KR20190086085A - Composition for improving liver function comprising gypenoside compound isolated from gynostemma pentaphyllum - Google Patents

Abstract

The present invention relates to a gypenoside compound isolated from Gynostemma pentaphyllum, wherein a composition for improving liver functions containing the gypenoside compound isolated from Gynostemma pentaphyllum has excellent liver damage inhibition, anti-inflammatory, and antioxidant effects. A problem to be solved by the present invention is to provide a method for improving liver function, protecting a liver, and inhibiting liver damage by using the composition for improving liver function containing the gypenoside compound.

Description

[0001] The present invention relates to a composition for improving liver function comprising a glyphosate compound isolated from the outside,

The present invention relates to a composition for improving liver function comprising a ziphenocide compound isolated from the outside as an active ingredient.

The liver plays an important role in detoxifying all the foods absorbed by the body, storing the nutrients in the liver, and releasing them like bile to regulate blood flow. If liver function deteriorates, GOT (Glutamate Oxaloacetate Transaminase or Aspartate Aminotransferase), GPT (Glutamate Pyruvate Transaminase or ALT (Alanine Aminotransferase)) and γ-GTP (γ-glutamyltranspeptidase) It is not discharged, and various complications (hepatic coma, ascites, esophageal varices, thrombocytopenia) can come, and anorexia, abdominal fullness (headache) and gum bleeding occur.

In the development of medicines, beverages and health functional food materials with liver function improving agent and liver effect, product development has been mainly focused on simple extracts of herbal medicines whose liver function improvement has been proved. Representative materials include Hovenia dulcis extract, Extract, Milk Seed Extract (Silymarin Formulation), Bokbunja Extract and Lactobacillus fermented Sea Tangle Extract have been developed and used as individual recognized health functional foods. However, many kinds of such compositions for improvement of liver function and for eliminating hangover are commercialized and sold, but they have not achieved great efficacy.

According to the data released by the National Statistical Office in 2016 (Statistics on the Cause of Death in 2016, National Statistical Office), liver disease is the eighth leading cause of death in Korea. Of the three causes of death by age, liver disease was reported to be the leading cause of death after cancer and suicide among the aged 30 ~ 60 generations.

Therefore, it is urgent to develop a material that can effectively treat liver disease together with liver protection by improving liver function.

Accordingly, the present inventors have conducted intensive studies to solve the problems of health functional foods including simple extracts of herbal medicines, and as a result, it has been found that there is an effect of improving the liver function of the zifenoid compound isolated from the outside And completed the present invention.

Accordingly, an object of the present invention is to provide a composition for improving liver function comprising at least one ziphenocide compound represented by the following formula (I).

Another object of the present invention is to provide a method for preparing a composition for improving liver function comprising the ziphenocide compound.

Another object of the present invention is to provide a method for improving liver function, liver protection, and liver damage using a composition for improving liver function comprising the above-described zifenoid compound.

In order to solve the above problems, the present invention provides a composition for improving liver function comprising at least one ziphenocide compound represented by the following formula (I)

(I)

In this formula,

R ₁ is the same or different sugar chain-linked polysaccharide selected from the group consisting of rhamnose, xylose, glucose and arabinose,

R ₂ is methyl (CH 3) or aldehyde (CHO)

,

,

,

,

,

또는

이다.R ₃ is

,

,

,

,

,

or

to be.

Polysaccharide refers to a saccharide in which two or more monosaccharides are linked by a single molecule through a glycosidic bond. The molecular weight of the polysaccharide ranges from several thousands to millions. The polysaccharide may be a polysaccharide such as starch or glycogen and a polysaccharide such as cellulose nicholine Polysaccharides and the like may be included, but are not limited thereto.

The monosaccharide has the simplest structure that is no longer hydrolyzed by acids, bases, enzymes, etc., such as rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose Carbohydrate basic unit, and the like can be included, but are not limited thereto.

The glycosidic linkage refers to a chemical bond in which the link between the carbon of one sugar molecule and the carbon of another sugar molecule is linked by an oxygen atom. A sugar containing an aldehyde or a ketone forms a cyclic hemiacetal or hemiketal structure in a molecule. The hydroxyl group newly emerging from the formation of this structure is referred to as an anomeric hydroxyl group, and a bond generated by substituting the anomeric hydroxy group Is a glycosidic bond. The glycoside bond derived from aldehyde is referred to as an aldose bond, and the glycoside bond derived from a ketone is referred to as a ketose bond.

The ziphenocide compound may be a ziphenocide compound that is isolated from the outside.

The ziphenocide compound may be a ziphenocide compound characterized by being Zifenoside UL4 as defined below.

In one embodiment of the invention, the ziphenocide compound is selected from the group consisting of ziphenoside UL1, ziphenoside UL2, ziphenoside UL3, ziphenoside UL4, ziphenoside UL5, ziphenoside UL6 and zifenoid And UL7. The present invention also provides a composition for improving liver function.

The ziphenoside UL1 was prepared from (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-damsar-24-en-3-O- [α-L-rhamnopyranosyl (1? 2)] -? - L-arabinopyranoside ((23ξ) -3 ?, 20 ?, 21? -Trihydroxy-19-oxo-21,23-epoxy- dammar- α-L-rhamnopyranosyl (1 → 2)] - α-L-arabinopyranoside.

In the above, ziphenoside UL2 was synthesized from (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-damsar-24-en-3-O- [β-D-glucopyranosyl (1? 3)] -? - L-arabinopyranoside ((23ξ) -3 ?, 20 ?, 21? -Trihydroxy-19-oxo-21,23-epoxy- dammar- β-D-glucopyranosyl (1 → 3)] - α-L-arabinopyranoside.

In the above, ziphenoside UL3 was synthesized from (23ξ) -3β, 20β, 21α-trihydroxy-21,23-epoxy-damsar-24-en-3-O- [α-L-rhamnopyranosyl )] [[beta] -D-xylopyranosyl (1 → 3)] - α-L-arabinopyranoside ((23ξ) -3β, 20β, 21α-trihydroxy- ene-3-O- [alpha -L-rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)] - α-L-arabinopyranoside.

In the above, ziphenoside UL4 was synthesized from (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-dammar- (1? 2)] [? -D-xylopyranosyl (1? 3)] -? - L-arabinopyranoside ((23ξ) -3 ?, 20 ?, 21? -Trihydroxy- 23-epoxy-dammar-24-ene-3-O- [α-L-rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)] - α-L-arabinopyranoside.

Wherein the ziphenoside UL5 is (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-damsar- (1? 2)] [? -D-glucopyranosyl (1? 3)] -? - L-arabinopyranoside ((23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23 -epoxy-dammar-24-ene-3-O- [α-L-rhamnopyranosyl (1 → 2)] [β-D-glucopyranosyl (1 → 3)] - α-L-arabinopyranoside.

In the above, ziphenoside UL6 is 3?, 20S, 21-trihydroxy-19-oxo-dimarb-24-ene-3-O- [? -L-rhamnopyranosyl (1? 2)] {[? D-xylopyranosyl (1 → 6)] [β-D-glucopyranosyl (1 → 3)]) - α-L-arabinopyranoside-21-O- β-D-glucopyranoside [Beta] -D-xylopyranosyl (1- > 6)] [[beta], 20S, 21-trihydroxy-19-oxo-dammar- -D-glucopyranosyl (1? 3)] -? - L-arabinopyranoside-21-O-? -D-glucopyranoside.

In the above, ziphenoside UL7 is a 3?, 20S-dihydroxy-21-carboxyl-damsar-24-en-3-O- [? -L-rhamnopyranosyl -X-L-arabinopyranoside-21-O-? -D-glucopyranoside (1? 3)] -? - L-arabinopyranoside 3β, 20S-dihydroxy-21-carboxyl-dammar-24-ene-3-0- [α-L-rhamnopyranosyl (1 → 2)] {[β-D-xylopyranosyl glucopyranosyl (1? 3)] -? - L-arabinopyranoside-21-O-? -D-glucopyranoside.

The structural formulas of the above-mentioned zifenoids UL1, ziphenoside UL2, ziphenoside UL3, ziphenoside UL4, ziphenoside UL5, ziphenoside UL6, and ziphenoside UL7 are as shown in FIG.

In one embodiment of the present invention, the composition for improving liver function may contain the ziphenocide compound in an amount of 0.01 to 100% by weight based on the weight of the total composition. More specifically, the composition for improving liver function may contain 1.0 to 90% by weight of the ziphenocide compound based on the weight of the total composition. When the content of the ziphenocidal compound is less than 0.01% by weight, the effect of improving the liver function is not exhibited. When the content of the ziphenocidal compound is more than 90% by weight, the effect of improving the liver function by the increase of the content is insignificant and not economical.

In another embodiment of the present invention, the composition for improving liver function may contain 0.2 to 30% by weight of ziphenoside UL4 relative to the weight of the total composition. When the content of zifenoid UL4 is less than 0.2% by weight, the effect of improving liver function is not exhibited. When it exceeds 30% by weight, the effect of improving the liver function by increasing the content is insignificant and not economical.

In one embodiment of the invention, the ziphenocide compound may be isolated from the extraterrestrial. The ziphenocidal compound separated from the outside is abbreviated as extramarginal ziphenocide in the following.

In one embodiment of the invention, the composition may additionally comprise a flavonoid compound.

The flavonoid compound may be synthesized or extracted / isolated from plants, or may be isolated from the outside. The flavonoid compound may be a novel flavonoid compound and may be a known flavonoid compound such as rutin or ombuoside. The flavonoid compound separated from the outside is abbreviated as extracellular flavonoid in the following.

In one embodiment of the invention, the composition may be a mixture of the ziphenocide compound and the flavonoid compound.

In one embodiment of the present invention there is provided a pharmaceutical composition comprising a compound selected from the group consisting of the defined ziphenoside UL1, ziphenoside UL2, ziphenoside UL3, ziphenoside UL4, ziphenoside UL5, ziphenoside UL6 and ziphenoside UL7 An extrinsic ziffenide fraction comprising at least one ziphenocide compound.

In one embodiment of the present invention, the composition for improving liver function may contain an extraordinary ziphenocide fraction in an amount of 0.01 to 100% by weight based on the weight of the total composition. More specifically, the off-axis ziphenocide fraction may be included in an amount of 20.0 wt% to 90 wt% based on the weight of the total composition. When the content of the fraction is less than 0.01% by weight, the effect of improving the liver function is not exhibited. When the content of the fraction is more than 90% by weight, the degree of increase of the liver function improving effect on the increase of the content is insignificant, It is not economical.

In one embodiment of the present invention, the composition for improving liver function may comprise a mixture comprising extracellular flavonoids and extrapolated ziphenocides as defined above.

The mixture may be a mixture of an extracellular flavonoid fraction and an extrinsic zyphenoid fraction, and the extracellular extracellular fraction may be an extracellular flavonoid and an extrinsic zyphenoid.

The mixture may be an extracellular flavonoid (or a fraction thereof) and an extraphenol diphenocide (or a fraction thereof) in a volume ratio of 9: 1 to 1: 9.

The liver function improvement may be hepatitis, fatty liver, liver fibrosis improvement function, or the like.

In one embodiment of the invention, the ziphenocide compound is selected from the group consisting of

(i) extracting the dried ground surface material other than the stones with a mixed solvent of water and alcohol, and concentrating the extract to obtain an extralogy of surface area extract;

(ii) filtering or centrifuging the solution obtained by dissolving the extralogy of surface extract in a mixed solvent of water and alcohol to obtain filtrate or supernatant; And

(iii) passing the filtrate or supernatant through an ion exchange resin column to purify, and concentrating and drying the eluate.

The step (iii) may further include the step of eluting the eluate with a mixed solvent of chloroform and alcohol in a column chromatography.

In step (i), the ground-portion dried material other than the stone is extracted at room temperature with a mixed solvent of water and alcohol mixed at a volume ratio of 9: 1 to 1: 9, and then heated and extracted with a reflux cooling method at 60 to 95, After cooling, the mixture is filtered under reduced pressure, and the filtrate is concentrated under reduced pressure to obtain an extralorgeal part-area extract.

In the step (ii), the extralipidial surface extract may be dissolved in a mixed solvent of water and alcohol mixed at a volume ratio of 9: 1 to 1: 2.

In the above step (i), a mixed solvent of purified water and ethanol corresponding to 5 to 20 times the mass / volume ratio of dried / cut off-shore ground parts (leaves and stems) is stirred and extracted at 50 to 95, Crude extract can be obtained.

In the step (ii), the ethanol crude extract obtained above is adsorbed on a column packed with a porous polymer synthetic adsorbent resin, washed with purified water and ethanol mixed solvent, and eluted, and then the eluate obtained is dried to obtain an extraneous zifenoid fraction .

The extraction solvent in step (i) and the washing and eluting solvent in step (ii) can be one or more of purified water, alcohol, C1 to C4 lower alcohol, ethyl acetate, butanediol, propylene glycol have.

The porous polymer synthetic adsorbent resin in step (ii) may be selected from the group consisting of Diaion HP20, HP21, and HP2MG resins.

The ziphenoside fraction eluted through the column was concentrated under reduced pressure to evaporate the solvent, followed by lyophilization to obtain a powdery form or an aqueous solution in which the solid content was kept constant, thereby preparing a composition for improving liver function Lt; / RTI >

However, the method for producing the ziphenocide compound according to the present invention is not limited thereto, and the ziphenocide compound may be extracted from plants other than the extraterrestrial, or may be synthesized by a general chemical synthesis method.

The extraneous raw material for obtaining the above-mentioned ziphenocide compound may be cultivated in Ulleung County, Gyeongbuk Province, but is not limited thereto.

The composition for improving liver function according to the present invention may be administered orally (for example, by taking or inhalation) or parenterally (for example, by injection, percutaneous absorption, rectal administration) Intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. The pharmaceutical composition according to the present invention may be formulated into tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, Can be formulated. The various forms of the pharmaceutical composition according to the present invention can be prepared by a known technique using a pharmaceutically acceptable carrier commonly used in each formulation. Examples of pharmaceutically acceptable carriers are excipients such as excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, encapsulating agents, sweeteners, solubilizers, bases, , Suspending agents, stabilizers, coloring agents and the like.

The composition for improving liver function according to the present invention may be a pharmaceutical composition.

The pharmaceutical composition may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt and / or a buffer for controlling osmotic pressure, and other therapeutically useful substances, Can be formulated in the form of an administration agent or a parenteral administration agent. In the case of parenteral administration, it can be administered by topical application to the skin, intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like.

In the above pharmaceutical composition, the content of the composition for improving liver function varies depending on the form of the pharmaceutical composition, but is about 0.01 to 100% by weight. The pharmaceutical composition of the present invention may be for prevention and treatment of liver disease.

The composition for improving liver function according to the present invention may be a food additive added to foods.

There is no particular limitation on the type of the food, and it may be in the form of an oral preparation such as a powder, granule, tablet, capsule, suspension, emulsion or syrup, or in the form of an oral preparation such as candy, sweets, gum, ice cream, Can be added.

The food may be prepared by appropriately using a filler, an extender, a binder, a wetting agent, a disintegrant, a sweetener, a fragrance, a preservative, a surfactant, a lubricant,

In the food, the content of the composition for improving liver function varies depending on the form of the health functional food, but is about 0.01 to 100% by weight. Since the health functional food of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

The above-mentioned "gynostemma pentaphyllum", which is also called "chrysanthemum pentaphyllum", is a perennial vine plant belonging to the cucurbitaceae native to southern part of Korea, the islands such as Jeju Island and Ulleungdo, and China and Japan. The roots grow sideways, the white hairs on the nodes, the tangles grow up, but they climb up with their tendrils. It is known that tea made from dried leaves is called vine tea. It restores the function of organs, exhibits antistress, antioxidant effect, and is effective for asthma, bronchitis and ulcer.

The above-mentioned "ziphenocide or ziphenocide compound" may be differently referred to as dammarane type saponin, triterpene saponin, gynosaponin and the like, Other names of the Nosides are also included in the scope of the present invention.

The "flavonoid or flavonoid compound" is a kind of secondary metabolites of plants or fungi. Chemically, the flavonoid has a general structure of 15 carbon skeletons composed of two phenyl rings (A and B) and a heterocyclic ring (C) Have. This carbon structure can be abbreviated as C6-C3-C6. The flavonoids include novel flavonoid compounds and may be modified in a variety of ways based on the isoflavonoid, neoflavonoid, flavone or isoflavone structure (e.g., glycoside or dimer Etc.) flavonoid compounds. Other names of flavonoids commonly used in the art are also included within the scope of the present invention.

The term "extract " used in the present invention means an extract obtained by subjecting a specific animal or animal or mixture thereof to an extraction method, a diluted solution or a concentrate of the extract, a dried product obtained by drying the extract, And extracts of all the formulations that can be formed using the extract. The method for extracting the above extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hydrothermal extraction, ultrasonic extraction, filtration, and reflux extraction. These may be performed alone or in combination with two or more methods.

The term "fraction" as used herein is a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. The fractionation method for obtaining the fraction is not particularly limited and may be carried out according to a method commonly used in the art. As a non-limiting example of the above-mentioned fractionation method, a method of treating a mixed extract of the present invention with a predetermined solvent to obtain a fraction from the above extract.

As used herein, the term " liver function improvement "means prevention of liver function deterioration through inhibition of liver cell death or promotion of activity. Specifically, it helps improve hunger, fatigue, anorexia, abdominal distension, headache, and reduces the risk of diseases such as jaundice, hepatitis, hepatic coma, ascites, thrombocytopenia, alcoholic fatty liver, nonalcoholic fatty liver, liver fibrosis Function.

As used herein, the term "significant" means statistically significant at the 99 or 95% level.

The composition for improving liver function comprising a ziphenocide compound isolated from the outside of the present invention has excellent liver injury inhibition, anti-inflammation, and antioxidant efficacy.

Figure 1 shows the structural formulas of the ziphenosides UL1 to UL7.
2 is a chromatogram of a test substance, (a) ziphenoside UL4, (b) extracellular flavonoid fraction, (c) extrapolated ziphenocide fraction, (d) extracellular flavonoid and ziphenocide mixture by HPLC-UVD .
FIG. 3 shows the results of measuring the activity of AST and ALT in serum of C57BL / 6 mice administered with D-GalN / LPS.
FIG. 4 shows the results of measuring the levels of inflammatory cytokines TNF-.alpha. And IL-6 in liver tissue of C57BL / 6 mice administered with D-GalN / LPS.
FIG. 5 shows the results of measurement of antioxidant enzyme activities and lipid peroxide contents in liver tissue of D5-GalN / LPS-administered C57BL / 6 rats.
FIG. 6 shows the results of measuring the activity of AST and ALT in serum of MCD rats.
FIG. 7 shows the results of measuring gene expression levels of inflammatory factors in liver of MCD mice.
FIG. 8 shows the results of measurement of the amount of inflammatory cytokine expression in rat liver cells.
FIG. 9 shows the result of measurement of antioxidant enzyme activities in liver tissue of MCD rats.
10 shows the H & E staining liver tissue of MCD rats.
Fig. 11 shows the result of measurement of the amount of triglyceride in liver tissue of MCD rats.
Figure 12 shows the Sirius red staining of MCD mice.
FIG. 13 shows the results of measurement of gene expression levels of hepatic fibrosis in liver of MCD mice.
FIG. 14 shows the results of measurement of expression amounts of Sirt 1 to 7 protein in MCD mice.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, it is to be understood that the examples and experimental examples are provided only for the understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.

Example  One. Outside  Isolated Zifenoid  Preparation of compounds

(1) Production of an outpatient extract

Weigh out 24.0 kg and add to 336.0 L of 40% ethanol, extract at 80 ~ 90 ℃ for 4 hours, and then extract the extract to obtain the first extract. 240.0 L of 40% ethanol is added to the extractor in which the extracted residue is left, and the mixture is extracted at 80 to 90 ° C for 2 hours and then discharged to obtain a secondary extract. The primary and secondary extracts are filtered and concentrated under reduced pressure to obtain a concentrate.

(2) Preparation of out-of-column fraction

The extracorporeal extract obtained in Example 1- (1) was packed with a porous synthetic adsorbent, Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), and 20 L of 95% ethanol was added thereto. And 40 L of purified water was poured to reduce the alcohol concentration of the resin to 15%. Ethanol is added to the extracellular concentrate to increase the solubility of the sample and make the concentration of ethanol in the concentrate 15%. The concentrate is then added to the column for adsorption. The column was eluted with 15% ethanol to remove the inorganic matter and eluted with 45% ethanol to obtain the extracellular flavonoid fraction, followed by elution with 80% ethanol to obtain the extrapolated ziphenocide fraction.

In addition, 15% ethanol was flowed through the column-derived extracellular extract obtained in Example 1- (1), followed by elution with 80% ethanol to obtain a mixture of flavonoids and ziphenocides.

Each eluate was concentrated under reduced pressure to evaporate the solvent and then lyophilized to obtain a powdery fraction.

(3) Separation and Identification of Components in Extraordinary Ziphenocide Fractions

The component separation from the extraordinary ziphenocide fractions obtained in Example 1- (2) was carried out as follows. The extrapolated ziphenoside fraction obtained in Example 1- (2) was taken in a column (diameter 4.6 cm x height 31 cm) using 323 g of silica gel, and then eluted with chloroform: methanol = 8: 1 mixed solvent (v / v ) 2 L; 5: 1 mixed solvent (v / v) 3 L; 3: 1 mixed solvent (v / v) 2 L; Chromatography was carried out using 3 L of 1: 1 mixed solvent (v / v) sequentially to obtain Fr. A (1.56 g); Fr. B (5.99 g); Fr. C (3.12 g); Fr. D (8.15 g). Each fraction was chromatographed on MCI-gel, increasing the aqueous concentration of acetonitrile to 5, 10, 15, 20, and 25%. A, zifenoid UL1 and zifenoid UL2 were obtained, and Fr. B, zifenoid UL3 and zifenoid UL4 were obtained, and Fr. C to Zifenoside UL5, Fr. ≪ RTI ID = 0.0 > UL6 < / RTI > and ziphenoside UL7.

The structural formulas of the above-mentioned zifenoids UL1, ziphenoside UL2, ziphenoside UL3, ziphenoside UL4, ziphenoside UL5, ziphenoside UL6, and zifenoxide UL7 are as shown in FIG.

(4) Yield evaluation

When the yield was evaluated, the yield of extramaradial zifenoid UL4 was low and the yield of the extracellular flavonoid and ziphenocide mixture was higher than that of the single extracellular flavonoid fraction or the single extruded zyphenoid fraction. Yield can be a factor in product development in both economic and commercial terms.

Example  2. Outside column Fraction  Chromatogram analysis

(A) ziphenoside UL4 obtained in Example 1, (b) extracellular flavonoid fractions, (c) extramarginal zephnoside fractions, (d) extracellular flavonoids and ziphenocide mixtures were analyzed by HPLC. HPLC-UVD was analyzed at 203 nm using a C18 column with a diameter of 4.6 mm and a length of 25 cm under conditions of gradient of acetonitrile and distilled water. The chromatogram is shown in FIG. The content of UL4 of the ziphenocide of (a) ziphenoside UL4, (b) extracellular flavonoid fractions, (c) extraneous ziphenocide fractions, (d) extracellular flavonoids and ziphenocide mixture (b) the off-flavonoid fraction is 1.65%, (c) the off-flavophosphorus fraction is 12.23%, and (d) the off-flavonoid and ziphenocide mixture contains 7.35%.

Experimental Example  One. Outside  Identification of liver damage inhibition effect by extract

D-galactosamine (D-GalN) is a type of aminosulfate that inhibits mRNA and protein synthesis through metabolic disturbance of galactose in the liver, thus causing liver damage. Lipopolysaccharide (LPS) is a major component of Gram-negative bacteria and plays an important role in the initiation phase of endotoxic damage and activates inflammatory cytokines, resulting in damage to liver tissue. Thus, the D-GalN / LPS-induced acute liver injury model induces typical hepatocyte necrosis and apoptosis, and is widely used for liver health-related studies.

Experimental animals were matched for 1 week under a certain condition (temperature 22 ± 1, humidity 55 ± 3%, light period 12 hours) in male rat C57BLS / J-m (Samtako, Osan, Korea) at 6 weeks of age. D-GalN (700 mg / kg) and LPS (1 μg / kg) were dissolved in physiological saline and injected intraperitoneally to give the rat model acute liver injury. The experimental group was divided into 7 groups; (D-GalN / LPS + silymarin 200 mg / kg, or less), a control group (D-GalN / LPS, hereinafter referred to as a negative control group) and a silymarin administration control group (D-GalN / LPS + extracellular flavonoid fractions 200 mg / kg, hereinafter, referred to as Flavonoid group) were administered to the group of the external flavonoid fractions (D-GalN / LPS plus Zifenoid UL4 40 mg / (D-GalN / LPS + extracellular flavonoid fractions, 200 mg / kg or less, ziphenoside group), extracorporeal flavonoids and ziphenocide mixture administration groups (D-GalN / LPS + extracellular flavonoids and lipid Noside mixture 200 mg / kg, below, mixture group). The rats of the 7 experimental groups were sacrificed under anesthesia and blood was collected from the abdominal aorta. Blood was centrifuged and the supernatant was used for AST and ALT measurements. The liver was removed, washed with physiological saline, and stored at -80 ° C.

1) Determination of liver damage inhibition effect by measuring activity of AST and ALT

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are used as indicators of hepatocyte necrosis because of the increase in AST and ALT levels in the blood, have. AST and ALT were analyzed using separate assay kits (Asan, Pharmaceutical, Seoul, Korea).

The results of measuring the AST and ALT activities in serum of the 7 experimental groups are shown in FIG. AST was significantly increased in the control group as compared with the normal group, and UL4 group, flavonoid group, ziphenoside group, and mixture group were significantly decreased as compared with the negative control group. In addition, ALT was significantly increased in the control group as compared with the normal group, and the UL4 group, the ziphenocide group, and the mixture group were significantly decreased as compared with the negative control group, and it was found to be similar to that of the silymarin group .

2) Determination of anti-inflammatory effect by measurement of IL-6 and TNF-α content

The contents of inflammatory cytokines in liver tissues were measured to confirm the anti - inflammatory effect of each experimental group. The liver tissues were ground with PBS and centrifuged and supernatant was used. The contents of IL-6 and TNF-α, which are inflammatory cytokines in liver tissues, were measured by ELISA assay kit (CUSABIO BIOTECH CO, Wuhan, China).

The results of measurement of IL-6 and TNF-? Contents in liver tissues are shown in Fig. The levels of IL-6 and TNF-α in the liver tissues were significantly increased in the control group compared to the control group, and the UL4, ziphenoside, and mixture groups were significantly decreased compared with the control group. As shown in Fig.

3) SOD, catalase and GSH - Px  Active measurement and MDA  Through content measurement Oxidative  Check for damage protection

The reactive oxygen species (ROS) produced by oxidative stress are mostly caused by antioxidant enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) However, if an excessive amount of ROS causes an imbalance in the antioxidant defense system, various diseases will occur and promote aging. Lipid peroxidation is a lipid peroxidated by the addition of oxygen to unsaturated fatty acids, a lipid component. This means that cells or tissue membranes are damaged from free radicals. Lipid peroxidation leads to the production of MDA (malondialdehyde) as a by-product by chain reaction with free radicals in polyunsaturated fatty acids and lipoproteins of cell membranes, and MDA is used as an index of lipid peroxidation.

The results of measuring the activity of SOD, catalase and GSH-Px and MDA content in liver tissue are shown in FIG. The activity of SOD, catalase and GSH-Px was significantly decreased in the control group and the content of MDA was significantly increased. SOD activity was significantly increased in UL4 group, flavonoid group, ziphenoside group, and mixture group compared with control group. Catalase activity was significantly increased in the flavonoid, ziphenocide, and mixture groups compared to the control. GSH-Px activity was significantly increased in the ziphenocide group and the mixture group as compared with the control group. MDA content was significantly decreased in the ziphenocide group and the mixed group compared to the control group.

4) Sintering

Based on the results of Examples 1) to 3) of Examples of the present invention and Experimental Example 1 described above, the inventors of the present invention have found that the extracellular ziphenoside UL4, the extracellular flavonoid fraction, the extramarginal zephnoside fraction and the extracellular flavonoid and ziphenocide mixture It was confirmed that the effect of improving liver function was excellent, and it was confirmed that it could be usefully used for improvement, prevention or treatment of liver disease.

Experimental Example  2. Outside  Flavonoids and Zifenoid  The effect of suppressing liver damage of the mixture powder (hereinafter referred to as mixture powder)

Methionine choline deficient (MCD) C57BLS / J-m (Samtako, Osan, Korea) mouse model, which causes metabolic disturbances due to methionine and choline deficiency, is widely used in nonalcoholic fatty liver tests. The MCD rats are deficient in nutrients, which increase the number of active oxygen species in the liver and accumulate triglyceride (TG).

The MCD mice were adapted for one week under constant conditions. The MCD rats were orally administrated daily with the powder dissolved in water, and the experiment was carried out after 4 weeks. The experimental group was classified into 6 groups; (MCD diet + silymarin 200 mg / kg, positive control group), 50 mg of the mixture powder (normal control group, normal group, negative control group, MCD diet group and negative control group) kg / kg group (MCD diet + mixture powder 50 mg / kg) mixture powder 100 mg / kg group (MCD diet + mixture powder 100 mg / kg) and mixture powder 200 mg / kg group (MCD diet + mixture powder 200 mg / kg) .

1) ALT and AST  Activity, SOD and catalase concentration, inflammation related gene expression level and serum inflammatory cytokine Expression level  Inhibition of Liver Damage by Measurement, Oxidative  Damage protection, and anti-inflammatory effects

After 4 weeks, the activity of ALT and AST in serum, the antioxidative enzymes SOD and catalase concentration, and the expression level of inflammation-related genes were measured in the MCD rats.

The degree of inflammation-related gene expression was measured by RT-PCR using the genes of Adgre1, Cd11c, Ccr2, Ccl2, Icam1 and TNF-α, IL-6 and IL1b as inflammatory cytokines.

As shown in FIG. 6, when the mixture powder and MCD diet were administered for 4 weeks, the mixture powder treated group had a lower concentration of ALT and AST in the serum than the negative control. Through this, it was confirmed that a mixture of extracellular flavonoid and ziphenocide inhibited liver damage.

As shown in FIG. 7, the expression of the inflammation-related gene in the mixture powder was similar to that of the positive control. In addition, the level of inflammation - related gene expression was inhibited in a concentration - dependent manner. In addition, as shown in FIG. 8, the mixture powder decreased the levels of inflammatory cytokines TNF-a, IL-6 and MCP-1 in a concentration-dependent manner. 7 and FIG. 8, it was confirmed that the extracellular flavonoids and the extramyogliphenocide mixture had an anti-inflammatory effect.

As shown in FIG. 9, the mixture powder increased the concentrations of SOD and catalase, which are antioxidative enzymes that can lower the oxidative reactive oxygen species concentration induced by the MCD diet, in a concentration-dependent manner. Through this, it was confirmed that a mixture of extracellular flavonoids and ziphenocides protected the oxidative damage.

2) Histological analysis of liver tissue and liver tissue Amount of triglyceride  Determination of fatty liver inhibition effect by measurement

The MCD rats were orally administered with the mixture powder dissolved in water, and after 4 weeks, the liver tissues of the MCD mice were euthanized and treated with 10% formalin (HCHO, formalin). The formalin-treated tissues were fixed on paraffin (Paraffin), hardened, and then sliced into 5 μm on the slide. The sections were stained with H & E (Hematoxylin & eosin), and analyzed by a microscope (Eclipse E600 polarizing microscope; Nikon, Tokyo, Japan) Respectively.

As shown in FIG. 10, the mixture powder lowered the concentration of MCD-induced fatty liver in a concentration-dependent manner. It was confirmed that the mixture of extracellular flavonoids and ziphenocides had an effect of inhibiting fatty liver.

As shown in FIG. 11, the mixture powder lowered the triglyceride induced by the MCD diet in a concentration-dependent manner. It was confirmed that a mixture of extracellular flavonoids and ziphenocides inhibited the accumulation of triglycerides.

3) Histopathologic analysis of liver and determination of gene expression level related to liver fibrosis

The MCD rats were orally administered with the mixture powder dissolved in water, and after 4 weeks, the liver tissues of the MCD mice were euthanized and treated with 10% formalin (HCHO, formalin). The formalin-treated tissues were fixed on paraffin (Paraffin), hardened, and then sliced into 5 μm on the slide. After the sections were stained with Sirius red, the tissues were analyzed with a microscope (Eclipse E600 polarizing microscope; Nikon, Tokyo, Japan) and the expression level of the tissue fibrosis related genes in liver cells was measured.

The level of gene expression related to liver fibrosis was measured by Acta2, Col1a, and Tgfb genes, and the amount of mRNA expression was measured by RT-PCR.

As shown in FIG. 12, the mixture powder lowered the liver tissue fibrosis induced by the MCD diet in a concentration-dependent manner. It was confirmed that a mixture of extracellular flavonoids and zifenoside inhibits hepatic fibrosis.

As shown in FIG. 13, the mixture powder inhibited the degree of hepatic fibrosis-related gene expression, which was increased by the MCD diet, in a concentration-dependent manner. It was confirmed that a mixture of extracellular flavonoids and zifenoside inhibits hepatic fibrosis.

4) Identification of protective effect of oxidative damage by measurement of Sirt protein expression level

As shown in Fig. 14, the expression levels of Sirt proteins (Sirt 1 to sirt 7) in the normal group, negative control group and 200 mg / kg of mixture powder were compared and Sirt 1 and 6 were relatively decreased in the negative control group Sirt 1 and Sit 6 were increased in Sirt 1 and Sirt 6 200 mg / kg group compared with negative control group and 200 mg / kg group powder.

Sitr 6 is known to act as an activator of the Nrf2 signaling system, which regulates antioxidant enzyme expression. Therefore, the mixture powder can increase the antioxidant enzyme expression by activating the Nrf2 signaling system by increasing the Sirt 6 protein expression level.

Through this, it was confirmed that a mixture of extracellular flavonoids and ziphenocides protected the oxidative damage.

Experimental Example  3. Outside  Flavonoids and Zifenoid  Stability of the mixture powder (hereinafter referred to as mixture powder)

In order to confirm the stability of the mixture powder, it was put in a 20 mL airtight container and stored in a stability chamber for 12 months at 25 ° C and a relative humidity of 60 ± 5% for 12 months. Acceleration conditions of 40 ± 2 ° C. and relative humidity of 75 ± 5% For 6 months. In the above test, the change in the weight loss and the change in the index component were observed through HPLC analysis, and the results are shown in Tables 1 and 2. As shown in Tables 1 and 2, it was confirmed that the mixture powder was stable at both room temperature and acceleration conditions.

Claims (10)

A composition for improving liver function comprising at least one ziphenocide compound represented by the following formula (I) as an active ingredient:
(I)

In this formula,
R ₁ is the same or different sugar chain-linked polysaccharide selected from the group consisting of rhamnose, xylose, glucose and arabinose,
R ₂ is methyl (CH ₃ ) or aldehyde (CHO)
R ₃ is

,

,

,

,

,

or

to be.
The method of claim 1, wherein the ziphenocide compound is selected from the group consisting of ziphenoside UL1, ziphenoside UL2, ziphenoside UL3, ziphenoside UL4, ziphenoside UL5, ziphenoside UL6, and ziphenoside UL7 A composition for improving liver function comprising one of the group selected from the group consisting of:
In the above,
Zifenoside UL1 was synthesized from (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-damsar-24-en-3-O- [α-L-rhamnopyranosyl - (2)] -? - L-arabinopyranoside ((23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-dammar- L-rhamnopyranosyl (1- > 2)] - [alpha] -L-arabinopyranoside);
Zifenoside UL2 was prepared from (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-damsar-24-en-3-O- [β-D-glucopyranosyl - 3 -] -? - L-arabinopyranoside ((23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-dammar- D-glucopyranosyl (1 → 3)] - α-L-arabinopyranoside);
Zifenoside UL3 is a (23?) - 3?, 20?, 21? -Trihydroxy-21,23-epoxy-damsar-24-en-3-O- [? -L-raminopyranosyl [? -D-xylopyranosyl (1? 3)] -? - L-arabinopyranoside ((23ξ) -3β, 20β, 21α-trihydroxy-21,23-epoxy- dammar- 3-O- [alpha -L-rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)] - α-L-arabinopyranoside;
Zifenoside UL4 was prepared from (23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy-damsar-24-en-3-O- [α-L-rhamnopyranosyl (2?) -? - D-xylopyranosyl (1? 3)] -? - L-arabinopyranoside ((23ξ) -3 ?, 20 ?, 21? -Trihydroxy-19- epoxy-dammar-24-ene-3-O- [alpha -L-rhamnopyranosyl (1 → 2)] [β-D-xylopyranosyl (1 → 3)] - α-L-arabinopyranoside;
Zifenoside UL5 is a (23?) - 3 ?, 20 ?, 21? -Trihydroxy-19-oxo-21,23-epoxy-damsar-24-en-3-O- [? -L-rhamnopyranosyl -2?)] [? - D-glucopyranosyl (1? 3)] -? - L-arabinopyranoside ((23ξ) -3β, 20β, 21α-trihydroxy-19-oxo-21,23-epoxy - dammar-24-ene-3-O- [alpha -L-rhamnopyranosyl (1 → 2)] [β-D-glucopyranosyl (1 → 3)] - α-L-arabinopyranoside;
Zifenoside UL6 is a 3?, 20S, 21-trihydroxy-19-oxo-dimercurine-24-en-3-O- [? -L-rhamnopyranosyl (1? 2)] {[? Glucopyranosyl (1? 3)] -? - L-arabinopyranoside-21-O-? -D-glucopyranoside (3? , 20S, 21-trihydroxy-19-oxo-dammar-24-ene-3-O- [α-L-rhamnopyranosyl (1 → 2)] {[β-D-xylopyranosyl -glucopyranosyl (1 → 3)] - α-L-arabinopyranoside-21-O- β-D-glucopyranoside);
Zifenoside UL7 is a 3?, 20S-dihydroxy-21-carboxyl-damsar-24-ene-3-O- [? -L-rhamnopyranosyl (1? 2)] {[? (1? 3)] -? - L-arabinopyranoside-21-O-? -D-glucopyranoside (3β, D-xylopyranosyl (1 → 6)] [β-D-glucopyranosyl ((1 → 2) 1? 3)] -? - L-arabinopyranoside-21-O-? -D-glucopyranoside;
Zifenoside XLVIII is a 3?, 20S, 21-trihydroxy-19-oxo-dimarb-24-en-3-O- [? -L-rhamnopyranosyl (1 → 2)] [ (1? 3)] -? - L-arabinopyranoside-21-O-? -D-glucopyranoside (3?, 20S, 21-trihydroxy-19-oxo-dammar-24-ene -3-O- [alpha -L-rhamnopyranosyl (1 → 2)] [β-D-glucopyranosyl (1 → 3)] - α-L-arabinopyranoside-21-O-β-D-glucopyranoside.
The composition for improving liver function according to claim 1, wherein the ziphenocide compound is contained in an amount of 0.01 to 100% by weight based on the weight of the total composition.
The composition for improving liver function according to claim 1, wherein the ziphenocide compound is ziphenoside UL4.
5. The composition for improving liver function according to claim 4, wherein the ziphenocide compound comprises ziphenoside UL4 in an amount of 0.2 to 30% by weight based on the weight of the total composition.
2. The composition for improving liver function according to claim 1, wherein the ziphenocidal compound is isolated from the outside.
7. The composition for improving liver function according to claim 6, further comprising a flavonoid compound.
The composition for improving liver function according to claim 7, wherein the mixing ratio of the ziphenocide compound and the flavonoid compound is 9: 1 to 1: 9.
The composition for improving liver function according to claim 1, wherein the liver function improvement is improvement of hepatitis, fatty liver, or liver fibrosis.
The method of claim 1, wherein the ziphenocide compound is selected from the group consisting of
(i) extracting the dried ground surface material other than the stones with a mixed solvent of water and alcohol, and concentrating the extract to obtain an extralogy of surface area extract;
(ii) filtering or centrifuging the solution obtained by dissolving the extralogy of surface extract in a mixed solvent of water and alcohol to obtain filtrate or supernatant; And
(iii) concentrating and drying the filtrate or supernatant liquid.

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