KR20200012306A - Pharmaceutical composition comprising extracts of Pinus koraiensis, Saururus chinensis or Lycium chinense for preventing or treating liver disease - Google Patents

Abstract

The present invention relates to a composition for preventing and treating liver diseases containing a complex extract of Pinus koraiensis leaves, Saururus chinensis, and Lycium chinense. The complex extract, in which Pinus koraiensis leaves, Saururus chinensis, and Lycium chinense are mixed, of the present invention decreases activities of AST and ALT, which are liver damage figures, more than each single extract due to the interaction of each component, significantly increases antioxidant enzymes, and has an excellent effect on prevention and treatment of liver diseases.

Description

Pharmaceutical composition comprising extracts of Pinus koraiensis, Saururus chinensis or Lycium chinense for preventing or treating liver disease}

The present invention relates to a pharmaceutical composition for preventing and treating liver disease, including a complex extract of pine needles, triticale and goji berry.

The liver is an important organ that stores and circulates blood, metabolizes lipids, excretes bile, stores various nutrients, regulates blood flow and detoxifies. However, the liver is overloaded as our body is exposed to various pollutants and toxic substances, and serious damage is caused by mental stress, heavy drinking, smoking, and the like.

Because the liver is the primary defense against damage caused by the ingestion of foreign substances, severe disease can occur due to viruses or various drugs due to decreased liver function. This can cause abnormalities in the immune system and cause other diseases. In particular, acute or chronic disorders occur due to various causes such as excessive intake of foods or alcohol containing fat components, viral infections, harmful substances such as various medicines, and malnutrition, resulting in fatty liver, hepatitis, jaundice, cirrhosis, liver cancer, etc. May be caused. In addition, excessive fat intake or excessive alcohol intake causes fatty liver in which lipids accumulate in liver tissue, and GOT (AST: Aspartic aminotransferase), GPT (ALT: Alanin aminotransferase), and γGPT (γ-glutamytransferase) Will increase.

Liver diseases are classified into viral liver disease, alcoholic liver disease, drug toxic liver disease, fatty liver, autoimmune liver disease, metabolic liver disease and other liver diseases depending on the cause of the disease. Since the liver is a large buffering capacity, the disease is often not recognized in the early stages of the disease, and since the disease is found only when the disease progresses, it is the cause of death not only in Korea but also in the world.

As described above, the loss of liver function is insensitive and loses the defense and detoxification effect of the human body, causing a lot of problems in the human body, the development of drugs and health functional foods that have a liver protection effect that is manufactured as a safe formulation without side effects It is necessary.

Catalase, on the other hand, is present in the peroxisome and acts on the oxidation of the alcohol with water. Hepatocellular injury of overdose and chronic alcohol intake is attracting attention due to excessive oxidative stress caused by CYP2E1 metabolism due to alcohol intake. In other words, the generation of free radicals and antioxidant activity caused by this process has been reported as a major cause of liver damage. The method of inhibiting hepatic cell damage caused by free radicals suggests two mechanisms that directly regulate antioxidant enzymes and remove free radicals, and indirectly produce highly reactive intermediates to regulate the activity of cytochrome P450 family enzymes. .

In view of the above, the present inventors investigated natural products having a new hangover resolution or liver protection effect, and completed the present invention by measuring the effect of the complex extract of pine needles, triticale and goji.

Republic of Korea Patent Publication No. 10-2017-0075250

The present invention provides a pharmaceutical composition for preventing and treating liver disease, which comprises a complex extract of pine needles, triticale and goji berry as an active ingredient.

The present invention provides a food composition for preventing and improving liver disease, which comprises a complex extract of pine needles, triticale and goji berry as an active ingredient.

The inventors have found that the complex extracts of pine needles, triticale and goji berries reduced liver damage levels AST and ALT activity and increased antioxidant enzymes than each single extract and completed the present invention. Hereinafter, the present invention will be described in detail.

It is common to review AST and ALT activity levels as an assessment of hepatoprotective effects. Damage to hepatocytes results in a change in transport function and membrane permeability, resulting in a large release of enzymes such as AST and ALT from the cells into the circulation, indicating damage to liver tissue during the toxicization process. Therefore, reducing the AST and ALT activity of the complex extract of the present invention means inhibiting damage to liver tissue and preventing and treating liver disease.

In addition, when antioxidant enzymes increase, free radicals which cause liver damage are removed. Therefore, increasing the sulfated enzyme of the complex extract of the present invention removes free radicals that cause liver damage, and means preventing and improving liver damage.

The present invention provides a pharmaceutical composition for preventing and treating liver disease, which comprises a complex extract of pine needles, triticale and goji berry as an active ingredient.

The "pine leaf" is a leaf of the pine tree ( Pinus koraiensis ), not only has a thick wax layer on the leaf surface, but also has a strong astringent taste. In oriental medicine, it is also called white leaf.

Saururus chinensis is a perennial herb belonging to the Saururaceae family, which is native to Jeju Island, and is also cultivated in the southern coastal region.

Lycium chinense is the fruit of the wolfberry tree.

The complex extract of pine needles, triticale and wolfberry of the present invention can be obtained through the steps of washing, drying, cutting and grinding, extracting and concentrating, and ripening pine needles, triticale and wolfberry.

In addition, the complex extract may be extracted from water, alcohol or a mixed solvent thereof. The alcohol is preferably a lower alcohol of C1 ~ C4, more preferably using methanol or ethanol, most preferably 50% ethanol.

Extraction method may be a shaking extraction, Soxhlet extraction or reflux extraction method, but is not limited thereto. It is preferable that extraction temperature is 40-100 degreeC, and it is more preferable that it is 60-80 degreeC. Moreover, it is preferable that extraction time is 2 to 24 hours, and it is preferable that extraction frequency is 1 to 5 times.

The complex extract may be mixed with 1 to 5 parts by weight, 1 to 3 parts by weight and 1 to 5 parts by weight of Goji berries. Preferably, pine needles: three hundred seconds: gojigi may be a mixture of 2: 1: 2 by weight.

The liver disease may be any one or more selected from the group consisting of acute hepatitis, alcoholic fatty liver, nonalcoholic fatty liver, jaundice, liver abscess, hemangioma, liver fibrosis, cirrhosis and liver cancer, preferably liver fibrosis, cirrhosis or liver cancer It may be, and more preferably liver fibrosis, but the present invention is not limited thereto.

As used herein, the term "prevention" refers to any action that inhibits or delays liver disease by administration of a composition according to the invention. The term "treatment" in the present invention refers to any action that improves or advantageously changes the symptoms of liver disease by administration of the composition according to the present invention. "Improvement" in the present invention means any action to improve the bad state of liver disease by administering or ingesting the composition of the present invention to a subject.

According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing and treating liver disease comprising a complex extract of pine needles, triticale and goji berry as an active ingredient.

The pharmaceutical compositions of the present invention can be formulated in a variety of formulations, including solutions, suspensions, emulsions, lotions, ointments, lyophilizers, etc., in accordance with conventional methods.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form pharmaceutical formulation suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and the formulation may be effectively administered by one or several administrations. Contains the amount. Suitable formulations for this purpose are parenteral, injectables, injectables and the like. In addition, the pharmaceutical composition for preventing or treating liver disease may include a pharmaceutically acceptable conventional inert carrier and diluent.

Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical compositions of the invention include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, and the like. Binders such as, alginate, and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, polysorbates, cetyl alcohol, and glycerol Active agents include, but are not limited to. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the composite extract and the recipient to receive it. Diluents include, but are not limited to, saline, aqueous buffers, solvents and / or dispersion media. In addition, for example, in the case of injectables, a preservative, a painless agent, a solubilizer or a stabilizer may be included, and in the case of a topical administration agent, a base, an excipient, a lubricant, or a preservative may be further included. The compositions of the present invention can be used unfrozen or frozen for future use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife® cell freezing medium (Cascade Biologics)) can be added to the cell population prior to freezing.

The dosage may vary depending on the concentration, but may be administered from 1 g / day to 10 g / day once or in several divided doses based on 60 kg.

However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore the dosage may be It is not intended to limit the scope of the present invention in terms of aspects.

In another aspect, the present invention provides a food composition for preventing and improving liver disease, comprising a complex extract of pine needles, three hundred seconds and goji berries as an active ingredient.

The present invention can be generally used as a commonly used food.

The food composition of the present invention can be used as a dietary supplement. The term "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the law on health functional foods, and "functionality" refers to the structure and function of the human body. It means the ingestion for the purpose of obtaining a useful effect for health use such as nutrient control or physiological action.

The food composition of the present invention may include a conventional food additive, and the suitability as the "food additive" is applicable according to the General Regulations and General Test Method of the Food Additives approved by the Ministry of Food and Drug Safety unless otherwise specified. It is judged by the standard and the standard about the item.

Examples of the items listed in the "Food Additive Revolution" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, Mixed preparations, such as a sodium L- glutamate preparation, an alkali addition additive, a preservative preparation, and a tar pigment preparation, are mentioned.

The food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and / or ameliorating diseases caused by liver damage.

For example, hard capsules of the health functional food in the form of capsules can be prepared by filling a mixture with additives such as complex extracts and excipients according to the present invention in a conventional hard capsules, soft capsules according to the present invention A mixture with an additive such as a food composition and an excipient may be prepared by filling a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.

The term definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents and the like are described in the literature known in the art and include those having the same or similar functions (Korean Pharmacopoeia, Munseongsa, Korea Pharmacy University Council, 5th edition, pp. 33-48, 1989).

There is no particular limitation on the type of food, and includes all of the health functional foods in the ordinary sense.

The present invention provides a method for preventing and treating liver disease, comprising administering a complex extract of pine needles, triticale and goji berries to a subject.

In the present invention, the terms "composite extract", "liver disease", "administration" and the like are the same as described above.

The subject refers to an animal, and may be a mammal, which may typically exhibit a beneficial effect by treatment with a complex extract of pine needles, triticale and goji berries of the present invention. Preferred examples of such subjects may include primates, such as humans. In addition, such subjects may include all subjects with or at risk of having symptoms of liver disease.

The present invention also provides a composition comprising a complex extract of pine needles, triticale and goji berries for use in the treatment of liver disease.

The present invention also provides the use of a complex extract of pine needles, triticale and goji berries for the treatment of liver disease.

The complex extract of pine needles, triticale and goji berry of the present invention decreases AST and ALT activity, a significant increase in antioxidant enzymes, significantly increases antioxidant enzymes, and prevents liver disease, due to the interaction of each component. Has an excellent effect on treatment.

Figure 1 is a HepG2 cells by treating the complex extracts by concentration, and confirmed the cytotoxicity by MTT assay.
Figure 2 is confirmed by Western blot analysis of AST and ALT changes in HepG2 cells treated with pine needle extract, triticale extract, goji berry extract and complex extract.
Figure 3 confirms the changes in AST and ALT in HepG2 cells treated with complex extract by enzyme immunoassay.
Figure 4 confirms the change in the amount of sulfate enzyme expression in HepG2 cells treated with the composite extract.

Examples and preparation examples are provided to aid the understanding of the present invention. The following examples and preparations are merely provided to more easily understand the present invention, but the contents of the present invention are not limited by the examples and preparations.

Preparation Example Preparation of Complex Extract

Pine needles were collected from Pinus Koraiensis , a Korean pine tree, for more than 30 years in Hongcheon-gun, Gangwon-do, Korea. The Gugija purchased Cheongyang, Chungcheong-do, and Sambaekcho, which are native to Yeongcheon, Gyeongsangbuk-do. Pine needles, wolfberry and triticale were dried and ground. 10 g of pine needles, 5 g of 300 seconds and 10 g of goji berry (2: 1: 2 ratio) were placed in 1 L of 50% ethanol and extracted for 3 days. Ethanol extraction was repeated twice. The extract was concentrated using a rotary evaporator (Eyela, Tokyo, Japan) and lyophilized to give a composite extract in the form of a powder. The obtained composite extract was 5.14 g (yield: 20.5%).

Example 1 Cytotoxicity Evaluation

In order to evaluate the cytotoxicity of the composite extract obtained at all times the preparation, MTT assay was measured in hepatocytes.

HepG2 cell line, a hepatocyte cell line, was cultured in an incubator containing 37 ° C. and 5% CO ₂ using DMEM medium containing 10% fetal calf serum, penicillin (100 U / mL) and streptomycin (100 mg / mL).

HepG2 cells were incubated in 96well plates for 24 hours by 1x10 ⁵ cells / ml each, and the complex extracts were treated by concentration. After 24 h, the cultures were discarded and 50 μl of MTT solution (1 mg / ml in PBS) was added and incubated for 2 hours. After the culture plate was washed with PBS, 100 μl of DMSO was added and left at room temperature for 20 minutes. Using a microplate reader, absorbance was measured at a wavelength of 570 nm (FIG. 1).

As shown in Figure 1, it was confirmed that the survival rate of hepatic cell at all concentrations of more than 80%, the results suggest that the composite extract does not show cytotoxicity to HepG2 cells.

Example 2 Effect of Complex Extract on Hepatic Damage Indices in Hepatocytes

Western blot analysis revealed changes in aspartate aminotransferase (AST) and alanine aminotransferase (AST), which are indicators of damage induced by carbon tetrachloride (CCl ₄ ) in HepG2 cells.

Enzymes used as indicators to confirm liver damage caused by carbon tetrachloride are AST, ALT, alanine transamidase, lactate dehydrogenase, ornitine carbonyl transferase, etc. (Sipes et al., 1997). Activity is widely used in clinical trials as an indicator of liver damage.

Specifically, HepG2 cells were dispensed in 6 well plates to have 10 ⁶ cells per well, and after 24 hours, the complex extracts were treated by concentration. The cells were washed with PBS and then lysis solution (20 mM sucrose, 1 mM EDTA, 20 μM Tris-CL, pH7.2, 1 mM DTT, 10 mM KCL, 1.5 mM MgC2, 5 μg / mL pepstatin A, 10 μg / eluting with the addition of ml leupetin and 2 μg / mL aprotinin). After centrifugation, the supernatant was collected and the protein concentration was measured. An aliquot containing 30 μg was separated on an SDS-polyacrylamide gel using a bio-rad protein assay kit and conjugated with a pooxidase using the indicated primary antibody. Each secondary antibody was used and analyzed by ECL detection system (FIG. 2).

Protein bands were quantified based on β-actin bands. Error bars represent standard errors (n = 3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test. (### p <0.001 verus Non-treated control. *** p <0.001 verus CCl4-treated. $ p <0.05, $$ p <0.01 verus 50% ethanol layer mono-treated).

As shown in FIG. 2, the complex extract inhibits AST and ALT, which are indicators of liver damage induced by carbon tetrachloride, compared to pine needles, triticale and goji berry single extract.

Example 3 Effect of Complex Extract on Hepatic Damage Indices in Hepatocytes 2

Enzyme-immunoassays confirmed changes in AST and ALT, markers of damage induced by carbon tetrachloride (CCl ₄ ), in HepG2 cells.

Specifically, HepG2 cells were divided into 6 well plates so that the number of cells was 10 ⁶ cells per well. After 24 hours, the complex extract was treated by concentration (100 μg / ml and 200 μg / ml), and 24 hours in a CO ₂ incubator. Incubated for 2 hours. After incubation, the plate was centrifuged at 2000 rpm for 5 minutes to collect the culture supernatant. The amounts of AST and ALT were measured by enzyme immunoassay (Kits, BD Pharmigen, USA) (FIG. 3).

Error bars represent standard errors (n = 3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test. (* p <0.05, ** p <0.01 verus CCl ₄ -treated).

As shown in Figure 3, the complex extract can be seen to inhibit the liver damage indicators AST and ALT.

Example 4 Effect of Complex Extracts on Antioxidant Enzymes in Hepatocytes

Western blot analysis was performed to determine the expression levels of the antioxidant enzymes Glutathione peroxidase and catalase in HepG2 cells. Specific Western blot method was carried out in the same manner as in Example 2.

Protein bands were quantified based on β-actin bands. Error bars represent standard errors (n = 3). Statistical significance was analyzed by one-way ANOVA with Tukey's multiple comparison test. (### p <0.001, ## p <0.01 verus Non-treated control. *** p <0.001, ** p <0.01 verus CCl4-treated.)

As shown in Figure 4, the complex extract can be seen to increase the expression of Glutathione peroxidase and catalase reduced by carbon tetrachloride.

According to the present invention, the complex extracts of pine needles, goji berry and three hundred gree reduced the liver damage level increased by carbon tetrachloride and increased the expression level of antioxidant enzymes compared to each single extract. This suggests that the complex extract has a better effect on liver damage than a single extract.

From the above results, it was confirmed that the composition containing the complex extract according to the present invention as an active ingredient has an effect of preventing and treating liver damage, and the composition of the present invention is applied as a pharmaceutical agent and a functional food for preventing and treating liver disease. Suggests that

Claims (10)

Pharmaceutical composition for liver disease prevention and treatment comprising a complex extract of pine needles, three hundred seconds and goji berries as an active ingredient. The method of claim 1,
The complex extract is water, C1 ~ C4 alcohol or a mixture of them is a liver disease prevention and treatment pharmaceutical composition. The method of claim 2,
Wherein the C1 ~ C4 alcohol is ethanol liver pharmaceutical composition for preventing and treating diseases. The method of claim 1,
The complex extract is mixed with 1 to 5 parts by weight of pine needles, 1 to 3 parts by weight of three hundred seconds and 1 to 5 parts by weight wolfberry liver composition for preventing and treating pharmaceutical composition. The method of claim 1,
The liver disease is at least one selected from the group consisting of acute hepatitis, alcoholic fatty liver, non-alcoholic fatty liver, jaundice, liver abscess, hemangioma, liver fibrosis, cirrhosis and liver cancer. Liver disease prevention and improvement food composition comprising a complex extract of pine needles, three hundred seconds and goji berry as an active ingredient. The method of claim 6,
The complex extract is water, C1 ~ C4 alcohol or food composition for preventing and improving liver disease that is extracted with a mixed solvent thereof. The method of claim 7, wherein
The C1 ~ C4 alcohol is ethanol, liver disease prevention and improvement food composition. The method of claim 6,
The complex extract is mixed with 1 to 5 parts by weight, 1 to 3 parts by weight and 1 to 5 parts by weight of pine nuts 1 to 5 parts by weight of liver disease preventing and improving food composition. The method of claim 6,
The liver disease is at least one selected from the group consisting of acute hepatitis, alcoholic fatty liver, non-alcoholic fatty liver, jaundice, liver abscess, hemangioma, liver fibrosis, cirrhosis and liver cancer.

Images