PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING MALE SEXUAL DYSFUNCTION

Abstract

The present invention relates to a composition and health functional food for preventing or treating male sexual dysfunction, which include, as an active ingredient, oil separated from a Pinus densiflora extract, oil separated from an Abies holophylla Maxim extract, or a mixture thereof. The composition according to the present invention has an excellent ability to enhance penile erectility, and may be usefully applied to a health food for alleviating erectile dysfunction symptoms or a therapeutic agent for erectile dysfunction.

Claims

1. A pharmaceutical composition for preventing or treating male sexual dysfunction, the pharmaceutical composition comprising, as an active ingredient, oil separated from a Pinus densiflora extract, oil separated from an Abies holophylla Maxim extract, or a mixture thereof.

2. The pharmaceutical composition of claim 1, wherein the oil is obtained from a pine needle extract or a fir needle extract.

3. The pharmaceutical composition of claim 1, wherein the composition comprises a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 1, wherein the composition is for oral administration.

5. The pharmaceutical composition of claim 1, wherein the oil is included in an amount of 0.1 wt % to 15 wt % with respect to a total weight of the composition.

6. The pharmaceutical composition of claim 1, wherein the extract is obtained by subjecting pine needles or fir needles to steam distillation.

7. The pharmaceutical composition of claim 1, wherein the male sexual dysfunction is caused by alcohol ingestion.

8. The pharmaceutical composition of claim 1, wherein the oil is obtained by the following processes: (S1) collecting pine needles, fir needles, or a mixture thereof; and (S2) contacting the collected pine needles, the collected fir needles, or the collected mixture thereof with steam to obtain an extract thereof.

9. The pharmaceutical composition of claim 8, wherein the contacting comprises injecting steam at 1 atm to 8 atm into a reactor comprising pine needles, fir needles, or a mixture thereof.

10. The pharmaceutical composition of claim 8, further comprising separating oil from the obtained extract.

11. The pharmaceutical composition of claim 1, wherein the male sexual dysfunction comprises erectile dysfunction, premature ejaculation, or prostatic hyperplasia.

12. The pharmaceutical composition of claim 1, wherein the composition is in the form of a liquid, a suspension, powder, granules, a tablet, a capsule, a pill, or an extract.

13. A health functional food for preventing or treating male sexual dysfunction, the health functional food comprising, as an active ingredient, oil separated from a Pinus densiflora extract, oil separated from an Abies holophylla Maxim extract, or a mixture thereof.

14. A formulation comprising: oil separated from a Pinus densiflora extract, oil separated from an Abies holophylla Maxim extract, or a mixture thereof; and β-cyclodextrin.

15. The formulation of claim 14, wherein the β-cyclodextrin comprises any one of 2,6-dimethyl-β-cyclodextrin, 2-hydroxyethyl-β-cyclodextrin, and 2-hydroxypropyl-β-cyclodextrin.

16. The formulation of claim 14, wherein the formulation comprises a β-cyclodextrin inclusion compound.

17. The formulation of claim 16, wherein the β-cyclodextrin inclusion compound is a compound in which oil separated from a Pinus densiflora extract, oil separated from an Abies holophylla Maxim extract, or a mixture thereof is encapsulated in an internal cavity of β-cyclodextrin.

18. A health functional food comprising: oil separated from a Pinus densiflora extract, oil separated from an Abies holophylla Maxim extract, or a mixture thereof; and β-cyclodextrin.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0091] FIG. 1 is a schematic conceptual view illustrating a method of separating oil from an extract through steam distillation.

[0092] FIG. 2 is a graph showing the results of evaluating cyclic GMP content in corpus cavernosum smooth muscles.

MODE FOR THE INVENTION

[0093] Hereinafter, configurations and effects of the present invention will be described in further detail with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0094] In addition, reagents and solvents, which will be described below, were purchased from Sigma-Aldrich unless otherwise specifically mentioned herein.

EXAMPLES

<Example 1> Preparation of Oil Separated from Pinus Densiflora Extract

[0095] 5 kg of pine needles (Collection place: Yeongcheon, Gyeongbuk) were taken, washed with 1.5 L of distilled water, dried in a dry oven at 60° C. for 5 hours, cut into an average length of 2 mm, and put into an extraction tank (capacity: 30 L). The pine needles were brought into contact with steam for 3 hours while steam was applied thereto at 120° C. and a pressure of 2 atm through a steam generator, thereby obtaining a Pinus Densiflora extract.

[0096] Oil positioned at an upper layer portion of the obtained Pinus Densiflora extract was separated, and then allowed to sequentially pass through a granular activated carbon column (manufacturer: Calgon Mitsubishi Chemical Corp., Japan) and a ultrafiltration device (0.2 μm, Ministart, Satorius Stedim Biotech, Germany) to obtain pure oil from which impurities were removed. [0097] Produced amount: 160 g [0098] Yield: 3.2% w/w [0099] Specific gravity: 0.93 g/cm.sup.3

<Example 1-1> Preparation of Test Solution Including Oil Separated from Pinus Densiflora Extract

[0100] 2 g of polysorbate 80 as a surfactant was added to 100 mL of distilled water, and 1 g of the oil separated from the Pinus densiflora extract obtained according to Example 1 was added to the mixed solution and stirred at 3,000 rpm for 30 minutes, and 400 mL of distilled water was added thereto to adjust the total amount to 500 mL such that the oil separated from the Pinus densiflora extract was included in an amount of 2 mg with respect to 1 mL of the mixed solution, thereby completing the preparation of a test solution.

<Example 2> Preparation of Oil Separated from Abies Holophylla Maxim Extract

[0101] 5 kg of fir needles (Collection place: Pyeongchang, Gangwon-do) were taken, washed with 1.5 L of distilled water, dried in a dry oven at 60° C. for 5 hours, cut into an average length of 2 mm, and put into an extraction tank (capacity: 30 L). The fir needles were brought into contact with steam for 3 hours while steam was applied thereto at 120° C. and a pressure of 2 atm through a steam generator, thereby obtaining an Abies Holophylla Maxim extract.

[0102] Oil positioned at an upper layer portion of the obtained Abies Holophylla Maxim extract was separated, and then allowed to sequentially pass through a granular activated carbon column (manufacturer: Calgon Mitsubishi Chemical Corp., Japan) and a ultrafiltration device (0.2 μm, Ministart, Satorius Stedim Biotech, Germany) to obtain pure oil from which impurities were removed. [0103] Produced amount: 125 g [0104] Yield: 2.5% w/w [0105] Specific gravity: 0.9 g/cm.sup.3

<Example 2-1> Preparation of Test Solution

[0106] 2 g of polysorbate 80 as a surfactant was added to 100 mL of distilled water, and 1 g of the oil separated from the Abies holophylla Maxim extract obtained according to Example 2 was added to the mixed solution and stirred at 3,000 rpm for 30 minutes, and 400 mL of distilled water was added thereto to adjust the total amount to 500 mL such that the oil separated from the Abies holophylla Maxim extract was included in an amount of 2 mg with respect to 1 mL of the mixed solution, thereby completing the preparation of a test solution.

[0107] Test Methods

[0108] <Method 1> Specific Gravity Measurement Method

[0109] The specific gravity of each of the oils separated from Pinus densiflora and Abies holophylla Maxim was measured according to the first method (measurement method using a pycnometer) of the specific gravity and density measurement method among general test methods of the Korean Pharmacopoeia eleventh edition.

[0110] <Method 2> Setup of Experimental Groups and Test Solution Administration Method

[0111] As experimental animals, 6-week-old male Sprague-Dawley (SD) white mice weighing about 250 g were used, and the mice were divided into a normal control, an erectile dysfunction-induced control, and experimental groups in which erectile dysfunction was induced and then the test solutions prepared according to Examples 1-1 and 1-2 were respectively administered, and 10 mice were assigned to each group.

[0112] A 20 v/v % aqueous ethanol solution was orally administered daily to the controls and the experimental groups at a dose of 1 mL for 30 days to induce erectile dysfunction due to chronic alcoholism.

[0113] In addition, 5 mg/kg of the test solutions of Example 1-1 (test solution including the oil separated from the Pinus densiflora extract) and Example 2-1 (test solution including the oil separated from the Abies holophylla Maxim extract) were respectively administered to the experimental groups once a day for 30 days using a zonde for oral administration (Oral zonde, 15 G, Samwoo Science, Korea) from the date at which the 20 v/v % aqueous ethanol solution started to be administered.

[0114] <Method 3> Preparation Method of Enzyme Source

[0115] Six-week-old male SD white mice were placed in an anesthesia chamber (JD-C-107R, 245×395×250 mm, Jeung-Do Bio & Plant Co., Ltd., Korea), saturated with ether, and subjected to inhalation anesthesia for 15 minutes, and then the lower abdomen of each mouse was incised about 3 cm, about 7 mL of blood was collected from the abdominal aorta, and then the penile tissue was separated under the pubic bone, peeled off to the entry site, and extracted. The corpus cavernosum was isolated from the extracted penile tissues, washed with physiological saline, and the remaining foreign matter and blood were removed by filter paper.

[0116] Subsequently, 0.1 M potassium phosphate buffer (pH 7.4) in a four-fold amount per 1 g of the corpus cavernosum tissue was added thereto, and homogenized using a homogenizer (ULTRA TURRAX®IKA®T18basic) at 6,000 rpm per minute to thereby prepare a ground homogenate.

[0117] The ground homogenate was centrifuged at 600×g for 10 minutes using a refrigerated centrifuge (Hanil Union 32R) to collect a supernatant, the supernatant was used as a nitrite content measurement source and centrifuged again at 10,000×g for 30 minutes to collect a supernatant, which was then used as an enzyme source for measuring the activity of nitric oxide synthase.

[0118] In addition, a 0.25 M sucrose solution (containing 0.02M Tris HCl buffer (pH 4.0), 1 mM ethylenediamine tetra acetic acid (EDTA), and 10 mM 2-mercaptoethanol) was added in a 5-fold amount per 1 g of the corpus cavernosum tissue and homogenized using a homogenizer (ULTRA TURRAX®IKA®T18basic) at 6,000 rpm per minute to thereby prepare a ground homogenate. The ground homogenate was centrifuged using a refrigerated centrifuge (Hanil Union 32R) at 8,000×g for 20 minutes to collect a supernatant, which was then used as an enzyme source for measuring the activity of guanylate cyclase and a source for measuring the content of cyclic-GMP nitrite.

[0119] <Method 4> Nitrite Content Measurement Method

[0120] To measure the content of nitrite, 200 μL of a Griess reagent [1% (w/v) sulfanilamide, 5% (v/v) phosphoric acid, and 0.1% (w/v) naphthylethylene diamine dihydrochloride] was added to 200 μL of the enzyme source, a reaction was allowed to occur therebetween at room temperature for 10 minutes, and then absorbance at 550 nm was measured (Genesys 20 Thermo Scientific, USA). The content of nitrite was calculated based on a standard curve of sodium nitrite, and the amount of nitrite per 1 g of tissue was converted to μmole.

[0121] <Method 5> Method of Evaluating Nitric Oxide Synthase Activity

[0122] Nitric oxide synthase activity was measured by colorimetric assay using a NADPH diaphorase activity measurement method. To 50 μL of the tissue enzyme source of each experimental animal were added a 50 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES, pH 7.4) solution, 25 μM of L-arginine, 1 mM nicotinamide adenine dinucleotide phosphate (NADPH, reduced form), 2 mM ethylenediamine tetra acetic acid (EDTA), 1.5 mM CaCl.sub.2, 1 mM dithiothreitol, 2 μg of calmodulin, and 1 mM nitroblue tetrazolium (NBT), a reaction was allowed to react therebetween at 37° C. for 5 minutes, and a change in absorbance at 585 nm was measured using Genesys 20 Thermo Scientific. The enzymatic activity was calculated as a value obtained by dividing the absorbance measured at a wavelength of 585 nm using Genesys 20 Thermo Scientific by the content of a used protein.

[0123] <Method 6> Method of Measuring Guanyl Cyclase Activity and Cyclic-GMP Content

[0124] For the measurement of guanyl cyclase activity, 60 nmole of [3H] guanosine 5′-triphosphate (GTP) (0.02 μCi/nmole), cyclic-GMP (0.3 μmole), manganese chloride (MnCl.sub.2, 0.3 mole), and Tris HCl buffer (pH 7.7, 30 μmole) were added to a standard reaction solution, and then an enzyme solution was added thereto to a final volume of 0.15 mL and mixed well, and a reaction was allowed to occur therebetween at 30° C. for 20 minutes while being stirred at 50 rpm in a thermostatic shaking incubator (BR-300LR, Taitec, Japan). After the reaction, the resulting solution was heated in boiling water for 2 minutes and cooled with iced water to terminate the reaction. The amount of cyclic-GMP produced after the enzymatic reaction was measured and subjected to aluminum oxide column chromatography. The reaction solution was added to a column, and then 5 mL of 0.05 M Tris HCl buffer (pH 7.4) was added thereto to perform perfusion, and a cyclic-GMP-containing perfusate collected through the column was perfused again with a 0.05 N formic acid solution and a 0.2 M ammonium formate solution containing 4 N formic acid through a Dowex 1-X2 column to collect the perfusate, which was then used to measure radioactivity to calculate the content. Radioactivity was measured using a liquid scintillation spec-trometer (Beckman, LS-233). Enzyme activity was determined by measuring radioactivity trapped by cyclic-GMP in a nonradioactive form from [3H] guanosine 5′-triphosphate (GTP) and expressed as cpm. Meanwhile, for the measurement of GMP concentration of the corpus cavernosum tissue, experiments were conducted in the same manner as in the above experiments, and then the unit of content was converted to pmole/mg protein/min.

[0125] <Method 7> Method of Evaluating Penile Erectility According to Change in Pressure of Corpus Cavernosum

[0126] For anesthesia, pentobarbital sodium was injected into the abdominal cavity of each white mouse at a dose of 3 mg/kg, and then the lower abdomen of each mouse was incised to a size of about 3 cm, about 7 mL of blood was collected from the abdominal aorta, and then the penile tissue was separated under the pubic bone and peeled off to the entry site, and the penile cavernous nerves were isolated therefrom. For neural stimulation, a platinum electrode was installed in the corpus cavernosum nerve and connected to an electrical stimulator (SEN-7103, Nihon Kohden, Japan).

[0127] In addition, the penile epithelium was incised to expose the corpus cavernosum, and for the measurement of an intracavernosal pressure, a 26 G needle was placed in the corpus cavernosum, which was then connected to a differential amplifier (DA 100, Biopac System, USA) through Sorenson Transpac cable (Abbott Critical Care System, USA) and the intracavernosal pressure was measured using Data Acquisition (MP 100, Biopac Systems, USA), and measurement values were recorded and analyzed using a data analysis program (Acqknowledge 1.5.5 program, Biopac Systems, USA). Intermittent perfusion was performed with heparinized saline (5,000 IU/ml) to prevent blood coagulation in a pressure transmission pipe.

[0128] For penile erectility observation, a penile erection was caused by applying cavernous nerve stimulation to each animal (Frequency: 1 Hz, Intensity: 3-5 V, Pulse duration: 1 msec) for 1 minute to set a standard for a normal penile erection. Thereafter, the intracavernosal pressure was lowered to the baseline, and after 15 minutes, the same intensity of electrical stimulation was applied to measure penile erectility.

[0129] <Method 8> Method of Evaluating Penile Erectility Using L-NAME

[0130] For the measurement of penile erectility using L-NAME, 10 minutes after the intracavernosal pressure was lowered to the baseline, L-NAME was injected into the corpus cavernosum according to concentration, and after 15 minutes, a penile erection was caused with electrical stimulation to observe penile erectility according to the concentration of a drug.

[0131] Hereinafter, the present invention will be described in further detail with reference to the following Experimental Examples. These Experimental Examples are intended to aid in the understanding of implementation of the present invention and are not intended to limit the scope of the present invention.

Experimental Examples

<Experimental Example 1> Content Measurement of Nitrite (NO.SUB.2.)

[0132] As a result of content measurement, the nitrite content of the corpus cavernosum tissue of the normal control was 0.57±0.04 μmole/g, whereas the control administered ethanol exhibited a significant decrease in nitrite content, i.e., 0.35±0.06 μmole/g, compared to the normal control, and the experimental groups, i.e., the group administered the oil separated from the Pinus densiflora extract and the group administered the oil separated from the Abies holophylla Maxim extract, exhibited significant increases in nitrite content, i.e., 0.49±0.07 μmole/g and 0.51±0.09 μmole/g, respectively, compared to the control.

<Experimental Example 2> Measurement of Nitric Oxide Synthase Activity

[0133] As a result of conducting a test for the measurement of nitric oxide synthase activity, the nitric oxide synthase activity of the normal control was 1.42±0.18 ΔOD/mg, whereas the control exhibited significantly inhibited nitric oxide synthase activity, i.e., 0.96±0.08 ΔOD/mg, compared to the normal control. In contrast, the experimental groups, i.e., the group administered the oil separated from the Pinus densiflora extract and the group administered the oil separated from the Abies holophylla Maxim extract, exhibited significant increases in nitric oxide synthase activity, i.e., 1.25±0.05 ΔOD/mg and 1.20±0.07 ΔOD/mg, respectively, compared to the control.

[0134] <Experimental Example 3> Measurement of Guanyl Cyclase Activity and Cyclic-GMP Content

[0135] As a result of conducting the experiment for guanyl cyclase activity, the normal control exhibited a guanyl cyclase activity of 103.7±7.5 cpm, whereas the control exhibited significantly inhibited guanyl cyclase activity, i.e., 77.3±6.2 cpm, compared to the normal control. In contrast, the experimental groups, i.e., the group administered the oil separated from the Pinus densiflora extract and the group administered the oil separated from the Abies holophylla Maxim extract, exhibited significant increases in guanyl cyclase activity, i.e., 93.2±7.1 cpm and 95.3±4.9 cpm, respectively, compared to the control.

[0136] As a result of conducting the experiment for cyclic-GMP content, the normal control exhibited a c-GMP content of 0.150±0.008 pmole/mg, whereas the control exhibited a significant decrease in c-GMP content, i.e., 0.082±0.012 pmole/mg, compared to the normal control. However, the experimental groups, i.e., the group administered the oil separated from the Pinus densiflora extract and the group administered the oil separated from the Abies holophylla Maxim extract, exhibited significant increases in c-GMP content, i.e., 0.115±0.006 pmole/mg and 0.125±0.007 pmole/mg, respectively, compared to the control.

[0137] From the result that the experimental groups respectively administered the oil separated from the Pinus densiflora extract and the oil separated from the Abies holophylla Maxim extract exhibited an increase in c-GMP content in the corpus cavernosum smooth muscle, it can be seen that the oil separated from the Pinus densiflora extract and the oil separated from the Abies holophylla Maxim extract affect the generation of nitric oxide (see FIG. 2).

<Experimental Example 4> Penile Erectility Evaluation

[0138] The internal pressure of the corpus cavernosum of the normal control was 90.9±7.8 mmHg, whereas the control administered an ethanol solution exhibited a significant decrease in the internal pressure thereof, i.e., 66.7±4.3 mmHg, compared to the normal control. In contrast, the respective experimental groups administered the oil separated from the Pinus densiflora extract and the oil separated from the Abies holophylla Maxim extract while ethanol was administered exhibited significant increases in the internal pressure of the corpus cavernosum, i.e., 82.1±4.8 mmHg and 80.1±5.2 mmHg, respectively, compared to the control.

<Experimental Example 5> Changes in Nitric Oxide Content and c-GMP Content after L-NAME Treatment

[0139] In the present invention, the term “L-NAME” is an abbreviation of N′-nitro-L-arginine-methyl ester hydrochloride and refers to a compound which is rep-resented by Formula 1 below, has the molecular formula of C.sub.7H.sub.16ClN.sub.5O.sub.4 (number average molecular weight: 269.69 g/mol), and functions as a nitric oxide synthase inhibitor.

##STR00001##

[0140] After L-NAME treatment, the nitric oxide content of the corpus cavernosum tissue was evaluated.

[0141] Specifically, normalization treatment to lower the intracavernosal pressure of each white mouse to the baseline was performed, and 20 minutes after L-NAME was injected into the corpus cavernosum according to concentration, the penile cavernous tissues were extracted, and the nitric oxide content was observed. The content of nitric oxide in the corpus cavernosum tissues of the normal control was 0.56±0.04 μmole, but as the amount of injected L-NAME increased, the content of nitric oxide decreased, i.e., 0.51±0.08 μmole at the concentration of injected L-NAME of 10.sup.−7 M, 0.48±0.05 mole at 10.sup.6M, and 0.41±0.03 mole at 10.sup.5M.

[0142] In addition, after the normalization treatment to lower the intracavernosal pressure of each white mouse to the baseline, the cyclic-GMP content of the corpus cavernosum tissue was 0.106±0.005 pmole/mg. When observing the content of cyclic-GMP after L-NAME was injected at various concentrations under the same conditions, the content of cyclic GMP decreased as the amount of L-NAME injected into the corpus cavernosum increased, i.e., 0.097±0.008 pmole/mg at 10.sup.7M, 0.092±0.005 pmole/mgat10.sup.6M, and 0.085±0.004 pmole/mgat10.sup.5M.

<Experimental Example 6> Evaluation of Penile Erectility by L-NAME Pre-treatment

[0143] The intracavernous pressure of the normal control was 90.9±7.8 mmHg, and was significantly reduced when treated with L-NAME, such as 89.9±6.5 mmHg at a concentration of L-NAME of 10.sup.7M, 86.6±8.3 mmHg at 10.sup.6M, 84.2±8.8 mmHg at 10.sup.5M, and 70.8±6.6 mmHg at 10.sup.4M. However, the intracavernous pressures of the group administered the oil separated from the Pinus densiflora extract and the group administered the oil separated from the Abies holophylla Maxim extract were 82.5±8.8 mmHg at a concentration of injected L-NAME of 10.sup.4M and 86.6±4.7 mmHg at a concentration of injected L-NAME of 10.sup.4M, respectively, showing restoration to almost anormal level.

[0144] Hereinafter, the present invention will be described in further detail with reference to the following Preparation Examples. These Preparation Examples are provided to aid in understanding of the practice of the present invention and are not intended to limit the scope of the present invention.

Preparation Examples

<Preparation Example 1> Preparation of Liquid

[0145] An inclusion compound was prepared by physically kneading β-cyclodextrin and the oil separated from a Pinus densiflora extract. Specifically, hydroxypropyl β-cyclodextrin was added to a reactor, and the oil separated from the Pinus densiflora extract was added thereto, followed by mixing and stirring for 30 minutes using mortars to thereby prepare an inclusion compound of the oil separated from the Pinus densiflora extract and hydroxypropyl β-cyclodextrin.

[0146] Subsequently, 60 mL of distilled water was added to the inclusion compound to dissolve the compound therein, xylitol and cherry flavor powder were added thereto, the resulting solution was stirred at 3,000 rpm for 20 minutes using a homomixer, thereby preparing a total amount of 100 mL of a liquid containing the oil separated from the Pinus densiflora extract. Used raw materials are the same as shown in Table 1 below.

TABLE-US-00001 TABLE 1 Raw materials Added amount Oil seperated from Pinus densiflora extrtact 0.3 g Hydroxypropyl β-cyclodextrin 3 g Xylitol 30 g Cherry flavor powder 0.05 g Distilled water 75 mL

<Preparation Example 2> Preparation of Liquid

[0147] A liquid containing the oil separated from the Abies holophylla Maxim extract was prepared using the same method as that used in Preparation Example 1, except that 0.3 g of the oil separated from the Abies holophylla Maxim extract was added instead of the oil separated from the Pinus densiflora extract.

<Preparation Example 3> Preparation of Granules

[0148] Hydroxypropyl β-cyclodextrin was added to a reactor, and the oil separated from the Pinus densiflora extract was added thereto, followed by stirring at 1,000 rpm for 30 minutes using a mixer to thereby prepare an inclusion compound of the oil separated from the Pinus densiflora extract and hydroxypropyl β-cyclodextrin.

[0149] Separately, dextrose, lactose, and microcrystalline cellulose were added to a speed mixer, and the inclusion compound of the oil separated from Pinus densiflora extract and hydroxypropyl β-cyclodextrin was added thereto, and 100 mL of distilled water was added thereto, followed by stirring at 800 rpm for about 1 hour. The stirred mixture was dried in a dryer at 70° C. for 5 hours, and then magnesium stearate was added thereto to thereby prepare granules containing the oil separated from the Pinus densiflora extract through a granulator. Used raw materials are the same as shown in Table 2 below.

TABLE-US-00002 TABLE 2 Raw materials Added amount Oil seperated from Pinus densiflora extrtact 30 g Hydroxypropyl β-cyclodextrin 300 g Dextrose 200 g Lactose 200 g g Microcrystalline cellulose 300 g Magnesium stearate 2 g Distilled water 100 mL

<Preparation Example 4> Preparation of Granules

[0150] Granules containing the oil separated from the Abies holophylla Maxim extract was prepared using the same method as that used in Preparation Example 3, except that 30 g of the oil separated from the Abies holophylla Maxim extract was added instead of the oil separated from the Pinus densiflora extract.

<Preparation Example 5> Preparation of Chewable Tablets

[0151] The oil separated from the Pinus densiflora extract and β-cyclodextrin were added to a mixer and stirred at 1,000 rpm for 30 minutes to thereby prepare an inclusion compound of the oil separated from the Pinus densiflora extract and hydroxypropyl β-cyclodextrin.

[0152] Separately, maltodextrose, xylitol, and microcrystalline cellulose were added to a speed mixer, and the inclusion compound of the oil isolated from Pinus densiflora extract and hydroxypropyl β-cyclodextrin was added thereto, and 130 mL of distilled water was added thereto, followed by stirring at 800 rpm for about 1 hour. The stirred mixture was dried in dryer at 70° C. for 5 hours and granulated in a granulator, and then magnesium stearate and yogurt flavor powder were added thereto and mixed to thereby prepare 1,000 mg/tablet of chewable tablets through a tablet press. Used raw materials are the same as shown in Table 3 below.

TABLE-US-00003 TABLE 3 Raw materials Added amount Oil seperated from Pinus densiflora extrtact 40 g Hydroxypropyl β-cyclodextrin 400 g Maltodextrin 100 g Microcrystalline cellulose 500 g Xylitol 300 g Yogurt flavor powder 3 g Magnesium stearate 3 g Distilled water 130 mL

<Preparation Example 6> Preparation of Chewable Tablets

[0153] Chewable tablets containing the oil separated from the Abies holophylla Maxim extract was prepared using the same method as that used in Preparation Example 5, except that 40 g of the oil separated from the Abies holophylla Maxim extract was added instead of the oil separated from the Pinus densiflora extract.