Composition for antioxidation, anti-inflammation, or osteoclast differentiation inhibition

Abstract

The present invention relates to a composition for antioxidation, anti-inflammation, or osteoclast differentiation inhibition and a composition for arthritis treatment, each of the compositions containing a fraction of a complex extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas as an active ingredient. The compositions of the present invention can be effectively used for osteoclast differentiation inhibition, antioxidation, or anti-inflammation, and arthritis treatment.

Claims

1. A method for alleviation or treatment of a bone disease, the method comprising: administering to a subject an effective amount of a composition comprising 50 to 100 μg/mL of an ethyl acetate fraction of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas.

2. The method of claim 1, wherein bone disease is selected from the group consisting of bone damage, osteoporosis, periodontal disease, Paget's disease, multiple myeloma and metastatic cancer.

3. The method of claim 1, wherein the ethyl acetate fraction is obtained through a process of fractionating the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas sequentially using hexane, ethyl acetate, butanol and water.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the measurement results of DPPH radical scavenging activity according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(2) FIG. 2 shows the measurement results of ABTS radical scavenging activity according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(3) FIG. 3 shows the measurement results of oxygen radical absorbance capacities of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(4) FIG. 4 shows the measurement results of nitric oxide (NO) production inhibitory activity according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(5) FIG. 5 shows the measurement results of osteoclast differentiation inhibitory activity according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(6) FIG. 6 illustrates images showing osteoclast differentiation inhibitory activities according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(7) FIG. 7 shows the measurement results of prostaglandin E.sub.2 (PGE.sub.2) production inhibitory effect according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

(8) FIGS. 8A and 8B show the measurement results of inflammatory cytokine (IL-1β and IL-6) production inhibitory effects according to the concentration of a complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and fractions thereof.

DETAILED DESCRIPTION

(9) Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Examples

Example 1: Preparation of Complex Hot-Water Extract of Cynanchum Wilfordii, Phlomis Umbrosa, and Angelica gigas

(10) The raw herbal medicines, which were prepared by separately drying the natural medicinal herbs Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas as they are and then finely cutting the medicinal herbs, were used. 125 g of Cynanchum wilfordii, 125 g of Phlomis umbrosa, and 135 g of Angelica gigas were mixed, and then subjected to hot-water extraction under reflux with water, of which the weight was 10 times (w/v, 1/10) the weight of the mixed medicinal herbs, for 8 hours. Thereafter, a filtrate obtained by filtration under reduced pressure using an ultrafiltration membrane having a molecular weight cut-off of 10 μm was concentrated to 20-40 Brix under reduced pressure and then freeze-dried, thereby obtaining 172 g of a powdered complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas.

Example 2: Preparation of Water and Organic Solvent Fractions

(11) 2-1. Hexane Fraction

(12) After 100 g of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas, prepared in the above step, was completely suspended in 500 mL of water, 500 mL of hexane was added and extraction was repeated four times. Then, the hexane fraction fluid was collected, and the solvent was completely removed by concentration under reduced pressure, thereby obtaining 0.5 g of a hexane fraction.

(13) 2-2. Ethyl Acetate Fraction

(14) After 500 ml of ethyl acetate was added to the remaining layer (lower layer fluid) excluding the hexane fraction of 2-1 above, extraction was repeated four times, and then the ethyl acetate fraction fluid was collected, and the solvent was completely removed by concentration under reduced pressure, thereby obtaining 3.2 g of an ethyl acetate fraction.

(15) 2-3. Butanol Fraction

(16) After 500 ml of butanol was added to the remaining layer (lower layer fluid) excluding the ethyl acetate fraction of 2-2 above, extraction was repeated four times, and then the butanol fraction fluid was collected, and the solvent was completely removed by concentration under reduced pressure, thereby obtaining 7.0 g of a butanol fraction.

(17) 2-4. Water Fraction

(18) The remaining layer (lower layer fluid) excluding the butanol fraction of 2-3 above was concentrated under reduced pressure to completely remove the solvent, thereby obtaining 89.3 g of a water fraction.

Example 3: Measurement of DPPH Radical Scavenging Activity

(19) The free radical scavenging activity, that is, antioxidant activity was measured by investigating ability to reduce or offset the free radical DPPH, that is, oxidation inhibitory activity. DPPH is a relatively stable free radical with a dark purple color, which is reduced and decolored by a sample, and thus DPPH was used to measure antioxidant activity. Specifically, 180 μL of a 100 μM DPPH solution was added to 20 μL of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, followed by incubation for 30 minutes in a dark room, and then the absorbance was measured at 517 nm to check the DPPH radical scavenging activity according to the concentration. Vitamin C was used as a positive control substance, and the free radical scavenging activity was expressed as a percentage compared with a sample non-added group.

(20) As a result, the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, when compared with vitamin C, showed somewhat low DPPH radical scavenging activities, which were dependent on the concentration thereof. Especially, the ethyl acetate fraction showed high radical scavenging activity at all the concentrations when compared with the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the other fractions, and the ethyl acetate fraction showed DPPH radical scavenging activity similar to that of vitamin C (FIG. 1).

Example 4: Measurement of DPPH Radical Scavenging Activity

(21) The 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) radical scavenging activity was measured by using a method in which ABTS free radicals generated by reaction with 2,2′-azinobis(2-amidinopropane) dihydrochloride (AAPH) is removed by an antioxidant substance of a sample to result in the decoloration of blue color as a unique color of the radical. Specifically, 1.0 mM AAPH was added to 2.5 mM ABTS solution, followed by incubation in a constant-temperature water tank at 70° C. for 30 minutes.

(22) Thereafter, 180 μL of ABTS solution was added to 20 μL of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, followed by incubation for 10 minutes, and then the absorbance was measured at 734 nm to check the ABTS radical scavenging activity according to the concentration. Vitamin C was used as a positive control substance, and the free radical scavenging activity was expressed as a percentage compared with a sample non-added group.

(23) As a result, the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, when compared with vitamin C, showed somewhat low ABTS radical scavenging activities, which were dependent on the concentration thereof. Especially, the ethyl acetate fraction showed high radical scavenging activity at all the concentrations when compared with the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the other fractions, and the ethyl acetate fraction at 500 and 1,000 μg/mL and the butanol fraction at 1,000 μg/mL showed ABTS radical scavenging activities similar to that of vitamin C (FIG. 2).

Example 5: Measurement of Oxygen Radical Absorbance Capacity (ORAC)

(24) Oxygen radical absorbance capacity (ORAC) assay is a test technique for antioxidant capacity, standardized by the US department of Agriculture, and according to the assay, a buffer, a sample, and a fluorescein solution were mixed, and a free radical initiator solution was added, and then it was determined whether a fluorescence quenching reaction is inhibited by the free radical initiator solution.

(25) Specifically, 25 μL of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof was added in a 96-well plate, and then 150 μL of an 8.16 nM fluorescein solution was added in each well, followed by incubation at 37° C. for 10 minutes. Then, 25 μL of 153 mM AAPH solution was added in each well, followed by well mixing, and then the reduction of fluorescence was measured at 37° C. for 100 minutes at intervals of 1 minute by using a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Vitamin C was used as a control substance, and all the results are expressed as vitamin C equivalent (VCE).

(26) As a result, the ethyl acetate fraction showed the highest ORAC value, 1954.0 mg VCE/g, and the butanol extract showed the next highest ORAC value, 1090.3 mg VCE/g (FIG. 3).

Example 6: Measurement of Anti-Inflammatory Effect Through Inhibition of Nitric Oxide (NO) Production

(27) 6-1. Confirmation of Cell Viability by Treatment with Complex Hot-Water Extract of Cynanchum wilfordii, Phlomis Umbrosa, and Angelica gigas and Fractions Thereof

(28) To investigate cytotoxicity of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, cell viability was evaluated using MTT assay before a test on nitric oxide production inhibitory ability. Specifically, RAW 264.7 cells were seeded into a 96-well plate at 5×10.sup.4 cells/well, and then incubated at 37° C. and 5% CO.sub.2 for 24 hours.

(29) Then, the media were exchanged with cell media containing the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof diluted by concentrations were exchanged, followed by incubation for 24 hours. After the incubation, 20 μL of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) solution was added in each well, followed by incubation for 4 h, and then the supernatant were removed. The formazan thus formed was dissolve in 100 μL of dimethyl sulfoxide (DMSO) solution, and the absorbance was measured at 540 nm by using a microplate reader. The cell survival rate was converted into a percentage by comparison of the measurements with the absorbance of a control group.

(30) As a result, the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof showed cell survival rates of 95% or more at concentrations of 50, 100, 200 μg/m L, confirming little cytotoxicity.

(31) 6-2. Measurement of Nitric Oxide (NO) Production Inhibitory Effect

(32) To investigate anti-inflammatory effects of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, a test on nitric oxide (NO) production inhibitory ability was conducted by GRIESS assay using RAW264.7 cells.

(33) Specifically, RAW264.7 cells were seeded into a 96-well plate at 5×10.sup.4 cells/well, followed by incubation for 24 hours. Then, the media were exchanged with cell media containing the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof diluted to 50, 100, and 200 μg/m L. The cells were treated together with celecoxib as a positive control, followed by pre-incubation for 3 h, and treated with 500 ng/mL of lipopolysaccharide (LPS) as a stimulus, followed by incubation for 24 hours. After the incubation, 100 μL of the supernatant was taken and transferred into a 96-well plate, and 100 μL of GRIESS solution was added, followed by incubation for 10 minutes. Thereafter, the absorbance was measured at 540 nm using a microplate reader to determine the NO production inhibitory effect.

(34) As a result, the NO production was reduced depending on the concentration in the hexane, ethyl acetate, and butanol fractions. Especially, the ethyl acetate fraction showed reductions in NO production to 38.2% and 8.6% at concentrations of 100 and 200 μg/mL, respectively, indicating that the NO production inhibitory effect of the ethyl acetate fraction was excellent considering 46.7% at 12.5 μM of the positive control celecoxib. In addition, the hexane fraction showed a reduction in NO production to 21.5% at a concentration of 200 μg/mL, indicating that the hexane fraction showed the next highest NO production inhibitory effect (FIG. 4).

Example 7: Measurement of Osteoclast Differentiation Inhibitory Effect

(35) 7-1. Confirmation of Cell Viability by Treatment with Complex Hot-Water Extract of Cynanchum wilfordii, Phlomis Umbrosa, and Angelica gigas and Fractions Thereof

(36) To investigate cytotoxicity of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, cell viability was evaluated using MTT assay before a test on osteoclast differentiation inhibitory effects. Specifically, RAW 264.7 cells were seeded into a 96-well plate at 5×10.sup.3 cells/well, and then incubated at 37° C. and 5% CO.sub.2 for 24 hours.

(37) Then, the media were exchanged with cell media containing the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof diluted by concentrations and 50 ng/μL RANKL, a differentiation factor, were exchanged, followed by incubation for 24 hours. After the incubation, 20 μL of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbrom ide (MTT) solution was added in each well, followed by incubation for 4 hours, and then the supernatant were removed. The formazan thus formed was dissolve in 100 μL of dimethyl sulfoxide (DMSO) solution, and the absorbance was measured at 540 nm by using a microplate reader. The cell survival rate was converted into a percentage by comparison of the measurements with the absorbance of a control group.

(38) As a result, the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof showed a cell survival rate of 93% or more at concentrations of 10, 50, 100 μg/m L, confirming little cytotoxicity.

(39) 7-2. Measurement of Osteoclast Differentiation Inhibitory Effect

(40) To investigate the effects of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof on osteoclast differentiation, Raw 264.7 cells were treated with RANKL to induce osteoclast differentiation, and then treated with the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, and the differentiated osteoclasts were counted.

(41) Specifically, RAW 264.7 cells were seeded into a 96-well plate at 5×10.sup.3 cells/well, and then incubated at 37° C. and 5% CO.sub.2 for 24 hours. Then, the media were exchanged with cell media containing 10, 50, and 100 μg/mL the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof and 50 ng/mL RANKL, and the cells were treated together with the bone resorption inhibitor alendronate as a positive control, followed by incubation for 4 days. The media were removed after the incubation, followed by washing with PBS, and then the cells were fixed and stained using a leukocyte acid phosphatase kit (Sigma). After the staining, the osteoclasts were counted using a microscope, and the osteoclast count in a sample was expressed as a percentage relative to the control.

(42) As a result, the ethyl acetate fraction inhibited 78.3% and 97.7% osteoclast differentiation at concentrations of 50 and 100 μg/mL, respectively, showing higher osteoclast differentiation inhibitory effects than the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the other fractions, and the ethyl acetate fraction showed an osteoclast differentiation inhibitory effect higher than 72.2% at 10 mM alendronate as a positive control (FIGS. 5 and 6).

Example 8: Measurement of Prostaglandin E.SUB.2 .(PGE.SUB.2.) Production Inhibitory Ability

(43) To measure PGE.sub.2 production inhibitory ability of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the fractions thereof, an ELISA kit was used for assay. RAW 264.7 cells were seeded into a 96-well plate at 5×10.sup.4 cells/well, and then incubated for 24 hours. Then, the media were exchanged with cell media containing the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the butanol and water fractions thereof diluted to 50, 100, and 200 μg/mL, and after 3 hours, the cells were treated with 1,000 ng/mL LPS, followed by incubation for 24 hours. Thereafter, the cell culture supernatant was taken, and quantified and analyzed by a method described in the user's manual of the ELISA kit.

(44) As a result, the butanol fraction showed a reduction in PGE.sub.2 production depending on the concentration thereof and, especially, showed reductions in PGE.sub.2 production to 54.1% and 36.9% at concentrations of 100 and 200 μg/mL, indicating a PGE.sub.2 production inhibitory effect higher than those of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the water fraction thereof (FIG. 7).

Example 9: Measurement of Inflammatory Cytokine (IL-1β and IL-6) Production Inhibitory Abilities

(45) To investigate effects of the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the butanol and water fractions thereof on the production of inflammatory cytokines, RAW 264.7 cells were pretreated with the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the butanol and water fractions thereof for 3 hours before stimulation with 1.000 ng/mL LPS. After the cells were stimulated with LPS for 24 hours, tests were conducted using the cell culture supernatant according to the manual of the ELISA kit, for cytokine measurement.

(46) As a result, the butanol fraction showed a reduction in IL-1β production to 21.8% and 14.0% and a reduction in IL-6 production to 65.4% and 34.9% at concentrations of 100 and 200 μg/mL, indicating that the butanol fraction significantly inhibited the production of inflammatory cytokines depending on the concentration, compared with the complex hot-water extract of Cynanchum wilfordii, Phlomis umbrosa, and Angelica gigas and the water fraction (FIGS. 8A-8B).