COMPOSITION COMPRISING CLEMATIS MANDSHURICA EXTRACT AS ACTIVE INGREDIENT FOR COGNITIVE FUNCTION IMPROVEMENT

Abstract

The present invention relates to a composition for improving cognitive function, including a Clematis terniflora var. mandshurica extract as an active ingredient. The composition for improving cognitive function according to an exemplary embodiment of the present invention can provide a composition which is effective for stress relief, mood improvement, antidepressant and cognitive function improvement by utilizing an extract derived from natural products. Further, the functional food composition of the present invention can be provided as a functional food composition with high preference while exhibiting effects of stress relief, antidepressant, and cognitive function improvement.

Claims

1. A composition for improving cognitive function, comprising a Clematis terniflora var. mandshurica extract as an active ingredient, the composition comprising the Clematis terniflora var. mandshurica extract as a first natural extract, and a second natural extract selected from the group consisting of a dandelion extract, a monocotyledon extract, an Indigofera pseudotinctoria Matsum extract, a Hydrangea serrata extract, a castanea crenata shell extract, a plantain extract, a Ligularia fischeri extract, a Ligularia stenocephala extract, and a mixture thereof.

2. The composition of claim 1, wherein the first natural extract and the second natural extract are extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 10 carbon atoms, and a mixed solvent thereof.

3. The composition of claim 1, further comprising a Capsicum annuum extract.

4. The composition of claim 1, wherein the cognitive function is learning ability, memory ability, or concentration.

5. The composition of claim 1, wherein the composition improves the deterioration of brain or cognitive function accompanied by a cerebral nervous disease.

6. The composition of claim 1, wherein the composition exhibits stress relief, mood improvement and antidepressant effects.

7. A pharmaceutical composition for improving cognitive function, comprising the composition according to claim 1.

8. A functional food composition for improving cognitive function, comprising the composition according to claim 1.

Description

DESCRIPTION OF DRAWINGS

[0086] FIG. 1 relates to a graph illustrating effects of the mixtures of the natural composite extracts of the present invention on the cerebral infarction site caused by ischemia.

[0087] FIG. 2 relates to a graph illustrating anti-depressant effects when the mixtures of the natural composite extracts of the present invention are acutely treated once.

[0088] FIG. 3 relates to a graph illustrating anti-depressant effects when the mixtures of the natural composite extracts of the present invention are repeatedly treated for a long period of time.

[0089] FIG. 4 relates to a graph illustrating amounts of cortisol in blood when the mixtures of the natural composite extracts of the present invention are repeatedly treated for a long period of time.

[0090] FIG. 5 is a graph for experiments of measuring dopamine of the natural composite extract of the present invention.

[0091] FIG. 6 is a graph for experiments of measuring serotonin of the natural composite extract of the present invention.

BEST MODE

[0092] Hereinafter, the Examples of the present invention will be described in detail such that a person skilled in the art to which the present invention pertains can easily carry out the present invention. However, the present invention can be implemented in various different forms, and is not limited to the Examples described herein.

Preparation Example 1: Preparation of Natural Extract

[0093] 1. Preparation of Clematis terniflora Var. Mandshurica (E1) Extract

[0094] 1) Extraction of Sample

[0095] After dried Clematis terniflora var. mandshurica was leached at room temperature for 48 hours by using 70% ethanol, a sample was filtered and concentrated.

[0096] 2) Acquisition of Fraction (E2)

[0097] After 1 g of the Clematis terniflora var. mandshurica extract extracted with 70% ethanol was dissolved in 30 ml of distilled water, fractionation was performed three times using hexane, and the separated aqueous layer was again fractionated three times. In the three-time fractionation step, the Clematis terniflora var. mandshurica extract was fractionated by using methyl chloride. After the fractionation three times, the separated aqueous layer was again fractionated three times by using ethyl acetate, the separated aqueous layer was again fractionated three times by using n-butanol, and each fraction was secured by concentrating the obtained solution.

[0098] 2. Preparation of Natural Extract

[0099] A concentrate was prepared from dandelion (E3), monocotyledon (E4) Indigofera pseudotinctoria Matsum (E5), Hydrangea serrata (E6), castanea crenata shell (E7), plantain (E8), Ligularia fischeri (E9), Ligularia stenocephala (E10), red hot pepper fruit (E11), and red hot pepper fruit stalk (E12) by using the same method as in that of the Clematis terniflora var. mandshurica extract. Thereafter, a natural extract was prepared by dissolving 1 g of each concentrate in 30 ml of distilled water.

Preparation Example 2: Preparation of Composition for Improving Cognitive Function

[0100] According to the composition as in the following Table 1, compositions for improving cognitive function of S1 to S7 were prepared by mixing the constituent ingredients.

TABLE-US-00001 TABLE 1 S1 S2 S3 S4 S5 S6 S7 E1 100  100  100  100  — — — E2 — — — — 100  100  100  E3 40 50 80 120  50 100  100  E4 30 40 50 80 40 60 60 E5 30 40 50 80 40 60 60 E6 30 40 50 80 40 60 60 E7 30 40 50 80 40 60 60 E8 30 40 50 80 40 60 60 E9 30 40 50 80 40 60 60 E10 30 40 50 80 40 60 60 E11 — — — — — — 40 E12 20 30 40 60 30 40 —

[0101] (Unit Parts by Weight)

Experimental Example 1: Experiment of Ischemic Animal Model

[0102] In order to confirm the effects of the compositions including the natural extract prepared in the Preparation Examples on the area of cerebral infarction caused by ischemia, the following experiment was performed.

Experimental Example 1-1: Local Ischemia-Induced Animal Model

[0103] A. Treatment with Drug and Construction of Local Ischemia-Induced Animal Model

[0104] A local ischemia-induced animal model was constructed using a male white rat (Sprague-Dawley rat) weighing 200 to 250 g. As a drug, 1 g/kg of each of Experimental Nos. S1 to S7 was orally administered once 1 hour before the induction of ischemia or once 1 hour after the induction of ischemia (oral administration group, n=6). After the experimental animal was anesthetized by injecting 30 to 40 mg/kg of ketamine intramuscularly to the animal, the skin of the neck was incised in the supine position, and the common carotid artery, the external carotid artery, and the internal carotid artery were located and separated from surrounding tissues. First, the superior parathyroid gland artery and the posterior fossa artery, which are branches of the external carotid artery, and the pterygopalatine artery, which is a branch of the internal carotid artery, were electrocauterized, and the external carotid artery were cut. Occlusion of origin of middle cerebral artery was performed by incorporating 4-0 nylon thread (ETHICON, INC) into the internal carotid artery through the external carotid artery, and then placing 16 to 18 mm of nylon thread within the branch of the common carotid artery.

[0105] B. Experiments of Effects of Ischemia on Reduction of Cerebral Infarction Site

[0106] Brains were removed by decapitating the animal 6 hours after induction of ischemia. The removed brains were cut at an interval of 2 mm from anterior pole, and reacted with a 2% triphenyl tetrazolium chloride (TTC, Sigma) solution at 37° C. for 30 minutes, followed by fixing in a 4% paraformaldehyde (Sigma) solution. After stained tissue sections were photographed, the area of a region that turned white due to the occurrence of cerebral infarction was measured unlike the red-stained normal region by using MCD image processing system (Imaging Research Inc.) for the photographs, the ratio of the cerebral infarction area to the area of the entire brain section was obtained, and then the average value was calculated. Further, the ratio of the volume occupied by the cerebral infarction site in the entire volume from each brain section was measured.

[0107] The composition including the natural composite extract of the present invention is reducing the cerebral infarction site caused by ischemia to a level significant to the control group. According to FIG. 1, as a result of measuring the ratio of the volume of the cerebral infarction to the volume of the entire brain section, pretreatment of S2, S3, S5, and S6 showed a significant effect on the reduction of cerebral infarction site caused by local ischemia.

[0108] In particular, it was confirmed that S5 and S6 showed beneficial results. In addition, through the experiment, it was confirmed that the case where the red hot pepper fruit stalk was used was excellent in effect as compared to the case where the red hot pepper fruit was used (S7). In the accompanying FIG. 1, each measured value shows the mean±standard deviation.

Experimental Example 2: Experiment of Depression Animal Model

Experimental Example 2-1: Preparation of Experimental Animals

[0109] Experimental animals were male white rats (Sprague Dawley, 140 to 180 g, Korea Experimental Animal Center), and these animals were adapted to the environment by placing four animals per breeding box for a week in a predetermined environment (indoor temperature 25±1° C., relative humidity 60±10%) and allowing the animals to uptake water and feed without limitation, and then used for the next experiment. Among the white rats, the animals which showed little movement and a deteriorated developmental situation in a general breeding state and an abnormal stereotyped behavior or markedly reduced swimming ability in forced swimming were excluded, and 50 animals in total were used for the present experiment.

Experimental Example 2-2: Experiment of Depression Animal Model

[0110] In the present experiment, a forced swimming test (FST), which is a standardized test method also called a behavioral despair test, was used. Ecologically, white rats dislike water, and thus, when exposed to water, will exhibit characteristics behaviors to get out of the environment. That is, experimental animals will forcibly swim to get out of water and will no longer swim after a certain period of time, and by using this principle, the forced swimming test method is known as a basic experiment which will search for antidepressant effects during the drug development. The FST process was performed as follows according to the method of Porsolt et al., who were the first proposers (Porsolt et al., Eur. J. Pharmacol. 51(3), 291-294, 1978).

[0111] First, a transparent acrylic cylindrical water tank with a height of 40 cm and a diameter of 18 cm was filled with water at 25° C. to a height of 15 cm, and the white rats in Experimental Example 2-1 were forced to swim the water tank, and then left for 15 minutes. Although the rat violently tried to go out of the tank for the first several minutes, the immobilization time increased as time went by, and the rat maintained its body in the almost immobilization state for the last several minutes. The typical immobilization state refers to a state in which a white rat floats on the water with only a part of its face on the surface of the water, with only a slight movement to keep the body balanced. After the forced swimming, the white rat body's moisture was wiped off, the body was dried with a 37° C. dryer for 30 minutes, and the animal was returned to the breeding box.

[0112] 24 hours after the first forced swimming, the second forced swimming was performed. In the present experiment, for the second forced swimming, the white rats were allowed to stay in the water bath for only 5 minutes under the same condition as the first forced swimming test, and the total immobilization time during this period was measured. During the second forced swimming, as a result of learned helplessness, most of the white rats show more immobilization time than the first forced swimming. Since the increase in immobilization state, which is regarded as a symptom indicator of depression in the forced swimming model, is reduced again by the treatment with the antidepressant agents, the decrease in immobilization state is considered to be related to the action of the antidepressant agents. Since some drugs do not show the effect during an acute treatment with the drug, but show the effect during a long-term treatment in some cases in the forced swimming test, in the present experiment, treatment with the drug was performed for 7 days, and then the second forced swimming was attempted. This method is often used in forced swimming tests because the antidepressant effect of antidepressants is generally obtained after drug administration for at least 2 weeks. The immobilization state was measured by video recording the entire process during the second forced swimming, and the immobilization times between the control group and the experimental group were compared and evaluated by measuring the immobilization time. Through video screen analysis, three trained evaluators obtained an average value among the evaluators by counting the immobilization time with a second clock, and used the average value as an analysis data.

[0113] Administration Method

[0114] Experimental Nos. S1 to S7 of the present invention were prepared as 100 mg/ml solutions, and thus orally administered. All the samples were dissolved in distilled water and administered orally, and administered 1 hour before the second forced swimming at the time of single administration, and when administered repeatedly for a long period of time for 7 days, administered 30 minutes after the first forced swimming, and then repeatedly administered for 7 days.

[0115] Antidepressant Effect During Acute Treatment Once

[0116] During the acute treatment with S1 to S7 of the present invention once (1 g/kg), the antidepressant effect was confirmed. The administration amount was set at 20 mg/kg during administration once with reference to the results of existing studies (Eur. J. Pharmacol. 138(3), 413-416, 1987; Neuropharmacology 28(3), 229-233, 1989). The experimental results are illustrated in FIG. 2.

[0117] As illustrated in FIG. 2, it can be seen that the average immobilization time caused by the acute treatment is significantly reduced in the experimental group to which S2, S3, S5, and S6 were administered (**, P<0.05). In the accompanying FIG. 3, each measured value shows the mean±standard deviation.

[0118] Antidepressant effect during long-term repeated treatment (for 7 days)

[0119] During the long-term repeated treatment with S1 to S7 of the present invention, the antidepressant effect was confirmed. The same experiment as the single treatment was performed by orally administering S1 to S7 at 50 mg/kg once daily for 7 days. The experimental results are illustrated in FIG. 3.

[0120] As illustrated in FIG. 3, the average immobilization time caused by the long-term treatment was reduced by administration of S2, S3, S5, and S6 (P<0.1). In the accompanying FIG. 3, each measured value shows the mean±standard deviation.

Experimental Example 3: Effects of Preventing and Improving Stress

Experimental Example 3-1: Preparation of Experimental Animals

[0121] 4-week-old ICR male mice {SAMTAKO Bio Korea, Korea} were subjected to an adaptation time of 1 week, and then bred in a space where lighting is turned on and off repeatedly in a unit of 12 hours, and an indoor temperature of 18 to 23° C. and a humidity of 60% were maintained. As a feed, a solid feed was supplied, and there was no restriction on the feed or grade other than the process of inducing stress.

Experimental Example 3-2: Stress Induction

[0122] The stress-inducing method is used by modifying the method of Wilner et al. (Reduction of sucrose preference by chronic unpredictable mild stress, 1987), and specifically, stress is induced by creating a variety of unexpected mentally stressful situations such as fasting, dietary restriction after fasting (feeding a small amount of feed), suspension of water supply, provision of empty jugs after suspension of water supply, tilted breeding farm, breeding of a number of experimental animals in a breeding farm, sparkling light, cold room, and continuous light.

[0123] 1. Comparison of Amount of Cortisol in Blood

[0124] The amounts of cortisol in blood were measured by administering the compositions for improving cognitive function of S1 to S7 to the experimental animals prepared above.

[0125] Administration Method

[0126] Experimental Nos. S1 to S7 of the present invention were orally administered at 50 mg/kg once daily for 7 days. All the samples were dissolved in distilled water and orally administered, and repeatedly administered for a long term for 7 days.

[0127] Amount of cortisol in blood during long-term repeated treatment (for 7 days)

[0128] During the long-term repeated treatment with S1 to S7 of the present invention, the amount of cortisol in blood was confirmed.

[0129] For the experimental results, as illustrated in FIG. 4, it can be seen that the average cortisol amount is significantly decreased in the experimental group to which S2, S3, S5, and S6 are administered.

Each measured value exhibits the average±standard deviation.

[0130] 2. Effects of Preventing Inhibition of Dopamine

[0131] Dopamine is a precursor of norepinephrine as a neurotransmitter in the central nervous system, and is involved in cognition and attention concentration, reward, regulation of motor function, and the like. Since it is known that dopamine secretion is inhibited in a chronic stress situation, the stress prevention and improvement effects caused by the composition of the present invention were confirmed through a dopamine measurement experiment.

[0132] As a result of measuring the dopamine concentration using the high performance liquid chromatography with electrochemical detection (HPLC-ECD) method, the value (% of control) compared with the dopamine concentration of the normal control group was shown as an average and standard deviation. The administration groups of the compositions for improving cognitive function of S1 to S7 and the stress control group were compared by verifying the significance with a statistical analysis using a Student's T-test, and p<0.05 was considered to be significant and displayed. The results are illustrated in FIG. 5.

[0133] Referring to FIG. 5, it can be seen that the concentration of dopamine measured from the hippocampus of the test animals subjected to stress is remarkably lower than that in the normal control group. By the way, it can be seen that the concentration of dopamine is maintained at higher levels in the groups to which the compositions for improving cognitive function of S1 to S7 are administered than in the stress control group.

[0134] In particular, it may mean that in the experimental groups to which S2, S3, S5, and S6 are administered, the effect of inhibiting reduction of dopamine is excellent.

[0135] 3. Effect of Improving Serotonin

[0136] As a result, the value (% of control) compared with the serotonin concentration of the normal control group was expressed as the mean and standard deviation. The administration groups of the compositions for improving cognitive function of S1 to S7 and the stress control group were compared by verifying the significance with a statistical analysis using a Student's T-test, and p<0.05 was considered to be significant and displayed. The results are illustrated in FIG. 6.

[0137] Referring to FIG. 6, it can be seen that the concentration of serotonin measured from the hippocampus of the test animals subjected to stress is remarkably lower than that in the normal control group. By the way, it can be seen that the concentration of serotonin was maintained at higher levels in the groups to which the compositions for improving cognitive function of S1 to S7 than in the stress control group.

[0138] In particular, it may mean that in the experimental groups to which S2, S3, S5, and S6 were administered, the effect of inhibiting reduction of serotonin is excellent.

[0139] Therefore, in the case of the above range, it can be seen that the effect of relieving stress is shown by inhibiting the reduction of serotonin caused by stress.

Experimental Example 4: Preference Test

[0140] 1. Sensory Evaluation Test

[0141] Tea beverages were prepared by diluting the compositions for improving cognitive function of S1 to S7. The tea beverages were sampled by 10 tasters, and the taste and aroma were expressed by an index of 1 to 10, and the average value (applied to a rounding of 0.05) is shown in the following Table 2. The higher the number of the index is, the higher the preference is.

TABLE-US-00002 TABLE 2 S1 S2 S3 S4 S5 S6 S7 Taste 6.0 6.5 6.5 6.0 7.0 7.5 6.0 Flavor 6.0 6.5 6.5 6.5 7.5 7.5 6.5 Overall 6.0 6.5 6.5 6.3 7.3 7.5 6.3 preference (average)

[0142] (Unit: Index)

[0143] Referring to Table 2, it can be seen that in the case of the compositions of improving the cognitive function of S2, S3, S5, and S6, the preference is enhanced. Therefore, in the case of using the compositions for improving cognitive function of S2, S3, S5, and S6, it is possible to provide a functional food for improving cognitive function, which is excellent in stress relief and antidepressant effects with flavor and taste of higher preference.

[0144] Although preferred Examples of the present invention have been described in detail hereinabove, the right scope of the present invention is not limited thereto, and it should be understood that many variations and modifications of those skilled in the art using the basic concept of the present invention, which is defined in the following claims, will also fall within the right scope of the present invention.