KR20100054675A - Composition extracted from marine zooplankton metabolite for improving liver function and process thereof - Google Patents

Abstract

PURPOSE: A composition containing a metabolite extract of rotifer for treating and preventing cancer and improving liver function is provided. CONSTITUTION: A method for manufacturing an active ingredient for improving liver function from marine plankton culture medium comprises: a step of treating culture medium of rotifer with methanol to obtain a crude extract; a step of fractioning and extracting crude extract with hexane or ethylacetate; and a step of isolating an extract through column chromatogranpy. A composition for improving liver function and/or preventing cancer contains the active ingredient. Rotifer belongs to the Brachionus. Rotifer is Brachionus rotundiformis, Brachionus plicatilis, Brachionus calyciflorus, or Brachionus rubens.

Description

Composition for improving liver function using a metabolite in marine animal plankton culture as an active ingredient, and a method for preparing the same {Composition Extracted from Marine zooplankton metabolite for Improving Liver Function and Process Thereof}

The present invention relates to a composition for improving liver function, and more particularly, to a composition for improving liver function extracted from a metabolite in a culture solution of zooplankton and a method for producing the same.

As problems such as increased resistance to antibiotics, which have been prepared through conventional chemical synthesis methods, have arisen, in recent years, efforts to obtain substances having pharmacological activity from natural substances have continued in the United States, Japan, and Europe. It is becoming. Therefore, in each country, it can be used for various uses such as antibiotics (antibiotics), anticancer drugs (anticancer drugs) useful for the human body, as well as pesticides, insecticides, pesticides such as herbicides, health supplements, food materials such as enzymes. In order to search for new substances, research on natural organisms, especially microorganisms that are easy to reproduce and cultivate, is being actively conducted.

In particular, marine animal plankton and freshwater zooplankton contain a large amount of various body components according to their types, and thus, they can be used as health functional product materials by converting these body components into high value-added materials. It is becoming.

Among them, marine zooplankton is not only easy to mass-produce, but also has a high added value, thus occupying an important position in the biological material industry, and various researches on such zooplankton have been made. For example, in Korean Patent No. 374748, animal plankton is cultivated using a safety frame and a barrier, and thus, animal plankton is grown using a process of inducing the cultured animal plankton to feed algae, and a water purification method using the same In Korean Patent Laid-Open Publication No. 2003-0019554, a method for culturing an animal plankton through a process of removing a growth inhibitory substance of the animal plankton from a culture solution containing an animal plankton with a solid-liquid separator and a solid-liquid separation membrane A zooplankton culture apparatus is proposed. On the other hand, Korean Patent No. 349426 discloses an anti-wrinkle cosmetic composition containing Artemia salina extract, a larvae of shellfish inhabiting seawater, in an amount of 0.01 to 50% by weight based on the total weight of the cosmetic composition.

However, given the potential of marine zooplankton, which can be easily cultured and contains a variety of body constituents, studies to obtain compositions that can improve human health, such as drugs or dietary supplements, from zooplankton It is constantly needed.

The present invention has been proposed to solve the above problems, and an object of the present invention is to improve liver function and / or cancer prevention effect containing the extract of the marine animal plankton metabolites properly treated with an organic solvent as an active ingredient It is to provide a composition having.

It is another object of the present invention to provide a composition having an effect of improving liver function and / or preventing cancer, comprising as an active ingredient an extract obtained from the metabolite of zooplankton obtained through the above method.

Other advantages and objects of the present invention will become more apparent from the following detailed description of the invention and the accompanying drawings.

According to an aspect of the present invention having the above object, the step of obtaining a crude extract by treating the culture solution of rotifer (rotifer), which is a marine animal plankton; The present invention provides a method for preparing an active ingredient for improving liver function from marine zooplankton culture medium comprising fractionating and extracting a crude methanol extract using hexane or ethyl acetate.

In this case, before the methanol crude extract is fractionated and extracted by hexane or ethyl acetate, the methanol crude extract may include mixing with methanol-water mixed solvent.

On the other hand, according to another aspect of the present invention provides a composition comprising an active ingredient for improving liver function from marine animal plankton culture obtained through the above-described method.

At this time, the composition has a liver cancer prevention effect, in particular, according to the embodiment of the present invention induces the activity of quinone reductase.

Preferably rotifers as marine zooplankton may be, for example, five beuraki Taunus in (Brachionus), an active ingredient for improving the liver function is characterized in that included at a concentration of 10 ~ 200 mM.

In the present invention, the extract obtained through relatively simple organic solvent extraction from the metabolite contained in the culture medium of rotifer, a marine animal plankton that can be easily cultured in large quantities, has liver function improvement and / or cancer prevention effect. A composition can be obtained.

Therefore, according to the purity of the active substance by the separation and purification process can be used as medicines and health supplements, it is expected that its value will be very high when applied to the food industry and the pharmaceutical industry, including health functional foods.

In other words, the extract obtained from the metabolite of marine zooplankton according to the present invention has been found to have an effect of improving liver function and preventing cancer, and thus can be expected to be utilized as a functional material using the extract as an active ingredient.

The present inventors have completed the present invention by studying the use as a bioactive component in a pharmacological composition or food using marine animal plankton, the present invention will be described in detail with reference to the accompanying drawings.

1. Extraction method of active substance improving liver function from animal plankton

Marine zooplankton has a well-established mass culture technology, which not only facilitates industrial supply and demand as a biomaterial, but also provides continuous supply. In particular, rotifer, which is a marine animal plankton, has been widely used as a food organism for fry, and since it is easy to mass-produce, industrial application is possible if it can secure economic feasibility through material development by high hatching value. Thus, in the present invention, fractions were extracted and extracted from the rotifer culture medium (metabolites), which is easy to mass-cultivate and easy to purchase in marine animal plankton using an organic solvent. A method of extracting an active ingredient having activity will be described with reference to FIG. 1.

First, methanol is added to the rotifer culture solution (metabolite) to obtain a crude methanol extract. Rotifer is a zooplankton, also called rotifer, which can be cultured in harsh conditions such as temperature and photosaline. Its reproductive power is very high, so it can be cultured in high density as well as high density culture (more than 10,000 individuals / ml). A very small (150-350 µm) zooplankton. Examples of rotifers selectable according to the invention include, for example, rotifers which inhabit seawater or inhabit freshwater, preferably belonging to the genus Brachius. Such as rotifers, for example beuraki five Taunus mainly live in the subtropics Rotundiformis ( Brachionus rotundiformis, type S, SS type), beuraki mainly inhabit the temperate zone five Taunus Plymouth car Antilles (Brachionus plicatilis , L type) as well as brachionus When Carly flow Ruth (Brachionus calyciflorus) or beuraki O Taunus Ruben's (such as the rotifer Brachionus rubens) are preferred, particularly preferably O beuraki Taunus Rotun a depot or Miss Bridal Kio Taunus Plymouth car Antilles.

Of course, known rotifer cultures and culturing methods can be used to culture them according to the type of rotifer selected. For example, the rotifer culture according to the present invention, as well as rotifer droppings and dead bodies, contains chlorella, protozoa, and the like of rotifers, and a high density culture method by batch culture and semi-continuous culture. It can be carried out as. According to the rotifer culture method, the difference in the composition of the culture medium does not occur, but the difference in the number of protozoa in the culture medium occurs depending on the temperature of the culture conditions. Although a large amount of rotifers can be cultured at a temperature of 30 ° C. or higher in a short time, typical culture conditions are about 25 to 30 ° C.

In the present invention, first, the body composition analysis of the rotifers described above confirms that the constituents of the rotifers contain the highest amount of protein. Subsequently, in the present invention, about 2-5 times (in the example, 3 times) methanol is added to the rotifer-separated and lyophilized culture solution, followed by extraction, filtration and concentration to obtain a crude methanol extract. Subsequently, the obtained methanol crude extract was again dissolved in methanol, and then mixed with water of about 0.5: 1 to 2: 1 (1: 1 in the example) based on methanol, and then using hexane and ethyl acetate, respectively. Separated. In particular, it is a support for fractionating extracts by molecular weight in extracts separated by ethyl acetate, and includes lipids, hormones, vitamins, and other small biomolecules in polar organic solvents or aqueous solvent mixtures. Column chromatography analysis using 'LH-20 Sephadex', an efficient column filler for the separation and extraction of compounds, confirmed that the extract contained in the ethyl acetate layer was divided into three fractional components. It was.

2. Effect of Animal Plankton Extract on the Liver Function

Through the above-described process, the effect of improving liver function on organic solvent extracts, especially ethyl acetate extract and hexane extract, obtained from the culture solution of rotifer which is an zooplankton is measured. To this end, after culturing a representative liver cancer cell line 'Hepa1c1c7', the organic solvent extract obtained from the rotifer culture solution obtained in the present invention is treated to a predetermined concentration, and then quinone reductase, a prophylactic cancer prevention indicator enzyme, is used as an effect of improving liver function. Induction of activity of quinone reductase) was measured.

Quinone reductase is one of the representative detoxifying enzymes, especially known as carcinogen detoxifying agents. In other words, when DNA is damaged, such as carcinogens and reactive oxygen species, tumor formation proceeds. Quinone reductase, one of Phase II detoxification enzymes, is also called vitamin K reductase. By inhibiting the action of carcinogens or mutagens, these tumor-producing substances interact with the DNA to block damage to the DNA. In other words, quinone reductase catalyzes the reduction of quinones using NAD (P) H to form hydroquinone, a stable metabolite, to block the generation of active oxygen species. In this way, quinone reductase exerts specific cytotoxicity against various cancer cells and inhibits DNA damage caused by carcinogens and reactive oxygen species.

According to the present invention, the ethyl acetate and / or hexane extracts of the rotifers were treated in liver cancer cell lines at a concentration of 10-200 mM (μg / ml), more preferably 20-100 mM (μg / ml). Quinone reductase activity is increased approximately 2-3 times. Accordingly, in the case of the extract obtained by treating the organic solvent from the rotifer culture (metabolic product) according to the present invention, for example, it exhibits an anticancer effect by inhibiting the tumorigenization process in tumor cells such as liver cancer cells. It has been shown to induce hepatic function improving activity. In particular, when the ethyl acetate fraction component was analyzed by column chromatography, three peaks were obtained. In particular, the second and third fraction components were found to be excellent in improving liver function and tumor suppression effect (FIG. 4).

Accordingly, the composition containing the organic solvent extract obtained from the culture solution of the rotifers according to the present invention as an active ingredient may be used in a pharmacological composition or a dietary supplement composition having liver improving effects and / or anticancer effects depending on the degree of purification thereof. have. In this case, if the organic solvent extract obtained from the rotifer culture of the present invention is used as a pharmacological composition, it may be prepared in a form suitable for human administration by using a liquid or a solid carrier (carrier) together.

The pharmacological composition containing the organic solvent extract of the rotifer culture of the present invention as an active ingredient can be administered orally, parenterally (intramuscularly or intravenously), enteric, rectally or topically (locally), and other general pharmacological compositions As in solutions, powders, tablets, capsules, ointments (creams or gels), suppositories. Suitable excipients for such formulations include liquid or solid fillers, extenders, solvents, emulsions, lubricants, flavors, colorants and / or buffers conventionally used in medicaments. Other auxiliary substances also include magnesium carbonate, titanium dioxide, mannitol and other sugars or sugar alcohols, milk proteins, gelatin, starch, cellulose and their derivatives, animal and vegetable oils (e.g. fish liver oil, sunflower oil, peanut oil, polyethylene glycol) And solvents (sterile water, mono or polyhydric alcohols such as glycerol).

Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments, but the present invention is not limited by the following examples.

Example  1: Component analysis of marine zooplankton

Marine zooplankton has been analyzed for its general composition in marine zooplankton belonging to rotifer, which is relatively easy to mass cultivate and easy to purchase due to the large difference in body composition according to the species. Among the rotifers, S-type seawater rotifer Brachionus rotundiformis was purchased from Aquanet in Tongyeong, Gyeongsangnam-do. The purchased rotifers were separated by filtration of the crude body and the culture solution (metabolite), and then lyophilized and stored at -50 ° C in powder form.

The general constituents of rotifers were analyzed according to the AOAC (1990) method known as Henneberg-Stohman quantitative method, moisture was 110 ℃ heat drying method, fat was Soxhlet method, crude protein was semi-micro Kjeldahl method , Ash was measured by dry calcification method, carbohydrate quantification was measured by phenol sulfuric acid method, general for the rotifer S-type Brachionus rotundiformis selected according to this embodiment The component analysis results are shown in Table 1 below.

Table 1. General component analysis of rotifers

Example  2: derived from the culture medium of marine animal plankton Crude extract  Produce

In this example, a crude extract obtained by extracting a metabolite in a culture solution of S-type Brachionus rotundiformis , a rotifer analyzed in Example 1, with an organic solvent. First, three times the amount of methanol (300 ml) was added to 100 g of the culture (metabolite) of the S-type Brachionus rotundiformis , a rotifer which is a lyophilized marine animal plankton, and then stirred twice every 6 hours. After filtration and concentration to give a crude methanol extract. Subsequently, the obtained crude methanol extract was dissolved again in 50 ml of methanol, mixed with water in the same ratio, and extracted according to the extraction procedure as shown in FIG. 1. First, 100 ml of hexane in the same ratio was taken to extract 100 ml of a mixture of methanol extract and water, and the hexane layer was separated. 100 ml of ethyl acetate was extracted from the remaining methanol / water layer, and the ethyl acetate layer was extracted. Each extraction was repeated twice. The methanol / water layer remaining after extraction was lyophilized, and the hexane and ethyl acetate layers were concentrated under reduced pressure and the residue was weighed. The yields for the methanol, hexane and ethyl acetate fractions obtained according to this example were 1.78%, 0.56%, and 0.89%, respectively, as shown in Table 2 below.

Table 2. Yield of Organic Solvent Extract of Metabolite of Animal Plankton Rotifer

Example  3: Isolation of Animal Plankton Organic Solvent Extract

In this example, the extract obtained by treating the metabolite of rotifer, which is an animal plankton obtained in Example 2 with an organic solvent, was separated using chromatography. First, the crude methanol extract obtained from the metabolite of rotifer was re-separated into hexane extract and ethyl acetate extract using liquid-liquid separation. Subsequently, the ethyl acetate extract was dissolved in methanol, and then filled with 'LH-20 Sephadex' resin as a column as a support for separating organic solvent extracts according to molecular weight, and the active material was separated using methanol as an eluent.

Among them, as shown in the Examples to be described later, the ethyl acetate extract having the best hepatic function improving activity was added to the prepared LH-20 Sephadex resin, eluted with methanol, and fractionated by molecular weight through column chromatography to obtain three fraction peaks (FIG. 2). The LH-20 Sephadex resin was filled in a resin of (10 × 350 mm) column, and ethyl acetate extract diluted to 100 mg / ml concentration was separated by flowing 100% methanol at a flow rate of 1.0 ml / min for 100 minutes. 5 ml of each fraction was measured at an absorbance of 215 nm.

Example  4: measurement of liver function improvement effect of the extract

In this embodiment, the organic solvent extract obtained from the rotifer metabolite was injected into the liver cancer cell line, and then the liver cancer cell line (Hepa1c1c7) of rats was measured to measure the effect of quinone reductase activity, a cancer prevention indicator enzyme, as a function of improving liver function. , Korea Cell Line Bank). First, a-minimum essential medium (MEM) / 10% FBS (fetal bovine serum) solution was mixed with liver cancer cell culture medium to prepare a cell number of 1 × 10 ⁵ cells / ml, and then 96 100 μl of the well plate was incubated for 24 hours at 5% carbon dioxide and 37 ° C. After the cells were stabilized, the ethyl acetate extract and the hexane extract obtained from the rotifer culture through Example 2 were treated at 7 concentrations of 3.125 to 200 µg / ml with a 2-fold gradient, and 5% carbon dioxide and 37 ° C. for 24 hours. The culture was carried out under the conditions of.

After the incubation, the cells were washed with phosphate buffered saline (PBS) solution, and then the cell membrane was lysed with 80 µl of a solution containing 0.08% digitonin and 2 mM EDTA to obtain a protein solution. 50 μl of the protein solution and reaction solution A (49 mL of 25 mM Tris buffer, 34 mg of BSA, 0.34 mL of 1.5% Tween-20 solution, 0.34 mL of coenzyme solution, 100 units of glucose-6-phosphate dehydrogena) 200 μl of Aze, 15 mg MTT, 50 μl of 50 mM menadione) was mixed and the absorbance increase was measured at 610 nm with a microplate reader, and the amount of protein was measured at 595 nm using a Bradford solution ( Biotech). The crude enzyme solution in the reaction solution A was composed of 150 mM glucose-6-phosphate, 4.5 mM NADP, 0.75 mM FAD, and the MTT was 3- (4,5-dimethylthiazo-2-yl). -2,5-diphenyltetrazolium bromide. The quinone reductase activity was calculated by the following equation.

Equation  One

(Equation 1, A is nmol MTT / min (absorbance increase rate at 610 nm of intracellular quinone reductase treated with the test sample) and B is mg protein (amount of protein in the whole cell treated with the test sample) )

The relative activity ratio for quinone reductase in liver cancer cell lines treated with hexane extract (HEX) and ethyl acetate extract (EA) in a predetermined concentration for the rotifer culture according to the present embodiment is shown in the graph of FIG. 3. As shown in FIG. 3, especially when the ethyl acetate fraction was treated at a concentration of 50 mM (μg / ml), the quinone reductase activity was increased by 2.6 times compared to the control group, and the nucleic acid fraction was the same. It was confirmed that the 1.9-fold increase in concentration.

Subsequently, Chemoprevetion lndex (CI) for each organic solvent extract obtained from the rotifer culture was calculated using Equation 2 below.

Equation  2

In Equation 2, A is IC ₅₀ (the concentration of the sample at which the cell treated with the test sample has a 50% survival rate), and B is the CD (the concentration of the quinone reductase activity of the cell treated with the test sample is doubled. Concentration).

The calculation results of the cancer prevention table using Equation 2 are shown in Table 3 below.

As shown in Table 3, the cancer prevention tables for the hexane extract and the ethyl acetate extract obtained from the rotifer cultures were 21.19 and 23.84, respectively, which were similar to the sulforaphane (CI = 25.16) known as a cancer prevention functional food material. According to the present invention it was confirmed that there is a very good cancer prevention effect of the organic solvent extract obtained from the rotifer culture.

Table 3. Cancer Prevention Indicators (CI) for Organic Solvent Extracts from Rotifer Cultures

Example  5: measurement of liver function improvement effect on fraction components

In this embodiment, using the components fractionated through column chromatography in Example 3 of the ethyl acetate extract obtained from the rotifer metabolite, according to the same procedure and method as in Example 4 to improve the liver function for the fraction component The effect was measured. Quinone reductase activity assay results for the three fractions (peak 1, peak 2, peak 3) obtained in Example 3 are shown in FIG. 4. As shown in FIG. 4, when fraction 3 (peak 3) was treated at a concentration of 50 mM (μg / ml), the quinone reductase activity increased nearly threefold, and fraction 2 (peak 2) was increased to 100. It can be seen that the quinone reductase activity was increased approximately 2.4-fold even when treated at the concentration of mM (μg / ml).

In the above, the present invention has been described in detail based on the preferred embodiments of the present invention, but the present invention is not limited thereto. Rather, those skilled in the art will be able to easily make various modifications and changes based on the embodiments described above. However, it will be more apparent through the appended claims that such variations and modifications fall within the scope of the present invention.

1 is a block diagram schematically illustrating a process for preparing an extract having an effect of improving liver function from a metabolite which is a culture solution of a rotifer which is a marine animal plankton according to the present invention;

Figure 2 is a graph showing the peak for each fraction eluted by column chromatography of the ethyl acetate extract obtained through the process of Figure 1;

Figure 3 is a graph of the relative activity of quinone reductase after the hexane extract and ethyl acetate extract obtained through the process of Figure 1 was added to the liver cancer cell line at a predetermined concentration.

Figure 4 is a graph of the relative activity of quinone reductase after the fraction fraction of the ethyl acetate extract shown in FIG.

Claims (11)

Treating crude culture of rotifers which are animal plankton with methanol to obtain crude extracts; Method for preparing an active ingredient for improving liver function from marine animal plankton culture medium comprising the step of fractionating, extracting the crude methanol extract using hexane or ethyl acetate. The method of claim 1, wherein the methanol crude extract is mixed with a methanol-water mixed solvent to prepare an active ingredient for improving liver function from marine animal plankton culture. The method of claim 1, further comprising the step of separating the extract obtained by hexane or ethyl acetate by column chromatography to obtain a fraction component. The method of claim 3, wherein the active ingredient for improving liver function is to prepare an active ingredient for improving liver function, characterized in that the second fraction or the third fraction of the components separated by the column chromatography Way. A composition comprising an active ingredient for improving liver function extracted from a culture of marine animal plankton. The composition according to claim 5, wherein the composition is obtained by the process according to any one of claims 1 to 4. According to claim 5, wherein the composition has a composition for preventing liver cancer. The composition of claim 5, wherein the composition induces the activity of quinone reductase. 6. The composition of claim 5, wherein the rotifer belongs to Brachionus . 6. The method of claim 5 or 9, wherein the rotifer is beuraki O Taunus rotun depot Miss (Brachionus rotundiformis), beuraki O Taunus replicon car subtilis (Brachionus plicatilis), beuraki O Taunus potassium loose during flow (Brachionus calyciflorus) or beuraki O Taunus Ruben's (Brachionus rubens) composition is selected from the. 6. The composition of claim 5, wherein the active ingredient for improving liver function is contained at a concentration of 10 to 200 mM.

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