KR20200134445A - A composition for the improvement hours of sleep comprising Passiflora incarnata extract - Google Patents

Abstract

The present invention relates to a composition for improving hours of sleep, which contains an extract of Passiflora incarnata as an active ingredient, and thus can be used for the treatment of insomnia by improving eye closure time.

Description

A composition for the improvement hours of sleep comprising Passiflora incarnata extract}

The present invention relates to a composition capable of treating insomnia by improving eye closure time by containing an extract of Passionflower (Passiflora incarnata) as an active ingredient.

As a result of a survey that most modern people have experience with sleep disorders, the number of people suffering from sleep disorders is increasing due to the social atmosphere or environment in which stress has increased rapidly.

These sleep disorders are also called sleep arousal disorders, and are classified into insomnia, hypersomnia, sleep awakening time disorder, and abnormal behaviors during sleep. Insomnia refers to a quantitative abnormality in which the tendency to sleep decreases, such as a sleep disorder that makes it difficult to fall asleep, a sleep disorder that does not allow you to sleep even once you fall asleep, and a sensational disorder that wakes up early in the morning. have. Hypersomnia refers to an increase in sleep tendency, and there are symptoms such as dehydration seizures, hallucinations at the time of elevation, dreams, sleep, paralysis, sleep seizures, and a periodicity that repeats the specular phase for about a week. There is also a mild sleep disorder. Sleep awakening time disorder refers to jet lag or sleep disturbance of night shift workers, and abnormal behavior during sleep causes sleep disorders due to diseases such as sleepwalking, night terrorism, and nocturia (夜尿症).

Among them, insomnia is the most common symptom, and most are primary insomnia, which is not caused by psychiatric problems, medical or neurological problems, or the use or discontinuation of drugs. When evaluating symptoms of insomnia, doctors generally use ① latency to sleep, ② duration of sleep, ③ unstable patterns of sleep, i.e., frequent nocturnal wakening events, and ④ sleep. It is evaluated by categorizing it into residual hangover effect, such as drowsiness, ⑤ impairment of cognitive and motor function, that occurs when waking from

It is thought that the cause of insomnia is excessive arousal, and it is thought that the activity of the upward reticular formation system is excessively activated even during sleep. In addition, it is thought that the formation of negative conditions for sleep causes primary insomnia, especially psychophysiological insomnia. This means that negative experiences that are difficult to fall asleep are repeated and learned, resulting in insomnia.

In the initial treatment for insomnia, central nervous system inhibitors such as barbiturate were commonly used. However, these compounds are known to have side effects such as lethargy, confusion and depression. Another drug treatment is zodiazepines sedative-hypnotic. However, this chemical species also has side effects such as development of resistance due to repeated administration and rebound insomnia due to discontinuation of administration. Other chemical treatments for insomnia include pyrazolopyrimidine-based hypnotics, zolpidem and zaleflon. However, most of the chemical drug treatments for insomnia developed up to now have side effects, and in particular, have a problem of causing serious side effects such as addiction symptoms to drugs when taken for a long time.

Therefore, there is a demand for a composition group of natural materials that can improve sleep time without side effects.

Korean Patent Registration No. 1961974 Republic of Korea Patent Publication No. 2019-0024593

It is an object of the present invention to provide a composition capable of treating insomnia by improving eye closure time by containing an extract of Passionflower (Passiflora incarnata) as an active ingredient.

The composition for improving sleep time of the present invention for achieving the above object may contain Passionflower (Passiflora incarnata) extract as an active ingredient.

The passionflower extract may be a mixture of passionflower leaves and passionflower fruit in a weight ratio of 1:0.1-0.8.

The passion flower extract may be extracted by mixing passionflower and water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof in a volume ratio of 1:1 to 100.

The mixed solvent may be an aqueous solution of 20 to 80% by volume of methanol, ethanol, butanol or propanol.

The passion flower extract may be administered once or repeatedly at 200 to 3000 mg/kg.

The composition for inducing sleep containing the passiflora extract of the present invention as an active ingredient is effective in improving, preventing or treating insomnia by increasing immobility time and eye closure time, so that it can be used as a functional food or pharmaceutical use.

Figure 1A is a graph showing the body weight of the control, PI 250 group and PI 500 group mice, Figure 1B is a graph showing the food intake of the control, PI 250 group, and PI 500 group mice, Figure 1C is a control group, PI 250 group and It is a graph showing the water intake of PI 500 group mice.
FIG. 2 is a diagram illustrating a measurement of a site of major action in the brain due to administration of distilled water, PI 250 mg/kg and PI 500 mg/kg by performing immunohistochemical staining with c-fos antibody.
Figure 3A is a fluorescence intensity showing the intracellular ROS production level when cultured with distilled water and PI extract at 6, 0.6 and 0.06 μg/mL, and Figure 3B is C6 rat glioma cells treated with distilled water and PI extract for 6 hours 3C is a graph measuring the cell viability of C6 rat glioma cells treated with distilled water and PI extract for 6 hours, and FIG. 3D is a graph measuring the cell viability of C6 rat glioma cells treated with distilled water and PI extract for 6 hours. This is a graph quantifying the expression of GABA receptor mRNA by PCR in C6 rat glioma cells.
Figure 4A is a graph showing melatonin levels after a single administration of distilled water, PI extract 250 mg/kg and PI extract 500 mg/kg, and Figure 4B is a graph showing melatonin levels after repeated administration of distilled water and PI extract 250 mg/kg .
Figure 5A is a graph showing the immobilization score after a single administration of distilled water, 250 mg/kg of PI extract and 500 mg/kg of PI extract, and FIG. 5B is an immobility after repeated administration of distilled water and 250 mg/kg of PI extract for 5 days. It is a graph showing the score, and FIG. 5C is a graph showing the numerical value of FIG. 5B as a curved area (AUC).
Figure 6A is a graph showing the eye closure time after a single administration of distilled water, PI extract 250 mg / kg and PI extract 500 mg / kg, Figure 6B is after repeated administration of distilled water and PI extract 250 mg / kg for 3 days It is a graph showing the eye closure time, and FIG. 6C is a graph showing the value of FIG. 6B as a curved area (AUC).
7 is a graph measured by HPLC of a PI extract extracted according to Example 1 of the present invention.

The present invention relates to a composition capable of treating insomnia by improving eye closure time by containing an extract of Passionflower (Passiflora incarnata) as an active ingredient.

Hereinafter, the present invention will be described in detail.

The composition for improving sleep time of the present invention contains Passion Flower (Passiflora incarnata) extract as an active ingredient.

The Passion Flower (Passiflora incarnata) is an evergreen perennial vine plant and may use Passion Flower flowers, leaves, stems, roots, and fruits. Native Americans used whole plants for swollen or irritated eyes, and the roots are commonly used as a tonic. In addition, leaves prevent rapid pulse, reduce high blood pressure, are non-toxic and are used as anxiety sedatives, as well as relieve asthma muscle spasms, epilepsy, nervous irritability, and colonic syndrome, and are used as poultices to soften burns and skin inflammation.

Passion flower extract of the present invention is an extract in which passionflower leaves and passionflower fruits are mixed in a weight ratio of 1:0.1-0.8, preferably 1:0.2-0.5. When the content of the passionflower fruit is less than the lower limit based on the passionflower leaf, the effect of improving the sleeping time cannot be expected, and when the content of the passionflower fruit exceeds the upper limit, the effect of the present invention may not be expressed at all and toxicity may be induced.

When the passionflower leaves and passionflower fruits are not used together, when used alone, the efficacy is 1.5 to 20 times lower than that of using the two substances together; When passionflower flowers, stems, and roots are used, the efficacy is 5 to 50 times lower than when the two substances are used together.

The mixture of passionflower, for example passionflower leaves and passionflower fruit, is mixed with an extraction solvent in a weight ratio of 1: 1 to 100, preferably 1: 5 to 20, and extracted at 70 to 100°C to prepare an extract. When the weight ratio of the mixture of the passionflower leaves and the passionflower fruit and the extraction solvent is out of the above range, the active ingredient of the passionflower leaf and passionflower fruit mixture may be extracted in a small amount in the extract.

The extraction solvent for extracting the extract is water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The extraction solvent is not particularly limited, but an extract extracted with 20 to 80 vol% of methanol, ethanol, butanol or propanol aqueous solution, preferably 20 to 80 vol% of ethanol exhibits excellent effects.

The term'extract' used in referring to the passion flower in the present specification includes not only the crude extract obtained by treating the extraction solvent, but also the processed product of the passion flower extract. For example, the passion flower extract may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the passion flower extract of the present invention has a meaning in a broad sense to include a processed product of a passion flower, such as a passion flower powder, formulated to administer a passion flower to an animal. Although the experiment was conducted with the passion flower extract in the present invention, it will be expected by those skilled in the art that the desired effect can be achieved even in the same form as the processed product of the passion flower.

Meanwhile, in the present specification, the term "containing as an active ingredient" means including an amount sufficient to achieve the efficacy or activity of the passion flower extract. For example, the passion flower extract is used at a concentration of 200 to 3000 mg/kg, preferably 200 to 2500 mg/kg, in a single or repeated administration. Passion flower extract is a natural product and does not have side effects on the human body even if it is administered in an excessive amount. Therefore, the upper limit of the quantity of the passion flower extract contained in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

The composition of the present invention may be used as a pharmaceutical composition for improving sleep time, and may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may be an excipient, boron Releases, sweeteners, binders, coatings, expanding agents, lubricants, lubricants or flavoring agents may be used.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Furthermore, it can be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration. .

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually, the skilled practitioner can readily determine and prescribe the dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention may be prepared in a unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a food composition for inducing sleep containing the passionflower extract as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gum, ice cream, vitamin complexes, health supplements. Etc.

The food composition of the present invention may contain not only the passionflower extract as an active ingredient, but also ingredients commonly added during food production, for example, protein, carbohydrates, fats, nutrients, seasonings and flavoring agents. . Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts, in addition to the passion flower extract of the present invention. .

The present invention provides a health functional food comprising a food composition for improving sleep time comprising the passion flower extract as an active ingredient. Health functional foods are foods prepared by adding passionflower extract to food materials such as beverages, teas, spices, gums, confectionery, or encapsulated, powdered, or suspension. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of passionflower extract added in such health functional foods cannot be uniformly regulated depending on the type of health functional food to be targeted, but can be added within the range that does not damage the original taste of the food. It is in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In addition, the present invention provides a use of a passionflower extract for the manufacture of a drug or food for improving sleep time. As described above, the passion flower extract can be used for improving sleep time.

In addition, the present invention provides a method for improving sleep time comprising administering an effective amount of passionflower extract to a mammal.

The term "mammal" as used herein refers to a mammal that is the subject of treatment, observation or experimentation, and preferably refers to a human.

The term "effective amount" as used herein refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician, Includes an amount that induces relief of symptoms of the disease or disorder. The effective amount and the number of administrations for the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. It can be adjusted according to various factors including condition, sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used simultaneously. In the prophylaxis, treatment or improvement method of the present invention, in the case of an adult, it is preferable to administer the passionflower extract at a dose of 200 to 1500 mg/kg when administered once to several times a day.

In the treatment method of the present invention, the composition containing the passionflower extract as an active ingredient is a conventional method through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. Can be administered.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention, but the following examples are only illustrative of the present invention, and it is obvious to those skilled in the art that various changes and modifications are possible within the scope and spirit of the present invention, It is natural that such modifications and modifications fall within the appended claims.

Example 1. Passion flower leaves: Passion fruit = 1: 0.25 weight ratio extract (PI extract)

A mixture of passionflower leaves and passionflower fruits in a weight ratio of 1:0.2 was mixed with 50% ethanol, extracted for 4 hours at 80°C, filtered, concentrated, and spray dried to obtain passionflower extract (PI extract). (Fig. 7).

<Test Example>

Mouse preparation

In order to verify the effect as a composition for improving sleep time, animals were used because there are limitations in studies involving humans.

As experimental animals, 24 7-week-old ICR male mice (weight 24 to 27 g) were purchased (Saeron Bio, Korea). The mice are free of specific diseases, pathogens or genetic defects.

After acclimating for 1 week before the start of the experiment, they were reared at room temperature (22±1 ℃) and 60% humidity under a 12 hour light cycle from 7:00-19:00. The animals were provided with free access to normal food (2018S; Harlan, USA) and water. The mice were sacrificed on the 2nd or 6th day after starting to administer the test substance.

All test substances used in this experiment were administered orally at the same time every day, and all procedures related to the mice were performed according to the ethical standards of the Institutional Animal Care and Use Committee of Soonchunhyang University (IACUC approval no.SCH16-0037).

-3 groups of mice-

Control group (Veh): distilled water (DW) 700 mg/kg _weight /day diet group 8 animals

PI 250 group (PI 250): Passion flower extract of Example 1 250 mg/kg _weight /day 8 mice in single dose diet group

PI 500 group (PI 500): Passion flower extract of Example 1 500 mg/kg _weight /day repeated administration diet group 8 mice

The ICR mice had too small eyes to measure the eye closure time, so SD (Sprague-Dawely) mice were used instead of the mice.

As for the experimental animals, 24 SD male mice (weight 175-225 g) of 7 weeks old were purchased (Saeron Bio, Korea), and were stored under the same conditions as ICR mice in other rooms.

All test substances used in this experiment were administered orally at the same time every day, and all procedures related to the mice were performed according to the ethical standards of the Institutional Animal Care and Use Committee of Soonchunhyang University (IACUC approval no.SCH16-0037).

In the present invention, single administration means that the following concentration is administered at once, and repeated administration means that the following concentration is administered for 5 days.

-3 groups of mice-

Control group (Veh): distilled water (DW) 700 mg/kg _weight /day diet group 8 animals

PI 250 group (PI 250): Passion flower extract of Example 1 250 mg/kg _weight /day administration 8 mice in the diet group

PI 500 group (PI 500): Passion flower extract of Example 1 500 mg/kg _weight /day administration diet group 8 mice

HPLC analysis

Passion flower extract powder (1 g) of Example 1 was dissolved in 50 ml of a 50% ethanol aqueous solution, sonicated for 10 minutes, and then filtered through a 0.45 μm syringe filter. HPLC was performed using an Agilent 1200 system equipped with a model G1312A binary LC pump, auto sampler and diode array detector. A C-18 (Waters, SunFire TM C18) column (250 mm X 4.6 mm id and 5 μm particle size) was selected, and the standard was purchased from Sigma Chemicals (St. Louis, MO, USA).

Chromatographic separation was performed with a mobile phase composed of 0.1% phosphoric acid prepared in nanopure water (87%) and 100% acetonitrile (13%) for 60 minutes, and the injection volume was 20 μL. The mobile phase flow rate is 1 ml min-1, the oven temperature is 35° C., and the detection wavelength is 360 nm.

Immunohistochemical staining of active regions in the brain after administration of PI extract with c-fos antibody

Immunohistochemical staining was performed using conventionally used protocols. Briefly, sections were treated with PBS containing 0.3% hydrogen peroxide for 30 minutes at room temperature (RT) and sequentially with 10% normal goat or rabbit serum under 0.05 M PBS at room temperature for 30 minutes. Then, goat anti-c-fos antibody (1:500, Santa Cruz biotechnology, CA, USA) diluted at 4°C was incubated overnight, followed by biotinylated goat anti-goat IgG and streptavidin peroxidase complex (1:200 dilution). , Vector, Burlingame, CA, USA) at 25° C. and incubated for 2 hours.

Sections were rabbit anti-COX-2 antibody (1:200, Cayman, Ann Arbor, MI, USA) or mouse phospho-IκBα (1:500; SantaCruz Biotechnology, Santa Cruz, CA, USA) at 4° C. for 48 hours. Incubated overnight. Then, the sections were incubated for 2 hours at 25° C. in a biotinylated goat anti-rabbit IgG or anti-mouse IgG and streptavidin-peroxidase complex (1:200, Vector, Burlingame, CA, USA). Subsequently, the sections were visualized by staining with 3, 3'-dinobenzidine in 0.1M Tris-HCl buffer (pH 7.2).

After dehydration, the sections were placed on a gelatin-coated slide with Canada Balsam (Wako, Tokyo, Japan).

Cell culture and reagents

Rat C6 glioma cell line (C6, ATCC-CCl-107) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and C6 glioma cell line was 10% (v/v) heat-inactivated fetus. Dulbecco's modified eagle medium high glucose (DMEM, 4.5 g/L glucose; Corning, NY, USA) with bovine serum (Gemcell, GEMINI Bioproducts, USA) and 1% (v/v) penicillin-streptomycin solution ( Thermo Fisher Scientific, Waltham, MA, USA) in a CO ₂ incubator (Thermo Fisher Scientific; 95% air and 5% CO ₂ ) at 37°C.

Cell viability analysis

Cell viability was measured using a water-soluble tetrazolium salt-1 (WST-1) assay kit (DoGenBio, Seoul, Korea). Rat C6 glioma cells were inoculated in 100 mL medium at a density of 3X10 ⁵ cells/well in a 96 well plate (SPL Life Sciences, Pocheonsi, Gyeonggi-do, Korea). The cells were incubated overnight at 37° C. in a 5% CO2 incubator, and then treated with PI extract at a concentration of 0.06 μg/mL for 24 hours. Then, 10 mL of EZ cytox cell viability measurement reagent was added to each well and incubated in a 5% CO2 incubator at 37°C for 2 hours. Finally, the optical density was measured at 450 nm using a micro plate reader (SunriseTM, Tecan). All experiments were repeated at least 3 times.

Rat C6 glioma cells were cultured with PI extracts of different concentrations (6, 0.6 and 0.06 μg/mL), and a concentration with 50% reduction in cell viability was calculated. Inhibitory concentration (IC50) was measured with GraphPad PRISM Version 7.0.1 (GraphPad Software, San Diego, CA, USA).

Measurement of intracellular ROS

Intracellular ROS levels were measured using the ROS indicator fluorogenic dichlorofluorescein diacetate (H2DCFDA, Invitrogen, Carlsbad, CA). Rat C6 glioma cells were cultured in a 37° C., 5% CO2 incubator for 6 hours, and then treated with PI extracts at concentrations of 6, 0.6, and 0.06 μg/mL for 6 hours. DW was used as a vehicle.

Cells were obtained, washed twice with PBS, analyzed using Guava EasyCyte Flow Cytometer (Millipore, Temecula, CA), and median fluorescence intensity (MFI) using FlowJo Version 10.0.7.2 (TreeStar, Ashland, OR, USA). ) Was measured. All experiments were repeated 3 times.

Rat C6 glioma cells were plated and treated with PI extract as in flow cytometry, and 10 μM H2DCFDA was added to each plate and incubated for 30 minutes at 37° C. and 5% CO2. After washing three times with PBS, cells were visualized using a DMi8 fluorescence microscope (Leica, Deerfield, IL, USA). Images were acquired with LAS X (Leica) and Photoshop CC 2017 program (Adobe system).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Rat C6 glioma cells were coated on a 60 mm plate and incubated for 6 hours in a 5% CO2 incubator at 37°C. Cells were treated with PI extract at a concentration of 6 μg/ml for 6 hours.

Isol-RNA Lysis reagent (2302700, SPRIME, Gaithersburg, MD, USA) was used to isolate total RNA, and MMLV reverse transcriptase (RT001, Enzynomics, Yuseong-gu, Daejeon, Korea) was added to RNA (11 μg), 1 μL of 10 pM oligo(dT) primer, 1 μL of dNTP mixture (2 mM each), followed by generation of standard supplement DNA (cDNA), 0.1% (v/v) DEPC-treated water (DB0154, BioBasic, Markham, ON, Canada) was added to a 200 μL PCR tube (Gunster biotech, USA) in a final volume of 6.5 μL. The mixture was incubated at 42° C. for 5 minutes and immediately cooled on ice, and then 10 μl MMLV-RT buffer 2 μl, MMLV reverse transcriptase 1 μl and RNase inhibitor (M007, Enzynomics) 0.5 μL were added.

Finally, SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA) was used to incubate at 42° C. for 50 minutes and then at 95° C. for 5 minutes. For qRT-PCR, SYBR green (DQ385-40H, Biofact, Yuseong-gu, Daejeon, Korea), designed to detect Gabra4, Gabbr1, Gabbr2, Gabrg2, GAT1, GAT3, Slc32a1, and GAD1 mRNA, 3 μL DW, 2 x real-time PCR Master Mix containing cDNA (10 μg) and 1 μL of primer (containing 10 μM each of the forward and reverse primers) was added to an optical 96-plate (Applied Biosystems). In order to amplify the PCR material, pre-denaturation was performed by treatment 40 times at 95°C for 10 minutes, 95°C for 15 seconds and 60°C for 1 minute, and PCR was performed using the StepOne Plus real-time PCR system (Applied Biosystems).

The primer sequences used in the present invention are shown in [Table 1]. All experiments were repeated at least three times, and relative gene expression levels were analyzed using the 2 ^-△△Ct method described by Livaket et al.

[Table 1]

Immobilization time for single and repeated administration of PI extract

PI extract was orally administered to 7-week-old ICR (Saeron Bio, Korea) mice, and the immobility time of the experimental animals was measured. Veh (control), PI 250 mg/kg (PI 250 group), or PI 500 mg/kg (PI 500 group) was orally administered to mice in each group as a single or repeated administration. 30 minutes after oral administration, the immobility time of the mouse was recorded for 1 hour, and recording was performed using a digital camcorder (HDR-CX405, SONY). After video recording, the immobility time of the mouse was separated by 30 seconds and analyzed visually. For objectivity, two observers performed measurements at random.

If there is no movement for 30 seconds, 10 seconds will score 1 point, if 20 seconds or 30 seconds, 2 or 3 points will be scored respectively.

Eye closure time according to single and repeated administration of PI extract

Since there was a technical problem that the movement of the eyes of ICR mice could not be easily recognized by video taking, PI extract was administered once or repeatedly to SD mice to measure the eye closure time. In order to gain additional confidence in the administration of PI to animals, SD mice were repeatedly administered PI extract for 3 days and then the eye closure time was compared with the control group.

Veh (control), PI 250 mg/kg (PI 250 group), or PI 500 mg/kg (PI 500 group) was orally administered to mice in each group as a single or repeated administration. 30 minutes after oral administration of the PI extract, the limb movements of SD mice were recorded for 10 minutes. Recording was performed using a digital camcorder (HDR-CX405, SONY), and the eye closure time of each SD mouse after video recording was visually analyzed at 10 second intervals. For objectivity, two observers performed measurements at random.

Data analysis

To ensure objectivity, two observers per experiment under the same conditions performed all conditions as blind conditions. For quantification of the immune response, the degree of staining was measured using 5 sections per each experimental group. C-fos immune response structure images were taken using a BX53 optical microscope (Olympus, Japan) equipped with a digital camera (DP71, Olympus) connected to a computer and a monitor.

Data shown represent the experimental mean ± standard error (SE) for each experimental group. Differences between the mean values were analyzed by one-way ANOVA, where appropriate, t-test and/or unpaired t-test.

Statistical analysis was performed using JMP9 (SAS Institute, USA) and Prism7 (GraphPad Software, San Diego, CA, USA). P values (*: <0.05, **: <0.005, ***: <0.0005) were considered statistically significant.

Test Example 1. Measurement of physiological changes after administration of PI extract

Figure 1A is a graph showing the body weight of the control, PI 250 group and PI 500 group mice, Figure 1B is a graph showing the food intake of the control, PI 250 group, and PI 500 group mice, Figure 1C is a control group, PI 250 group and It is a graph showing the water intake of PI 500 group mice.

For safety as a functional food, PI extract should not affect food intake or weight change other than the effect of improving the sleeping time of animals.

1A to 1C, it was confirmed that the control group, the PI 250 group, and the PI 500 group did not change their body weight, food intake, or water consumption. These results did not show any significant difference between the PI 250 group and the PI 500 group and the control group.

Therefore, PI extract does not affect the basic functions of animals.

Test Example 2. After administration of PI extract, check the active area in the brain by immunohistochemical staining using c-fos antibody

FIG. 2 is a diagram illustrating a measurement of a site of major action in the brain due to administration of distilled water, PI 250 mg/kg and PI 500 mg/kg by performing immunohistochemical staining with c-fos antibody.

The black square represents the RMM of MB (MB: Mamillary body; RMM: Retromammillary nu medial). The RMM is known to be highly related to sleep, and it appears that after the administration of PI extract, primary activity occurs at the RMM site, allowing the animal to sleep.

The entire brain tissue was incised at 20 μm intervals, and immunohistochemical staining was performed with anti-c-fos. Images of the control group, the PI 250 group, and the PI 500 group were obtained around the MB (brain diagram, right view) from bregma -2.54 mm to bregma -2.80 mm.

As shown in FIG. 2, in the PI 250 group and the PI 500 group, c-fos-positive cells were found around the MB (mammillary body), while the control group confirmed that there was no c-fos signal around the MB.

A number of c-fos positive cells were observed around MB in the PI 250 group and PI 500 group, but not in the control group.

Test Example 3. ROS production and cell viability measurement after treating rat C6 glioma cells with PI extract

3A is a fluorescence intensity showing the intracellular ROS production level when distilled water and PI extract are cultured at 6, 0.6 and 0.06 μg/mL (fluorescence intensity is measured by flow cytometry), and FIG. 3B is with distilled water and PI extract. It is a graph quantifying the change in DCF fluorescence intensity of C6 rat glioma cells treated for 6 hours, Figure 3C is a graph measuring the cell viability of C6 rat glioma cells treated for 6 hours with distilled water and PI extract (WST- 1 use of analysis).

The ROS detector H2DCFDA can penetrate the non-fluorescent cell membrane, and when it enters the cell through the cell membrane, it reacts with an esterase or HO- and is hydrolyzed to DCFH to show non-fluorescence. Glioma cells were treated with PI extract for 6 hours, and then the intracellular ROS level was measured.

Cell viability was measured using an assay based on WST-1 that produces an orange formazan product that can be detected by a spectrophotometer according to mitochondrial dehydrogenase activity, and at this time, rat C6 glioma cells were cultured with PI extract for 6 hours. I did.

3A and 3B, the relative ROS production (DCX-MFI) value when the concentration of the PI extract of the cells treated with the PI extract was 6 and 0.6 μg/mL was significantly increased compared to the control cells. .

In addition, as shown in Figure 3C, when the concentration of the PI extract 0.06 to 6 ㎍ / ㎖ it was confirmed that does not significantly affect the cell viability. Specifically, the survival rate of C6 glioma cells significantly decreased after treatment with the PI extract at a concentration of 60 μg/ml, and treatment with other concentrations did not show any significance.

Test Example 4. Quantification of GABAa receptor and related gene mRNA expression by real-time polymerase chain reaction (PCR)

3D is a graph quantifying GABA receptor mRNA expression by PCR in C6 rat glioma cells treated with distilled water and PI extract (0.6 μg/mL) for 6 hours.

There are several different subtypes of GABA receptors, including Gabra1 and Gabra4 as alpha subtypes, Gabbr1 and Gabbr2 as beta subtypes, GAT1, GAT3 and Slc32a1 as GABA transporters, and GAD as GABA synthase.

As shown in FIG. 3D, in the PI extract-treated group, the mRNA expression levels of Gabra1 and Gabra4 (α-subtype) were lower than that of the control group, and Gabbr1 and Gabbr2 (beta-subtype) were significantly increased compared to the control group. In addition, in the PI extract-treated group, the expression of Gabrg2 mRNA, a gamma-subtype, was significantly increased compared to the control group.

However, GAT3 expression was significantly different between the PI extract-treated group and the control group, but the mRNA expression of GAT1 and Slc32al GABA transporter was not significantly different between the PI extract-treated group and the control group.

In addition, GABA synthase GAD1 showed a significant difference between the PI extract treatment group and the Veh treatment group.

As a result, it can be seen that the PI extract increases the mRNA expression of GABA receptors and related genes, and shows the ability to inhibit neuronal activity. Interestingly, it has been reported that GABA activity is greatly affected by ROS expression, and the increase or decrease of ROS expression is not only related to cell damage or expression of intracellular cytokines, but also acts as an important factor required for the activity of GABA.

The above results confirmed that the PI extract decreased the α-subtype of the GABA receptor, but increased the β and γ-subtypes, and increased GABA transport GAT1 and GAT3. The expression of Slc32al was not affected by the PI extract.

The GABA transporter plays a role in promoting GABA synthesis by increasing GAD1, so PI extract enhances the receptor β and γ-subtypes as ligands rather than α-subtype receptors and activates GAD1 through some GABA transporters (GAT1 and GAT3). It is expected to exert its efficacy by promoting GABA synthesis through. The β and γ-subtypes of ligand receptors contribute greatly to synaptic transmission due to the GABA-induced sleep-inducing effect on the hypothalamus.

Test Example 5. Measurement of serum melatonin level after oral administration of PI extract once or repeatedly

Figure 4A is a graph showing the melatonin level after a single administration of distilled water, PI extract 250 mg / kg and PI extract 500 mg / kg, Figure 4B is a graph showing the melatonin level after repeated administration of distilled water and PI extract 250 mg / kg .

As shown in Figures 4A and 4B, it was confirmed that the serum melatonin level was 7.32±0.612 in the control group or 8.37±0.858 in the PI 250 group, and 10.92±0.694 in the PI 500 group after administration of a single administration of the control or PI extract. That is, it was confirmed that there was a significant difference between the control group and the PI 500 group.

In addition, it was confirmed that the serum melatonin levels were 7.82±0.585 and 12.00±1.600, respectively, after administration of the control group and the PI extract at 250 mg/kg repeatedly. That is, it was confirmed that there was a significant difference between the control group and the PI 250 group.

Test Example 6. After orally administering PI extract once or repeatedly Dead time measurement

Figure 5A is a graph showing the immobilization score after a single administration of distilled water, 250 mg/kg of PI extract and 500 mg/kg of PI extract, and FIG. 5B is an immobility after repeated administration of distilled water and 250 mg/kg of PI extract for 5 days. It is a graph showing the score, and FIG. 5C is a graph showing the numerical value of FIG. 5B as a curved area (AUC).

As shown in Figure 5A, it was confirmed that the immobility time of mice after single oral administration of the PI extract at a concentration of 250 mg/kg (PI 250 group) or 500 mg/kg (PI 500 group) was extended compared to the control group. I did. However, compared with the control group, the PI 250 group did not significantly extend, the PI 500 group significantly extended, and the PI 500 group's immobility score was significantly longer than the PI 250 group.

In addition, as shown in FIG. 5B, it was confirmed that the immobility score when the PI extract was repeatedly orally administered at a concentration of 250 mg/kg (PI 250 group) for 5 days was continuously higher than that of the control group from the start of administration.

In addition, as shown in Figure 5C, the AUC showed a significant difference between the PI 250 group and the control group.

These results are the same as those of the following eye closure time test.

Test Example 7. Measurement of eye occlusion time after oral administration of PI extract once or repeatedly

Figure 6A is a graph showing the eye closure time after a single administration of distilled water, PI extract 250 mg / kg and PI extract 500 mg / kg, Figure 6B is after repeated administration of distilled water and PI extract 250 mg / kg for 3 days It is a graph showing the eye closure time, and FIG. 6C is a graph showing the numerical value of FIG. 6B as a curved area (AUC).

The cumulative minutes of eye occlusion time of SD rats were measured from 30 minutes to 1 hour after oral administration of PI extract and distilled water.

As shown in Figure 6A, after a single oral administration of the PI extract at a concentration of 250 mg/kg (PI 250 group) or 500 mg/kg (PI 500 group), the eye occlusion time of SD rats was longer than that of the control group. Confirmed. Moreover, it was confirmed that the eye closure time of the PI 500 group was significantly higher than that of the PI 250 group.

In addition, as shown in Figs. 6B and 6C, it was confirmed that the group to which the PI extract was repeatedly orally administered for 3 days at a concentration of 250 mg/kg (PI 250 group) had a higher eye occlusion time of SD rats compared to the control group. From the 2nd day, it was confirmed that the eye closure time gradually increased in the PI 250 group than in the control group.

The curve part (AUC) between the PI 250 group and the control group also showed a significant difference.

The present invention confirmed that it is impossible to explain all the mechanisms by which PI extract causes sleep, but it is closely related to the regulation of GABA, which is the most basic mechanism, and that the active site is limited to RMM of the maternal fluid of the brain. Repeated administration of PI 250 mg/kg (PI 250 group) did not cause significant changes in animal body weight or abnormal feeding behavior.

In addition, single or repeated administration of PI 250 mg/kg (PI 250 group) can increase sleep time, and PI 500 mg/kg (PI 500 group) can increase sleep time in mice with a single administration.

Hereinafter, examples of the formulation of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit this, but is intended to be described in detail.

Formulation Example 1. Preparation of powder

500 mg of extract powder obtained in Example 1

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2. Preparation of tablet

300 mg of extract powder obtained in Example 1

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3. Preparation of Capsule

200 mg of extract powder obtained in Example 1

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

Formulation Example 4. Preparation of injection

600 mg of extract powder obtained in Example 1

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above ingredients per ampoule according to a conventional injection preparation method.

Formulation Example 5. Preparation of liquid formulation

4 g of extract powder obtained in Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of liquid preparation, add each ingredient to purified water to dissolve it, add lemon zest, mix the above ingredients, add purified water and add purified water to adjust the total to 100g, then fill in a brown bottle for sterilization. To prepare a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of extract powder obtained in Example 1

Vitamin mixture right amount

Vitamin A acetate 70 ㎍

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

1.7 mg of nicotinic acid amide

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

1.75 mg ferrous sulfate

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for granules, but it is possible to arbitrarily modify the mixing ratio. After mixing the above ingredients according to a conventional granule preparation method, granules And can be used to prepare a health functional food composition according to a conventional method.

Formulation Example 7. Preparation of functional beverage

1,000 mg of extract powder obtained in Example 1

1,000 mg citric acid

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

Total 900 mL with purified water

After mixing the above ingredients according to a normal health drink manufacturing method, stirring and heating the resulting solution at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and stored in a refrigerator. It is used to prepare the functional beverage composition of the invention.

The composition ratio is composed of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences, such as the demand class, the country of demand, and the purpose of use.

Claims (5)

Passion flower (Passiflora incarnata) composition for improving sleep time, characterized in that it contains the extract as an active ingredient. The composition for improving sleep time according to claim 1, wherein the passionflower extract is a mixture of passionflower leaves and passionflower fruits in a weight ratio of 1:0.1-0.8. The composition for improving sleep time according to claim 1, wherein the passion flower extract is extracted by mixing a passion flower, water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof in a volume ratio of 1:1 to 100 . The composition for improving sleep time according to claim 1, wherein the mixed solvent is an aqueous solution of 20 to 80% by volume of methanol, ethanol, butanol or propanol. The composition for improving sleep time according to claim 1, wherein the passionflower extract is administered once or repeatedly at 200 to 3000 mg/kg.

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