JP2009500446A - Pharmaceutical composition having an effect of preventing and treating liver diseases, comprising an extract of keyonomi - Google Patents

Abstract

An object of the present invention is to provide a pharmaceutical composition having an effect of preventing and treating liver diseases, which comprises an extract of a key flea.
[MEANS FOR SOLVING PROBLEMS] The present invention relates to a pharmaceutical composition having an effect of preventing and treating liver diseases, comprising an extract of keyonomi (Lonicera caerulea L. var. Edulis), the composition comprising hepatitis, liver sclerosis Or has preventive and therapeutic effects on diseases such as fatty liver.
[Selection] Figure 5

Description

  The present invention relates to a pharmaceutical composition having an effect of preventing and treating liver diseases, comprising an extract of keyonomi (Lonicera caerulea L. var. Edulis).

  The liver is located between the digestive system and the systemic circulatory system in the human body, and is responsible for the important functions of protecting the whole body from toxic substances coming from outside and responsible for the metabolism of in vitro substances. . Since in vitro substances that have entered the body pass through the liver, the liver is more likely to be exposed to many toxic substances in addition to nutrients. .

  Liver diseases include toxic liver diseases caused by excessive use of alcohol or the like according to the cause of induction, and viral liver diseases caused by viral infection. Viral liver diseases are caused by infections such as hepatitis B and C viruses, and recently, toxic liver diseases caused by food, medicine, Chinese medicine, alcohol, etc. are increasing. Liver disease is difficult to diagnose early because subjective symptoms do not appear well. When subjective symptoms appear, significant liver damage has occurred. Liver tissue is a tissue having a greater recovery function than other tissues, but recovery is difficult when liver cells are already deformed.

  To date, no clear therapeutic agent has been developed for the treatment of chronic hepatitis and cirrhosis. Although interferon and nucleic acid analog rabibudin are used as therapeutic agents for chronic hepatitis, these drugs are drugs that exhibit the activity of suppressing viral activity and are not drugs that restore the function of hepatocytes. To date, silymarin is the most widely known agent for restoring hepatocyte function throughout the world. Silymarin is extracted from a herb with the scientific name of the medicinal plant Carduus marianus Linne Silybum marianum, and has been known as an important drug in the western world, including ancient Greece since BC. Since being introduced to the Republic of Korea as a liver treatment agent in the 1970s, many drugs based on this component have been put on the market. In the case of preparations containing silymarin as the main component, it has already been widely applied clinically to the treatment of liver diseases, but the main component silymarin is a poorly soluble substance that hardly dissolves in water. It has the disadvantage of low water absorption. Currently, silymarin preparations are only used as an adjuvant therapy for the treatment of liver diseases such as toxic liver disease, chronic hepatitis, and liver sclerosis, and therefore, the development of drugs that can quickly restore hepatocyte function. The situation is still required.

  As a study on the liver function improving effect useful from natural products, Patent Document 1 discloses fermented milk useful for maintaining and improving liver function and a method for producing the same, and Patent Document 2 discloses an extract of kemponashi fruit. Is disclosed as a functional food composition having an effect of improving liver function and drunkenness as an active ingredient, and Patent Document 3 discloses the use of a functional food coated with an extract of Ezokogi. Patent Document 4 discloses a therapeutic agent for hepatitis B containing an extract of Komikanso and a method for producing the same, and Patent Document 5 discloses a composition for treating a viral liver disease containing an extract of Indica periwinkle. To do.

In addition to the existing therapeutic compositions described above, as part of a study to obtain a substance having excellent liver disease therapeutic activity from natural products, the present inventor has applied an extract of keyonomi to a damaged hepatocyte cell line. When added, the growth of hepatocytes is promoted, and this confirms that GOT and GPT values used as biochemical indicators of liver function are remarkably reduced by restoring damaged liver function. The present invention was completed by clarifying that the composition containing the extract has excellent preventive and therapeutic effects on liver diseases such as hepatitis, liver sclerosis, or fatty liver.
Korean Patent Registration No. 80759 Republic of Korea Patent Publication No. 2003-0027615 Korean Patent Publication No. 2003-0011818 Korean Patent Publication No. 2003-0063308 Korean Patent Publication No. 2004-0018733

  An object of the present invention is to provide a pharmaceutical composition having an effect of preventing and treating liver diseases, which includes an extract of a keyonomi.

  In one embodiment of the present invention, the present invention relates to a pharmaceutical composition having an effect of preventing and treating liver disease, which comprises an extract of keyonomi.

  According to the present invention, since the composition containing the extract of keyonomi has no side effects such as toxicity and has excellent liver function recovery and growth recovery ability, it can be effectively used for the prevention and treatment of liver diseases. it can.

  The term “extract” in the present invention means an active ingredient separated from a natural product, and here is a substance separated from a key flea and has an effect of promoting hepatocyte growth. The extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes an extract, a dry powder thereof, or all forms formed using the extract. .

  In the present invention, the term “keyonic flea (Lonicera caerulea L. var. Edulis)” is meant to include all organs of a natural, hybrid, or variant key flea, such as roots, branches, stems, leaves, flowers, and fruits. Preferably, it is a fruit of a key flea. Keyronomid (Lonicera caerulea L. var. Edulis) is a dicotyledonous plant of the Lepidoptera honeysuckle family, which is a deciduous shrub that reaches 1.5 m in height, and has many branches that form a shield in the form of small branches. There is a bamboo leaf, and the part inside the trunk of the tree is white. The leaves grow oppositely, are needle-shaped or elliptical, are not sharp or pointed at both ends, have no teeth, short hair on the edges and surface, and a lot of villi on the back. Flowers are generally short in flower pattern and hang under the leaves, and the corolla is funnel-shaped, yellowish white and blooms in summer. Endena is divided into five, the corolla is yellowish-white, cylindrical bell-shaped, 1.2-1.5 cm long, with some hair. The stamens are shorter than the stamens, have no hair, the two ovaries are combined, and are actually oval or nearly circular, ripen purple-black in July-August and covered with white powder. It is a cold zone plant distributed in Siberia, Sakhalin, northern China, Tibet, North Korea, etc. Prior to the present invention, the promotion of growth of hepatocytes by the keyfish and the effect of improving the liver function by this were not investigated.

  The liver function improving effect is objectively determined by the degree to which cell growth is promoted in hepatocytes damaged by administration of the composition and liver enzyme values, that is, AST (Aspartate Aminotransferase: GOT) and ALT (Alanine Aminotransferase: GPT). Can be evaluated. ALT and AST are enzymes in hepatocytes, and when hepatocytes are damaged or destroyed, they are shed and increase in blood concentration. Therefore, ALT and AST values are liver diseases caused by hepatocyte damage. Used as a biochemical indicator.

  In a specific embodiment of the present invention, a control group in which an extract of keyonomi is added to the HepG2 cell line, which is a hepatocyte of a human damaged by ethyl alcohol, and the degree of hepatocyte growth promotion is not added to the extract of keyonomi, As a result of comparison, growth was promoted in proportion to the concentration. Addition of cayo flea extract at a concentration of 0.25 mg / ml promoted cell growth of about 27% and addition of 0.5 mg / ml resulted in about 54% cell growth. Moreover, as a result of observing whether or not liver function could be improved by measuring ALT and AST levels, administration of an extract of keyonomi decreased the ALT and AST values in the HepG2 cell line. When the extract of keyonomi was administered, the ALT and AST values appeared to be more than 25% more recoverable than when the existing drug silymarin was administered. The above results demonstrate that the extract of the keyronomia of the present invention has an excellent liver function improving effect by promoting the growth of hepatocytes, and thus has a liver disease therapeutic activity.

  The term “liver disease” in the present invention means all diseases that induce a decrease in liver function, such as viruses (eg, A, B, C, D, or E viruses), alcohol, drugs (tuberculosis drugs, Aspirin, antibiotics, anesthetics, antihypertensives, oral contraceptives, etc.) and inborn errors of metabolism. Specific examples of liver diseases include hepatitis, liver sclerosis, and fatty liver. Hepatitis includes all chronic and acute hepatitis.

  The term “prevention” in the present invention means all actions to suppress or delay the decrease in liver function by administration of the composition. The term “treatment” in the context of the present invention refers to any act of restoring or beneficially changing liver function and growth by administration of the composition.

  Administration of the composition of the present invention can prevent and treat not only diseases such as hepatitis, liver sclerosis, and fatty liver, but also symptoms or complications induced thereby. For example, liver disease symptoms that can be prevented and treated with an extract of keyonomi include fatigue, vomiting, loss of appetite, abdominal pain, jaundice, and complications include swelling, ascites, gastrointestinal bleeding, esophageal varices bleeding, liver Including encephalopathy (hepatic coma).

  In the present invention, the extract of the keyonomi is produced by a method of extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used immediately, or concentrated and / or dried.

  When extracting using an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate dichloromethane, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) 1,3-butylene glycol, propylene glycol, or an organic solvent that is a mixed solvent thereof, is used at room temperature or warming under the minimized conditions that the active ingredient of the herbal medicine is not destroyed. Can be extracted. Depending on the organic solvent to be extracted, there may be a difference in the degree of extraction and loss of the active ingredient of the drug, so a suitable organic solvent is selected and used. The extraction method is not particularly limited, and examples include immersion extraction, ultrasonic extraction, and reflux extraction.

  The solvent extract may further include filtering the extract to remove floating solid particles. Particles may be filtered using cotton, nylon, etc., and ultrafiltration, refrigeration filtration, centrifugation, etc. may be used, but are not limited thereto.

  For the concentration of the extract, methods such as reduced pressure concentration and reverse osmotic pressure concentration can be used. The drying step after the concentration includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, foam drying, high frequency drying, infrared drying and the like. Optionally, the method may further include grinding the final dried extract.

  Also, the extract can perform an additional fractionation process. For example, the extract is suspended in distilled water, and a nonpolar solvent-soluble layer is extracted and separated with a nonpolar organic solvent such as nucleic acid, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof. And can be used after concentration and / or drying.

  In one specific embodiment, the extract of the keyonomies of the present invention is water, more preferably 5 to 25 times, preferably 7 to 15 times the dry weight (kg) of the fruit, more preferably 7 to 15 times, C4 lower alcohol, or a mixed solvent thereof, preferably water or a mixed solvent of water and ethyl alcohol, at an extraction temperature of 20 to 100 ° C., preferably 60 to 100 ° C., for 0.5 hour to 2 days, preferably 1 It was obtained by continuous extraction 1 to 5 times, preferably 2 to 3 times, by hot water extraction, cold soaking extraction, ultrasonic extraction, reflux extraction, preferably reflux extraction method for a period of time to 1 day. Further, the extract was filtered through a filter paper, and the filtrate was concentrated under reduced pressure at 20 to 100 ° C., preferably 50 to 70 ° C. with a rotary vacuum concentrator, and then dried to produce an extract of the key yonom of the present invention in powder form. . The extract of keyonomi in powder form can be used as it is or dissolved in a solvent at a constant concentration.

Since the extract of the above-mentioned keyonomi contains an ingredient obtained from a natural product as an active ingredient, it is safe and does not cause toxicity, side effects and tolerance, and thus has an advantage that it can be taken for a long time. In fact, it was determined that no toxicity was induced as a result of mouse acute experiments. In addition, the composition can be used not only for humans but also for livestock such as cattle, horses, sheep, pigs, goats, rabbits, antelopes and dogs that can cause liver diseases.
The above-mentioned extract of keyonomi contains 0.01 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the pharmaceutical composition for preventing and treating liver diseases. In addition, the above composition does not increase the medicinal effect, but can be used in a pharmaceutical composition, and can contain additional components that can improve odor, taste, vision, and the like. In addition, the composition includes inorganic and organic additives such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folic acid pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, and mixtures thereof. Additional objects can be included. The composition may be used alone or may additionally contain a substance having therapeutic activity for an existing liver disease.

  The composition comprises a pharmaceutically acceptable carrier and can be formulated for human or veterinary use. According to the purpose, it can be formulated in various forms of preparations suitable for normal oral use, parenteral use, topical administration, etc. in the pharmaceutical field. For solid preparations for oral administration such as powders, granules, tablets, capsules, etc., binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents , Pigments, fragrances and the like can be used. Liquid oral preparations include suspensions, internal solutions, oils, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, wetting agents, sweeteners, and fragrances , And preservatives. In the case of an injection, a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer and the like can be mixed and used. Such compositions can be presented in unit-volume (single dose) or multi-volume (several dose) containers, such as enclosed ampoules and vials, and are ready for use as a sterile liquid carrier, such as It can be stored under freeze-dry conditions requiring only the addition of an injectable number. Extemporaneous injection solutions and suspensions can be prepared from sterile acid preparations, granules, and tablets.

In yet another embodiment, the present invention relates to a method for preventing and treating liver disease by administering to a patient a composition comprising an extract of keyonomi.
In the present invention, the term “patient” refers to a human having a disease in which liver function is reduced or damaged, such as hepatitis, liver sclerosis, or fatty liver, and horse, sheep, pig, goat, rabbit, antelope, It means animals such as dogs, and symptoms can be improved by administration of the composition of the present invention. By administering to a patient a composition containing the extract of the keyronomia of the present invention, the aforementioned diseases can be effectively prevented and treated. The composition of the present invention can be administered in parallel with existing therapeutic agents for liver diseases.

  The term “administration” in the present invention means introducing a given substance into a patient by any appropriate method, and the administration route of the composition of the present invention is any general route as long as the target tissue can be reached. It can be administered orally or parenterally. The composition can also be administered by any device that allows the active agent to migrate to the target cells.

  The composition of the present invention is administered in a pharmaceutically effective amount.

  The term “pharmacologically effective amount” in the present invention means a reasonable amount of benefit / risk ratio applicable to medical treatment, sufficient amount to treat the disease, and the effective volume level is the patient Gender, age, severity, drug activity, drug sensitivity, administration time, route of administration, and excretion ratio, treatment period, factors that include drugs used at the same time, and other factors well known in the medical field Can depend on. The compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Single or multiple doses can be administered. In view of all of the above factors, it is important to administer an amount that produces the maximum effect in a minimum amount without side effects and can be readily determined by one skilled in the art. The administration method of the extract produced according to the production method of the present invention is preferably oral administration or intravenous administration. In general, the effective dose is from 1 to 1 at a time based on a normal adult in the case of oral administration. 20 mg / kg is preferable, and in the case of intravenous administration, 1 to 20 mg / kg is preferable. The dosage volume level for a particular patient can vary according to gender, age, health status, diet, administration time, administration method, drug mix, and severity of the disease.

  Hereinafter, the present invention will be described in detail with reference to embodiments and experimental examples.

  However, the following embodiment exemplifies the present invention, and the content of the present invention is not limited to the following embodiment.

Embodiment 1: Manufacture of a fruit extract of a key flea (1-1) A hot water extraction method using a water solvent The fruit of a wild key flea harvested directly in Yanbian area and Mt. Shirayo in China was dried and used for experiments. . After adding 100 g of crushed keyonomi seeds to 1 liter of distilled water and stirring well, the mixture was reflux extracted for 3 hours at an extraction temperature maintaining 90 to 95 ° C., and the filtrate was separated. The herbal extract was concentrated under reduced pressure at 0 ° C. and then freeze-dried to obtain 21.2 g of herbal composition powder extract.

(1-2) Hot Water Extraction Method Using Water-Alcohol Mixed Solvent After adding 1 liter of 25% ethyl alcohol to 100 g of crushed kehonomi fruit and stirring well as in Embodiment 1-1 above The extract was refluxed for 3 hours at an extraction temperature maintained at 80 to 90 ° C. by applying heat, the filtrate was separated, the herbal extract was concentrated under reduced pressure at 55 to 65 ° C., and then freeze-dried to obtain a crude drug composition 19.5 g of product powder X was obtained.

Embodiment 2: Measurement of effect on growth of hepatic cell line by extract of fruit of fruitfish HepG2 cells are put in each well of 96 well plate so that the number of cells becomes 1 × 10 4, and fetal bovine serum (fetal bovine The cells were cultured for 16 hours in a cell culture medium containing 10% serum. A 96-well plate was washed with physiological saline, and a solution obtained by diluting the fruit extract of kehonomi in a new cell culture solution at a concentration of 0, 0.125 mg, 0.25 mg, 0.5 mg, 1 mg / ml was added to each well. Added to. After culturing for 48 hours, the number of cells in each well was measured by the SRB method.
Forty-eight hours later, the culture medium containing the extract of the fruit was removed from the 96-well plate, the 96-well plate was washed with physiological saline, and the cells in each well were fixed with 70% acetone for 20 minutes. The fixed cells were dried and then stained with SRB solution for 30 minutes, and the unstained solution was washed 5 times with 1% acetic acid solution. When the washing was finished, it was dried again, 10 mM tris solution was added to dissolve the SRB bound to the cell protein, and the absorbance at 562 nm was measured with a spectrophotometer. The results were expressed as a percentage by comparing the absorbance of each well treated with the extract of the fruit of the keyronomid, with the well not treated with the fruit extract of the keyronomid as the control group (FIG. 1). From this result, it can be seen that the fruit extract of the keyakinomi has the same or slightly increased cell number up to a concentration of 0.5 mg / ml compared to the well not treated with the fruit extract of the keyakinomi. It shows that there is almost no impact on growth.

Embodiment 3. FIG. Measurement of the effect of ethyl alcohol on the growth of hepatic cell lines HepG2 cells were placed in each well of a 96-well plate so that the number of cells was 1 × 10 4 and contained 10% of fetal bovine serum. Cultured in cell culture for 6 hours. Wash the 96-well plate with saline and dilute ethyl alcohol in cell culture to a concentration of 0, 0.5, 1.0, 1.5, 2.0% (v / v) This was administered to each well of a 96-well plate in which HepG2 was cultured. After inducing cell damage with ethyl alcohol for 24 hours, the culture solution containing ethyl alcohol was removed, a new culture solution was added, and after culturing for 48 hours, the number of cells in each well was determined by the SRB method. It was measured by.

  The culture solution was removed, the 96-well plate was washed with physiological saline, and the cells in each well were fixed with 70% acetone for 20 minutes. The fixed cells were dried and then stained with SRB solution for 30 minutes, and the unstained solution was washed 5 times with 1% acetic acid solution. When the washing was finished, it was dried again, 10 mM tris solution was added to dissolve the SRB bound to the cell protein, and the absorbance at 562 nm was measured with a spectrophotometer. The results were expressed as a percentage by comparing the absorbance of each well treated with ethyl alcohol using the wells not treated with ethyl alcohol as a control group (FIG. 2). From this result, it was found that ethyl alcohol promoted cell growth up to a concentration of 1%, but the number of cells was decreased by ethyl alcohol at a concentration of 1.5% or more. I understood.

[Embodiment 4] Growth recovery ability measurement by administration of an extract of a fruit of fruit tree in cells in which cell growth by ethyl alcohol is suppressed and induced The number of HepG2 cells is 1 × 10 4 in each well of a 96-well plate Then, the cells were cultured for 16 hours in a cell culture medium containing 10% of fetal bovine serum. The 96-well plate is washed with physiological saline, ethyl alcohol is diluted to a cell culture solution to a concentration of 1.5% (v / v), and administered to each well of the 96-well plate in which HepG2 is cultured. did. After inducing cell damage with ethyl alcohol for 24 hours, the culture medium containing ethyl alcohol was removed and new cells were added at concentrations of 0 mg, 0.125 mg, 0.25 mg, 0.5 mg, 1 mg / ml. A solution obtained by diluting the fruit extract of keyonomi was added to each well. After culturing for 48 hours, the culture solution was removed, the 96-well plate was washed with physiological saline, and the cells in each well were fixed with 70% acetone for 20 minutes. The fixed cells were dried and then stained with SRB solution for 30 minutes, and the unstained solution was washed 5 times with 1% acetic acid solution. When the washing was finished, it was dried again, 10 mM tris solution was added to dissolve the SRB bound to the cell protein, and the absorbance at 562 nm was measured with a spectrophotometer. As a result, the wells that were not treated with ethyl alcohol and keyonomi fruit extract were used as a control group, and the absorbance in wells treated with ethyl alcohol alone was added to each well treated with ethyl alcohol. The absorbance in the well was comparatively measured (FIG. 3).

  The 1.5% ethyl alcohol concentration resulted in a 22% decrease in absorbance compared to wells that did not receive ethyl alcohol. However, when the fruit of the fruit was treated in wells that received ethyl alcohol, 0.5 mg / ml Up to the concentration, the absorbance increased in proportion to the increase in the concentration of the fruit extract of the keyronomid. Therefore, it can be seen that the cell growth was recovered from the damage caused by the ethyl alcohol in the well treated with the extract of the fruit of the keyronomid.

  Therefore, in the cells in which cell damage was induced by ethyl alcohol, the degree to which the cell growth was recovered by the administration of the extract of the fruit of the chironomid was obtained by Equation 1 and shown in Table 1.

  In the results of Table 1, in cells in which cell damage was induced by the administration of ethyl alcohol (1.5% concentration condition), the concentration of the fruit extract of keyonomi was 20.45 ± 7.72% at a concentration of 0.125 mg / ml. 27.14 ± 8.14% at 0.25 mg / ml concentration and 54.77 ± 3.37% damaged cells recovered at 0.5 mg / ml concentration. Therefore, it has been found that the extract of the fruit of a key flea in hepatocyte damage acts as a drug that restores the function of liver tissue by promoting hepatocyte growth.

Embodiment 5: Hepatic cell line damaged by administration of ethyl alcohol and recovery of liver function by extract of keyonomi fruit Hep3B cells, which are a kind of human hepatocytes, are placed at 2 × 10 4 in each well of a 96-well plate. After culturing for 12 hours to allow the cells to adhere to the bottom of the plate, the culture supernatant was removed and washed with a phosphate buffer solution. A culture solution in which ethyl alcohol was added to a new culture solution so that the final concentration of ethyl alcohol was 5% was placed in each well and cultured for 12 hours to induce cell damage. The culture broth was removed from each well, and the culture broth (0.3 mg / ml) containing the fruit extract of keyonomi and the broth not containing each were added to wells in which ethyl alcohol was added to induce cell damage. In order to measure ALT and AST in wells with and without ethyl alcohol, SPOTCHEM ™ II strip was purchased from Array Japan and used in this experiment. SPOTCHEM ™ II strip can measure all of ALT (GOT), AST (GPT), blood urea nitrogen, glucose total cholesterol, and total bilirubin in one strip. In this strip, the portion for measuring the amount of ALT and AST is a rice-colored zone, but when the amount of ALT and AST increases in the sample, it changes to a deep blue color. After 48 hours, the culture supernatant of each well was taken and dropped on a SPOTCHEM ™ II strip to confirm color development (FIG. 4).

  In FIG. 4, the control group is the color development result of the culture solution of the well in which Hep3B cells are not treated with all the extract of ethyl alcohol and keyonomi fruit. A slight amount of ALT and AST in the serum changed the rice color to a light blue color. However, in the culture medium of the wells to which ethyl alcohol was administered at a concentration of 5%, the color change appeared in a deep blue color, indicating that the AST and ALT activities increased rapidly. It is similar to the ALT and AST values of the control group representing a light blue color. After inducing cell damage with ethyl alcohol, the results of treatment with the fruit of the fruit of the fruit (0.3 mg / ml) were similar to the result of adding only the fruit of the fruit of the fruit (0.3 mg / ml), It can be seen that the ALT and AST values were reduced when compared with the culture medium of wells that were developed in a shallow blue color and treated with ethyl alcohol and were not treated with the fruit extract of keyonomi.

Embodiment 6: Toxicity test The hot water extract or hot water alcoholic beverage extract produced in the above embodiment 1 was dissolved in distilled water, and this was administered to mice (10 per group) at 500 mg / kg each. Observation for 7 days showed no cytotoxicity in view of the absence of dead mice.

Embodiment 7: Administration Effect of Extract of Keyonomi Fruit in Acute Hepatitis-Induced Mice In the mouse induced with acute hepatitis, the following experiment was conducted to analyze the ability of liver function to be restored by administration of the extract of keyonomi fruit. went. Forty mice (ICR species) were divided into four groups of A, B, C and D, 10 mice per group. Group A did not receive any drug, Group B was fed only with olive oil, and Group C was silymarin purchased from Sigma and dissolved in olive oil to 20 mg / ml. 100 ul / mouse was orally administered for 3 days, and Group D was prepared by dissolving the extract of the fruit of the fleafish obtained in Embodiment 1 in distilled water to 500 mg / ml, and dissolving this solution per mouse. 100 ul was orally administered for 3 days. During the period of drug administration, no food was administered in groups A, B, C, and D, and only water was supplied. After carbon tetrachloride was mixed with olive oil to 1%, 100 ul was injected into the abdominal cavity of mice in groups B, C and D. Then, after 18 hours, blood was collected from the mice of groups A, B, C, and D, and the activity of AST and ALT in the mouse blood was measured by the method as in Embodiment 5, and the average AST of each group, The activity value of ALT was compared. As a result, the AST and ALT average activity values of the A group without carbon tetrachloride injection were 23 units and 37 units, and the AST and ALT values of the B group injected with only carbon tetrachloride were 1, 023 units and 1,129 units, which represent a phenomenon in which acute hepatitis was induced. On the other hand, the AST and ALT values of group C pre-administered silymarin were 337 units and 446 units, and the AST and ALT average activity value of group D pre-administered the extract of keyonomi was 107 units. The average AST and ALT values of the extract of keyonomi decreased by 230 units and 207 units, respectively, compared to mice administered silymarin. From the AST and ALT recovery ratios, silymarin had a liver function recovery ability of 63.8%, and the liver fruit recovery ability of the liver fruit extract was 89.0%. It was shown that the recovery ability of the product was 25% or more.

[Embodiment 8] Comparison of histochemical results of a fruit of a key flea and silymarin in an acute hepatitis-induced mouse In Embodiment 7, in each experimental group A, B, C, and D, mice representing the average values of AST and ALT Histochemical analysis was performed on the liver tissue. Liver tissue was sliced from each group of mice, fixed in 10% formalin, dehydrated by immersion in different concentrations of ethyl alcohol, and finally embedded in paraffin. The paraffin-embedded tissue was sectioned into 4-5 μm and adhered to a slide glass, then stained with hematoxylin and eosin and observed with an optical microscope (FIG. 5). In the liver tissue of mice injected with carbon tetrachloride, there are mysterious deaths and many parts that are not stained with hematoxylin and eosin are scattered (Fig. 5-A), but silymarin and keyonomi extract are administered, and carbon tetrachloride is administered. The liver tissues of the injected mice (FIGS. 5-B and 5-C) can be seen as compared with normal liver tissues (FIG. 5-D), and almost no mortality reaction occurred. In particular, when comparing the liver tissues of mice administered with the extract of keyonomim and mice administered with silymarin, it was confirmed that the mice administered with the extract of keyonomi had fewer dead cells than silymarin. .

Embodiment 9. FIG. Liver function recovery effect clinical experiment with keyonomi fruit extract Normally, three volunteers with hepatitis symptoms were treated with the keyonomi fruit extract (1 g) produced in Embodiment 1 for 10 days. Or it was taken twice a day for 20 days, and ALT and AST, which are enzymes in hepatocytes, were examined. All three participants in the study reported a decrease in liver values after taking 10 or 20 days.

Volunteer 1: Male, 40-year-old pre-symptoms: I have had difficulty with hepatitis B for 8 years.

After taking: After taking for 10 days, it decreased from AST32 to 11.1 and from ALT73 to 14.3.

Thereafter, the dose decreased to AST10.3 and ALT10.6 after 20 days without taking them.

Applicant 2: Male, 35-year-old pre-symptoms: He was ill with liver disease, and he had a high liver value every time he was examined.

After taking: After taking for 20 days, it decreased from AST53 to 2.25 and ALT96 to 35.

Thereafter, the dose decreased to AST 2.25 and ALT 5.75 after 60 days without taking them.

Volunteer 3: Female, 35-year-old before taking symptoms: All the people in the house are hepatitis patients. After taking: 20 days after taking, decreased from AST 69 to 26, ALT 72 to 29.

Embodiment 10: Production of pharmaceutical composition Production Example 1-Production of powder and capsules 100 mg of the hot water extract obtained in Embodiment 1 above was mixed with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. It was. Using a suitable apparatus, the mixture is Packed into 5 gelatin capsules.

  The components of the powder and capsules are as follows.

Active ingredient 100mg
Lactose 14.8mg
Crystalline cellulose 3mg
Magnesium stearate 0.2mg
Production Example 2 Production of Injection Solution An injection solution containing 100 mg of the hot water extract obtained in Embodiment 1 was produced by the following method.

  100 ml was prepared by dissolving 100 mg of the hot water extract obtained in Embodiment 1 above, 600 mg of sodium chloride, and 100 mg of ascorbic acid in distilled water. This solution was placed in a bottle and sterilized by heating at 120 ° C. for 30 minutes.

  The components of the injection solution are as follows.

Active ingredient 1000mg
Sodium chloride 6000mg
Ascorbic acid 1000mg
Quantitative determination of distilled water Production Example 3-Production of Acid Agents Among the Korean Pharmacy General Formulations, the following ingredients are produced according to the acid agent production method.

1) Extract of active ingredient in one package (dry powder) 100 milligrams lactose 100 milligrams talc 10 milligrams 2) Water-soluble fraction of active ingredient in one package 100 milligrams lactose 100 milligrams talc 10 milligrams Production Example 4-Tablet production Korea It is manufactured with the following ingredient content according to the manufacturing method of a tablet among the pharmaceutical formulation general rules.

1) One tablet of active ingredient extract (dry powder) 100 milligrams corn starch 100 milligrams lactose 100 milligrams magnesium stearate 2 milligrams 2) One tablet of water-soluble ingredients 100 milligrams corn starch 100 milligrams lactose 100 milligrams stearin 2 mg of magnesium oxide

Production Example 5-Manufacture of Capsules Of the general rules for preparations of Korean pharmaceuticals, the capsules are produced with the following content according to the capsule production method.

1) 1 capsule of active ingredient extract (dry powder) 100 milligrams corn starch 100 milligrams lactose 100 milligrams magnesium stearate 2 milligrams 2) Water-soluble component of 100 milligrams corn starch 100 milligrams lactose 100 milligrams stearin Magnesium acid 2 milligrams Production Example 6-Production of injections Among the Korean Pharmacopoeia General Formulations, production is carried out according to the method for producing injections with the following component contents.

1) Inside one ampoule (2ml)
Active ingredient extract (dry powder) 50 milligrams of sterile distilled water for injection Appropriate amount pH adjuster Appropriate amount 2) In 1 ampoule (2 ml)
50 mg of sterile distilled water for injection Appropriate amount of pH adjusting agent Appropriate amount Production Example 7-Manufacture of liquid agent According to the manufacturing method of liquid preparations, it is manufactured with the following component contents.

1) 100 ml medium active ingredient extract (dry powder) 1 milligram isomerized sugar 10 gram mannitol 5 gram purified water 2) 100 ml medium active ingredient water soluble fraction 100 milligram isomerized sugar 10 gram mannitol 5 gram purified water

It is the result of having analyzed the influence which it has on the cell growth by administration of the extract of a fruit of a flea tree in HepG2 cell. It is the result of having analyzed the influence which it has on the cell growth by the administration of ethyl alcohol in HepG2 cell. Absorbance in wells treated with ethyl alcohol alone and wells treated with ethyl alcohol alone in wells not treated with ethyl alcohol and keony fruit extract and in wells treated with ethyl alcohol in wells treated with ethyl alcohol extract This is a result of comparative measurement of absorbance. It is the result explaining the liver function recovery phenomenon by administration of the extract of a fruit of a key flea on SPOTCHEMTM II strip. It is the result which demonstrated the effect of the extract of a key flea fruit and silymarin in the acute hepatitis induction mouse | mouth by the histochemical analysis.

Claims (6)

  A pharmaceutical composition characterized by having an effect of preventing and treating liver diseases, comprising an extract of Keyonomi (Lonicera caerulea L. var. Edulis).   The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an extract of a fruit of a sea bream.   The pharmaceutical composition according to claim 1, which is extracted using water, an organic solvent, or a mixed solvent thereof.   The pharmaceutical composition according to claim 1, which has a preventive and therapeutic effect on hepatitis, liver sclerosis, or fatty liver.   The pharmaceutical composition according to claim 1, characterized in that the extract of keyonomi has a weight ratio of 0.01 to 100% of the total composition.   The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is manufactured in a powder, granule, tablet, capsule, or injectable form.

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