Pharmaceutical composition for treating or preventing neuropsychiatric disease, containing flavone-6-C-glucose derivatives as active ingredients

Abstract

The present invention provides a pharmaceutical composition or a food composition comprising, as active ingredients, flavone-6-C-glucose derivatives or galenical extracts containing flavone-6-C-glucose derivatives. The composition of the present invention shows a functional effect of effectively treating or preventing cognitive dysfunction disorders such as delirium, dementia or amnesia, stroke, palsy, attention disorders, anxiety disorders or sleep disorders.

Claims

1. A method for treating cognitive disorders, stroke, palsy, attention disorders, anxiety disorders or sleep disorders, comprising administering a pharmaceutical composition comprising a purified swertisin to a subject in need thereof.

2. The method of claim 1, wherein the swertisin is administered with at least one herbal extract selected from a group consisting of Swertia japonica, Swertia pseudochinensis, Enicostemma hyssopifolium, Swertia mussotii Franch., Enicostemma hyssopifolium, Swertia franchetiana, Gentianella austriaca (Gentianaceae), Machaerium hirtum Vell. (Fabaceae), Aleurites moluccana L. Willd. (Euforbiaceae), Zizyphus spinosa (Rhamnaceae), Belamcanda chinensis (Iridaceae), Wilbrandia ebracteata, Cayaponia tayuya (Cucurbitaceae), Commelina communis L. (Commelinaceae) and Aquilegia oxysepala Trautv. et Mey (Ranunculoideae).

3. The method of claim 1, wherein the cognitive disorder is due to stroke, palsy, delirium, dementia or amnesia.

4. The method of claim 3, wherein the dementia is Alzheimer's disease, vascular dementia, dementia caused by attention deficit hyperaction disorder, alcoholic dementia, traumatic dementia and dementia due to aftereffects of Parkinson's disease.

5. The method of claim 3, wherein the dementia is Alzheimer's disease or vascular dementia.

6. The method of claim 3, wherein the stroke or palsy is dementia caused by cerebral infarction or cerebral ischemia.

7. The method of claim 1, wherein the stroke or palsy is a cranial nerve cell damage caused by cerebral infarction or cerebral ischemia.

8. The method of claim 1, wherein the anxiety disorder is panic disorder, obsessive compulsive disorder or post-traumatic stress disorder.

9. The method of claim 1, wherein the sleep disorder is hypersomnia.

10. A method for treating cognitive disorders, stroke, palsy, attention disorders, anxiety disorders or sleep disorders, comprising administering a food composition comprising a purified swertisin to a subject in need thereof.

11. The method of claim 10, wherein the swertisin is administered with at least one extract of an herbal drug selected from the group consisting of Swertia japonica, Swertia pseudochinensis, Enicostemma hyssopifolium, Swertia mussotii Franch., Enicostemma hyssopifolium, Swertia franchetiana, Gentianella austriaca (Gentianaceae), Machaerium hirtum Vell. (Fabaceae), Aleurites moluccana L. Willd. (Euforbiaceae), Zizyphus spinosa (Rhamnaceae), Belamcanda chinensis (Iridaceae), Wilbrandia ebracteata, Cayaponia tayuya (Cucurbitaceae), Commelina communis L. (Commelinaceae) and Aquilegia oxysepala Trautv. et Mey (Ranunculoideae).

12. A method for treating a cognitive disorder, stroke, palsy or an attention disorder, comprising administering a pharmaceutical composition comprising a purified isoorientin to a subject in need thereof.

13. A method for treating cognitive disorders, stroke, palsy or attention disorders, comprising administering a food composition comprising a purified isoorientin to a subject in need thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates effect of the pharmaceutical composition comprising swertisin, which is one of the flavone-6-C--D-glucose derivatives, in enhancing memory and learning abilities as per Alzheimer's disease.

(2) FIG. 2 illustrates effect of the pharmaceutical composition comprising isoorientin, which is one of the flavone-6-C--D-glucose derivatives, in enhancing memory and learning abilities as per Alzheimer's disease.

(3) FIG. 3 illustrates effect of the pharmaceutical composition comprising the Swertia japonica extract comprising the flavone-6-C--D-glucose derivatives in enhancing memory and learning abilities as per Alzheimer's disease.

(4) FIG. 4 illustrates effect of the pharmaceutical composition comprising the Swertia pseudochinensis extract comprising the flavone-6-C--D-glucose derivatives in enhancing memory and learning abilities as per Alzheimer's disease.

(5) FIGS. 5 and 6 illustrate effect of swertisin, which is one of the flavone-6-C--D-glucose derivatives, in enhancing memory and learning abilities as per Alzheimer's disease.

(6) FIGS. 7 and 8 illustrate effect of the pharmaceutical composition comprising the Swertia japonica extract comprising the flavone-6-C--D-glucose derivatives in enhancing memory and learning abilities as per vascular dementia.

(7) FIG. 9 illustrates effect of the pharmaceutical composition comprising the Swertia japonica extract comprising the flavone-6-C--D-glucose derivatives in protecting cranial nerve cells as per stroke and palsy.

(8) FIGS. 10 and 11 illustrate effect of swertisin, which is one of the flavone-6-C--D-glucose derivatives, in enhancing memory and learning abilities as per vascular dementia.

(9) FIG. 12 illustrates effect of swertisin, which is one of the flavone-6-C--D-glucose derivatives, in protecting cranial nerve cells as per stroke and palsy.

(10) FIG. 13 illustrates anti-anxiety effect of swertisin, which is one of the flavone-6-C--D-glucose derivatives.

(11) FIG. 14 illustrates awakening effect of swertisin, which is one of the flavone-6-C--D-glucose derivatives.

DESCRIPTION OF EMBODIMENTS

(12) The present disclosure will be described more fully hereinafter with reference to the accompanying examples. However, the present disclosure may be embodied in many different forms, and should not be construed as being limited to the embodiments set forth herein.

(13) In addition, reagents and solvents used hereinafter were purchased from Sigma and optical rotatory power was measured by using the JASCO P-1020 polarimeter unless otherwise said. UV was measured by using the Hitachi JP/U3010, IR was measured by using the JASCO FT/IR-5300, NMR was measured by using the Avance 400 (Bruker, 400 MHz) and FAB Mass spectrum was measured by using the JEOL JMS-700 mass spectrometer.

Example 1. Preparation of 70% Ethanol Extract of Swertia japonica Containing Flavone-6-C--D-glucose Derivatives

(14) Swertia japonica (40 g) was grinded by using a grinder and divided into extraction bottles. 70% ethanol was added to the residue until being higher than the sample surface. The same was extracted twice at 60 C. for 2 hours, and then filtered and concentrated under reduced pressure.

Example 2. Preparation of 70% Ethanol Extract of Swertia pseudochinensis Comprising Flavone-6-C--D-glucose Derivatives

(15) The extract was prepared in the same manner as Example 1 except that Swertia pseudochinensis (40 g) was used herein instead of Swertia japonica.

Example 3. Separation and Purification of Swertisin and Isoorientin

(16) A column chromatography ( 6.534.9 cm) was conducted on the 70% ethanol extract of Swertia japonica (10.66 g) prepared in Example 1 with CH.sub.2Cl.sub.2-MeOHH.sub.2O mixed solvent (9:1:0.1.fwdarw.8.5:1.5:0.1.fwdarw.4:1:0.1, v/v, last 100% MeOH) as a mobile phase, and silica gel (70-230 mesh) as a stationary phase. The same was divided into 12 subfractions. MPLC (C.sub.18 column 130 g, MeOHH.sub.2O gradient) was conducted on the subfraction 9 (860 mg) to separate compound 1 (100 mg). The fraction 10 (560 mg) was divided into 5 subfractions via Sephadex LH-20 column chromatography ( 3.438.5 cm) by using a 100% MeOH solvent. MPLC(C.sub.18, 48 g, MeOHH.sub.2O gradient) was conducted on the subfraction 10-4 (180 g) to further separate the compound 1 (50 mg). The subfraction 12 (4.19 g) was divided into 5 subfractions via Sephadex LH-20 column chromatography ( 3.433.5 cm) by using a MeOHH.sub.2O mixed solvent (4:1, v/v). MPLC(C.sub.18, 26 g, MeOHH.sub.2O gradient) was conducted on the subfraction 12-4 (80 mg) to separate compound 2 (30 g). The structures of the compounds 1 and 2 were identified as swertisin and isoorientin respectively through .sup.1H-NMR and .sup.13C-NMR data analyses, and comparative analysis with the data disclosed in the related document.

(17) [G. Cheng, Y. Bai, Y. Zhao, J. Tao, Y. Liu, G. Tu, L. Ma, N. Liao and X. Xu. Flavonoids from Ziziphus jujuba Mill var. spinosa. Tetrahedron 2000, 56, 8915-8920.]

(18) [Y. Li, Y. Suo, Z. Liao, L. Ding, The glycosides from Lomatogonium rotatum. Natural Product Research 2008, 22-3, 198-202.]

(19) Swertisin: Yellow powder. .sup.1H-NMR (400 MHz DMSO-d.sub.6), (splitting caused by rotational isomerism): 7.97 (2H, d, J=8.4 Hz, 2, 6-H); 6.93 (2H, d, J=8.0 Hz, 3, 5-H); 6.88, 6.86 (1H, s, 8-H); 6.85, 6.84 (1H, s, 3-H), 4.61 (1H, d, J=8.0 Hz, Glc 1-H), 3.87 (3H, s, OCH.sub.3). .sup.13C-NMR (100 MHz DMSO-d.sub.6), (splitting caused by rotational isomerism): Table

(20) Isoorientin: Yellow needle. .sup.1H-NMR (500 MHz DMSO-d.sub.6), (splitting caused by rotational isomerism): 7.43 (1H, d, J=8.5 Hz, 6-H); 7.41 (1H, s, 2-H); 6.91 (1H, d, J=8.0 Hz, 5-H) 6.67 (1H, s, 3-H) 6.48 (1H, s, 8-H), 4.60 (1H, d, J=10.0 Hz, Glc 1-H). .sup.13C-NMR (100 MHz DMSO-d.sub.6), (splitting caused by rotational isomerism):

(21) TABLE-US-00002 TABLE .sup.13C-NMR Spectral Data for 1 and 2 (in DMSO-d.sub.6) .sub.c 1 Position Rotamer 1 Rotamer 2 2 2 163.7 163.9 163.5 3 103.0 103.0 102.6 4 181.9 182.2 181.8 5 159.5 160.3 156.2 6 109.6 109.7 108.9 7 164.9 164.9 163.6 8 90.2 91.0 93.5 9 156.7 156.8 160.7 10 104.1 104.6 103.2 .sup.1 120.8 120.9 118.9 .sup.2 128.5 128.5 113.2 3, 116.0 116.0 145.8 .sup.4 161.4 161.4 149.8 .sup.5 116.0 116.0 76.0 .sup.6 128.5 128.5 121.2 OCH.sub.3 56.2 56.5 glc 1 70.8 70.9 73.0 2 72.5 72.8 70.6 3 79.0 79.1 78.9 4 69.6 70.2 70.1 5 81.7 81.9 81.6 6 61.7 61.7 61.4

Experimental Example 1. Observation of Memory-Enhancing Effect in Alzheimer's Disease

(22) An experiment was conducted to confirm effects of swertisin or isoorientin, and extracts of Swertia pseudochinensis or Swertia japonica containing the same prepared in Examples 1 to 3 in treating dementia by using a model of memory impairment induced by scopolamine. Detailed method follows.

1) Preparation of Laboratory Animals

(23) Six-week old ICR mice in about 26 g to 28 g (Orientbio Inc, Republic of Korea) were received water and feed without constraint and adapted for 5 days under an environment having about 231 C. of temperature, about 6010% of humidity and 12-hour light/dark cycle (animal laboratory at College of Pharmacy, Kyung-Hee University), and then used for the experiment.

2) Statistics Process

(24) Every experiment result was processed by using ANOVA (one-way analysis of variance) and a significance test was conducted at a level of p<0.05 or below by using Student-Newman-Keuls Test when significance was recognized as exists.

3) Experimental Example 1-1: Passive Avoidance Test 1

(25) An apparatus for measuring passive avoidance reaction was prepared. The apparatus has 2 separated chambers (the first and second chambers) and there is a guillotine-shaped passage that connects the first chamber and the second chamber. The first chamber was maintained brightly by using a light, and the second chamber was maintained darkly. A grid was installed on the floor of the second chamber. The grid generates 0.5 mA of electric shock for 3 seconds when the laboratory animal moved into the dark chamber.

(26) About 30 minutes later, 1 mg/Kg of scopolamine, which was dissolved in distilled water, was intraperitoneally administered to the drug administration groups 1 to 4 and controls 1 and 2 (Ebert, U. et al., Eur. J. Clin. Invest., 28, pp 944-949, 1998), and 0.9% of saline solution was intraperitoneally administered to the control 3. After 30 minutes, passive avoidance learning was conducted on the drug administration groups 1-4 and controls 1-3. In detail, the mice were located in the first chamber (i.e. bright space) and the passage was open after about 20 seconds of observation time, and then latency time of the mice moving to the second chamber (i.e. dark space) was measured. The mice that did not move to the second chamber (i.e. dark space) after 60 seconds from opening of the guillotine-shaped passage were excluded.

(27) Twenty-four hours after the passive avoidance learning, a main experiment was conducted on the drug administration groups 1-4 and controls 1-3. Latency time of the mice of each group moving their 4 legs into the dark space after 10 seconds of observation time from opening of the guillotine-shaped passage was measured up to 300 seconds. It means that the passive avoidance learning and memory are better if the latency time is longer.

(28) The passive avoidance test was conducted on the drug administration groups 1-4 and controls 1-3 in the same manner as the method explained above except that swertisin, which is one of the flavone-6-C--D-glucose derivatives prepared in Example 3, was dissolved in 10% Tween 80 and administered to the drug administration groups 1-4 in amounts of 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg and 10 mg/kg respectively.

(29) Average latency time of the mice of each group at the passive avoidance learning and the main experiment were shown in Table 2 and FIG. 1.

(30) TABLE-US-00003 TABLE 2 Passive Avoidance Main Experiment Group Learning (sec.) (sec.) Drug Admin. Gr. 1 (1.25 mg/kg) 30.20 14.53 67.13 26.08 Drug Admin. Gr. 2 (2.5 mg/kg) 19.10 10.37 82.11 35.20 Drug Admin. Gr. 3 (5 mg/kg) 20.40 10.11 117.3 44.16 Drug Admin. Gr. 4 (10 mg/kg) 29.70 18.76 117.7 52.28 Control 1 (Donepezil) 25.60 10.83 138.7 34.03 Control 2 22.70 14.76 35.30 19.04 Control 3 30.22 20.24 257.7 45.02

(31) As shown in Table 2 and FIG. 1, latency time of moving to the second chamber of the drug administration groups 1-4, wherein swertisin prepared in Example 3 was administered, were remarkably extended compared to that of the control 2, wherein scopolamine was administered. In detail, latency time of the drug administration group 1, wherein 1.25 mg/Kg of swertisin was administered, was about 2-fold extended compared to that of the control 2; latency time of the drug administration group 2, wherein 2.5 mg/Kg of swertisin was administered, was about 2.5-fold extended compared to that of the control 2; latency time of the drug administration group 3, wherein 5 mg/Kg of swertisin was administered was about 3-fold extended compared to that of the control 2; and latency time of the drug administration group 4, wherein 10 mg/Kg of swertisin was administered, was about 3-fold extended compared to that of the control 2.

(32) Accordingly, it was confirmed that swertisin prepared in Example 3 was effective for preventing or treating cognitive impairment such as dementia.

4) Experimental Example 1-2: Passive Avoidance Test 2

(33) The passive avoidance test was conducted on the drug administration groups 1-4 and controls 1-3 in the same manner as Experimental Example 1-1 except that isoorientin, which is one of the flavone-6-C--D-glucose derivatives prepared in Example 3, was dissolved in 10% Tween 80 and administered to the drug administration groups 1-4 in amounts of 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg and 10 mg/kg respectively.

(34) Average latency time of the mice of each group at the passive avoidance learning and the main experiment were shown in Table 3 and FIG. 2.

(35) TABLE-US-00004 TABLE 3 Passive Avoidance Main Experiment Group Learning (sec.) (sec.) Drug Admin. Gr. 1 (1.25 mg/kg) 43.60 5.340 41.00 7.988 Drug Admin. Gr. 2 (2.5 mg/kg) 40.00 5.499 80.90 19.44 Drug Admin. Gr. 3 (5 mg/kg) 35.20 5.236 119.3 73.43 Drug Admin. Gr. 4 (10 mg/kg) 26.80 5.085 102.8 15.89 Control 1 (Donepezil) 21.30 4.964 127.8 11.43 Control 2 23.00 6.481 47.22 5.354 Control 3 13.30 3.180 265.2 20.12

(36) As shown in Table 3 and FIG. 2, latency time of moving to the second chamber of the drug administration groups 1-4, wherein isoorientin prepared in Example 3 was administered, were remarkably extended compared to that of the control 2, wherein scopolamine was administered. In detail, latency time of the drug administration group 2, wherein 2.5 mg/Kg of isoorientin was administered, was about 2-fold extended compared to that of the control 2; latency time of the drug administration group 3, wherein 5 mg/Kg of isoorientin was administered, was about 3-fold extended compared to that of the control 2; and latency time of the drug administration group 4, wherein 10 mg/Kg of isoorientin was administered, was about 2.5-fold extended compared to that of the control 2.

(37) Accordingly, it was confirmed that isoorientin prepared in Example 3 was effective for preventing or treating cognitive impairment such as dementia.

5) Experimental Example 1-3: Passive Avoidance Test 3

(38) The passive avoidance test was conducted on the drug administration groups 1-3 and controls 1-3 in the same manner as Experimental Example 1-1 except that a 70% ethanol extract of Swertia japonica prepared in Example 1, was dissolved in 10% Tween and administered to the drug administration groups 1-3 in amounts of 100 mg/kg, 200 mg/kg and 400 mg/kg respectively.

(39) Test result therefrom was shown in Table 4 and FIG. 3.

(40) TABLE-US-00005 TABLE 4 Passive Avoidance Group Learning Main Experiment Drug Admin. Gr. 1 (100 mg/kg) 38.20 20.82 87.20 70.09 Drug Admin. Gr. 2 (200 mg/kg) 36.70 14.97 136.3 81.70 Drug Admin. Gr. 3 (400 mg/kg) 38.30 17.93 100.4 76.23 Control 1 (Donepezil) 26.40 16.23 169.6 79.98 Control 2 31.00 19.28 40.90 25.78 Control 3 20.78 13.11 246.1 56.98

(41) As shown in Table 4 and FIG. 3, latency time of moving to the second chamber of the drug administration groups 1-3, wherein the Swertia japonica extract containing swertisin prepared in Example 1 was administered, were remarkably extended compared to that of the control 2, wherein scopolamine was administered. In detail, latency time of the drug administration group 1, wherein 100 mg/Kg of the Swertia japonica extract containing swertisin was administered, was about 2-fold extended compared to that of the control 2; latency time of the drug administration group 2, wherein 200 mg/Kg of the Swertia japonica extract containing swertisin was administered, was about 3-fold extended compared to that of the control 2; and latency time of the drug administration group 3, wherein 400 mg/Kg of the Swertia japonica extract containing swertisin was administered, was about 2.5-fold extended compared to that of the control 2.

(42) Accordingly, it was confirmed that the Swertia japonica extract containing swertisin prepared in Example 1 was effective for preventing or treating cognitive impairment such as dementia.

6) Experimental Example 1-4: Passive Avoidance Test 4

(43) The passive avoidance test was conducted on the drug administration groups 1-3 and controls 1-3 in the same manner as Experimental Example 1-1 except that a 70% ethanol extract of Swertia pseudochinensis prepared in Example 2, was dissolved in 10% Tween 80 and administered to the drug administration groups 1-3 in amounts of 100 mg/kg, 200 mg/kg and 400 mg/kg respectively.

(44) Test result therefrom was shown in Table 5 and FIG. 4.

(45) TABLE-US-00006 TABLE 5 Passive Avoidance Group Learning Main Experiment Drug Admin. Gr. 1 (100 mg/kg) 26.50 15.67 76.50 37.67 Drug Admin. Gr. 2 (200 mg/kg) 28.10 8.724 111.5 34.24 Drug Admin. Gr. 3 (400 mg/kg) 26.50 19.78 64.00 27.28 Control 1 (Donepezil) 28.10 19.02 159.6 65.51 Control 2 29.80 14.18 40.44 20.27 Control 3 28.20 18.16 216.5 64.47

(46) As shown in Table 5 and FIG. 4, latency time of moving to the second chamber of the drug administration groups 1-3, wherein the Swertia pseudochinensis extract containing swertisin prepared in Example 2 was administered, were remarkably extended compared to that of the control 2, wherein scopolamine was administered. In detail, latency time of the drug administration group 1, wherein 100 mg/Kg of the Swertia pseudochinensis extract containing swertisin was administered, was about 2-fold extended compared to that of the control 2; latency time of the drug administration group 2, wherein 200 mg/Kg of the Swertia pseudochinensis extract containing swertisin was administered, was about 3-fold extended compared to that of the control 2; and latency time of the drug administration group 3, wherein 400 mg/Kg of the Swertia pseudochinensis extract containing swertisin was administered, was about 2-fold extended compared to that of the control 2.

(47) Accordingly, it was confirmed that the Swertia pseudochinensis extract containing swertisin prepared in Example 2 was effective for preventing or treating cognitive impairment such as dementia.

7) Experimental Example 5: Y-Maze Test

(48) A Y-maze was prepared for the experiment. The Y-maze has 3 branches and each branch has 42 cm of length, 3 cm of width and 12 cm of height. Angle between any two arms is 120 degrees and the arms were made of black polyvinyl resin.

(49) The mice prepared in the above 1) were separated into 5 groups (10 mice per each group) comprised of drug administration groups 1 and 2, and controls 1 to 3.

(50) The swertisin prepared in Example 3 (8 mg) was dissolved in 4 mL of 10% Tween 80 (Polyoxyethylene sorbitan monooleate: Sigma, U.S.A.), and administered to the drug administration groups 1 and 2 in an amounts of 5 mg/Kg and 10 mg/Kg respectively. Meanwhile, 5 mg/Kg of donepezil was administered to the control 1, and 0.15 mL of 10% Tween 80 was administered to the controls 2 and 3 respectively.

(51) About 30 minutes later, 1 mg/Kg of scopolamine, which was dissolved in distilled water, was intraperitoneally administered to the drug administration groups 1 and 2, and controls 1 and 2 (Ebert, U. et al., Eur. J. Clin. Invest., 28, pp. 944-949, 1998), and 0.9% of saline solution was intraperitoneally administered to the control 3.

(52) After 30 minutes, mice of the drug administration groups 1 and 2, and the controls 1-3 were carefully located on each branches of the Y-maze, which was divided as A, B and C respectively, and let the mice move freely for 8 minutes, and then recorded branches where mice have entered. The entrance was recorded when the entire body (i.e. including the tail) has completely entered. It was also recorded when the mice entered again into the branch that the mice have entered before.

(53) One point was scored when the mouse sequentially entered into 3 different branches (actual alternation). Alternation behavior is defined as sequentially entering into every 3 branch, and it was converted into % via Equation 1 below (Sarter, M. et al., Psychopharmacology., 94, pp. 491-495, 1998).

(54) $\begin{array}{cc}\mathrm{Alternation}\mathrm{Behavior}\left(\%\right)=\frac{\mathrm{Actual}\mathrm{Alternation}}{\mathrm{Maximum}\mathrm{Alternation}}s100\left(\mathrm{Maximum}\mathrm{Alternation}\text{:}\mathrm{Total}\mathrm{Entrance}-2\right)& \left[\mathrm{Equation}1\right]\end{array}$

(55) Alternation behavior converted by Equation 1 was shown in Table 6 and FIG. 5, and the total entry, which means total number of entry to each branch, was shown in Table 7 and FIG. 6.

(56) TABLE-US-00007 TABLE 6 Group Alternation Behavior (%) Drug Admin. Gr. 1 (5 mg/kg) 56.22 12.29 Drug Admin. Gr. 2 (10 mg/kg) 59.36 5.653 Control 1 (Donepezil) 61.49 5.447 Control 2 46.93 10.41 Control 3 75.32 6.167

(57) TABLE-US-00008 TABLE 7 Group Total Entry Drug Admin. Gr. 1 (5 mg/kg) 27.10 7.795 Drug Admin. Gr. 2 (10 mg/kg) 28.88 4.155 Control 1 (Donepezil) 29.30 4.296 Control 2 33.11 3.822 Control 3 27.30 3.335

(58) As shown in Table 6 and FIG. 5, alternation behavior of the drug administration groups 1 and 2, wherein swertisin prepared in Example 3 was administered, was increased compared to that of the control 2, wherein scopolamine was administered, and specifically, the drug administration group 2 exhibited similar level of alternation behavior to that of the control 1, wherein donepezil was administered.

(59) Also, as shown in Table 7 and FIG. 6, the total entry of the drug administration groups 1 and 2, and the controls 1-3 were shown as having a similar value. Therefore, it can be known that change of mouse activity was irrelevant to increase of the alternation behavior. Thus, it was confirmed that learning, memory and attention abilities of the mice of the drug administration groups 1 and 2 were remarkably improved by swertisin.

(60) Accordingly, it was confirmed that swertisin, which is one of the flavone-6-C--D-glucose derivatives prepared in Example 2 was effective in preventing or treating cognitive impairment such as Alzheimer's disease, and attention disorders.

Experimental Example 2: Observation of Enhancing Effect on Memory in Vascular Dementia and Protective Effect on Cranial Nerve Cells in Stroke and Palsy

1) Preparation of Laboratory Animals

(61) Seven-week old ICR mice in about 30 g to 35 g (Orientbio Inc, Republic of Korea) were received water and feed without constraint and adapted for 5 days under an environment having about 231 C. of temperature, about 6010% of humidity and 12-hour light/dark cycle (animal laboratory at College of Pharmacy, Kyung-Hee University), and then used for the experiment.

2) Statistics Process

(62) Every experiment result was processed by using ANOVA (one-way analysis of variance) and a significance test was conducted at a level of p<0.05 or below by using Student-Newman-Keuls Test when significance was recognized as exists.

3) Preparation of Mouse Model of Stroke

(63) The laboratory animal in 1) was located in an operating room, adapted for 60 minutes, and anesthetized in a space providing anaesthetic gasses (nitrous oxide: 70%; oxygen: 30%; isoflurane: 2.0%). Pain reflex was examined to confirm the anesthesia. The laboratory animal was lain down straight on an operating table, and fixed its upper limb and head. Skin from the spot, where the upper limb and the center line meet, was incised as 1.5 cm to expose the both common carotid arteries by using tweezers while not damaging the tissues. Tissues and nerves attached to the exposed carotid arteries were separated and fixed with an aneurysm clip. The clip was removed after 15 minutes and reperfused, and the incised area was sutured with sutures. After the suture, the laboratory animal was immediately moved to a recovery room (32-33 C., O.sub.2 enrichment condition) and kept until it recovers from anesthesia. Observation on its weight and status was conducted once a day for 4 days after the operation.

4) Experimental Example 2-1: Y-Maze Test

(64) Administration of the Swertia japonica extract prepared in Example 1 was initiated to the mice prepared in the 1) immediately after the operation 3), and kept administering at the same time for 7 days. Five groups (5 mice per each group) were prepared as drug administration groups 1, 2 and 3, wherein the Swertia japonica extract prepared in Example 1 was administered thereto; and controls 1 and 2. 50 mg/kg, 100 mg/kg and 200 mg/kg of the Swertia japonica extract was administered to the drug administration groups 1, 2 and 3 respectively; and 0.15 mL of Tween 80 was administered to the controls 1 and 2 respectively. At the 7th day from the initial administration, the Y-maze test was conducted for the drug administration groups 1 to 3 and the controls 1 to 2 in the same manner as Experimental Example 1-5.

(65) After 60 minutes, mice of the drug administration groups 1-3 and the controls 1-2 were carefully located on each branches of the Y-maze, which was divided as A, B and C respectively, and let the mice move freely for 8 minutes, and then recorded branches that mice have entered. The entrance was recorded when the entire body (i.e. including the tail) has completely entered. It was also recorded when the mice entered again into the branch that the mice have entered before. The result was converted into % via Equation 1.

(66) Alternation behavior converted by Equation 1 was shown in FIG. 7 and Table 8, and the total entry, which means total number of entry to each branch, was shown in FIG. 8 and Table 9.

(67) TABLE-US-00009 TABLE 8 Group Alternation Behavior (%) Drug Admin. Gr. 1 (50 mg/kg) 53.16 11.36 Drug Admin. Gr. 2 (100 mg/kg) 66.28 9.827 Drug Admin. Gr. 3 (200 mg/kg) 70.53 12.11 Control 1 (Stroke-induced Group) 46.62 10.01 Control 2 77.96 17.95

(68) TABLE-US-00010 TABLE 9 Group Total Entry Drug Admin. Gr. 1 (50 mg/kg) 23.40 4.450 Drug Admin. Gr. 2 (100 mg/kg) 20.50 8.888 Drug Admin. Gr. 3 (200 mg/kg) 23.00 3.162 Control 1 (Stroke-induced Group) 20.00 5.050 Control 2 24.20 2.168

(69) As shown in Table 8 and FIG. 7, alternation behavior of the drug administration groups 1-3, wherein the Swertia japonica extract prepared in Example 1 was administered, was increased compared to that of the control 1, wherein stroke was induced, and the drug administration group 3 exhibited similar level of alternation behavior to the control 2, which is a normal group.

(70) Also, as shown in Table 9 and FIG. 8, the total entry of the drug administration groups 1-3, and the controls 1-2 were shown as having a similar value. Therefore, it can be known that change of mouse activity was irrelevant to increase of the alternation behavior. Thus, it was confirmed that learning and memory abilities of the mice of the drug administration groups 1-3 were remarkably improved by the Swertia japonica extract.

(71) Meanwhile, upon termination of the Y-maze test, a 4% paraformaldehyde fixation was conducted on the drug administration groups 1-3 and the control 2, and brain was excised. Paraformaldehyde-fixed brain sample was washed and section having 30 m of thickness was prepared therefrom. The Nissl staining, which can stain a native region of brain cells in normal status, was performed. Brain cell damage in the hippocampal region of the drug administration groups 1-3, wherein the Swertia japonica extract prepared in Example 1 was administered, and the controls 1 and 2 was assessed through a neurological scoring. Pertaining to the evaluation standard, 0 point was scored when there was no damage on the hippocampal region; 1 point for 0-30% of damage; 2 points for 30-60% of damage; and 3 points for 60-100% of damage. The points were scored by 3 people who have no knowledge of the drug administration groups and the controls. Average point was used to calculate the degree of damage. The neurological score according to the evaluation standard was shown in FIG. 9 and Table 10.

(72) TABLE-US-00011 TABLE 10 Group Neurological Score Drug Admin. Gr. 1 (50 mg/kg) 1.9170 0.8333 Drug Admin. Gr. 2 (100 mg/kg) 1.6670 0.8165 Drug Admin. Gr. 3 (200 mg/kg) 0.8889 0.1925 Drug Admin. Gr. 1 (Stroke-induced Group) 2.3330 0.4714 Control 2 0.0000 0.0000

(73) As shown in Table 10 and FIG. 9, the neurological score of the drug administration groups 1-3, wherein the Swertia japonica extract prepared in Example 1 was administered, was decreased compared to that of the control 1, wherein stroke was induced, and specifically, the drug administration group 3 exhibited significant result to the stroke-induced group. When considering that the neurological score reflects the damaged brain cells of the hippocampal region in the brain, it was confirmed that the damaged brain cells of the mouse model of stroke in drug administration groups 1-3 were remarkably protected by the Swertia japonica extract.

5) Experimental Example 2-2: Y-Maze Test

(74) Administration of swertisin prepared in Example 3 was initiated to the mice prepared in 1) immediately after the operation 3), and kept administering at the same time for 7 days. Five groups (5 mice per each group) were prepared as drug administration groups 1, 2 and 3, wherein swertisin prepared in Example 3 was administered thereto; and controls 1 and 2. 50 mg/kg, 100 mg/kg and 200 mg/kg of swertisin was administered to the drug administration groups 1, 2 and 3 respectively; and 0.15 mL of Tween 80 was administered to the controls 1 and 2 respectively. At the 7th day from the initial administration, the Y-maze test was conducted for the drug administration groups 1 to 3 and the controls 1 to 2 in the same manner as Experimental Example 1.

(75) After 60 minutes, mice of the drug administration groups 1-3 and the controls 1-2 were carefully located on each branches of the Y-maze, which was divided as A, B and C respectively, and let the mice move freely for 8 minutes, and then recorded branches that mice have entered. The entrance was recorded when the entire body (i.e. including the tail) has completely entered. It was also recorded when the mice entered again into the branch that the mice have entered before. The result was converted into % via Equation 1.

(76) Alternation behavior converted by Equation 1 was shown in FIG. 10 and Table 11, and the total entry, which means total number of entry to each branch, was shown in FIG. 11 and Table 12.

(77) TABLE-US-00012 TABLE 11 Group Alternation Behavior (%) Drug Admin. Gr. 1 (50 mg/kg) 56.10 7.431 Drug Admin. Gr. 2 (100 mg/kg) 57.58 12.86 Drug Admin. Gr. 3 (200 mg/kg) 70.46 15.74 Control 1(Stroke-induced Group) 51.84 11.00 Control 2 77.69 4.804

(78) TABLE-US-00013 TABLE 12 Group Total Entry Drug Admin. Gr. 1 (50 mg/kg) 24.14 2.478 Drug Admin. Gr. 2 (100 mg/kg) 28.50 6.364 Drug Admin. Gr. 3 (200 mg/kg) 22.00 5.958 Control 1(Stroke-induced Group) 23.33 3.445 Control 2 25.20 6.017

(79) As shown in Table 11 and FIG. 10, alternation behavior of the drug administration groups 1-3, wherein swertisin prepared in Example 3 was administered, was increased compared to that of the control 2, wherein stroke was induced, and specifically, the drug administration group 3 exhibited similar level of alternation behavior to the control 2, which is a normal group.

(80) Also, as shown in Table 12 and FIG. 11, the total entry of the drug administration groups 1-3, and the controls 1-2 were shown as having a similar value. Therefore, it can be known that change of mouse activity was irrelevant to increase of the alternation behavior. Thus, it was confirmed that learning and memory abilities of the mouse model of stroke in the drug administration groups 1-3 were remarkably improved by swertisin.

(81) Upon termination of the Y-maze test, the drug administration groups 1-3 and the control 2 were sacrificed to prepare the brain section, and the Nissl staining was conducted to evaluate brain cell damage in the hippocampal region of the drug administration groups 1-3, wherein swertisin prepared in Example 3 was administered, and the controls 1 and 2 through the neurological scoring. The neurological score according to the evaluation standard was shown in FIG. 12 and Table 13.

(82) TABLE-US-00014 TABLE 13 Group Neurological Score Drug Admin. Gr. 1(50 mg/kg) 1.8130 0.3750 Drug Admin. Gr. 2(100 mg/kg) 1.2500 0.2500 Drug Admin. Gr. 3(200 mg/kg) 1.1670 0.5204 Control 1(Stroke-induced Group) 2.5630 0.2577 Control 2 0.0000 0.0000

(83) As shown in Table 13 and FIG. 12, the neurological score of the drug administration groups 1-3, wherein swertisin prepared in Example 3 was administered, was decreased compared to that of the control 1, wherein stroke was induced, and specifically, the drug administration group 3 exhibited significant result to the stroke-induced group. When considering that the neurological score reflects the damaged brain cells of the hippocampal region in the brain, it was confirmed that the damaged brain cells of the mouse model of stroke in drug administration groups 1-3 were remarkably protected by swertisin.

Experimental Example 3: Observation of Alleviating Effect in Anxiety Symptoms

1) Preparation of Laboratory Animals

(84) Six-week old ICR mice in about 26 g to 28 g (Orientbio Inc, Republic of Korea) were received water and feed without constraint and adapted for 5 days under an environment having about 231 C. of temperature, about 6010% of humidity and 12-hour light/dark cycle (animal laboratory at College of Pharmacy, Kyung-Hee University), and then used for the experiment.

2) Statistics Process

(85) Every experiment result was processed by using ANOVA (one-way analysis of variance) and a significance test was conducted at a level of p<0.05 or below by using Student-Newman-Keuls Test when significance was recognized as exists.

3) Experimental Example 3-1: Marble Burying Test

(86) As a method of measuring alleviation of anxiety symptoms in a normal status, the marble burying test was conducted based on the nature of mice that they dig when feel anxiety. Inside of a cage in size of 40 cm s 27 cm s 18 cm was covered with straw to the height of 4 cm, and 25 marbles having same pattern were arranged at intervals of 3 cm. Two groups (10 mice per each group) were prepared as the drug administration group 1, wherein swertisin, which is one of the flavone-6-C--D-glucose derivatives prepared in Example 3, was administered, and the control 1. Swertisin (10 mg/kg) was administered to the drug administration group 1, and 0.15 mL of 10% Tween 80 was administered to the control 1.

(87) After 60 minutes of administration, mice of the drug administration group 1 and the control 1 were carefully located in the cage having the marbles and the mice were allowed to move freely for 30 minutes. After 30 minutes, the number of buried marbles under straw was counted. At this time, 1 point was given when the marble was completely buried and invisible; and 0.5 point was given when only half of the marble was buried. The more the number of buried marbles, it was assessed that the laboratory animal feels anxiety severer (Broekkamp, C L. et al., Eur J Pharmacol., 126, pp 223-229, 1986).

(88) Result of the test is shown in Table 14 and FIG. 13.

(89) TABLE-US-00015 TABLE 14 Group Number of Buried Marbles Drug Admin. Gr. 1 (10 mg/kg) 21.78 1.856 Control 1 18.78 3.563

(90) As shown in Table 14 and FIG. 13, the number of the buried marbles in the drug administration group 1, wherein swertisin prepared in Example 3 was administered, was decreased compared to that of the control 1. Therefore, it was confirmed that the anxiety symptoms that the control 1 in a normal status feels were remarkably decreased by swertisin of the drug administration group 1.

Experimental Example 4: Observation of Awakening Effect in Hypersomnia Symptoms

1) Preparation of Laboratory Animals

(91) Six-week old ICR mice in about 26 g to 28 g (Orientbio Inc, Republic of Korea) were received water and feed without constraint and adapted for 5 days under an environment having about 231 C. of temperature, about 6010% of humidity and 12-hour light/dark cycle (animal laboratory at College of Pharmacy, Kyung-Hee University), and then used for the experiment. Foods were blocked at 24 hours before the test.

2) Statistics Process

(92) Every experiment result was processed by using ANOVA (one-way analysis of variance) and a significance test was conducted at a level of p<0.05 or below by using Student-Newman-Keuls Test when significance was recognized as exists.

3) Experimental Example 4-1: Sleep-Inducing Test

(93) The mice prepared in 1) were divided into 2 groups (10 mice per each group) including the drug administration group 1, wherein swertisin was administered, and the controls 1 and 2. Swertisin prepared in Example 3 was dissolved in 10% Tween 80 (Polyoxyethylene sorbitan monooleate: Sigma, U.S.A.) and administered to the drug administration group 1 in an amount of 10 mg/kg. Meanwhile, 0.15 mL of 10% Tween 80 was administered to the control 1.

(94) After 1 hour of administration, pentobarbital was dissolved in a saline solution and intraperitoneally administered to the mouse in an amount of 60 mg/kg. The mouse was located in the cage. At this time, straw was laid in the cage only enough to cover the floor, and only one mouse was located in the cage for observation. When there was no righting reflex (i.e. a reflex that corrects the orientation of the body when the mouse is taken out of its normal upright position) after administration of pentobarbital, the time taken after the administration was regarded as the time taken for sleep induction. When the mouse showed righting reflex and corrected its orientation of the body thereafter, the mouse was deemed as awaken and sleep duration was calculated.

(95) Result of the test is shown in Table 15 and FIG. 14 below.

(96) TABLE-US-00016 TABLE 15 Group Total Sleep Time (min.) Drug Admin. Gr. 1 (10 mg/kg) 94.47 21.83 Control 1 122.6 7.348

(97) As shown in Table 15 and FIG. 14, total sleep time of the drug administration group 1, wherein swertisin was administered, was remarkably decreased compared to the control 1. Therefore, it was observed that administration of swertisin, which is one of the flavone-6-C--D-glucose derivatives, showed an awakening effect in normal status.

(98) Preparation of Formulation and Food Composition

(99) Preparation of Powders

(100) The Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 (20 mg), lactose (100 mg) and talc (10 mg) were mixed. A sealed pouch was filled with the same to prepare the powders.

(101) Preparation of Tablets

(102) Tablets were prepared by using the Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 and ingredients listed in Table 16.

(103) TABLE-US-00017 TABLE 16 Ingredient Content (mg) Swertia japonica extract or 10 Swertia pseudochinensis extract (Example 1 or 2) Corn Starch 100 Lactose 100 Magnesium Stearate 2

(104) The above ingredients were mixed and compressed according to conventional tablet manufacturing methods to prepare the tablets.

(105) Preparation of Capsules

(106) Capsules were prepared by using the Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 and ingredients listed in Table 17.

(107) TABLE-US-00018 TABLE 17 Ingredient Content (mg) Swertia japonica extract or 10 Swertia pseudochinensis extract (Example 1 or 2) Crystalline Cellulose 100 Lactose 100 Magnesium Stearate 2

(108) The above ingredients were mixed and a gelatin capsule was filled with the same according to conventional capsule manufacturing methods to prepare the capsules.

(109) Preparation of Injections

(110) Injections were prepared by using the Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 and ingredients listed in Table 18.

(111) TABLE-US-00019 TABLE 18 Ingredient Content (mg) Swertia japonica extract or 10 Swertia pseudochinensis extract (Example 1 or 2) Mannitol 180 Sterile Distilled Water for 2974 Injection Na.sub.2HPO.sub.4, 12H.sub.2O 26

(112) A solution was prepared by mixing the above ingredients according to the conventional methods. A 2 ml ample was filled with the solution. The same was sterilized to prepare the injections.

(113) Preparation of Liquid Solutions

(114) Liquid solutions were prepared by using the Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 and ingredients listed in Table 19.

(115) TABLE-US-00020 TABLE 19 Ingredient Content Swertia japonica extract or 20 mg Swertia pseudochinensis extract (Example 1 or 2) Isomerose 10 g Mannitol 5 g Purified Water 85 ml

(116) A solution (100 ml) was prepared by adding and dissolving each ingredient listed in Table 19 to purified water. Lemon scent was added thereto. Purified water was added thereto again. A brown bottle was filled with the solution. The same was sterilized to prepare the liquid solutions.

(117) Preparation of Health Food Composition

(118) The health food composition was prepared by using the Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 and ingredients listed in Table 20.

(119) TABLE-US-00021 TABLE 20 Ingredient Content Swertia japonica extract or 1000 mg Swertia pseudochinensis extract (Example 1 or 2) Nicotinic Acid Amide 1.7 mg Folate 50 g Calcium Pantothenate 0.5 mg Ferrous Sulfate 1.75 mg Zinc Oxide 0.82 mg Magnesium Carbonate 25.3 mg Monopotassium Phosphate 15 mg Dicalcium Phosphate 55 mg Potassium Citrate 90 mg Calcium Carbonate 100 mg Vitamin Complex q.s Mineral Complex q.s

(120) The above ingredients were mixed according to the conventional health food manufacturing methods, and granules were prepared to prepare the health food composition according to the conventional methods.

(121) Preparation of Health Beverage

(122) The health beverage was prepared by using the Swertia japonica extract or the Swertia pseudochinensis extract comprising swertisin prepared in Example 1 or 2 and ingredients listed in Table 21.

(123) TABLE-US-00022 TABLE 21 Ingredient Content Swertia japonica extract or 1000 mg Swertia pseudochinensis extract (Example 1 or 2) Citric Acid 1000 mg Oligosaccharides 10 0 g Plum Concentrate 2 g Taurine 1 g Purified Water 900 mL in total

(124) The ingredients listed in Table 21 were dissolved in purified water according to the conventional health beverage manufacturing methods to prepare a solution (900 ml). The solution was stir heated at 85 C. for 1 hour. The solution was then filtered and obtained in a 2 l container. The same was seal sterilized and kept refrigerated to prepare the health beverage.

INDUSTRIAL APPLICABILITY

(125) The composition of the present disclosure comprising flavone-6-C--D-glucose derivatives as active ingredients can prevent, improve or treat cognitive disorders, stroke, palsy, attention disorders, anxiety disorders or sleep disorders.