KR101166354B1 - Lipase inhibitor containing the extracts from oceanic life species for anti-obesity - Google Patents

Abstract

The present invention relates to an anti-obesity lipase inhibitor comprising marine organism extracts, and more particularly, sargassum confusum ), poached seaweed ( agarum cribrosum ), sargassum horneri ) and an anti-obesity lipase inhibitor comprising any one marine organism extract selected from the group consisting of zostera marina and the lipase comprising the marine organism extract, crystalline cellulose, magnesium stearate and silicon dioxide. It relates to an inhibitor capsule formulation.

Lipase inhibitors comprising the marine life extract of the present invention and capsules prepared using the same, by inhibiting the activity of the digestive enzymes to enable the role of anti-obesity functional food to those who are overweight or obese findings, We can contribute to national life health.

Egg mother's nest, poached seaweed, hoesan mother's nest, horse skin, anti-obesity, lipase inhibitor

Description

Lipase inhibitor containing the extracts from oceanic life species for anti-obesity}

The present invention relates to an anti-obesity lipase inhibitor comprising marine organism extracts, and more particularly, sargassum confusum ), poached seaweed ( agarum cribrosum ), sargassum horneri ) and an anti-obesity lipase inhibitor comprising any one marine organism extract selected from the group consisting of zostera marina and the lipase comprising the marine organism extract, crystalline cellulose, magnesium stearate and silicon dioxide. It relates to an inhibitor capsule formulation.

Obesity is a risk of 5D (disfigurement, discomfort, disability, disease, death), causing various complications and eventually cause death, and should be prevented if possible, and should be treated if obese. Obese people are low in productivity due to various symptoms and become inconvenient, which adversely affects the quality of life and is not cost-effective.

According to the results of the National Nutrition Survey, 27.3% of Koreans are judged to be obese people with a BMI of 25 or more and are expected to increase to 40% within the next five years. Obesity is a problem in itself, but because it causes various complications that lead to adult diseases such as diabetes, hyperlipidemia, and vascular disease, the Korean Medical Association specifies that obesity is a disease and should be treated. . Obesity is more affluent than developed countries and developed countries, and since most of the world's human problems, developed countries around the world have been leading the way in the development of medicines for the treatment of this disease. As a result, Xenical and Reductil have recently been developed and sold. Currently, the global obesity drug market is estimated at $ 2.9 billion (2004 estimate) and is expected to expand to $ 8 billion within the next 10 years.

On the other hand, obese people feel social prejudice and inequality in their bodies, and are embarrassed by women and may show anxiety, depression, adaptation disorder, personality disorder and hysteria. They feel socially inferior and feel guilty about their lack of control over weight loss. In other words, in the current trend that emphasizes appearance, the scale of related industries is rapidly increasing as obesity suppression or weight loss has become one of the appearance management industries, reflecting the needs of people who want to become thinner.

In view of the severity of the problem of obesity, so many research funds, researchers and time have been invested around the world so far, various proposals have been proposed based on diet, exercise, and behavioral therapy, but the failure rate is 95%. There is no cool solution. Therefore, there is a need to renovate existing research or to try new solutions. If a new solution is proposed, either locally or fundamentally, to solve the problem of obesity, it will not only improve the quality of life of individuals but also have a great economic and social impact.

An object of the present invention is to provide anti-obesity enzyme inhibitors from obese patients from natural marine organisms that have been proven safe, and to develop health functional foods using the same.

In order to solve the above problems, the present invention is a sarcosum confusum , agarum seaweed cribrosum ), sargassum Provided is an anti-obesity lipase inhibitor comprising any one marine organism extract selected from the group consisting of horneri ) and zostera marina . In another aspect, the present invention is any one marine organisms extract selected from the group consisting of alveolar moss, persimmon seaweed, hoesaeng mamo and horseshoe skin; Crystalline cellulose; Magnesium stearate; It provides a capsule for lipase inhibition comprising; and silicon dioxide.

Lipase inhibitors comprising the marine life extract of the present invention and capsules prepared using the same, by inhibiting the activity of the digestive enzymes to enable the role as a health functional food for anti-obesity to those who are overweight or obese We can contribute to national life health.

The present invention is a sarcoma ( sargassum) confusum ), agarum cribrosum , sargassum Provided is an anti-obesity lipase inhibitor comprising any one marine organism extract selected from the group consisting of horneri ) and zostera marina .

In the present invention, the extract may be extracted using purified water or ethanol.

In the present invention, the extract is pulverized the marine life and filtered through a sieve of 20 to 60 mesh, the filtered sample and purified water or ethanol in a content ratio (w / v) of 1: 3 to 10 to obtain an extract while stirring It may be prepared by filtration and concentration under reduced pressure.

The present invention is also any one of the marine life extract selected from the group consisting of albino cap, poachol seaweed, boksae cap, and horse skin; Crystalline cellulose; Magnesium stearate; It provides a capsule for lipase inhibition comprising; and silicon dioxide.

In the present invention, the capsule preparation may further include any one or more natural pigments selected from the group consisting of gardenia green, safflower yellow pigment and red yeast red pigment.

In addition, in the present invention, the capsule preparation is mixed with the concentrate of the marine extract and crystalline cellulose in a weight ratio of 4: 6 to 6: 4, 400 to 500mg each may be filled in pale yellow capsules.

Hereinafter, the anti-obesity lipase inhibitor and lipase inhibitory capsule preparation of the present invention will be described in more detail.

In order to prevent obesity, an object of the present invention is to provide an anti-obesity lipase inhibitor that can lower body fat by inhibiting the lipase, a digestive enzyme of fat.

The anti-obesity enzyme inhibitor of the present invention is prepared using extracts of marine organisms, and it is easy to supply raw materials and is safe when used as a health functional food, and simultaneously provides various nutrients such as iron and minerals contained in extracts of marine organisms. can do.

The present invention provides an anti-obesity lipase inhibitor comprising any one marine organism extract selected from the group consisting of alveolar moth, perilla seaweed, algae moss and horseshoe skin.

Preferably the present invention provides an anti-obesity lipase inhibitor comprising an extract of Alzheimer's disease.

In the present invention, the extract may be extracted using purified water or ethanol, preferably the extract may be extracted using ethanol.

In the present invention, the extract is pulverized the marine life and filtered through a sieve of 20 to 60 mesh, the filtered sample and purified water or ethanol in a content ratio (w / v) of 1: 3 to 10 to obtain an extract while stirring It may be prepared by filtration and concentration under reduced pressure.

Preferably in the present invention, the extract is used as a sample by pulverizing the marine organisms with a commercially available pulverizer, a sieve of 40 mesh, mixed the sample and ethanol in a content ratio (w / v) of 1: 5 and room temperature The extract was obtained by stirring at a speed of 120 rpm for 24 hours, and the supernatant was taken by centrifugation at 10,000 rpm at 4 ° C. and stored at −20 ° C. until analysis.

The present invention is also any one of the marine life extract selected from the group consisting of albino cap, poachol seaweed, boksae cap, and horse skin; Crystalline cellulose; Magnesium stearate; It provides a capsule for lipase inhibition comprising; and silicon dioxide.

In the present invention, the capsule formulation may further include any one or more natural pigments selected from the group consisting of Gardenia green, safflower yellow pigment and red yeast red pigment, and in the present invention, the capsule formulation may be concentrated and crystallized from marine extracts. The cellulose may be mixed in a weight ratio of 4: 6 to 6: 4, and may be filled in 400 to 500 mg of pale yellow capsules.

Preferably, the present invention extracts almond mojaban with ethanol, and then concentrated to 35Brix and kneaded by mixing the crystalline cellulose 101 in a weight ratio of 5: 5 to solids and kneading until the moisture is 2.3% at 60 ℃ After drying, it is filtered through an 18 mesh sieve, mixed with magnesium stearate and silicon dioxide, and may be filled with 450 mg of a pale yellow capsule.

Hereinafter, examples and experimental examples of the present invention will be described. However, the following examples and the like are intended to explain the present invention in more detail, and the rights of the present invention are not intended to be limited thereto.

Example 1 Extraction of Lipase Inhibitor Sample from Marine Life

Samples for measuring enzyme inhibitory activity were obtained from the market in Gangneung, Korea, or collected from the East Sea by mobilizing divers (algae, seaweed, seaweed, seaweed, seaweed, seaweed, kelp, horsetail, violet leaf). ) Was used as a sample by pulverizing with a grinder and sieving with a 40 mesh sieve. The extraction method used each sample and water in a ratio of 1: 5 (w / v) and each sample and ethanol in a ratio of 1: 5 (w / v), and stirred for 24 hours at a speed of 120 rpm at room temperature. After extraction, the supernatant was collected by centrifugation at 10,000 rpm at 4 ° C. and stored at −20 ° C. until analysis.

Example 2 Preparation of Capsules Containing Marine Life Extract

A capsule formulation was prepared using the marine organism extract of Example 1.

As a representative raw material was used Al 쏭 ma ban, processing process of the capsule formulation containing al 쏭 ma ban extract was as follows. The extract obtained by extracting Almex caps with ethanol (alcohol) was suspended in 60% ethanol and centrifuged. The supernatant thus obtained was concentrated to obtain an alginimoban concentrate, then mixed with crystalline cellulose 101 in a 1: 1 ratio and kneaded, and dried at 60 ° C. until the moisture became 2.3%. 100 g (primary granules) of powdered alkalescent mother powder were dropped on an 18-mesh sieve, 42 g of 50% fructooligosaccharide solution was added, kneaded, dried, and then lowered to a 24-mesh sieve (secondary granules). To the secondary granules of Algin mother jar, 5 mg of magnesium stearate and 5 mg of silicon dioxide were added and mixed, followed by filling 450 mg of pale yellow capsules. Placebo is mixed with crystalline cellulose 101 and purified water, and then mixed with 5 mg of natural pigments, gardenia green, safflower yellow, and red yeast red pigment, and then kneaded and dried (placebo granules). Magnesium stearate and 5 mg of silicon dioxide are added in a fixed ratio. After that, 450 mg each was filled in a pale yellow capsule.

Experimental Example 1 Lipase Inhibitory Activity Extracted from Marine Life

1. Screening enzyme inhibitors from marine organism extracts

Lipase inhibitory activity was measured by the turbidity method. 100 μl of each marine organism extract was mixed with 1.5 ml of substrate 0.04% olive oil (Tris-deoxycholate buffer, pH 7.7), 1.5 ml of enzyme solution was added, and immediately absorbed at 340 nm using a UV-spectrophotometer for 2 minutes. Recorded. The activity of the inhibitor was expressed as a relative value of the enzyme activity in the presence of the inhibitor, with the enzyme activity of 100 when no inhibitor was added. As a result of lipase inhibitory activity, more than 70% inhibition rate was found in the extracts of Almex algae, Hoengseng moss, persimmon seaweed and horseshoe skin. In the purified water extract, Hoengsama moss extract was 36.1% higher than other extracts. Was the highest (see Table 1 below).

[Table 1] Inhibition activities of extracts from various oceanic life species against lipase

sample Scientific name Lipase Extract ethanol Purified water Egg Alma Hat Sargassum confusum 77.6 5.8 Aunt hat Sargassum horneri 100 36.1 Seaweed Undaria pinnatifida 43.7 0.6 Iron seaweed Costaria costata 38.3 0.1 Boiled seaweed Agarum cribrosum 85.2 21.4 Kelp Laminaria japonica 57.1 0.5 Horseback Zostera marina 98.9 2.5 Purple leaves Polyneura hilliae 32.7 1.7

2. IC ₅₀ value (mg / ml) measurement

1) After diluting the sample prepared in Example 1 to a constant concentration, the enzyme inhibitory effect was measured, and then the regression curves for these concentrations were calculated to determine the IC ₅₀ value. The rare curve of the lipase was shown by calculating the amount (mg / ml) required to inhibit 50% of the enzyme by adding a straight line by adding 2.5, 5 and 10 mg, respectively, to investigate the inhibition rate (see Table 2 and FIG. 1 below). . On the basis of these results, a final selection of the alpine mother's bed showing high inhibitory activity against lipase was performed.

[Table 2] IC ₅₀ value (mg / ㎖) measurement results

enzyme sample IC ₅₀ (mg / ml) ethanol Lipase
(Lipase) Egg Alma Hat 37.8 Aunt hat 42.2 Boiled seaweed 28.9 Horseback 133.7

2) Inhibitory Activity against Human Origin Digestive Enzyme

Inhibitory activity against digestive enzymes was measured after diluting marine extracts to a constant concentration (10 mg / ml). Digestive enzymes were used as porcine and human origin, and IC ₅₀ values were measured for the human origin lipase of the alginimozaban extract, which showed the highest inhibitory activity against porcine origin enzyme. As a result, Almemozaban extract showed a 12 to 24 times higher inhibitory activity than human origin lipase, showing a strong effect on the inhibition of obesity in humans (see Table 3 below).

Table 3 Results of IC ₅₀ Value (mg / mL) of Human Origin Digestive Enzymes

enzyme IC ₅₀ (mg / ml) porcine human Lipase 8.227 0.345

Experimental Example 2 Experiment of Inhibiting Proliferation of Cellular Cells

1. Cell Culture

The culture medium of 3T3-L1 cells was Dulbecco's modified Eagle's medium (DMEM), and various concentrations of fetal bovine serum (FBS), penicillin (100 units / ml), and streptomycin (100 ㎍ / ml) were used. And the like were added. 3T3-L1 was washed with Dulbecco's phosphate buffered saline A (DPBS-A) solution at intervals of 3-4 days of culture, and then treated with 1 ml of 0.25% trypsin solution per 50 ml flask for 1 minute at room temperature. The trypsin solution was discarded and stored at 37 ° C. for 5 minutes to detach and passage cells. The desorbed cells and then 1 to a new culture vessel which 10% FBS was added to the DMEM rich (DMEM / FBS10) 10 ㎖: transferred to a split ratio of 20 CO ₂ The cells were cultured in an incubator (37 ° C., 5% CO ₂ ). Under these passage conditions, 3T3-L1 is known to remain preadipocyte.

2. Measurement of Cell Proliferation Rate by MTT Method

The proliferation rate of 3T3-L1 cells was measured by applying the method of Skaper et al. MTT used for cell proliferation rate was dissolved in DPBS-A at a concentration of 5 mg / ml and filtered and sterilized. Cells were seeded at a concentration of 3.1 × 10 ⁴ cells / well in a 24-well plate and the final volume of the culture was 1 ml. MTT solution was added 4 hours before the end of cell culture (100 μl / well). After the end of the culture, the cell culture solution was removed and 400 μl of DMSO was added to dissolve the formazan of blue crystals produced by the cells. The absorbance of each well of the culture plate in which Fomazan was dissolved was measured at 570 nm with an ELISA reader. After the MTT solution was added in the above process, the culture plate was wrapped with silver foil and cultured in a state of blocking light to minimize the reduction of MTT by light.

3. Culture Conditions for Measuring Cell Proliferation Rate

3T3-L1 cells on the third day of culture in subculture were detached by trypsin treatment in the same manner as described above, and suspended in DMEM medium to which 10% FBS was added according to experimental conditions. 100 μl of cell suspension was dispensed into each well of a 24-well plate to inoculate 3.1 × 10 ⁴ cells per well, and then cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 24 hours to attach cells. Then, 50 μl of the sample or culture diluted in the well was added to make the final volume 1 ml. After treatment as described above, the effect of the sample on the proliferation rate of 3T3-L1 cells by culturing for 3 days in a CO ₂ incubator.

4. Effect of Sample Solution on 3T3-L1 Cell Proliferation

Al capsillan extract was diluted in culture medium at various concentrations, filtered and sterilized, and then treated with attached 3T3-L1 cells in the same manner as mentioned above. The effect of the sample solution on the proliferation rate of the cells was calculated by converting the absorbance of the experimental group into the percentage of the absorbance of the control group measured by the MTT method. As a result, at the concentration of 2.5 ㎍ / ㎖ in the algae extracts than the control without any treatment, up to about 80% or more proliferation inhibition rate was shown (see Fig. 2).

Experimental Example 3 Isolation and Purification of Bioactive Substances from Marine Biological Extracts

Thin layer chromatography

Identification and purification of the extract was made by TLC (silica gel 60 F254, Merck). The development of TLC proceeded with Hexane: ethylacetate: Methanol (= 10: 3: 0.5) ZZA as a developing solvent, and the band was confirmed by burning on a hotplate after sparing 10% sulfuric acid. In order to identify the enzyme inhibitory activity band using TLC, samples were developed by TLC, each band was scratched, extracted with ethanol, and enzyme inhibitory activity was measured.

2. Isolation and Purification of Active Substances

100 g of Algin mother jar was crushed with a grinder to form a powder, and then extracted with 95% ethanol (500 ml) at 75 ± 5 ° C. for 8 hours. After extraction, the filtrate was concentrated using a rotary evaporator. At this time, about 17.6 g of the concentrated ethanol extract was obtained. 17.6 g of ethanol extracts were all taken up, dissolved in 80% methanol, centrifuged to remove precipitates, and the solvent was partitioned into n-hexane in the supernatant to remove nonpolar material. The extra active fraction 80% methanol layer was further divided into 30% methanol and chloroform layers (see FIG. 3). At this time, the concentrated amount of the chloroloam layer was about 7.11 g. Enzyme inhibitory activity of each purification step was expressed as IC ₅₀ value for the standardization of the product (see Table 4 below).

Table 4. The inhibitory effect of crude extract and purify extracts against lipase

Figure 3 (Fraction) Inhibition
(IC ₅₀ , mg / ml) ① 6.78 ② - ③ - ④ 2.51 ⑤ - ⑥ 1.97

Experimental Example 4 Efficacy Evaluation Experiment Using Animal Experiment

As the consumption of animal saturated fat increases due to the improvement of the economic level, various health disorders are caused by the nutritional imbalance such as the incidence of circulatory diseases such as hypertension, arteriosclerosis, and heart disease. In particular, obesity is a problem in itself, but it is a bigger problem because it causes various complications leading to adult diseases such as diabetes, hyperlipidemia, and vascular disease, and the Korean Medical Association has to classify obesity as a disease and treat it. It is specified as the target. Obesity is more serious in rich and developed countries, and is becoming a problem for most of the world. Modern obese people to treat obesity is to control the body's fat metabolism, diet, exercise, surgery, etc. are being performed.

Recently, studies have been attempted using seaweed extracts in Japan or France to reduce body weight and body fat without any side effects. In Korea, polysaccharides in seaweed have long been effective in reducing blood cholesterol and triglyceride content (Kang Jung-ho, 1968). There is also a report showing a decrease in total cholesterol content in blood (Kim Eun-ju, 1983). In addition, the effect of ingestion of seaweed (seaweed, seaweed) on lipid metabolism in rats was not different in the blood glucose or plasma triglycerides by the addition of fiber (pectin, cellulose, seaweed, seaweed), but the total lipid content was different. The results showed a significant decrease in the group of seaweed and laver added to the addition group (Kyung, 1991), which is very positive for the obese and those in the field.

In this experiment, white rats were fed high-fat diet to induce obesity, and then Sargassum confusum belonging to Phaeophyceae was collected and extracted, filtered and concentrated with ethanol to high-fat diet. The purpose of this study was to examine the effect of reducing body weight and body fat.

Obesity Induction Animal Testing

1) Materials and Methods

① Animal experiment

Thirty male 6-week-old Sprague-Dawley male rats were purchased from the Central Animal Laboratory and used for one week after feeding with regular feed. To determine the induction of obesity, two groups of 10 normal diets and 20 high-fat diet (HFD) supplements replacing 40% of the calories in the diet were tested. It was performed for a week.

② Dietary preparation

Diet was prepared in the normal group based on AIN-93G (398.8 kcal / 100 g), the experimental group was prepared with a high fat diet (477.8 kcal / 100 g) to induce obesity. Dietary intake and body weight were measured once a week, and the diet was prepared and made into a solid, dried and stored at -20 ° C and supplied frozen. And water was freely supplied without restriction.

2) results

The dietary intake, weight change, and dietary efficiency of the rats fed the normal diet and the high fat diet are shown in Table 5 and FIG. 4 below. Obesity induction period of the experimental animals was 5 weeks. The average feed group consumed 23.92 g daily and had a feed efficiency of 0.20. The HFD group had a low feed intake, but had an average daily weight gain of 5.77 g and a high dietary efficiency of 0.28. In five weeks, the average weight gain of the high-fat diet group was 38.5 g (16%) more than the normal diet group. As a result of statistical treatment, the weight of HFD group was significantly higher than that of general dietary group, and it was judged that obesity was induced.

[Table 5] Body weight, body weight gain, food intake and food efficiency ratio in rats fed experimental diet for the period of inducing obesity

Group Body weight (g) Body weight gain (g / day) Food intake
(g / day) Food efficiency ratio ^{1 )} Initial Final ND 236.4 ± 6.27 399.8 ± 20.45 ^b 4.67 ± 0.51 ^b 23.92 ± 1.33 ^a 0.20 ± 0.02 ^b HFD 236.3 ± 8.03 438.2 ± 29.82 ^a 5.77 ± 0.86 ^a 20.53 ± 1.45 ^b 0.28 ± 0.03 ^a

Values are mean ± SD of 10 rats per group.

¹⁾ Food efficiency ratio: body weight gain / food intake for 5 weeks

ND: normal diet, HFD: high fat diet

^{a- b} Data are significantly different by one-way ANOVA followed Duncan's multiple range test at the 0.05 level of significance.

2. Obesity Inhibition Animal Experiment

1) Materials and Methods

① Animal experiment

Thirty male 6-week-old Sprague-Dawley male rats were purchased from the Central Experimental Animal Co., Ltd. and adapted to a regular diet for one week. Weekly obesity was induced, and the diet was prepared by feeding 3% alginus mavan extract powder into the high-fat diet to examine the anti-obesity effect of alginus mavan extract from 6 to 11 weeks. The experimental group was divided into a normal diet group (ND), a control group (high fat diet group), and a 3% HFD + lipase inhibitor group (lipase inhibitor, LI group), and each group had 10 randomized complete block designs. Classified as Two rats were kept per cage, and the temperature and humidity of the cage were adjusted to 22 ± 0.5 ℃ and 55 ± 5%, respectively, and the contrast period was adjusted to 12 hours.

② Dietary preparation

Diet was prepared based on AIN-93G diet of the normal group (398.8 kcal / 100 g), experimental group was prepared with a high fat diet (477.8 kcal / 100 g) to induce obesity (Table 6). The diet of each experimental group (HFD, LI) ingesting a high fat diet was prepared to have the same energy level. The dietary supply was adjusted according to the average intake of the high-fat diet group to adjust the energy level to be almost identical between the experimental groups. Dietary intake and body weight were measured once a week, and the diet was prepared and made into a solid, dried and stored at -20 ° C and supplied frozen. And water was freely supplied without restriction.

Table 6 Composition of experiment diets (g / kg diet)

Ingredients Experimental Groups ^{2 )} ND (n = 10) HFD (n = 10) LI (n = 10) Casein 200 200 200 Sucrose 100 100 100 Corn starch 397.486 254.5 249.5 Soybean oil 70 70 70 Dexrose 132 132 132 Lard - 150 150 Cellulose 50 50 25 L-cystine 3 3 3 AIN-93-mineral mixture 35 35 35 AIN-93-vitamin mixture 10 10 10 Choline bitrate 2.5 2.5 2.5 t-Butylhydroquinone 0.014 0.014 0.014 Sargassum confusum
extract powder - - 30

¹⁾ The diet was prepared to be isocaloric, 477.8 kcal / 100 g, among experimental groups except normal diet (398.8 kcal / 100 g) according to # 110700 AIN-93G guide line.

²⁾ ND: normal diet, HFD: high-fat diet, LI: HFD + Sargassum confusum extract powder 3%

2) results

After obesity induction, dietary intake, weight gain, and dietary efficiency are shown in Table 7, and the change in weight during the experiment period is shown in FIG. The daily weight gain was the highest in the ND group (2.19g / day) <LI group (2.36g / day) <HFD group (2.96g / day) in order to increase the HFD group fed only high-fat diet. This result shows that the high-fat diet with Almex algae extract has the effect of inhibiting weight gain. This suggests that the high fat diet suggests the feed intake of rats. In addition, the dietary efficiency of HFD group was 0.15, but the ND and LI groups were 0.09 and 0.13, respectively, which showed lower feed efficiency than HFD group. This is the result that the high-fat diet has a high calorie density to increase the dietary efficiency, and the dietary efficiency is slightly decreased in the LI group compared to the HFD group. It is considered that this is reduced. In other words, even if you eat the same high-fat diet, it is interpreted to mean that some nutrients are not absorbed and excreted by Alzheimer's maternal extract. This can be said to have the effect of reducing the weight gain by the same principle as the obesity treatment drug Xenical. As a preliminary study of this study, it was confirmed in vitro as a kind of Digestive Enzyme Inhibitor (DEI), which is a lipase inhibitory activity and the 3T3-L1 cells, which are fat animal cells. the evaluation of the growth inhibition by the case of alcoholic extract by IC ₅₀ values showed a Lipase inhibition rate to 6.78 mg / mL, growth inhibition rate of 35.25% fat cells are seen in 2.5 ug / mL concentrations alssong of the hat room extract Lipase inhibitor (LI) function was confirmed. Therefore, it is not possible to digest some fats of the high fat diets that the experimental animals ingested by feed eggs, and eventually lower the absorption rate of fats, resulting in a reduction of calorie intake, which is believed to have the effect of reducing the weight gain.

[Table 7] Body weight, body weight gain, food intake and food efficiency ratio in rats fed experimental diet

Group Body weight (g) Body weight gain (g / day) Food intake
(g / day) Food efficiency ratio ^{1 )} Initial Final ND 399.82 ± 20.45 ^b 467.66 ± 21.41 ^b 2.19 ± 0.17 ^b 24.99 ± 2.11 ^a 0.09 ± 0.01 ^c HFD 438.30 ± 26.01 ^a 530.13 ± 22.54 ^a 2.96 ± 0.31 ^a 19.80 ± 1.01 ^b 0.15 ± 0.01 ^a LI 437.99 ± 28.97 ^a 511.30 ± 22.87 ^a 2.36 ± 0.24 ^b 18.89 ± 0.48 ^b 0.13 ± 0.01 ^b

Values are mean ± SD of 10 rats per group.

¹⁾ Food efficiency ratio: body weight gain / food intake for 5 weeks

ND: normal diet, HFD: high fat diet, LI: HFD + Sargassum confusum extract powder 3%

^{a- c} Data are significantly different by one-way ANOVA followed Duncan's multiple range test at the 0.05 level of significance.

Experimental Example 5 Human Effect Experiment

1. Survey Subject and Test Method

1) Subjects of Alzheimer's disease preparations were taken four capsules of the formulation (capsules prepared in Example 2) with 200 ml of water three times a day 30 minutes before meals. If not eaten before meals, it is recommended to eat at least 30 minutes after eating. The study was conducted for a total of 12 weeks from the time the subjects consumed the preparation.

2) Test contents and method

① Body measurement and body composition measurement

Anthropometric measurements were taken four times before the start of the study (week 0), 4 weeks, 8 weeks, and 12 weeks. The height was measured once by 0.1 cm with an electronic extensometer (Fanics, Korea) in a naturally upright position with bare feet. Body weight and body composition analysis were measured using the body composition analyzer In Body 2.0 (Biospace Co., Korea) with minimal clothes, weight (kg, weight), body mass index (kg / m ² ) and lean body mass. Lean body mass (kg), Body fat mass (kg), Percent Body Fat (%), Waist-Hip circumference Ratio, BMR (Basal Metabolic Rate, kcal), Arm cir (Arm circumference (cm) was used as a study result. The waist circumference was measured at the midpoint between the lowermost part of the rib and the iliac bone in the upright position, and the hip circumference was calculated by measuring the widest circumference of the great trochanter.

② Blood collection and pretreatment

Blood sampling and analysis were performed four times before starting the test (week 0), 4 weeks, 8 weeks, and 12 weeks. The subjects fasted for 12 hours before blood collection and on the day of testing, the clinician collected a total of 12 ml of blood from the brachial vein. Among them, 5 ml of blood was left at room temperature for 30 minutes to measure C-reactive protein and free fatty acid, followed by centrifugation at 3000 rpm (4 ° C.) for 8 minutes to obtain serum. The remaining 5 ml of blood was immediately centrifuged (Combi-514R, Hanil Co.) for 10 minutes at 3000 rpm (4 ° C) for determination of serum lipids, protein, mineral levels, and enzyme activity related to liver function. Store at ° C. 2 ml of whole blood was dispensed into anticoagulant tubes for DNA protection and hemoglobin measurement of leukocytes in the blood and used for analysis within 3 hours after blood collection.

③ biochemical analysis of blood

Blood analysis was performed to evaluate the anti-obesity effect, the health risks and nutritional status of the mother extract.

Triglyceride

Serum triglyceride concentrations were measured using a kit reagent for measuring triglycerides from Stanbio Laboratory Inc. (TX, USA) using Wahlefeld's method. The measuring principle is as follows. After triglyceride in serum is decomposed into glycerol and fatty acid by lipase, glycerol phosphorylates glycerol with glycerol kinase in the presence of ATP. Glycerol-3-phosphate is oxidized by glycerylphosphate oxidase (GPO) to break down into hydrogen peroxide and dihydroxyacetone phospate. Hydrogen peroxide reacts with 4-chlorophenol and 4-aminopyrine to form a red quinoneimnie complex. It was colorimetrically quantified at 500 nm in a blood autoanalyzer (ARCO, Biotechnical, Italy).

-Free fatty acid (FFA) measurement

Serum free fatty acid concentration was measured using Wako's NEFA HR kit reagent using ACS-ACOD method. Free fatty acids in serum are produced by acyl CoA, AMP and pyrrolic (PPI) when AC and magnesium are acted on as coenzymes of CoA and ATP. ACOD is applied to the resulting Acyl CoA to produce 2,3-Trans-enoyl-CoA and hydrogen peroxide. CoA remaining after ACS reaction was precipitated with N-ethyl malate and removed by oxidizing hydrogen peroxide and POD to form a purple quinone substance. Colorimetric determination was performed at 555 nm using a blood analyzer (Hitachi7180, Japan).

Leptin measurement

Serum leptin concentrations were measured using kit reagent (cat no .: DY398) for measuring human leptin from R & D systems using antigen-antibody reactions. Attach the capture antibody to the bottom of the 96-well plate to which serum is applied and leave at room temperature for 12 hours. After 12 hours, the suspended solids were washed and blocked with PBS (137mM NaCl, 2.7mM KCl, 8.1mM Na ₂ HPO ₄ , 1.5mM KH ₂ PO ₄ , pH 7.2-7.4) containing 1% bovine serum albumin. Leave on for 1 hour before washing. 100 μl of serum was applied thereto, and after washing for 2 hours at room temperature, 100 μl of detective antibody was attached to the well and washed after 2 hours. 100 μl of streptavidin-HRP was added thereto and left at room temperature for 20 minutes in the dark, followed by washing. 100 μl of the coloring reagent was added and left in the dark for 20 minutes, and then the reaction was stopped with 100 μl of sulfuric acid (2N). The absorbance was measured at 450nm wavelength in ELISA reader.

④ Survey of bowel movement and water intake

During the study period, the defecation status of the study subjects should be described together with the number of bowel movements (times / day) and the form when preparing the meal journal. If the type of stool was different from before the start of the test, the cause of the problem was improved by analyzing the questionnaire and meal journal. The water intake was also included when preparing a meal journal, and the intake unit was cup (200 ml) or liter (ℓ).

⑤ Statistical method

All data analysis and experimental results were expressed as percentage or mean ± standard deviation using SPSS / Windows 14.0. Comparison of factors for general characteristics between the two groups was analyzed using independent t-test and chi-square test. . The difference between the changes before and after the ingestion of each product was measured by paired t-test, and the difference between the mean value and the change between groups by measurement time point was tested by an independent t-test.

2. Experimental results

1) Body measurement and body composition evaluation

The measurement results of the body measurements before and after the ingestion of the preparations of the study subjects are shown in Table 8, and the results of the body composition measurements are shown in Table 9. Anthropometric and body composition measurements before ingestion of the formulation were not significantly different between the two groups. After 12 weeks of ingestion, group C significantly decreased body weight, body mass index, body fat mass, body fat percentage (p <0.05), arm circumference (p <0.01), waist / hip circumference ratio (p <0.001).

In the SC group, body weight, body mass index (p <0.01), arm circumference, waist circumference, W / H ratio, body fat mass and body fat percentage (p <0.001) were significantly decreased after 12 weeks of ingestion. Although there was a tendency to decrease, there was no significant difference compared to before preparation. At 4 weeks after ingestion of the preparation, both groups showed similar decreases in body measurements and body composition, but at 12 weeks, the SC group showed a decrease in all body measurements and body composition except for waist / hip circumference. Appeared to be larger. These results suggest that fat absorption is suppressed by the intake of the Maban extract, resulting in a decrease in body fat despite the increase in caloric intake in the SC group. In addition, the significant decrease in waist circumference in the SC group at 12 weeks was a result of abdominal obesity. Except for waist circumference, there were no significant differences among the groups in the changes of body measurements and body composition measurements.

[Table 8] Measurement results of body measurements

[Table 9] Body composition measurement results

2) Biochemical Evaluation of Blood

Blood lipid levels

Triglycerides and free fatty acids are the main markers of blood lipids for evaluating anti-obesity effects, but cholesterol, LDL-cholesterol, and HDL-cholesterol are also used as site markers. It is known to be high. Serum triglyceride levels were significantly decreased in the SC group (week 0: 115.71 ± 61.11 mg / dL, 12 weeks: 69.03 ± 25.83 mg / dL, p <0.01). Blood triglyceride levels in group C showed a significant decrease 8 weeks after ingestion of the preparation, but increased to the initial level at the end of the study. Serum total cholesterol levels were not significantly different in both groups during the period of intake of the formulation despite the decrease in body fat mass. LDL-cholesterol showed a significant decrease in group C at 12 weeks after ingestion of medication (week 0: 91.79 ± 21.14 mg / dL, week 12: 77.19 ± 15.0 mg / dL, p <0.01). There was a tendency to decrease (week 0: 101.43 ± 32.52 mg / dL, 12 weeks: 91.09 ± 23.50 mg / dL). HDL-cholesterol increased significantly in both groups at 12 weeks of drug intake (Group C week: 69.97 ± 19.69 mg / dL, week 12: 87.67 ± 17.11 mg / dL, SC group 0: 69.63 ± 20.72 mg / dL). At 12 weeks: 96.24 ± 19.0 mg / dL, p <0.001)

Free fatty acid, a metabolic factor that predicts the degree of lipolysis according to weight loss, was decreased in group C at 12 weeks after ingestion (0 weeks: 506.07 ± 218.73mg / ㎗, 12 weeks: 429.93 ± 225.15mg / ㎗). In the SC group, free fatty acid decreased significantly (week 0: 495.75 ± 252.13 mg / dL, week 12: 325.47 ± 248.76 mg / dL, p <0.01) at 12 weeks (see Table 10 below).

2) Biochemical Evaluation of Blood

Blood lipid levels

Triglycerides and free fatty acids are the main markers of blood lipids for evaluating anti-obesity effects, but cholesterol, LDL-cholesterol, and HDL-cholesterol are also used as site markers. It is known to be high. Serum triglyceride levels were significantly decreased in the SC group (week 0: 115.71 ± 61.11 mg / dL, 12 weeks: 69.03 ± 25.83 mg / dL, p <0.01). Blood triglyceride levels in group C showed a significant decrease 8 weeks after ingestion of the preparation, but increased to the initial level at the end of the study. Serum total cholesterol levels were not significantly different in both groups during the intake period. LDL-cholesterol showed a significant decrease in group C at 12 weeks after ingestion of formulation (week 0: 91.79 ± 21.14 mg / dL, week 12: 77.19 ± 15.0 mg / dL, p <0.01). There was a tendency to decrease (week 0: 101.43 ± 32.52 mg / dL, 12 weeks: 91.09 ± 23.50 mg / dL). HDL-cholesterol increased significantly in both groups at 12 weeks of drug intake (Group C week: 69.97 ± 19.69 mg / dL, week 12: 87.67 ± 17.11 mg / dL, SC group 0: 69.63 ± 20.72 mg / dL). At 12 weeks: 96.24 ± 19.0 mg / dL, p <0.001)

Free fatty acid, a metabolic factor that predicts the degree of lipolysis according to weight loss, was decreased in group C at 12 weeks after ingestion (0 weeks: 506.07 ± 218.73mg / ㎗, 12 weeks: 429.93 ± 225.15mg / ㎗). In the SC group, free fatty acid decreased significantly (week 0: 495.75 ± 252.13 mg / dL, week 12: 325.47 ± 248.76 mg / dL, p <0.01) at 12 weeks (see Table 10 below).

[Table 10] Blood Lipid Measurement Results

Blood C-reactive protein and leptin levels

As mentioned in the introduction, leptin is a hormone synthesized and secreted from adipose tissue that acts on the hypothalamus to control appetite and obesity, and obese people have higher levels of leptin resistance than normal people. The mean concentrations of leptin before the intake of the study subjects were 15.4 ± 8.7ng / ml in the C group and 19.3 ± 7.5ng / ml in the SC group, and there was no difference between the groups. It was decreased (p <0.05) while there was no change in group C. At week 12, there were significant differences in leptin change between groups (C group: 10.68 ± 21.65ng / ml increase, SC group: 3.56 ± 12.63ng / ml decrease, p <0.05). This is because the amount of leptin secreted by adipose tissue decreases and the leptin resistance is alleviated according to the decrease of body fat in SC group. Therefore, ingestion of Alzheimer's mother extract can induce lowering of leptin in the blood and can be expected to inhibit obesity (see Table 11 below).

Table 11 Results of C-reactive protein and leptin measurement in blood

- bowel conditions and water intake assessment

The number of bowel movements and water intake before and after the preparation of the study subjects are shown in Table 12 below. The frequency of bowel movement before ingestion of the preparation was 0.93 ± 0.18 times / day in the C group and 0.87 ± 0.5 times / day in the SC group. After 12 weeks of ingestion, the frequency of defecation in group C was 0.90 ± 0.45 times / day, which tended to decrease. In the SC group, the number of bowel movements was 1.19 ± 0.48 times / day at 12 weeks after the intake of the preparation, which was significantly increased compared to before the intake of the preparation. The SC group showed symptoms of thin stools for 1 ~ 2 days after the ingestion of the preparation, which was considered as a cause of thin stools due to the excretion of fat which was not absorbed by the lipohydrolytic enzyme inhibitory activity of Maban extract. By limiting the intake of foods high in fat content it was intended to normalize bowel activity. This symptom disappeared within 1 week of the ingestion and returned to normal bowel movement.

The water intake before the preparation was 4.57 ± 3.46 cups (200ml) / day in the C group and 3.38 ± 1.46 cups (200ml) / day in the SC group. After 12 weeks of ingestion, the water intake of group C increased to 5.12 ± 3.55 cups (200ml) / day, but there was no significant difference. The intake was increased 5.09 ± 2.29 cups (200ml) / day significantly compared to before the formulation intake, but there was no difference between the groups.

There are many factors that help defecation, and in case of inhibiting fat absorption, as in the case of Maban extract used in this study, the defecation may be smoothed by the fat that has not been absorbed, and the water intake may increase due to the side effect of ingestion of the preparation. When stool excretion is smooth. Therefore, although the amount of fat excreted in the feces was not measured, the ability of the maternal extract to inhibit the hydrolase could be helpful for reducing body fat by smoothing the bowel movement in obese people.

[Table 12] Defecation status and water intake measurement results

Extracts of Alzheimer's disease and the like of the present invention can be used for the purpose of preventing and treating obesity, which is a modern disease of obesity, by inhibiting the activity of the digestive enzyme lipase, and ultimately will contribute to national health and industrial development of the country. It is expected.

1 is a graph showing the degree of inhibition of the proliferation rate of 3T3-L1 cells according to the concentration of alginosa mother extract as absorbance values.

Figure 2 is a photograph showing the degree of inhibition of proliferation rate of 3T3-L1 cells according to the concentration of Almemojapan extract.

Figure 3 is a schematic diagram of a method for extracting the enzyme inhibitory active material from the egg Alma mother.

Figure 4 is a graph showing the change in weight of the rats (normal diet pay group and high-fat diet pay group).

Figure 5 is a graph showing the weight change of the rats fed the diet to which dietary supplements, high fat diet, high fat diet was added.

Claims (6)

(i) a first step of grinding horseshoe blood and filtering through a sieve of 20 to 60 mesh; (ii) a second step of mixing purified water or ethanol in a content ratio (w / v) of 1: 3 to 10 after the first step and obtaining an algal skin extract while stirring; And (iii) a third step of filtration and concentration under reduced pressure the horseshoe skin extract; Method for producing an anti-obesity lipase inhibitor comprising a. delete delete delete delete delete

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