KR20200001977A - A composition for improving preventing and treating of cognitive and failure of ones memory comprising Passiflora incarnata L. extract - Google Patents

Abstract

The present invention relates to a composition for alleviating, preventing or treating hypomnesis and forgetfulness, wherein the composition contains a Passiflora caerulea extract as an active component, to prevent, treat or alleviate symptoms such as hypomnesis, induction of forgetfulness and the like caused by lack of sleep.

Description

Composition for improving, preventing and treating of cognitive and failure of one's memory comprising Passiflora incarnata L. extract

The present invention relates to a composition capable of improving, preventing or treating memory loss and forgetfulness by containing Passiflora incarnata L. extract as an active ingredient.

Sleeping is an essential activity for all living things, and humans spend about a third of their time sleeping. You may think that when humans sleep, all organs stop working and rest, but in reality they do not. During sleep, our brains work to consolidate memories from waking, to relax, or to analyze potential risks.

Most adults get 7-8 hours of sleep each day to rest, but each person has their own individual differences. For example, some people can sleep 4 hours a day and live their lives freely, but some people can sleep 8 hours a day to achieve this level of living.

Age is an important factor in determining the amount of sleep needed, and newborns sleep 16-18 hours a day. However, as the sleep time gradually decreases, the age of two requires 12 hours, and the age of 13 requires about 8 hours. The amount of sleep required continues to decrease, reaching an average of about six hours of sleep per day at age 50 (Durand, 1998).

There are two main stages of sleep: shallow sleep and deep sleep, depending on whether you have rapid eye movement or not, and rapid eye movement sleep and non-rapid eye. movement sleep, quiet sleep). When we measure the brain waves during REM sleep, Beta waves appear when we are usually awake. The pupils under the eyelids move, and heart rate, blood pressure, and breathing are very irregular. Also, if you dream at this stage, you can easily remember the contents of the dream.

In this non-REM sleep, there is almost no rhythm and the heart rate is stable. The brain activity at this time is also very slow and regular. At this time, the pupils of the eyelids are hardly moved and breathing is slow and regular with comfortable facial expressions.

During sleep, non-REM and REM sleep activity alternates over time, but as people get older, the percentage of REM sleep gradually decreases (de Weed & van den Bossche, 2003).

Wilson at MIT University in the United States records mouse hippocampus activity by plugging an electric needle into the hippocampus (which plays a crucial role in the processing of learning information) and training the mouse to find its way through a complex maze during the waking day. It was. They recorded the hippocampus' activity while the mouse was sleeping. As a result, the two activities showed similar patterns. In other words, the pattern of the hippocampus activity while the mouse was sleeping was as if the mouse remembered the daytime situation, and then dreamed at night and quickly repeated the hippocampus activity during the night. REM) appeared only during sleep.

Considering the results of the above-described conventional studies, sleep disorders that result in long REM sleep activity may cause fatigue accumulation problems, and if the accumulation of fatigue continues, not only memory may be reduced, but amnesia symptoms may be aggravated.

Therefore, there is a need for a natural material that can prevent, treat or improve symptoms such as memory loss and forgetfulness caused by lack of sleep.

Republic of Korea Patent No. 111439 Republic of Korea Patent No. 1725979

It is an object of the present invention to provide a pharmaceutical composition capable of preventing and treating memory loss and forgetfulness by containing an extract of Passiflora incarnata L. as an active ingredient.

In addition, another object of the present invention is to provide a food composition that can improve or prevent memory loss and forgetfulness by containing the extract (Passiflora incarnata L.) as an active ingredient.

The pharmaceutical composition capable of preventing and treating memory loss and forgetfulness of the present invention for achieving the above object may contain the Passage (Passiflora incarnata L.) extract as an active ingredient.

The passion flower extract may be mixed with the passion flower leaves and passion fruit 1: 1 weight ratio of 0.1-0.8.

The passion flower extract may be extracted by mixing the passion flower 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 20 to 80% by volume of methanol, ethanol, butanol or propanol aqueous solution.

The pharmaceutical composition may be usefully used for memory loss and forgetfulness caused by lack of sleep.

In addition, the food composition capable of improving or preventing memory loss and forgetfulness of the present invention for achieving the above-described other object may contain the extract of Passionflower (Passiflora incarnata L.) as an active ingredient.

The composition of the present invention is very effective in improving, preventing or treating symptoms such as memory loss and forgetfulness caused by lack of sleep, and also can be taken in the form of food because it is not toxic.

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

The present invention relates to a composition capable of improving, preventing or treating memory loss and forgetfulness by containing Passiflora incarnata L. extract as an active ingredient.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The composition capable of improving, preventing or treating memory loss and forgetfulness of the present invention contains Passiflora incarnata L. extract as an active ingredient.

The passion flower (Passiflora incarnata L.) can be used as an evergreen perennial plant, flowers, leaves, stems, roots, berries of passion flower. Native Americans used the entire plant for swelling or inflammation, and roots are commonly used as tonics. In addition, the leaf prevents rapid pulse, reduces high blood pressure, is used as a sedative of non-inhibiting and non-inhibiting insomnia and anxiety, relieves asthma, muscle spasms, epilepsy, nervousness, colon syndrome, and is used as a poultice agent for burns and skin Softens inflammation.

The passion flower extract of the present invention is an extract in which the passion leaf and passion fruit are mixed in a weight ratio of 1: 0.1-0.8, preferably in a weight ratio of 1: 0.2-0.5. If the content of passion fruit on the basis of the passion flower leaves below the lower limit can not improve, prevent or treat memory loss and forgetfulness, if the upper limit is exceeded, the effect of the present invention is not expressed at all and toxicity may be induced. have.

When used alone without using the passion flower leaves and passion fruit fruit shows 1.5 to 20 times lower efficacy than when using the two materials together; Passive flowers, stems, roots when using a combination of the two materials show a 5 to 50 times lower efficacy.

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

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

The term 'extract' as used herein to refer to the passion flower includes not only the crude extract obtained by treating the extraction solvent, but also a processed product of the passion flower extract. For example, the passion flower extract may be prepared in powder form by an additional process 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 broadly including a processed material of the passion flower, for example, passion flower powder, which is formulated to administer the passion flower to the animal. Although the experiment with the passion flower extract in the present invention, it will be expected to those skilled in the art that the desired effect can be achieved in the same form as the processed material of the passion flower.

On the other hand, the term 'containing as an active ingredient' herein means containing an amount sufficient to achieve the efficacy or activity of the passion flower extract. In one example, the passion flower extract is used at a concentration of 10 to 1500 μg / ml, preferably 100 to 1000 μg / ml. Since the passion flower extract has no adverse effects on the human body even when excessively administered as a natural product, the upper limit of the quantity of the passion flower extract contained in the composition of the present invention can be selected and performed by those 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 adjuvant may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, Lubricants, flavors and the like can be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration.

Formulation forms 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, nontoxic, 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 include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance 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 in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, 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. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

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, and preferably, oral administration. .

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dose form or formulated into pharmaceutically acceptable carriers and / or excipients or incorporated into multi-dose containers. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

In another aspect, the present invention provides a food composition for improving or preventing memory loss and forgetfulness containing the extract as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Examples of the food to which the composition of the present invention may be added include beverages, alcoholic beverages, confectionary, diet bars, dairy products, meat, chocolates, pizza, ramen noodles, other noodles, gums, ice creams, vitamin complexes, and health supplements. Etc.

The food composition of the present invention may include not only passion flower extract as an active ingredient, but also components commonly added in food production, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. . Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example 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 prepared with a drink and a beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included 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 or preventing memory loss and forgetfulness including the passion flower extract as an active ingredient. Health functional food is a food prepared by adding passion flower extract to food materials such as beverages, teas, spices, gums, confectionery, encapsulated, powdered, suspensions, etc. This means, unlike general medicines, there is no side effect that can occur when taking long-term use of medicines as food raw materials. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. Although the addition amount of the passion flower extract in such a dietary supplement cannot be uniformly prescribed | regulated depending on the kind of the dietary supplement which is target, what is necessary is just to add in the range which does not impair the original taste of a food, and it is normal 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of a health functional food 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 dietary supplement of the present invention may be in the form of pills, tablets, capsules or beverages.

The present invention also provides the use of a passion flower extract for the manufacture of a medicament or food for the improvement, prevention or treatment of memory loss and forgetfulness. As mentioned above, the passion flower extract may be used for the purpose of improving, preventing or treating memory loss and forgetfulness.

The present invention also provides a method for improving, preventing or treating memory loss and forgetfulness comprising administering to a mammal an effective amount of a passion flower extract.

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

As used herein, the term “effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, doctor or other clinician, Amounts that induce alleviation of the symptoms of the disease or disorder. The effective amount and frequency of administration for the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes 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, general health of the patient. It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously. In the prophylaxis, treatment or improvement method of the present invention, for adults, it is preferable to administer the passion flower extract at a dose of 0.001 g / kg to 10 g / kg once to several times a day.

In the therapeutic method of the present invention, the composition comprising the passion flower extract as an active ingredient may be administered in a conventional manner through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. Can be administered.

Hereinafter, preferred examples are provided to aid the understanding of the present invention, but the following examples are merely for exemplifying the present invention, and it will be apparent to those skilled in the art that various changes and modifications can be made within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the appended claims.

Example 1. Passion Leaves: Passion Fruit = 1: 0.25 weight ratio extract (PF extract)

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

<Test Example>

Mouse ready

Many studies have been conducted to verify the effect as a composition for improving, preventing or treating memory loss and forgetfulness, but most studies have used animals because of limitations in human studies. The test was performed using DBA / 2 mice, among which known to be essentially deficient in sleep function.

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

One week before the start of the experiment, the animals were bred at room temperature (22 ± 1 ° C.) and 60% humidity under a 12 hour light cycle (light cycle from 7: 00-19: 00). The animals were provided with free access to normal food (2018S; Harlan, USA) and water, and daily oral administration of distilled water or clockwort extract for 4 days from the start of day (day 1).

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 in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Soonchunhyang University (IACUC approval no. SCH16-0037).

4 groups of mice

Sham group: ICR mice, 5 dietary groups _weighing 100 mg / kg distilled water (DW)

Control: DBA / 2 mice, 5 dietary groups _weighing 100 mg / kg distilled water (DW)

PF 10: DBA / 2 mice, 5 mg diet _weight group of the Passion Flower Extract of Example 1

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

The mice were sacrificed on day 4 of dosing, applied to the brain with 4% paraformaldehyde solution and fixed with the same fixative.

For aquatic lab studies, Sprague-Dawely (SD) mice were used instead of DBA / 2 mice.

The experimental animals purchased 24 male SD mice (weight 175-225 g) of 7 weeks old (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 mice were performed in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Soonchunhyang University (IACUC approval no. SCH16-0037).

2 groups of mice

Control group: SD mice, 5 dietary groups _weighing 100 mg / kg of distilled water (DW)

PF 100: SD mouse, 5 mg diet _weight group of the clock flower extract of Example 1

To determine whether administration of chronic PF extracts affected uptake, activity, and metabolic changes, eight-week-old C57BL / 6 mice were purchased from the Charles River and analyzed for total behavioral and metabolic changes. The experimental animals 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 mice were performed in accordance with the ethical standards of the Institutional Animal Care and Use Committee of Soonchunhyang University (IACUC approval no. SCH16-0037).

2 groups of mice

Control: C57BL / 6 mice, distilled water (DW) 8 mg diet _weight group 8

PF 100: C57BL / 6 mice, 8 mg dietary group 100 mg / kg _weight diet of Example 1

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 physiological saline group and COX-2 inhibitor (celecoxib) treated group (each group n = 5).

Saline (1 ml / 100g body weight) and an equal volume of 20 mg / kg celecoxib (prescription formulation; Pfizer Inc.) were administered orally to mice daily using needles two weeks before sacrifice. Subsequently, the brain was removed and immunohistochemistry was performed with chromosome anti-DCX and mouse anti-phospho-IκBα to confirm the role of COX-2 in neurogenesis with hippocampal dentate gyrus, and the amount of SGZ It is expressed as the ratio between the number of cells and the number of tertiary branches of each neuroblast dendrites.

Cytotoxicity by MTT Assay

Eight PF extracts (using 10 mg / kg _{weight of} passion flower extract) with varying concentrations from 2 to ² mg / ml to ^2-5 mg / ml were tested. Passion flower extract was dissolved in α-MEM medium (WELGEN, Gyeongsan-si, Korea) to 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. The reagents were then diluted with α-MEM medium to a final concentration of 0.5 mg / ml and then the solution was stored at 4 ° C. and protected from light.

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

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

C. elegans culture

Wild-type N2 strains were purchased from the Pretty Nematode (Worm, 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 were incubated in nematode growth medium (NGM) agar plates (1.7% agar, 2.5 mg / mL peptone, 25 mM NaCl, 50 mM KH 2 PO 4 pH 6.0, 5 μg / mL cholesterol, 1 mM CaCl 2, 1 mM MgSO 4) at 20 ° C. Escherichia coli OP50 was supplied as a nutrient source to the NGM plates.

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

Oxidative Stress Resistance Analysis Using Worms

Age synchronous adult worms were treated with different dilutions of PF extracts (50 mg / L, 100 mg / L and 500 mg / L) in NGM plates for 24 hours. Diluted PF (100 μL each) was sprinkled onto solid NGM plates (5 mL). Subsequently, 12.5 mg / L of 5-fluoro-2'-deoxyuridiene (5-fluoro-2'-deoxyuridiene, Sigma-Aldrich, St. Louis, USA) was added to the NGM plate to prevent internal incubation. Added. Next, the worms were exposed to S-bsal 2 mM hydrogen peroxide (5 L of sterilized distilled water, 5.85 g of sodium chloride, 1 g of potassium dihydrogen phosphate, and 6 g of potassium phosphate) without cholesterol.

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

Life Analysis Using Worms

Simultaneous worms at age 60 were treated with different concentrations of PF extracts (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 the log-rank test.

Thermal Shock Stress Resistance Measurement Using Worm

Three-day-old worms with age concurrency were picked out of NGM plates and transferred to NGM plates containing 500 mg / L PF. After 24 hours the worms were left in a 35 ° C. incubator for 10 hours to induce heat shock stress and then the worms were transferred back to 20 ° C.

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

How to process tissue in mouse

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. Brains were removed and fixed in the same fixative at 25 ° C. for 12 hours and then preserved by infiltrating brain tissue with 30% sucrose overnight. Brain sections were sequentially cut off from the coronal plane to a thickness of 30 μm using a cryostat (Leica, Wetzlar, Germany) and collected on 6-well plates containing PBS for further processing.

The sections were treated under the same conditions so that immunohistochemical data could be compared between groups. The section shows a mouse brain atlas (Franklin and Paxinos, 2008) between rat brain maps (Paxinos and Watson, 2006) and Bregma posterior -3.00 to -4.08 mm relative to Bregma posterior -1.82 to -2.46 mm. Was selected in connection with. Ten portions spaced 90 μm apart were sequentially treated with 0.3% hydrogen peroxide (H 2 O 2) in PBS for 30 minutes and 5% normal goat serum in 0.05 M PBS at 25 ° C. for 30 minutes.

Immunohistochemical Staining for Double Cortin (DCX), Ki67, COX-2 and pIkappaβ

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

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

Neuroblasts (DCX positive cells) and Ki67 positice cells in the granulation zone (SGZ) were calculated and 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, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 48 hours at 4 ° C. The sections were then 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. . The sections were then visualized by staining with 3, 3'-dinobenzidine in 0.1 M Tris-HCl buffer, pH 7.2. Sections were placed on gelatin coated slides with Canada Balsam (Wako, Tokyo, Japan) after dehydration.

Immunohistochemical Staining on Microglia (Iba-1)

Freshly prepared brain sections were treated with 0.3% hydrogen peroxide (H 2 O 2) in PBS for 30 minutes and then sequentially with 10% normal goat serum in 0.05 M 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'-dinobenzidine (3, 3'-daminobenzidine) in 0.1 M Tris-HCl buffer, pH 7.2. Sections were placed on gelatin coated slides with Canada Balsam (Wako, Tokyo, Japan) after dehydration.

Western Blot

Hippocampus and hypothalamus fragments were removed from the skull of SD mice and homogenized with lysed buffer (iNtRon Biotechnology, Seoul, Korea). Protein concentration was measured by BCA kit (iNtRon Biotechnology), total protein (20 μg / sample) was placed in each lane of 12% SDS-PAGE, electrophoresed and transferred to PVDF membrane (Bio-Rad Laboratories, CA, USA). The membrane was then blocked with TBST [100 mM Tris-HCl (pH 7.6), 0.8% NaCl and 0.1% Tween-20] containing 10% skim milk (BD Biosciences, CA, USA). The membrane is the 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 ℃. The membrane was then washed and incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Vector, CA, USA).

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

Monitor behavioral and metabolic changes by indirect calorimetry

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

Phenotypic measurements of animals were performed using Mini-spec LF50 (Bruker Biospins, The Woodlands, TX) followed by body composition (delicate tissue, fat and fluid in mice on benchtop platforms).

Measure with Morris underwater maze

The experiment was conducted by applying the existing Morris method. First, the mice were transferred to the behavior observation room and stabilized 1 hour before the start of the experiment. Maze specifications are 90 cm in diameter, 32.5 cm in height, and the white platform is 5 cm in diameter. The perimeter of the underwater maze kept constant space cues, such as video systems and computer systems and water temperature controls. The maze was then filled with water and placed below 1 cm of water height so that the mouse could not see the platform. The maze was divided into four quadrants using four markers and divided into northeast (NE), northwest (NW), southeast (SE), and southwest (SW), and a platform was installed in one quadrant of the maze. . Morris's maze test runs for five days, on the first day each mouse is allowed to swim freely in the maze for one minute to adjust to the water, with no platform installed, and the fourth day from the second day. Up to four mice per day were allowed to swim in the maze for one minute at ten minute intervals four times a day. One test method for 4 days from the 2nd day, the mouse that has been raised for 1 second within 1 minute on the platform already installed in the labyrinth does not find the platform within 1 minute after finishing the experiment or 10 seconds on the platform. The mouse was not artificially placed on the platform for one second after the end of the experiment and the experiment was terminated, and the position of the platform was fixed at the same position. On the fifth day, the platform was removed from the labyrinth, and the time the mouse stayed in the house where the platform was was measured. To perform the memory test on day 5, the last day of Morris's maze test, free swimming was performed for 60 seconds after the platform was removed, and the learning curves of One way ANOVA analyzed on day 2-4 were compared. On day 6, 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 were conducted under blind conditions under the same conditions. For quantification of the immune response, the degree of staining was measured using 5 sections for each experimental group. Images of c-fos immune response structures were taken using a BX53 optical microscope (Olympus, Japan) equipped with a digital camera (DP71, Olympus) connected to a computer and monitor.

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

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

FIG. 1A is a DCX positive cell image in the hippocampus dental gyrus of the Sham group, control group, PF 10 group and PF 50 group (3 days of oral administration) (upper image of the hippocampus of each group). 1B is an enlarged image of the black box of FIG. 1A (scale bar = 100 μm).

1C shows Ki67 in hippocampal teeth gyrus of Sham group, control group, PF 10 group and PF 50 group (oral administration 3 days) Positive cell image, 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, GR protein expression in the hippocampus and GR protein expression in the hypothalamus treated with the control and PF 50 groups, respectively.

As shown in FIGS. 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 extracts (10 mg / kg and 50 mg / kg) were administered. . DBA / 2 (control), which received only distilled water, had very low expression of DCX-positive cells and very poor dendrites in the subgranular zone (SGZ) of the dental gyrus.

DCX positive neuroblasts in PF treated mice were not highly expressed or developed as in the Sham group in SGZ (right magnified image in FIG. 1A). Specifically, each dendritic dendritic spine in the pituitary zone (SGZ) was highly branched (secondary and tertiary branching) in the Sham, PF 10 and PF 50 groups. However, the control group was found to exhibit a low number of neuroblastoma and branching patterns in the pituitary region (SGZ) of the tooth gyrus (FIG. 1A).

DCX and Ki67 positive cells in the PF treatment group was found to be significantly higher than the vehicle-treated Sham group and the control group.

In addition, as shown in FIGS. 1E to 1G, neuroblasts (DCX) of the hippocampus were compared with a glucocorticoid receptor (GR) known as a marker for HPA axis regulation in the hippocampus and the hypothalamus, and mice treated with PF extracts. It was confirmed that neuroblastoma and GR protein expression in E. coli significantly different from the control group.

Test Example 2 Cytotoxicity

Compared with the control group, the PF 10 and PF 50 groups were diluted from 4 mg / ml to 3.125 × 10 ⁻² mg / ml by a 2-fold dilution method. Results showed that PF 10 and PF 50 groups were not cytotoxic at all concentrations and significantly increased cell proliferation (P <0.0001), further increasing cell viability.

Test Example 3 Confirmation of Increased Resistance to Oxidative Stress Induced by PF Extract in C. elegans

To investigate the effect of PF extract on the response to oxidative stress, the survival of worms (pretty nematodes) was observed under oxidative stress conditions (6 hours exposure to H 2 O 2) depending on the presence of PF extract.

As a result, the PF extract (50 mg / L, 100 mg / L and 500 mg / L) was confirmed to increase the survival rate under oxidative stress compared to the control (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 Life Extension by Treating PF Extraction

We investigated whether PF extract could affect the lifespan of C. elegans. Treatment of PF extracts significantly extended the average and maximum life of C. elegans.

The treatment of 500 mg / L out of the three different PF concentrations (PF 50 mg / L, 100 mg / L and 500 mg / L) analyzed showed that the life extension effect was better than 50 mg / L and 100 mg / L. . The average life span of the pretty nymph nematode in the control group not treated with PF was 17.6 days and the maximum life value was 27 days, whereas the pretty nymph nematode treated with PF 500 mg / L extended the life expectancy up to 24.2 days. Was found to increase to 34 days.

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

Test Example  5. PF  Induction of Aging-related Genes by Extracts

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 lifespan of pretty little nematodes.

It was investigated whether hsp-16.2 expression was increased in long-life pretty nematodes treated with 250 mg / L PF extract. As a result, the hsp-16.2 :: GFP reporter was markedly treated by 250 mg / L PF extract treatment. It was confirmed that it is induced. Specifically, the GFP fluorescence intensity was 3.130X10 ⁴ ± 1.378X10 ³ in the PF-treated pretty nymph, which was higher than that of the control group 1.372X10 ⁴ ± 0.898X10 ³ (mean ± SEM of 20 individual pretty nymphs).

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

Test Example 6 Body Weight and Feeding Behavior of Mice During PF Treatment

 Figure 2A is a graph showing the change in body weight of the control and PF 100 group, Figure 2B is a graph showing the number of dietary intakes of the control and PF 100 group (using the BioDAQ® system running program), Figure 2C is the control and PF 100 group Cumulative dietary intake was measured (using the BioDAQ® System Execution Program), and FIG. 2D is a graph showing daily water intake in the control and PF 100 groups.

As shown in FIGS. 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, cumulative dietary intake, and daily water consumption were also significant. 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.

As shown in FIGS. 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 they were ± 0.282 pg / mL (control, Veh) and 3.841 ± 0.217 pg / mL (PF 100 group).

Serotonin levels in the PF 100 group were 32.0% higher and melatonin levels were 31.0% higher than the control group.

Test Example 8 Automated Phenotypic Analysis of Behavioral, Physiological and Metabolic Data Due to Administration of PF Extracts

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

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

As shown in FIGS. 3A-3E, the dietary intake (Kcal / kg / h) was 20.11 ± 0.808 (control, Veh) and 17.84 ± 0.465 (PF), respectively, and the water intake (mL / kg / h) was 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 the activities are 745.5 ± 50.45 (Veh) and 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 ratios are 0.8114 ± 0.0219 (Veh) and 0.8024 ± 0.215 (PF), respectively.

All data measured automatically during the 12-hour day and night cycle (Zeitgeber time (ZT)) was examined to confirm that there was no significant difference between the control group (Veh) and the PF 100 group.

In addition, as shown in Figure 3F to 3H, it was confirmed that there is 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 of Neural Environment in Hippocampus by Administration of PF Extract

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

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

As shown in FIG. 4A, similar to the results of DBA / 2 mice (FIG. 1), DCX was significantly increased in mice and significantly increased PaV, GABA neutrophil markers, BDNF, and neuronal flexibility markers. . Melatonin levels increased significantly in serum and MelaR1 also increased significantly in hippocampus.

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

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

In addition, as shown in FIGS. 4C to 4E, microglia in the control group (Veh) had activated, rounded and swollen cell bodies, while microglia in the PF treated group were confirmed to be less activated and thin body. Microglia expanded to other groups around the hippocampus, including the black lines of the rectangular CA1, CA3 and dental gyrus (DG).

Test Example 10 Iba-1 Immunohistochemistry

Figure 5A is a diagram staining the immune tissue of Iba-1 in the hippocampus of the control group and PF 100 group, Figure 5B is a diagram staining CA1, CA3 and DG of the control group and PF 100 group, Figure 5C is a control group and PF 100 It is a graph measuring the ratio between 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) had activated, rounded and swollen cell bodies, whereas microglia in the PF treated group were identified as less activated and thinner. Microglia expanded to other groups around the hippocampus, including the black, rectangular CA1, CA3, and dental gyrus (DG).

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

Test Example 11 DCX Expression in Hippocampus Dentate Gyrus by COX-2 Inhibition

FIG. 6A is a graph showing DCX positive neuroblasts and pIkappaB positive cells in hippocampus dentate gyrus of SD mice treated with the control group (Veh) and COX-2 inhibitors, and FIG. Figure 6C is a graph showing the ratio of cell number of neuroblasts in SGZ, Figure 6C is a graph showing the three or more branched dendritic dendrite in the SGZ of the control group (Veh) and COX-2 inhibitor-treated group tooth.

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

The expression level of Iba-1 in the PF treated group was low, but the expression of the inflammatory marker COX-2 was higher than the control (Veh). However, when COX-2 inhibitors were treated in normal mice, they showed significantly lower levels of neural tissue development than the control group. COX-2 is not necessarily involved in neuronal inflammation but shows a high neural tissue signal.

Therefore, the PF extract can inhibit neuronal inflammation, and therefore, it is believed that the PF treated group increases brain plasticity by inhibiting the activity of microglia in the brain without inducing neuroinflammation by COX-2.

In addition, as shown in Figures 6B and 6C, the number of neuroblastoma cells and tertiary branch dendritic projections in the SGZ of the dentate gyrus showed a significant difference between the control group (Veh) and the COX-2 inhibitor-treated group. .

Test Example 12. Morris Water Maze Test

FIG. 7A is a graph showing time to find a target platform for the control group and the PF 100 group for each measurement period, and FIG. 7B is a graph showing the distance to the platform for which the control group and the PF 100 group are targeted 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 time spent in the platform quadrant with the control and PF 100 group.

As shown in FIG. 7, on day 1 there was no difference between the control and PF 100 groups at the time of finding the platform (FIG. 7A) and the distance to the platform (FIG. 7B), and both groups had similar driving and visual capabilities. Have Days 2 to 4 showed differences in the time to find the platform between each group (FIG. 7A) and the distance to the platform (FIG. 7B), and on day 4, the PF 100 group was found to be significantly superior to the control group.

In addition, on day 5, the last day, the number of seconds the mouse stayed in the place where the removed platform was placed was found to be significantly superior to the PF 100 group compared to the control on day 4 (FIGS. 7C and 7D).

By measuring the hippocampal dependent learning, including the acquisition of spatial memory and long-term spatial memory by the passion flower extract of Example 1, when coagulation of the passion flower extract of Example 1 is significantly increased, neurocoagulation function is increased and sleep function is essential. As a result, it was confirmed that the memory of the deficient animal could be substantially improved.

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

Formulation Example 1 Preparation of Powder

500 mg of extract powder obtained in Example 1

Lactose 100 mg

Talc 10 mg

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

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

200 mg of extract powder obtained in Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation Example 4 Preparation of Injection

600 mg of extract powder obtained in Example 1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule.

Formulation Example 5 Preparation of Liquid

4 g extract powder obtained in Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a lemon flavor, and then mixing the above ingredients, adding purified water to adjust the total to 100g by adding purified water, and then filling into a brown bottle to sterilize To prepare a liquid solution.

Formulation Example 6 Preparation of Granules

1,000 mg of extract powder obtained in Example 1

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is a composition which is relatively suitable for granules in a preferred embodiment, the composition ratio may be arbitrarily modified, and the above components are mixed according to a conventional granulation method and then granulated. It can be prepared and used in the manufacture of 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

Citric Acid 1,000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 mL of purified water

After mixing the above components in accordance with the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare functional beverage compositions of the invention.

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Claims (6)

A pharmaceutical composition for the prevention and treatment of memory loss and forgetfulness, which comprises an extract of Passiflora incarnata L. as an active ingredient. The pharmaceutical composition for preventing and treating memory loss and forgetfulness according to claim 1, wherein the passion flower extract is mixed with passion flower leaves and passion fruit in a weight ratio of 1: 0.1-0.8. According to claim 1, The passion flower extract is memory loss and forgetfulness, characterized in that extracted by mixing the passion flower and water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof in a volume ratio of 1: 1 to 100 Prophylactic and therapeutic pharmaceutical compositions. The pharmaceutical composition for preventing and treating memory loss and forgetfulness according to claim 3, wherein the mixed solvent is 20 to 80% by volume of an aqueous methanol, ethanol, butanol or propanol solution. The pharmaceutical composition for preventing and treating memory loss and forgetfulness according to claim 1, wherein the pharmaceutical composition is useful for memory loss and forgetfulness caused by lack of sleep. A food composition for improving or preventing memory loss and forgetfulness, which comprises an extract of Passiflora incarnata L. as an active ingredient.

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