WO2017086536A1 - Anti-obesity composition containing pheophorbide a separated from gelidium amansii extract as active component, and method for preparing same - Google Patents

Abstract

The present invention relates to an anti-obesity composition containing, as an active component, Pheophorbide A separated from Gelidium amansii extract. Pheophorbide A separated from Gelidium amansii extract according to the present invention has an excellent lipid storage suppression effect, thereby exhibiting an anti-obesity effect, and thus may be utilized in anti-obesity foods, cosmetics, or pharmaceuticals.

Description

Anti-obesity composition containing pheophobide A isolated from the extract of ummu fern as an active ingredient and method for preparing the same

The present invention relates to a composition for anti-obesity containing the pheophorbide A (Pheophorbide A) isolated from the extract of the wormwood as an active ingredient and a method for producing the same.

Obesity is a term used to refer to the overweight of a person. Pathologically refers to a case in which the so-called body weight index (BMI) of the World Health Organization (WHO) is 30 or more. In Korea, over 25 diagnoses obesity. In most cases, it means that your weight is higher than normal, but even if you don't weigh too much, your body's constituents are high in fat and are said to be obese. Obesity may not be an issue on its own, but the social complications caused and the secondary complications caused by excess fat are severe. For example, obesity can significantly increase the incidence of hyperlipidemia, hypertension, arteriosclerosis, diabetes, fatty liver, joint abnormalities, and the like.

Currently, dietary therapy, exercise therapy, behavioral therapy, etc. are used to correct lifestyle, drug treatment, and surgical treatment. However, diet pills such as appetite suppressants and fat digestion inhibitors are used for cardiovascular system such as hypertension. It can cause side effects such as disease and stroke, and if you stop taking it, you will often gain weight again. In addition, in the case of severe obesity, surgery can be performed, such as surgery to remove fat or to limit the amount of food that the body can digest satellite or gastric band insertion is performed.

Recently, however, with the recognition of the importance of vegetable compounds, which are useful ingredients in plants, and the finding of materials that can be used as anti-obesity functional compositions, benefiting the general health by using natural products derived from natural products, rather than synthetic drugs that may cause side effects. Researches on their mechanism of action are being actively conducted.

Conventional patents that implement the anti-obesity effect by using a natural product as a substitute for synthetic drugs as described above, Republic of Korea Patent Publication No. 10-1369060 'Anti-obesity composition containing acacia bark derivative', Republic of Korea Patent Korean Patent Publication No. 10-1482718 'Anti-obesity composition containing red ginseng by-product as an active ingredient and its preparation method', Republic of Korea Patent Publication No. 10-1445966 'Anti-obesity composition comprising Baekdu-gu extract', Republic of Korea Patent Publication No. 10 -0783204 'anti-obesity or anti-hyperlipidemic composition comprising the Nuruk extract' and the like.

On the other hand, Gelidium amansii is a perennial seaweed of the algae belonging to the Umugasariaceae. After the summer breeding season, the upper part of the main body melts away and only the lower part remains, and sprouts grow again in the following spring. Distributed on the east coast, the south coast and the outlying islands of the Yellow Sea, it is most produced on the south coast of the East Sea. It grows attached to the rock 20 ~ 30m deep in the sea. It faces the outer sea and its bottom is sandy, and it lives in a place where seawater communicates well. Wash the umami fern with fresh water, and dry it in the sun to filter the residue and cool it. This daikon has been used since ancient times as a soft drink. There are three main bodies in the Umu-sagari, namely the male and female spores, the male and female spores. These are very similar in appearance, and cannot be distinguished from each other without examining reproductive organs or nuclear images. There are three generations of male and female spouse generation, small spore spore generation and quadrant spore generation, which live in the three generations.

The umaga saga is a representative seaweed that has been widely used as food additives, cosmetics, medicines, livestock feed and industrial raw materials for a long time, and is the main raw material of agar. However, only less than 10% of the total production of agar, simply processed and used as a cheap raw material is a large amount of neglected situation. Therefore, it is a very important task to try to improve the added value through the development of new use of agar, which is mass-produced on the coast of Korea.

Therefore, the inventors of the present invention, as a result of searching for a natural product with excellent anti-obesity effect in marine biological material, it was confirmed that the pheophorbide A isolated from the extract of the wormwood has excellent anti-obesity effect, The invention was completed.

The main object of the present invention is to provide a composition for anti-obesity that can exhibit an excellent anti-obesity effect by containing as a active ingredient, Pheophorbide A (Pheophorbide A) isolated from the extract.

It is another object of the present invention to provide a method for separating Pheophorbide A from cow radish with high yield.

In order to achieve the above object, the present invention provides a composition for anti-obesity containing Pheophorbide A (Pheophorbide A), which is isolated from the extract of the wormwood.

In a preferred embodiment of the present invention, the pheophorbide A is a high performance liquid chromatography using a mixture of water and trifluoroacetic acid as the first solvent and acetonitrile as the second solvent It is characterized by the compound isolate | separated and obtained by HPLC).

In a preferred embodiment of the invention, the pheophorbide A (Pheophorbide A) is characterized by having a lipid accumulation inhibitory effect.

In a preferred embodiment of the present invention, the pheophorbide A is characterized in that it comprises 5 to 15% by weight based on the total weight of the anti-obesity composition.

In another embodiment, the present invention provides a food comprising such an anti-obesity composition.

In another embodiment, the present invention provides a cosmetic comprising the composition for anti-obesity as described above.

In another embodiment, the present invention provides a medicament comprising such an anti-obesity composition.

The present invention, in another embodiment, the step of extracting a radish of a radish with a solvent to prepare a extract of radish; And separating the pheophorbide A by high performance liquid chromatography (HPLC) using the mixed solution of water and trifluoroacetic acid as the first solvent and acetonitrile as the second solvent as the mobile phase. It provides a method for producing an anti-obesity composition, characterized in that.

In a preferred embodiment of the present invention, the solvent is characterized in that at least one member selected from the group consisting of alcohol, methanol, chloroform and purified water.

In a preferred embodiment of the present invention, the mixture of water and trifluoroacetic acid is characterized in that consisting of 99.9% water (v / v) and 0.1% (v / v) trifluoroacetic acid.

In a preferred embodiment of the present invention, the first solvent and the second solvent is characterized in that the step gradient (100: 0, 50:50, 20:80, 0: 100).

In a preferred embodiment of the invention, the mobile phase is characterized in that acetonitrile.

Pheophorbide A (Pheophorbide A) isolated from the extract of Gelidium amansii according to the present invention is excellent in lipid accumulation inhibitory effect, can be usefully used as an active ingredient of the composition for anti-obesity, anti-obesity food, Application as a cosmetic or a medicine is possible.

1 is a photograph of the ummu saga used as a raw material of the present invention.

Figure 2 is a manufacturing process of the extract of the radish of the present invention.

Figure 3 is a graph showing the cytotoxicity results of the extract of radish.

Figure 4 is a photograph showing the lipid accumulation inhibitory effect of the extract.

Figure 5 is a chromatogram showing the results of HPLC analysis by wavelength (230 nm, 400 nm) for the extract of radish.

FIG. 6 is a chromatogram showing the results of HPLC analysis for Pheophorbide A isolated from umaga.

Figure 7 is a data showing the cytotoxicity results for the pheophorbide A (Pheophorbide A) isolated from the woogami.

FIG. 8 is a photograph showing the effect of inhibiting lipid accumulation on Pheophorbide A isolated from the larvae.

FIG. 9 is a UV-Spectrum graph for Pheophorbide A isolated from umaga.

FIG. 10 is a graph showing the results of HR / Mass analysis of pheophorbide A isolated from umaga.

FIG. 11 is a graph showing ¹ H NMR spectral data of Pheophorbide A separated from umaga.

FIG. 12 is a graph showing ¹³ C NMR spectral data of Pheophorbide A isolated from umaga.

FIG. 13 is a graph showing COSY spectral data of Pheophorbide A separated from umaga.

FIG. 14 is a graph showing HMBC spectral data of Pheophorbide A isolated from umaga.

FIG. 15 is a graph showing HSQC-DEPT spectral data of Pheophorbide A isolated from umaga.

FIG. 16 shows the structure of Pheophorbide A isolated from umaga.

In the present invention, we tried to confirm that the extract of the wormwood and pheophorbide A isolated from it has an anti-obesity effect.

In the present invention, after extracting the wormwood with solvent, it was confirmed that there is a lipid accumulation inhibitory effect, and separated using high performance liquid chromatography (HPLC), the anti-obesity active ingredient is a pheophorbide A (Pheophorbide A) I could confirm it.

Therefore, in one aspect, the present invention relates to a composition for anti-obesity containing Pheophorbide A isolated from Gelidium amansii as an active ingredient.

The pheophorbide A (Pheophorbide A) is a compound which is separated and obtained by a high performance liquid chromatography (HPLC) using a mixed solution of water and trifluoroacetic acid as a first solvent and acetonitrile as a second solvent as a mobile phase It is characterized by.

Pheophorbide A (Pheophorbide A) isolated from the wormwood ( Gelidium amansii ) as described above has an anti-obesity effect due to its excellent lipid storage inhibitory effect.

According to a preferred embodiment of the present invention, the pheophorbide A may include 5 to 15% by weight based on the total weight of the anti-obesity composition. If the pheophorbide A compound is less than 5% by weight based on the total weight of the anti-obesity composition, since the anti-obesity effect cannot be sufficiently obtained, the function as the anti-obesity composition is lowered and exceeds 15%. It is uneconomical because a sufficient anti-obesity effect can be achieved without adding more content.

In another aspect, the present invention may provide food, cosmetics, and medicines including the anti-obesity composition.

In another aspect, the present invention comprises the steps of extracting the radish with a solvent to prepare a radish extract; And separating the pheophorbide A by high performance liquid chromatography (HPLC) using the mixed solution of water and trifluoroacetic acid as the first solvent and acetonitrile as the second solvent as the mobile phase. It relates to a method for producing an anti-obesity composition, characterized in that.

According to a preferred embodiment of the present invention, the radish extract is extracted with a solvent and then centrifuged and filtered to obtain a supernatant; And an extract prepared by concentrating the supernatant and then lyophilizing.

More detailed preparation method of the extract of daikon radish is as follows.

First, as shown in FIG. 1, the native radish larvae native to Jeju Island are chilled for 30 to 5 hours using a solvent. At this time, the solvent for the cold immersion treatment may be ethyl acetate, distilled water, methanol, etc. Among them, distilled water is preferable in terms of economics and safety. After immersed in the same manner as described above using the same solvent to clean one to five times and dried for 12 to 36 hours. At this time, the drying method is not particularly limited, but drying in a shaded place using a fan is preferable in terms of economy and convenience.

Next, the dried daikon fertilizer raw material is pulverized using various grinding methods such as a carter mill, and then the crushed daikon fertilizer is put into an extractor, and the solvent is injected into a tank to perform solvent extraction. In this case, the solvent may be one or more selected from the group consisting of alcohol, methanol, chloroform and purified water, but it is preferable to use 75-85% alcohol alcohol among them in terms of safety and economics. In addition, it is preferable to extract for 30 minutes to 24 hours at 60 to 120 ℃ during extraction in terms of improving the yield of the extract as much as possible.

When the above-described solvent extraction is completed, centrifugation for 5 to 30 minutes at 3,000 to 15,000 rpm, filtered using a paper filter paper of 15 to 25 ㎛, injected into the concentrator and then compared to the total volume at 40 to 80 ℃ 1 Concentrate to / 15 to 1/25 to give a concentrate. The concentrate can be lyophilized for long term storage.

According to a preferred embodiment of the present invention, the pheophorbide A is a high performance liquid chromatograph using the obtained extract of ummu fern as a mobile phase using a mixture of water and trifluoroacetic acid as a first solvent and acetonitrile as a second solvent. It can be separated by chromatography (HPLC).

The mixed solution of water and trifluoroacetic acid is preferably 99.9% (v / v) of water and 0.1% (v / v) of trifluoroacetic acid in terms of being able to separate pheophorbide A in high yield. Do.

In this case, the mobile phase may be separated using acetonitrile, more preferably, the first solvent and the second solvent are separated by a step gradient (100: 0, 50:50, 20:80, 0: 100). It is preferable in that it can be separated into high purity.

Daikon radish extract obtained from the manufacturing method as described above may have an anti-obesity effect due to the excellent lipid storage inhibitory effect.

As described above, the present invention is to provide a composition for implementing the anti-obesity effect based on the ummu fern, which is a marine biomaterial replacing the synthetic medicine, the natural origin in a situation where the safety of raw materials is emerging The use of natural materials can be useful as anti-obesity foods, cosmetics and medicines because it can ensure economic and safety and exhibit an excellent anti-obesity effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

1. Manufacture of Daikon Ferns Extract and Establish Optimal Production Process

1-1. Preparation and Yield of Daikon Radish Extract

The 300 kg of Umbrella purchased from Suhyup Jeju Island was washed three times with 100 kg each, followed by extraction, concentration and lyophilization.

First, 100 kg of umami fern samples were added to a two-ton washer, and 10 times purified water was added to the sample volume, followed by chilling for 1 hour. After that, three times of washing were performed in the same manner. Cold air dried. The cold-dried sample was pulverized using a Carter mill, and then 100 kg of the powder sample was added to a non-woven fabric in a two-ton extractor, followed by injecting one ton of 80% alcohol alcohol into a tank and extracting it at 70 ° C. for 12 hours. The extracted extract was separated for 20 minutes at 10,000 rpm using a continuous centrifuge, injected into the concentrator through a filtration process using a 20 μm paper filter paper, and concentrated to 1/20 of the total volume at 60 ° C., and frozen. It was dried, and repeated three times to prepare the final radish extract (Fig. 2).

As a result of repeated extraction three times under the same conditions as described above, 8.91 kg of the extract in the first extraction, 9.37 kg in the second extraction, 9.34 kg in the third extraction, 9.9 kg per 100 kg of the U.S. agar sample The dried extract was recovered. Therefore, the average yield of radish extract was found to be 9.2 ± 0.25%.

1-2. Component Analysis of Daikon Radish Extract

The contents of salinity, protein, total sugar, reducing sugar, total phenol and total flavonoid content, etc. were analyzed. For the analysis of the ingredients, the extract was used for analysis after the preparation to the concentration of 10 mg / ml in distilled water.

The salt content of the daikon radish extract was measured using an ES Meter machine, and the salt content of the daikon radish extract was calculated in terms of NaCl.

Protein content analysis was performed using the Bradford method. 0.5 ml of protein assay dye was added to 0.1 ml of radish fern extract, and the absorbance was measured at 595 nm. The protein concentration was calculated based on BSA.

Total sugar analysis was performed using Phenol-sulfuric acid method. 0.2 ml of 5% phenol was added to 0.2 ml of the sample solution, followed by stirring. 1 ml of sulfuric acid solution was slowly added dropwise to the reaction mixture, mixed and reacted at room temperature for 20 minutes, and the absorbance was measured at 490 nm. The calibration curve was calculated using) -glucose and the total equivalent weight was calculated.

The reduction stage was analyzed using the DNS method. 0.5 ml of the DNS solution was added to 0.5 ml of the extract, and boiled for 5 minutes, and then cooled by adding 5 ml of distilled water. After that, the absorbance was measured at 546 nm, and a reduction curve was calculated after preparing a calibration curve using D (+)-glucose as a reference.

Total flavonoid analysis was performed using Flavonoid-AlCl ³⁺ method. The yellow color produced by the reaction between flavonoids and AlCl ³⁺ was added to 0.5 ml of extract, 0.5 ml of 2% AlCl ³⁺ , reacted at room temperature for 1 hour, and the absorbance was measured at 500 nm. acid was used.

Total polyphenol analysis was performed using the Dewanto method. 0.2 ml of distilled water and 0.2 ml of Folin-ciocalteu's phenol reagent were added to 0.1 ml of the sample, followed by reaction at room temperature for 3 minutes, and 2 ml of 20% NaCO ₃ for 1 hour at room temperature, and then absorbance at 765 nm. Gallic acid was used.

The results of the component analysis of the extract of radish are shown in Table 1 below.

Table 1

division	Water content (%)	Salinity (%)	% Per total	Reducing Sugar (%)
Daikon Fermented Extract	4.13 ± 0.1	0.07 ± 0.01	13.1 ± 0.2	11.7 ± 0.2
	Total protein (%)	Polyphenol (%)	Flavonoids (%)	Other (%)
	6.6 ± 0.1	2.6 ± 0.8	15.5 ± 1.6	46.3 ± 0.05

Table 1 shows 13.1 ± 0.2% of total sugar, 11.7 ± 0.2% of reducing sugar, 6.6 ± 0.1% of total protein, 2.6 ± 0.8% of polyphenol, and 15.5 ± 1.6% of flavonoids.

The higher the content of the polyphenols can be used as a functional material with excellent physiological efficacy. Flavonoids are a pigment compound mainly distributed in plants and present in glycosides in combination with various sugars, and are known to have antimicrobial, anticancer, antiviral and anti-inflammatory effects, and show little toxicity. In particular, it is known to lower the content of LDL cholesterol in the blood to prevent various blood lipid-related diseases and to suppress obesity by weight loss.

As such, the extracts of daikon radish contain a large amount of flavonoids and polyphenols in addition to sugars and proteins. It was judged that.

2. Evaluation of Anti-obesity Activity of Daikon Radish Extract

2-1. Cytotoxicity Assessment (MTT assay)

In order to confirm the cytotoxicity of the extract prepared from 1-1, MTT assay was performed using 3T3L-1, 3T3-F442A and C3HT101 / 2clone8 cells.

First, the cells were placed in a 6-well plate, incubated at 37 ° C. for 24 hours, and then samples were added to each well by concentration, followed by incubation at 37 ° C. for 2 days, and then culture was removed from each well, 100 μl of MTT solution (a solution of tetrazolium dye MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)) was added thereto, followed by incubation for 2 hours, followed by MTT The solution was removed. 100 μl of dimethyl sulfoxide was added to each well, followed by reaction for 15 to 20 minutes, and the absorbance was measured at an optical density of 540 nm.

As a result, as can be seen in Figure 3, when treated with a concentration of 50 μg / ml of the extract, the three kinds of mast cells confirmed that the cell viability is 100%, there is no cytotoxicity, 100 μg / Only when the ml treatment was confirmed to have a 70 ~ 80% survival rate.

2-2. Lipid accumulation inhibitory effect (Oil-Red O)

Oil-Red O staining using 3T3L-1, 3T3-F442A, and C3HT101 / 2clone8 cells was performed to confirm the intracellular lipid accumulation inhibitory ability of the extract of Bovine fern from 1-1.

First, the number of cells was measured and added to a 6-well plate to be 0.8 × 10 ⁵ / well, followed by 2 days of culture to allow 100% of the cells to be cultured, and 2 days of further culture was inhibited to inhibit cell differentiation. , The culture was removed. Next, 2 ml of differentiation culture medium containing MDI (1M dexamethasone), 0.5 mM isobutylmethylxanthine (IBMX: isobutylmethylxanthine) and 10 µg / ml insulin (insulin) were added, followed by incubation for 3 days. . From this time, samples were treated by concentration (50 μg / ml, 100 μg / ml). After 3 days, the culture solution was removed, and 2 ml of the differentiated culture solution to which insulin was added and the sample were added and cultured for 2 days. Thereafter, 1 ml of the culture solution was removed at 2 days intervals, and 1 ml of the fresh culture solution and the sample were added thereto, and then cultured for about 10 days. After inducing differentiation of adipocytes, the cell culture was removed and washed by adding 1 ml of PBS. After 200% of 10% formaldehyde was treated and fixed at room temperature for 1 hour, formaldehyde was removed, and 60% Isopropanol was washed with 200 μl and dried completely. 1 ml each of Oil red O working solution was added and incubated at room temperature for 30 minutes. Then, after removing the supernatant, and washed twice with PBS, Isopropanol 1ml was added to confirm the effect of inhibiting lipid accumulation after re-dissolution.

As a result, as can be seen in Figure 4, it was confirmed that the lipid accumulation in the cytotoxicity evaluation results in 50 ㎍ / ml which does not have any cytotoxic effect when treated with the extract.

3. Isolation of anti-obesity substance from extract

3-1. Establishment of Peak Condition for Separation of Substances from Daikon Fern Extract

In order to isolate the anti-obesity substance from the prepared U. fern extract, A (RT, 10-30 minutes), B (RT, 30-50 minutes), C (RT, 50-70) at wavelengths of 230 nm and 400 nm After dividing by HPLC into the area), the effect of inhibiting lipid accumulation of 3T3L-1 cells was confirmed.

At this time, HPLC analysis conditions are as follows.

Solvents used: A: DW + 0.1% (v / v) TFA, B: Acetonitrile (ACN)

TABLE 2

Retention time (RT)	A solvent (%)	B solvent (%)
0	100	0
15	50	50
45	20	80
55	0	100
60	0	100
62	100	0
70	100	0

Sample concentration: 33.3 mg / ml

Injection volume: 100 μl

Detector: wavelength 400nm, wavelength 230nm

-Column: UG120 5㎛ size 4.6mmI.D X 250mm shiseido Co., JAPAN

Flow rate: 0.7 ml / min

As shown in FIG. 5, based on HPLC results analyzed at 230 nm and 400 nm wavelengths, the aliquots were divided into A, B, and C regions, and the U. aureus aliquots from which the samples of each region were collected were collected into 3T3L-1 cells. The purpose of this study was to analyze the efficacy of lipid accumulation inhibition.

First, add 20 µl of agar (0-20 minutes, 20-30 minutes, 30-40 minutes, 40-50 minutes, 50-60 minutes, 60-70 minutes) to DMEM medium to which 10% FBS was added. It was. Next, in order to confirm the anti-obesity activity of the aliquot, the experiment was performed by Oil red O staining using 3T3L-1 cells.

TABLE 3

sample	Average(%)	error(%)	% Inhibition
Before processing (DMSO)	100.00	0.65	0
05-20 minutes Aliquot Concentration 10PPM	77.85	0.65	22.15
20-30 minutes Aliquot Concentration 10PPM	69.53	0.33	30.47
30-40 minutes Aliquot Concentration 10PPM	67.78	0.84	32.22
40-50 minutes Aliquot Concentration 10PPM	62.31	3.09	37.69
50-60 minutes Aliquot Concentration 10PPM	59.02	1.85	40.98
60-70 minutes Aliquot Concentration 10PPM	77.57	2.57	22.43

As a result, as shown in Table 3, it was confirmed that the sample collected between 50-60 components was the best lipid storage inhibitory effect at 10ppm.

Therefore, based on the above results, it was determined that there was a substance having excellent anti-obesity effect among the peaks of 50-60 components, and it was decided to separate the peak of this portion.

3-2. Separation and purification of indicator substances

Based on the results of 3-1, 50-60 component aliquots, which were aliquoted from the radish fern, were loaded and purified at 400 nm wavelength using prep-LC, and assayed as follows.

-Solvent Condition: A: DW + 0.1% TFA B: ACN

Table 4

Retention time (RT)	A solvent (%)	B solvent (%)
0	100	0
15	50	50
45	20	80
55	0	100
60	0	100
62	100	0
70	100	0

Sample concentration: 33.3ml / mg

Injection volume: 3 ml

Detector: wavelength 400 nm

-Column: ODS-H80 250X20mm S-4um, 8nm, YMC, JAPAN

Flow rate: 20 ml / min

As a result, as shown in FIG. 5, when the extract of the U. fern extract was separated and purified at the wavelength of 400 nm, the peak of the 56-component peak appeared to be the highest.

HPLC analysis was carried out to identify peaks of 56 components of the isolated and purified Wu-mu-Li aliquots.

At this time, HPLC analysis conditions are as follows.

Solvent conditions: A: DW + 0.1% (v / v) TFA, B: Acetonitrile (ACN)

Table 5

Retention time (RT)	A solvent (%)	B solvent (%)
0	100	0
15	50	50
45	20	80
55	0	100
60	0	100
62	100	0
70	100	0

-Sample concentration: 56 minutes aliquot

Injection volume: 100 μl

Detector: wavelength 400nm

-Column: UG120 5㎛ size 4.6mmI.D X 250mm shiseido Co., JAPAN

Flow rate: 0.7 ml / min

As a result of confirming the peaks of the aliquots fractionated from the extract of the U.S. agar from the HPLC analysis conditions of Table 5, the peaks were identified at 56 components as shown in FIG. 6. As a result of the cytotoxicity test of this preparative peak, it was confirmed that there is no cytotoxicity as shown in FIG.

3-3. Inhibition of Lipid Accumulation of 56-Minute Peak Aliquots Aliquoted from Daikon Fern Extract

In order to confirm whether the 56-minute peak fraction separated and purified from 3-2 inhibits the accumulation of lipids in the cells, lyophilization of the aliquot, 3T3L-1, C3HT101 / 2clone8, 3T3-F442A in the same manner as 2-2 Oil-Red O staining was used to determine the effect of lipid accumulation.

As a result, as shown in FIG. 8, it was confirmed that lipid accumulation was inhibited at a concentration of 50 μg / ml of the 56-minute peak aliquot. Therefore, it was confirmed that the 56-minute aliquot has excellent anti-lipid effect and anti-obesity effect.

3-4. Identification of Substances from 56-Minute Peak Aliquots Aliquoted from Daikon Fern Extract

UV-spectrum (FIG. 9), HR / Mass (FIG. 10), ¹ H-NMR (FIG. 11), ¹³ C-NMR (FIG. 12), COSY spectrum (56) of 56-component peak aliquots aliquoted from the extract ), Molecular weight and structure were confirmed using the HMBC spectrum (FIG. 14) and the HSQC-DEPT spectrum (FIG. 15).

As a result, as shown in the UV-spectrum analysis, as shown in Figure 9, the 56-minute peak aliquot was the highest absorption wavelength at 411nm confirmed that the 56-minute peak aliquot has the potential as an anti-obesity substance, such as HR / Mass, ^{As a result of 1} H-NMR and ¹³ C NMR analysis, it was confirmed that the molecular formula was C ₃₅ H ₃₆ N ₄ O ₅ , which was a substance having a molecular weight of 592.27. As a result of structural analysis of the substance having a molecular weight of 592.27 through 2D-NMR (COSY, HMBC, HSQC-DEPT) analysis, the substance which gives anti-obesity effect isolated from the extract of ummu fern was Pheophorbide A. It was confirmed (Fig. 16).

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Pheophorbide A (Pheophorbide A) isolated from the extract of Gelidium amansii according to the present invention exhibits anti-obesity effect due to its excellent lipid accumulation inhibitory effect, and thus can be applied as an anti-obesity food, cosmetic or pharmaceutical product. .

Claims (12)

1. Anti-obesity composition containing Pheophorbide A (Pheophorbide A) isolated from the extract of Umbrella.
2. According to claim 1, The pheophorbide A (Pheophorbide A) is a high performance liquid chromatography (HPLC) using a mixture of water and trifluoroacetic acid as a first solvent and acetonitrile as a second solvent as a mobile phase It is a compound isolated and obtained, The composition for anti-obesity characterized by the above-mentioned.
3. According to claim 1, wherein the pheophorbide A (Pheophorbide A) is an anti-obesity composition, characterized in that it has a lipid accumulation inhibitory effect.
4. The anti-obesity composition according to claim 1, wherein the pheophorbide A comprises 5 to 15 wt% based on the total weight of the anti-obesity composition.
5. Food comprising the anti-obesity composition of any one of claims 1 to 4.
6. Cosmetics containing the anti-obesity composition of any one of claims 1 to 4.
7. A pharmaceutical product comprising the anti-obesity composition of any one of claims 1 to 4.
8. Preparing the extract of radish by extracting the radish with a solvent; And separating the pheophorbide A by high performance liquid chromatography (HPLC) using the mixed solution of water and trifluoroacetic acid as the first solvent and acetonitrile as the second solvent as the mobile phase. Method for producing an anti-obesity composition, characterized in that.
9. The method of claim 8, wherein the solvent is at least one selected from the group consisting of alcohol, methanol, chloroform and purified water.
10. The method of claim 8, wherein the mixed solution of water and trifluoroacetic acid comprises 99.9% (v / v) of water and 0.1% (v / v) of trifluoroacetic acid.
11. The method of claim 8, wherein the first and second solvents are graded gradient (100: 0, 50:50, 20:80, 0: 100).
12. The method of claim 8, wherein the mobile phase is acetonitrile.

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