KR101404286B1 - Method for preparing the extract fortified with chalcones from by-product of Angelica keiskei juice - Google Patents

Abstract

The present invention relates to a method for producing an extract containing chalcone at a high concentration in a fresh chestnut foil. More particularly, the present invention relates to a method for producing an extract of chrysanthemum chinensis extract with a solvent selected from the group consisting of alcohols having 1 to 6 carbon atoms and a water- Which comprises concentrating the extract to obtain an insoluble precipitate, and a purification step of removing the water-soluble component from the insoluble precipitate, and a chalcone-containing extract prepared by the method. The method of the present invention is effective for extracting chalcone from by-products obtained in the conventional green juice production. In addition, the extract of the present invention contains chalcone at a high concentration and is excellent in liver function protection, blood glucose control, anti-cancer or antioxidant effect.

Chinese herb, hydroxyderisin,

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing an extract of chalcone from an extract of green tea,

The present invention relates to a method for producing an extract containing chalcone in a high concentration in a fresh chestnut which is a by-product of fresh chestnut green juice, and more particularly to a method for producing an extract containing chalcone in a high concentration, A method for preparing a chalcogenol-containing extract, which comprises extracting with a solvent, concentrating the extract and obtaining an insoluble precipitate, and a purification step of removing the water-soluble component from the insoluble precipitate, Containing extract.

Angelica keiskei Koidzumi is a sub tropical tropical perennial herb that belongs to the subfamily, and contains various vitamins and minerals, various flavonoids such as physiologically active substances, coumarin and saponin, and is currently used as a raw material for green juice and reproductive in Korea. It is a green vegetable that is attracting attention as food.

It is also known as Shinchungcho. It grows for vegetables and is about 1 meter tall. The stem grows straight and branches. Leaves are dark green and shiny. A small flower of a light yellow color is bloomed in a bloom in the autumn, and the smell is unique, and it feels good when it smells. When you cut the stem or leaves, yellow juice comes out. If you pick up the stem of a leaf, it grows enough to have a young leaf the next day and it is also called 'Myeongilbye'. It contains carotene, vitamin C and iron, and is said to have a tonic effect.

Chrysanthemum japonica is mainly grown in the subtropical region. In Korea in the 1970s, it has been widely used as green juice in Korea. For a long time, chrysanthemum has been used as a folk remedy for hypertension, liver disease and neuralgia, and its physiological activities such as antihyperlipidemia, hypotension, anticancer activity and inhibition of gastric acid secretion have been reported.

Recently, Takara Bio Bio Co., Ltd. of Japan has developed two kinds of chalcone (4-hydroxyderricin, xanthoangelol) contained in Angelica keiskei to promote the differentiation of adipocytes in progenitor adipocytes and the activity to promote adipocyte glucose uptake The activity of chalcone in chinese cabbage has been studied.

Recently, as the public interest in health has increased, studies on functional food and functional food material processing technology that can prevent disease through daily intake of food have been actively carried out. Among them, greenish-yellow vegetables contain a large amount of vitamins such as beta-carotene, ascorbic acid, and tocopherol and various polyphenol compounds. These ingredients effectively prevent free radicals, which cause aging, It is known to inhibit. Green juice, which is a green juice made by cutting and grinding the greenish-yellow vegetables with physiological activity without heating, is originally produced so that the nutrients of greenish-yellow vegetables can be easily digested and absorbed in the body.

Many studies have been carried out on the physiological activity of green juice by many researchers until now, and there have been a lot of researches on the use of green juice which is being abolished or being discarded.

Among the various green juice products currently being produced by the green juice experts, the most abundant produce is the fresh goose. In addition, the amount of green vegetable byproducts produced in the green juice may vary depending on the raw materials and season, but 30 to 70% of the used plants are usually used, and industrials have been trying to solve these green vegetable byproducts.

However, there has been no report on a method for extracting an additional effective ingredient from a freshly picked papaya juice which has been juiced until now. Especially, an extract containing a high concentration of chalcone, which is a representative active ingredient of papaya extract, There has been no report on the method of preparation.

The inventors of the present invention found that the chalcone of chrysanthemum morifolium was present in a large amount in the water-insoluble component while studying the application method of the green chrysanthemum powder and extracted the green juice, And then concentrated to remove the water-soluble fraction. Thus, an extract containing chalcone at a high concentration can be prepared, thereby completing the present invention.

Therefore, an object of the present invention is to provide a process for producing a water-soluble extract, comprising the steps of: a) extracting a fresh persimmon leaf with a solvent selected from the group consisting of a mixed solvent of an alcohol having 1 to 6 carbon atoms and water, (b) step; And (c) a purification step of removing the water-soluble component from the insoluble precipitate obtained in the above.

Another object of the present invention is to provide a chalcone-containing extract prepared by the above method.

In order to attain the above object, the present invention provides a process for preparing a mixture of (a) extracting a fresh persimmon leaf with a solvent selected from the group consisting of a mixed solvent of an alcohol having 1 to 6 carbon atoms and water, (b) concentrating the extract Obtaining an insoluble precipitate; And (c) a purification step of removing the water-soluble component from the insoluble precipitate obtained in the above.

In order to achieve another object of the present invention, there is provided a chalcone-containing extract prepared by the above method.

Hereinafter, the present invention will be described in detail.

Angelica keiskei Koidzumi is a sub-tropical perennial vegetation of lepidoptera, also known as myeonil. It is rich in vitamins and minerals. It contains various flavonoids, coumarin and saponin which are physiologically active substances. It is a green vegetable that has been attracting attention as a health food.

The present invention is a by-product which is mainly discarded as a solid component remaining after juice-making a fresh plant. The fresh persimmon leaves of the present invention can be used after being dried, washed, or the like, though the present invention is not limited thereto. As the drying method, it is possible to use any of the methods of biped, shade, hot air drying and natural drying. Preferably, it may be hot-air drying, thereby preventing degeneration and discoloration of fresh-cut pods.

The present invention relates to a process for producing a polyvinyl alcohol solution, comprising the steps of: (a) extracting a fresh persimmon leaf with a solvent selected from the group consisting of a mixed solvent of an alcohol having 1 to 6 carbon atoms and water; (b) concentrating the extract and obtaining an insoluble precipitate; And (c) a purification step of removing the water-soluble component from the insoluble precipitate obtained in the above.

In the step (a), the fresh persimmon leaves are extracted with a solvent.

The fresh persimmon leaves of the present invention can be extracted by a solvent extraction method known in the art. Examples of the extraction solvent include any one selected from organic solvents such as water, alcohols having 1 to 6 carbon atoms such as ethanol and methanol, acetone, ethyl acetate, n-nucleic acid, diethyl ether, acetone and benzene, A solvent may be used. Preferably, it may be extracted with any one solvent selected from the group consisting of a mixed solvent of an alcohol having 1 to 6 carbon atoms and water.

Most preferably, the extraction solvent may be ethanol. When water is used as a solvent, the yield is very low as compared with the case where a mixture of ethanol or ethanol and water is used as a solvent. When a mixture of ethanol and water is used as a solvent, the higher the ethanol ratio, the better the extraction yield, and preferably a mixture of ethanol having a ratio of 95% or more can be used as the solvent.

As the extraction method, ordinary extraction methods such as cold-rolling, warm-up, heating and the like can be used by using the above-mentioned solvent. When extracting with ethanol, the ratio of fresh persimmon leaf to ethanol is not particularly limited, but ethanol may be added to the fresh persimmon leaf in an amount of 1 to 10 times by weight. Preferably, to increase the extraction efficiency, ethanol may be added twice to four times to the acacia leaves.

In one experimental example of the present invention, fresh persimmon leaves were extracted according to the method of Example 1 with different extraction solvents. As a result, it was confirmed that the highest efficiency was obtained when 100% ethanol was extracted.

The temperature at the time of extraction is not particularly limited so long as the extraction component does not break down, and may be, for example, from 4 ° C to 120 ° C. Preferably 50 ° C to 75 ° C, and most preferably 50 ° C to 60 ° C.

The extraction time may vary depending on the extraction temperature and the extraction solvent, but may be, for example, 0.5 to 48 hours, preferably 10 to 15 hours, when the extraction is performed at 50 to 60 ° C in a 100% ethanol solvent Most preferably from 12 hours to 13 hours.

In one experimental example of the present invention, the fresh persimmon leaves were extracted at different extraction times. As a result, the extraction efficiency decreased with increasing extraction time for 15 hours or longer, and it was found to be most effective when extracted for 12 hours to 13 hours.

In the step (b), the extract is concentrated to obtain an insoluble precipitate.

In the step (c), the water-soluble component is removed from the insoluble precipitate obtained in the above step and purified.

Concentration refers to removing the solvent in the solution and increasing the concentration of the solute. The extract may be concentrated by a concentration apparatus or a concentration method known in the art and may be, for example, precipitation concentration, evaporation concentration, reduced pressure concentration, ultrafiltration, reverse osmosis, centrifugation. The chalcone has high heat stability, but it can be concentrated under reduced pressure at 40 to 55 ° C, preferably for safe use of ethanol alcohol.

The concentration ratio can be preferably from 1/2 to 1/10, and most preferably from 1/4 to 1/5. In case of concentrating to 1/10 or more, loss of chalcone component occurs in the process of recovery and filtration.

In one experimental example of the present invention, the extract was concentrated by varying the concentration ratio. As a result, it was confirmed that when concentrated to a maximum, loss of the chalcone component occurs at the time of recovery, and when it is concentrated at a ratio of 1/5, it is suitable for recovery and filtration.

The purification may be any known purification process as long as it removes water-soluble components from the concentrate to enhance the chalcone content of the present invention. For example, the purification may be performed by, for example, separating the precipitate using a filter paper and setting a constant molecular weight cut- Separation using an ultrafiltration membrane having a hydrophilic or hydrophobic property, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), and decolorization process. Preferably, the precipitate precipitated in the concentration step is filtered with a chisel filter to obtain a precipitate, which is then mixed with water and then filtered again to obtain a precipitate.

In chrysanthemum, chalcone is contained in a large amount in the water-insoluble component.

In one experimental example of the present invention, the ethanol extract of Angelica gigas Nakai was concentrated to separate the water-soluble component and the non-water-soluble component, and the content of each chalcone was measured.

As a result, it was found that the water-soluble part contained 0.44 mg / g of chalcone, while the water-insoluble part contained 3.26 mg / g of chalcone, and the water-insoluble part contained about 7.4 times more chalcone than the water- Respectively.

This fact was first discovered by the present inventors.

In order to further increase the chalcone content of the extract of the present invention, the water-soluble component may be removed using the water, and the precipitate may be further dissolved in an alcohol having 1 to 6 carbon atoms and filtered. As described above, the chalcone component is contained in the water-insoluble component so that when dissolved in alcohol, the chalcone component is dissolved in alcohol and passed through the filter. Thus, the filtered filtrate is recovered, concentrated and lyophilized to obtain an extract having a higher chalcone ratio.

Thus, the production method of the present invention produces an extract containing chalcone at a high concentration in a fresh chestnut foil.

In one embodiment of the present invention, 300 weight% ethanol was added to the fresh persimmon leaf, heat was applied thereto while stirring, and the extract solution was extracted at 55 ° C for 12 hours. After filtration, the extract was concentrated and the precipitate was recovered with a chitosan filter. Water was added, After removing the water - soluble components by vacuum drying, extracts containing high concentration of chalcone were prepared.

According to the method of the present invention, the extract containing 209.2 mg / g of chalcone was able to be extracted. However, in the case of the extract extracted by the usual chalcone extraction method, about 15 mg / g of chalcone was extracted (see Example 1-2).

The chalcone contained in the extract prepared by the method of the present invention may also be xanthoangelol or 4-hydroxyderricin.

In one embodiment of the present invention, AK-C20 was prepared by pulverizing the extract prepared by the method of the present invention, and the kind and content thereof were measured by NMR and HPLC. As a result, the chalcone of the extract prepared by the method of the present invention was mainly composed of zanthone and hydroxyderisin, and its content was 131.1 mg / g in case of azoxyanuride and 78.1 mg / g in case of hydroxyderisin (See Example 1).

The present invention also provides chalcogen-containing extracts prepared by the above method.

The extract of the present invention is characterized in that chalcone is contained at a high concentration.

Since the extract of the present invention contains chalcone at a high concentration, it exhibits excellent liver function protection, inhibition of blood glucose, anticancer or antioxidant activity, and this function is far superior to that of the conventional herbal extract.

In one embodiment of the present invention, an animal experiment or a cell experiment was conducted on liver function protection, blood glucose control, anti-cancer or anti-oxidative effect of the composition of the present invention. As a result, the composition of the present invention inhibits blood glucose levels in the mice, suppresses GOT and GPT levels in mice that induce hepatotoxicity, decreases the survival rate of cancer cells, has excellent electron donating ability, inhibits lipid peroxidation and SOD activity, Was superior to the conventional herbal extract (see Example 2).

The extract of the present invention not only exhibits the above-described efficacy but also is harmless to the human body since it is extracted from plants used for edible and medicinal purposes, and does not cause side effects due to long-term use.

Accordingly, the present invention provides a method for producing an extract containing chalcone at a high concentration in a fresh chestnut foil, and an extract containing the chalcone produced by the above method. The method of the present invention is effective for preparing an extract containing a high concentration of chalcone from a by-product obtained from the conventional green juice production. In addition, the extract of the present invention contains chalcone at a high concentration and is excellent in liver function protection, blood glucose control, anti-cancer or antioxidant effect.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Experimental Example 1 >

1. Extraction solvent and time

1) Extraction efficiency test according to extraction solvent

To compare the extraction efficiency according to the extraction solvent, extraction was carried out according to the method of Example 1 with purified water, 30% ethanol, 60% ethanol and 100% ethanol.

As a result, it was found that the yield of purified water was much lower than that of ethanol extract. In case of ethanol extraction, 100% ethanol was the most efficient.

2) Extraction efficiency test according to extraction time

In order to compare the extraction efficiency according to the extraction time, extraction was carried out according to the method of Example 1, but the extraction time was 5 hours, 10 hours, 15 hours and 20 hours.

As a result, the increase was the largest between 10 hours and 15 hours.

Therefore, the extraction time was increased from 1 hour to 15 hours.

As a result, it was shown that extraction from 12 to 13 hours was the most efficient.

3) Extraction efficiency test according to extraction temperature

In order to compare the extraction efficiency according to the extraction temperature, extraction was carried out according to the method of Example 1, and extraction was performed at room temperature, 25 ° C and 75 ° C.

As a result, the content of solids increased as the temperature increased, but it was found that 50 ° C to 60 ° C was suitable because the process was difficult to perform because the stability was above 75 ° C.

2. Concentration and Filtration

1) In order to establish the most efficient concentration, the extracted extracts were concentrated at 1/2, 1/5, and maximal concentration.

As a result, solidification is increased when the concentration is increased, but when the concentration is maximized, loss is generated at the time of recovery.

2) In order to optimize the filtration process for the recovery of active ingredients, filtration was performed using a 20 mesh filter net or a microfilter, centrifugation and filtration using a separatory funnel.

As a result, the recovery efficiency is high in the case of centrifugation, but a large amount of loss occurs at the time of recovery, and the operation is troublesome. Separation funnels have a long separation time, small capacity, and difficulty in precision separation. In the case of using the filter net, it was confirmed that the recovery rate of the 20 mesh filter net was low and the recovery efficiency was high and the operation efficiency was high when the micro filter was used.

3. Separation Tablets

1) Purification of NaOH, water and ethanol were performed to increase the purity of the surface components.

As a result, the purity of NaOH was decreased and the purification efficiency of water purification was low. In the case of ethanol purification, it was confirmed that the purity was increased by removing the water - soluble component incorporated in the solid matter.

2) The ethanol-extracted extract is concentrated by concentration under reduced pressure to recover the insoluble precipitate, which is then dissolved in alcohol, filtered through a filter paper, and concentrated to dryness to obtain a water-insoluble component. The insoluble precipitate is recovered and concentrated. The water-soluble component and the water-insoluble component were separated in the same manner as that obtained.

 As a result of measuring the chalcone content by HPLC quantitative analysis, it was confirmed that the water-soluble component contained about 0.44 mg / g and the non-water-soluble component contained about 3.26 mg / g of chalcone (see FIG. As a result, it was confirmed that the chalcone component of the ethanol extract of chrysanthemum morifolium was mostly contained in the water - insoluble part.

≪ Example 1 >

Production of chalcone extract by the method of the present invention

<1-1> Production of AK-C20

300% by weight of ethanol was added to the by-product after the green juice extract of the kuzuchishi, and heat was applied thereto while stirring, followed by extraction at 55 ° C for 12 hours.

The extract was filtered from 1 mesh to 150 mesh in the transfer tube while transferring through the transfer tube. The ethanol component was removed by low-temperature condensation with 40 brix using an evaporator, and the ethanol component was removed therefrom. The ethanol component was further concentrated to 60 brix at 55 ° C to precipitate chalcone.

 The components precipitated in the solution were passed through a chisel filter at a rate of 85 ml / min to filter and collect the precipitate. Recovery The immersed component is determined as crude chalcone, and dehydration reaction is carried out at 85 ° C for 6 hours to remove water and obtain a chalcone component.

In order to increase the chalcone content in the recovered precipitate, 10 times as much water as the precipitate was added, and the mixture was heated and stirred at 80 ° C for 2 hours, and then cooled to remove water and freeze-dried to remove the water-soluble component.

To increase the chalcone ratio of the extract, ethanol (95%) was added to 15 times of the precipitate after the above method, and the solution was stirred with heating (40 ° C for 1 hour), filtered using a chisel filter, The solids dried by lyophilization were made into powder. It was named AK-C20.

<1-2> Analysis of ingredients and content

The analysis of the components and contents of the chalcone - reinforced materials was carried out by HPLC analysis. The conditions of the analysis were as follows: Column: Shiseido CAPCELL PAK C18, UG120, 5 (4.6 mm ID x 250 mm) Temperature 35 ° C Flow rate 1500 l / min (15.0 MPa) 20, mobile phase composition: 1% acetic acid + methanol: distilled water = 8: 2 (v / v%) and measuring time: 15 min (7-10 min; sample).

The two active components from the HPLC analysis were fractionated and the structure was analyzed to identify them (see Examples 1-3 below).

The sample to be analyzed was quantitatively analyzed, and then methanol was added thereto, and the resultant was ultrasonically extracted for 10 minutes. The solution was filtered through a membrane (0.45 μm) to be used as a test solution.

The content of the sample analyzed by HPLC was calculated by calorimetric analysis obtained by analyzing the surface composition of the chalcone, and the chalcone content in the final sample was calculated by applying the sample amount and the dilution concentration.

As a result, as shown in FIG. 1A, it was confirmed that AK-C20 contained a high concentration of chalcone.

It was confirmed that the chalcone component of AK-C20 was contained at 209.2 mg / g, and the concentration thereof was 78.1 mg / g in the case of hydroxyderisin and 131.1 mg / g in case of zanthone angerole.

As a control group, the extract was extracted by a general method of extracting with 300% by weight ethanol from fresh persimmon leaves, followed by filtration, concentration and drying. The content of chalcone was measured by the same method and it was confirmed that chalcone was contained at about 15 mg / g.

As a result, it was confirmed that the method of the present invention can produce an extract containing chalcone at a high concentration from the fresh chestnut which is a by-product of green juice production.

<1-3> Structural analysis

The active ingredient of AK-C20 of the present invention was analyzed through EI-MS and NMR spectrum analysis. EI-MS used JMS-AX505WA (JEOL), and NMR used Advance 600 spectrometer (Bruker). As a result, an active ingredient of AK-C20 was identified as a hydroxyderisin having a structure represented by the following formulas (1) and (2)

The molecular weight of the compound represented by the formula (1) was determined to be 392 from the molecular ion peak ([M] ⁺ m / z 392) in the EI / MS spectrum. ^{1 H-NMR (600MHz, CDCl} 3) in the spectrum δ 1.59 (3H, s, 7 '' - CH 3), 1.63 (3H, s, 8 '' - CH 3), 1.85 (3H, s, 3 '' _{-CH 3), 2.08 (2H,} m, 4 '' - H), 2.10 (2H, m, 5 '' - H), 3.49 (1H, d, J = 7.1 Hz, 1 '' - H), 5.05 (1H, d, J = 8.9 Hz, 5'-H), 6.88 (2H, (2H, d, J = 8.5 Hz, 2,6-H), 7.72 (1H, d, J = ¹³ C-NMR (150 MHz, d, J = 8.9 Hz, 6'-H), 7.83 (1H, d, J = 15.4 Hz, CDCl ₃₎ δ 16.5 in spectrum _{(3 '' - C H 3} ), 17.9 (7 '' - C H 3), 21.9 (1 '' - C), 25.9 (8 '' - C H 3), 26.6 ( C), 114.9 (3'-C), 116.2 (3, 5'-C), 39.9 C), 118.2 (? -C), 121.2 (2? -C), 123.9 (6? -C), 127.9 C), 144.4 (? -C), 162.1 (4,2'-C), 164.1 (4'-C) and 192.5 .

The molecular weight of the compound represented by the formula (2) was determined from the molecular ion peak ([M] ⁺ m / z 338) to 338 in the EI / MS spectrum. ^{1 H-NMR (600MHz, CDCl} 3) in the spectrum δ 1.60 (3H, s, 4 '' - CH 3), 1.71 (3H, s, 5 '' - CH 3), 3.31 (1H, d, J = 7.0 Hz, 1 '' - H) , 3.82 (3H, s, 4-OCH 3), 5.30 (1H, t, J = 7.0 Hz, 2 '' - H), 6.41 (1H, d, J = 9.0 Hz, (2H, d, J = 9.4 Hz, 2 H), 7.39 (2H, d, J = , 6-H), 7.70 (1H, d, J = 9.4 Hz, 6'-H), 7.72 (1H, d, J = 15.4 Hz, b), 13.38 ^{were, 13 C-NMR (150MHz,} CDCl 3) δ 18.0 in the spectrum _{(4 '' - CH 3)} , 21.9 (1 '' - C), 26.0 (5 '' - CH 3), 56.0 (4-OCH 3 ), 102.4 (5'-C), 108.1 (5 ', 3''-C), 114.8 (1'-C), 114.3 C), 122.2 (2 ' -C), 127.7 (1-C), 129.4 (6 ' -C), 130.8 , 163.5 (4'-C), 163.5 (4'-C), 158.3 (4-C) It was identified as Derridine.

&Lt; Example 2 >

AK-C20 functional efficacy verification

<2-1> Liver function protection

In order to examine the protection effect against hepatotoxicity caused by carbon tetrachloride, ICR mice were orally administered AK-C20 at a dose of 200 mg / kg, 400 mg / kg and 600 mg / kg for 7 days and then intraperitoneally administered carbon chloride (CCl ₄ ) Serum was analyzed.

As a result, as shown in Table 1, GOT (glutamic-oxaloacetic transference aminase) and GPT (glutamic-pyruvic transaminase) were significantly lower than those in the induced group.

Serum GOT, GPT after 7 days oral administration Enzyme activity sGOT sGPT Induced group (CCl4 administration) 1163.8 ± 381 3007.5 + - 622.8 CCl4 + silymarin (100 mg / kg) 620.5 ± 212.0 ^* 1959 ± 515.7 ^* CCl4 + AK-C20 (200 mg / kg) 735 + 247.1 ^* 2068.7 ± 504.4 ^* CCl4 + AK-C20 (400 mg / kg) 764 + 214.3 ^* 1905.3 ± 426.2 ^* CCl4 + AK-C20 (600 mg / kg) 497.5 ± 78.3 ^* 1628.2 + - 384.7 ^*

* p <0.01 vs induced group

The mice were then implanted under ether anesthesia to cut the liver tissue, fixed overnight in 10% formalin, embedded in paraffin, and cut with 4-5 μm tissue samples with a microtome to prepare slides. From these slides, hepatocyte and eosin (H & E) staining were performed to observe liver damage.

As shown in FIG. 2, it was confirmed that the degree of cell necrosis and inflammation penetration caused by carbon tetrachloride in the liver tissue of the mice was greatly improved by the AK-C20 of the present invention.

&Lt; 2-2 >

Fasting blood glucose was measured in ICR mice that were fasted the previous day. After the measurement, glucose and glucose and AK-C20 were administered and blood glucose levels were measured again after 30 minutes.

Results As shown in Table 2, it was confirmed that the blood glucose level of the group to which glucose and AK-C20 were orally administered together was lower than that of the group administered only glucose.

Blood glucose measurement after oral glucose administration Blood glucose measurement Before oral administration (fasting glucose) 30 minutes after oral administration Glucose administration (10 g / kg) 50.6 ± 7.0 243 ± 24.3 Glucose + AK-C20 (1.25 g / kg) 52.2 ± 5.9 223.6 ± 12.7 Glucose + AK-C20 (2.5 g / kg) 53 ± 5.7 210.8 ± 10.1 ^* Glucose + AK-C20 (5 g / kg) 52.7 ± 5.6 142.6 + - 12.6 ^*

* p <0.01 vs glucose administration group (30 minutes elapsed)

<2-3> Anticancer effect

The HT-29 cell line, a colon cancer cell, was cultured and treated with various concentrations of AK-C20 (0, 5, 25, 50, 100, 200 ug / ml) The survival rate of cancer cells was measured.

As a result, as shown in FIG. 3, the survival rate of cancerous green juice did not significantly affect the survival rate of cancer cells, and the cell viability of HT-29 cells was decreased at the concentrations of 100 ug / ml and 200 ug / ml for AK-C20. As shown in FIG. 4, it was confirmed that the proliferation of HT-29 cells was inhibited with the passage of time when AK-C20 was administered.

<2-4> Antioxidant activity - Electron donating ability, inhibition of lipid peroxidation, SOD activity

To verify the antioxidant effect of ethanol extract powder, electron donating abilities (EDA), inhibition of lipid peroxidation and SOD activity were measured.

As a test substance, fresh frozen green lyophilized powder (1 mg / ml) and AK-C20 (1 mg / ml) were used and vitamin C (1 mg / ml) was used as a positive control.

The DPPH solution was prepared by dissolving 20 mg of DPPH (1,1-diphenyl-2-picryl hydrazyl) in 150 ml of ethanol. 0.5 ml of the DPPH solution and 0.5 ml of the prepared sample were shaken and reacted at room temperature for 30 minutes. The absorbance at 517 nm was measured and the absorbance difference for the control group was calculated to calculate the electron donating ability.

As shown in FIG. 5, DPPH was found to have an improved electron donating ability of 23.1% and AK-C20, which was 200% higher than that of the conventional green juice green juice, and 63% .

The inhibitory effect of thiobarbituric acid reactive substances (TBARS) was measured using linoleic acid as a substrate to verify the ability of AK-C20 to protect against lipid peroxidation.

After adding 50 mM Tris buffer and 0.1 mM EDTA to 0.8% sodium lauryl sulfate containing 0.1% (w / v) linoleic acid, the sample was added, We irradiate it for 90 minutes in ultraviolet lamp. After the irradiation, 0.44M trichloroacetic acid (TCA) and 0.8% 2-thiobarbituric acid were mixed. The mixture was developed at 100 ° C for 15 minutes, immediately cooled, and absorbance was measured at 532 nm. The inhibition of lipid peroxidation was calculated by the following equation.

(%) = (1 - sample absorbance / control absorbance) x 100

As shown in FIG. 6, it was confirmed that lipid peroxidation inhibition was shown to be 11.5%, AK-C20 32.3%, and vitamin C 50.9% in the fresh chestnut green juice (p <0.01) .

SOD (superoxide dismutase) is an enzyme that converts superoxide to hydrogen peroxide and oxygen molecule. It is one of the important enzymes in the body antioxidant system. The degree of inhibition of superoxide generation by xanthine oxdiase was evaluated by SOD assay kit (Dojindo , Japan).

As shown in FIG. 7, AK-C20 was found to inhibit the formation of superoxide anion, which is an oxygen radical, with 57.9% of green juice, 64.4% of AK-C20 and 94.1% of vitamin C, 68%, respectively.

In conclusion, AK-C20 was found to be excellent in liver protection, blood glucose control, anti-cancer and antioxidant functions.

As described above, the present invention provides a method for producing an extract containing chalcone at a high concentration in a fresh chestnut foil, and an extract containing the chalcone produced by the method. The method of the present invention is effective for preparing an extract containing a high concentration of chalcone from a by-product obtained from the conventional green juice production. In addition, the extract of the present invention contains chalcone at a high concentration, and thus has excellent potential for industrial use because it has excellent liver function protection, blood glucose control, anticancer or antioxidant effect.

Figure 1 shows the HPLC analysis of the extract. Figure 1A shows the HPLC analysis results of the extract (AK-C20) prepared by the method of the present invention. Figure IB is the HPLC analysis of the water soluble fraction from the extraction process of the present invention.

FIG. 2 shows experimental results of liver function improvement by ingestion of AK-C20.

FIG. 3 is a result of measuring the cell survival rate by using an MTT assay to measure the anticancer effect of AK-C20 and the known green tea juice (AK-C20: extract prepared by the method of the present invention).

FIG. 4 is a photograph showing the state of colon cancer cells treated with AK-C20 according to the passage of time. FIG.

FIG. 5 is a graph showing the electron donating ability of AK-C20 and the known green algae green juice (GJ: freeze-dried powder of green juice green liquor (1 mg / ml); AK-C20: AK-C20 (1 mg / ml); C: Vitamin C (1 mg / ml)).

6 is a comparative graph of the inhibition of lipid peroxidation of AK-C20 and known green algae green juice (GJ: freeze-dried powder of fresh green goat juice (1 mg / ml); AK-C20: AK-C20 C: Vitamin C (1 mg / ml)).

FIG. 7 is a graph showing the SOD activity of AK-C20 and the known green juice green juice (GJ: freeze-dried powder of fresh green goat juice (1 mg / ml); AK-C20: AK-C20 (1 mg / ml); C: Vitamin C (1 mg / ml)).

Claims (5)

(a) extracting an acacia leaves with ethanol; (b) concentrating the extract and obtaining an insoluble precipitate; And (c) a purification step of removing a water-soluble component from the insoluble precipitate obtained above; And (d) dissolving the purified product obtained in (c) with ethanol and filtering A method for producing an extract containing chalcone. delete delete delete A chalcone-containing mulberry leaf extract prepared by the method of claim 1.

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