KR20160008425A - Composition for improvement of liver function comprising exopolysaccharide derived from ceriporia lacerata culture broth extracts as an active ingredient - Google Patents

Abstract

The present invention relates to the use of Ceriporia < RTI ID = 0.0 > The present invention relates to a composition for improving liver function and preventing or treating liver diseases, which comprises an exopolysaccharide derived from a mycelial liquid culture extract as an active ingredient. The extracellular polysaccharide derived from the culture medium of the mycelium of the present invention can be applied not only as a composition for preventing or treating liver diseases, but also as a material for related foods due to an excellent liver function improving effect.

Description

Technical Field [0001] The present invention relates to a composition for improving liver function comprising an extracellular polysaccharide derived from an extract of a culture medium of Sicilia lacarata as an effective ingredient. [0002] The present invention relates to a composition for enhancing liver function,

The present invention relates to the use of Ceriporia < RTI ID = 0.0 > The present invention relates to a composition for improving liver function or an agent for preventing or treating liver disease, which comprises an extracellular polysaccharide derived from a mycelial culture liquid extract as an active ingredient.

The liver is an organ that plays an important role in defending the body from toxic substances introduced from the outside and taking charge of the metabolism of the in vitro substances. It converts the various nutrients ingested into the form necessary for the living body, And also performs a variety of functions such as switching the state of the living body foreign body into an easy-to-excrete state. Since the in vivo material passes through the liver once, the liver is more likely to be exposed to many toxic substances than nutrients, and is more likely to be damaged than other organs. The liver is an organ with excellent regeneration ability and is sufficiently restored to some damage. However, if it is continuously damaged by excessive stress, smoking, exposure to chemicals due to environmental pollution, alcohol and virus infections, bile secretion, etc., not only the function is deteriorated but also the part of the liver tissue is completely destroyed and the damaged part is normal Resulting in unrecoverable consequences. Finally, liver fibrosis can lead to fatal liver cirrhosis, and liver cirrhosis can develop into liver cancer. In addition, liver disease is not found at the initial stage of pain or subjective symptoms, but it is not found until the end of the term, so it is difficult to treat at the appropriate time and the mortality rate is high. Thus, despite the seriousness of liver disease, there is no effective treatment for liver disease.

Antiviral drugs are used in the case of hepatitis caused by hepatitis virus, but their side effects are serious. In recent years, toxic substances caused by environmental pollution have not yet been effectively treated. Therefore, development of drugs that can treat and prevent liver damage is urgently needed.

Currently, clinically widely used treatments for liver diseases include dimethyldethoxy biphenylate, an artificial synthetic material similar to silymarin and schizandrin, a component of Schizandrin, isolated from milk sac (Silybum marianum) Ursodeoxycholic acid (ursodeoxy cholic acid) and vitamin B complex group. There are not many kinds of therapeutic agents compared with the severity of the disease and side effects are frequently reported. In this regard, the natural efficacy of natural products has been proven for a long time and generally has less side effects than chemical substances, so the development of therapeutic agents for liver diseases from natural products is very significant.

Ceriporia lacerata is a type of white rot fungus which is known to carry out co-metabolism of lignin decomposition in order to utilize carbon sources such as cellulose, hemicellulose, other polysaccharides and glycerol in the ecosystem. However, since the existence of the serpolyla lucerata was first reported to academia in 2002, research on its industrialization is still insignificant. Specifically, the present inventors have filed a patent application entitled "Method for Producing Extract of Culturia Lacera rata for Prevention and Treatment of Diabetes Mellitus, Korean Patent No. 10-1031605 on " Culture Extracts " The extent to which the therapeutic effect of the culture extract on diabetes is disclosed.

Accordingly, the inventors of the present invention have continued intensive studies on the culture solution of mycelium lacticera mycelium, and as a result, (Exopolysaccharide; EPS) derived from a culture supernatant has an effect of improving liver function, thereby completing the present invention.

KR 10-1031605 B1

It is an object of the present invention to provide a composition for improving liver function or preventing or treating liver disease by containing a natural substance derived from an extract of cultured Mycelia lacticera mycelium as an effective ingredient.

Another object of the present invention is to provide a food for improving liver function, which contains a natural substance derived from an extract of a culture medium of the mycelium of ceriplor larcosera.

In order to achieve the above object, the present invention provides a composition for improving liver function, comprising an extracellular polysaccharide derived from a culture medium of a mycelium of ceriplora lacera, as an active ingredient.

In order to accomplish the above other objects, the present invention provides a composition for preventing or treating liver disease, which comprises an extracellular polysaccharide derived from a culture solution of a mycelial fungus Lactacera mycelium as an active ingredient.

The extracellular polysaccharide may contain about 40 to 60% by weight of sugar and about 30 to 40% by weight of protein and have a molecular weight of about 100 to 150 kDa.

The liver disease may be liver fibrosis, liver cirrhosis, hepatitis or liver cancer.

In order to accomplish the above-mentioned further object, the present invention provides a food for improving liver function, which contains an extracellular polysaccharide derived from a culture solution of mycelia lacticera mycelium.

The composition containing the extracellular polysaccharide derived from the culture medium of the mycelium of ceriplora lactaclor according to the present invention as an effective ingredient can be effectively used for improving liver function or preventing or treating liver disease.

FIG. 1 is a graph showing the molecular weight of EPS according to the purification of culture.

Hereinafter, the present invention will be described in detail.

The present invention relates to the use of Ceriporia < RTI ID = 0.0 > The present invention provides a composition for improving liver function, comprising an exopolysaccharide (EPS) derived from a mycelial culture liquid extract as an active ingredient.

In addition, the present invention provides a composition for preventing or treating liver disease, which comprises an extracellular polysaccharide derived from a culture medium of a mycelium of cerporolactaceras as an active ingredient.

In the composition according to the invention, the extracellular polysaccharide comprises about 40 to 60% by weight of sugar, about 30 to 40% by weight of protein, about 40 to 50% by weight of sugar and about 32 to 38% About 43 to about 47 weight percent sugar, about 33 to about 36 weight percent protein, and preferably about 45 weight percent sugar and about 34 weight percent protein.

The sugar may contain mannose, galactose and glucose.

The extracellular polysaccharide may have a molecular weight of about 100-150 kDa, about 110-140 kDa, or about 115-125 kDa, and preferably about 120 kDa.

In one preferred embodiment, the extracellular polysaccharide is produced by: (a) liquid culturing mycelia lacticera mycelium to prepare a culture of mycelium lacticera mycelium, (b) culturing mycelia lacticera mycelium (C) extracting the culture medium of the cultured Mycelium lacticus mycelium with a solvent, filtering it, and concentrating it under reduced pressure.

The medium for liquid culture of the mycelium of seripositive Lactacera in the step (a) is selected from the group consisting of sugar, glucose, starch, water, barley, soybean powder, magnesium sulfate (MgSO ₄ ), potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) and water, and the hydrogen ion concentration may be pH 4.5 to 6.0.

In one preferred embodiment, the culture medium contains 1 to 2% by weight of sugar, 0.2 to 1% by weight of glucose, 0.2 to 1% by weight of starch, 0.1 to 0.5% by weight of water, 0.1 to 0.5% 0.2 ~ 4 weight%, magnesium sulfate _{(MgSO 4) 0.05 ~ 0.25%} , 1 potassium phosphate by weight (KH ₂ PO _{4) 2} potassium phosphate 0.05 ~ 0.25% by _{weight, (K 2 HPO 4) 0.05} ~ 0.25 wt% and water 92 To 98% by weight.

The liquid culture in the step (a) can be performed under a blue LED light source, and can be performed by maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm.

At this time, the liquid culture is maintained at 20 to 25 ° C with a hydrogen ion concentration (pH) of 4.5 to 6.0, a light source of blue LED and an illuminance of 0.5 LUX, air is injected at 0.5 to 1.5 kgf / cm ² , To 2,000 ppm and can be carried out for 8 to 13 days, and it is preferable that the extracellular polysaccharide content is high because it is carried out at 22 ° C, pH 5, 1.0 kgf / cm ² , and 1,500 ppm for 10 days.

The parent strains in step (a) were incubated in a shaking incubator at 25 ° C using a PDB (Potato dextrose agar) medium and one well of a well-preserved strain stored in a 4-well culture flask. And cultured for 7 to 9 days. At this time, the amount of the mycelium to be added to the inoculum is preferably about 0.5% based on the amount of the solution to be cultured. Since the content of extracellular polysaccharide is not high as the amount of mycelium (% / 100 ml) is high, the composition of the medium is not the most favorable nutritional ratio and environmental condition for growth of mycelium, Is preferably applied.

The culture solution can be separated and purified into mycelium and an aqueous solution. The separated tablets can be purified by repeatedly removing the mycelium from the mycelium with a centrifugal separator (Multi-Sheet Filter Press) and a vibrating centrifugal separator (PALLSEP), and then irradiating ultraviolet rays (UV) for 1 minute. In addition, it is necessary to store oxygen after removing oxygen, because when the hyphae are present in the solution, the content of the active ingredient is changed by the growth of hyphae.

In the step (b), the mycelial culture solution prepared in the step (a) may be pulverized by vacuum drying or freeze-drying. The drying is preferably carried out at a low temperature of 40 DEG C or lower, preferably 30 DEG C or lower, for 48 to 96 hours in order to prevent the disappearance of the active substance. The drying in the step (b) is preferably performed using a vacuum freeze dryer rather than a vacuum dryer in which the evaporation temperature is set to a relatively high level because the change in effective substance content is minimized.

In step (c), the extracellular polysaccharide, which is an active ingredient of the composition of the present invention, is isolated and extracted by extracting the mycelial culture dried product obtained in step (b) with a solvent. This procedure was performed by adding 100 mL of distilled water to 5 g of the dried powder, suspending it well, centrifuging (8,000 rpm, 20 min), adding cold alcohol corresponding to 2 to 3 times its amount to the supernatant, ) And allowed to stand for 12 hours. A crude extracellular polysaccharide can be prepared by centrifuging the supernatant alone (8,000 rpm, 20 min) and collecting the precipitate. The extract is preferably vacuum-freeze-dried at 30 ° C or lower.

The composition for improving liver function and preventing or treating liver disease containing an extracellular polysaccharide derived from an extract of a culture medium of a ceriplora lactamase according to the present invention as an active ingredient may further contain suitable carriers, excipients and diluents conventionally used have.

The extracellular polysaccharide may be contained in an amount of 0.1 to 80% by weight, preferably 0.1 to 50% by weight, based on the total weight of the composition. However, the effective content of the most preferred extract may be suitably adjusted depending on the method and purpose of use of the composition.

Each of the compositions according to the present invention can be formulated and used according to a conventional method. Suitable formulations include, but are not limited to, tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories and the like.

The composition according to the present invention can be prepared into a suitable formulation using a pharmaceutically inert organic or inorganic carrier. That is, when the formulations are tablets, coated tablets, dragees and hard capsules, lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or salts thereof may be used. In the case of soft capsules, vegetable oils, waxes, fats, semi-solid and liquid polyols can be used. Water, polyol, glycerol, vegetable oil, etc. may also be used when the formulation is in the form of a solution or syrup.

The composition according to the present invention may further contain a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizer, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant and the like in addition to the above carrier.

The method of administration of the composition according to the present invention can be easily selected according to the formulation, and can be administered orally or parenterally. The dosage may vary depending on the patient's age, sex, weight, degree of pathology, route of administration, but generally amounts to 5 to 500 mg / kg, preferably 100 to 250 mg / It can be administered in divided doses. However, the dosages do not in any way limit the scope of the invention.

The composition according to the present invention not only provides excellent liver function improving effect but also has little toxicity and side effects due to drugs and can be used safely not only for the purpose of treating liver disease but also for long term administration for preventive purpose.

The liver disease may be liver fibrosis, liver cirrhosis, hepatitis, or liver cancer, but is not limited thereto.

In addition, the present invention provides a food for improving liver function, which contains an extracellular polysaccharide derived from a culture solution of mycelia lacticera mycelium.

The food according to the present invention may be in the form of a powder, a granule, a tablet, a capsule or a drink, and may be a candy, a chocolate, a beverage, a gum, a tea, a vitamin complex, a health supplement or a health functional food.

At this time, the extracellular polysaccharide according to the present invention in the food may generally be contained in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, of the total food, and 0.02 to 10 g , Preferably 0.3 to 1 g.

The food product may further comprise a food-acceptable food-aid additive together with the extracellular polysaccharide of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

Manufacturing example  One. Three Li Pori Lacerata  Derived from the culture medium extract Of exopolysaccharide (EPS)  Produce

1.1 Three Li Pori Lacerata  Production of culture medium

The bacterium cultivated by subculture was stored frozen at -80 ℃ and stored in a PDA (Potato dextrose agar) medium (87 plastic culture (Difco, Becton Dickinson and Company) were used to store a sufficient number of complete strains in a refrigerator at 4 ° C for 2 to 3 times. Then, 600 ml of PDB (Potato dextrose broth) medium (Difco, Becton Dickinson and Company) was added to the Erlenmeyer flask, and one PDA culture was added and cultured for 8 days with shaking. Then, sugar 1.5%, glucose 0.5% potato starch 0.5% by weight, can water 0.25 weight%, barley minutes, 0.25 wt%, 0.75 wt% soybean meal, magnesium sulfate (MgSO ₄₎ 0.05% by weight, 1 potassium phosphate ( KH ₂ PO ₄ ) 0.05% by weight potassium diphosphate (K ₂ HPO ₄ ) 0.05% by weight and water 96.85% by weight was sterilized in an 800 L fermenter at 121 ° C. and 1.5 kgf / cm ² for 20 minutes , 600 ml of a PDB culture strain to be used as a starter was inoculated in a state of being cooled to 23 ° C and air was ventilated at 0.5 to 1.5 kgf / cm ² , and the concentration of carbon dioxide was 1,000 to 2,000 ppm, and the mycelium of Seripolyla cerera was replaced with 23 Lt; 0 > C for 10 days to prepare a culture medium of Sera lipolactacera mycelium.

1.2 Three Li Pori Lacerata  From the culture broth extract EPS Manufacturing

The cultured Mycelium lacticera mycelium was lyophilized at 25 ° C for 72 hours using a vacuum freeze dryer to be powdered. To 5 g of the dried powder, add 100 ml of distilled water, suspend it well, and centrifuge it (8,000 rpm, 20 minutes). Add 2 ~ 3 times the amount of cold alcohol to the supernatant and add to the refrigerator (4 ℃) Time. After the supernatant was centrifuged again (8,000 rpm, 20 minutes), the precipitate was recovered and crude EPS was extracted. The crude EPS was dried in a freeze dryer for 72 hours to obtain complete EPS.

Example  One. EPS Characterization of

1.1 Gel permeation  Chromatography ( Come Permeation Chromatography , GPC ) EPS Molecular weight measurement

The EPS prepared in Preparation Example 1 was dissolved in 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ (PH adjusted to 4 with glacial acetic acid) solution. After centrifugation, the supernatant was filtered with a 0.45 m syringe filter and analyzed by GPC.

The analytical conditions were a refractive index as a detector and a GPC column using a OHpak SB 805 HQ (Shodex, Japan). The mobile phase was eluted with 0.1 M Na ₂ SO ₄ /0.05 M NaN ₃ (adjusted to pH 4 with glacial acetic acid) Was flowed at a rate of 1.0 mL / min. Standard curves were prepared using dextran (American Polymer Corporation, USA) with different molecular weights (130, 400, 770 and 1200 kDa) and refractive index (RI) Knauer K-2310 And the molecular weight of EPS was measured (Table 1).

As a result, the molecular weight of EPS was found to be about 120 kDa (Fig. 1).

1.2 EPS Of sugar and protein content

EPS was secondarily purified and treated with protein hydrolyzing enzyme to measure sugar and protein content.

Specifically, the first purified EPS was dissolved in distilled water and centrifuged (8,000 rpm, 20 minutes) to separate the supernatant. The cooled supernatant was added with 2 ~ 3 times the amount of cold alcohol, 4 ° C) and allowed to stand for 12 hours. After centrifugation (8,000 rpm, 20 minutes) of the supernatant alone in the above-mentioned filtrate, the precipitate was recovered to obtain a second purified EPS. Next, the protein hydrolyzing enzyme alcalase was treated at a concentration of 0.5% at 50 ° C for 30 minutes.

The sugar content was measured by the phenol-sulfuric acid method. 25 μL of 80% phenol was added to 1 mL of the diluted sample. 2.5 mL of sulfuric acid was added, and the solution was cooled at room temperature and absorbance was measured at 465 nm. Protein content was determined by the BCA method (Smith PK, meat al ., Analytical Biochemistry , 150 (1): 76-85 (1985)) and bovine serum albumin was used as a standard.

As a result, as shown in Table 2 below, the sugar content was 45 to 51% by weight and the protein content was 33 to 34% by weight.

Also, as a result of the sugar composition analysis of EPS, it was found that EPS mainly contains mannose, galactose and glucose.

Example  2. EPS Of liver function improvement

In order to investigate the effect of enhancing the liver function of EPS derived from the culture medium of Cryptolaria lucerata, EPS prepared in Preparation Example 1 and C57BL / Ksj (BL / Ls) homozygous diabetic ( db / db ) Respectively. The db / db The mouse was a 6-week-old male of about 30 to 40 g, produced by Japan SLC Inc. and supplied through a central laboratory animal. After about 7 days of quarantine purification and adaptation period, body weight and blood glucose were measured A total of 30 healthy animals were selected that were appropriate and free from common symptoms.

Experimental animals were divided into two groups: negative control ("DM group"), EPS low dose group (150 mg / kg, "DM-EXO150 group"), EPS high dose group (300 mg / kg, "DM- EXO300 group" (metformin) -300 mg / kg, "DM-MET300 group") were randomly divided into groups and fed for 6 weeks. Six normal control ("NC group") were also bred for 6 weeks under the same conditions. All test substances and positive control substances were orally administered at the same time every day, and the normal and negative controls were orally administered with water. The experimental animals were fed a commercial animal feed (Samtaco co. Ltd., Korea), and water was fed ad libitum. The breeding conditions of the animal breeding room were adjusted to 12 hours of light intensity (8 am to 8 pm illumination), temperature 23 ± 3, and relative humidity 50 ± 10%.

ALT and AST contents were measured to examine the liver damage of db / db mice after 6 weeks of EPS administration (Ji-Eun Kim, et al . , Food Science and Biotechnology 21 (6): 1685-1693 (2012)). Alanine transaminase (ALT) and aspartate transaminase (AST), which are generally known to be increased by toxic administration, liver cirrhosis, liver metabolic changes such as hepatitis and liver cancer, Can be used as a marker in determining the extent of liver damage.

As a result, ALT and AST showed a remarkable decrease of about 25 and 14%, respectively, in the DM-EXO300 group treated with 300 mg / kg EPS compared with the DM group treated with negative control (Table 3). This confirms that EPS protects the liver from db / db mice.

Claims (13)

Ceriporia A composition for improving liver function comprising an extracellular polysaccharide derived from a mycelial culture liquid extract as an active ingredient.
A composition for preventing or treating liver disease, comprising an extracellular polysaccharide derived from an extract of a culture medium of a serpia lacrocerata mycelia as an active ingredient.
3. The method according to claim 1 or 2,
Wherein the extracellular polysaccharide comprises 40-60 wt% sugar and 30-40 wt% protein and has a molecular weight of 100-150 kDa.
The method of claim 3,
Wherein the extracellular polysaccharide comprises 43-47 wt% sugar and 33-36 wt% protein and has a molecular weight of 115-125 kDa.
The method of claim 3,
Wherein the sugar contains mannose, galactose and glucose.
3. The method according to claim 1 or 2,
The extracellular polysaccharide can be produced by: (a) liquid culturing mycelia lacticera mycelium to prepare a culture medium of a seriposita lactamera mycelium; (b) drying a culture medium of the mycelium lacticarata mycelium; And (c) extracting the culture medium of the cultivated mycelium lacticarata with a solvent, followed by filtration and concentration under reduced pressure.
The method according to claim 6,
The medium for the liquid culture may be selected from the group consisting of sugar, glucose, starch, water, barley, soybean powder, magnesium sulfate (MgSO ₄ ), potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ) Wherein the hydrogen ion concentration is pH 4.5 to 6.0.
The method according to claim 6,
Wherein the liquid culture is performed under a blue LED light source and is performed by maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm.
3. The method according to claim 1 or 2,
Wherein the extracellular polysaccharide is present in an amount of 0.1 to 80% by weight based on the total weight of the composition.
3. The method of claim 2,
Wherein the liver disease is liver fibrosis, liver cirrhosis, hepatitis or liver cancer.
A food for improving liver function, comprising an extracellular polysaccharide derived from a culture solution of mycelium lacticera mycelium.
12. The method of claim 11,
Wherein the food is in the form of a powder, a granule, a tablet, a capsule or a beverage.
12. The method of claim 11,
The food for improving liver function, wherein the food is candy, chocolate, beverage, gum, tea, a vitamin complex, a health supplement, or a health functional food.

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