KR101826109B1 - Food composition for improvement of osteoporosis and pharmaceutical compositions for prevention or treatment of osteoporosis with gossypetin - Google Patents

Abstract

The present invention relates to a food composition for improving osteoporosis and a pharmaceutical composition for preventing or treating osteoporosis. The gossypetin of the present invention inhibits the deacidase II (CA II) which causes acidification between osteoclast and bone and Ae 2 and ClC-7 which are involved in chlorine migration, And exhibits the effect of inhibiting the expression of MMP-9 and cathepsin K, which are involved in the expression of LC3 Ⅱ, Rab7 and bone resorption, which are involved in bone resorption area.
In addition, gossypetin inhibits bone resorption of osteoclasts and improves osteoporosis and exerts therapeutic effects.

Description

TECHNICAL FIELD The present invention relates to a food composition for improving osteoporosis and a pharmaceutical composition for preventing or treating osteoporosis,

The present invention relates to a food composition for improving osteoporosis and a pharmaceutical composition for preventing or treating osteoporosis.

Bones are dynamic tissues in which formation and absorption take place continuously and respond to various kinds of stimuli. This metabolism of bones destroys, absorbs or forms new bones, depending on the degree of activity of osteoblasts and osteoclasts.

Osteoblasts play a role in bone formation, and osteoclasts play a role in bone destruction. Osteoblast differentiates from osteoblast progenitor cells. Osteoprogenitor cells are transformed into osteoblasts by various factors such as Bone Morphogenetic Protein (BMP) and transforming growth factor-β (TGF-β) Differentiated. The differentiated osteoblasts secrete protein and non-protein components and induce minerals to exist as bone cells.

On the other hand, osteoblasts produce macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL), which are essential for osteoclast differentiation. At this time, M-CSF and RANKL stimulate osteoclast precursor cells having one nucleus, and differentiate into osteoclasts, which are macromolecular cells, through continuous differentiation process. The differentiated osteoclasts secrete proteases and the like on the surface of the bones and induce the acidification of the bones surface, thereby breaking down proteins and minerals, which are constituents of the bones, and destroying the bones.

When metabolism of osteoclasts and osteoclasts is normal, bone remodeling occurs by interaction. However, in women, after menopause, sudden bone loss occurs and balance is easy to break. In addition, as the osteogenesis function deteriorates due to aging, bone loss persists and osteoporosis occurs. Osteoporosis progresses rapidly in the early stages, bone loss due to bone resorption may occur, and osteogenic precursor cells may not be able to differentiate into osteoblasts, resulting in insufficient osteogenesis.

The World Health Organization (WHO) defines osteoporosis as "a skeletal disease that increases the risk of fractures due to weakened bone strength." The bone strength is determined by bone mass and bone quality. In the mid-twenties or early 30's, the maximum bone mass is formed during the lifetime, and thereafter the bone loss progresses with age. Between the ages of 30s and 50s, bone mass is generally maintained, and it is maintained by the homeostasis of bone formation that removes old bone and bone formation that forms new bone.

As an agent for the prevention and treatment of osteoporosis, the invention of bone resorption inhibitor has been actively carried out, but its effect is still insufficient. Estrogen therapy is effective for the treatment of osteoporosis in postmenopausal women, but it has been reported to increase the incidence of breast cancer and increase the risk of arteriosclerosis. In addition, osteoporosis medications that require long-term administration are known to be highly likely to cause side effects.

Therefore, in recent years, there have been active researches to develop an osteoporosis improvement agent or therapeutic agent containing no harmful or harmful natural products or ingredients derived therefrom.

On the other hand, gossypetin is a type of flavonoid having the formula C ₁₅ H ₁₀ O ₈ , molecular weight 318. Gossyphetine exists in the form of dark yellow needle crystals. Melting point is 311 ~ 313 ℃, and it has good melting property in ethanol and methanol. It is almost insoluble in water and precipitates from the alkaline solution to yellow but soon turns green to blue and dark brown. It is light reddish in concentrated sulfuric acid, and precipitates in dark red (in ethanol) in lead acetate.

However, a composition for improving and treating osteoporosis containing gossypetin has not been reported.

Korean Patent Registration No. 10-0814652 (Registration Date: Mar. 12, 2008) discloses a pharmaceutical composition comprising component A containing one or more flavonol glycosides, component B containing one or more tetrahydrofolic acid compounds, a component containing one or more calcium supplements C, and one or more magnesium supplements, and describes a composition for the treatment and / or prevention of osteoporosis and / or inflammatory joint disease. Korean Patent Registration No. 10-0849253 (registered on Jul. 23, 2008) discloses a composition for treating osteoporosis containing Rhizoma Drynariae extract and a composition comprising naringin and a method for treating osteoporosis . ≪ / RTI >

The present invention relates to a composition for inhibiting the process of causing acidification between osteoclasts and bones and containing a component inhibiting the formation of wrinkles related to the osteosynthesis area so as to reduce osteoporosis and thereby improve or prevent osteoporosis And to provide them.

In order to achieve the above object, the present invention provides a food composition for osteoporosis improvement, which comprises gossypetin as an active ingredient.

In the osteoporosis-improving food composition of the present invention, it is preferable that the goshi-petun has a concentration of 1 to 50 μM. More preferably 1 to 20 [mu] M. When the above range is satisfied, it is possible to inhibit the expression of deacid dehydratase II (CA II), Ae2, ClC-7, LC3 II, Rab7, MMP-9 and cathepsin K and remarkably excellently improve osteoporosis .

The food composition for improving osteoporosis according to the present invention may be used as a food composition for osteoporosis improvement such as meat, cereal, caffeinated beverage, general beverage, chocolate, bread, snack, confectionery, candy, pizza, jelly, noodle, dairy, ice cream, , A vitamin complex, and other health supplement foods. However, the present invention is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises gossypetin as an active ingredient.

In the pharmaceutical composition for preventing or treating osteoporosis of the present invention, it is preferable that the concentration of goshi fetin is 1 to 50 μM. More preferably 1 to 20 [mu] M. When the above range is satisfied, it is possible to inhibit the expression of deacid dehydratase II (CA II), Ae2, ClC-7, LC3 II, Rab7, MMP-9 and cathepsin K, Excellent effect can be exhibited.

The pharmaceutical composition for preventing or treating osteoporosis of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Examples of usable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and one or more selected from among them may be used.

Meanwhile, the pharmaceutical composition for preventing or treating osteoporosis according to the present invention may be in a form suitable for the method of use, and may be formulated to be capable of providing rapid, sustained or delayed release of the active ingredient after administration to a mammal, It is recommended to adopt the method of formulation. Examples of specific formulations include, but are not limited to, PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, POWDERS, SYRUPS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, DECOCTIONS, (TABLETS), SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, CAPSULES, CREAMS, TROCHES, TINCTURES, Pastes (PASTES), pills (PILLS), soft or hard gelatin capsules.

On the other hand, the dosage of the pharmaceutical composition for preventing or treating osteoporosis of the present invention is preferably determined in consideration of the administration method, the age, sex, weight, and severity of the disease. For example, the pharmaceutical composition for preventing or treating osteoporosis of the present invention may be administered at least once per day at a dose of 0.00001 to 100 mg / kg (body weight) based on the active ingredient. However, the dosage is only an example and may be changed by a doctor's prescription depending on the condition of the recipient.

The gossypetin of the present invention has no adverse effect on human body and has anion exchanger (Ae2), chloride channel ClC-7 which is involved in deoxidation dehydratase II (CA II) causing acidification between osteoclast and bone And inhibits the expression of MMP-9 and cathepsin K, which are involved in the expression of LC3 Ⅱ, Rab7 and bone resorption, which are involved in the bone resorption area. Thus, gossypetin inhibits bone resorption of osteoclasts and improves osteoporosis and exerts therapeutic effects.

Figure la is the Western blot results of deacid dehydratase II. Figure 1b shows the results of anion exchanger Ae2 RT-PCR. 1C is the result of Western blotting of the anion exchanger Ae2. FIG. 1 d shows RT-PCR results of the chloride channel ClC-7.
FIG. 2A is a fluorescence microscopy photograph showing lysosome formation of oocystatin-treated osteoclasts. Figure 2b is the Western blot results of LC3I and LC3II. Figure 2C shows the Western blot results of Rab7.
FIG. 3A shows fluorescence analysis results of goshi-petun-treated osteoclasts. Figure 3B shows the RT-PCR result of MMP-9. FIG. 3c is a MMP-9 secretion inhibition result (western blot) of gossyphetin using yymography. Figure 3D is the result of RT-PCR of the channeled channel cathepsin K (Cathepsin K).

Hereinafter, the present invention will be described in more detail with reference to the following examples or experimental examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Experimental Example  One: Koshi Petun  Osteoclast acidification Inhibition  Experiment]

< Experimental Example  1-1: Deacidyl dehydratase II Western blot >

In this experiment, Western blotting was performed in order to measure the activity of deoxidase II (carbonic anhydrase II, CA II) which helps osteoclast bone absorption and acidification.

Mouse macrophages (RAW 264.7) were purchased from ATCC (USA) and primary cultured. RAW 264.7 was incubated in Dulbecco's Modified Eagles's Medium (DMEM, Sigma co., St. Louis, Mo.) supplemented with 10% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin , USA) medium at 37 ° C and 5% CO ₂ .

In order to differentiate these cells into osteoclasts, RANKL (50 ng / ml) was treated with a receptor activator of nuclear factor-kB ligand and treated with 1, 10 and 20 μM gossypetin. The Western blot was then performed on 10% SDS-PAGE.

Protein bands obtained by electrophoresis were transferred to a nitrocellulose membrane and blocked in 5% skim milk for 3 hours to prevent non-specific binding. The deacidase II protein primary antibody Abcam (Cambridge, UK) was diluted 1,000-fold and reacted with stirring, followed by reaction with secondary antibody for 1 hour.

After the reaction, the deacylated dehydratase II protein on the nitrocellulose membrane was detected with Supersignal West pico chemiluminescence (Pierce Biotech. NJ, USA), and X-ray film (Konica Co., Tokyo , Japan).

< Experimental Example  1-2: Anion exchanger Of Ae2  RT- PCR >

In this experimental example, RT-PCR of anion exchanger Ae2 was performed.

In order to differentiate into osteoclasts, RANKL at a concentration of 50 ng / ml was treated and treated with gossypetin at a concentration of 1, 10 and 20 μM, respectively. The osteoclasts were treated with trypsin for 5 days. After the mRNA was isolated using trisole, cDNA was synthesized using 5 μg RNA, 200 unit reverse transcriptase, and 0.5 mg / ml Oligo- (dT).

Then, 25 μl of a reaction master mix containing 5 × PCR buffer, 25 mM MgCl ₂ , 10 mM dNTP, 10 pmol primer and 5 units of Taq DNA polymerase and cDNA were used to amplify the cDNA using ' RT-PCR was performed on My Cycler ™ Thermal Cycler from Bio-Rad Laboratories (Berkeley, Calif.). The PCR conditions are shown in Table 1 below.

forward reverse size Ae2  5'-GAC AGC GAG AAG GGC TAA GA-3 ' 5'-GGA CTG GCG GTG GTA TTC AA-3 ' 255 bp β-actin 5'-GAC TAC CTC ATG AAG ATC-3 ' 5'-GAT CCA CAT CTG CTG GAA-3 ' 500 bp

< Experimental Example  1-3: Anion exchanger Of Ae2 Western Blat >

In this experiment, Western blotting was performed to measure anion exchanger Ae2 to help osteoclast bone resorption and to make acidification.

Mouse macrophages (RAW 264.7) were purchased from ATCC (USA) and primary cultured. RAW 264.7 was incubated in Dulbecco's Modified Eagles's Medium (DMEM, Sigma co., St. Louis, Mo.) supplemented with 10% FBS, 2 mM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin , USA) medium at 37 ° C and 5% CO ₂ .

To differentiate into osteoclasts, RANKL at a concentration of 50 ng / ml was treated and treated with 1, 10 and 20 μM gossypetin, respectively. The Western blot was then performed on 10% SDS-PAGE.

Protein bands obtained by electrophoresis were transferred to a nitrocellulose membrane and blocked in 5% skim milk for 3 hours to prevent nonspecific binding. Ae2 protein primary antibody Novus Biologicals (Littleton, CO) was diluted 1,000-fold and reacted with stirring, followed by reaction with secondary antibody for 1 hour.

Ae2 protein was detected with Supersignal West pico chemiluminescence (Pierce Biotech, NJ, USA) and photographed with X-ray film (Konica Co., Tokyo, Japan).

Experimental Example 1-4: RT-PCR of Chloride Channel ClC-7 &gt;

In this experiment, we performed RT-PCR to measure ClC-7, which helps bone resorption and acidification of osteoclasts.

Mouse macrophages (RAW 264.7) were purchased from ATCC (USA) and primary cultured. After primary culture, RAW 264.7 was incubated in Dulbecco's Modified Eagles's Medium (DMEM, Sigma co., St. Louis, MO) supplemented with 10% FBS and 2 mM glutamine, 100 U / ml penicillin, 100 mg / ml streptomycin. MO, USA) medium at 37 ° C and 5% CO ₂ .

To differentiate these cells into osteoclasts, RANKL at a concentration of 50 ng / ml was treated and treated with 1, 10, and 20 μM gossypetin, respectively. The osteoclasts were treated with trypsin for 5 days. After the mRNA was isolated using trisole, cDNA was synthesized using 5 μg RNA, 200 unit reverse transcriptase, and 0.5 mg / ml Oligo- (dT). Then, 25 μl of a reaction master mix containing 5 × PCR buffer, 25 mM MgCl ₂ , 10 mM dNTP, 10 pmol primer and 5 units of Taq DNA polymerase and cDNA were used to amplify the cDNA using ' RT-PCR was performed on My Cycler ™ Thermal Cycler from Bio-Rad Laboratories (Berkeley, Calif.). At this time, the PCR conditions are shown in Table 2 below.

forward reverse size ClC-7  5'-CGA GAT CGC GAT GAG GA-3 ' 5'-TCA CCC CAT TGA GGA AGC AC-3 ' 607 bp GAPDH 5'-AAC TTT GGC ATT GTG GAA GGG-3 ' 5'-GAC ACA TTG GGG GTA GGA ACA C-3 ' 224 bp

< Experimental Example  1-5: Experimental Results>

As a result, deacid dehydratase II, Ae2, and ClC-7 were all inhibited depending on the concentration of psichene (FIG. 1). Figure la is the Western blot results of deacid dehydratase II. Figure 1b shows the results of anion exchanger Ae2 RT-PCR. 1C is the result of Western blotting of the anion exchanger Ae2. Figure 1D shows the RT-PCR result of the chloride channel ClC-7.

From these results, it can be confirmed that gossyphetin is effective in inhibiting the acidification process between osteoclasts and bone.

[ Experimental Example  2: Koshi Petun  Lysosomes Wrinkle film  Fusion process Inhibition  Experiment]

< Experimental Example  2-1: Koshi Petun  After treatment, lysosome staining>

In this experimental example, the effect of the inhibition of lysosome and wrinkle formation of goshiethene was investigated.

RANKL at 50 ng / ml was treated with osteoclast precursor cells (Raw 264.7), treated with 1, 10 and 20 μM gossypetin, respectively. The osteoclasts were fixed with 4% formaldehyde for 20 minutes and then stained with Alexa Fluor 488 Phalloidin (Life Technologies) and LysoTracker® Red DND-99 (Life Technologies) And photographed using a fluorescence microscope.

< Experimental Example  2-2: LC3 I, LC3 II Western Blat >

In this experimental example, Western blotting of LC3I and LC3II was performed in order to measure the lysosome formation process.

RANKL at 50 ng / ml was treated with osteoclast precursor cells (Raw 264.7), treated with 1, 10 and 20 μM gossypetin, respectively. The Western blot was then performed on 10% SDS-PAGE. Protein bands obtained by electrophoresis were transferred to a nitrocellulose membrane and blocked in 5% skim milk for 3 hours to prevent nonspecific binding.

Thereafter, the primary antibody (Novus Biologicals (Littleton, CO)) of LC3I and LC3II proteins was diluted 1,000-fold and reacted with stirring, followed by reaction with secondary antibody for 1 hour.

LC3I and LC3II proteins were detected with Supersignal West pico chemiluminescence (Pierce Biotech, NJ, USA) and photographed with X-ray film (Konica Co., Tokyo, Japan).

< Experimental Example  2-3: Rab7  of Western Blat >

In this experiment, Western blotting of Rab7 was performed in order to measure Rab7 protein expressed during the fusion process of lysosome and wrinkle membrane.

RANKL at a concentration of 50 ng / ml and gossypetin at a concentration of 1, 10 and 20 μM were treated with osteoclast precursor cells (raw 264.7) for 5 days, respectively. The Western blot was then performed on 10% SDS-PAGE.

Protein bands obtained by electrophoresis were transferred to a nitrocellulose membrane and blocked in 5% skim milk for 3 hours to prevent nonspecific binding.

Rab7 protein was detected by Supersignal West pico chemiluminescence (Pierce Biotech., NJ, USA) and then incubated with primary antibody (Novus Biologicals (Littleton, CO) diluted 1,000-fold and reacted with secondary antibody for 1 hour. And photographed with an X-ray film (Konica Co., Tokyo, Japan).

< Experimental Example  2-4: Experimental Results>

As a result of the experiment, LC3 Ⅰ protein was converted into LC3 Ⅱ and activated by autolysosome when autophagy was formed, and LC3 Ⅱ was found to decrease the activation depending on the concentration of pheophytin. In addition, it was shown that goshi-fetin inhibits Rab7 in a concentration-dependent manner (Fig. 2). FIG. 2A shows fluorescence microscopy photographs of osteoclast-treated osteoclasts. Figure 2b is the Western blot results of LC3I and LC3II. Figure 2C shows the Western blot results of Rab7.

These results suggest that goshi-pectin is effective in osteoclast formation and inhibition of wrinkle formation.

[ Experimental Example  3: Koshi Petun  Bone resorption Inhibition  Experiment]

< Experimental Example  3-1: Using 'Resorption kit' Koshi Petun  Bone resorption Inhibition  Check>

In this experiment, a 'resorption kit' was used to confirm osteoclast bone resorption.

The oocyte cells treated with calcium and phosphorus-coated plates were treated with 1, 10, and 20 μM of goshiheptin, respectively, for 5 days, and the degree of calcium and phosphorus degradation was checked. nm fluorescence spectrometry to confirm 'resorption activity'.

< Experimental Example  3-2: Goshi Petin  by MMP -9 RT- PCR >

In this experimental example, RT-PCR of MCP-9 with goshi fetin was measured.

In order to differentiate into osteoclasts, RANKL at a concentration of 50 ng / ml was treated and treated with gossypetin at a concentration of 1, 10 and 20 μM, respectively. The osteoclasts were treated with trypsin for 5 days. After the mRNA was isolated using trisole, cDNA was synthesized using 5 μg RNA, 200 unit reverse transcriptase, and 0.5 mg / ml Oligo- (dT).

Then, 25 μl of a reaction master mix containing 5 × PCR buffer, 25 mM MgCl ₂ , 10 mM dNTP, 10 pmol primer and 5 units of Taq DNA polymerase and cDNA were used to amplify the cDNA using ' RT-PCR was performed on My Cycler ™ Thermal Cycler from Bio-Rad Laboratories (Berkeley, Calif.). The PCR conditions are shown in Table 3 below.

forward reverse size MMP-9  5'-AGC CGA CTT TTG TGG TCT TCC-3 ' 5'-TCG TCG TCG AAA TGG GCA TC-3 ' 547 bp GAPDH 5-AAC TTT GGC ATT GTG GAA GGG-3 5'-GAC ACA TTG GGG GTA GGA ACA C-3 ' 224 bp

< Experimental Example  3-3: Zymography  Used Koshi Petun MMP -9 secretion Inhibition  Check>

In this experiment, MMP-9 secretion that breaks down bone collagen was examined for bone resorption.

The osteoclasts differentiated for 4 days were supplemented with media without FBS and treated with 1, 10, and 20 μM gossypetin for 1 day, respectively. Then, the culture medium was collected and gel containing 0.1% gelatin was electrophoresed. The gel was incubated with 2.5% Triton X-100 for 1 hour at 37 ° C in a shaking incubator, and the cells were suspended in 50 mM Tris-HCl buffer pH 7.5).

After washing, the cells were incubated in 50 mM Tris-HCl buffer (pH 7.5), 10 mM CaCl ₂ , 0.05% Brij-35, 200 mM NaCl buffer for 16 hours. After that, the cells were stained with a buffer containing 0.1% Coomassie brilliant blue R-250, 10% acetic acid and 45% methanol, and then stained with 0.7% acetic acid, 40% After destaining with methanol, it was confirmed.

< Experimental Example  3-4: Intra-osteoclast cathepsin ( Cathepsin ) K's RT- PCR >

In this experiment, RT-PCR was performed in order to measure cathepsin K degrading substrate protein such as type 1 collagen, which is a protease that is highly expressed in osteoclasts.

 The medium was changed every two days for 5 days, and gossypetin was treated at 1, 10, and 20 μM concentration once. RANKL was treated with 50 ng / ml of medium. The osteoclast-treated osteoclasts were separated by mRNA and then cDNA was synthesized using 5 μg RNA, 200 unit reverse transcriptase, and 0.5 mg / ml Oligo- (dT).

Then, 25 μl of a reaction master mix containing 5 × PCR buffer, 25 mM MgCl ₂ , 10 mM dNTP, 10 pmol of a primer, 5 units of Taq DNA polymerase and cDNA RT-PCR was performed using a My Cycler ™ Thermal Cycler from Bio-Rad Laboratories (Berkeley, Calif.). The PCR conditions are shown in Table 4 below.

forward reverse size Cathepsin K  5'-AGG CGG CTA TAT GAC CAC TG-3 ' 5'-CCG AGC CAA GAG AGC ATA TC-3 407 bp GAPDH 5'-AAC TTT GGC ATT GTG GAA GGG-3 5'-GAC ACA TTG GGG GTA GGA ACA C-3 '' 224 bp

< Experimental Example  3-5: Experimental results>

As a result of the experiment, the fluorescence analyzer confirmed that the degree of bone resorption was decreased depending on the concentration of goethethene. In addition, the bone resorption-related enzymes MMP-9 and Cathepsin K were also inhibited in dependence on goshi fetin concentration (Fig. 3). FIG. 3A shows fluorescence analysis results of goshi-petun-treated osteoclasts. Figure 3B shows the RT-PCR result of MMP-9. FIG. 3c is a MMP-9 secretion inhibition result (western blot) of gossyphetin using yymography. Figure 3D is the result of RT-PCR of the channeled channel cathepsin K (Cathepsin K).

From these results, it can be confirmed that goshihepatene inhibits bone resorption and is effective in improving and treating osteoporosis.

[ Example  1: manufacture of food composition for osteoporosis improvement]

In this Example, a food composition for osteoporosis improvement was prepared as follows.

(1) Manufacturing of wire

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then the mixture was prepared into powder having a particle size of 60 mesh by a pulverizer. Black beans, black sesame seeds and perilla seeds were each steamed and dried by known methods, and then power distribution and pulverization were carried out to prepare powder having a particle size of 60 mesh. Thereafter, 30% by weight of brown rice, 15% by weight of yulmu, 20% by weight of barley, 9% by weight of glutinous rice, 7% by weight of perilla seeds, 8% by weight of black beans, 7% by weight of black sesame seeds, 0.5 wt% were mixed to prepare an electric wire.

(2) Production of chewing gum

Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 76.9% by weight of sugar, 1% by weight of a flavor, 2% by weight of water and 0.1%

(3) Candy manufacturing

Candies were prepared in a conventional manner by mixing 60% by weight of sugar, 39.8% by weight of starch syrup, 0.1% by weight of fragrance and 0.1% by weight of gossyphetine.

(4) Manufacturing of biscuits

By weight of starch, 0.77% by weight of glucose, 0.78% by weight of glucose, 11.78% by weight of palm shortening, 1.54% by weight of ammonia, 0.17% by weight of sodium bicarbonate, 0.16% by weight of sodium bisulfite 1.45 wt% of rice flour, 0.0001 wt% of vitamin B, 0.0001 wt% of vitamin B, 0.04 wt% of milk fractions, 20.6998 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% 0.29% by weight of spray salt, and 7.27% by weight of spray oil and 1% by weight of goshi petin were blended to prepare a biscuit by a conventional method.

(5) health drink manufacturing

0.0001 wt.% Of niacinamide, 0.0001 wt.% Of sodium riboflavin, 0.0001 wt.% Of pyridoxine hydrochloride, 0.001 wt.% Of inositol, 0.002 wt.% Of orthoacetic acid, 98.7362 wt.% Of water, 1% by weight was added to prepare a health drink.

(6) Production of sausages

The sausage was mixed with 65.18 wt% of pork, 25 wt% of chicken meat, 3.5 wt% of starch, 1.7 wt% of soybean protein, 1.62 wt% of salt, 0.5 wt% of glucose, 1.5 wt% of glycerin and 1 wt% .

(7) Health supplement manufacturing

A mixture of 55% by weight of spirulina, 10% by weight of guar gum enzyme hydrolyzate, 0.01% by weight of vitamin B hydrochloride, 0.01% by weight of vitamin B6 hydrochloride, 0.23% by weight of DL-methionine, 0.7% by weight of magnesium stearate, And 10% by weight of glycoprotein were mixed to prepare a refillable health supplement food by a conventional method.

[ Example  2: Osteoporosis For prevention or treatment  Preparation of pharmaceutical composition]

In this Example, a pharmaceutical composition for preventing or treating osteoporosis was prepared as follows.

(1) Manufacture of powders

2 g of lactose was mixed with 1 g of gossyphein, and filled in an airtight container to prepare a powder.

(2) Preparation of tablets

100 mg of corn starch, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and tableted according to a conventional tablet preparation method.

(3) Manufacture of capsules

100 mg of corn starch, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed and filled in gelatin capsules to prepare capsules.

(4) Injection preparation

100 mg of goshiheptin was dissolved in an appropriate amount of distilled water for injection, the pH was adjusted to about 7.5, and the solution was filled and sterilized in a 2 ml volume ampoule to prepare an injection.

Claims (4)

A composition for inhibiting bone resorption of osteoclasts, which comprises gossypetin at a concentration of 20 μM as an active ingredient and inhibits osteoclast bone resorption.
A gossypetin concentration of 20 [mu] M was added,
Wherein the gossypetin at a concentration of 20 [mu] M inhibits bone resorption of osteoclasts. delete delete

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