WO2006004358A1

WO2006004358A1 - Dicoumarol-removed extract of artemisia, preparation and pharmaceutical compositions thereof

Info

Publication number: WO2006004358A1
Application number: PCT/KR2005/002095
Authority: WO
Inventors: Jong Oh Kim; Tae Young Oh; Byoung Ok Ahn; Jeong Hoon Kim; Jong Won Kwon; Won Bae Kim; Seul Min Choi; Kyung Ku Kang
Original assignee: Dong-A Pharm. Co., Ltd.
Priority date: 2004-07-03
Filing date: 2005-07-01
Publication date: 2006-01-12
Also published as: KR20060002639A; KR101000951B1

Abstract

The present invention relates to an extract of Artemisia (A. asiatica, A. mongolica, A. princeps, and A. argyi) from which harmful ingredients showing blood coagulation inhibitory action were removed selectively without loss of eupatilin, jaceosidin and artemicapin C contents, a method to prepare said Artemisia extract, a therapeutic or preventive pharmaceutical composition for gastrointestinal disease containing said Artemisia extract as an effective ingredient, a therapeutic or preventive pharmaceutical composition for gastrointestinal disease containing artemicapin C of a constituent of said Artemisia extract as an effective ingredient, and a pharmaceutical composition of Artemisia extract comprising said Artemisia extract, surfactant and co-solvent.

Description

DICOUMAROL-REMOVED EXTRACT OF ARTEMISIA, PREPARATION AND PHARMACEUTICAL COMPOSITIONS

THEREOF

Technical Field

[1] The present invention relates to an extract of Artemisia (A. asiatica, A. mongolica,

A. princeps, and A. argyi) from which harmful ingredients showing blood coagulation inhibitory action were removed selectively without loss of eupatilin, jaceosidin and artemicapin C contents, a method to prepare said Artemisia extract, a therapeutic or preventive pharmaceutical composition for gastrointestinal disease containing said Artemisia extract as an effective ingredient, a therapeutic or preventive pharmaceutical composition for gastrointestinal disease containing artemicapin C of a constituent of said Artemisia extract as an effective ingredient, and a pharmaceutical composition of Artemisia extract comprising said Artemisia extract, surfactant and co-solvent.

[2]

Background Art

[3] Among many of digestive troubles, gastritis and gastric ulcer are most frequently happening diseases and mean the status that GI tract mucosa has been digested by gastric acid to form an ulcer. It is reported that about 10% of total population experience gastritis or gastric ulcer once in their lives. While stomach wall consists of mucous layer, submucous layer, muscle layer and serosa, gastritis means a status when the mucosa has been damaged and gastric ulcer is a case when even the submucous layer and muscle layer have been damaged. While the gastritis and gastric ulcer occur very frequently, their decisive reasons are still unidentified. But, imbalance between offensive factor and defensive factor, that is to say, increase of offensive factor or weakening of defensive factor is suggested as a major reason. Increased secretion of acid and pepsin can be suggested as an increasing factor of offensive factor; loss in structure or form of gastric mucosa, decrease of mucous secretion, decrease of bi¬ carbonate secretion, or lowered prostaglandin production can be suggested weakening factors of defensive factor; and there is a report that contamination by Helicobacter pylori causes gastric ulcer.

[4] One of major reasons for gastric ulcer is among others hyper-secretion of gastric acid, which is a high concentration of hydrochloric acid secreting from gastric mucosa, activates pepsin for breakdown of protein, and does a fatal action on damaged stomach wall. Gastric acid secretes from cell of stomach wall; there are three receptors involving gastric acid secretion in membrane of stomach wall cell. These receptors secrete gastric acid in reaction to stimuli of histamine, gastrin, and acetylcholine. Therapeutic medicines currently used for gastritis and gastric ulcer are mainly promoting agents of defensive factor, for example, antacids that can neutralize already- produced acid with no effect on secretion quantity, gastric secretion inhibitory agent, prostaglandin seretagogue, and gastric wall coating agent. Recently, noted methods to inhibit gastric secretion include histamine-H antagonist inhibiting action of histamine that promotes gastric secretion, anticholinergic that decreases gastric secretion by inhibiting parasympathomimetics, or gastrin antagonist. More particularly, ranitidine, cimetidine, famotidine for histamine-H antagonist and omeprazole for H+/K+ ATPase are remarkably used.

[5] However, in case when these medicines are used, problems would accompany, for example, relapse rate is high, and long-term administration is needed. Actually, six weeks administration can heal ulcer, but it was reported that the disease would relapse after one year or so in most of these patients. Moreover, there are repeated reports that the case of long-term administration of ranitidine, cimetidine, or famotidine for gastric ulcer treatment caused stomach tumor, and the case of long-term administration of omeprazole caused neuroendocrine cell tumor. Therefore, it is sincerely required to develop a therapeutic medicine for gastritis and gastric ulcer that is safe for human body (DN & P 4(5), 1991. 6).

[6] But, gastritis and gastric ulcer can occur by various complicate reasons, and accurate reason of disease has not been identified yet, so there is not an absolute therapeutic method established.

[7] By the way, inflammatory bowel disease is a chronic and obstinate disease of idiopathic high-relapse rate; ulcerative colitis and Crohn s disease are included therein. Ulcerative colitis and Crohn s disease are recognized to be different diseases in pathogenic mechanism, immunological background and pathological findings. But both of the two diseases occur easily in young people of twenties and are accompanied by complicating diseases such as dermatitis, ophthalmia, arthritis, pancreatitis, cholangitis, etc. and nutrition disorder, and above all their differential diagnosis is difficult, therefore they are called altogether inflammatory bowel diseases. Although inflammatory bowel disease is not a commonplace disease, it shows chronic history and requires prolonged treatment; its morbidity is high in young patients whose pro¬ ductivity is highest; and recent number of patients is drastically increasing. From these points, therefore, inflammatory bowel disease is being noticed.

[8] The reason for inflammatory bowel disease is not disclosed yet, but oxygen free radical is known to have an important role in pathology, which can be recognized by chemiluminescence showing that oxygen free radicals of neutrophils at inflamed colic area are produced excessively, and by a study report showing that the inflammation is improved by superoxide dismutase which is a representing antioxidant. Therapeutic method for inflammatory bowel disease should vary according to status and location of the lesions, so there is no absolute therapeutic method established yet. In addition to medicinal therapy applied to a number of patients, operation is also needed. Therefore, current therapeutic goal on inflammatory bowel disease is to alleviate the severity, to inhibit the relapse, and to control the clinical symptoms and complications.

[9] As medicinal therapy, anti-inflammatory agents such as aminosalicylates and corti¬ costeroid, immunosuppressants such as azathiopurine and cyclosporin A, and an¬ tibiotics such as metronidazole are used; the result is however unsatisfactory. Moreover, most medicines accompany with side effects, so they cannot be used for a long period, which constitutes an important problem of theirs. In case of mesalazine that is used most popularly for inflammatory bowel disease, for instance among others, it has high transfer rate to fetus through placenta even at its low concentration, so it is restricted in use for pregnant women. A constitutional hypersensitiveness and a convulsive nephritis were reported as its side effects. The convulsive nephritis occurs at about 1% of patients, and can be recognized only by a periodical test on creatinidine value in blood. Administration of mesalazine for more than 18 months after occurrence of kidney disorder causes a permanent loss in the kidney's function, so a periodical kidney function test through blood test is required. In addition, corticosteroid preparations such as prednisone and prednisolone are useful as a short-term palliative therapy, but a constitutional severe side effect follows in case of their long-term ad¬ ministration, so they are used only at a short- term therapy. In consideration of such problems, there are a sincere requirement to develop a new medicine for inflammatory bowel disease that has a high effectiveness and safety.

[10]

[11] By the way, Artemisia is a perennial herbaceous plant that belongs to Compositae family and has a unique flavor and taste. Its young leaves are edible and have been used for rice-and-mugwort cake. Due to its warming action on uterus according to Korean traditional medicine, it has been known to have effectiveness for uterus bleeding and bleeding during pregnancy and used for blood supplementing, gyne¬ cological disease and anti-diarrheal (Dong-Eui-Bo-Gam).

[12] As a pharmacological action of the Artemisia, it was reported that the extract of

Artemisia prolongs blood coagulation time (J. Pharm. Soc. Korea, 28(2): 69-77, 1984). The flavone extracted from Artemisia reportedly has effect of anticancer activity, platelet aggregation inhibition (Zhongguo Zhongyao Zazhi, 17(6): 353, 1992) and antifungal activity, and does anti body-complementary action to help protection system of host. So, it is effective for allergy or inflammation (Chem. Pharm. Bull, 33(5): 2028-2034, 1985), and does blood pressure lowering action and tranquilizing action. [13] In addition, eupatilin was separated and identified from Artemisia, and its uses for antitumor agent (WO 88/03805), anti-psoriasis (WO 88/03800), antiallergenic agent (JP 84155314) and anti-aphthous ulcer agent (CA 2065496 AA) was applied for patents. On the other hand, Artemisia capillaris that is a same genus plant as the Artemisia reportedly shows therapeutic effect of colitis {Korean J. of Gastroenterology , 28: 224, 1996). The Artemisia capillaris is quite different, on morphological and pharmacological base, from the Artemisia of the present invention, and due to its action of wet febrile jaundice has been used from the past as an herb medicine for therapeutic agent for jaundice (βon-Cho-Hak 366, 1995, Korean Pharmacists Soc, Seoul, Korea).

[14] As pharmacological ingredients in the Artemisia, isocoumarin, coumarin, diter- penlactone, flavonoid, phellandrene, cuprol, cardinene and so on were disclosed ( Plants Med. 60, 437, 1994; J. Nat. Prod. 44(5), 586-7, 1981; Herba Hungarica, 1985, Tom. 24 No. 2-3).

[15] Out of the various ingredients of Artemisia genus, coumarins may cause, when overdosed, bleeding from skin and mucous areas including GI tract (Thrombosis and haemostasis, 66(1), 153-159, 1991); and hepatotoxicity may occur (Toxicology, 88(1-3), 113-125, 1994; International Journal of Experimental Pathology, 77(2), 79-82, 1996).

[16] The said coumarins are benzopyrene derivatives. About 1,300 kinds of derivatives were already reported from natural plants, germs and bacteria; and a variety of coumarins are contained also in the Artemisia. It was reported that in addition to dicoumarol and isocoumarin, coumarin, dracunculin, scopoletin, isofraxidin, fraxidin, daphnoretin, herniarine, scoparone and so on were contained.

[17] It is known that dicoumarol inhibits the synthesis of vitamin-K dependent antico- agulating factors II, VII, IX and X in the liver; prolongs APTT (activated partial thromboplastin time) and PT (prothrombin time) in dog and decreases the number of blood platelets. Since dicoumarol was separated from spoiled sweet clover by Levine in 1985, it was disclosed to be contained in Conioselinum Univittatum at about 0.44 mg/dl. The same researchers also found the existence of dicoumarol at about 0.58, 1.86 and 6.00 mg/dl respectively in alcoholic herb extracts sold in Thailand according to different kinds, and raised a possibility that reason for idiopathic vitamin K deficiency that infants from 2 weeks to 2 months old caught mostly is the breast milk of mothers who took the herb extracts. Based on the test result of hepatotoxicity of coumarin on rats, its use at food spice was banned in America in 1954; and the use of coumarin in England was also banned in 1965.

[18] So far, there were not sufficient studies done on side effects that are probable by blood coagulation inhibitory ingredients contained in Artemisia when the Artemisia is used as a medicine, so there were not studies either on methods to remove these blood coagulation inhibitory ingredients selectively.

[19] In Oriental countries like Korea, China and Japan, the Artemisia extracts have been used in various dosage forms such as hot water extracts, pill form, powder form, or smashed then squeezed juice form. However, there were not systematic analyses studied on effective ingredients or on blood coagulation inhibitory ingredients in Artemisia extract, and their contents at dosage were not so high. With development of extracting methods made nowadays, however, above blood coagulation inhibitory in¬ gredients as well as effective ingredients at dosage are contained as high concentration, so probability of side effect according to long-term administration of medicines containing Artemisia extract is mostly increased.

[20] Moreover, gastritis, gastric ulcer and inflammatory bowel disease, due to their char¬ acteristics, are easy to be accompanied by bleeding, which may remarkably decrease therapeutic effect of Artemisia extract on those diseases. Whereas chronic diseases such as gastritis, gastric ulcer and inflammatory bowel disease need medicine ad¬ ministered for a long period, minimization of above side effects by the Artemisia extract is as important as pharmacological effect of Artemisia extract. In view of de¬ velopment of a medicine with high effectiveness and stability, therefore, a process de¬ velopment of Artemisia extraction method is sincerely required wherein the effective ingredients are still maintained and only the blood coagulation inhibitory ingredients were removed selectively.

[21] Due to its insolubility, the Artemisia extract when dosed into human body is low in solubility and elution rate in gastric juice during absorption progress, so its absorption is delayed and its bioavailability is lowered, too, which constitutes another drawback. Therefore, various formulation methods that can improve elution rate or solubilization of insoluble medicines including the Artemisia extract should be searched.

[22] By the way, there are a number of ingredients known so far that are contained in

Artemisia asiatica as Artemisia genus and its extract, which include scopletin, sesamine, eupafolin, matricarin, β-sitosterol, α-amyrin, capillin, capillarin, monoterpenoids, isoscopoletin, artepillin A, C, aromatic compounds and so on, but pharmacological activities of each ingredient are not identified yet.

[23] The Artemisia extracts contain, in addition to ingredients known so far, many other unknown ingredients, and a portion of these ingredients show pharmacological action beneficial for human body. However, efforts have been concentrated so far on possibility of medical usage of the Artemisia extract as it is which was extracted by use of water or ethanol and other various solvents. The present inventors have tested a lot of experiments to recognize that the Artemisia extracts have therapeutic effect on gastritis and peptic ulcer, or even stronger therapeutic effect than the case when purified eupatilin or jaceosidin is used. Thus, present inventors could have a conclusion that unknown ingredients still unidentified do synergic action with eupatilin or jaceosidin.

[24]

[25] Therefore, present inventors have studied further in order to maximize therapeutic effect and to minimize side effects in use of Artemisia extracts as preventive or therapeutic agents for GI tract disease or inflammatory bowel disease. After such di¬ versified studies and efforts made so far, present inventors recognized that artemicapin C among ingredients of said Artemisia extract has an excellent therapeutic effect in healing of gastritis and peptic ulcer together with eupatilin, jaceosidin; and succeeded in removing selectively dicoumarol which is fatal for GI tract disease or inflammatory bowel disease without loss of eupatilin, jaceosidin and artemicapin C content from Artemisia extract. The present inventors also solubilized insoluble Artemisia extract or improved its elution rate through pharmaceutical composition comprising said Artemisia extract, surfactant and co-solvent; thereby completed the present invention.

[26]

Disclosure of Invention Technical Problem

[27] It is an objective of the present invention to provide an extract of Artemisia

(Artemisia asiatica, A. mongolica, A. princeps, and A. argyi) containing eupatilin, jaceosidin and artemicapin C, from which only harmful ingredient showing blood co¬ agulation inhibitory action was removed selectively, and to provide a production method of said Artemisia extract.

[28] It is another objective of the present invention to provide a therapeutic or preventive pharmaceutical composition for gastrointestinal disease containing artemicapin C as an effective ingredient of said Artemisia extract.

[29] It is another objective of present invention to provide a pharmaceutical composition comprising said Artemisia extract, surfactant and co-solvent in order to increase its bioavailability remarkably by rising in solubility and elution rate of Artemisia extract as insoluble medicine, and to provide an oral administrative preparation including the same.

[30]

Technical Solution

[31] In order to achieve above objectives, the present invention provides an extract of

Artemisia (Artemisia asiatica, A. mongolica, A. princeps, A. argyi) containing eupatilin, jaceosidin and artemicapin C, from which blood coagulation inhibitory ingredient only was removed selectively. Particularly, the present invention provides an extract of Artemisia from which dicoumarol as blood coagulation inhibitory ingredient was removed selectively.

[32] The present invention also provides a method to prepare said extract of Artemisia.

[33] The present invention further provides a therapeutic or preventive pharmaceutical composition containing said extract of Artemisia as an effective ingredient, and an oral administrative preparation including the same.

[34]

[35] The present invention will be described hereinafter in detail.

[36] The extract of Artemisia according to the present invention is obtained out of an extract of Artemisia already made by extraction with alcohol or alcohol- water solution, containing eupatilin, jaceosidin and artemicapin C that are effective for therapy or prevention of GI tract disease or inflammatory bowel disease, from which blood co¬ agulation inhibitory ingredient that is harmful for therapy or prevention of GI tract disease or inflammatory bowel disease is removed. The Artemisia extract obtained by extraction of Artemisia with alcohol or alcohol-water solution has various pharma¬ cological ingredients, wherein representative pharmacological ingredients include eupatilin, jaceosidin and artemicapin C and representative blood coagulation inhibitory ingredients include dicoumarol (Table 1).

[37]

[38] [Table 1]

[39] Ingredient Content in Extract of Artemisia Extracted with Alcohol or Alcohol-

Water Solution

[40]

[41]

[42] Table 2 shows that, in Artemisia extract obtained by extraction method of the present invention, however, contents of eupatilin and jaceosidin do not change, but dicoumarol as harmful ingredient was removed (Table 2).

[43] [Table 2] [44]

[45]

[46] Furthermore, the present invention provides a method to prepare extract of

Artemisia where said harmful ingredient was removed.

[47] More particularly, the present invention provides a method to prepare extract of

Artemisia, comprising:

[48] Step 1 : Artemisia leaves are extracted by alcohol or alcohol- water solution;

[49] Step 2: Alkaline solution is added into the extract obtained from above step 1, and the whole reacts at 80°C for an hour or more;

[50] Step 3: Acid solution is added into the extract obtained from above step 2 to neutralize; and

[51] Step 4: The extract obtained from above step 3 is concentrated by vacuum, and then lyophilized.

[52] In above step 1, dried Artemisia leaves are extracted at room temperature with alcohol or alcohol- water solution by 1 or 2 times of cold precipitation for 10 to 36 hours, ultrasonic extraction for 3 to 8 hours, or 2 to 3 times of reflux extraction for 4 to 10 hours, then dried to obtain an Artemisia extract containing high concentration of eupatilin and jaceosidin. At this time, it is characterized by that said alcohol or alcohol- water solution is selected from the group consisting of 10 to 100% of ethyl alcohol and 10 to 100% of methyl alcohol, and more preferably 70 to 100% of ethyl alcohol.

[53] In the step 2, KOH-water solution is added into the Artemisia extract obtained from above step 1 for adjusting at pH 10 ~ 11. Then, the whole is reacted in a thermostat at 80°C for an hour to prepare an Artemisia extract containing high concentration of eupatilin and jaceosidin, from which dicoumarol as a harmful ingredient was removed.

[54] In the step 3, one or more selected from the group consisting of hydrochloric acid, citric acid and malic acid are added so that the Artemisia extract is neutralized to pH 6.5 to 7.5.

[55] In the last step 4, the Artemisia extract, after all reactions have finished, is vacuum concentrated and lyophilized to prepare solid-type medicine material.

[56] [57] According to the preparing method of Artemisia extract of the present invention, carboxyl group (-COOH) of dicoumarol (formula 4) is decarboxylatively hydrolyzed by above said reaction process, but carboxyl group does not exist in eupatilin (formula 1) and jaceosidin (formula 2), and artemicapin C (formula 3) does not go through in- activation reaction by hydrolysis of carboxyl group at above said reaction conditions. Therefore, the Artemisia extract preparation method of the present invention can se¬ lectively remove dicoumarol as a harmful ingredient without loss of eupatilin, jaceosidin and artemicapin C contents as effective ingredients in Artemisia extract.

[58] [Formula 1] [59]

[60]

[61] [Formula 2]

[62]

[63] [64] [Formula 3] [65]

[66] [67] [Formula 4] [68]

[69] [70] Moreover, the present invention provides a therapeutic or preventive phar¬ maceutical composition for gastrointestinal disease containing an effective ingredient as artemicapin C which is a constituent of said Artemisia extract.

[71] Artemicapin C of the present invention can be administered in general phar¬ maceutical composition forms such as tablet, granule, capsule, powder, syrup, ointment, suppository, and subcutaneous, intramuscular, intravenous injections or in- travenous drips and so on, and at one to several times by 10 to 1000 mg a day.

[72] Therapeutic effect of artemicapin C of the present invention on gastritis and peptic ulcer was tested by submersion-confinement stress gastric ulcer model, hydrochloric acid-ethanol (HCl-EtOH) gastritis model and indomethacin gastric ulcer model. The result showed an inhibitory effect on gastric ulcer lesions together with promotion of prostaglandin biosynthesis, thereby an excellent therapeutic effect for gastritis and peptic ulcer.

[73]

[74] Furthermore, the present invention provides a therapeutic and preventive phar¬ maceutical composition for gastrointestinal diseases containing said Artemisia extract as an effective ingredient. The pharmaceutical composition containing Artemisia extract according to the present invention as effective ingredient is suitable as a preventive or therapeutic agent for GI tract disease or inflammatory bowel disease. Preferably, above GI tract disease is gastritis or gastric ulcer. Preferably, above in¬ flammatory bowel disease is ulcerative colitis or Crohn's disease.

[75] In order to study effect of Artemisia extract of the present invention from which harmful ingredient was removed for GI tract disease, its protective effect on acetate-in duced gastric lesions was considered. According to the result, Artemisia extract of the present invention from which dicoumarol was removed showed a significant protective effect on gastric lesions in comparison to control group.

[76] In order to study further the effect of Artemisia extract of the present invention from which harmful ingredient was removed for inflammatory bowel disease, its protective effect on inflammatory bowel disease induced by trinitrobenzene sulfonic acid (TNBS) was considered. According to the result, Artemisia extract of the present invention from which dicoumarol was removed showed a significant protective effect on inflammatory bowel disease in comparison to control group.

[77] The Artemisia extract of the present invention from which dicoumarol was removed particularly showed a protective effect on GI tract disease or inflammatory bowel disease similar to the case of Artemisia extract from which dicoumarol was not removed; however, the dicoumarol-removed extract of Artemisia according to the present invention showed far lowered occurrence probability of side effect through prolonged blood coagulation time at long-term administration in comparison to Artemisia extract from which dicoumarol was not removed.

[78]

[79] Artemisia extract of the present invention has an excellent therapeutic effect for gastrointestinal disease, gastritis and gastric ulcer, and can be administered in general pharmaceutical composition forms such as tablet, granule, capsule, powder, syrup, ointment, suppository, and subcutaneous, intramuscular, intravenous injections or in- travenous drips and so on, and at one to several times by 10 to 1000 mg a day.

[80]

[81] The pharmaceutical composition containing Artemisia extract according to the present invention as effective ingredient may further consist of surfactant and co- solvent.

[82] When the Artemisia extract as an insoluble medicine is exposed in water solution and human body fluids, said surfactant helps forming of automatic micelle drug transfer system by which micelles form automatically, thereby maximizes the solubility and elution rate of above said insoluble medicine, and has a role in keeping physical and chemical stability. In the present invention, minimum quantity of said surfactant is used for easy administration.

[83] As for said surfactants, various kinds of surfactants may be used including anionic, cationic, nonionic, and bi-functional surfactant as far as they are pharmaceutically permitted. More particularly, one or more selected from group consisting of poly- oxyethylene sorbitan fatty acid ester (tween), sorbitan fatty acid ester (span), poly- oxy ethylene fatty acid ester (Myrj), poly oxy ethylene polyoxypropylene block copolymer (poloxamer), polyoxyethylene polyoxypropylene copolymer (pluronic), reactant of natural or hydrogenated vegetable oil and ethylene glycol (cremophor, hereinafter called as cremophor), dioctylsulfosuccinic acid sodium salt, lauryl sulfonic acid sodium salt, phospholipid, propylene glycol mono- or di- fatty acid ester (Miglyol 840), trans-esterification reactant of natural vegetable oil triglyceride and polyalkylene polyol (labrafil M), mono-, di- or mono/di glyceride (imbitol), sterol and derivatives thereof may be used. Preferably, said surfactants selected from the group consisting of polyoxyethylene polyoxypropylene block copolymer, reactant of natural or hyd rogenated vegetable oil and ethylene glycol, and polyoxyethylene sorbitan fatty acid ester are used.

[84] Above co-solvent selected from the group consisting of propylene carbonate, propylene glycol, ethanol, diethyleneglycol monoethyl ether (transcutol), glycofurol, polyethylene glycol, dimethyl isosorbide and N-methyl pyrrolidone may be used. Preferably, co-solvent selected from the group consisting of diethyleneglycol monoethyl ether, propylene glycol, and polyethylene glycol is used. The composition of the present invention is prepared by mixing of Artemisia extract, surfactant and co- solvent by weight ratio of 1 : 0.1-50 : 0.1-50, preferably 1 : 0.1-20 : 0.1-20.

[85] Above said composition may further comprises commonly available pharma¬ cological materials within a range where no bad influence is expected for medicine ef¬ fectiveness, which may increase solubility of Artemisia extract and absorption in GI tract, and may disperse and emulsify with water on oral administration to increase elution and to improve bioavailability. These materials include most widely used additives like fatty acids and fatty acid alcohols, saccharides such as white sugar, malt ion taffy, purified white sugar, gelatin, sugar and starch syrup, lubricating agents like talc, magnesium stearate, excipients such as micro crystalline cellulose, phosphorous hydro calcium, starch, mannitol, antioxidants to prevent oxidation of medicine, flavoring agents, antiseptics, fragrance agents, sweetening agents, pigments, pH controlling agents and viscosity controlling agents, which may preferably be added into the Artemisia extract at generally acceptable quantity.

[86] The fatty acids and fatty acid alcohols that may be used in the composition of the present invention may include citric acid, oleic acid, stearyl alcohol, myristic acid, linoleic acid, lauric acid, capric acid, caprylic acid or caproic acid, without being limited by these. The antioxidants that may be used in the composition of the present invention may include butylated hydroxytoluene, sodium bisulfite, α-tocopherol, vitamin C (ascorbic acid), β-carotin, tocopherol acetate, fumaric acid, nalic acid, butylated hydroxyanisole, propyl galate, or sodium ascorbate, but are not restricted therein.

[87] The flavoring agents that may be used in the composition of the present invention may include mixed fruit flavor, apple flavor, strawberry flavor, cherry flavor, mint flavor, vanilla flavor, yogurt flavor, or drink flavor, but are not restricted therein.

[88] The antiseptics that may be used in the composition of the present invention may include benzoic acid, sodium benzoate, ethyl parabene, methyl parabene, or propyl parabene, but are not restricted therein.

[89] The fragrance agents that may be used in the composition of the present invention may include menthol, mint oil, orange oil, clove oil, cinnamon oil, strawberry essence, and other commonly used fruit fragrance or plant essences, but are not restricted therein.

[90] The sweetening agents that may be used in the composition of the present invention may include pure white sugar, glucose, fructose, aspartame, stebiocide, sorbitol, mannitol, oligo saccharide, starch syrup, malt ion taffy, but are not restricted therein.

[91] The pigments that may be used in the composition of the present invention may include Green No. 3, Red No. 2, Red No. 3, Blue No. 1, Blue No. 2, Yellow No. 4, Yellow No. 5, water soluble mannitol, caramel, titanium oxide or ferric oxide, but are not restricted therein.

[92] The pH controlling agents that may be used in the composition of the present invention may include sodium carbonate, sodium hydroxide, potassium hydroxide, tri- ethanolamine or monoethanolamine, but are not restricted therein.

[93] The viscosity controlling agents that may be used in the composition of the present invention may include hydroxypropylcellulose (HPC), hydroxypropylmethyl cellulose (HPMC), hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, acacia, bentonite, alginic acid, propylene glycol alginate, polyvinyl pyrolidon, polyvinyl alcohol, carbopol, polycarbopil, tragacanth, or xanthan gum, but are not restricted therein.

[94]

[95] The present invention provides an oral administrative preparation including phar¬ maceutical composition containing said Artemisia extract.

[96] Above oral administrative preparation may be tablet, pill, powder, hard capsule, gelatin banded hard capsule or soft capsule, caramel-type or jelly-type chewing tablet and other oral administrative liquid that contains water solution. Preferably, a soft capsule type containing said composition is provided. As for said composition according to the present invention, Artemisia extract, surfactants and co-solvents are mixed, heated at 80°C to be solved, and then can be formulated by a common method to get an oral administrative preparation.

[97]

Advantageous Effects

[98] As was described in detail in this specification, the Artemisia extract of the present invention, that is to say, Artemisia extract from which harmful ingredient was se¬ lectively removed without loss of eupatilin, jaceosidin, and artemicapin C contents shows an excellent preventive or therapeutic effect for gastrointestinal disease or in¬ flammatory bowel disease. Specifically, dicoumarol-removed extract of Artemisia according to the present invention shows same or higher effect for GI tract disease or inflammatory bowel disease when compared with existing Artemisia extracts, and a further safer medicine where occurrence possibility of side effect according to long- term administration was cut off, because of selective removal of harmful ingredients. Artemicapin C of the present invention described as above promotes biosynthesis of prostaglandin having cell protecting effect together with inhibitory effect on gastric ulcer lesions, so shows an excellent therapeutic effect for gastritis and peptic ulcer. In addition, a pharmaceutical composition of Artemisia extract according to the present invention and an oral administrative preparation including the same can increase solubility and elution rate of Artemisia extract as an insoluble drug, so remarkably improve bioavailability.

[99]

Brief Description of the Drawings

[100] The drawings appended hereto constitute a part of this specification, illustrate examples of the invention, and together with the description serve to explain the principles of the invention.

[101] In the drawings : [102] FlG 1 confirms existence of eupatilin, dicoumarol, and artemicapin C by a liquid chromatography of Artemisia that was extracted with alcohol or alcohol- water solution, but did not pass removing process of harmful ingredients;

[103] FlG 2 confirms existence of eupatilin, dicoumarol, and artemicapin C by a liquid chromatography of Artemisia that was extracted with alcohol or alcohol- water solution, and passed removing process of harmful ingredients;

[104] FlG 3 is a graph showing compared elution rate vs. time after stirring of a soft capsule preparation (test preparation) of the present invention containing 60 mg of the Artemisia extract in pH 1.2 buffer solution, and a preparation (comparative preparation) according to Comparative example, as a result of Experimental example 10;

[105] FIG 4 is a graph showing compared elution rate vs. time after stirring of a soft capsule preparation (test preparation) of the present invention containing 60 mg of the Artemisia extract in pH 6.8 buffer solution, and a preparation (comparative preparation) according to Comparative example, as a result of Experimental example 10; and

[106] FlG 5 is a graph showing compared in-blood concentration vs. time after oral ad¬ ministration into rat of a soft capsule preparation (test preparation) of the present invention containing 20 mg of eupatilin, and a preparation (comparative preparation) obtained from Comparative example, as a result of Experimental example 11.

[107]

Best Mode for Carrying Out the Invention

[108] The following examples realize the present invention in detail; but the present invention is not limited within these examples.

[109]

[110] Example 1

[111] lOOg of finely-cut leaves of Artemisia was cold-precipitated two times at room temperature with IL of 70% ethanol for 24 hours; remnant liquid was vacuum- concentrated, and lyophilized to get a solid of 10.8g. In this solid, eupatilin content was 0.449%, jaceosidin content was 0.277%, artemicapin C content was 0.067%, and dicoumarol content was 0.004%.

[112]

[113] Example 2

[114] lOOg of finely-cut leaves of Artemisia was reflux-extracted two times with IL of

70% ethanol for 4 hours; remnant liquid was vacuum-concentrated, and lyophilized to get a solid of 16.2g. In this solid, eupatilin content was 0.412%, jaceosidin content was 0.225%, artemicapin C content was 0.062%, and dicoumarol content was 0.004%. [115]

[116] Example 3

[117] lOOg of finely-cut leaves of Artemisia was ultrasonic-extracted two times at room temperature with IL of 70% ethanol for 4 hours; remnant liquid was vacuum-con¬ centrated, and lyophilized to get a solid of 12.8g. In this solid, eupatilin content was 0.443%, jaceosidin content was 0.252%, artemicapin C content was 0.066%, and dicoumarol content was 0.004%.

[118]

[119] Example 4

[120] lOOg of finely-cut leaves of Artemisia was cold-precipitated two times at room temperature with IL of 90% ethanol for 24 hours; remnant liquid was vacuum- concentrated, and lyophilized to get a solid of 7.2g. In this solid, eupatilin content was 0.734%, jaceosidin content was 0.513%, artemicapin C content was 0.110%, and dicoumarol content was 0.007%.

[121]

[122] Example 5

[123] lOOg of finely-cut leaves of Artemisia was reflux-extracted two times with IL of

90% ethanol for 4 hours; remnant liquid was vacuum-concentrated, and lyophilized to get a solid of 13.4g. In this solid, eupatilin content was 0.823%, jaceosidin content was 0.493%, artemicapin C content was 0.123%, and dicoumarol content was 0.006%.

[124]

[125] Example 6

[126] lOOg of finely-cut leaves of Artemisia was ultrasonic-extracted two times at room temperature with IL of 90% ethanol for 4 hours; remnant liquid was vacuum-con¬ centrated, and lyophilized to get a solid of 9.7g. In this solid, eupatilin content was 0.896%, jaceosidin content was 0.487%, artemicapin C content was 0.134%, and dicoumarol content was 0.007%.

[127]

[128] Example 7

[129] lOOg of finely-cut leaves of Artemisia was cold-precipitated two times at room temperature with IL of 95% ethanol for 24 hours; remnant liquid was vacuum- concentrated, and lyophilized to get a solid of 5.0g. In this solid, eupatilin content was 1.010%, jaceosidin content was 0.640%, artemicapin C content was 0.151%, and dicoumarol content was 0.008%.

[130]

[131] Example 8

[132] lOOg of finely-cut leaves of Artemisia was reflux-extracted two times with IL of

95% ethanol for 4 hours; remnant liquid was vacuum-concentrated, and lyophilized to get a solid of 10.3g. In this solid, eupatilin content was 0.994%, jaceosidin content was 0.565%, artemicapin C content was 0.149%, and dicoumarol content was 0.009%.

[133]

[134] Example 9

[135] lOOg of finely-cut leaves of Artemisia was ultrasonic-extracted two times at room temperature with IL of 95% ethanol for 4 hours; remnant liquid was vacuum-con¬ centrated, and lyophilized to get a solid of 9.7g. In this solid, eupatilin content was 0.987%, jaceosidin content was 0.534%, artemicapin C content was 0.148%, and dicoumarol content was 0.008%.

[136]

[137] Example 10

[138] lOOg of finely-cut leaves of Artemisia was cold-precipitated two times at room temperature with IL of 100% ethanol for 24 hours; remnant liquid was vacuum- concentrated, and lyophilized to get a solid of 5.2g. In this solid, eupatilin content was 1.284%, jaceosidin content was 0.649%, artemicapin C content was 0.192%, and dicoumarol content was 0.009%.

[139] In the Artemisia extract obtained as above, the existence of eupatilin, jaceosidin, and dicoumarol was confirmed by a high-functional liquid chromatography (FlG. 1). As for analysis conditions, Inertsil ODS II column was used with ultraviolet wavelength 330 nm to confirm (flow rate: 1.0 ml/min). As for moving bed, 5mM sodium methane sulfonate (CH SO Na) and 1OmM potassium phosphate (KH PO ) vs. acetonitril were mixed and used at 5 : 5 (retention time 0-15 min) and 6 : 4 (retention time 15-40 min).

[140]

[141] Example 11

[142] lOOg of finely-cut leaves of Artemisia was reflux-extracted two times at room temperature with IL of 100% ethanol for 4 hours; remnant liquid was vacuum- concentrated, and lyophilized to get a solid of 8.9g. In this solid, eupatilin content was 1.129%, jaceosidin content was 0.638%, artemicapin C content was 0.169%, and dicoumarol content was 0.008%.

[143]

[144] Example 12

[145] lOOg of finely-cut leaves of Artemisia was ultrasonic-extracted two times at room temperature with IL of 100% ethanol for 4 hours; remnant liquid was vacuum- concentrated and lyophilized to get a solid of 7.1g. In this solid, eupatilin content was 0.978%, jaceosidin content was 0.541%, artemicapin C content was 0.146%, and dicoumarol content was 0.008%.

[146] [147] Example 13

[148] lOOg of finely-cut leaves of Artemisia was cold-precipitated at room temperature with 800ml of 95% ethanol for 24 hours; and remnant liquid was vacuum-concentrated at 1/150, then lactose 7.Og and corn starch 3.0g were added to combine. The whole was passed through 20 mesh sieve and granulized, then hot-air dried at 80°C to get a solid of 14.8g. In this solid, eupatilin content was 0.429%, jaceosidin content was 0.231%, and artemicapin C content was 0.064%.

[149]

[150] Example 14

[151] 10Og of finely-cut leaves of Artemisia was cold-precipitated at room temperature with 800ml of 95% ethanol for 24 hours; and remnant liquid was vacuum-concentrated at 1/100, then dextrin 0.5g was uniformly dispersed. The whole was spray-dried to get a solid of 13.9g. In this solid, eupatilin content was 0.417%, jaceosidin content was 0.227%, and artemicapin C content was 0.062%.

[152]

[153] Example 15

[154] lOOg of finely-cut leaves of Artemisia was cold-precipitated two times at room temperature with 800ml of 95% ethanol for 24 hours; and remnant liquid was vacuum- concentrated and lyophilized to get a solid of 5.0g. In this solid, eupatilin content was 1.3127%, jaceosidin content was 0.667%, and artemicapin C content was 0.196%.

[155]

[156] Hereinafter in this specification, various methods to prepare dicoumarol-removed extract of Artemisia out of the Artemisia extract obtained from above Examples 1 to 12 will be described in the following Examples in detail.

[157]

[158] Example 16

[159] Dicoumarol-removed extract of Artemisia

[160] Into the Artemisia extract obtained in Example 1, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished extract of Artemisia was vacuum-concentrated and lyophilized to get a solid of 10.3g. In this solid, eupatilin content was 0.441%, jaceosidin content was 0.269%, artemicapin C content was 0.063%, and dicoumarol content was 0%.

[161]

[162] Example 17

[163] Into the Artemisia extract obtained in Example 2, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 15.8g. In this solid, eupatilin content was 0.410%, jaceosidin content was 0.219%, artemicapin C content was 0.057%, and dicoumarol content was 0%.

[164]

[165] Example 18

[166] Into the Artemisia extract obtained in Example 3, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 12.6g. In this solid, eupatilin content was 0.437%, jaceosidin content was 0.246%, artemicapin C content was 0.062%, and dicoumarol content was 0%.

[167]

[168] Example 19

[169] Into the Artemisia extract obtained in Example 4, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 7.1g. In this solid, eupatilin content was 0.729%, jaceosidin content was 0.509%, artemicapin C content was 0.107%, and dicoumarol content was 0%.

[170]

[171] Example 20

[172] Into the Artemisia extract obtained in Example 5, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 12.8g. In this solid, eupatilin content was 0.818%, jaceosidin content was 0.487%, artemicapin C content was 0.118%, and dicoumarol content was 0%.

[173]

[174] Example 21

[175] Into the Artemisia extract obtained in Example 6, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 9.3g. In this solid, eupatilin content was 0.887%, jaceosidin content was 0.473%, artemicapin C content was 0.129%, and dicoumarol content was 0%.

[176]

[177] Example 22

[178] Into the Artemisia extract obtained in Example 7, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 4.8g. In this solid, eupatilin content was 0.998%, jaceosidin content was 0.613%, artemicapin C content was 0.146%, and dicoumarol content was 0%.

[179]

[180] Example 23

[181] Into the Artemisia extract obtained in Example 8, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 10. Ig. In this solid, eupatilin content was 0.983%, jaceosidin content was 0.541%, artemicapin C content was 0.143%, and dicoumarol content was 0%.

[182]

[183] Example 24

[184] Into the Artemisia extract obtained in Example 9, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 9.5g. In this solid, eupatilin content was 0.973%, jaceosidin content was 0.523%, artemicapin C content was 0.145%, and dicoumarol content was 0%.

[185]

[186] Example 25

[187] Into the Artemisia extract obtained in Example 10, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 4.9g. In this solid, eupatilin content was 1.236%, jaceosidin content was 0.621%, artemicapin C content was 0.188%, and dicoumarol content was 0%.

[188] In the dicoumarol-removed extract of Artemisia obtained as above, existence of eupatilin, jaceosidin, and dicoumarol was confirmed by high functional liquid chro¬ matography (FTG X). As for analysis conditions, Inertsil ODS II column was used, ul¬ traviolet wavelength was 330nm, and moving bed of 5mM sodium methane sulfonate and 1OmM potassium phosphate vs. acetonitrile by mixing ratio of 5 : 5 was used.

[189]

[190] Example 26

[191] Into the Artemisia extract obtained in Example 11, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 8.5g. In this solid, eupatilin content was 1.121%, jaceosidin content was 0.629%, artemicapin C content was 0.166%, and dicoumarol content was 0%.

[192]

[193] Example 27

[194] Into the Artemisia extract obtained in Example 12, aqueous solution of potassium hydroxide was added to regulate to pH 10 ~ 11, then the whole was kept in a thermostat at 80°C for an hour to react; one or more of hydrochloric acid, citric acid and malic acid were used to neutralize to pH of 6.5 ~ 7.5. Reaction-finished Artemisia extract was vacuum-concentrated and lyophilized to get a solid of 6.8g. In this solid, eupatilin content was 0.967%, jaceosidin content was 0.537%, artemicapin C content was 0.141%, and dicoumarol content was 0%.

[195]

[196] Example 28

[197] Oral administrative preparation including oral administrative composition of

Artemisia extract

[198] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 40mg and polyoxyethylene polyoxypropylene block copolymer 40mg were mixed together. Into this mixed solution, cremophor RH40 and diethyleneglycol monoethyl ether lOOmg were added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get a preparation.

[199]

[200] Example 29

[201] Oral administrative preparation including oral administrative composition the Artemisia extract [202] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 40mg and polyoxyethylene polyoxypropylene block copolymer 40mg were mixed together. Into this mixed solution, propylene carbonate lOOmg was added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get a preparation.

[203]

[204] Example 30

[205] Oral administrative preparation including oral administrative composition of

Artemisia extract

[206] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 30mg and polyoxyethylene polyoxypropylene block copolymer 40mg were mixed together. Into this mixed solution, diethyleneglycol monoethyl ether lOOmg and polyethylene glycol 400 50mg were added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get a preparation.

[207]

[208] Example 31

[209] Oral administrative preparation including oral administrative composition of

Artemisia extract

[210] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 80mg and polyoxyethylene polyoxypropylene block copolymer 40mg were mixed together. Into this mixed solution, polyethylene glycol 400 80mg and propylene glycol 5mg were added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get a preparation.

[211]

[212] Example 32

[213] Oral administrative preparation including oral administrative composition of

Artemisia extract

[214] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene poly¬ oxypropylene block copolymer 40mg and cremophor RH40 60mg were mixed together. Into this mixed solution, dimethyl isosorbide lOOmg was added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get a preparation.

[215]

[216] Example 33

[217] Oral administrative preparation including oral administrative composition of Artemisia extract

[218] 60mg of the Artemisia extract obtained in Example 22, poly oxy ethylene sorbitan fatty acid ester 80mg and cremophor EL 80mg were mixed together. Into this mixed solution, diethyleneglycol monoethyl ether 95mg was added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get a preparation.

[219]

[220] Example 34

[221] Oral administrative preparation including oral administrative composition of

Artemisia extract

[222] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 40mg, and cremophor RH 4040mg were mixed together. Into this mixed solution, N-methyl pyrrolidone lOOmg was added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. This composition was filled into soft capsule to get formulation.

[223]

[224] Example 35

[225] Oral administrative preparation including oral administrative composition of

Artemisia extract

[226] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 200mg, and polyoxyethylene polyoxypropylene block copolymer 200mg were mixed together. Into this mixed solution, polyethylene glycol 400 150mg and propylene glycol 600mg were added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. Into this composition, white sugar 10,000mg, citric acid 30mg, and mixed fruit flavor 5mg were added; the whole was solved, and then sterilized by application of a proper quantity of purified water. After completion of sterilization, this was filled in a bottle to form a liquid preparation.

[227]

[228] Example 36

[229] Oral administrative preparation including oral administrative composition of

Artemisia extract

[230] 60mg of the Artemisia extract obtained in Example 22, cremophor RH 40 20mg, and polyoxyethylene polyoxypropylene block copolymer lOmg were mixed together. Into this mixed solution, micro crystalline cellulose 90mg, polyvinyl pyrolidon (Colidon CL) 30mg, magnesium stearate 2.5mg, and colloidal silicondioxide (Aerosil 200) 2mg were added; the whole was mixed then formed into tablets.

[231] [232] Example 37

[233] Oral administrative preparation including oral administrative composition of

Artemisia extract

[234] 60mg of the Artemisia extract obtained in Example 22, polyoxyethylene sorbitan fatty acid ester 350mg, and polyoxyethylene polyoxypropylene block copolymer 200mg were mixed together. Into this mixed solution, polyethylene glycol 400 380mg and propylene glycol 20mg were added; then the whole was heated at 80°C to solve until it got transparent; thereby a composition was obtained. Into this composition, malt ion taffy l,000mg, purified white sugar l,000mg, gelatin lOOmg, sugar 500mg, starch syrup lOOmg, strawberry essence 5mg, and citric acid 5mg were added; the whole was formed into caramel-type chewing tablet.

[235]

[236] Comparative Example

[237] Lactose 8kg and cornstarch 4kg were put together in a high-speed mixer and uniformly mixed. This mixture and 3kg of the Artemisia extract obtained by same method as Example 1 were mixed again and combined in a combiner, then sifted through a No. 20 sieve. The same was hot-air dried at 60°C, then sifted through a No. 20 sieve to get granules. These granules were filled in an automatic capsule filler to get granule preparations.

[238]

[239] Hereinafter in this specification, in order to know therapeutic effects of Artemisia extract obtained from above Examples, eupatilin, and artemicapin C for gastritis and peptic ulcer, medicine effects were tested and compared through an submersion- confinement stress gastric ulcer model, HCl-EtOH gastritis model, and indomethacin gastric ulcer model. In addition, mucosa-protecting effects of Artemisia extract, eupatilin, and artemicapin C were investigated to identify their action mechanism.

[240]

[241] Experimental Example 1

[242] Anti-ulcer effect of Artemisia extract, eupatilin, and artemicapin C in submersion-confinement stress gastric ulcer model

[243] In accordance with the method of Takagi, et al. (Jap. J. Pharmacol. 18: 9-18, 1968), rat was fast for 24 hours, then oral-administered with the Artemisia extract of Example 22, eupatilin quantity equivalent to this Artemisia extract, and artemicapin C quantity equivalent to this Artemisia extract respectively. 30 minutes later, it was put in a stress cage, and kept in water so that xiphoid was immersed at water temperature 21+1°C for 8 hours. Then, the animal was killed under etherization; its stomach was taken out, injected into by 13 ml of 1% formalin, and put in 1% formalin solution to fix for an hour. The fixed stomach was incised in line with expansion part and stretched out. The area of gastric lesion was measured by a microscope (XlO), and inhibitory rate of gastric lesion was calculated according to the following Equation 1. 50% effective dose (ED ) was calculated through regression analysis.

[244]

[245] Equation 1

[246] Lesion inhibitory rate (%)

[247]

_ {gastric lesion area of control group (mm²) - gastric lesion area of medicine-dosed group (mm²)} -χ -, QQ gastric lesion area of control group (mm²)

[248]

[249] Table 3 shows anti-ulcer effects of said Artemisia extract of Example 22, eupatilin quantity equivalent to this Artemisia extract, and artemicapin C quantity equivalent re¬ spectively. According to test results, all the test-material-dosed groups except control group showed anti-ulcer effect on gastric ulcer induced by submersion-confinement stress. While the ulcer area of control group was 30.5+2.7mm ; quantity-dependent anti-ulcer effect was observed in Artemisia extract dosed group in 25mg, 50mg, and 100mg/kg dosed groups.

[250]

[251] [Table 3]

[252] Anti-ulcer effect of Artemisia extract, eupatilin, and artemicapin C in submersion-confinement stress gastric ulcer model

[253]

[254] *: shows significant difference compared with control group (p<0.05) [255] [256] In the group where artemicapin C content corresponding to the same content in Artemisia extract was administered, remarkable anti-ulcer effect was not observed. In case of the group where eupatilin only was administered, anti-ulcer effects could be recognized in all quantity groups, but their strengths were remarkably weaker than the case in test group where Artemisia extract was administered. On the other hand, in test groups where both eupatilin and artemicapin C were administered simultaneously, anti-ulcer effect was observed too; their strengths were stronger than the case where only one of eupatilin or artemicapin C was dosed, but weaker than the case of test group where Artemisia extract was dosed. These test results mean that anti-ulcer effect of Artemisia extract was not dependent on single eupatilin, but caused by synergism of artemicapin C together with other various unknown ingredients.

[257] [258] Experimental Example 2 [259] Effect of Artemisia extract, eupatilin, and artemicapin C in HCl-EtOH induced gastritis model [260] Rat was fast for 24 hours, then oral-administered with the Artemisia extract of

Example 22, eupatilin quantity equivalent to this Artemisia extract content, and artemicapin C quantity equivalent to this Artemisia extract content respectively in accordance with the method of Mizui, et al. (Jap. J. Pharmacol. 33: 939-945, 1983). One hour later, a solution of 60% ethanol dissolved in 15OnM HCl was oral- administered at 1.5 ml per each. One hour later, the animal was killed under etherization; its stomach was taken out, injected into by 13 ml of 1% formalin, and put in 1% formalin solution to fix for an hour. The fixed stomach was incised in line with expansion part and stretched out. The area of gastric lesion was measured by a microscope (XlO), and inhibitory rate of gastric lesion and 50% effective dose (mg/kg) were calculated according to the same method as submersion-confinement stress model.

[261] Table 4 shows effects of Artemisia extract obtained in the Example 22, eupatilin quantity equivalent to this Artemisia extract content, and artemicapin C quantity equivalent to this Artemisia extract content respectively in HCl-EtOH induced gastritis model. While the ulcer area of control group was 53.2+8.2mm , dosage-dependent anti-gastritis effect was observed in Artemisia extract dosed group for 25mg, 50mg, 100mg/kg dosed groups, and in case of 100 mg/kg administered group, inhibitory rate of gastric lesion was 82.5%.

[262] In the group where single eupatilin or single artemicapin C was administered, a weak gastric lesion inhibitory effect was observed, but the strength was relatively weaker in comparison to Artemisia administered group. In the group where both of eupatilin and artemicapin C were administered simultaneously, anti-gastritis effect was stronger than that of respective single-administered group: lesion inhibitory rate was 60 to 70%; however, it was weaker than that of Artemisia administered group. Therefore, it was concluded that anti-ulcer effect of Artemisia extract was caused by synergism of various ingredients contained in the extract.

[263]

[264] [Table 4]

[265] Effect of Artemisia extract, eupatilin, and artemicapin C in HCl-EtOH induced gastritis model

[266]

[267] *: shows significant difference compared with control group (p<0.05) [268] [269] Experimental Example 3 [270] Effect of Artemisia extract, eupatilin, and artemicapin C in indomethacin gastric ulcer model

[271] In accordance with the method of Urushidani, et al. {Jap. J. Pharmacol. 27: 316-319, 1977), rat was fast for 24 hours, then oral-administered with the Artemisia extract of Example 22, eupatilin quantity equivalent to this Artemisia extract content, and artemicapin C quantity equivalent to this Artemisia extract content respectively. One hour later, indomethacin 40 mg/kg was oral-administered. Six hours later, the animal was killed under etherization; its stomach was taken out, injected into by 13 ml of 1% formalin, and put in 1% formalin solution to fix for an hour. The fixed stomach was incised in line with expansion part and stretched out. The of gastric lesion area (mm²) was measured by a microscope (XlO), and inhibitory rate of gastric lesion and 50% effective dose (mg/kg) were calculated according to the same method as submersion-confinement stress model.

[272] [273] [Table 5]

[274] Effect of Artemisia extract, eupatilin, and artemicapin C in indomethacin gastric ulcer model

[275]

[276] *: shows significant difference compared with control group (p<0.05) [277] [278] As was obvious from above Table 5, gastric ulcer induced by indomethacin was decreased dosage-dependently when Artemisia extract was administered. In the group where single eupatilin or single artemicapin C was administered, their respective anti¬ ulcer effect was weaker than that of Artemisia extract; maximum lesion inhibitory rate in eupatilin-administered group was about 54%. In the case where both of eupatilin and artemicapin C having their respective contents equivalent to the Artemisia extract content were administered simultaneously, observed lesion inhibitory effect was about 50 to 60%; however, it was relatively weaker than that of Artemisia extract itself.

[279] [280] Experimental Example 4 [281] Gastric mucosa protective effect of artemicapin C in alcohol-induced gastric mucosa lesion model [282] Rat was fast for 24 hours, then oral-administered with high-purity alcohol of 99% or more at the quantity of 4g/kg to induce lesion on gastric mucosa. At an hour or earlier prior to inducement of gastric mucosa lesion, the Artemisia extract of Example 22, eupatilin, and artemicapin C were oral-administered with the dosage as shown in Table 6 respectively. Autopsy was done at 30 minutes or later after inducement of gastric mucosa lesion; prostaglandin E content in gastric mucosa was measured by EIA method (Enzyme Immunoassay Kit, Assay Designs, Inc.).

[283] As the results were shown in Table 6, decrease of prostaglandin due to gastric mucosa damage induced by alcohol was recovered statistical-significantly by single administration of Artemisia extract, eupatilin, and artemicapin C. Specifically, combined-administered group of eupatilin, and artemicapin C showed about 90% or more of normal animal group. Such results mean that the artemicapin contained in Artemisia extract has gastric mucosa protective effect.

[284]

[285] [Table 6]

[286] Prostaglandin (PGE ) content change in gastric mucosa

[287]

[288] *: shows significant difference in comparison to control group (p<0.05) [289] [290] The results described as above mean that anti-ulcer effect by Artemisia extract is not a sole action of eupatilin only, but caused by a complicated action of eupatilin and artemicapin C, or other unknown ingredients; specifically, eupatilin and artemicapin C can promote prostaglandin E production in gastric mucosa, so show gastric mucosa protective effect.

[291] [292] Hereinafter, the effects of pharmaceutical composition containing dicoumarol- removed extract of Artemisia obtained as in Example 22 on therapy quality of gastritis and peptic ulcer in artificially induced gastritis and peptic ulcer animal model will be described in detail by the following experimental examples. Experimental examples of the present invention were given here only to illustrate possible advantageous effects of the present invention; and overall contents of the present invention were not limited therein. [293]

[294] Experimental Example 5

[295] Protective action on acetate-induced gastric lesion

[296] The effect of pharmaceutical composition containing dicoumarol-removed extract of Artemisia obtained as in Example 22 on acetate-induced gastric lesion which is most widely used as an experimental sub-chronic ulcer model was studied. Test groups included normal group where acetic acid was not administered; control group where only acetic acid was administered; a group where Artemisia extract of Example 7 was administered; a group where Artemisia extract of Example 22, from which dicoumarol was removed was administered; and a group where ranitidine widely used in gastric ulcer treatment was administered.

[297] In accordance with standard test method (Gastroenterology 95: 636, 1988), 6- week old rat was etherized, laparotomy-operated, contacted to acetic acid in its GI serosa, and then sewn to prepare an acetate-induced gastric ulcer model. One day after gastric ulcer inducement and on, each medicine was administered once a day for 4 weeks. At 2 weeks and at 4 weeks after beginning of administration, a portion of each group was done autopsy; its stomach was taken out; and ulcer-healing grade was assessed ( Gas- troenterologica japonica 17(6): 530, 1982, 7. Clin Gastroenterol 13(suppl. 1): S42, 1991). Furthermore, Kruskal-Wallis dispersion was analyzed, and then multiple- comparison was done through Dunn test. Tables 7 and 8 illustrate the test results, wherein dicoumarol-removed extract of Artemisia obtained in Example 22 of the present invention showed same ulcer healing effect as the Artemisia extract obtained in Example 7.

[298]

[299] [Table 7]

[300] Ulcer healing effect at 2-week administration

[301]

[302] [303] [Table 8] [304] Ulcer healing effect at 4-week administration [305]

[306]

[307] As can be known in above Table 7 for 2 week administration, in Pas-Alsian Blue

(PAS-AB) dyeing and sub-ulcer fibrosis compared with control group, both groups where Artemisia extract obtained in Example 7 was administered and dicoumarol- removed extract of Artemisia obtained in Example 22 was administered respectively showed a significant recovery in terms of mucosa compactness and fibrosis degree; and in Pas-Alsian Blue dyeing that represents maturity of reproduced mucosa, they did not have significance, but had stronger Pas dyeing property than control group, which meant high maturity.

[308] As can be known furthermore in above Table 8, in tissue finding assessed after 4 week administration, fibrosis degree (cell compactness) of healed sub-ulcer did not show differences according to each group; but in vascularization degree of sub-ulcer and normalization of reproduced mucosa, both groups where Artemisia extract obtained in Example 7 was administered and dicoumarol-removed extract of Artemisia obtained in Example 22 was administered respectively showed a significant recovery when compared with control group.

[309] The results described as above helped to confirm therapeutic effect of the Artemisia extract of the present invention where harmful ingredient was selectively removed, which means it is possible to introduce safe and useful therapy on peptic ulcer that reduced side effect of existing Artemisia extract.

[310]

[311] Now, various uses of Artemisia extract as preventive or therapeutic medicine for in¬ flammatory bowel disease will be described as below in detail according to Ex¬ perimental Examples.

[312] In order to test therapeutic effect of dicoumarol-removed extract of Artemisia obtained in Example 22 for inflammatory bowel disease, medicine effect of Artemisia extract was investigated in inflammatory bowel disease model induced by trini- trobenzene sulfonic acid (TNBS) according to following methods.

[313]

[314] Experimental Example 6

[315] Medicine effect on inflammatory bowel disease

[316] At first, test groups comprised a normal group with no administration of TNBS, a control group administered with TNBS only, a group administered with the Artemisia extract of Example 7, a group administered with the dicoumarol-removed extract of Artemisia of Example 22, and a mesalazine-administered group where mesalazine is used widely for treatment of inflammatory bowel disease. In accordance with the method of Shibata, et al. (Dig. Endosc. 5: 13, 1993), 7-week old SPF SD he-rat was fast for a day, and then etherized. A rubber canula (inner diameter of 2mm) was inserted up to 8cm from the anus, and TNBS (TCl, lot No. HEOl) dissolved in 30% ethanol was administered with a quantity of 20mg/ml per each. Then, it was washed a time with 2ml of physiological saline. Each medicine was oral-administered every day from first day to 14th day after inducement. On 7th day and 15th day of the test, all animals were done autopsy; lesion formation at large intestine mucosa was assessed according to Wallace standard (Table 9, Can. J. Physiol. Pharmacol. 66: 422, 1988). Number of animals per each group was 8 or more. Lesion score of an animal was transformed to rank by Sigma-Stat method that is a common statistic-processing program, and then analyzed by Kruscal-Wallis non-parametric ANOVA. Then, multiple-comparison was done by Student-Newman-Keuls method. In case where p< 0.05, inter-group significance was recognized.

[317] [318] [Table 9] [319] Lesion index of large intestine mucosa [320]

[321]

[322] Table 10 shows medicine effect of dicoumarol-removed extract of Artemisia of

Example 22 according to test method of afore said Shibata, et al. (Dig. Endosc. 5: 13,

1993). [323]

[324] [Table 10]

[325] Medicine effect in TNBS-induced inflammatory bowel disease model

[326]

[327] *: shows significant difference in comparison to control group (p<0.05) [328] [329] According to the results, all test groups showed on 7th day a significant inhibitory effect at large intestine mucosa lesion, including mesalazine group at 25mg/kg only, which is equivalent to clinical dosage quantity of mesalazine that is most widely used for inflammatory bowel disease. On the other hand, at 15th test day when chronic in¬ flammatory finding appears, all test groups of Artemisia extract, dicoumarol-removed extract of Artemisia, and mesalazine showed excellent therapeutic effect in comparison to control group; but significant differences could be recognized only in the 25mg/kg dosage groups of Artemisia extract, and dicoumarol-removed extract of Artemisia.

[330] [331] Experimental Example 7 [332] Oxygen free radical elimination [333] Even though cause of inflammatory bowel disease was not completely understood yet, it was disclosed recently that oxidative stress by oxygen free radical has an important role in pathologic physiology as was mentioned herein before. So, present inventors studied chemiluminescence of neutrophils as an indication of oxygen free radical elimination and effect on peroxy-lipid content in large intestine mucosa by use of dicoumarol-removed extract of Artemisia obtained in Example 22.

[334] [335] 1) Effect on chemiluminescence of neutrophils [336] Human neutrophils were separated according to method of Grisham, et al. (J. Immunol. Methods 82: 315, 1985); and lxlθ⁶cell/ml suspension was prepared. The chemiluminescence that appears as activation of human neutrophils by Helicobacter pylori (H. pylori) was measured according to method of Suematsu, et al. (J. Clin. Lab. Immunol. 24: 125, 1987). Prepared neutrophils solution IOOL KRP buffer 0.89mL were mixed, and luminol 5L was added as chemiluminescence indication material. Then, this mixture was put in a chemiluminometer (Berthold Model LB 9505) at 37°C; 5 minutes later, H. pylori liquid (1.2xl0⁸cfu/mL) 2OL was added therein then mixed well, and the chemiluminescence was measured for 30 minutes. As to control group, a 2:1 mixture of lactose vs. starch as adjunctive of Artemisia extract was added into the neutrophils mixed solution to get 10D/mL. The peak total area was considered as chemiluminescence strength. The chemiluminescence inhibitory effect of dicoumarol- removed extract of Artemisia obtained in Example 22 was calculated as percentage to peak area of control group. Table 11 shows the results.

[337] [338] [Table 11] [339] Inhibition of Artemisia extract on chemiluminescence of human neutrophils by Helicobacter pylon

[340]

[341] *: shows significant difference in comparison to control group (p<0.05, Fisher's Exact test)

[342] [343] According to the results, dicoumarol-removed extract of Artemisia obtained in Example 22 inhibited chemiluminescence of neutrophils dosage-dependently as in the case of Artemisia extract obtained in Example 7. At concentration of 20D/mL or more, significance was recognized, and at a higher concentration of lOOD/mL, inhibitory rate was 81.6% and 83.2% respectively. This means that both Artemisia extract and dicoumarol-removed extract of Artemisia significantly inhibit production of oxygen free radical and activation of neutrophils due to external stimuli like Η. pylori, etc.

[344] [345] 2) Peroxy-lipid in colon mucosa [346] Peroxy-lipid content was measured for damaged colon mucosa reserved at -70°C in separation during aforesaid effect test on TNBS-induced inflammatory bowel disease. In accordance with the method of Ohkawa, et al. (Anal. Biochem. 95: 351, 1979), into a certain amount of polished homogeneous solution of the colon mucosa tissue, 8.1% sodium dodesyl sulfate, 20% acetate buffer (pH 3.5), and 0.8% thiobarbituric acid were added. The whole was heated at 95°C for an hour to react, and cooled to room temperature. Then, produced red-color thiobarbituric acid reactant was passed with n- butanol vs. pyridine (15:1) mixed solution, and absorbance change at wavelength 532nm was measured to quantify. Peroxy-lipid content was represented as n mole quantity of MDA (malonic dialdehyde) per lmg protein.

[347] Test results were shown in Table 12; detailed discussions were as follows. In the control group of 7th test day where only TNBS was applied, increase of peroxy-lipid production was observed significantly in comparison to no-treatment normal group; but in all quantity groups administered with 5 and 25mg/kg of Artemisia extract and dicoumarol-removed extract of Artemisia after TNBS treatment, any significant difference was not observed in comparison to no-treatment normal group. In addition, in all quantity groups where Artemisia extract and dicoumarol-removed extract of Artemisia were administered, significant inhibitory effects on peroxy-lipid production were observed more or lees in comparison to control group although.

[348] [349] [Table 12] [350] P Prroodduuction of peroxy-lipid at colon mucosa damage lesion induced by TNBS (7 test day)

[351]

[352] *: shows significant difference in comparison to normal group (no-treatment) (p<

0.01, Dunnett's t-test) [353] +: shows significant difference in comparison to control group (p<0.01, Dunnett's t- test) [354]

[355] Above test results mean that the dicoumarol-removed extract of Artemisia shows a medicine effect on inflammatory bowel disease because it can isolate oxygen free radical, activate neutrophils against oxygen stress, which is one of major pathologic mechanisms on inflammatory bowel disease, and thereby prevent lipid per oxidation efficiently. The dicoumarol-removed extract of Artemisia according to the present invention showed same effect on inflammatory disease as currently existing Artemisia extract.

[356]

[357] Experimental Example 8

[358] Acute toxicity test on oral-administered rat

[359] 6-week old specific pathogen-free (SPF) SD origin he-rats and she-rats were tested for acute toxicity on Artemisia extract obtained in Example 7 and Artemisia extract obtained in Example 22. Experiment animals were supplied from Biogenomics Company; after gross appearance examination done at acquisition of animals, seven days or more passed by during acclimation breeding, then only healthy animals were selected and used for the experiment. Whole experimental procedure was done within SPF Clean Animal Breeding Zone at R&D Center, Dong-A Pharmaceutical Co., Ltd., where experimental conditions were set at temperature 23+2°C, relative humidity 60+10%, ventilation 15-20 times/hr, illumination 150-600 Lux, illuminating time for 12 hours (07:00 on - 19:00 off) per day. Radiation exposed (2 rad) solid feed for ex¬ perimental animal was fed freely; sterilized tap water was fed freely as drinking water. Feed composition sheet and general germ test were given by supplier; and any factors to cause effects on test were not observed in feed or drinking water.

[360] Experimental groups were designed to comprise quantity groups of lg/kg, 2g/kg, and 5g/kg of extract of Artemisia to each 5 animals by each sex per group; experiment material was suspended in 5% hydroxypropylmethyl cellulose (HPMC) solution, then oral-administered one time. After administration of test material, whether the animal was dead or not, or clinical symptoms were observed once a day for 14 days. On first, third, seventh, and, 14th day after administration of test material, animals were weighed. After completion of such experiment, blood was collected from abdominal main vein under etherization; hematological test (automatic blood cell tester, Mascot 850) and blood coagulation activity test (automatic blood coagulation tester) were carried out. On the other hand, abdominal and chest organs were observed by gross ex¬ amination. According to test results, in all animals administered with Artemisia extract obtained in Example 7 and Artemisia extract obtained in Example 22 during experiment period, any especially remarkable clinical symptoms or weight changes were not observed; no animal was dead. Tables 13 - 15 show the results. [361]

[362] [Table 13]

[363] Death rate of rats when Artemisia extract according to the present invention was oral-administered

[364]

[365]

[366] [Table 14]

[367] Change in clinical symptoms of rats when Artemisia extract according to the present invention was oral-administered

[368]

[369]

[370] [Table 15]

[371] Weight change of rats when Artemisia extract according to the present invention was oral-administered

[372]

[373] [374] Furthermore, in hematological test and gross examinational autopsy findings, any toxicologically special change was not observed.

[375] [376] [377] [Table 16] [378] Hematological change of rats when Artemisia extract according to the present invention was oral-administered

[379]

[380] [381] However, as presented in Table 17, Artemisia extract obtained in Example 7 prolonged prothrombin time (PT) as indication of blood coagulation activity, and activated partial thromboplastin time (APTT) in comparison to control group. Even though such change did not show a statistically significant difference when compared to control group, it means probability of adverse action due to prolonged blood co¬ agulation time when dosed for a long time. On the other hand, the dicoumarol-removed extract of Artemisia obtained in Example 22 showed PT and APTT levels similar to control group. [382]

[383] [Table 17]

[384] Effect on blood coagulation activity

[385]

[386] [387] Above results mean: synthesis of coagulation factors as vitamin K dependent anti¬ coagulation element was inhibited by dicoumarol contained in extract of Artemisia obtained in Example 7, and in case of extract of Artemisia according to Example 22, prolonged effect of blood coagulation has disappeared due to removal of dicoumarol. In the results as above, the dicoumarol-removed extract of Artemisia obtained in Example 22 did not show toxicity change including change in blood coagulation activity up to 5g/kg, and were recognized as a safe material of which 50% lethal dose (LD ) on oral administration is 5g/kg or more.

50

[388] [389] Hereinafter, Experimental examples were given to consider solubility, elution rate and in-blood concentration in oral administrative preparation of Artemisia extract according to the present invention by automatic micelle drug transfer system.

[390] [391] Experimental example 9 [392] Compared solubility experiment [393] Solubility of the oral administrative preparation containing oral administrative composition of Artemisia extract according to the present invention by automatic micelle drug transfer system was measured in comparison to that of oral administrative preparation containing oral administrative composition of Artemisia extract without such system.

[394] Specifically, soft capsule including oral administrative composition of Artemisia extract obtained from Example 22 and granule including oral administrative composition of Artemisia extract obtained from Comparative example were measured in terms of drug solubility under pH condition in GI tract (pH 1.2) and under pH condition in small intestine and large intestine (pH 6.8).

[395] Procedure of experiment was as follows: [396] Each quantity equivalent to Artemisia extract 60mg in oral administrative preparations obtained from Example 22 and Comparative example was taken and added into buffer solution of pH 1.2 (Korean Pharmacopoeia, No. 8 Rev.: into sodium chloride 2.Og, hydrochloric acid 7.0ml and water were added and mixed together to make total volume of IL.) and into buffer solution of pH 6.8 (Korean Pharmacopoeia, No. 8 Rev.: into 0.2mol/L potassium dihydrogen phosphate solution 250ml, 0.2mol/L sodium hydroxide solution 118ml and water were added and mixed together to make total volume of IL.) 150ml respectively. This solution was stirred for an hour at 37°C, and then solubility was measured. The solubility was measured by use of HPLC after centrifugation. More particularly, Hichrom RPB (250*4.6mm, particle size: 5D) was used as column, analysis wavelength was 350nm, and 0.5% acetate buffer with ace- tonitrile mixed by 58: 42 volume ratio was used as moving bed. Flow rate of moving bed was lml/min, and injected sample was 2OD.

[397] Results were shown in Table 18. [398] [399] [Table 18] [400] Compared solubility at pH 1.2 and pH 6.8 [401]

[402] [403] As shown in above Table 18, solubility of oral administrative preparation obtained from Example 22 of the present invention was 8.3D/ml and 8.6D/ml at pH 1.2 and pH 6.8 respectively, while solubility of oral administrative preparation obtained from Comparative example was 1.5D/ml and 3.4D/ml respectively. When each solubility was compared respectively, solubility of oral administrative preparation according to the present invention was higher by 5.5 times and 2.5 times respectively at pH 1.2 and pH 6.8. It is concluded, therefore, that oral administrative preparation by automatic micelle drug transfer system according to the present invention has very high solubility under pH condition of small intestine and large intestine in GI tracts when compared to preparations made by usual methods; it dissolves stably even at various pH conditions.

[404]

[405] Experimental example 10

[406] Compared elution rate experiment

[407] Elution rates of oral administrative preparation including oral administrative composition of Artemisia extract by automatic micelle drug transfer system according to the present invention and of oral administrative preparation including oral admin¬ istrative composition of Artemisia extract without such system were measured re¬ spectively and compared one another.

[408] Procedure of experiment was as follows:

[409] Each quantity equivalent to Artemisia extract 60mg in oral administrative preparation including oral administrative composition obtained from Example 22 and Comparative example was taken and added into 500ml buffer solution of pH 1.2 and of pH 6.8 prepared by same method as above Experimental example 9; then the whole was stirred at lOOrpm. At stirring time of 10 min, 20min, 30min, 40min, 50min, and 60min by paddle method, sample was taken and Artemisia extract content extracted from the same was measured. While the content of Artemisia extract contained in oral administrative preparation is counted as 100, elution rate was calculated in terms of % by use of Artemisia extract measured from above sample,

[410] FIG. 3 is a graph showing the elution rate measured at pH 1.2, and FIG. 4 is a graph showing the elution rate measured at pH 6.8

[411] As shown in FIG. 3, oral administrative preparation (test preparation) obtained from

Example 22 of the present invention showed elution rate of 60% or more in first 10 minutes, and stable high elution rate for 20 to 60 minutes. On the other hand, oral ad¬ ministrative preparation (comparative preparation) obtained from Comparative example showed lower elution rate than oral administrative preparation obtained from Example 1 at every stirring time measured. Therefore it is concluded that oral admin¬ istrative preparation according to the present invention has high elution rate at pH 1.2 buffer solution, and oral administrative preparation according to the present invention shows a rapid increase of elution rate for first 10 minutes in stirring time.

[412] With reference to FIG. 4, oral administrative preparation (test preparation) obtained from Example 22 of the present invention showed elution rate of 60% or more in 10 minutes, and stable high elution rate for 20 to 60 minutes. On the other hand, oral ad¬ ministrative preparation obtained from Comparative example (comparative preparation) showed lower elution rate than oral administrative preparation obtained from Example 1 at every stirring time measured. Therefore it is concluded that oral ad¬ ministrative preparation according to the present invention has high elution rate at pH 6.8 buffer solution.

[413] As shown in FIG. 3 and FIG. 4, it is concluded that oral administrative preparation according to the present invention has higher elution rate at pH 1.2, and at pH 6.8 in comparison to oral administrative preparation obtained from Comparative example; and elution rate profiles resemble each other, which means stable high elution rate at various pH conditions.

[414]

[415] Experimental example 11

[416] Compared in-blood concentration experiment

[417] Concentration in blood was measured and compared when oral administrative preparation according to the present invention and oral administrative preparation obtained from Comparative example were administered to rats.

[418] Experimental procedures were as follows:

[419] Experimental-use white he-mice (Sprague-Dawely origin) with weight of 250-31Og bought from Biogenomics were adapted for about 1 to 2 weeks, and then used in the experiment. Prior to beginning of experiment, mouse was fast for 24 hours, then etherized, cannulated at left femoral aorta; a tube connected to a syringe filled with 50 IU/ml heparin was inserted therein. Around two hours later when the mouse awoke from etherization, soft capsule preparation made from Example 22 and preparation made from Comparative example were administered by use of oral zonde as quantity of eupatilin 20mg/kg. At 0, 5min, lOmin, 30min, 1 hr, 2hr, 4hr, 6hr, and 8hr after ad¬ ministration made, blood was taken out from left femoral aorta, centrifuged at 3500rpm for 10 minutes. So, blood plasma was separated and reserved at -20°C until analyzed.

[420] Eupatilin content in blood was quantified with HPLC. At first, blood 300D was put in micro tube; acetate buffer IOOD and glucuronidase 2OD were added therein; and the whole was kept at 37°C to react for 2 hours. Biochainin A IOOD as internal standard material and ether 1.5ml were added therein, and the whole was vortex stirred for two minutes, centrifuged at 12,000rpm for two minutes, and then supernatant was separated out and evaporated under nitrogen gas. Moving bed of 130D was added in and the whole was vortex stirred for a minute, and then IOOD was taken and injected to HPLC. As for analysis conditions, column was Hichrom RPB (250*4.6mm, particle size: 5D), analysis wavelength was 350nm, moving bed was 0.5% acetate buffer and acetonitrile mixed at 50 : 50 volume ratio. Row rate of moving bed was lml/min Injected sample was IOOD. Eupatilin was quantified as area ratio to internal standard material.

[421] The results were shown in FIG. 5. [422] With reference to FlG. 5, in-blood concentration when soft capsule preparation (test preparation) of the present invention obtained from Example 22 was administered shows higher value than in-blood concentration when preparation (comparative preparation) obtained from comparative example was administered. The soft capsule preparation according to the present invention has two times or more of maximum in- blood concentration as high as preparation made from Comparative example, and ten times or more of in-blood concentration area under curve (AUC).

[423] Furthermore, in-blood concentration according to soft capsule preparation of the present invention became zero when 8 hours passed by after administration made into mouse while in-blood concentration according to preparation made from Comparative example became zero after 4 hours. Therefore, it is concluded that soft capsule preparation according to the present invention remains longer in blood. Through this experiment, it is known that soft capsule preparation according to the present invention has higher value of bioavailability than preparation made from Comparative example.

Claims

[I] A therapeutic or preventive pharmaceutical composition for gastrointestinal disease comprising artemicapin C as an effective ingredient.

[2] A therapeutic or preventive pharmaceutical composition for gastrointestinal disease according to claim 1, wherein said artemicapin C is extracted from

Artemisia. [3] A method of removing blood coagulation inhibitory ingredient from Artemisia extract, which comprise: extracting from Artemisia leaves by alcohol or alcohol-water solution; adjusting to pH 10 ~ 11 by alkaline solution; reacting at 80°C for an hour or more; and neutralizing to pH 6.5 ~ 7.5 by acid solution to hydrolyze blood coagulation inhibitory ingredient. [4] A method according to claim 3, wherein said alkaline solution is aqueous potassium hydroxide solution. [5] A method according to claim 3, wherein said acid solution is one or more selected from the group consisting of hydrochloric acid, citric acid, and malic acid. [6] Extract of Artemisia (Artemisia asiatica, A. mongolica, A. princeps, and A. argyi) which comprises eupatilin, jaceosidin, and artemicapin C, and wherein blood coagulation inhibitory ingredient is removed by a method according to claim 3. [7] Extract of Artemisia according to claim 6, wherein said blood coagulation inhibitory ingredient is dicoumarol. [8] A therapeutic or preventive pharmaceutical composition for gastrointestinal disease comprising extract of Artemisia according to claim 6 as an effective ingredient. [9] A therapeutic or preventive pharmaceutical composition according to claim 8, wherein said gastrointestinal disease is gastritis, gastric ulcer, or inflammatory bowel disease. [10] A pharmaceutical composition of Artemisia extract comprising the extract of

Artemisia according to claim 6, surfactant, and co-solvent.

[II] A pharmaceutical composition according to claim 10, wherein said surfactant is one or more selected from the group comprising polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene fatty acid ester, poly¬ oxyethylene polyoxypropylene block copolymer, polyoxyethylene poly- oxypropylene copolymer, reactant of natural or hydrogenated vegetable oil and ethylene glycol, dioctylsulfosuccinic acid sodium salt, lauryl sulfonic acid sodium salt, phospholipid, propylene glycol mono- or di- fatty acid ester, trans- esterification reactant of natural vegetable oil triglyceride and polyalkylene polyol, mono-, di- or mono/di glyceride, sterol, and derivatives thereof.

[12] A pharmaceutical composition according to claim 10, wherein said co-solvent is one or more selected from the group comprising propylene carbonate, propylene glycol, ethanol, diethyleneglycol monoethyl ether, glycofurol, polyethylene glycol, dimethyl isosorbide, and N-methyl pyrrolidone.

[13] A pharmaceutical composition according to claim 10, wherein said composition is prepared by mixing of Artemisia extract, surfactant and co-solvent at weight ratio of 1: 0.1 to 50: 0.1 to 50.

[14] An oral administrative preparation comprising the pharmaceutical composition mentioned in claim 10.

[15] An oral administrative preparation according to claim 14, wherein said oral ad¬ ministrative preparation is selected from the group comprising tablet, pill, powder, hard capsule, gelatin banded hard capsule, soft capsule, caramel-type or jelly-type chewing tablet and other oral administrative liquid that contains water solution.