KR101091641B1 - Efficient isolating method of ellagic acid from pomegranate and functional cosmetics composition with ellagic acid extracts - Google Patents

Abstract

The present invention relates to an efficient separation method of ellagic acid (ellagic acid), which is a whitening substance from the pomegranate rind, and to a functional cosmetic composition containing the ellagic acid extract separated by the above method. Method for increasing the extraction efficiency by increasing the solubility of ellagic acid by adding acetonitrile after extraction to separate the ellagic acid from the tannin polymer by ultrasonic extraction and acid hydrolysis treatment, and the ellagic acid extract separated by the above method. It relates to a functional cosmetic composition containing. In addition, ellagic acid or ellagic acid-containing extract extracted by the above method showed a higher antilipidic acid effect (25% increase), anti-wrinkle effect (23% increase) and similar whitening effect than the existing whitening substance arbutin.

Pomegranate, Ellagic Acid, Whitening, Cosmetics, Functionality

Description

Efficient isolating method of ellagic acid from pomegranate and functional cosmetics composition with ellagic acid extracts}

The present invention relates to a method for efficiently separating ellagic acid from pomegranate, and more specifically, to remove ellagic acid from tannin polymer through ultrasonic extraction and acid hydrolysis by dissolving the pomegranate skin in 50% ethanol before reflux extraction. After acetonitrile is added to increase the solubility of ellagic acid to increase the extraction efficiency.

Pomegranate (Punica granatum Linne ) is a deciduous broad-leaved leaf belonging to the family Punicaceae, and has been widely used for diseases such as astringents, dysentery, repellents, or ulcers from ancient times for medicinal purposes. In particular, pomegranate peel (used in terms of pericarp, peel, rind, hul, etc.) accounts for 30% of the fruit and is known to be particularly effective for respiratory diseases and skin diseases.

Pomegranate skin has a lot of tannins, and it has been widely used as astringent placebo. They are mainly ellagitannin, and HHDP (hexahydroxydiphenic acid) is connected by glucose or quinic acid structure and ester bond. Hydrolyzed upon exposure to strong acids, it is divided into punicalagin, a polymer-size, water-soluble phenolic structure, which in turn is either punicalin (4,6- (SS) -gallagyl D-glucose) and ellagic acid (or hexahydroxydiphenic acid) consisting of four rings of polyphenols. (Zhou et al., 2008, Phytochem.Anal. 19: 86-89). In particular, ellagic acid is contained in about 45 kinds of fruits and nuts, such as strawberries, grapes, pomegranates, raspberries (raspberries), bilberry (blueberries), and walnuts. Red raspberry contains 1.50 mg, strawberry contains 0.64 mg and walnut contains 0.59 mg (Yoshimura et al., 2005, Biosci. Biotechnol. Biochem. 69: 2373-2373).

It is reported that ellagic acid is a powerful antioxidant that is effective in skin whitening and antiwrinkle formation. It is strongly attached to copper, which is an important component of tyrosinase activity, a melanogenesis enzyme (Kaur et al., 1997, Indian J. Exp. Biol. 35: 478-482). Yoshimura et al. (2005) conducted experiments with human skin cells and mice to show that ellagic acid protects the skin by preventing the production of enzymes that cause collagen destruction (MMP) and by reducing the expression of inflammatory substances (ICAM). Reported. Yoshimura et al. (2005) reported that it prevents skin wrinkles and pigmentation due to ultraviolet rays (UV) even when applying ellagic acid or ellagic acid-containing pomegranate extract to the skin.

In addition to skin protection, ellagic acid is known to have antioxidant, antiviral, antimutagenic and anticancer functions (Goldberg et al., 1999, J. Agric. Food. Chem. 47: 3978-3985). Ellagic acid is known to induce apoptosis of cancer cells, that is, to cause cancer cells to die as normal cells. In particular, ellagic acid is known to inhibit cancer cell activity in the breast and esophagus, skin, colon, prostate and pancreas. Cell growth was reduced and growth of cells infected with HPV virus, which causes cervical cancer, was found to be inhibited, and the degradation of leukemia cells in vitro was confirmed (Ancos et al., 2000, J. Agric. Food Chem. 48: 4565-4570).

So far, methods for extracting ellagic acid have been tried at various angles and have shown low extraction efficiencies due to the difficulty of using expensive analytical systems such as HPLC and large amounts of by-products generated through the decomposition of ellagitannins polymers (Hayeshi et al. al., 2007, Food and Chemical Toxicology 45: 286-295). Recently, attempts were made to ferment in a solid medium using Lentinus edodes in cranberry concentrate. Aspergillus niger ) alone, or a mixed culture of Candida utilis and Aspergillus niger , attempted to produce ellagic acid through the α-glucosidase enzyme reaction, but the efficiency and time required for bioprocessing were too long.

Korean Patent Publication No. 2007-33236 discloses a liver function protecting agent containing ellagic acid, ellagitannin, and a natural plant extract including the same as an active ingredient, and Korean Patent Publication No. 2003-0055950 contains pomegranate extract. An anti-aging cosmetic composition is disclosed, and Korean Patent Registration No. 0873090 discloses a method for preparing pearl leaf extract containing ellagic acid and a method for separating ellagic acid.

The present invention has been made in accordance with the above requirements, the present invention has developed a method for extracting ellagic acid known to have excellent skin whitening and anticancer efficacy from discarded pomegranate skin with low cost and high efficiency, containing ellagic acid The present invention was completed by preparing a functional cosmetic using the extract.

In order to solve the above problems, the present invention provides a method for efficiently separating ellagic acid from pomegranate.

The present invention also provides a cosmetic composition containing ellagic acid or ellagic acid extract separated by the above method.

The present invention also provides a pharmaceutical composition for preventing or treating atopic dermatitis containing ellagic acid or ellagic acid extract separated by the above method.

According to the present invention, it is possible to extract ellagic acid from pomegranate skin at low cost and high efficiency. In addition, the separated ellagic acid or ellagic acid extract may be usefully used in the manufacture of functional cosmetics.

In order to achieve the object of the present invention,

(a) a solvent mixing step of adding water, ethanol, methanol, ethyl acetate, n-hexanoic acid or a mixture thereof to the dried and ground pomegranate skin;

(b) treating the ultrasound of 20-100 mHz intensity at 25-45 ° C. for 10 minutes to 1 hour;

(c) adding 0.1-1 M HCl;

(d) reflux extraction at 60-100 ° C. for 30 minutes-6 hours or at 200 ° C. for 1-3 hours with a Soxhlet apparatus;

(e) adding acetonitrile to the extract; And

(f) separating the upper layer through centrifugation;

It provides a method for efficiently separating the ellagic acid (ellagic acid) from the pomegranate comprising.

In the method of the present invention, the pomegranate skin of step (a) is dried and ground, preferably lyophilized and ground. The solvent is not particularly limited, but may be water, ethanol, methanol, ethyl acetate, n-hexane or a mixture thereof, preferably 50% ethanol or 50% methanol.

In the method of the present invention, the ellagic acid can be separated from the ellagic tannin polymer through the sonication of step (b).

In the process of the present invention, the ellagic acid, which was not separated in the sonication step, is separated from the polymer by acid hydrolysis by reflux extraction at high temperature with 0.1 to 1 M concentration of HCl in steps (c) and (d). . The reflux extraction may be performed at 60 to 100 ° C. for 30 minutes to 6 hours or at a temperature of 200 ° C. for 1 to 3 hours with a soxhlet device. Instead of acid hydrolysis, Aspergillus niger may be substituted or combined with β-glucosidase enzyme treatment. Here, the enzyme hydrolysis is allowed to react at 37 ° C. for 18 hours or more after adding 0.2 M acetic acid buffer solution (pH 5.0). The elajitannin polymer consists of punicalagins linked to glucose, and glucose-linked punicalins and ellagic acid are separated from punicalagins during acid hydrolysis or enzymatic treatment.

In the method of the present invention, acetonitrile is added to the extract in step (e), because acetonitrile increases the solubility of the separated ellagic acid.

In the method of the present invention, it is preferable to repeat the steps (d) to (f) three times.

In the method of the present invention, after the step (f) may further comprise the step of passing the supernatant through a size chromatography column.

After filling the XAD-2010 (AXT-204) hydrophobic resin with an empty glass column using 30% ethanol, the solution (f) filtered through a 0.45 um filter is passed through the column. 100% ethanol or extract solution (ethanol: water: acetone = 6: 3: 1 v / v / v /) after washing the column with 50% methanol, 50% ethanol, or 50% acetone in three column volumes Separate the ellagic acid.

The method of the invention is preferably, after step (f)

(g) concentrating the supernatant;

(h) filtering the concentrate with a glass filter;

(i) separating the filtrate through a C18 reversed phase column;

(j) adding HCl to the separation solution and then stirring for 1 to 3 hours in a 75 to 85 ° C. hot water bath;

(k) adding acetonitrile;

(l) separating the upper layer through centrifugation; And

(m) removing the solvent of the supernatant;

.

In the method of the present invention, the supernatant concentration in the step (g) is to reduce the volume of the filtrate loaded to the C18 reversed phase column.

In the method of the present invention, the separation solvent of step (i) is preferably 40 to 60% methanol.

In the method of the present invention, the HCl added in the step (j) is 0.1 ~ 1M, preferably 0.1M, it is preferable to stir for 2 hours in an 80 ℃ hot water bath.

In the method of the present invention, the solvent removal of the supernatant in the step (m) may use a rotary evaporator, but is not limited thereto.

The method of the invention is preferably, after step (f)

(g) concentrating the supernatant;

(h) filtering the concentrate with a glass filter;

(i) separating the filtrate through a size chromatography column;

(j) adding acetonitrile to the separation solution;

(k) separating the upper layer through centrifugation; And

(l) removing the solvent of the supernatant;

.

In the method of the present invention, step (i) is the step of separating the filtrate through a size chromatography column, which specifically comprises XAD-2010 (AXT-204) hydrophobic resin with an empty glass column. After filling with 30% ethanol, the (h) filtrate filtered through a 0.45 um filter is passed through a column. 100% ethanol or extract solution (ethanol: water: acetone = 6: 3: 1 v / v / v /) after washing the column with 50% methanol, 50% ethanol, or 50% acetone in three column volumes Separate the ellagic acid.

The present invention also provides a cosmetic composition containing ellagic acid or ellagic acid extract separated by the method of the present invention. The ellagic acid of the present invention has an antioxidant effect, a whitening effect, a lipid acid suppression effect, an anti-wrinkle effect, and the addition amount of the ellagic acid-containing extract was 0.1 to 1% by weight based on the total weight of the cosmetic composition. .

Components included in the cosmetic composition of the present invention may include components commonly used in cosmetic compositions in addition to the ellagic acid or ellagic acid-containing extract as an active ingredient, for example, stabilizers, solubilizers, vitamins, pigments and Conventional adjuvants such as perfumes and carriers.

The anti-aging cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, emulsion, cream, lotion, pack, foundation, lotion, essence, hair cosmetics and the like.

Specifically, the cosmetic composition of the present invention skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, Formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

In the case of the solution or emulsion of the present invention, a solvent, a solvent or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolenic derivatives or ethoxylated glycerol fatty acid esters.

The present invention also provides a pharmaceutical composition for preventing or treating atopy containing ellagic acid or ellagic acid extract separated by the method of the present invention. Since ellagic acid is known to be effective in treating atopy (Petersen et al., 1996, Allergy Clin. Immunol. 97: 672-679; Choi and Yan, 2009. Biol. Pharm. Bull. 32: 1118-1121) The ellagic acid extract isolated by the method of the invention can be used as a pharmaceutical composition for preventing or treating atopy.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Experimental material

Ellagic acid, arbutin, Folin-Denis, Griess reagent, aluminum nitrate, ferrous tartarte, MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolimbromide ], Tyrosinase, L-DOPA, DPPH (1,1-diphenyl-2-picrylhydrazyl) and other reagents are Sigma. purchased from co.

2. Ellagic acid  extraction

Freeze-dried and pulverized pomegranate skin for ellagic acid extraction, diluted 50 times with 50% ethanol, treated with ultrasonic wave for 30 minutes (40mHz), 1N HCl added, and refluxed at 200 ℃ using Soxhlet machine for 3 hours Extracted. At this time, acetonitrile was added to increase the solubility. The supernatant was recovered by centrifugation and then the solvent was removed using a solvent evaporator concentrator.

3. Ellagic acid  Separation and Extraction Efficiency TLC  And C18 Inverse phase HPLC )

After extracting ellagic acid (EA) from the pomegranate skin, it was confirmed and quantified by TLC. 1, 5, 10, 50, 100, 200, 300 mM concentrations of ellagic acid standards or 1 μl of the final extract were added dropwise and then developed on Whatman K5 TLC plate (silica gel 60 F254 TLC plate; Merck Co.) solvent acetonitrile Three times in / water (85:15, v / v) or twice in an ethyl acetate / acetic acid / water (3: 1: 1, v / v / v) solvent system. Color development was confirmed by dipping in 0.3% (w / v) N- (1-naphthyl) -ethylenediamine + 5% (v / v) H ₂ SO ₄ containing methanol and baking for 10 minutes in a 121 ℃ oven. Instead of sulfuric acid development, irradiating with UV 245nm to identify the nonpolar material.

Separation through a C18 open column was passed through the sample 0.22㎛ filter, separation conditions were separated step by step by increasing methanol by 20% from 0% to 100%. HPLC conditions were performed using U-bondapak (C18) (7.8 x 300 mm), A solvent containing 1% formic acid and B solvent using 1% formic acid containing acetonitrile. Solvent B was separated at a flow rate of 0.8 mL / min and 245 nm with gradient up to 7-100% for 30 minutes.

4. Ellagic acid  or Ellagic acid  Efficacy Analysis of Pomegranate Extract

1) Tannin, Flavonoid, Total Phenolic Level

Flavonoid content was determined based on rutin, Phenolics content based on tannin, and protoanthocyanin content based on catechin. Total phenol was incubated in the dark for 45 minutes after adding 0.8 mL of sodium carbonate (7.5%) with 1 mL of Folin-Denis reagent to 200 μl of the sample. Absorbance was measured at 760 nm using a spectrophotometer. Flavonoid content was diluted 0.8 mL of each sample in 0.9 mL of 80% ethanol, taken in 0.1 mL and mixed in 4.3 mL of 80% ethanol containing 10% aluminum nitrate and 1M potassium acetate, and left at room temperature for 40 minutes. Absorbance was measured. Protoanthocyanin The content was quantified by the Sarni-Manchado et al. (2000) method. 10 mL of the sample was placed in a centrifuge tube (25 mL), pH was adjusted to 7.0 with 1N NaOH, 5 mL of Na ₂ HP0 ₄ and 2.5 mL cinchonic sulate solution were added, and the mixture was left at room temperature for 20 minutes. After centrifugation (8,000xg, 20 minutes), the precipitate was dissolved in 5 mL ethanolic hydrochloic acid (10 mL hydrochloric acid in 90 mL 90% EtOH). A 0.1 mL sample was placed in a test tube, and 8 mL of distilled water, 0.5 mL of a Folin-Ciocaltea reagent, and 1.5 mL of a 20% Na ₂ CO ₃ solution were sequentially added, left at room temperature for 2 hours, and the absorbance value was measured at 760 nm.

2) Electron donating ability

Antioxidant efficacy was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) to determine the radical scavenging effect. Sample solution diluted by concentration to 100 μM DPPH ethanol solution was added to a 96-well plate and reacted at 37 ° C. for 30 minutes, and then absorbance was measured at 515 nm using VERSAmax, a microplate reader (Molecular Devices, USA). Antioxidant efficacy was determined by examining Vit C concentrations (0, 0.1, 0.5, 1, 5, 10, 20mM) and repeated data three times using 10 mg of arbutin, ellagic acid, and ellagic acid extract, whitening standards, were used. It was.

3) Measurement of nitrite sacvenging ability

Nitrite scavenging ability was measured by Marcocco et al. 0.5 mL of 0.01 M phosphate buffer (pH 7.4) and 0.5 mL of sample solution were added to 2 mL of 10 mM sodium nitroprusside aqueous solution, and the mixture was reacted at 25 ° C. for 100 minutes. 1 mL of the reaction solution was mixed with 1 mL of sulfanilic acid (0.33% in 20% acetic acid), and left for 5 minutes. 1 mL of 0.1% (w / v) naphthylethylene-diamine dihydrochloride solution (in 20% acetic acid) was added thereto, and the absorbance was measured at 540 nm after standing for 30 minutes. Arbutin, ellagic acid, and ellagic acid extracts (10 mg) were used as whitening standards to obtain the scavenging efficiency in%.

4) Lipid Acid Suppression Effect

The ARA-L kit containing luminol + free radicals from HP-CLA (Tohoku, Japan) and ABCD GmbH (berlin, Germany), which is an antioxidant measuring device using chemiluminescence, was used. The luminol + free radicals in the ampoule react to delay the formation of photons until the antilipidal oxidation of the sample is consumed. At this time, the lag time (second) of the generated graph determined the antioxidant power of the sample. To obtain the antioxidant standard curve, the anti-lipid acid efficacy was determined by examining Trolox concentrations (0, 1, 10, 20, 30, 40 uM), using 10 mg of whitening standards, arbutin, ellagic acid, and ellagic acid extract. The data were obtained by repeated experiments.

5) whitening effect

Melanin produced by adding melanin-producing enzyme Tyrosinase (from mushroom) (1U / mL) to 100mM phosphate buffer (pH7.0) and 1mM L-DOPA (3- (3,4-dihydroxyphenyl) -L-alanine) Was measured at 475 nm. The degree of inhibition of melanin production was Vit C 0.1 or 1 mM, arbutin, ellagic acid, ellagic acid 1 mg / mL of extract was added to calculate the degree of enzyme inhibition in%. As a positive control, Vit C 0.1 mM or 1 mM, and arbutin whitening substance 1 mg / mL were used. Each sample was measured three times to obtain an average and standard deviation.

6) Wrinkle suppression effect

HS68 (ATCC CRL 1635) was incubated in a 5% CO ₂ chamber at 37 ° C. in DMEM with 1% antibiotic-antimycotic, 10% FBS added to a 100 mm cell culture dish. Cells were cultured by diluting 1: 5 subculture at 80% confluency. Serum-starved confluent cells were washed twice with PBS buffer and irradiated with UV (UVB, 100 mJ / cm ² ) immediately after 24 hours after serum-free medium was fed. Before UVB treatment, cells were treated with arbutin (10 ug / ml), a standard whitening material, and ellagic acid and ellagic acid extract (10 ug / ml). The control was used without UVB irradiation or with UVB irradiation treatment without whitening material. Wrinkle-inducing MMP-1 (Matrix metalloproteinase-1) content was measured using an ELISA kit (TPI Inc., Lynnwood, WA, USA).

5. Investigate the safety of skin cells

Growth inhibition of HS68 cells (human newborn foreskin fibroblast) was measured using MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolimbromide]. Incubate 1.0 × 10 ⁴ HS68 cells per 100 μl in a 96 well plate for 24 hours, followed by arbutin, ellagic acid, and ellagic acid. The containing extracts were treated for 24 hours at concentrations of 0, 0.1, 0.5, 1, 5, 10, 25 and 50 mg, respectively. After washing with PBS and treated with MTT reagent 1 mg / ml concentration for 3 hours, dissolved in DMSO, the absorbance was measured at 570nm using a microplate reader.

6. Ellagic acid  Cosmetic preparation using pomegranate extract

0.1% of ellagic acid-containing pomegranate peel extract was added to prepare a lotion (skin lotion), nourishing lotion, nourishing cream, massage cream, and nourishing essence to prevent whitening and wrinkle formation. Here, the formulation of the cosmetic containing the ellagic acid-containing pomegranate extract liposome is a formulation example and the comparative formulation example is an example in which the pomegranate extract liposome is not added.

7. Ellagic acid  Investigation of Skin Stability of Contained Cosmetics

To investigate the skin irritation and protective effect and skin stability of cosmetics containing ellagic acid (skin or nutrition lotion, nutrition cream), phototoxicity and human body in the forearm of 40 healthy adults (20 males, 20 females) The presence or absence of irritation was confirmed.

Example  1. from pomegranate rind Ellagic acid  extraction

First, the ellagic acid from the pomegranate skin is divided into three different ways: It separated by the liquid-liquid separation method extraction, 2) hydrolysis extraction method, and 3) solid-phase extraction method. The pomegranate skin was lyophilized and then ground and mixed with the solvent to extract.

1) Extraction by liquid separation method is as follows.

50% ethanol was added to the dried and pulverized pomegranate skin, and ultrasonic waves of 40 mHz intensity were treated at 40 ° C. for 30 minutes. The mixture was refluxed at 200 ° C. for 3 hours using a Soxhlet apparatus, the extract was separated by centrifugation, and the solvent was removed using a rotary evaporator.

There is no particular limitation on the extraction solvent, preferably 50% ethanol, or 50% methanol, and ethyl acetate or hexane may be added if necessary. The ellagic acid was separated from the ellag tannin polymer by sonication and then recovered through reflux extraction. The reflux step and the phase separation step are preferably repeated three times.

The extract extracted by the above-mentioned method from 1 kg of dried pomegranate skin and solvent removed contained 210 mg of viscous residue and contained 68 mg / g (56.7% recovery) of ellagic acid.

For reference, pomegranate skin contains 0.3% to 12% of ellagic acid (Food Chemistry, 2008. 1007-11).

2) Extraction using hydrolysis is as follows.

50% ethanol was added to the dried and pulverized pomegranate skin, and ultrasonic waves of 40 mHz intensity were treated at 40 ° C. for 30 minutes. 1 M HCl was added, reflux extraction was performed at 200 ° C. for 3 hours using a Soxhlet instrument, acetonitrile was added to the extract, the top layer was separated by centrifugation, and the solvent was removed using a rotary evaporator.

The reflux step and the phase separation step are preferably repeated three times. HCl was extracted with reflux at high temperature using a concentration of 0.1-1 M to remove ellagic acid, which could not be separated in the sonication step, from the polymer through acid hydrolysis. Instead of acid hydrolysis, Aspergillus niger may be substituted or combined with β-glucosidase enzyme treatment. Here, the enzyme hydrolysis is allowed to react at 37 ° C. for 18 hours or more after adding 0.2 M acetate buffer solution (pH 5.0). Elastagnin polymers consist of punicalagins linked to glucose, and glucose-linked punicalins and ellagic acid are separated from punicalagins during acid hydrolysis or enzymatic treatment. The separated ellagic acid is added to the solubility of the added acetonitrile.

The extract extracted by the above method from 1 kg of dried pomegranate peel and solvent removed contained 285 g of viscous residue containing 97 mg / g (66.4% recovery) of ellagic acid.

3) The extraction steps by solid phase extraction are as follows.

50% ethanol was added to the dried and pulverized pomegranate skin, and ultrasonic waves of 40 mHz intensity were treated at 40 ° C. for 30 minutes. 1 M HCl was added, reflux extraction was performed at 200 ° C. for 3 hours using a Soxhlet instrument, acetonitrile was added to the extract, and the upper layer was separated by centrifugation. The supernatant was concentrated using a rotary evaporator, the concentrate was filtered through a glass filter, and the filtrate was separated by passing through a C18 reversed phase column. 0.1 M HCl was added to the separation solution, followed by stirring for 2 hours in an 80 ° C. hot water bath, acetonitrile was added, the upper layer was separated by centrifugation, and the solvent of the supernatant was removed using a rotary evaporator.

Extracted by the above-mentioned method from 1 kg of dried pomegranate peel and solvent removed, 155 g of the extracted viscous residue contained 113 mg / g (95% recovery) of ellagic acid.

From 1 kg of pomegranate skin, 14.3g was extracted by liquid solution, 27.6g by acid hydrolysis, and 19.1g by solid phase extraction. The highest amount of ellagic acid was extracted by acid hydrolysis, and solid phase extraction through C18 open column was performed. The recovery of ellagic acid in the sample was high.

Example  2. Extracted from Pomegranate Peel Ellagic acid  Quantification and Purification

1 is confirmed by TLC after extraction of ellagic acid (EA) from the pomegranate skin (left) and antioxidant measurement results (right). The dried and pulverized pomegranate skin was subjected to Soxhlet extraction using 50% ethanol and then ultrasonically pulverized, and the sample refluxed three times was quantified by TLC. Arrows indicate EA. TLC confirmed that the glucose of the ellagitannin polymer was separated during acid hydrolysis and sonication.

The whitening material in pomegranate skin was recovered more than 95% by sonication, acid hydrolysis and soxhlet extraction. The ellagic acid was present as a polymer so that reflux extraction was repeated three times under hydrolysis conditions with 1N HCl after sonication (30 min, 40 ° C.). The recovery rates were 71 mg / g for the first extraction, 25.2 mg / g for the second extraction, and 0.8 mg / g for the third extraction. As a result of reaction with purple DPPH solution to confirm the location of ellagic acid known to have antioxidant power, purple was turned yellow due to the antioxidant activity of the expected spot at the bottom as indicated by the arrow.

2 shows the results of ellagic acid separation and TLC analysis from a pomegranate skin using a C18 reversed phase column or HPLC. C18 reverse phase column separation conditions were separated by stages by increasing methanol by 20% from 0% to 100%. HPLC conditions were U-bondapak (C18) (7.8 x 300 mm), A solvent 1% formic acid containing, B solvent 1% formic acid containing acetonitrile. Solvent B was separated at 245 nm at 0.8 mL / min flow rate with gradient to 7-100% for 30 min.

It was confirmed that the ellagic acid was separated in 60% methanol when purified by a C18 reversed-phase open column and separated in 35-40% acetonitrile (containing 1% formic acid) upon decomposition by C18-reverse phase HPLC. Ellagic acid standards were dissolved in methanol or DMSO at 1 mg / mL. Pure ellagic acid was obtained using a C18 reversed-phase open column.

Example  3. Ellagic acid  Investigation of ingredients and functionalities of extracts

Efficacy of ellagic acid extract (using solid extract) from arbutin, ellagic acid (EA), or pomegranate was compared to human epithelial cells after antioxidant, nitrite scavenging, antilipidic, whitening and antiwrinkle Their safety was also confirmed.

1) Total phenol, flavonoid, catechin content investigation

Table 1.

Extraction method Flavonoids
[mg / g] Protoanthocyanin
[mg / g] Phenolics
[mg / g] Liquid separation extraction 46.50 25.06 221.0 Acid Hydrolysis Extraction 41.32 21.77 354.4 Solid phase extraction 53.71 33.58 207.9

Flavonoid content was determined based on rutin, Phenolics content based on tannin, and protoanthocyanin content based on catechin. The content of anthocyanin based on flavonoid and catechin was the highest in solid phase extraction of 53.7mg / g and 33.58mg / g, respectively, and the total phenolic amount representing tannins was the highest in acid hydrolysis extraction. Total phenol content was 4 ~ 8.5 times higher than flavonoid content and anthocyanin content was about half of flavonoid.

2) antioxidant effect

Arbutin (Arbutin), ellagic acid (EA), the antioxidant power was measured using the extract of 10 mg, showed 15mM, 12mM, 5.9mM vitC equivalent antioxidant power (Fig. 3). Arbutin showed 20% higher antioxidant activity than ellagic acid, and ellagic acid extract from pomegranate showed lower antioxidant activity than ellagic acid itself.

3) nitrite scavenging effect

Nitrite scavenging activity showed that when vitC 1mM and 10mM showed 23% and 90% scavenging activity, arbutin (10mg) exhibited 59% scavenging activity and 17% lower scavenging activity than ellagic acid (10mg) showing 75.9%. The extract (10 mg) was 67% higher than arbutin but slightly lower than ellagic acid.

4) Antilipid acid effect

Anti-lipid acidity measurement, an important factor for skin whitening, showed that the anti-lipid acidity of arbutin, ellagic acid, and ellagic acid extracts, such as the whitening standards, were 16uM, 20.2uM, and 21.2uM trolox equivalent. In other words, ellagic acid was better than arbutin, and antilipidic acid was superior to ellagic acid extract than ellagic acid.

5) whitening effect

As a result of measuring the degree of inhibition of tyrosinase, which causes melanin production, which is an important factor for skin whitening, it showed 80.2% inhibition rate with vitC (1mM) treatment, 48.2% for arbutin, 40.21% for ellagic acid, and 35.60% for ellagic acid extract. Effect. Ellagic acid showed a 83% whitening effect of arbutin and the extract was slightly lower than ellagic acid, but showed a whitening effect of 35%.

6) Anti-wrinkle effect

In order to see the effect of wrinkles on the skin, we measured the amount of MMP-1 (matrix metallo proteinase) that induces wrinkles after irradiating ultraviolet light (UVB) to HS68 cells, which are human epithelial cells. Based on the amount at 100%, arbutin treatment was 86%, ellagic acid was 75%, ellagic acid extract was 63%, elagic acid was 11% higher than arbutin, and the extract was 23% higher in preventing wrinkle formation.

Example  4. Intracellular safety investigation

MTT analysis using human epithelial cells (HS68 cells) on the safety of the samples showed that arbutin (IC ₅₀ = 15.38 mg / mL), ellagic acid-containing pomegranate extract (IC ₅₀ = 5.20 mg / mL), In order of ellagic acid (IC ₅₀ = 3.89 mg / mL). Therefore, arbutin showed 5 times more intracellular safety than ellagic acid, and ellagic acid-containing pomegranate extract was 25% safer than ellagic acid itself.

Example  5. Ellagic acid  Containing whitening products

One. Ellagic acid  Contains lotion (skin lotion)

Table 2. Preparation of skin lotion (skin lotion) according to the present invention

number Raw material Formulation Example 1 (%) Comparative Formulation Example 1 (%) One Pomegranate extract containing ellagic acid
Liposomes (Example 1) 0.1 - 2 glycerin 3.0 3.0 3 Butylene glycol 2.0 2.0 4 Propylene glycol 2.0 2.0 5 Polyoxyethylene (60) Cured Castor Oil 1.0 1.0 6 ethanol 10.0 10.0 7 Triethanolamine 0.1 0.1 8 antiseptic a very small amount a very small amount 9 Pigment a very small amount a very small amount 10 Spices a very small amount a very small amount 11 Purified water Balance Balance

Formulation Example 1: Raw materials 2, 3, 4, and 8 were sequentially added to the raw material 11, and then dissolved by stirring. The raw material 5 was heated to about 60 ° C. to dissolve. Input. Finally, the raw materials 1, 6, 7, 9 are added and sufficiently stirred, and then passed through a high pressure homogenizer and aged.

Comparative Formulation Example 1: Raw materials 2, 3, 4, and 8 were added to raw material 11 in order, and then dissolved. The raw materials 5 were heated and dissolved at about 60 ° C., and then raw material 10 was added and stirred. Put it in. Finally, the raw materials 6, 7, 9 are added and sufficiently stirred, and then aged through a high pressure homogenizer.

2. Ellagic acid  Contains Nutritional Lotion

Table 3. Preparation of nutrition lotion according to the present invention

number Raw material Formulation Example 2 (%) Comparative Formulation Example 2 (%) One Ellagic acid or pomegranate extract
Liposomes (Example 1) 0.1 - 2 Cetearyl Alcohol 1.7 1.7 3 Stearic acid 1.5 1.5 4 Ceramide 0.7 0.7 5 Polysorbate 80 1.2 1.2 6 cholesterol 1.5 1.5 7 Dicetyl phosphate 0.4 0.4 8 glycerin 5.0 5.0 9 Jojoba Seed Oil 10.0 10.0 10 Carboxy Vinyl Polymer 0.2 0.2 11 Xanthan Gum 0.3 0.3 12 antiseptic a very small amount a very small amount 13 Spices a very small amount a very small amount 14 Purified water Balance Balance

Formulation Example 2: Raw materials 2, 3, 4, 5, and 6 are homogenized at a constant temperature to be referred to as nonionic amphiphilic lipids. The nonionic amphiphilic lipid and the raw materials 1, 7, 8, and 14 are mixed and homogenized at a constant temperature and passed through a high pressure homogenizer, and then the raw material 9 is gradually added and homogenized at a constant temperature, and then passed through the high pressure homogenizer again. Let's do it. Then, the raw materials 10, 11, 12, 13 are added to disperse to stabilize and mature.

Comparative Formulation Example 2: The raw materials 2, 3, 4, 5 and 6 are homogenized at a constant temperature to be referred to as nonionic amphiphilic lipids. The nonionic amphiphilic lipid and the raw materials 7, 8 and 14 are mixed and homogenized at a constant temperature to pass through a high pressure homogenizer, and then the raw material 9 is gradually added at a constant temperature to homogenize and then passed again to the high pressure homogenizer. Then, the raw materials 10, 11, 12, 13 are added to disperse to stabilize and mature.

3. Ellagic acid  Nutritional Cream

Table 4. Preparation of nutrition cream according to the present invention

number Raw material Formulation Example 3 (%) Comparative Formulation Example 3 (%) One Pomegranate extract containing ellagic acid
Liposomes (Example 1) 0.1 - 2 Cetearyl Alcohol 4.0 4.0 3 Stearic acid 3.0 3.0 4 Ceramide 0.7 0.7 5 Polysorbate 80 2.0 2.0 6 cholesterol 3.0 3.0 7 Dicetyl phosphate 0.4 0.4 8 glycerin 5.0 5.0 9 Jojoba Seed Oil 22.0 22.0 10 Carboxy Vinyl Polymer 0.5 0.5 11 Triethanolamine 0.5 0.5 12 antiseptic a very small amount a very small amount 13 Spices a very small amount a very small amount 14 Purified water Balance Balance

Formulation Example 3: Raw materials 2, 3, 4, 5 and 6 are homogenized at a constant temperature to be referred to as nonionic amphiphilic lipids. The nonionic amphiphilic lipid and the raw materials 1, 7, 8, and 14 are mixed and homogenized at a constant temperature and passed through a high pressure homogenizer, and then the raw material 9 is gradually added and homogenized at a constant temperature, and then passed through the high pressure homogenizer again. Let's do it. Then, the raw materials 10, 11, 12, 13 are added to disperse to stabilize and mature.

Comparative Formulation Example 3 The raw materials 2, 3, 4, 5, and 6 were homogenized at a constant temperature to be referred to as nonionic amphiphilic lipids. The nonionic amphiphilic lipid and the raw materials 7, 8 and 14 are mixed and homogenized at a constant temperature to pass through a high pressure homogenizer, and then the raw material 9 is gradually added at a constant temperature to homogenize and then passed again to the high pressure homogenizer. Then, the raw materials 10, 11, 12, 13 are added to disperse to stabilize and mature.

The pomegranate extract containing 78% ellagic acid was used to prepare skin, lotion, nourishing cream, and essence.

Example  6. Ellagic acid  Investigation of Skin Stability of Contained Cosmetics

1.Check skin irritation and protective effect of ellagic acid-containing skin lotion and nutrition lotion

Apply a lotion (10cm / 5cm ² ) of skin lotion or nutrition lotion prepared in the present invention to the forearm of 40 healthy men and women (20 men, 20 women) and shield the rest with aluminum foil for 30 minutes. Afterwards, a long wavelength ultraviolet ray was irradiated at a dose of 10.2 J / cm ² using an ultraviolet irradiator, and skin conditions (erythema, edema, papules, etc.) were observed after 24 hours. As a result, it was confirmed that the skin lotion or the nutrient lotion does not have phototoxicity and has a skin protection effect because no side effects such as erythema occur on the skin.

2. Confirm the skin irritation and protective effect of nourishing cream containing ellagic acid

40 minutes of healthy adult men and women (20 men, 20 women) apply the nourishing cream prepared in the present invention to the forearm by a predetermined amount (10cm / 5cm ² ) and shield the rest with aluminum foil 30 minutes later Long-wavelength ultraviolet rays were irradiated at a dose of 10.2 J / cm ² , and skin conditions (erythema, edema, papules, etc.) were observed after 24 hours. As a result of the observation, no side effects such as erythema occurred on the skin, and thus the nourishing cream had no phototoxicity and had a skin protective effect.

3. Skin stability test of ellagic acid-containing lotion and cream

A human intelligence test was conducted on the forearm of 40 healthy adult men and women (20 men, 20 women) with the lotion and cream prepared in the present invention. The forearm of the test subject was wiped well with 70% ethanol, and then the pin chamber containing 20 μl of the lotion and cream prepared in the present invention in a conventional cosmetic base was closed and attached for 24 hours using an adhesive tape. After 24 hours, the adhesive tape and the pin chamber were removed, and the sample remaining on the skin with a gauze was lightly removed. Experimental results As shown in Table 5, the human intelligence test to confirm the skin irritation of the lotion and cream according to the present invention did not show a positive response was confirmed that there is no irritation to the human skin.

Table 5. Skin stability test results of ellagic acid-containing lotions and creams

sample After 24 hours (n = 40) After 48 hours (n = 40)  Remarks Skin lotion  0/40 0/40 Number of reaction protons /
Total number of people Nutrition lotion 0/40 0/40 Nutrition cream 0/40 0/40

1 is confirmed by TLC after extraction of ellagic acid (EA) from the pomegranate skin (left) and antioxidant measurement results (right).

2 shows the results of ellagic acid separation and TLC analysis from a pomegranate skin using a C18 reversed phase column or HPLC.

Figure 3 is the result of measuring the antioxidant power using arbutin (Arbutin), ellagic acid (EA), ellagic acid extract 10mg.

Figure 4 is the result of measuring the nitrite scavenging ability using Arbutin (Arbutin), ellagic acid (EA), ellagic acid extract 10mg.

5 is a result of measuring the anti-lipid acid septicity using Arbutin (Arbutin), ellagic acid (EA), ellagic acid extract 10mg.

6 is a result of measuring the degree of Tyrosinase inhibition using Arbutin (Arbutin), ellagic acid (EA), ellagic acid extract 10mg.

Figure 7 shows the production amount of MMP-1 (matrix metallo proteinase) to induce wrinkles after irradiating ultraviolet (UVB) to HS68 cells, human epithelial cells using Arbutin (Arbutin), ellagic acid (EA), ellagic acid extract 10mg It is a result of a measurement.

8 is a result of MTT analysis using the human epithelial cells (HS68 cells) for the safety of the human skin of the samples.

Claims (8)

(a) a solvent mixing step of adding water, ethanol, methanol, ethyl acetate, n-hexane or a mixture thereof to the dried and ground pomegranate skin; (b) treating the ultrasound of 20-100 mHz intensity at 25-45 ° C. for 10 minutes to 1 hour; (c) adding 0.1-1 M HCl; (d) reflux extraction at 60-100 ° C. for 30 minutes-6 hours or at 200 ° C. for 1-3 hours with a Soxhlet apparatus; (e) adding acetonitrile to the extract; And (f) separating the upper layer through centrifugation; Method for efficiently separating ellagic acid (ellagic acid) from the pomegranate comprising a. The method of claim 1, wherein the steps (d) to (f) are repeated three times. The method of claim 1, wherein the solvent of step (a) is 50% ethanol or 50% methanol. The method of claim 1, further comprising passing the supernatant through a size chromatography column after step (f). The method of claim 1, wherein after step (f) (g) concentrating the supernatant; (h) filtering the concentrate with a glass filter; (i) separating the filtrate through a C18 reversed phase column; (j) adding HCl to the separation solution and then stirring for 1 to 3 hours in a 75 to 85 ° C. hot water bath; (k) adding acetonitrile; (l) separating the upper layer through centrifugation; And (m) removing the solvent of the supernatant; Method further comprising a. The method of claim 1, wherein after step (f) (g) concentrating the supernatant; (h) filtering the concentrate with a glass filter; (i) separating the filtrate through a size chromatography column; (j) adding acetonitrile to the separation solution; (k) separating the upper layer through centrifugation; And (l) removing the solvent of the supernatant; Method further comprising a. delete delete

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