KR20140140346A - Composition for anti-obesity effect comprising Liriodendron tulipifera L. extract - Google Patents

Abstract

An effect of controlling fat accumulation of acetylacetate extract of Liriodendron tulipifera L. leaf was tested. The cell survival rate of cells exposed to a Liriodendron tulipifera L. extract (25-200 μg/ml) for 72 hours was not influenced when compared with an untreated control group. The Liriodendron tulipifera L. extract inhibited fat accumulation of 3T2-L1 fat cells in concentration of 100-200 μg/ml relying on dose. The expression of PPARγ and C/EBPα which are main markers of adipogenesis was significantly reduced in the case where the cells were treated with a Liriodendron tulipifera L. extract for eight days, compared with the untreated control group (p<0.005). Disclosed in the result is usage of a Liriodendron tulipifera L. extract as a natural anti-obesity substance for a treating agent.

Description

[0002] The present invention relates to an anti-obesity composition comprising a tree lily leaf extract and a composition comprising an anti-obesity effect comprising Liriodendron tulipifera L. extract,

The present invention relates to an anti-obesity composition containing a tree lily leaf extract, wherein the tree lily extract inhibits fat accumulation of adipocytes in a dose-dependent manner, and when the cellulase extract is treated for 8 days, The expression of the markers PPARgamma and C / EBPa was significantly reduced when compared to the untreated control group.

Obesity has increased significantly since the 1950s. Currently, over 30% of adults are obese (Lankford et al ., 2013). There is a strong correlation between obesity and various life-threatening diseases such as cardiovascular disease, type 2 diabetes, hyperlipidemia and arthritis (Visscher and Seidell, 2001). Currently, cardiovascular disease is the most common cause of death in the West (Kim et al ., 2012). The causes of obesity are energy inequalities due to overeating and lack of exercise (Reaven et al ., 2004). Therefore, a healthy diet and appropriate physical activity are very important for obesity management. Several drugs have been used to treat obesity (Kolanowski, 1999). However, only orlistat is currently approved for the long-term use of obesity treatment because it has no serious side effects (Zhang and Huang, 2012). Therefore, new attempts to treat obesity are required.

Adipose tissue functions as a main site for energy supply by releasing fatty acids by hydrolyzing stored triglycerides (neutral fat) and serves as a storage site for dietary fat (Stapleton et al ., 2013). The triglyceride level in adipose tissue plays an important role in the balance between lipogenesis and lipolysis, which is closely related to the amount of fat cells (Marques et al ., 1998). Fat tissue enlargement is attributed to increased fat cell size and increased fat cell count (Jung et &lt; RTI ID = 0.0 &gt; al ., 2011).

Peroxisome proliferator-activated receptor-γ (PPARγ) and C / EBPα (CCAAT / enhancer binding protein-α) have been reported as major transcription factors in adipocyte production (Tang et al ., 2003). The amount of adipose tissue can be reduced by inhibiting adipocyte production through regulation of these major transcription factors (Kong et al ., 2010).

Synthetic drugs for the treatment of obesity are expensive and have serious side effects (Kolanowski, 1999). To overcome these problems, studies on medicinal plants and natural products have been conducted (Vasudeva et al ., 2010).

Liliodendron tulipifera L. is originated in North America and is known as tulip tree (TT) or yellow poplar (Graziose et al ., 2011). Peels have traditionally been used by Native Americans as tonic and malaria treatments (Graziose et al., 2011). The bark is called anti-malaria (Graziose et al ., 2011), antioxidant (Xu et al ., 2011) and anticancer effects (Xu et al ., 2011) have been reported. In addition, the leaf is an anti-bacterial (Bae et al ., 1990), anti-tumor (Moon et al ., 2007) and anti-malarial effects (Graziose et al ., 2011) have been reported to have some efficacy. However, there have been few studies on antiobesity effects.

The present invention aims to provide an anti-obesity composition which inhibits adipocyte production without side effects. The anti-obesity composition represents a pharmaceutical composition or food.

The present inventors screened anti-adipocyte production activity against dozens of natural products, selected natural products having high activity among them, and selected a tree lily leaf extract having no cytotoxicity and high anti-obesity effect, And its applicability to food.

For adipocyte differentiation Tree lily  Effect of leaf extract on anti-adipocyte formation

3T3-L1 adipocyte precursor cells were used for anti-adipocyte production activity screens for dozens of natural products, treated with 100 μg / ml of a tree lily leaf extract and its fraction extracts (hexane, ethyl acetate and water) , The ethyl acetate fraction showed significant inhibitory activity on adipocyte formation. The inhibitory effect of the tree lily leaf extract on adipocyte production was observed with oil red O staining. As shown in the morphological observation, fat accumulation in the tree lily leaf extract treated cells was very low as compared to untreated control cells (Fig. 1a). The cellulase treatment of the tree lily leaf extract significantly decreased the fat accumulation as compared to the untreated control cells (Fig. 1B). The results showed that the tree lily leaf extract at concentrations of 100 and 200 ug / ml inhibited fat accumulation by 23 and 49%, respectively, of untreated control cells.

However, in the experiment using differentiated adipocytes, the inhibition effect of the tree lily leaf extract was hardly shown (data not shown). These results suggest that tree lily leaf extract has anti - adipogenic effect.

For cell survival rate Tree lily  Effect of leaf extract

In order to investigate the effect of tree lily leaf extract on 3T3-L1 cell survival in early stages of adipocyte differentiation, differentiated 3T3-L1 cells were seeded at various concentrations (0, 25, 50, 100 and 200 / / Lily leaf extract for 72 hours. Cells treated with tree lily leaf extract had no effect on cell viability as compared to control cells (Figure 2).

PPAR gamma and C / EBP for α expression Tree lily  Effect of leaf extract

PPARy and C / EBPa are major transcription factors during adipocyte differentiation (Lee et < RTI ID = 0.0 &gt; al ., 2011). Therefore, the present inventors analyzed the major transcription markers involved in adipocyte production to examine the molecular mechanism of the effect of the tree lily leaf extract on anti-adipocyte production. After 3T3-L1 adipose precursor cells were exposed to the woody lily leaf extract for 8 days, we measured the PPARγ and C / EBPα protein levels. Cells treated with the tree lily leaf extracts significantly inhibited the expression of these proteins compared to the untreated control cells (FIG. 3). PPARγ expression was significantly reduced (p <0.005, p <0.005) compared to untreated control cells at 100 and 200 μg / ml of tree lily leaf extract for 8 days (Fig. 3a). The expression of C / EBPα was statistically significantly reduced (p <0.005, p <0.005) (Fig. 3b) when exposed to 100 and 200 ㎍ / ㎖ of tree lily leaf extract for 8 days compared to untreated control cells.

PPARy has also been reported as a major regulator of inflammation and immune responses (Ricote and Glass, 2007). In general, obesity is associated with low levels of chronic inflammation in adipose tissue (Xu et al ., 2003), PPARgamma has been attracting attention as a new target of therapeutic agents for the treatment of chronic inflammatory diseases such as obesity-induced insulin resistance, atherosclerosis and neurodegenerative diseases (Kostadinova et &lt; RTI ID = al ., 2005).

In conclusion, L. lily leaf extract significantly inhibited adipocyte production without affecting cell survival by controlling the expression of PPARγ and C / EBPα. Therefore, the tree lily leaf extract can be used as an agent for preventing and / or treating obesity.

The present invention relates to an anti-obesity pharmaceutical composition comprising a tree lily leaf extract.

The present invention is characterized in that the tree lily leaf extract is extracted with a solvent selected from the group consisting of hot water, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, butanol, acetone, aqueous acetone solution, dichloromethane, ethyl acetate, do.

The present invention also relates to an anti-obesity food comprising a tree lily leaf extract.

The food may be prepared in various forms such as beverage form, health functional food, bread, pastry, and the like.

The present invention also relates to a method of extracting the above-mentioned tree lily leaf extract with a solvent selected from the group consisting of hot water, methanol, methanol aqueous solution, ethanol, aqueous ethanol solution, butanol, acetone, aqueous acetone solution, dichloromethane, ethyl acetate, &Lt; / RTI &gt;

One embodiment of a method for obtaining a tree lily leaf extract is as follows.

The lily blossom leaves are shaded, and the organic solvent is added in an amount of 2 to 200 times, preferably 10 to 30 times the volume of the three-lipped lily blossom. The mixture is heated at 10 to 30 DEG C for 1 to 20 days, To 10 days, filtered, and concentrated under reduced pressure to prepare a tree lily leaf extract. The extraction solvent may be selected from the group consisting of methanol, aqueous methanol, ethanol, aqueous ethanol, butanol, acetone, aqueous acetone, dichloromethane, ethyl acetate, and mixtures thereof. It is also possible to extract hot water at 70 ~ 100 ℃.

Extracting the tree lily leaf extract obtained by extracting with the organic solvent in the production of the tree lily leaf extract, at least one step of re-extracting with a solvent selected from the group consisting of hexane, dichloromethane, ethyl acetate and butanol, Leaf extract can be prepared.

The pharmaceutical composition comprising the tree lily leaf extract of the present invention may be mixed with a pharmaceutically acceptable carrier or excipient or diluted with a diluent to prepare a pharmaceutical composition having the above-mentioned function. Examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltodol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition containing the tree lily leaf extract may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The pharmaceutical compositions of the present invention may be formulated using methods well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to mammals such as rats, livestock, humans, and the like. The formulations herein may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders and the like.

The pharmaceutical composition of the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. A typical daily dose of the pharmaceutical composition of the present invention is 10 mg / kg body weight to 100 mg / kg body weight, preferably 10 mg / kg body weight to 30 mg / kg body weight, based on the active ingredient It is a range of body weight and can be administered once or several times. However, the actual dosage of the active ingredient should be determined in light of various relevant factors, such as the route of administration, the age, sex, and weight of the patient, and the severity of the disease, and accordingly, It does not.

The present invention includes an anti-obesity food comprising a tree lily leaf extract.

The present invention can provide a health food or beverage composition capable of preventing or alleviating obesity and obesity complications, including a tree lily leaf extract.

Examples of foods to which the tree lily leaf extract of the present invention can be added include various foods, beverages, confectionery, gum, ice cream, tea, instant tea, granules, flavorings, vitamin complexes and other health supplement foods , But is not limited thereto.

The above-described tree lily leaf extract of the present invention may be added to the raw material during the production of the food or may be appropriately mixed with the cooked food to produce the food for health promotion or beverage. In this case, The content of the extract may range from 0.01% to 30% by weight.

The pharmaceutical composition of the present invention, or the food or drink for health promotion, may further include other herbal medicines or extracts thereof that are pharmaceutically acceptable for elevating or supplementing the desired effect. Representative examples of such herbal medicines include, Angelica, angelica, and angiosperm. The herbal medicine may be used in an amount of 0.01 wt% to 50 wt% based on the total weight of the composition.

The composition comprising the tree lily leaf extract of the present invention is excellent in the effect of inhibiting adipocyte differentiation, inhibiting fat accumulation and inhibiting adipocyte formation, and thus being useful for prevention and treatment of obesity.

Figure 1 shows the effect of the tree lily leaf extract on fat accumulation in adipocyte differentiation. 3T3-L1 preadipocytes were treated with samples during the entire differentiation process (0-8 days). Representative microscopic morphological images of adipocytes were treated with 0, 25, 50, 100 and 200 μg / ml of tree lily leaf extract, followed by dyeing with oil red O 8 days later, and microscopic photographs were taken at 200 times (A). The absorbance measured from the elution of isopropanol from the oil red O-stained cells is shown in (B). Values were expressed as mean ± standard deviation of three independent experiments. * P <0.05, *** P <0.005 compared with untreated control group. "TT" means tree lily leaf extract.
Figure 2 shows the effect of tree lily leaf extract on survival of differentiated adipocytes. 3T3-L1 cells were fully cultured and then cultured for 72 hours with adipogenic cocktail and various concentrations of woody lily leaf extract (0, 25, 50, 100 and 200 ug / ml). CCK-8 assays to test cell viability were performed 72 hours after treatment. Values are expressed as mean ± standard deviation for three independent experiments. "TT" means tree lily leaf extract.
Figure 3 shows the effect of the tree lily leaf extract on protein expression associated with adipocyte formation by Western blot analysis. The results show that PPARγ (A) and C / EBPα (B) expression levels were decreased in 3T3-L1 cells treated with woody lily leaf extract on the eighth day of differentiation. These signals were normalized to β-actin. Values are expressed as mean + standard deviation for three independent experiments. * P <0.05, *** P <0.005 compared with the untreated control group. "TT" means tree lily leaf extract.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of the embodiments.

Reagents and samples

3T3-L1 adipose precursor cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). 3-isobutyl-1-methylxanthine, oily red O solution and dexamethasone were purchased from Sigma-Aldrich Chemical (St. Louis, Mo., USA), DMEM (Dulbecco's modified Eagle's medium), beta-actin antibody, insulin. FBS, NCS (newborn calf serum), 25% trypsin-EDTA, antibiotics and antimicrobial agents were purchased from Gibco (Rockville, MD, USA). Cellular Factor Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Quant-iT protein assay kit was purchased from Invitrogen (Auckland, New Zealand), and antibodies against PPAR and C / EBP were obtained from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Tree lily  Extract preparation

Liliodendron tulipifera L. ) leaves were provided by Kangwon Provincial Forest Development Institute. The guarantee sample (G-11-15) was deposited in the Gangwon-do Forest Research Institute Plant Specimens Room. The tree lily blades were pulverized and extracted twice with 70% acetone (2 × 10 L) at room temperature for 3 days.

Cell culture

3T3-L1 adipose precursor cells were treated with 5% CO ₂ in DMEM containing 10% NCS, 100 IU / ml penicillin and 100 ug / ml streptomycin And kept at 37 째 C until the cells were sufficiently grown. To induce adipocyte formation, the cells were cultured until the next two days after full growth, and then induced by adding a differentiation-promoting mixture containing 5 / / ml insulin, 0.5 袖 M dexamethasone and 0.5 袖 M IBMX to DMEM containing 10% FBS. The medium was then changed once a day with 10% FBS / DMEM containing 5 μg / ml insulin. Samples were treated with 0, 25, 50, 100, and 200 ㎍ / ㎖ of tree lily leaf extract at the start of differentiation, and each medium was changed every 2 days and treated for a total of 8 days.

oil Red  O dyeing

Oil Red O staining was performed as follows. The differentiated adipocytes were washed with PBS and fixed with PBS containing 10% formalin for 60 minutes at room temperature. The fixed cells were washed with PBS three times, then stained with filtered Oil Red O solution for 30 minutes at room temperature, and then the cells were washed twice with distilled water. Cell morphology was confirmed with an inverted microscope (ZEISS, Oberkochen, Germany) and images were taken using a digital camera (Nikon, Tokyo, Japan). Dyed oil droplets were dissolved in isopropanol and quantitated at 450 nm using a fluorescence microplate reader (Molecular Devices). Values were calculated as percentage compared to untreated control and expressed as mean ± standard deviation.

Cell survival analysis

Cell viability was determined using CCK-8. Briefly, 3T3-L1 cells were stimulated with adipocyte-forming promoting cocktail in the absence or presence of 0, 25, 50, 100, and 200 ug / ml thyme lily leaf extracts, 5,000 cells per well) for 72 hours. After each indicated time, the DMEM containing the test substance was replaced with DMEM containing 20 μl of CCK-8 solution and cultured in the dark for two hours. Survival amount of dehydrogenase activity in the cells was detected by measuring the absorbance at 450 ㎚ a fluorescent micro-plate reader ^{(Spectramax / M2 e, Molecular Devices} , Sunnyvale, CA, USA). Each analysis was performed three times.

Western Blat  analysis

Cells were seeded in 100 mm dish. Adipocyte differentiation and tree lily leaf extract treatment were performed as described above. Cell extracts were prepared by adding protein extraction solution (PRO-PREP, Intron Biotechnology, Sungnam, Korea). The protein content in the cell lysate was determined by the Bradford method. Protein (20 μg) was separated by 10% SDS-PAGE and then blocked with 5% skim milk powder in TBS containing 0.1% Tween 20. Blots were over-night incubated at 4 ° C with goat anti-mouse IgG-HRP secondary antibody in primary antibody and blocking solution containing PPARγ (1: 200) and C / EBPα (1: 5000) Target proteins were detected with ECL Western blotting detection reagent (GE Healthcare, Seoul, South Korea).

Statistical analysis

Data are presented as mean ± standard deviation. Data were analyzed using student's t test to determine statistical significance. p <0.05 was considered statistically significant.

&Lt; Preparation Example 1 >

100 mg of a tree lily leaf extract, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and filled in gelatin capsules according to a conventional capsule preparation method to prepare capsules.

&Lt; Preparation Example 2 >

100 mg of tree lily leaf extract was dissolved in distilled water and adjusted to pH 7.5 with a pH regulator. Then, 10 ml of water for injection and 5 mg of a pH adjuster were added to distilled water for injection to prepare a 2 ml volume ampoule And sterilized to prepare an injection.

&Lt; Production Example 3 >

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then powdered into a powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame, and perilla were steamed and dried by a known method, and then powdered into 60 mesh powder by a grinder. The tree lily leaf extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried with a spray hot air dryer, and pulverized to a size of 60 mesh with a pulverizer to obtain a dried powder of tree lily leaf extract. The granules were prepared by blending dry powders of the above-prepared grains, seeds and tree lily leaves extract in the following proportions.

Cereals: 30% by weight of brown rice, 15% by weight of yulmu, 20% by weight of barley, 9% by weight of glutinous rice,

Seeds: 7% by weight of perilla, 8% by weight of black beans, 7% by weight of black sesame,

Tree lily leaf extract 4%

&Lt; Preparation Example 4 >

0.0001 wt% of nicotinic acid amide, 0.0001 wt% of sodium riboflavin hydrochloride, 0.0001 wt% of pyridoxine hydrochloride, 0.001 wt% of inositol, 0.002 wt% of orthoacetic acid and 98.7362 wt% of water, And 1% by weight of a tree lily leaf extract were mixed to prepare a health drink.

&Lt; Preparation Example 5 >

A mixture of 55% by weight of Spirulina, 10% by weight of guar gum enzyme hydrolyzate, 0.01% by weight of vitamin B1 hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, And 10% by weight of the tree lily leaf extract of the present invention were mixed to prepare a refillable health supplement.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as defined in the appended claims. It can be understood that

Claims (5)

An anti-obesity pharmaceutical composition comprising a tree lily leaf extract.
The method according to claim 1,
Wherein the tree lily leaf extract is extracted with a solvent selected from the group consisting of hot water, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, butanol, acetone, aqueous acetone solution, dichloromethane, ethyl acetate and mixtures thereof. A pharmaceutical composition.
An anti-obesity food containing a tree lily leaf extract.
The method of claim 3,
Wherein said food is in the form of a drink.
The method of claim 3,
Wherein the tree lily leaf extract is extracted with a solvent selected from the group consisting of hot water, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, butanol, acetone, aqueous acetone solution, dichloromethane, ethyl acetate and mixtures thereof. food.

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