KR101291042B1 - Composition for preventing and treating alcoholic liver disease comprising hesperidin - Google Patents

Abstract

The present invention relates to a composition for preventing alcoholic liver disease containing flavonoids, and more particularly, to a method for preparing flavonoid derivatives by glycosylation and hydrolysis of glycosides.
The alcoholic liver disease prevention composition according to the present invention can be extracted with high purity to reduce the bitter taste of narirutin and provide an alcoholic liver disease prevention pharmaceutical composition and food composition of which the water solubility of hesperidin is increased.

Description

Alcoholic liver disease prevention composition containing hesperidin {COMPOSITION FOR PREVENTING AND TREATING ALCOHOLIC LIVER DISEASE COMPRISING HESPERIDIN}

The present invention relates to a composition for preventing alcoholic liver disease containing flavonoids.

Flavonoids have a variety of physiological activities, including antioxidant activity, antimicrobial activity, antiviral activity, anti-inflammatory activity, anti-cancer activity, and also has the effect of preventing the atherosclerosis by inhibiting the oxidation of low-density lipids in plasma. There are thousands of flavonoids, and important flavonoids are flavonols, flavones, flavaones, flavanones, flavanones, flavanols, and ansocyanidins. ), Isoflavone, dihydroflavonl and chalcone. Narirutin, narigenin, hesperedin, and the like belong to flavanone compounds. Furthermore, in the medical field, as the new flavonoids are extracted, diseases to be treated are diversified, such as liver disease, diabetes, rheumatoid arthritis, various cancers, analgesic, viral diseases, allergic diseases, arteriosclerosis, cardiovascular diseases, stress, It has been found that there is pharmacological activity in depression.

On the other hand, flavonoids derived from citrus peels are known to have various pharmacological activities, for example, lipid metabolism-improving effects, anticancer and antiviral effects associated with cardiovascular disease. In particular, hesperidin and hesperetin present in citrus rinds are capillary potentiated, reduced permeability, lowered blood pressure, lowered blood cholesterol, antiplatelet aggregation, anti-inflammatory and antiviral action Meyer, OC, Angiology, 45, 579-584 (1994); Struckmann JR, et al., Angiol., 45, 419-428 (1994); Matsubara, Y., et al., Japan Organic Synthesis Chem. Association Journal., 52, 318-327 (1994, Mar.); Galati EM, et al., Farmaco., 51 (3), 219-221 (1996, Mar.); Monforte MT., Et al , Parmaco., 50 (9), 595-599 (1995, Sep.); JP 95-86929; JP 95-86930; Chung, MI, et al., Chin. Pharm. J. (Taipei)., 46 429-437 (1994, Nov.); Galati, EM, et al., Farmaco., 40 (11), 709-712 (1994, Nov.); and Emim JA, et al., J. Pharm.Pharmacol , 46 (2), 118-122 (1994)), and other bioflavonoids are known to have antioxidant, hypotensive, anticancer and antiviral effects (Saija, A., et a L., Free Raical Biol. Med., 19, 481-486 (1995); Matsubara, Y., et al., Japan Organic Synthesis Chem. Association Journal., 52, 318-327 (1994, Mar.); Galati EM, et al., Parmaco., 51 (3), 219-221 (1996, Mar.), Felicia V., et al., Nutr. Cancer, 26, 167-181 (1996); EP 0352147 A2 (1990.1.25); and Kaul, TN, et al., J. Med. Virol., 15, 71-79 (1985)), and also naringin and naringenin are known to act as cholesterol lowering, anticancer and anti-ulcer (protecting from alcohol) (Monforte MT., Et al. Farmaco., 50 (9), 595-599 (1995, Sep.); JP 95-86929; JP 95-86930; Felicia V., et al., Nutr. Cancer, 26, 167-181 (1996); EP 0352147 A2 (1990.1.24); and Martin MJ, et al., Pharmacol., 49, 144-150 (1994)).

The present inventors have completed the present invention by focusing on the fact that citrus peel-derived flavonoids are hydrolyzed or glycosylated to show more dramatic effects on improving liver function.

An object of the present invention is to extract a flavonoid with high purity to provide an alcoholic liver disease prevention composition.

In one embodiment, to achieve the above object, a method of preparing flavonoid derivatives by glycosylation or hydrolysis of flavonoids is provided. In another embodiment, a flavonoid provides a method of making a flavonoid derivative, characterized in that any one selected from the group consisting of hesperidin, hesperetin, naringin, naringenin and narirutin. In another embodiment, the glycosylation is provided by the addition of a sugar substrate and the sugar transfer enzyme CGTase (Cyclodextrin glucanotransferase) provides a method for producing a flavonoid derivative. In another embodiment, a method for preparing a flavonoid derivative wherein the sugar substrate is beta-cyclodextrin is provided. In another embodiment, the hydrolysis provides a method for producing a flavonoid derivative, characterized in that using a hesperidinase enzyme. In another embodiment, the flavonoid derivative provides a method for producing a flavonoid derivative, characterized in that when hydrolyzed hesperetin-7-O-glucoside or naringenin-7-O-glucoside. In another embodiment, the flavonoid derivative provides a method for producing a flavonoid derivative, characterized in that when glycosylated G-naurritin or G- hesperidin.

In one embodiment, the present invention provides a pharmaceutical composition for preventing and treating alcoholic liver disease, including glycosylated or hydrolyzed flavonoid derivatives. In another embodiment, the flavonoid derivative is any one selected from the group consisting of hesperidin, hesperetin, naringin, naringenin and narirutin Provided is a pharmaceutical composition for preventing and treating alcoholic liver disease. In another embodiment, the flavonoid derivative when he hydrolyzed provides a pharmaceutical composition for the prevention and treatment of alcoholic liver disease, characterized in that hesperetin-7-O-glucoside or naringenin-7-O-glucoside. . In another embodiment, the flavonoid derivatives when the glycosylation provides a pharmaceutical composition for preventing and treating alcoholic liver disease, characterized in that G- naurritin or G- hesperidin. In another embodiment, hesperetin-7-O-glucoside vs. naringenin-7-O-glucoside is 4: 1 to provide a pharmaceutical composition for preventing and treating alcoholic liver disease. In another embodiment, G-naruritin versus G-hesperidin provides a pharmaceutical composition for preventing and treating alcoholic liver disease, characterized in that 4: 1.

In one embodiment, there is provided a food composition for preventing and ameliorating alcoholic liver disease, including glycosylated or hydrolyzed flavonoid derivatives. In another embodiment, the flavonoid derivative provides a food composition for preventing and improving alcoholic liver disease, characterized in that any one selected from the group consisting of hesperidin, hesperetin, naringin, naringenin and narirutin. In another embodiment, the flavonoid derivative is a hesperetin-7-O-glucoside or naringenin-7-O-glucoside when hydrolyzed, characterized in that the food composition for preventing and preventing alcoholic liver disease to provide. In another embodiment, the flavonoid derivatives, when glycosylated, provides a food composition for preventing and improving alcoholic liver disease, characterized in that G-naurritin or G-hesperidine. In another embodiment, hesperetin-7-O-glucoside vs. naringenin-7-O-glucoside is 4: 1, which provides a food composition for preventing and improving alcoholic liver disease. In another embodiment, G-naruritin versus G-hesperidin provides a food composition for preventing and ameliorating alcoholic liver disease, characterized in that 4: 1.

The alcoholic liver disease prevention composition according to the present invention can be extracted with high purity to reduce the bitter taste of narirutin and provide an alcoholic liver disease prevention pharmaceutical composition and food composition of which the water solubility of hesperidin is increased.

1 shows a process for glycosylating flavonoids.
Figure 2 shows the process of hydrolyzing the flavonoids.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Example  1. Extraction of Flavonoids

Jeju-free pesticide citrus ( citrus unshiu ) was purchased through Ilhae Co., Ltd. to separate the pericarp, dried in a 50 ° C. dry oven, pulverized with a grinder, and stored at -18 ° C. or lower until used for experiments. 30 times the capacity (w / v) of 70% ethanol was added to the dried citrus peel powder, and extracted using ultrasonic apparatus (ultrasonic processor, VCX-750, sonic & materials, Inc.) for 10 minutes at 30 ℃. After extraction, the solid and the supernatant were separated by centrifugation (8000 rpm, 10 minutes), and the same amount of hexane was added to the extract to remove fat-soluble components such as carotenoids. The fat-soluble component was removed and concentrated at 40 ° C. using a rotary evaporator to leave 70% ethanol.

Example  2. Hesperidin ( hesperidin ) And Narirutin  detach

The solvent of Example 1 was removed and the remaining extract was reextracted with water to elute the water-soluble narirutin, where insoluble hesperidin was isolated. The separated hesperidin was suspended in distilled water, adjusted to pH 11.3 by adding NaOH, and the dried solid was completely dissolved. HCl was added again to adjust pH to 7.0, and cooled to 12 ℃ filtered. The remaining solids were filtered and lyophilized to obtain a hesperidin extract having a purity of 90% or more of citrus peels. Ethyl acetate (ethyl acetate) was added to the water extract in which nadiroutin was dissolved to separate the ethyl acetate layer and the water layer. The solvent of the obtained ethyl acetate layer was removed with a vacuum condenser, and lyophilized to obtain a narirutin extract having a purity of about 70%.

Example  3. Hesperidin  And Narirutin  Enzyme treatment to enhance body availability

Flavonoids are mainly bound to plants in the form of glycosides, and narirutin is bound to rutinose at position C ₇ to naringenin, an aglycon, and hesperidin Hesperetin, an aglycon, contains rutinose C ₇ Is coupled to the location. Hesperidin from citrus peel is particularly in In vitro experiments have reported antioxidant, hypolipidemic, and anti-inflammatory effects, but glycosides are not water soluble, which makes them difficult to apply to foods and has some drawbacks for their absorption in the body. On the other hand, narirutin is water soluble, so it is advantageous for food application, but it is too bitter and strong, so any type of treatment is needed to reduce bitter taste. First, in order to facilitate the use of food materials by improving solubility, a glycosylation was induced, and second, a deshamnosylation that removes rhamnose from lutinose using hesperidinase. It was.

3-1. Hesperidin  And Narirutin Glycosylation

Glycosylated flavonoids were prepared using cyclodextrin and CGTase for the acceptance of hesperidin and narirutin from citrus peel and enhancement of body absorption. 1 g each of hesperidin and narirutin extract isolated from citrus peels was suspended in 1 L of distilled water, and then 3 g of beta-cyclodextrin was added and warmed to 70 ° C. to dissolve beta-cyclodextrin. In the case of the hesperidin extract, the mixture was adjusted to pH 4.9 by adding HCl to the hesperidin-containing solution encapsulated with beta-cyclodextrin, and filtered after cooling to 12 ° C. The precipitate formed by cooling was filtered off to obtain a filtrate. The resulting filtrate was adjusted to pH 5.5 by adding 15% NaOH solution, and 0.1 ml of Toruzyme ^® , a kind of cyclodextrin transferase (CGTase), was added to induce a sugar transfer reaction at 60 ° C. overnight. In the case of narirutin extract, water solubility is omitted, so the process of increasing the solubility through pH adjustment was omitted, and 1 ml of toruzyme was added and reacted for 3 hours at 60 ° C. to induce a sugar transfer reaction. After the reaction was completed, the reaction solution was heated to a temperature of 90 ℃ or more for 5 minutes to inactivate the enzyme. Amberlite (Amberite) XAD-7HP, a porous adsorption resin, was used to remove the remaining cyclodextrin that was not used in the sugar transfer reaction.

3-2. Hesperidin  And Narirutin  Hydrolysis derhamnosylation )

An enzyme reaction product using hesperidinase was prepared as a method for enhancing body absorption of hesperidin and narirutin extract. Hesperidin extract was dissolved in DW adjusted to pH 3.8 at a concentration of 1 mg / ml and then reacted with 0.0625 mg / ml of hesperidinase. In the case of narirutin extract, it was dissolved in DW adjusted to pH 3.8 at a concentration of 2 mg / ml and then reacted with hesperidinase of 0.0625 mg / ml to obtain a result (hesperetin-7-O-glucoside and narine Xenin-7-O-glucoside). Enzyme reactions were obtained after 1 h of reaction at 40 ° C., respectively. After the reaction, the reaction solution was heated to a temperature of 90 ° C. or higher for 5 minutes to inactivate the enzyme.

Example  4. Hesperidin  And Narirutin  Determination of Alcoholic Liver Disease Inhibitory Effect

Hesperidin and Narirutin extract obtained through this experiment, G-hesperidin and G-narirutin obtained through the saccharification process, and three groups to investigate the inhibitory effect of alcoholic liver disease according to the degree of body absorption of enzyme-treated hesperidin and narirutin The animal experiment was conducted to divide the fatty liver production. The treated hesperidin and narirutin extracts were mixed in a ratio of 4: 1 and used in animal experiments.

One) Animal diet

The diet used in the animal experiment was a liquid fatty liver induction diet [Central Laboratory Animal Co., Ltd.] recommended by the American Institute of Nutrition (AIN). The Lieber-DeCarli diet was up-regulated with the addition of corn oil to effectively induce fatty liver. The liquid diet contains calories at 1kcal / mL by default, which is 41.36% from fat, 4.8% from carbohydrates, 18.07% from protein and 36% from ethanol in the experimental group. The final dietary table for each experimental group is shown in Table 1 below.

Component Amount (g / L) Control group ethanol Ethanol +
Flavonoids Ethanol +
G-flavonoids Ethanol +
D-flavonoids casein 41.4 41.4 41.4 41.4 41.4 L-cystine 0.5 0.5 0.5 0.5 0.5 DL-methionine 0.3 0.3 0.3 0.3 0.3 Corn oil 15.7 15.7 15.7 15.7 15.7 olive oil 28.4 28.4 28.4 28.4 28.4 Safflower oil 2.7 2.7 2.7 2.7 2.7 Maltose dextrin 99.2 8.7 8.7 8.7 8.7 Cellulose 10.0 10.0 10.0 10.0 10.0 Mineral mixtures 8.8 8.8 8.8 8.8 8.8 Vitamin mixture 2.5 2.5 2.5 2.5 2.5 Choline Bitarrate 0.5 0.5 0.5 0.5 0.5 Xanthan gum 3.0 3.0 3.0 3.0 3.0 Ethanol (mL) - 41 41 41 41 Dried sample - - 60 60 60

2) Animal Breeding

The animals used in the experiment were 8 weeks old (weight 279.2 ± 6.9g) male Sprague-Dawley, Inc. (Central Experimental Animal) and purchased for 6 weeks in the animal breeding room (room temperature 22 ± 2). At room temperature, 55 ± 5% of relative humidity and 12 h light-dark cycle, distilled water freely fed into individual cages was fed to the Lieber-DeCarli liquid diet. The body weight was divided into four groups of nine dogs and adapted to the Lieber-DeCarli liquid standard diet for about seven days. The control group was the animal group fed with the Lieber-DeCarli liquid standard diet only. The animal group fed with the same liquid diet as the control group was supplemented with the calorie supplemented with ethanol instead of the carbohydrate (maltose dextrin) of the control standard diet. ethanol), glycosylated hesperidin, narirutin extract group, enzyme-treated hesperidin, narirutin extract group. Under these conditions, a Lieber-DeCarli liquid diet was fed to all animals for six weeks at 70 mL each day until the end of the experiment.

3) Weight gain measurement and serum and tissue sampling

In order to measure the weight change of the animals, the weight of each animal was measured every week over 6 weeks at a fixed time. And using retroorbital sinus bleeding to measure changes in the fats in the blood, just before feeding the Lieber-DeCarli liquid diet (week 0), and at the third week and finally at the expense of animals. At week point blood was drawn from all animals. After the blood was collected, the serum sample was separated using a centrifuge (2,000 xg for 10 min). The separated serum was immediately analyzed for HDL-cholesterol, and the rest of the serum was stored at −70 ° C. until analysis of other components. The blood was fasted for about 12 hours, at which time only water was supplied. At 6 weeks, immediately after blood collection, major organs of each animal were extracted to determine the effect of glycation, enzyme treatment with hesperidin and narirutin extract on organ weight.

4) Major markers of serum fat and fatty liver ( biomarkers ) analysis

The serum triglycerides concentration was calculated by measuring the absorbance at 500 nm after color development using a kit (Asan Pharmaceuticals) using enzyme colorimetric method. Non HDL-cholesterol calculations were calculated as total cholesterol minus HDL-cholesterol. The concentrations of GOT and GPT present in serum were measured using kit (Asan Co., Ltd.) using enzyme colorimetric method (Reitman-Frankel method), and the absorbance was measured by adding the reaction reagent prepared in 0.10 mL of serum sample. Alternatively, the GPT concentration was measured under the same conditions as the sample using the standard curve solution, and the absorbance was measured and the concentration was calculated (Asan Pharmaceutical Co., Ltd., Asan Pharmaceutical Co., Ltd.).

5) Analysis through liver tissue photography hepatic histology )

Liver tissue photographs were taken at the Anatomy Room, College of Medicine, Yeungnam University. At week 6, the liver of one animal closest to the mean weight in each animal group was used for tissue analysis. The liver tissue was first frozen and then sectioned about 5-10 μm thick. Sections were stained with hepatoxylin, eosin, oil red O reagent.

6) alpha between the extract and the liver tissue of the lipid-tocopherol (α- tocopherol) and measuring total cholesterol

Liver lipids were extracted from approximately 500 mg homogenized liver samples by chloroform: methanol (v / v, BHT 151.3 μmol / L) mixed organic solvent method, and after organic solvent removal (N-EVAPTM 111, Organomation Associates Inc., MA, USA) and total lipids were measured. Directly use the extracted liver tissue fat to evaporate the organic solvent at 40 ° C using a nitrogen concentrator and chloroform: methanol mixture (1: 5, v / v) 500 μL was added and homogenized, followed by HPLC analysis. The HPLC column used was a reversed phase column (Alltima C18, 5 μm, 4.6 × 150 mm, Alltech Associates, Inc., IL, USA) maintained at 40 ° C., Beckman System Gold software (Beckman Instruments, Inc., Fullerton, CA, USA). The mobile phase was 100% methanol (2 mL / min) and analyzed at UV 295 nm.

7) Statistical processing and result processing

The results of the experiments were expressed as mean and standard deviation (means ± SD). After verifying with ANOVA using SPSS package program software, Duncan's multiple range test at p <0.05 level. ) To compare and analyze.

Example  5. Hesperidin  And Narirutin  Results of alcoholic liver disease inhibition effect

1) Weight gain measurement result

Six weeks of dietary intake of Liber-DeCali, an alcohol-induced fatty liver-derived diet, was carried out using SD rats, and the diet containing alcohol was higher than that of the non-alcoholic Liber-DeCarli diet. In the group, body weight began to decrease from the beginning, and the intake of flavonoids did not affect weight loss.

2) major markers of serum fat and fatty liver ( biomarkers ) Analysis

The effects of flavonoids on the change of concentrations of GOT, GPT, ALP, and GTP, which are serum markers of fatty liver, related to liver tissue damage for 6 weeks, are shown in Table 2. It was. In the group ingesting flavonoids with alcohol, these indicator concentrations were significantly lower than those ingesting only ethanol, and especially in the G-flavonoid and flavonoid-7-glc group, the liver damage was weaker than the flavonoid group. The flavonoid modification process is thought to be effective in alleviating damage to liver tissue (Table 2).

Serum biomarker Control group ethanol Flavonoid G-flavonoids Flavonoid-7-glc GOT, U / L 38.18 ± 5.58 ^d 90.52 ± 9.35 ^a 54.78 ± 9.84 ^b 51.15 ± 10.62 ^bc 43.01 ± 6.20 ^cd GPT, U / L 6.79 ± 2.12 ^c 29.93 ± 8.06 ^a 27.29 ± 5.17 ^a 18.09 ± 3.50 ^b 18.13 ± 3.78 ^b ALP, K-A 30.15 ± 4.0 ^b 46.88 ± 8.87 ^a 38.86 ± 8.31 ^ab 31.52 ± 5.18 ^b 30.34 ± 4.0 ^b GTP, U / L 2.14 ± 0.46 ^b 4.72 ± 1.47 ^a 3.84 ± 0.92 ^ab 3.43 ± 0.79 ^ab 3.49 ± 0.49 ^ab

3) Analysis through liver tissue photography hepatic histology )

Lipid droplets stained with red were clearly observed in the ethanol intake group, and in the group in which hesperidin and narirutin were ingested with ethanol, the formation of fat globules was less active than in the ethanol group. . Fat globule formation was not yet observed in the flavonoid ingested group after glycosylation, and fat globule was observed in the flavonoid ingested group after the derhamnosidation process, but the size of fat globules was reduced compared to the ethanol group. It can be seen that. These results suggest that the glycosylation or de-rhamnosidation process is considered to be useful for the improvement of the alcoholic fatty liver production inhibitory effect of flavonoids, as shown in the comparison of fatty acid content and fatty acid content of liver tissue.

4) Extraction of Liver Lipids and α- of Liver Tissue tocopherol And total cholesterol

The results of ingestion of G-hesperidin / narirutin and hesperidin / narigenin-7-glc on alcoholic fatty liver formation are shown in the table. As a result, the total cholesterol and total lipid contents were significantly increased in the liver tissues of the ethanol diet group, although the liver weight was decreased compared to the control group (P <0.05). In addition, when comparing ethanol and flavonoid diet group and ethanol diet group, there was no significant difference in total cholesterol by ingesting flavonoids, but total lipid content was significantly decreased. When he ingested glycosylated flavonoids or flavonoids treated with sidde-rhamnosidation, hesperidin and narirutin produced through these modifications were significantly reduced when ingested glycosylated flavonoids or flavonoids treated with ethanol together with ethanol. The alcoholic fatty liver formation was found to be more effective.

Table 4 shows the fatty acid content present in the fat layer of liver tissue. As can be seen from the content of liver lipids, the content of fatty acids present in cholesterol, TG, and phospholipids is significantly increased in the alcohol diet group compared to the diet diet group. In general, the content of these fatty acids was lower in the group ingesting flavonoids after glycosylation or de-rhamnosidation than hesperidin and narirutin.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (9)

delete delete delete A pharmaceutical composition for preventing and treating alcoholic liver disease, comprising hesperetin-7-O-glucoside and naringenin-7-O-glucoside in a weight ratio of 4: 1, respectively. delete delete Hesperetin-7-O-glucoside and naringenin-7-O-glucoside, each comprising a weight ratio of 4: 1, alcoholic liver disease prevention and improvement food composition.

delete delete

Images