KR20050079431A - Method for separating an oligomeric proanthocyanidins from an extract of wild grape seeds - Google Patents

Abstract

The present invention relates to a method for separating proanthocyanidin oligomers from extracts of wild grapes, and more specifically, after extracting hot water using a mixed solvent of alcohol and water after fine grinding of wild grapes, to purify high purity antioxidants. The present invention relates to a method for separating proanthocyanidin oligomers from extracts of the melanocytes economically and rapidly by applying column chromatography packed with a macroabsorbent resin.

According to the method of the present invention, antioxidants can be separated from the melon seeds with high purity and high yield, and the separated antioxidants are functional foods containing a multifunctional natural antioxidant that prevents rancidity and aging of foods caused by free radicals or It can be usefully used as an additive of food.

Description

Method for separating an oligomeric proanthocyanidins from an extract of wild grape seeds}

The present invention relates to a method for separating proanthocyanidin oligomers from extracts of wild grapes, and more specifically, after extracting hot water using a mixed solvent of alcohol and water after fine grinding of wild grapes, to purify high purity antioxidants. The present invention relates to a method for separating proanthocyanidin oligomers from extracts of the melanocytes economically and rapidly by applying column chromatography packed with a macroabsorbent resin.

, ^- radicals superoxide radicals (superoxide radical, O ₂₎ by the like (active oxygen) is vivo enzyme system as unsafe oxygen with electron unpaired, reduced metabolism, chemicals, various physical and chemical factors such as pollutants, It is made from highly reactive oxygen species such as hydroxyl radicals (HO ·) and singlet oxygen (O ₂ ) (Mates, JM and Sanchez-Jimenez, FM, INT. J. BIOCHEM. CELL, 32, 157-170, 2000). These free radicals damage cell lipid membranes, intracellular proteins and DNA, and are known to cause various diseases such as cancer, stroke, Parkinson's, brain diseases, heart disease, arteriosclerosis, skin disease, autoimmune diseases, and aging. (Cross, EE, Halliwell, BB, Borish, ET, Rryor, WA, Ames, BN, Saul, RL and McCord, JM Ann.Intern.Med ., 107, 526-545, 1987; Kim Young-gon, Kim Young-kyun, Free Radical , Yeomungak (1997).

Antioxidant activity refers to an activity that prevents the production of free radicals in the body and prevents oxidative phenomena that cause irreparable damage to cells. Antioxidants give the active hydrogen atoms of the antioxidant molecule itself against various active oxygen, making the radicals a stable compound with no activity, and at the same time, they are transformed into resonance-reactive radicals, stabilized by resonance Again by interaction with other radicals it acts with a reaction mechanism that is not a radical but a stable compound (Halliwell, B. and Gutteridge, JMC (ed.), Pp. 86-187, 1989; Frei, B. , Stocker, R. and Ames, BN, Proc. Natl. Acad. Sci. USA, 85, 9748-9752, 1988).

Antioxidants that have been developed and used as a treatment for diseases caused by these free radicals include BHT (tert-butylhydroxytoluene) and BHA (tert-butylhydroxyanisol), but these are synthetic phenolic compounds that are toxic in vivo and are safe for allergies and tumors. It is controversial (Xin Dong Hwa, Food Science and Industry, 30, 1, 1997).

Therefore, in recent years, interest in natural antioxidants having an antioxidant effect has increased from plants that humans have eaten safely for a long time. (Hocman, G., Comp. Biochem.Physiol ., 93B, 201-212, 1989; Jongpyeong Kim, Yeongdong-dong, New Material Exploration, Free Academy, pp 325-343, 1996). However, natural antioxidants have the advantage of being safe to the living body compared to synthetic antioxidants, but their disadvantages are weak.

On the other hand, proanthocyanidins, which are known to be safe and excellent among natural antioxidants developed until recently (Shahidi, F. and Wanasundara, PKJ, Crit. Rev. Food Sci. Nutr. .32 , 67-103, 1992). Proanthocyanidins are one of the polyphenols, catechin, epicatechin, catechin gallate, epicatechin gallate, gallocatechin gallate or epigallo. It is a generic name for oligomers and polymers of dimers or more that are based on catechin gallate (Epigallocatechin gallate) (Rice-Evans, CA, Miller, NJ and Paganda, G., Free) rad. Biol. Med. , 20, 933-956, 1996; Salah, N., Miller, NJ, Paganga, G., Tijburg, L., Bolwell, GP and Rice-Evans, C., Arch. , 322, 339-346, 1995). Proanthocyanidins have excellent free radical scavenging ability, and are known to be carcinogenic, anti-arteriosclerosis, antiviral and analgesic (Boukharta, M., Girardin, M. and Metche, M., J. Chromatography , 455, 406, 1988; Ricardo da Silva, JM, Rigaud, J., Cheynier, V., Cheminat, A. and Moutounet, M., Phytochemistry , 30 (4), 1259, 1991).

As for the separation and purification of proanthocyanidins having excellent natural antioxidant capacity, studies have been conducted on various plants such as grape seeds, pine bark, ginkgo biloba, peanuts and cacao beans (German, JB, Nutritional studies of flavonoids in wine). In: Rice-Evans, CA, Packer, L. (Eds.), Flavonoids in Health and Disease.Marcel Dekker, New York, Pp. 343-358, 1997; Teel, RW, Phytother.Res. 6, 251-. 254, 1992; Pekic, B., Kovac, V., Alonso, E. and Revilla, E., Food Chem. , 61, 201-206, 1997). Examples of industrial extraction of proanthocyanidins from various plants including grape seeds include grape seeds (Japanese Patent Laid-Open No. 3-200781, U.S. Patent No. 5,484,594) and pine bark (U.S. Patent No. 4,698,360). Extraction in patent WO 97/44407).

In addition, the results of studies with the proanthocyanidin purification method include the counter-current liquid-liquid distribution method (Japanese Patent Laid-Open No. 61-16982), and the solid-liquid extraction method (Japanese Patent Laid-Open No. 8-176137, published in Europe). Patent 0707005), a method using column chromatography (Japanese Patent Application Laid-Open No. 7-62014), and the like are known.

Murru, on the other hand, belongs to the vine family and is a vine-grown tree plant that grows in the foothills of the altitude of 100-1,300m above sea level. However, there are no studies on extracts containing oligomeric proanthocyanidins and natural antioxidants from the melanoma seeds. This is because the production of wild grapes is wild or cultivated in some localized areas of Japan, China, and Manchuria. However, research on the seeds of wild grapes by domestic and foreign scholars is still insufficient. There is a situation.

Accordingly, the present invention has found that the seedlings of the wild grapes contain a large amount of natural antioxidants such as oligomeric proanthocyanidins, and they are also rapidly used as a water-ethanol mixed solvent using a large air absorption resin composed of cheap alkyl benzene-based polymers. The optimum conditions which can be separated easily were found, and the present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a method for economically and rapidly separating an extract having natural antioxidant function from a melon seedling in high yield and high purity.

Separation method of the proanthocyanidin oligomer from the extract of the melon seed yarn of the present invention for achieving the above object is the step of grinding the melon seed yarn; Extracting the pulverized myrhea seed extract by concentrating by extracting the pulverized seedling seeds at 50-80 ° C. for 8 to 12 hours using a hydrous 50-80% alcohol mixed extract solvent; And suspending the melon seedling extract in a mixed solvent of water and ethanol, and placing the extract in a large air absorption resin composed of an alkyl benzene-based polymer, and then mixing a mixed solvent of water and ethanol having a concentration gradient of 10%, 70% and 95%. And eluting the proanthocyanidin compound sequentially.

Looking at the present invention in more detail as follows.

In the present invention, in order to obtain a natural antioxidant such as oligomeric proanthocyanidins, the fruit seedlings from which the pulp of the fruit fruit was removed were used. This grape seedling has the advantage that it can be easily obtained without steaming process when removing the fruit pulp unlike grape seedlings.

According to the present invention, referring to Figure 1, after first crushing the seedling seeds after leaching (400rpm) for more than 8 hours at 50 ~ 80 ℃ in a 50 ~ 80% alcohol mixed extract solvent (mixed solvent of water and ethanol), Filter using a vacuum pump and concentrate to about 70% of the filtrate. The concentrated extract was placed on a column filled with a macroabsorbent resin, followed by concentration gradients of 10%, 70%, and 95% ethanol (a mixed solvent of water and ethanol with ethanol concentrations of 10%, 70%, and 95%). Received the fraction layer sequentially with a mixed solvent having a 70% fraction containing the most oligomeric proanthocyanidins of it was concentrated under reduced pressure at about 50 ℃ to obtain an extract of the melon seeds.

Furthermore, in the present invention, as a result of experimenting the hot water extraction conditions of the crushed yam seedlings for optimal production of natural antioxidants from the buckwheat seedlings, leaching at about 80 ℃ using a hydrous 70% alcohol extraction solvent is the most It was found to be an optimal condition for economical and rapid extraction.

In addition, in the present invention, in order to obtain an extract containing a large amount of more purified oligomeric proanthocyanidins, an alkyl benzene-based air absorption number is used rather than using a silica gel adsorption column which is most widely used for natural materials. Paper was used. This is because silica gel may be difficult to effectively separate the glycoside and the water-soluble substance even if the polarity is high when the polar glycoside is separated according to natural products. In this case, if an alkyl benzene-based air absorption resin column is used instead of a silica gel column and eluted with water and alcohol, water-soluble substances are separated into water elution fractions, and glycosides are effectively separated into water-containing alcohol fractions having lower polarity than water. Ancillary resins are used to separate glycosides.

Proanthocyanidins are less polar than glycosides. Glycosides are usually separated on silica gel columns when using butanol or hydrous butanol as eluents, while proanthocyanidins contain ethyl acetate, which is less polar than butanol. A substance that separates when used as an eluent. However, the method of separating proanthocyanidins using silica gel column is difficult to apply industrially since ethyl acetate, which is harmful to human body, must be used as the eluent. However, using the air absorption resin, proanthocyanidins can be separated using a hydrous ethanol eluent having a lower polarity than the eluent suitable for glycoside separation.

Such air-absorbing resin can be used to purchase a commercially available product, in the present invention used AB-8 air-absorbing resin (manufactured by Nanchang University of China). In addition, the AB-8 macroadhesive resin can be reused with simple and inexpensive operation compared to Sephadex LH-20, cellulose or celite.

The hydrothermal extract of hot water extracted according to the present invention was placed on AB-8 air-absorbent resin, and eluted with 10% ethanol to obtain water-soluble substances having higher polarity than oligomeric proanthocyanidins such as free sugar, water-soluble protein, and water-soluble carbohydrate. After eluting first, the oligomeric proanthocyanidins were eluted with 70% ethanol. Finally eluted with 95% ethanol to elute lipids and nonpolar substances to wash the AB-8 macroabsorbent resin, and 100% distilled water flowed to regenerate the resin.

Finally, the antioxidant activity of the 70% ethanol eluate fraction was twice as good as that of the artificial antioxidant BHA, and the antioxidant meringue containing a large amount of oligomeric proanthocyanidins of 1.78 g / (100 g meringue). An extract could be obtained.

Antioxidant capacity of the extract of the melanoma seeds prepared according to the present invention is DPPH (1,1-Diphenyl-2-picryl hydrazyl) (Chen, CW and Ho CT, J. Food Lipids , 2, 35-46, 1995; Kikuzaki, H , Hisamoto, MI, Sampietro, AR and Vattuone, MA, J. Ethnopharmacol. , 50, 2161-2168, 2002) and TAP (Total antioxidant potential) (Singleton, VL and Rossi, JAJR, AM.J. Enol. Viticult ., 16, 144-153, 1965; Compodonico, P., Barbieri, E., Pizarro, M., Sotomayor, CP and Lissi, EA, Boletin de la Sociedad Chilena de Quimica, 43 (3), 281-285, Quantitative analysis was performed using the Folin-Ciocalteu method (Minussi, RC, Rossi, M., Bologna, L., Cordi, L., Rotilio, D., Pastore, GM and Duran, N., Food Chem., 82, 409-416, 2003).

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

Example 1

Effect of Extraction Yield and Activity on Mixing Composition of Extraction Solvents for Extracting Hot Water from Crushed Maroon Seeds

In order to confirm the composition of the mixed solvent which shows the optimum yield and activity when extracting the mulberry seed with the mixed solvent of water and ethanol, the mixed solvent of water and alcohol with 50-80% (EtOH) content of 50-80% (v / v)) was tested according to the change of the mixed solvent composition. The seedlings were purchased in Jinan, Jeollabuk-do, and 100g of the dried seedlings were washed and dried with 400 ml of mixed solvent and stirred once at 80 rpm for 12 hours at 400 ° C.

The Folin-Ciocalteu method was used for the quantitative analysis of the melon seed extract. As a standard, a calibration curve was indicated using (+)-catechin (Sigma). First, after appropriately diluting the extract of Murussac in ethanol, 100 µl was injected into a test tube in 10 ml, and 1 ml of a 10-fold diluted Pauline-Ciocaltu reagent (Sigma) and 7.5% sodium carbonate ( 0.8 ml of Na ₂ CO ₃ , sigma) was added thereto, followed by reaction at room temperature for 30 minutes, followed by measurement of absorbance at 765 nm. Experimental results showed that the yield of the extract of the wild seedling was 0.32g higher when the mixed solvent was 70% than the 60%, and the yield was almost similar in the mixed solvent of 70% and 80%.

In addition, the DPPH (1,1-Diphenyl-2-picryl hydrazyl, Sigma) radical scavenging activity was measured in order to see the antioxidant activity of the extract of the myrtle seed in each extraction solvent. After extracting the sample and 0.3mM DPPH solution for each concentration in the cuvettes (Cuvette) for 30 minutes at room temperature, absorbance was measured at 517nm. The DPPH solution was calibrated so that the absorbance of the reaction solution was 0.8 when measuring the light absorbance. IC ₅₀ values were calculated with sample concentration controlling 50% DPPH radicals.

Experimental results The antioxidant activity in each extraction solvent is shown in Table 1 below. All of the extracts showed antioxidant activity in 50-80% of the extraction solvent, which was confirmed to be superior to BHA, a phosphate oxidizing agent.

Antioxidant Activity of DPPH by 50 ~ 80% Alcoholic Mixed Solvents Solvent Extraction Solvent According to Alcohol Concentration (v / v) IC ₅₀ ^a (Μg / ml) 50% 25.71 60% 23.20 70% 22.30 80% 22.31 BHA 32.90

^a IC ₅₀ : DPPH free radical scavenging activity Example 2

Effect of Extraction Yield and Activity on Hot Water Extraction from Crushed Maroon Seeds according to Temperature Change

In order to confirm the temperature condition showing optimum yield and activity in the first extraction process after grinding the seedling seeds, experiments were carried out depending on the extraction temperature of 50 ~ 90 ℃. 100 g of the dried seedlings were washed and dried with 400 ml of mixed solvent, and extracted once with stirring at 400 rpm for 12 hours at a temperature ranging from 50 to 90 ° C. Quantitative and activity analysis of the melon seedling extract was measured in the same manner as in Example 1.

Experimental results showed that the yield of the extract increased as the temperature increased. The maximum yield was increased to 0.24g between 60 ℃ and 70 ℃ but only 0.06g was increased between 80 ℃ and 90 ℃.

In addition, the analysis of antioxidant activity showed the lowest activity at 50 ℃, the highest activity at 80 ℃ it was found that the activity decreased rather than at 90 ℃. Therefore, as the temperature increases, the yield increases, but after 80 ° C., the increase in yield slows down and the activity decreases.

Antioxidant Activity of DPPH According to Extraction Temperature Extraction temperature (℃) IC ₅₀ ^a (Μg / ml) 50 30.40 60 24.20 70 22.80 80 22.32 90 23.01. BHA 32.90

^a IC ₅₀ : DPPH free radical scavenging activity Example 3

Effect of Extraction Yield and Activity According to Extraction Time on Extracting Hot Water from Crushed Maroon Seeds

After grinding the seedling seeds, experiments were conducted to determine the extraction operation time, which shows the optimum yield and activity in the first extraction process. 100 g of the dried seedlings were washed and dried, and then extracted twice with 400 ml of a mixed solvent at 80 ° C. for 12 hours at 400 rpm. Quantitative and activity analysis of the melon seedling extract at each time was measured in the same manner as in Example 1.

As shown in FIG. 2, the yield of the extract increased as time passed, and the yield increased by 0.12 g on average from 4 hours to 8 hours, but only slightly increased after 8 hours. Appeared.

In addition, DPPH activity increased most significantly between 1 and 2 hours, and gradually increased after 2 hours, and then increased again from 6 to 8 hours. Could confirm.

Therefore, the most economical and rapid extraction of antioxidants from the melon seed was most effective by stirring extraction for about 8 hours at about 80 ℃ using a hydrous about 70% alcohol mixed extract solvent.

Example 4

Antioxidant Activity in Extracting Crushed Mulberry Seed at Room Temperature

After pulverizing the seedling seeds, the extract was extracted with 100% distilled water and 70% ethanol at room temperature without applying heat in the first extraction process to analyze the antioxidant activity and yield. 100 g of the dried seedlings were washed and dried with 400 ml of mixed solvent at room temperature and stirred at 400 rpm for 24 hours. Quantitative and activity analysis of the melon seedling extract at each time was measured in the same manner as in Example 1.

Experimental results showed that the extraction yield was very low when using 100% distilled water extraction solvent, but the antioxidant capacity was superior to BHA. In addition, the extraction yield of more than twice that of 100% distilled water was obtained in case of using the hydrous 70% alcohol extraction solvent, and the antioxidant capacity was also excellent.

Therefore, the antioxidant material could be extracted even when only 100% distilled water was used at room temperature when extracting the antioxidant from the melon seed chamber. In order to extract the antioxidant more effectively, it is preferable to use a hydrous alcohol rather than 100% distilled water. In addition, antioxidants can be extracted from the melon seedling at room temperature, but extraction with heat is more effective.

Extraction Yield and DPPH Activation Ability According to Extraction Solvent in Primary Extraction Process at Room Temperature Extraction solvent Yield (g / 100g beech seed) IC ₅₀ ^a (Μg / ml) 100% distilled water 0.50 24.31 70% Alcohol (EtOH) 1.32 22.90

^a IC ₅₀ : DPPH free radical scavenging activity Example 5

DPPH Antioxidant Search

In order to measure the radical scavenging activity of DPPH (1,1-Diphenyl-2-picryl hydrazyl), 100 g of dried and dried Murusa seeds were stirred with 400 ml of 70% mixed solvent at 400 rpm for 8 hours at 80 ° C. And extracted twice. The extract was concentrated under reduced pressure at 50 ° C. and placed on a column filled with AB-8 macroabsorbent resin and eluted with 10%, 70%, and 95% hydrous ethanol eluent. The fraction of 70% eluent containing the most oligomeric proanthocyanidins was concentrated under reduced pressure to obtain the final melon seed extract.

Figure 3 is a graph showing the analysis of the DPPH antioxidant activity of the BHAs widely used as a primary extract (crude extracts), the first extract of the myrtle seed through the column (thru column) and phosphorus oxidizing agent. The sample of the fraction obtained by passing the first extract through the column showed 1.46 times better DPPH activity than the first extract, and the antioxidant ability was more than two times better than BHA. The results are shown in Table 4 below.

Antioxidant Activity of DPPH According to Extraction Stage of Extract Kinds IC ₅₀ ^a (Μg / ml) Crude extracts of the flowering seeds 22.9 Thru column obtained by passing the primary extract through the column 15.7 BHA 32.9

^a IC ₅₀ : DPPH free radical scavenging activity Example 6

Total antioxidant potential (TAP) antioxidant search

In order to detect TAP antioxidant activity, 14 mM ABTS (2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), Sigma) and 9 mM potassium persulphate (Sigma) were 1: 1. After mixing with (v / v) and left for 12 hours at room temperature in the dark to prepare ABTS radicals. After extracting the ABTS radical prepared by concentration in Cuvettes (Cuvette) for 30 minutes in a dark room at room temperature, the absorbance was measured at 734 nm. The ABTS radical solution was calibrated so that the absorbance of the reaction solution was 0.7 when measuring the light absorbance. IC ₅₀ values were calculated as sample concentrations controlling 50% ABTS radicals.

The sample sample was prepared in the same manner as in Example 5 and the fraction obtained by passing the primary extract of the melon seeds and the extract through the column filled with the air-absorbent resin, artificial antioxidant (BHA) was compared and analyzed TAP antioxidant capacity, In order to more easily compare the experimental results of Figure 4 is shown in Table 5 as IC ₅₀ value.

The final extract obtained by passing the first extract through the column showed DPPH antioxidant capacity about 2 times better than BHA, and the TAP antioxidant activity showed similar tendency to that of DPPH.

Antioxidant Activity of TAP by Different Extraction Stages of Extract Kinds IC ₅₀ ^a (Μg / ml) Crude extracts of the flowering seeds 40.7 Thru column obtained by passing the primary extract through the column 31.6 BHA 72.0

^a IC ₅₀ : ABTS free radical scavenging activity Example 7

In the same manner as in Example 5, a sample sample prepared by separating and purifying an antioxidant substance from the melon seed chamber was analyzed by thin layer chromatography (hereinafter referred to as TLC). TLC uses a TLC plate (Silica gel, Merck) as a stationary phase and a mixed solvent of toluene: acetone: acetic acid (3: 3: 1, (v / v)) (Sun, B., Leandro, C., Ricardo) as a mobile phase. da Silva, J. and Spranger, I., J. Agr.FOOD Chem. , 46 (4), 1390-1396, 1998).

As shown in FIG. 5, the fraction (Cw) obtained by passing the first melon seed extract (Tw) through a column filled with the AB-8 macroabsorbent resin is effectively purified at the same position as the catechin (S) as a standard. Could be confirmed.

Table 6 below is a graph showing the yield and antioxidant capacity in each process of separating and purifying the extract having antioxidant capacity from the seedlings. The yield of the final extract containing the final oligomeric proanthocyanidin from the melon seeds was obtained 1.78g / (100g melon seeds), the antioxidant ability was confirmed that more than two times better than the artificial antioxidant BHA.

Yield and Antioxidant Capacity According to Extraction Stage Extraction step Yield (g / 100g melon seed) DPPH (IC ₅₀ , μg / ml) TAP (IC ₅₀ , μg / ml) Crude extracts 2.05 22.9 40.7 Thru column 1.78 15.7 31.6

As described above, the method of the present invention can separate the antioxidants from the melon seeds with high purity and high yield, and the separated antioxidants are multifunctional natural antioxidants that prevent rancidity and aging of food caused by free radicals. It may be usefully used as a functional food or additives containing the food.

1 is a process flow diagram illustrating a process of extracting an antioxidant substance (proanthocyanidin oligomer) from the melon seed extract according to the present invention.

2 is a graph showing the extraction yield and activity according to the extraction time change during the extraction of antioxidants according to the present invention.

Figure 3 is a graph showing the DPPH antioxidant activity of the first extract and column chromatography fraction of the melon seedling obtained in accordance with the present invention.

Figure 4 is a graph showing the TAP antioxidant activity of the first seed extract and column chromatography fractions obtained according to the present invention.

5 is a photograph of the first extract and column chromatography fraction of the melon seed yarn obtained in accordance with the present invention after thin-film chromatography and analyzed by UV-lamp.

Claims (3)

Grinding the melon seed yarn; Extracting the pulverized myrhea seed extract by concentrating by extracting the pulverized seedling seeds at 50-80 ° C. for 8 to 12 hours using a hydrous 50-80% alcohol mixed extract solvent; And Suspension of the melon seedling extract in a mixed solvent of water and ethanol and placed in a large absorbing resin consisting of alkyl benzene-based polymer, and then mixed with a mixture of water and ethanol having a concentration gradient of 10%, 70% and 95% Elution of the proanthocyanidin-based compound in a sequential order; Separation method of the proanthocyanidin oligomer from the extract. The method of claim 1, wherein the pulverized melon seed yarn is extracted for 8 hours at 80 ° C. using a hydrous 70% alcohol mixed extract solvent. The method of claim 1, wherein the concentrated melon seed extract is concentrated to about 70%.