KR20150066729A - A Composition for preventing and treating fatty liver comprising Houttuynia cordata extract - Google Patents

Abstract

The present invention relates to a composition for preventing or treating fatty liver, containing a Huttuynia cordata extract as an active ingredient and, more specifically to a composition for preventing or treating fatty liver, containing a Huttuynia cordata ethyl acetate (HC-EA) extract as an active ingredient.

Description

[0001] The present invention relates to a composition for prevention or treatment of fatty liver comprising an extract of Aspergillus oryzae as an active ingredient,

The present invention relates to a composition for prevention or treatment of fatty liver comprising an extract of Aspergillus oryzae as an active ingredient, and more particularly to a composition for prevention or treatment of fatty liver comprising an extract of Aspergillus ethyl acetate (HC-EA) as an active ingredient.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in developing countries and has become a major challenge in health care systems. Once progressed, NAFLD results in non-alcoholic steatohepatitis (NASH) causing cirrhosis, hepatocellular carcinoma and eventually liver failure [Ludwig, J .; Viaggiano, TR; McGill, DB; Oh, BJ Mayo ClinProc . 1980 , 55, 434-8; Farrell, GC; Okuda L. J. Gastroenterol . Hepatol . 2003 , 18, 124-138]. NAFLDs are classified as Teeth with Types I and II (Matteoni, CA; Younossi, ZM; Gramlich, T .; Boparai, N .; Liu, YC; McCullough, AJ. Gastroenterology 1999 , 116, 1413-1419]. Disease progression from type II to III is associated with oxidative stress leading to lipid peroxidation and increased inflammation by activation of pro-inflammatory cytokines [TeSligte, K .; Bourass, I .; Sels, JP; Driessen, A .; Stockbrugger, RW; Koek, GH Eur . J. Intern . Med . 2004 , 15, 10-21]. Lipid accumulation in liver bearing NAFLD disease has been identified in both clinical and experimental animal models [Ayyad, C .; Andersen, T. Obes . Rev. 2000 , 1, 113-119, 2000]. Thus, alleviating serum and liver lipid levels is important for the prevention of NAFLD.

Activation of liver AMPK results in increased fatty acid oxidation and inhibition of hepatic lipid production, cholesterol synthesis and glucose production [Hiquchi, N .; Kato, M .; Tanaka, M .; Miyazaki, M .; Takao, S .; Kohjima, M .; Kotoh, K .; Enjoji, M .; Nakamuta, M .; Takayanagi, R. Exp. Ther . Med . 2011 , 2, 1077-1081]. In the course of energy shortage, AMPK inhibits the synthesis of de novo fatty acids by inactivation of ACC (acetyl-CoA carboxylase) [Viollet, B .; Guigas, B .; Leclerc, J .; Lantier, L .; Mounier, R .; Andreelli, F .; Foretz, M. Acta Physiol . ( Oxf ) 2009 , 196, 81-98]. AMPK plays an important role in glucose and lipid metabolism and plays an important role in the regulation of metabolic diseases such as diabetes, obesity and cancer. Thus, AMPK is known as a therapeutic target for metabolic diseases including fatty liver [Zhang, YL; 7. J. Hepatol . 2009 , 51, 29-38; Musso, G .; Gambino, R .; Cassader, M. Annu . Rev. Med . 2010 , 61, 375-392].

Houttuynia cordata ; H. cordata ) is a name given because of the fishy smell of leaves. Hwasungcho is excellent for diarrhea and drainage. It is used for coughing caused by lung abscesses, pneumonia, pneumonia, acute bronchitis, enteritis, urinary tract infections, boils, and it is used when you can not see urine.

[Prior Patent Literature]

Korean Patent Publication No. 1020060045096

The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a novel composition for prevention and treatment of fatty liver.

In order to achieve the above object, the present invention provides a composition for preventing or treating fatty liver comprising an extract of Aspergillus oryzae as an active ingredient.

In one embodiment of the present invention, the extract of Rhododendron koreanum is preferably an extract of Rhodochrosensis ethyl acetate, but is not limited thereto.

The present invention also provides a food composition for alleviating fatty liver comprising an extract of Aspergillus as an active ingredient.

The present invention also provides a composition for prevention or treatment of fatty liver comprising lutein, isoquercitrin, quercitrin and quercetin.

The present invention also provides a composition for preventing or treating fatty liver comprising quercitrin as an active ingredient.

The present invention also provides a food composition for alleviating fatty liver comprising quercitrin as an active ingredient.

The pharmaceutical composition comprising the extract or the compound of the present invention contains the above compound in an amount of 0.1 to 50% by weight based on the total weight of the composition.

The pharmaceutical compositions comprising the extracts or compounds of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts or compounds of the present invention may also be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in suitable aggregates.

The pharmaceutical composition containing the extract or the compound according to the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories, Can be used. Examples of carriers, excipients and diluents that may be contained in the composition containing the extract or compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. The base of the suppository may be witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like, and it is preferably an ointment agent, a warning agent, a lotion agent, a liniment agent, Or a pharmaceutical composition in the form of a topical dermatological agent of a cataplasma.

The preferred dose of the extract or compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides a health functional food composition comprising said extract or compound and a pharmaceutically acceptable food-aid additive. The food composition according to the present invention is useful as a health functional food product including various foods such as chewing gum, caramel product, candy, ice cream, confectionery, beverage such as soft drink, mineral water and alcoholic drink, vitamins and minerals .

In this case, the amount of the extract or the compound in the food may be 0.001 to 99.9% by weight of the whole food, and the beverage may be added in a proportion of 0.001 to 0.1 g, more preferably 0.05 to 0.1 g, .

The beverage containing the extract or the compound of the present invention is not particularly limited to other components other than those containing the extract or the compound as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, such as ordinary beverages, .

In addition to the above, the health functional food composition of the present invention may further contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the functional food compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described.

The present invention investigates the effects of Horseradish ethyl acetate (HC-EA) extracts on high fat (HFD) -induced hepatic steatosis and describes the molecular mechanisms involved in the animal model of nonalcoholic fatty liver disease (NASH) He said. After 2 weeks of feeding HFD, HC-EA extract (100, 200, or 300 mg / kg) was orally administered once daily for 8 weeks. HC-EA extract induced a dose-dependent reduction in plasma lipid levels, reduced total liver lipid accumulation and improved HFD-induced liver tissue lesions. The morphological changes observed with Hematoxylin-Eosin and Oil-Red O staining in HFD-fed rat hepatocytes were reversed by HC-EA treatment. The mechanism of action is that HC-EA extract increases the phosphorylation of AMPK (AMP-activated protein kinase) in the liver of HFD-fed rats and its primary downstream target enzyme acetyl-CoA carboxylase, and the expression of carnitine palmitoyl transferase 1 And regulate the sterol regulatory element binding protein 1, fatty acid synthase, and glutamate pyruvate transaminase protein levels. In addition, increased expression of liver cytochrome P450 (CYP) compositions such as CYP2E1 and CYP4A was significantly inhibited by HC-EA extract (100 and 300 mg / kg).

Hereinafter, the present invention will be described in detail.

HC - EA  High performance liquid chromatography of the extract ( HPLC ) Fingerprint analysis and quantitative evaluation of key ingredients

In order to identify the major active ingredients contained in the HC-EA extract, we performed HPLC fingerprint analysis. Data showed that HC-EA extracts contained polyphenolic compounds such as rutin (1.21%), isoquercitrin (1.53%), quercitrin (5.86%) and quercetin (1.16%). The major polyphenolic compound of the HC-EA extract was identified as quercitrin (Figure 1A, peak 2). To quantify quercitrin contained in HC-EA, the extract was dissolved in ethanol and filtered through a 0.45 um polytetrafluoroethylen (PTFE) syringe filter (Advantec Toyo Roshi Kaisha Ltd., Tokyo, Japan). HPLC was performed using a Waters 2695 Alliance system equipped with a system controller, auto injector, column oven and a Waters 2998 photodiode array detector. HC-EA (10 [mu] L) was injected at 30 [deg.] C on a Sunfire RP 18 column (250 x 4.6 mm ID; 5 um). The mobile phase consisted of ethanol (A) and water (B) and the gradient elution program was as follows: a 30 minute gradient was started using 60% A, linearly increased to 100% And finally returned to the initial conditions and maintained at a flow rate of 0.8 mL / min for 4 minutes. Quercitrin was detected at 450 nm; Its spectral range is from 200 to 600 nm. The calibration curves of quercitrin analysis were analyzed using Waters Empower 2 chromatography software. The amount of quercitrin in HC-EA extract was 4.38 ± 0.035 mg / g (FIG. 1B).

Food intake and body weight for HFD fed rats ( BW Effects of HC-EA extract

BW increase was significantly higher in HFD-fed rats compared to normal saline control rats. These results indicate that the HFD-fed rat model has been successfully established. After 8 weeks of administration of HC-EA (300 mg / kg / day), the BW increase in the rats was significantly lower in the HFD group than in the rats (p <0.001). There was no significant difference between daily food and water intake between groups during the experiment (Table 1)

Table 1 shows changes in body weight, food and water intake in HFD-fed rats treated with HC-EA for 8 weeks. The vehicle (distilled water) used in the preparation of the HC-EA extract was given the same volume. Values (mean ± SEM) were obtained from 8 animals in each group after 8 weeks of the experiment. ^a P < 0.05 Comparisons with values of chew fed rats treated with vehicle in each group. ^b P < 0.05 compared to the values of vehicle-treated HFD-fed rats in each group.

For the lipid parameters HC - EA  Effect of extract

HFD induced an increase in plasma total cholesterol (TC), triglyceride (TG), and low-density lipoprotein-cholesterol (LDL-C) levels in rats (Table 2). Treatment with HC-EA extract (300 mg / kg / day) reduced plasma levels of TC, TG, and LDL-C in HFD-fed rats by 34.8%, 31.1%, and 51.4%, respectively, compared to their vehicle counterparts. Plasma levels of high-density lipoprotein-cholesterol (HDL-C) in HFD-fed rats were lowered by 56.3% compared to the normal diet group. After 8 weeks of treatment with HC-EA (300 mg / kg / day), lower plasma HDL-C levels in HFD-fed rats increased to nearly the same level as the normal diet group. In addition, plasma free fatty acids (FFAs) were significantly higher in HFD-fed rats receiving vehicle alone. After 8 weeks of treatment, plasma FFA was 32.4% lower in HC-EA (300 mg / kg / day) -treated HFD-fed rats compared to the vehicle counterpart. In HFD-fed rats, both LDL-C / HDL-C and TC / HDL-C were significantly higher than those observed in the normal diet group (p <0.001). HC-EA treatment induced a dose-dependent reduction in LDL-C / HDL-C and TC / HDL-C in HFD-fed rats relative to their vehicle treated counterparts (Table 2). Liver TC levels were 2-fold higher in HFD-fed rats than in the normal feed group, but they were 42.1% lower in the 8 week HC-EA (300 mg / kg / day) -treated group (FIG. Liver TG levels were 2.2 times higher in HFD-fed rats than in the normal feed group. HFD-fed rats treated with HC-EA (300 mg / kg / day) for 8 weeks significantly lowered liver TG levels by 56.1% compared to their vehicle treated counterparts (FIG. 2).

Table 2 shows the changes in plasma lipid parameters in HFD-fed rats treated with HC-EA extract for 8 weeks. The vehicle (distilled water) used in the preparation of the HC-EA extract was given the same volume. Values (mean ± SEM) were obtained from 8 animals in each group after 8 weeks of the experiment. ^a P < 0.05, ^b P < 0.01 Comparisons with values of chew fed rats treated with vehicle in each group. ^c P < 0.05, ^d P < 0.01 compared to the values of vehicle-treated HFD-fed rats in each group.

Morphological changes in hepatocytes

No histological abnormalities were observed in the normal diet group (Figure 3), but HFD-fed rats for 8 weeks developed a high degree of steatosis. HFD-fed rats treated with HC-EA for 8 weeks significantly reduced long-term lipid accumulation compared to their vehicle-treated counterparts, particularly the HC-EA treated group at 300 mg / kg, (Fig. 3, left panel and Table 3). In the Oil Red O-stained (ORO) section, the rats in the HFD group showed abundant and larger ORO + lipid droplets compared to the normal diet group. HFD rats treated with 300 mg / kg of HC-EA showed lower and smaller ORO + lipid droplets compared to the HFD group (Fig. 3, right panel).

Table 3 shows the pathological grade of hepatic steatosis. The vehicle (distilled water) used in the preparation of the HC-EA extract was given the same volume. Each group consists of 8 rats and the table shows the number of rats per grade. ^a P < 0.05, ^b P < 0.01 compared to the values of normal chew fed rats in each group. ^c P < 0.05, ^d P < 0.01 compared to the values of vehicle-treated HFD-fed rats in each group.

In liver cells AMPK  And For phosphorylation and protein expression of ACC  HC-EA extract Effect of

As shown in Fig. 4, HFD reduced the degree of phosphorylation of AMPK and protein levels compared to normal diet-fed rats. Similarly, both the degree of phosphorylation and protein levels of liver ACC in HFD-fed rats were down regulated relative to normal diet fed rats (Fig. 4A). In addition, HFD significantly reduced the phospho-AMPK / AMPK and phospho-ACC / ACC ratios by 50.2% and 51.8%, respectively. HFD-induced down-regulation in the ratio of phospho-AMPK / phospho-ACC / ACC to phospho-ACC / ACC was significantly reversed compared to the vehicle treated vehicle after 8 weeks of HC-EA extract (300 mg / kg / day) (73.6% and 87.8% increase respectively) (Fig. 4B).

Effect of HC-EA Extract on Protein Expression of SREBP-1, FAS, and CPT-1 in HFD-Fed Rat Hepatocytes

As shown in Figure 5, protein expression levels of SREBP-1 and FAS in the liver of HFD-fed rats were higher than in normal diet fed rats. In liver of HFD-fed rats treated with HCE (300 mg / kg / day) for 8 weeks, the level of SREBP-1 protein expression was reduced by 54.4% compared to the expression level of vehicle treated HFD fed rats (FIG. HFD-induced increases in hepatic FAS protein expression were reversed by HCE treatment (300 mg / kg / day) (41.2% reduction). Protein levels of CPT-1 in the liver of HFD-fed rats were 45.5% lower than in normal diet-fed rats. Administration of HCE (300 mg / kg / day) to HFD-fed rats upregulated liver CPT-1 protein levels by two-fold compared to vehicle-treated HFD-fed rats (Figure 5).

Liver cells CYP2E1  And On CYP4A1 expression Effect of HC-EA extract

As shown in FIG. 6, protein levels of liver CYP2E1 and CYP4A1 expression were significantly increased in the liver of HFD-fed rats compared to normal diet-fed rats. However, treatment with HCE (300 mg / kg / day) significantly recovered compared to vehicle-treated HFD-fed rats.

The present invention suggests that HC-EA extract acts by regulating AMPK-dependent signaling pathways and related mediators. In summary, HC-EA extracts could be developed to prevent and treat obesity-related nonalcoholic fatty liver disease.

Figure 1 shows an HPLC fingerprint analysis. (A) HPLC chromatogram of HC-EA extract, 1: Rutin, 2: quercitrin, 3: isoquercitrin and 4: Quercetin. (B) Structure and standard curve of quercitrin. HC-EA: H. cordata ethyl acetate.
Figure 2 shows the effect of Hc-EA extract on hepatic levels of TC and TG in HFD-fed rats. Control rats were given the same volume of vehicle (distilled water) used to make the HC-EA extract. HC-EA (100 mg / kg / day) -treated HFD-fed rats, HC-EA (200 mg / kg / day) -treatment of liver lipid with vehicle-treated normal autologous rats, vehicle treated HFD-fed rats HFD-fed rats, and HC-EA (300 mg / kg / day) -treated HFD-fed rats. Values (mean ± SEM) were obtained from 8 animals in each group after 8 weeks of the experiment. ^# P <0.05 compared with the value of each group of normal writing (chew) benefit from vehicle treated rats, and * p <0.05, ** p < 0.01 and *** p <0.001, the value of the vehicle treated rats and benefit HFD- compare. HFD: high fat diet; HC-EA: Acetic acid ethyl acetate.
Figure 3 shows histopathological findings in the liver of HFD-fed rats treated with HCE for 8 weeks. Control rats were given the same volume of vehicle (distilled water) used in the preparation of HC-EA extract. (100 mg / kg / day) -treated HFD-fed rats, HC-EA (200 mg / kg / day) -treated HFD- fed rats, vehicle treated HFD-fed rats, Fed rats, and HC-EA (300 mg / kg / day) -treated HFD-fed rats. Shoot at 400x magnification. High-fat free diets show macrovesicular steatosis in the periportal region. The pathological grade of hepatic steatosis is shown in Table 3. Hematoxylin and eosin staining (left panel), and oil Red O staining (right panel).
Figure 4 shows the effect of HC-EA extract on the degree of phosphorylation and / or protein levels of AMPK and ACC in HFD-fed rats. Control rats were given the same volume of vehicle (distilled water) used to make the HC-EA extract. (100 mg / kg / day) -treated HFD-fed rats, HC-EA (200 mg / kg / day) -treated HFD- fed rats, vehicle treated HFD-fed rats, (HC-EA 300-HFD) treated rats and HC-EA (300 mg / kg / day) -treated HFD-fed rats. Similar results were obtained in an additional 4 replicates. Protein levels (mean ± SEM) were obtained from each group (n = 5). # p < 0.05, compared to the value of a normal chew-fed rat vehicle treated in each group. * p < And cp < 0.05 and dp < 0.01 vs. Vehicle-treated HFD-fed rats.
Figure 5: Effect of HC-EA extract on protein levels of SREBP-1, FAS, and CPT-1 in HFD-fed rats. Control rats were given the same volume of vehicle (distilled water) used to make the HC-EA extract. (100 mg / kg / day) -treated HFD-fed rats, HC-EA (200 mg / kg / day) -treated HFD-fed rats, and HC-EA (300 mg / kg / day) -treated HFD-fed rats. Use beta-actin as an internal control. (B) Quantitative graph of protein levels (mean ± SEM) was obtained from each group (n = 5). # &Lt; 0.05 Comparisons with values of normal chew fed rats in each group. * P < 0.05, ** p < 0.01 and *** p < 0.001 compared to values of vehicle treated HFD-fed rats. HFD: high fat diet; HC-EA: Acetic acid ethyl acetate.
Figure 6 shows the effect of HC-EA extract on hepatic CYP2E1 and CYP4A1 expression in HFD-fed rats. Control rats were given the same volume of vehicle (distilled water) used to make the HC-EA extract. (100 mg / kg / day) -treated HFD-fed rats, HC-EA (200 mg / kg / day) -treated HFD-fed rats, and HC-EA (300 mg / kg / day) -treated HFD-fed rats. Use beta-actin as an internal control. (B) Quantitative graph of protein levels (mean ± SEM) was obtained from each group (n = 5). # &Lt; 0.05 Comparisons with values of normal chew fed rats in each group. * P < 0.05, ** p < 0.01 and *** p < 0.001 compared to values of vehicle treated HFD-fed rats. HFD: high fat diet; HC-EA: Acetic acid ethyl acetate.

The present invention will now be described in more detail by way of non-limiting examples.

Example 1: Plant material and extraction

The dried whole plant material of H. cordata was purchased from the Kyungdong market, Seoul, Korea, and received confirmation from professor Kim Rong-ho of Konkuk University. The voucher sample (HC-KU2012) was stored in our plant specimen room for future reference. To obtain H. cordata extract, the dried plant material was ground with a mixer and degreased 3 times with 3 volumes of ethanol. The residue was extracted with a 1:10 ratio (w / v) at 70-80 ° C in ethanol for 2 hours in a heating mantle. The supernatant was filtered and concentrated by rotary evaporation at 50 &lt; 0 &gt; C. For further fractionation, the alcohol extract (50 g) was fractionated with hexane, ethyl acetate (EA), and n-butanol fractions to 0.53, 8.72 and 38.25 g, respectively. The EA fraction of H. cordata (HC-EA, 76.5%) was lyophilized and refrigerated at -20 ° C until further use. Other reagents used in the present invention were usually the highest available.

Example 2: HPLC  Fingerprint analysis

A Shimadz system with a binary pump, an in-line degasser, an autosampler, and a PAD detector was used. UV spectra were recorded between 200 and 450 nm and UV traces were measured at 360 nm. A Symmetry C18 analytical column (250 X 4.6 mm i.d .; particle size, 5 um) equipped with a Novapack RP C18 guard column (Waters, Millford, Mass.) Was used for analytical purposes. The mobile phase consisted of formic acid (A) in 0.5% deionized water and 0.5% formic acid (B) in acetonitrile: methanol (50:50, v / v). The gradient program is as follows: 25% B at 40% B for 20 min at 1 mL min-1 flow rate, 50% B for 5 min at 40% B, 5 min at 100% B for 5 min and 50% at 100% B for 5 min. A 10-minute post-run returning to the starting condition for reconditioning. Keep the column oven temperature at 40 ° C. The injection volume is 10 μl.

Example  3: Animals

8 week old male Wistar rats were purchased from Nara Biotech Co., Ltd. (Seoul, South Korea). Animals were adapted for one week in a specific temperature controlled room (25 ± 1 ° C) and maintained at 12:12 person-cancer cycle (light at 06:00 h), with free feeding of water and feed at the Konkuk University Animal Center. All animal testing procedures were carried out in accordance with Guidelines for the Welfare of Animals and Guidelines for the Protection and Use of Konkuk University Experimental Animals.

Experimental Example 4: Experimental Protocol

The rats were fed a high fat diet containing 45% kcal fat, mainly containing lard, for 2 weeks used to induce a rapid increase in body weight and obesity (11% kcal fat # LM-485, Teklad, Madison, (HFD) (# D12451, Research Diets, New Brunswick, NJ). After feeding HFD for 2 weeks, the rats were orally administered once daily with HC-EA extract for 8 weeks at a dose of 100, 200, and 300 mg / kg in a volume of 2 ml / kg of distilled water. Another group of HFD- and control diet-fed rats were similarly treated with distilled water instead of HC-EA extract for the same treatment period. Water consumption, food intake and body weight were measured once daily at the same time daily throughout the experiment. After 8 weeks of treatment, rats were weighed and anesthetized with sodium pentobarbital (30 mg / kg, ip). Blood samples were collected from the tail vein. The collected blood samples were centrifuged at 2,000 x g for 10 minutes at 4 ° C. Plasma was then removed and placed in aliquots for each assay determination. The liver was removed from blood collection, washed with physiological saline, weighed and stored immediately at -70 ° C. Each experimental group contained 8 rats.

Example 5: Blood analysis

A diagnostic kit for determining plasma levels of TC and TG was purchased from Cayman Chemical Company (Michigan, USA). Diagnostic kits for determining plasma levels of HDL-C and LDL-C were purchased from Bio-Quant Diagnostics (CA, USA) and calculated using the Friedewald equation [Friedewald, WT; Levy, R .; Fredrickson, DS Clin . Chem. 1972, 18, 499-502]. Plasma levels of FFAs were measured using Abcam plc. (MA, USA). &Lt; / RTI &gt;

Example 6: Liver essay

To measure liver lipids, frozen liver samples were thawed on ice and homogenized with deionized water. Extraction and separation of lipids were performed using the Folch technique [Folch, J .; Lees, M .; Sloane-Stanley, GH J. Biol . Chem . 1957 , 226, 497-506]. Cholesterol and TG levels in lipid extracts were measured by colorimetry using a commercial kit (Thermo Electron Corporation).

Example  7: Histology

The formalin-fixed, paraffin-filled liver sections were stained with haematoxylin and eosin (H & E) to identify the entire structure and collagen bundle, respectively, and performed by the van Gieson method [vanGoor, H .; Gerrits PO; Grond, J. Histochemistry 1986 , 85, 251-253]. The frozen liver was continuously sectioned to a thickness of 4 mu m with a cryostat, placed on a slide, and dried at 37 DEG C for 15 minutes. The sections were then immobilized in neutral buffer 10% formalin for 10 minutes. To detect neutral lipid accumulation, the sections were stained with Red Oil 'O' (ORO) for 10 minutes. Lipid staining and fatty liver grading were determined using frozen sections reacted with ORO. (1 grade, 0-25%, grade 2, 26-50%, grade 3, 51-75%, grade 4, 76-100%) were classified according to the macrovesicular fat present in hepatocytes [Ramirez-Zacarias, JL; Castro-Munozledo, F .; Kuri-Harcuch, W. Histochemistry 1992 , 97, 493-497

].

Example  8. Preparation of liver fraction

Liver tissues were incubated with a solution containing 137 mM / l NaCl, 20 mM / l Tris-HCl, 1% Tween 20, 10% glycerol, 1 mM / l PMSF (phenylmethylsulfonyl fluoride), and a protease inhibitor in DMSO solution And homogenized with cold lysis buffer (pH 7.4). The homogenate was then centrifuged at 2,000 x g for 10 minutes at 4 ° C. Protein concentrations of each fraction were determined using a commercial kit (Bio-Rad Laboratories, Hercules, Calif., USA).

Example  9. Western Blot  analysis

The liver tissues were cultured in chilled buffer containing lysis buffer (1 x RIPA (Boston Scientific Company, MA), 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, and 1 ug / ml aprotinin, pepstatin, and leupeptin] After homogenization of the samples, the homogenized cells were washed with cold PBS, collected and centrifuged. The cell pellet was resuspended in lysis buffer and incubated on ice for 1 hour. Subsequently, the adipose tissue and homogenized cell debris were removed by centrifugation and protein concentration in the lysate was determined using Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Hercules, Calif.). Subsequently, the disruption solution was electrophoresed on a 10% polyacrylamide gel containing sodium dodecyl sulfate and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 0.1% Tween-20 solution in Tris-buffered saline containing 5% BSA for 1 hour at room temperature. After incubation with primary antibody at 4 ° C overnight, the membrane was incubated with horseradish peroxidase-attached primary antibody for 1 hour at room temperature. Immunodetection was performed using an ECL Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ). The homogenate was separated by SDS-polyacrylamide gel electrophoresis and Western blot analysis was performed using acetyl-CoA carboxylase (Santa Cruz Biotechnology, Inc., CA, USA), pACC (phospho-ACC at Ser 79; Signaling Technology, MA, USA), AMPKα (Cell Signaling Technology), phospho-AMPKα (pAMPKα Cell Signaling Technology), CPT-1 (carnitine palmitoyl transferase 1), SREBP-1 (sterol regulatory element binding protein 1; Santa Cruz Biotechnology, Inc.), FAS (fatty acid synthase; Cell Signaling Technology); And anti-rat antibodies that bind to beta-actin (Santa Cruz Biotechnology, Inc.). The blots were incubated with the appropriate peroxidase-conjugated secondary antibody. The membrane was then washed three times in TBST (20 mmol / l Tris-HCl, pH 7.5), 150 mmol / l NaCl, and 0.05% Tween 20 and diluted in an enhanced chemiluminescence detection system (Amersham Corp., Braunschweig, Germany ) Was used to visualize on an X-ray film. Dendrites analysis of the vans for protein expression was performed with Bio-Rad Densitograph Software (MA, USA).

Data of the present invention are expressed as means ± SEM (SEM). Statistical analysis was performed by one way analysis of variance (ANOVA). The Dunnett range post hoc comparison was used to determine the source of the appropriate significant differences. P values less than 0.05 were considered statistically significant. For histological studies, non-parametric Kruskal-Wallis tests were performed and Mann-Whitney's U tests were used to compare group-to-group data. SigmaPlot (Version 11.0) software was used for statistical analysis.

Claims (7)

A composition for prevention or treatment of fatty liver comprising an extract of Aspergillus as an active ingredient. The composition for prevention or treatment of fatty liver according to any one of claims 1 to 3, wherein the extract of Allium cepa is Ethyl acetate extract. A food composition for alleviating fatty liver comprising an extract of Aspergillus as an active ingredient. 4. The food composition for alleviating fatty liver disease according to claim 3, wherein the herbal extract is Ethyl Acetate Extract. A composition for prevention or treatment of fatty liver comprising lutein, isoquercitrin, quercitrin and quercetin. A composition for prevention or treatment of fatty liver comprising quercitrin as an active ingredient. A food composition for alleviating fatty liver comprising quercitrin as an active ingredient.

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