ANTI-OBESITY FUNCTIONAL FEED COMPOSITION CONTAINING HIBISCUS MANIHOT L. EXTRACT AS ACTIVE INGREDIENT FOR COMPANION ANIMALS

Abstract

Proposed is an anti-obesity functional feed composition for companion animals, the composition including Geumhwagyu (Hibiscus manihot L.) extract as an active ingredient. The Geumhwagyu (Hibiscus manihot L.) extract induces the degradation of CEBPα (CCAAT/enhancer-binding protein-α), thereby inhibiting the differentiation of preadipocytes. In addition, the Geumhwagyu (Hibiscus manihot L.) extract inhibits the expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein-α (CEBPα), perilipin-1, adiponectin, fatty acid binding protein-4 (FABP4), etc., thereby inhibiting the accumulation of lipids in adipocytes. Thus, the Geumhwagyu (Hibiscus manihot L.) extract shows anti-obesity activity by lowering the content of triacylglycerol (TG).

Claims

1. A method of treating obesity for companion animals, comprising: administering an anti-obesity functional feed composition containing Geumhwagyu (Hibiscus manihot L.) extract or a fraction thereof as an active ingredient to a subject.

2. The method of claim 1, wherein the extract is extract of Geumhwagyu (Hibiscus manihot L.) flowers, stems, leaves, or roots.

3. The method of claim 1, wherein a solvent used to obtain the extract is water, hot water, C.sub.1-4 alcohol, or a mixed solution thereof.

4. The method of claim 1, wherein the extract has an effect of inhibiting expression of Peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein-α (CEBPα), Perilipin-1, Adiponectin, or FABP4 (Fatty acid binding protein-4 which is a protein associated with lipid accumulation in adipocytes.

5. A method of treating obesity for companion animals, comprising: administering an additive for an anti-obesity functional feed composition containing Geumhwagyu (Hibiscus manihot L.) extract or a fraction thereof as an active ingredient to a subject.

6. The method of claim 5, wherein the extract is extract of Geumhwagyu (Hibiscus manihot L.) flowers, stems, leaves, or roots.

7. The method of claim 5, wherein a solvent used to obtain the extract is water, hot water, C.sub.1-4 alcohol, or a mixed solution thereof.

8. The method of claim 5, wherein the extract has an effect of inhibiting expression of Peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein-α (CEBPα), Perilipin-1, Adiponectin, or FABP4 (Fatty acid binding protein-4 which is a protein associated with lipid accumulation in adipocytes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 includes Oil Red O-stained cell images showing lipid formation when treated with flower extract (HMF), leaf extract (HMF), stem extract (HMF), and root extract (HMF) of Geumhwagyu (Hibiscus manihot L.) and includes a graph showing the optical density of each cell lysate;

[0023] FIG. 2 includes Oil Red O-stained cell images and Western blot test images of Geumhwagyu (Hibiscus manihot L.) leaf extract (HMF), the images showing the optical density, TG content, and control of expression of lipid formation-related proteins (PPARγ, CEBPα, perilipin-1, adiponectin, FABP4) etc.) of cell lysates;

[0024] FIG. 3 shows the results of analysis of the effect of Geumhwagyu (Hibiscus manihot L.) leaf extract (HMF) on the lipid formation of adipocytes according to the treatment time, and includes Oil Red O-stained cell images and Western blot test results showing lipid accumulation in cells, the optical density of the cell lysates, and the expression of lipid formation-related proteins;

[0025] FIG. 4 shows the lipid accumulation inhibitory effect of Geumhwagyu (Hibiscus manihot L.) leaf extract (HMF) extracted with water and an ethanol aqueous solution, the effect being observed through the Oil Red O staining; and

[0026] FIG. 5 shows the lipid accumulation inhibitory effect of Geumhwagyu (Hibiscus manihot L.) leaf extract (HMF) extracted with water and a methanol aqueous solution, the effect being observed through Oil Red O staining.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Hereinafter, preferred embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments are provided so that this disclosure will be thorough and complete and will fully convey the spirit of the invention to those skilled in the art.

Example 1: Preparation of Geumhwagyu (Hibiscus Manihot L.) Extract

[0028] Flower, leaf, stem, and root extracts of Geumhwagyu (Hibiscus manihot L.) were prepared through a method described below. First, the flowers, leaves, stems, and roots of Geumhwagyu (Hibiscus manihot L.) plants grown by E-Farm Co., Ltd., which is an agricultural company located in Punggi, Gyeongsangbuk-do, Korea, were washed with distilled water and dried. For each of the dried flowers, leaves, stems, and roots, 10 g of the product pulverized plant part was taken and immersed in water at 40° C. for 12 hours to obtain extracts of the flowers, leaves, stems, and roots of Geumhwagyu (Hibiscus manihot L.). In this case, the volume of the water was about 20 times the volume of the dried pulverized plant part. Thereafter, each of the extracts was filtered and then lyophilized to obtain the final extracts of Geumhwagyu (Hibiscus manihot L.) flowers (HMF), leaves (HMF), stems (HMF), and roots (HMF).

Example 2: Cytotoxicity Assay

[0029] In order to check whether the Geumhwagyu (Hibiscus manihot L.) extracts of the present disclosure obtained in Example 1 were toxic to cells, cytotoxicity was analyzed in vitro.

[0030] To this end, 3T3-L1, which is a preadipocyte, was purchased from KCLB and used. After the cells were dispensed in a cell culture flask, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/ml) were added to a DMEM medium, and the cells were cultured in an incubator maintained at 37° C., 5% CO.sub.2, and 95% humidity conditions, and then used. For cytotoxicity assay, the MTT assay was used. The method uses a principle that dehydrogenases in mitochondria of cells that are normal in their metabolism reduce a yellow water-soluble of tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltrazolium bromide] (MTT) to non-soluble dark purple MTT formazan crystals. The optical density of the crystals was measured at an appropriate wavelength, for example, mainly 500 to 600 nm, for the evaluation of cytotoxicity.

[0031] First, the preadipocytes (3T3-L1) were dispensed into 96 wells at a concentration of 1×10.sup.5 cells/well and then cultured for 24 hours. Next, the cultured cells were treated with each of the Geumhwagyu (Hibiscus manihot L.) flower, leave, and stem extracts prepared in Example 1, at various concentrations including 0, 50, 100, and 200 μg/mL, and the treated cells were cultured for 24 hours. Next, the MTT was added at a concentration of 1 mg/mL and, and the reaction was performed in an incubator at 37° C. for 2 hours. After the reaction, dimethyl sulfoxide (DMSO) was added, and the optical density was measured at 570 nm using a microplate reader.

[0032] As a result of the analysis, it was confirmed that all of the Geumhwagyu (Hibiscus manihot L.) extracts had no cytotoxicity, and then an experiment described below was performed. The results of only the leave extract are shown in FIG. 2D, but the results of the other extracts are not shown.

Example 3: Checking Activity of Lipid Accumulation Inhibitory Activity, Triglyceride (TG) Formation Inhibitory Activity, and Inhibitory Activity for Expression of Lipid-Accumulation-Related Proteins

[0033] 3T3-L1 cells, which are preadipocytes, were dispensed into 96 wells at a concentration of 1×10.sup.5 cells/well and cultured for 3 days. Next, the cultured cells were differentiated into an MDI medium (adipogenic medium). As the MDI medium, 10% FBS/DMEM treated with 0.5 mM IBMX, 1 μM dexamethasone, and 1 μg/ml insulin was used. This time point was regarded as Day 0 of differentiation, and after 2 days of differentiation, the used medium was replaced with 1 μg/ml of insulin medium. After 2 days of culture, the used medium was replaced with 10% FBS medium, and this condition was maintained until Day 8. The Geumhwagyu (Hibiscus manihot L.) extract was added on Day 0 of differentiation so that the cells were treated with the Geumhwagyu (Hibiscus manihot L.) for 8 days. After 8 days of differentiation, the degree of differentiation of adipocytes and the degree of inhibition of differentiation by the Geumhwagyu (Hibiscus manihot L.) extract were analyzed through Oil Red O staining. After the completion of the differentiation of adipocytes and the treatment with the Geumhwagyu (Hibiscus manihot L.) extract, the cells were washed with 1× phosphate-buffered saline (PBS), followed by addition of 10% (w/v) formalin was, and were fixed at room temperature for 1 hour. After removing the formalin and washing each well used to culture the cells with 60% (v/v) isopropanol, the isopropanol was completely removed under a hood. An Oil Red O solution was added to the dried cell culture wells, and the wells left intact for 10 minutes and then washed with distilled water. Next, adipocyte differentiation was observed through microscopic imaging. The substances remaining in the stained cell culture wells were dissolved in 100% isopropanol, and the optical density of the obtained solution was measured at 500 nm. The amount of triglyceride (TG) in the adipocytes was measured using Triglyceride Assay Kit Quantification, ab65336, which is a commercially available TG assay kit.

[0034] For the analysis of proteins, 3T3-L1 cells, which are preadipocytes, were dispensed into 24 wells at a concentration of 1×10.sup.5 cells/well, cultured for 3 days, and differentiated with a MDI medium (adipogenic medium). This time point was regarded as Day 0 of differentiation, and after 2 days of differentiation, the used medium was replaced with 1 μg/ml of an insulin medium. After 2 days of culture, the used medium was replaced with a 10% FBS medium, and this condition was maintained until Day 8. The Geumhwagyu (Hibiscus manihot L.) extract was added on Day 0 of differentiation so that the cells were treated with the Geumhwagyu (Hibiscus manihot L.) for 8 days. After 8 days of differentiation, the cells were harvested and the expression of lipid accumulation-related proteins was investigated through Western blot analysis.

[0035] The test results are summarized in FIGS. 1 to 3.

[0036] FIG. 1 includes Oil Red O-stained cell images showing lipid formation when treated with flower extract (HMF), leaf extract (HMF), stem extract (HMF), and root extract (HMF) of Geumhwagyu (Hibiscus manihot L.) and includes a graph showing the optical density of each cell lysate. Referring to FIG. 1, it is confirmed that the extracts of Geumhwagyu (Hibiscus manihot L.) roots, stems, leaves, and flowers inhibit lipid formation.

[0037] FIG. 2 includes Oil Red O-stained cell image for showing lipid accumulation when treated with an extract of Geumhwagyu (Hibiscus manihot L.) leaves, graphs showing the optical density and TG content of each cell lysate, and Western blot test images showing control of expression of lipid formation-related proteins. Referring to FIG. 2, it is confirmed that the Geumhwagyu (Hibiscus manihot L.) leaf extract inhibits the formation of lipid, the accumulation of TG, and the expression of PPARγ, CEBPα, Perilipin-1, Adiponectin, FABP4, etc., which are proteins associated with lipid formation, in a concentration-dependent manner.

[0038] FIG. 3 shows the results of analysis of the effect of Geumhwagyu (Hibiscus manihot L.) leaf extract on the lipid formation of adipocytes according to the treatment time. FIG. 3 includes Oil Red O-stained cell images and Western blot test results showing lipid accumulation, the optical density of each cell lysate, and the expression of lipid formation-related proteins. The Geumhwagyu (Hibiscus manihot L.) leaf extract inhibited lipid accumulation, TG content, and expression of PPARγ, CEBPα, Perilipin-1, Adiponectin, and FABP4, which are proteins associated with lipid accumulation, in a treatment time-dependent manner.

[0039] From these results, it was confirmed that when cells were treated with Geumhwagyu (Hibiscus manihot L.) extract after differentiation of the cells from preadipocytes to adipocytes, the lipid accumulation inhibitory effect was about 60% or more, preferably about 60% to 80% at a concentration of 50 μg/ml or more with respect to the adipogenic cells. In addition, the suppression of TG content and the suppression of the expression of lipid formation-related proteins prove that the Geumhwagyu (Hibiscus manihot L.) extract has anti-obesity activity even in the late stage of lipid accumulation in adipocytes.

[0040] On the other hand, the effect of the Geumhwagyu (Hibiscus manihot L.) leaf extract was compared among the ethanol aqueous solution extract, the methanol aqueous solution extract, and the water extract, and the comparison results are shown in FIGS. 4 and 5. The ethanol aqueous solution extract and the methanol aqueous solution extract were prepared through extraction using an extraction solvent instead of water, followed by distillation under reduced pressure.

[0041] Referring to FIGS. 4 and 5, the lipid accumulation inhibitory effect was about 60% or more and preferably about 60% to 70% (v/v) when 50 μg/ml of the water extract was used. That is, the water extract exhibited the remarkable anti-obesity effect. On the other hand, the ethanol 30% to 70% (v/v) aqueous solution extract and the methanol 30% to 70% (v/v) aqueous solution extract exhibited only about 10% of the lipid accumulation inhibitory effect at the same concentration, so the effect was insignificant.

<Example 4: Checking Weight Loss Efficacy

[0042] Three 1-year-old male Pomeranians (having an average weight of about 4 kg) which were neutered at 6 months of age, were fed Royal Canin mini (in-door) feed in an amount according to their body weight for 1 month. In this case, 100 mg of Geumhwagyu (Hibiscus manihot L.) water extract powder was sprinkled on the feed once a day. As a result, the appropriate weight loss effect was confirmed without negatively affecting the health of the test animals during the test period.