KR20090097440A - METHOD FOR PREPARING CAFFEIC ACID IMPROVING gamma-GCS ACTIVITY, THE CAFFEIC ACID PREPARED THEREFROM, AND A FOOD COMPOSITION AND A PHARMACEUTICAL COMPOSITION COMPRISING THE CAFFEIC ACID - Google Patents

Abstract

A method for producing caffeic acid improving gamma-GCS activity from green perilla leaves is provided to secure an improvement to liver and antioxidant effect at a little cost. A method for producing caffeic acid improving gamma-GCS activity from green perilla leaves comprises: a first step of reflux-extracting dried green perilla leaves in distilled water; a second step of centrifuging the extract to obtain supernate from it; a third step of filtering the supernate and vacuum-concentrating and freeze-drying the filtrate in order to obtain powder; a fourth step of extracting the powder using a plurality of extraction solvents in sequential order; and a fifth step of separating and refining the fractions showing high gamma-GCS activity. In the first step, 100g of the dried green perilla leaves are extracted in 0.8-1.2L of distilled water at 100-110°C for 3-4 hours twice. In the second step, the centrifugation is performed at 4°C and 7500rpm for 25-30 minutes. The extraction solvents are a first, second, third and fourth extraction solvent.

Description

Method for preparing caffeic acid to increase the activity of γ-UCS from perilla leaves, caffeic acid prepared therefrom, and a food composition and pharmaceutical composition comprising the caffeic acid {Method for preparing caffeic acid improving γ-GCS activity, the caffeic acid prepared therefrom, and a food composition and a pharmaceutical composition comprising the caffeic acid}

 The present invention relates to a method for producing caffeic acid which increases the activity of γ-GCS from perilla leaves, caffeic acid prepared therefrom, and a food composition and a pharmaceutical composition comprising the caffeic acid, and more particularly, a strong antioxidant from perilla leaves. It is effective in protecting liver function by increasing the activity of γ-GCS, thus producing caffeic acid which can be widely used for preventing and treating liver function deterioration, caffeic acid prepared therefrom, and prepared by the above method. It relates to a food composition and a pharmaceutical composition comprising caffeic acid.

Perilla is a herbaceous herb that belongs to labiatae, and sesame leaves broadly refer to all the leaves of perilla. Perilla is a crop that is grown to squeeze oil. It is sesame leaves that harvest and edible leaves during growth. Perilla leaves contain aromatic essential oils such as perillaaldehyde, limonene, and perylketone. In addition, wild perilla leaves are eaten as a side dish such as namul, pickles, and sesame leaf kimchi, or they are used as spices in radish or tang. In the past, perilla has been cultivated mainly for the purpose of harvesting seed oil, but the recent growth of meat consumption, the development of the food culture, and the rapid growth of the ssam vegetable consumption market caused by the well-being craze have allowed the development of varieties for perilla and its production all year round. Therefore, it is expected that industrial value will be great if it reveals the medical function of perilla leaves and applies them to food and pharmaceuticals in various ways.

Perilla aldehydes, rosemary acid, anthocyanin caffeic acid, and the like have been reported as a substance indicating the functionality of perilla leaves. Since these components have high water solubility, high intake rates can be expected when ingested, and especially when caffeic acid is ingested, the small intestine absorption rate is 95% or more. Caffeic acid is known to have strong antioxidant activity, especially in large amounts in vegetables and fruits. However, there have been no reports on the anticancer effect of caffeic acid.

Domestic mortality from liver disease and liver cancer accounts for 10% of all deaths. According to the 2005 cause of death statistics released by the National Statistical Office, liver disease is ranked 6th among the causes of death in Korea, and 5-8% of the total population is known to be a hepatitis B virus, threatening national health. to be. However, hepatic medicines prescribed in hospitals or on the market are usually those that have preventive and therapeutic effects on acute liver injury in animals or clinical trials, most of which are synthetic chemicals that reduce the damage to liver cells rather than treat the disease fundamentally. It only serves as an adjuvant treatment in the sense of making it a substitute, and is not a substitute for life management or regular checkups. Therefore, there is a need for the development of a technique for finding a liver protection and liver function enhancing therapeutic agent from natural substances other than synthetic chemicals. In accordance with these needs, various techniques for treating liver cancer and liver disease have been developed using conventional plant extracts, such as mushroom extracts, garlic extracts, and herb extracts. The prior art relates to the manufacture of medicines or functional foods using food extracts that have a hepatoprotective effect as an active ingredient, and these food extracts ultimately increase liver ALT, AST, CT and γ-GCS levels, thereby ultimately protecting the liver. Indicates.

On the other hand, glutathione (Glutathione) is a representative liver protection enzyme, plays an important role in liver detoxification, oxidation and reduction reactions, and controlling the amount of this enzyme is the γ-glutamylcysteine synthetase (γ) -GCS). However, conventional methods for extracting effective ingredients for treating liver cancer from plants include disadvantages of requiring expensive equipment, complicated methods, high cost, and long analysis time. There is no research showing that the plant extract itself as a mixture is effective in protecting the liver or preventing liver disease, and the natural single substance alone is useful for liver disease by increasing the activity of γ-GCS.

In addition, methods of separating active substances from plants include manure, fractional crystallization, extraction, distillation, fractional distillation, and separation of mixtures by chromatography. Refers to the operation of dissolving and separating specific substances. Usually, extraction is carried out using an organic solvent (chloroform, dichloromethane, ethyl acetate: non-aqueous solvent) and water. When the organic solvent and water are mixed with each other, the layers of the two solvents split and the density of the organic solvent is larger than that of water. The water layer is above and the organic solvent layer is below. Therefore, after separating the mixture by taking the lower layer using a separatory funnel, the organic material and water used as a solvent are distilled to obtain a pure material. This is commonly used to separate water-soluble and non-aqueous materials (solubles in organic solvents). In addition, chromatography is a separation method capable of separating similar compounds constituting a complex compound, consisting of a stationary phase and a mobile phase (mobile phase). The material to be separated is dissolved by dissolving in a suitable solvent to move along the mobile phase or adsorbed on the fixed phase. This is because the degree of distribution of each component of the material to be separated varies depending on the affinity of the stationary and mobile phases. That is, the components strongly bound to the stationary phase are present in the stationary phase and the weakly bound components pass through the stationary phase along the mobile phase, and as a result, each component moves at a different speed and is separated. This process is called resolution and uses a rate of flow (Rf) to represent the travel distance.

[Rf = distance traveled by each sample / distance traveled by solvent (solvent line)]

Chromatography is classified into gas chromatography (Gas Chromatography, GC), in which the mobile phase is a gas, and liquid chromatography (LC), in which the mobile phase is a liquid. Column chromatography.

The above-mentioned separation method of the mixture uses different physical and chemical characteristics of the mixture, and a suitable method should be selected according to the characteristics and conditions of the mixture. Therefore, even if the mixture separation method widely used in the prior art, to separate and purify only the specific material desired in a specific mixture, the type of separation method, the reagent used, the separation order, separation conditions and the like must be carefully selected.

The present invention has been made to solve the problems of the prior art, the first problem to be solved by the present invention is to provide a method for producing caffeic acid to increase the activity of γ-GCS from the perilla leaves in a simple and inexpensive way .

The second problem to be solved by the present invention is to provide a caffeic acid prepared by the above method, by increasing the activity of γ-GCS and having a liver function improving and liver protective activity.

The third and fourth tasks to be solved by the present invention is to provide a food composition and a pharmaceutical composition comprising the caffeic acid.

In order to achieve the first object of the present invention,

Performing reflux cooling with distilled water on the dried perilla leaves;

Centrifuging the extract obtained from the reflux cooling extraction step to obtain a supernatant;

Obtaining perilla leaf extract powder by concentrating the filtrate obtained by filtering the supernatant under reduced pressure and lyophilization;

Preparing fractions by sequentially extracting the perilla leaf extract powder using a plurality of extracting solvents; And

It provides a method for producing caffeic acid to increase the activity of γ-GCS from perilla leaves comprising the step of separating and purifying the fraction showing the highest γ-GCS activity among the fractions.

According to one preferred embodiment of the present invention, the reflux cooling extraction step is repeated twice the extraction process for 3 hours to 4 hours at 100 ℃ to 110 ℃ temperature distilled water of 0.8 liter to 1.2 liter volume per 100g of dried perilla leaves This can be done by.

According to another preferred embodiment of the present invention, the centrifugation step may be performed for 25 to 30 minutes at a temperature of 7,500rpm at a temperature of 4 ℃.

According to another preferred embodiment of the present invention, the fraction preparation step may be performed using four extraction solvents consisting of a first extraction solvent, a second extraction solvent, a third extraction solvent and a fourth extraction solvent.

According to another preferred embodiment of the present invention, the fraction manufacturing step,

Dissolving the perilla leaf extract powder in the first extraction solvent, followed by stirring to separate the first organic solvent layer and the first distilled water layer to obtain a first fraction from the first organic solvent layer;

Mixing the first distilled water layer with the second extraction solvent and then stirring to separate the second organic solvent layer and the second distilled water layer to obtain a second fraction from the second organic solvent layer;

Mixing the second distilled water layer with the third extraction solvent and then stirring to separate the third organic solvent layer and the third distilled water layer to obtain a third fraction from the third organic solvent layer; And

After mixing the third distilled water layer with the fourth extraction solvent and stirring to separate the fourth organic solvent layer and the fourth distilled water layer to obtain a fourth fraction from the fourth organic solvent layer and from the fourth distilled water layer Obtaining a fifth fraction.

According to another preferred embodiment of the present invention, the first, second, third and fourth extraction solvent may be a mixed solution of distilled water and hexane, chloroform, ethyl acetate and butanol, respectively.

According to another preferred embodiment of the present invention, the first extraction solvent may be a mixed solution in which 100 to 120 parts by weight of hexane is mixed with respect to 100 parts by weight of distilled water.

According to another preferred embodiment of the present invention, the mixing ratio of the first distilled water layer and the second extraction solvent, the second distilled water layer and the third extraction solvent and the third distilled water layer and the fourth extraction solvent, respectively, The distilled water layer, the second distilled water layer and the third distilled water layer may be 100 to 120 parts by weight of the second extraction solvent, the third extraction solvent and the fourth extraction solvent.

According to another preferred embodiment of the present invention, the separation and purification step may be performed using a column chromatography apparatus.

According to another preferred embodiment of the present invention, the separation and purification step,

A first separation step of performing column chromatography on the fractions using a polystyrene-divinylbenzene copolymer resin as a stationary phase and an aqueous methanol solution as a mobile phase;

A secondary separation step of performing the column chromatography using the hydroxypropylated dextran resin as a stationary phase and an aqueous methanol solution as the mobile phase to the results of the primary separation step; And

Selecting and purifying a product showing the highest γ-GCS activity among the results of the secondary separation step may include.

According to another preferred embodiment of the present invention, the primary separation step is performed by using five kinds of mobile phases, each having a weight ratio of distilled water: methanol of 100: 0, 75:25, 50:50, 25:75 and 0: 100, respectively. Can be performed.

According to another preferred embodiment of the present invention, the secondary separation step may be performed using a mobile phase having a weight ratio of distilled water: methanol of 30:70.

In order to achieve the second object of the present invention,

It provides caffeic acid prepared by the above method.

According to a preferred embodiment of the present invention, the caffeic acid may have hepatoprotective efficacy by increasing the activity of γ-GCS.

The present invention to achieve the third object,

It provides a food composition comprising the caffeic acid.

In addition, the present invention to achieve the fourth object,

It provides a pharmaceutical composition comprising the caffeic acid.

According to the present invention, a simple and inexpensive method to increase the activity of γ-GCS from perilla leaves to improve the function of the liver and can be produced caffeic acid having a strong antioxidant effect, food composition and pharmaceutical composition comprising the prepared caffeic acid It can be widely used to prevent and treat liver failure.

Hereinafter, the present invention will be described in more detail.

Caffeic acid contained in perilla leaves is known to have an anti-cancer effect because the absorption rate of the small intestine is more than 95% when consumed, and the caffeic acid has a strong antioxidant activity. In addition, the present invention perilla leaves ( Perilla Caffeic acid, isolated from frutescens , has been shown to increase the activity of γ-GCS, which enhances GSH, a hepatoprotective enzyme. It is intended to provide a method of preparation.

Specifically, the present invention,

Performing reflux cooling with distilled water on the dried perilla leaves; Centrifuging the extract obtained from the reflux cooling extraction step to obtain a supernatant; Obtaining perilla leaf extract powder by concentrating the filtrate obtained by filtering the supernatant under reduced pressure and lyophilization; Preparing fractions by sequentially extracting the perilla leaf extract powder using a plurality of extracting solvents; And separating and purifying the fraction showing the highest γ-GCS activity among the fractions.

The reflux cooling extraction step uses a reflux cooler, which is a device that cools water vapor and returns it to the bottom container in order to capture vaporized water vaporized during hot water extraction. Therefore, the hot water extraction step and the reflux cooling extraction step are the same process. The reflux cooling extraction step is preferably carried out by repeating the extraction process for 3 hours to 4 hours at 100 ℃ to 110 ℃ temperature distilled water of 0.8 liter to 1.2 liter volume of dried perilla leaves, the distilled water of If the volume is less than 0.8 liters, the volume of the extract obtained from the reflux cooling extraction process is too small to facilitate the subsequent process, and the specific heat of the mixture of perilla leaves and distilled water becomes large, so that the heating temperature becomes high or the heating time becomes long, and reflux There is a problem that the time and cost of the cooling extraction takes a lot, and if the volume of the distilled water exceeds 1.2 liters, the final powder yield from the extract is not high, and the heating time is unnecessarily long, which increases the process cost. Not desirable

In addition, the extraction time is preferably from 3 hours to 4 hours, when the heating time is less than 3 hours there is a fear that the components constituting the perilla leaves do not separate and extracts are not produced from perilla leaves, when heated for 4 hours or more There is a fear that the active ingredient is destroyed or deteriorated. In addition, it is preferable to repeat the above two times to maximize the yield of the reflux cooling extraction process. If the reflux cooling extraction process is carried out once, the active ingredient is not completely extracted from the perilla leaves, and the first reflux cooling extract recovers the perilla leaves from which the extract is removed, and then the second reflux cooling extraction process is carried out during the first reflux cooling extraction. 60% of the weight of the extracted extract can be obtained, but it is not preferable to proceed any further because the yield is lowered when reflux cooling extraction is performed three or more times. The reflux cooling extraction may be performed at a temperature of 100 ° C. to 110 ° C., and at a temperature below 100 ° C., the heat provided is used only to increase the temperature of the mixture of dried perilla leaves and distilled water, and thus the liquid active ingredient of perilla leaves is not vaporized. The process does not proceed, and the active ingredient of perilla leaves may cause chemical changes or destruction at temperatures of 110 ℃ or more.

The centrifugation step may be performed for 25 to 30 minutes at a temperature of 7,500rpm at a temperature of 4 ℃. This is in accordance with the regulations set forth in the Standard Operating Procedures (SOPs).

In addition, the fraction preparation step may be performed using four extraction solvents consisting of a first extraction solvent, a second extraction solvent, a third extraction solvent and a fourth extraction solvent.

In the step of preparing a fraction, the perilla leaf extract powder is dissolved in the first extraction solvent and stirred to separate the first organic solvent layer and the first distilled water layer to obtain a first fraction from the first organic solvent layer. ; Mixing the first distilled water layer with the second extraction solvent and then stirring to separate the second organic solvent layer and the second distilled water layer to obtain a second fraction from the second organic solvent layer; Mixing the second distilled water layer with the third extraction solvent and then stirring to separate the third organic solvent layer and the third distilled water layer to obtain a third fraction from the third organic solvent layer; And mixing the third distilled water layer with the fourth extraction solvent and then stirring to separate the fourth organic solvent layer and the fourth distilled water layer to obtain a fourth fraction from the fourth organic solvent layer, and the fourth distilled water. Obtaining a fifth fraction from the layer. Hereinafter, the step of extracting using an organic solvent will be described in detail.

The first, second, third and fourth extraction solvents use a mixed solution of distilled water and hexane, chloroform, ethyl acetate and butanol, respectively. The first extraction solvent is a solution in which 100 to 120 parts by weight of hexane is mixed with respect to 100 parts by weight of distilled water. The polarity of the organic solvent is shown in Table 1 below.

Organic solvent Polarity index ( Polarity Index ) Hexane 0 Butanol 4 chloroform 4.1 Ethyl acetate 4.4 water 9

The polarity index means that the larger the size, the greater the degree of polarity. Accordingly, according to Table 1, water has a strong polarity, and hexane, chloroform, ethyl acetate and butanol may be referred to as relatively nonpolar materials. When a mixture of polar and nonpolar materials is dissolved in a polar solvent and a nonpolar solvent, the polar material is mainly dissolved in the polar solvent and the nonpolar material is mainly dissolved in the nonpolar solvent. The mixture can be separated using this property, and the present invention separates the caffeic acid mixture from the reflux extract of perilla leaves using the extraction method using such an organic solvent.

In Figure 1, according to a preferred embodiment of the present invention, a hydrothermal extract (hereinafter hot water) using a mixed solvent of water and hexane, a mixed solvent of water and chloroform, a mixed solvent of water and ethyl acetate and a mixed solvent of water and butanol Extract means a mixture of perilla leaves active ingredient extracted through reflux cooling extraction process). For example, Perilla leaves ( Perilla frutescens ) Fractionation using the above four organic solvents from hydrothermal extract (PLE-0), showing a high γ-GCS activity at the concentration of 500 μg / mL compared to the PLE-III (ethyl acetate layer, untreated extract). 133.1 ± 4.5% increase).

Mixing ratios of the first distilled water layer and the second extraction solvent, the second distilled water layer and the third extraction solvent, and the third distilled water layer and the fourth extraction solvent are respectively the first distilled water layer, the second distilled water layer, and the third It is preferable that the second extraction solvent, the third extraction solvent and the fourth extraction solvent are 100 to 120 parts by weight based on 100 parts by weight of the distilled water layer. When the second extraction solvent, the third extraction solvent and the fourth extraction solvent are mixed at a ratio of less than 100 parts by weight with respect to 100 parts by weight of the first distilled water layer, the second distilled water layer and the third distilled water layer, the polarity is large. Since the ratio of distilled water becomes large, the extraction solvent is generally polarized, which makes it difficult to separate nonpolar substances from the hydrothermal extracts to be separated, and when mixed in excess of 120 parts by weight, the extraction solvent is nonpolar and the polarity of the hydrothermal extracts. There is a problem that the separation process does not proceed because the material is not dissolved in the extraction solvent.

The separation and purification steps can be carried out using a column chromatography apparatus. In particular, the separation and purification using the column chromatography apparatus, the first separation step of performing the column chromatography using the polystyrene-divinylbenzene-based copolymer resin as a stationary phase, the aqueous solution of methanol as a mobile phase for the fractions ; A secondary separation step of performing the column chromatography using the hydroxypropylated dextran resin as a stationary phase and an aqueous methanol solution as the mobile phase to the results of the primary separation step; And selecting and purifying the product showing the highest γ-GCS activity among the results of the secondary separation step. The column chromatography is a method used when the separation is difficult because the physical properties of the mixture is similar, filling about 2/3 of the polystyrene-divinylbenzene copolymer resin of the stationary phase in a transparent tube (column) of about 50-100cm. Then, an aqueous methanol solution is poured to make the inside gel. In this case, it should be noted that the solvent should always contain more than the top of the stationary phase. When the mixture is dissolved in the mobile phase of methanol and introduced at the top of the column, the dissolved methanol is slowly moved down by gravity. In this case, it is preferable that the mobile phase select a substance capable of dissolving all of the mixture, and for this reason, methanol is used as the mobile phase in the present invention. The mobile phase conditions are 100% distilled water → 100% methanol (polarity 5.1), eluting at least 5 times the column capacity of the mobile phase with increasing methanol concentration (25% increments) (see FIG. 1). That is, the weight ratio of distilled water: methanol may be performed using five kinds of mobile phases of 100: 0, 75:25, 50:50, 25:75 and 0: 100, respectively. Depending on the weight ratio of distilled water and methanol, the degree of dissolution of the mixture (PLE-III fraction) is different, and in the case of 50:50, since the dissolution is best, the mixture can be purified. Elution is performed until the absorbance of the eluted material is less than or equal to 0.3 at 280 nm for accurate separation at each step. After concentration and drying of each eluted fraction, the activity of 50% distilled water: 50% methanol PLE-III-C fraction increased by 135.6 ± 7.4% at a concentration of 500 μg / mL.

Using this fraction as a stationary phase, the mobile phase is eluted under conditions of 30% distilled water: 70% methanol using a hydroxypropylated dextran resin such as Sephadex LH-20 resin. The degree of adsorption of the compounds in the mixture (PLE-III-C fraction) to the hydroxypropylated dextran resin, which is a stationary phase, differs slightly.The mixture is separated as it descends to obtain a total of four fractions. Can be. Of these four fractions, the PLE-III-C-3 fraction (see Figure 2; 171.4 ± 13.2% increase) is caffeic acid (see Figure 1). The separated solution can be placed in a rotary vacuum evaporator to blow off only the mobile phase to obtain pure material.

The method of distinguishing caffeic acid among the four fractions (PLE-III-C-1, PLE-III-C-2, PLE-III-C-3, PLE-III-C-4) of the secondary separation step is As shown in FIG. 2, the method having the highest γ-GCS activity is selected. PLE-III-C-1 fraction and PLE-III-C-2 fraction have no effect of increasing γ-GCS activity, and PLE-III-C-3 fraction increases 75% of γ-GCS activity. It is effective, and PLE-III-C-4 fractions also increase γ-GCS activity but are less than 25%. Therefore, the γ-GCS activity is measured and the fraction having a distinctly high activity is judged to be separated by caffeic acid. Activity of the enzyme is Ray (Ray et al., ( 1999). Free Radic Biol Med 27, 1346-56) and the like can be referred to, and to measure the activity of γ-GCS as the formation of ADP using two enzymes, lactate dehydrogenase and pyruvate kinase. Can be. Specifically, the enzyme reaction solution (Tris-HCl buffer 0.1M pH 8.0, 10 mM L-glutamate, 10 mM L-α-aminobutylate, 5 mM ATP, 20 mM MgCl2, 150 mM KCl, 2 mM EDTA) at 37 ° C. in advance for 5 minutes. , 2 mM phosphoenolpyruvate, 0.2 mM NADH) was preheated, followed by the addition of 17 μg of pyruvate phosphatase and lactic acid dehydrogenase, and the oxidizing rate of NADH was measured for 3 minutes at a wavelength of 340 nm, and the amount of protein was corrected to adjust γ The control group, expressed as the activity of -GCS and not treated with the extract, is based on 100%.

On the other hand, the present invention provides a caffeic acid prepared by the above method. Caffeic acid prepared according to this step has already been shown to exhibit a strong antioxidant effect, in addition to increasing the activity of γ-GCS has been shown to add an effect on the protection of liver function. Glutathione is a representative hepatoprotective enzyme and plays an important role in liver detoxification, oxidation, and reduction reaction. It is γ-GCS that regulates the amount of glutathione enzyme, and caffeic acid is the level of γ-GCS. This is because it increases significantly.

The present invention also provides a food composition containing the caffeic acid.

The food composition according to the present invention can be prepared in various forms such as canned food, bottled food, dried processed foods, fermented foods, and various kinds of additives can be mixed. The additive may be one commonly used in this kind of nutritional food. Therefore, such additives include various types of vitamins, minerals, synthetic flavoring substances and natural flavors, concentrates, natural sweeteners (sormatin, stevia, etc.), synthetic sweeteners (saccharin, stevia extract) , Aspartame and the like), colorants, flavors (cheese, chocolate, etc.) and vegetable fibers such as polydextrose, peptic acid and salts thereof, alginic acid and salts thereof, and the like, and these additives may be used singly or in combination.

Caffeic acid of the present invention can be added to a variety of foods, such as soft drinks, candy, snacks, ice cream, etc., these foods are filled in a suitable container and distilled sterilization (120 ℃, 20 minutes) has a sufficient shelf life. Furthermore, in order to enhance the taste, known additives include natural flavors such as rose, lemon, vanilla or peppermint, natural pigments such as chlorophyllin and flavonoid, and sweeteners such as glucose, honey and sugar. It is also possible to use mixed.

Finally, the present invention provides a pharmaceutical composition comprising the caffeic acid.

Pharmaceutical compositions of the invention may include pharmaceutically acceptable additives. The additives may optionally be used one or two or more of conventional excipients, disintegrants, binders, lubricants, suspending agents and the like. For example, when the composition of the present invention is prepared in a solid form such as tablets or hard capsules, microcrystalline cellulose, lactose, low-substituted hydroxycellulose and the like can be used as excipients, sodium starch glycolate, anhydrous as a disintegrant. Calcium monohydrogen phosphate may be used. As the binder, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose and the like can be used. The lubricant can be selected from magnesium stearate, silicon dioxide, talc and the like. In addition, as the suspending agent, surfactants (eg, sorbitan esters, polysorbates, etc.) commonly used in the pharmaceutical field may be used.

The compositions of the present invention can be formulated in a variety of forms, including solid and liquid forms, which include tablets, capsules (hard or soft), solutions, suspensions, emulsions, syrups and the like. It may preferably have liquid formulations such as liquids, suspensions, emulsions, and syrups.

The composition of the present invention may be administered by various methods of administration, including oral or parenteral, and may be preferably administered orally. The dosage may be adjusted to an appropriate amount depending on the condition such as age, age and sex and the severity of the disease, etc., and can be easily adjusted by those skilled in the art who are clinically engaged in the treatment or prevention of the patient.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the Examples are not intended to limit the scope of the present invention, which will be construed as to help the understanding of the present invention.

Perilla leaves ( Perilla frutescens ) From γ- GCS  Isolation and Purification of Indicators for Increased Activity (see Figure 1)

Dried Perilla Leaves ( Perilla frutescens ) 1 kg of reflux-cooled extraction with 15 L of distilled water for 3 hours, and then centrifuged at 7,500 rpm, 30 minutes, 4 ℃ to obtain a supernatant. In order to remove insoluble matters that may remain in the centrifuged supernatant, the supernatant was again replaced by whatman No. Filter using 41. The filtered supernatant was concentrated under reduced pressure and then lyophilized to obtain perilla leaves extract (PLE-0) in powder form. The PLE-0 extract thus obtained was dissolved in water, and the polarity of the organic solvent was sequentially changed, and hexane, chloroform, ethyl acetate, and butanol fractions were first separated according to polarity. Ethyl acetate fraction with the highest γ-GCS activity was continuously adsorbed onto diaion HP-20 resin (manufactured by Mitsubish Chemical Co., Japan) and then eluted in 50% distilled water: 50% methanol solution by varying the concentration of the mixed distilled water: methanol solution. It was. The eluted fractions were separated again by using a column filled with sephadex LH-20 (manufactured by Amersham Bio Sciences, Sweden) to separate caffeic acid, a hepatoprotective indicator of perilla leaves.

PLE - III -C-3 confirms that the fraction is caffeic acid Experimental Example  (Figure 3 To  8)

Structural analysis was carried out through NMR analysis on a single substance of PLE-III-C-3 fraction. The functional groups of most organic compounds absorb electromagnetic waves in the ultraviolet region. Saturated organic molecules have no absorption in near and visible light (200-800 nm), but chromospheres such as multiple bonds such as C = C, CO, etc. Is present, absorption occurs. Therefore, UV spectrum scanning of this material was performed to investigate the structural characteristics of the purified PLE-III-C-3 fraction, and it was confirmed that it had high absorbance near 320 nm. The characteristic of this wavelength band material is the structural characteristic which has a hydroxyl group in a phenol ring.

In addition, the molecular weight was measured using an electron impact mass spectrum (EI-MS), Perilla The molecular weight of the PLE-III-C-3 material obtained from frutescens ) was confirmed (see FIG. 3). From the experimental results, it was confirmed that the molecular weight of the liver-improving substance of PLE-III-C-3 fraction obtained from Perilla frutescens hydrothermal extract was 180.

¹ H-NMR shows the tendency of the oxidation of the substance, the number of methoxy groups, the presence and location of sugars, and the like.In general, aromatic hydrogen is detected at 6 ppm or more and aliphatic hydrogen at 5 ppm or less. Perilla Referring to the ¹ H-NMR spectrum (Table 2) of the PLE-III-C-3 fraction obtained from frutescens ), it can be seen that the five peaks shown between 6.19 ppm and 7.54 ppm are olefin hydrogens.

δ ¹³ C δ ^One H HMBC One 127.95 2 115.23 7.03 (1H, d, 1.5) C4, C6, C7 3 147.16 4 149.59 5 116.63 6.93 (1 H, d, 8.0) C1, C3 6 122.98 6.77 (1H, dd, 8.0, 1.5) C2, C4, C7 7 146.78 7.54 (H, d, 16) C1, C2, C6, C9 8 115.69 6.22 (H, d, 16) C1, C9 9 171.17

( ¹ H and ¹³ H NMR measured at 500 MHz)

Next, referring to the ¹³ C NMR data shown in FIG. 5, it can be seen that the eight peaks between 113.87 ppm and 148.34 ppm are methane carbons, and the peak represented by single at 178.39 ppm is the peak of carboxylic acid carbon. It can be seen, thereby confirming that the number of carbon is nine. 2D NMR COSY (FIG. 6) and HMBC (FIG. 7) analysis were also performed in parallel.

Based on the spectral analysis data, the perilla leaf (Perilla frutescensThe liver function enhancing substance of the PLE-III-C-3 fraction obtained by finding γ-GCS activity from) was found to be caffeic acid having the chemical formula shown in FIG. 8.

t- BHP Perilla leaves in liver function impairment Perilla frutescens Protection effect

Six rats as experimental animals were set as a group, and the perilla leaf extract and t -butylhydroperoxide ( tert -butylhydroperoxide ( t -BHP) according to the present invention were not treated with the control group, the group treated with t- BHP only, The perilla leaf extract 1000 ㎍ / kg according to the invention was added to the diet and treated with t -BHP, and the perilla leaf extract according to the present invention was added to the diet and then treated with t -BHP. Four groups were set up. t- BHP was used for the purpose of damaging the liver of rats. After controlling diet for 5 days, 0.2 mmol / kg of t- BHP was treated in the group except the untreated control group, and then dissected after 18 hours, and the liver tissue was taken out to measure GSH and GSSG.

Table 3 shows the evaluation results for each group. Referring to Table 3 below, the ratio of GSH / GSSG in the group treated with 0.2 mmol / kg of t- BHP was decreased compared to the group without treatment, but the perilla leaf extract 1000 and 3000 mg / kg were treated according to the present invention.

In the group it can be seen that the ratio is increased, and thus the perilla leaf extract according to the present invention can be seen to have an excellent liver protective effect against liver damage caused by t- BHP treatment.

Treatment GSH (nmol / mg protein) GSSG (nmol / mg protein) GSH Of GSSG Untreated control 67.7 ± 4.7 16.5 ± 1.4 4.1 ± 0.1 t - BHP (0.2 nmol / kg) 57.0 ± 1.6 ^d 36.7 ± 8.0 ^d 1.6 ± 0.4 ^b PLE ^a (mg / kg) 1000 75.1 ± 11 ^e 14.5 ± 4.3 ^e 5.5 ± 1.5e 3000 99.2 ± 15 ^{c, d} 17.3 ± 4.2 ^e 5.8 ± 0.7 ^e

^a Values are expressed as mean ± standard deviation (n = 6).

^b p <0.01 compared to untreated control.

^c p <0.01 (n = 6) compared to t- BHP alone treatment group.

^d p <0.05 compared to untreated control.

^e p <0.05 compared to t- BHP alone group (n = 6).

Figure 1 is a schematic process flow diagram for a method for producing caffeic acid to increase the activity of γ-GCS from perilla leaves according to a preferred embodiment of the present invention.

Figure 4 shows four fractions (PLE-III-C-1, PLE-III-C-2, PLE-III-) obtained from a PLE-III-C fraction obtained by the method according to the invention via a Sephadex LH-20 column. Γ-GCS activity of C-3, PLE-III-C-4) is shown.

FIG. 3 shows the electron impact mass spectrum (EI-MS) results for PLE-III-C-3 fractions obtained by the method according to the invention.

Fig. 4 shows the ¹ H-NMR spectral results of 1D NMR for PLE-III-C-3 fractions obtained by the method according to the present invention.

Fig. 5 shows the ¹³ C-NMR spectral results of 1D NMR for PLE-III-C-3 fractions obtained by the method according to the present invention.

Fig. 6 shows the COSY spectral results of 2D NMR on PLE-III-C-3 fractions obtained by the method according to the invention.

Fig. 7 shows the HMBC spectrum results of 2D NMR on PLE-III-C-3 fractions obtained by the method according to the present invention.

8 is a diagram showing the chemical formula for caffeic acid increasing the activity of γ-GCS obtained by the method according to the present invention.

Claims (16)

Performing reflux cooling with distilled water on the dried perilla leaves; Centrifuging the extract obtained from the reflux cooling extraction step to obtain a supernatant; Obtaining perilla leaf extract powder by concentrating the filtrate obtained by filtering the supernatant under reduced pressure and lyophilization; Preparing fractions by sequentially extracting the perilla leaf extract powder using a plurality of extracting solvents; And Separating and purifying the fraction showing the highest γ-GCS activity among the fractions Method for producing caffeic acid to increase the activity of γ-GCS from perilla leaves.       According to claim 1, wherein the reflux cooling extraction step is carried out by repeating the extraction process for 3 to 4 hours at a temperature of 100 ℃ to 110 ℃ with distilled water of 0.8 liter to 1.2 liter volume per 100g of dried perilla leaves A method for producing caffeic acid, which increases the activity of γ-GCS from perilla leaves.        According to claim 1, wherein the centrifugation step is a method for producing caffeic acid to increase the activity of γ-GCS from perilla leaves, characterized in that carried out for 25 minutes to 30 minutes at a speed of 7,500rpm at a temperature of 4 ℃ . The gamma -GCS from perilla leaves according to claim 1, wherein the fraction preparation step is performed using four extraction solvents consisting of a first extraction solvent, a second extraction solvent, a third extraction solvent, and a fourth extraction solvent. Method for preparing caffeic acid to increase the activity of. The method of claim 4, wherein the fraction manufacturing step, Dissolving the perilla leaf extract powder in the first extraction solvent, followed by stirring to separate the first organic solvent layer and the first distilled water layer to obtain a first fraction from the first organic solvent layer; Mixing the first distilled water layer with the second extraction solvent and then stirring to separate the second organic solvent layer and the second distilled water layer to obtain a second fraction from the second organic solvent layer; Mixing the second distilled water layer with the third extraction solvent and then stirring to separate the third organic solvent layer and the third distilled water layer to obtain a third fraction from the third organic solvent layer; And After mixing the third distilled water layer with the fourth extraction solvent and stirring to separate the fourth organic solvent layer and the fourth distilled water layer to obtain a fourth fraction from the fourth organic solvent layer and the fourth distilled water layer Obtaining a fifth fraction from the Method for producing caffeic acid from perilla leaves, characterized in that it comprises a. The method of claim 5, wherein the first, second, third and fourth extraction solvents are a mixed solution of distilled water and hexane, chloroform, ethyl acetate and butanol, respectively. The method of claim 6, wherein the first extraction solvent is a mixed solution of 100 to 120 parts by weight of hexane with respect to 100 parts by weight of distilled water. The mixing ratio of the first distilled water layer and the second extraction solvent, the second distilled water layer and the third extraction solvent, and the third distilled water layer and the fourth extraction solvent, respectively. Caffeic acid increasing the activity of γ-GCS from perilla leaves, characterized in that 100 to 120 parts by weight of the second, third and fourth extraction solvents, based on 100 parts by weight of the distilled water layer and the third distilled water layer. How to prepare. The method of claim 1, wherein the separating and purifying are performed using a column chromatography apparatus. The method of preparing caffeic acid, which increases the activity of γ-GCS from perilla leaves. The method of claim 9, wherein the separating and purifying step, A first separation step of performing column chromatography on the fractions using a polystyrene-divinylbenzene copolymer resin as a stationary phase and an aqueous methanol solution as a mobile phase; A secondary separation step of performing the column chromatography using the hydroxypropylated dextran resin as a stationary phase and an aqueous methanol solution as the mobile phase to the results of the primary separation step; And Selecting and purifying the product showing the highest γ-GCS activity among the results of the secondary separation step Method for producing caffeic acid to increase the activity of γ-GCS from perilla leaves, characterized in that it comprises a. The method of claim 10, wherein the first separation step is characterized in that the weight ratio of distilled water: methanol is carried out using five kinds of mobile phases of 100: 0, 75:25, 50:50, 25:75 and 0: 100, respectively Method of producing caffeic acid to increase the activity of γ-GCS from perilla perilla. The method of claim 10, wherein the secondary separation step is a method for producing caffeic acid to increase the activity of γ-GCS from perilla leaves, characterized in that carried out using a mobile phase having a weight ratio of distilled water: methanol of 30:70. Caffeic acid which increases the activity of γ-GCS prepared by the method according to any one of claims 1 to 12. The caffeic acid of claim 13, wherein the caffeic acid has hepatoprotective effect by increasing the activity of γ-GCS. A food composition comprising caffeic acid according to claim 13. A pharmaceutical composition comprising caffeic acid according to claim 13.

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