KR20090109007A - Ethanol extracts and caffeic acid from Ocnanthe javanica DC. having hepatoprotective effect, the method for preparing thereof and functional foodstuff comprising the same - Google Patents

Abstract

PURPOSE: A method for extracting caffeic acid is provided for use in producing health-oriented foods. CONSTITUTION: A method for extracting caffeic acid comprises: a first step of extracting Oenanthe javanica DC in ethanol; a second step of filtering the extract and decompression-concentrating the filtrate; a third step of freeze-drying the concentrate to obtain a first Oenanthe javanica DC extract; a fourth step of dipping the freeze-dried first Oenanthe javanica DC extract in an organic solvent to obtain a second Oenanthe javanica DC extract; and a fifth step of chromatographing the second Oenanthe javanica DC extract.

Description

Method for extracting caffeic acid having hepatoprotective and hepatic damage prevention effect from firewood and functional health-oriented foods comprising the same {Ethanol extracts and caffeic acid from Ocnanthe javanica DC. having hepatoprotective effect, the method for preparing pretty and functional foodstuff comprising the same}

The present invention relates to a method for extracting caffeic acid from Oenanthe javanica DC and to a functional health food comprising the same, and more particularly, to a method for extracting isolated caffeic acid, which is effective in protecting liver cells and preventing liver damage from the light parsley. And it relates to a functional health-oriented food comprising the same.

The liver functions to protect the whole body from in vitro exogenous substances, and the risk of exposure to other toxic substances as well as nutrients that are beneficial to health is higher than that of other organs. The liver is a long-term excellent regeneration, but if the damage is sustained, part of the liver tissue is completely destroyed, the function is degraded, it becomes difficult to function as a normal liver, and related diseases include liver fibrosis, cirrhosis, liver cancer, etc. have. Most of these chronic liver diseases are asymptomatically before the onset of symptoms, and most of the damage to the degree of liver damage is a symptomatic symptom and the treatment is so difficult to treat and cure. Accordingly, efforts to find a functional material capable of preventing liver cells and liver damage that can prevent chronic liver damage in advance while having safety without burdening the liver are necessary.

Carbon tetrachloride (CCl ₄ ) is a sample that damages the liver, and is widely used to study liver damage mainly by direct administration to rodents such as mice. When carbon tetrachloride (CCl ₄ ) is reacted by a metabolic enzyme such as cytochrome P ₄₅₀ to produce the free radical CCl ₃ , it causes oxidation of hepatic triglycerides and membrane phospholipids, which combine with oxygen to form lipid peroxides. In addition, these peroxides destroy several functions of the liver and cause damage to liver tissue (Chang. JM et. Al., Drug and chemical toxicology, 6. 443-453, 1983).

Flax is a perennial herb that is a genus of perennial herb, widely cultivated and used mainly in food, and its leaves and stems are medicinal plants used for the treatment of blood pressure drop, fever, diuresis, blood, hemostasis, tonic, poison and pneumonia in oriental medicine. It is one (Chinese Medicine Dictionary, Shanghai Science and Technology Press, p1332-1333, 1981, Ph.D., Plant Physics, Jinmyung Publisher, p306, 1980).

Buttercup methanol extract has been reported to have a mitigating effect on liver function damage caused by carbon tetrachloride in rabbits (Seohwajung et al., J. Korean Soc. Food Nur., 14, 72-76, 1985). It has been shown to prevent carbon tetrachloride-induced acute liver tissue damage (Lee, SI et. Al., J. Korean Soc. Food Nur., 22, 392-397, 1993). In addition, parsley extracts of the same apiaceae have been patented for the prevention and treatment of liver damage (published patent 2002-0090248).

Caffeic acid, one of the active ingredients of honey propolis extract, has been used as a medicinal herb as an antibacterial, anti-inflammatory, and anticancer agent (Banskota et al., Phytotherapy Research, 15, 561-571, 2001: Ozen et al., Journal of Applied Toxicology, 23, 27-35, 2004), reported as a pharmacologically safe ingredient that stimulates lipid peroxidation inhibition and antioxidant enzyme activity (Zhao, WX et al., World Journal of Gastroenterology 9, 1278-1281, 2003).

The functional food material having a known liver cell protection and liver damage prevention effect is extremely limited, and according to the recent health-oriented recent trend, the need for the discovery of the active substance having the above-mentioned efficacy from the food material has emerged.

In addition, the active ingredient has not yet been found to function to protect the liver cells and prevent liver damage of the radish, has not been used as a functional health-oriented food material such as beverages, and has not been disclosed.

In order to solve the above problems of the prior art of the present invention, one object of the present invention is to provide a method for extracting caffeic acid having hepatocellular protection and liver damage prevention effect from the radish.

It is another object of the present invention to provide a functional health-oriented food having hepatic cell protection and hepatic damage prevention physiological activity, which includes the fluoride-derived caffeic acid prepared by the above method.

In order to achieve the above object, the present invention has excellent liver protection action, excellent safety ( Oenanthe javanica DC) provides a method for extracting caffeic acid derived from.

The present invention is characterized by extracting the firewood by the extraction method ( Oenanthe javanica DC) extract.

The present invention is Oenanthe javanica DC) provides functional health foods containing caffeic acid.

Oenanthe according to the present invention Caffeic acid derived from javanica DC) is effective in the production of functional health-oriented foods, such as powders, beverages, processed foods, etc., because the caffeic acid derived from liver cells protects and prevents liver damage.

The present invention provides an excellent hepatoprotective action, and extracts the caffeic acid of Oenanthe javanica DC excellent safety.

In the present invention, the fraction obtained by primary reflux extraction of Oenanthe javanica DC with ethanol and secondary extraction with ethyl acetate is subjected to analytical thin layer chromatography and reverse phase high performance liquid chromatography to obtain a compartment corresponding to caffeic acid. It is characterized in that the extraction of caffeic acid by performing collecting thin layer chromatography and reverse phase high performance liquid chromatography.

First, in order to extract the caffeic acid, the leaves and stems of dry radish are powdered, and then 80% ethanol is added in a volume of 10 to 20 times the weight of the radish powder, and extracted by stirring at 200 to 300 ° C. for 2 to 4 hours. It is preferable. The extract is preferably filtered to obtain a supernatant, concentrated under reduced pressure using a rotary evaporator, and dried to obtain a powdery radish primary extract.

5% (W / V) distilled water was added to the primary extract of the parsley, 1 to 2 volumes of nucleic acid was mixed to remove the nucleic acid fractions eluted to the nucleic acid, and 1 to 2 volumes of chloroform was mixed with the remaining aqueous layer to chloroform eluted with chloroform. It is preferable to remove the fractions, mix 1 to 2 volumes of ethyl acetate in the remaining aqueous layer, separate the ethyl acetate fraction eluted from ethyl acetate from the aqueous layer, filter with 0.3 µm filter paper, and concentrate under reduced pressure to obtain a fluoride secondary extract. .

In addition, it is preferable to identify the fluoride-derived caffeic acid by dissolving the bulgeul secondary extract with methanol, performing analytical thin layer chromatography and reversed phase high performance liquid chromatography.

It is preferable that the collected liquid is separated by depressurizing and evaporating to dryness using the above-mentioned reverse phase high performance liquid chromatography method.

The present invention provides a firewood extract, characterized in that by extracting the firewood by the extraction method.

In the present invention, cell experiments and animal experiments were conducted to investigate whether or not feminine derived caffeic acid exhibited hepatocellular protection and liver damage prevention effects. According to a preferred embodiment of the present invention, it can be seen that the fluoride-derived caffeic acid of the present invention protects liver cell damage induced by hydrogen peroxide and prevents liver damage caused by carbon tetrachloride.

In addition, the present invention can be utilized as a material of functional health-oriented foods containing a calculus-derived caffeic acid.

The functional health-oriented food has functions such as protecting liver cells and preventing liver damage, and examples thereof include beverages, pills, granules, processed foods, and the like, but are not limited thereto.

In the present invention, the beverage containing caffeic acid preferably contains any one or more selected from white sugar, liquid fructose, citric acid, sodium citrate, maltodextrin, calcium lactate, sodium chloride, potassium chloride, magnesium chloride, white claudi and carbon dioxide.

The beverage containing caffeic acid preferably contains 5-100 mg of caffeic acid per 100 ml.

In addition, the functional health food can be prepared by a conventional method to eat 10-40 mg / kg, preferably 30 to 40 mg / kg, once or several times a day from the fire radish-derived caffeic acid. However, it should be understood that the actual content should be determined in view of various related factors such as the type and weight of the health food, and thus the content does not limit the scope of the present invention in any aspect.

By the following examples will be described in detail the present invention. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the examples.

Example 1 Preparation of Caffeic Acid Derived from Flakes

Oenanthe javanica DC was supplied from Hanwoo Co., Ltd., Hwasun, and used in the present invention. 50 g of dried and powdered buttercups were added with 20-fold volume of ethanol, stirred at 250 ° C. for 3 hours, filtered using a 0.3 μm filter, and the supernatant was concentrated under reduced pressure at 45 ° C. for 30 minutes to obtain 10 ^−4. Torr, lyophilized under a condition of 72 hours to obtain a powder of the bulrush primary extract.

200 ml of distilled water was added to 10 g of the primary extract of Burberry to completely dissolve the primary extract powder, and 200 ml of nucleic acid was added thereto to mix and fractionated. The nucleic acid fraction eluted in the nucleic acid layer of the fraction was removed and performed once more in the same manner. 200 ml of chloroform was added to the separated aqueous layer and the nucleic acid fractions were mixed and fractionated. The chloroform fraction eluted in the chloroform of the fraction was removed and subjected to the same method once more. To the aqueous layer separated from the chloroform fractions, 200 ml of ethyl acetate was added, mixed and fractionated. At this time, the ethyl acetate fraction separated from the aqueous layer was collected in a collection container, and once more carried out in the same manner, collected in the same collection container, filtered through a 0.3 μm filter paper, and then filtered through a 0.3 μm filter solution and a nitrogen gas solvent evaporation method. An extract (ethyl acetate fraction) was obtained.

The secondary extract was again dissolved in methanol for thin layer chromatography for analysis (ethyl acetate: liquid acid: water: formic acid = 40:20: 5: 5 (v / v / v / v)) and reversed phase high performance liquid chromatography (70%). Methanol, 0.1% phosphoric acid) was isolated from single fractions of vulgaris and identified as caffeic acid by structural analysis by ¹³ C / ¹ H-NMR Fluorides were identified by using thin membrane chromatography for collection and reversed-phase high-pressure liquid chromatography. The derived caffeic acid was collected.

Example 2 Measurement of Hepatocellular Protective Effect of Flax-Derived Caffeic Acid

In order to confirm the hepatocyte protective effect of the caffeic acid collected and collected in Example 1, the HepG 2 cell line was treated with fluoride-derived caffeic acid, and then cultured by using hydrogen peroxide to induce hepatocellular damage and then using the XTT method. Hepatoprotective effect of was measured.

HepG 2 cell line was prepared in a 80 well confluence state in a 24 well culture vessel and incubated in a 37 ° C. incubator with a DMEM medium, followed by 10, 20, 30, 40, and 50 ㎍ / mL concentration of caffeic acid derived from vulgaris for 24 hours. Treat and incubate. After removal of the culture medium, 2mM hydrogen peroxide was used to induce hepatic cell damage, and forma formed in living hepatocytes using the XTT cell viability evaluation method (Rochem NW et al., J. Immunol. Methods 142: 257-265, 1991). The glass was measured for absorbance at 450 nm wavelength to obtain a value.

As shown in FIG. 2, it was confirmed that fluoride-derived caffeic acid protects hepatocyte damage induced by hydrogen peroxide from 20 μg / ml or more.

Example 3 Measurement of Prevention of Liver Damage by Carbon Tetrachloride of Caffeic Acid Derived from Flakes

In order to measure the protective effect of hepatic decay-derived caffeic acid, male oral rats were orally administered with the heparin-derived caffeic acid prepared in Example 1, induce liver damage by carbon tetrachloride, and GOT (Glutamic Oxalacetic Transaminase) in serum. GPT (Glutamic Pyruvic Transaminase) amount was measured. In addition, liver tissue flakes according to each administration group were evaluated in comparison with the control group.

 500-600 g of male rats were kept for 1 week in the laboratory environment and fed with sufficient feed and water. A total of 24 male rats were divided into 4 groups of 6 rats each, and 1% volume of body weight was orally administered as shown in <Table 1>. On day 15, carbon tetrachloride was dissolved in corn oil at a concentration of 20%, 1% volume of body weight was orally administered once, dissected after 24 hours to obtain serum and liver tissue, and the serum was stored at -20 ° C until the experiment. . Serum of each group was evaluated using GOT and GPT Kit (Asan Pharmaceutical), and group 2 was statistically significantly higher than group 1, and similar to group 1 in groups 3 and 4 treated with flameless caffeic acid. By evaluating the results of the two enzymes GOT and GPT, which are increased due to liver damage, it was confirmed that there was an effect of preventing liver damage <Table 2>.

In addition, as a result of comparing the tissue flakes of each group, it was confirmed that the liver damage induced by carbon tetrachloride was prevented by the previously administered caffeic acid (40 mg / kg / day) (FIGS. 3A and 3B).

<Table 1> Classification of laboratory animals for the prevention of liver damage of caffeic acid isolated from firewood

Group 1 to 14 days 15th Group 1 (n = 6) Corn oil group Group 2 (n = 6) Corn oil group 20% carbon tetrachloride Group 3 (n = 6) 10 mg / kg / day dissolved caffeic acid in corn oil 20% carbon tetrachloride Group 4 (n = 6) 40 mg / ㎏ / day dissolved caffeic acid in corn oil 20% carbon tetrachloride

<Table 2> Effect of Caffeic Acid Isolated from Flame Butter on Preventing Liver Damage

Group GOT (Karmen / mL) GPT (Karmen / mL) Group 1 108.56 ± 16.87 ^a 32.08 ± 6.01 ^a Group 2 156.14 ± 22.98 ^b 65.89 ± 8.17 Group 3 117.49 ± 6.97 ^a 29.15 ± 3.63 ^a 4th group 113.53 ± 14.40 ^a 34.53 ± 15.53 ^a

Characters such as ^{a and b do} not show statistical significance at 95% confidence

Example 4 Preparation of Functional Drink Containing Flaky Derived Caffeic Acid

The present invention is prepared in Example 1 Buttercup ( Oenanthe javanica DC) A functional beverage having a physiological activity of protecting liver cells and preventing liver damage, containing ethanol extract or flax-derived caffeic acid, was prepared.

(1) 100 mg of purified water ethanol extract 20 mg, 50 mg and 100 mg were mixed, respectively, 100 mg per white sugar, 10 mg liquid fructose, 1.0 mg citric acid, and 0.3 mg sodium citrate, followed by 3% carbon dioxide. To prepare a carbonated beverage containing a firewood ethanol extract.

(2) 180 mg of purified water was mixed with 20 mg, 50 mg, and 100 mg of ethanol extract, respectively, 5 mg per white, maltodextrin 100 mg, 0.1 mg anhydrous citric acid, 0.03 mg anhydrous sodium citrate, 0.03 mg calcium lactate, sodium chloride An ion beverage containing flaky ethanol extract added with 0.01 mg, 0.01 mg potassium chloride, 0.001 mg magnesium chloride, and 0.02 mg white clady was prepared.

 (3) 100 mg of purified water ethanol extract 20 mg, 50 mg and 100 mg were mixed, respectively, 100 mg per white sugar, 10 mg liquid fructose, 1.0 mg citric acid and 0.3 mg sodium citrate, followed by 3% carbon dioxide. The carbonated beverage containing caffeine-derived caffeic acid was prepared by adding thereto.

(4) 180 mg of purified water was mixed with 10 mg and 40 mg of flax-derived caffeic acid, respectively, 5 mg per white, maltodextrin 100 mg, 0.1 mg anhydrous citric acid, 0.03 mg anhydrous sodium citrate, 0.03 mg calcium lactate, 0.01 mg sodium chloride. , Ion chloride containing 0.01 mg of potassium chloride, 0.001 mg of magnesium chloride, and 0.02 mg of white Claudia was prepared.

As described above, those skilled in the art have been described with reference to the preferred embodiments of the present invention, various modifications and changes of the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. It will be appreciated that it can be changed.

Figure 1 shows a reverse phase high performance liquid chromatography graph of caffeic acid isolated from fluoride ethanol extract according to the present invention.

Figure 2 is a graph showing the hepatocellular protective effect of the caffeic acid isolated from the radish according to the present invention.

CON: no hydrogen peroxide treatment group,

2 mM HPO: hydrogen peroxide treatment group,

CA 10 to CA 50: 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml treatment group derived from firewood

^* Statistically significant difference in 95% confidence interval compared with hydrogen peroxide treatment group>

Figure 3a shows a photograph of the tissue flakes for confirming the prevention of liver damage by administration of caffeic acid isolated from the bulge according to the present invention.

Experimental group

A: corn oil group (14 days) + saline solution

B: Corn oil-treated group (14 days) + 20% carbon tetrachloride prepared and administered to physiological saline (the last day of the experiment)

C: A group in which 40 mg / kg / day of disintegrated caffeic acid in corn oil was administered (14 days) + 20% carbon tetrachloride was prepared and administered in physiological saline (the last day of the experiment).

Figure 3b is a chart showing the evaluation of the liver tissue preventive tissue flakes by administration of the caffeic acid isolated from the bulge according to the present invention.

<Determine the level of tissue damage according to the following>

1) Normal (-), Light (+), Severe (++), Severe (+++)

2) 1 ~ 6: number of individuals in each group>

Claims (7)

Ethanol extraction of Oenanthe javanica DC; Filtering the flameless ethanol extract, and then concentrating the filtrate under reduced pressure and freeze drying to obtain a primary flame extract; Treating the freeze-dried radish primary extract with an organic solvent to obtain a radish secondary extract; And extracting caffeic acid having hepatoprotective and hepatic damage prevention effects comprising the step of chromatography the bulrush secondary extract. The method for extracting caffeic acid according to claim 1, wherein the ethanol extraction comprises adding 20 to 25 times the volume of 70 to 100% ethanol to Oenanthe javanica DC and stirring the mixture at 200 to 300 ° C. for 2 to 4 hours. . The organic solvent treatment of claim 1, wherein hexane, chloroform, and ethylacetate are sequentially added to the freeze-dried fluoride primary extract powder to obtain a fluoride secondary extract, which is an ethyl acetate fraction. Caffeic acid extraction method, characterized in that. The extracting method of caffeic acid according to claim 1, wherein the chromatographic step is performed by dissolving the fluoride secondary extract in methanol, collecting thin membrane chromatography, and reversed phase high performance liquid chromatography. Claim 1 to 4, characterized in that extracted by the extraction method of any one selected from claim 1 ( Oenanthe javanica DC) extract. Bulminari claim having a protective and damage prevention bioactive liver by the method of claim 1 extracted (Oenanthe Functional health food containing caffeic acid derived from javanica DC). The functional health food according to claim 6, wherein the functional health food is any one selected from liquid, powder, pill and granules.

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