KR101315888B1 - Composition comprising an extact of cirsium japonicum and apigenin isolated thereform for the improvement and remedial of insomnia - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for improving insomnia and treatment using apigenin as an active ingredient, and more particularly, containing apigenin isolated from thistle extract having an insomnia-improving effect as an active ingredient, and frequently during insomnia, grapefruit, and sleep. The present invention relates to a pharmaceutical composition that can be used (used) as an improving or therapeutic agent such as awakening, sleeplessness, and dizziness of the head.

Description

COMPOSITION COMPRISING AN EXTACT OF CIRSIUM JAPONICUM AND APIGENIN ISOLATED THEREFORM FOR THE IMPROVEMENT AND REMEDIAL OF INSOMNIA} Thistle Extract or Apigenin Isolated from It

The present invention relates to a method for the treatment of cirsium japonicum ) extract or a pharmaceutical composition for improving insomnia as an active ingredient comprising apigenin isolated therefrom, More specifically, thistle extract having an insomnia improvement effect or apigenin isolated from it as an active ingredient It relates to a pharmaceutical composition and health functional food for improving and treating insomnia, sleepiness, frequent waking up during sleep, sleeplessness, dizziness, and the like.

Primary insomnia is insomnia that is not caused by other primary sleep disorders, psychiatric problems, medical or neurological problems, or the use or withdrawal of drugs. It is also estimated that hyperarousal or ascending reticular formation system is caused by excessive activity during sleep as well as during awakening. Primary insomnia includes psycho-physiologic insomnia, sleep statemisperception, inadequate sleephygiene, and idiopathic insomnia, the most common of which is psychophysiological insomnia Primary insomnia (Hauri PJ, Fischer J. Persistent psychophysiologic (learned) insomnia. Sleep 1986; 9: 38-53.).

Psychophysiological insomnia is associated with internal mental activity or external stimuli through repeated and learned negative experiences that are difficult to sleep. Conditional association with external stimuli occurs when sleep-related situations or behaviors are frequently associated with insomnia, resulting in conditioned hyperawakeness just before sleep (Bonnet MH, Arand DL. Hyperarousal and insomnia. Sleep Med Rev 1997 ; 1: 97-108.). If this is repeated several times, lying on the bed will result in a conditional hyperawakening, and bedtime-related behaviors, such as brushing teeth, preparing bedclothes, or extinguishing fire, also cause hyperawakening. These people sleep well in unfamiliar surroundings that are not associated with insomnia. The learned linkages to internal mental activity are largely due to the conditional excess of worrying about poor sleep. These excessive worries can not sleep, and because you can not sleep because you try to sleep more and more awakening, causing a vicious cycle that can not sleep. Sleep polysomnography results in longer sleep latency, increased total sleep time, stage 1 and stage 2 berem sleep time, and alpha involvement (Sateia MJ.The international classification of sleep disorders. 2 ^nd d.:Diagnosticandcodingmanual.Westchester, Illinois: American Academy of Sleep Medicine, 2005: 6-8.).

Psychophysiological insomnia is found in 1-2% of the total population and is diagnosed in about 12-15% of insomnia patients referred to sleep centers (Buysse DJ, Reynolds III CF, Kupfer DJ, et al. Clinical diagnoses in 216 insomnia patients using the International Classification of Sleep Disorders (ICSD), DSM-IV, and ICD-10 categories: Areport from the APA / NIMH DSM-IV field trial.Slee 1994; 17: 630-637.). It is more common in women than men, and is usually invented between 20 and 40 years.

Sleep status cognitive impairment is diagnosed when subjectively complains of insomnia, but objective sleep test results show no problems with starting or maintaining sleep (Carskadon MA, Dement WC, Mitler MM, et al. Self-reports versus sleep laboratory findings in 122 drug-free subjects with complaints of chronic insomnia.Am J Psychiat 1976; 133: 1382-1388). The prevalence is unknown, but it is thought to be less than 5% of insomnia patients who visit outpatients. There is no theory explaining exactly the difference between subjective abnormalities and objective outcomes, but most often these patients experience objective problems that are not known by standard sleep polysomnography.

Inadequate sleep hygiene is caused by habits and daily activities that prevent you from sleeping well at night and staying awake during the day (Nau SD, Lichstein KL. Insomnia: causes and treatments.In:Carney PR, Berry RB, Geyer JD.edc , Clinical sleep disorders.Philadelphia: Lippincott Williams & Wilkins. 2005: 157-190). The exact prevalence of inadequate sleep hygiene is unknown, but it is thought that this abnormality will occur in 1-2% of the total population, and 5-10% of people who visit sleep centers have this diagnosis. In chronic insomnia, this is a very common cause and persistence of insomnia, and if it is diagnosed as a primary or secondary cause, more than 30% of sleep center patients are involved.

Idiopathic insomnia is very rare and does not provide adequate sleep throughout life (Hauri PJ, Olmstead EM. Childhood onset insomnia. Sleep 1980: 3: 59-65.). Typically, it begins at birth or begins at early puberty. Some have family history (Bastien CH, Morin CM. Familial incidence of insomnia. J Sleep Res 2000: 9: 49-54.). This is presumed to be the cause of neuromodulation of sleep. The prevalence is unknown, but it is estimated to be 0.7% in puberty and 1% in early adulthood, with similar prevalence thereafter. Less than 10% of patients who have had insomnia at sleep centers get this diagnosis.

Cirsium japonicum ) is a perennial herb belonging to the Asteraceae family, distributed in Korea, Japan and northeast China. Thistle has diuretic, detoxification, hemostasis and tonic effects, and is used to treat menstrual irregularities, uterine fibroids, anemia, bleeding, and bleeding. Thistle plant contains about 78 kinds of flavonoids (apigenin, luteolin, myricetin, kaemferol, etc.) with excellent physiological activity, and has anticancer, antimutagenic, and immunostimulating activity. Physiological activities such as antifungal and antibacterial activity (Chung MS, Um Hj, Kim CK, Kim GH (2007) .Development of functional tea product using circium japonicum . Korean J. Food Culture 22 (2): 261-265).

Thistle also inhibits lipids and oxidation and promotes alcohol detoxification, which has a protective effect on the liver. Decreases the concentration of total cholesterol and triglycerides in the liver, thus delaying liver damage and reducing serum lipid content in hyperlipidemia. Thistle leaf extract has an antirheumatic effect.

However, no disclosure or teaching has been made about the insomnia-improving effect of thistle extract or apigenin isolated from thistle.

Accordingly, the present inventors continued their efforts to develop an insomnia improver using natural products. As a result, the present inventors confirmed that the extract obtained using thistle and the active substance apigenin, which was purely separated therefrom, had an improvement in insomnia and a therapeutic effect. The invention was successfully completed.

After all, the main object of the present invention is to provide a pharmaceutical composition for improving insomnia and treatment with thistle extract having an effect of improving insomnia or apigenin isolated therefrom as an active ingredient.

In order to achieve the above object, the present invention provides an extract of thistle having an insomnia effect.

In the present invention, the thistle extract is characterized in that the crude extract of thistle or non-polar solvent soluble extract.

The milk thistle crude extract of the present invention is collected by drying thistle and powdered with a crusher, C1 such as water, methanol, ethanol, butanol, etc. in volumes of about 1 to 20 times, preferably 3 to 10 times the weight of the sample. To a polar solvent of C4 to lower alcohol or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably 60 to 100% ethanol, at about 0.1 to 12 hours at 60 to 100 ° C, preferably By using extraction methods such as stirring extraction, hot water extraction, cold extraction, warm extraction, reflux cooling extraction or ultrasonic extraction for 1 to 6 hours, preferably the extract obtained after stirring extraction is obtained by filtration, concentration under reduced pressure or drying. Can be.

In addition, the milk thistle non-solvent soluble extract of the present invention is a solvent selected from thistle crude extract obtained from hexane, chloroform, methylene chloride, dichloromethane, ethyl acetate, glycerin, propylene glycol, butylene glycol or ether, preferably chloroform or Ethyl acetate, more preferably, ethyl acetate may be added, followed by extraction to obtain silica gel column chromatography.

Accordingly, the present invention provides a new use of thistle extract prepared by the above method or apigenin isolated therefrom for improving and / or treating insomnia.

Specifically, the present invention provides a pharmaceutical composition for improving and treating insomnia, which contains the thistle extract or apigenin isolated therefrom as an active ingredient.

In the present invention, the apigenin may be represented by the following Chemical Formula 1, and the apigenin may be used both as a chemically synthesized compound and a commercially available compound, as well as the purified and purified from thistle.

[Formula 1]

The pharmaceutical composition for improving and treating insomnia of the present invention comprises 0.0001 to 99.99% by weight, preferably 0.001 to 50% by weight of the thistle extract or thistle isolated from the total weight of the composition.

In addition, the pharmaceutical compositions according to the invention may further comprise suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinyriphyllolidon, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the pharmaceutical compositions according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injections, respectively, according to a conventional method. Can be used.

When formulated, diluents or excipients such as fillers, weights, binders, humectants, sealants, and surfactants are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. This solid preparation includes the compound is prepared by mixing at least one excipient, for example, starch calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple rare agents, water and liquid paraffin. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base material of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention may be administered by varying the dose depending on the progress of the disease, the age of the patient, sex, physical condition, administration period, the method of administration, the weight of the patient, the meal, the rate of discharge. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered parenterally or orally in the range of 0.001 to 100 µg / kg, more preferably in the range of 0.01 to 100 µg / kg. Administration can be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In addition, the pharmacological dosage forms of thistle or apigenin isolated therefrom can be used in the form of their pharmaceutically acceptable salts, and can also be used alone or in combination with other pharmaceutically active substances, as well as in suitable collections. Can be.

The present invention also provides a health functional food for insomnia improvement and treatment containing the thistle extract or apigenin isolated therefrom as an active ingredient.

The health functional food of the present invention may further comprise a food acceptable additive in addition to the thistle extract or apigenin isolated therefrom, and may be suitably used according to a conventional method, and the mixed amount of the active ingredient may be It may be appropriately determined depending on the purpose of use, for example, prophylactic, health or therapeutic treatment.

An effective amount of thistle extract or apigenin isolated from the dietary supplement may be used according to the effective amount of the pharmaceutical composition, but for long-term intake for health and hygiene purposes or for health control purposes, It may be less than the above range, it is clear that the active ingredient can be used in an amount above the above range because there is no problem in terms of stability.

In addition, there are no particular restrictions on the type of health functional food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups , Beverages, tea, drink, alcoholic beverages and vitamin complexes, health functional foods, and the like.

It may also be added to foods or beverages for the purpose of improving insomnia and therapeutic effects. At this time, the amount of thistle extract or apigenin isolated from the food or beverage may be added to 0.01 to 30% by weight, preferably 0.3 to 15% by weight of the total food weight, the health beverage composition based on 100ml 0.01 to 5 g, preferably 0.03 to 1 g may be added.

According to the present invention as described above, it can be seen that the apigenin, the main ingredient of thistle, increases the total sleep time by confirming a significant response in the pentobarbital induced sleep prolongation effect.

In addition, since thistle of the present invention is approved as a health functional food, apigenin isolated from thistle extract of the present invention extracted therefrom also has no problems such as toxicity and side effects, so there are no drugs or health functional foods for the treatment and prevention of obesity. It can be usefully used.

1 shows a high performance analyzer (HPLC) profile of an active material according to the invention.
Figure 2 shows the nuclear magnetic resonance ( ¹ H NMR) spectrum of the active material according to the present invention.
Figure 3 shows the nuclear magnetic resonance ( ¹³ C NMR) spectrum of the active material according to the present invention.
Figure 4 shows the pentobarbital-induced sleep prolongation effect by the active material according to the present invention.
5 is a graph showing the change in chloride influx by the active material according to the present invention.
Figure 6 is a graph of the expression of glutamic acid decarboxylase (GAD) by the active material according to the present invention.
Figure 7 shows GABA receptor subunit (GABA _{A) with} active substance according to the invention graph of the expression of receptor subunits.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Isolation and Identification of Active Substances

Thistle ( circium japonicum ) was collected and dried in the mountainous region of Chungbuk. What was purchased at Gyeongdong Market (Seoul, Korea) was used after drying. The dried thistle ( circium japonicum ) was extracted by adding 95% alcohol and concentrated in a reduced pressure concentrator to prepare a crude alcohol extract.

The crude crude extract was dissolved in distilled water and ethyl acetate, mixed at a ratio of 1: 1 (v / v), and repeated three times. The ethyl acetate extract was decompressed, concentrated, and dried.

The dried product was subjected to silica gel flash chromatography (solvent type: chloroform / ethyl acetate = 2: 1), and the active fractions were dried under reduced pressure and dried again, and then dissolved in a small amount of methanol to be saturated with Millipore (0.45 nm). Filtration was used to fractionate the active material with a high performance analyzer (HPLC, column: ODS 20 × 250 nm; 80% methanol).

1 to 3 show the structure of the active material, high performance analyzer (HPLC) profile, nuclear magnetic resonance ( ¹ H and ¹³ C NMR) spectra of the present invention.

From the above results, it was confirmed that the active material according to the present invention was apigenin (4 ′, 5,7-trihydroxyflavone).

Example  2. Experimental animals

20-25g male mice (ICR mice, SAMTAKO, Korea) were used in animal experiments with 10-12 mice per group. The experimental animals were kept for one week to adapt to the new environment in an independent cage with an acrylic cage (45 × 60 × 25 cm), artificially adjusted to a 12-h light / dark cycle and maintained at an appropriate temperature (22 ± 2 ° C). . In addition, all animal experiments were conducted between 10:00 and 17:00 and were conducted in accordance with the guidelines of animal testing of the National Institutes of Health.

Example  3. Cell culture

Primary cultures of cerebellar granulocytes were prepared from the cerebellum of Sprague-Dawley rats. After 8 days of culture, the cells show the function of GABA receptors, and the expression pattern is similar to that seen in the cerebellum of postnatal development, but different from the cerebellum of mature mice. Cells were incubated at 1 × 10 5 / well in 96 microplates. Cytosine arabinofuranoside (final concentration, 10) was added at 18-24 h after culture to inhibit proliferation of non-neuronal cells.

Experimental Example  One. Pentobarbital  Induced Sleep Extension

Pentobarbital sodium is dissolved in 0.9% saline and intraperitoneally injected (i.p.) in each mouse to induce sleep. Test samples are dissolved in physiological saline and orally administered (p.o.) to mice (0.1 ml / 10 g). 10-12 rats were used for each dosing group, and all experiments took place between 1:00 and 5:00. Animals are fasted for 24 hours before the experiment and pentobarbital is injected 1 hour after administration of apigenin. Animals that stopped moving within 15 minutes after pentobarbital injection were immediately transferred to another box and judged to have slept the subjects that had not moved for more than 3 minutes. The latency co onset of sleep represents the time elapsed until the animal loses its reflexes to lean back and infuse pentobarbital from the infusion. Record the time of waking up, continually observing the animal and correcting its posture. Sleep time is the time when the animal shows spontaneous movement again and moves to another box. Animals that fail to fall asleep within 15 minutes of pentobarbital administration are excluded from the experiment.

Apitonin induced pentobarbital-induced sleep prolongation showed a significant response at 50 mg / kg. Non-premature mice were used as a positive control group and were administered 15 minutes before pentobarbital administration, and showed a significant response at sleep induction time.

Experimental Example  2. Intracellular chloride influx ( chloride influx ) Measure

The concentration of chloride ion (Cl-) in cerebellar neuron cells is measured using a fluorescent reagent sensitive to chloride ion (Cl-). The cells are cultured overnight in medium containing 10 mmol fluorescent reagent. After loading, cells are washed three times with buffers containing chloride ions (Cl-) as appropriate. Then replace the wash buffer to the presence of compounds or controls. Use repeated fluorescence measurements. Chloride ion [Cl-] i in cultured cerebellar neuron cells was 12.6 ± 3.0 mM, and chloride ion [Cl-] i in cerebellar neural cells treated with apigenin (12.5-100) increased significantly. Pentobarbital (10) also increased the influx of chloride ions (Cl-) into the cerebellar New Year cells.

Experimental Example  3. Gava ( GABAA ) Receptor subunit and glutamic acid Carboxy  Removal enzymes ( GAD  65/67) expression

Apigenin (50 mg / kg, i.p.) and pentobarbital sodium (40 mg / kg, i.p.) are treated in mice for 3 days, respectively. For Western blot analysis, mice are killed and the hippocampus portion of the brain is removed and treated with lysis buffer. The samples are each ground with an ultrasonic tissue grinder under an appropriate amount of lysis buffer. Each mill is centrifuged for 20 minutes at a rate of 20,000X g. Equal amounts of protein are aliquoted into polyacrylamide (SDS / 12%) and transferred to nitrocellulose membranes (Hyboud ECL, American Pharmacia Biotech Inc., Piscataway, NJ, USA).

The membrane is incubated at 1: 1000 in a rabbit polyclonal antibody (Santa Cruz Biotechnology Inc.) specific for the subunit of the GABAA receptor or stored at 4 ° C. for 16 hours in a glutamic acid decarboxylase (GAD 65/67). do. Check in the observation system for ECL western blotting.

All statistical analyzes were performed using SigmaStat software. The ANOVA procedure was used to analyze the data.

To see the effect on mouse brain hippocampus, pentobarbital (40 mg / kg) and apigenin (50 mg / kg) were administered for 3 days. Apigenin significantly increased glutamic acid carboxylase (GAD) protein levels. No effect was seen in the remaining alpha, beta, and gamma subunits.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Apigenin isolated from thistle extract, characterized in that it is prepared by the following production method having the effect of inducing sleep through the interaction of Pentobarbital-induced sleep behavior, GABA receptors and GABAergic system 0.001 to A pharmaceutical composition for improving or treating insomnia, containing 50% by weight.
(a) drying thistle ( circium japonicum );
(b) adding 95% alcohol to the milk thistle prepared by step (a), extracting it, and concentrating under reduced pressure to obtain a crude crude extract;
(c) dissolving the crude extract obtained in step (b) in distilled water, repeating the above-described partitioning three times by mixing ethyl acetate, and then concentrating and drying the ethyl acetate extract under reduced pressure to obtain an ethyl acetate extract. step;
(d) performing silica gel flash chromatography on the ethyl acetate extract obtained in step (c), obtaining fractions and drying under reduced pressure; And
(e) adding methanol to the fraction obtained in step (d), filtering and performing high performance liquid chromatography (HPLC) to fractionate apigenin, an active substance represented by the following Chemical Formula 1 .
[Formula 1]

delete Apigenin isolated from thistle extract, characterized in that it is prepared by the following production method having the effect of inducing sleep through the interaction of Pentobarbital-induced sleep behavior, GABA receptors and GABAergic system 0.001 to A dietary supplement for the improvement of insomnia symptoms containing 50% by weight.
(a) drying thistle ( circium japonicum );
(b) adding 95% alcohol to the milk thistle prepared by step (a), extracting it, and concentrating under reduced pressure to obtain a crude crude extract;
(c) dissolving the crude extract obtained in step (b) in distilled water, repeating the above-described partitioning three times by mixing ethyl acetate, and then concentrating and drying the ethyl acetate extract under reduced pressure to obtain an ethyl acetate extract. step;
(d) performing silica gel flash chromatography on the ethyl acetate extract obtained in step (c), obtaining fractions and drying under reduced pressure; And
(e) adding methanol to the fraction obtained in step (d), filtering and performing high performance liquid chromatography (HPLC) to fractionate apigenin, an active substance represented by the following Chemical Formula 1 .
[Formula 1]

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