KR101754149B1 - Anti-Obesity Food Composition Containing Solidago Virgaurea Extract and Method for Preparing the Same - Google Patents

Abstract

The present invention relates to an anti-obesity food composition containing an undiluted extract and a preparation method thereof. More specifically, the present invention relates to a food composition containing an undiluted extract or fraction having an excellent anti-obesity effect and a method for producing the same.

Description

[0001] The present invention relates to an anti-obesity food composition containing an extract of anchovy,

The present invention relates to an anti-obesity food composition containing an undiluted extract and a preparation method thereof. More specifically, the present invention relates to a food composition containing an undiluted extract or fraction having an excellent anti-obesity effect and a method for producing the same.

Recently, with the recent economic development and the high growth of science and technology, advanced age-type aging and adult diseases are increasing rapidly. The cause of this is the westernization of the stress and dietary habits of modern people, excessive nutrition and decreased physical activity, and the death rate of heart and cerebrovascular disease, which is a complication accompanied by actual obesity, is 1 or 2, Has been suggested as a cause of various adult diseases.

As the metabolic syndrome accompanying obesity is concentrated on interest, adipose tissue is being studied not only as a fat reservoir but also as an endocrine organ that regulates metabolism. Adipokines such as adiponectin, leptin, resistin, adipicin, and visfatin, which are produced and secreted from adipose tissue, have been found in many studies.

Currently, the domestic obesity treatment market is estimated to be over 80 billion won, which is growing by about 10% every year. Anti-obesity Functional food products have been scientifically recognized for their functionality. Pyrus Pusan and CLA (Conjugated Linolic Acid) are found in the market, and CLA induces the suicide mechanism of fat cells to reduce the number of fat cells. CLA's patent rights are sold in the market in foreign countries, and the products are produced and sold by paying royalties to foreign countries.

At one time, the advent of anti-obesity drugs, such as Xenical and Reductil, led to the formation of a 50 billion-dollar market, but sales were banned due to cardiovascular disease, damage to the large intestine mucosa and side effects.

Pancreatic lipase acts as a key enzyme that breaks down triglyceride into 2-monoacyl glycerol and fatty acids. Representative pancreatic lipase inhibitors include Streptomyces is effective as a tetrahydrolipstatin derivative of lipstatin derived from toxitricini to inhibit about 30% of the fat ingested. Although it is currently on the market as a pharmaceutical product, it can not be used for the treatment of gastrointestinal disorders, hypersensitivity, There are side effects.

Accordingly, there is an increasing demand for the development of an anti-obesity medicament or a food composition which is safe even with long-term use and has no side effects, yet has the effect of preventing obesity or reducing body fat.

Solidago virgaurea subsp . asiatica Kitam. ex Hara) is a chrysanthemum which is sometimes called "jiwanghwa", "kwangwon", "jinhwanghwa", "dumemi", and pigs. Origin is Korea and Japan. The young leaves are edible as herbs, and are used in the private sector as dry foods, diuretics, and so on.

It is known that Ulleungdo and Jeonnam Goheung produce most of the domestic distribution. Among them, Ulleungdo is known to be cultivated much more than Keunheung in Jeonnam with annual average of 200M / T from 70ha area (Source: Technology Center, Goheung County Agricultural Technology Center). In the case of Gohung, Jeollanam-do, it is cultivated and distributed along with anchovy and ginger in the insects.

In the present study, the effects of anti-melanogenesis inhibition, antiinflammatory effect, and osteoblast activation were investigated. However, the anti-obesity-related effects Have not been reported.

The researchers are natural Solidago Virgaurea (Solidago virgaurea subsp . asiatica Kitam. ex Hara extract extracts and fractions were found to be effective for the prevention of obesity or body fat reduction and to establish the optimum conditions for the unstable extraction of the active ingredients of the undiluted extracts based on the results of anti-obesity activity. It was completed.

It is an object of the present invention to provide an anti-obesity food composition containing an undiluted extract and a process for producing the same.

It is another object of the present invention to provide a food composition containing fractions of an undiluted extract and a process for their preparation.

It is still another object of the present invention to provide a food composition containing fractions of an undiluted extract having an excellent anti-obesity or body fat reducing effect and a process for producing the same.

In one embodiment of the present invention for achieving the above object, there is provided an anti-obesity functional food composition containing an unstable extract or fraction as an active ingredient.

In one embodiment of the present invention, there is provided a process for preparing an anti-obesity functional food composition comprising grinding undesired spots, extracting the extract, and concentrating the extract at reduced pressure to obtain a concentrate do.

In one embodiment of the present invention, there is provided a method for producing a microorganism, which comprises the steps of crushing undescended eggs including outposts and flowers, leaching the pulverized material with an organic solvent, filtering and drying the sample, There is provided a method for preparing an anti-obesity functional food composition comprising re-leaching, filtering and drying the sample, leaching with water, and filtering.

In one embodiment of the present invention, there is provided a method for preparing an anti-obesity functional food composition comprising fractionating an undiluted extract extracted with an organic solvent using an organic solvent.

Hereinafter, the present invention will be described in detail.

The present invention provides an anti-obesity functional food composition containing an undiluted extract or fraction as an active ingredient.

In the present invention, the term "extract" means an active ingredient isolated from a natural product.

The anti-obesity effect of the composition of the present invention can be confirmed by inhibiting the differentiation of 3T3-L1 cells for verifying fat accumulation inhibitory function.

In one embodiment of the present invention, the method for producing the extract may be a conventional extraction method such as an ultrasonic extraction method, a filtration method, and a reflux extraction method. Preferably, the extract may be an extract obtained by extracting the undesired substance from which the foreign substance is removed by washing and drying with water, C _1-4 alcohol, methylene chloride, ethyl acetate, hexane or a mixed solvent thereof. It may be an extract.

The extraction solvent may be used in an amount of 2 to 50 times, preferably 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 72 hours for leaching in the extraction solvent, preferably for 24 to 48 hours.

After extraction, the extract can be fractionated by sequentially applying a fresh fraction solvent. The fraction solvent used for fractionation is water, C _1-4 alcohol, methylene chloride, ethyl acetate, or hexane, and ethyl acetate and butanol are preferred.

After the extract or fraction is obtained, a method such as concentration or freeze-drying can be additionally used.

In one embodiment of the invention, the food composition further comprises additives selected from the group consisting of flavors, flavors, colorants, fillers, stabilizers, natural carbohydrates, nutrients, vitamins, thickeners, pH adjusting agents, preservatives and mixtures thereof As shown in FIG.

In one embodiment of the present invention, the functional food composition of the present invention can be manufactured by common formulations such as tablets, pills, granules, powders, liquids, hard capsules, soft capsules, etc., , A cookie, a tea, a drink, a vitamin complex, a meat, a sausage, a candy, a cotton, a jelly and the like.

To prepare the various formulations or forms as described above, a pharmaceutically acceptable carrier or excipient such as the excipients described above can be used, and it is known to those skilled in the art that the formulation Any carrier or additive can be used.

According to the present invention, there is provided a food composition containing an unstable extract or fraction having an excellent obesity-preventing or fat-reducing effect and a method for producing the same.

Figure 1 shows the HPLC results of 70% EtOH extract.
Figure 2 shows the HPLC results of the EtOAc and n-BuOH fractions fractionated from 70% EtOH extract.
Fig. 3 is a chart comparing efficacy confirmed after non-averages fractionation.
Figure 4 is a graph showing the cytotoxicity assay (MTT) of the undersaturated n-BuOH fraction.
FIG. 5 is a graph showing changes in body weight of a C57BL / 6 mouse group administered with a high-fat diet and at the same time with an undescended extract for 8 weeks.
FIG. 6 is a graph showing changes in body weight of a C57BL / 6 mouse group administered with a high-fat diet and at the same time with an undiluted extract for 8 weeks.
FIG. 7 is a graph showing the blood index for a group of rats after a high fat diet was administered to a C57BL / 6 mouse group and at the same time, an undiluted extract was administered for 8 weeks.
FIG. 8 is a graph showing the weight change of major organs for the mouse group after administering a high fat diet to the C57BL / 6 mouse group and at the same time administering the undiluted extract for 8 weeks.
FIG. 9 is a graph showing the results of western blot for the verification of fat accumulation inhibitory function in vivo and the results thereof.
FIG. 10 shows the result of treatment with an undiluted extract after 3T3-L1 cells were exchanged with a culture solution, followed by staining with oil red O. FIG.
Fig. 11 shows the results of the expression of PPAR-r and C / EBP-a, which are known as differentiation mechanisms in 3T3-L1 cells.
Fig. 12 shows the results of expression of ACC known as a target gene of AMPK and AMPK, which are known as differentiation mechanisms in 3T3-L1 cells.
FIG. 13 shows the results of the expression of FAS known as the differentiation mechanism in 3T3-L1 cells and FABP5 (aP2) used as a differentiation marker.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

[Example 1]

Preparation of undrawn fraction

Production Example 1: Extraction method

Seedlings containing crushed flowers and flowers were crushed and leached at room temperature for 48 hours using dichloromethane. The samples were filtered and then dried. The dried sample was leached at 70% EtOH at room temperature for 48 hours. The sample was filtered, dried, and then leached at room temperature for 48 hours using water, followed by filtration and concentration.

Production Example 2: Fractionation method

Fifteen fractions were dissolved in 500 ml of DW by dissolving 15 g of the unstained extract of 70% EtOH in EtOAC. The separated water layer was fractionated three times using n-BuOH and the obtained fractions were concentrated to obtain fractions.

[Example 2]

Evaluation of fat accumulation inhibitory function

Experimental Example 1: Inhibition of differentiation of 3T3-L1 cells for inhibiting lipid accumulation

3T3-L1 preadipocyte cells are purchased from the American Type Culture Collection (ATCC). Cell culture was performed in Dulbecco's modified Eagle's medium (DMEM) medium (Gibco) containing 10% fetal bovine serum (FBS), penicillin (100 U / ml) and streptomycin And cultured in CO ₂ incubator at 37, 5% CO ₂ . 3T3-L1 cells were seeded on a 24-well plate and the cells were post-confluent. The cells were treated with 0.5 mM dexamethasone, 10 g / ml insulin, ) And treated with 0.5 mM IBMX. After 4 days, medium was replaced with insulin (5 g / ml), and after 2 days, the medium was changed into normal medium to induce differentiation into adipocytes. Samples were treated at adipocyte differentiation. To confirm the inhibition of adipogenesis of each reagent, the lipid differentiation medium and each of the reagents were treated at the same time. After 8 days, the media of the differentiated cells were discarded and washed in 1X PBS for 3 times And fixed in 10% formalin reagent. After 1 hour, dye is stained with 0.6% Oil Red O reagent and the relative OD value of TG is measured again. (Final concentration of all samples is 10 ug / ml)

As a result of the evaluation, as shown in FIG. 3, the anti-obesity activity test for the EtOAC and n-BuOH fractions obtained by solvent fractionation of the undiluted 70% EtOH extract showed that the n-BuOH fraction contained an effective substance .

In addition, as shown in FIG. 4, the fractions did not show toxicity even at 10 ug / ml, which is an effective effect showing efficacy, and 50 ug / ml, which corresponds to 5 times of the effective efficacy.

Experimental Example 2: Evaluation of anti-obesity efficacy by animal experiment (weight change, blood index and major organ weight change)

Seventy (70) five-week-old C57BL / 6 mice were purchased from BioLink and purified for 1 week. After that, we checked the weight every week. 6-week-old rats were randomly divided into 5 groups (12 rats) and 1 group (10 rats). The normal control group (10 rats) was fed a normal diet and the remaining 5 groups (60 rats) Weekly. One group of the groups that received high fat diets did not receive anything other than high fat diets (control group). Three groups out of the other four groups determined the respective sample concentration of the off-flavored extract (the effective concentration conferred in the previous experiment) and used daily (8 weeks * 7 days = 56 days) Lt; / RTI > The other group administered 500 gm / kg of garnia.

After 8 weeks of treatment, the mice were anesthetized with cardiac hemorrhage and liver, pancreas, spleen, heart, muscle and adipose tissue were collected. Lipid metabolism enzyme activity and factors in liver, pancreas, spleen, heart, muscle, adipose tissue and blood were analyzed and related biomarkers such as plasma lipid and cholesterol were analyzed in the blood.

45% of high fat diets were used instead of 60% high fat diets. The purpose of this study was to investigate the effect of drugs containing anhydrous extracts on obesity in obese subjects.

According to the evaluation results of the weight change of the rats shown in FIG. 5 and FIG. 6, there was no significant difference compared to the normal control group until the 2nd to 3rd weeks, but from the 4th week onwards, compared with the normal control group, (S100 mg / kg, S500 mg / kg and S1000 mg / kg), and the amount of change was also decreased. Compared with the group administered with Garcinia (G500mg / kg), which is known to be effective for anti-obesity, it was confirmed that the weight of the rats in the group treated with anastrozole extract was low and the rate of change of body weight was small from the third week onwards.

(S100 mg / kg, S500 mg / kg and S1000 mg / kg) compared to the control group in the results of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) The liver is not toxic. BUN (blood urea nitrogen) and CREA (creatinine) were also confirmed to be free of renal toxicity. Results for HDL-cholesterol and LDL-cholesterol, although not showing elevated HDL-cholesterol. LDL-cholesterol concentration-dependently. As a result of TG (triglyceride), the value of the group (S1000 mg / kg) administered with an averaged extract was significantly lower than that of the control (Con-Fat).

According to Fig. 8 and the following Table 1, which is the result of the evaluation of the weight change of the major organs of rats,

Heart liver Jira kidney Fat Normal control group 2.04 15.9 0.94 3.09 2.59 Control group 2.14 15.1 1.01 3.16 4.53 Garcinia 2.28 14.4 1.05 3.22 3.75 100 wrestling 2.04 14.2 0.98 3.13 3.39 Unstructured 500 2.12 13.5 1.02 3.00 2.71 Unsteady 1000 2.18 13.6 1.059 3.05 2.57

As shown in Table 1 and Fig. 8, the weight of fats and fats in both groups decreased compared to the control group. However, the weight of the fat was decreased in a dose - dependent manner in the group treated with the untreated group than the group of the Gardinian.

Experimental Example 3: Western assay for inhibiting lipid accumulation in vivo Bloom Lot (western blot)

Proteins were homogenized using adipose tissue and then centrifuged at 13500 rpm for 15 minutes at 4 ° C. The supernatant was taken and the supernatant protein was quantified. Proteins were quantitated using Bio-Rad protein quantification reagent (Bio-Rad, Hercules, CA, USA) and analyzed using Laemmli sample buffer (Bio-Rad) and mercaptomethanol ) Were mixed to prepare a sample. The protein sample thus quantified was separated by electrophoresis using SDS-polyacrylamide gel, and the acrylamide gel was transferred to a nitrocellulose membrane. TBS-T containing 5% skin milk was dipped, blocked at room temperature for 1 hour, and washed 3 times with TBS-T every 5 minutes. The prepared membrane was treated with primary antibody and incubated at room temperature for 2 hours or overnight at 4 ° C and then washed 3 times with TBS-T every 3 minutes. After ECL solution was applied, protein expression was analyzed by immersing in BioMax MR film in the dark room.

According to the evaluation result of FIG. 9, the fat-related biomarkers in the fat tissue were significantly decreased in the biomarkers related to lipogenesis, and the protein expression of the CREB, which is an upper regulatory factor, was decreased in the undisturbed group.

Experimental Example 4: Western assay for inhibiting fat accumulation in vitro Western blot

The 3T3-L1 preadipocyte cells were unstained ( Solidago virga I have aurea . gigantea) were treated and cultured. The treated cells were washed with PBS, treated with trypsin, allowed to float, and centrifuged to collect the cells. Lysis buffer was added to the obtained cells, and the cells were scraped and centrifuged at 13500 rpm for 15 minutes at 4 ° C. The supernatant was taken to quantify the proteins in the supernatant. Proteins were quantitated using Bio-Rad protein quantification reagent (Bio-Rad, Hercules, CA, USA) and analyzed using Laemmli sample buffer (Bio-Rad) and mercaptomethanol ) Were mixed to prepare a sample. The thus-quantified protein sample was separated by electrophoresis using SDS-polyacrylamide gel, and the acrylamide gel was transferred to a nitrocellulose membrane. After immersing in TBS-T containing 5% skin milk, the cells were blocked for 1 hour at room temperature and then washed three times every 5 minutes with TBS-T. The prepared membrane was treated with a primary antibody After incubation at room temperature for 2 hours or overnight at 4 ° C, TBS-T was washed three times per minute. After ECL solution was applied, protein expression was analyzed by immersing in BioMax MR film in the dark room.

According to the evaluation result of FIG. 10, 3T3-L1 cells were treated with an undifferentiated extract after exchange with a differentiation culture medium and then stained with oil red O, and the control and the group treated with Garcinia were stained However, it was confirmed that the reddish-stained part was hardly found in the group treated with the anthocyanin extract, and the quantitative graph also confirmed that the effect of the group treated with the anthocyanin extract was excellent.

11, the expression of PPAR-r and C / EBP-a, known as the differentiation mechanism in 3T3-L1 cells, was reduced by the undrained extract, and according to Fig. 12, The expression of ACC, known as the target gene of AMPK and AMPK, known as the mechanism, was reduced due to an undesired extract. According to FIG. 13, FAS known as the differentiation mechanism in 3T3-L1 cells and FABP4 aP2) was decreased due to the anhydrous extract.

Claims (9)

1) crushing the underside;
2) an extraction step of extracting the pulverized product;
3) dissolving the extract of step 2) in ultrapure water (DW), and then fractionating the extract using EtOAC;
4) separating only the water layer in the step of fractionating in step 3), and performing fractionation using n-BuOH; And
5) A method for producing an anti-obesity functional food composition comprising an n-BuOH fraction as an active ingredient, which comprises concentrating the fraction of step 4) under reduced pressure to obtain a concentrate.
The method according to claim 1,
The method for producing an anti-obesity functional food composition according to the above 1), wherein the n-BuOH fraction is an effective ingredient.
The method according to claim 1,
The step 2) comprises: 2-1) leaching the pulverized material using an organic solvent;
2-2) filtering and drying the sample;
2-3) re-leaching the dried sample with an organic solvent;
2-4) filtering and drying the sample;
2-5) leaching with water; And
2-6) A process for producing an anti-obesity functional food composition comprising an undegraded n-BuOH fraction containing an effective component.
The method according to claim 1,
Wherein the step 2) is carried out by one or more extraction methods selected from the group consisting of ultrasonic extraction, filtration and reflux extraction, and the n-BuOH fraction as an active ingredient.
A functional food composition for an anti-obesity food comprising an n-BuOH fraction prepared by the production method according to claim 1 as an active ingredient.
6. The method of claim 5,
Wherein the functional food composition for an anti-obesity further comprises an additive selected from the group consisting of flavors, flavors, colorants, fillers, stabilizers, natural carbohydrates, nutrients, vitamins, thickeners, pH adjusters, preservatives and mixtures thereof ≪ / RTI >
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