Health Functional Food Composition For Preventing And Alleviating Depression, Containing Vaccinium Bracteatum Thunb. Fruit Extract

Abstract

The present invention provides a health functional food composition and a pharmaceutical composition for preventing and alleviating depression, both compositions using an extract of Vaccinium bracteatum Thunb. fruits, which are Korean natural resources, so as to be safely usable without toxicity and side effects.

Claims

1. A health functional food composition containing a Vaccinium bracteatum Thunb. fruit extract as an active ingredient for prevention or relief of depression.

2. The health functional food composition of claim 1, wherein the Vaccinium bracteatum Thunb. fruit extract is extracted with at least one solvent selected from the group consisting of water, ethanol, methanol, propanol, isopropanol, and butanol.

3. A health functional food for prevention or relief of depression, the health functional food comprising the health functional food composition of claim 1 at an amount of 0.01-99.9 wt %.

4. The health functional food of claim 3, wherein the health functional food is manufactured in the form of any one selected from a tablet, a capsule, a soft capsule, granules, and a liquid preparation.

5. A method for prevention or relief of depression comprising: administering, to a subject, a composition including comprising a Vaccinium bracteatum Thunb. fruit extract as an active ingredient.

6. The method of claim 5, wherein the Vaccinium bracteatum Thunb. fruit extract is extracted with at least one solvent selected from the group consisting of water, ethanol, methanol, propanol, isopropanol and butanol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows a schematic diagram illustrating a Vaccinium bracteatum Thunb. fruit extract.

[0014] FIG. 2 shows a schematic diagram illustrating the fractions of a Vaccinium bracteatum Thunb. Fruit extract.

[0015] FIG. 3 shows a graph illustrating the comparison of immobility time, swimming time, and climbing time in the forced swimming test among white mice administered with Vaccinium bracteatum Thunb. fruit extracts of the present invention for 6 days, control group mice, and escitalopram oxalate administration group mice.

[0016] FIG. 4 shows graphs illustrating the comparison of the increase or decrease in the neurotransmitter and the stress hormone in the blood among mice administered with the Vaccinium bracteatum Thunb. fruit extracts of the present invention, control group mice, and escitalopram oxalate administration group mice.

[0017] FIG. 5 shows a graph illustrating the cytotoxicity degree of the Vaccinium bracteatum Thunb. fruit extract of the present invention on SH-SY5Y cells (neuroblastoma, human dopaminergic neuronal cells).

[0018] FIGS. 6A and 6B show graphs illustrating the neuronal protective effect of the Vaccinium bracteatum Thunb. fruit extract against oxidative stress induced through the treatment with hydrogen peroxide.

[0019] FIGS. 7A and 7B show graphs illustrating the intracellular stress induction through the treatment with the mental stress hormone corticosterone and the neuronal protective effect of the Vaccinium bracteatum Thunb. fruit extract according to the concentration.

[0020] FIG. 8 shows a graph illustrating the effect of the Vaccinium bracteatum Thunb. fruit extract of the present invention to increase the 5-HT-induced intracellular Ca.sup.2+ increase in a dose-dose-dependent manner in the 5-HT.sub.1A receptor gene-expressed cell line.

[0021] FIG. 9 shows a graph illustrating the effect of the Vaccinium bracteatum Thunb. fruit extract of the present invention to inhibit the 5-HT-induced intracellular Ca.sup.2+ increase in a dose-dependent manner in the 5-HT.sub.2A receptor gene-expressed cell line.

[0022] FIG. 10 shows a graph illustrating the effect of the fractions of the Vaccinium bracteatum Thunb. fruit extract of the present invention to inhibit the 5-HT-induced intracellular Ca.sup.2+ increase in the 5-HT.sub.2A receptor gene-expressed cell line.

DETAILED DESCRIPTION

[0023] 1. Preparation of Vaccinium bracteatum Thunb. Fruit Extract

[0024] FIG. 1 shows a procedure of obtaining a Vaccinium bracteatum Thunb. fruit extract. 30 L of distilled water was added to 2.0 kg of Vaccinium bracteatum Thunb. fruits, followed by heating and extraction using a reflux extractor for 3 hours after the temperature rose to 100 C. or higher. The obtained extract was subjected to filtration under reduced pressure and then concentration under reduced pressure, thereby obtaining 107.02 g of a concentrate.

[0025] 2. Preparation of Polar Solvent- and Nonpolar Solvent-Soluble Fractions of Vaccinium bracteatum Thunb. Fruits

[0026] As shown in FIG. 2, the preparation of fractions of the Vaccinium bracteatum Thunb. fruit extract by performing systemic fraction from the Vaccinium bracteatum Thunb. fruit extract will be specifically described below. The concentrate was sufficiently suspended in water and subjected to primary solvent carryover three times using a 2-fold volume of hexane, leading to separation into a water layer and a hexane ext. (41.8 mg). The water layer was again subjected to secondary solvent carryover three times using a 2-fold volume of chloroform, thereby securing a CHCl.sub.3 ext. (79.9 mg). Tertiary solvent carryover using ethyl acetate was performed three times, thereby securing an EtOAc ext. (312.4 mg). Last, quaternary solvent carryover using n-butanol was performed three times, thereby securing an n-BuOH ext. (5 g).

[0027] 3. Animals and Breeding

[0028] As test animals for determining an anti-depressive effect of the Vaccinium bracteatum Thunb. fruit extract, 6-week-old ICR male mice were purchased from SAMTACO (Korea), and acclimated for 1 week in an animal breeding room in predetermined conditions (temperature: 222 C., humidity: 505%, 12 hr-light/dark cycle) before use.

[0029] 4. Animal Behavior Test on Anti-Depression Using Vaccinium bracteatum Thunb. Fruit Extracts

[0030] The ICR male mice were orally administered with the Vaccinium bracteatum Thunb. fruit extract of the present invention. For a comparative group, escitalopram oxalate used an anti-depressant was orally administered. These were administered for 6 days according to the test.

[0031] Before drug administration, for the pre-test (pre-swim), tap water around 25 C. was poured into a cylindrical water tank (20 cm in diameter, 40 cm in height) from the bottom of the cylinder to 15 cm, and then the mice were put into the water tank, forced to swim for 15 minutes, taken out from the water, wiped with a dry towel, and then returned to breeding boxes. After 6 days of drug administration, the present test (post-swim) was carried out. The mice were put into the cylinder, and forced to swim for 6 minutes under videotaping. The recorded image was analyzed excluding the first minute therefrom, and animal behavior during the last 5 minutes was classified into 3 types: immobility behavior (the mouse only floated near the surface of the water, slightly moving while only a part of the upper body including the face was above water), swimming behavior (the mouse moved horizontally around the cylinder moving the front and hind legs in a swimming motion), and climbing behavior (the mouse scratched the wall, somewhat violently kicking the front extremities to move remain above water). The duration of each behavior type was measured.

[0032] FIG. 3 shows a graph illustrating the comparison of immobility time, swimming time, and climbing time in the forced swimming test among white mice administered with Vaccinium bracteatum Thunb. fruit extracts of the present invention for 6 days, control group mice, and escitalopram oxalate administration group mice. As shown in FIG. 3, during the forced swimming after the administration of each drug for 6 days, the immobility time was 169.657.15 seconds for escitalopram oxalate as the comparison group, and 114.989.26 seconds and 118.296.35 seconds for Vaccinium bracteatum Thunb. fruit extracts (100 and 200 mg/kg p.o), showing significant reductions, compared with the control group (245.484.05 seconds) (P<0.01 and P<0.001). The swimming time was 128.166.65 seconds for escitalopram oxalate as the comparison group, and 177.079.79 seconds and 172.236.72 seconds for Vaccinium bracteatum Thunb. fruit extracts (100 and 200 mg/kg p.o), showing significant increases, compared with the control group (51.823.38 seconds) (P<0.01 and P<0.001).

[0033] As seen above, it was confirmed that the Vaccinium bracteatum Thunb. fruit extract of the present invention decreased the immobility time during which the limbs do not move, showing an excellent anti-depressive effect compared with escitalopram oxalate as the comparison group.

[0034] 5. Measurement of Norepinephrine (NE) and Corticosterone (COR) in Plasma Using Vaccinium bracteatum Thunb. Fruit Extract

[0035] The effects on concentration changes of the neurotransmitter NE associated with depression and the stress hormone COR in normal conditions were measured by isolating plasma from the mice after 6 day-administration of each drug and using the Abnova ELISA kit according to the instruction of the manufacturer.

[0036] FIG. 4 shows graphs illustrating the comparison of the increase or decrease in the neurotransmitter and the stress hormone in the blood among mice administered with the Vaccinium bracteatum Thunb. fruit extracts of the present invention, control group mice, and escitalopram oxalate administration group mice. The anti-depressive effects of the Vaccinium bracteatum Thunb. fruit extract on the variations of NE and COR in normal conditions were investigated. The reason for a feeling of sadness is due to a small amount of serotonin or norepinephrine (noradrenaline) secreted externally from brain neurons to the outside. As a result of investigating an increase or decrease in the neurotransmitter norepinephrine through the Vaccinium bracteatum Thunb. fruit extract, as shown in FIG. 4(A), when normal animals were administered with the Vaccinium bracteatum Thunb. fruit extracts (100 and 200 mg/kg) and escitalopram oxalate (10 mg/kg), the Vaccinium bracteatum Thunb. fruit extracts (100 and 200 mg/kg) administration groups showed an increase tendency of NE concentration compared with the control group. These results indicate that the Vaccinium bracteatum Thunb. fruit extract is effective as an anti-depressant by increasing the concentration of NE.

[0037] In addition, as shown in FIG. 4(B), the level of the stress hormone corticosterone was significantly reduced in the Vaccinium bracteatum Thunb. fruit extract (100 and 200 mg/kg) administration groups compared with the control group, and the comparative group of escitalopram oxalate showed a decrease tendency of corticosterone compared with the control group, with no significance. Therefore, considering that the corticosterone levels are known to increase when an individual feels stressed, these results suggest that the Vaccinium bracteatum Thunb. fruit extract would also show an anti-depressive effect in stress depression models.

[0038] 6. Investigation of Stress Relieving Activity in Brain Neurons Using Vaccinium bracteatum Thunb. Fruit Extract

[0039] SH-SY5Y cells (neuroblastoma, human dopaminergic neuronal cells) were incubated in MEM medium containing 1% antimycotics/antibiotics and 10% FBS. The cells were seeded at a density of 10.sup.5 cell/ml in a 96-well plate, and then incubated for 24 hours.

[0040] To investigate the cytotoxicity of the Vaccinium bracteatum Thunb. fruit extract on neurons, the cells were treated with the extract according to the concentration. Cell viability was evaluated using an MTT assay 24 hours after the treatment with the Vaccinium bracteatum Thunb. fruit extract.

[0041] FIG. 5 shows a graph illustrating the cytotoxicity degree of the Vaccinium bracteatum Thunb. fruit extract of the present invention on SH-SY5Y cells (neuroblastoma, human dopaminergic neuronal cells).

[0042] The cytotoxicity of the Vaccinium bracteatum Thunb. fruit extract was measured using MTT to determine the degree of cytotoxicity thereof, thereby setting the maximum safety range of use concentration thereof. The cell line used was SH-SY5Y cells, and the cells were treated with the Vaccinium bracteatum Thunb. fruit hot-water extract at concentrations of 1, 3, 10, 30, and 100 g/ml to investigate cytotoxicity. The Vaccinium bracteatum Thunb. fruit extract at 1, 3, 10, and 30 g/ml showed no significant difference compared to the control (0 g/ml). However, the Vaccinium bracteatum Thunb. fruit extract at 100 g/ml showed a reduced cell viability, although no significant difference was shown compared to the control. From the above results, tests were performed within 30 g/ml.

[0043] To determine the effect of Vaccinium bracteatum Thunb. fruit extract on neuronal apoptosis caused by oxidative stress, the use concentration of hydrogen peroxide was first determined. For the determination, the cells were treated with hydrogen peroxide at different concentrations for 24 hours to investigate cell cytotoxicity, and the concentration of Vaccinium bracteatum Thunb. fruit extract showing 50-60% cytotoxicity was determined and used in the test. For the test to investigate the neuronal protective effect of the Vaccinium bracteatum Thunb. fruit extract, the test cell line (SH-SY5Y cell line) was dispensed in a 96-well microplate, incubated for 24 hours, treated with the Vaccinium bracteatum Thunb. fruit extract for 2 hours, treated with 10 M H.sub.2O.sub.2, and incubated for 24 hours. After 24 hours, cell viability was determined using an MTT assay.

[0044] FIG. 6 shows graphs illustrating the neuronal protective effect of the Vaccinium bracteatum Thunb. fruit extract against oxidative stress induced through the treatment with hydrogen peroxide. In FIG. 6A, for the determination of the use concentration of hydrogen peroxidase, the cell line (SH-SY5Y cells) used was treated with H.sub.2O.sub.2 according to the concentration to investigate cytotoxicity, and as a result, H.sub.2O.sub.2 at 10 M showed a cell viability of 50-60%, indicating significant cytotoxicity, compared with the control (0 g/ml), and thus were used for the test. FIG. 6B confirmed that in the treatment with the Vaccinium bracteatum Thunb. fruit extract together with 10 M hydrogen peroxide (H.sub.2O.sub.2), the Vaccinium bracteatum Thunb. fruit hot-water extract at 0.3, 1, 3, and 10 g/ml showed cell viability of 54.752.85%, 52.185.05%, 69.011.48%, and 80.241.14%, respectively, indicating significant increases (P<0.05 or P<0.01).

[0045] To investigate the resistance activity of the Vaccinium bracteatum Thunb. fruit extract against mental stress at the cellular level, the test was conducted to investigate the protective effect on neuronal damages caused by the stress hormone corticosterone. The cells were treated with corticosterone at different concentrations for 24 hours to investigate cell cytotoxicity, and the concentration of corticosterone showing 50-60% cytotoxicity was determined, and used in the test. For the test to investigate the protective effect of the Vaccinium bracteatum Thunb. fruit extract, the test cell line (SH-SY5Y cell line) was dispensed in a 96-well microplate, incubated for 24 hours, treated with the Vaccinium bracteatum Thunb. fruit extract for 2 hours, treated with 1 M corticosterone, and incubated for 24 hours. After 24 hours, cell viability was determined using an MTT assay.

[0046] FIG. 7 shows graphs illustrating the intracellular stress induction through the treatment with the mental stress hormone corticosterone and the neuronal protective effect of the Vaccinium bracteatum Thunb. fruit extract according to the concentration. For the determination of the use concentration of the stress hormone corticosterone, SH-SY5Y cells were treated with corticosterone at 50, 100, 300, 500, and 1000 M to investigate cytotoxicity, and cell viability was reduced in corticosterone at 300, 500, and 1000 M compared with the control (0 g/ml), and 1 mM corticosterone showing 50-60% cytotoxicity was used for the test. FIG. 7B confirmed that in the treatment with the Vaccinium bracteatum Thunb. fruit extract together with corticosterone, the Vaccinium bracteatum Thunb. fruit hot-water extract at 0.3, 1, 3, and 10 g/ml showed cell viability of 63.280.42%, 66.552.21%, 86.914.27%, and 89.085.47%, respectively, indicating significant increases (P<0.05 or P<0.01).

[0047] 7. Measurement of Intracellular Ca.sup.2+ Activity Using Vaccinium bracteatum Thunb. Fruit Extract in 5-HT.sub.1A or 5-HT.sub.2A Receptor Gene-Expressed Cell Line

[0048] CHO-K1 cell line (Chinese Hamster Ovary Cells) was purchased from ATCC, and dispensed using a culture liquid (RPMI 1640, fetal bovine serum 10%, penicillin 100 IU/ml, streptomycin 100 g/ml) in a 96-well black wall/clear bottom (BD Falcon), and then incubated in 37 C., 5% CO.sub.2 incubator for 24 hours. The cells, in which serotonin 1A receptor (5-HT.sub.1A receptor) or serotonin 2A receptor (5-HT.sub.2A receptor) cDNA was transiently expressed in cells using the plasmid transfection reagent lipofectamine 2000 for 48 hours, were used for the test.

[0049] Before fluorescent staining, the cells were washed once with HEPES-buffered solution (155 mM NaCl, 2 mM CaCl.sub.2), 1 mM MgCl.sub.2, 3 mM KCl, 10 mM HEPES, 10 mM Glucose, pH 7.4), treated with Fura-2/AM at a final concentration of 5 M, which is a fluorescent dye for measuring intracellular calcium concentration, and then incubated in the 37 C., 5% CO.sub.2 incubator for 60 minutes while the light was blocked. After 60 minutes, the cells were washed once with HEPES-buffered solution, and then the intracellular calcium concentration was determined by the selective exposure to the light at 340/380 nm using a high-throughput system (HTS) system and then the measurement of the 340/380 nm ratio analyzed using a digital fluorescence analyzer. The Fura-2/AM present in cells is excited at 340 nm in the state combined with Ca.sup.2+, and excited at 380 nm in the unbound state, and therefore, an increase in the 340/380 nm ratio value means that fura-2/AM combined with intracellular Ca.sup.2+ is increased.

[0050] The value obtained by the treatment with 100 M serotonin (5-HT) was used for a control, and the change in amount of intercellular calcium was investigated after the cells were pre-treated with the Vaccinium bracteatum Thunb. fruit extract at 1, 3, 10, 30, and 100 g/ml for 1 minute and then 100 M 5-HT. In FIGS. 8 and 9, the number on the error bar of each item means the number of cells used in the test.

[0051] FIG. 8 shows a graph illustrating the effect of the Vaccinium bracteatum Thunb. fruit extract of the present invention to increase the 5-HT-induced intracellular Ca.sup.2+ increase in a dose-dose-dependent manner in the 5-HT.sub.1A receptor gene-expressed cell line. In serotonin-induced intracellular Ca.sup.2+ in the 5-HT.sub.1A receptor, the Vaccinium bracteatum Thunb. fruit extract activated the intracellular Ca.sup.2+ compared with serotonin, and thus the effect of the Vaccinium bracteatum Thunb. fruit extract as a partial agonist of the 5-HT.sub.1A receptor was confirmed. As shown in FIG. 8, the intracellular Ca.sup.2+ induced by 5-HT.sub.1A receptor-specific serotonin was activated by 10.316.86%, 47.451.45%, 62.732.37%, 67.081.36%, and 88.093.50%, using the Vaccinium bracteatum Thunb. fruit extract at 1, 3, 10, 30, and 100 g/ml, in a dose-dependent manner, compared with the control treated with 100 M serotonin.

[0052] 8. Measurement of Intracellular Ca.sup.2+ Activity in 5-HT.sub.2A Receptor Gene-Expressed Cell Line Using Solvent Fractions of Vaccinium bracteatum Thunb. Extract

[0053] CHO-K1 cell line (Chinese Hamster Ovary Cells) was purchased from ATCC, and dispensed using a culture liquid (RPMI 1640, fetal bovine serum 10%, penicillin 100 IU/ml, streptomycin 100 g/ml) in a 96-well black wall/clear bottom (BD Falcon), and then incubated in 37 C., 5% CO.sub.2 incubator for 24 hours. The cells, in which serotonin 2A receptor (5-HT.sub.2A receptor) cDNA was transiently expressed in cells using the plasmid transfection reagent lipofectamine 2000 for 48 hours, were used for the test.

[0054] Before fluorescent staining, the cells were washed once with HEPES-buffered solution (155 mM NaCl, 2 mM CaCl.sub.2), 1 mM MgCl.sub.2, 3 mM KCl, 10 mM HEPES, 10 mM Glucose, pH 7.4), treated with Fura-2/AM at a final concentration of 5 M, which is a fluorescent dye for measuring intracellular calcium concentration, and then incubated in the 37 C., 5% CO.sub.2 incubator for 60 minutes while the light was blocked. After 60 minutes, the cells were washed once with HEPES-buffered solution, and then the intracellular calcium concentration was determined by the selective exposure to the light at 340/380 nm using a high-throughput system (HTS) system and then the measurement of the 340/380 nm ratio analyzed using a digital fluorescence analyzer. The Fura-2/AM present in cells is excited at 340 nm in the state combined with Ca.sup.2+, and excited at 380 nm in the unbound state, and therefore, an increase in the 340/380 nm ratio value means that fura-2/AM combined with intracellular Ca.sup.2+ is increased.

[0055] The treatment with 100 M serotonin (5-HT) was used as a control, and the change in amount of intercellular calcium was investigated after the cells were pre-treated with the hexane, chloroform, ethyl acetate, butanol, and water fractions of the Vaccinium bracteatum Thunb. fruit extract and then 100 M 5-HT.

[0056] FIG. 9 shows a graph illustrating the effect of the Vaccinium bracteatum Thunb. fruit extract of the present invention to inhibit the 5-HT-induced intracellular Ca.sup.2+ increase in a dose-dependent manner in the 5-HT.sub.2A receptor gene-expressed cell line. It was confirmed in FIG. 9 that, in serotonin-induced intracellular Ca.sup.2+ in the 5-HT.sub.2A receptor, the Vaccinium bracteatum Thunb. fruit extract inhibited the intracellular Ca.sup.2+, compared with serotonin, in a dose-dependent manner. As shown in FIG. 9, the intracellular Ca.sup.2+ concentration induced by 5-HT.sub.2A receptor-specific serotonin was inhibited by 29.150.81%, 37.530.49%, 42.090.92%, 54.390.55%, and 59.030.48%, using the Vaccinium bracteatum Thunb. fruit extract at 1, 3, 10, 30, and 100 g/ml, in a dose-dependent manner, compared with the control treated with 100 M serotonin.

[0057] FIG. 10 shows a graph illustrating the effect of the fractions of the Vaccinium bracteatum Thunb. fruit extract of the present invention to inhibit the 5-HT-induced intracellular Ca.sup.2+ increase in the 5-HT.sub.2A receptor gene-expressed cell line. In FIG. 10, the number on the error bar of each item means the number of cells used in the test. FIG. 10 shows the results on the intracellular Ca.sup.2+ concentration inhibitory activity of the solvent fractions of the Vaccinium bracteatum Thunb. fruit extract, compared with serotonin, in the serotonin-induced Ca.sup.2+ increase in the 5-HT.sub.2A receptor. As shown in FIG. 10, among the solvent fractions, such as hexane, chloroform, ethyl acetate, butanol, and water fractions, of the Vaccinium bracteatum Thunb. fruit extract, the ethyl acetate and butanol fractions showed the highest inhibitory activity on the serotonin-induced intracellular Ca.sup.2+ increase in the 5-HT.sub.2A receptor.

[0058] 9. Manufacturing of Medicines or Health Foods Using Vaccinium bracteatum Thunb. Fruit Extract

[0059] A pharmaceutical composition or health food composition containing a Vaccinium bracteatum Thunb. fruit extract as an active ingredient for immunity enhancement may be manufactured in the form of a tablet, a capsule, a soft capsule, granules, or a liquid preparation. According to another proper embodiment, the composition may be manufactured as a drink additive. The medicine or health food for immunity enhancement may be manufactured by formulation into a powder, granules, a tablet, a capsule, a suspension, an emulsion, a syrup, an aerosol, a percutaneous medicine, a suppository, or a sterile injection solution such that the composition containing the Vaccinium bracteatum Thunb. fruit extract as an active ingredient is contained at 0.01-99.9 wt %.

[0060] The sterile injection solution can be manufactured by containing 0.01-99.9 wt % of the pharmaceutical composition and mixing the pharmaceutical composition with 99.9-0.01 wt % of purified water or glucose. The capsule may be manufactured by containing 0.01-99.9 wt % of the pharmaceutical composition after freeze drying and mixing the pharmaceutical composition with 99.9-0.01 wt % of a vitamin or calcium agent.

[0061] The daily dose of the prepared pharmaceutical composition corresponds to a content in which the extract is contained at 10-2000 mg/kg body weight. Furthermore, a health functional food containing 0.01-99.9 wt % of the pharmaceutical composition for prevention or relief of depression symptoms can also be manufactured.

INDUSTRIAL APPLICABILITY

[0062] The present invention provides a health functional food composition containing a Vaccinium bracteatum Thunb. fruit extract as an active ingredient for relief of depression causes and symptom diseases, and thus can replace the raw materials for a plant growing in nature, thereby expecting the reduction of manufacturing and production costs and the import substitution and export effects through industrialization, so that the present invention is industrially applicable.