KR20130044286A - Pharmaceutical composition for sedation or sleeping induction comprising aster glehni extract as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for inducing relaxation or sleep contains Aster glehni extract as an active ingredient and is provided to be used as a health functional food and a medical substance that prevents or improves sleeplessness, epilepsy, and spasm reactions. CONSTITUTION: A pharmaceutical composition for inducing relaxation or sleep comprises Aster glehni extract as an active ingredient. The extract is a leaf extract of Aster glehni. The extract is extracted with water, low quality alcohol of C1-C4, or a mixed solvent of the same. The extract is extracted with a mixed solvent of water and ethanol in a ratio of 6:4 to 8:2. The composition is injected into an individual that needs to relax or needs induced sleep with an effective dose of 250-500 mg/kg.

Description

Pharmaceutical composition for sedation or sleeping induction comprising Aster glehni extract as an active ingredient

The present invention relates to a soothing or sleep-inducing pharmaceutical composition comprising the wormwood extract as an active ingredient.

Mung-namul refers to a characteristic group of wild vegetables belonging to the Asteraceae family in Korea and covers 1/13 of the area of Korean wild vegetables grown. It is one of the wild vegetables widely used in Korea and is used as a side dish when young. The genus of the plant belongs to the genus Aster, Ligularia genus, Solidago genus, Saussurea genus and Ainsliaea genus.

Aster glehni , native to Korea's Ulleungdo Island, is about 1 m high, with stem hairs, and rhizomes growing sideways. Leaves are staggered and when the flowers are bloomed, the leaves on the bottom are blurred. Stems are alternate, long oval 13-19cm long, 4-6cm butterfly. In addition, there are irregular sawtooth at the edge, hairs on both sides, preemption on the back, flowers bloom in August-October, white, about 1.5 cm in diameter, and hang in the end of main stems and branches.

Hangeul Bogam describes that worms are effective in removing wind, fever and detoxification, removing phlegm, and coughing. It is also known to treat colds, tonsillitis, bronchitis, spear, venom, snake bites, and bees.

On the other hand, considering the rapidly increasing traffic accidents and the accident caused by industrial accidents, skull injuries, electronic amusements, and addiction due to abuse of drugs cause epileptic seizures, the number of patients with epilepsy is expected to increase gradually do.

Amino acids that act as excitatory or inhibitory neurotransmitters in the central nervous system have been of interest since the recent disappearance of balance between excitatory and inhibitory neurotransmitters in the central nervous system has been suggested as a cause of seizures. In particular, research reports suggest that impairment of functional balance between glutamic acid, which is an excitatory neurotransmitter, and gamma-aminobutyric acid, an inhibitory transporter, plays an important role in seizure mechanisms. The development of therapeutics is mainly based on this research (Crawford et al., 1963; Braughman et al., 1980; Croucher et al., 1982).

Looking at the development of drugs for treating or preventing seizures, there is a barbiturate drug of the early 1900s developed as a modern sedative. Although it has been introduced and used as an effective medication for anxiety treatment, it has low stability index due to low treatment index and has not solved side effects such as addiction.

In 1957, benzodiazepine-based drugs such as chlordiazepoxide were developed and used as sedatives, and many derivatives related to these have been developed as sedatives. Although these drugs are relatively safe and have anti-anxiety effects that alleviate anxiety symptoms, they are also used as anticonvulsants, sedative sleeping pills, muscle relaxants, etc. In combination with alcohol or barbiturate system, there are some problems to be solved, such as enhanced central nervous system suppression, and rarely cause side effects such as worsening anxiety and causing hostility. (J. Clin. Psychiat. 42, 1981, MA Schuckit, Current therapeutic options in the management of anxiety, pp. 15-24).

In addition, buspirone, an azaspirodecanedione-based drug that has been developed since the 1970s, unlike other anti-anxiety agents, does not bind to the benzodiazepine receptor and does not directly affect the GABA (γ-aminobutyric acid) nervous system. Since it acts on dopamine receptors and serotonin receptors in the hippocampus, it is known that there are fewer side effects such as movement disorders than the existing benzodiazepines. However, the pharmacological effect is delayed by two or three weeks, and there is a possibility of side effects on the dopamine system. Ambien (Monsanto), which currently occupies 80% of the US sleeping pill market, has reported side effects in the morning, lasting eight hours.

Accordingly, the present inventors made an effort to develop a drug having an anticonvulsant effect without any side effects. As a result, the present invention was confirmed that the spasm was suppressed when the extract of the wormwood extract was administered to a mouse having an ankylosing spasm response, and the sleep extension was induced. It was completed.

It is an object of the present invention to provide a pharmaceutical composition for soothing or sleeping induction.

In order to achieve the above object, the present invention as one embodiment is worm worm ( Aster glehni ) provides a soothing or sleep-inducing pharmaceutical composition comprising the extract as an active ingredient.

In the present invention, the wormwood worms can be used to buy and use commercially available, or those collected or grown in nature.

The extract can be extracted from a variety of organs, for example, can be extracted from the roots, famine, stems, leaves, berries, etc. Among them, it is preferable to extract from the wormwood leaves.

The wormwood extract included in the composition of the present invention may be extracted by a known extraction method, and the method is not limited. For example, the wormwood extract is an extract of wormwood dried material obtained by crushing the leaves of wormwood, which has been removed by washing and drying, with water, a lower alcohol of C ₁ to C ₄ , or a mixed solvent thereof. Can be. Preferably, the solvent may be water, methanol, ethanol, a solvent mixed with water and ethanol, and most preferably, extracted with a mixed solvent mixed with water and ethanol. The extract using an organic solvent contains a large amount of non-polar terpene-based compound, but the extract using a mixed solvent of water and ethanol contains a high content of phenolic compound. Also preferably, the water and ethanol are mixed and extracted in a range of 6: 4 to 8: 2. As a specific embodiment, the present invention confirms the yield of the extract according to the mixing ratio of water and ethanol and the anticonvulsant effect of the present invention, water and ethanol in the range of 6: 4 to 8: 2, preferably 7: 3 The highest yield of the extract and the highest phenolic compound were contained when mixed in the range of.

In the present invention, the extract may include any one of an extract obtained by an extraction treatment, a diluent or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof.

The extraction method is not particularly limited and may be extracted at room temperature or warmed under conditions where the active ingredient is not destroyed or minimized. More specifically, the method for obtaining the wormwood worm extract in the present invention is as follows. Sapphire is used as the elution solvent with a volume of water, ethanol or a mixed ratio of about 6: 4 to 8: 2 of the volume up to about 2 to 20 times the dry weight, the extraction temperature is 20 to 100 ℃, Preferably at 40 ° C., the extraction period is extracted using extraction methods such as shaking extraction, hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for about 3 hours to 10 days, preferably 4-8 hours.

The wormwood extract of the present invention contains a large amount of a phenolic compound caffeoylquinic acid (caffeoylquinic acid), the caffeoyl quinic acid compound is 3,4-di-O-dicafeoyl quinic acid (3, 4-Di-O-dicaffeoylquinic acid), 3,5-di-O-dicaffeoylquinic acid (3,5-Di-O-dicaffeoylquinic acid), 4,5-di-O-dicafeoylquinic acid ( 4,5-Di-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, and 3-O - p - it has to be at least one compound selected from the group consisting of ilkwi acid (3-O- p -coumaroylquinic acid) Kumar. In addition, the wormwood wormwood extract contains a flavonoid compound astragalin (astragalin) and kempferol (kaempferol).

On the other hand, the wormwood worm fraction of the present invention can be obtained by fractionation from the wormwood extract, more specifically, the wormwood extract can be obtained by fractionation using a solvent such as ethyl acetate, hexane, chloroform, butanol, etc. have. Fractions can be obtained by methods known in the art, for example, after suspension of the wormwood methanol extract in distilled water, about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, Ethyl acetate or chloroform, butanol and the like may be added to extract and separate the solvent-soluble layer from 1 to 10 times, preferably 2 to 5 times. It is also possible to carry out further conventional fractionation processes.

Thereafter, the wormwood fractions may be separated and purified by silica gel chromatography to separate the compounds. When performing silica gel chromatography for separating the compound, it is preferable to use a mixed solvent of chloroform, methanol and water as the mobile phase, and a mixed solvent of methanol and water may be used in the additional chromatography. The chromatography may be performed once or several times until a single compound is purified, and may be concentrated and recrystallized as necessary.

The isolated compound is a flavonoid compound, preferably, kaempferol 3-O-α-L-rhamnopyranoisde, camphorol 3-O-β-D -Galactopyranoside (Kaempferol 3-O-β-D-galactopyranoside) and camphorol 3-O-β-D-glucopyranoside (kaempferol 3-O-β-D-glucopyranoisde) It may be one or more compounds.

Thus, in another aspect, the present invention is a compound isolated from the wormwood extract or fractions thereof 3,4-di-O-dicafeoylquinic acid (3,4-Di-O-dicaffeoylquinic acid), 3,5-di 3,5-Di-O-dicaffeoylquinic acid, 4,5-Di-O-dicaffeoylquinic acid, 5-O café five days quinone acid (5-O-caffeoylquinic acid) , 3-O- cafe five days quinone acid (3-O-caffeoylquinic acid) , 3-O- p - into Kumano ilkwi acid (3-O- p -coumaroylquinic acid ), Camphorol 3-O-α-L-lamnopyranoside (kaempferol 3-O-α-L-rhamnopyranoisde), camphorol 3-O-β-D-galactopyranoside (Kaempferol 3-O- anti-convulsant composition comprising at least one compound selected from the group consisting of β-D-galactopyranoside) and camphorol 3-O-β-D-glucopyranoside (kaempferol 3-O-β-D-glucopyranoisde) To provide.

In addition, the compounds of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the invention may be prepared by conventional methods, for example by dissolving the compounds in an excess of an aqueous acid solution and precipitating the salts using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile .

In addition, the compounds of the present invention can be used in the form of a pharmaceutically acceptable metal salt using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (eg, silver nitrate).

In a specific embodiment of the present invention was administered to the mouse wormwood extract extract to confirm the effect of sleep extension (Example 3). In addition, after injecting pentylenetetrazole (PTZ) to cause cramps in the mouse, it was confirmed that the stiffness spasm is inhibited when the wormwood extract of the present invention is administered (Example 4).

Therefore, the pharmaceutical composition of the present invention can be used for anticonvulsant use.

As used herein, the term "anticonvulsant" means inhibiting convulsive action, a phenomenon in which the systemic or local skeletal muscle is involuntarily rapidly contracted, including sleep enhancement and sedation.

Diseases that can be prevented or treated by the anticonvulsant effect include anxiety, manic, depression, phobia, aggression, subarachnoid hemorrhage, abnormalities associated with nervous system shock, abuse agents such as cocaine, nicotine, alcohol and benzodiazepines Symptoms associated with withdrawal in children, epilepsy including posttraumatic epilepsy, Parkinson's disease, psychosis, migraine, cerebral anemia, Alzheimer's disease, Huntington's chorea, premature dementia, obsessive-compulsive disorder (OCD), neuropathic defects associated with AIDS, sleep Neurological health in disorders such as disorders (including insomnia and sleep attacks), convulsions such as Gilles de la Tourette's syndrome, traumatic cerebral injury, tinnitus, neuralgia, neurological pain, toothache, cancer pain, and diabetes Inadequate neuronal activity, multiple sclerosis (MS), motor neuron disease, ataxia, myopathy (convulsiveness), temporomandibular joint dysfunction, causing poor May be one or more diseases selected from the group consisting of atrophic lateral sclerosis (ALS), it is not limited.

As used herein, the term "prevention" means any action that inhibits convulsions or delays the onset by administration of a composition.

As used herein, the term "treatment" means any action that improves or advantageously alters convulsions by administration of the composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

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

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type and severity of the subject, the severity, age, sex and activity of the drug. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. However, for the desired effect, the wormwood extract of the present invention or a fraction thereof is preferably administered at 50 to 1,000 mg / kg, preferably at 250 to 500 mg / kg.

The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents that have anticonvulsive effects and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition of the present invention does not have toxicity because it is an edible natural product extract as an active ingredient, and is harmless to the human body.

In another aspect, the present invention provides an anxiety, manic depression, phobia comprising administering the anticonvulsant composition to a subject having the onset or possibility of developing a disease that can be prevented or treated through anticonvulsant activity in a pharmaceutically effective amount. , Aggression, subarachnoid hemorrhage, abnormalities associated with nervous system shock, symptoms associated with withdrawal of abuse substances such as cocaine, nicotine, alcohol and benzodiazepines, epilepsy including posttraumatic epilepsy, Parkinson's disease, psychosis, migraine , Cerebral anemia, Alzheimer's disease, Huntington's chorea, premature dementia, obsessive compulsive disorder (OCD), neuropathic defects associated with AIDS, sleep disorders (including insomnia and sleep attacks), Gilles de la Tourette's syndrome Nerve health in diseases such as cramps, traumatic cerebral injury, tinnitus, neuralgia, nervous pain, toothache, cancer pain, diabetes Prevention of one or more diseases selected from the group consisting of inadequate neuronal activity, multiple sclerosis (MS), motor neuron disease, ataxia, myopathy (spasm), temporal mandibular joint dysfunction and muscular dystrophy (ALS) Or provide a method of treatment.

As used herein, the term "individual" means any animal including a human who has already developed or can develop a disease that can be prevented or treated through anticonvulsant activity, and refers to a composition comprising an extract, fraction or compound of the present invention. By administering to an individual, the disease can be effectively prevented and treated.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered as desired, but is not limited to intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration. The composition may also be administered by any device capable of transferring the active agent to the target cell.

In another aspect, the present invention provides an anticonvulsant food composition comprising an Aster glehni extract, a fraction thereof, or a compound separated therefrom. The composition may include a food supplement acceptable food additives in addition to the active ingredient.

The extract is preferably extracted by mixing water and ethanol in the range of 6: 4 to 8: 2. More preferably, the mixture is extracted in the range of 7: 3.

The term "food supplementary additive " in the present invention means a component which can be added to foods in a supplementary manner, and it can be appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, Although pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like are included, the examples of the food additives of the present invention are not limited by the above examples.

A health functional food may be included in the food composition of the present invention. The term "health functional food" in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and circles by using raw materials and components having useful functions in the human body. The term "functional" as used herein means that the structure and function of the human body have a beneficial effect on health uses such as controlling nutrients or physiological actions. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, unlike general medicines, there is an advantage that there is no side effect that may occur when a medicine is taken for a long time by using food as a raw material, and it is excellent in portability. Therefore, the health functional food of the present invention can be used as an adjuvant Is possible.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, during the preparation of food, the wormwood extract, wormwood fraction or compound separated therefrom of the present invention is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight of the raw material composition. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient as well as ordinary foods. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In still another aspect, the present invention provides an anticonvulsant quasi-drug composition comprising a wormwood extract, a fraction thereof, or a compound separated therefrom.

The wormwood extract, fraction or compound may be added as it is, or used with other quasi-drugs or quasi-drug components, and may be appropriately used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Preferably, the quasi-drug composition may be used for the preparation of antiseptic cleaners, shower foams, gagrins, wet wipes, detergent soaps, hand washes, humidifier fillers, masks, ointments or filter fillers.

In another aspect, the present invention comprises the steps of washing and drying the wormwood and then pulverizing with a grinder; Extracting the pulverized wormwood wormwood extract by using a solvent selected from water, a lower alcohol of C ₁ -C ₄ or a mixed solvent thereof; And 3,4-di-O-dicafeoylquinic acid (3,4-Di-O-dicaffeoylquinic acid), 3,5-di-O-di, comprising performing silica gel chromatography on the extract. 3,5-Di-O-dicaffeoylquinic acid, 4,5-Di-O-dicaffeoylquinic acid, 5-O-cafeoylqui acid (5-O-caffeoylquinic acid) , 3-O- cafe five days quinone acid (3-O-caffeoylquinic acid) , 3-O- p - in ilkwi acid (3-O- p -coumaroylquinic acid) , Astra ground Kumar It provides a process for the preparation of at least one compound selected from the group consisting of (astragalin) and kempferol.

In another aspect, the present invention comprises the steps of washing and drying the wormwood and then pulverizing with a grinder; Extracting the pulverized wormwood wormwood extract by using a solvent selected from water, a lower alcohol of C ₁ -C ₄ or a mixed solvent thereof; Obtaining the butanol fraction of the wormwood extract by fractionating the wormwood extract with butanol; And subjecting the fractions to silica gel chromatography, kempferol 3-O-α-L-rhamnopyranoisde, kemperol 3-O-β- From the group consisting of D-galactopyranoside (Kaempferol 3-O-β-D-galactopyranoside) and camphorol 3-O-β-D-glucopyranoside (kaempferol 3-O-β-D-glucopyranoisde) Provided are methods for the preparation of one or more compounds selected.

Since the composition containing the wormwood extract of the present invention exhibits the effect of anticonvulsions, it can be usefully used as a therapeutic agent for insomnia, epilepsy and convulsions. It can also be used as a dietary supplement and quasi-drug that can prevent or improve insomnia, epilepsy and convulsive reactions.

Figure 1 shows the extraction yield of the wormwood ivy according to water, ethanol and a mixed solvent thereof. Among them, when water and ethane were mixed at 7: 3, the yield was confirmed to be the highest.
Figure 2 shows the compound identified on the HPLC chromatogram of wormwood extract. Specifically, 3,4-di-O-dicaffeoylquinic acid, 3,5-di-O-dicafeoylquinic acid (3,5-Di-O -dicaffeoylquinic acid), 4,5-Di-O-dicaffeoylquinic acid, 5-O-caffeoylquinic acid, 3- O- cafe five days quinone acid (3-O-caffeoylquinic acid) and 3-O- p - was ilkwi acid (3-O- p -coumaroylquinic acid) in Kumasi.
Figure 3 shows the content of the compound identified on the HPLC chromatogram when extracting the wormwood using different extraction solvents.

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

Reference Example  1: Instrument and Reagents Used

The instrument used for HPLC analysis was a Varian HPLC system consisting of a Prostar 210 pump and a Prostar 325 UV-Vis detector. The column for HPLC used was a Shiseido (Chuoku) Capcell Pak C18 column (5 μm, 4.6 mm (i.d.) × 250 mm). The mobile phase used in the analysis was J.T. Baker's solvent for HPLC was purchased and used. Seven standard products used in the experiment were provided by Professor Lee Kang-no of Sungkyunkwan University.

In addition, used for the anticonvulsant effect experiments pentylene tetrazole (pentylenetetrazole), para-Kumar acid (p -coumaric acid) and caffeic acid (caffeic acid) was used as a Sigma Corp..

Example  One: Grumpy  Extraction, fractionation and separation

(500g, Ulleungdo Agricultural Technology Center) was crushed and extracted with methanol (MeOH) while refluxing three times. The extract was filtered, concentrated under reduced pressure, and lyophilized to obtain a methanol extract (73 g). This was partitioned and extracted three times with 800 ml each of chloroform (CHCl ₃ ) and water (H ₂ O). The chloroform soluble part was concentrated under reduced pressure to obtain 26.7 g of a chloroform fraction. The remaining aqueous layer was partitioned and extracted three times with 800 ml of butanol (BuOH) in a separatory funnel to obtain a butanol soluble part, and then concentrated under reduced pressure to obtain 10.5 g of butanol fraction.

10 g of butanol fraction was developed in a silica gel column with chloroform-methanol-water (CHCl ₃ -MeOH-H ₂ O) (7: 3: 1, lower layer), and fractionated in 60 ml portions. Subfraction 28 fractions were concentrated to Fr. B, concentrate 1454 was Fr. C. Fr. B was developed in an ODS column with developing solvent methanol-water (MeOH-H ₂ O) (1: 1), 20 ml aliquots were concentrated, and small fractions 7-13 were concentrated. Recrystallized from methanol, Compound 1 was obtained as a pale yellow powder. Got it. In the same manner as in the purification of Compound 1 from Fr.B, Fr.C was developed on an ODS column to obtain 3-4 small fractions and 13-18 small fractions, which were then concentrated and recrystallized from methanol. 110 mg) and compound 3 (150 mg) were obtained. Various physical functions and spectroscopic results of the compounds 1 to 3 are shown below. rhamnopyranoisde (afzelin)), camphorol 3-O-β-D-galactopyranoside, camphorol 3-O-β-D-glucopyranoside Astragalin (kaempferol 3-O-β-D-glucopyranoisde (astragalin)) was identified.

Compound 1 ( Kaempferol  3-O-α-L- rhamnopyranoside , afzelin )

A pale yellow powder was obtained from methanol-water (1: 1). mp 173-178 ° C., UV λ _max (MeOH) nm: 268, 357; IR ν _max cm ^-1 : 3400-3100 (broad, OH), 1655 (α, β-unsaturated ketone), 1605 (aromatic C = C), 1355, 1170, 1100- 1000 (glycosidic CO). ¹ H-NMR (DMSO-d ₆ , 500 MHz) δ: 0.81 (3H, d, J = 5.6 Hz, rha-CH ₃ ), 5.32 (1H, d, J = 1.47 Hz, H-1 ''), 6.21 (1H, d, J = 2.0 Hz, H-6), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.92 (2H, d, J = 8.8 Hz, H-3 ', 5' ), 7.76 (2H, doublet, 8.8 Hz, H-3 ', 5'); ¹³ C-NMR (DMSO- d ₆ , 125 MHz) δ: kaempferol-93.8 (C-8), 98.8 (C-6), 104.2 (C-10), 115.5 (C-3 ', 5 '), 120.7 (C-1'), 130.6 (C-2 ', 6'), 134.3 (C-3), 156.6 (C-9), 157.3 (C-2), 160.0 (C-4 ') , 161.4 (C-5), 164.3 (C-7), 177.8 (C-4), L-Rha-101.8 (C-1``), 74.2 (C-2 ''), 76.4 (C-3 ''), 69.9 (C-4``), 77.4 (C-5''), 60.9 (C-6'').

Compound 2 ( Kaempferol  3-O-β-D- galactopyranoside )

Yellow powder. mp 220-224 ° C., IR ν _max (KBr, cm ⁻¹ ): 3457 (OH), 1659 (α, β-unsaturated ketone), 1605, 1560, 1491 (aromatic C = C), 1362, 1262, 1183, 1062 (glycosidic CO). UV λ _max (MeOH) nm: 265, 356; ¹ H-NMR (500 MHz, DMSO- d ₆ ) δ: 8.06 (2H, d, J = 8.8 Hz, H-2 ', 6'), 6.85 (2H, d, J = 8.8 Hz, H-3 ' , 5 '), 6.42 (1H, d, J = 1.9 Hz, H-8), 6.18 (1H, d, J = 1.9 Hz, H-6), 5.39 (1H, d, J = 7.5 Hz, H- 1 ''; ¹³ C-NMR (DMSO- d ₆ , 125 MHz) δ: Camphorol-93.7 (C-8), 99.4 (C-6), 104.0 (C-10), 115.1 (C-3 ', 5 '), 120.9 (C-1'), 131.0 (C-2 ', 6'), 133.3 (C-3), 156.4 (C-2), 156.4 (C-9), 160.0 (C-4 ' ), 161.2 (C-5), 164.2 (C-7), 177.6 (C-4), D-gal-60.2 (C-6``), 67.9 (4 ''), 71.2 (C-2 '' ), 73.1 (C-3``), 75.6 (C-5 ''), 101.7 (C-1 '').

Compound 3 ( Kaempferol  3-O-β-D- glucopyranoside  ( astragalin ))

mp 230-233 ° C. FeCl ₃ , Mg-HCl, Zn-HCl, Molish tests: positive; IR ν _max cm ^-1 : 3,619-3,000 (broad, OH), 1,655 (α, β-unsaturated ketone), 1,606, 1,562, 1506 (aromatic C = C), 1360, 1,291, 1,179, 1,056, 1,011 (glycosi Dick CO), 799; UV λ _max (MeOH) nm: 267, 300 (sh), 352; ¹ H-NMR (DMSO-d ₆ , 500 MHz) δ: 5.54 (1H, d, J = 7.2 Hz, H-1 of D-Glc), 6.21 (1H, d, J = 2.1 Hz, H-6) , 6.43 (1H, d, J = 2.1 Hz, H-8), 6.89 (2H, d, J = 8.8 Hz, H-3 ', 5'), 8.04 (2H, d, J = 8.8 Hz, H- 2 ', 6'); ¹³ C-NMR (DMSO- d ₆ , 125 MHz) δ: camphorol-156.3 (C-2), 133.2 (C-3), 177.4 (C-4), 161.2 (C-5), 98.7 (C- 6), 164.2 (C-7), 93.6 (C-8), 156.3 (C-9), 103.9 (C-10), 120.9 (C-1), 130.7 (C-2), 115.0 (C-3 ), 159.9 (C-4), 115.0 (C-5), 130.8 (C-6) D-Glc-100.9 (C-1), 74.2 (C-2), 76.4 (C-3), 69.9 (C-4), 77.4 (C-5), 60.8 (C-6).

Example  2: HPLC  Extract Preparation for Analysis

Extraction solvents were water, ethanol (EtOH) and mixed solvents of 400 ml of water-ethanol (7: 3), water-ethanol (5: 5) and water-ethanol (3: 7). Accurately weigh 20 g of finely chopped worms and place them in an Erlenmeyer flask containing 400 ml of each solvent. These were extracted by ultrasonication at 40 ° C. for 6 hours. Thereafter, the filtrate was concentrated in a vacuum concentrator, and each extract obtained by freeze drying was used as a sample for HPLC analysis.

HPLC analysis was carried out under the same conditions as known methods, and the content was calculated using a calibration curve from the peak area obtained from the experimental results. Briefly describing the method, the sample and the standard compound were dissolved in 80% methanol, filtered through a syringe filter and then scanned, and the UV detector fixed wavelength was 246 nm. 0.05% phosphoric acid (solvent A) and methanol (solvent B) were used as the mobile phase, and the solvent gradient was {0-10 min, 60% A: 40% B; 10-20 minutes, 50% A: 50% B; 20-30 minutes, 40% A: 60% B; 30-35 minutes, 60% A: 40% B} and the flow rate was 1.00 ml / min.

Astragalin and kaempferol were used as indicators to analyze the flavonoid components under the same HPLC analysis conditions. Astragaline peaked at 14.4 min at t _{R and} 24.3 min for cemprolol under these conditions. A calibration curve was prepared at concentrations of 50, 100, and 200 μg / ml to obtain astragalin y = 27.10x + 50.50 (R ² = 0.998), and camphorol at y = 54.85x + 287.01 (R ² = 0.999). Where x is the concentration (μg / ml) y is the peak area (counts). 1.000 mg / g of sample was prepared and injected into HPLC to obtain the peak area, and the content was evaluated by calculation.

Extraction yield was 17.2%, 19.4%, 17.2%, 8.1% when extracted with W, WE (7: 3), WE (3: 7), E solvent (W: water; WE: water-ethanol; E: ethanol). The extraction yield was lowest when the E solvent was extracted, and the WE (7: 3) solvent showed the highest extraction yield. Seven kinds of CQ (caffeoylquinic acid) standards were used for HPLC analysis. In order to examine the extraction effect in more detail after this experiment, we extracted from WE 5: 5 ratio to 9: 1 ratio. As a result, the extraction yield was high at about 19% in 6: 4, 7: 3, and 8: 2 solvents, but the WE 7: 3 solvent showed the highest extraction rate. The results are shown in FIG.

Compounds identified on the HPLC chromatogram were 3-Op-coumaroylquinic acid (3-pCQ), 3,4-di-O-dicafeoylquinic acid (3,4-Di-O -dicaffeoylquinic acid, 3,4-DQ), 5-O-caffeoylquinic acid (5-CQ), 3-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 3- CQ) and 3,5-di-O-dicafeoylquinic acid (3,5-Di-O-dicaffeoylquinic acid, 3,5-DQ) (FIG. 2). Of these, 3,4-DQ was found only in ethanol extracts and 4,5-DQ was only found in W-E (7: 3), while 3,5-DQ was found in all four extracts. In particular, W extract showed only 3,5-DQ out of three CQs, so that isomerization was expected between 3,4-DQ and 4,5-DQ depending on the extraction solvent. As shown in FIG. 3, the HPLC column used in this experiment is a reversed phase column using a fixed-phase octadecylsilane (ODS), and thus, a compound having a short retention time (a compound having a relatively high polarity) has a relatively high polarity. It can be said that a long compound has a small polarity. In the W extract and the W-E (7: 3) extract, the contents of 5-CQ and 3-pCQ were particularly high. In particular, 3-pCQ reached 46.10mg / g in W-E (7: 3) extract, while it was very low in 6.39mg / g in E extract. The total CQ of the W-E (7: 3) extract was 38.7%, whereas the E extract was about 20.7%. W and W-E (3: 7) extracts also had higher CQ content but lower than W-E (7: 3) extracts.

In addition, the flavonoid compound, camphorol and its glycoside, astragalin, was used in the analysis, which was much lower than the CQ compound. The highest content of the extract was W-E (3: 7) extract, which showed 13.2 mg / g of astragalin and 0.23 mg / g of camphorol (Table 1).

compound Extraction solvent Water (W) Ethanol (E) W-E (7: 3) W-E (3: 7) 3,4-DQ ND 1.61 ± 0.02 ^a ND ND 3,5-DmQ ND ND ND ND 3,5-DQ 2.02 ± 0.11 2.17 ± 0.33 4.88 ± 0.98 4.43 ± 0.11 4,5-DQ ND ND 1.63 ± 0.51 ND 5-CQ 9.87 ± 0.11 4.76 ± 0.17 13.34 ± 0.24 13.69 ± 0.18 3-CQ 2.06 ± 0.11 1.74 ± 0.11 4.93 ± 0.40 3.25 ± 0.05 3-pCQ 45.97 ± 0.61 6.39 ± 0.67 46.10 ± 4.22 34.06 ± 0.80 Total (mg / g) 59.92 ± 0.83 16.67 ± 0.53 70.89 ± 2.56 55.43 ± 0.11 % of extract 34.94 ± 0.49 20.70 ± 0.66 38.74 ± 1.40 30.04 ± 0.59 Astragaline 1.43 ± 0.19 4.08 ± 0.23 8.18 ± 0.56 13.2 ± 0.48 Camphorol 0.15 ± 0.01 0.16 ± 0.02 0.20 ± 0.01 0.23 ± 0.03

^a means the mean value of experiment 3, ND means not detected

Example  3: Grub  Determine the effect of extract on sleeping time

As the experimental animals, male ICR mice of 4 to 5 weeks old weighing about 20 ± 2 g were used and were purchased from Hyochang Science (Daegu). Methanol extract was used by dissolving in Tween 80 solution [Twin 80: saline (1: 4)] to the test animals at a dose of 100, 200 mg / kg. Lorazepam and pentobarbital were used as control drugs.

Mice were experimented with 5 mice per group. 15 minutes after the administration of lorazepam, oral administration of each sample 30 minutes after the intraperitoneal injection of 30 mg / kg of pentobarbital salt, righting reflex: Time from loss to recovery) was measured.

The results of the sleep extension effect on the mice are shown in Table 3. Induction of sleep with pentobarbital alone averaged 50 minutes of sleep. As a control drug, lorazepam was extended to 5.38 times at 5 mg / kg, and p -coumaric acid and caffeic acid were 3.36 times and 2.74 times at 20 mg / kg, respectively. Wormwood WE (7: 3) extracts prolonged sleep by 1.25 and 1.57 times at 100 mg / kg and 200 mg / kg, respectively.

group Dose (mg / kg) Time (min) Pentobarbital 50.0 ± 3.9 ^g 30% EtOH Extract 100 62.5 ± 8.8 ^g 200 78.5 ± 9.5 ^ef p -coumaric acid 10 109.9 ± 12.3 ^d 20 167.8 ± 23.6 ^b Caffeic acid 10 89.5 ± 9.4 ^e 20 137.2 ± 19.5 ^c Lorazepam 5 269.0 ± 29.1 ^a

Example  4: Grub  Confirmation of Anticonvulsant Effect of Extracts

Mice were experimented with 5 mice per group. After oral administration of the sample for one week and 60 minutes after the last administration, pentylenetetrazole (PTZ) (70 mg / kg, sc) was injected in accordance with Sohn's method to observe the presence of convulsions and death. . Pentylenetetrazole (PTZ) is a central nervous system stimulant and has been reported to inhibit the action of GABA on chloride ion conduction and to increase the excitatory transporter glutamate (Metcalf 1979).

Table 3 shows the effect of the treatment group on PTZ-induced mouse cramps. Onset time, stiffness spasm at 10 mg / kg and 20 mg / kg for p -coumaric acid and caffeic acid, and 100 mg / kg and 200 mg / kg for WE (7: 3) extract Examination of tonic extensive and mortality significantly inhibited tonic spasm (Table 3).

A group administered only pentobarbital was used as a control.

process Effective amount Onset time T.E. Mortality (mg / kg) Inc (%) Lat (sec) Inc (%) Lat (sec) Inc (%) Lat (sec) Control group 100 45.1 ± 6.14 100 159.3 ± 16.3 100 191.5 ± 18.5 30% EtOH ext. 100 100 53.4 ± 6.42 100 187.7 ± 19.7 100 218.2 ± 17.9 200 100 61.6 ± 6.01 100 196.9 ± 21.4 100 234.4 ± 21.4 p -coumaric acid 10 100 63.2 ± 5.97 100 241.8 ± 18.6 100 289.9 ± 19.8 20 100 74.2 ± 6.33 100 306.8 ± 21.5 100 374.2 ± 20.1 Caffeic acid 10 100 61.9 ± 5.42 100 198.9 ± 18.0 100 236.8 ± 17.5 20 100 67.0 ± 6.19 100 256.4 ± 19.9 100 305.2 ± 18.2

(T.E means tonic extensive, Inc., incidence, and Lat means latency after pentylenetetrazole treatment.)

Claims (5)

Sprout Aster glehni ) A soothing or sleep-inducing pharmaceutical composition comprising an extract as an active ingredient.
The method of claim 1,
The extract is a calming or sleep-inducing pharmaceutical composition that is a leaf extract of wormwood.
The method of claim 1,
The extract is calm, or sleep-inducing pharmaceutical composition that is extracted with water, C ₁ ~ C ₄ Lower alcohol or a mixed solvent thereof.
The method of claim 3,
The extract is a soothing or sleep-inducing pharmaceutical composition that is extracted with a mixed solvent of water and ethanol mixed in the range of 6: 4 to 8: 2.
The method of claim 1,
The composition is a calming or sleep-inducing pharmaceutical composition that is administered to an individual in need of sedation or sleep in an effective amount of 250 to 500mg / kg.

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