COMPOSITION FOR AMELIORATING COGNITIVE FUNCTION CONTAINING PROCESSED POLYGONI MULTIFLORI RADIX EXTRACT

The present invention is described more particularly by the next in the embodiment. In the embodiment for the present invention is which exemplify these, these range number by one of the present invention looked into immediately after being turned off.

Example available bit

1 attainments number: number pleuropterus multflorus extract number bath method

3 year it buys pleuropterus multflorus dry powder (chiri Mt.sewage five farming combination legal entity) strain line extractor method number 320 rpm by adding 10 kg and methanol 20 L 12 in a process for the extraction of periodical repetition time 0 and 3. The filtrate after 45 μm filter to filter the aggregate of decompressing the concentrator (EYELA, Tokyo, Rikakikai Co. , Tokyo, Japan) using a method number 50 °C reduced in pressure by the concentrated extract pleuropterus multflorus 380 g are obtained.

2 attainments number: number identifying method number pleuropterus multflorus derived active substance reservoir and structure

Number number number number number after suspension in 100 g extract prepared by the method 1 attainments pleuropterus multflorus atrough-building, using the minute nuclear the atmosphere which it will peel hexene fractions, chloro gun ROM fractions and methanol fraction after seeds, methanol fractions again fractions A (methanol), B (gun ROM chloro), C (the nuclear it counted), D (butanol), E sequentially after seeds (acetonitrile), (water: methanol=100:0) (water: methanol=0:100) again fractions A1 - A9, B1 - B7 (methanol: chloro gun ROM=100:0) (methanol:=0:100 gun ROM chloro), C1 - C7 (methanol: hexene=100:0) (methanol: hexene=0:100), D1 - D5 (methanol: butanol=100:0) (methanol: butanol=0:100), E1 - E4 (water: acetonitrile=100:0) (water: acetonitrile=0:100) concentration gradient fraction to his (also 1).

A1 - E4 positive number circulation using high performance liquid chromatography fractions 2, 3, 5, 4 ' - tetra - 2 a-O-a beta-Dthe low id rock hour steal it cutswriting base nose side, lane, obtaining diacerein and cleaning sofa glow separation was a positive number: analysis column is Lichrophore 100 RP C18 column (4. 6 × 250 mm, 5 μm, Merck & Co. , Kenilworth, NJ, USA) deflection, a furnace solvent 1. 0% glacial acetic acid (solution A) to 100% acetonitrile (solution B) have analyzed, 20, 10, 10 and 10 minutes respectively mid conditions reference solvent B 10%, 20%, 25% and 35% with respect to the maintained. Injecting sample is maintained in mid rate 1 ml/min 30 °C 20 μL fractions have the 280 nm absorbance of physiologically active substance have diode array UV detector (Agilent 1200 series, Agilent co.) quantifying in him.

HPLC analysis result, 2, 3, 5, 4 ' - tetra the low id rock hour steal it cutswriting base nose side (compound 1) - 2 a-O-a beta-D, lane (compound 2), obtaining diacerein (compound 4) (compound 3) and cleaning sofa glow diethyl ether (also 2), 1 the table in terms of the content of each component to quantitative mistletoe.

In addition nucleonanalysis method spectroscopic device using the specified pores 2, 3, 5, 4 - 2 - 2 a-O-a beta-Dthe low id rock hour steal it cutswriting base nose side, lane, obtaining diacerein and cleaning formula sofa glow identifying result to 3 also when the mistletoe.

Test example: pleuropterus multflorus derived composition method number cognitive function effect test

In the present invention according to 4 species active ingredients including said number 1 number attainments pleuropterus multflorus extract concentrated powder prepared by the method number number 10, 25 and 50 μg/mL each methanol concentration 100 μg/mL (H100) and high pressure liquid coolant and concentration to 200 μg/mL (H200) dissolving sequential test method number pleuropterus multflorus derived composition sample at night.

Test example 1: pleuropterus multflorus neuron protecting effect derived composition number method

Test method number (glial) cells derived composition pleuropterus multflorus neuron protecting effect cross nerve C6 hydrogen peroxide (H₂ O₂ ) Or A β_{25 - 35} Processing cells to oxidative stress induced by assaying protective effect test for him.

C6 100 units/ml of penicillin-resistant Streptococcus pneumoniae Streptococcus feed and 10% FBS DMEM added cross nerve cells and 37 °C and adhesive, 5% CO₂ Him as in incubator.

The cells are cultured in 1 - 2 cell differentiation by [...] type (refeeding) all at once one reaches maximum PBS (phosphate buffer saline) cut into trypsin - EDTA solution (0. 05% trypsin and 0. 02% EDTA containing) attached cells after the separation of the centrifugal separator and an organic solvent and then mixing the iron passaging cells was used.

<Oxidative stress induced by hydrogen peroxide>

C6 cells (confluent) state 96 well plate 5 x 10 when a front cross nerve growth⁴ Cells/ml to the cell inoculation (seeding) to thereby deposit the threshold H oxidation stress₂ O₂ (100 μm) processing time 24 culturing substrate. The method of the present invention is then ready to lay in a number derived from said sample by pleuropterus multflorus concentration (10, 25, 50 μg/mL) regulated to finish each handling a test group (Sigma) 5 mg/ml MTT solution in each well after the injection time re 4 37 °C culture in a formazan (formazan) DMSO (Sigma) for 1 to 540 nm were measured absorbance. The C6 cells also causes oxidative stress as well as normal group in the same manner as measured absorbance cross nerve, cell survival rate calculating contrast have normal group, also 4a mistletoe to result.

Also as shown in 4a, normal cell survival rate 100% contrast H group₂ O₂ In processing the controls (control) 42. 81% survival rates were confirmed indicating damage to neuronal cells has been subjected. The method of the present invention concentration pleuropterus multflorus number derived from each sample (10, 25, 50 μg/mL) 51 each handling a test group respectively. 06, 51. 35, 55. 69% cell survival by oxygen kind activities occurring in human body showing a number/number neuron protecting effect can be beat generation billion industry.

(ROS) separate number while nerve intracellular activity oxygen kind billion effect using dCF provided dA C6 nerve cross cells analysis (Byun et al. , 2009) conducting.

Specifically C6 (confluent) state 96 well plate 5 x 10 when a front cross nerve cell growth⁴ Cells/well 2 days time is reduced oxidative stress particularly when the cells following are cultured in 37 °C threshold H₂ O₂ A (100 μm) him as time processing 24. The causes after oxidative stress, said method according of the present invention derived from each sample number ready to lay in a pleuropterus multflorus concentration (10, 25, 50 μg/mL) test group each handling finish after culturing a regulated time 24, 80 μm in 30 minutes material injected into each well 37 °C culture of dCFH-a dA solution after fluorescent reader in (ex) excitation wavelength 480 nm, have -535 nm emission wavelength (em) in detecting the fluorescence intensity, as well as oxidative stress causes normal group in the same manner as the C6 nerve cross cells also fluorescent measures the strength, also 4b shown to result.

Also 4b (left graph) from, the group consisting of processing measured 60 minutes ROS hydrogen peroxide generating amount over time and to selectively added instead of oxidative stress is increased, as compared to production in test group number can be wire strands are regulated active oxygen kind billion. 4b (right graph) also from, based on 100% normal group (normal) which gives a 60 minutes 94 is regulated. 24% has been show, each of the generated rate lower than regulated test group (control) and viscoelasticity. The composition of the present invention H₂ O₂ A lever effect due to toxicity protection are disclosed.

Β<A_{25 - 35} Oxidative stress induced by>

Instead A β hydrogen peroxide_{25 - 35} (Amyloid (25 - 35) Peptide) 25 μm using oxidative stress causing the under the outside C6 nerve cross cells and made of the same number or physiological measuring survival rates also result of generating ROS are symmetrically 5a to 5b mistletoe.

As also shown in 5a, cell survival rates by 100% normal group (normal), the method of the present invention concentration pleuropterus multflorus number derived from each sample (10, 25, 50 μg/mL) test group each handling when comparing the finish controls (control), controls in 56. 07% discharges filtered survival of neuronal cells to confirm that the machining, 81 test group respectively. 72, 93. 67, 90. 72% cell survival rate to controls much higher than shown.

Also 5b (left graph) from, the group consisting of processing measured 60 minutes ROS hydrogen peroxide generating amount over time can be added instead of oxidative stress is increased, as compared to production in test group number can be wire strands are regulated active oxygen kind billion. 5b (right graph) from also, based on the normal group (normal) 60 minutes which gives a 100% 91 is regulated. 68% has been show, each of the generated rate lower than regulated test group (control) and viscoelasticity. The composition of the present invention A β_{25 - 35} A lever effect due to toxicity protection are disclosed.

The 2, 3, 5, 4 ' - tetra - 2 a-O-a beta-Dthe low id rock hour steal it cutswriting base nose side, lane, obtaining diacerein and cleaning method due to oxidative stress-induced neuronal number sofa glow pleuropterus multflorus extract containing and protecting effect leads to the creation of a ROS can be identify.

Test example 2: enhancing the method number pleuropterus multflorus derived composition (In vivo) Effect cognitive ability

Mouse model of the present invention composition using cognitive enhancing effect was assayed for ability.

5 mouse week zero ICR mouse (25 g - 30 g, male, system: CrljOri: CDI, orient (main)) a deflection, unrestricted dietary water was supplied. Breeding chamber 12 when the night cycle time interval maintaining low -, 20 ± 2 °C temperature, humidity of 50 ± 10% in his rearing. Feed include [...] (Korean, trillion onlyhundred qualities 21. 1%, crude 3 room. 5%, 5 trillion fibers. 0%, inquiry minutes 8. 0%, calcium 0. 6%, phosphorus 0. 6%) supplying solid feed water supply when the integer into drinking water with him.

Normal mouse group (Normal), controls (A β_{25 - 35} Processing group; Control), test group (A β_{25 - 35} The present invention according to method after pleuropterus multflorus derived composition sample number (100, 200 μg/mL concentration)With such a nucleotide-administration group (H100) group (H200)) into his database.

Induced memory impairment (A β_{25 - 35} Processing) is established ether anesthesia Belknap method and then A β according to Laursen_{25 - 35} Conducting injection. Specifically A β_{25 - 35} After dissolving in a physiological saline solution at a concentration of 5 nm 72 37 °C culture nor post-incubation time, combined with a 26 - gauge needle to 10 μl syringe hamilton microliters per to each mouse the crown of the head [bregma (two total bone measuring point) caudal direction (toward the tail) in 0. 5 mm, 1 right at the midline (canonical line). 0 mm] to 2. Prepared A β 2 mm depth_{25 - 35} 5 μl of his slowly about a 30 seconds. The same procedure A β at the normal group of mouse_{25 - 35} Such as injecting amount of saline instead of his.

Normal group and is regulated drinking water (100 μl/one), the present invention according to method ionite pleuropterus multflorus derived composition sample test group number (100, 200 μg/mL) after each melting rate and amount of one syringe 14 is using 100 μl/treatment with respect oral administered.

Mouse 3 the weight is re-same time intervals were measured.

Immediately after an overcurrent flows to the long-term extraction is established ether anesthesia to structure of blood brain an aqueous saline good for a stand-alone reports a number after the following weight and water to the number found stand-alone were measured. Extraction organ rubbed for keeping pack sealingly -80 °C as well as experiments are included in the refrigeration [...] dishes.

Object memory power injury treatment effect><- whether test

To test whether the tubes no visible object number it became work box (40 x 40 x 40 cm) embodiment in been. (A1, A2) into a box shape and size two unable to an, starting in the center of each group of mouse box 10 minutes and recording the number of times a mouse leaving two objects. 24 time after one of the next two object after changing new object, an object existing catch and numerializes the recording times have new objects, also 6 shown to result.

As also shown in 6, A β_{25 - 35} The member controls into the new object object recognizing ratio is smaller 41. 9% and 58. 1% new object recognizing ratio of about 16 next to an object. A normal group differences in 2% (26. 4%) lower than can be confirmed. The contrast of the group consisting of mouse A β_{25 - 35} Memory impairment by new object due without noticing that resulting in not shown. Test group (group and H200 H100 group) while mouse hybridoma new object 66 ratio is smaller. 3%, 69. 5% due to contact an end portion of the curiosity of new media object can be confirmed.

The potentiating effect cognitive ability - space><T-a labeling test

T-a labeling method of conducting test Montagomery according. Exterior to T-shaped invisible black acryloyl it became work number T-a labeling is set out box, left arm, right arm (width 12 cm, height 20 cm, starting part 76 cm, each sight the left arm each 31. 5 cm) configuration to right arm passage portion is installed is separated from an open door. The thermal blocking door to inhibit programmed with each group of mouse right arm training sorbing period to 10 minutes left into recording the starting number. 24 right after extraction has been completed blocking of a stand-alone number, enabling a starting point in the left arm and right arm each army mouse to enter new way too soon for recording the number of times a ratio curiosity, 7 also have shown to result.

As shown in fig. 7, a group of normal way of access to 40 degree. 0%, access to new way 60 degree. As shown by the 1% point, controls are way 53 degree of access. 4%, access to new way 46 degree. 6% as compared to a normal group access to a larger difference between two way generates not way of higher degree as shown in space as well as rather access to new road is considered to recognize a memory power sensing signal is received. While in the case of access to new way test group (group and H200 H100 group) mouse 57 each degree. 1% and 59. 0% represents a normal group similar identifying higher spatial ability stamp were.

The potentiating effect - space memory power><Morris water maze test

Morris water maze test through space memory for offering crowd. Morris water maze learning its memory space in order to test animal experimental to, training quadrant through space in the cues stored along finding experiments metals are disclosed.

Specifically diameter of 150 cm, height 30 cm 8 cm diameter 1 cm of pro-duction of metals with respect to the stand 4 if minute 120,130 if minute sleep positioned below. To maintain a temperature of 22 ± 1 °C water-white dye (Poster color, ShinHan) each group of mouse when swimming metals cannot be directly burnt so as to output the user control system reporting. Each if minuteeach army mouse using different metals is clue (visual clue) marker is configured to receive user space to stand. 2 experiments was performed with a step. In the first step the user is learning step observation along metals 3 have, in the second step the 4 and contains the memory inspection was the third embodiment. Experiments into a water bath along wall each group of mouse begins too soon, lateral central wall when water programmed with each quadrant, the quadrant and metals capable of randomly selects a defined starting position not changing during experimental without order. Each group of mouse at day 3 (embodiment 4 at known time intervals of 12 hours), metals in quadrant 3 to 60 seconds along the user during training axis with the starting recording time. 60 seconds elapsed after 15 seconds in mouse experimental autoimmune metals to metals does not stands derived metals to metals on cognitive cues to a location space 60 seconds when moving in exchange for compensation 15 seconds on metals to metals stands to relax his reactions.

The third embodiment experiments 3 and 4 contains is his. The first is a white metals for producing burned after 60 seconds using brake control system invisible space cues reaches of time change recording have metals, metals and a second control system detects the frequency (%) after 60 seconds quadrant were consideration metals have measuring flows, not hidden in tank for producing a second white cells exposed metals to other metals metals and measuring the time for the user 60 seconds when the supporting stand, each result also 8, 9a and 9b also is also shown.

Figure 8 shows a variation where the rise time is mouse are also each group metals, A β_{25 - 35} Memory impairment due to administration by training showed inducing wavelength so that it has been confirmed that the metals can be reduced time to reach the brake but, controls are most long brake time to reach the kind of metals other experiments was compared. In test group while the last blade at each time of arrival to normal group (group H100 and H200) metals faster sources rather than cream-gel, a group of shorter arrival times in particular H200 improving ability to confirm whether the most space in the trench can be. The result is the administration of the present invention according to A β_{25 - 35} Protection against damage by cognitive ability recognize the position capable of metals undergo mouse the user time though, finally mouse space at or protecting effect by revealing the ability whether damage.

The control system detects the quadrant 60 seconds or more metals and metals also 9a provides mouse are searched (%) recorded in the flows in the chemical formula, in the case of recorded in the time ratio of 25% rest on the quadrant provides normal group metals, controls are 9. Up to 3%. The troop test while in consideration has been significantly increased to 22 rest on the quadrant H200 metals group time. Up to 7%.

Wherein the meanings of mouse remain on the pasted to the four quadrant quadrant of memory metals and metals which is to recognize and indicia provides control system so as to find a quadrant quadrant flows, to improve the ability to memory cues space confirming that the present invention according to composition (learning and pharmaceutical compositions) can be.

The motion control system metals also 9b is whether the user thus, whether the visual capability by, or spatial memory due to its ability whether hidden metals exposed electrode embodiment as separate metals representing the result of tests are disclosed. 9b also appears in the field, when metals embodiment as hidden, test group metals are much faster than normal group along the user time can be confirmed. When the metals from the group consisting of metals exposed while control system finding embodiment experiments all significant differences between the time at which control system user cannot. The two result value possible visual capacity due to damage of the difference voltages having different levels to non-pharmaceutical compositions containing ability stamp space door number predetermined position can take place.

As a result the present invention according to composition to damage and cognitive ability is diffused, is even improve memory can be cylindrical.

Test example 3: NO erase effect in brain tissue

A β in brain tissue_{25 - 35} In accordance with a generation number caused by injecting NO billion was tested for their effectiveness.

Schmidt number measurement is organized according to a method of generating NO billion in determining the content of NO, specifically in the brain tissue 10 minutes further comprise a 3,000 rpm on mixing 1% after 130 μl 150 μl supernatant after centrifuging the distilled water containing 5% phosphoric acid (sulfanilic acid) phenylsulfanyl 0. 1% N - (1 - naphthyl) ethylene d amide D high draw claw idbefore solution to 1:1 (v/v) grease to prevent cracks and 15 minutes at room temperature by reacting a reagent (griess reagent) through the 540 nm absorbance was measured in terms of concentration. The NaNO standard curve₂ According to the number as well as a bath concentration. 10 also result the mistletoe.

10 is also shown, in the case of a regulated amount 4 NO in the brain. 9 μmol/L/mg protein, 2 normal population. 6 μmol/L/mg protein exhibits A β_{25 - 35} The NO implantation is 1. 8 times increase in his car. The test group (group H100 and H200) compared 4 respectively. 1, 4. 2 μmol/L/mg protein in normal brain tissue is also used for reducing NO group even amount can be compared. The composition of the present invention with the effect of the brain tissue NO generation billion in number and, in addition NO can be improve by oxidative stress in brain tissue.

Test example 4: inhibition of lipid peroxidation in the brain tissue generating billion number effect

The measurement of the number of lipid peroxide generating billion Ohkawa brain tissue using MDA (Malondialdehyde) content conducting method measures.

Each group of mouse brain tissue after 10 minutes in saline using centrifuge 3,000 rpm to crab age me homo further comprise a multilayer tissue supernatants as well as phosphoric acid 1% 0. 67% thio barbituric acid (thiobarbituric acid; TBA) by mixing 2 ml 1 - butanol in 45 minutes 95 °C by reacting 10 minutes by adding cool 3,000 rpm in a supernatant after centrifuging the 535 nm, 520 nm absorbance in a long time. MDA concentration using a high pressure liquid coolant according to a standard curve number obtained by calculating degrees of inhibition of lipid peroxidation, mistletoe to 11 also result.

The also 11, 26 normal group concentration of MDA. 5 nmol/mg protein while, in regulated 37. A β 2 nmol/mg protein_{25 - 35} The lipid peroxide 1 is implanted. 4 times more generated can not inserted. The test group (group H100 and H200) compared 27 respectively. 4, 30. 2 nmol/mg protein lipid peroxide number effect billion starting point, in the case of comparable levels of lipid peroxidation billion was normal group in particular H100 group number. The composition of the present invention generating billion number effect can be belief that inhibition of lipid peroxidation in the brain tissue.