KR20120086919A - Preparation method of germinated perilla seed oil - Google Patents

Abstract

PURPOSE: A producing method of germinated perilla seed oil is provided to economically produce the perilla seed oil containing massive amount of immunity enhancing components. CONSTITUTION: A producing method of germinated perilla seed oil comprises the following steps: soaking perilla seeds in water at 25-35 deg C for 12-18 hours; germinating the water soaked perilla seeds at 25-35 deg C for 42-54 hours in the humidity of 55-65%; drying the germinated perilla seeds; roasting the dried perilla seeds at 195-205 deg C for 15-25 minutes; and compressing the roasted perilla seeds to obtain the germinated perilla seed oil.

Description

Preparation method of germinated perilla oil {PREPARATION METHOD OF GERMINATED PERILLA SEED OIL}

The present invention relates to germinated perilla oil with improved anti-cancer, immune activity, and anti-allergic effects, and more particularly, after roasting perilla and roasting at 195 to 205 ° C.

According to the present invention, unlike the common perilla oil, promotes the induction of apoptosis (apoptosis) of human leukemia cells (Molt-4 cells) showing an anticancer / immune activity enhancing effect and increases the phagocytic activity of Tc lymphocytes and macrophages in the thymus, Germinated sesame oil having an effect of suppressing a systemic anaphylactic reaction having an anti-allergic effect can be obtained.

In the late twentieth century, the life expectancy of modern people has been prolonged due to the development of medicine, but various stresses and westernized food cultures lead to various diseases as they age. As a result, due to the recent aging of the population, increased interest in income and well-being, the health functional food market has reached about KRW 2.3 trillion (as of 2010), which is continuing to grow.

In particular, modern people are easily exposed to various stresses and environmental pollution, so they can be easily affected by various diseases. Therefore, the treatment of the disease itself is also important, but coping with stress such as various stresses and environmental pollution is very helpful in preventing the disease.

As a food and food material used for research on immune function, general vegetable material, seafood material, mushroom material, microbial material such as lactic acid bacteria, chitosan material, etc. have been used (necessary identification of the activation of forests and natural materials, monthly Food, 2, 11, 90-96 (2003). However, the immuno-active food material using the above-described material has a problem that the efficacy does not meet expectations or the toxicity and industrial economics are relatively low.

On the other hand, perilla oil is one of the foods that are known as health foods of the elderly because it has a function of anti-arteriosclerosis, anti-cancer, and cognitive ability as a domestic seed oil with high linolenic acid content. When perilla oil is produced after germinating to a certain level, perilla oil having excellent anti-cancer / immune enhancing effect and anti-allergic effect is produced.

It is an object of the present invention to economically provide perilla oil containing a large amount of factors that enhance immune activity using perilla oil, which is ingested in our daily life.

Other objects and advantages of the invention will be described below and will be appreciated by the embodiments of the invention. In addition, the objects and advantages of the present invention can be realized by means and combinations indicated in the claims.

One aspect of the present invention is to immerse the perilla at 25 ~ 35 ℃ 12 hours to 18 hours; Germinating the soaked perilla at a humidity of 55-65% and a temperature of 25-35 ° C. for 42-54 hours; Drying the germinated perilla; Roasting the dried germinated sesame seeds at 195 to 205 ° C. for 15 to 25 minutes; And squeezing the roasted sesame seed to extract the germinated sesame oil.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, wherein the germinated sesame oil is an active ingredient that induces apoptosis of human leukemia cells (Molt-4 cells).

In addition, another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, wherein the germinated sesame oil enhances the activity of cytotoxic T cells and macrophages.

In addition, another aspect of the present invention provides a pharmaceutical composition for immediately preventing or treating allergy having the germinated sesame oil as an active ingredient.

In addition, another aspect of the present invention provides an immune enhancing food composition comprising the germinated sesame oil as an active ingredient.

The content of the present invention will be described in detail as follows.

After immersing perilla at 12 ~ 18 hours at room temperature 25 ~ 35 ℃, spread perilla on gauze 1 ~ 2cm, germinating at 55 ~ 65% humidity, 25 ~ 35 ℃ for 42 ~ 54 hours, and washing and drying. The dried germinated sesame seeds roasted for 15-25 minutes at 195 ~ 205 ℃ and extracted germinated sesame oil using a compression extractor.

Anti-cancer activity is in charge of the human immune system, so the stronger the immune system, the better the anti-cancer activity is known. In order to evaluate the cytotoxicity of cancer cells by preparing germinated perilla oil, cytotoxicity against human acute T lymphoblastic leukemia cell line (Molt-4 cells) and normal human renal epithelial cell line (293T) (MTT assay) We examined the apoptosis-related gene expression patterns (RT-PCR analysis) and related protein expression patterns (Western blotting) for Molt-4 cells, and to evaluate the activity of immune cells. The effects of lymphocyte subpopulations (B, T, Th, Tc, NK cells) on spleen and thymus cells (flow cytometry) and phagocytic activity using peritoneal macrophages (NBT assay) were observed. In addition, the antiallergic effect was measured by the compound 48/80 induced systemic anaphylaxis (acute allergic reaction) and degranulation induction in mast cells using RBL-2H3 (mast cell line).

According to the present invention, unlike general immunoactive activators, germinated perilla oil can be obtained as a general food and can be relatively excellent in immunological activity. In addition, it can be seen that the anticancer activity and anti-allergic efficacy is also improved.

1 is a graph showing the results of measuring cytotoxicity by the MTT assay method for the normal human kidney epithelial cell line (293T) treated with each sample.
Figure 2 is a graph showing the results of measuring cytotoxicity by the MTT assay method for human acute T lymphocytic leukemia cell line (Molt-4) treated with each sample.
FIG. 3 shows electrophoresis images of apoptosis-related gene expression patterns of human acute T lymphoblastic leukemia cell lines (Molt-4) treated with each sample by RT-PCR.
4 is a result of Western blotting of apoptosis-related protein expression patterns of human acute T lymphoblastic leukemia cell line (Molt-4) treated with each sample.
Figure 5 is a graph showing the phagocytic activity (NBT assa) using the peritoneal macrophages of mice ingested each sample.
Figure 6 is a graph showing the effect on degranulation in RBL-2H3 (mast cell line) after each sample ingested anaphylactic mice.

Hereinafter, preferred embodiments of the present invention will be described in detail. Prior to this, terms or words used in the present specification and claims should not be construed as being limited to the common or dictionary meanings, and the inventors should properly explain the concept of terms in order to best explain their own invention. Based on the principle that can be defined, it should be interpreted as meaning and concept corresponding to the technical idea of the present invention. Therefore, the configurations shown in the embodiments described herein are only the most preferred embodiments of the present invention and do not represent all of the technical idea of the present invention, and various equivalents may be substituted for them at the time of the present application. It should be understood that there may be variations.

(Example 1)

After immersing perilla at 30 ° C. for 12 hours or more, perilla was placed on gauze for 1-2 cm, germinated at 60% humidity and temperature of 30 ° C. for 48 hours, followed by washing and drying. The dried germinated sesame seeds were roasted for 20 minutes at 200 ° C. and extracted with germinated sesame oil using a compression extractor.

(Experimental Example 1)

To explore anticancer activity in vitro The experiments were carried out to observe the cytotoxicity of normal cells through the human kidney epithelial cell line (293T) culture system and to observe the anticancer activity against human acute T lymphoblastic leukemia cell line (Molt4). The gene expression pattern was analyzed by PCR, and the related protein expression characteristics were analyzed by Western blotting.

Negative control: no addition

Comparative Example 1: Corn oil

Comparative Example 2: Non-Germinated Perilla Oil

Example: Germinated Perilla Oil

Experimental Example 1-1 Cytotoxicity Test on Normal Cells

Each sample was added to the normal human kidney epithelial cell line (293T) by concentration (1, 10, 100 µg / ml) by MTT assay and CO ₂ at 37 ° C. for 48 hours. Incubated in the incubator, 20 uL of MTT solution diluted with 5 mg / mL of D-PBS (pH 7.4) was added to each well 4 hours before the end of the incubation, dissolved in 100 uL of 10% SDS dissolved in 0.1 N HCl for 18 hours. The light is shaded. The absorbance of each color well was measured at 570 nm using an ELISA reader and compared with the absorbance of the control group, the cell viability was converted into percentage. The result is shown in FIG. Indicated.

As shown in FIG. 1, no cytotoxicity was observed for normal cells in all samples.

Experimental Example 1-2 Cytotoxicity Test on Human Acute T Lymphoblastic Leukemia Cell Line (Molt-4)

Each sample was added to each human acute T lymphoblastic leukemia cell line (Molt-4) by MTT assay (1, 10, 100 µg / ml) and cultured in a CO ₂ incubator at 37 ° C. for 48 hours. 4 hours before the end of the culture, 20 μL of MTT solution diluted with 5 mg / mL of D-PBS (pH 7.4) was added to each well, dissolved in 100 μL of 10% SDS dissolved in 0.1 N HCl, and then shaded for 18 hours. The absorbance of each color well was measured at 570 nm using an ELISA reader and compared with the absorbance of the control group, the cell viability was converted into percentage. The result is shown in FIG. Indicated.

As shown in Figure 2, Comparative Example 1 (jade oil) had no effect on cytotoxicity against human acute T lymphoblastic leukemia cell line (Molt-4), Comparative Example 2 (perilla oil), Example (germination) In perilla oil, cytotoxicity (anticancer activity) against leukemia cells was observed, and in particular, in Example (germinated perilla oil), it was more effective.

Experimental Example 1-3 Cell Death Induction Experiment on Human Acute T Lymphoblastic Leukemia Cell Line (Molt-4)

To examine the effect of inducing apoptosis on Molt-4 cells, the relevant gene expression patterns were analyzed by RT-PCR. Molt-4 cells were treated with 10 ㎍ / ㎖ of Comparative Example 1 (fermented oil), Comparative Example 2 (perilla oil) and Example (germinated perilla oil), and then cultured for 24 hours, RNA was isolated from each cell. p53, NFkB (p105), Bax, Bcl-xl, iNOS genes were identified. Total RNA was prepared according to the manufacturer's method using Trizol reagent. cDNA was synthesized using M-MLV reverse transcriptase and random hexamer as primers and amplified for 30 cycles in Takara PCR thermocycler (Dice) using Taq DNA polymerase. PCR products were confirmed by electrophoresis on 1% agarose gel and stained with EtBr. The photoelectrophoresis on the agarose gel is shown in FIG.

As shown in FIG. 3, the expression of p53 (tumor suppressor gene), NFkB (transcription factor; p105), and Bax (apoptosis induction gene) in perilla oil (10 μg / mL) were compared with that of Comparative Example 1 (Okbae oil). On the contrary, the expression of BcL-xL (apoptosis inhibitory gene) was decreased and the NONO gene iNOS gene was not expressed. p53 and NFkB gene expression of Comparative Example 2 (unseeded oil), Bax and BcL-xL gene activity of Example (seeded oil) was good.

Experimental Example 1-4 Protein Expression Observation Experiment on Human Acute T Lymphoblastic Leukemia Cell Line (Molt-4)

Western Blotting was performed to examine the pattern of protein expression in Molt-4 cells treated with 10 μg / ml of each sample for 48 hours. Each sample was treated with 10 μg / mL in Molt-4 cells and incubated for 24 hours, followed by 1% NP-40 lysis buffer (250 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8.0, 1% NP-40, 0.6). Proteins were isolated by mM PMSF, 10 μg / mL aprotinin, 1 μg / mL pepstatin A), quantified using Bradford method using Coomassie blue R-250, and 20 μg of protein was analyzed by 12.5% SDS- for Western blot analysis. After PAGE, transfer to nitrocellulose membrane, and then blocked with 5% non-fat dry milk for 2 hours, p53 rabbit polyclonal IgG antibody (1: 1000), PCNA rabbit polyclonal IgG antibody (1: 1000) 1 After staining for 1 hour at 4 ℃, and secondary staining with goat anti-rabbit IgG-HRP conjugated antibody was reacted in the same manner to the X-ray film by the enhanced chemiluminescence (ECL) method. The results are shown in FIG.

As shown in Figure 4, in the Molt-4 cells treated with Example (germinated sesame oil), the expression of p53 protein known as a representative tumor suppressor gene product was significantly increased compared to Comparative Example 2 (unseed sesame oil). Expression of proliferating cell nuclear antigen (PCNA), a proliferating cell nuclear antigen protein, was inhibited.

Through the results of Experimental Example 1-4, it can be seen that the so-called sesame oil has an excellent anti-cancer effect of inhibiting the proliferation of leukemia cells.

(Experimental Example 2)

The main immune cells are T, B lymphocytes and phagocytes. T lymphocytes are the central cell population that controls the immune system as a whole. Important markers of differentiation of thymic lymphocytes are CD4 and CD8 antigens. CD4 ⁺ CD8 ^- T lymphocytes are the most important immune cells as T _H cells, and CD4 ^- CD8 ⁺ T lymphocytes are Tc cells that destroy target cells. Do it. B lymphocytes produce antibodies and perform various actions. In addition, phagocytic systems include neutrophils and macrophages, which phagocytize the invading foreign body. To evaluate the activity of these immune cells, lymphocyte subpopulations (B, T, Th, Tc and NK cells) were analyzed by flow cytometer and phagocytosis using mouse peritoneal macrophages to evaluate phagocytic activity. Activity (NBT assay) was observed.

Experimental Example 2-1

The mice were orally administered for 3 weeks (500mg / kg) and cervical spine was used to extract the spleen and thymus, and then the spleen and thymic cell suspensions were prepared and PE / FITC conjugated-anti B220 and Thy in 1 × 10 ⁶ cells / well. -1, double-stained with NK antibody and PE-anti CD4 / FITC-anti CD8 monoclonal antibody (1:30 dilution) and reacted at 4 ° C for 30 minutes and flow cytometer (excitation 488nm, emission 525nm / FITC, 575nm / PE) Subpopulations of lymphocytes in each cell were measured. The results are shown in Table 1 below.

My spleen and thymus lymphocyte subset analysis (in vivo )
Cell type
Splenocyte-spleen (%) Thymocyte-thymus (%) NK cell (%) B cell T cell T _H T _C T _H T _C Control 28.7 ± 3.2 21.7 ± 2.1 17.2 ± 2.5 5.0 ± 0.4 0.8 ± 0.1 18.4 ± 1.6 5.0 ± 0.5 Comparative Example 1 (C-O) 30.0 ± 3.4 24.3 ± 1.5 14.1 ± 1.5 5.0 ± 0.3 0.9 ± 0.2 18.0 ± 1.3 7.7 ± 0.7 Comparative Example 2 (N-P) 27.1 ± 2.8 25.7 ± 2.8 15.2 ± 0.9 8.3 ± 0.6 ^*

0.7 ± 0.1 18.9 ± 1.5 7.8 ± 0.8 Example (G-P) 25.0 ± 2.9 25.3 ± 2.2 13.8 ± 2.1 6.7 ± 0.5 0.7 ± 0.2 17.1 ± 1.6 6.4 ± 0.7

As shown in Table 1, immune cell activity (lymphocyte subpopulation) was increased in a subpopulation of thymic Tc (cytotoxic T) cells, especially in perilla oil fed group.

Experimental Example 2-2

In order to evaluate the activity of the phagocytes of Experimental Example 2-1, the phagocytic activity (NBT assay) using the mouse peritoneal macrophages was observed.

To determine the activity of macrophages, macrophages were taken from the abdominal cavity of the mice to which the samples were administered, and the supernatant of the macrophages was taken and the amount of superoxide anion was measured at 517 nm by NBT reduction. The reaction solution was prepared with 50uL of each sample, 125uM NADH and 63uM of NBT in 150uL of PBS (pH8.4), and the absorbance of Superoxide anion was expressed as inhibition activity (%) compared to the control without the sample. The results are shown in Fig.

As can be seen in Figure 5, the effect on the phagocytic activity (NBT assay) using the mouse peritoneal macrophages, especially in the group treated with Example (germinated sesame oil) significantly increased the phagocytic activity compared to the control.

This result indicates that perilla oil has an effect of regulating immunity by enhancing the activity of cytotoxic T cells and macrophages. These Tc cells and macrophages are known to be important cells closely related to anticancer activity.

Experimental Example 3

Anaphylaxis shock is an immediate type of allergy and refers to a violent and immediate shock condition that occurs when a specific antigen is contacted and then later contacts the antigen.Histamines in granules are caused by degranulation when the antigen is invaded by this cause. This secretion causes the blood vessels to expand, causing a sudden drop in blood pressure, which prevents the heart from supplying enough blood, and a severe shock condition persists and eventually becomes fatal (Phoenicin shock). In order to measure the anti-allergic effect, ICR mice were administered Compound 48/80 to induce systemic anaphylaxis, and the effects of each sample were evaluated. The mast cell line (RBL-2H3) was used to induce degranulation with Compound 48/80. After the treatment of each sample, degranulation inhibition was observed.

Experimental Example 3-1

8-week-old ICR mice were injected ip ( ip ) with compound 48/80 (8 mg / kg BW), known as degranulation of mast cells, in the peritoneal cavity of mice (8 mice / group 1). The sample of Example (500 mg / kg) was orally administered 1 hour before compound 48/80 injection, and second oral administration 5-10 minutes after compound 48/80 injection. The mortality was measured for 1 hour after inducing anaphylactic shock by compound 48/80. The results are shown in Table 2.

Experimental group Total Dosage (Marine) Lethal population (mari) Survival rate (%) Control group (CON) 8 7 12.5 Comparative Example 1 (C-O) 8 6 25 Comparative Example 2 (N-P) 8 4 50 Example (G-P) 8 3 62.5

As shown in Table 2 above, administration of Compound 48/80 induced systemic anaphylaxis, and the administration of each sample resulted in the inhibition of anaphylaxis of Comparative Example 2 and Example (Perilla oil) as compared to Comparative Example 1 (Pear oil). Was observed to be more than 2 times, and it can be seen that the Example (germinated sesame oil) (62.5%) is more effective in inhibiting systemic anaphylaxis than Comparative Example 2 (unseed sesame oil) (50%).

Experimental Example 3-2

Degranulation was induced by using a mast cell line (RBL-2H3) with Compound48 / 80, and each sample was treated. In detail, the Wistar-based basophillic (basophilic configuration) cell line, RBL-2H3 was used as a cell line, Mg ^{2 +,} and a Ca ^{2 +} is washed with not included PBS, collected by treatment with 0.25% trypsin tyroid buffer (137mM NaCl, 2.7 mM KCl, 1.8 mM CaCl ₂ , 1.1 mM MgCl ₂ , 11.9 mM NaHCO ₃ , 0.4 mM NaH ₂ PO ₄ , 5.6 mM glucose, pH 7.2) and then adjust to 1 × 10 ⁶ cells / ml Was dispensed on. Each sample (100 μg / mL) was added thereto, and the resultant was allowed to stand in a 37 ° C. incubator for 10 minutes, and then Compound 48/80 was added so as to have a final concentration of 5 μg / mL and left in the incubator for 10 minutes. The reaction was terminated by treatment at 4 ° C. for 10 minutes to recover the supernatant. The reaction was terminated and then measured at wavelengths of 360 nm and 450 nm using a UV-spectrophotometer. The results are shown in FIG.

As shown in Figure 6, it can be seen that in Comparative Example 2 (unseated sesame oil) and Example (seed sesame oil) inhibit the degranulation of mast cells of 15% and 13%, respectively. These results demonstrate that (germinated) perilla oil is particularly effective against histamine-mediated immediate allergy.

As mentioned above, although this invention was demonstrated by the limited embodiment, this invention is not limited by this and it will be described below by the person of ordinary skill in the art, and the following. Of course, various modifications and variations are possible within the scope of the claims.

Claims (5)

Soaking perilla at 25-35 ° C. for 12-18 hours;
Germinating the soaked perilla at a humidity of 55-65% and a temperature of 25-35 ° C. for 42-54 hours;
Drying the germinated perilla;
Roasting the dried germinated sesame seeds at 195 to 205 ° C. for 15 to 25 minutes; And
Compressing the roasted sesame seeds and extracting germinated sesame oil. A pharmaceutical composition for the prevention or treatment of cancer, comprising the germinated sesame oil of claim 1 which induces apoptosis of human leukemia cells (Molt-4 cells). A pharmaceutical composition for preventing or treating cancer comprising the germinated sesame oil of claim 1 which enhances the activity of cytotoxic T cells and macrophages. A pharmaceutical composition for immediate allergy prophylaxis or treatment comprising the germinated sesame oil of claim 1 as an active ingredient. The food composition for immune enhancement which uses the germination of claim 1 as an active ingredient.

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