TRANQUILIZING COMPOSITION

Abstract

The present invention relates to a composition for improving, preventing or treating an anxiety disorder, and a tranquilizing composition.

Claims

1-10. (canceled)

11. A method for preventing or treating an anxiety disorder, comprising: administering to a subject in need thereof a composition comprising at least one extract selected from the group consisting of Cynanchum wilfordii, Phlomis umbrosa and Angelica gigas.

12. The method of claim 11, wherein the anxiety disorder is separation or isolation anxiety disorder, paranoia, panphobia, generalized obsessive-compulsive disorder, anxiety neurosis or panic disorder.

13. The method of claim 11, wherein the extract is a crude extract obtained by extraction with at least one solvent selected from the group consisting of water and a linear or branched alcohol having 1 to 4 carbon atoms.

14. The method of claim 11, wherein the extract is a solvent fraction obtained by extracting a crude extract obtained by extraction with at least one solvent selected from the group consisting of water and a linear or branched alcohol having 1 to 4 carbon atoms with at least one solvent selected from the group consisting of ethyl ether, ethyl acetate and butanol.

15. The method of claim 11, wherein the composition is a pharmaceutical composition.

16. The method of claim 11, wherein the composition is a food composition.

17. A method for tranquilizing, comprising: administering to a subject in need thereof a composition comprising at least one extract selected from the group consisting of Cynanchum wilfordii, Phlomis umbrosa and Angelica gigas.

18. The method of claim 17, wherein the method is for treating or preventing bipolar disorder, neurasthenia or schizophrenia.

19. The method of claim 17, wherein the extract is a crude extract obtained by extraction with at least one solvent selected from the group consisting of water and a linear or branched alcohol having 1 to 4 carbon atoms.

20. The method of claim 17, wherein the extract is a solvent fraction obtained by extracting a crude extract obtained by extraction with at least one solvent selected from the group consisting of water and a linear or branched alcohol having 1 to 4 carbon atoms with at least one solvent selected from the group consisting of ethyl ether, ethyl acetate and butanol.

21. The method of claim 17, wherein the composition is a pharmaceutical composition.

22. The method of claim 17, wherein the composition is a food composition.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0069] FIG. 1 is a schematic diagram of preparation of the fraction according to an embodiment of the present invention;

[0070] FIG. 2 is a graph showing the measurement result of tranquilization induction time of the crude extract according to an embodiment of the present invention;

[0071] FIG. 3 is a graph showing the measurement result of tranquilization induction time of the butanol fraction according to an embodiment of the present invention;

[0072] FIG. 4 is a graph showing the measurement result of tranquilization duration of the crude extract according to an embodiment of the present invention;

[0073] FIG. 5 is a graph showing the measurement result of tranquilization duration of the butanol fraction according to an embodiment of the present invention; and

[0074] FIG. 6 is a graph showing the evaluation result of the crude extract and butanol fraction against anxiety disorders according to an embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0075] A pharmaceutical composition for preventing or treating an anxiety disorder, comprising at least one extract selected from the group consisting of Cynanchum wilfordii, Phlomis umbrosa and Angelica gigas.

MODES FOR CARRYING OUT THE INVENTION

[0076] Hereinafter, the present invention will be described in detail with reference to the following embodiments. However, the embodiments are only for exemplifying the present invention, and the present invention is not limited to the embodiments.

Example 1. Preparation of Crude Extract

[0077] After mixing natural medicinal herbs, the roots of Cynanchum wilfordii, the roots of Phlomis umbrosa and the roots of Angelica gigas, in a weight ratio of 1:1:1.08, the mixture was heated with 10 fold water at a temperature of 95 to 105° C. for 8 hours and extracted. Then, the filtrate obtained by filtration was lyophilized at −80° C., to obtain a crude extract in powder form.

Example 2. Preparation of Butanol Fraction of Crude Extract

[0078] 2 kg of the crude extract in powder form of Example 1 were dissolved with water to have 10 to 15 brix, fed into a large-scale stirrer, and stirred at 400 rpm for 6 hours, to obtain an aqueous solution of the crude extract. Then, n-butanol in an amount corresponding to 2 fold (v/v) of the aqueous solution of the crude extract was fed thereinto, stirred at about 400 rpm for 2 hours, and left standing at room temperature for 24 hours or more, to perform a liquid-liquid fraction. After the liquid-liquid fraction, the top clear n-butanol layer was carefully collected, and the liquid-liquid fraction and collection process was repeated twice for the bottom water layer. Thereafter, only the n-butanol layer was collected and the solvent was completely removed using a vacuum concentrator to have 30 to 33 brix, and then the concentrate was dissolved sufficiently with distilled water to have 13 to 15 brix, followed by lyophilization. The powders obtained after completion of lyophilization were ground with a blender and sifted through a 500 mesh sieve for homogenization, and then left in a desiccator under reduced pressure (25° C., −0.76 mbar) for 8 hours or more, to be double packaged and stored.

Experimental Example 1. Measurement of Tranquilization Induction Time

[0079] 1-1. Crude Extract

[0080] The efficacy was proved through the pentobarbital-induced tranquilization model in mice. Specifically, after 1 week acclimation, the composition of Example 1 was repeatedly administered once daily for 4 weeks in total. The composition of the example was taken in an amount of 100 or 300 mg/kg. After 30 minutes from the last intake, the tranquilization inducer pentobarbital 40 mg/kg was administered, and the tranquilization induction time was measured. The result was shown in FIG. 2 and Table 1.

TABLE-US-00001 TABLE 1 Group Tranquilization induction time (sec.) Control group 553.25 ± 33.67  Example 1 (100 mg/kg)  379.38 ± 44.30** Example 1 (300 mg/kg) 403.00 ± 54.96* Statistically significant control group *p < 0.05, **p < 0.01

[0081] As can be confirmed from FIG. 2 and Table 1, it was confirmed that when compared with the control group, the tranquilization induction time was statistically significantly reduced in both of the two dose groups of the composition of Example 1 (100 mg/kg intake group: p<0.01, 300 mg/kg intake group: p<0.05).

[0082] 1-2. Butanol Fraction

[0083] The efficacy was proved through the pentobarbital-induced tranquilization model in mice. Specifically, after 1 week acclimation, the composition of Example 2 was repeatedly administered once daily for 2 weeks in total. The composition of Example 2 was taken in an amount of 200 or 400 mg/kg. After 45 minutes from the last intake, the tranquilization inducer pentobarbital 45 mg/kg was administered, and the tranquilization induction time was measured. The result was shown in FIG. 3 and Table 2.

TABLE-US-00002 TABLE 2 Group Tranquilization induction time (min.) Control group 3.926 ± 0.442018   Example 2 (200 mg/kg)  3.31 ± 0.221359436* Example 2 (400 mg/kg) 2.902 ± 0.364032966* Statistically significant control group *p < 0.05, ** p < 0.01

[0084] As can be confirmed from FIG. 3 and Table 2, it was confirmed that when compared with the control group, the tranquilization induction time was statistically significantly reduced in both of the dose groups 200 mg/kg and 400 mg/kg of Example 2 (p<0.05).

Experimental Example 2. Measurement of Tranquilization Duration

[0085] 2-1. Crude Extract

[0086] After inducing tranquilization in Experimental Example 1-1, the time in which nerves are activated was measured for each group, and the duration of tranquilization was measured. The result was shown in FIG. 4 and Table 3.

TABLE-US-00003 TABLE 3 Group Tranquilization duration (sec.) Control group 727.63 ± 65.73  Example 1 (100 mg/kg) 2274.50 ± 404.42** Example 1 (300 mg/kg) 2207.13 ± 418.52** Statistically significant control group * p < 0.05, **p < 0.01

[0087] As can be confirmed from FIG. 4 and Table 3, it was confirmed that the duration of tranquilization was statistically significantly increased in both of the two dose groups of the composition of the example (p<0.01).

[0088] 2-2. Butanol Fraction

[0089] After inducing tranquilization in Experimental Example 1-2, the time in which nerves are activated was measured for each group, and the duration of tranquilization was measured. The result was shown in FIG. 5 and Table 4.

TABLE-US-00004 TABLE 4 Group Tranquilization duration (min.) Control group 50.73 ± 6.845363   Example 2 (200 mg/kg) 76.862 ± 5.359339512* Example 2 (400 mg/kg) 83.456 ± 1.945412553* Statistically significant control group *p < 0.05, ** p < 0.01

[0090] As can be confirmed from FIG. 5 and Table 4, it was confirmed that the duration of tranquilization was statistically significantly increased in both of the two dose groups of Example 2 (p<0.05).

Experimental Example 3. Evaluation of Tranquilization Induction at Titer or Less

[0091] The tranquilization inducer pentobarbital 30 mg/kg that is smaller than or equal to the titer was administered, to evaluate the rate of tranquilization induction in the tranquilization borderline state. The result was shown in Table 5.

TABLE-US-00005 TABLE 5 Dose Tranquilization rate Tranquilization time Group (mg/kg) (%) (min.) Control group — 20 6.52 Example 10 30 10.37 100 60 13.02 200 50 11.27 400 50 10.59 Tranquilization rate (%) = number of subjects with activated nerves/total number of subjects × 100

[0092] As can be confirmed from Table 5, the tranquilization induction rate was about 20% in the non-treated group, whereas the tranquilization induction rate was about 60 to 50% in the groups administered with the composition of the example in an amount of 100 mg/kg or more, and particularly better effect was shown in increasing the duration of tranquilization. Thus, it was confirmed that the composition of the example has the effect of inducing tranquilization.

[0093] Consequently, regarding the improvement of anxiety disorders, it was confirmed that the composition of the example statistically significantly improves both of the tranquilization induction time and the tranquilization duration, which are the associated markers, in the pentobarbital-induced tranquilization animal model, and has the excellent effect of improving anxiety disorders, when compared with the control group.

Experimental Example 4. Evaluation of Mechanism of Action Associated with Tranquilizing (GPCR)

[0094] In order to define the mechanism of action on improvement of anxiety disorders, the pattern of GPCR activity associated with tranquilizing was analyzed, and a profiling assay was performed to find the mechanism of action point showing the effect.

[0095] GPCRs, target proteins of different ligands, are the group of receptors responsive to various signal transmitters such as neurotransmitters, hormones, etc., and play an important role in regulation of physiological functions such as reproduction, metabolism, immunity, psychological processes, etc., in human. Thus, the GPCR profiling was performed to screen the GPCRs which are affected by the composition of the example, assess the dose response on adenosine A1, beta-3 adrenergic receptor, adenosine A2A and GABAA ion channel, which are known as the mechanism of action associated with tranquilizing, and perform the tracking evaluation of the mechanism of action.

[0096] Specifically, in order to see the dose-response mechanism of the extracts, the test was carried out by serial dilution with 8 concentrations including the highest concentration 100 ug/ml. When the effective maximum value of a reference agonist for each known receptor is calculated as 100 percent, a GPCR having 50% or higher activity obtained by converting the activity compared therewith into percent (%), may be determined to have activity as an agonist. For the adenosine A1 receptor, a calcium fluorimetry assay was performed in human recombinant BA/F3 cells in which the stable expression of A1 receptor is induced, and the activity thereof was evaluated by comparison with when the effective maximum value of the adenosine A1 receptor agonist CPA is calculated as 100 percent. The result was shown in FIG. 6 and Table 6.

TABLE-US-00006 TABLE 6 Concentration (ug/ml) Example 1 (%) Example 2 (%) 0.03 −0.1 ± 1.2 −0.8 ± 1.6 0.1  0.5 ± 1.1  9.5 ± 2.1 0.3   5 ± 2.5 42.8 ± 0.7 1 28.7 ± 5.2 .sup. 72 ± 2.9 3 .sup. 52 ± 5.3 83.4 ± 0.6 10 63.5 ± 5.3 93.7 ± 9.1 30  75.4 ± 16.5 94.7 ± 1.6 100 87.9 ± 4.6 102.9 ± 1.08

[0097] As can be seen from FIG. 6 and Table 6, as the concentrations of the extracts of Example 1 and the butanol fractions thereof of Example 2 increase to 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 ug/ml, the extracts and the butanol fractions thereof showed the concentration dependent agonist activity. The extracts of Example 1 at the concentration of 3 ug/ml or more and the extracts of Example 2 at the concentration of 1 ug/ml or more showed high activity of 50% or higher. It was confirmed that among the mechanisms of action associated with tranquilizing, the composition of the example induces the activity of concentration dependent adenosine A1 agonist to improve anxiety disorders, and that other adenosine A2A, beta-3 adrenergic receptor and GABAA ion channel are not associated therewith. Accordingly, the mechanism of action on improvement of anxiety disorders was obtained. In addition, the strong agonist activity for adenosine A1 receptor was confirmed, and the 50% effective concentration (EC50) was exhibited in the composition at 2.15 ug/ml of Example 1 and the composition at 0.33 ug/ml of Example 2.

INDUSTRIAL APPLICABILITY

[0098] The present invention relates to a composition for improving, preventing or treating an anxiety disorder, and a tranquilizing composition.