KR101972694B1 - Method for Preparing a Composition Comprising Allium ampeloprasum L. Extract and the Composition for Preventing or Improving Obesity Comprising the Same - Google Patents

Abstract

The present invention relates to a method for preparing an Allium ampeloprasum L. extract and a composition for preventing or alleviating obesity comprising the extract. The method comprises the steps of: obtaining homogenized Allium ampeloprasum L.; and obtaining an Allium ampeloprasum L. extract by mixing the homogenized Allium ampeloprasum L. and a solvent in a weight ratio of 1:1.

Description

Method for preparing a composition Comprising Allium ampeloprasum L. Extract and the Composition for Preventing or Improving Obesity Comprising the Same}

The present invention relates to a method for producing Ungung garlic extract and a composition for preventing or improving obesity comprising the same.

Garlic, an indispensable ingredient in Korean diet, has traditionally been used as a folk remedy with various pharmacological effects such as anticancer, antioxidant and antibacterial as well as spices. In addition, allicin, the main active ingredient of garlic, is known to have various physiological effects such as blood pressure strengthening, blood lipid lowering and blood sugar reducing effect. However, in the case of raw garlic, it is difficult to continuously consume sufficient amount to exhibit a physiological activity effect due to its unique smell and aroma, and it may irritate the stomach wall and cause tissue damage when excessively ingested. On the other hand, black garlic heated by garlic has been reported not only to decrease the spicy taste and aroma of garlic, but also to increase the sweet taste, and to increase the specific bioactive substances such as S-allylcysteine due to chemical transformation during the manufacturing process. have. Due to the difference in the content of these bioactive substances, black garlic is known to have superior antioxidant, arteriosclerosis, and lipid enhancement effects than normal garlic.

Elephant garlic (Allium ampeloprasum L.) is a plant belonging to the allium with less smell and about 10 times larger scale than general garlic (Allium sativum L.). In particular, the garlic garlic has a superior texture and sweetness compared to general garlic, making it suitable for use as a variety of food materials.

Conventional Patent No. 10-1446528 discloses a vinegar composition using black garlic and a method for preparing the same, but a composition using male garlic has not been studied until now. The raw material of the registered patent is garlic (Allium sativum L.), the raw material used in the present invention is the elephant garlic (Allium ampeloprasum L.), there is a difference in the raw material. Until recently, domestic research has been limited to the research on the growth of Ungung garlic, and the research on the biological activity effect using the same is not sufficient. In the case of the existing registered patents, only the case containing garlic is disclosed, and studies using allium garlic (Allium ampeloprasum L.) have not been disclosed at all.

Accordingly, the present invention is to provide a composition for preventing or improving obesity having an excellent effect using the male garlic.

Republic of Korea Patent No. 10-1446528

The present invention is to provide a composition for preventing or improving obesity, which can be ingested at any time due to the occurrence of odor less by using the male garlic with less side effects even when ingested raw.

The present invention provides a composition for the prevention or improvement of obesity, including the male extract.

In addition, the present invention comprises the steps of homogenizing the male garlic to obtain a homogenized male garlic; And it provides a method for producing Ungung garlic extract comprising the step of dissolving the homogenized Ungung garlic with a solvent to obtain a Ungung garlic extract.

An object of the present invention is to achieve an excellent obesity prevention or treatment effect by including male garlic.

1 is a road related to the effect of lively and black female garlic on the differentiation of 3T3-L1 adipocytes, (A) the effect of URG and UBG (butanol soluble fraction) on the lipid accumulation of 3T3-L1 adipocytes Lipid droplets were stained with Oil Red O solution and photographed at 100-fold magnification (lower right picture, taken with a microscope at 400x magnification, with preadipocytes as negative control, Fully differentiated adipocytes were positive controls, (B) is a diagram related to the absorbance of stained cells measured at 495 nm, and (C) is REG or SEG (ethanol soluble fraction) for lipid accumulation of 3T3-L1 adipocytes It is a diagram regarding the effect of.
Figure 2 is a diagram showing the effect of REG or SEG on the mRNA expression of genes involved in the adiogenesis process, (A) is treated with SEG or REG during the differentiation period, 8 days after differentiation, Total RNA is isolated and the expression of fat-associated genes by RT-PCR, (B) is a diagram for the quantification of gene expression using a quantitation program (image J).
3 is a diagram showing the effect of REG or SEG on the protein expression involved in adiogenesis process, (A) lysing the cells treated with SEG or REG during the differentiation period and extract the total protein, PPARγ and C / Road analysis of protein expression of EBPα was confirmed by Western blot, confirming the equivalent amount loading with β-actin. (B) is a diagram of quantified protein expression.

The present invention provides a composition for preventing or improving obesity, including a male extract garlic.

In the present specification, the male garlic is a large bulbous plant belonging to the genus Liliaceae, and may also be called elephant garlic, and the scientific name refers to a plant of Allium ampeloprasum .

In the present invention, the extract may include one or more solvents selected from the group consisting of butanol, hexane and ethyl alcohol, in that the butanol as a solvent may be excellent in inhibiting the adipocyte differentiation effect It may be more desirable.

In the present invention, the male and female garlic extract may include one or more selected from the group consisting of raw male garlic, black female garlic and black female garlic hot water extract, and fat cell differentiation without toxicity when the male black garlic is included. It may be more preferable in that it can be better in that it can exhibit an anti-obesity effect by suppressing.

The present invention comprises the steps of homogenizing the male garlic to obtain a homogenized male garlic; And

It provides a method for producing Ungung garlic extract comprising the step of dissolving the homogenized Ungung garlic with a solvent to obtain a Ungung garlic extract.

In the present invention, the solvent may be at least one solvent selected from the group consisting of butanol, hexane and ethyl alcohol, butanol may be more preferred.

In the present invention, the male and female garlic may be one or more selected from the group consisting of raw male garlic, black female garlic and black female garlic hot water extract.

In the present invention, the composition described above may be mixed 1: 1 to 1: 100 of the solvent, based on the weight part with respect to the male garlic extract.

In the present invention, the black female garlic may include a step of aging 6 hours to 72 hours at 80 to 90 ℃ after heating for 12 hours to 56 hours at 80 to 90 ℃ using a steamer, prepared through the process One black male garlic is preferable in that it has anti-obesity effect without cytotoxicity. Black horseradish garlic of the present invention was heated at 86 ° C. for 24 hours using a steamer (manufacturer: red ginseng book family, model name: HK-9600), and aged at 86 ° C. for 12 hours using steam.

The composition comprising the male extract of the present invention may be a food composition or a functional food composition as a composition for preventing or improving obesity.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the embodiments according to the present disclosure may be modified in various other forms, and the scope of the present specification is not to be interpreted as being limited to the embodiments described below. The embodiments of the present specification are provided to more fully describe the present specification to those skilled in the art. In addition, "%" and "part" which show content below are a basis of weight unless there is particular notice.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples indicated as preferred examples. The male garlic (Elephant garlic) has shown a significant increase in the total pyruvic acid content in fermented male black garlic compared to normal garlic, and male female garlic has been known to contain scopoletin, which has anti-obesity activity.

Production Example  1. Preparation of solvent-specific samples

In this study, the cultivated and harvested Ungyeom garlic in Gyeongsangbuk-do, Uigun-gun was supported by the Army Technical Technology Center. Three kinds of elephant garlic extracts were prepared. In the case of raw female garlic (REG), the skin was removed, washed, dried and ground homogenized. Black female garlic (SEG) and black female garlic hot water extract (SEGE) were aged for 12 hours after heating with 2 L of distilled water using a steamer. The aged black horse villages were homogenized by distilled water with or without distilled water in a 1: 1 ratio. In this study, they were named as black horse mackerel garlic and black horse mackerel garlic hot water extract. Each homogenized garlic was mixed in 70% hexane, 70% ethyl alchol, and butanol in a 1: 1 ratio (by weight) and then dissolved at 200 rpm for 30 hours using a shaker incubator. The dissolved extract was completely dried using a rotary vacuum concentrator, diluted to a constant concentration using DMSO, and used as a test sample for analysis of anticancer and anti-obesity effects after sterilization with a membrane filter (0.22-um pore size).

Experimental Example  1. Cell Culture

After 3T3-L1 adipocytes were distributed from Korea Cell Line Bank (KCLB), they were cultured in a 37 ° C. incubator supplied with 5% CO ₂ in DMEM medium containing 10% Newborn Calf Serum and 1% antibiotics.

Experimental Example  2. Induction of adipocyte differentiation

 In order to induce differentiation of 3T3-L1 preadipoycytes into mature adipocytes, after dispensing on a 6-well plate, when the cells became 100% confluent, MDI solution (0.5 mM IBMX, 0.25 μM DEX, 167 nM insulin, and 100 μM indomethacin) were replaced with DMEM medium containing 10% FBS. Two days after the induction of differentiation, the medium was replaced with a medium containing insulin of 167 nM insulin. The medium of the cells in culture was replaced with a medium containing fresh insulin every two days. In order to observe the inhibitory effect of adipocyte differentiation of male garlic, REG, SEG or SEGE was treated at a concentration of 2, 4 mg / ml during the differentiation period (total 8 days).

Experimental Example  3. Intracellular Lipid Staining (Oil Red O staining, ORO )

 To identify lipid droplets in the cells of mature 3T3-L1 adipocytes that were differentiated, ORO staining was performed. Differentiated 3T3-L1 cells were fixed with 0.6% Glutaraldehyde at room temperature for 1 hour, washed with 60% isopropyl alcohol and stained with 0.4% ORO soultion for 20 minutes at room temperature. Stained cells were photographed and quantified by measuring the absorbance at 570 nm by dissolving ORO solution with 100% isopropyl alcohol.

Experimental Example  4. Intracellular mRNA  Expression level measurement

  After intracellular total RNA was isolated using RNAiso Plus reagent (TaKaRa Bio, Japan), complimentary DNA (cDNA) synthesis was performed according to the manufacturer's manual using Prime-Script RT reagent Kit (TaKaRa Bio, Japan). . PCR conditions were repeated 30-35 times with 30 seconds (denaturation) at 94 ℃, 30 seconds (annealing) at 60 ℃, 45 seconds (extension) at 72 ℃ and the resulting product was separated by electrophoresis on 1.2% agarose gel And stained with ethidium bromide and visualized.

Experimental Example  5. Measurement of Protein Expression Level in Cells

The cells were dissolved in lysis buffer ((Biosesang, Seongnam, Korea) and left for 30 minutes at 4 ° C, and then the cell solution was centrifuged at 12,000 rpm for 15 minutes.The same amount of protein (30μg) was purified by 10% SDS polyacrylamide gel. The protein in the gel was transferred to Nitrocellulose Membrane (Amersham Protran Premium 0.2 μm NC, GE Healthcare Life Sciences, Germany) and blocked with 5% skim milk for 1 hour, after which the primary antibody was kept at 4 ° C. overnight. After the reaction, the secondary antibody was reacted for 1 hour at room temperature After the reaction, an enhanced chemiluminescence reagent (GEHealthcare, Buckinghamshire, UK) was applied to the membrane and detected using a FUSION Solo apparatus (Vilber Lourmat).

Experimental Example  6. Statistical Processing

The results of the experiments repeated three times indicated the mean and standard deviation, and the statistical significance between groups was analyzed by one-way batch analysis using SPSS Inc. (SPSS Inc.) to determine the significance (* P <0.05, ** P <0.01).

Experimental Example  7. REG , SEG  And SEGE  3T3- L1 fat precursor cells  Impact on Differentiation

Lipid droplet is one of the most characteristic features of Adipogenesis in which the progenitor cells differentiate into mature adipocytes. Lipid droplets are mainly composed of triglyceride (TG), a triglyceride. TG is an important energy source such as glucose, but when excessively ingested, it is absorbed and stored in fat cells, which causes obesity. In this study, we investigated whether REG, SEG and SEGE affect the accumulation of fat droplets accumulated in cells during differentiation of 3T3-L1 adipocytes.

 For the first time, elephant garlic extracts extracted from three different solvents (hexane, 70% ethyl alchol, and butanol) were treated with 3T3-L1 cells at the same concentration. As a result, the three extracts showed the best inhibitory effect of adipocyte differentiation in Butanol fractionation layer (data not shown). Therefore, Butanol fractionation layer was used alone in the lower study.

Next, REG and SEG extracted from the Butanol layer were treated with 2, 4 mg / ml throughout the period of differentiation of cells and ORO staining was performed. As a result of the experiment, it was confirmed that both REG and SEG suppressed fat accumulation of adipocytes in a concentration-dependent manner (see FIGS. 1A and B). As a result of quantitative analysis of intracellular ORO stained adipose cells, REG and SEG treatment at 4 mg / ml concentration inhibited 57.3% and 64.8% adipocyte differentiation, respectively, compared to the control (PC) (see FIG. 1B).

However, when confirming the appearance of undifferentiated cells that are not stained ORO, the cells treated with REG were found to cause cell damage as the cell shape was modified. Therefore, the effect of REG on the accumulation of fat droplets is presumed to be caused by induction of cell death by external stimulation and not by inhibition of fat differentiation process. In addition, SEG and SEGE extracted from ethanol were lower in adipocyte differentiation inhibitory activity than each extract extracted from butanol shown in the above results, and SEGE was found to be lower in differentiation inhibitory activity than SEG (see FIG. 1C).

Based on the above results, we confirmed that the SEG of butanol fractionation layer has the best adipocyte differentiation inhibitory ability among elephant garlic extracts cooked in various ways and extracted from various solvents.

Experimental Example  8. SEG  Of genes involved in fat production mRNA  And influence on protein expression

Peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT / enhancer, major transcriptional regulators involved in adipose formation, to investigate the effects of REG and SEG on the expression of 3T3-L1 adipocyte differentiation factors in adipocyte differentiation. mRNA expression levels of -binding protein alpha (C / EBPalpha), adiponectin (adipocytokine), atypic acid-binding proteins (p2), and low density lipoprotein (LDL), differentiation markers, were compared. Treatment of SEG compared to the control inhibited the expression of PPARgamma, C / EBPalpha, ap2 and LPL (see FIGS. 2A and B). On the other hand, REG did not inhibit or suppress the expression of various adipocyte differentiation-related factors below SEG. Reduction of mRNA expression levels of PPARgamma and C / EBPalpha by SEG and REG showed the same results at the protein level (see FIGS. 3A and B).

 That is, raw male garlic, black female garlic, and black female garlic hot water extract have anti-obesity effects, and in particular, black female garlic can have an in vitro anti-obesity effect by effectively inhibiting the differentiation of 3T3-L1 adipocytes. Confirmed. Therefore, when the ungna garlic extract provided by the present invention, it can be utilized as health functional foods and medicines having a weight control function.

However, as in black female garlic, live female garlic had a fat cell differentiation inhibitory effect, but this may be due to cytotoxic effect. Thus, this toxic effect is caused by tissue damage in vivo when ingested raw female garlic for weight control purposes. And normal cell destruction may be accompanied by side effects, but it was confirmed that the effect of improving the prevention of obesity is excellent.

The above description is merely illustrative of the present invention, and those skilled in the art to which the present invention pertains may various modifications without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed herein are not intended to limit the present invention but to describe the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The scope of protection of the present invention should be construed by the claims, and all descriptions within the equivalent scope should be construed as being included in the scope of the present invention.

Claims (10)

delete delete delete delete Homogenizing the male garlic to obtain a homogenized male garlic; And
Comprising the homogenized Ungung garlic with a solvent in a 1: 1 weight ratio to dissolve to obtain a Ungung garlic extract,
The male garlic is black female garlic, the solvent is butanol,
Obtaining the homogenized male garlic,
Heating the male garlic for 12 to 56 hours at 80 to 90 ° C., and then for 6 to 72 hours at 80 to 90 ° C .; And
Homogenizing the aged male garlic; including,
64.8% inhibition of adipocyte differentiation, obesity prevention or improvement for manung garlic extract manufacturing method. delete delete delete delete delete

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