JP7541292B2 - Bone metabolism improving composition and method for producing same - Google Patents

Description

The present invention relates to a bone metabolism improving composition and a method for producing the same, and in particular to a bone metabolism improving composition extracted from ginger that has a bone resorption inhibitory effect and a method for producing the same.

The number of people suffering from osteoporosis in Japan has reached over 10 million, and it has become a problem that has a major impact on society. In the bone tissue of the human body, bone formation and bone resorption are constantly taking place. The balance between bone formation and bone resorption is disrupted due to aging and other factors, causing bone resorption to predominate. If this state continues for a long period of time, bone mass decreases, and bone strength decreases, leading to osteoporosis, which is defined as a bone disorder in which the risk of fracture increases. Bone resorption is performed by osteoclasts, and the more significant the differentiation and activation of osteoclasts, the higher the bone resorption rate. On the other hand, bone formation is performed by osteoblasts, and the more significant the differentiation and activation of osteoblasts, the higher the bone formation rate. Therefore, if a composition having a bone formation promoting effect that increases the bone formation rate and/or a bone resorption inhibiting effect that decreases the bone resorption rate can be obtained, it will be effective for osteoporosis.

Collagen peptides containing, as an essential dipeptide, a dipeptide having a Hyp-Gly structure obtained by enzymatically treating collagen that can be obtained from the bones and skin of mammals and the bones, skin, and scales of fish, are known as components effective in preventing and improving osteoporosis (Patent Document 1). However, although collagen peptides have a bone formation promoting effect, they do not have a bone resorption inhibiting effect.
Also, a solvent extract of the fruit, peel, and pericarp of Unshu mandarin orange is known as a composition for preventing and improving osteoporosis (Patent Document 2).Furthermore, a bone metabolism improving composition containing as an active ingredient a ginger extract containing 6-gingerol and 6-shogaol, which are the pungent components of ginger (Patent Document 3), and a precursor osteoblast differentiation inducer containing as an active ingredient an extract of one or more plants selected from the group consisting of sweet basil, holy basil, common thyme, Italian large leaf, and fennel (Patent Document 4) are known.

Of the plants known as the raw material for the osteoporosis treatment drugs mentioned above, ginger is widely used as a spice and folk medicine around the world. In Asia in particular, it is eaten raw, pickled, and used as an ingredient in curry powder, while in Western and Arab countries, it is dried and used in cooking and in bread and sweets. In traditional Chinese medicine, it is called shokyo (ginger) and is said to have the effect of improving metabolic function, and is used for vomiting, pain relief, appetite stimulation, etc.

In Japan, ginger is widely used in condiments and sweets, as well as in ginger tea, which is believed to be effective in preventing colds. Harvested ginger is stored at low temperatures as a fresh produce and supplied to the market, while the juice squeezed out for processing is used in beverages and other products. The residue left over after squeezing is an unused resource that is usually disposed of, so there is a need to make effective use of it.

JP 2010-106003 A JP 2006-83151 A JP 2014-43416 A Patent No. 6560471

The bone metabolism improving composition of Patent Document 3 is a ginger extract containing 6-gingerol and 6-shogaol obtained by extraction using normal hexane. This extract contains other components in addition to 6-gingerol and 6-shogaol, but the involvement of these other components in the bone metabolism improving effect has not been examined.

The present invention aims to provide a bone metabolism improving composition that has a bone resorption inhibitory effect in order to prevent and improve osteoporosis.

The bone metabolism improving composition of the present invention is characterized by containing three or more of the following active ingredients: 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione.

The bone metabolism improving composition is characterized in that the three or more components contained therein are derived from ginger extract.

The bone metabolism improving composition is characterized by not containing 6-gingerol.

The method for producing a bone metabolism improving composition of the present invention is characterized in that the bone metabolism improving composition contains, as active ingredients, any three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione derived from ginger extract, and the three or more ingredients are obtained by extracting ginger extract using normal hexane, and then eluting the ginger extract with a mixed solvent of normal hexane and acetone by silica gel column chromatography.

The bone metabolism improving composition of the present invention contains three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione as active ingredients, and therefore exhibits excellent bone resorption inhibitory effects and is effective in preventing and improving osteoporosis.

The above three or more ingredients contained in the bone metabolism improving composition are derived from ginger extract, so the resulting bone metabolism improving composition is safer for the human body and easier to ingest.

The method for producing a bone metabolism improving composition of the present invention comprises, as active ingredients, any three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione derived from ginger extract, and the above three or more ingredients are obtained by extracting ginger extract using normal hexane and then eluting the ginger extract with a mixed solvent of normal hexane and acetone by silica gel column chromatography, so that a desired combination of ingredients derived from ginger extract can be obtained by a simple operation.

HPLC of normal hexane extract of ginger pomace. FIG. 1 is a diagram showing a procedure for fractionation using a silica gel column. HPLC of fraction A and fraction B. HPLC of fraction C and fraction D. HPLC of fractions E and F. HPLC of fraction G and fraction H. HPLC of fraction I and fraction J. FIG. 1 shows TRAP staining images of each fraction. FIG. 1 shows the results of TRAP staining of osteoclast numbers. The structures of the components contained in fractions F to H are shown. These are the results of evaluation of cytotoxicity. FIG. 1 shows a TRAP staining image. 1 shows the results of quantitative analysis of TRAP staining images. This is a TRAP stained image of zebrafish scales. This is a TRAP stained image of zebrafish scales. 1 shows the results of quantitative analysis of TRAP staining images.

The inventors conducted a search for active ingredients that exert the osteoclast differentiation inhibitory effect of ginger extract, and found that when ginger extract contains three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione, it has a significant bone resorption inhibitory effect. The present invention is based on this finding.

The following describes an embodiment of the present invention. 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione contained in the bone metabolism improving composition of the present invention can be prepared by chemical synthesis, or by extracting them from natural products and purifying them using a silica gel column or the like. Alternatively, commercially available products may be used.

When extracting the above-mentioned components from natural products, the whole or a part of the plant containing them (e.g., whole plant, leaves, roots, rhizomes, stems, root bark, or flowers) is used as is, or they are extracted using a part that has been simply processed (e.g., dried, cut, or powdered) (e.g., herbal medicine).

In the present invention, the rhizome of ginger (Zingiber officinale), a perennial plant of the Zingiberaceae family native to tropical Asia, can be used as the natural product.
Raw materials for obtaining the extract include fresh ginger, dried ginger, and processed products thereof. For example, the pomace obtained by squeezing juice for use in beverages can be preferably used. In particular, this pomace is discarded as an unused resource, and can be effectively utilized.

Examples of methods for extracting ginger include organic solvent extraction, mixed solvent extraction with organic solvent and water, and supercritical extraction. Examples of organic solvents include nitrous oxide, acetone, ethanol, ethyl methyl ketone, glycerin, ethyl acetate, methyl acetate, diethyl ether, cyclohexane, dichloromethane, edible oils and fats, 1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, carbon dioxide, 1-butanol, 2-butanol, butane, 1-propanol, 2-propanol, propane, propylene glycol, normal hexane, water, and methanol. Among these, normal hexane, which is approved for use as an extraction solvent in the production of oily foods, is preferred as a solvent for low-temperature extraction from ginger pomace, which will be described later. An example of a solvent for supercritical extraction is _CO2 .

The ginger extract is preferably purified using a solid-phase adsorbent or the like. As the solid-phase adsorbent, silica gel is preferable, and a commercially available one can be used. The average particle size of the silica gel can be, for example, 5 μm to 500 μm.

When performing silica gel column chromatography, any solvent can be used as the elution solvent, so long as the solvents do not separate from each other and are not irreversibly adsorbed to the silica gel. For example, normal hexane, acetone, ethanol, ethyl methyl ketone, ethyl acetate, diethyl ether, cyclohexane, etc. can be used as the elution solvent. Only one type of elution solvent can be used, or a mixed solvent of two or more types can be used.

To obtain the bone metabolism improving composition of the present invention, it is preferable to use normal hexane and acetone as the elution solvent. Specifically, after elution for a predetermined time with only normal hexane (100% hexane), elution for a predetermined time with a mixed solvent consisting of normal hexane and acetone. The ratio of this mixed solvent is, for example, normal hexane:acetone=(98:2 to 70:30) by volume. Within this range, it is preferable to perform gradient elution in which the ratio of acetone is increased stepwise or continuously. The ratio of this mixed solvent is more preferably normal hexane:acetone=(98:2 to 80:20) by volume, and even more preferably (98:2 to 85:15).

Each fraction obtained by elution is developed by thin layer chromatography using normal hexane-acetone as a developing solvent, and similar fractions are combined to obtain a fraction containing three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione. This fraction is concentrated to remove the solvent, yielding a brown, transparent, oil-soluble liquid.

As described above, the bone metabolism improving composition of the present invention can be prepared by directly using the fraction obtained by elution and separation from ginger extract by silica gel column chromatography. It may also be used after diluting with an appropriate solvent, or it may be processed into a powder form using an adsorbent material or used in a paste form.

The bone metabolism improving composition of the present invention can be applied to foods for specified health uses, supplements, medicines, etc. When the bone metabolism improving composition of the present invention is used as a food such as an agent for preventing or improving osteoporosis, the composition can be in the form of various foods such as bread, cakes, noodles, confectionery, jellies, frozen foods, ice cream, dairy products, and beverages.
To prepare foods of various forms, the bone metabolism improving composition of the present invention can be used alone or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc.

As the ginger raw material, ginger pomace (water content about 75% by mass) from which the juice was squeezed was prepared. 60 kg of this pomace was frozen at -10°C or lower. This frozen raw material was added with about twice the volume of normal hexane (extraction solvent), stirred and crushed, and then immersed at 0°C or lower for 15 minutes. Thereafter, the normal hexane solution (layer) was collected, and normal hexane was removed from it at a temperature below room temperature using a centrifugal thin-film concentrator and a reduced-pressure distillation apparatus. The obtained ginger extract was a brown, transparent, oil-soluble liquid having a refreshing aroma after grating ginger and a spicy component specific to ginger.
The components of the obtained ginger extract were analyzed by high performance liquid chromatography (hereinafter, including high performance liquid chromatogram, referred to as HPLC). The measurement conditions for HPLC were as follows: column: Wakosil 11-5C18 φ4.6×250 mm; mobile phase: hydrous acetonitrile; gradient from 30% by mass acetonitrile to 90% by mass acetonitrile (0-20 min) + 90% by mass acetonitrile (20-40 min); detector: 228 nm ultraviolet absorbance detector (column temperature: 40° C.). Analysis was performed at a mobile phase flow rate of 0.6 mL/min.
The results of the HPLC are shown in FIG.

1, from the MS of each peak and the HPLC of the standard sample, the peak indicated by _Pa was identified as 6-gingerol, a hot ingredient contained in ginger, the peak indicated by _Pb as neral, a monoterpene, the peak indicated by _Pc as geranial, an isomer of neral, the peak indicated by _Pd as phellandrene, the peak indicated by _{Pe as} farnesene, and the peak indicated by _{Pf as zingiberene. From these results, it was found that, calculated from the total peak area of peaks Pa to Pf ,} these components account for the majority of the components in the ginger extract.

Next, to separate each component in the ginger extract, fractionation was performed using a silica gel open column (Sogo Rikagaku Glass Manufacturing, φ20 x 300 mm). The eluent was initially hexane alone, then changed to a mixed solvent of hexane and acetone, and the acetone ratio was gradually increased to increase the polarity of the mixed solvent, and finally the ginger extract was eluted with ethanol alone. During elution, 30-200 mL per fraction was collected sequentially, and 52 fractions were finally collected. Details of the elution conditions are shown in Figure 2.

The ginger extract (GHE) was applied to a silica gel column equilibrated with hexane. As shown in FIG. 2, fractions 1 to 13 were collected by elution with hexane alone, fractions 14 to 20 were collected by elution with hexane:acetone (98:2), fractions 21 to 36 were collected by elution with hexane:acetone (93:7), fractions 37 to 46 were collected by elution with hexane:acetone (88:12), fractions 47 to 50 were collected by elution with hexane:acetone (85:15), fraction 51 was collected by elution with ethanol:acetone (50:50), and fraction 52 was collected by elution with ethanol alone. Each fraction was divided into 10 fractions (fractions A to J) based on the chromatogram of thin layer chromatography, and concentrated to obtain fractions for testing.

Figures 3 to 7 show the HPLC results of fractions A to J. The HPLC measurement conditions were as follows: column: Wakosil II-5C18 φ4.6 × 250 mm; mobile phase: hydrous acetonitrile; gradient from 30% acetonitrile to 90% acetonitrile (0-20 min) + 90% acetonitrile (20-40 min); and detector: 228 nm ultraviolet absorbance detector (column temperature: 40°C). Analysis was performed at a mobile phase flow rate of 0.6 mL/min.

As shown in Figures 3 and 4, fractions A to D mainly contained low-polarity terpene compounds such as zingiberene, farnesene, phellandrene, neral, and geranial.

As shown in Figures 5 and 6, fraction E contained 8-gingerol (peak P1), fraction F and fraction G contained 8-gingerol (peak P1), 10-gingerol (peak P2), 1-dehydro-6-gingerdione (peak P3), and 6-gingerdione (peak P4), and fraction H contained 8-gingerol (peak P1), 10-gingerol (peak P2), 1-dehydro-6-gingerdione (peak P3), and 6-gingerol. Fractions F and G did not contain 6-gingerol.

As shown in Figure 7, fraction I contains 6-gingerol, and the major components were hardly detected in fraction J. The components in each fraction were identified by comparison with HPLC and MS of authentic samples. The yields of each of fractions A to J are shown in Table 1.

The ginger extract (GHE) and each fraction (fraction A to fraction J) obtained above were used to evaluate the inhibitory effect on osteoclast differentiation by the following method.
<Evaluation of the effect of inhibiting osteoclast differentiation>
Mouse macrophage-derived osteoclast precursor cells (RAW264.7) were seeded at 3 x ¹⁰³ cells/well in a 24-well plate and cultured in α-MEM medium containing 10% by mass of fetal bovine serum (FBS) at 37°C in the presence of 5% by volume _CO2 . After 24 hours, a test sample was added and cultured for another 24 hours. Next, 100 ng/ml of RANKL was added to the medium and cultured for differentiation induction for 6 days. Thereafter, tartrate-resistant acid phosphatase (TRAP) staining was performed on the 6th day of differentiation induction. Cells that were TRAP stained positive and had 11 or more nuclei per cell were considered to be differentiated mature osteoclasts, and their number was counted.
The test samples used were a negative control (no addition), ginger extract (20 μg/ml), and fractions A to J (10 μg/ml). The test was repeated three times.

The images of each TRAP staining are shown in Figure 8, and the results for the number of osteoclasts are shown in Figure 9. As shown in Figures 8 and 9, the number of differentiated osteoclasts was significantly reduced in the samples to which fractions F to H were added, compared to the negative control. The number of differentiated osteoclasts was reduced in the samples to which fractions F to H were added, to a degree equal to or greater than that of ginger extract.

As shown in Figure 10, when we look at the components of fractions F to H, we see that fractions F to H contain three or more of the components 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione, and as a result, they are found to have an osteoclast differentiation inhibitory effect equal to or greater than that of ginger extract. Fraction E, which contains 8-gingerol as the main component, and fraction I, which contains 6-gingerol as the main component, also exhibited differentiation inhibitory effects, but the effects were lower than those of fractions F to H.

As shown in the HPLC chart in Figure 1, the peaks for 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione are small and are trace components in ginger extract. Nevertheless, fractions containing three or more of these components showed a high inhibitory effect on osteoclast differentiation as described above, indicating that these components are effective in inhibiting osteoclast differentiation.

From the above, it has been confirmed that a composition containing three or more of the components 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione inhibits bone resorption, which reduces bone mass, and is considered to be effective in preventing and improving osteoporosis.

Next, the inhibitory effect on osteoclast differentiation was evaluated for each component (8-gingerol, 10-gingerol) that is commonly present in fractions F to H and is thought to be effective in inhibiting osteoclast differentiation, as well as for the homologous 6-gingerol. The inhibitory effect on osteoclast differentiation was evaluated after checking the cytotoxicity of the above three gingerols.

<Evaluation of cytotoxicity>
The evaluation results of cytotoxicity are shown in Figure 11. As a result of treating RAW264.7 cells with each gingerol, as shown in Figure 11, there was no difference in the degree of cell proliferation at any concentration (0.6 μM, 2.5 μM, 10 μM), and no strong cytotoxicity was observed.

<Evaluation of the effect of inhibiting osteoclast differentiation>
Mouse macrophage-derived osteoclast precursor cells (RAW264.7) were seeded at ^1x103 cells/well in a 96-well plate and cultured in α-MEM medium containing 10% by mass of fetal bovine serum (FBS) at 37°C in the presence of 5% by volume _CO2 . After 24 hours, a test sample was added and cultured for another 24 hours. Next, 100ng/ml of RANKL was added to the medium and cultured for differentiation induction for 6 days. Thereafter, tartrate-resistant acid phosphatase (TRAP) staining was performed on the 6th day of differentiation induction. Cells that were TRAP stained positive and had 11 or more nuclei per cell were considered to be differentiated mature osteoclasts, and their number was counted.
The test samples used were four types: no addition as a negative control, 6-gingerol, 8-gingerol, and 10-gingerol (each at three concentrations of 0.6, 2.5, and 10 μM). The test was repeated six times.

The TRAP staining images are shown in Figure 12. As shown in Figure 12, the sample with 6-gingerol added showed a slight inhibitory effect compared to the negative control, while the samples with 8-gingerol and 10-gingerol added showed a significant inhibition of the proliferation of differentiated osteoclasts. Figure 12(a) is a microscopic image observed at 200x magnification, and Figure 12(b) is a microscopic image observed at 20x magnification. In the 200x magnification image, the frequency of appearance of large cells with many nuclei was low, especially in the sample with 10-gingerol added.

Figure 13 shows the results of quantitative analysis of the TRAP stained images. After photographing the entire well at 20x magnification, quantitative evaluation of the degree of staining was performed. Figure 13(a) shows the number of mature osteoclasts of a certain size or more. Figure 13(b) shows the area of the TRAP stained region quantified by analysis with ImageJ, and Figure 13(c) shows the degree of staining (shade). Compared to the negative control, 6-gingerol, 8-gingerol, and 10-gingerol each showed a tendency to reduce the number of osteoclasts and the area of the TRAP stained region, confirming the effect of inhibiting the proliferation of differentiated osteoclasts. In particular, 10-gingerol showed small values for not only the number of osteoclasts and the TRAP stained area, but also the degree of staining, and showed a high effect of inhibiting the proliferation of differentiated osteoclasts even with a small amount added. It was found that the inhibitory effect on the differentiation and maturation of osteoclasts becomes stronger as the methylene group of gingerol becomes longer, and the effect of 10-gingerol in particular is strong.

In addition, the ginger extract obtained above and 10-gingerol contained in the ginger extract were used to evaluate the bone metabolism improving effect in individual animals by the following method.
<Individual animal testing>
Individual animal tests were carried out using zebrafish (AB Strain Male). Scales were removed from around the tail of the zebrafish in advance, and the zebrafish were raised for 8 days under the conditions shown in Test 1 to Test 8 below. Then, the scales regenerated around the tail of the zebrafish were collected, fixed with paraformaldehyde, and stained with TRAP on the 9th day.
Test 1: No addition Test 2: Addition of 25 μM prednisolone (steroid osteoporosis inducer) Test 3: Addition of 25 μM prednisolone + 30 μM alendronate (osteoporosis treatment drug) Test 4: Addition of 25 μM prednisolone + 1 ppm ginger extract Test 5: Addition of 25 μM prednisolone + 10 ppm ginger extract Test 6: Addition of 25 μM prednisolone + 0.001 ppm 10-gingerol Test 7: Addition of 25 μM prednisolone + 0.01 ppm 10-gingerol Test 8: Addition of 25 μM prednisolone + 0.1 ppm 10-gingerol

Test 1 was performed as a negative control, and Test 2 was performed as a positive control. The number of test repetitions was 5 to 6. Each drug and test sample was exposed to water. Ginger extract in Tests 4 and 5, and 10-gingerol in Tests 6 to 8 were added uniformly as liposome preparations using lecithin, and the final concentrations in each test were as described. Specifically, the liposome preparations used were a 1% emulsion of ginger extract using phosphatidylcholine (PC70) and a 0.1% emulsion of 10-gingerol using phosphatidylcholine (PC70) that had been prepared in advance. The zebrafish scales (bone tissue) after TRAP staining were observed under an optical microscope at 40x magnification, and the shape of the scales and the inhibitory effect on osteoclast differentiation and maturation (degree of staining) were evaluated.

14 shows the TRAP stained images of the scales of each zebrafish. The scales of the zebrafish given only prednisolone in Test 2 were disintegrated and stained strongly, confirming an increase in osteoclasts. The scales of the zebrafish given alendronate in Test 3 were stained less than those in Test 2, indicating inhibition of osteoclasts.
On the other hand, in the case of the zebrafish scales exposed to ginger extract (Tests 4 and 5), the degree of staining was reduced (reduction in osteoclasts) and the shape was maintained, as in Test 3. This result was in a good condition equivalent to that of untreated zebrafish scales not exposed to prednisolone. In addition, the ginger extract concentrations of 1 ppm and 10 ppm both showed similar good results.

Figure 15 shows TRAP staining images of zebrafish scales exposed to 10-gingerol. The degree of staining of the three test samples (Test 6, Test 7, Test 8) with different concentrations of 10-gingerol was reduced to the same level or less than that of the scales exposed to ginger extract in Tests 4 and 5. This result was in better condition than that of untreated zebrafish scales that were not exposed to prednisolone. Furthermore, for 10-gingerol concentrations of 0.001 ppm, 0.01 ppm, and 0.1 ppm, the degree of staining was small and the shape of the scales was maintained, showing good results.

Figure 16 shows the quantitative analysis results of the TRAP staining images analyzed with ImageJ. Quantification was performed by measuring the area intensity of the stained area. As for IntDen, which indicates the degree of staining, the intensity of TRAP staining was reduced in all groups from Test 3 to Test 8 compared to the steroid prednisolone group (Test 2), confirming the effect of inhibiting osteoclast activation. Specifically, the prednisolone group (Test 2) showed an IntDen value that was about three times that of the negative control Test 1, while the alendronate group (Test 3) showed an IntDen value smaller than that of Test 2, showing a certain improvement effect. In contrast, the IntDen values of the ginger extract groups Tests 4 and 5 were reduced to values almost equivalent to those of Test 1, showing a greater effect of inhibiting osteoclast activation than Test 3. In addition, it was confirmed that the IntDen values of the 10-gingerol group in Tests 6 to 8 were even smaller than those of Tests 4 and 5. As mentioned above, ginger extract and 10-gingerol showed significant effects even in small amounts, demonstrating that they also exert an excellent effect on improving bone metabolism in the living body.

The bone metabolism improving composition of the present invention has a bone resorption inhibitory effect due to the inclusion of specific ingredients, and can therefore be used to prevent and improve osteoporosis.

Claims (3)

A composition for improving bone metabolism, comprising as active ingredients three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione , and not containing 6-gingerol . The bone metabolism improving composition according to claim 1, characterized in that the three or more components contained in the bone metabolism improving composition are derived from ginger extract. A method for producing a bone metabolism improving composition, comprising:
The bone metabolism improving composition contains, as active ingredients, any three or more of 8-gingerol, 10-gingerol, 1-dehydro-6-gingerdione, and 6-gingerdione derived from a ginger extract, and does not contain 6-gingerol;
A method for producing a bone metabolism improving composition, characterized in that the three or more components are obtained by extracting ginger extract using normal hexane, and then eluting the ginger extract with a mixed solvent of normal hexane and acetone by silica gel column chromatography.