WO2006109996A1 - Composition comprising the extract from melandryum firmum for improvement of liver function and treatment of liver diseases - Google Patents

Composition comprising the extract from melandryum firmum for improvement of liver function and treatment of liver diseases Download PDF

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Publication number
WO2006109996A1
WO2006109996A1 PCT/KR2006/001353 KR2006001353W WO2006109996A1 WO 2006109996 A1 WO2006109996 A1 WO 2006109996A1 KR 2006001353 W KR2006001353 W KR 2006001353W WO 2006109996 A1 WO2006109996 A1 WO 2006109996A1
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Prior art keywords
extract
group
takesimensis
silene
layer
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PCT/KR2006/001353
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French (fr)
Inventor
Deug-Yong Shin
Tae-Saeng Choi
Dong-Hoon Jin
Min-Kyung Cho
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Gencross Co.
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Publication of WO2006109996A1 publication Critical patent/WO2006109996A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/36—Caryophyllaceae (Pink family), e.g. babysbreath or soapwort
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B43—WRITING OR DRAWING IMPLEMENTS; BUREAU ACCESSORIES
    • B43K—IMPLEMENTS FOR WRITING OR DRAWING
    • B43K29/00—Combinations of writing implements with other articles
    • B43K29/06—Combinations of writing implements with other articles with sharpening devices
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A45—HAND OR TRAVELLING ARTICLES
    • A45D—HAIRDRESSING OR SHAVING EQUIPMENT; EQUIPMENT FOR COSMETICS OR COSMETIC TREATMENTS, e.g. FOR MANICURING OR PEDICURING
    • A45D29/00—Manicuring or pedicuring implements
    • A45D29/11—Polishing devices for nails
    • A45D29/12—Polishing devices for nails manually operated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B43—WRITING OR DRAWING IMPLEMENTS; BUREAU ACCESSORIES
    • B43K—IMPLEMENTS FOR WRITING OR DRAWING
    • B43K25/00—Attaching writing implements to wearing apparel or objects involving constructional changes of the implements
    • B43K25/02—Clips

Definitions

  • the present invention relates to a pharmaceutical composition and health functional food for improving liver function and treating liver diseases. More particularly, the present invention relates to a pharmaceutical composition and health functional food for improving liver function and treating liver diseases comprising as an active ingredient at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract that can inhibit AST and ALT activity and suppress collagen, alpha-smooth-muscle actin, and TGF- ⁇ .
  • a liver is an organ where enzyme reactions and energy metabolism constantly takes place and which plays a central role in metabolizing materials existing in or entering a living body. In addition, it is located between the digestive system and the circulatory system and protects the body from external non-bodily materials. Since all non-bodily materials pass through the liver once they enter the body, the liver is more likely to be exposed to various toxic materials besides nutriments and also, more likely to be damaged than other organs. Since the liver has an excellent ability to recover, it can easily recover from minor damages back to its normal state. However, when the liver is constantly damaged, and some of the tissues become irreparably destroyed, such tissues cannot recover back to its normal state, leading to over-accumulation of connective tissues.
  • liver damage is chronic, it can aggravate into hepatic fibrosis and cirrhosis regardless of the cause.
  • Hepatic fibrosis or cirrhosis can be caused by excessive consumption of alcohol, abuse of chemical drugs, viral hepatitis, interruption of bile secretion, and the like. They are hardly ever diagnosed in the initial stages due to a lack of pain or felt symptoms, and usually discovered in the final stages, which makes their treatment harder and increases mortality, leading to a widespread problem.
  • Hepatic fibrosis is a part of the reaction of a living body in adapting to hepatitis such as chronic hepatitis, and refers to a condition where damaged liver tissues are not restored to normal liver cells but changed into fibrous tissues like collagen.
  • hepatic fibrosis is an adaptive reaction of a living body during the process of restoration of damaged tissues, it necessarily results in deterioration of liver function, because it replaces liver tissues with fibrous tissues which cannot perform vital functions of the liver such as metabolism and bile secretion.
  • hepatic fibrosis can be accompanied by necrosis or inflammation, because it occurs as a result of over-accumulation of connective tissue in liver tissue, when the balance between synthesis and decomposition of connective tissue is lost. (Popper, Leber Magen Darm, 8, 65, 1978; Schuppan et al., Z. Gastro., 26 Suppl. 3, 28, 1988). Appearance of stellate cells is an important characteristic of any type of liver damage.
  • stellate cells are activated during hepatic fibrosis, which increases the expression of alpha-smooth-muscle actin ( ⁇ -SMA). Accordingly, present development of drugs for treating hepatic fibrosis focuses on inhibiting the activity of stellate cells. Drugs for inhibiting hepatic fibrosis are reported to include penicillamine,
  • 16,16-dimethylprostaglandins E2 biphenyl dimethyl dicarboxylic acid, colchicine, glucocorticoid, malotilate, gamma interferon, pentoxifylline, pyridine-2,4-dicarboxylic- diethylamide, pyridine-2,4-dicarboxylic-di (2-methoxyethyl)amide, and the like.
  • they are reported to have minimal effectiveness or have severe side effects when clinically applied.
  • TGF- ⁇ transforming growth factor-beta
  • TGF- ⁇ activity is interrupted by antibodies against TGF- ⁇ , antisense RNA, or variation in the cellular receptors of TGF- ⁇
  • hepatic fibrosis is reported to be sharply inhibited.
  • such research is still experimental, and there is yet no drug that has been clinically applied.
  • the present invention provides a pharmaceutical composition and health functional food including as an active ingredient at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract that can effectively inhibit hepatic fibrosis and protect the liver without toxicity.
  • An embodiment of the present invention provides a pharmaceutical composition for treating and preventing liver diseases, which contains at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract.
  • Another embodiment of the present invention provides a pharmaceutical composition for treating and preventing liver diseases, which contains at least one fraction selected from the group consisting of hexane, butanol, and water fractions of at least one selected from the group consisting of Melandryum firmum and Silene takesimensis.
  • Another embodiment of the present invention provides a health functional food for improving liver function which contains at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract as an active ingredient.
  • Another embodiment of the present invention provides a health functional food for improving liver function which includes at least one fraction selected from the group consisting of hexane, butanol, and water fractions of at least one selected from the group consisting of Melandryum firmum and Silene takesimensis.
  • FIG. 1 shows the effect of Melandryum firmum extract of the present invention on stromal cells and hepatic fibrosis-inducing cells.
  • FIG. 2 shows the effect of Silene takesimensis extract of the present invention on stromal cells and hepatic fibrosis-inducing cells.
  • FIG. 3 shows the effect of Melandryum firmum extract of the present invention on alpha-smooth-muscle actin.
  • FIG. 4 shows the effect of the extract of Experimental Example 2-1 in the present invention on GOT and GPT.
  • FIG. 5 shows the effect of the extract of Experimental Example 2-2 in the present invention on collagen accumulated in liver tissues.
  • FIG. 6 shows the effect of the extract of Experimental Example 2-2 in the present invention on the degree of hepatic fibrosis in liver tissues.
  • FIG. 7 shows the effect of the extract of Experimental Example 2-3 in the present invention on alpha-smooth-muscle actin.
  • FIG. 8 shows the effect of the extract of Experimental Example 3-2 in the present invention on the degree of hepatic fibrosis in liver tissues.
  • FIG. 9 shows the effect of the extract of Experimental Example 3-2 in the present invention on collagen expression.
  • FIG. 10 shows the effect of the extract of Experimental Example 3-3 in the present invention on alpha-smooth-muscle actin.
  • FIG. 11 is shows the effect of the extract of Experimental Example 3-4 in the present invention on TGF- ⁇ mRNA.
  • the present invention relates to Melandryum firmum extract, Silene takesimensis extract, or mixed extracts thereof that can inhibit activity of AST and ALT and suppress collagen, alpha-smooth-muscle actin, and TGF- ⁇ , and thereby improve liver function and treat liver diseases.
  • Melandryum firmum is a biannual herb belonging to the pink Caryophyllaceae Family, and its leaf and stem are used as medicinal material when it blooms in spring and summer. It is called Saponaria vaccaria L. in oriental medicine. It has been known not only to increase blood circulation but also been used to treat menstrual disorders, menstrual pains, lack of milk secretion, and the like. Particularly, it is effective against lack of menstruation due to problems in female sex hormone secretion and menstrual pains, and when it is used in conjunction with pangolin, it can promote milk secretion in women
  • Silene takesimensis is a perennial plant belonging to the same pink family as Melandryum firmum and grows on rocks in Uleung Island, Korea. However, no medicinal properties of Silene takesimensis has been reported up to now, and have been cultivated only for decorative use (Changbok Lee, 1797, Korean illustrated plant book, Hyangmoon Printing Co.; Woocheol Lee, 1996, Standard color-pictorial plant book, Academy Printing Co.).
  • Herb extracts of the present invention can include other herb extracts originated from plants of the same genus as Melandryum firmum, and Silene takesimensis, such as Melandryum firmum Rohrb. and Melandryum firmum for. pubescens Ohwi.
  • Melandryum firmum or Silene takesimensis extract of the present invention can be prepared in conventional methods of preparing plant extracts, and preferably in the hydrothermal extraction method, solvent extraction, or ultrasound extraction.
  • the extract of the present invention can be obtained from the whole plant, stem, or leaf of Melandryum firmum or Silene takesimensis, and preferably from their stem and leaf. More specifically, Melandryum firmum or Silene takesimensis can be extracted after being minced or ground fresh or dried. When dried, Melandryum firmum or Silene takesimensis should preferably contain less than 5% of moisture. Then, they are minced or ground to a powder, and less than 0.6mm particles thereof are selected to prepare an extract.
  • Solvent used for the extraction can include water, organic solvent, or mixed solvents thereof.
  • the organic solvent can include alcohol, hexane, chloroform, dichloromethane, or ethyl acetate.
  • the alcohol can include C1 to C4 alcohol such as ethanol, methanol, butanol, isopropanol, and propanol. More specifically, Melandryum firmum or Silene takesimensis can be extracted as follows; taking the fresh herb or drying it first; mincing and grinding it; mixing the minced or ground herb with water, organic solvent, or mixed solvents thereof, which is mixed in a ratio (kg/L) ranging from 1 :0.1 to 1:10 and preferably, 1:0.2 to 1:5.
  • the amount of solvent used is about 5 to 25 times and preferably, 5 to 10 times as much as the minced or ground herb; obtaining a soluble extract with the water, organic solvent, or mixed solvents thereof at 10 to 50 0 C and preferably, 20 to 30 0 C for about one hour to one day in a solvent extraction or an ultrasound extraction method; and concentrating the resulting extract under reduced pressure and lyophilizing it.
  • hydrothermal extraction can be applied to obtain the extract as follows; taking the fresh herb or drying it first, mincing or grinding it, adding thereto water in an amount which is 5 to 10 times as much as the minced or ground herb, and performing an extraction at 90 to 100 0 C and preferably, at 90 to 95 °C for 1 to 5 hours and preferably, 1 to 3 hours.
  • the resulting product is referred to as a Melandryum firmum or Silene takesimensis extract, and an organic solvent can be added to this extract to further fractionate it, in order to produce a fractionated extract.
  • the fractionated extract may be obtained as follows: Melandryum firmum or Silene takesimensis is minced or ground, and then added to methanol to obtain a crude extract.
  • the crude extract is put into an extractor containing water and hexane and separated into a first water layer and a hexane layer.
  • the first water layer is separated into ethyl acetate and a second water layer.
  • the resulting second water layer is separated into a butanol layer and a third water layer.
  • the hexane layer, butanol layer, and the first to third water layers may be used as an active ingredient.
  • mixed extracts of Melandryum firmum or Silene takesimensis can be prepared by mixing the minced or ground Melandryum firmum or Silene takesimensis, and thereafter performing an extraction therefrom, or by respectively extracting each minced or ground Melandryum firmum or Silene takesimensis, and then mixing them.
  • the mixing ratio also has no particular limit.
  • the present invention provides a Melandryum firmum extract, a Silene takesimensis extract, or mixed extracts thereof prepared in the aforementioned methods and effective in improving liver function and treating liver diseases.
  • a Melandryum firmum and Silene takesimensis extract were prepared by a method of hydrothermal extraction and ethanol extraction, and their medicinal effects were examined and verified by using animal models with liver diseases.
  • the Melandryum firmum extract, Silene takesimensis extract, or mixed extract thereof achieved recovery of fibrotic liver damage induced by carbon tetrachloride, through inhibition of the activity of ALT and AST enzymes in the liver.
  • the extracts inhibited hepatic fibrosis without affecting liver stromal cells and to decreased the amount of collagen, alpha-smooth-muscle actin, and TGF- ⁇ accumulated inside the liver tissues due to hepatic fibrosis.
  • the above medicinal effects of the extracts were enhanced in a concentration-dependent manner.
  • the extracts sharply reduced the amount of GOT and GPT in liver cells of hepatitis-induced mouse and inhibited the spread and growth of fibrosis cells inside the liver tissues.
  • a Melandryum firmum extract, Silene takesimensis extract, or mixed extract thereof can be effectively used for improving liver function and treating liver diseases.
  • the liver diseases include acute chronic hepatitis, hepatic fibrosis, cirrhosis, and hepatoma, but are not limited thereto.
  • composition of the present invention includes one or more from the group consisting of Melandryum firmum extract and
  • Silene takesimensis extract as an active ingredient and provides a pharmaceutical composition that can effectively treat and prevent liver diseases.
  • a pharmaceutical composition for treating and preventing liver diseases includes 0.1 to 80wt% and preferably, 1 to 50wt% of at least either of a Melandryum firmum extract or a Silene takesimensis extract based on the total weight of the composition.
  • the amount of the extract is less than 0.1wt%, it has little effect on the liver diseases, while when the amount of the extract is more than 80wt%, its administration can be costly compared to its effects.
  • the most preferable active amount of the extract can be suitably controlled depending on the usage and purpose of the composition.
  • a pharmaceutical composition can further include other herb extracts that is pharmaceutically allowed other than the Melandryum firmum extract, Silene takesimensis extract, or mixed extract thereof.
  • the pharmaceutical composition comprising either a Melandryum firmum extract or a Silene takesimensis extract can be respectively formulated by conventional methods.
  • a suitable formulation of the pharmaceutical composition may include, but is not limited to, tablets, pills, powders, granules, sugar-coated tablets, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and so on.
  • the formulation of the pharmaceutical composition can use an organic or inorganic carrier, which is pharmaceutically inactive.
  • an organic or inorganic carrier which is pharmaceutically inactive.
  • a coated tablet, a sugar-coated tablet, or a hard capsule lactose, sucrose, starch or a derivative thereof, talc, and stearic acid or salts thereof, can be used.
  • a soft capsule vegetable oil, wax, lipid, semisolid, and liquid polyol can be used.
  • water, polyol, glycerol, and vegetable oil and so on can be used.
  • natural oil or hydrogenated oil, wax, lipid, liquid polyol, and so on can be used.
  • the pharmaceutical agent may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizer, a flavoring, a colorant, an osmotic pressure regulator, an antioxidant, and so on.
  • the administration method can be selected depending upon the preparation thereof, and it includes oral or parenteral administration.
  • the dose of the pharmaceutical composition is preferably from 5 to 500 mg/kg per day, and preferably 100 to 250 mg/kg in the case of a healthy male adult, and it may vary depending on the age, gender, and body weight of the individual patient, the severity of the patient's symptom, and administration route. It may be administered once to three times per day.
  • the administration dose does not limit the scope of the present invention.
  • the present invention provides a pharmaceutical composition including a Melandryum firmum extract, a Silene takesimensis extract, or mixed extracts thereof that has an excellent effect of inhibiting hepatic fibrosis and protecting liver tissues, so that it is not only effective in inhibiting progression of hepatic fibrosis and cirrhosis but also can be safely used in long term administration for preventing as well as treating liver diseases without toxicity or side effects.
  • Another embodiment of the present invention provides a health functional food containing either Melandryum firmum extract, Silene takesimensis extract, or both, which can improve liver function.
  • the health functional food containing the extract includes medicinal products, foods, and beverages having a purpose of improvement of liver function.
  • Foods which can contain the extract of the present invention includes, for example, various kinds of foods, candy, chocolate, a beverage , gum, tea, multiple vitamin formulas, health foods, and the like and can be manufactured as a powder, granules, tablets, capsules, or a beverage.
  • the amount of the extract included in the food or beverage is generally in a range of 0.01 to 50wt% and preferably, in a range of 0.1 to 20wt% based on the entire weight of the food or beverage.
  • the extract can be included in a ratio ranging from 0.02 to 10g and preferably, from 0.3 to 1g based on 100ml of the entire composition.
  • the health functional food can further include a food additive, which is sitologically allowed, in addition to the extract of the present invention.
  • a composition for the health functional drink has no particular limit to its liquid components except that the extract is included in a predetermined ratio as a necessary component, and can also include diverse flavoring agents or natural carbohydrates added to conventional beverages.
  • the natural carbohydrate can include monosaccharide such as glucose and the like; disaccharide such as fructose and the like; polysaccharide such as maltose, sucrose, and the like; sugar such as dextrin, cyclodextrin, and the like; and sugar-alcohol such as xylitol, sorbitol, erythrytol, and the like.
  • the flavouring agent can include natural flavouring agents such as thaumatin, Stevia extract (for example, baudioside A, glycyrrhizin, and the like) and synthetic flavouring agents such as saccharin, aspartame, and the like.
  • natural flavouring agents such as thaumatin, Stevia extract (for example, baudioside A, glycyrrhizin, and the like) and synthetic flavouring agents such as saccharin, aspartame, and the like.
  • the natural carbohydrate is included generally in a ratio ranging from 1 to 2Og and preferably, from 5 to 12g based on 100ml of the composition of the present invention.
  • composition for the health functional food can include various nutrients, vitamins, minerals (an electrolyte), flavouring agents such as synthetic flavouring agents and natural flavouring agents, colorants and fillers (cheese, chocolate, and so on), pectic acid and its salt, alginic acid and its salt, organic acid, protective colloid thickener, pH controlling agents, stabilizers, preservatives, glycerine, alcohol, carbonating agent used for carbonated beverages, and the like.
  • it can also include fruit pulp for manufacturing natural fruit juices, fruit juice beverages, and vegetable beverages.
  • These components can be included independently or in combination.
  • the present invention has no particular limit in the added amount of the additives, but they can be generally included in a ratio ranging from 0.01 to 20 parts by weight based on 100 parts by weight of a health functional food. The following examples illustrate the present invention in more detail.
  • Example 1 Preparation of a Melandryum firmum extract
  • Melandryum firmum and Silene takesimensis mixture was performed, and the extract was freeze dried, to obtain 100mg of a mixed extract powder of Melandryum firmum and Silene takesimensis.
  • Example 4 Preparation of a fractionated extract 10g of a Melandryum firmum Rohrbach powder was added to 10OmL of methanol to obtain a crude extract.
  • the crude extract was put into an extractor including water and hexane and separated into a water layer and a hexane layer.
  • the water layer was fractionated three times with ethyl acetate to obtain ethyl acetate and a water layer.
  • the resulting water layer is fractionated with butanol to obtain a butanol layer and a water layer.
  • Reagents such as carbon tetrachloride (CCI 4 ), olive oil, Sirius-red dye, and
  • ALT serum alanine amino transferase
  • AST asparate amino transferase
  • mice Eight to ten week-old BAIb/c mice, which weigh 25 to 3Og, were used for experiments. They were allowed to freely take fodder (Hanla fodder Co.) and water. Their cage was set at a temperature of 21 to 24 0 C and relative humidity of 40 to 80%. In addition, light in the cage was controlled to repeat day and night every 12 hours.
  • Each experimental group was assigned to have 8 to 10 mice.
  • Example 1 liver stromal cells and hepatic fibrosis-inducing cells. Liver stromal cells were used as
  • (A) shows the experimental results of Control group 1
  • (B) shows the results of the hepatic stromal cells and hepatic fibrosis in Experimental group 1 after cell treatment with the extract of the present invention was performed.
  • FIG. 2 shows the experimental result of Control group 2
  • (B) shows the results of the hepatic stromal cells and hepatic fibrosis in Experimental group 2 after cell treatment with the extract of Example 2 was performed.
  • Hepatic fibrosis cells express alpha-smooth-muscle actin protein.
  • Control group 1 which had no treatment
  • Comparative group 1 treated with 0.1 ⁇ M of pirferidonem
  • Comparative group 2 treated with 1 ⁇ M of pirferidonem
  • Experimental group 1 treated with 20ug/ml of the extract of Example 1.
  • treatment with an extract of the present invention decreased expression of alpha-smooth-muscle actin.
  • the extract did not cause cell death but induced trans-differentiation of the cell property.
  • mice Eight to ten week-old male Balb/c mice were assigned to experimental groups. Each experimental group had 8 to 10 mice. The mice were administered with the toxic substance, carbon tetrachloride (CCI 4 ) once a week for two weeks to induce hepatitis and hepatic fibrosis. From the second day after the administration, distilled water, the extract of Example 1 , and a silymarine solution were respectively administered by mouth for 12 days. The mice were sacrificed in the afternoon of the 12 th day, and their blood and liver tissues were sampled to examine the degrees of hepatitis and hepatic fibrosis.
  • CCI 4 carbon tetrachloride
  • GOT Glutamyl oxaloacetic transminase
  • GPT Glutamyl pyruvic transminase
  • Negative Control group 3 was not administered with anything. Positive control group 3 was treated with carbon tetrachloride (CCI 4 ) and thereafter, with distilled water. In addition, Experimental group 3 was administered with 40mg of an extract of Example 1 per mouse. Comparative group 3 was administered with 30mg/kg of silymarine per mouse.
  • CCI 4 carbon tetrachloride
  • GOT and GPT respectively indicate numerical values for the level of aspartic acid degradation enzyme and alanine decomposition enzyme.
  • a liver When a liver has a problem and its cells are damaged, these components enter the blood in large amounts and can be identified therein, is a sensitive indicator of liver cell death.
  • Zero to 40Ul of GOT and GPT is normal, and 40 to 60Ul is still regarded as a normal range, while 60 to 300Ul indicates chronic inflammation, and 300Ul shows destruction of liver cells.
  • GOT and GPT values decreased in mice of Experimental group 3 treated with an extract of the present invention. Accordingly, the extract can effectively treat hepatitis.
  • FIG. 5 shows the effect of an extract of the present invention on collagen accumulation in a hepatitis-induced mouse.
  • A is a tissue photograph of Negative Control group 3
  • B is a tissue photograph of Negative Control group 3 right after treatment with carbon tetrachloride
  • C is a tissue photograph of Negative Control group 3 after treatment with distilled water following treatment of carbon tetrachloride
  • D is a tissue photograph of Comparative group 3
  • (E) is a tissue photograph of Experimental group 3.
  • FIG. 6 shows the effect of an extract of the present invention on hepatic fibrosis of liver cells (the content of collagen) in a hepatic fibrosis-induced mouse.
  • Experimental group 3 administered with the extract of Example 1 had sharply decreased hepatic fibrosis compared with Positive control group 3 and Comparative group 3 when hepatic fibrosis of mouse liver tissues were examined by staining collagen.
  • FIG. 7 shows the effect of an extract of the present invention on alpha- smooth-muscle actin in a hepatitis and hepatic fibrosis-induced mouse.
  • Positive control group 3 treated with carbon tetrachloride and Experimental group 3 treated with an extract of Example 1 were examined for the amount of alpha-smooth-muscle actin by Western blotting. A band was hardly identifiable for Experimental group 3.
  • mice Eight to ten week-old male Balb/c mice (8 to 10 mice per experimental group) was administered by mouth with 100 ⁇ l of a mixed solution, which was prepared by mixing the toxic substance, carbon tetrachloride (CCI 4 ) and olive oil in a ratio of 1:1 , once a week for a total of five times to induce hepatic fibrosis.
  • a mixed solution which was prepared by mixing the toxic substance, carbon tetrachloride (CCI 4 ) and olive oil in a ratio of 1:1 , once a week for a total of five times to induce hepatic fibrosis.
  • CCI 4 carbon tetrachloride
  • olive oil olive oil
  • Example 1 or 2 and a silymarine solution were respectively administered by mouth for 5 days as described in Table 1.
  • the mice were sacrificed in the afternoon of the 5 th day, and their blood and liver tissues were sampled to examine the extent of hepatic fibrosis.
  • Negative control group 4 a group administered with nothing was designated as Negative control group 4, and Positive control group 4 was administered with distilled water after administration of carbon tetrachloride (CCI 4 ).
  • Experimental groups 4 to 7 were respectively administered with extracts of Examples 1 and 2 in the amounts of 10mg and 40mg per mouse. Comparative group 4 was administered with 50mg/kg of silymarine per mouse. Table 1
  • Positive control group 4 was identified to have 29 ⁇ g/mg of collagen deposit in tissues.
  • Experimental groups 4 to 7 administered with the extract of Example 1 or 2 resulted in a lower collagen content deposited in tissues, which showed a decrease in manner that is dependent on the concentration of the extract.
  • FIG. 9 shows the effect of an extract of the present invention on collagen expression in hepatic fibrosis-induced mice.
  • A shows liver cell tissues of a normal mouse (Negative control group 4),
  • B Positive control group 4,
  • C Experimental group 4,
  • D Experimental group 5,
  • E Experimental group 6, and
  • F Experimental group 7.
  • Experimental groups 4 to 7 treated with an extract of Example 1 or 2 turned out to have decreased hepatic fibrosis compared with Positive control group 4 when hepatic fibrosis in mouse liver tissues was examined by collagen staining.
  • the amount of alpha-smooth-muscle actin was determined by Western blotting. The amount of alpha-smooth-muscle actin was compared by signals detected on the light-sensitive film. The results are provided in Table 3 and FIG. 10.
  • mice normal mice not treated with carbon tetrachloride (Negative control group 4), other mice treated with tetrachloride (Positive control group 4), and
  • the Negative control group had a value of 439, but Positive control group 4 had an increased value of 695 due to treatment with carbon tetrachloride. This increased difference was found to decrease in a manner dependent on the concentration of the extract during treatment.
  • Experimental group 5 recovered to 454, which is very close to the normal value of 439, when it was treated with 40mg of the extract of Example 1.
  • the amount of mRNA was shown to decrease in a manner dependent on the concentration of the extract in the group treated with the extract of the present invention.
  • mice Joongang experiment animal Co. weighing 25 ⁇ 5g and SPF Sprague-Dawley rats (Biogenomics Co.) weighing 235 ⁇ 10g were assigned to 3 groups with 3 per group. Each group was respectively administered by mouth with
  • the 9 groups displayed no externally-identifiable differences from the Control group in terms of weight gain, intake amount of fodder, and the like. This shows that Melandryum firmum extract of the present invention is safe.
  • an extract can be made into a variety of formulations for administration.
  • the following formulations are exemplary in the present invention, but it is understood that the present invention is not limited thereto.
  • Formulation 1 Tablets extract of Example 1 200mg lactose 100mg starch 10Omg magnesium stearate a suitable amount The above components were mixed and formulated into tablets by a conventional method.
  • Formulation 2 Capsules extract of Example 2 10Omg lactose 50mg starch 50mg talc 2mg magnesium sterarate a suitable amount
  • Formulation 3 Liquid extract of Example 1 500mg extract of Example 2 500mg sugar 20mg isomerized sugar 20mg lemon flavor a suitable amount
  • a health beverage composition was prepared by mixing the above components by a conventional method, agitating and heating the mixture at 85 °C for one hour, filtering and filling it in a sterilized 2L container, sealing and sterilizing the container, and storing it in a refrigerator.
  • Formulation 6 Health Food extract of Example 2 10OOmg vitamin mixture a suitable amount vitamin A acetate 70 ⁇ g vitamin E 1.0mg vitamin B 1 0.13mg vitamin B 2 0.15mg vitamin B 6 O. ⁇ mg vitamin B 12 0.2 ⁇ g vitamin C 10mg biotin 10 ⁇ g
  • a granule-type health food was formulated by mixing the above components by a conventional method.
  • the vitamin and mineral mixture were included in a composition ratio which is known to be preferable for health foods, but the composition ratio can be optionally varied.
  • Melandryum firmum extract or Silene takesimensis extract of the present invention can inhibit activity of AST and ALT and also, the amount of collagen, alpha-smooth-muscle actin, and TGF- ⁇ accumulated due to hepatic fibrosis in liver tissues, and accordingly be effectively used as a drug or health functional food for improving liver function and preventing and treating liver diseases.

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Abstract

The present invention relates to a pharmaceutical composition and a health functional food including one or more from the group consisting of Melandryum firmum extract and Silene takesimensis extract as an active ingredient for improving liver function and treating liver diseases. Since the Melandryum firmum and Silene takesimensis extract of the present invention can not only inhibit AST and ALT activity but also suppress the amount of collagen, alpha-smooth-muscle actin, and TGF-βaccumulated in liver tissues caused by hepatic fibrosis, they can be effectively used in pharmaceutical compositions and health functional foods for treating and preventing liver diseases.

Description

TiTLE OF THE INVENTION
COMPOSITION COMPRISING THE EXTRACT FROM MELANDRYUM FIRMUM FOR IMPROVEMENT OF LIVER FUNCTION AND TREATMENT OF LIVER DISEASES
BACKGROUND OF THE INVENTION (a) Field of the Invention
The present invention relates to a pharmaceutical composition and health functional food for improving liver function and treating liver diseases. More particularly, the present invention relates to a pharmaceutical composition and health functional food for improving liver function and treating liver diseases comprising as an active ingredient at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract that can inhibit AST and ALT activity and suppress collagen, alpha-smooth-muscle actin, and TGF-β. (b) Description of the Related Art
A liver is an organ where enzyme reactions and energy metabolism constantly takes place and which plays a central role in metabolizing materials existing in or entering a living body. In addition, it is located between the digestive system and the circulatory system and protects the body from external non-bodily materials. Since all non-bodily materials pass through the liver once they enter the body, the liver is more likely to be exposed to various toxic materials besides nutriments and also, more likely to be damaged than other organs. Since the liver has an excellent ability to recover, it can easily recover from minor damages back to its normal state. However, when the liver is constantly damaged, and some of the tissues become irreparably destroyed, such tissues cannot recover back to its normal state, leading to over-accumulation of connective tissues. Accordingly, scar tissue is formed in the liver, and it cannot return to its normally functioning state. When liver damage is chronic, it can aggravate into hepatic fibrosis and cirrhosis regardless of the cause. Hepatic fibrosis or cirrhosis can be caused by excessive consumption of alcohol, abuse of chemical drugs, viral hepatitis, interruption of bile secretion, and the like. They are hardly ever diagnosed in the initial stages due to a lack of pain or felt symptoms, and usually discovered in the final stages, which makes their treatment harder and increases mortality, leading to a widespread problem.
Hepatic fibrosis is a part of the reaction of a living body in adapting to hepatitis such as chronic hepatitis, and refers to a condition where damaged liver tissues are not restored to normal liver cells but changed into fibrous tissues like collagen. Although hepatic fibrosis is an adaptive reaction of a living body during the process of restoration of damaged tissues, it necessarily results in deterioration of liver function, because it replaces liver tissues with fibrous tissues which cannot perform vital functions of the liver such as metabolism and bile secretion. In addition, hepatic fibrosis can be accompanied by necrosis or inflammation, because it occurs as a result of over-accumulation of connective tissue in liver tissue, when the balance between synthesis and decomposition of connective tissue is lost. (Popper, Leber Magen Darm, 8, 65, 1978; Schuppan et al., Z. Gastro., 26 Suppl. 3, 28, 1988). Appearance of stellate cells is an important characteristic of any type of liver damage.
Particularly, stellate cells are activated during hepatic fibrosis, which increases the expression of alpha-smooth-muscle actin (α-SMA). Accordingly, present development of drugs for treating hepatic fibrosis focuses on inhibiting the activity of stellate cells. Drugs for inhibiting hepatic fibrosis are reported to include penicillamine,
16,16-dimethylprostaglandins E2, biphenyl dimethyl dicarboxylic acid, colchicine, glucocorticoid, malotilate, gamma interferon, pentoxifylline, pyridine-2,4-dicarboxylic- diethylamide, pyridine-2,4-dicarboxylic-di (2-methoxyethyl)amide, and the like. However, they are reported to have minimal effectiveness or have severe side effects when clinically applied.
Recently, TGF-β (transforming growth factor-beta), which is a cytokine isolated from Kupffer macrophages and fat-storing cells, has been reported as an important medium of hepatic fibrosis. In addition, when TGF-β activity is interrupted by antibodies against TGF-β, antisense RNA, or variation in the cellular receptors of TGF-β, hepatic fibrosis is reported to be sharply inhibited. However, such research is still experimental, and there is yet no drug that has been clinically applied. SUMMARY OF THE INVENTION
The present invention provides a pharmaceutical composition and health functional food including as an active ingredient at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract that can effectively inhibit hepatic fibrosis and protect the liver without toxicity.
An embodiment of the present invention provides a pharmaceutical composition for treating and preventing liver diseases, which contains at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract. Another embodiment of the present invention provides a pharmaceutical composition for treating and preventing liver diseases, which contains at least one fraction selected from the group consisting of hexane, butanol, and water fractions of at least one selected from the group consisting of Melandryum firmum and Silene takesimensis. Another embodiment of the present invention provides a health functional food for improving liver function which contains at least one from the group consisting of Melandryum firmum extract and Silene takesimensis extract as an active ingredient.
Another embodiment of the present invention provides a health functional food for improving liver function which includes at least one fraction selected from the group consisting of hexane, butanol, and water fractions of at least one selected from the group consisting of Melandryum firmum and Silene takesimensis. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effect of Melandryum firmum extract of the present invention on stromal cells and hepatic fibrosis-inducing cells.
FIG. 2 shows the effect of Silene takesimensis extract of the present invention on stromal cells and hepatic fibrosis-inducing cells.
FIG. 3 shows the effect of Melandryum firmum extract of the present invention on alpha-smooth-muscle actin. FIG. 4 shows the effect of the extract of Experimental Example 2-1 in the present invention on GOT and GPT.
FIG. 5 shows the effect of the extract of Experimental Example 2-2 in the present invention on collagen accumulated in liver tissues.
FIG. 6 shows the effect of the extract of Experimental Example 2-2 in the present invention on the degree of hepatic fibrosis in liver tissues.
FIG. 7 shows the effect of the extract of Experimental Example 2-3 in the present invention on alpha-smooth-muscle actin.
FIG. 8 shows the effect of the extract of Experimental Example 3-2 in the present invention on the degree of hepatic fibrosis in liver tissues.
FIG. 9 shows the effect of the extract of Experimental Example 3-2 in the present invention on collagen expression. FIG. 10 shows the effect of the extract of Experimental Example 3-3 in the present invention on alpha-smooth-muscle actin.
FIG. 11 is shows the effect of the extract of Experimental Example 3-4 in the present invention on TGF-β mRNA. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Melandryum firmum extract, Silene takesimensis extract, or mixed extracts thereof that can inhibit activity of AST and ALT and suppress collagen, alpha-smooth-muscle actin, and TGF-β, and thereby improve liver function and treat liver diseases.
Melandryum firmum is a biannual herb belonging to the pink Caryophyllaceae Family, and its leaf and stem are used as medicinal material when it blooms in spring and summer. It is called Saponaria vaccaria L. in oriental medicine. It has been known not only to increase blood circulation but also been used to treat menstrual disorders, menstrual pains, lack of milk secretion, and the like. Particularly, it is effective against lack of menstruation due to problems in female sex hormone secretion and menstrual pains, and when it is used in conjunction with pangolin, it can promote milk secretion in women
(Bonchogangmok).
Silene takesimensis is a perennial plant belonging to the same pink family as Melandryum firmum and grows on rocks in Uleung Island, Korea. However, no medicinal properties of Silene takesimensis has been reported up to now, and have been cultivated only for decorative use (Changbok Lee, 1797, Korean illustrated plant book, Hyangmoon Printing Co.; Woocheol Lee, 1996, Standard color-pictorial plant book, Academy Printing Co.).
Herb extracts of the present invention can include other herb extracts originated from plants of the same genus as Melandryum firmum, and Silene takesimensis, such as Melandryum firmum Rohrb. and Melandryum firmum for. pubescens Ohwi.
Melandryum firmum or Silene takesimensis extract of the present invention can be prepared in conventional methods of preparing plant extracts, and preferably in the hydrothermal extraction method, solvent extraction, or ultrasound extraction. The extract of the present invention can be obtained from the whole plant, stem, or leaf of Melandryum firmum or Silene takesimensis, and preferably from their stem and leaf. More specifically, Melandryum firmum or Silene takesimensis can be extracted after being minced or ground fresh or dried. When dried, Melandryum firmum or Silene takesimensis should preferably contain less than 5% of moisture. Then, they are minced or ground to a powder, and less than 0.6mm particles thereof are selected to prepare an extract.
Solvent used for the extraction can include water, organic solvent, or mixed solvents thereof. The organic solvent can include alcohol, hexane, chloroform, dichloromethane, or ethyl acetate. The alcohol can include C1 to C4 alcohol such as ethanol, methanol, butanol, isopropanol, and propanol. More specifically, Melandryum firmum or Silene takesimensis can be extracted as follows; taking the fresh herb or drying it first; mincing and grinding it; mixing the minced or ground herb with water, organic solvent, or mixed solvents thereof, which is mixed in a ratio (kg/L) ranging from 1 :0.1 to 1:10 and preferably, 1:0.2 to 1:5. Here, the amount of solvent used is about 5 to 25 times and preferably, 5 to 10 times as much as the minced or ground herb; obtaining a soluble extract with the water, organic solvent, or mixed solvents thereof at 10 to 500C and preferably, 20 to 300C for about one hour to one day in a solvent extraction or an ultrasound extraction method; and concentrating the resulting extract under reduced pressure and lyophilizing it. In addition, hydrothermal extraction can be applied to obtain the extract as follows; taking the fresh herb or drying it first, mincing or grinding it, adding thereto water in an amount which is 5 to 10 times as much as the minced or ground herb, and performing an extraction at 90 to 1000C and preferably, at 90 to 95 °C for 1 to 5 hours and preferably, 1 to 3 hours.
The resulting product is referred to as a Melandryum firmum or Silene takesimensis extract, and an organic solvent can be added to this extract to further fractionate it, in order to produce a fractionated extract.
The fractionated extract may be obtained as follows: Melandryum firmum or Silene takesimensis is minced or ground, and then added to methanol to obtain a crude extract. The crude extract is put into an extractor containing water and hexane and separated into a first water layer and a hexane layer. The first water layer is separated into ethyl acetate and a second water layer. The resulting second water layer is separated into a butanol layer and a third water layer. The hexane layer, butanol layer, and the first to third water layers may be used as an active ingredient.
Next, mixed extracts of Melandryum firmum or Silene takesimensis can be prepared by mixing the minced or ground Melandryum firmum or Silene takesimensis, and thereafter performing an extraction therefrom, or by respectively extracting each minced or ground Melandryum firmum or Silene takesimensis, and then mixing them. The mixing ratio also has no particular limit.
The present invention provides a Melandryum firmum extract, a Silene takesimensis extract, or mixed extracts thereof prepared in the aforementioned methods and effective in improving liver function and treating liver diseases.
According to one embodiment of the present invention, a Melandryum firmum and Silene takesimensis extract were prepared by a method of hydrothermal extraction and ethanol extraction, and their medicinal effects were examined and verified by using animal models with liver diseases.
As a result, the Melandryum firmum extract, Silene takesimensis extract, or mixed extract thereof achieved recovery of fibrotic liver damage induced by carbon tetrachloride, through inhibition of the activity of ALT and AST enzymes in the liver. The extracts inhibited hepatic fibrosis without affecting liver stromal cells and to decreased the amount of collagen, alpha-smooth-muscle actin, and TGF-β accumulated inside the liver tissues due to hepatic fibrosis. In addition, the above medicinal effects of the extracts were enhanced in a concentration-dependent manner.
Furthermore, the extracts sharply reduced the amount of GOT and GPT in liver cells of hepatitis-induced mouse and inhibited the spread and growth of fibrosis cells inside the liver tissues. As a result, a Melandryum firmum extract, Silene takesimensis extract, or mixed extract thereof can be effectively used for improving liver function and treating liver diseases. The liver diseases include acute chronic hepatitis, hepatic fibrosis, cirrhosis, and hepatoma, but are not limited thereto.
In addition, the pharmaceutical composition of the present invention includes one or more from the group consisting of Melandryum firmum extract and
Silene takesimensis extract as an active ingredient and provides a pharmaceutical composition that can effectively treat and prevent liver diseases.
According to an embodiment of the present invention, a pharmaceutical composition for treating and preventing liver diseases includes 0.1 to 80wt% and preferably, 1 to 50wt% of at least either of a Melandryum firmum extract or a Silene takesimensis extract based on the total weight of the composition. When the amount of the extract is less than 0.1wt%, it has little effect on the liver diseases, while when the amount of the extract is more than 80wt%, its administration can be costly compared to its effects. However, the most preferable active amount of the extract can be suitably controlled depending on the usage and purpose of the composition.
According to an embodiment of the present invention, a pharmaceutical composition can further include other herb extracts that is pharmaceutically allowed other than the Melandryum firmum extract, Silene takesimensis extract, or mixed extract thereof. Preferably, an extract selected from the group consisting of
Dioscoreaceae, lacquer tree, Korean lettuce, Hovenia dulcis, arrowroot flower, red- bean flower, Rhynchosia Nulubilis, mulberry leaves, Moms fruit, red bean, mung bean, iris, and mixtures thereof, which are known to be effective in improving liver function, can be included therein. The pharmaceutical composition comprising either a Melandryum firmum extract or a Silene takesimensis extract can be respectively formulated by conventional methods. A suitable formulation of the pharmaceutical composition may include, but is not limited to, tablets, pills, powders, granules, sugar-coated tablets, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories, and so on. The formulation of the pharmaceutical composition can use an organic or inorganic carrier, which is pharmaceutically inactive. For formulation into a tablet, a coated tablet, a sugar-coated tablet, or a hard capsule, lactose, sucrose, starch or a derivative thereof, talc, and stearic acid or salts thereof, can be used. Furthermore, for formulation into a soft capsule, vegetable oil, wax, lipid, semisolid, and liquid polyol can be used. For formulation into a solution or a syrup, water, polyol, glycerol, and vegetable oil and so on can be used. As the carrier in suppository formulations, natural oil or hydrogenated oil, wax, lipid, liquid polyol, and so on can be used.
The pharmaceutical agent may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizer, a flavoring, a colorant, an osmotic pressure regulator, an antioxidant, and so on. The administration method can be selected depending upon the preparation thereof, and it includes oral or parenteral administration. The dose of the pharmaceutical composition is preferably from 5 to 500 mg/kg per day, and preferably 100 to 250 mg/kg in the case of a healthy male adult, and it may vary depending on the age, gender, and body weight of the individual patient, the severity of the patient's symptom, and administration route. It may be administered once to three times per day. The administration dose does not limit the scope of the present invention.
Therefore, the present invention provides a pharmaceutical composition including a Melandryum firmum extract, a Silene takesimensis extract, or mixed extracts thereof that has an excellent effect of inhibiting hepatic fibrosis and protecting liver tissues, so that it is not only effective in inhibiting progression of hepatic fibrosis and cirrhosis but also can be safely used in long term administration for preventing as well as treating liver diseases without toxicity or side effects.
Another embodiment of the present invention provides a health functional food containing either Melandryum firmum extract, Silene takesimensis extract, or both, which can improve liver function.
The health functional food containing the extract includes medicinal products, foods, and beverages having a purpose of improvement of liver function. Foods which can contain the extract of the present invention includes, for example, various kinds of foods, candy, chocolate, a beverage , gum, tea, multiple vitamin formulas, health foods, and the like and can be manufactured as a powder, granules, tablets, capsules, or a beverage.
Herein, the amount of the extract included in the food or beverage is generally in a range of 0.01 to 50wt% and preferably, in a range of 0.1 to 20wt% based on the entire weight of the food or beverage. As for the health beverage composition, the extract can be included in a ratio ranging from 0.02 to 10g and preferably, from 0.3 to 1g based on 100ml of the entire composition.
The health functional food can further include a food additive, which is sitologically allowed, in addition to the extract of the present invention.
A composition for the health functional drink has no particular limit to its liquid components except that the extract is included in a predetermined ratio as a necessary component, and can also include diverse flavoring agents or natural carbohydrates added to conventional beverages. The natural carbohydrate can include monosaccharide such as glucose and the like; disaccharide such as fructose and the like; polysaccharide such as maltose, sucrose, and the like; sugar such as dextrin, cyclodextrin, and the like; and sugar-alcohol such as xylitol, sorbitol, erythrytol, and the like. The flavouring agent can include natural flavouring agents such as thaumatin, Stevia extract (for example, baudioside A, glycyrrhizin, and the like) and synthetic flavouring agents such as saccharin, aspartame, and the like. The natural carbohydrate is included generally in a ratio ranging from 1 to 2Og and preferably, from 5 to 12g based on 100ml of the composition of the present invention. In addition, the composition for the health functional food can include various nutrients, vitamins, minerals (an electrolyte), flavouring agents such as synthetic flavouring agents and natural flavouring agents, colorants and fillers (cheese, chocolate, and so on), pectic acid and its salt, alginic acid and its salt, organic acid, protective colloid thickener, pH controlling agents, stabilizers, preservatives, glycerine, alcohol, carbonating agent used for carbonated beverages, and the like. Furthermore, it can also include fruit pulp for manufacturing natural fruit juices, fruit juice beverages, and vegetable beverages. These components can be included independently or in combination. The present invention has no particular limit in the added amount of the additives, but they can be generally included in a ratio ranging from 0.01 to 20 parts by weight based on 100 parts by weight of a health functional food. The following examples illustrate the present invention in more detail.
However, it is understood that the present invention is not limited by these examples.
Example 1 : Preparation of a Melandryum firmum extract
1Og of a Melandryum firmum Rohrbach powder was added to 10OmL of ethanol, solvent extraction was performed for the Melandryum firmum mixture at room temperature for 24 hours, and the extract was filtered and freeze-dried to obtain 120mg of a Melandryum firmum extract powder.
Example 2: Preparation of a Silene takesimensis extract
10g of Silene takesimensis powder was added to 10OmL of distilled water, and hydrothermal extraction was performed for the Silene takesimensis mixture at 95°C for 3 hours, and the extract was filtered and freeze dried to obtain 240mg of
Silene takesimensis extract powder.
Example 3: Preparation of mixed extracts of Melandryum firmum and Silene takesimensis
5g of Melandryum firmum Rohrbach powder and 5g of Silene takesimensis powder were added to 10OmL of ethanol. Organic solvent extraction for the
Melandryum firmum and Silene takesimensis mixture was performed, and the extract was freeze dried, to obtain 100mg of a mixed extract powder of Melandryum firmum and Silene takesimensis.
Example 4: Preparation of a fractionated extract 10g of a Melandryum firmum Rohrbach powder was added to 10OmL of methanol to obtain a crude extract. The crude extract was put into an extractor including water and hexane and separated into a water layer and a hexane layer. The water layer was fractionated three times with ethyl acetate to obtain ethyl acetate and a water layer. The resulting water layer is fractionated with butanol to obtain a butanol layer and a water layer.
Reference Example 1 : Experimental animals and Treatments
1) Reagents Reagents such as carbon tetrachloride (CCI4), olive oil, Sirius-red dye, and
Fast Green FCF were made by Sigma Chemical Co., and alpha-smooth-muscle actin antibody was made by Santa Cruz Co. In addition, serum alanine amino transferase (ALT) and asparate amino transferase (AST) were measured by using a kit for ALT/ AST serum analysis made by Yongdong Pharmaceutical Co.
2) Animals
Eight to ten week-old BAIb/c mice, which weigh 25 to 3Og, were used for experiments. They were allowed to freely take fodder (Hanla fodder Co.) and water. Their cage was set at a temperature of 21 to 240C and relative humidity of 40 to 80%. In addition, light in the cage was controlled to repeat day and night every 12 hours.
Each experimental group was assigned to have 8 to 10 mice.
Experimental Example 1 : In vitro treatment effects of Melandryum firmum extract or Silene takesimensis extract on hepatic fibrosis
Following experiments were performed to examine treatment effects of the extracts prepared in Examples 1 and 2.
1-1) Inhibition effects against growth of hepatic fibrosis-inducing cells
20/ig/ml of the extract of Example 1 was treated with hepatic stromal cells
(Chang cell, ATCC) and hepatic fibrosis cells (Ito cell, health Science Research
Resource Bank, Japan) to examine how the extract of Example 1 affects liver stromal cells and hepatic fibrosis-inducing cells. Liver stromal cells were used as
Control group 1. The experimental results are shown in FIG. 1.
Referring to FIG. 1, (A) shows the experimental results of Control group 1 , and (B) shows the results of the hepatic stromal cells and hepatic fibrosis in Experimental group 1 after cell treatment with the extract of the present invention was performed.
Compared with the experimental result of Control group 1 , Experimental group 1 treated with the extract had no effect on hepatic stromal cells but had some effect on hepatic fibrosis cells, in which hepatic fibrosis had already progressed. The results show that the extract of the present invention inhibits the growth of only hepatic fibrosis cells. Furthermore, normal hepatic stromal cells treated with a great dosage of the extract did not die nor change, showing that an extract of the present invention does not have cell toxicity. The effects of Silene takesimensis extract on stromal cells and hepatic fibrosis-inducing cells were examined in the same method as above except that an extract of Example 2 was used
The result is provided in FIG. 2. Referring to FIG. 2, (A) shows the experimental result of Control group 2, and (B) shows the results of the hepatic stromal cells and hepatic fibrosis in Experimental group 2 after cell treatment with the extract of Example 2 was performed.
The result turned out to be the same as that using the Melandryum firmum extract.
1-2) Effects on transformation of hepatic fibrosis-inducing cells (HSC)
Hepatic fibrosis cells express alpha-smooth-muscle actin protein. The effect of the extract of the present invention on alpha-smooth-muscle actin, which is a hepatic fibrosis-inducing material, was examined. Western blotting was performed on protein samples from the cells of
Experiment 1-1 to identify alpha-smooth-muscle actin. Each comparative group 1 and 2 was respectively treated with 0.1 μM and 1 μM of pirferidonem, which is currently known to inhibit hepatic fibrosis of liver tissues. The result is provided in FIG. 3. Referring to FIG. 3, (A) shows effects on alpha-smooth-muscle actin in
Control group 1 which had no treatment, (B) Comparative group 1 treated with 0.1 μM of pirferidonem, (C) Comparative group 2 treated with 1μM of pirferidonem, and (D) Experimental group 1 treated with 20ug/ml of the extract of Example 1.
As shown in FIG. 3, treatment with an extract of the present invention decreased expression of alpha-smooth-muscle actin. Herein, the extract did not cause cell death but induced trans-differentiation of the cell property.
Experimental Example 2: Treatment effects of Melandryum firmum extract or Silene takesimensis extract on hepatitis and hepatic fibrosis
The following experiment was performed to examine treatment effects of an extract of Examples 1 and 2 on hepatitis and hepatic fibrosis.
Eight to ten week-old male Balb/c mice were assigned to experimental groups. Each experimental group had 8 to 10 mice. The mice were administered with the toxic substance, carbon tetrachloride (CCI4) once a week for two weeks to induce hepatitis and hepatic fibrosis. From the second day after the administration, distilled water, the extract of Example 1 , and a silymarine solution were respectively administered by mouth for 12 days. The mice were sacrificed in the afternoon of the 12th day, and their blood and liver tissues were sampled to examine the degrees of hepatitis and hepatic fibrosis.
2-1) Measurement of GOT and GPT
Hepatitis symptoms were examined by measuring GOT (Glutamyl oxaloacetic transminase) and GPT (Glutamyl pyruvic transminase) in serum. The result is provided in FIG. 4.
Negative Control group 3 was not administered with anything. Positive control group 3 was treated with carbon tetrachloride (CCI4) and thereafter, with distilled water. In addition, Experimental group 3 was administered with 40mg of an extract of Example 1 per mouse. Comparative group 3 was administered with 30mg/kg of silymarine per mouse.
GOT and GPT respectively indicate numerical values for the level of aspartic acid degradation enzyme and alanine decomposition enzyme. When a liver has a problem and its cells are damaged, these components enter the blood in large amounts and can be identified therein, is a sensitive indicator of liver cell death. Zero to 40Ul of GOT and GPT is normal, and 40 to 60Ul is still regarded as a normal range, while 60 to 300Ul indicates chronic inflammation, and 300Ul shows destruction of liver cells.
As shown in FIG. 4, GOT and GPT values decreased in mice of Experimental group 3 treated with an extract of the present invention. Accordingly, the extract can effectively treat hepatitis.
2-2) Comparison of the amount of collagen in tissues and tissue staining
Liver tissues were stained with sirius red to analyze collagen accumulation and to identify the extent of treatment of hepatitis and hepatic fibrosis. The results are provided in FIGS. 5 and 6. FIG. 5 shows the effect of an extract of the present invention on collagen accumulation in a hepatitis-induced mouse. Herein, (A) is a tissue photograph of Negative Control group 3, (B) is a tissue photograph of Negative Control group 3 right after treatment with carbon tetrachloride, (C) is a tissue photograph of Negative Control group 3 after treatment with distilled water following treatment of carbon tetrachloride, (D) is a tissue photograph of Comparative group 3, and (E) is a tissue photograph of Experimental group 3. In addition, FIG. 6 shows the effect of an extract of the present invention on hepatic fibrosis of liver cells (the content of collagen) in a hepatic fibrosis-induced mouse.
As shown in FIGS. 5 and 6, Experimental group 3 administered with the extract of Example 1 had sharply decreased hepatic fibrosis compared with Positive control group 3 and Comparative group 3 when hepatic fibrosis of mouse liver tissues were examined by staining collagen.
2-3) Comparison of the amount of alpha-smooth-muscle actin in tissues A Western blot was performed to determine the amount of alpha-smooth- muscle actin was compared by signals detected on the light-sensitive film. The result is provided in FIG. 7.
FIG. 7 shows the effect of an extract of the present invention on alpha- smooth-muscle actin in a hepatitis and hepatic fibrosis-induced mouse. Positive control group 3 treated with carbon tetrachloride and Experimental group 3 treated with an extract of Example 1 were examined for the amount of alpha-smooth-muscle actin by Western blotting. A band was hardly identifiable for Experimental group 3.
Experimental Example 3: Treatment of hepatic fibrosis by Melandryum firmum extract or Silene takesimensis extract.
Eight to ten week-old male Balb/c mice (8 to 10 mice per experimental group) was administered by mouth with 100μl of a mixed solution, which was prepared by mixing the toxic substance, carbon tetrachloride (CCI4) and olive oil in a ratio of 1:1 , once a week for a total of five times to induce hepatic fibrosis. When administration of carbon tetrachloride was complete, distilled water, an extract of
Example 1 or 2, and a silymarine solution were respectively administered by mouth for 5 days as described in Table 1. The mice were sacrificed in the afternoon of the 5th day, and their blood and liver tissues were sampled to examine the extent of hepatic fibrosis.
3-1) Comparison of blood levels of ALT and AST Serum levels of ALT (Alanine aminotransferase) and AST (Aspartate aminotransferase) enzymes were measured. The results are provided in the following Table 1.
Herein, a group administered with nothing was designated as Negative control group 4, and Positive control group 4 was administered with distilled water after administration of carbon tetrachloride (CCI4). In addition, Experimental groups 4 to 7 were respectively administered with extracts of Examples 1 and 2 in the amounts of 10mg and 40mg per mouse. Comparative group 4 was administered with 50mg/kg of silymarine per mouse. Table 1
Figure imgf000017_0001
Referring to the experimental results in Table 1 , Experimental groups 4 to 7 treated with the extracts of Examples 1 and 2 resulted in a decreased level of AST and ALT in blood. The extent of AST and ALT decrease was related to the amount of the extract used in a concentration-dependent manner.
3-2) Comparison of collagen content in tissues and tissue staining Liver tissues were stained with sirius red to measure the quantity of collagen in tissues, and the extent of treatment of hepatic fibrosis was examined based on the collagen measurement. The results are provided in Table 2 and FIG. 8. Table 2
Figure imgf000018_0001
Referring to Table 2 and FIG. 8, Positive control group 4 was identified to have 29μg/mg of collagen deposit in tissues. On the other hand, Experimental groups 4 to 7 administered with the extract of Example 1 or 2 resulted in a lower collagen content deposited in tissues, which showed a decrease in manner that is dependent on the concentration of the extract.
FIG. 9 shows the effect of an extract of the present invention on collagen expression in hepatic fibrosis-induced mice. Herein, (A) shows liver cell tissues of a normal mouse (Negative control group 4), (B) Positive control group 4, (C) Experimental group 4, (D) Experimental group 5, (E) Experimental group 6, and (F) Experimental group 7.
Referring to FIG. 9, Experimental groups 4 to 7 treated with an extract of Example 1 or 2 turned out to have decreased hepatic fibrosis compared with Positive control group 4 when hepatic fibrosis in mouse liver tissues was examined by collagen staining.
3-3) Comparison of the amount of alpha-smooth-muscle actin in tissues
The amount of alpha-smooth-muscle actin was determined by Western blotting. The amount of alpha-smooth-muscle actin was compared by signals detected on the light-sensitive film. The results are provided in Table 3 and FIG. 10.
Herein, normal mice not treated with carbon tetrachloride (Negative control group 4), other mice treated with tetrachloride (Positive control group 4), and
Experimental groups 4 to 7 treated with the extract at various concentrations were examined in regard to the amount of alpha-smooth-muscle actin by Western blotting, and the intensity of bands appearing on the film were comparatively estimated.
Table 3
Figure imgf000020_0001
As shown in Table 3 and FIG. 10, the Negative control group had a value of 439, but Positive control group 4 had an increased value of 695 due to treatment with carbon tetrachloride. This increased difference was found to decrease in a manner dependent on the concentration of the extract during treatment. In particular, Experimental group 5 recovered to 454, which is very close to the normal value of 439, when it was treated with 40mg of the extract of Example 1.
3-4) Comparison of the amount of TGF-β mRNA in tissues mRNA was taken from liver tissues, and RT-PCR was performed thereto to examine the amount of TGF-β. The result is provided in Table 4 and FIG. 11. Table 4
Figure imgf000021_0001
As shown in Table 4 and FIG. 11 , the amount of mRNA was shown to decrease in a manner dependent on the concentration of the extract in the group treated with the extract of the present invention.
Experimental Example 4: Toxicity Experiments
Animal testing was carried out to examine the toxicity of the extracts of Examples 1 to 3.
ICR-based mice (Joongang experiment animal Co.) weighing 25±5g and SPF Sprague-Dawley rats (Biogenomics Co.) weighing 235±10g were assigned to 3 groups with 3 per group. Each group was respectively administered by mouth with
20mg/kg, 10mg/kg, and 1mg/kg of the extract of Example 1 and its toxicity was observed for 24 hours.
As a result, no animal died in any of the 9 groups. The 9 groups displayed no externally-identifiable differences from the Control group in terms of weight gain, intake amount of fodder, and the like. This shows that Melandryum firmum extract of the present invention is safe.
According to the present invention, an extract can be made into a variety of formulations for administration. The following formulations are exemplary in the present invention, but it is understood that the present invention is not limited thereto. Formulation 1 : Tablets extract of Example 1 200mg lactose 100mg starch 10Omg magnesium stearate a suitable amount The above components were mixed and formulated into tablets by a conventional method.
Formulation 2: Capsules extract of Example 2 10Omg lactose 50mg starch 50mg talc 2mg magnesium sterarate a suitable amount
The above components were mixed and filled into gelatin capsules to prepare capsules by a conventional method. Formulation 3: Liquid extract of Example 1 500mg extract of Example 2 500mg sugar 20mg isomerized sugar 20mg lemon flavor a suitable amount
The entire amount of a liquid formulation was made up to be 1000ml by adding purified water. The above components were mixed by a conventional method of preparing liquid formulations, and the resulting product was filled in a brown bottle and sterilized. Formulation 4: Injection extract of Example 2 10Omg sodium metabisulfite 3.0mg methylparaben O.δmg propylparaben 0.1mg sterilized distilled water for injection a suitable amount
An injection formulation was prepared by mixing the above components to make up a final volume of 2ml, then filling a 2ml volume ample with the mixed components, and sterilizing the ample by a conventional method. Formulation 5: Health Beverage extract of Example 1 IOOOmg citric acid IOOOmg oligosaccharide 10Og concentrated Japanese apricot juice 2g taurine 1g purified water to make up a final volume of 900ml
A health beverage composition was prepared by mixing the above components by a conventional method, agitating and heating the mixture at 85 °C for one hour, filtering and filling it in a sterilized 2L container, sealing and sterilizing the container, and storing it in a refrigerator. Formulation 6. Health Food extract of Example 2 10OOmg vitamin mixture a suitable amount vitamin A acetate 70μg vitamin E 1.0mg vitamin B1 0.13mg vitamin B2 0.15mg vitamin B6 O.δmg vitamin B12 0.2μg vitamin C 10mg biotin 10μg
Nicotinic acid amide 1.7mg folic acid 50μg calcium pantothenate O.δmg vitamin E 1.0mg mineral mixture a suitable amount ferrous sulfate 1.75mg zinc oxide 0.82mg magnesium carbonate 25.3mg potassium phosphate monobasic 15mg potassium phosphate bibasic 55mg potassium citrate 90mg calcium carbonate 100mg magnesium chloride 24.8mg A granule-type health food was formulated by mixing the above components by a conventional method. Herein, the vitamin and mineral mixture were included in a composition ratio which is known to be preferable for health foods, but the composition ratio can be optionally varied.
Melandryum firmum extract or Silene takesimensis extract of the present invention can inhibit activity of AST and ALT and also, the amount of collagen, alpha-smooth-muscle actin, and TGF-β accumulated due to hepatic fibrosis in liver tissues, and accordingly be effectively used as a drug or health functional food for improving liver function and preventing and treating liver diseases.
While this invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A pharmaceutical composition for treating and preventing liver diseases comprising at least one selected from the group consisting of a Melandryum firmum extract and a Silene takesimensis extract as an active ingredient.
2. The pharmaceutical composition of claim 1 , wherein the extract is contained in a ratio ranging from 0.1 to 80 wt% based on the total weight of the composition.
3. The pharmaceutical composition of claim 1 , wherein the extract is prepared by applying water, an organic solvent, or a mixed solvent thereof to at least one selected from the group consisting of Melandryum firmum and Silene takesimensis .
4. The pharmaceutical composition of claim 1 , wherein the extract contains a fractionated extract selected from the group consisting of a hexane layer, butanol layer, and the first to third water layers which are obtained by the method comprising: separating a crude extract of Melandryum firmum or Silene takesimensis into a first water layer and a hexane layer; separating the first water layer into ethyl acetate and a second water layer; and separating the second water layer into a butanol layer and a third water layer.
5. The pharmaceutical composition of claim 3, wherein the organic solvent is one or more selected from the group consisting of a 1 to 4-carbon alcohol, hexane, chloroform, dichloromethane, ethyl acetate, and combinations thereof.
6. The pharmaceutical composition of claim 1 , wherein the extract is prepared by hydrothermally extracting one or more from the group consisting of Melandryum firmum or Silene takesimensis.
7. The pharmaceutical composition of claim 1, wherein the liver diseases are selected from the group consisting of acute chronic hepatitis, hepatic fibrosis, cirrhosis, and hepatoma.
8. The pharmaceutical composition of claim 1 , wherein the composition is formulated as one selected from the group consisting of a tablet, a pill, a powder, granules, a coated tablet, a sugar-coated tablet, a hard or soft capsule, a solution, a suspension, an injection, and a suppository.
9. A health functional food for improving liver function comprising at least one selected from the group consisting of a Melandryum firmum extract and a Silene takesimensis extract as an active ingredient.
10. The health functional food of claim 9, wherein the extract contains a fractionated extract selected from the group consisting of a hexane layer, butanol layer, and the first to third water layers which are obtained by the method comprising: separating a crude extract of Melandryum firmum or Silene takesimensis into a first water layer and a hexane layer; separating the first water layer into ethyl acetate and a second water layer; and separating the second water layer into a butanol layer and a third water layer.
11. The health functional food of claim 10, wherein the food can be formulated as one selected from the group consisting of a powder, granules, a tablet, a capsule, and a beverage.
PCT/KR2006/001353 2005-04-12 2006-04-12 Composition comprising the extract from melandryum firmum for improvement of liver function and treatment of liver diseases WO2006109996A1 (en)

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