KR20070068837A - Extracts from the seed of rosa multiflora for inhibiting the differentiation of adipocytic cells - Google Patents

Abstract

A composition for inhibiting differentiation of adipocytic cells comprising the fruit extract of Rosa multiflora var. platyphylla is provided to inhibit increase of body weight by inhibiting differentiation of preadipocytic cells into adipocytic cells, and reduce side effects or toxicity of its application. The composition for inhibiting differentiation of adipocytic cells, and preventing and inhibiting obesity comprises the fruit extract of Rosa multiflora var. platyphylla which is prepared by (a) extracting fruits of Rosa multiflora var. platyphylla with an organic solvent selected from ethanol, methanol, propanol, butanol, ethylacetate, chloroform and dichloromethane, (b) filtering and concentrating the crude extract under reduced pressure, and (c) selectively removing the organic solvent, wherein the composition is a pharmaceutical composition or a food composition.

Description

Extract fraction from the seed of rosa multiflora for inhibiting the differentiation of adipocytic cells

1 is a photograph comparing the effect of the extraction and concentration of Youngsil extract with solvents such as ethyl acetate, ethanol and distilled water on the differentiation of NIH3T3-L1 precursor fat cells by Oil red O staining method.

Figure 2 is the same as Figure 1 is a photograph showing an enlarged microscopic view of the adipocytes treated with each extract.

FIG. 3 is a photograph showing that Youngsil ethanol extract is treated to NIH3T3-L1 cell line, which is a progenitor cell, and expression of the adipocyte differentiation-specific gene PPARγ is inhibited upon differentiation induction.

4 is a graph showing that Youngsil ethanol extract inhibits weight gain in mice.

Figure 5 is a photograph showing the inhibition of mouse abdominal adipose tissue increase Youngsil ethanol extract.

The active fraction according to the present invention exhibits an obesity prevention and suppression effect by inhibiting adipocyte differentiation.

Obesity is a condition in which excessive accumulation of fat in body fat tissue adversely affects health. Excessive calorie intake compared to consumption is the main cause of obesity, in addition to obesity due to endocrine disorders, lack of exercise, genetic factors, etc. Obesity is known as a cause of various adult diseases such as hypertension, type 2 diabetes, hyperlipidemia and arteriosclerosis to the extent that the World Health Organization defines it as a disease that needs treatment. According to the World Health Organization, as of 2004, obesity is responsible for 46% of all diseases and 59% of all deaths. In 2006, it is expected to increase rapidly to 60% of various diseases and 73% of all deaths, indicating the seriousness of obesity in modern society. In Korea, childhood obesity is gradually increasing due to the rapid change in diet, and the obesity population has increased by 3% every year for the past 5 years. As of 2004, 3 out of 10 adult populations exceeded normal weight. The government-led measures are required.

Obesity treatment includes methods of improving lifestyles such as exercise therapy, diet and behavioral therapy, and medication and body fat removal surgery. In order to recover to normal weight, lifestyle modifications such as exercise and diet control should be preceded, but this alone is limited in weight loss. Many drugs are being used or under development based on new mechanisms of action for the purpose of suppressing obesity. Anti-obesity drugs can be divided into drugs that affect the central nervous system and affect appetite and drugs that inhibit absorption by acting on the gastrointestinal tract. Drugs that suppress appetite by acting on the central nervous system include fenfluramine and dexfenfluramine, which inhibit serotonin (5-HT) reuptake, ephedrine and caffeine, which inhibits noradrenaline reuptake, and sibutramine, which simultaneously inhibits serotonin and noradrenaline absorption. There is this. Drugs that act on the gastrointestinal tract and inhibit obesity include orlistat, which inhibits the activity of lipases produced in the pancreas and reduces the absorption of fatty acids. However, some of these anti-obesity drugs have serious side effects and require careful prescription. Fenfluramine, an appetite suppressant, is not only classified as a drug but has been found to cause side effects such as primary pulmonary hypertension and heart valve lesions. Its use is limited for patients. Orlistat, which is expected to have no side effects because it acts in the intestine as an anti-lipase inhibitor, has been reported to cause gastrointestinal side effects such as bloating, frequent farts, fatty stools, and sudden changes, causing discomfort and inhibiting absorption of fat-soluble vitamins. Orlistat is also questioning the efficacy of weight loss in carbohydrate-based dieters.

One of the alternatives for reducing the side effects of each of the above drugs and for preventing and suppressing more basic obesity is adipocyte differentiation inhibitor. Until the early 1990s, adipose tissue was composed mainly of fat cells, and it was considered as an energy store that stores energy for the human body as triglyceride and decomposes and uses it when necessary. Since the mid-1990s, however, it has been found that adipose tissue is not just an energy store, but also secretes substances that affect other tissues. Currently secreted substances include TNF-α, IL-6, IL-8, plasminogen activator inhibitor-1, angiotensin-II, leptin and adiponectin, which are known to cause various metabolic and cardiovascular diseases [Proc . Nutl. Soc., 64, 163-169 (2005)]. Energy storage and endocrine secretion in adipose tissue are closely related to the differentiation and growth of adipocytes and co-accumulation process occurs. Adipocyte differentiation is the process of proliferating progenitor cells and differentiating them into new adipocytes, which accumulate triglycerides.Insulin, insulin like growth factor-1 (IGF-1), and growth hormone act on the proliferative cells as CCAAR enhancers. Transcription factors such as the binding protein (C / EBP) family and peroxisome-activated receptor γ (PPARγ) are activated. The growth of adipocytes results in the formation of non-large adipocytes through the accumulation of triglycerides in the already differentiated adipocytes without any change in adipocyte count. Obesity due to the increase of hypertrophy fat cells is mainly seen in adults and can be controlled by diet, but it is important to suppress the differentiation of fat cells because obesity caused by differentiation of adipocytes, which are mainly found in children and high obesity, is difficult to control by diet Do. Therefore, the research and development of drugs that can control the number of fat cells produced by inhibiting the differentiation of fat cells, and accordingly to control the excess energy accumulated is being actively progressed. However, the development of drugs as drugs requires a lot of cost and time for effective drug search and synthesis. On the contrary, since the herbal medicines used in traditional medicine have been used for a long time, there is a merit that there is little concern about side effects and toxicity, and there is a high possibility of discovering new active ingredients based on the confirmed drug efficacy.

In this regard, the present inventors explored the inhibitory effect of differentiation of progenitor cells, NIH3T3L-1 adipocytes, into the adipocytes of herbal medicines, which have been used in traditional Korean medicine, including consent, and prevented and inhibited obesity in high-fat dietary ICR mice. Observation of the vines ( Rosa multiflora var . Youngsil ethanol extract, a fruit of platyphylla ) , specifically inhibited adipocyte differentiation and confirmed a significant effect in a test for inhibiting obesity using a mouse, thus completing the present invention.

Honeysuckle is commonly used to treat arthritis and toothache. Petals are used as a lotion, and its fruit, Yeongsil, is known as an excellent medicine for treating women's menstrual pain, menstrual disorders, constipation, nephritis, cystitis, each species, and several species.

Accordingly, an object of the present invention is to provide a method for extracting an extract containing an active ingredient of Youngsil, which inhibits adipocyte differentiation and shows effectiveness in preventing and suppressing obesity, with a composition having the highest activity, and a herbal medicine using the extract. .

The present invention is characterized by an active fraction composition having both adipocyte differentiation inhibitory effect and obesity inhibitory effect obtained in Youngsil.

In the present invention, Youngsil is extracted with an organic solvent such as ethanol, and then concentrated under reduced pressure. The concentrated extract is suspended and distilled with distilled water to remove the water-soluble substance, and then a precipitate is obtained and freeze-dried. The organic solvent used to extract Youngsil may be selected from ethanol, methanol, propanol, butanol, ethyl acetate, chloroform, dichloromethane, and the like, but is not limited thereto.

On the other hand, in the present invention, in the preparation of Youngsil extract, Youngsil is extracted and concentrated with an organic solvent such as ethanol and suspended in distilled water, which is then again extracted and concentrated with a solvent such as ethyl acetate, chloroform, dichloromethane, etc. It further includes.

The present invention includes those used as an anti-obesity inhibitor and the like as the active ingredient extract according to the extraction method.

The present invention is described in more detail as follows.

The present invention relates to an extract of Youngsil, which shows the effect of inhibiting the differentiation of fat cells and preventing and inhibiting obesity. That is, the extract obtained from Youngsil inhibits adipocyte differentiation and inhibits obesity induction for the first time through a mouse by a high fat diet, thereby developing an active composition having an obesity prevention and suppression effect.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to Examples.

Example 1 Inhibitory Effect of Youngsil Extracts on Adipocyte Differentiation

Distilled water, 95% ethanol and ethyl acetate were compared to determine the best solvent for the extraction of active fractions that inhibit adipocyte differentiation from Youngsil. The dried Youngsil was extracted with distilled water (heated at 50 ° C.), 95% ethanol, and 100% methanol corresponding to 10 times the mass after pulverization for at least 72 hours. Each extract was filtered using 3M filter paper, and the distilled water extract and the 95% ethanol extract were concentrated under reduced pressure immediately. The methanol extract was concentrated under reduced pressure, suspended with distilled water of the same volume as methanol, and then added with the same volume of ethyl acetate, and mixed vigorously with a stirrer. The ethyl acetate layer was recovered and concentrated under reduced pressure. Each concentrate was then dissolved in 100 mg / ml in DMSO and tested for inhibiting adipocyte differentiation. Induction of differentiation of NIH3T3L-1 cells, which are progenitor cells, was as follows. When NIH3T3L-1 was incubated in DMEM containing 10% fetal bovine serum (FBS) and the cell density was about 90%, dexamethasone, IBMX, and insulin were treated for 48 to 72 hours to induce adipocyte differentiation. Was exchanged with fresh medium containing FBS and insulin. Adipocyte differentiation inhibitory activity was measured by treating 95% ethanol and ethyl acetate extracts with NIH3T3L-1 adipocytes at 25 ㎍ / ml from the beginning of differentiation induction for 7 days and then inhibiting the differentiation of adipocytes by Oil red O staining. Investigation was performed (FIGS. 1 and 2). Differentiation inhibitory effect test was performed in the following examples in the same manner. Distilled water extract showed no differentiation inhibitory effect, and ethyl acetate extract had the best differentiation inhibitory effect. Although the ethanol extract was weaker in activity than the ethyl acetate extract, it showed that the active ingredient was also extracted with ethanol because it showed the effect of inhibiting differentiation. Since the possibility of extracting ethanol from the active ingredient suggests the possibility of using it as a food in future, 95% ethanol will be used to extract the active ingredient. However, the selection of 95% ethanol as the extraction solvent does not limit the solvent selection of the present invention. If higher extraction efficiency and extraction solvent are required, ethyl acetate can be used as the extraction solvent.

Example 2 Preparation of Youngsil Ethanol Extract Sample

Distilled water was added to the ethanol extract and concentrate to remove impurities and remove water-soluble substances from the Youngsil ethanol extract to increase the concentration of the active ingredient. Distilled water corresponding to 1/10 of the ethanol use volume was added to the concentrate, and then suspended in a homogeneous manner, followed by centrifugation at 10,000 rpm for 10 minutes at room temperature to separate the supernatant and the precipitate. Inhibition of the differentiation of adipocytes in the supernatant and sediment of the aqueous layer did not show differentiation inhibitory effect in the aqueous layer, but only in the non-aqueous sediment. In the subsequent efficacy test using a mouse, ethanol extraction, concentration and subsequent removal of water-soluble substances were freeze-dried to obtain a powdery sample.

Example 3: Inhibition of Adipocyte Differentiation of Youngsil Ethanol Extract IC ₅₀ decision

Adipocyte differentiation inhibitory activity was measured by dissolving the active fractions extracted from Youngsil in DMSO and then treating them to concentrations of 12.5, 6.25, 3.1, 1.5 ㎍ / ml from the initial stage of induction of adipocyte differentiation and then differentiate them for 7 days. The degree of differentiation was observed by O staining. After microscopic observation, 1 mL of DMSO was added to the stained cells to extract the adsorbed Oil red O, and the degree of differentiation was quantitatively analyzed by measuring the absorbance at 505 nm. The degree of differentiation of each fraction treated group was expressed as a percentage compared to the absorbance of the control group. Youngsil ethanol extract inhibited more than 50% of adipocyte differentiation at 12.5 μg / ml or more (Table 1.).

 Example 4 Measurement of Inhibitory Effect of Adipocyte Specific Gene Expression

In order to confirm that Youngsil ethanol extract inhibits adipocyte differentiation, the effect of PPARγ on the expression regulated genes during adipocyte differentiation was confirmed by Western blot. After treatment of the adipocyte differentiation with 25 ㎍ / ㎖ youngsil ethanol extract PPARγ expression according to the differentiation time was compared with DMSO treatment group (Fig. 3). In the control group, the expression of PPARγ was increased from the first day of induction, whereas the increase in expression was delayed by one day when the Youngsil ethanol extract was treated. The above results show that Youngsil ethanol extract inhibits fat differentiation by controlling gene expression related to differentiation of adipocytes.

Example 5: ICR  Obesity Prevention and Suppression Effects in Mice

When ICR mice consume high-fat diets containing 40% or more fat, their body weight rapidly increases, and after three months, their body weight is more than doubled compared to mice fed a normal diet. To investigate the effect of Youngsil on obesity, the test was performed by mixing Youngsil ethanol extract with high fat diet and free feeding to 5 weeks old mice weighing 25g. Ten mice were tested divided into five groups in one group. Each group was fed a normal diet control diet, high fat diet containing 3% casein in high fat diet, Youngsil 0.5% test group containing 0.5% Youngsil ethanol extract and 2.5% casein, Youngsil 3% containing 3% Youngsil ethanol extract In the test group and 3% of casein, the JENAL group included 0.024% of XENAL, a drug that inhibited lipolysis and absorption. Mice of each test group were measured once a week body weight and feed intake. There was no significant difference in feed intake between all groups fed free-fat diets, but weight gain showed a decrease in weight gain in young-sil fed groups. As a result of comparative analysis of body weight of the high fat control group including 3% test group and 3% casein, the body weight was significantly lower in the 3% test group compared to high fat control group after 2 weeks. Compared to the average high-fat control body weight after 4 weeks of 34 ± 1.3 g (mean ± standard error), the Youngsil 3% test group weighed 29.9 ± 0.5 g and had a weight suppression effect of about 12% (p <0.02). It was observed that Youngsil ethanol extract inhibited obesity at about the same level as the weight measurement in the normal diet control group (FIG. 4). Abdominal fat change was observed by sacrifice of mice treated with Youngsil ethanol extract for 5 weeks (FIG. 5). The Youngsil treated 3% test group had less abdominal adipose tissue and was similar to the normal diet control group compared to the high fat diet control group. It was confirmed that Youngsil ethanol extract inhibits the increase of abdominal adipose tissue.

Example 6 Preparation of Feed

High-fat diet containing more than 40% fat mixed Youngsil ethanol extract 0.5%, 3%, JENNIAL 0.024%, casein 3%. It was prepared in the form of feed of a constant size and stored in a refrigerator until use. The composition of the feed is shown in Table 2.

As described above, the active fraction composition of Youngsil obtained in the present invention is not known the exact mechanism of action, but inhibits adipocyte differentiation, and can be used as an active ingredient containing it as an active ingredient because it has an excellent effect of preventing and inhibiting obesity in animal tests. .

Claims (7)

Youngsil extract having adipocyte differentiation inhibitory activity, anti-obesity or anti-obesity effect. A) extracting an organic solvent from Youngsil to obtain a crude extract, b) filtering the crude extract and then concentrating (decompression) and optionally c) removing the solvent. The method of claim 2, wherein the organic solvent is selected from ethanol, methanol, propanol, butanol, ethyl acetate, chloroform and dichloromethane. According to claim 2, Extraction and concentration with alcohols having 1 to 4 carbon atoms suspended in distilled water and then again extracted and concentrated with a solvent selected from ethyl acetate, chloroform, dichloromethane of the Youngsil extract Manufacturing method Youngsil extract having adipocyte differentiation inhibitory activity, obesity prevention or obesity inhibitory effect obtained by the method of any one of claims 2 to 4. A pharmaceutical composition comprising a Youngsil extract having adipocyte differentiation inhibitory activity, anti-obesity or anti-obesity effect, and a pharmaceutically acceptable carrier or excipient. Edible beverages, foods or food additives containing Youngsil extracts having an adipocyte differentiation inhibitory activity, anti-obesity or anti-obesity effect.

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