KR101097799B1 - The preparing method of Ulva pertusa kjellman extracts having improved immunity-activity - Google Patents

Abstract

The present invention is characterized in that it comprises the step of extracting for 1 ~ 5 minutes under the conditions of 120 ~ 150Mpa, 110 ~ 120 ℃ each of the forked leek extract with improved immune activity.

The high temperature and high pressure liquefaction extraction is performed by sealing the dried perforated seaweed with water in a container in a high temperature and high pressure liquefier, and then performing the extraction process for 1 to 5 minutes under the conditions of 120 to 150 Mpa and 110 to 120 ° C using a high pressure liquefier. Then, the extract is filtered through 20 μm filter paper, concentrated and lyophilized.

The present invention is easy to elute the useful components by the high-temperature, high-temperature short-time extraction of poached greens, the bonds such as sulfuric acid group bonds, hydrogen bonds are separated and the cytotoxic substances are destroyed and the immune activity enhancing substance is eluted in large quantities.

 Poached greens, immune activity, sulfuric acid group bond, high temperature ultrahigh pressure extraction

Description

 The preparation method of Ulva pertusa kjellman extracts having improved immunity-activity}

The present invention relates to a green onion extract, and to a method for producing a green onion extract with a large increase in yield, and the active ingredient is effectively eluted by extracting the small green onion at high temperature and high pressure short time.

Jeju Mt. Brown is the main culprit in the devastation of Jeju fisheries, so that more than 30% of the fishery in Jeju coastal villages can be proceeded to mud recording. Jeju Island now collects huge holes every year with huge funds. Therefore, the situation that the treatment method of browning blue is urgently required. According to the researches so far, algae have a balanced distribution of trace elements such as vitamins and minerals to improve metabolism, intestinal action by low-calorie polysaccharide dietary fiber and removal of toxic substances. It is also known to be effective in normalizing blood pressure and cholesterol and preventing cancer. Like this, the green onions are rich in nutrients including vitamins A, B, and C. Therefore, although the possibility of developing them as functional food materials is high, they are not used for food because they are considered as hazardous resources in Korea. to be. However, in Europe, the green onions are already used for food (salad & soup), and in Europe, there is a lack of biological activity and food research. In addition, due to the acidic polysaccharides present in a significant amount of green onion is expected to have the effect of anti-tumor, anti-viral, immune enhancement, blood anticoagulant action, but there is little systematic research for use as a functional material. Therefore, in order to increase the utilization of the nookal green onions in Korea, we tried to develop an effective extraction technology for enhancing the anti-aging immune activity.

However, perforated green onion contains a large amount of polysaccharides containing sulfate groups, unlike land plants, and is a jelly component that contains a large amount of water in the cell wall and is separated from the circular body and connected to the cytoplasm, so that the cell wall is thicker than the land plants. Extraction of algae cannot destroy cell walls and effectively elute useful components. In other words, the simple hot water extraction of perforated seaweed not only shows low yield but also requires long time heating for more than 24 hours at high temperature of 100 or more, and polysaccharides and active ingredients are destroyed by such high temperature long time heating, which makes it difficult to expect great physiological activity. As a result, it is difficult to expect efficient elution of poached greens containing large amounts of polysaccharides and active ingredients.

In addition, the use of ultra high pressure treatment, a new technology for overcoming these problems, has been used. However, the existing ultra high pressure treatment produces ultra high pressure extraction at a high temperature near 100 ° C and generates toxic substances that can harm the human body such as HMF (Hydroxymethyl furfural). Only low temperature process is below 50 ℃, and even this low temperature process has low extraction efficiency, high energy consumption, and breakdown of many useful components due to heat, protein variation, loss of components, and solubility. The shortcomings of hot water extraction, such as extraction and unstable against heat, have been improved. However, the extraction of perforated lees does not decompose the binding of sulfated polysaccharides, which is a characteristic of perforated lees, and thus does not perform effective extraction of substances useful for immune function. For extraction, perform hot water extraction after low temperature ultra high pressure treatment. This leads to waste of time and resources.

In order to solve the conventional problems as described above, the present invention has a purpose to provide a method for producing a green onion extract with improved immune activity by extracting the green onion for a short time by high temperature and high pressure liquefaction technology.

In the high temperature and high pressure liquefaction extraction process of the present invention, the process of repetition of hot water extraction from the high temperature and high pressure extraction is unified, and the process proceeds quickly. It can be extracted inside, and the extract is almost free of impurities, and a high purity single component can be easily obtained.

In addition, the high temperature and high pressure liquefaction extraction process of the present invention facilitates the elution of useful components due to the appropriate destruction of the cell tissue to be extracted, the bonds such as sulfate group bonds, hydrogen bonds are separated to destroy the cytotoxic substances and enhance immune activity The material is eluted in large quantities.

The high temperature ultrahigh pressure extraction of the present invention is an optimum extraction condition in the greenery as described above, even if the extraction under the high temperature ultra-high pressure due to the structure of the green shreds does not cause rapid cell destruction.

 The present invention is characterized in that it comprises a step for extracting for 1 ~ 5 minutes under the conditions of 120 ~ 150Mpa, 110 ~ 120 as a method for producing a green onion extract with enhanced immune activity.

The high temperature and high pressure liquefaction extraction is sealed by putting dried dry brown seaweed together with water in the high temperature and high pressure liquefier container, and performing the extraction process for 1 ~ 5 minutes under the condition of 120 ~ 150Mpa, 110 ~ 120 using a high pressure liquefier. After that, the extract was filtered through 20㎛ filter paper, concentrated and lyophilized.

The present invention is easy to elute the useful components in a short time by being extracted at high pressure and high temperature at the same time, the bonds such as sulfuric acid group bonds, hydrogen bonds are separated so that a large amount of immune activity enhancing substance is eluted.

  In order to confirm this, the present invention was to examine the effect of enhancing the immune activity of the process by measuring the extraction yield and immune activity of the greenish blue after the extraction process.

Although it is expected to have many activities such as antitumor, antiviral, immune enhancing, and anticoagulant action of blood, it is rarely used due to the recognition of environmental hazards. Therefore, through the high temperature and ultra-high pressure treatment proposed in the present invention to study the functional food material utilization method of the green onions can be obtained the yield increase and immune activity enhancement extract of the green onions, and proposed a method to solve such problems In other words, the economic and ecological effects of creating added value of perforated litter treated as waste can be achieved.

In addition, the introduction of high temperature ultra-high pressure liquefaction process technology solves the problems of the existing extraction process and at the same time improves the yield and improves the immunological activity through relatively high immunity. There is a small advantage that this extraction process can be applied to the extraction process of functional food material of other algae or terrestrial resources with a structure similar to perforated seagrass.

The specific extraction method of the greenish green of the present invention is as follows.

The dried perforated seaweed is sealed with water in a container of a high temperature and high pressure liquefier, and then extracted using a high pressure liquefier for 1 to 5 minutes under conditions of 120 to 150 Mpa and 110 to 120 ° C. Filtrate in 20㎛ filter paper, concentrate and freeze-dry.

Example 1.High temperature and high pressure extraction

1) Raw material: Ulva perusa ( Ulva pertusa kjellman ) was washed and dried.

    2) High temperature and high pressure extraction: The extraction process was carried out for 5 minutes under conditions of 150Mpa, 110 ℃ using a high temperature, high pressure liquefier (Fig. 1).

    3) Concentration: Each extract obtained in the above section was filtered under reduced pressure filter (20㎛ filter paper) and concentrated under reduced pressure to a temperature lower than the extraction temperature.

    4) Freeze drying: The concentrates obtained by the above method were used by freeze drying.

Example 2 Hot Water Extraction

The dried perforated green hot water was extracted for 24 hours at 100 ℃.

The extract obtained above was used by concentrated freeze-drying under reduced pressure as in Example 1.

Example 3 Low Temperature High Pressure Extraction

After the dried perforated seaweed was sealed with water in a plastic container to prevent air from entering, ultra-high pressure extraction was performed at a temperature of 50 ° C. for 15 minutes at a pressure of 150 Mpa using an ultra-high pressure extraction device.

The extract obtained above was used by concentrated freeze-drying under reduced pressure as in Example 1.

Experimental Example Comparison of Immune Activity of Extracts from Forge Extracts by Different Extraction Methods

Yields and cytotoxic activity were compared for the greenish green extracts obtained in Examples 1, 2, and 3.

1) yield

Sample High-Pressure (Mpa) temperature (℃) yields
(%, w / w) Ulva pertusa kjellman
- 100 4.98 150 50 8.31 150 110 12.28

The extraction yield of the greenish green laver is as follows. Among all conditions, the highest yield was obtained at the high temperature and high pressure liquefied extract. Extraction yield was 2.5 times higher than that of 100 ℃ general hot water extraction. In addition, the extraction yield is 1.5 times higher than the low temperature and high pressure extract. This is due to the high temperature and high pressure liquefaction extraction of the thick cell walls of the brownish brown easily breaks the existing material, the amount of elution and new material is eluted seems to be high yield.

2) Anti Aging Activity Comparison

  ① Immunostimulating effect of Natural Killer cell (NK cell) (Fig. 2)

In the NK cell-92MI in D-MEM medium 2 mM L-glutamine, 0.2 mM myoinositol, 20 mM folic acid, 10-4M 2-mercaptoethanol, 12.5% FBS and 12.5% horse serum (Myelocult) 2 × 10 7 cells / Diluted to a concentration of ml and used. Human T cells were cultured in T-25 Flask, and the extracts of Examples 1,2 and 3 were administered, followed by 3-4 passages while observing the degree of proliferation. Dispense NK-92MI cells into 4 well 5 × 10 ⁴ cells / into 24 well plates at 900 μl. After 24 hours, 100 μl of supernatant of T cells was applied to each plate for 48 days after incubation for NK-92MI. The activity of the cell was measured by using a cell counter, and the activity of the NK-92MI cell was measured using the number of living cells.

As a result of measuring the activity of NK cells for 6 days, growth was increased under all treatment conditions.

On day 6, high temperature and high pressure extract showed 11.8 × 10 ² cells / ml, showing the highest activity. It is believed that immuno-active substances are more eluted than conventional extraction methods due to the proper destruction of tissues and cells through high-temperature ultrahigh pressure treatment.

 ② Measurement of nitric oxide production ability in macrophages (FIG. 3)

J774.1 macrophage (mouse), cell growth for 48 hours at 5 ℃ CO ₂ , 37 ℃ conditions in a concentration of 4 ~ 5 × 104 cells / ㎖ in a 24well plate using RPMI1640 medium containing 10% FBS Were used for the experiments. The amount of nitric oxide generated in macrophage was determined by quantifying the amount of nitric accumulated in activated macrophage cultures using a microplate assy.

First, the extracts of Examples 1, 2, and 3 were added, and the cells were cultured for 48 hours, and 50 µl of the supernatant was taken. The same volume of Griesstldir (1% sulfanilamide / 0.1%, N- (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% H3PO4 ) Was added and reacted at room temperature for 10 minutes, and the absorbance of 540 nm was measured using an ELISA reader (Molecular Devices, USA). Sodium nitrite was used as a standard for nitrite, and the concentration was calculated by comparing with a standard curve obtained by diluting twice with RPMI 1640 medium from 32 μm to 0.25 μm. LPS (lipopolysaccharide) was used as a positive control material of NO production ability.

The results of confirming the NO producing ability using macrophages are shown as above. The nitric oxide syntase (NOS) of macrophages is not always present, but because the expression of NOS gene is induced by the effects of various cytokines such as TNF-a and intracellular bacterial toxins such as E. coli derived lipopolysaccharide (LPS). In the experiment, the extracts of Examples 1, 2 and 3 were administered together with LPS to confirm NO production ability.

Figure 3 shows the results of confirming the NO production ability using macrophages.

As shown in the results of Fig. 3, the extracts of Examples 1, 2, and 3 were cultured in LPS and J774.1 cell lines, and the NO concentrations were measured in the culture medium. It was confirmed. In particular, the high temperature and high pressure liquefied extract of the present invention showed the highest 30.0㎛. The mass production of NO, which plays an important role as an immune mediator, means that the immune active function is excellent. Therefore, the elution amount of the immunoactive component through the high temperature and high pressure liquefaction process is higher than that of the general extract and the low temperature and high pressure extract.

③ Comparison of cell morphology in dermal fibroblast cells of rind extract (Fig. 4)

2 x 10 ⁵ fibroblasts (CCD-986sk) were placed in a 6-well plate containing DMEM and 10% FBS in each well, and each extract was treated at a concentration of 1.0 mg / mL. Morphological observations of cells incubated under CO ₂ conditions are shown. The above results show that all extracts show active cell differentiation, which means that there is no large toxicity (high survival rate) at the cellular level, which also means that HMF (Hydroxymethyl furfural) can be a problem in high temperature and high pressure liquefaction. ) Means no toxic substances that can harm the human body. In particular, high-temperature, high-pressure liquefied extract shows high cell activity. It also appears that the cell activity due to the high yield of useful bioactive substances is higher than other extraction processes through high temperature and high pressure liquefaction process.

 ④ HPLC peak comparison of extracts obtained by process (Fig. 5)

 In order to compare the produced immunoactive substances extracted by the conditions, high-performance liquid chromatography (HPLC) analysis was used to obtain peaks of perilla extract through each extraction process. Inter-process analysis was performed. Separation conditions, the extracts of Examples 1, 2, 3 were filtered through a 0.2 ㎛ syringe filter in analytical water and prepared at a concentration of 100 ppm, respectively, and measured by injection volume of 20 μl. The HPLC instrument was a BIO-TEK 522 controller pump of BIO-TEK instrument (Italy) HPLC 500 series and BIO-TEK HPLC 535 UV Detector (254 nm) .The column was Prevail C18 (5㎛, 4.6 × 250) from Alltech. Mm) was used. The mobile phase used a mixed solution of water and methanol (50: 50, v / v), the flow rate was flowed at 0.50ml / min.

As a result of HPLC analysis of the extract obtained from the process, the peaks of more substances were confirmed in the high temperature and high pressure liquefied extract. This effectively destroys the thick cell walls of the perforated greens, and it seems that more bioactive substances were extracted from other processes (100 ℃ hot water extraction, low temperature, high pressure).

1 is a photograph of a high temperature high pressure liquefier used in the present invention.

Figure 2 is a graph comparing the immunostimulating effect of the natural Killer cells (NK cells) of the green rind extract.

Figure 3 is a graph showing the nitric oxide production ability of macrogallium extract in macrophages.

Figure 4 is a comparison of cell morphology in dermal fibroblast cells of the green rind extract.

Figure 5 is a graph comparing the HPLC peak of the green rind extract.

Claims (1)

A method for producing an immune activity-enhanced perilla extract, characterized in that it comprises the step of extracting for 1 to 5 minutes under the conditions of 120 ~ 150Mpa, 110 ~ 120 ℃ the pericarp.

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