added.

Example 7. Investigation of optimal yield through central synthesis planning method and surface reaction analysis method

1) Punicalin

In order to statistically investigate the optimal punicalin yield, 20 combinations of three important factors, pomegranate skin content (%, w/v), high temperature pressing time (minutes), and ultrasonic treatment time (minutes), are determined using the central synthesis plan. It was investigated using.

The experimental plans for the three independent variables are shown in Table 6 below.

Punicalin -1.682 -One 0 One 1.682 Skin content (%, w/v) One 5 10 15 20 High temperature pressing time (min) 60 90 180 240 300 Sonication time (minutes) 5 10 20 30 40

The 20 combinations of the central synthesis plan are shown in Table 7 below.

Run No. Codded value Actual value Skin content
(%, w/v) High temperature pressing time (min) Sonication time (min) Skin content
(%, w/v) High temperature pressing time (min) Sonication time (min) One -One -One -One 5.0 90.0 10.0 2 One -One -One 15.0 90.0 10.0 3 -One One -One 5.0 240.0 10.0 4 One One -One 15.0 240.0 10.0 5 -One -One One 5.0 90.0 30.0 6 One -One One 15.0 90.0 30.0 7 -One One One 5.0 240.0 30.0 8 One One One 15.0 240.0 30.0 9 -1.682 0 0 1.6 165.0 20.0 10 1.682 0 0 18.4 165.0 20.0 11 0 -1.682 0 10.0 38.9 20.0 12 0 1.682 0 10.0 291.1 20.0 13 0 0 -1.682 10.0 165.0 3.2 14 0 0 1.682 10.0 165.0 36.8 15 0 0 0 10.0 165.0 20.0 16 0 0 0 10.0 165.0 20.0 17 0 0 0 10.0 165.0 20.0 18 0 0 0 10.0 165.0 20.0 19 0 0 0 10.0 165.0 20.0 20 0 0 0 10.0 165.0 20.0

In addition, the formula leaked from the three 3D graphs by the response surface analysis method disclosed in FIG. 6 is as shown in Equation 1 below, F value is 6.842, R ² is 798.7, Prob> F is 0.0014, which is a statistically significant model. Proved to be.

[Equation 1]

Y=-135.06+11.96X ₁ +0.63X ₂ +5.91X ₃ -0.007X ₁ ² -0.06X ₂ ² -0.003X ₃ ² -3.22X ₁ X ₂ -0.0013X ₁ X ₃ -0.09X ₂ X ₃

Y= Punicalin (mM)

X ₁ = Skin content (%, w/v)

X ₂ = High temperature pressing time (min)

X ₃ = Sonication time (min)

Based on the above results, the optimum condition for the production of punicalin is that after 5 seconds of irradiation at an intensity of 50% (700W, 20 kHz) after pressing a pomegranate peel with a content of 10% (w/v) at 121°C for 165 minutes, 1 It was confirmed that the second stopping process was ultrasonicated for a total of 20 minutes, and 65.93 mg/g of punicalin was produced under the above conditions.

2) Ellagic acid

In order to statistically investigate the optimal yield of ellagic acid, the central synthesis plan was used to determine the three important factors: pomegranate skin content (%, w/v), sonication time (minutes) and enzyme treatment concentration (%, v/v). It was investigated using 20 combinations.

The experimental plans for the three independent variables are shown in Table 8 below.

Ellagic acid -1.682 -One 0 One 1.682 Skin content (%, w/v) One 5 10 15 20 Sonication time (minutes) 5 10 20 30 40 Enzyme treatment concentration (%, v/v) 0.06 0.3 One 3 4

The 20 combinations of the central synthesis plan are shown in Table 9 below.

Run No. Codded value Actual value Skin content
(%, w/v) Sonication time (min) Enzyme treatment concentration (%, v/v) Skin content
(%, w/v) Sonication time (min) Enzyme treatment concentration (%, v/v) One -One -One -One 5.0 10.0 0.30 2 One -One -One 15.0 10.0 0.30 3 -One One -One 5.0 30.0 0.30 4 One One -One 15.0 30.0 0.30 5 -One -One One 5.0 10.0 3.00 6 One -One One 15.0 10.0 3.00 7 -One One One 5.0 30.0 3.00 8 One One One 15.0 30.0 3.00 9 -1.682 0 0 1.6 20.0 1.65 10 1.682 0 0 18.4 20.0 1.65 11 0 -1.682 0 10.0 3.2 1.65 12 0 1.682 0 10.0 36.8 1.65 13 0 0 -1.682 10.0 20.0 0.62 14 0 0 1.682 10.0 20.0 3.92 15 0 0 0 10.0 20.0 1.65 16 0 0 0 10.0 20.0 1.65 17 0 0 0 10.0 20.0 1.65 18 0 0 0 10.0 20.0 1.65 19 0 0 0 10.0 20.0 1.65 20 0 0 0 10.0 20.0 1.65

In addition, the formula leaked from the three 3D graphs by the response surface analysis method disclosed in FIG. 7 is as shown in Equation 2 below, and F valuesms 9.72, Prob>F is 0.0014, which proved to be a statistically significant model.

[Equation 2]

Y=-138.96+19.25X ₁ +7.88X ₂ +52.21X ₃ -0.01X ₁ ² -1.18X ₂ ² -0.03X ₃ ² -0.69X ₁ X ₂ -0.16X ₁ X ₃ -10.40X ₂ X ₃

Y=Ellagic acid (mM)

X ₁ = skin content (%, w/v)

X ₂ = Sonication time (minutes)

X ₃ = Enzyme treatment concentration (%, v/v)

Based on the above results, the optimum condition for ellagic acid generation was to use a pomegranate peel with a content of 10% (w/v), irradiation for 5 seconds at an intensity of 50% (700W, 20 kHz), and then stop for 1 second for a total of 20 It was confirmed that, when ultrasonic treatment was performed for minutes and the enzyme (1.25 U/ml) was treated at a concentration of 1.65% (v/v), 110 mg/g of ellagic acid was produced.

Example 8. Ellagic acid Verification of functionality of pomegranate extract with enhanced content

1) Analysis of total phenol, flavonoid, and proanthocyanin content by extraction condition

Changes in the total phenol, flavonoid, and proanthocyanin content in the pomegranate extract according to the respective preparation methods of the present invention were analyzed. Flavonoid content was measured based on rutin, total phenol content was measured based on tannin, and protoanthocyanin content was measured based on catechin.

As a result of the measurement, as disclosed in Table 10, each component increased as the treatment process was added. In particular, the content of proanthocyanin increased by 2-3 times compared to hot water extraction during high temperature pressurization. When ultrasonic treatment was added, the component content slightly increased compared to before treatment, and when enzyme treatment was added, it was confirmed that the most excellent content was improved.

Total phenol Flavonoids Proanthocyanin (mg/g dry weight) Water extraction 121 ^d ±9.25 63.8 ^d ±9.1 23.77 ^e ±2.16 High temperature pressure 237.7 ^c ±20.44 102.9 ^c ±9.13 51.3 ^d ±3.20 High temperature pressurization + ultrasound 291.1 ^c ±10.71 120.4 ^bc ±12.25 61.4 ^c ±3.55 High temperature pressure+ Ultrasound+
Enzyme treatment (1h) 308.9 ^bc ±11.71 132.9 ^b ±11.09 80.1 ^b ±7.55 High temperature pressure+ Ultrasound+
Enzyme treatment (6h) 337.0 ^a ±21.71 152.2 ^a ±12.21 91.8 ^a ±9.55

The letters a to e in the column mean that there is a significant difference between them, and p<0.05.

2) antioxidant effect

DPPH (1,1-diphenyl-2-picrylhydrazyl) was used for the antioxidant efficacy of pomegranate extract with enhanced ellagic acid content after sonication after high temperature pressurization with the best total phenol, flavonoid, and proanthocyanin content. This was confirmed by measuring the radical scavenging effect.

As a result, it was confirmed that the pomegranate extract having an enhanced ellagic acid content of the present invention as disclosed in FIG. 8 has excellent antioxidant effect.

3) Brain nerve cell protective effect

It was confirmed the protective effect of cranial nerve cells in damage to cranial nerve cells by glutamate of the pomegranate extract having an enhanced ellagic acid content subjected to enzyme treatment for 6 hours after sonication after high temperature pressure of the present invention.

SH-SY5Y cells (KCLB 22266, Korean Cell Line Bank, Seoul, Korea) were used in the experiment to confirm the neuronal protective effect. The viability of cells was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis.

Brain nerve cells (SH-SY5Y) (neuroblastoma, human dopaminergic neuronal cells) were cultured in RPMI-1640 medium containing 1% (v/v) antifungal/antibiotic and 10% (v/v) FBS, and 96-well plates After dispensing so that the number of cells was 10 ⁴ ~ 10 ⁶ cells/ml, the pomegranate extract with enhanced ellagic acid content was treated by concentration after 24 hours. Then, 100mM glutamate was treated to induce stress, and then absorbance was measured at 570nm wavelength using MTT analysis to confirm cytotoxicity. As a positive control, 1-10 μM theanine, which has known brain sedation, was used.

As a result, it was confirmed that the pomegranate extract having an enhanced ellagic acid content of the present invention as disclosed in FIG. 9 has an effect of protecting cranial nerve cells from cytotoxicity caused by glutamate.

4) Inhibitory effect of increased cortisol secretion by glutamate

The content of cortisol, a stress hormone, was measured in order to confirm the anti-stress effect of the pomegranate extract with enhanced ellagic acid content subjected to enzyme treatment for 6 hours after high-temperature pressure and ultrasonic treatment.

After centrifuging the cell culture solution at 4° C. and 10,000×g for 10 minutes, the supernatant was taken and used in the experiment. The protein content of the supernatant was examined with a BCA kit, and cortisol content was analyzed using a cortisol ELISA kit (Calbiotech) according to the manufacturer's protocol.

As a result, it was confirmed that the pomegranate extract having an enhanced ellagic acid content of the present invention as disclosed in FIG. 10 reduced the cortisol content increased by glutamate in a concentration-dependent manner.

5) AChE activity inhibition effect increased by glutamate

AChE breaks down acetylcholin, a neurotransmitter in the brain, and is known to cause the formation of amyloid plaques or neurofibrillary tangles. Therefore, by treating the brain nerve cells (SH-SY5Y) with 100 mM glutamate, the cells were subjected to high-temperature pressure and ultrasonic treatment of the present invention, and then the pomegranate extract with enhanced ellagic acid content was treated for 6 hours by concentration. The culture medium was centrifuged at 10,000×g for 10 minutes, and then the supernatant was taken, and the degree of AChE inhibition using the supernatant was measured according to the manufacturer's protocol with an AChE inhibition assay kit to confirm the anti-dementia improvement effect.

As a result, as disclosed in FIG. 11, it was confirmed that the inhibition rate of AChE activity reduced by glutamate increased when the pomegranate extract with enhanced ellagic acid content of the present invention was treated.

Claims (10)

delete delete delete delete delete delete delete 1) High-temperature pressurization of pomegranate skin powder at 110-130° C. for 10 to 200 minutes;
2) After step 1) above, after dissolving the pomegranate peel powder subjected to high-temperature pressurization in water, irradiation at 600-800W, 15-25 kHz for 3-7 seconds, stopping for 0.5-1.5 seconds for a total of 10-30 minutes Performing ultrasonic treatment during the process; And
3) treating the high-temperature pressurized and sonicated product of step 2) with glucanase and arabinase derived from Aspergillus niger for 1 to 6 hours; including Method for efficiently extracting ellagic acid from pomegranate. delete delete

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