KR20210040340A - A composition for improving, preventing and treating of neurological disorders comprising Passiflora incarnata L. extract - Google Patents

Abstract

The present invention relates to a composition for alleviating, preventing or treating neurological diseases, and by containing an extract of a mixture of Passiflora caerulea leaves and Passiflora caerulea fruits in a weight ratio of 1 : 0.1 to 0.8 as an active component, it is possible to prevent, treat or alleviate neurological diseases such as Alzheimer's disease, Parkinson's disease, etc.

Description

A composition for improving, preventing and treating of neurological disorders comprising Passiflora incarnata L. extract}

The present invention relates to a composition capable of improving, preventing or treating neurological diseases by containing the Passion Flower (Passiflora incarnata L.) extract as an active ingredient.

Sleeping is an essential activity for all living things to live, and humans spend about a third of the day sleeping. You might think that when humans sleep, all organs stop working and take a break, but in reality this is not the case. During sleep, our brain consolidates our waking memories, makes our body relax, and analyzes potential risk factors.

Most adults sleep 7-8 hours a day to rest, but there are individual differences from person to person. For example, some people sleep only 4 hours a day without difficulty in doing their daily activities and can live a lively life, but some people sleep 8 hours a day to achieve this level of life.

Age is an important factor in determining how long you need to sleep, and newborns sleep 16-18 hours a day. However, the sleep time gradually decreases, and 12 hours is required for 2 years old and 8 hours for 13 years old. The amount of sleep required continues to decrease, and by the age of 50, an average of about 6 hours of sleep per day is required (Durand, 1998).

There are two main stages of sleep, shallow sleep and deep sleep, which are rapid eye movement sleep (active sleep) and non-rapid eye sleep, depending on whether there is rapid eye movement or not. It is divided into movement sleep, quiet sleep, and quiet sleep). When measuring the brain waves during REM sleep, beta waves appear when we are awake. At this time, the pupils under the eyelids move, and the heart rate, blood pressure, and breathing are very irregular. Also, if you dream at this stage, you can easily remember the content of your dreams.

In this non-REM sleep, there is almost no motion and the heart rate is stable. In addition, brain activity at this time is also very slow and regular. At this time, the pupils in the eyelids hardly move, and breathing is slow and regular, along with a comfortable facial expression.

During sleep, non-REM sleep and REM sleep activities continue to alternate, but the rate of REM sleep gradually decreases with age (de Weed & van den Bossche, 2003).

Professor Wilson at the University of MIT in the United States recorded the hippocampus activity of the mouse while inserting an electrocutting needle into the mouse's hippocampus (which plays a critical role in the processing of learning information) and training the mouse to navigate through a complex maze during the waking day. I did. And even while the mouse was sleeping, I recorded the activity of the hippocampus. Then, it was confirmed that the two activities showed similar patterns. In other words, the activity pattern of the hippocampus while the mouse is sleeping is as if the mouse remembers the daytime situation and then quickly repeats the activity of the hippocampus during the day while dreaming at night. REM) appeared only in sleep state.

Considering the results of the previous studies as described above, when a sleep disorder that increases REM sleep activity occurs, a problem of accumulation of fatigue occurs, and if accumulation of fatigue persists, not only memory decreases but also forgetfulness symptoms may become severe.

Therefore, there is a need for a natural substance capable of preventing, treating or improving symptoms such as memory loss and forgetfulness caused by lack of sleep.

Korean Registered Patent No. 1141439 Korean Patent Registration No. 1725979

An object of the present invention is to provide a pharmaceutical composition capable of preventing and treating neurological diseases by containing an extract of Passionflower (Passiflora incarnata L.) as an active ingredient.

In addition, another object of the present invention is to provide a food composition capable of improving or preventing neurological diseases by containing an extract of Passionflower (Passiflora incarnata L.) as an active ingredient.

The pharmaceutical composition capable of preventing and treating neurological diseases of the present invention for achieving the above object may contain an extract of a mixture in which passionflower leaves and passionflower fruits are mixed in a weight ratio of 1:0.1-0.8 as an active ingredient. .

The extract increases the protein expression of lba-1, DCX, and BDNF in vitro, and can reduce the expression of corticotropin secretion factor (CRF) and glucocorticoid receptor (GR).

The extract may be extracted by mixing a mixture with 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 neurological diseases include Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, schizophrenia, obsessive-compulsive disorder, rat syndrome, dementia, anorexia nervosa, anorexia nervosa, obesity, cerebral ischemic disease, neurodegenerative disease, or It may be selected from the group consisting of diabetic neuropathy.

In addition, the food composition capable of improving or preventing neurological diseases of the present invention for achieving the other objects described above contains an extract of a mixture of passionflower leaves and passionflower fruits in a weight ratio of 1:0.1-0.8 as an active ingredient. can do.

The composition of the present invention is very effective in improving, preventing or treating neurological diseases such as Alzheimer's disease and Parkinson's disease, and is not toxic, so it can be consumed in the form of food.

1A is an image of DCX-positive cells in the hippocampal tooth gyrus of Sham group, control group, PF 10 group and PF 50 group, FIG. 1B is an enlarged image of the black box of FIG. 1A, and FIG. 1C is Sham group, control group, PF Ki67 in the hippocampal tooth gyrus of group 10 and PF 50 Positive cell images, Figure 1D is an enlarged image of the black box of Figure 1C, Figures 1E, 1F, and 1G are DCX protein expression in the hippocampus treated with the control group and PF 50 group, respectively, GR protein in the hippocampus It is a graph showing the expression and the expression of the GR protein in the hypothalamus.
2A is a graph showing the change in body weight of the control group and PF 100 group, FIG. 2B is a graph showing the number of dietary intakes of the control group and the PF 100 group, and FIG. 2C is a graph measuring the accumulated dietary intake of the control group and the PF 100 group. 2D is a graph showing the daily water intake of the control group and the PF 100 group, FIG. 2E is a graph showing the serum 5-HT level of the control group and the PF 100 group, and FIG. 2F shows the melatonin level of the control group and the PF 100 group. This is the graph shown.
Figure 3A is a graph showing the dietary intake of the control and PF 100 group C57BL/6 mice, Figure 3B is a graph showing the water intake of the control and PF 100 group, Figure 3C is a graph showing the calorific value of the control and PF 100 group , Figure 3D is a graph showing the RER of the control group and PF 100 group, Figure 3E is a graph showing the activity of the control group and PF 100 group, Figures 3F to 3H are fat, body fluids, lean (Lean ) Is a graph.
4A is a Western blot showing the expression of DCX, COX-2, Iba-1, PaV, BDNF and Mela R1 in the hippocampus of the control group and PF 100 group, and the right graph is a graph of the expression of each protein.
4B is a Western blot showing the expression of CRF and GR in the hypothalamus of the control group and the PF 100 group, and the right graph is a graph showing the expression of each protein.
FIG. 5A is a view of staining the immune tissues of Iba-1 in the hippocampus of the control group and PF 100 group, FIG. 5B is a view of staining CA1, CA3 and DG of the control group and PF 100 group, and FIG. 5C is a control and PF 100 group. It is a graph measuring the ratio between the active and inactive forms of the group.
6A is a graph showing DCX-positive neuroblasts and pIkappaB-positive cells in the hippocampal gyrus of SD mice, which are control (Veh) and COX-2 inhibitor-administered groups, and FIG. 6B is a control (Veh) and COX-2 inhibitor-administered group tooth gyrus It is a graph showing the ratio of the number of neuroblasts in the SGZ of, and FIG. 6C is a graph showing the third or more branched dendrites in the SGZ of the control (Veh) and the COX-2 inhibitor-administered dentate gyrus.
FIG. 7A is a graph showing the time for the control group and the PF 100 group to find the target platform for each measurement period, FIG. 7B is a graph showing the distance to the target platform for the control group and the PF 100 group for each measurement period, and FIG. 7C is a control group. And PF 100 group is a graph showing the time spent in the platform quadrant, Figure 7D is a graph showing the ratio of the time spent in the platform quadrant of the control group and PF 100 group.

The present invention relates to a composition capable of improving, preventing or treating neurological diseases by containing an extract of a mixture in which passionflower leaves and passionflower fruits are mixed in a weight ratio of 1:0.1-0.8 as an active ingredient.

Hereinafter, the present invention will be described in detail.

The composition capable of improving, preventing or treating neurological diseases of the present invention contains an extract of a mixture of passionflower leaves and passionflower fruits in a weight ratio of 1:0.1-0.8 as an active ingredient.

The Passion Flower (Passiflora incarnata L.) 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, and are used as a non-toxic and non-inhibitory sedative of insomnia and anxiety, as well as relieve muscle spasms of asthma, epilepsy, nervous irritability, and colonic syndrome. It softens the inflammation.

The 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, neurological diseases cannot be improved, prevented or treated, and when the content of the passionflower fruit exceeds the upper limit, the effect of the present invention is not expressed at all and toxicity may be induced.

When the passionflower leaves and passionflower fruits are not used together, but 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 leaf 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 extract of the present invention has a meaning in a broad sense to include a processed product of a passion flower, for example, a passion flower powder, which is formulated so that the passion flower can be administered to an animal. Although the experiment was conducted with the passion flower extract in the present invention, it will be foreseen 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 "contained as an active ingredient" means including an amount sufficient to achieve the efficacy or activity of the extract. As an example, the extract of the present invention is used in a concentration of 10 to 1500 ㎍ / ㎖, preferably 100 to 1000 ㎍ / ㎖. Since the extract of the present invention is a natural product and does not have side effects on the human body even when administered in an excessive amount, the upper limit of the quantity of the 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 pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, Lubricating agents or flavoring agents can 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, trackcacanth 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.

Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and biocompatible, and include 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 injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Further, 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 a 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 unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by incorporating it into a multi-dose container. In this case, 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 improving or preventing memory loss and forgetfulness, containing a 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, gums, ice creams, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include not only the passionflower extract as an active ingredient, but also ingredients commonly added during food production, for example, protein, carbohydrates, fats, nutrients, flavoring agents 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, in addition to the passion flower extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included. .

The present invention provides a health functional food comprising a food composition for improving or preventing memory loss and forgetfulness 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 medicine or food for improvement, prevention or treatment of memory loss and forgetfulness. As described above, the passion flower extract may be used for improvement, prevention or treatment of memory loss and forgetfulness.

In addition, the present invention provides a method for improving, preventing or treating memory loss and forgetfulness, comprising administering an effective amount of passionflower extract to a mammal.

The term "mammal" as used herein refers to a mammal that is an object of treatment, observation or experiment, 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, which Includes an amount that induces relief of symptoms of the disease or disorder. The effective amount and frequency of administration for the active ingredient of the present invention may be changed according to 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 content 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 rate of secretion of the composition, duration of treatment, and drugs used concurrently. 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 0.001 g/kg to 10 g/kg when administered once to several times a day.

In the treatment method of the present invention, the composition comprising 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. It can be administered as.

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

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

A mixture of passionflower leaves and passionflower fruits in a weight ratio of 1: 0.25 was mixed with 60% ethanol, extracted for 2 hours at 80°C, filtered, concentrated, and spray dried to obtain passionflower extract (PF extract). .

<Test Example>

Mouse preparation

Many studies are being conducted to verify the effect as a composition for improving, preventing, or treating memory loss and forgetfulness, but most of the studies are using animals because there are limitations in studies involving humans. Among them, the test was performed using DBA/2 mice known to be essentially deficient in sleep function.

As experimental animals, 15 8-week-old DBA/2 male mice (average weight 22 g) and 8-week-old ICR male mice (average weight 24 g) were purchased from Samtako (Korea).

After acclimation 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 (light cycle from 7:00-19:00). The animals were provided to freely use normal food (2018S; Harlan, USA) and water, and distilled water or passionflower extract was orally administered daily for 4 days from the start of administration (day 1).

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

-4 groups of mice-

Sham group: ICR mice, distilled water (DW) 100 mg/kg _weight diet group 5 mice

Control: DBA/2 mice, distilled water (DW) 100 mg/kg _weight diet group 5 mice

PF 10: DBA/2 mice, Passion flower extract of Example 1 10 mg/kg _weight Diet group 5 mice

PF 50: DBA/2 mice, 50 mg/kg _weight diet group of Passion Flower extract of Example 1, 5 mice

The mice were sacrificed on the 4th day of administration, applied to the brain with a 4% paraformaldehyde solution, and fixed with the same fixative before use.

For the aquatic maze study, SD (Sprague-Dawely) mice were used instead of DBA/2 mice.

As 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 DBA/2 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 mouse were performed according to the ethical standards of the Institutional Animal Care and Use Committee (IACUC approval no. SCH16-0037) of Soonchunhyang University.

-2 groups of mice-

Control: SD mice, distilled water (DW) 100 mg/kg _weight diet group 5 mice

PF 100: SD mouse, Passion flower extract of Example 1 100 mg / kg _weight 5 mice in the diet group

To confirm whether administration of chronic PF extract affects intake, activity and metabolic changes, 8-week-old C57BL/6 mice were purchased from Charles River and analyzed for total behavioral and metabolic changes. Experimental animals were kept under the same conditions as DBA/2 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 mouse were performed according to the ethical standards of the Institutional Animal Care and Use Committee (IACUC approval no. SCH16-0037) of Soonchunhyang University.

-2 groups of mice-

Control: C57BL/6 mice, 8 mice in the diet group _weighing 100 mg/kg of distilled water (DW)

PF 100: C57BL/6 mice, Passion flower extract of Example 1 100 mg/kg _weight Diet group 8 mice

COX-2 inhibitor

To elucidate the correlation between COX-2 and neuroblast differentiation, experimental animals were randomly divided into two groups. It was divided into a physiological saline group and a COX-2 inhibitor (celecoxib) treatment group (each group n=5).

Saline (1 ml/100 g body weight) and the same volume of 20 mg/kg celecoxib (prescription formulation; Pfizer Inc.) were orally administered to mice using a needle daily from 2 weeks before sacrifice. Then, the brain was removed and immunohistochemistry was performed with chromosomal anti-DCX and mouse anti-phospho-IκBα to confirm the role of COX-2 in neurogenesis with hippocampal dentate gyrus. It was expressed as the ratio between the number of cells in and the number of tertiary branches of each neuroblastic dendrites.

Cytotoxicity by MTT assay

2 Eight PF extract solutions (using Passion flower extract 10 mg/kg _weight ^{) having various concentrations of 2} mg/ml to 2 ^-5 mg/ml were tested. Passion flower extract was dissolved in α-MEM medium (WELGEN, Gyeongsan-si, Korea) to obtain a maximum solubility of about 24 mg/ml and filtered through a 0.2 μm syringe filter (Corning, Germany).

The MTT solution was completely dissolved in 10 mg of MTT (Thiazolyl blue tetrazolium bromide, Sigma-Aldrich, MO, USA) in 2 ml of PBS pH 7.4 and filtered through a 0.2 μm syringe filter. Subsequently, the reagent was diluted with α-MEM medium to a final concentration of 0.5 mg/ml, and the solution was stored at 4° C. and protected from light.

In addition, after collecting bone marrow cells from the femur and tibia of the mouse and measuring the number of ^{cells, 2x10 5} cells/200 μl α-MEM medium was inoculated into each well of a 96-well plate for 48 hours under conditions of 37°C and 5% CO2. During incubation. The medium was aspirated, and 200 μL (control) of various concentrations of PF solution and PF-free α-MEM medium (control) were dispensed twice to each well. After incubation for 24 hours, all PF solutions were completely removed and cells were incubated with 100 μl of MTT solution for 4 hours in dark conditions.

When the purple precipitate was observed under a microscope, 100 μl of dimethyl sulfoxide (DMSO) was dispensed into each well to stop MTT activity, and the optical density was measured with Multi SKAN (Thermo Fisher, CA, USA) at 570 nm.

Worm culture (C. elegans culture)

The wild-type N2 strain was purchased from the C. elegans genetics center (CGC, Minneapolis/St. Paul, USA). Green fluorescent protein (GFP)-expressing strain, CL2070 (dvIs70 [Phsp-16.2:: GFP, rol-6]) was also purchased from CGC.

Worms are cultured on a nematode growth medium (NGM) agar plate (1.7% agar, 2.5 mg/mL peptone, 25 mM NaCl, 50 mM KH2PO4 pH 6.0, 5 μg/mL cholesterol, 1 mM CaCl2, 1 mM MgSO4) at 20°C. And Escherichia coli OP50 was supplied as a nutrient source to NGM plates.

By allowing 5 groups of young adult (L4) worms to lay eggs on fresh NGM plates at 20° C., worms with synchronicity of the same age were obtained. After 4 hours, all adult worms were removed from the plate and the eggs were kept at 20° C. for 3 days.

Oxidative stress resistance analysis using worms

Adult worms with age synchronicity were treated with different dilutions of PF extract (50 mg/L, 100 mg/L and 500 mg/L) in NGM plates for 24 hours. Diluted PF (100 μL each) was sprayed onto a solid NGM plate (5 mL). Thereafter, 12.5 mg/L of 5-fluoro-2'-deoxyuridiene (Sigma-Aldrich, St. Louis, USA) was added to the NGM plate to prevent internal hatching. Added. Next, the worms were exposed to cholesterol-free S-bsal 2 mM hydrogen peroxide (sodium chloride 5.85 g in 1 L of sterile distilled water, potassium dihydrogen phosphate 1 g, potassium phosphate 6 g).

The number of dead nematodes was measured after 6 hours exposure to hydrogen peroxide, and three independent repeated experiments were performed, and differences between groups were tested and analyzed using one-way ANOVA.

Life analysis using worms

Concurrent worms at the age of 60 were treated with PF extracts of different concentrations (50 mg/L, 100 mg/L, 500 mg/L) and 5-fluoro-2'-deoxyduridiene 12.5 mg/L. Transferred to NGM plate. Live worms were then transferred to fresh NGM plates every 2-3 days and counted daily until all worms died.

The lifetime of worms exposed to PF was compared with that of untreated control worms using a log-rank test.

Thermal shock stress resistance measurement using worms

Three-day-old worms with synchronicity by age were picked from NGM plates and transferred to NGM plates containing 500 mg/L PF. After 24 hours, in order to induce thermal shock stress, the worm was left in an incubator at 35° C. for 10 hours, and then the worm was transferred to 20° C. again.

The survival rate after 24 hours at 20 ℃ was compared in the control and N-acetylcysteine (NAC) treated worms. We used the standard twotailed Student's t-test for statistical analysis.

How to treat tissue in mice

For histology, mice were anesthetized with 1 g/kg urethane (Sigma-Aldrich, St. Louis, MO, USA) and percutaneously perfused with 0.1 M PBS (pH 7.4). After removing and fixing the brain in the same fixative at 25° C. for 12 hours, the brain tissue was preserved by infiltrating the brain tissue in 30% sucrose overnight. Sequentially, brain sections were cut from the coronal plane to a thickness of 30 μm using a cryostat (Leica, Wetzlar, Germany) and collected in a 6-well plate containing PBS for further processing.

The sections were processed under the same conditions so that immunohistochemical data could be compared between groups. The section is a rat brain map for each animal (Paxinos and Watson, 2006) and a mouse brain atlas between -3.00 to -4.08 mm post Bregma based on -1.82 to -2.46 mm posterior to Bregma (Franklin and Paxinos, 2008). Was chosen in relation to Ten portions separated by 90 μm from each other were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 minutes and 5% normal goat serum in 0.05 M PBS at 25° C. for 30 minutes.

Immunohistochemical staining for doublecortin (DCX), Ki67, COX-2 and pIkappaβ

Immunohistochemical staining was performed under the same conditions for each group to check whether the staining degree was correct. Immunohistochemical staining was performed using the protocol described in our previous study (Yi et al., 2011, 2010).

Sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 minutes and 10% normal goat or rabbit serum in 0.05M PBS for 30 minutes, and then diluted overnight at 4°C with goat anti-DCX antibody (1:500). , Santa Cruz Biotechnology, CA, USA) and rabbit anti-Ki67 (1:1000, Abcam, Cambridge, UK) after incubation with biotinylated rabbit anti-goat IgG (diluted 1:200, Vector, Burlingame, CA, USA) ), biotinylated goat anti-rabbit IgG (diluted 1:200, Vector, Burlingame, CA, USA) and streptavidin peroxidase complex (1:200 dilution, Vector, Burlingame, CA) at 25°C for 2 hours I did.

Neuroblasts (DCX positive cells) and Ki67 positice cells were counted in the granulation area (SGZ), and these are shown as bar graphs in FIGS. 1A and 1B.

Sections were obtained from rabbit anti-COX-2 antibody (1:200, Cayman, Ann Arbor, MI, USA) or mouse phospho-IκBα (1:500, SantaCruz Biotechnology, Santa Cruz, CA, USA) for 48 hours at 4°C. After culture, the sections were treated with biotinylated goat anti-rabbit IgG or anti-mouse IgG and streptavidin-peroxidase complex (1:200, Vector, Burlingame, CA) at 25° C. for 2 hours. . Subsequently, sections were visualized by staining with 3, 3'-dinobenzidine in 0.1 M Tris-HCl buffer (pH 7.2). After dehydration, the sections were placed on a gelatin-coated slide with Canada Balsam (Wako, Tokyo, Japan).

Immunohistochemical staining for microglial cells (Iba-1)

Freshly prepared brain sections were treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 minutes and then sequentially treated with 10% normal goat serum in 0.05M PBS for 30 minutes.

Treated with rabbit anti-Iba-1 (1:1,000, Chemicon, Temecula, CA) diluted overnight at 4°C and biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (1:200, Vector, Burlingame, CA). Sections were visualized using 3, 3'-daminobenzidine (3, 3'-daminobenzidine) in 0.1 M Tris-HCl buffer (pH 7.2). After dehydration, the sections were placed on a gelatin-coated slide with Canada Balsam (Wako, Tokyo, Japan).

Western blot

The hippocampus and hypothalamus fragments were removed from the skull of SD mice and homogenized with dissolved buffer (iNtRon Biotechnology, Seoul, Korea). Protein concentration was measured with a BCA kit (iNtRon Biotechnology), and total protein (20 μg/sample) was placed in each lane of 12% SDS-PAGE, electrophoresed, and then transferred to a PVDF membrane (Bio-Rad Laboratories, CA, USA). The membrane was then blocked with TBST [100mM Tris-HCl (pH 7.6), 0.8% NaCl and 0.1% Tween-20] containing 10% skim milk (BD Biosciences, CA, USA). The membrane is a primary antibody goat anti-DCX (Doublecortin, 1:200, SantaCruz Biotechnology, TX, USA), rabbit anti-glucocorticoid receptor (GR) (1:1,000, SantaCruz Biotechnology, TX, USA), rabbit anti-COX -2(1:1000, Cayman chemical, MI, USA), rabbit anti-CRF(1:200, ABBIOTEC, SD, USA), rabbit anti-melatonin receptor 1A(Mela R1A) (1:1,000, abcam), rabbit anti-parvalbumin (1:3,000, swant, Switzerland), rabbit anti-c-Fos (1:500, SantaCruz Biotechnology, TX, USA), rabbit anti-BDNF (1:3000, Proteintech, USA), rabbit anti-Iba1 (1:2,000, Wako, Osaka, Japan), mouse anti-tau (1:500, Calbiochem), rabbit antiphospho-tau (1:1,000, Cell Signaling Technology, MA, USA) and rabbit anti-GAPDH (glyceraldehyde 3- phosphate dehydrogenase, 1:5,000, Cell Signaling Technology, MA, USA) was incubated overnight at 4°C. Next, the membrane was washed and incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (Vector, CA, USA).

Immune response signals were detected through improved chemiluminescence (Abclon, Seoul, Korea) and recorded with the MicroChemi 4.2 system.

Monitoring behavioral and metabolic changes by indirect calorimetry

Metabolic performance parameters (energy intake and energy expenditure) were studied using Phenomaster®, an automated synthetic indirect calorimetry system (TSE System GmBH, Bad Homburg, Germany). Before the experiment, rats were acclimated for 2 days in a metabolic chamber containing food and water, and then oxygen consumption (VO2), carbon dioxide production (VCO2), and food intake were measured for 3 days. The room temperature for all metabolic studies was maintained at 22° C. with a 12 hour light and dark cycle. The respiratory rate (RER, VCO2/VO2) was calculated using standard in-house software.

Animal phenotypic measurements were performed using a Mini-spec LF50 (Bruker Biospins, The Woodlands, TX), followed by body composition (delicate tissue, fat, and body fluids of mice on a benchtop platform).

Measured with Maurice's underwater maze

The experiment was conducted by applying the existing Morris method. First, 1 hour before the start of the experiment, the mice were transferred to a behavioral observation room and allowed to stabilize. The dimensions of the maze are 90 cm in diameter and 32.5 cm in height, and the diameter of the white platform is 5 cm. Around the underwater maze, spatial cues such as a computer system connected to a video camera and a water temperature control device were kept constant. Then, the maze was filled with water and installed 1 cm below the height of the water so that the mouse could not see the platform. The maze was divided into four divisions using four markers, divided into northeast (NE), northwest (NW), southeast (SE), and southwest (SW), and a platform was installed in one quadrant of the maze. . The Morris underwater maze test is conducted for 5 days.On the first day, each mouse is allowed to swim freely in the maze for 1 minute so that they can adapt to the water.At this time, the platform was not installed, and on the 4th day from the second day. Until, each mouse was allowed to swim in the maze for 1 minute at 10 minute intervals 4 times a day. In the experiment method once for 4 days from the second day to the 4th day, a mouse that is already on the platform installed in the maze for 1 second within 1 minute finishes the experiment and cannot find the platform within 1 minute or does not climb on the platform for 10 seconds. After the end of the experiment, the mouse was artificially placed on the platform for 1 second and the experiment was terminated, and the position of the platform was fixed at the same place. On the 5th day, after the platform was removed from the maze, the time the mouse stayed in the branch where the platform was located was measured. To perform the memory test on the 5th day, the last day of the Morris underwater maze test, free swimming was performed for 60 seconds after removing the platform, and the learning curves of the one way ANOVA analyzed on days 2-4 were compared. On the 6th day, the percentage of time spent in the previously acquired platform quadrant was compared, and the higher the percentage of time spent in the platform quadrant, the higher the level of memory retention.

Data analysis

In order 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. The c-fos immune response structure image was 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 means 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. Improved DCX- and Ki67-positive cell expression in DBA/2 mice by short-term (3 days) administration of PF extract

1A is an image of DCX-positive cells in the hippocampal tooth gyrus of the Sham group, the control group, the PF 10 group and the PF 50 group (oral administration 3 days) (the upper image is a foreground of the hippocampal tooth gyrus of each group), 1B is an enlarged image of the black box of Fig. 1A (scale bar = 100 μm).

1C is Ki67 in the hippocampal tooth gyrus of the Sham group, the control group, the PF 10 group and the PF 50 group (oral administration 3 days). This is an image of positive cells, and FIG. 1D is an enlarged image of the black box of FIG. 1C.

1E, 1F, and 1G are graphs showing DCX protein expression in the hippocampus treated with the control and PF 50 groups, GR protein expression in the hippocampus, and GR protein expression in the hypothalamus, respectively.

1A to 1D, it was confirmed that DCX-positive neuroblasts and Ki67-positive cells (proliferative cells) were highly expressed in DBA/2 to which PF extract (10 mg/kg and 50 mg/kg) was administered. . DBA/2 (control) treated with distilled water only showed very low expression of DCX-positive cells, and the dendrites were not very good in the subgranular zone (SGZ) of the dentate gyrus.

DCX-positive neuroblasts in PF-treated mice were highly expressed or not developed in SGZ as in the Sham group (right enlarged image of FIG. 1A). Specifically, each cell dendritic process in the pituitary gland (SGZ) was highly branched (secondary and tertiary branching) in the Sham group, the PF group 10, and the PF group 50. However, it was confirmed that the control group showed a low number of neuroblasts and branching patterns in the pituitary region (SGZ) of the tooth gyrus (FIG. 1A).

It was confirmed that DCX and Ki67-positive cells in the PF-treated group were significantly higher than the vehicle-treated Sham group and the control group.

In addition, as shown in Figures 1E to 1G, as a result of comparing the neuroblastic cells (DCX) of the hippocampus with the glucocorticoid receptor (GR) known as a marker for regulating the HPA axis in the hippocampus and hypothalamus, mice treated with PF extract It was confirmed that there was a significant difference in the expression of neuroblasts and GR protein in the control group.

Test Example 2. Cytotoxicity

Compared to the control group, PF 10 group and PF 50 group were ^{diluted from 4 mg/ml to 3.125×10 -2} mg/ml by a 2-fold dilution method. According to the results, the PF 10 group and PF 50 group had no cytotoxicity at all concentrations, and significantly increased cell proliferation (P <0.0001), resulting in further increase in cell viability.

Test Example 3. Confirmation of increased resistance to oxidative stress induced by PF extract in pretty little nematode (C. elegans)

To investigate the effect of the PF extract on the response to oxidative stress, the survival of worms (pretty little nematodes) under oxidative stress conditions (exposed to H2O2 for 6 hours) according to the presence or absence of the PF extract was observed.

As a result, it was confirmed that the PF extract (50 mg/L, 100 mg/L, and 500 mg/L) increased the survival rate under oxidative stress compared to the control group (53.32±10.17 (PF 50), 56.63±20.28 (PF 100)). ) And 65.56±5.56 (PF 500) vs 42.22±6.19 (control)).

Test Example 4. Extending the lifespan according to PF extract treatment

Whether the PF extract can affect the lifespan of C. elegans was investigated. Treatment of PF extract significantly extended the average and maximum lifespan of C. elegans.

It was confirmed that the life extension effect when treated with 500 mg/L among the three different PF concentrations analyzed (PF 50 mg/L, 100 mg/L and 500 mg/L) was superior to 50 mg/L and 100 mg/L. . The average lifespan value of the pretty little nematode in the control group that was not treated with PF was 17.6 days, and the maximum life value was 27 days, whereas the average lifespan of the pretty little nematode treated with PF 500 mg/L was extended to 24.2 days, and the maximum lifespan. It was confirmed that the increase was increased to 34 days.

These results indicate that the concentration of the most effective PF extract on the oxidative stress and lifespan of C. elegans is 500 mg/L.

Test Example 5. Induction of aging-related genes by PF extract

Expression of the GFP reporter bound to the hsp-16.2 promoter correlates with the lifespan of the organism (Rea et al., 2005). This suggests that hsp-16.2 can be used as a molecular marker for predicting the lifespan of nematodes.

It was investigated whether the expression of hsp-16.2 was increased in pretty little nematodes treated with 250 mg/L of PF extract, and as a result, hsp-16.2::GFP reporter was remarkable by treatment with 250 mg/L of PF extract. It was confirmed that it was induced. More specifically, GFP fluorescence intensity was confirmed to be higher than that (mean ± SEM of 20 individual Caenorhabditis elegans) PF in the Caenorhabditis elegans treated with 3.130X10 ⁴ ± 1.378X10 ³ 1.372X10 ⁴ ± ³ 0.898X10 the control group.

Based on this result, if the control group was set to 100%, the PF treatment group (250 mg/L) was as high as 162.3%.

Test Example 6. Weight and feeding habits of mice during PF treatment

Figure 2A is a graph showing the change in body weight of the control group and PF 100 group, Figure 2B is a graph showing the number of dietary intakes of the control group and the PF 100 group (using the BioDAQ® system execution program), Figure 2C is the control group and the PF 100 group It is a graph measuring the accumulated dietary intake (using the BioDAQ® system execution program), and FIG. 2D is a graph showing the daily water intake of the control group and the PF 100 group.

2A to 2D, no significant difference in body weight was observed in the control group (Veh) and the PF 100 group even after 12 days, and the number of dietary intakes, accumulated dietary intake, and daily water consumption were also significant differences. Was not observed.

Test Example 7. Serum 5-HT and melatonin levels

Figure 2E is a graph showing the serum 5-HT levels of the control group and PF 100 group, Figure 2F is a graph showing the melatonin levels of the control group and PF 100 group.

2E and 2F, serum serotonin (5-HT) in mice was 954.0±88.31 ng/mL (control, Veh) and 1259.0±75.97 ng/mL (PF 100 group), respectively, and melatonin was 2.931, respectively. It was confirmed that it was ±0.282 pg/mL (control group, Veh) and 3.841±0.217 pg/mL (PF 100 group).

Compared to the control group, the serotonin level of the PF 100 group was 32.0% higher and the melatonin level was 31.0% higher.

Test Example 8. Automated phenotypic analysis of behavioral, physiological and metabolic data resulting from administration of PF extract

Figure 3A is a graph showing the dietary intake of the control and PF 100 group C57BL/6 mice, Figure 3B is a graph showing the water intake of the control and PF 100 group, Figure 3C is a graph showing the calorific value of the control and PF 100 group , Figure 3D is a graph showing the RER of the control group and PF 100 group, Figure 3E is a graph showing the activity of the control group and PF 100 group, Figures 3F to 3H are fat, body fluids, lean (Lean ) Is a graph.

8-week-old male C57BL/6 was acclimated in an automatic metabolic lung cage (TSE systems, Germany) for a week before the start of recording, and mice were measured and recorded every 5 minutes continuously for 12 days. The respiratory rate (RER) was estimated by calculating the ratio of VCO2/VO2 (RER; respiratory rate).

3A to 3E, the dietary intake (Kcal/kg/h) is 20.11±0.808 (control, Veh) and 17.84±0.465 (PF), respectively, and the water intake (mL/kg/h) is 5.843, respectively. ±0.441(Veh) and 6.235±0.750(PF), calorific value (Kcal/kg/h) is 17.10±1.093(Veh) and 17.84±0.465(PF), respectively, and activity is 745.5±50.45(Veh) and respectively, respectively. 756.3±84.49 (PF), VO2 (ml/kg/h) is 3525.0±208.8 (Veh) and 3324.0±107.3 (PF), respectively, and VCO2 (mL/kg/h) is 2894.0±243.2 (Veh) and 2696.0±147.2(PF), and the VCO2/VO2 ratio is 0.8114±0.0219(Veh) and 0.8024±0.215(PF), respectively.

As a result of examining all data automatically measured in the day and night cycle (Zeitgeber time; ZT) for 12 hours, it was confirmed that there was no significant difference between the control group (Veh) and the PF 100 group.

In addition, as shown in FIGS. 3F to 3H, it was confirmed that there was no significant difference in body composition for 12 days in the PF 100 group and the control group.

Test Example 9. Changes in protein expression in the neural environment in the hippocampus by administration of PF extract

4A is a Western blot showing the expression of DCX, COX-2, Iba-1, PaV, BDNF and Mela R1 in the hippocampus of the control group and PF 100 group, and the right graph is a graph of the expression of each protein.

4B is a Western blot showing the expression of CRF and GR in the hypothalamus of the control group and the PF 100 group, and the right graph is a graph showing the expression of each protein.

As shown in Fig. 4A, similar to the result of DBA/2 mice (Fig. 1), DCX significantly increased in mice, and PaV, GABA-related endonerve markers, BDNF, neuronal flexibility markers were significantly increased. . Melatonin levels were significantly increased in serum, and MelaR1 was also significantly increased in the hippocampus.

Although not statistically significant, the tendency of Iba-1, a microglial marker, was contrary to the level of COX-2, which plays an important role in the inflammatory process. For example, in the PF 100 group, COX-2 expression was increased, but Iba-1 expression was decreased.

In addition, as shown in Fig. 4B, it was confirmed that the expression of corticotropin secretory factor (CRF) and glucocorticoid receptor (GR) was significantly reduced in the hypothalamus of the PF 100 group.

In addition, as shown in Figs. 4C to 4E, microglial cells in the control group (Veh) are activated and have rounded and swollen cell bodies, whereas microglial cells in the PF-treated group are less activated and were identified as thin bodies. Microglia enlarged in other groups around the hippocampus, including rectangles CA1, CA3, and dentate gyrus (DG) with black lines.

Test Example 10. Iba-1 Immunohistochemistry

FIG. 5A is a view of staining the immune tissues of Iba-1 in the hippocampus of the control group and PF 100 group, FIG. 5B is a view of staining CA1, CA3 and DG of the control group and PF 100 group, and FIG. 5C is a control and PF 100 group. It is a graph measuring the ratio between the active and inactive forms of the group.

Microglial marker Iba-1 was subjected to immunohistochemical staining to measure microglial activity.

As shown in FIG. 5A, microglia in the control group (Veh) were activated and had round and swollen cell bodies, whereas microglia in the PF-treated group were less activated and were identified as thin bodies. Microglia enlarged in other groups around the hippocampus, including rectangles CA1, CA3, and dentate gyrus (DG) with black lines.

This ratio was proved by the bar graph of FIG. 5C, and it was confirmed that the control group and the PF-treated group showed a significant difference.

For example, it was confirmed that the protein expression of lba-1, DCX and BDNF was increased, and the expression of the corticotropin secretion factor (CRF) and glucocorticoid receptor (GR) was decreased.

Test Example 11. DCX expression in hippocampal tooth gyrus by COX-2 inhibition

FIG. 6A is a graph showing DCX-positive neuroblasts and pIkappaB-positive cells in the hippocampal gyrus of SD mice, which are control (Veh) and COX-2 inhibitor-administered groups, and FIG. 6B is a control (Veh) and COX-2 inhibitor-administered group. It is a graph showing the ratio of the number of neuroblasts in the SGZ of, and FIG. 6C is a graph showing the third or more branched dendrites in the SGZ of the control (Veh) and the COX-2 inhibitor-administered dentate gyrus.

As shown in Fig. 6A, DCX-positive neuroblasts and their dendrites were well developed in the control group (Veh), but not in the COX-2 inhibitor-administered group. In addition, pIkappaB positive cells were not seen in both the control group (Veh) and the COX-2 inhibitor administration group.

In the PF-treated group, the expression level of Iba-1 was low, but the expression of the inflammation marker COX-2 was higher than that of the control group (Veh). However, it was confirmed that COX-2 inhibitors showed significantly lower levels of neural tissue development than the control group when treated with normal mice, and COX-2 does not necessarily participate in neuronal inflammation, but shows a signal necessary for high neural tissue development.

Therefore, since PF extract can inhibit neuronal inflammation, it is judged that the PF-treated group increases cerebral cerebrality by inhibiting the activity of microglia in the brain without inducing neuroinflammation by COX-2.

In addition, as shown in Figs. 6B and 6C, it was confirmed that the ratio of the number of neuroblasts in the SGZ of the tooth gyrus and the branched dendrites of the third order or more showed a significant difference between the control group (Veh) and the COX-2 inhibitor administration group. .

Test Example 12. Water Maze Test (Morris Water Maze Test)

FIG. 7A is a graph showing the time for the control group and the PF 100 group to find the target platform for each measurement period, FIG. 7B is a graph showing the distance to the target platform for the control group and the PF 100 group for each measurement period, and FIG. 7C is a control group. And PF 100 group is a graph showing the time spent in the platform quadrant, Figure 7D is a graph showing the ratio of the time spent in the platform quadrant of the control group and PF 100 group.

As shown in FIG. 7, there was no difference between the control group and the PF 100 group in the time to find the platform (FIG. 7A) and the distance to the platform (FIG. 7B) on the first day, and both groups showed similar motive power and visual ability. Have. On the 2nd to 4th days, there was a difference in the time to find the platform between each group (FIG. 7A) and the distance to the platform (FIG. 7B), and on the 4th day, it was confirmed that the PF 100 group was significantly better than the control group.

In addition, on the 5th day, the last day, it was confirmed that the number of times (seconds) the mouse stayed at the place where the removed platform was placed was significantly superior to the control group on the 4th day (FIGS. 7C and 7D).

By measuring the acquisition of spatial memory by the passiflora extract of Example 1 and hippocampal-dependent learning including long-term spatial memory, when the passiflora extract of Example 1 is administered to an animal, neuroaggregation function is significantly increased and sleep function is essentially It was confirmed that the memory of deficient animals can be substantially improved.

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 tablets

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 manufacturing 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 in the amount of 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 formulations, add and dissolve each component in purified water, add an appropriate amount of lemon scent, mix the above ingredients, add purified water and add purified water to adjust the total to 100 g, then fill in a brown bottle for sterilization. To prepare a liquid formulation.

Formulation Example 6. Preparation of granules

1,000 mg of extract powder obtained in Example 1

The right amount of vitamin mixture

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 ㎍

10 mg of vitamin C

Biotin 10 ㎍

1.7 mg of nicotinic acid amide

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

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 also 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

900 mL total by adding purified water

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

The composition ratio is a mixture 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 (6)

A pharmaceutical composition for preventing or treating neurological diseases, characterized in that it contains an extract of a mixture of passionflower leaves and passionflower fruits in a weight ratio of 1: 0.1-0.8 as an active ingredient. The method of claim 1, wherein the extract increases the protein expression of lba-1, DCX, and BDNF in vitro, and decreases the expression of corticotropin secretion factor (CRF) and glucocorticoid receptor (GR). A pharmaceutical composition for preventing or treating neurological diseases, characterized in that. The pharmaceutical for preventing or treating neurological diseases according to claim 1, wherein the extract is extracted by mixing a mixture 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 Ever composition. The pharmaceutical composition for preventing or treating neurological diseases according to claim 3, wherein the mixed solvent is an aqueous solution of 20 to 80% by volume of methanol, ethanol, butanol, or propanol. The method of claim 1, wherein the neurological disease is Alzheimer's disease, Parkinson's disease, chronic stress-related depression, stroke, Huntington's chorea, schizophrenia, obsessive-compulsive disorder, rat syndrome, dementia, anorexia nervosa, anorexia nervosa, obesity, cerebral ischemic disease. , Neurodegenerative disease, or a pharmaceutical composition for preventing or treating neurological diseases, characterized in that selected from the group consisting of diabetic neuropathy. A food composition for improving or preventing neurological diseases, characterized in that it contains an extract of a mixture of passionflower leaves and passionflower fruits in a weight ratio of 1:0.1-0.8 as an active ingredient.

Images